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Sample records for arabidopsis adp-ribosylation factor

  1. Structure of Plasmodium falciparum ADP-ribosylation factor 1

    SciTech Connect

    Cook, William J.; Smith, Craig D.; Senkovich, Olga; Holder, Anthony A.; Chattopadhyay, Debasish

    2011-09-26

    Vesicular trafficking may play a crucial role in the pathogenesis and survival of the malaria parasite. ADP-ribosylation factors (ARFs) are among the major components of vesicular trafficking pathways in eukaryotes. The crystal structure of ARF1 GTPase from Plasmodium falciparum has been determined in the GDP-bound conformation at 2.5 {angstrom} resolution and is compared with the structures of mammalian ARF1s.

  2. An Entamoeba histolytica ADP-ribosyl transferase from the diphtheria toxin family modifies the bacterial elongation factor Tu.

    PubMed

    Avila, Eva E; Rodriguez, Orlando I; Marquez, Jaqueline A; Berghuis, Albert M

    2016-06-01

    ADP-ribosyl transferases are enzymes involved in the post-translational modification of proteins; they participate in multiple physiological processes, pathogenesis and host-pathogen interactions. Several reports have characterized the functions of these enzymes in viruses, prokaryotes and higher eukaryotes, but few studies have reported ADP-ribosyl transferases in lower eukaryotes, such as parasites. The locus EHI_155600 from Entamoeba histolytica encodes a hypothetical protein that possesses a domain from the ADP-ribosylation superfamily; this protein belongs to the diphtheria toxin family according to a homology model using poly-ADP-ribosyl polymerase 12 (PARP12 or ARTD12) as a template. The recombinant protein expressed in Escherichia coli exhibited in vitro ADP-ribosylation activity that was dependent on the time and temperature. Unlabeled βNAD(+), but not ADP-ribose, competed in the enzymatic reaction using biotin-βNAD(+) as the ADP-ribose donor. The recombinant enzyme, denominated EhToxin-like, auto-ADP-ribosylated and modified an acceptor from E. coli that was identified by MS/MS as the elongation factor Tu (EF-Tu). To the best of our knowledge, this is the first report to identify an ADP-ribosyl transferase from the diphtheria toxin family in a protozoan parasite. The known toxins from this family (i.e., the diphtheria toxin, the Pseudomonas aeruginosa toxin Exo-A, and Cholix from Vibrio cholerae) modify eukaryotic elongation factor two (eEF-2), whereas the amoeba EhToxin-like modified EF-Tu, which is another elongation factor involved in protein synthesis in bacteria and mitochondria. PMID:27234208

  3. Protein Poly(ADP-ribosyl)ation Regulates Arabidopsis Immune Gene Expression and Defense Responses

    PubMed Central

    Feng, Baomin; Liu, Chenglong; de Oliveira, Marcos V. V.; Intorne, Aline C.; Li, Bo; Babilonia, Kevin; de Souza Filho, Gonçalo A.; Shan, Libo; He, Ping

    2015-01-01

    Perception of microbe-associated molecular patterns (MAMPs) elicits transcriptional reprogramming in hosts and activates defense to pathogen attacks. The molecular mechanisms underlying plant pattern-triggered immunity remain elusive. A genetic screen identified Arabidopsis poly(ADP-ribose) glycohydrolase 1 (atparg1) mutant with elevated immune gene expression upon multiple MAMP and pathogen treatments. Poly(ADP-ribose) glycohydrolase (PARG) is predicted to remove poly(ADP-ribose) polymers on acceptor proteins modified by poly(ADP-ribose) polymerases (PARPs) with three PARPs and two PARGs in Arabidopsis genome. AtPARP1 and AtPARP2 possess poly(ADP-ribose) polymerase activity, and the activity of AtPARP2 was enhanced by MAMP treatment. AtPARG1, but not AtPARG2, carries glycohydrolase activity in vivo and in vitro. Importantly, mutation (G450R) in atparg1 blocks its activity and the corresponding residue is highly conserved and essential for human HsPARG activity. Consistently, mutant atparp1atparp2 plants exhibited compromised immune gene activation and enhanced susceptibility to pathogen infections. Our study indicates that protein poly(ADP-ribosyl)ation plays critical roles in plant immune gene expression and defense to pathogen attacks. PMID:25569773

  4. Endogenous ADP-ribosylation of elongation factor 2 in polyoma virus-transformed baby hamster kidney cells

    SciTech Connect

    Fendrick, J.L.; Iglewski, W.J. )

    1989-01-01

    Polyoma virus-transformed baby hamster kidney (pyBHK) cells were cultured in medium containing ({sup 32}P)orthophosphate and 105 (vol/vol) fetal bovine serum. A {sup 32}P-labeled protein with an apparent molecular mass of 97 kDa was immunoprecipitated from cell lysates with antiserum to ADP-ribosylated elongation factor 2 (EF-2). The {sup 32}P labeling of the protein was enhanced by culturing cells in medium containing 2% serum instead of 10% serum. The {sup 32}P label was completely removed from the protein by treatment with snake venom phosphodiesterase and the digestion product was identified as ({sup 32}P)AMP, indicating the protein was mono-ADP-ribosylated. HPLC analysis of tryptic peptides of the {sup 32}P-labeled 97-kDa protein and purified EF-2, which was ADP-ribosylated in vitro with diphtheria toxin fragment A and ({sup 32}P)NAD, demonstrated an identical labeled peptide in the two proteins. The data strongly suggest that EF-2 was endogenously ADP-ribosylated in pyBHK cells. Maximum incorporation of radioactivity in EF-2 occurred by 12 hr and remained constant over the subsequent 12 hr. It was estimated that 30-35% of the EF-2 was ADP-ribosylated in cells cultured in medium containing 2% serum. When {sup 32}P-labeled cultures were incubated in medium containing unlabeled phosphate, the {sup 32}P label was lost from the EF-2 within 30 min.

  5. ADP-ribosylation factor-like protein 4C (ARL4C) interacts with galectin-3 during oocyte development and embryogenesis in rainbow trout (Oncorhynchus mykiss)

    Technology Transfer Automated Retrieval System (TEKTRAN)

    ADP-ribosylation factor-like protein 4 (ARL4) is a GTP-binding protein which belongs to the ADP-ribosylation factor protein (ARF) superfamily of small GTPases. ARL4 has been shown to be mainly related to the development of male germ cells and embryogenesis in mouse. To investigate the role of ARL4 i...

  6. TFIIF, a basal eukaryotic transcription factor, is a substrate for poly(ADP-ribosyl)ation.

    PubMed Central

    Rawling, J M; Alvarez-Gonzalez, R

    1997-01-01

    We have examined the susceptibility of some of the basal eukaryotic transcription factors as covalent targets for poly(ADP-ribosyl)ation. Human recombinant TATA-binding protein, transcription factor (TF)IIB and TFIIF (made up of the 30 and 74 kDa RNA polymerase II-associated proteins RAP30 and RAP74) were incubated with calf thymus poly(ADP-ribose) polymerase and [32P]NAD+ at 37 degrees C. On lithium dodecyl sulphate/PAGE and autoradiography, two bands of radioactivity, coincident with RAP30 and RAP74, were observed. No radioactivity co-migrated with TATA-binding protein or TFIIB. The phenomenon was dependent on the presence of nicked DNA, which is essential for poly(ADP-ribose) polymerase activity. Covalent modification of TFIIF increased with time of incubation, with increasing TFIIF concentration and with increasing NAD+ concentration. High-resolution PAGE confirmed that the radioactive species associated with RAP30 and RAP74 were ADP-ribose polymers. From these observations, we conclude that both TFIIF subunits are highly specific substrates for covalent poly(ADP-ribosyl)ation. PMID:9164864

  7. Localization and characterization of the human ADP-ribosylation factor 5 (ARF5) gene

    SciTech Connect

    McGuire, R.E. |; Daiger, S.P.; Green, E.D.

    1997-05-01

    ADP-ribosylation factor 5 (ARF5) is a member of the ARF gene family. The ARF proteins stimulate the in vitro ADP-ribosyltransferase activity of cholera toxin and appear to play a role in vesicular trafficking in vivo. We have mapped ARF5, one of the six known mammalian ARF genes, to a well-defined yeast artificial chromosome contig on human chromosome 7q31.3. In addition, we have isolated and sequenced an {approximately}3.2-kb genomic segment that contains the entire ARF5 coding region, revealing the complete intron-exon structure of the gene. With six coding exons and five introns, the genomic structure of ARF5 is unique among the mammalian ARF genes and provides insight about the evolutionary history of this ancient gene family. 20 refs., 2 figs., 1 tab.

  8. MARTX effector cross kingdom activation by Golgi-associated ADP-ribosylation factors.

    PubMed

    Kim, Byoung Sik; Satchell, Karla J F

    2016-08-01

    Vibrio vulnificus infects humans and causes lethal septicemia. The primary virulence factor is a multifunctional-autoprocessing repeats-in-toxin (MARTX) toxin consisting of conserved repeats-containing regions and various effector domains. Recent genomic analyses for the newly emerged V. vulnificus biotype 3 strain revealed that its MARTX toxin has two previously unknown effector domains. Herein, we characterized one of these domains, Domain X (DmXVv ). A structure-based homology search revealed that DmXVv belongs to the C58B cysteine peptidase subfamily. When ectopically expressed in cells, DmXVv was autoprocessed and induced cytopathicity including Golgi dispersion. When the catalytic cysteine or the region flanking the scissile bond was mutated, both autoprocessing and cytopathicity were significantly reduced indicating that DmXVv cytopathicity is activated by amino-terminal autoprocessing. Consistent with this, host cell protein export was affected by Vibrio cells producing a toxin with wild-type, but not catalytically inactive, DmXVv . DmXVv was found to localize to Golgi and to directly interact with Golgi-associated ADP-ribosylation factors ARF1, ARF3 and ARF4, although ARF binding was not necessary for the subcellular localization. Rather, this interaction was found to induce autoprocessing of DmXVv . These data demonstrate that the V. vulnificus hijacks the host ARF proteins to activate the cytopathic DmXVv effector domain of MARTX toxin. PMID:26780191

  9. Modes of Action of ADP-Ribosylated Elongation Factor 2 in Inhibiting the Polypeptide Elongation Cycle: A Modeling Study

    PubMed Central

    Chen, Kevin C.; Xie, Honglin; Cai, Yujie

    2013-01-01

    Despite the fact that ADP-ribosylation of eukaryotic elongation factor 2 (EF2) leads to inhibition of protein synthesis, the mechanism by which ADP-ribosylated EF2 (ADPR•EF2) causes this inhibition remains controversial. Here, we applied modeling approaches to investigate the consequences of various modes of ADPR•EF2 inhibitory actions on the two coupled processes, the polypeptide chain elongation and ADP-ribosylation of EF2. Modeling of experimental data indicates that ADPR•EF2 fully blocks the late-phase translocation of tRNAs; but the impairment in the translocation upstream process, mainly the GTP-dependent factor binding with the pretranslocation ribosome and/or the guanine nucleotide exchange in EF2, is responsible for the overall inhibition kinetics. The reduced ADPR•EF2-ribosome association spares the ribosome to bind and shield native EF2 against toxin attack, thereby deferring the inhibition of protein synthesis inhibition and inactivation of EF2. Minimum association with the ribosome also keeps ADPR•EF2 in an accessible state for toxins to catalyze the reverse reaction when nicotinamide becomes available. Our work underscores the importance of unveiling the interactions between ADPR•EF2 and the ribosome, and argues against that toxins inhibit protein synthesis through converting native EF2 to a competitive inhibitor to actively disable the ribosome. PMID:23861744

  10. Poliovirus Proteins Induce Membrane Association of GTPase ADP-Ribosylation Factor

    PubMed Central

    Belov, George A.; Fogg, Mark H.; Ehrenfeld, Ellie

    2005-01-01

    Poliovirus infection results in the disintegration of intracellular membrane structures and formation of specific vesicles that serve as sites for replication of viral RNA. The mechanism of membrane rearrangement has not been clearly defined. Replication of poliovirus is sensitive to brefeldin A (BFA), a fungal metabolite known to prevent normal function of the ADP-ribosylation factor (ARF) family of small GTPases. During normal membrane trafficking in uninfected cells, ARFs are involved in vesicle formation from different intracellular sites through interaction with numerous regulatory and coat proteins as well as in regulation of phospholipase D activity and cytoskeleton modifications. We demonstrate here that ARFs 3 and 5, but not ARF6, are translocated to membranes in HeLa cell extracts that are engaged in translation of poliovirus RNA. The accumulation of ARFs on membranes correlates with active replication of poliovirus RNA in vitro, whereas ARF translocation to membranes does not occur in the presence of BFA. ARF translocation can be induced independently by synthesis of poliovirus 3A or 3CD proteins, and we describe mutations that abolished this activity. In infected HeLa cells, an ARF1-enhanced green fluorescent protein fusion redistributes from Golgi stacks to the perinuclear region, where poliovirus RNA replication occurs. Taken together, the data suggest an involvement of ARF in poliovirus RNA replication. PMID:15890959

  11. ADP ribosylation factor like 2 (Arl2) protein influences microtubule dynamics in breast cancer cells

    SciTech Connect

    Beghin, Anne . E-mail: anne.beghin@recherche.univ-lyon1.fr; Honore, Stephane; Messana, Celine; Matera, Eva-Laure; Aim, Jennifer; Burlinchon, Sandrine; Braguer, Diane; Dumontet, Charles

    2007-02-01

    ADP ribosylation factor like 2 (Arl2) protein is involved in the folding of tubulin peptides. Variants of the human adenocarcinoma line MCF7 cells with increased or reduced content of Arl2 protein were produced and characterized. Western blot analysis performed after separation of the different fractions of tubulins showed that the content in polymerizable soluble heterodimers was significantly increased in cells with the highest Arl2 expression level (MA+) and reduced in cells with the lowest Arl2 expression level (MA-) in comparison to control cells (MP). Microtubule dynamic instability, measured after microinjection of rhodamine-labelled tubulin in living cells, was significantly enhanced in MA+ cells and reduced in MA- cells. These alterations involved modifications of the microtubule growth and shortening rates, duration of attenuation phases, percentage of time spent in each phase (growth, shortening and attenuation) and catastrophe frequency. We also observed modifications in the expression level of the tumor suppressor protein phosphatase 2Ac, which has been shown to form a complex with Arl2. Finally, cell cycle progression was modified in these cells, particularly in regard to duration of telophase. In summary, alterations in Arl2 protein content were found to be associated with modifications in tubulin pools, microtubule dynamics as well as cell cycle progression.

  12. Small G proteins in peroxisome biogenesis: the potential involvement of ADP-ribosylation factor 6

    PubMed Central

    2009-01-01

    Background Peroxisomes execute diverse and vital functions in virtually every eukaryote. New peroxisomes form by budding from pre-existing organelles or de novo by vesiculation of the ER. It has been suggested that ADP-ribosylation factors and COPI coatomer complexes are involved in these processes. Results Here we show that all viable Saccharomyces cerevisiae strains deficient in one of the small GTPases which have an important role in the regulation of vesicular transport contain functional peroxisomes, and that the number of these organelles in oleate-grown cells is significantly upregulated in the arf1 and arf3 null strains compared to the wild-type strain. In addition, we provide evidence that a portion of endogenous Arf6, the mammalian orthologue of yeast Arf3, is associated with the cytoplasmic face of rat liver peroxisomes. Despite this, ablation of Arf6 did neither influence the regulation of peroxisome abundance nor affect the localization of peroxisomal proteins in cultured fetal hepatocytes. However, co-overexpression of wild-type, GTP hydrolysis-defective or (dominant-negative) GTP binding-defective forms of Arf1 and Arf6 caused mislocalization of newly-synthesized peroxisomal proteins and resulted in an alteration of peroxisome morphology. Conclusion These observations suggest that Arf6 is a key player in mammalian peroxisome biogenesis. In addition, they also lend strong support to and extend the concept that specific Arf isoform pairs may act in tandem to regulate exclusive trafficking pathways. PMID:19686593

  13. NO-Mediated [Ca2+]cyt Increases Depend on ADP-Ribosyl Cyclase Activity in Arabidopsis1[OPEN

    PubMed Central

    Hotta, Carlos T.; Davey, Matthew P.; Dodd, Antony N.

    2016-01-01

    Cyclic ADP ribose (cADPR) is a Ca2+-mobilizing intracellular second messenger synthesized from NAD by ADP-ribosyl cyclases (ADPR cyclases). In animals, cADPR targets the ryanodine receptor present in the sarcoplasmic/endoplasmic reticulum to promote Ca2+ release from intracellular stores to increase the concentration of cytosolic free Ca2+ in Arabidopsis (Arabidopsis thaliana), and cADPR has been proposed to play a central role in signal transduction pathways evoked by the drought and stress hormone, abscisic acid, and the circadian clock. Despite evidence for the action of cADPR in Arabidopsis, no predicted proteins with significant similarity to the known ADPR cyclases have been reported in any plant genome database, suggesting either that there is a unique route for cADPR synthesis or that a homolog of ADPR cyclase with low similarity might exist in plants. We sought to determine whether the low levels of ADPR cyclase activity reported in Arabidopsis are indicative of a bona fide activity that can be associated with the regulation of Ca2+ signaling. We adapted two different fluorescence-based assays to measure ADPR cyclase activity in Arabidopsis and found that this activity has the characteristics of a nucleotide cyclase that is activated by nitric oxide to increase cADPR and mobilize Ca2+. PMID:26932235

  14. Differential interaction of ADP-ribosylation factors 1, 3, and 5 with rat brain Golgi membranes.

    PubMed Central

    Tsai, S C; Adamik, R; Haun, R S; Moss, J; Vaughan, M

    1992-01-01

    Six mammalian ADP-ribosylation factors (ARFs) identified by cDNA cloning were expressed as recombinant proteins (rARFs) that stimulated cholera toxin ADP-ribosyltransferase activity. Microsequencing of soluble ARFs I and II (sARFs I and II), purified from bovine brain, established that they are ARFs 1 and 3, respectively. Rabbit antibodies (IgG) against sARF II reacted similarly with ARFs 1, 2, and 3 (class I) on Western blots. ARFs 1 and 3 were distinguished by their electrophoretic mobilities. Antiserum against rARF 5 cross-reacted partially with rARF 4 but not detectably with rARF 6 and minimally with class I ARFs. Guanosine 5'-O-(3-thiotriphosphate) (GTP[gamma S]) increased recovery of ARF activity and immunoreactivity in organelle fractions separated by density gradient centrifugation, after incubation of rat brain homogenate with ATP and a regenerating system. ARF 1 accumulated in microsomes plus Golgi and Golgi fractions, whereas ARF 5 seemed to localize more specifically in Golgi; the smaller increment in ARF 3 was distributed more evenly among fractions. On incubation of Golgi with a crude ARF fraction, GTP[gamma S], and an ATP-regenerating system, association of ARF activity with Golgi increased with increasing ATP concentration paralleled by increases in immunoreactive ARFs 1 and 5 and, to a lesser degree, ARF 3. Golgi incubated with GTP[gamma S] and purified ARF 1 or 3 bound more ARF 1 than ARF 3. Based on immunoreactivity and assay of ARF activity, individual ARFs 1, 3, and 5 appeared to behave independently and selectively in their GTP-dependent association with Golgi in vitro. Images PMID:1409634

  15. Differential interaction of ADP-ribosylation factors 1, 3, and 5 with rat brain Golgi membranes.

    PubMed

    Tsai, S C; Adamik, R; Haun, R S; Moss, J; Vaughan, M

    1992-10-01

    Six mammalian ADP-ribosylation factors (ARFs) identified by cDNA cloning were expressed as recombinant proteins (rARFs) that stimulated cholera toxin ADP-ribosyltransferase activity. Microsequencing of soluble ARFs I and II (sARFs I and II), purified from bovine brain, established that they are ARFs 1 and 3, respectively. Rabbit antibodies (IgG) against sARF II reacted similarly with ARFs 1, 2, and 3 (class I) on Western blots. ARFs 1 and 3 were distinguished by their electrophoretic mobilities. Antiserum against rARF 5 cross-reacted partially with rARF 4 but not detectably with rARF 6 and minimally with class I ARFs. Guanosine 5'-O-(3-thiotriphosphate) (GTP[gamma S]) increased recovery of ARF activity and immunoreactivity in organelle fractions separated by density gradient centrifugation, after incubation of rat brain homogenate with ATP and a regenerating system. ARF 1 accumulated in microsomes plus Golgi and Golgi fractions, whereas ARF 5 seemed to localize more specifically in Golgi; the smaller increment in ARF 3 was distributed more evenly among fractions. On incubation of Golgi with a crude ARF fraction, GTP[gamma S], and an ATP-regenerating system, association of ARF activity with Golgi increased with increasing ATP concentration paralleled by increases in immunoreactive ARFs 1 and 5 and, to a lesser degree, ARF 3. Golgi incubated with GTP[gamma S] and purified ARF 1 or 3 bound more ARF 1 than ARF 3. Based on immunoreactivity and assay of ARF activity, individual ARFs 1, 3, and 5 appeared to behave independently and selectively in their GTP-dependent association with Golgi in vitro. PMID:1409634

  16. Effects of Site-Directed Mutagenesis of Escherichia coli Heat-Labile Enterotoxin on ADP-Ribosyltransferase Activity and Interaction with ADP-Ribosylation Factors

    PubMed Central

    A. Stevens, Linda; Moss, Joel; Vaughan, Martha; Pizza, Mariagrazia; Rappuoli, Rino

    1999-01-01

    Escherichia coli heat-labile enterotoxin (LT), an oligomeric protein with one A subunit (LTA) and five B subunits, exerts its effects via the ADP-ribosylation of Gsα, a guanine nucleotide-binding (G) protein that activates adenylyl cyclase. LTA also ADP-ribosylates simple guanidino compounds (e.g., arginine) and catalyzes its own auto-ADP-ribosylation. All LTA-catalyzed reactions are enhanced by ADP-ribosylation factors (ARFs), 20-kDa guanine nucleotide-binding proteins. Replacement of arginine-7 (R7K), valine-53 (V53D), serine-63 (S63K), valine 97 (V97K), or tyrosine-104 (Y104K) in LTA resulted in fully assembled but nontoxic proteins. S63K, V53D, and R7K are catalytic-site mutations, whereas V97K and Y104K are amino acid replacements adjacent to and outside of the catalytic site, respectively. The effects of mutagenesis were quantified by measuring ADP-ribosyltransferase activity (i.e., auto-ADP-ribosylation and ADP-ribosylagmatine synthesis) and interaction with ARF (i.e., inhibition of ARF-stimulated cholera toxin ADP-ribosyltransferase activity and effects of ARF on mutant auto-ADP-ribosylation). All mutants were inactive in the ADP-ribosyltransferase assay; however, auto-ADP-ribosylation in the presence of recombinant human ARF6 was detected, albeit much less than that of native LT (Y104K > V53D > V97K > R7K, S63K). Based on the lack of inhibition by free ADP-ribose, the observed auto-ADP-ribosylation activity was enzymatic and not due to the nonenzymatic addition of free ADP-ribose. V53D, S63K, and R7K were more effective than Y104K or V97K in blocking ARF stimulation of cholera toxin ADP-ribosyltransferase. Based on these data, it appears that ARF-binding and catalytic sites are not identical and that a region outside the NAD cleft may participate in the LTA-ARF interaction. PMID:9864224

  17. Effects of site-directed mutagenesis of Escherichia coli heat-labile enterotoxin on ADP-ribosyltransferase activity and interaction with ADP-ribosylation factors.

    PubMed

    Stevens, L A; Moss, J; Vaughan, M; Pizza, M; Rappuoli, R

    1999-01-01

    Escherichia coli heat-labile enterotoxin (LT), an oligomeric protein with one A subunit (LTA) and five B subunits, exerts its effects via the ADP-ribosylation of Gsalpha, a guanine nucleotide-binding (G) protein that activates adenylyl cyclase. LTA also ADP-ribosylates simple guanidino compounds (e.g., arginine) and catalyzes its own auto-ADP-ribosylation. All LTA-catalyzed reactions are enhanced by ADP-ribosylation factors (ARFs), 20-kDa guanine nucleotide-binding proteins. Replacement of arginine-7 (R7K), valine-53 (V53D), serine-63 (S63K), valine 97 (V97K), or tyrosine-104 (Y104K) in LTA resulted in fully assembled but nontoxic proteins. S63K, V53D, and R7K are catalytic-site mutations, whereas V97K and Y104K are amino acid replacements adjacent to and outside of the catalytic site, respectively. The effects of mutagenesis were quantified by measuring ADP-ribosyltransferase activity (i.e., auto-ADP-ribosylation and ADP-ribosylagmatine synthesis) and interaction with ARF (i.e., inhibition of ARF-stimulated cholera toxin ADP-ribosyltransferase activity and effects of ARF on mutant auto-ADP-ribosylation). All mutants were inactive in the ADP-ribosyltransferase assay; however, auto-ADP-ribosylation in the presence of recombinant human ARF6 was detected, albeit much less than that of native LT (Y104K > V53D > V97K > R7K, S63K). Based on the lack of inhibition by free ADP-ribose, the observed auto-ADP-ribosylation activity was enzymatic and not due to the nonenzymatic addition of free ADP-ribose. V53D, S63K, and R7K were more effective than Y104K or V97K in blocking ARF stimulation of cholera toxin ADP-ribosyltransferase. Based on these data, it appears that ARF-binding and catalytic sites are not identical and that a region outside the NAD cleft may participate in the LTA-ARF interaction. PMID:9864224

  18. Molecular cloning and characterization of an ADP-ribosylation factor 6 gene (ptARF6) from Pisolithus tinctorius.

    PubMed

    Wang, Liling; Li, Haibo; Zhou, Yifeng; Qin, Yuchuan; Wang, Yanbin; Liu, Bentong; Qian, Hua

    2016-05-01

    ADP-ribosylation factor 6 (ARF6) is an evolutionarily conserved molecule that has an essential function in intracellular trafficking and organelle structure. To better understand its role during presymbiosis between plant roots and compatible filamentous fungi, the full-length cDNA sequence of ARF6 from Pisolithus tinctorius was cloned and a variety of bioinformatics analyses performed. The full-length sequence was 849 bp long and contained a 549 bp open reading frame encoding a protein of 182 amino acids. A phylogenetic analysis showed that ptARF6 was the ortholog of the ADP ribosylation factor 6/GTPase SAR1 gene from the white-rot basidiomycete Trametes versicolor. A domain architecture analysis of the ARF6 protein revealed a repeat region, which is a common feature of ARF6 in other species. Recombinant ARF6 protein was expressed with an N-terminal 6×His tag and purified using Ni(2+)-NTA affinity chromatography. The molecular mass of the recombinant protein was estimated by SDS-PAGE to be 25 kDa. The recombinant ARF6 protein bound strongly to 18:1 and 18:2 phosphatidic acids. Thus, ARF6 may participate in the signaling pathways involved in membrane phospholipid composition. The intracellular distribution of ptADP6 in HEK239T cells also indicates that ptADP6 may function not only in plasma membrane events but also in endosomal membranes events. Real-time quantitative PCR revealed that the differential expression of ptARF6 was associated with the presymbiotic stage. ptARF6 may be induced by presymbiosis during the regulation of mycorrhizal formation. PMID:26928195

  19. A presynaptic role for the ADP ribosylation factor (ARF)-specific GDP/GTP exchange factor msec7-1

    PubMed Central

    Ashery, Uri; Koch, Henriette; Scheuss, Volker; Brose, Nils; Rettig, Jens

    1999-01-01

    ADP ribosylation factors (ARFs) represent a family of small monomeric G proteins that switch from an inactive, GDP-bound state to an active, GTP-bound state. One member of this family, ARF6, translocates on activation from intracellular compartments to the plasma membrane and has been implicated in regulated exocytosis in neuroendocrine cells. Because GDP release in vivo is rather slow, ARF activation is facilitated by specific guanine nucleotide exchange factors like cytohesin-1 or ARNO. Here we show that msec7-1, a rat homologue of cytohesin-1, translocates ARF6 to the plasma membrane in living cells. Overexpression of msec7-1 leads to an increase in basal synaptic transmission at the Xenopus neuromuscular junction. msec7-1-containing synapses have a 5-fold higher frequency of spontaneous synaptic currents than control synapses. On stimulation, the amplitudes of the resulting evoked postsynaptic currents of msec7-1-overexpressing neurons are increased as well. However, further stimulation leads to a decline in amplitudes approaching the values of control synapses. This transient effect on amplitude is strongly reduced on overexpression of msec7-1E157K, a mutant incapable of translocating ARFs. Our results provide evidence that small G proteins of the ARF family and activating factors like msec7-1 play an important role in synaptic transmission, most likely by making more vesicles available for fusion at the plasma membrane. PMID:9927699

  20. Site of ADP-ribosylation and the RNA-binding site are situated in different domains of the elongation factor EF-2

    SciTech Connect

    Davydova, E.K.

    1987-01-01

    One of the proteins participating in the process of elongation of polypeptide chains - elongation factor 2 (EF-2) - can be ADP-ribosylated at a unique amino acid residue - diphthamide. Since the ADP-ribosylation of EF-2 at dipthamide leads to a loss of affinity of the factor for RNA while the presence of RNA inhibits the ADP-ribosylation reaction, it seemed probable to the authors that diphthamide participated directly in the binding of EF-2 to DNA. The experiments presented in this article showed that this was not the case: diphthamide and the RNA-binding site are situated on different domains of EF-2. Thus, ADP-ribosylation of factor EF-2 in one domain leads to a loss of the ability to bind to RNA in the other. The authors investigated the mutual arrangement of diphthamide and the RNA-binding site on the EF-2 molecule by preparing a factor from rabbit reticulocytes and subjecting it to proteolytic digestion with elastase. The factor was incubated with elastase for 15 min at 37/sup 0/C at an enzyme:substrate ratio of 1:100 in buffer solution containing 20 mM Tris-HCl, pH 7.6, 10 mM KCl, 1 mM MgCl/sub 2/, and 2 mM dithiothreitol. The reaction was stopped by adding para-methylsulfonyl fluoride to 50 micro-M. The authors obtained a preparation as a result of proteolysis and applied it on a column with RNA-Sepharose and separated into two fractions: RNA-binding and without affinity for RNA. The initial preparation and its fractions were subjected to exhaustive ADP-ribosylation in the presence of diphtheria toxin and (U-/sup 14/C) nicotinaide adenine dinucleotide ((/sup 14/C)NAD) (296 mCi/mmole). The samples were analyzed electrophoretically in a polyacrylamide gel gradient in the presence of sodium dodecyl sulfate. For the detection of (/sup 14/C) ADP-ribosylated components, the gels were dried and exposed with RM-V x-ray film.

  1. ADP-ribosylation Factor-related Protein 1 Interacts with NS5A and Regulates Hepatitis C Virus Propagation.

    PubMed

    Lim, Yun-Sook; Ngo, Huong T T; Lee, Jihye; Son, Kidong; Park, Eun-Mee; Hwang, Soon B

    2016-01-01

    The life cycle of hepatitis C virus (HCV) is tightly coupled to the lipid metabolism of host cells. In order to identify host factors involved in HCV propagation, we have previously screened a small interfering RNA (siRNA) library targeting host genes that control lipid metabolism and lipid droplet (LD) formation using cell culture-grown HCV (HCVcc)-infected cells. In this study, we selected and characterized the gene encoding ADP-ribosylation factor-related protein 1 (ARFRP1). ARFRP1 is essential for LD growth and is involved in the regulation of lipolysis. siRNA-mediated knockdown of ARFRP1 significantly inhibited HCV replication in both subgenomic replicon cells and HCVcc-infected cells. ARFRP1 interacted with NS5A and NS5A partially colocalized with LD. Silencing of ARFRP1 abrogated HCV-induced LD growth and viral protein expressions. Moreover, ARFRP1 recruited synaptosomal-associated protein 23 (SNAP23) to sites in close proximity to LDs in HCV-infected cells. Silencing of ARFRP1 ablated relocalization of SNAP23 to LD. These data indicate that HCV regulates ARFRP1 for LD growth to facilitate viral propagation and thus ARFRP1 may be a potential target for antiviral therapy. PMID:27550144

  2. ADP-ribosylation Factor-related Protein 1 Interacts with NS5A and Regulates Hepatitis C Virus Propagation

    PubMed Central

    Lim, Yun-Sook; Ngo, Huong T. T.; Lee, Jihye; Son, Kidong; Park, Eun-Mee; Hwang, Soon B.

    2016-01-01

    The life cycle of hepatitis C virus (HCV) is tightly coupled to the lipid metabolism of host cells. In order to identify host factors involved in HCV propagation, we have previously screened a small interfering RNA (siRNA) library targeting host genes that control lipid metabolism and lipid droplet (LD) formation using cell culture-grown HCV (HCVcc)-infected cells. In this study, we selected and characterized the gene encoding ADP-ribosylation factor-related protein 1 (ARFRP1). ARFRP1 is essential for LD growth and is involved in the regulation of lipolysis. siRNA-mediated knockdown of ARFRP1 significantly inhibited HCV replication in both subgenomic replicon cells and HCVcc-infected cells. ARFRP1 interacted with NS5A and NS5A partially colocalized with LD. Silencing of ARFRP1 abrogated HCV-induced LD growth and viral protein expressions. Moreover, ARFRP1 recruited synaptosomal-associated protein 23 (SNAP23) to sites in close proximity to LDs in HCV-infected cells. Silencing of ARFRP1 ablated relocalization of SNAP23 to LD. These data indicate that HCV regulates ARFRP1 for LD growth to facilitate viral propagation and thus ARFRP1 may be a potential target for antiviral therapy. PMID:27550144

  3. Differential expression during development of ADP-ribosylation factors, 20-kDa guanine nucleotide-binding protein activators of cholera toxin.

    PubMed

    Tsai, S C; Adamik, R; Tsuchiya, M; Chang, P P; Moss, J; Vaughan, M

    1991-05-01

    Cholera toxin exerts its effects on cells in large part through the ADP-ribosylation of guanine nucleotide-binding proteins. Toxin-catalyzed ADP-ribosylation is enhanced by approximately 20-kDa guanine nucleotide-binding proteins termed ADP-ribosylation factors (ARFs), which are allosteric activators of the toxin catalytic unit. Rabbit antiserum against a purified bovine brain ARF (sARF II) reacted on immunoblots with two approximately 20-kDa ARF-like proteins (sARF I and II) in tissue extracts from bovine, rat, frog, and chicken. Levels of ARF were higher in brain than in non-neural tissues. In rat brain, on the second postnatal day, amounts of sARF I and II were similar. By the 10th postnatal day and thereafter, sARF II predominated. Relative levels of ARF determined by immunoreactivity were in agreement with levels assessed in functional assays of cholera toxin-catalyzed ADP-ribosylation. Based on nucleotide and deduced amino acid sequences of human and bovine cDNAs, there appear to be at least six different ARF-like genes. Northern blots of rat brain poly(A)+ RNA were hybridized with cDNA and oligonucleotide probes specific for each of the human and bovine ARF genes. From the second to the 27th postnatal day, ARF 3 mRNA increased, whereas mRNAs for ARFs 2 and 4 decreased; and those for ARFs 1, 5, and 6 were apparently unchanged. Partial amino acid sequence of sARF II is consistent with it being either the ARF 1 or 3 gene product. The developmental changes in rat brain ARF parallel neuronal maturation and synapse formation. PMID:1902473

  4. Structure, organization and evolution of ADP-ribosylation factors in rice and foxtail millet, and their expression in rice

    PubMed Central

    Muthamilarasan, Mehanathan; Mangu, Venkata R.; Zandkarimi, Hana; Prasad, Manoj; Baisakh, Niranjan

    2016-01-01

    ADP-ribosylation factors (ARFs) have been reported to function in diverse physiological and molecular activities. Recent evidences also demonstrate the involvement of ARFs in conferring tolerance to biotic and abiotic stresses in plant species. In the present study, 23 and 25 ARF proteins were identified in C3 model- rice and C4 model- foxtail millet, respectively. These proteins are classified into four classes (I–IV) based on phylogenetic analysis, with ARFs in classes I–III and ARF-like proteins (ARLs) in class IV. Sequence alignment and domain analysis revealed the presence of conserved and additional motifs, which may contribute to neo- and sub-functionalization of these proteins. Promoter analysis showed the presence of several cis-regulatory elements related to stress and hormone response, indicating their role in stress regulatory network. Expression analysis of rice ARFs and ARLs in different tissues, stresses and abscisic acid treatment highlighted temporal and spatial diversification of gene expression. Five rice cultivars screened for allelic variations in OsARF genes showed the presence of allelic polymorphisms in few gene loci. Altogether, the study provides insights on characteristics of ARF/ARL genes in rice and foxtail millet, which could be deployed for further functional analysis to extrapolate their precise roles in abiotic stress responses. PMID:27097755

  5. Poly(ADP-ribosyl)ation of Apoptosis Antagonizing Transcription Factor Involved in Hydroquinone-Induced DNA Damage Response.

    PubMed

    Ling, Xiao Xuan; Liu, Jia Xian; Yun, Lin; DU, Yu Jun; Chen, Shao Qian; Chen, Jia Long; Tang, Huan Wen; Liu, Lin Hua

    2016-01-01

    The molecular mechanism of DNA damage induced by hydroquinone (HQ) remains unclear. Poly(ADP-ribose) polymerase-1 (PARP-1) usually works as a DNA damage sensor, and hence, it is possible that PARP-1 is involved in the DNA damage response induced by HQ. In TK6 cells treated with HQ, PARP activity as well as the expression of apoptosis antagonizing transcription factor (AATF), PARP-1, and phosphorylated H2AX (γ-H2AX) were maximum at 0.5 h, 6 h, 3 h, and 3 h, respectively. To explore the detailed mechanisms underlying the prompt DNA repair reaction, the above indicators were investigated in PARP-1-silenced cells. PARP activity and expression of AATF and PARP-1 decreased to 36%, 32%, and 33%, respectively, in the cells; however, γ-H2AX expression increased to 265%. Co-immunoprecipitation (co-IP) assays were employed to determine whether PARP-1 and AATF formed protein complexes. The interaction between these proteins together with the results from IP assays and confocal microscopy indicated that poly(ADP-ribosyl)ation (PARylation) regulated AATF expression. In conclusion, PARP-1 was involved in the DNA damage repair induced by HQ via increasing the accumulation of AATF through PARylation. PMID:26822515

  6. Structure, organization and evolution of ADP-ribosylation factors in rice and foxtail millet, and their expression in rice.

    PubMed

    Muthamilarasan, Mehanathan; Mangu, Venkata R; Zandkarimi, Hana; Prasad, Manoj; Baisakh, Niranjan

    2016-01-01

    ADP-ribosylation factors (ARFs) have been reported to function in diverse physiological and molecular activities. Recent evidences also demonstrate the involvement of ARFs in conferring tolerance to biotic and abiotic stresses in plant species. In the present study, 23 and 25 ARF proteins were identified in C3 model- rice and C4 model- foxtail millet, respectively. These proteins are classified into four classes (I-IV) based on phylogenetic analysis, with ARFs in classes I-III and ARF-like proteins (ARLs) in class IV. Sequence alignment and domain analysis revealed the presence of conserved and additional motifs, which may contribute to neo- and sub-functionalization of these proteins. Promoter analysis showed the presence of several cis-regulatory elements related to stress and hormone response, indicating their role in stress regulatory network. Expression analysis of rice ARFs and ARLs in different tissues, stresses and abscisic acid treatment highlighted temporal and spatial diversification of gene expression. Five rice cultivars screened for allelic variations in OsARF genes showed the presence of allelic polymorphisms in few gene loci. Altogether, the study provides insights on characteristics of ARF/ARL genes in rice and foxtail millet, which could be deployed for further functional analysis to extrapolate their precise roles in abiotic stress responses. PMID:27097755

  7. Identification of a brefeldin A-insensitive guanine nucleotide-exchange protein for ADP-ribosylation factor in bovine brain.

    PubMed Central

    Tsai, S C; Adamik, R; Moss, J; Vaughan, M

    1994-01-01

    ADP-ribosylation factors (ARFs) are approximately 20-kDa guanine nucleotide-binding proteins that participate in vesicular transport in the Golgi and other intracellular compartments and stimulate cholera toxin ADP-ribosyltransferase activity. ARFs are active in the GTP-bound form; hydrolysis of bound GTP to GDP, possibly with the assistance of a GTP hydrolysis (GTPase)-activating protein results in inactivation. Exchange of GDP for GTP and reactivation were shown by other workers to be enhanced by Golgi membranes in a brefeldin A-sensitive reaction, leading to the proposal that the guanine nucleotide-exchange protein (GEP) was a target of brefeldin A. In the studies reported here, a soluble GEP was partially purified from bovine brain. Exchange of nucleotide on ARFs 1 and 3, based on increased ARF activity in a toxin assay and stimulation of binding of guanosine 5'-[gamma-[35S]thio]triphosphate, was dependent on phospholipids, with phosphatidylserine being more effective than cardiolipin. GEP appeared to increase the rate of nucleotide exchange but did not affect the affinity of ARF for GTP. Whereas the crude GEP had a size of approximately 700 kDa, the partially purified GEP behaved on Ultrogel AcA 54 as a protein of 60 kDa. With purification, the GEP activity became insensitive to brefeldin A, consistent with the conclusion that, in contrast to earlier inferences, the exchange protein is not itself the target of brefeldin A. PMID:8159707

  8. Arfaptin 1, a putative cytosolic target protein of ADP-ribosylation factor, is recruited to Golgi membranes.

    PubMed

    Kanoh, H; Williger, B T; Exton, J H

    1997-02-28

    ADP-ribosylation factors (ARFs) have been implicated in vesicle transport in the Golgi complex. Employing yeast two-hybrid screening of an HL60 cDNA library using a constitutively active mutant of ARF3 (ARF3.Q71L), as a probe, we have identified a cDNA encoding a novel protein with a calculated molecular mass of 38.6 kDa, which we have named arfaptin 1. The mRNA of arfaptin 1 was ubiquitously expressed, and recombinant arfaptin 1 bound preferentially to class I ARFs, especially ARF1, but only in the GTP-bound form. The interactions were independent of myristoylation of ARF. Arfaptin 1 in cytosol was recruited to Golgi membranes by ARF in a guanosine 5'-O-(3-thiotriphosphate)-dependent and brefeldin A-sensitive manner. When expressed in COS cells, arfaptin 1 was localized to the Golgi complex. The yeast two-hybrid system yielded another clone, which encoded a putative protein, which we have named arfaptin 2. This consisted of the same number of amino acids as arfaptin 1 and was 60% identical to it. Arfaptin 2 was also ubiquitously expressed and bound to the GTP-, but not GDP-liganded form of class I ARFs, especially ARF1. These results suggest that arfaptins 1 and 2 may be direct target proteins of class 1 ARFs. Arfaptin 1 may be involved in Golgi function along with ARF1. PMID:9038142

  9. ADP Ribosylation Factor 6 (ARF6) Promotes Acrosomal Exocytosis by Modulating Lipid Turnover and Rab3A Activation*

    PubMed Central

    Pelletán, Leonardo E.; Suhaiman, Laila; Vaquer, Cintia C.; Bustos, Matías A.; De Blas, Gerardo A.; Vitale, Nicolas; Mayorga, Luis S.; Belmonte, Silvia A.

    2015-01-01

    Regulated secretion is a central issue for the specific function of many cells; for instance, mammalian sperm acrosomal exocytosis is essential for egg fertilization. ARF6 (ADP-ribosylation factor 6) is a small GTPase implicated in exocytosis, but its downstream effectors remain elusive in this process. We combined biochemical, functional, and microscopy-based methods to show that ARF6 is present in human sperm, localizes to the acrosomal region, and is required for calcium and diacylglycerol-induced exocytosis. Results from pulldown assays show that ARF6 exchanges GDP for GTP in sperm challenged with different exocytic stimuli. Myristoylated and guanosine 5′-3-O-(thio)triphosphate (GTPγS)-loaded ARF6 (active form) added to permeabilized sperm induces acrosome exocytosis even in the absence of extracellular calcium. We explore the ARF6 signaling cascade that promotes secretion. We demonstrate that ARF6 stimulates a sperm phospholipase D activity to produce phosphatidic acid and boosts the synthesis of phosphatidylinositol 4,5-bisphosphate. We present direct evidence showing that active ARF6 increases phospholipase C activity, causing phosphatidylinositol 4,5-bisphosphate hydrolysis and inositol 1,4,5-trisphosphate-dependent intra-acrosomal calcium release. We show that active ARF6 increases the exchange of GDP for GTP on Rab3A, a prerequisite for secretion. We propose that exocytic stimuli activate ARF6, which is required for acrosomal calcium efflux and the assembly of the membrane fusion machinery. This report highlights the physiological importance of ARF6 as a key factor for human sperm exocytosis and fertilization. PMID:25713146

  10. Cloning of an ADP-ribosylation factor gene from banana (Musa acuminata) and its expression patterns in postharvest ripening fruit.

    PubMed

    Wang, Yuan; Wu, Jing; Xu, Bi-Yu; Liu, Ju-Hua; Zhang, Jian-Bin; Jia, Cai-Hong; Jin, Zhi-Qiang

    2010-08-15

    A full-length cDNA encoding an ADP-ribosylation factor (ARF) from banana (Musa acuminata) fruit was cloned and named MaArf. It contains an open reading frame encoding a 181-amino-acid polypeptide. Sequence analysis showed that MaArf shared high similarity with ARF of other plant species. The genomic sequence of MaArf was also obtained using polymerase chain reaction (PCR). Sequence analysis showed that MaArf was a split gene containing five exons and four introns in genomic DNA. Reverse-transcriptase PCR was used to analyze the spatial expression of MaArf. The results showed that MaArf was expressed in all the organs examined: root, rhizome, leaf, flower and fruit. Real-time quantitative PCR was used to explore expression patterns of MaArf in postharvest banana. There was differential expression of MaArf associated with ethylene biosynthesis. In naturally ripened banana, expression of MaArf was in accordance with ethylene biosynthesis. However, in 1-methylcyclopropene-treated banana, the expression of MaArf was inhibited and changed little. When treated with ethylene, MaArf expression in banana fruit significantly increased in accordance with ethylene biosynthesis; the peak of MaArf was 3 d after harvest, 11 d earlier than for naturally ripened banana fruits. These results suggest that MaArf is induced by ethylene in regulating postharvest banana ripening. Finally, subcellular localization assays showed the MaArf protein in the cytoplasm. PMID:20435371

  11. Centaurin-alpha 1, an ADP-ribosylation factor 6 GTPase activating protein, inhibits beta 2-adrenoceptor internalization.

    PubMed

    Lawrence, Joanna; Mundell, Stuart J; Yun, Hongruo; Kelly, Eamonn; Venkateswarlu, Kanamarlapudi

    2005-06-01

    The small GTP-binding protein ADP ribosylation factor 6 (ARF6) has recently been implicated in the internalization of G protein-coupled receptors (GPCRs), although its precise molecular mechanism in this process remains unclear. We have recently identified centaurin alpha(1) as a GTPase activating protein (GAP) for ARF6. In the current study, we characterized the effects of centaurin alpha(1) on the agonist-induced internalization of the beta(2)-adrenoceptor transiently expressed in human embryonic kidney (HEK) 293 cells. Using an enzyme-linked immunosorbent assay as well as confocal imaging of cells, we found that expression of centaurin alpha(1) strongly inhibited the isoproterenol-induced internalization of beta(2)-adrenoceptor. On the other hand, expression of functionally inactive versions of centaurin alpha(1), including an R49C mutant, which has no catalytic activity, and a double pleckstrin homology (PH) mutant (DM; R148C/R273C), which has mutations in both the PH domains of centaurin alpha(1), rendering it unable to translocate to the cell membrane, were unable to inhibit beta(2)-adrenoceptor internalization. In addition, a constitutively active version of ARF6, ARF6Q67L, reversed the ability of centaurin alpha(1) to inhibit beta(2)-adrenoceptor internalization. Finally, expression of centaurin alpha(1) also inhibited the agonist-induced internalization of beta(2)-adrenoceptor endogenously expressed in HEK 293 cells, whereas the R49C and DM mutant versions of centaurin alpha(1) had no effect. Together, these data indicate that by acting as an ARF6 GAP, centaurin alpha(1) is able to switch off ARF6 and so inhibit its ability to mediate beta(2)-adrenoceptor internalization. Thus, ARF6 GAPs, such as centaurin alpha(1), are likely to play a crucial role in GPCR trafficking by modulating the activity of ARF6. PMID:15778454

  12. Molecular cloning, characterization, and expression of human ADP-ribosylation factors: two guanine nucleotide-dependent activators of cholera toxin.

    PubMed Central

    Bobak, D A; Nightingale, M S; Murtagh, J J; Price, S R; Moss, J; Vaughan, M

    1989-01-01

    ADP-ribosylation factors (ARFs) are small guanine nucleotide-binding proteins that enhance the enzymatic activities of cholera toxin. Two ARF cDNAs, ARF1 and ARF3, were cloned from a human cerebellum library. Based on deduced amino acid sequences and patterns of hybridization of cDNA and oligonucleotide probes with mammalian brain poly(A)+ RNA, human ARF1 is the homologue of bovine ARF1. Human ARF3, which differs from bovine ARF1 and bovine ARF2, appears to represent a newly identified third type of ARF. Hybridization patterns of human ARF cDNA and clone-specific oligonucleotides with poly(A)+ RNA are consistent with the presence of at least two, and perhaps four, separate ARF messages in human brain. In vitro translation of ARF1, ARF2, and ARF3 produced proteins that behaved, by SDS/PAGE, similar to a purified soluble brain ARF. Deduced amino acid sequences of human ARF1 and ARF3 contain regions, similar to those in other G proteins, that are believed to be involved in GTP binding and hydrolysis. ARFs also exhibit a modest degree of homology with a bovine phospholipase C. The observations reported here support the conclusion that the ARFs are members of a multigene family of small guanine nucleotide-binding proteins. Definition of the regulation of ARF mRNAs and of function(s) of recombinant ARF proteins will aid in the elucidation of the physiologic role(s) of ARFs. Images PMID:2474826

  13. Guanine nucleotide-binding proteins that enhance choleragen ADP-ribosyltransferase activity: nucleotide and deduced amino acid sequence of an ADP-ribosylation factor cDNA.

    PubMed Central

    Price, S R; Nightingale, M; Tsai, S C; Williamson, K C; Adamik, R; Chen, H C; Moss, J; Vaughan, M

    1988-01-01

    Three (two soluble and one membrane) guanine nucleotide-binding proteins (G proteins) that enhance ADP-ribosylation of the Gs alpha stimulatory subunit of the adenylyl cyclase (EC 4.6.1.1) complex by choleragen have recently been purified from bovine brain. To further define the structure and function of these ADP-ribosylation factors (ARFs), we isolated a cDNA clone (lambda ARF2B) from a bovine retinal library by screening with a mixed heptadecanucleotide probe whose sequence was based on the partial amino acid sequence of one of the soluble ARFs from bovine brain. Comparison of the deduced amino acid sequence of lambda ARF2B with sequences of peptides from the ARF protein (total of 60 amino acids) revealed only two differences. Whether these are cloning artifacts or reflect the existence of more than one ARF protein remains to be determined. Deduced amino acid sequences of ARF, Go alpha (the alpha subunit of a G protein that may be involved in regulation of ion fluxes), and c-Ha-ras gene product p21 show similarities in regions believed to be involved in guanine nucleotide binding and GTP hydrolysis. ARF apparently lacks a site analogous to that ADP-ribosylated by choleragen in G-protein alpha subunits. Although both the ARF proteins and the alpha subunits bind guanine nucleotides and serve as choleragen substrates, they must interact with the toxin A1 peptide in different ways. In addition to serving as an ADP-ribose acceptor, ARF interacts with the toxin in a manner that modifies its catalytic properties. PMID:3135549

  14. Cholix Toxin, a Novel ADP-ribosylating Factor from Vibrio cholerae

    SciTech Connect

    Jorgensen, Rene; Purdy, Alexandra E.; Fieldhouse, Robert J.; Kimber, Matthew S.; Bartlett, Douglas H.; Merrill, A. Rod

    2008-07-15

    The ADP-ribosyltransferases are a class of enzymes that display activity in a variety of bacterial pathogens responsible for causing diseases in plants and animals, including those affecting mankind, such as diphtheria, cholera, and whooping cough. We report the characterization of a novel toxin from Vibrio cholerae, which we call cholix toxin. The toxin is active against mammalian cells (IC50 = 4.6 {+-} 0.4 ng/ml) and crustaceans (Artemia nauplii LD50 = 10 {+-} 2 {mu}g/ml). Here we show that this toxin is the third member of the diphthamide-specific class of ADP-ribose transferases and that it possesses specific ADP-ribose transferase activity against ribosomal eukaryotic elongation factor 2. We also describe the high resolution crystal structures of the multidomain toxin and its catalytic domain at 2.1- and 1.25-{angstrom} resolution, respectively. The new structural data show that cholix toxin possesses the necessary molecular features required for infection of eukaryotes by receptor-mediated endocytosis, translocation to the host cytoplasm, and inhibition of protein synthesis by specific modification of elongation factor 2. The crystal structures also provide important insight into the structural basis for activation of toxin ADP-ribosyltransferase activity. These results indicate that cholix toxin may be an important virulence factor of Vibrio cholerae that likely plays a significant role in the survival of the organism in an aquatic environment.

  15. GTP but not GDP analogues promote association of ADP-ribosylation factors, 20-kDa protein activators of cholera toxin, with phospholipids and PC-12 cell membranes.

    PubMed

    Walker, M W; Bobak, D A; Tsai, S C; Moss, J; Vaughan, M

    1992-02-15

    ADP-ribosylation factors (ARFs) are a family of approximately 20-kDa guanine nucleotide-binding proteins initially identified by their ability to enhance cholera toxin ADP-ribosyltransferase activity in the presence of GTP. ARFs have been purified from both membrane and cytosolic fractions. ARF purified from bovine brain cytosol requires phospholipid plus detergent for high affinity guanine nucleotide binding and for optimal enhancement of cholera toxin ADP-ribosyltransferase activity. The phospholipid requirements, combined with a putative role for ARF in vesicular transport, suggested that the soluble protein might interact reversibly with membranes. A polyclonal antibody against purified bovine ARF (sARF II) was used to detect ARF by immunoblot in membrane and soluble fractions from rat pheochromocytoma (PC-12) cell homogenates. ARF was predominantly cytosolic but increased in membranes during incubation of homogenates with nonhydrolyzable GTP analogues guanosine 5'-O-(3-thiotriphosphate), guanylyl-(beta gamma-imido)-diphosphate, and guanylyl-(beta gamma-methylene)-diphosphate, and to a lesser extent, adenosine 5'-O-(3-thiotriphosphate). GTP, GDP, GMP, and ATP were inactive. Cytosolic ARF similarly associated with added phosphatidylserine, phosphatidylinositol, or cardiolipin in GTP gamma S-dependent fashion. ARF binding to phosphatidylserine was reversible and coincident with stimulation of cholera toxin-catalyzed ADP-ribosylation. These observations may reflect a mechanism by which ARF could cycle between soluble and membrane compartments in vivo. PMID:1737779

  16. Mechanism of activation of cholera toxin by ADP-ribosylation factor (ARF): both low- and high-affinity interactions of ARF with guanine nucleotides promote toxin activation.

    PubMed

    Bobak, D A; Bliziotes, M M; Noda, M; Tsai, S C; Adamik, R; Moss, J

    1990-01-30

    Activation of adenylyl cyclase by cholera toxin A subunit (CT-A) results from the ADP-ribosylation of the stimulatory guanine nucleotide binding protein (GS alpha). This process requires GTP and an endogenous guanine nucleotide binding protein known as ADP-ribosylation factor (ARF). One membrane (mARF) and two soluble forms (sARF I and sARF II) of ARF have been purified from bovine brain. Because the conditions reported to enhance the binding of guanine nucleotides by ARF differ from those observed to promote optimal activity, we sought to characterize the determinants influencing the functional interaction of guanine nucleotides with ARF. High-affinity GTP binding by sARF II (apparent KD of approximately 70 nM) required Mg2+, DMPC, and sodium cholate. sARF II, in DMPC/cholate, also enhanced CT-A ADP-ribosyltransferase activity (apparent EC50 for GTP of approximately 50 nM), although there was a delay before achievement of a maximal rate of sARF II stimulated toxin activity. The delay was abolished by incubation of sARF II with GTP at 30 degrees C before initiation of the assay. In contrast, a maximal rate of activation of toxin by sARF II, in 0.003% SDS, occurred without delay (apparent EC50 for GTP of approximately 5 microM). High-affinity GTP binding by sARF II was not detectable in SDS. Enhancement of CT-A ADP-ribosyltransferase activity by sARF II, therefore, can occur under conditions in which sARF II exhibits either a relatively low affinity or a relatively high affinity for GTP. The interaction of GTP with ARF under these conditions may reflect ways in which intracellular membrane and cytosolic environments modulate GTP-mediated activation of ARF. PMID:2111167

  17. The Structure of RalF, an ADP-Ribosylation Factor Guanine Nucleotide Exchange Factor from Legionella pneumophila, Reveals the Presence of a Cap over the Active Site

    SciTech Connect

    Amor,J.; Swails, J.; Zhu, X.; Roy, C.; Nagai, H.; Ingmundson, A.; Cheng, X.; Kahn, R.

    2005-01-01

    The Legionella pneumophila protein RalF is secreted into host cytosol via the Dot/Icm type IV transporter where it acts to recruit ADP-ribosylation factor (Arf) to pathogen-containing phagosomes in the establishment of a replicative organelle. The presence in RalF of the Sec7 domain, present in all Arf guanine nucleotide exchange factors, has suggested that recruitment of Arf is an early step in pathogenesis. We have determined the crystal structure of RalF and of the isolated Sec7 domain and found that RalF is made up of two domains. The Sec7 domain is homologous to mammalian Sec7 domains. The C-terminal domain forms a cap over the active site in the Sec7 domain and contains a conserved folding motif, previously observed in adaptor subunits of vesicle coat complexes. The importance of the capping domain and of the glutamate in the 'glutamic finger,' conserved in all Sec7 domains, to RalF functions was examined using three different assays. These data highlight the functional importance of domains other than Sec7 in Arf guanine nucleotide exchange factors to biological activities and suggest novel mechanisms of regulation of those activities.

  18. Regulation of Bone Morphogenetic Protein Signaling by ADP-ribosylation.

    PubMed

    Watanabe, Yukihide; Papoutsoglou, Panagiotis; Maturi, Varun; Tsubakihara, Yutaro; Hottiger, Michael O; Heldin, Carl-Henrik; Moustakas, Aristidis

    2016-06-10

    We previously established a mechanism of negative regulation of transforming growth factor β signaling mediated by the nuclear ADP-ribosylating enzyme poly-(ADP-ribose) polymerase 1 (PARP1) and the deribosylating enzyme poly-(ADP-ribose) glycohydrolase (PARG), which dynamically regulate ADP-ribosylation of Smad3 and Smad4, two central signaling proteins of the pathway. Here we demonstrate that the bone morphogenetic protein (BMP) pathway can also be regulated by the opposing actions of PARP1 and PARG. PARG positively contributes to BMP signaling and forms physical complexes with Smad5 and Smad4. The positive role PARG plays during BMP signaling can be neutralized by PARP1, as demonstrated by experiments where PARG and PARP1 are simultaneously silenced. In contrast to PARG, ectopic expression of PARP1 suppresses BMP signaling, whereas silencing of endogenous PARP1 enhances signaling and BMP-induced differentiation. The two major Smad proteins of the BMP pathway, Smad1 and Smad5, interact with PARP1 and can be ADP-ribosylated in vitro, whereas PARG causes deribosylation. The overall outcome of this mode of regulation of BMP signal transduction provides a fine-tuning mechanism based on the two major enzymes that control cellular ADP-ribosylation. PMID:27129221

  19. Nuclear ADP-Ribosylation Reactions in Mammalian Cells: Where Are We Today and Where Are We Going?

    PubMed Central

    Hassa, Paul O.; Haenni, Sandra S.; Elser, Michael; Hottiger, Michael O.

    2006-01-01

    Since poly-ADP ribose was discovered over 40 years ago, there has been significant progress in research into the biology of mono- and poly-ADP-ribosylation reactions. During the last decade, it became clear that ADP-ribosylation reactions play important roles in a wide range of physiological and pathophysiological processes, including inter- and intracellular signaling, transcriptional regulation, DNA repair pathways and maintenance of genomic stability, telomere dynamics, cell differentiation and proliferation, and necrosis and apoptosis. ADP-ribosylation reactions are phylogenetically ancient and can be classified into four major groups: mono-ADP-ribosylation, poly-ADP-ribosylation, ADP-ribose cyclization, and formation of O-acetyl-ADP-ribose. In the human genome, more than 30 different genes coding for enzymes associated with distinct ADP-ribosylation activities have been identified. This review highlights the recent advances in the rapidly growing field of nuclear mono-ADP-ribosylation and poly-ADP-ribosylation reactions and the distinct ADP-ribosylating enzyme families involved in these processes, including the proposed family of novel poly-ADP-ribose polymerase-like mono-ADP-ribose transferases and the potential mono-ADP-ribosylation activities of the sirtuin family of NAD+-dependent histone deacetylases. A special focus is placed on the known roles of distinct mono- and poly-ADP-ribosylation reactions in physiological processes, such as mitosis, cellular differentiation and proliferation, telomere dynamics, and aging, as well as “programmed necrosis” (i.e., high-mobility-group protein B1 release) and apoptosis (i.e., apoptosis-inducing factor shuttling). The proposed molecular mechanisms involved in these processes, such as signaling, chromatin modification (i.e., “histone code”), and remodeling of chromatin structure (i.e., DNA damage response, transcriptional regulation, and insulator function), are described. A potential cross talk between nuclear

  20. ADP-Ribosylation Factor 6 Acts as an Allosteric Activator for the Folded but not Disordered Cholera Toxin A1 Polypeptide

    PubMed Central

    Banerjee, Tuhina; Taylor, Michael; Jobling, Michael G.; Burress, Helen; Yang, ZhiJie; Serrano, Albert; Holmes, Randall K.; Tatulian, Suren A.; Teter, Ken

    2014-01-01

    Summary The catalytic A1 subunit of cholera toxin (CTA1) has a disordered structure at 37°C. An interaction with host factors must therefore place CTA1 in a folded conformation for the modification of its Gsα target which resides in a lipid raft environment. Host ADP-ribosylation factors (ARFs) act as in vitro allosteric activators of CTA1, but the molecular events of this process are not fully characterized. Isotope-edited Fourier transform infrared spectroscopy monitored ARF6-induced structural changes to CTA1, which were correlated to changes in CTA1 activity. We found ARF6 prevents the thermal disordering of structured CTA1 and stimulates the activity of stabilized CTA1 over a range of temperatures. Yet ARF6 alone did not promote the refolding of disordered CTA1 to an active state. Instead, lipid rafts shifted disordered CTA1 to a folded conformation with a basal level of activity that could be further stimulated by ARF6. Thus, ARF alone is unable to activate disordered CTA1 at physiological temperature: additional host factors such as lipid rafts place CTA1 in the folded conformation required for its ARF-mediated activation. Interaction with ARF is required for in vivo toxin activity, as enzymatically active CTA1 mutants that cannot be further stimulated by ARF6 fail to intoxicate cultured cells. PMID:25257027

  1. ADP-ribosylation factor arf6p may function as a molecular switch of new end take off in fission yeast

    SciTech Connect

    Fujita, Atsushi

    2008-02-01

    Small GTPases act as molecular switches in a wide variety of cellular processes. In fission yeast Schizosaccharomyces pombe, the directions of cell growth change from a monopolar manner to a bipolar manner, which is known as 'New End Take Off' (NETO). Here I report the identification of a gene, arf6{sup +}, encoding an ADP-ribosylation factor small GTPase, that may be essential for NETO. arf6{delta} cells completely fail to undergo NETO. arf6p localizes at both cell ends and presumptive septa in a cell-cycle dependent manner. And its polarized localization is not dependent on microtubules, actin cytoskeletons and some NETO factors (bud6p, for3p, tea1p, tea3p, and tea4p). Notably, overexpression of a fast GDP/GTP-cycling mutant of arf6p can advance the timing of NETO. These findings suggest that arf6p functions as a molecular switch for the activation of NETO in fission yeast.

  2. ADP-ribosylation factor 1 expression regulates epithelial-mesenchymal transition and predicts poor clinical outcome in triple-negative breast cancer

    PubMed Central

    Schlienger, Sabrina; Campbell, Shirley; Pasquin, Sarah; Gaboury, Louis; Claing, Audrey

    2016-01-01

    Metastatic capacities are fundamental features of tumor malignancy. ADP-ribosylation factor (ARF) 1 has emerged as a key regulator of invasion in breast cancer cells. However, the importance of this GTPase, in vivo, remains to be demonstrated. We report that ARF1 is highly expressed in breast tumors of the most aggressive and advanced subtypes. Furthermore, we show that lowered expression of ARF1 impairs growth of primary tumors and inhibits lung metastasis in a murine xenograft model. To understand how ARF1 contributes to invasiveness, we used a poorly invasive breast cancer cell line, MCF7 (ER+), and examined the effects of overexpressing ARF1 to levels similar to that found in invasive cell lines. We demonstrate that ARF1 overexpression leads to the epithelial-mesenchymal transition (EMT). Mechanistically, ARF1 controls cell–cell adhesion through ß-catenin and E-cadherin, oncogenic Ras activation and expression of EMT inducers. We further show that ARF1 overexpression enhances invasion, proliferation and resistance to a chemotherapeutic agent. In vivo, ARF1 overexpressing MCF7 cells are able to form more metastases to the lung. Overall, our findings demonstrate that ARF1 is a molecular switch for cancer progression and thus suggest that limiting the expression/activation of this GTPase could help improve outcome for breast cancer patients. PMID:26908458

  3. ADP Ribosylation Factor 6 Regulates Neuronal Migration in the Developing Cerebral Cortex through FIP3/Arfophilin-1-dependent Endosomal Trafficking of N-cadherin.

    PubMed

    Hara, Yoshinobu; Fukaya, Masahiro; Hayashi, Kanehiro; Kawauchi, Takeshi; Nakajima, Kazunori; Sakagami, Hiroyuki

    2016-01-01

    During neural development, endosomal trafficking controls cell shape and motility through the polarized transport of membrane proteins related to cell-cell and cell-extracellular matrix interactions. ADP ribosylation factor 6 (Arf6) is a critical small GTPase that regulates membrane trafficking between the plasma membrane and endosomes. We herein demonstrated that the knockdown of endogenous Arf6 in mouse cerebral cortices led to impaired neuronal migration in the intermediate zone and cytoplasmic retention of N-cadherin and syntaxin12 in migrating neurons. Rescue experiments with separation-of-function Arf6 mutants identified Rab11 family-interacting protein 3 (FIP3)/Arfophilin-1, a dual effector for Arf6 and Rab11, as a downstream effector of Arf6 in migrating neurons. The knockdown of FIP3 led to impaired neuronal migration in the intermediate zone and cytoplasmic retention of N-cadherin in migrating neurons, similar to that of Arf6, which could be rescued by the coexpression of wild-type FIP3 but not FIP3 mutants lacking the binding site for Arf6 or Rab11. These results suggest that Arf6 regulates cortical neuronal migration in the intermediate zone through the FIP3-dependent endosomal trafficking. PMID:27622210

  4. ADP Ribosylation Factor 6 Regulates Neuronal Migration in the Developing Cerebral Cortex through FIP3/Arfophilin-1-dependent Endosomal Trafficking of N-cadherin

    PubMed Central

    Hara, Yoshinobu; Fukaya, Masahiro

    2016-01-01

    Abstract During neural development, endosomal trafficking controls cell shape and motility through the polarized transport of membrane proteins related to cell–cell and cell–extracellular matrix interactions. ADP ribosylation factor 6 (Arf6) is a critical small GTPase that regulates membrane trafficking between the plasma membrane and endosomes. We herein demonstrated that the knockdown of endogenous Arf6 in mouse cerebral cortices led to impaired neuronal migration in the intermediate zone and cytoplasmic retention of N-cadherin and syntaxin12 in migrating neurons. Rescue experiments with separation-of-function Arf6 mutants identified Rab11 family-interacting protein 3 (FIP3)/Arfophilin-1, a dual effector for Arf6 and Rab11, as a downstream effector of Arf6 in migrating neurons. The knockdown of FIP3 led to impaired neuronal migration in the intermediate zone and cytoplasmic retention of N-cadherin in migrating neurons, similar to that of Arf6, which could be rescued by the coexpression of wild-type FIP3 but not FIP3 mutants lacking the binding site for Arf6 or Rab11. These results suggest that Arf6 regulates cortical neuronal migration in the intermediate zone through the FIP3-dependent endosomal trafficking. PMID:27622210

  5. ADP-ribosylation factor 1 expression regulates epithelial-mesenchymal transition and predicts poor clinical outcome in triple-negative breast cancer.

    PubMed

    Schlienger, Sabrina; Campbell, Shirley; Pasquin, Sarah; Gaboury, Louis; Claing, Audrey

    2016-03-29

    Metastatic capacities are fundamental features of tumor malignancy. ADP-ribosylation factor (ARF) 1 has emerged as a key regulator of invasion in breast cancer cells. However, the importance of this GTPase, in vivo, remains to be demonstrated. We report that ARF1 is highly expressed in breast tumors of the most aggressive and advanced subtypes. Furthermore, we show that lowered expression of ARF1 impairs growth of primary tumors and inhibits lung metastasis in a murine xenograft model. To understand how ARF1 contributes to invasiveness, we used a poorly invasive breast cancer cell line, MCF7 (ER+), and examined the effects of overexpressing ARF1 to levels similar to that found in invasive cell lines. We demonstrate that ARF1 overexpression leads to the epithelial-mesenchymal transition (EMT). Mechanistically, ARF1 controls cell-cell adhesion through ß-catenin and E-cadherin, oncogenic Ras activation and expression of EMT inducers. We further show that ARF1 overexpression enhances invasion, proliferation and resistance to a chemotherapeutic agent. In vivo, ARF1 overexpressing MCF7 cells are able to form more metastases to the lung. Overall, our findings demonstrate that ARF1 is a molecular switch for cancer progression and thus suggest that limiting the expression/activation of this GTPase could help improve outcome for breast cancer patients. PMID:26908458

  6. Nuclear localization and molecular partners of BIG1, a brefeldin A-inhibited guanine nucleotide-exchange protein for ADP-ribosylation factors.

    PubMed

    Padilla, Philip Ian; Pacheco-Rodriguez, Gustavo; Moss, Joel; Vaughan, Martha

    2004-03-01

    Brefeldin A-inhibited guanine nucleotide-exchange protein 1 (BIG1) is an approximately 200-kDa brefeldin A-inhibited guanine nucleotide-exchange protein that preferentially activates ADP-ribosylation factor 1 (ARF1) and ARF3. BIG1 was found in cytosol in a multiprotein complex with a similar ARF-activating protein, BIG2, which is also an A kinase-anchoring protein. In HepG2 cells growing with serum, BIG1 was primarily cytosolic and Golgi-associated. After incubation overnight without serum, a large fraction of endogenous BIG1 was in the nuclei. By confocal immunofluorescence microscopy, BIG1 was localized with nucleoporin p62 at the nuclear envelope (probably during nucleocytoplasmic transport) and also in nucleoli, clearly visible against the less concentrated overall matrix staining. BIG1 was also identified by Western blot analyses in purified subnuclear fractions (e.g., nucleoli and nuclear matrix). Antibodies against BIG1, nucleoporin, or nucleolin coimmunoprecipitated the other two proteins from purified nuclei. In contrast, BIG2 was not associated with nuclear BIG1. Also of note, ARF was never detected among proteins precipitated from purified nuclei by anti-BIG1 antibodies, although microscopically the two proteins do appear sometimes to be colocalized in the nucleus. These data are consistent with independent intracellular movements and actions of BIG1 and BIG2, and they are also evidence of the participation of BIG1 in both Golgi and nuclear functions. PMID:14973189

  7. GB virus type C E2 protein inhibits human immunodeficiency virus type 1 Gag assembly by downregulating human ADP-ribosylation factor 1

    PubMed Central

    Wang, Chenliang; Timmons, Christine L.; Shao, Qiujia; Kinlock, Ballington L.; Turner, Tiffany M.; Iwamoto, Aikichi; Zhang, Hui; Liu, Huanliang; Liu, Bindong

    2015-01-01

    GB virus type C (GBV-C) glycoprotein E2 protein disrupts HIV-1 assembly and release by inhibiting Gag plasma membrane targeting, however the mechanism by which the GBV-C E2 inhibits Gag trafficking remains unclear. In the present study, we identified ADP-ribosylation factor 1 (ARF1) contributed to the inhibitory effect of GBV-C E2 on HIV-1 Gag membrane targeting. Expression of GBV-C E2 decreased ARF1 expression in a proteasomal degradation-dependent manner. The restoration of ARF1 expression rescued the HIV-1 Gag processing and membrane targeting defect imposed by GBV-C E2. In addition, GBV-C E2 expression also altered Golgi morphology and suppressed protein traffic through the secretory pathway, which are all consistent with a phenotype of disrupting the function of ARF1 protein. Thus, our results indicate that GBV-C E2 inhibits HIV-1 assembly and release by decreasing ARF1, and may provide insights regarding GBV-C E2's potential for a new therapeutic approach for treating HIV-1. PMID:26675377

  8. ARNO3, a Sec7-domain guanine nucleotide exchange factor for ADP ribosylation factor 1, is involved in the control of Golgi structure and function

    PubMed Central

    Franco, Michel; Boretto, Joëlle; Robineau, Sylviane; Monier, Solange; Goud, Bruno; Chardin, Pierre; Chavrier, Philippe

    1998-01-01

    Budding of transport vesicles in the Golgi apparatus requires the recruitment of coat proteins and is regulated by ADP ribosylation factor (ARF) 1. ARF1 activation is promoted by guanine nucleotide exchange factors (GEFs), which catalyze the transition to GTP-bound ARF1. We recently have identified a human protein, ARNO (ARF nucleotide-binding-site opener), as an ARF1-GEF that shares a conserved domain with the yeast Sec7 protein. We now describe a human Sec7 domain-containing GEF referred to as ARNO3. ARNO and ARNO3, as well as a third GEF called cytohesin-1, form a family of highly related proteins with identical structural organization that consists of a central Sec7 domain and a carboxy-terminal pleckstrin homology domain. We show that all three proteins act as ARF1 GEF in vitro, whereas they have no effect on ARF6, an ARF protein implicated in the early endocytic pathway. Substrate specificity of ARNO-like GEFs for ARF1 depends solely on the Sec7 domain. Overexpression of ARNO3 in mammalian cells results in (i) fragmentation of the Golgi apparatus, (ii) redistribution of Golgi resident proteins as well as the coat component β-COP, and (iii) inhibition of SEAP transport (secreted form of alkaline phosphatase). In contrast, the distribution of endocytic markers is not affected. This study indicates that Sec7 domain-containing GEFs control intracellular membrane compartment structure and function through the regulation of specific ARF proteins in mammalian cells. PMID:9707577

  9. ADP-Ribosylation: Activation, Recognition, and Removal

    PubMed Central

    Li, Nan; Chen, Junjie

    2014-01-01

    ADP-ribosylation is a type of posttranslational modification catalyzed by members of the poly(ADP-ribose) (PAR) polymerase superfamily. ADP-ribosylation is initiated by PARPs, recognized by PAR binding proteins, and removed by PARG and other ADP-ribose hydrolases. These three groups of proteins work together to regulate the cellular and molecular response of PAR signaling, which is critical for a wide range of cellular and physiological functions. PMID:24552704

  10. The family of bacterial ADP-ribosylating exotoxins.

    PubMed Central

    Krueger, K M; Barbieri, J T

    1995-01-01

    Pathogenic bacteria utilize a variety of virulence factors that contribute to the clinical manifestation of their pathogenesis. Bacterial ADP-ribosylating exotoxins (bAREs) represent one family of virulence factors that exert their toxic effects by transferring the ADP-ribose moiety of NAD onto specific eucaryotic target proteins. The observations that some bAREs ADP-ribosylate eucaryotic proteins that regulate signal transduction, like the heterotrimeric GTP-binding proteins and the low-molecular-weight GTP-binding proteins, has extended interest in bAREs beyond the bacteriology laboratory. Molecular studies have shown that bAREs possess little primary amino acid homology and have diverse quaternary structure-function organization. Underlying this apparent diversity, biochemical and crystallographic studies have shown that several bAREs have conserved active-site structures and possess a conserved glutamic acid within their active sites. PMID:7704894

  11. Characterization of ADP ribosylation factor 1 gene from Exopalaemon carinicauda and its immune response to pathogens challenge and ammonia-N stress.

    PubMed

    Duan, Yafei; Li, Jian; Zhang, Zhe; Li, Jitao; Liu, Ping

    2016-08-01

    ADP ribosylation factors (Arf), as highly conserved small guanosine triphosphate (GTP)-binding proteins, participates in intracellular trafficking and organelle structure. In this study, a full-length cDNA of Arf1 (designated EcArf1) was cloned from Exopalaemon carinicauda by using rapid amplification of cDNA ends (RACE) approaches. The full-length cDNA of EcArf1 was 1428 bp, which contains an open reading frame (ORF) of 549 bp, encoding a 182 amino-acid polypeptide with the predicted molecular weight of 20.69 kDa and estimated isoelectric point was 7.24. Sequence analysis revealed that the conserved Arf protein family signatures were identified in EcArf1. The deduced amino acid sequence of EcArf1 shared high identity (95%-98%) with that of other species and clustered together with Arf1 of other shrimp in the NJ phylogenetic tree, indicating that EcArf1 should be a member of the Arf1 family. Quantitative real-time RT-qPCR analysis indicated that EcArf1 was expressed in hemocytes, hepatopancreas, gills, muscle, ovary, intestine, stomach and heart, and the most abundant level was in hemocytes and gills, which were also the two main target tissues of pathogen infection and environmental stress. After Vibrio parahaemolyticus challenge, EcArf1 transcripts level significantly increased in hemocytes and hepatopancreas at 3 h and 6 h, respectively. The expression of EcArf1 in hemocytes and hepatopancreas significantly up-regulated at 12 h and 6 h respectively, and down-regulated at 72 h and 48 h, respectively. EcArf1 expression in hepatopancreas and gills both significantly increased at 6 h and decreased at 24 h under ammonia-N stress. The results suggested that EcArf1 might be involved in immune responses to pathogens (V. parahaemolyticus and WSSV) challenge and ammonia-N stress in E. carinicauda. PMID:27231192

  12. ADP-Ribosyltransferases and Poly ADP-Ribosylation

    PubMed Central

    Liu, Chao; Yu, Xiaochun

    2016-01-01

    Protein ADP-ribosylation is an important posttranslational modification that plays versatile roles in multiple biological processes. ADP-ribosylation is catalyzed by a group of enzymes known as ADP-ribosyltransferases (ARTs). Using nicotinamide adenine dinucleotide (NAD+) as the donor, ARTs covalently link single or multiple ADP-ribose moieties from NAD+ to the substrates, forming mono ADP-ribosylation or poly ADP-ribosylation (PARylation). Novel functions of ARTs and ADP-ribosylation have been revealed over the past few years. Here we summarize the current knowledge on ARTs and PARylation. PMID:25938242

  13. Wnt pathway activation by ADP-ribosylation

    PubMed Central

    Yang, Eungi; Tacchelly-Benites, Ofelia; Wang, Zhenghan; Randall, Michael P.; Tian, Ai; Benchabane, Hassina; Freemantle, Sarah; Pikielny, Claudio; Tolwinski, Nicholas S.; Lee, Ethan; Ahmed, Yashi

    2016-01-01

    Wnt/β-catenin signalling directs fundamental processes during metazoan development and can be aberrantly activated in cancer. Wnt stimulation induces the recruitment of the scaffold protein Axin from an inhibitory destruction complex to a stimulatory signalosome. Here we analyse the early effects of Wnt on Axin and find that the ADP-ribose polymerase Tankyrase (Tnks)—known to target Axin for proteolysis—regulates Axin's rapid transition following Wnt stimulation. We demonstrate that the pool of ADP-ribosylated Axin, which is degraded under basal conditions, increases immediately following Wnt stimulation in both Drosophila and human cells. ADP-ribosylation of Axin enhances its interaction with the Wnt co-receptor LRP6, an essential step in signalosome assembly. We suggest that in addition to controlling Axin levels, Tnks-dependent ADP-ribosylation promotes the reprogramming of Axin following Wnt stimulation; and propose that Tnks inhibition blocks Wnt signalling not only by increasing destruction complex activity, but also by impeding signalosome assembly. PMID:27138857

  14. Wnt pathway activation by ADP-ribosylation.

    PubMed

    Yang, Eungi; Tacchelly-Benites, Ofelia; Wang, Zhenghan; Randall, Michael P; Tian, Ai; Benchabane, Hassina; Freemantle, Sarah; Pikielny, Claudio; Tolwinski, Nicholas S; Lee, Ethan; Ahmed, Yashi

    2016-01-01

    Wnt/β-catenin signalling directs fundamental processes during metazoan development and can be aberrantly activated in cancer. Wnt stimulation induces the recruitment of the scaffold protein Axin from an inhibitory destruction complex to a stimulatory signalosome. Here we analyse the early effects of Wnt on Axin and find that the ADP-ribose polymerase Tankyrase (Tnks)--known to target Axin for proteolysis-regulates Axin's rapid transition following Wnt stimulation. We demonstrate that the pool of ADP-ribosylated Axin, which is degraded under basal conditions, increases immediately following Wnt stimulation in both Drosophila and human cells. ADP-ribosylation of Axin enhances its interaction with the Wnt co-receptor LRP6, an essential step in signalosome assembly. We suggest that in addition to controlling Axin levels, Tnks-dependent ADP-ribosylation promotes the reprogramming of Axin following Wnt stimulation; and propose that Tnks inhibition blocks Wnt signalling not only by increasing destruction complex activity, but also by impeding signalosome assembly. PMID:27138857

  15. Analysis of Chromatin ADP-Ribosylation at the Genome-wide Level and at Specific Loci by ADPr-ChAP.

    PubMed

    Bartolomei, Giody; Leutert, Mario; Manzo, Massimiliano; Baubec, Tuncay; Hottiger, Michael O

    2016-02-01

    Chromatin ADP-ribosylation regulates important cellular processes. However, the exact location and magnitude of chromatin ADP-ribosylation are largely unknown. A robust and versatile method for assessing chromatin ADP-ribosylation is therefore crucial for further understanding its function. Here, we present a chromatin affinity precipitation method based on the high specificity and avidity of two well-characterized ADP-ribose binding domains to map chromatin ADP-ribosylation at the genome-wide scale and at specific loci. Our ADPr-ChAP method revealed that in cells exposed to oxidative stress, ADP-ribosylation of chromatin scales with histone density, with highest levels at heterochromatic sites and depletion at active promoters. Furthermore, in growth factor-induced adipocyte differentiation, increased chromatin ADP-ribosylation was observed at PPARγ target genes, whose expression is ADP-ribosylation dependent. In combination with deep-sequencing and conventional chromatin immunoprecipitation, the established ADPr-ChAP provides a valuable resource for the bioinformatic comparison of ADP-ribosylation with other chromatin modifications and for addressing its role in other biologically important processes. PMID:26833088

  16. Proteomics Approaches to Identify Mono(ADP-ribosyl)ated and Poly(ADP-ribosyl)ated proteins

    PubMed Central

    Vivelo, Christina A.; Leung, Anthony K. L.

    2015-01-01

    ADP-ribosylation refers to the addition of one or more ADP-ribose units onto protein substrates and this protein modification has been implicated in various cellular processes including DNA damage repair, RNA metabolism, transcription and cell cycle regulation. This review focuses on a compilation of large-scale proteomics studies that identify ADP-ribosylated proteins and their associated proteins by mass spectrometry using a variety of enrichment strategies. Some methods, such as the use of a poly(ADP-ribose)-specific antibody and boronate affinity chromatography and NAD+ analogues, have been employed for decades while others, such as the use of protein microarrays and recombinant proteins that bind ADP-ribose moieties (such as macrodomains), have only recently been developed. The advantages and disadvantages of each method and whether these methods are specific for identifying mono(ADP-ribosyl)ated and poly(ADP-ribosyl)ated proteins will be discussed. Lastly, since poly(ADP-ribose) is heterogeneous in length, it has been difficult to attain a mass signature associated with the modification sites. Several strategies on how to reduce polymer chain length heterogeneity for site identification will be reviewed. PMID:25263235

  17. Proteomics approaches to identify mono-(ADP-ribosyl)ated and poly(ADP-ribosyl)ated proteins.

    PubMed

    Vivelo, Christina A; Leung, Anthony K L

    2015-01-01

    ADP-ribosylation refers to the addition of one or more ADP-ribose units onto protein substrates and this protein modification has been implicated in various cellular processes including DNA damage repair, RNA metabolism, transcription, and cell cycle regulation. This review focuses on a compilation of large-scale proteomics studies that identify ADP-ribosylated proteins and their associated proteins by MS using a variety of enrichment strategies. Some methods, such as the use of a poly(ADP-ribose)-specific antibody and boronate affinity chromatography and NAD(+) analogues, have been employed for decades while others, such as the use of protein microarrays and recombinant proteins that bind ADP-ribose moieties (such as macrodomains), have only recently been developed. The advantages and disadvantages of each method and whether these methods are specific for identifying mono(ADP-ribosyl)ated and poly(ADP-ribosyl)ated proteins will be discussed. Lastly, since poly(ADP-ribose) is heterogeneous in length, it has been difficult to attain a mass signature associated with the modification sites. Several strategies on how to reduce polymer chain length heterogeneity for site identification will be reviewed. PMID:25263235

  18. Quantitative site-specific ADP-ribosylation profiling of DNA-dependent PARPs.

    PubMed

    Gagné, Jean-Philippe; Ethier, Chantal; Defoy, Daniel; Bourassa, Sylvie; Langelier, Marie-France; Riccio, Amanda A; Pascal, John M; Moon, Kyung-Mee; Foster, Leonard J; Ning, Zhibin; Figeys, Daniel; Droit, Arnaud; Poirier, Guy G

    2015-06-01

    An important feature of poly(ADP-ribose) polymerases (PARPs) is their ability to readily undergo automodification upon activation. Although a growing number of substrates were found to be poly(ADP-ribosyl)ated, including histones and several DNA damage response factors, PARPs themselves are still considered as the main acceptors of poly(ADP-ribose). By monitoring spectral counts of specific hydroxamic acid signatures generated after the conversion of the ADP-ribose modification onto peptides by hydroxylamine hydrolysis, we undertook a thorough mass spectrometry mapping of the glutamate and aspartate ADP-ribosylation sites onto automodified PARP-1, PARP-2 and PARP-3. Thousands of hydroxamic acid-conjugated peptides were identified with high confidence and ranked based on their spectral count. This semi-quantitative approach allowed us to locate the preferentially targeted residues in DNA-dependent PARPs. In contrast to what has been reported in the literature, automodification of PARP-1 is not predominantly targeted towards its BRCT domain. Our results show that interdomain linker regions that connect the BRCT to the WGR module and the WGR to the PRD domain undergo prominent ADP-ribosylation during PARP-1 automodification. We also found that PARP-1 efficiently automodifies the D-loop structure within its own catalytic fold. Interestingly, additional major ADP-ribosylation sites were identified in functional domains of PARP-1, including all three zinc fingers. Similar to PARP-1, specific residues located within the catalytic sites of PARP-2 and PARP-3 are major targets of automodification following their DNA-dependent activation. Together our results suggest that poly(ADP-ribosyl)ation hot spots make a dominant contribution to the overall automodification process. PMID:25800440

  19. HPF1/C4orf27 Is a PARP-1-Interacting Protein that Regulates PARP-1 ADP-Ribosylation Activity.

    PubMed

    Gibbs-Seymour, Ian; Fontana, Pietro; Rack, Johannes Gregor Matthias; Ahel, Ivan

    2016-05-01

    We report the identification of histone PARylation factor 1 (HPF1; also known as C4orf27) as a regulator of ADP-ribosylation signaling in the DNA damage response. HPF1/C4orf27 forms a robust protein complex with PARP-1 in cells and is recruited to DNA lesions in a PARP-1-dependent manner, but independently of PARP-1 catalytic ADP-ribosylation activity. Functionally, HPF1 promotes PARP-1-dependent in trans ADP-ribosylation of histones and limits DNA damage-induced hyper-automodification of PARP-1. Human cells lacking HPF1 exhibit sensitivity to DNA damaging agents and PARP inhibition, thereby suggesting an important role for HPF1 in genome maintenance and regulating the efficacy of PARP inhibitors. Collectively, our results demonstrate how a fundamental step in PARP-1-dependent ADP-ribosylation signaling is regulated and suggest that HPF1 functions at the crossroads of histone ADP-ribosylation and PARP-1 automodification. PMID:27067600

  20. HPF1/C4orf27 Is a PARP-1-Interacting Protein that Regulates PARP-1 ADP-Ribosylation Activity

    PubMed Central

    Gibbs-Seymour, Ian; Fontana, Pietro; Rack, Johannes Gregor Matthias; Ahel, Ivan

    2016-01-01

    Summary We report the identification of histone PARylation factor 1 (HPF1; also known as C4orf27) as a regulator of ADP-ribosylation signaling in the DNA damage response. HPF1/C4orf27 forms a robust protein complex with PARP-1 in cells and is recruited to DNA lesions in a PARP-1-dependent manner, but independently of PARP-1 catalytic ADP-ribosylation activity. Functionally, HPF1 promotes PARP-1-dependent in trans ADP-ribosylation of histones and limits DNA damage-induced hyper-automodification of PARP-1. Human cells lacking HPF1 exhibit sensitivity to DNA damaging agents and PARP inhibition, thereby suggesting an important role for HPF1 in genome maintenance and regulating the efficacy of PARP inhibitors. Collectively, our results demonstrate how a fundamental step in PARP-1-dependent ADP-ribosylation signaling is regulated and suggest that HPF1 functions at the crossroads of histone ADP-ribosylation and PARP-1 automodification. PMID:27067600

  1. A Clickable Aminooxy Probe for Monitoring Cellular ADP-Ribosylation

    PubMed Central

    Morgan, Rory K.; Cohen, Michael S.

    2015-01-01

    ADP-ribosylation is essential for cell function, yet there is a dearth of methods for detecting this post-translational modification in cells. Here, we describe a clickable aminooxy alkyne (AO-alkyne) probe that can detect cellular ADP-ribosylation on acidic amino acids following Cu-catalyzed conjugation to an azide-containing reporter. Using AO-alkyne, we show that PARP10 and PARP11 are auto-ADP-ribosylated in cells. We also demonstrate that AO-alkyne can be used to monitor stimulus-induced ADP-ribosylation in cells. Functional studies using AO-alkyne support a previously unknown mechanism for ADP-ribosylation on acidic amino acids, wherein a glutamate or aspartate at the initial C1′-position of ADP-ribose transfers to the C2′ position. This new mechanism for ADP-ribosylation has important implications for how glutamyl/aspartyl-ADP-ribose is recognized by proteins in cells. PMID:25978521

  2. ADP ribosylation factor 1 mutants identify a phospholipase D effector region and reveal that phospholipase D participates in lysosomal secretion but is not sufficient for recruitment of coatomer I.

    PubMed Central

    Jones, D H; Bax, B; Fensome, A; Cockcroft, S

    1999-01-01

    The small GTP-binding protein, ADP-ribosylation factor 1 (ARF1) is essential for the formation of coatomer-coated vesicles from the Golgi and is also an activator of phospholipase D (PLD). Moreover, ARF1-regulated PLD is part of the signal-transduction pathway that can lead to secretion. In this study, substitution and deletion mutants of ARF1 were tested for their ability to activate PLD. These map the PLD effector region of ARF1 to the alpha2 helix, part of the beta2-strand and the N-terminal helix and its ensuing loop. ARF mutants with an increased or decreased ability to activate PLD showed similar characteristics when tested for their ability to stimulate secretion from HL60 cells. ARF1, deleted of the N-terminal 17 amino acid residues (Ndel17), did not support PLD activity or secretion, and neither did it inhibit the activity of wild-type myristoylated ARF1 (myrARF1). In contrast, Ndel17 effectively competed with wild-type myrARF1 to prevent coatomer binding to membranes. This appears to define a structural role for Ndel17, as it can bind a high-molecular mass complex in cytosol. In addition, ethanol has no effect on recruitment of coatomer to membrane. We conclude that the function of ARF-regulated PLD is in the signal-transduction pathway leading to secretion of lysosomal granules, and not as an essential component of ARF1-mediated coatomer binding. PMID:10377261

  3. Structure-based Mechanism of ADP-ribosylation by Sirtuins

    SciTech Connect

    Hawse, William F.; Wolberger, Cynthia

    2009-12-01

    Sirtuins comprise a family of enzymes found in all organisms, where they play a role in diverse processes including transcriptional silencing, aging, regulation of transcription, and metabolism. The predominant reaction catalyzed by these enzymes is NAD{sup +}-dependent lysine deacetylation, although some sirtuins exhibit a weaker ADP-ribosyltransferase activity. Although the Sir2 deacetylation mechanism is well established, much less is known about the Sir2 ADP-ribosylation reaction. We have studied the ADP-ribosylation activity of a bacterial sirtuin, Sir2Tm, and show that acetylated peptides containing arginine or lysine 2 residues C-terminal to the acetyl lysine, the +2 position, are preferentially ADP-ribosylated at the +2 residue. A structure of Sir2Tm bound to the acetylated +2 arginine peptide shows how this arginine could enter the active site and react with a deacetylation reaction intermediate to yield an ADP-ribosylated peptide. The new biochemical and structural studies presented here provide mechanistic insights into the Sir2 ADP-ribosylation reaction and will aid in identifying substrates of this reaction.

  4. Arginine-Specific Mono ADP-Ribosylation In Vitro of Antimicrobial Peptides by ADP-Ribosylating Toxins

    PubMed Central

    Castagnini, Marta; Picchianti, Monica; Talluri, Eleonora; Biagini, Massimiliano; Del Vecchio, Mariangela; Di Procolo, Paolo; Norais, Nathalie; Nardi-Dei, Vincenzo; Balducci, Enrico

    2012-01-01

    Among the several toxins used by pathogenic bacteria to target eukaryotic host cells, proteins that exert ADP-ribosylation activity represent a large and studied family of dangerous and potentially lethal toxins. These proteins alter cell physiology catalyzing the transfer of the ADP-ribose unit from NAD to cellular proteins involved in key metabolic pathways. In the present study, we tested the capability of four of these toxins, to ADP-ribosylate α- and β- defensins. Cholera toxin (CT) from Vibrio cholerae and heat labile enterotoxin (LT) from Escherichia coli both modified the human α-defensin (HNP-1) and β- defensin-1 (HBD1), as efficiently as the mammalian mono-ADP-ribosyltransferase-1. Pseudomonas aeruginosa exoenzyme S was inactive on both HNP-1 and HBD1. Neisseria meningitidis NarE poorly recognized HNP-1 as a substrate but it was completely inactive on HBD1. On the other hand, HNP-1 strongly influenced NarE inhibiting its transferase activity while enhancing auto-ADP-ribosylation. We conclude that only some arginine-specific ADP-ribosylating toxins recognize defensins as substrates in vitro. Modifications that alter the biological activities of antimicrobial peptides may be relevant for the innate immune response. In particular, ADP-ribosylation of antimicrobial peptides may represent a novel escape mechanism adopted by pathogens to facilitate colonization of host tissues. PMID:22879887

  5. ADP-ribosylation of proteins: Enzymology and biological significance

    SciTech Connect

    Althaus, F.R.; Richter, C.

    1987-01-01

    This book presents an overview of the molecular and biological consequences of the posttranslational modification of proteins with ADP-ribose monomers and polymers. Part one focuses on chromatin-associated poly ADP-ribosylation reactions which have evolved in higher eukaryotes as modulators of chromatin functions. The significance of poly ADP-ribosylation in DNA repair, carcinogenesis, and gene expression during terminal differentiation is discussed. Part two reviews mono ADP-ribosylation reactions which are catalyzed by prokaryotic and eukaryotic enzymes. Consideration is given to the action of bacterial toxins, such as cholera toxin, pertussis toxin, and diphtheria toxin. These toxins have emerged as tools for the molecular probing of proteins involved in signal transduction and protein biosynthesis.

  6. PARPs and ADP-Ribosylation: Fifty Years… and Counting

    PubMed Central

    Kraus, W. Lee

    2015-01-01

    Summary Over 50 years ago, the discovery of poly(ADP-ribose) (PAR) set a new field of science in motion - the field of poly(ADP-ribosyl) transferases (PARPs) and ADP-ribosylation. The field is still flourishing today. The diversity of biological processes now known to require PARPs and ADP-ribosylation was practically unimaginable even two decades ago. From an initial focus on DNA damage detection and repair in response to genotoxic stresses, the field has expanded to include the regulation of chromatin structure, gene expression, and RNA processing in a wide range of biological systems, including reproduction, development, aging, stem cells, inflammation, metabolism, and cancer. This special focus issue of Molecular Cell includes a collection of three Reviews, three Perspectives, and a SnapShot, which together summarize the current state of the field and suggest where it may be headed. PMID:26091339

  7. Intracellular Mono-ADP-Ribosylation in Signaling and Disease

    PubMed Central

    Bütepage, Mareike; Eckei, Laura; Verheugd, Patricia; Lüscher, Bernhard

    2015-01-01

    A key process in the regulation of protein activities and thus cellular signaling pathways is the modification of proteins by post-translational mechanisms. Knowledge about the enzymes (writers and erasers) that attach and remove post-translational modifications, the targets that are modified and the functional consequences elicited by specific modifications, is crucial for understanding cell biological processes. Moreover detailed knowledge about these mechanisms and pathways helps to elucidate the molecular causes of various diseases and in defining potential targets for therapeutic approaches. Intracellular adenosine diphosphate (ADP)-ribosylation refers to the nicotinamide adenine dinucleotide (NAD+)-dependent modification of proteins with ADP-ribose and is catalyzed by enzymes of the ARTD (ADP-ribosyltransferase diphtheria toxin like, also known as PARP) family as well as some members of the Sirtuin family. Poly-ADP-ribosylation is relatively well understood with inhibitors being used as anti-cancer agents. However, the majority of ARTD enzymes and the ADP-ribosylating Sirtuins are restricted to catalyzing mono-ADP-ribosylation. Although writers, readers and erasers of intracellular mono-ADP-ribosylation have been identified only recently, it is becoming more and more evident that this reversible post-translational modification is capable of modulating key intracellular processes and signaling pathways. These include signal transduction mechanisms, stress pathways associated with the endoplasmic reticulum and stress granules, and chromatin-associated processes such as transcription and DNA repair. We hypothesize that mono-ADP-ribosylation controls, through these different pathways, the development of cancer and infectious diseases. PMID:26426055

  8. ADP-ribosylation of transducin by pertussis toxin

    SciTech Connect

    Watkins, P.A.; Burns, D.L.; Kanaho, Y.; Liu, T.Y.; Hewlett, E.L.; Moss, J.

    1985-11-05

    Transducin, the guanyl nucleotide-binding regulatory protein of retinal rod outer segments that couples the photon receptor, rhodopsin, with the light-activated cGMP phosphodiesterase, can be resolved into two functional components, T alpha and T beta gamma. T alpha (39 kDa), which is (TSP)ADP-ribosylated by pertussis toxin and (TSP)NAD in rod outer segments and in purified transducin, was also labeled by the toxin after separation from T beta gamma (36 kDa and approximately 10 kDa); neither component of T beta gamma was a pertussis toxin substrate. Labeling of T alpha was enhanced by T beta gamma and was maximal at approximately 1:1 molar ratio of T alpha : T beta gamma. Limited proteolysis by trypsin of T alpha in the presence of guanyl-5'-yl imidodiphosphate (Gpp(NH)p) resulted in the sequential appearance of proteins of 38 and TS kDa. The amino terminus of both 38- and TS-kDa proteins was leucine, whereas that of T alpha could not be identified and was assumed to be blocked. The TS-kDa peptide was not a pertussis toxin substrate. Labeling of the 38-kDa protein was poor and was not enhanced by T beta gamma. Trypsin treatment of (TSP)ADP-ribosyl-T alpha produced a labeled 37-38-kDa doublet followed by appearance of radioactivity at the dye front. It appears, therefore, that, although the 38-kDa protein was poor toxin substrate, it contained the ADP-ribosylation site. Without rhodopsin, labeling of T alpha (in the presence of T beta gamma) was unaffected by Gpp(NH)p, guanosine 5'-O-(thiotriphosphate) (GTP gamma S), GTP, GDP, and guanosine 5'-O-(thiodiphosphate) (GDP beta S) but was increased by ATP. When photolyzed rhodopsin and T beta gamma were present, Gpp(NH)p and GTP gamma S decreased (TSP)ADP-ribosylation by pertussis toxin. Thus, pertussis toxin-catalyzed (TSP)ADP-ribosylation of T alpha was affected by nucleotides, rhodopsin and light in addition to T beta gamma.

  9. Pierisins and CARP-1: ADP-ribosylation of DNA by ARTCs in butterflies and shellfish.

    PubMed

    Nakano, Tsuyoshi; Takahashi-Nakaguchi, Azusa; Yamamoto, Masafumi; Watanabe, Masahiko

    2015-01-01

    The cabbage butterfly, Pieris rapae, and related species possess a previously unknown ADP-ribosylating toxin, guanine specific ADP-ribosyltransferase. This enzyme toxin, known as pierisin, consists of enzymatic N-terminal domain and receptor-binding C-terminal domain, or typical AB-toxin structure. Pierisin efficiently transfers an ADP-ribosyl moiety to the N(2) position of the guanine base of dsDNA. Receptors for pierisin are suggested to be the neutral glycosphingolipids, globotriaosylceramide (Gb3), and globotetraosylceramide (Gb4). This DNA-modifying toxin exhibits strong cytotoxicity and induces apoptosis in various human cell lines, which can be blocked by Bcl-2. Pierisin also produces detrimental effects on the eggs and larvae of the non-habitual parasitoids. In contrast, a natural parasitoid of the cabbage butterfly, Cotesia glomerata, was resistant to this toxin. The physiological role of pierisin in the butterfly is suggested to be a defense factor against parasitization by wasps. Other type of DNA ADP-ribosyltransferase is present in certain kinds of edible clams. For example, the CARP-1 protein found in Meretrix lamarckii consists of an enzymatic domain without a possible receptor-binding domain. Pierisin and CARP-1 are almost fully non-homologous at the amino acid sequence level, but other ADP-ribosyltransferases homologous to pierisin are present in different biological species such as eubacterium Streptomyces. Possible diverse physiological roles of the DNA ADP-ribosyltransferases are discussed. PMID:25033755

  10. Transcutaneous Immunization with Bacterial ADP-Ribosylating Exotoxins, Subunits, and Unrelated Adjuvants

    PubMed Central

    Scharton-Kersten, Tanya; Yu, Jian-mei; Vassell, Russell; O'Hagan, Derek; Alving, Carl R.; Glenn, Gregory M.

    2000-01-01

    We have recently described a needle-free method of vaccination, transcutaneous immunization, consisting of the topical application of vaccine antigens to intact skin. While most proteins themselves are poor immunogens on the skin, we have shown that the addition of cholera toxin (CT), a mucosal adjuvant, results in cellular and humoral immune responses to the adjuvant and coadministered antigens. The present study explores the breadth of adjuvants that have activity on the skin, using diphtheria toxoid (DTx) and tetanus toxoid as model antigens. Heat-labile enterotoxin (LT) displayed adjuvant properties similar to those of CT when used on the skin and induced protective immune responses against tetanus toxin challenge when applied topically at doses as low as 1 μg. Interestingly, enterotoxin derivatives LTR192G, LTK63, and LTR72 and the recombinant CT B subunit also exhibited adjuvant properties on the skin. Consistent with the latter finding, non-ADP-ribosylating exotoxins, including an oligonucleotide DNA sequence, as well as several cytokines (interleukin-1β [IL-1β] fragment, IL-2, IL-12, and tumor necrosis factor alpha) and lipopolysaccharide also elicited detectable anti-DTx immunoglobulin G titers in the immunized mice. These results indicate that enhancement of the immune response to topical immunization is not restricted to CT or the ADP-ribosylating exotoxins as adjuvants. This study also reinforces earlier findings that addition of an adjuvant is important for the induction of robust immune responses to vaccine antigens delivered by topical application. PMID:10948159

  11. Rapid Evolution of PARP Genes Suggests a Broad Role for ADP-Ribosylation in Host-Virus Conflicts

    PubMed Central

    Daugherty, Matthew D.; Young, Janet M.; Kerns, Julie A.; Malik, Harmit S.

    2014-01-01

    Post-translational protein modifications such as phosphorylation and ubiquitinylation are common molecular targets of conflict between viruses and their hosts. However, the role of other post-translational modifications, such as ADP-ribosylation, in host-virus interactions is less well characterized. ADP-ribosylation is carried out by proteins encoded by the PARP (also called ARTD) gene family. The majority of the 17 human PARP genes are poorly characterized. However, one PARP protein, PARP13/ZAP, has broad antiviral activity and has evolved under positive (diversifying) selection in primates. Such evolution is typical of domains that are locked in antagonistic ‘arms races’ with viral factors. To identify additional PARP genes that may be involved in host-virus interactions, we performed evolutionary analyses on all primate PARP genes to search for signatures of rapid evolution. Contrary to expectations that most PARP genes are involved in ‘housekeeping’ functions, we found that nearly one-third of PARP genes are evolving under strong recurrent positive selection. We identified a >300 amino acid disordered region of PARP4, a component of cytoplasmic vault structures, to be rapidly evolving in several mammalian lineages, suggesting this region serves as an important host-pathogen specificity interface. We also found positive selection of PARP9, 14 and 15, the only three human genes that contain both PARP domains and macrodomains. Macrodomains uniquely recognize, and in some cases can reverse, protein mono-ADP-ribosylation, and we observed strong signatures of recurrent positive selection throughout the macro-PARP macrodomains. Furthermore, PARP14 and PARP15 have undergone repeated rounds of gene birth and loss during vertebrate evolution, consistent with recurrent gene innovation. Together with previous studies that implicated several PARPs in immunity, as well as those that demonstrated a role for virally encoded macrodomains in host immune evasion, our

  12. Arsenite induced poly(ADP-ribosyl)ation of tumor suppressor P53 in human skin keratinocytes as a possible mechanism for carcinogenesis associated with arsenic exposure

    SciTech Connect

    Komissarova, Elena V.; Rossman, Toby G.

    2010-03-15

    Arsenite is an environmental pollutant. Exposure to inorganic arsenic in drinking water is associated with elevated cancer risk, especially in skin. Arsenite alone does not cause skin cancer in animals, but arsenite can enhance the carcinogenicity of solar UV. Arsenite is not a significant mutagen at non-toxic concentrations, but it enhances the mutagenicity of other carcinogens. The tumor suppressor protein P53 and nuclear enzyme PARP-1 are both key players in DNA damage response. This laboratory demonstrated earlier that in cells treated with arsenite, the P53-dependent increase in p21{sup WAF1/CIP1} expression, normally a block to cell cycle progression after DNA damage, is deficient. Here we show that although long-term exposure of human keratinocytes (HaCaT) to a nontoxic concentration (0.1 muM) of arsenite decreases the level of global protein poly(ADP-ribosyl)ation, it increases poly(ADP-ribosyl)ation of P53 protein and PARP-1 protein abundance. We also demonstrate that exposure to 0.1 muM arsenite depresses the constitutive expression of p21 mRNA and P21 protein in HaCaT cells. Poly(ADP-ribosyl)ation of P53 is reported to block its activation, DNA binding and its functioning as a transcription factor. Our results suggest that arsenite's interference with activation of P53 via poly(ADP-ribosyl)ation may play a role in the comutagenic and cocarcinogenic effects of arsenite.

  13. Chemical reporters for exploring ADP-ribosylation and AMPylation at the host-pathogen interface

    PubMed Central

    Westcott, Nathan P.; Hang, Howard C.

    2014-01-01

    Bacterial pathogens secrete protein toxins and effectors that hijack metabolites to covalently modify key host proteins and interfere with their function during infection. Adenosine metabolites, such as nicotinamide adenine dinucleotide (NAD) and adenosine triphosphate (ATP), have in particular been co-opted by these secreted virulence factors to reprogram host pathways. While some host targets for secreted virulence factors have been identified, other toxin and effector substrates have been elusive, which require new methods for their characterization. In this review, we focus on chemical reporters based on NAD and ATP that should facilitate the discovery and characterization of adenosine diphosphate (ADP)-ribosylation and adenylylation/AMPylation in bacterial pathogenesis and cell biology. PMID:25461386

  14. 50Years of poly(ADP-ribosyl)ation.

    PubMed

    Virág, László

    2013-12-01

    The seminal paper published in 1963 by Chambon, Weil and Mandel reporting a new NAD-dependent protein modification now known as poly(ADP-ribosyl)ation (PARylation) marked the launch of a new era in both protein research and cell biology. In the coming decades, the identity, biochemical characteristics and regulation of enzymes responsible for the synthesis and degradation of protein-bound poly(ADP-ribose) have been discovered and the surprisingly multifarious biological roles of PARylation have not ceased to amaze cell and molecular biologists ever since. The review series on PARylation following this preface is comprised of ten papers written by great experts of the field and aims to provide practicing physicians and basic scientists with the state-of-the-art on the "writers, readers and erasers" of poly(ADP-ribose), some recent paradigm shifts of the field and its translational potential. PMID:23727362

  15. Aryl Hydrocarbon Receptor Activation by Dioxin Targets Phosphoenolpyruvate Carboxykinase (PEPCK) for ADP-ribosylation via 2,3,7,8-Tetrachlorodibenzo-p-dioxin (TCDD)-inducible Poly(ADP-ribose) Polymerase (TiPARP)*

    PubMed Central

    Diani-Moore, Silvia; Zhang, Sheng; Ram, Payal; Rifkind, Arleen B.

    2013-01-01

    Effects of the environmental toxin and carcinogen 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD, dioxin) include a wasting syndrome associated with decreased gluconeogenesis. TCDD is a potent activator of the aryl hydrocarbon receptor (AHR), a ligand activated transcription factor. The relationship between gene activation by the AHR and TCDD toxicities is not well understood. We recently identified a pathway by which the AHR target gene TiPARP (TCDD-inducible poly(ADP-ribose) polymerase) contributes to TCDD suppression of transcription of phosphoenolpyruvate carboxykinase (PEPCK), a key regulator of gluconeogenesis, by consuming NAD+ and decreasing Sirtuin 1 activation of the peroxisome proliferator-activated receptor γ coactivator 1α (PGC1α), a transcriptional activator of PEPCK. We report here that TCDD-induced TiPARP also targets PEPCK for ADP-ribosylation. Both cytosolic and mitochondrial forms of PEPCK were found to undergo ADP-ribosylation. Unexpectedly, AHR suppression also enhanced ADP-ribosylation and did so by a poly(ADP-ribose) polymerase-independent mechanism. This report 1) identifies ADP-ribosylation as a new posttranslational modification for PEPCK, 2) describes a pathway by which transcriptional induction of TiPARP by the AHR can lead to a downstream posttranslational change in a TCDD target protein (PEPCK), and 3) reveals that the AHR exerts complex, previously unidentified modulatory effects on ADP-ribosylation. PMID:23770670

  16. The Promise of Proteomics for the Study of ADP-ribosylation

    PubMed Central

    Daniels, Casey M.; Ong, Shao-En; Leung, Anthony K. L.

    2015-01-01

    ADP-ribosylation is a post-translational modification where single units (mono-ADP-ribosylation) or polymeric chains (poly-ADP-ribosylation) of ADP-ribose are conjugated to proteins by ADP-ribosyltransferases. This post-translational modification and the ADP-ribosyltransferases (also known as PARPs) responsible for its synthesis have been found to play a role in nearly all major cellular processes, including DNA repair, transcription, translation, cell signaling and cell death. Furthermore, dysregulation of ADP-ribosylation has been linked to diseases including cancers, diabetes, neurodegenerative disorders and heart failure, leading to the development of therapeutic PARP inhibitors, many of which are currently in clinical trials. The study of this therapeutically important modification has recently been bolstered by the application of mass spectrometry-based proteomics, arguably the most powerful tool for the unbiased analysis of protein modifications. Unfortunately, progress has been hampered by the inherent challenges that stem from the physicochemical properties of ADP-ribose which as a post-translational modification is highly charged, heterogeneous (linear or branched polymers, as well as monomers), labile, and found on a wide range of amino acid acceptors. In this perspective, we discuss the progress that has been made in addressing these challenges, including the recent breakthroughs in proteomics techniques to identify ADP-ribosylation sites, and future developments to provide a proteome-wide view of the many cellular processes regulated by ADP-ribosylation. PMID:26091340

  17. Activation of exocytosis by cross-linking of the IgE receptor is dependent on ADP-ribosylation factor 1-regulated phospholipase D in RBL-2H3 mast cells: evidence that the mechanism of activation is via regulation of phosphatidylinositol 4,5-bisphosphate synthesis.

    PubMed Central

    Way, G; O'luanaigh, N; Cockcroft, S

    2000-01-01

    The physiological stimulus to exocytosis in mast cells is the cross-linking of the high-affinity IgE receptor, FcepsilonR1, with antigen. We demonstrate a novel function for ADP-ribosylation factor 1 (ARF1) in the regulation of antigen-stimulated secretion using cytosol-depleted RBL-2H3 mast cells for reconstitution of secretory responses. When antigen is used as the stimulus, ARF1 also reconstitutes phospholipase D activation. Using ethanol to divert the phosphatidic acid (the product of phospholipase D activity) to phosphatidylethanol causes inhibition of ARF1-reconstituted secretion. In addition. ARF1 causes an increase in phosphatidylinositol 4,5-bisphosphate (PIP(2)) levels at the expense of phosphatidylinositol 4-monophosphate. The requirement for PIP(2) in exocytosis was confirmed by using phosphatidylinositol transfer protein (PITPalpha) to increase PIP(2) levels. Exocytosis, restored by either ARF1 or PITPalpha, was inhibited when PIP(2) levels were depleted by phospholipase Cdelta1. We conclude that the function of ARF1 and PITPalpha is to increase the local synthesis of PIP(2), the function of which in exocytosis is likely to be linked to lipid-protein interactions, whereby recruitment of key components of the exocytotic machinery are targeted to the appropriate membrane compartment. PMID:10657240

  18. Poly(ADP-Ribosyl)ation Affects Histone Acetylation and Transcription

    PubMed Central

    Verdone, Loredana; La Fortezza, Marco; Ciccarone, Fabio; Caiafa, Paola; Zampieri, Michele; Caserta, Micaela

    2015-01-01

    Poly(ADP-ribosyl)ation (PARylation) is a posttranslational protein modification catalyzed by members of the poly(ADP-ribose) polymerase (PARP) enzyme family. PARylation regulates a wide variety of biological processes in most eukaryotic cells including energy metabolism and cell death, maintenance of genomic stability, chromatin structure and transcription. Inside the nucleus, cross-talk between PARylation and other epigenetic modifications, such as DNA and histone methylation, was already described. In the present work, using PJ34 or ABT888 to inhibit PARP activity or over-expressing poly(ADP-ribose) glycohydrolase (PARG), we show decrease of global histone H3 and H4 acetylation. This effect is accompanied by a reduction of the steady state mRNA level of p300, Pcaf, and Tnfα, but not of Dnmt1. Chromatin immunoprecipitation (ChIP) analyses, performed at the level of the Transcription Start Site (TSS) of these four genes, reveal that changes in histone acetylation are specific for each promoter. Finally, we demonstrate an increase of global deacetylase activity in nuclear extracts from cells treated with PJ34, whereas global acetyltransferase activity is not affected, suggesting a role for PARP in the inhibition of histone deacetylases. Taken together, these results show an important link between PARylation and histone acetylation regulated transcription. PMID:26636673

  19. Role of NAD+ and ADP-Ribosylation in the Maintenance of the Golgi Structure

    PubMed Central

    Mironov, Alexander; Colanzi, Antonino; Silletta, Maria Giuseppina; Fiucci, Giusy; Flati, Silvio; Fusella, Aurora; Polishchuk, Roman; Mironov, Alexander; Tullio, Giuseppe Di; Weigert, Roberto; Malhotra, Vivek; Corda, Daniela; Matteis, Maria Antonietta De; Luini, Alberto

    1997-01-01

    We have investigated the role of the ADP- ribosylation induced by brefeldin A (BFA) in the mechanisms controlling the architecture of the Golgi complex. BFA causes the rapid disassembly of this organelle into a network of tubules, prevents the association of coatomer and other proteins to Golgi membranes, and stimulates the ADP-ribosylation of two cytosolic proteins of 38 and 50 kD (GAPDH and BARS-50; De Matteis, M.A., M. DiGirolamo, A. Colanzi, M. Pallas, G. Di Tullio, L.J. McDonald, J. Moss, G. Santini, S. Bannykh, D. Corda, and A. Luini. 1994. Proc. Natl. Acad. Sci. USA. 91:1114–1118; Di Girolamo, M., M.G. Silletta, M.A. De Matteis, A. Braca, A. Colanzi, D. Pawlak, M.M. Rasenick, A. Luini, and D. Corda. 1995. Proc. Natl. Acad. Sci. USA. 92:7065–7069). To study the role of ADP-ribosylation, this reaction was inhibited by depletion of NAD+ (the ADP-ribose donor) or by using selective pharmacological blockers in permeabilized cells. In NAD+-depleted cells and in the presence of dialized cytosol, BFA detached coat proteins from Golgi membranes with normal potency but failed to alter the organelle's structure. Readdition of NAD+ triggered Golgi disassembly by BFA. This effect of NAD+ was mimicked by the use of pre–ADP- ribosylated cytosol. The further addition of extracts enriched in native BARS-50 abolished the ability of ADP-ribosylated cytosol to support the effect of BFA. Pharmacological blockers of the BFA-dependent ADP-ribosylation (Weigert, R., A. Colanzi, A. Mironov, R. Buccione, C. Cericola, M.G. Sciulli, G. Santini, S. Flati, A. Fusella, J. Donaldson, M. DiGirolamo, D. Corda, M.A. De Matteis, and A. Luini. 1997. J. Biol. Chem. 272:14200–14207) prevented Golgi disassembly by BFA in permeabilized cells. These inhibitors became inactive in the presence of pre–ADP-ribosylated cytosol, and their activity was rescued by supplementing the cytosol with a native BARS-50–enriched fraction. These results indicate that ADP-ribosylation plays a role in the

  20. State of the art of protein mono-ADP-ribosylation: biological role and therapeutic potential.

    PubMed

    Fabrizio, Gaia; Scarpa, Emanuele Salvatore; Di Girolamo, Maria

    2015-01-01

    Mono-ADP-ribosylation is a post-translational modification that was discovered more than five decades ago, and it consists of the enzymatic transfer of ADP-ribose from NAD⁺ to acceptor proteins. In viruses and prokaryotes, mono-ADP-ribosylation is mainly, but not exclusively, a mechanism used to take control of the host cell. In mammals, mono-ADP-ribosylation serves to regulate protein functions, and it is catalysed by two families of toxin-related cellular ADP-ribosyltransferases: ecto-enzymes that modify various cell-surface proteins, like integrins and receptors, and intracellular enzymes that act on a variety of nuclear and cytosolic proteins. These two families have been recently renamed the ARTCs (clostridia toxin like) and ARTDs (diphtheria toxin like), depending on their conserved structural features, and in terms of their relationships to the bacterial toxins. In addition, two members of the structurally non-related sirtuin family can also modify cellular proteins by mono-ADP-ribosylation. Recently, new examples of ADP-ribosylation of proteins involved in signal transduction and intracellular trafficking have been discovered, thus opening the route to the better molecular understanding of this reaction and of its role in human cell physiology and pathology. PMID:25553458

  1. ADP-ribosylation of membrane components by pertussis and cholera toxin

    SciTech Connect

    Ribeiro-Neto, F.A.P.; Mattera, F.; Hildebrandt, J.D.; Codina, J.; Field, J.B.; Birnbaumer, L.; Sekura, R.D.

    1985-01-01

    Pertussis and cholera toxins are important tools to investigate functional and structural aspects of the stimulatory (N/sub s/) and inhibitory (N/sub i/) regulatory components of adenylyl cyclase. Cholera toxin acts on N/sub s/ by ADP-ribosylating its ..cap alpha../sub s/ subunit; pertussis toxin acts on N/sub i/ by ADP-ribosylating its ..cap alpha..; subunit. By using (/sup 32/P)NAD/sup +/ and determining the transfer of its (/sup 32/P)ADP-ribose moiety to membrane components, it is possible to obtain information on N/sub s/ and N/sub i/. A set of protocols is presented that can be used to study simultaneously and comparatively the susceptibility of N/sub s/ and N/sub i/ to be ADP-ribosylated by cholera and pertussis toxin.

  2. Identification of a Class of Protein ADP-Ribosylating Sirtuins in Microbial Pathogens.

    PubMed

    Rack, Johannes Gregor Matthias; Morra, Rosa; Barkauskaite, Eva; Kraehenbuehl, Rolf; Ariza, Antonio; Qu, Yue; Ortmayer, Mary; Leidecker, Orsolya; Cameron, David R; Matic, Ivan; Peleg, Anton Y; Leys, David; Traven, Ana; Ahel, Ivan

    2015-07-16

    Sirtuins are an ancient family of NAD(+)-dependent deacylases connected with the regulation of fundamental cellular processes including metabolic homeostasis and genome integrity. We show the existence of a hitherto unrecognized class of sirtuins, found predominantly in microbial pathogens. In contrast to earlier described classes, these sirtuins exhibit robust protein ADP-ribosylation activity. In our model organisms, Staphylococcus aureus and Streptococcus pyogenes, the activity is dependent on prior lipoylation of the target protein and can be reversed by a sirtuin-associated macrodomain protein. Together, our data describe a sirtuin-dependent reversible protein ADP-ribosylation system and establish a crosstalk between lipoylation and mono-ADP-ribosylation. We propose that these posttranslational modifications modulate microbial virulence by regulating the response to host-derived reactive oxygen species. PMID:26166706

  3. Identification of a Class of Protein ADP-Ribosylating Sirtuins in Microbial Pathogens

    PubMed Central

    Rack, Johannes Gregor Matthias; Morra, Rosa; Barkauskaite, Eva; Kraehenbuehl, Rolf; Ariza, Antonio; Qu, Yue; Ortmayer, Mary; Leidecker, Orsolya; Cameron, David R.; Matic, Ivan; Peleg, Anton Y.; Leys, David; Traven, Ana; Ahel, Ivan

    2015-01-01

    Summary Sirtuins are an ancient family of NAD+-dependent deacylases connected with the regulation of fundamental cellular processes including metabolic homeostasis and genome integrity. We show the existence of a hitherto unrecognized class of sirtuins, found predominantly in microbial pathogens. In contrast to earlier described classes, these sirtuins exhibit robust protein ADP-ribosylation activity. In our model organisms, Staphylococcus aureus and Streptococcus pyogenes, the activity is dependent on prior lipoylation of the target protein and can be reversed by a sirtuin-associated macrodomain protein. Together, our data describe a sirtuin-dependent reversible protein ADP-ribosylation system and establish a crosstalk between lipoylation and mono-ADP-ribosylation. We propose that these posttranslational modifications modulate microbial virulence by regulating the response to host-derived reactive oxygen species. PMID:26166706

  4. Yeast Golgi-localized, gamma-Ear-containing, ADP-ribosylation factor-binding proteins are but adaptor protein-1 is not required for cell-free transport of membrane proteins from the trans-Golgi network to the prevacuolar compartment.

    PubMed

    Abazeed, Mohamed E; Fuller, Robert S

    2008-11-01

    Golgi-localized, gamma-Ear-containing, ADP-ribosylation factor-binding proteins (GGAs) and adaptor protein-1 (AP-1) mediate clathrin-dependent trafficking of transmembrane proteins between the trans-Golgi network (TGN) and endosomes. In yeast, the vacuolar sorting receptor Vps10p follows a direct pathway from the TGN to the late endosome/prevacuolar compartment (PVC), whereas, the processing protease Kex2p partitions between the direct pathway and an indirect pathway through the early endosome. To examine the roles of the Ggas and AP-1 in TGN-PVC transport, we used a cell-free assay that measures delivery to the PVC of either Kex2p or a chimeric protein (K-V), in which the Vps10p cytosolic tail replaces the Kex2p tail. Either antibody inhibition or dominant-negative Gga2p completely blocked K-V transport but only partially blocked Kex2p transport. Deletion of APL2, encoding the beta subunit of AP-1, did not affect K-V transport but partially blocked Kex2p transport. Residual Kex2p transport seen with apl2Delta membranes was insensitive to dominant-negative Gga2p, suggesting that the apl2Delta mutation causes Kex2p to localize to a compartment that precludes Gga-dependent trafficking. These results suggest that yeast Ggas facilitate the specific and direct delivery of Vps10p and Kex2p from the TGN to the PVC and that AP-1 modulates Kex2p trafficking through a distinct pathway, presumably involving the early endosome. PMID:18784256

  5. Yeast Golgi-localized, γ-Ear–containing, ADP-Ribosylation Factor-binding Proteins Are but Adaptor Protein-1 Is Not Required for Cell-free Transport of Membrane Proteins from the Trans-Golgi Network to the Prevacuolar Compartment

    PubMed Central

    Abazeed, Mohamed E.

    2008-01-01

    Golgi-localized, γ-Ear–containing, ADP-ribosylation factor-binding proteins (GGAs) and adaptor protein-1 (AP-1) mediate clathrin-dependent trafficking of transmembrane proteins between the trans-Golgi network (TGN) and endosomes. In yeast, the vacuolar sorting receptor Vps10p follows a direct pathway from the TGN to the late endosome/prevacuolar compartment (PVC), whereas, the processing protease Kex2p partitions between the direct pathway and an indirect pathway through the early endosome. To examine the roles of the Ggas and AP-1 in TGN–PVC transport, we used a cell-free assay that measures delivery to the PVC of either Kex2p or a chimeric protein (K-V), in which the Vps10p cytosolic tail replaces the Kex2p tail. Either antibody inhibition or dominant-negative Gga2p completely blocked K-V transport but only partially blocked Kex2p transport. Deletion of APL2, encoding the β subunit of AP-1, did not affect K-V transport but partially blocked Kex2p transport. Residual Kex2p transport seen with apl2Δ membranes was insensitive to dominant-negative Gga2p, suggesting that the apl2Δ mutation causes Kex2p to localize to a compartment that precludes Gga-dependent trafficking. These results suggest that yeast Ggas facilitate the specific and direct delivery of Vps10p and Kex2p from the TGN to the PVC and that AP-1 modulates Kex2p trafficking through a distinct pathway, presumably involving the early endosome. PMID:18784256

  6. Poly(ADP-ribosyl)ation reactions in the regulation of nuclear functions.

    PubMed Central

    D'Amours, D; Desnoyers, S; D'Silva, I; Poirier, G G

    1999-01-01

    Poly(ADP-ribosyl)ation is a post-translational modification of proteins. During this process, molecules of ADP-ribose are added successively on to acceptor proteins to form branched polymers. This modification is transient but very extensive in vivo, as polymer chains can reach more than 200 units on protein acceptors. The existence of the poly(ADP-ribose) polymer was first reported nearly 40 years ago. Since then, the importance of poly(ADP-ribose) synthesis has been established in many cellular processes. However, a clear and unified picture of the physiological role of poly(ADP-ribosyl)ation still remains to be established. The total dependence of poly(ADP-ribose) synthesis on DNA strand breaks strongly suggests that this post-translational modification is involved in the metabolism of nucleic acids. This view is also supported by the identification of direct protein-protein interactions involving poly(ADP-ribose) polymerase (113 kDa PARP), an enzyme catalysing the formation of poly(ADP-ribose), and key effectors of DNA repair, replication and transcription reactions. The presence of PARP in these multiprotein complexes, in addition to the actual poly(ADP-ribosyl)ation of some components of these complexes, clearly supports an important role for poly(ADP-ribosyl)ation reactions in DNA transactions. Accordingly, inhibition of poly(ADP-ribose) synthesis by any of several approaches and the analysis of PARP-deficient cells has revealed that the absence of poly(ADP-ribosyl)ation strongly affects DNA metabolism, most notably DNA repair. The recent identification of new poly(ADP-ribosyl)ating enzymes with distinct (non-standard) structures in eukaryotes and archaea has revealed a novel level of complexity in the regulation of poly(ADP-ribose) metabolism. PMID:10455009

  7. The inhibitory G protein G(i) identified as pertussis toxin-catalyzed ADP-ribosylation.

    PubMed

    Katada, Toshiaki

    2012-01-01

    Pertussis toxin (PTX) produced by Bordetella pertussis was first introduced by Ui and his colleagues in research on signal transduction under the name islet-activating protein in 1979, when the mechanism of toxin-induced stimulation of insulin release from pancreatic islets was reported in the rat. The stimulatory effect of PTX in vivo results from the blockage of α(2)-adrenergic receptor-mediated inhibition of insulin release. The receptor-induced inhibition of cAMP formation was also abolished in pancreatic islets isolated from PTX-treated rats, suggesting that the toxin caused uncoupling of adenylyl cyclase inhibition from receptor stimulation. The action of PTX on isolated membranes required a cytosolic factor, nicotinamide adenine dinucleotide (NAD), and the uncoupling induced by PTX was shown to be due to the toxin-catalyzed ADP-ribosylation of a 41-kDa protein with NAD as another substrate. The 41-kDa PTX substrate was soon identified and purified as the α-subunit of the inhibitory G protein that transmits an inhibitory signal from membrane receptors to adenylyl cyclase. After demonstration of the molecular mechanism of PTX, the toxin was widely utilized as a probe for identifying and analyzing major αβγ-trimeric G proteins. Thus, PTX-sensitive G proteins appeared to carry positive and negative signals from many membrane receptors to a variety of effectors other than adenylyl cyclase. PMID:23207763

  8. Poly-ADP-ribosylation of HMGB1 regulates TNFSF10/TRAIL resistance through autophagy

    PubMed Central

    Yang, Minghua; Liu, Liying; Xie, Min; Sun, Xiaofang; Yu, Yan; Kang, Rui; Yang, Liangchun; Zhu, Shan; Cao, Lizhi; Tang, Daolin

    2015-01-01

    Both apoptosis ("self-killing") and autophagy ("self-eating") are evolutionarily conserved processes, and their crosstalk influences anticancer drug sensitivity and cell death. However, the underlying mechanism remains unclear. Here, we demonstrated that HMGB1 (high mobility group box 1), normally a nuclear protein, is a crucial regulator of TNFSF10/TRAIL (tumor necrosis factor [ligand] superfamily, member 10)-induced cancer cell death. Activation of PARP1 (poly [ADP-ribose] polymerase 1) was required for TNFSF10-induced ADP-ribosylation of HMGB1 in cancer cells. Moreover, pharmacological inhibition of PARP1 activity or knockdown of PARP1 gene expression significantly inhibited TNFSF10-induced HMGB1 cytoplasmic translocation and subsequent HMGB1-BECN1 complex formation. Furthermore, suppression of the PARP1-HMGB1 pathway diminished autophagy, increased apoptosis, and enhanced the anticancer activity of TNFSF10 in vitro and in a subcutaneous tumor model. These results indicate that PARP1 acts as a prominent upstream regulator of HMGB1-mediated autophagy and maintains a homeostatic balance between apoptosis and autophagy, which provides new insight into the mechanism of TNFSF10 resistance. PMID:25607248

  9. Microtubule protein ADP-ribosylation in vitro leads to assembly inhibition and rapid depolymerization

    SciTech Connect

    Scaife, R.M. ); Wilson, L. ); Purich, D.L. )

    1992-01-14

    Bovine brain microtubule protein, containing both tubulin and microtubule-associated proteins, undergoes ADP-ribosylation in the presence of ({sup 14}C)NAD{sup +} and a turkey erythrocyte mono-ADP-ribosyltransferase in vitro. The modification reaction could be demonstrated in crude brain tissue extracts where selective ADP-ribosylation of both the {alpha} and {beta} chains of tubulin and of the high molecular weight microtubule-associated protein MAP-2 occurred. In experiments with purified microtubule protein, tubulin dimer, the high molecular weight microtubule-associated protein MAP-2, and another high molecular weight microtubule-associated protein which may be a MAP-1 species were heavily labeled. Tubulin and MAP-2 incorporated ({sup 14}C)ADP-ribose to an average extent of approximately 2.4 and 30 mol of ADP-ribose/mol of protein, respectively. Assembly of microtubule protein into microtubules in vitro was inhibited by ADP-ribosylation, and incubation of assembled steady-state microtubules with ADP-ribosyltransferase and NAD{sup +} resulted in rapid depolymerization of the microtubules. Thus, the eukaryotic enzyme can ADP-ribosylate tubulin and microtubule-associated proteins to much greater extents than previously observed with cholera and pertussis toxins, and the modification can significantly modulate microtubule assembly and disassembly.

  10. The natural history of ADP-ribosyltransferases and the ADP-ribosylation system.

    PubMed

    Aravind, L; Zhang, Dapeng; de Souza, Robson F; Anand, Swadha; Iyer, Lakshminarayan M

    2015-01-01

    Catalysis of NAD(+)-dependent ADP-ribosylation of proteins, nucleic acids, or small molecules has evolved in at least three structurally unrelated superfamilies of enzymes, namely ADP-ribosyltransferase (ART), the Sirtuins, and probably TM1506. Of these, the ART superfamily is the most diverse in terms of structure, active site residues, and targets that they modify. The primary diversification of the ART superfamily occurred in the context of diverse bacterial conflict systems, wherein ARTs play both offensive and defensive roles. These include toxin-antitoxin systems, virus-host interactions, intraspecific antagonism (polymorphic toxins), symbiont/parasite effectors/toxins, resistance to antibiotics, and repair of RNAs cleaved in conflicts. ARTs evolving in these systems have been repeatedly acquired by lateral transfer throughout eukaryotic evolution, starting from the PARP family, which was acquired prior to the last eukaryotic common ancestor. They were incorporated into eukaryotic regulatory/epigenetic control systems (e.g., PARP family and NEURL4), and also used as defensive (e.g., pierisin and CARP-1 families) or immunity-related proteins (e.g., Gig2-like ARTs). The ADP-ribosylation system also includes other domains, such as the Macro, ADP-ribosyl glycohydrolase, NADAR, and ADP-ribosyl cyclase, which appear to have initially diversified in bacterial conflict-related systems. Unlike ARTs, sirtuins appear to have a much smaller presence in conflict-related systems. PMID:25027823

  11. Agonist-induced ADP-ribosylation of a cytosolic protein in human platelets

    SciTech Connect

    Bruene, B.; Molina Y Vedia, L.; Lapetina, E.G. )

    1990-05-01

    {alpha}-Thrombin and phorbol 12,13-dibutyrate stimulated the mono(ADP-ribosyl)ation of a 42-kDa cytosolic protein of human platelets. This effect was mediated by protein kinase C activation and was inhibited by protein kinase C inhibitor staurosporine. It also was prevented by prostacyclin, which is known to inhibit the phospholipase C-induced formation of 1,2-diacylglycerol, which is one of the endogenous activators of protein kinase C. On sodium dodecyl sulfate/polyacrylamide gel electrophoresis, the 42-kDa protein that is ADP-ribosylated by {alpha}-thrombin was clearly distinct from the {alpha} subunits of membrane-bound inhibitory and stimulatory guanine nucleotide-binding regulatory proteins, respectively G{sub i{alpha}} and G{sub s{alpha}}; the 47-kDa protein that is phosphorylated by protein kinase C in platelets; and the 39-kDa protein that has been shown to be endogenously ADP-ribosylated by agents that release nitric oxide. This information shows that agonist-induced activation of protein kinase leads to the ADP-ribosylation of a specific protein. This covalent modification might have a functional role in platelet activation.

  12. ADP ribosylation of human neutrophil peptide-1 regulates its biological properties.

    PubMed

    Paone, Gregorino; Wada, Akihiro; Stevens, Linda A; Matin, Abul; Hirayama, Toshiya; Levine, Rodney L; Moss, Joel

    2002-06-11

    In human airways, epithelial cells lining the lumen and intraluminal cells (e.g., polymorphonuclear cells) participate in the innate immune response. These cells secrete or express on their surfaces arginine-specific ADP ribosyltransferases. Defensins, antimicrobial proteins secreted by immune cells, are arginine-rich, leading us to hypothesize that ADP ribosylation could modify their biological activities. We found that an arginine-specific ADP ribosyltransferase-1 present on airway epithelial cells modifies Arg-14 of alpha defensin-1. ADP-ribosylated defensin-1 had decreased antimicrobial and cytotoxic activities but still stimulated T cell chemotaxis and IL-8 release from A549 cells. Further, ADP-ribosylated defensin-1 inhibited cytotoxic and antimicrobial activities of unmodified defensin-1. We identified ADP-ribosylated defensin-1 in bronchoalveolar lavage fluid from smokers but not from nonsmokers, confirming its existence in vivo. Thus, airway mono-ADP-ribosyltransferases could have an important regulatory role in the innate immune response through modification of alpha defensin-1 and perhaps other basic molecules, with alteration of their biological properties. PMID:12060767

  13. Radiolabelling of bovine myristoylated alanine-rich protein kinase C substrate (MARCKS) in an ADP-ribosylation reaction.

    PubMed

    Chao, D; Severson, D L; Zwiers, H; Hollenberg, M D

    1994-01-01

    In an ADP-ribosylation reaction, we have observed the radiolabelling of a protein in a crude bovine brain homogenate, which upon two-dimensional gel electrophoresis migrated with an acidic pI (< 4.5) and an apparent molecular mass (80-90 kDa) consistent with the properties of the myristoylated, alanine-rich, protein kinase C substrate protein termed MARCKS. To establish the identity of this radiolabelled constituent in brain homogenates, we first purified bovine brain MARCKS using calmodulin-Sepharose affinity chromatography and we then supplemented the crude ADP-ribosylation reaction mixture with this purified MARCKS fraction. Concordant increases in radiolabelling and silver staining of the same protein component from the MARCKS-supplemented ADP-ribosylation reaction, as compared with the ADP-ribosylated crude homogenate, established the identity of this constituent as MARCKS. The radiolabelling of MARCKS was lower in comparison with the ADP-ribosylation of the related neuronal protein B-50/GAP-43 under identical reaction conditions. The potential functional consequences of the ADP-ribosylation of MARCKS are discussed and the possibility is raised that other members of the MARCKS family, such as the F52/MacMARCKS/MRP protein, may also be subject to ADP-ribosylation. PMID:7605610

  14. NADP/sup +/ enhances cholera and pertussis toxin-catalyzed ADP-ribosylation of membrane proteins

    SciTech Connect

    Kawai, Y.; Whitsel, C.; Arinze, I.J.

    1986-05-01

    Cholera or pertussis toxin-catalyzed (/sup 32/P)ADP-ribosylation is frequently used to estimate the concentration of the stimulatory (Ns) or inhibitory (Ni) guanine nucleotide regulatory proteins which modulate the activity of adenylate cyclase. With this assay, however, the degradation of the substrate, NAD/sup +/, by endogenous enzymes such as NAD/sup +/-glycohydrolase (NADase) present in the test membranes can influence the results. In this study the authors show that both cholera and pertussis toxin-catalyzed (/sup 32/P)ADP-ribosylation of liver membrane proteins is markedly enhanced by NADP/sup +/. The effect is concentration dependent; with 20 ..mu..M (/sup 32/P)NAD/sup +/ as substrate maximal enhancement is obtained at 0.5-1.0 mM NADP/sup +/. The enhancement of (/sup 32/P)ADP-ribosylation by NADP/sup +/ was much greater than that by other known effectors such as Mg/sup 2 +/, phosphate or isoniazid. The effect of NADP/sup +/ on ADP-ribosylation may occur by inhibition of the degradation of NAD/sup +/ probably by acting as an alternate substrate for NADase. Among inhibitors tested (NADP/sup +/, isoniazid, imidazole, nicotinamide, L-Arg-methyl-ester and HgCl/sub 2/) to suppress NADase activity, NADP/sup +/ was the most effective and, 10 mM, inhibited activity of the enzyme by about 90%. In membranes which contain substantial activities of NADase the inclusion of NADP/sup +/ in the assay is necessary to obtain maximal ADP-ribosylation.

  15. Structural basis of actin recognition and arginine ADP-ribosylation by Clostridium perfringens ι-toxin

    PubMed Central

    Tsuge, Hideaki; Nagahama, Masahiro; Oda, Masataka; Iwamoto, Shinobu; Utsunomiya, Hiroko; Marquez, Victor E.; Katunuma, Nobuhiko; Nishizawa, Mugio; Sakurai, Jun

    2008-01-01

    The ADP-ribosylating toxins (ADPRTs) produced by pathogenic bacteria modify intracellular protein and affect eukaryotic cell function. Actin-specific ADPRTs (including Clostridium perfringens ι-toxin and Clostridium botulinum C2 toxin) ADP-ribosylate G-actin at Arg-177, leading to disorganization of the cytoskeleton and cell death. Although the structures of many actin-specific ADPRTs are available, the mechanisms underlying actin recognition and selective ADP-ribosylation of Arg-177 remain unknown. Here we report the crystal structure of actin-Ia in complex with the nonhydrolyzable NAD analog βTAD at 2.8 Å resolution. The structure indicates that Ia recognizes actin via five loops around NAD: loop I (Tyr-60–Tyr-62 in the N domain), loop II (active-site loop), loop III, loop IV (PN loop), and loop V (ADP-ribosylating turn–turn loop). We used site-directed mutagenesis to confirm that loop I on the N domain and loop II are essential for the ADP-ribosyltransferase activity. Furthermore, we revealed that Glu-378 on the EXE loop is in close proximity to Arg-177 in actin, and we proposed that the ADP-ribosylation of Arg-177 proceeds by an SN1 reaction via first an oxocarbenium ion intermediate and second a cationic intermediate by alleviating the strained conformation of the first oxocarbenium ion. Our results suggest a common reaction mechanism for ADPRTs. Moreover, the structure might be of use in rational drug design to block toxin-substrate recognition. PMID:18490658

  16. Phosphoproteomic Approach to Characterize Protein Mono- and Poly(ADP-ribosyl)ation Sites from Cells

    PubMed Central

    2015-01-01

    Poly(ADP-ribose), or PAR, is a cellular polymer implicated in DNA/RNA metabolism, cell death, and cellular stress response via its role as a post-translational modification, signaling molecule, and scaffolding element. PAR is synthesized by a family of proteins known as poly(ADP-ribose) polymerases, or PARPs, which attach PAR polymers to various amino acids of substrate proteins. The nature of these polymers (large, charged, heterogeneous, base-labile) has made these attachment sites difficult to study by mass spectrometry. Here we propose a new pipeline that allows for the identification of mono(ADP-ribosyl)ation and poly(ADP-ribosyl)ation sites via the enzymatic product of phosphodiesterase-treated ADP-ribose, or phospho(ribose). The power of this method lies in the enrichment potential of phospho(ribose), which we show to be enriched by phosphoproteomic techniques when a neutral buffer, which allows for retention of the base-labile attachment site, is used for elution. Through the identification of PARP-1 in vitro automodification sites as well as endogenous ADP-ribosylation sites from whole cells, we have shown that ADP-ribose can exist on adjacent amino acid residues as well as both lysine and arginine in addition to known acidic modification sites. The universality of this technique has allowed us to show that enrichment of ADP-ribosylated proteins by macrodomain leads to a bias against ADP-ribose modifications conjugated to glutamic acids, suggesting that the macrodomain is either removing or selecting against these distinct protein attachments. Ultimately, the enrichment pipeline presented here offers a universal approach for characterizing the mono- and poly(ADP-ribosyl)ated proteome. PMID:24920161

  17. A novel Hsp70 inhibitor prevents cell intoxication with the actin ADP-ribosylating Clostridium perfringens iota toxin

    PubMed Central

    Ernst, Katharina; Liebscher, Markus; Mathea, Sebastian; Granzhan, Anton; Schmid, Johannes; Popoff, Michel R.; Ihmels, Heiko; Barth, Holger; Schiene-Fischer, Cordelia

    2016-01-01

    Hsp70 family proteins are folding helper proteins involved in a wide variety of cellular pathways. Members of this family interact with key factors in signal transduction, transcription, cell-cycle control, and stress response. Here, we developed the first Hsp70 low molecular weight inhibitor specifically targeting the peptide binding site of human Hsp70. After demonstrating that the inhibitor modulates the Hsp70 function in the cell, we used the inhibitor to show for the first time that the stress-inducible chaperone Hsp70 functions as molecular component for entry of a bacterial protein toxin into mammalian cells. Pharmacological inhibition of Hsp70 protected cells from intoxication with the binary actin ADP-ribosylating iota toxin from Clostridium perfringens, the prototype of a family of enterotoxins from pathogenic Clostridia and inhibited translocation of its enzyme component across cell membranes into the cytosol. This finding offers a starting point for novel therapeutic strategies against certain bacterial toxins. PMID:26839186

  18. Fanconi anemia protein FANCD2 inhibits TRF1 polyADP-ribosylation through tankyrase1-dependent manner

    PubMed Central

    2011-01-01

    Background Fanconi anemia (FA) is a rare autosomal recessive syndrome characterized by developmental abnormalities, progressive bone marrow failure, and predisposition to cancer. The key FA protein FANCD2 crosstalks with members of DNA damage and repair pathways that also play a role at telomeres. Therefore, we investigated whether FANCD2 has a similar involvement at telomeres. Results We reveal that FANCD2 may perform a novel function separate to the FANCD2/BRCA pathway. This function includes FANCD2 interaction with one of the telomere components, the PARP family member tankyrase-1. Moreover, FANCD2 inhibits tankyrase-1 activity in vitro. In turn, FANCD2 deficiency increases the polyADP-ribosylation of telomere binding factor TRF1. Conclusions FANCD2 binding and inhibiting tankyrase-1PARsylation at telomeres may provide an additional step within the FA pathway for the regulation of genomic integrity. PMID:21314979

  19. Calcium-dependent ADP-ribosylation of high-mobility-group I (HMGI) proteins.

    PubMed Central

    Giancotti, V; Bandiera, A; Sindici, C; Perissin, L; Crane-Robinson, C

    1996-01-01

    Micrococcal nuclease digestion of nuclei from mouse Lewis lung carcinoma cells releases a protein mixture into the supernatant that lacks histone H1 and contains a full complement of high-mobility-group I (HMGI) proteins (i.e. I, Y and I-C). This implies that all three HMGI proteins are localized at the nuclease-sensitive regions of active chromatin. It is also shown that if Ca2+ ions are present in the nuclear incubation buffer (with or without exogenous nuclease), all three HMGI proteins become ADP-ribosylated. We propose that this modification of HMGI family proteins is part of the general poly(ADP-ribosyl)ation that accompanies DNA damage in apoptosis and other processes. PMID:8760375

  20. Auto ADP-ribosylation of NarE, a Neisseria meningitidis ADP-ribosyltransferase, regulates its catalytic activities.

    PubMed

    Picchianti, Monica; Del Vecchio, Mariangela; Di Marcello, Federica; Biagini, Massimiliano; Veggi, Daniele; Norais, Nathalie; Rappuoli, Rino; Pizza, Mariagrazia; Balducci, Enrico

    2013-12-01

    NarE is an arginine-specific mono-ADP-ribosyltransferase identified in Neisseria meningitidis that requires the presence of iron in a structured cluster for its enzymatic activities. In this study, we show that NarE can perform auto-ADP-ribosylation. This automodification occurred in a time- and NAD-concentration-dependent manner; was inhibited by novobiocin, an ADP-ribosyltransferase inhibitor; and did not occur when NarE was heat inactivated. No reduction in incorporation was evidenced in the presence of high concentrations of ATP, GTP, ADP-ribose, or nicotinamide, which inhibits NAD-glycohydrolase, impeding the formation of free ADP-ribose. Based on the electrophoretic profile of NarE on auto-ADP-ribosylation and on the results of mutagenesis and mass spectrometry analysis, the auto-ADP-ribosylation appeared to be restricted to the addition of a single ADP-ribose. Chemical stability experiments showed that the ADP-ribosyl linkage was sensitive to hydroxylamine, which breaks ADP-ribose-arginine bonds. Site-directed mutagenesis suggested that the auto-ADP-ribosylation site occurred preferentially on the R(7) residue, which is located in the region I of the ADP-ribosyltransferase family. After auto-ADP-ribosylation, NarE showed a reduction in ADP-ribosyltransferase activity, while NAD-glycohydrolase activity was increased. Overall, our findings provide evidence for a novel intramolecular mechanism used by NarE to regulate its enzymatic activities. PMID:23964075

  1. Detection and Quantification of ADP-Ribosylated RhoA/B by Monoclonal Antibody

    PubMed Central

    Rohrbeck, Astrid; Fühner, Viola; Schröder, Anke; Hagemann, Sandra; Vu, Xuan-Khang; Berndt, Sarah; Hust, Michael; Pich, Andreas; Just, Ingo

    2016-01-01

    Clostridium botulinum exoenzyme C3 is the prototype of C3-like ADP-ribosyltransferases that modify the GTPases RhoA, B, and C. C3 catalyzes the transfer of an ADP-ribose moiety from the co-substrate nicotinamide adenine dinucleotide (NAD) to asparagine-41 of Rho-GTPases. Although C3 does not possess cell-binding/-translocation domains, C3 is able to efficiently enter intact cells, including neuronal and macrophage-like cells. Conventionally, the detection of C3 uptake into cells is carried out via the gel-shift assay of modified RhoA. Since this gel-shift assay does not always provide clear, evaluable results an additional method to confirm the ADP-ribosylation of RhoA is necessary. Therefore, a new monoclonal antibody has been generated that specifically detects ADP-ribosylated RhoA/B, but not RhoC, in Western blot and immunohistochemical assay. The scFv antibody fragment was selected by phage display using the human naive antibody gene libraries HAL9/10. Subsequently, the antibody was produced as scFv-Fc and was found to be as sensitive as a commercially available RhoA antibody providing reproducible and specific results. We demonstrate that this specific antibody can be successfully applied for the analysis of ADP-ribosylated RhoA/B in C3-treated Chinese hamster ovary (CHO) and HT22 cells. Moreover, ADP-ribosylation of RhoA was detected within 10 min in C3-treated CHO wild-type cells, indicative of C3 cell entry. PMID:27043630

  2. Detection and Quantification of ADP-Ribosylated RhoA/B by Monoclonal Antibody.

    PubMed

    Rohrbeck, Astrid; Fühner, Viola; Schröder, Anke; Hagemann, Sandra; Vu, Xuan-Khang; Berndt, Sarah; Hust, Michael; Pich, Andreas; Just, Ingo

    2016-04-01

    Clostridium botulinum exoenzyme C3 is the prototype of C3-like ADP-ribosyltransferases that modify the GTPases RhoA, B, and C. C3 catalyzes the transfer of an ADP-ribose moiety from the co-substrate nicotinamide adenine dinucleotide (NAD) to asparagine-41 of Rho-GTPases. Although C3 does not possess cell-binding/-translocation domains, C3 is able to efficiently enter intact cells, including neuronal and macrophage-like cells. Conventionally, the detection of C3 uptake into cells is carried out via the gel-shift assay of modified RhoA. Since this gel-shift assay does not always provide clear, evaluable results an additional method to confirm the ADP-ribosylation of RhoA is necessary. Therefore, a new monoclonal antibody has been generated that specifically detects ADP-ribosylated RhoA/B, but not RhoC, in Western blot and immunohistochemical assay. The scFv antibody fragment was selected by phage display using the human naive antibody gene libraries HAL9/10. Subsequently, the antibody was produced as scFv-Fc and was found to be as sensitive as a commercially available RhoA antibody providing reproducible and specific results. We demonstrate that this specific antibody can be successfully applied for the analysis of ADP-ribosylated RhoA/B in C3-treated Chinese hamster ovary (CHO) and HT22 cells. Moreover, ADP-ribosylation of RhoA was detected within 10 min in C3-treated CHO wild-type cells, indicative of C3 cell entry. PMID:27043630

  3. Brefeldin A-induced ADP-ribosylation in the structure and function of the Golgi complex.

    PubMed

    Colanzi, A; Mironov, A; Weigert, R; Limina, C; Flati, S; Cericola, C; Di Tullio, G; Di Girolamo, M; Corda, D; De Matteis, M A; Luini, A

    1997-01-01

    Brefeldin A (BFA) is a fungal metabolite that exerts generally inhibitory actions on membrane transport and induces the disappearance of the Golgi complex. Previously we have shown that BFA stimulates the ADP-ribosylation of two cytosolic proteins of 38 and 50 KD. The BFA-binding components mediating the BFA-sensitive ADP-ribosylation (BAR) and the effect of BFA on ARF binding to Golgi membranes have similar specificities and affinities for BFA and its analogues, suggesting that BAR may have a role in the cellular effects of BFA. To investigate this we used the approach to impair BAR activity by the use of BAR inhibitors. A series of BAR inhibitors was developed and their effects were studied in RBL cells treated with BFA. In addition to the common ADP-ribosylation inhibitors (nicotinamide and aminobenzamide), compounds belonging to the cumarin (novobiocin, cumermycin, dicumarol) class were active BAR inhibitors. All BAR inhibitors were able to prevent the BFA-induced redistribution of a Golgi marker (Helix pomatia lectin) into the endoplasmic reticulum, as assessed in immunofluorescence experiments. At the ultrastructural level, BAR inhibitors prevented the tubular-vesicular transformation of the Golgi complex caused by BFA. The potencies of these compounds in preventing the BFA effects on the Golgi complex were similar to those at which they inhibited BAR. Altogether these data support the hypothesis that BAR mediates at least some of the effects of BFA on the Golgi structure and function. PMID:9193673

  4. Roles of Asp179 and Glu270 in ADP-Ribosylation of Actin by Clostridium perfringens Iota Toxin

    PubMed Central

    Belyy, Alexander; Tabakova, Irina; Lang, Alexander E.; Jank, Thomas; Belyi, Yury; Aktories, Klaus

    2015-01-01

    Clostridium perfringens iota toxin is a binary toxin composed of the enzymatically active component Ia and receptor binding component Ib. Ia is an ADP-ribosyltransferase, which modifies Arg177 of actin. The previously determined crystal structure of the actin-Ia complex suggested involvement of Asp179 of actin in the ADP-ribosylation reaction. To gain more insights into the structural requirements of actin to serve as a substrate for toxin-catalyzed ADP-ribosylation, we engineered Saccharomyces cerevisiae strains, in which wild type actin was replaced by actin variants with substitutions in residues located on the Ia-actin interface. Expression of the actin mutant Arg177Lys resulted in complete resistance towards Ia. Actin mutation of Asp179 did not change Ia-induced ADP-ribosylation and growth inhibition of S. cerevisiae. By contrast, substitution of Glu270 of actin inhibited the toxic action of Ia and the ADP-ribosylation of actin. In vitro transcribed/translated human β-actin confirmed the crucial role of Glu270 in ADP-ribosylation of actin by Ia. PMID:26713879

  5. Distribution of protein poly(ADP-ribosyl)ation systems across all domains of life

    PubMed Central

    Perina, Dragutin; Mikoč, Andreja; Ahel, Josip; Ćetković, Helena; Žaja, Roko; Ahel, Ivan

    2014-01-01

    Poly(ADP-ribosyl)ation is a post-translational modification of proteins involved in regulation of many cellular pathways. Poly(ADP-ribose) (PAR) consists of chains of repeating ADP-ribose nucleotide units and is synthesized by the family of enzymes called poly(ADP-ribose) polymerases (PARPs). This modification can be removed by the hydrolytic action of poly(ADP-ribose) glycohydrolase (PARG) and ADP-ribosylhydrolase 3 (ARH3). Hydrolytic activity of macrodomain proteins (MacroD1, MacroD2 and TARG1) is responsible for the removal of terminal ADP-ribose unit and for complete reversion of protein ADP-ribosylation. Poly(ADP-ribosyl)ation is widely utilized in eukaryotes and PARPs are present in representatives from all six major eukaryotic supergroups, with only a small number of eukaryotic species that do not possess PARP genes. The last common ancestor of all eukaryotes possessed at least five types of PARP proteins that include both mono and poly(ADP-ribosyl) transferases. Distribution of PARGs strictly follows the distribution of PARP proteins in eukaryotic species. At least one of the macrodomain proteins that hydrolyse terminal ADP-ribose is also always present. Therefore, we can presume that the last common ancestor of all eukaryotes possessed a fully functional and reversible PAR metabolism and that PAR signalling provided the conditions essential for survival of the ancestral eukaryote in its ancient environment. PARP proteins are far less prevalent in bacteria and were probably gained through horizontal gene transfer. Only eleven bacterial species possess all proteins essential for a functional PAR metabolism, although it is not known whether PAR metabolism is truly functional in bacteria. Several dsDNA viruses also possess PARP homologues, while no PARP proteins have been identified in any archaeal genome. Our analysis of the distribution of enzymes involved in PAR metabolism provides insight into the evolution of these important signalling systems, as well as

  6. Regulation of chromatin structure by poly(ADP-ribosyl)ation

    PubMed Central

    Beneke, Sascha

    2012-01-01

    The interaction of DNA with proteins in the context of chromatin has to be tightly regulated to achieve so different tasks as packaging, transcription, replication and repair. The very rapid and transient post-translational modification of proteins by poly(ADP-ribose) has been shown to take part in all four. Originally identified as immediate cellular answer to a variety of genotoxic stresses, already early data indicated the ability of this highly charged nucleic acid-like polymer to modulate nucleosome structure, the basic unit of chromatin. At the same time the enzyme responsible for synthesizing poly(ADP-ribose), the zinc-finger protein poly(ADP-ribose) polymerase-1 (PARP1), was shown to control transcription initiation as basic factor TFIIC within the RNA-polymerase II machinery. Later research focused more on PARP-mediated regulation of DNA repair and cell death, but in the last few years, transcription as well as chromatin modulation has re-appeared on the scene. This review will discuss the impact of PARP1 on transcription and transcription factors, its implication in chromatin remodeling for DNA repair and probably also replication, and its role in controlling epigenetic events such as DNA methylation and the functionality of the insulator protein CCCTC-binding factor. PMID:22969794

  7. The ADP-ribosyl cyclases--the current evolutionary state of the ARCs.

    PubMed

    Ferrero, Enza; Lo Buono, Nicola; Horenstein, Alberto L; Funaro, Ada; Malavasi, Fabio

    2014-01-01

    The major ADP-ribosylating enzyme families are the focus of this special issue of Frontiers in Bioscience . However, there is room for another family of enzymes with the capacity to utilize nicotinamide adenine dinucleotide (NAD): the ADP-ribosyl cyclases (ARCs). These unique enzymes catalyse the cyclization of NAD to cyclic ADP ribose (cADPR), a widely distributed second messenger. However, the ARCs are versatile enzymes that can manipulate NAD, NAD phosphate (NADP) and other substrates to generate various bioactive molecules including nicotinic acid adenine dinucleotide diphosphate (NAADP) and ADP ribose (ADPR). This review will focus on the group of well-characterized invertebrate and vertebrate ARCs whose common gene structure allows us to trace their origin to the ancestor of bilaterian animals. Behind a facade of gene and protein homology lies a family with a disparate functional repertoire dictated by the animal model and the physical trait under investigation. Here we present a phylogenetic view of the ARCs to better understand the evolution of function in this family. PMID:24896331

  8. Common features of the NAD-binding and catalytic site of ADP-ribosylating toxins.

    PubMed

    Domenighini, M; Magagnoli, C; Pizza, M; Rappuoli, R

    1994-10-01

    Computer analysis of the three-dimensional structure of ADP-ribosylating toxins showed that in all toxins the NAD-binding site is located in a cavity. This cavity consists of 18 contiguous amino acids that form an alpha-helix bent over a beta-strand. The tertiary folding of this structure is strictly conserved despite the differences in the amino acid sequence. Catalysis is supported by two spatially conserved amino acids, each flanking the NAD-binding site. These are: a glutamic acid that is conserved in all toxins, and a nucleophilic residue, which is a histidine in the diphtheria toxin and Pseudomonas exotoxin A, and an arginine in the cholera toxin, the Escherichia coli heat-labile enterotoxins, the pertussis toxin and the mosquitocidal toxin of Bacillus sphaericus. The latter group of toxins presents an additional histidine that appears important for catalysis. This structure suggests a general mechanism of ADP-ribosylation evolved to work on different target proteins. PMID:7830559

  9. Poly-ADP-ribosylation-mediated degradation of ARTD1 by the NLRP3 inflammasome is a prerequisite for osteoclast maturation.

    PubMed

    Wang, C; Qu, C; Alippe, Y; Bonar, S L; Civitelli, R; Abu-Amer, Y; Hottiger, M O; Mbalaviele, G

    2016-01-01

    Evidence implicates ARTD1 in cell differentiation, but its role in skeletal metabolism remains unknown. Osteoclasts (OC), the bone-resorbing cells, differentiate from macrophages under the influence of macrophage colony-stimulating factor (M-CSF) and receptor-activator of NF-κB ligand (RANKL). We found that M-CSF induced ADP-ribosyltransferase diphtheria toxin-like 1 (ARTD1) auto-ADP-ribosylation in macrophages, a modification that marked ARTD1 for cleavage, and subsequently, for degradation upon RANKL exposure. We established that ARTD1 proteolysis was NLRP3 inflammasome-dependent, and occurred via the proteasome pathway. Since ARTD1 is cleaved at aspartate(214), we studied the impact of ARTD1 rendered uncleavable by D214N substitution (ARTD1(D214N)) on skeletal homeostasis. ARTD1(D214N), unlike wild-type ARTD1, was resistant to cleavage and degradation during osteoclastogenesis. As a result, ARTD1(D214N) altered histone modification and promoted the abundance of the repressors of osteoclastogenesis by interfering with the expression of B lymphocyte-induced maturation protein 1 (Blimp1), the master regulator of anti-osteoclastogenic transcription factors. Importantly, ARTD1(D214N)-expressing mice exhibited higher bone mass compared with controls, owing to decreased osteoclastogenesis while bone formation was unaffected. Thus, unless it is degraded, ARTD1 represses OC development through transcriptional regulation. PMID:27010854

  10. Poly-ADP-ribosylation-mediated degradation of ARTD1 by the NLRP3 inflammasome is a prerequisite for osteoclast maturation

    PubMed Central

    Wang, C; Qu, C; Alippe, Y; Bonar, S L; Civitelli, R; Abu-Amer, Y; Hottiger, M O; Mbalaviele, G

    2016-01-01

    Evidence implicates ARTD1 in cell differentiation, but its role in skeletal metabolism remains unknown. Osteoclasts (OC), the bone-resorbing cells, differentiate from macrophages under the influence of macrophage colony-stimulating factor (M-CSF) and receptor-activator of NF-κB ligand (RANKL). We found that M-CSF induced ADP-ribosyltransferase diphtheria toxin-like 1 (ARTD1) auto-ADP-ribosylation in macrophages, a modification that marked ARTD1 for cleavage, and subsequently, for degradation upon RANKL exposure. We established that ARTD1 proteolysis was NLRP3 inflammasome-dependent, and occurred via the proteasome pathway. Since ARTD1 is cleaved at aspartate214, we studied the impact of ARTD1 rendered uncleavable by D214N substitution (ARTD1D214N) on skeletal homeostasis. ARTD1D214N, unlike wild-type ARTD1, was resistant to cleavage and degradation during osteoclastogenesis. As a result, ARTD1D214N altered histone modification and promoted the abundance of the repressors of osteoclastogenesis by interfering with the expression of B lymphocyte-induced maturation protein 1 (Blimp1), the master regulator of anti-osteoclastogenic transcription factors. Importantly, ARTD1D214N-expressing mice exhibited higher bone mass compared with controls, owing to decreased osteoclastogenesis while bone formation was unaffected. Thus, unless it is degraded, ARTD1 represses OC development through transcriptional regulation. PMID:27010854

  11. PARP-2 regulates cell cycle-related genes through histone deacetylation and methylation independently of poly(ADP-ribosyl)ation

    SciTech Connect

    Liang, Ya-Chen; Hsu, Chiao-Yu; Yao, Ya-Li; Yang, Wen-Ming

    2013-02-01

    Highlights: ► PARP-2 acts as a transcription co-repressor independently of PARylation activity. ► PARP-2 recruits HDAC5, 7, and G9a and generates repressive chromatin. ► PARP-2 is recruited to the c-MYC promoter by DNA-binding factor YY1. ► PARP-2 represses cell cycle-related genes and alters cell cycle progression. -- Abstract: Poly(ADP-ribose) polymerase-2 (PARP-2) catalyzes poly(ADP-ribosyl)ation (PARylation) and regulates numerous nuclear processes, including transcription. Depletion of PARP-2 alters the activity of transcription factors and global gene expression. However, the molecular action of how PARP-2 controls the transcription of target promoters remains unclear. Here we report that PARP-2 possesses transcriptional repression activity independently of its enzymatic activity. PARP-2 interacts and recruits histone deacetylases HDAC5 and HDAC7, and histone methyltransferase G9a to the promoters of cell cycle-related genes, generating repressive chromatin signatures. Our findings propose a novel mechanism of PARP-2 in transcriptional regulation involving specific protein–protein interactions and highlight the importance of PARP-2 in the regulation of cell cycle progression.

  12. Poly(ADP-ribosylation) regulates chromatin organization through histone H3 modification and DNA methylation of the first cell cycle of mouse embryos

    SciTech Connect

    Osada, Tomoharu; Rydén, Anna-Margareta; Masutani, Mitsuko

    2013-04-26

    Highlights: •Histone modification of the mouse pronuclei is regulated by poly(ADP-ribosylation). •Hypermethylation of the mouse female pronuclei is maintained by poly(ADP-ribosylation). •Parp1 is physically interacted with Suz12, which may function in the pronuclei. •Poly(ADP-ribosylation) affects ultrastructure of chromatin of the mouse pronucleus. -- Abstract: We examined the roles of poly(ADP-ribosylation) in chromatin remodeling during the first cell cycle of mouse embryos. Drug-based inhibition of poly(ADP-ribosylation) by a PARP inhibitor, PJ-34, revealed up-regulation of dimethylation of histone H3 at lysine 4 in male pronuclei and down-regulation of dimethylation of histone H3 at lysine 9 (H3K9) and lysine 27 (H3K27). Association of poly(ADP-ribosylation) with histone modification was suggested to be supported by the interaction of Suz12, a histone methyltransferase in the polycomb complex, with Parp1. PARP activity was suggested to be required for a proper localization and maintenance of Suz12 on chromosomes. Notably, DNA methylation level of female pronuclei in one-cell embryos was robustly decreased by PJ-34. Electron microscopic analysis showed a frequent appearance of unusual electron-dense areas within the female pronuclei, implying the disorganized and hypercondensed chromatin ultrastructure. These results show that poly(ADP-ribosylation) is important for the integrity of non-equivalent epigenetic dynamics of pronuclei during the first cell cycle of mouse embryos.

  13. Two novel human members of an emerging mammalian gene family related to mono-ADP-ribosylating bacterial toxins

    SciTech Connect

    Koch-Nolte, F.; Haag, F.; Braren, R.

    1997-02-01

    Mono-ADP-ribosylation is one of the posttranslational protein modifications regulating cellular metabolism, e.g., nitrogen fixation, in prokaryotes. Several bacterial toxins mono-ADP-ribosylate and inactivate specific proteins in their animal hosts. Recently, two mammalian GPI-anchored cell surface enzymes with similar activities were cloned (designated ART1 and ART2). We have now identified six related expressed sequence tags (ESTs) in the public database and cloned the two novel human genes from which these are derived (designated ART3 and ART4). The deduced amino acid sequences of the predicted gene products show 28% sequence identity to one another and 32-41% identity vs the muscle and T cell enzymes. They contain signal peptide sequences characteristic of GPI anchorage. Southern Zoo blot analyses suggest the presence of related genes in other mammalian species. By PCR screening of somatic cell hybrids and by in situ hybridization, we have mapped the two genes to human chromosomes 4p14-p15.l and 12q13.2- q13.3. Northern blot analyses show that these genes are specifically expressed in testis and spleen, respectively. Comparison of genomic and cDNA sequences reveals a conserved exon/intron structure, with an unusually large exon encoding the predicted mature membrane proteins. Secondary structure prediction analyses indicate conserved motifs and amino acid residues consistent with a common ancestry of this emerging mammalian enzyme family and bacterial mono(ADP-ribosyl)transferases. It is possible that the four human gene family members identified so far represent the {open_quotes}tip of an iceberg,{close_quote} i.e., a larger family of enzymes that influences the function of target proteins via mono-ADP-ribosylation. 35 refs., 4 figs.

  14. Poly(ADP-ribosyl)ation of Methyl CpG Binding Domain Protein 2 Regulates Chromatin Structure.

    PubMed

    Becker, Annette; Zhang, Peng; Allmann, Lena; Meilinger, Daniela; Bertulat, Bianca; Eck, Daniel; Hofstaetter, Maria; Bartolomei, Giody; Hottiger, Michael O; Schreiber, Valérie; Leonhardt, Heinrich; Cardoso, M Cristina

    2016-03-01

    The epigenetic information encoded in the genomic DNA methylation pattern is translated by methylcytosine binding proteins like MeCP2 into chromatin topology and structure and gene activity states. We have shown previously that the MeCP2 level increases during differentiation and that it causes large-scale chromatin reorganization, which is disturbed by MeCP2 Rett syndrome mutations. Phosphorylation and other posttranslational modifications of MeCP2 have been described recently to modulate its function. Here we show poly(ADP-ribosyl)ation of endogenous MeCP2 in mouse brain tissue. Consequently, we found that MeCP2 induced aggregation of pericentric heterochromatin and that its chromatin accumulation was enhanced in poly(ADP-ribose) polymerase (PARP) 1(-/-) compared with wild-type cells. We mapped the poly(ADP-ribosyl)ation domains and engineered MeCP2 mutation constructs to further analyze potential effects on DNA binding affinity and large-scale chromatin remodeling. Single or double deletion of the poly(ADP-ribosyl)ated regions and PARP inhibition increased the heterochromatin clustering ability of MeCP2. Increased chromatin clustering may reflect increased binding affinity. In agreement with this hypothesis, we found that PARP-1 deficiency significantly increased the chromatin binding affinity of MeCP2 in vivo. These data provide novel mechanistic insights into the regulation of MeCP2-mediated, higher-order chromatin architecture and suggest therapeutic opportunities to manipulate MeCP2 function. PMID:26772194

  15. How to kill tumor cells with inhibitors of poly(ADP-ribosyl)ation.

    PubMed

    Mangerich, Aswin; Bürkle, Alexander

    2011-01-15

    Poly(ADP-ribosyl)ation is a post-translational modification catalyzed by the enzyme family of poly(ADP-ribose) polymerases (PARPs). PARPs exhibit pleiotropic cellular functions ranging from maintenance of genomic stability and chromatin remodeling to regulation of cell death, thereby rendering PARP homologues promising targets in cancer therapy. Depending on the molecular status of a cancer cell, low-molecular weight PARP inhibitors can (i) either be used as monotherapeutic agents following the concept of synthetic lethality or (ii) to support classical chemotherapy or radiotherapy. The rationales are the following: (i) in cancers with selective defects in homologous recombination repair, inactivation of PARPs directly causes cell death. In cancer treatment, this phenomenon can be employed to specifically target tumor cells while sparing nonmalignant tissue. (ii) PARP inhibitors can also be used to sensitize cells to cytotoxic DNA-damaging treatments, as some PARPs actively participate in genomic maintenance. Apart from that, PARP inhibitors possess antiangiogenic functions, thus opening up a further option to inhibit tumor growth. In view of the above, a number of high-potency PARP inhibitors have been developed during the last decade and are currently evaluated as cancer therapeutics in clinical trials by several leading pharmaceutical companies. PMID:20853319

  16. Rifamycin Antibiotic Resistance by ADP-Ribosylation: Structure and Diversity of Arr

    SciTech Connect

    Baysarowich, J.; Koteva, K; Hughes, D; Ejim, L; Griffiths, E; Zhang, K; Junop, M; Wright, G

    2008-01-01

    The rifamycin antibiotic rifampin is important for the treatment of tuberculosis and infections caused by multidrug-resistant Staphylococcus aureus. Recent iterations of the rifampin core structure have resulted in new drugs and drug candidates for the treatment of a much broader range of infectious diseases. This expanded use of rifamycin antibiotics has the potential to select for increased resistance. One poorly characterized mechanism of resistance is through Arr enzymes that catalyze ADP-ribosylation of rifamycins. We find that genes encoding predicted Arr enzymes are widely distributed in the genomes of pathogenic and nonpathogenic bacteria. Biochemical analysis of three representative Arr enzymes from environmental and pathogenic bacterial sources shows that these have equally efficient drug resistance capacity in vitro and in vivo. The 3D structure of one of these orthologues from Mycobacterium smegmatis was determined and reveals structural homology with ADP-ribosyltransferases important in eukaryotic biology, including poly(ADP-ribose) polymerases (PARPs) and bacterial toxins, despite no significant amino acid sequence homology with these proteins. This work highlights the extent of the rifamycin resistome in microbial genera with the potential to negatively impact the expanded use of this class of antibiotic.

  17. Allosteric Activation of the RNF146 Ubiquitin Ligase by a Poly(ADP-ribosyl)ation Signal

    PubMed Central

    DaRosa, Paul A.; Wang, Zhizhi; Jiang, Xiaomo; Pruneda, Jonathan N.; Cong, Feng; Klevit, Rachel E.; Xu, Wenqing

    2014-01-01

    Protein poly(ADP-ribosyl)ation (PARylation) plays a role in diverse cellular processes such as DNA repair, transcription, Wnt signaling, and cell death1–6. Recent studies have shown that PARylation can serve as a signal for the polyubiquitination and degradation of several critical regulatory proteins, including Axin and 3BP2 (refs 7–9). The RING-type E3 ubiquitin ligase RNF146 (a.k.a. Iduna) is responsible for PARylation-dependent ubiquitination (PARdU)10–12. Here we provide a structural basis for RNF146 catalyzed PARdU and how PARdU specificity is achieved. First, we show that iso-ADPr, the smallest internal poly(ADP-ribose) (PAR) structural unit, binds between the WWE and RING domains of RNF146 and functions as an allosteric signal that switches the RING domain from a catalytically inactive state to an active one. In the absence of PAR, the RING domain is unable to efficiently bind and activate an E2. Binding of PAR/iso-ADPr induces a major conformational change that creates a functional RING structure. Thus RNF146 represents a new mechanistic class of RING E3 ligases whose activities are regulated by non-covalent ligand binding, which may provide a template for designing inducible protein-degradation systems. Second, we found that RNF146 directly interacts with the PAR polymerase tankyrase (TNKS). Disruption of the RNF146/TNKS interaction inhibits turnover of the substrate Axin in cells. Thus, both substrate PARylation and PARdU are catalyzed by enzymes within the same protein complex, and PARdU substrate specificity may be primarily determined by the substrate-TNKS interaction. We propose that maintenance of unliganded RNF146 in an inactive state may serve to maintain the stability of the RNF146-TNKS complex, which in turn regulates the homeostasis of PARdU activity in the cell. PMID:25327252

  18. Poly(ADP-ribosyl)ation of Methyl CpG Binding Domain Protein 2 Regulates Chromatin Structure*

    PubMed Central

    Becker, Annette; Zhang, Peng; Allmann, Lena; Meilinger, Daniela; Bertulat, Bianca; Eck, Daniel; Hofstaetter, Maria; Bartolomei, Giody; Hottiger, Michael O.; Schreiber, Valérie; Leonhardt, Heinrich; Cardoso, M. Cristina

    2016-01-01

    The epigenetic information encoded in the genomic DNA methylation pattern is translated by methylcytosine binding proteins like MeCP2 into chromatin topology and structure and gene activity states. We have shown previously that the MeCP2 level increases during differentiation and that it causes large-scale chromatin reorganization, which is disturbed by MeCP2 Rett syndrome mutations. Phosphorylation and other posttranslational modifications of MeCP2 have been described recently to modulate its function. Here we show poly(ADP-ribosyl)ation of endogenous MeCP2 in mouse brain tissue. Consequently, we found that MeCP2 induced aggregation of pericentric heterochromatin and that its chromatin accumulation was enhanced in poly(ADP-ribose) polymerase (PARP) 1−/− compared with wild-type cells. We mapped the poly(ADP-ribosyl)ation domains and engineered MeCP2 mutation constructs to further analyze potential effects on DNA binding affinity and large-scale chromatin remodeling. Single or double deletion of the poly(ADP-ribosyl)ated regions and PARP inhibition increased the heterochromatin clustering ability of MeCP2. Increased chromatin clustering may reflect increased binding affinity. In agreement with this hypothesis, we found that PARP-1 deficiency significantly increased the chromatin binding affinity of MeCP2 in vivo. These data provide novel mechanistic insights into the regulation of MeCP2-mediated, higher-order chromatin architecture and suggest therapeutic opportunities to manipulate MeCP2 function. PMID:26772194

  19. The ARTT motif and a unified structural understanding of substraterecognition in ADP ribosylating bacterial toxins and eukaryotic ADPribosyltransferases

    SciTech Connect

    Han, S.; Tainer, J.A.

    2001-08-01

    ADP-ribosylation is a widely occurring and biologically critical covalent chemical modification process in pathogenic mechanisms, intracellular signaling systems, DNA repair, and cell division. The reaction is catalyzed by ADP-ribosyltransferases, which transfer the ADP-ribose moiety of NAD to a target protein with nicotinamide release. A family of bacterial toxins and eukaryotic enzymes has been termed the mono-ADP-ribosyltransferases, in distinction to the poly-ADP-ribosyltransferases, which catalyze the addition of multiple ADP-ribose groups to the carboxyl terminus of eukaryotic nucleoproteins. Despite the limited primary sequence homology among the different ADP-ribosyltransferases, a central cleft bearing NAD-binding pocket formed by the two perpendicular b-sheet core has been remarkably conserved between bacterial toxins and eukaryotic mono- and poly-ADP-ribosyltransferases. The majority of bacterial toxins and eukaryotic mono-ADP-ribosyltransferases are characterized by conserved His and catalytic Glu residues. In contrast, Diphtheria toxin, Pseudomonas exotoxin A, and eukaryotic poly-ADP-ribosyltransferases are characterized by conserved Arg and catalytic Glu residues. The NAD-binding core of a binary toxin and a C3-like toxin family identified an ARTT motif (ADP-ribosylating turn-turn motif) that is implicated in substrate specificity and recognition by structural and mutagenic studies. Here we apply structure-based sequence alignment and comparative structural analyses of all known structures of ADP-ribosyltransfeases to suggest that this ARTT motif is functionally important in many ADP-ribosylating enzymes that bear a NAD binding cleft as characterized by conserved Arg and catalytic Glu residues. Overall, structure-based sequence analysis reveals common core structures and conserved active sites of ADP-ribosyltransferases to support similar NAD binding mechanisms but differing mechanisms of target protein binding via sequence variations within the ARTT

  20. Cellular regulation of ADP-ribosylation of proteins: 3. Selective augmentation of in vitro ADP-ribosylation of histone H3 in murine thymic cells after in vivo emetine treatment

    SciTech Connect

    Sooki-Toth, A.; Banfalvi, G.; Staub, M.; Antoni, F. ); Szoelloesi, J. ); Kirsten, E. ); Kun, E. )

    1989-09-01

    Thymic cells were isolated at intervals of between 0 and 144 h from mice that received one intraperitoneal injection of emetine, and thymus weight, incorporation of ({sup 14}C)leucine into proteins and ({sup 3}H)thymidine into DNA in intact thymic cells, as well as initial rates of protein ADP-ribosylation in permeabilized cells were simultaneously monitored. The effect of emetine as an inhibitor of protein synthesis corresponds to the induction of sequential cellular events, such as cell exit and remigration, by other antimitotic agents and produces an activation of proliferation of cells reentering into this organ. Proliferation, as demonstrated by a large increase in DNA synthesis and entrance into S phase, was kinetically related to an apparent increase in poly (ADP-ribose) polymerase activity in thymic cells and a highly significant in vitro ADP-ribosylation of histone H3. since no DNA fragmentation occurred in thymic cells, as tested by a fluorometric technique it is probable that a selective activation of poly (ADP-ribose) polymerase may have been induced in cells that undergo differentiation and proliferation while repopulating thymus.

  1. AmtB Is Necessary for NH4+-Induced Nitrogenase Switch-Off and ADP-Ribosylation in Rhodobacter capsulatus‡

    PubMed Central

    Yakunin, Alexander F.; Hallenbeck, Patrick C.

    2002-01-01

    Rhodobacter capsulatus possesses two genes potentially coding for ammonia transporters, amtB and amtY. In order to better understand their role in the physiology of this bacterium and their possible significance in nitrogen fixation, we created single-knockout mutants. Strains mutated in either amtB or amtY did not show a growth defect under any condition tested and were still capable of taking up ammonia at nearly wild-type rates, but an amtB mutant was no longer capable of transporting methylamine. The amtB strain but not the amtY strain was also totally defective in carrying out ADP-ribosylation of Fe-protein or the switch-off of in vivo nitrogenase activity in response to NH4+ addition. ADP-ribosylation in response to darkness was unaffected in amtB and amtBY strains, and glutamine synthetase activity was normally regulated in these strains in response to ammonium addition, suggesting that one role of AmtB is to function as an ammonia sensor for the processes that regulate nitrogenase activity. PMID:12107124

  2. Pertussis toxin-catalyzed ADP-ribosylation of a G protein in mouse oocytes, eggs, and preimplantation embryos: Developmental changes and possible functional roles

    SciTech Connect

    Jones, J.; Schultz, R.M. )

    1990-06-01

    G proteins, which in many somatic cells serve as mediators of signal transduction, were identified in preimplantation mouse embryos by their capacity to undergo pertussis toxin-catalyzed ADP-ribosylation. Two pertussis toxin (PT) substrates with Mr = 38,000 and 39,000 (alpha 38 and alpha 39) are present in approximately equal amounts. Relative to the amount in freshly isolated germinal vesicle (GV)-intact oocytes, the amount of PT-catalyzed ADP-ribosylation of alpha 38-39 falls during oocyte maturation, rises between the one- and two-cell stages, falls by the eight-cell and morula stages, and increases again by the blastocyst stage. The decrease in PT-catalyzed ADP-ribosylation of alpha 38-39 that occurs during oocyte maturation, however, does not require germinal vesicle breakdown (GVBD), since inhibiting GVBD with 3-isobutyl-1-methyl xanthine (IBMX) does not prevent the decrease in the extent of PT-catalyzed ADP-ribosylation. A biologically active phorbol diester (12-O-tetradecanoyl phorbol 13-acetate), but not an inactive one (4 alpha-phorbol 12,13-didecanoate, 4 alpha-PDD), totally inhibits the increase in PT-catalyzed ADP-ribosylation of alpha 38-39 that occurs between the one- and two-cell stage; TPA inhibits cleavage, but not transcriptional activation, which occurs in the two-cell embryo. In contrast, cytochalasin D, genistein, or aphidicolin, each of which inhibits cleavage of one-cell embryos, or alpha-amanitin or H8, each of which inhibits transcriptional activation but not cleavage of one-cell embryos, have little or inhibitory effects on the increase in PT-catalyzed ADP-ribosylation of alpha 38-39. Results of immunoblotting experiments using an antibody that is highly specific for alpha il-3 reveal the presence of a cross-reactive species of Mr = 38,000 (alpha 38) in the GV-intact oocyte, metaphase II-arrested egg, and one-, two-cell embryos.

  3. TCDD-inducible poly-ADP-ribose polymerase (TIPARP/PARP7) mono-ADP-ribosylates and co-activates liver X receptors.

    PubMed

    Bindesbøll, Christian; Tan, Susanna; Bott, Debbie; Cho, Tiffany; Tamblyn, Laura; MacPherson, Laura; Grønning-Wang, Line; Nebb, Hilde Irene; Matthews, Jason

    2016-04-01

    Members of the poly-ADP-ribose polymerase (PARP) family catalyse the ADP-ribosylation of target proteins and are known to play important roles in many cellular processes, including DNA repair, differentiation and transcription. The majority of PARPs exhibit mono-ADP-ribosyltransferase activity rather than PARP activity; however, little is known about their biological activity. In the present study, we report that 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD)-inducible poly-ADP-ribose polymerase (TIPARP), mono-ADP-ribosylates and positively regulates liver X receptor α (LXRα) and LXRβ activity. Overexpression of TIPARP enhanced LXR-reporter gene activity. TIPARP knockdown or deletion reduced LXR regulated target gene expression levels in HepG2 cells and inTiparp(-/-)mouse embryonic fibroblasts (MEFs) respectively. Deletion and mutagenesis studies showed that TIPARP's zinc-finger and catalytic domains were required to enhance LXR activity. Protein interaction studies using TIPARP and LXRα/β peptide arrays revealed that LXRs interacted with an N-terminal sequence (a.a. 209-236) of TIPARP, which also overlapped with a putative co-activator domain of TIPARP (a.a. 200-225). Immunofluorescence studies showed that TIPARP and LXRα or LXRβ co-localized in the nucleus.In vitroribosylation assays provided evidence that TIPARP mono-ADP-ribosylated both LXRα and LXRβ. Co-immunoprecipitation (co-IP) studies revealed that ADP-ribosylase macrodomain 1 (MACROD1), but not MACROD2, interacted with LXRs in a TIPARP-dependent manner. This was complemented by reporter gene studies showing that MACROD1, but not MACROD2, prevented the TIPARP-dependent increase in LXR activity. GW3965-dependent increases in hepatic Srebp1 mRNA and protein expression levels were reduced inTiparp(-/-)mice compared withTiparp(+/+)mice. Taken together, these data identify a new mechanism of LXR regulation that involves TIPARP, ADP-ribosylation and MACROD1. PMID:26814197

  4. The NarE protein of Neisseria gonorrhoeae catalyzes ADP-ribosylation of several ADP-ribose acceptors despite an N-terminal deletion.

    PubMed

    Rodas, Paula I; Álamos-Musre, A Said; Álvarez, Francisca P; Escobar, Alejandro; Tapia, Cecilia V; Osorio, Eduardo; Otero, Carolina; Calderón, Iván L; Fuentes, Juan A; Gil, Fernando; Paredes-Sabja, Daniel; Christodoulides, Myron

    2016-09-01

    The ADP-ribosylating enzymes are encoded in many pathogenic bacteria in order to affect essential functions of the host. In this study, we show that Neisseria gonorrhoeae possess a locus that corresponds to the ADP-ribosyltransferase NarE, a previously characterized enzyme in N. meningitidis The 291 bp coding sequence of gonococcal narE shares 100% identity with part of the coding sequence of the meningococcal narE gene due to a frameshift previously described, thus leading to a 49-amino-acid deletion at the N-terminus of gonococcal NarE protein. However, we found a promoter region and a GTG start codon, which allowed expression of the protein as demonstrated by RT-PCR and western blot analyses. Using a gonococcal NarE-6xHis fusion protein, we demonstrated that the gonococcal enzyme underwent auto-ADP-ribosylation but to a lower extent than meningococcal NarE. We also observed that gonoccocal NarE exhibited ADP-ribosyltransferase activity using agmatine and cell-free host proteins as ADP-ribose acceptors, but its activity was inhibited by human β-defensins. Taken together, our results showed that NarE of Neisseria gonorrhoeae is a functional enzyme that possesses key features of bacterial ADP-ribosylating enzymes. PMID:27465490

  5. Poly(ADP-Ribosyl)ation Is Required to Modulate Chromatin Changes at c-MYC Promoter during Emergence from Quiescence

    PubMed Central

    Battistelli, Cecilia; Ciotti, Agnese; Amati, Paolo; Maione, Rossella

    2014-01-01

    Poly(ADP-ribosyl)ation is a post-translational modification of various proteins and participates in the regulation of chromatin structure and transcription through complex mechanisms not completely understood. We have previously shown that PARP-1, the major family member of poly(ADP-ribose)polymerases, plays an important role in the cell cycle reactivation of resting cells by regulating the expression of Immediate Early Response Genes, such as c-MYC, c-FOS, JUNB and EGR-1. In the present work we have investigated the molecular mechanisms by which the enzyme induces c-MYC transcription upon serum stimulation of quiescent cells. We show that PARP-1 is constitutively associated in vivo to a c-MYC promoter region recognized as biologically relevant for the transcriptional regulation of the gene. Moreover, we report that serum stimulation causes the prompt accumulation of ADP-ribose polymers on the same region and that this modification is required for chromatin decondensation and for the exchange of negative for positive transcriptional regulators. Finally we provide evidence that the inhibition of PARP activity along with serum stimulation impairs c-MYC induction by preventing the proper accumulation of histone H3 phosphoacetylation, a specific chromatin mark for the activation of Immediate Early Response Genes. These findings not only suggest a novel strategy by which PARP-1 regulates the transcriptional activity of promoters but also provide new information about the complex regulation of c-MYC expression, a critical determinant of the transition from quiescence to proliferation. PMID:25047032

  6. Thromboxane-induced renal vasoconstriction is mediated by the ADP-ribosyl cyclase CD38 and superoxide anion

    PubMed Central

    Vogel, Paul A.; Kopple, Tayler E.; Arendshorst, William J.

    2013-01-01

    The present renal hemodynamic study tested the hypothesis that CD38 and superoxide anion (O2·−) participate in the vasoconstriction produced by activation of thromboxane prostanoid (TP) receptors in the mouse kidney. CD38 is the major mammalian ADP-ribosyl cyclase contributing to vasomotor tone through the generation of cADP-ribose, a second messenger that activates ryanodine receptors to release Ca2+ from the sarcoplasmic reticulum in vascular smooth muscle cells. We evaluated whether the stable thromboxane mimetic U-46619 causes less pronounced renal vasoconstriction in CD38-deficient mice and the involvement of O2·− in U-46619-induced renal vasoconstriction. Our results indicate that U-46619 activation of TP receptors causes renal vasoconstriction in part by activating cADP-ribose signaling in renal resistance arterioles. Based on maximal renal blood flow and renal vascular resistance responses to bolus injections of U-46619, CD38 contributes 30–40% of the TP receptor-induced vasoconstriction. We also found that the antioxidant SOD mimetic tempol attenuated the magnitude of vasoconstriction by U-46619 in both groups of mice, suggesting mediation by O2·−. The degree of tempol blockage of U-46619-induced renal vasoconstriction was greater in wild-type mice, attenuating renal vasoconstriction by 40% compared with 30% in CD38-null mice. In other experiments, U-46619 rapidly stimulated O2·− production (dihydroethidium fluorescence) in isolated mouse afferent arterioles, an effect abolished by tempol. These observations provide the first in vivo demonstration of CD38 and O2·− involvement in the vasoconstrictor effects of TP receptor activation in the kidney and in vitro evidence for TP receptor stimulation of O2·− production by the afferent arteriole. PMID:23884143

  7. Cholera toxin-induced ADP-ribosylation of a 46 kDa protein is decreased in brains of ethanol-fed mice

    SciTech Connect

    Nhamburo, P.T.; Hoffman, P.L.; Tabakoff, B.

    1988-01-01

    The acute in vitro effects of ethanol on cerebral cortical adenylate cyclase activity and beta-adrenergic receptor characteristics suggested a site of action of ethanol at Gs, the stimulatory guanine nucleotide binding protein. After chronic ethanol ingestion, the beta-adrenergic receptor appeared to be uncoupled (i.e., the form of the receptor with high affinity for agonist was undetectable), and stimulation of adenylate cyclase activity by isoproterenol or guanine nucleotides was reduced, suggesting an alteration in the properties of Gs. To further characterize this change, cholera and pertussis toxin-mediated /sup 32/P-ADP-ribosylation of mouse cortical membranes was assessed in mice that had chronically ingested ethanol in a liquid diet. /sup 32/P-labeled proteins were separated by SDS-PAGE and quantitated by autoradiography. There was a selective 30-50% decrease in cholera toxin-induced labeling of 46 kDa protein band in membranes of ethanol-fed mice, with no apparent change in pertussis toxin-induced labeling. The 46 kDa protein has a molecular weight similar to that of the alpha subunit of Gs, suggesting a reduced amount of this protein or a change in its characteristics as a substrate for cholera toxin-induced ADP-ribosylation in cortical membranes of ethanol-fed mice.

  8. Differences in the poly(ADP-ribosyl)ation patterns of ICP4, the herpes simplex virus major regulatory protein, in infected cells and in isolated nuclei.

    PubMed Central

    Blaho, J A; Michael, N; Kang, V; Aboul-Ela, N; Smulson, M E; Jacobson, M K; Roizman, B

    1992-01-01

    Infected-cell protein 4 (ICP4), the major regulatory protein in herpes simplex viruses 1 and 2, was previously reported to accept 32P from [32P]NAD in isolated nuclei. This modification was attributed to poly(ADP-ribosyl)ation (C. M. Preston and E. L. Notarianni, Virology 131:492-501, 1983). We determined that an antibody specific for poly(ADP-ribose) reacts with ICP4 extracted from infected cells, electrophoretically separated in denaturing gels, and electrically transferred to nitrocellulose. Our results indicate that all forms of ICP4 observed in one-dimensional gel electrophoresis are poly(ADP-ribosyl)ated. Poly(ADP-ribose) on ICP4 extracted from infected cells was resistant to cleavage by purified poly(ADP-ribose) glycohydrolase unless ICP4 was in a denatured state. Poly(ADP-ribose) added to ICP4 in isolated nuclei was sensitive to this enzyme. This result indicates that the two processes are distinct and may involve different sites on the ICP4 molecule. Images PMID:1328673

  9. Platelet cytosolic 44-kDa protein is a substrate of cholera toxin-induced ADP-ribosylation and is not recognized by antisera against the. alpha. subunit of the stimulatory guanine nucleotide-binding regulatory protein

    SciTech Connect

    Molina Y Vedia, L.M.; Reep, B.R.; Lapetina, E.G. )

    1988-08-01

    ADP-ribosylation induced by cholera toxin and pertussis toxin was studied in particulate and cytosolic fractions of human platelets. Platelets were disrupted by a cycle of freezing and thawing in the presence of a hyposmotic buffer containing protease inhibitors. In both fractions, the A subunit of cholera toxin ADP-ribosylates two proteins with molecular masses of 42 and 44 kDa, whereas pertussis toxin ADP-ribosylates a 41-kDa polypeptide. Two antisera against the {alpha} subunit of the stimulatory guanine nucleotide-binding regulatory protein recognize only the 42-kDa polypeptide. Cholera toxin-induced ADP-ribosylation of the 42- and 44-kDa proteins is reduced by pretreatment of platelets with iloprost, a prostacyclin analog. The 44-kDa protein, which is substrate of cholera toxin, could be extracted completely from the membrane and recovered in the cytosolic fraction when the cells were disrupted by Dounce homogenization and the pellet was extensively washed. A 44-kDa protein can also be labeled with 8-azidoguanosine 5{prime}-({alpha}-{sup 32}P)triphosphate in the cytosol and membranes. These finding indicate that cholera and pertussis toxins produced covalent modifications of proteins present in particulate and cytosolic platelet fractions. Moreover, the 44-kDa protein might be an {alpha} subunit of a guanine nucleotide-binding regulatory protein that is not recognized by available antisera.

  10. Exogenous nitric oxide (NO) generation or IL-1[beta]-induced intracellular NO production stimulates inhibitory auto-ADP-ribosylation of glyceraldehyde-3-phosphate dehydrogenase in RINm5F cells

    SciTech Connect

    Dimmeler, S.; Bruene, B. ); Ankarcrona, M.; Nicotera, P. )

    1993-04-01

    Nitric oxide (NO) stimulates the auto-ADP-ribosylation of the glycolytic enzyme glyceraldehyde-3-phosphate dehydrogenase (GAPDH) which results in the inhibition of enzyme activity. In the present work the authors show that addition of exogenous NO or IL-1[beta]-induced intracellular NO generation cause GAPDH ADP-ribosylation and inhibition of enzyme activity. Incubation of RINm5F cells with sodium nitroprusside (SNP) for 18 h caused a time- and dose-dependent inhibition of GAPDH activity. Half-maximal inhibition of GAPDH activity was observed with 80 [mu]M of the NO donor, with maximal inhibition after roughly 6 h of incubation. In parallel, SNP induced endogenous ADP-ribosylation of GAPDH measured by a decreased incorporation of [[sup 32]P]ADP-ribose from [[sup 32]P]NAD[sup +] in the cytosol of the SNP-treated cells. Stimulation of endogenous NO production by inducing the NO synthase by exposure to the cytokine IL-1[beta] results in decreased GAPDH activity. IL-1[beta] (10[sup [minus]9] M) inhibited GAPDH activity about 55%, compared with control values. Production of nitrite and inhibition of GAPDH was reversed by the NAD[sup +] synthease inhibitor N[sub G]-monomethyl-L-arginine, indicating the endogenous generated NO was the effective molecule. Again, GAPDH inhibition was associated with NO-stimulated endogenous ADP-ribosylation of the enzyme Western blot analysis of GAPDH excluded degradation of GAPDH by NO. NO-stimulated auto-ADP-ribosylation resulted in inhibition of the glycolytic enzyme GAPDH and may be relevant as a cytotoxic effect of NO. In concert with its inhibitory actions on iron-sulfur enzymes like aconitase and electron transport proteins of the respiratory chain, NO may mediate autocytotoxic effect in [beta]-cells. 40 refs., 7 figs.

  11. Identification of a GTP-binding protein. cap alpha. subunit that lacks an apparent ADP-ribosylation site for pertussis toxin

    SciTech Connect

    Fong, H.K.W.; Yoshimoto, K.K.; Eversole-Cire, P.; Simon, M.I.

    1988-05-01

    Recent molecular cloning of cDNA for the ..cap alpha.. subunit of bovine transducin (a guanine nucleotide-binding regulatory protein, or G protein) has revealed the presence of two retinal-specific transducins, called T/sub r/ and T/sub c/, which are expressed in rod or cone photoreceptor cells. In a further study of G-protein diversity and signal transduction in the retina, the authors have identified a G-protein ..cap alpha.. subunit, which they refer to as G/sub z/..cap alpha.., by isolating a human retinal cDNA clone that cross-hybridizes at reduced stringency with bovine T/sub r/ ..cap alpha..-subunit cDNA. The deduced amino acid sequence of G/sub z/..cap alpha.. is 41-67% identical with those of other known G-protein ..cap alpha.. subunits. However, the 355-residue G/sub z/..cap alpha.. lacks a consensus site for ADP-ribosylation by pertussis toxin, and its amino acid sequence varies within a number of regions that are strongly conserved among all of the other G-protein ..cap alpha.. subunits. They suggest that G/sub z/..cap alpha.., which appears to be highly expressed in neural tissues, represents a member of a subfamily of G proteins that mediate signal transduction in pertussis toxin-insensitive systems.

  12. Functional Characterization of an Extended Binding Component of the Actin-ADP-Ribosylating C2 Toxin Detected in Clostridium botulinum Strain (C) 2300 ▿

    PubMed Central

    Sterthoff, Charlott; Lang, Alexander E.; Schwan, Carsten; Tauch, Andreas; Aktories, Klaus

    2010-01-01

    Clostridium botulinum C2 toxin consists of the binding component C2II and the enzyme component C2I, which ADP-ribosylates G-actin of eukaryotic cells. Trypsin-activated C2II (C2IIa) forms heptamers that mediate cell binding and translocation of C2I from acidic endosomes into the cytosol of target cells. By genome sequencing of C. botulinum strain (C) 2300, we found that C2II from this strain carries a C-terminal extension of 129 amino acids, unlike its homologous counterparts from strains (C) 203U28, (C) 468, and (D) 1873. This extension shows a high similarity to the C-terminal receptor-binding domain of C2II and is presumably the result of a duplication of this domain. The C2II extension facilitates the binding to cell surface receptors, which leads to an increased intoxication efficiency compared to that of C2II proteins from other C. botulinum strains. PMID:20145093

  13. Immunochemical analysis of poly(ADP-ribosyl)ation in HaCaT keratinocytes induced by the mono-alkylating agent 2-chloroethyl ethyl sulfide (CEES): Impact of experimental conditions.

    PubMed

    Debiak, Malgorzata; Lex, Kirsten; Ponath, Viviane; Burckhardt-Boer, Waltraud; Thiermann, Horst; Steinritz, Dirk; Schmidt, Annette; Mangerich, Aswin; Bürkle, Alexander

    2016-02-26

    Sulfur mustard (SM) is a bifunctional alkylating agent with a long history of use as a chemical weapon. Although its last military use is dated for the eighties of the last century, a potential use in terroristic attacks against civilians remains a significant threat. Thus, improving medical therapy of mustard exposed individuals is still of particular interest. PARP inhibitors were recently brought into the focus as a potential countermeasure for mustard-induced pathologies, supported by the availability of efficient compounds successfully tested in cancer therapy. PARP activation after SM treatment was reported in several cell types and tissues under various conditions; however, a detailed characterization of this phenomenon is still missing. This study provides the basis for such studies by developing and optimizing experimental conditions to investigate poly(ADP-ribosyl)ation (PARylation) in HaCaT keratinocytes upon treatment with the monofunctional alkylating agent 2-chloroethyl ethyl sulfide ("half mustard", CEES). By using an immunofluorescence-based approach, we show that optimization of experimental conditions with regards to the type of solvent, dilution factors and treatment procedure is essential to obtain a homogenous PAR staining in HaCaT cell cultures. Furthermore, we demonstrate that different CEES treatment protocols significantly influence the cytotoxicity profiles of treated cells. Using an optimized treatment protocol, our data reveals that CEES induces a dose- and time-dependent dynamic PARylation response in HaCaT cells that could be completely blocked by treating cells with the clinically relevant pharmacological PARP inhibitor ABT888 (also known as veliparib). Finally, siRNA experiments show that CEES-induced PAR formation is predominantly due to the activation of PARP1. In conclusion, this study provides a detailed analysis of the CEES-induced PARylation response in HaCaT keratinocytes, which forms an experimental basis to study the

  14. 2-Azido-( sup 32 P)NAD+, a photoactivatable probe for G-protein structure: Evidence for holotransducin oligomers in which the ADP-ribosylated carboxyl terminus of alpha interacts with both alpha and gamma subunits

    SciTech Connect

    Vaillancourt, R.R.; Dhanasekaran, N.; Johnson, G.L.; Ruoho, A.E. )

    1990-05-01

    A radioactive and photoactivatable derivative of NAD+, 2-azido-(adenylate-32P)NAD+, has been synthesized and used with pertussis toxin to ADP-ribosylate Cys347 of the alpha subunit (alpha T) of GT, the retinal guanine nucleotide-binding protein. ADP-ribosylation of alpha T followed by light activation of the azide moiety of 2-azido-(adenylate-32P)ADP-ribose produced four crosslinked species involving the alpha and gamma subunits of the GT heterotrimer: an alpha trimer (alpha-alpha-alpha), and alpha-alpha-gamma crosslink, an alpha dimer (alpha-alpha), and an alpha-gamma crosslink. The alpha trimer, alpha-alpha-gamma complex, alpha dimer, and alpha-gamma complexes were immunoreactive with alpha T antibodies. The alpha-alpha-gamma and the alpha-gamma complexes were immunoreactive with antisera recognizing gamma subunits. No evidence was found for crosslinking of alpha T to beta T subunits. Hydrolysis of the thioglycosidic bond between Cys347 and 2-azido-(adenylate-32P)ADP-ribose using mercuric acetate resulted in the transfer of radiolabel from Cys347 of alpha T in the crosslinked oligomers to alpha monomers, indicative of intermolecular photocrosslinking, and to gamma monomers, indicative of either intermolecular crosslinked complexes (between heterotrimers) or intramolecular crosslinked complexes (within the heterotrimer). These results demonstrate that GT exists as an oligomer and that ADP-ribosylated Cys347, which is four residues from the alpha T-carboxyl terminus, is oriented toward and in close proximity to the gamma subunit.

  15. Sulfur and nitrogen mustards induce characteristic poly(ADP-ribosyl)ation responses in HaCaT keratinocytes with distinctive cellular consequences.

    PubMed

    Mangerich, Aswin; Debiak, Malgorzata; Birtel, Matthias; Ponath, Viviane; Balszuweit, Frank; Lex, Kirsten; Martello, Rita; Burckhardt-Boer, Waltraud; Strobelt, Romano; Siegert, Markus; Thiermann, Horst; Steinritz, Dirk; Schmidt, Annette; Bürkle, Alexander

    2016-02-26

    Mustard agents are potent DNA alkylating agents with mutagenic, cytotoxic and vesicant properties. They include bi-functional agents, such as sulfur mustard (SM) or nitrogen mustard (mustine, HN2), as well as mono-functional agents, such as "half mustard" (CEES). Whereas SM has been used as a chemical warfare agent, several nitrogen mustard derivatives, such as chlorambucil and cyclophosphamide, are being used as established chemotherapeutics. Upon induction of specific forms of genotoxic stimuli, several poly(ADP-ribose) polymerases (PARPs) synthesize the nucleic acid-like biopolymer poly(ADP-ribose) (PAR) by using NAD(+) as a substrate. Previously, it was shown that SM triggers cellular poly(ADP-ribosyl) ation (PARylation), but so far this phenomenon is poorly characterized. In view of the protective effects of PARP inhibitors, the latter have been proposed as a treatment option of SM-exposed victims. In an accompanying article (Debiak et al., 2016), we have provided an optimized protocol for the analysis of the CEES-induced PARylation response in HaCaT keratinocytes, which forms an experimental basis to further analyze mustard-induced PARylation and its functional consequences, in general. Thus, in the present study, we performed a comprehensive characterization of the PARylation response in HaCaT cells after treatment with four different mustard agents, i.e., SM, CEES, HN2, and chlorambucil, on a qualitative, quantitative and functional level. In particular, we recorded substance-specific as well as dose- and time-dependent PARylation responses using independent bioanalytical methods based on single-cell immuno-fluorescence microscopy and quantitative isotope dilution mass spectrometry. Furthermore, we analyzed if and how PARylation contributes to mustard-induced toxicity by treating HaCaT cells with CEES, SM, and HN2 in combination with the clinically relevant PARP inhibitor ABT888. As evaluated by a novel immunofluorescence-based protocol for the detection of

  16. Cellular regulation of poly ADP-ribosylation of proteins: II. Augmentation of poly(ADP-ribose) polymerase in SV40 3T3 cells following methotrexate-induced G1/S inhibition of cell cycle progression

    SciTech Connect

    Sooki-Toth, A.; Asghari, F.; Kirsten, E.; Kun, E. )

    1987-05-01

    SV40-3T3 cells were exposed in monolayer cultures to 5{times}10{sup {minus}7} M methotrexate (MTX), that inhibited thymidylate synthetase, arrested cell growth without cell killing in 24 h and did not induce single- (ss) or double-strand (ds) breaks in DNA. Following 24, up to 72 h, the poly(ADP-ribose) polymerase content of attached cells was induced by 5{times}10{sup {minus}7} MTX and the augmentation of the enzyme increased with the time of exposure to the drug. Inhibition of protein or RNA synthesis abolished augmentation of enzymatic activity; so too did the initiation of maximal cell growth by thymidine + hypoxanthine, by-passing the inhibitory site of MTX. Isolation of the ADP-ribosylated enzyme protein by gel electrophoresis identified poly(ADP-ribose) polymerase protein as the molecule that was induced by 5{times}10{sup {minus}7} M MTX. Under identical conditions, the poly(ADP-ribose) polymerase induction in 3T3 cells could not be demonstrated. A possible cell-cycle dependent biosynthesis of the enzyme protein is proposed in SV40 3T3 cells.

  17. LdARL-3A, a Leishmania promastigote-specific ADP-ribosylation factor-like protein, is essential for flagellum integrity.

    PubMed

    Cuvillier, A; Redon, F; Antoine, J C; Chardin, P; DeVos, T; Merlin, G

    2000-06-01

    The small G protein-encoding LdARL-3A gene, a homologue of the human ARL-3 gene, was isolated from Leishmania donovani, and its protein product characterised. It is unique in the Leishmania genome and expressed only in the extracellular promastigote insect form, but not in the intracellular amastigote mammalian form, as shown by northern blots and western blots developed with a specific anti-C terminus immune serum. Indirect immunofluorescence microscopy revealed distinct labelled spots regularly distributed on the plasma membrane, including the part lining the flagellum and the flagellar pocket. By transfection experiments, it was found that wild-type LdARL-3A-overexpressing promastigotes reached higher densities in culture, but released significantly less secreted acid phosphatase in the extracellular medium than the parental strain. When LdARL-3A blocked under the GDP-bound 'inactive' form or with an inactivated potential myristoylation site was overexpressed, the cells displayed an apparent wild-type phenotype, but died earlier in the stationary phase; in contrast to parental cells, they showed a diffuse pattern of fluorescence labelling in the cytoplasm and on the cell membrane. Strikingly, when a constitutively 'active' form of LdARL-3A (blocked under the GTP-bound form) was overexpressed, the promastigotes were immobile with a very short flagellum, a slow growth rate and a low level of acid phosphatase secretion; the length of the flagellum was inversely proportional to mutant protein expression. We concluded that LdARL-3A could be an essential gene involved in flagellum biogenesis; it may provide new approaches for control of the parasite at the insect stage. PMID:10806117

  18. ADP-ribosylation factor-like 4C (ARL4C), a novel ovarian cancer metastasis suppressor, identified by integrated genomics

    PubMed Central

    Su, Dan; Katsaros, Dionyssios; Xu, Shenhua; Xu, Haiyan; Gao, Yun; Biglia, Nicoletta; Feng, Jianguo; Ying, Lisha; Zhang, Ping; Benedetto, Chiara; Yu, Herbert

    2015-01-01

    Understanding the molecular mechanisms involving the initiation, progression, and metastasis of ovarian cancer is important for the prevention, detection, and treatment of ovarian cancer. In this study, two ovarian cancer cell lines, HO-8910 and its derivative HO-8910PM with highly metastatic potential, were applied to comparative genomic hybridization (CGH) analysis. We found 14 chromosome fragments with different copy numbers between the two cell lines, one (2q36.1-37.3) of which was confirmed to be one-copy loss in HO-8910PM by fluorescent in situ hybridization (FISH). Using the microarray data on gene expression profiles from these cell lines, 6 significantly expression-decreased genes located on 2q36.1-37.3 in HO-8910PM were identified. Of the 6 genes, ARL4C was identified as a novel ovarian cancer-related gene using integrated molecular and genomic analyses. ARL4C mRNA expression was validated by quantitative PCR to be markedly decreased in HO-8910PM cells, compared to that in HO-8910. Both overexpression and knockdown of ARL4C demonstrated that low ARL4C expression promotes the migration but not influences proliferation capability of ovarian cancer cells in vitro, indicating its specific role in ovarian cancer progression. Furthermore, ovarian cancer patients with medium and high expression of ARL4C mRNA had a favorable prognosis compared to those with low expression, suggesting the ARL4C could be a potential predictor for ovarian cancer prognosis. PMID:25901194

  19. BEX5/RabA1b Regulates trans-Golgi Network-to-Plasma Membrane Protein Trafficking in Arabidopsis[W

    PubMed Central

    Feraru, Elena; Feraru, Mugurel I.; Asaoka, Rin; Paciorek, Tomasz; De Rycke, Riet; Tanaka, Hirokazu; Nakano, Akihiko; Friml, Jiří

    2012-01-01

    Constitutive endocytic recycling is a crucial mechanism allowing regulation of the activity of proteins at the plasma membrane and for rapid changes in their localization, as demonstrated in plants for PIN-FORMED (PIN) proteins, the auxin transporters. To identify novel molecular components of endocytic recycling, mainly exocytosis, we designed a PIN1-green fluorescent protein fluorescence imaging–based forward genetic screen for Arabidopsis thaliana mutants that showed increased intracellular accumulation of cargos in response to the trafficking inhibitor brefeldin A (BFA). We identified bex5 (for BFA-visualized exocytic trafficking defective), a novel dominant mutant carrying a missense mutation that disrupts a conserved sequence motif of the small GTPase, RAS GENES FROM RAT BRAINA1b. bex5 displays defects such as enhanced protein accumulation in abnormal BFA compartments, aberrant endosomes, and defective exocytosis and transcytosis. BEX5/RabA1b localizes to trans-Golgi network/early endosomes (TGN/EE) and acts on distinct trafficking processes like those regulated by GTP exchange factors on ADP-ribosylation factors GNOM-LIKE1 and HOPM INTERACTOR7/BFA-VISUALIZED ENDOCYTIC TRAFFICKING DEFECTIVE1, which regulate trafficking at the Golgi apparatus and TGN/EE, respectively. All together, this study identifies Arabidopsis BEX5/RabA1b as a novel regulator of protein trafficking from a TGN/EE compartment to the plasma membrane. PMID:22773752

  20. HLB1 Is a Tetratricopeptide Repeat Domain-Containing Protein That Operates at the Intersection of the Exocytic and Endocytic Pathways at the TGN/EE in Arabidopsis.

    PubMed

    Sparks, J Alan; Kwon, Taegun; Renna, Luciana; Liao, Fuqi; Brandizzi, Federica; Blancaflor, Elison B

    2016-03-01

    The endomembrane system plays essential roles in plant development, but the proteome responsible for its function and organization remains largely uncharacterized in plants. Here, we identified and characterized the HYPERSENSITIVE TO LATRUNCULIN B1 (HLB1) protein isolated through a forward-genetic screen in Arabidopsis thaliana for mutants with heightened sensitivity to actin-disrupting drugs. HLB1 is a plant-specific tetratricopeptide repeat domain-containing protein of unknown function encoded by a single Arabidopsis gene. HLB1 associated with the trans-Golgi network (TGN)/early endosome (EE) and tracked along filamentous actin, indicating that it could link post-Golgi traffic with the actin cytoskeleton in plants. HLB1 was found to interact with the ADP-ribosylation-factor guanine nucleotide exchange factor, MIN7/BEN1 (HOPM INTERACTOR7/BREFELDIN A-VISUALIZED ENDOCYTIC TRAFFICKING DEFECTIVE1) by coimmunoprecipitation. The min7/ben1 mutant phenocopied the mild root developmental defects and latrunculin B hypersensitivity of hlb1, and analyses of ahlb1/ min7/ben1 double mutant showed that hlb1 and min7/ben1 operate in common genetic pathways. Based on these data, we propose that HLB1 together with MIN7/BEN1 form a complex with actin to modulate the function of the TGN/EE at the intersection of the exocytic and endocytic pathways in plants. PMID:26941089

  1. Activation of Telomerase by Ionizing Radiation: Differential Response to the Inhibition of DNA Double-Strand Break Repair by Abrogation of Poly(ADP-ribosyl)ation, by LY294002, or by Wortmannin

    SciTech Connect

    Neuhof, Dirk Zwicker, Felix; Kuepper, Jan-Heiner; Debus, Juergen; Weber, Klaus-Josef

    2007-11-01

    Purpose: Telomerase activity represents a radiation-inducible function, which may be targeted by a double-strand break (DSB)-activated signal transduction pathway. Therefore, the effects of DNA-PK inhibitors (Wortmannin and LY294002) on telomerase upregulation after irradiation were studied. In addition, the role of trans-dominant inhibition of poly(ADP-ribosyl)ation, which strongly reduces DSB rejoining, was assessed in comparison with 3-aminobenzamide. Methods and Materials: COM3 rodent cells carry a construct for the dexamethasone-inducible overexpression of the DNA-binding domain of PARP1 and exhibit greatly impaired DSB rejoining after irradiation. Telomerase activity was measured using polymerase chain reaction ELISA 1 h after irradiation with doses up to 10 Gy. Phosphorylation status of PKB/Akt and of PKC{alpha}/{beta}{sub II} was assessed by western blotting. Results: No telomerase upregulation was detectable for irradiated cells with undisturbed DSB rejoining. In contrast, incubation with LY294002 or dexamethasone yielded pronounced radiation induction of telomerase activity that could be suppressed by Wortmannin. 3-Aminobenzamide not only was unable to induce telomerase activity but also suppressed telomerase upregulation upon incubation with LY294002 or dexamethasone. Phospho-PKB was detectable independent of irradiation or dexamethasone pretreatment, but was undetectable upon incubations with LY294002 or Wortmannin, whereas phospho-PKC rested detectable. Conclusions: Telomerase activation postirradiation was triggered by different treatments that interfere with DNA DSB processing. This telomerase upregulation, however, was not reflected by the phosporylation status of the putative mediators of TERT activation, PKB and PKC. Although an involvement of PKB in TERT activation is not supported by the present findings, a respective role of PKC isoforms other than {alpha}/{beta}{sub II} cannot be ruled out.

  2. HLB1 Is a Tetratricopeptide Repeat Domain-Containing Protein That Operates at the Intersection of the Exocytic and Endocytic Pathways at the TGN/EE in Arabidopsis[OPEN

    PubMed Central

    Sparks, J. Alan; Renna, Luciana; Liao, Fuqi; Brandizzi, Federica

    2016-01-01

    The endomembrane system plays essential roles in plant development, but the proteome responsible for its function and organization remains largely uncharacterized in plants. Here, we identified and characterized the HYPERSENSITIVE TO LATRUNCULIN B1 (HLB1) protein isolated through a forward-genetic screen in Arabidopsis thaliana for mutants with heightened sensitivity to actin-disrupting drugs. HLB1 is a plant-specific tetratricopeptide repeat domain-containing protein of unknown function encoded by a single Arabidopsis gene. HLB1 associated with the trans-Golgi network (TGN)/early endosome (EE) and tracked along filamentous actin, indicating that it could link post-Golgi traffic with the actin cytoskeleton in plants. HLB1 was found to interact with the ADP-ribosylation-factor guanine nucleotide exchange factor, MIN7/BEN1 (HOPM INTERACTOR7/BREFELDIN A-VISUALIZED ENDOCYTIC TRAFFICKING DEFECTIVE1) by coimmunoprecipitation. The min7/ben1 mutant phenocopied the mild root developmental defects and latrunculin B hypersensitivity of hlb1, and analyses of a hlb1/ min7/ben1 double mutant showed that hlb1 and min7/ben1 operate in common genetic pathways. Based on these data, we propose that HLB1 together with MIN7/BEN1 form a complex with actin to modulate the function of the TGN/EE at the intersection of the exocytic and endocytic pathways in plants. PMID:26941089

  3. Insights into the Localization and Function of the Membrane Trafficking Regulator GNOM ARF-GEF at the Golgi Apparatus in Arabidopsis[W

    PubMed Central

    Naramoto, Satoshi; Otegui, Marisa S.; Kutsuna, Natsumaro; de Rycke, Riet; Dainobu, Tomoko; Karampelias, Michael; Fujimoto, Masaru; Feraru, Elena; Miki, Daisuke; Fukuda, Hiroo; Nakano, Akihiko; Friml, Jiří

    2014-01-01

    GNOM is one of the most characterized membrane trafficking regulators in plants, with crucial roles in development. GNOM encodes an ARF-guanine nucleotide exchange factor (ARF-GEF) that activates small GTPases of the ARF (ADP ribosylation factor) class to mediate vesicle budding at endomembranes. The crucial role of GNOM in recycling of PIN auxin transporters and other proteins to the plasma membrane was identified in studies using the ARF-GEF inhibitor brefeldin A (BFA). GNOM, the most prominent regulator of recycling in plants, has been proposed to act and localize at so far elusive recycling endosomes. Here, we report the GNOM localization in context of its cellular function in Arabidopsis thaliana. State-of-the-art imaging, pharmacological interference, and ultrastructure analysis show that GNOM predominantly localizes to Golgi apparatus. Super-resolution confocal live imaging microscopy identified GNOM and its closest homolog GNOM-like 1 at distinct subdomains on Golgi cisternae. Short-term BFA treatment stabilizes GNOM at the Golgi apparatus, whereas prolonged exposures results in GNOM translocation to trans-Golgi network (TGN)/early endosomes (EEs). Malformed TGN/EE in gnom mutants suggests a role for GNOM in maintaining TGN/EE function. Our results redefine the subcellular action of GNOM and reevaluate the identity and function of recycling endosomes in plants. PMID:25012191

  4. An early secretory pathway mediated by GNOM-LIKE 1 and GNOM is essential for basal polarity establishment in Arabidopsis thaliana

    PubMed Central

    Doyle, Siamsa M.; Haeger, Ash; Vain, Thomas; Rigal, Adeline; Viotti, Corrado; Łangowska, Małgorzata; Ma, Qian; Friml, Jiří; Raikhel, Natasha V.; Hicks, Glenn R.; Robert, Stéphanie

    2015-01-01

    Spatial regulation of the plant hormone indole-3-acetic acid (IAA, or auxin) is essential for plant development. Auxin gradient establishment is mediated by polarly localized auxin transporters, including PIN-FORMED (PIN) proteins. Their localization and abundance at the plasma membrane are tightly regulated by endomembrane machinery, especially the endocytic and recycling pathways mediated by the ADP ribosylation factor guanine nucleotide exchange factor (ARF-GEF) GNOM. We assessed the role of the early secretory pathway in establishing PIN1 polarity in Arabidopsis thaliana by pharmacological and genetic approaches. We identified the compound endosidin 8 (ES8), which selectively interferes with PIN1 basal polarity without altering the polarity of apical proteins. ES8 alters the auxin distribution pattern in the root and induces a strong developmental phenotype, including reduced root length. The ARF-GEF–defective mutants gnom-like 1 (gnl1-1) and gnom (van7) are significantly resistant to ES8. The compound does not affect recycling or vacuolar trafficking of PIN1 but leads to its intracellular accumulation, resulting in loss of PIN1 basal polarity at the plasma membrane. Our data confirm a role for GNOM in endoplasmic reticulum (ER)–Golgi trafficking and reveal that a GNL1/GNOM-mediated early secretory pathway selectively regulates PIN1 basal polarity establishment in a manner essential for normal plant development. PMID:25646449

  5. Insights into the localization and function of the membrane trafficking regulator GNOM ARF-GEF at the Golgi apparatus in Arabidopsis.

    PubMed

    Naramoto, Satoshi; Otegui, Marisa S; Kutsuna, Natsumaro; de Rycke, Riet; Dainobu, Tomoko; Karampelias, Michael; Fujimoto, Masaru; Feraru, Elena; Miki, Daisuke; Fukuda, Hiroo; Nakano, Akihiko; Friml, Jiří

    2014-07-01

    GNOM is one of the most characterized membrane trafficking regulators in plants, with crucial roles in development. GNOM encodes an ARF-guanine nucleotide exchange factor (ARF-GEF) that activates small GTPases of the ARF (ADP ribosylation factor) class to mediate vesicle budding at endomembranes. The crucial role of GNOM in recycling of PIN auxin transporters and other proteins to the plasma membrane was identified in studies using the ARF-GEF inhibitor brefeldin A (BFA). GNOM, the most prominent regulator of recycling in plants, has been proposed to act and localize at so far elusive recycling endosomes. Here, we report the GNOM localization in context of its cellular function in Arabidopsis thaliana. State-of-the-art imaging, pharmacological interference, and ultrastructure analysis show that GNOM predominantly localizes to Golgi apparatus. Super-resolution confocal live imaging microscopy identified GNOM and its closest homolog GNOM-like 1 at distinct subdomains on Golgi cisternae. Short-term BFA treatment stabilizes GNOM at the Golgi apparatus, whereas prolonged exposures results in GNOM translocation to trans-Golgi network (TGN)/early endosomes (EEs). Malformed TGN/EE in gnom mutants suggests a role for GNOM in maintaining TGN/EE function. Our results redefine the subcellular action of GNOM and reevaluate the identity and function of recycling endosomes in plants. PMID:25012191

  6. Crystal structure of the catalytic domain of Pseudomonas exotoxin A complexed with a nicotinamide adenine dinucleotide analog: implications for the activation process and for ADP ribosylation.

    PubMed Central

    Li, M; Dyda, F; Benhar, I; Pastan, I; Davies, D R

    1996-01-01

    The catalytic, or third domain of Pseudomonas exotoxin A (PEIII) catalyzes the transfer of ADP ribose from nicotinamide adenine dinucleotide (NAD) to elongation factor-2 in eukaryotic cells, inhibiting protein synthesis. We have determined the structure of PEIII crystallized in the presence of NAD to define the site of binding and mechanism of activation. However, NAD undergoes a slow hydrolysis and the crystal structure revealed only the hydrolysis products, AMP and nicotinamide, bound to the enzyme. To better define the site of NAD binding, we have now crystallized PEIII in the presence of a less hydrolyzable NAD analog, beta-methylene-thiazole-4-carboxamide adenine dinucleotide (beta-TAD), and refined the complex structure at 2.3 angstroms resolution. There are two independent molecules of PEIII in the crystal, and the conformations of beta-TAD show some differences in the two binding sites. The beta-TAD attached to molecule 2 appears to have been hydrolyzed between the pyrophosphate and the nicotinamide ribose. However molecule 1 binds to an intact beta-TAD and has no crystal packing contacts in the vicinity of the binding site, so that the observed conformation and interaction with the PEIII most likely resembles that of NAD bound to PEIII in solution. We have compared this complex with the catalytic domains of diphtheria toxin, heat labile enterotoxin, and pertussis toxin, all three of which it closely resembles. Images Fig. 1 Fig. 3 PMID:8692916

  7. PARP2 Is the Predominant Poly(ADP-Ribose) Polymerase in Arabidopsis DNA Damage and Immune Responses

    PubMed Central

    Song, Junqi; Keppler, Brian D.; Wise, Robert R.; Bent, Andrew F.

    2015-01-01

    Poly (ADP-ribose) polymerases (PARPs) catalyze the transfer of multiple poly(ADP-ribose) units onto target proteins. Poly(ADP-ribosyl)ation plays a crucial role in a variety of cellular processes including, most prominently, auto-activation of PARP at sites of DNA breaks to activate DNA repair processes. In humans, PARP1 (the founding and most characterized member of the PARP family) accounts for more than 90% of overall cellular PARP activity in response to DNA damage. We have found that, in contrast with animals, in Arabidopsis thaliana PARP2 (At4g02390), rather than PARP1 (At2g31320), makes the greatest contribution to PARP activity and organismal viability in response to genotoxic stresses caused by bleomycin, mitomycin C or gamma-radiation. Plant PARP2 proteins carry SAP DNA binding motifs rather than the zinc finger domains common in plant and animal PARP1 proteins. PARP2 also makes stronger contributions than PARP1 to plant immune responses including restriction of pathogenic Pseudomonas syringae pv. tomato growth and reduction of infection-associated DNA double-strand break abundance. For poly(ADP-ribose) glycohydrolase (PARG) enzymes, we find that Arabidopsis PARG1 and not PARG2 is the major contributor to poly(ADP-ribose) removal from acceptor proteins. The activity or abundance of PARP2 is influenced by PARP1 and PARG1. PARP2 and PARP1 physically interact with each other, and with PARG1 and PARG2, suggesting relatively direct regulatory interactions among these mediators of the balance of poly(ADP-ribosyl)ation. As with plant PARP2, plant PARG proteins are also structurally distinct from their animal counterparts. Hence core aspects of plant poly(ADP-ribosyl)ation are mediated by substantially different enzymes than in animals, suggesting the likelihood of substantial differences in regulation. PMID:25950582

  8. PARP2 Is the Predominant Poly(ADP-Ribose) Polymerase in Arabidopsis DNA Damage and Immune Responses.

    PubMed

    Song, Junqi; Keppler, Brian D; Wise, Robert R; Bent, Andrew F

    2015-05-01

    Poly (ADP-ribose) polymerases (PARPs) catalyze the transfer of multiple poly(ADP-ribose) units onto target proteins. Poly(ADP-ribosyl)ation plays a crucial role in a variety of cellular processes including, most prominently, auto-activation of PARP at sites of DNA breaks to activate DNA repair processes. In humans, PARP1 (the founding and most characterized member of the PARP family) accounts for more than 90% of overall cellular PARP activity in response to DNA damage. We have found that, in contrast with animals, in Arabidopsis thaliana PARP2 (At4g02390), rather than PARP1 (At2g31320), makes the greatest contribution to PARP activity and organismal viability in response to genotoxic stresses caused by bleomycin, mitomycin C or gamma-radiation. Plant PARP2 proteins carry SAP DNA binding motifs rather than the zinc finger domains common in plant and animal PARP1 proteins. PARP2 also makes stronger contributions than PARP1 to plant immune responses including restriction of pathogenic Pseudomonas syringae pv. tomato growth and reduction of infection-associated DNA double-strand break abundance. For poly(ADP-ribose) glycohydrolase (PARG) enzymes, we find that Arabidopsis PARG1 and not PARG2 is the major contributor to poly(ADP-ribose) removal from acceptor proteins. The activity or abundance of PARP2 is influenced by PARP1 and PARG1. PARP2 and PARP1 physically interact with each other, and with PARG1 and PARG2, suggesting relatively direct regulatory interactions among these mediators of the balance of poly(ADP-ribosyl)ation. As with plant PARP2, plant PARG proteins are also structurally distinct from their animal counterparts. Hence core aspects of plant poly(ADP-ribosyl)ation are mediated by substantially different enzymes than in animals, suggesting the likelihood of substantial differences in regulation. PMID:25950582

  9. Activation of Escherichia coli heat-labile enterotoxins by native and recombinant adenosine diphosphate-ribosylation factors, 20-kD guanine nucleotide-binding proteins.

    PubMed Central

    Lee, C M; Chang, P P; Tsai, S C; Adamik, R; Price, S R; Kunz, B C; Moss, J; Twiddy, E M; Holmes, R K

    1991-01-01

    Escherichia coli heat-labile enterotoxins (LT) are responsible in part for "traveler's diarrhea" and related diarrheal illnesses. The family of LTs comprises two serogroups termed LT-I and LT-II; each serogroup includes two or more antigenic variants. The effects of LTs result from ADP ribosylation of Gs alpha, a stimulatory component of adenylyl cyclase; the mechanism of action is identical to that of cholera toxin (CT). The ADP-ribosyltransferase activity of CT is enhanced by 20-kD guanine nucleotide-binding proteins, known as ADP-ribosylation factors or ARFs. These proteins directly activate the CTA1 catalytic unit and stimulate its ADP ribosylation of Gs alpha, other proteins, and simple guanidino compounds (e.g., agmatine). Because of the similarities between CT and LTs, we investigated the effects of purified bovine brain ARF and a recombinant form of bovine ARF synthesized in Escherichia coli on LT activity. ARF enhanced the LT-I-, LT-IIa-, and LT-IIb-catalyzed ADP ribosylation of agmatine, as well as the auto-ADP ribosylation of the toxin catalytic unit. Stimulation of ADP-ribosylagmatine formation by LTs and CT in the presence of ARF was GTP dependent and enhanced by sodium dodecyl sulfate. With agmatine as substrate, LT-IIa and LT-IIb exhibited less than 1% the activity of CT and LT-Ih. CT and LTs catalyzed ADP-ribosyl-Gs alpha formation in a reaction dependent on ARF, GTP, and dimyristoyl phosphatidylcholine/cholate. With Gs alpha as substrate, the ADP-ribosyltransferase activities of the toxins were similar, although CT and LT-Ih appeared to be slightly more active than LT-IIa and LT-IIb. Thus, LT-IIa and LT-IIb appear to differ somewhat from CT and LT-Ih in substrate specificity. Responsiveness to stimulation by ARF, GTP, and phospholipid/detergent as well as the specificity of ADP-ribosyltransferase activity are functions of LTs from serogroups LT-I and LT-II that are shared with CT. Images PMID:1902492

  10. NEVERSHED and INFLORESCENCE DEFICIENT IN ABSCISSION are differentially required for cell expansion and cell separation during floral organ abscission in Arabidopsis thaliana.

    PubMed

    Liu, Bin; Butenko, Melinka A; Shi, Chun-Lin; Bolivar, Jenny L; Winge, Per; Stenvik, Grethe-Elisabeth; Vie, Ane Kjersti; Leslie, Michelle E; Brembu, Tore; Kristiansen, Wenche; Bones, Atle M; Patterson, Sara E; Liljegren, Sarah J; Aalen, Reidunn B

    2013-12-01

    Floral organ shedding is a cell separation event preceded by cell-wall loosening and generally accompanied by cell expansion. Mutations in NEVERSHED (NEV) or INFLORESCENCE DEFICIENT IN ABSCISSION (IDA) block floral organ abscission in Arabidopsis thaliana. NEV encodes an ADP-ribosylation factor GTPase-activating protein, and cells of nev mutant flowers display membrane-trafficking defects. IDA encodes a secreted peptide that signals through the receptor-like kinases HAESA (HAE) and HAESA-LIKE2 (HSL2). Analyses of single and double mutants revealed unique features of the nev and ida phenotypes. Cell-wall loosening was delayed in ida flowers. In contrast, nev and nev ida mutants displayed ectopic enlargement of abscission zone (AZ) cells, indicating that cell expansion alone is not sufficient to trigger organ loss. These results suggest that NEV initially prevents precocious cell expansion but is later integral for cell separation. IDA is involved primarily in the final cell separation step. A mutation in KNOTTED-LIKE FROM ARABIDOPSIS THALIANA1 (KNAT1), a suppressor of the ida mutant, could not rescue the abscission defects of nev mutant flowers, indicating that NEV-dependent activity downstream of KNAT1 is required. Transcriptional profiling of mutant AZs identified gene clusters regulated by IDA-HAE/HSL2. Several genes were more strongly downregulated in nev-7 compared with ida and hae hsl2 mutants, consistent with the rapid inhibition of organ loosening in nev mutants, and the overlapping roles of NEV and IDA in cell separation. A model of the crosstalk between the IDA signalling pathway and NEV-mediated membrane traffic during floral organ abscission is presented. PMID:23963677

  11. Functional Analysis of Transcription Factors in Arabidopsis

    PubMed Central

    Mitsuda, Nobutaka; Ohme-Takagi, Masaru

    2009-01-01

    Transcription factors (TFs) regulate the expression of genes at the transcriptional level. Modification of TF activity dynamically alters the transcriptome, which leads to metabolic and phenotypic changes. Thus, functional analysis of TFs using ‘omics-based’ methodologies is one of the most important areas of the post-genome era. In this mini-review, we present an overview of Arabidopsis TFs and introduce strategies for the functional analysis of plant TFs, which include both traditional and recently developed technologies. These strategies can be assigned to five categories: bioinformatic analysis; analysis of molecular function; expression analysis; phenotype analysis; and network analysis for the description of entire transcriptional regulatory networks. PMID:19478073

  12. Molecular insights into plant cell proliferation disturbance by Agrobacterium protein 6b

    PubMed Central

    Wang, Meimei; Soyano, Takashi; Machida, Satoru; Yang, Jun-Yi; Jung, Choonkyun; Chua, Nam-Hai; Yuan, Y. Adam

    2011-01-01

    The Agrobacterium Ti plasmid (T-DNA) 6b proteins interact with many different host proteins implicated in plant cell proliferation. Here, we show that Arabidopsis plants overexpressing 6b display microRNA (miRNA) deficiency by directly targeting SERRATE and AGO1 via a specific loop fragment (residues 40–55). In addition, we report the crystal structures of Agrobacterium tumefaciens AK6b at 2.1 Å, Agrobacterium vitis AB6b at 1.65 Å, and Arabidopsis ADP ribosylation factor (ARF) at 1.8 Å. The 6b structure adopts an ADP-ribosylating toxin fold closely related to cholera toxin. In vitro ADP ribosylation analysis demonstrates that 6b represents a new toxin family, with Tyr 66, Thr 93, and Tyr 153 as the ADP ribosylation catalytic residues in the presence of Arabidopsis ARF and GTP. Our work provides molecular insights, suggesting that 6b regulates plant cell growth by the disturbance of the miRNA pathway through its ADP ribosylation activity. PMID:21156810

  13. Analysis of knockout mutants reveals non-redundant functions of poly(ADP-ribose)polymerase isoforms in Arabidopsis.

    PubMed

    Pham, Phuong Anh; Wahl, Vanessa; Tohge, Takayuki; de Souza, Laise Rosado; Zhang, Youjun; Do, Phuc Thi; Olas, Justyna J; Stitt, Mark; Araújo, Wagner L; Fernie, Alisdair R

    2015-11-01

    The enzyme poly(ADP-ribose)polymerase (PARP) has a dual function being involved both in the poly(ADP-ribosyl)ation and being a constituent of the NAD(+) salvage pathway. To date most studies, both in plant and non-plant systems, have focused on the signaling role of PARP in poly(ADP-ribosyl)ation rather than any role that can be ascribed to its metabolic function. In order to address this question we here used a combination of expression, transcript and protein localization studies of all three PARP isoforms of Arabidopsis alongside physiological analysis of the corresponding mutants. Our analyses indicated that whilst all isoforms of PARP were localized to the nucleus they are also present in non-nuclear locations with parp1 and parp3 also localised in the cytosol, and parp2 also present in the mitochondria. We next isolated and characterized insertional knockout mutants of all three isoforms confirming a complete knockout in the full length transcript levels of the target genes as well as a reduced total leaf NAD hydrolase activity in the two isoforms (PARP1, PARP2) that are highly expressed in leaves. Physiological evaluation of the mutant lines revealed that they displayed distinctive metabolic and root growth characteristics albeit unaltered leaf morphology under optimal growth conditions. We therefore conclude that the PARP isoforms play non-redundant non-nuclear metabolic roles and that their function is highly important in rapidly growing tissues such as the shoot apical meristem, roots and seeds. PMID:26428915

  14. Rapamycin inhibits poly(ADP-ribosyl)ation in intact cells

    SciTech Connect

    Fahrer, Joerg; Wagner, Silvia; Buerkle, Alexander; Koenigsrainer, Alfred

    2009-08-14

    Rapamycin is an immunosuppressive drug, which inhibits the mammalian target of rapamycin (mTOR) kinase activity inducing changes in cell proliferation. Synthesis of poly(ADP-ribose) (PAR) is an immediate cellular response to genotoxic stress catalyzed mostly by poly(ADP-ribose) polymerase 1 (PARP-1), which is also controlled by signaling pathways. Therefore, we investigated whether rapamycin affects PAR production. Strikingly, rapamycin inhibited PAR synthesis in living fibroblasts in a dose-dependent manner as monitored by immunofluorescence. PARP-1 activity was then assayed in vitro, revealing that down-regulation of cellular PAR production by rapamycin was apparently not due to competitive PARP-1 inhibition. Further studies showed that rapamycin did not influence the cellular NAD pool and the activation of PARP-1 in extracts of pretreated fibroblasts. Collectively, our data suggest that inhibition of cellular PAR synthesis by rapamycin is mediated by formation of a detergent-sensitive complex in living cells, and that rapamycin may have a potential as therapeutic PARP inhibitor.

  15. Molecular Screening Tools to Study Arabidopsis Transcription Factors

    PubMed Central

    Wehner, Nora; Weiste, Christoph; Dröge-Laser, Wolfgang

    2011-01-01

    In the model plant Arabidopsis thaliana, more than 2000 genes are estimated to encode transcription factors (TFs), which clearly emphasizes the importance of transcriptional control. Although genomic approaches have generated large TF open reading frame (ORF) collections, only a limited number of these genes is functionally characterized, yet. This review evaluates strategies and methods to identify TF functions. In particular, we focus on two recently developed TF screening platforms, which make use of publically available GATEWAY®-compatible ORF collections. (1) The Arabidopsis thaliana TF ORF over-Expression (AtTORF-Ex) library provides pooled collections of transgenic lines over-expressing HA-tagged TF genes, which are suited for screening approaches to define TF functions in stress defense and development. (2) A high-throughput microtiter plate based protoplast trans activation (PTA) system has been established to screen for TFs which are regulating a given promoter:Luciferase construct in planta. PMID:22645547

  16. Novel Disease Susceptibility Factors for Fungal Necrotrophic Pathogens in Arabidopsis

    PubMed Central

    García-Andrade, Javier; Angulo, Carlos; Neumetzler, Lutz; Persson, Staffan; Vera, Pablo

    2015-01-01

    Host cells use an intricate signaling system to respond to invasions by pathogenic microorganisms. Although several signaling components of disease resistance against necrotrophic fungal pathogens have been identified, our understanding for how molecular components and host processes contribute to plant disease susceptibility is rather sparse. Here, we identified four transcription factors (TFs) from Arabidopsis that limit pathogen spread. Arabidopsis mutants defective in any of these TFs displayed increased disease susceptibility to Botrytis cinerea and Plectosphaerella cucumerina, and a general activation of non-immune host processes that contribute to plant disease susceptibility. Transcriptome analyses revealed that the mutants share a common transcriptional signature of 77 up-regulated genes. We characterized several of the up-regulated genes that encode peptides with a secretion signal, which we named PROVIR (for provirulence) factors. Forward and reverse genetic analyses revealed that many of the PROVIRs are important for disease susceptibility of the host to fungal necrotrophs. The TFs and PROVIRs identified in our work thus represent novel genetic determinants for plant disease susceptibility to necrotrophic fungal pathogens. PMID:25830627

  17. Transcription Factor Arabidopsis Activating Factor1 Integrates Carbon Starvation Responses with Trehalose Metabolism1[OPEN

    PubMed Central

    Garapati, Prashanth; Feil, Regina; Lunn, John Edward; Van Dijck, Patrick; Balazadeh, Salma; Mueller-Roeber, Bernd

    2015-01-01

    Plants respond to low carbon supply by massive reprogramming of the transcriptome and metabolome. We show here that the carbon starvation-induced NAC (for NO APICAL MERISTEM/ARABIDOPSIS TRANSCRIPTION ACTIVATION FACTOR/CUP-SHAPED COTYLEDON) transcription factor Arabidopsis (Arabidopsis thaliana) Transcription Activation Factor1 (ATAF1) plays an important role in this physiological process. We identified TREHALASE1, the only trehalase-encoding gene in Arabidopsis, as a direct downstream target of ATAF1. Overexpression of ATAF1 activates TREHALASE1 expression and leads to reduced trehalose-6-phosphate levels and a sugar starvation metabolome. In accordance with changes in expression of starch biosynthesis- and breakdown-related genes, starch levels are generally reduced in ATAF1 overexpressors but elevated in ataf1 knockout plants. At the global transcriptome level, genes affected by ATAF1 are broadly associated with energy and carbon starvation responses. Furthermore, transcriptional responses triggered by ATAF1 largely overlap with expression patterns observed in plants starved for carbon or energy supply. Collectively, our data highlight the existence of a positively acting feedforward loop between ATAF1 expression, which is induced by carbon starvation, and the depletion of cellular carbon/energy pools that is triggered by the transcriptional regulation of downstream gene regulatory networks by ATAF1. PMID:26149570

  18. Transcription Factor Arabidopsis Activating Factor1 Integrates Carbon Starvation Responses with Trehalose Metabolism.

    PubMed

    Garapati, Prashanth; Feil, Regina; Lunn, John Edward; Van Dijck, Patrick; Balazadeh, Salma; Mueller-Roeber, Bernd

    2015-09-01

    Plants respond to low carbon supply by massive reprogramming of the transcriptome and metabolome. We show here that the carbon starvation-induced NAC (for NO APICAL MERISTEM/ARABIDOPSIS TRANSCRIPTION ACTIVATION FACTOR/CUP-SHAPED COTYLEDON) transcription factor Arabidopsis (Arabidopsis thaliana) Transcription Activation Factor1 (ATAF1) plays an important role in this physiological process. We identified TREHALASE1, the only trehalase-encoding gene in Arabidopsis, as a direct downstream target of ATAF1. Overexpression of ATAF1 activates TREHALASE1 expression and leads to reduced trehalose-6-phosphate levels and a sugar starvation metabolome. In accordance with changes in expression of starch biosynthesis- and breakdown-related genes, starch levels are generally reduced in ATAF1 overexpressors but elevated in ataf1 knockout plants. At the global transcriptome level, genes affected by ATAF1 are broadly associated with energy and carbon starvation responses. Furthermore, transcriptional responses triggered by ATAF1 largely overlap with expression patterns observed in plants starved for carbon or energy supply. Collectively, our data highlight the existence of a positively acting feedforward loop between ATAF1 expression, which is induced by carbon starvation, and the depletion of cellular carbon/energy pools that is triggered by the transcriptional regulation of downstream gene regulatory networks by ATAF1. PMID:26149570

  19. Positional distribution of transcription factor binding sites in Arabidopsis thaliana

    PubMed Central

    Yu, Chun-Ping; Lin, Jinn-Jy; Li, Wen-Hsiung

    2016-01-01

    Binding of a transcription factor (TF) to its DNA binding sites (TFBSs) is a critical step to initiate the transcription of its target genes. It is therefore interesting to know where the TFBSs of a gene are likely to locate in the promoter region. Here we studied the positional distribution of TFBSs in Arabidopsis thaliana, for which many known TFBSs are now available. We developed a method to identify the locations of TFBSs in the promoter sequences of genes in A. thaliana. We found that the distribution is nearly bell-shaped with a peak at 50 base pairs (bp) upstream of the transcription start site (TSS) and 86% of the TFBSs are in the region from −1,000 bp to +200 bp with respect to the TSS. Our distribution was supported by chromatin immunoprecipitation sequencing and microarray data and DNase I hypersensitive site sequencing data. When TF families were considered separately, differences in positional preference were observed between TF families. Our study of the positional distribution of TFBSs seems to be the first in a plant. PMID:27117388

  20. Subcellular Distribution of NTL Transcription Factors in Arabidopsis thaliana.

    PubMed

    Liang, Mingwei; Li, Hongjuan; Zhou, Fang; Li, Huiyong; Liu, Jin; Hao, Yi; Wang, Yingdian; Zhao, Heping; Han, Shengcheng

    2015-10-01

    NAC with a transmembrane (TM) motif1-like (NTL) transcription factors, containing three regions: the N-terminal NAC domain (ND), the middle regulation region (RR), and the C-terminal TM domain, belong to the tail-anchored proteins. Although these NTLs play numerous essential roles in plants, their subcellular distribution and the mechanism of translocation into the nucleus (NU) remain unclear. In this study, we found that most of the full-length NTLs were localized in the endoplasmic reticulum (ER), with the exception of NTL11 and NTL5, which were restricted to the NU. Furthermore, we found that NTL11 contains a TM domain, whereas NTL5 does not. The ND of all of the NTLs was responsible for nuclear localization in plants. After truncation of the TM domain, NTL8_NR, NTL10_NR and NTL13_NR localized in the cytoplasm (CT) and NU, and other NTL_NRs were only localized in the NU, suggesting that the RR of NTL8, NTL10 and NTL13 contains some inhibitory region to mask the nuclear localization signal sequence in the ND domain and permit their diffusion between CT and NU. Furthermore, the N-terminus of NTL11 was translocated to the NU, but the C-terminus was degraded in Arabidopsis mesophyll protoplasts. The chimeric construct of NTL11_ND with NTL10_RR and TM domain (11ND-10RT) was localized exclusively in the ER, and not in the NU. However, 10ND-11RT was found mainly in the NU. Our results indicated that the TM domain is essential for NTL targeting the ER and the N-terminal fragment, including ND and RR, is translocated into the NU after activation through proteolytic cleavage events upon stimulation by internal and external environmental signals. PMID:26201836

  1. Temperature as a determinant factor for increased and reproducible in vitro pollen germination in Arabidopsis thaliana

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Despite much effort, a robust protocol for in vitro germination of Arabidopsis thaliana pollen was still elusive. Here we show that controlled temperatures, a largely disregarded factor in previous studies, and a simple optimized medium, solidified or liquid, yielded pollen germination rates above 8...

  2. Dynamic Distribution and Interaction of the Arabidopsis SRSF1 Subfamily Splicing Factors.

    PubMed

    Stankovic, Nancy; Schloesser, Marie; Joris, Marine; Sauvage, Eric; Hanikenne, Marc; Motte, Patrick

    2016-02-01

    Ser/Arg-rich (SR) proteins are essential nucleus-localized splicing factors. Our prior studies showed that Arabidopsis (Arabidopsis thaliana) RSZ22, a homolog of the human SRSF7 SR factor, exits the nucleus through two pathways, either dependent or independent on the XPO1 receptor. Here, we examined the expression profiles and shuttling dynamics of the Arabidopsis SRSF1 subfamily (SR30, SR34, SR34a, and SR34b) under control of their endogenous promoter in Arabidopsis and in transient expression assay. Due to its rapid nucleocytoplasmic shuttling and high expression level in transient assay, we analyzed the multiple determinants that regulate the localization and shuttling dynamics of SR34. By site-directed mutagenesis of SR34 RNA-binding sequences and Arg/Ser-rich (RS) domain, we further show that functional RRM1 or RRM2 are dispensable for the exclusive protein nuclear localization and speckle-like distribution. However, mutations of both RRMs induced aggregation of the protein whereas mutation in the RS domain decreased the stability of the protein and suppressed its nuclear accumulation. Furthermore, the RNA-binding motif mutants are defective for their export through the XPO1 (CRM1/Exportin-1) receptor pathway, but retain nucleocytoplasmic mobility. We performed a yeast two hybrid screen with SR34 as bait and discovered SR45 as a new interactor. SR45 is an unusual SR splicing factor bearing two RS domains. These interactions were confirmed in planta by FLIM-FRET and BiFC and the roles of SR34 domains in protein-protein interactions were further studied. Altogether, our report extends our understanding of shuttling dynamics of Arabidopsis SR splicing factors. PMID:26697894

  3. Wheat Transcription Factor TaAREB3 Participates in Drought and Freezing Tolerances in Arabidopsis.

    PubMed

    Wang, Jingyi; Li, Qian; Mao, Xinguo; Li, Ang; Jing, Ruilian

    2016-01-01

    AREB (ABA response element binding) proteins in plants play direct regulatory roles in response to multiple stresses, but their functions in wheat (Triticum aestivum L.) are not clear. In the present study, TaAREB3, a new member of the AREB transcription factor family, was isolated from wheat. Sequence analysis showed that the TaAREB3 protein is composed of three parts, a conserved N-terminal, a variable M region, and a conserved C-terminal with a bZIP domain. It belongs to the group A subfamily of bZIP transcription factors. TaAREB3 was constitutively expressed in stems, leaves, florets, anthers, pistils, seeds, and most highly, in roots. TaAREB3 gene expression was induced with abscisic acid (ABA) and low temperature stress, and its protein was localized in the nucleus when transiently expressed in tobacco epidermal cells and stably expressed in transgenic Arabidopsis. TaAREB3 protein has transcriptional activation activity, and can bind to the ABRE cis-element in vitro. Overexpression of TaAREB3 in Arabidopsis not only enhanced ABA sensitivity, but also strengthened drought and freezing tolerances. TaAREB3 also activated RD29A, RD29B, COR15A, and COR47 by binding to their promoter regions in transgenic Arabidopsis. These results demonstrated that TaAREB3 plays an important role in drought and freezing tolerances in Arabidopsis. PMID:26884722

  4. REGIA, an EU project on functional genomics of transcription factors from Arabidopsis Thaliana.

    PubMed

    Paz-Ares, Javier

    2002-01-01

    Transcription factors (TFs) are regulatory proteins that have played a pivotal role in the evolution of eukaryotes and that also have great biotechnological potential. REGIA (REgulatory Gene Initiative in Arabidopsis) is an EU-funded project involving 29 European laboratories with the objective of determining the function of virtually all transcription factors from the model plant, Arabidopsis thaliana. REGIA involves: 1. the definition of TF gene expression patterns in Arabidopsis; 2. the identification of mutations at TF loci; 3. the ectopic expression of TFs (or derivatives) in Arabidopsis and in crop plants; 4. phenotypic analysis of the mutants and mis-expression lines, including both RNA and metabolic profiling; 5. the systematic analysis of interactions between TFs; and 6. the generation of a bioinformatics infrastructure to access and integrate all this information. We expect that this programme will establish the full biotechnological potential of plant TFs, and provide insights into hierarchies, redundancies, and interdependencies, and their evolution. The project involves the preparation of both a TF gene array for expression analysis and a normalised full length open reading frame (ORF) library of TFs in a yeast two hybrid vector; the applications of these resources should extend beyond the scope of this programme. PMID:18628849

  5. Wheat Transcription Factor TaAREB3 Participates in Drought and Freezing Tolerances in Arabidopsis

    PubMed Central

    Wang, Jingyi; Li, Qian; Mao, Xinguo; Li, Ang; Jing, Ruilian

    2016-01-01

    AREB (ABA response element binding) proteins in plants play direct regulatory roles in response to multiple stresses, but their functions in wheat (Triticum aestivum L.) are not clear. In the present study, TaAREB3, a new member of the AREB transcription factor family, was isolated from wheat. Sequence analysis showed that the TaAREB3 protein is composed of three parts, a conserved N-terminal, a variable M region, and a conserved C-terminal with a bZIP domain. It belongs to the group A subfamily of bZIP transcription factors. TaAREB3 was constitutively expressed in stems, leaves, florets, anthers, pistils, seeds, and most highly, in roots. TaAREB3 gene expression was induced with abscisic acid (ABA) and low temperature stress, and its protein was localized in the nucleus when transiently expressed in tobacco epidermal cells and stably expressed in transgenic Arabidopsis. TaAREB3 protein has transcriptional activation activity, and can bind to the ABRE cis-element in vitro. Overexpression of TaAREB3 in Arabidopsis not only enhanced ABA sensitivity, but also strengthened drought and freezing tolerances. TaAREB3 also activated RD29A, RD29B, COR15A, and COR47 by binding to their promoter regions in transgenic Arabidopsis. These results demonstrated that TaAREB3 plays an important role in drought and freezing tolerances in Arabidopsis. PMID:26884722

  6. REGIA, An EU Project on Functional Genomics of Transcription Factors From Arabidopsis Thaliana

    PubMed Central

    The REGIA Consortium

    2002-01-01

    Transcription factors (TFs) are regulatory proteins that have played a pivotal role in the evolution of eukaryotes and that also have great biotechnological potential. REGIA (REgulatory Gene Initiative in Arabidopsis) is an EU-funded project involving 29 European laboratories with the objective of determining the function of virtually all transcription factors from the model plant, Arabidopsis thaliana. REGIA involves: 1. the definition of TF gene expression patterns in Arabidopsis; 2. the identification of mutations at TF loci; 3. the ectopic expression of TFs (or derivatives) in Arabidopsis and in crop plants; 4. phenotypic analysis of the mutants and mis-expression lines, including both RNA and metabolic profiling; 5. the systematic analysis of interactions between TFs; and 6. the generation of a bioinformatics infrastructure to access and integrate all this information. We expect that this programme will establish the full biotechnological potential of plant TFs, and provide insights into hierarchies, redundancies, and interdependencies, and their evolution. The project involves the preparation of both a TF gene array for expression analysis and a normalised full length open reading frame (ORF) library of TFs in a yeast two hybrid vector; the applications of these resources should extend beyond the scope of this programme. PMID:18628849

  7. GOLDEN2-LIKE transcription factors coordinate the tolerance to Cucumber mosaic virus in Arabidopsis.

    PubMed

    Han, Xue-Ying; Li, Peng-Xu; Zou, Li-Juan; Tan, Wen-Rong; Zheng, Ting; Zhang, Da-Wei; Lin, Hong-Hui

    2016-09-01

    Arabidopsis thaliana GOLDEN2-LIKE (GLKs) transcription factors play important roles in regulation of photosynthesis-associated nuclear genes, as well as participate in chloroplast development. However, the involvement of GLKs in plants resistance to virus remains largely unknown. Here, the relationship between GLKs and Cucumber mosaic virus (CMV) stress response was investigated. Our results showed that the Arabidopsis glk1glk2 double-mutant was more susceptible to CMV infection and suffered more serious damages (such as higher oxidative damages, more compromised in PSII photochemistry and more reactive oxygen species accumulation) when compared with the wild-type plants. Interestingly, there was little difference between single mutant (glk1 or glk2) and wild-type plants in response to CMV infection, suggesting GLK1 and GLK2 might function redundant in virus resistance in Arabidopsis. Furthermore, the induction of antioxidant system and defense-associated genes expression in the double mutant were inhibited when compared with single mutant or wild-type plants after CMV infection. Further evidences showed that salicylic acid (SA) and jasmonic acid (JA) might be involved in GLKs-mediated virus resistance, as SA or JA level and synthesis-related genes transcription were impaired in glk1glk2 mutant. Taken together, our results indicated that GLKs played a positively role in virus resistance in Arabidopsis. PMID:27346129

  8. Expression of aberrant forms of AUXIN RESPONSE FACTOR8 stimulates parthenocarpy in Arabidopsis and tomato.

    PubMed

    Goetz, Marc; Hooper, Lauren C; Johnson, Susan D; Rodrigues, Julio Carlyle Macedo; Vivian-Smith, Adam; Koltunow, Anna M

    2007-10-01

    Fruit initiation in Arabidopsis (Arabidopsis thaliana) is generally repressed until fertilization occurs. However, mutations in AUXIN RESPONSE FACTOR8 (ARF8) uncouple fruit initiation from fertilization, resulting in the formation of seedless, parthenocarpic fruit. Here we induced parthenocarpy in wild-type Arabidopsis by introducing either the mutant genomic (g) Atarf8-4 sequence or gAtARF8:beta-glucuronidase translational fusion constructs by plant transformation. Silencing of endogenous AtARF8 transcription was not observed, indicating that the introduced, aberrant ARF8 transcripts were compromising the function of endogenous ARF8 and/or associated factors involved in suppressing fruit initiation. To analyze the role of ARF8 in tomato (Solanum lycopersicum) we initially emasculated 23 tomato cultivars to test for background parthenocarpy. Surprisingly, all had a predisposition to initiate fertilization-independent fruit growth. Expression of gAtarf8-4 in transgenic tomato ('Monalbo') resulted in a significant increase in the number and size of parthenocarpic fruit. Isolation of tomato ARF8 cDNA indicated significant sequence conservation with AtARF8. SlARF8 may therefore control tomato fruit initiation in a similar manner as AtARF8 does in Arabidopsis. Two SlARF8 cDNAs differing in size by 5 bp were found, both arising from the same gene. The smaller cDNA is a splice variant and is also present in Arabidopsis. We propose that low endogenous levels of the splice variant products might interfere with efficient formation/function of a complex repressing fruit initiation, thereby providing an explanation for the observed ovary expansion in tomato and also Arabidopsis after emasculation. Increasing the levels of aberrant Atarf8-4 transcripts may further destabilize formation/function of the complex in a dosage-dependent manner enhancing tomato parthenocarpic fruit initiation frequency and size and mimicking the parthenocarpic dehiscent silique phenotype found in

  9. Arabidopsis ribosomal proteins control developmental programs through translational regulation of auxin response factors

    PubMed Central

    Rosado, Abel; Li, Ruixi; van de Ven, Wilhelmina; Hsu, Emily; Raikhel, Natasha V.

    2012-01-01

    Upstream ORFs are elements found in the 5′-leader sequences of specific mRNAs that modulate the translation of downstream ORFs encoding major gene products. In Arabidopsis, the translational control of auxin response factors (ARFs) by upstream ORFs has been proposed as a regulatory mechanism required to respond properly to complex auxin-signaling inputs. In this study, we identify and characterize the aberrant auxin responses in specific ribosomal protein mutants in which multiple ARF transcription factors are simultaneously repressed at the translational level. This characteristic lends itself to the use of these mutants as genetic tools to bypass the genetic redundancy among members of the ARF family in Arabidopsis. Using this approach, we were able to assign unique functions for ARF2, ARF3, and ARF6 in plant development. PMID:23144218

  10. Regulation of Cell Fate Determination by Single-Repeat R3 MYB Transcription Factors in Arabidopsis

    SciTech Connect

    Wang, Shucai; Chen, Jay

    2014-01-01

    MYB transcription factors regulate multiple aspects of plant growth and development. Among the large family of MYB transcription factors, single-repeat R3 MYB are characterized by their short sequence (<120 amino acids) consisting largely of the single MYB DNA-binding repeat. In the model plant Arabidopsis, R3 MYBs mediate lateral inhibition during epidermal patterning and are best characterized for their regulatory roles in trichome and root hair development. R3 MYBs act as negative regulators for trichome formation but as positive regulators for root hair development. In this article, we provide a comprehensive review on the role of R3 MYBs in the regulation of cell type specification in the model plant Arabidopsis.

  11. ZmGRF, a GA regulatory factor from maize, promotes flowering and plant growth in Arabidopsis.

    PubMed

    Xu, Miaoyun; Lu, Yunming; Yang, Hongmei; He, Jingcheng; Hu, Zhiqiu; Hu, Xiaolong; Luan, Mingda; Zhang, Lan; Fan, Yunliu; Wang, Lei

    2015-01-01

    Transcription factors that act as positive regulators of gibberellin (GA) biosynthetic genes in plants are not well understood. A nuclear-localized basic leucine zipper transcription factor, ZmGRF, was isolated from maize. The core DNA sequence motif recognized for binding by ZmGRF was CCANNTGGC. ZmGRF overexpression in transgenic Arabidopsis plants promoted flowering, stem elongation, and cell expansion. Chromatin immunoprecipitation assays revealed that ZmGRF bound directly to the cis-element CCANNTGGC in the promoter of the Arabidopsis ent-kaurene oxidase (AtKO1) gene and promoted AtKO1 expression. GA4 content increased by 372-567% in transgenic Arabidopsis plants overexpressing ZmGRF compared to wild-type control plants. The GIBBERELLIN-INSENSITIVE DWARF1 gene, which encodes a GA receptor, was also upregulated and the growth-repressing DELLA protein gene GA INSENSITIVE was downregulated. Our results showed ZmGRF functioned through the GA-signaling pathway. PMID:25477078

  12. GCN2-dependent phosphorylation of eukaryotic translation initiation factor-2α in Arabidopsis

    PubMed Central

    Zhang, Yuhua; Wang, Yifei; Kanyuka, Kostya; Parry, Martin A. J.; Powers, Stephen J.; Halford, Nigel G.

    2008-01-01

    The yeast regulatory protein kinase, general control non-derepressible-2 (GCN2) plays a key role in general amino acid control. GCN2 phosphorylates the α subunit of the trimeric eukaryotic translation initiation factor-2 (eIF2), bringing about a decrease in the general rate of protein synthesis but an increase in the synthesis of GCN4, a transcription factor that promotes the expression of genes encoding enzymes for amino acid biosynthesis. The present study concerned the phosphorylation of Arabidopsis eIF2α (AteIF2α) by the Arabidopsis homologue of GCN2, AtGCN2, and the role of AtGCN2 in regulating genes encoding enzymes of amino acid biosynthesis and responding to virus infection. A null mutant for AtGCN2 called GT8359 was obtained and western analysis confirmed that it lacked AtGCN2 protein. GT8359 was more sensitive than wild-type Arabidopsis to herbicides that affect amino acid biosynthesis. Phosphorylation of AteIF2α occurred in response to herbicide treatment but only in wild-type Arabidopsis, not GT8359, showing it to be AtGCN2-dependent. Expression analysis of genes encoding key enzymes for amino acid biosynthesis and nitrate assimilation revealed little effect of loss of AtGCN2 function in GT8359 except that expression of a nitrate reductase gene, NIA1, was decreased. Analysis of wild-type and GT8359 plants infected with Turnip yellow mosaic virus or Turnip crinkle virus showed that AteIF2α was not phosphorylated. PMID:18603615

  13. Identification of novel transcription factors regulating secondary cell wall formation in Arabidopsis

    PubMed Central

    Cassan-Wang, Hua; Goué, Nadia; Saidi, Mohammed N.; Legay, Sylvain; Sivadon, Pierre; Goffner, Deborah; Grima-Pettenati, Jacqueline

    2013-01-01

    The presence of lignin in secondary cell walls (SCW) is a major factor preventing hydrolytic enzymes from gaining access to cellulose, thereby limiting the saccharification potential of plant biomass. To understand how lignification is regulated is a prerequisite for selecting plant biomass better adapted to bioethanol production. Because transcriptional regulation is a major mechanism controlling the expression of genes involved in lignin biosynthesis, our aim was to identify novel transcription factors (TFs) dictating lignin profiles in the model plant Arabidopsis. To this end, we have developed a post-genomic approach by combining four independent in-house SCW-related transcriptome datasets obtained from (1) the fiber cell wall-deficient wat1 Arabidopsis mutant, (2) Arabidopsis lines over-expressing either the master regulatory activator EgMYB2 or (3) the repressor EgMYB1 and finally (4) Arabidopsis orthologs of Eucalyptus xylem-expressed genes. This allowed us to identify 502 up- or down-regulated TFs. We preferentially selected those present in more than one dataset and further analyzed their in silico expression patterns as an additional selection criteria. This selection process led to 80 candidates. Notably, 16 of them were already proven to regulate SCW formation, thereby validating the overall strategy. Then, we phenotyped 43 corresponding mutant lines focusing on histological observations of xylem and interfascicular fibers. This phenotypic screen revealed six mutant lines exhibiting altered lignification patterns. Two of them [Bel-like HomeoBox6 (blh6) and a zinc finger TF] presented hypolignified SCW. Three others (myb52, myb-like TF, hb5) showed hyperlignified SCW whereas the last one (hb15) showed ectopic lignification. In addition, our meta-analyses highlighted a reservoir of new potential regulators adding to the gene network regulating SCW but also opening new avenues to ultimately improve SCW composition for biofuel production. PMID:23781226

  14. C4 protein of Beet severe curly top virus is a pathomorphogenetic factor in Arabidopsis.

    PubMed

    Park, Jungan; Hwang, Hyun-Sik; Buckley, Kenneth J; Park, Jong-Bum; Auh, Chung-Kyun; Kim, Dong-Giun; Lee, Sukchan; Davis, Keith R

    2010-12-01

    The Curtovirus C4 protein is required for symptom development during infection of Arabidopsis. Transgenic Arabidopsis plants expressing C4 from either Beet curly top virus or Beet severe curly top virus produced phenotypes that were similar to symptoms seen during infection with wild-type viruses. The pseudosymptoms caused by C4 protein alone were novel to transgenic Arabidopsis and included bumpy trichomes, severe enations, disorientation of vascular bundles and stomata, swelling, callus-like structure formation, and twisted siliques. C4 induced abnormal cell division and altered cell fate in a variety of tissues depending on the C4 expression level. C4 protein expression increased the expression levels of cell-cycle-related genes CYCs, CDKs and PCNA, and suppressed ICK1 and the retinoblastoma-related gene RBR1, resulting in activation of host cell division. These results suggest that the Curtovirus C4 proteins are involved actively in host cell-cycle regulation to recruit host factors for virus replication and symptom development. PMID:20960205

  15. Submergence Confers Immunity Mediated by the WRKY22 Transcription Factor in Arabidopsis[W

    PubMed Central

    Hsu, Fu-Chiun; Chou, Mei-Yi; Chou, Shu-Jen; Li, Ya-Ru; Peng, Hsiao-Ping; Shih, Ming-Che

    2013-01-01

    Transcriptional control plays an important role in regulating submergence responses in plants. Although numerous genes are highly induced during hypoxia, their individual roles in hypoxic responses are still poorly understood. Here, we found that expression of genes that encode members of the WRKY transcription factor family was rapidly and strongly induced upon submergence in Arabidopsis thaliana, and this induction correlated with induction of a large portion of innate immunity marker genes. Furthermore, prior submergence treatment conferred higher resistance to the bacterial pathogen Pseudomonas syringae in Arabidopsis. Among the WRKY genes tested, WRKY22 had the highest level of induction during the early stages of submergence. Compared with the wild type, WRKY22 T-DNA insertion mutants wrky22-1 and wrky22-2 had lower disease resistance and lower induction of innate immunity markers, such as FLG22-INDUCED RECEPTOR-LIKE KINASE1 (FRK1) and WRKY53, after submergence. Furthermore, transcriptomic analyses of wrky22-2 and chromatin immunoprecipitation identified several potential targets of WRKY22, which included genes encoding a TIR domain–containing protein, a plant peptide hormone, and many OLIGO PEPTIDE TRANSPORTER genes, all of which may lead to induction of innate immunity. In conclusion, we propose that submergence triggers innate immunity in Arabidopsis via WRKY22, a response that may protect against a higher probability of pathogen infection either during or after flooding. PMID:23897923

  16. Abiotic and biotic stress tolerance in Arabidopsis overexpressing the multiprotein bridging factor 1a (MBF1a) transcriptional coactivator gene.

    PubMed

    Kim, Min-Jung; Lim, Gah-Hyun; Kim, Eun-Seon; Ko, Chang-Beom; Yang, Kwang-Yeol; Jeong, Jin-An; Lee, Myung-Chul; Kim, Cheol Soo

    2007-03-01

    We conducted a genetic yeast screen to identify salt tolerance (SAT) genes in a maize kernel cDNA library. During the screening, we identified a maize clone (SAT41) that seemed to confer elevated salt tolerance in comparison to control cells. SAT41 cDNA encodes a 16-kDa protein which is 82.4% identical to the Arabidopsis Multiprotein bridging factor 1a (MBF1a) transcriptional coactivator gene. To further examine salinity tolerance in Arabidopsis, we functionally characterized the MBF1a gene and found that dehydration as well as heightened glucose (Glc) induced MBF1a expression. Constitutive expression of MBF1a in Arabidopsis led to elevated salt tolerance in transgenic lines. Interestingly, plants overexpressing MBF1a exhibited insensitivity to Glc and resistance to fungal disease. Our results suggest that MBF1a is involved in stress tolerance as well as in ethylene and Glc signaling in Arabidopsis. PMID:17234157

  17. Analysis of functional redundancies within the Arabidopsis TCP transcription factor family

    PubMed Central

    Danisman, Selahattin; de Folter, Stefan; Immink, Richard G. H.

    2013-01-01

    Analyses of the functions of TEOSINTE-LIKE1, CYCLOIDEA, and PROLIFERATING CELL FACTOR1 (TCP) transcription factors have been hampered by functional redundancy between its individual members. In general, putative functionally redundant genes are predicted based on sequence similarity and confirmed by genetic analysis. In the TCP family, however, identification is impeded by relatively low overall sequence similarity. In a search for functionally redundant TCP pairs that control Arabidopsis leaf development, this work performed an integrative bioinformatics analysis, combining protein sequence similarities, gene expression data, and results of pair-wise protein–protein interaction studies for the 24 members of the Arabidopsis TCP transcription factor family. For this, the work completed any lacking gene expression and protein–protein interaction data experimentally and then performed a comprehensive prediction of potential functional redundant TCP pairs. Subsequently, redundant functions could be confirmed for selected predicted TCP pairs by genetic and molecular analyses. It is demonstrated that the previously uncharacterized class I TCP19 gene plays a role in the control of leaf senescence in a redundant fashion with TCP20. Altogether, this work shows the power of combining classical genetic and molecular approaches with bioinformatics predictions to unravel functional redundancies in the TCP transcription factor family. PMID:24129704

  18. Two GATA transcription factors are downstream effectors of floral homeotic gene action in Arabidopsis.

    PubMed

    Mara, Chloe D; Irish, Vivian F

    2008-06-01

    Floral organogenesis is dependent on the combinatorial action of MADS-box transcription factors, which in turn control the expression of suites of genes required for growth, patterning, and differentiation. In Arabidopsis (Arabidopsis thaliana), the specification of petal and stamen identity depends on the action of two MADS-box gene products, APETALA3 (AP3) and PISTILLATA (PI). In a screen for genes whose expression was altered in response to the induction of AP3 activity, we identified GNC (GATA, nitrate-inducible, carbon-metabolism-involved) as being negatively regulated by AP3 and PI. The GNC gene encodes a member of the Arabidopsis GATA transcription factor family and has been implicated in the regulation of chlorophyll biosynthesis as well as carbon and nitrogen metabolism. In addition, we found that the GNC paralog, GNL (GNC-like), is also negatively regulated by AP3 and PI. Using chromatin immunoprecipitation, we showed that promoter sequences of both GNC and GNL are bound by PI protein, suggesting a direct regulatory interaction. Analyses of single and double gnc and gnl mutants indicated that the two genes share redundant roles in promoting chlorophyll biosynthesis, suggesting that in repressing GNC and GNL, AP3/PI have roles in negatively regulating this biosynthetic pathway in flowers. In addition, coexpression analyses of genes regulated by AP3, PI, GNC, and GNL indicate a complex regulatory interplay between these transcription factors in regulating a variety of light and nutrient responsive genes. Together, these results provide new insights into the transcriptional cascades controlling the specification of floral organ identities. PMID:18417639

  19. Inhibition of poly(ADP-ribosyl)ation in cancer: old and new paradigms revisited.

    PubMed

    Lupo, Barbara; Trusolino, Livio

    2014-08-01

    Inhibitors of poly(ADP-ribose) polymerases actualized the biological concept of synthetic lethality in the clinical practice, yielding a paradigmatic example of translational medicine. The profound sensitivity of tumors with germline BRCA mutations to PARP1/2 blockade owes to inherent defects of the BRCA-dependent homologous recombination machinery, which are unleashed by interruption of PARP DNA repair activity and lead to DNA damage overload and cell death. Conversely, aspirant BRCA-like tumors harboring somatic DNA repair dysfunctions (a vast entity of genetic and epigenetic defects known as "BRCAness") not always align with the familial counterpart and appear not to be equally sensitive to PARP inhibition. The acquisition of secondary resistance in initially responsive patients and the lack of standardized biomarkers to identify "BRCAness" pose serious threats to the clinical advance of PARP inhibitors; a feeling is also emerging that a BRCA-centered perspective might have missed the influence of additional, not negligible and DNA repair-independent PARP contributions onto therapy outcome. While regulatory approval for PARP1/2 inhibitors is still pending, novel therapeutic opportunities are sprouting from different branches of the PARP family, although they remain immature for clinical extrapolation. This review is an endeavor to provide a comprehensive appraisal of the multifaceted biology of PARPs and their evolving impact on cancer therapeutics. PMID:25026313

  20. Kinase-mediated changes in nucleosome conformation trigger chromatin decondensation via poly-ADP-ribosylation

    PubMed Central

    Thomas, Colin J.; Kotova, Elena; Andrake, Mark; Adolf-Bryfogle, Jared; Glaser, Robert; Regnard, Catherine; Tulin, Alexei V.

    2014-01-01

    SUMMARY Dynamically controlled post-translational modifications of nucleosomal histones alter chromatin condensation to regulate transcriptional activation. We report that a nuclear tandem kinase, JIL-1, controls gene expression by activating Poly(ADP-ribose) Polymerase 1 (PARP-1). JIL-1 phosphorylates the C-terminus of the H2Av histone variant, which stimulates PARP-1 enzymatic activity in the surrounding chromatin, leading to further modification of histones and chromatin loosening. The H2Av nucleosome has a higher surface representation of PARP-1 binding patch consisting of H3 and H4 epitopes. Phosphorylation of H2Av by JIL-1 restructures this surface patch leading to activation of PARP-1. Exposure of Val61 and Leu23 of the H4 histone is critical for PARP-1 binding on nucleosome and PARP-1 activation following H2Av phosphorylation. We propose that chromatin loosening and associated initiation of gene expression is activated by phosphorylation of H2Av in a nucleosome positioned in promoter regions of PARP-1 dependent genes. PMID:24508391

  1. Studies on protein poly(ADP-ribosylation) using high resolution gel electrophoresis.

    PubMed

    Boulikas, T

    1990-08-25

    Analysis of poly(ADP-ribose) synthesized in cellular lysates or in isolated nuclei on 100-cm-long thin gels of 20% polyacrylamide, 2.5 M urea permits determination of the exact size of poly(ADP-ribose) molecules using labeled oligonucleotides as molecular weight markers. The size and concentration of poly(ADP-ribose) molecules increase at time intervals during its synthesis. Differences in the concentration of poly(ADP-ribose) size classes among cell lines are also shown. Inhibition of poly(ADP-ribose) degradation by ethacridine that directly interacts with the polymer and inhibits its hydrolysis by poly(ADP-ribose) glycohydrolase shows a dramatic increase in both polymer size and concentration. Use of alkaline conditions for the hydrolysis of poly(ADP-ribose)-protein linkages reveals a specific shortening of all size classes of poly(ADP-ribose) compared with its size in preparations obtained by extensive digestion of nuclei with nucleases, RNases, and proteases. PMID:2167322

  2. Cuticular wax biosynthesis is up-regulated by the MYB94 transcription factor in Arabidopsis.

    PubMed

    Lee, Saet Buyl; Suh, Mi Chung

    2015-01-01

    The aerial parts of all land plants are covered with hydrophobic cuticular wax layers that act as the first barrier against the environment. The MYB94 transcription factor gene is expressed in abundance in aerial organs and shows a higher expression in the stem epidermis than within the stem. When seedlings were subjected to various treatments, the expression of the MYB94 transcription factor gene was observed to increase approximately 9-fold under drought, 8-fold for ABA treatment and 4-fold for separate NaCl and mannitol treatments. MYB94 harbors the transcriptional activation domain at its C-terminus, and fluorescent signals from MYB94:enhanced yellow fluorescent protein (eYFP) were observed in the nucleus of tobacco epidermis and in transgenic Arabidopsis roots. The total wax loads increased by approximately 2-fold in the leaves of the MYB94-overexpressing (MYB94 OX) lines, as compared with those of the wild type (WT). MYB94 activates the expression of WSD1, KCS2/DAISY, CER2, FAR3 and ECR genes by binding directly to their gene promoters. An increase in the accumulation of cuticular wax was observed to reduce the rate of cuticular transpiration in the leaves of MYB94 OX lines, under drought stress conditions. Taken together, a R2R3-type MYB94 transcription factor activates Arabidopsis cuticular wax biosynthesis and might be important in plant response to environmental stress, including drought. PMID:25305760

  3. Constitutively expressed ERF-VII transcription factors redundantly activate the core anaerobic response in Arabidopsis thaliana.

    PubMed

    Bui, Liem T; Giuntoli, Beatrice; Kosmacz, Monika; Parlanti, Sandro; Licausi, Francesco

    2015-07-01

    Plant adaptation to hypoxic conditions is mediated by the transcriptional activation of genes involved in the metabolic reprogramming of plant cells to cope with reduced oxygen availability. Recent studies indicated that members of the group VII of the Ethylene Responsive Transcription Factor (ERFs) family act as positive regulators of this molecular response. In the current study, the five ERF-VII transcription factors of Arabidopsis thaliana were compared to infer a hierarchy in their role with respect to the anaerobic response. When the activity of each transcription factor was tested on a set of hypoxia-responsive promoters, RAP2.2, RAP2.3 and RAP2.12 appeared to be the most powerful activators. RAP2.12 was further dissected in transactivation assays in Arabidopsis protoplasts to identify responsible regions for transcriptional activation. An ultimate C-terminal motif was identified as sufficient to drive gene transcription. Finally, using realtime RT-PCR in single and double mutants for the corresponding genes, we confirmed that RAP2.2 and RAP2.12 exert major control upon the anaerobic response. PMID:26025519

  4. Characterization of a novel arginine/serine-rich splicing factor in Arabidopsis.

    PubMed Central

    Lopato, S; Waigmann, E; Barta, A

    1996-01-01

    Many splicing factors in vertebrate nuclei belong to a class of evolutionarily conserved proteins containing arginine/serine (RS) or serine/arginine (SR) domains. Previously, we demonstrated the existence of SR splicing factors in plants. In this article, we report on a novel member of this splicing factor family from Arabidopsis designated atRSp31. It has one N-terminal RNA recognition motif and a C-terminal RS domain highly enriched in arginines. The RNA recognition motif shows significant homology to all animal SR proteins identified to date, but the intermediate region does not show any homology to any other known protein. Subsequently, we characterized two cDNAs from Arabidopsis that are highly homologous to atRSp31 (designated atRSp35 and atRSp41). Their deduced amino acid sequences indicate that these proteins constitute a new family of RS domain splicing factors. Purified recombinant atRSp31 is able to restore splicing in SR protein-deficient human S100 extracts. This indicates that atRSp31 is a true plant splicing factor and plays a crucial role in splicing, similar to that of other RS splicing factors. All of the three genes are differentially expressed in a tissue-specific manner. The isolation of this new plant splicing factor family enlarges the essential group of RS domain splicing factors. Furthermore, because no animal equivalent to this protein family has been identified to date, our results suggest that these proteins play key roles in constitutive and alternative splicing in plants. PMID:8989882

  5. Basic helix-loop-helix transcription factors and epidermal cell fate determination in Arabidopsis

    PubMed Central

    Zhao, Hongtao; Li, Xia; Ma, Ligeng

    2012-01-01

    Cell fate determination is an important process in multicellular organisms. Plant epidermis is a readily-accessible, well-used model for the study of cell fate determination. Our knowledge of cell fate determination is growing steadily due to genetic and molecular analyses of root hairs, trichomes, and stomata, which are derived from the epidermal cells of roots and aerial tissues. Studies have shown that a large number of factors are involved in the establishment of these cell types, especially members of the basic helix-loop-helix (bHLH) superfamily, which is an important family of transcription factors. In this mini-review, we focus on the role of bHLH transcription factors in cell fate determination in Arabidopsis. PMID:23073001

  6. Organelle RNA recognition motif-containing (ORRM) proteins are plastid and mitochondrial editing factors in Arabidopsis

    PubMed Central

    Shi, Xiaowen; Bentolila, Stephane; Hanson, Maureen R.

    2016-01-01

    ABSTRACT Post-transcriptional C-to-U RNA editing occurs at specific sites in plastid and plant mitochondrial transcripts. Members of the Arabidopsis pentatricopeptide repeat (PPR) motif-containing protein family and RNA-editing factor Interacting Protein (RIP, also known as MORF) family have been characterized as essential components of the RNA editing apparatus. Recent studies reveal that several organelle-targeted RNA recognition motif (RRM)-containing proteins are involved in either plastid or mitochondrial RNA editing. ORRM1 (Organelle RRM protein 1) is essential for plastid editing, whereas ORRM2, ORRM3 and ORRM4 are involved in mitochondrial RNA editing. The RRM domain of ORRM1, ORRM3 and ORRM4 is required for editing activity, whereas the auxiliary RIP and Glycine-Rich (GR) domains mediate the ORRM proteins' interactions with other editing factors. The identification of the ORRM proteins as RNA editing factors further expands our knowledge of the composition of the editosome. PMID:27082488

  7. Organelle RNA recognition motif-containing (ORRM) proteins are plastid and mitochondrial editing factors in Arabidopsis.

    PubMed

    Shi, Xiaowen; Bentolila, Stephane; Hanson, Maureen R

    2016-05-01

    Post-transcriptional C-to-U RNA editing occurs at specific sites in plastid and plant mitochondrial transcripts. Members of the Arabidopsis pentatricopeptide repeat (PPR) motif-containing protein family and RNA-editing factor Interacting Protein (RIP, also known as MORF) family have been characterized as essential components of the RNA editing apparatus. Recent studies reveal that several organelle-targeted RNA recognition motif (RRM)-containing proteins are involved in either plastid or mitochondrial RNA editing. ORRM1 (Organelle RRM protein 1) is essential for plastid editing, whereas ORRM2, ORRM3 and ORRM4 are involved in mitochondrial RNA editing. The RRM domain of ORRM1, ORRM3 and ORRM4 is required for editing activity, whereas the auxiliary RIP and Glycine-Rich (GR) domains mediate the ORRM proteins' interactions with other editing factors. The identification of the ORRM proteins as RNA editing factors further expands our knowledge of the composition of the editosome. PMID:27082488

  8. Peroxidation due to cryoprotectant treatment is a vital factor for cell survival in Arabidopsis cryopreservation.

    PubMed

    Ren, Li; Zhang, Di; Jiang, Xiang-Ning; Gai, Ying; Wang, Wei-Ming; Reed, Barbara M; Shen, Xiao-Hui

    2013-11-01

    Cryopreservation can be a safe and cost-effective tool for the long-term storage of plant germplasm. In Arabidopsis, the ability to recover from cryogenic treatment was lost as growth progressed. Growth could be restored in 48-h seedlings, whereas 72-h seedlings died after cryogenic treatment. Why seedling age and survival are negatively correlated is an interesting issue. A comparative transcriptomics was performed to screen differentially expressed genes between 48- and 72-h seedlings after exposure to cryoprotectant. Among differentially expressed genes, oxidative stress response genes played important roles in cryoprotectant treatment, and peroxidation was a key factor related to cell survival. Seedlings underwent more peroxidation at 72-h than at 48-h. A comprehensive analysis indicated that peroxidation injured membrane systems leading to photophosphorylation and oxidative phosphorylation damage. Furthermore, the apoptosis-like events were found in cryogenic treatment of Arabidopsis seedlings. 48- and 72-h seedlings underwent different degrees of membrane lipid peroxidation during cryoprotectant treatment, and reducing the injury of oxidative stress was an important factor to successful cryopreservation. This study provided a novel insight of genetic regulatory mechanisms in cryopreservation, and established an excellent model to test and evaluate the effect of exogenous antioxidants and conventional cryoprotectants in plant cryopreservation. PMID:24094052

  9. A Nuclear Factor Regulates Abscisic Acid Responses in Arabidopsis1[W][OA

    PubMed Central

    Kim, Min Jung; Shin, Ryoung; Schachtman, Daniel P.

    2009-01-01

    Abscisic acid (ABA) is a plant hormone that regulates plant growth as well as stress responses. In this study, we identified and characterized a new Arabidopsis (Arabidopsis thaliana) protein, Nuclear Protein X1 (NPX1), which was up-regulated by stress and treatment with exogenous ABA. Stomatal closure, seed germination, and primary root growth are well-known ABA responses that were less sensitive to ABA in NPX1-overexpressing plants. NPX1-overexpressing plants were more drought sensitive, and the changes in response to drought were due to the altered guard cell sensitivity to ABA in transgenic plants and not to a lack of ABA production. The nuclear localization of NPX1 correlated with changes in the expression of genes involved in ABA biosynthesis and ABA signal transduction. To understand the function of NPX1, we searched for interacting proteins and found that an ABA-inducible NAC transcription factor, TIP, interacted with NPX1. Based on the whole plant phenotypes, we hypothesized that NPX1 acts as a transcriptional repressor, and this was demonstrated in yeast, where we showed that TIP was repressed by NPX1. Our results indicate that the previously unknown protein NPX1 acts as a negative regulator in plant response to changes in environmental conditions through the control of ABA-regulated gene expression. The characterization of this factor enhances our understanding of guard cell function and the mechanisms that plants use to modulate water loss from leaves under drought conditions. PMID:19759343

  10. GROWTH REGULATING FACTOR5 Stimulates Arabidopsis Chloroplast Division, Photosynthesis, and Leaf Longevity1[OPEN

    PubMed Central

    Vercruyssen, Liesbeth; Tognetti, Vanesa B.; Gonzalez, Nathalie; Van Dingenen, Judith; De Milde, Liesbeth; Bielach, Agnieszka; De Rycke, Riet; Van Breusegem, Frank; Inzé, Dirk

    2015-01-01

    Arabidopsis (Arabidopsis thaliana) leaf development relies on subsequent phases of cell proliferation and cell expansion. During the proliferation phase, chloroplasts need to divide extensively, and during the transition from cell proliferation to expansion, they differentiate into photosynthetically active chloroplasts, providing the plant with energy. The transcription factor GROWTH REGULATING FACTOR5 (GRF5) promotes the duration of the cell proliferation period during leaf development. Here, it is shown that GRF5 also stimulates chloroplast division, resulting in a higher chloroplast number per cell with a concomitant increase in chlorophyll levels in 35S:GRF5 leaves, which can sustain higher rates of photosynthesis. Moreover, 35S:GRF5 plants show delayed leaf senescence and are more tolerant for growth on nitrogen-depleted medium. Cytokinins also stimulate leaf growth in part by extending the cell proliferation phase, simultaneously delaying the onset of the cell expansion phase. In addition, cytokinins are known to be involved in chloroplast development, nitrogen signaling, and senescence. Evidence is provided that GRF5 and cytokinins synergistically enhance cell division and chlorophyll retention after dark-induced senescence, which suggests that they also cooperate to stimulate chloroplast division and nitrogen assimilation. Taken together with the increased leaf size, ectopic expression of GRF5 has great potential to improve plant productivity. PMID:25604530

  11. Phytochrome-interacting transcription factors PIF4 and PIF5 induce leaf senescence in Arabidopsis.

    PubMed

    Sakuraba, Yasuhito; Jeong, Jinkil; Kang, Min-Young; Kim, Junghyun; Paek, Nam-Chon; Choi, Giltsu

    2014-01-01

    Plants initiate senescence to shed photosynthetically inefficient leaves. Light deprivation induces leaf senescence, which involves massive transcriptional reprogramming to dismantle cellular components and remobilize nutrients. In darkness, intermittent pulses of red light can inhibit senescence, likely via phytochromes. However, the precise molecular mechanisms transducing the signals from light perception to the inhibition of senescence remain elusive. Here, we show that in Arabidopsis, dark-induced senescence requires phytochrome-interacting transcription factors PIF4 and PIF5 (PIF4/PIF5). ELF3 and phytochrome B inhibit senescence by repressing PIF4/PIF5 at the transcriptional and post-translational levels, respectively. PIF4/PIF5 act in the signalling pathways of two senescence-promoting hormones, ethylene and abscisic acid, by directly activating expression of EIN3, ABI5 and EEL. In turn, PIF4, PIF5, EIN3, ABI5 and EEL directly activate the expression of the major senescence-promoting NAC transcription factor ORESARA1, thus forming multiple, coherent feed-forward loops. Our results reveal how classical light signalling connects to senescence in Arabidopsis. PMID:25119965

  12. ANAC005 is a membrane-associated transcription factor and regulates vascular development in Arabidopsis.

    PubMed

    Zhao, Jun; Liu, Jiang-Shu; Meng, Fu-Ning; Zhang, Zhen-Zhen; Long, Hao; Lin, Wen-Hui; Luo, Xiao-Min; Wang, Zhi-Yong; Zhu, Sheng-Wei

    2016-05-01

    Vascular tissues are very important for providing both mechanical strength and long-distance transport. The molecular mechanisms of regulation of vascular tissue development are still not fully understood. In this study we identified ANAC005 as a membrane-associated NAC family transcription factor that regulates vascular tissue development. Reporter gene assays showed that ANAC005 was expressed mainly in the vascular tissues. Increased expression of ANAC005 protein in transgenic Arabidopsis caused dwarf phenotype, reduced xylem differentiation, decreased lignin content, repression of a lignin biosynthetic gene and genes related to cambium and primary wall, but activation of genes related to the secondary wall. Expression of a dominant repressor fusion of ANAC005 had overall the opposite effects on vascular tissue differentiation and lignin synthetic gene expression. The ANAC005-GFP fusion protein was localized at the plasma membrane, whereas deletion of the last 20 amino acids, which are mostly basic, caused its nuclear localization. These results indicate that ANAC005 is a cell membrane-associated transcription factor that inhibits xylem tissue development in Arabidopsis. PMID:26178734

  13. Reduced tolerance to abiotic stress in transgenic Arabidopsis overexpressing a Capsicum annuum multiprotein bridging factor 1

    PubMed Central

    2014-01-01

    Background The pepper fruit is the second most consumed vegetable worldwide. However, low temperature affects the vegetative development and reproduction of the pepper, resulting in economic losses. To identify cold-related genes regulated by abscisic acid (ABA) in pepper seedlings, cDNA representational difference analysis was previously performed using a suppression subtractive hybridization method. One of the genes cloned from the subtraction was homologous to Solanum tuberosum MBF1 (StMBF1) encoding the coactivator multiprotein bridging factor 1. Here, we have characterized this StMBF1 homolog (named CaMBF1) from Capsicum annuum and investigated its role in abiotic stress tolerance. Results Tissue expression profile analysis using quantitative RT-PCR showed that CaMBF1 was expressed in all tested tissues, and high-level expression was detected in the flowers and seeds. The expression of CaMBF1 in pepper seedlings was dramatically suppressed by exogenously supplied salicylic acid, high salt, osmotic and heavy metal stresses. Constitutive overexpression of CaMBF1 in Arabidopsis aggravated the visible symptoms of leaf damage and the electrolyte leakage of cell damage caused by cold stress in seedlings. Furthermore, the expression of RD29A, ERD15, KIN1, and RD22 in the transgenic plants was lower than that in the wild-type plants. On the other hand, seed germination, cotyledon greening and lateral root formation were more severely influenced by salt stress in transgenic lines compared with wild-type plants, indicating that CaMBF1-overexpressing Arabidopsis plants were hypersensitive to salt stress. Conclusions Overexpression of CaMBF1 in Arabidopsis displayed reduced tolerance to cold and high salt stress during seed germination and post-germination stages. CaMBF1 transgenic Arabidopsis may reduce stress tolerance by downregulating stress-responsive genes to aggravate the leaf damage caused by cold stress. CaMBF1 may be useful for genetic engineering of novel

  14. Functions of heat shock transcription factors involved in response to photooxidative stresses in Arabidopsis.

    PubMed

    Yabuta, Yukinori

    2016-07-01

    Because plants are continually exposed to various environmental stresses, they possess numerous transcription factors that regulate metabolism to adapt and acclimate to those conditions. To clarify the gene regulation systems activated in response to photooxidative stress, we isolated 76 high light and heat shock stress-inducible genes, including heat shock transcription factor (Hsf) A2 from Arabidopsis. Unlike yeast or animals, more than 20 genes encoding putative Hsfs are present in the genomes of higher plants, and they are categorized into three classes based on their structural characterization. However, the multiplicity of Hsfs in plants remains unknown. Furthermore, the individual functions of Hsfs are also largely unknown because of their genetic redundancy. Recently, the developments of T-DNA insertion knockout mutant lines and chimeric repressor gene-silencing technology have provided effective tools for exploring the individual functions of Hsfs. This review describes the current knowledge on the individual functions and activation mechanisms of Hsfs. PMID:27095030

  15. Gene duplication of type-B ARR transcription factors systematically extends transcriptional regulatory structures in Arabidopsis

    PubMed Central

    Choi, Seung Hee; Hyeon, Do Young; Lee, ll Hwan; Park, Su Jin; Han, Seungmin; Lee, In Chul; Hwang, Daehee; Nam, Hong Gil

    2014-01-01

    Many of duplicated genes are enriched in signaling pathways. Recently, gene duplication of kinases has been shown to provide genetic buffering and functional diversification in cellular signaling. Transcription factors (TFs) are also often duplicated. However, how duplication of TFs affects their regulatory structures and functions of target genes has not been explored at the systems level. Here, we examined regulatory and functional roles of duplication of three major ARR TFs (ARR1, 10, and 12) in Arabidopsis cytokinin signaling using wild-type and single, double, and triple deletion mutants of the TFs. Comparative analysis of gene expression profiles obtained from Arabidopsis roots in wild-type and these mutants showed that duplication of ARR TFs systematically extended their transcriptional regulatory structures, leading to enhanced robustness and diversification in functions of target genes, as well as in regulation of cellular networks of target genes. Therefore, our results suggest that duplication of TFs contributes to robustness and diversification in functions of target genes by extending transcriptional regulatory structures. PMID:25425016

  16. Regulation of WRKY46 Transcription Factor Function by Mitogen-Activated Protein Kinases in Arabidopsis thaliana.

    PubMed

    Sheikh, Arsheed H; Eschen-Lippold, Lennart; Pecher, Pascal; Hoehenwarter, Wolfgang; Sinha, Alok K; Scheel, Dierk; Lee, Justin

    2016-01-01

    Mitogen-activated protein kinase (MAPK) cascades are central signaling pathways activated in plants after sensing internal developmental and external stress cues. Knowledge about the downstream substrate proteins of MAPKs is still limited in plants. We screened Arabidopsis WRKY transcription factors as potential targets downstream of MAPKs, and concentrated on characterizing WRKY46 as a substrate of the MAPK, MPK3. Mass spectrometry revealed in vitro phosphorylation of WRKY46 at amino acid position S168 by MPK3. However, mutagenesis studies showed that a second phosphosite, S250, can also be phosphorylated. Elicitation with pathogen-associated molecular patterns (PAMPs), such as the bacterial flagellin-derived flg22 peptide led to in vivo destabilization of WRKY46 in Arabidopsis protoplasts. Mutation of either phosphorylation site reduced the PAMP-induced degradation of WRKY46. Furthermore, the protein for the double phosphosite mutant is expressed at higher levels compared to wild-type proteins or single phosphosite mutants. In line with its nuclear localization and predicted function as a transcriptional activator, overexpression of WRKY46 in protoplasts raised basal plant defense as reflected by the increase in promoter activity of the PAMP-responsive gene, NHL10, in a MAPK-dependent manner. Thus, MAPK-mediated regulation of WRKY46 is a mechanism to control plant defense. PMID:26870073

  17. NAC Transcription Factor SPEEDY HYPONASTIC GROWTH Regulates Flooding-Induced Leaf Movement in Arabidopsis[W

    PubMed Central

    Rauf, Mamoona; Arif, Muhammad; Fisahn, Joachim; Xue, Gang-Ping; Balazadeh, Salma; Mueller-Roeber, Bernd

    2013-01-01

    In rosette plants, root flooding (waterlogging) triggers rapid upward (hyponastic) leaf movement representing an important architectural stress response that critically determines plant performance in natural habitats. The directional growth is based on localized longitudinal cell expansion at the lower (abaxial) side of the leaf petiole and involves the volatile phytohormone ethylene (ET). We report the existence of a transcriptional core unit underlying directional petiole growth in Arabidopsis thaliana, governed by the NAC transcription factor SPEEDY HYPONASTIC GROWTH (SHYG). Overexpression of SHYG in transgenic Arabidopsis thaliana enhances waterlogging-triggered hyponastic leaf movement and cell expansion in abaxial cells of the basal petiole region, while both responses are largely diminished in shyg knockout mutants. Expression of several EXPANSIN and XYLOGLUCAN ENDOTRANSGLYCOSYLASE/HYDROLASE genes encoding cell wall–loosening proteins was enhanced in SHYG overexpressors but lowered in shyg. We identified ACC OXIDASE5 (ACO5), encoding a key enzyme of ET biosynthesis, as a direct transcriptional output gene of SHYG and found a significantly reduced leaf movement in response to root flooding in aco5 T-DNA insertion mutants. Expression of SHYG in shoot tissue is triggered by root flooding and treatment with ET, constituting an intrinsic ET-SHYG-ACO5 activator loop for rapid petiole cell expansion upon waterlogging. PMID:24363315

  18. Identification of transcription factors linked to cell cycle regulation in Arabidopsis

    PubMed Central

    Dehghan Nayeri, Fatemeh

    2014-01-01

    Cell cycle is an essential process in growth and development of living organisms consists of the replication and mitotic phases separated by 2 gap phases; G1 and G2. It is tightly controlled at the molecular level and especially at the level of transcription. Precise regulation of the cell cycle is of central significance for plant growth and development and transcription factors are global regulators of gene expression playing essential roles in cell cycle regulation. This study has uncovered TFs that are involved in the control of cell cycle progression. With the aid of multi-parallel quantitative RT-PCR, the expression changes of 1880 TFs represented in the Arabidopsis TF platform was monitored in Arabidopsis synchronous MM2d cells during a 19 h period representing different time points corresponding to the 4 cell cycle phases after treatment of MM2d cells with Aphidicolin. Comparative TF expression analyses performed on synchronous cells resulted in the identification of 239 TFs differentially expressed during the cell cycle, while about one third of TFs were constitutively expressed through all time points. Phase-specific TFs were also identified. PMID:25482767

  19. Regulation of WRKY46 Transcription Factor Function by Mitogen-Activated Protein Kinases in Arabidopsis thaliana

    PubMed Central

    Sheikh, Arsheed H.; Eschen-Lippold, Lennart; Pecher, Pascal; Hoehenwarter, Wolfgang; Sinha, Alok K.; Scheel, Dierk; Lee, Justin

    2016-01-01

    Mitogen-activated protein kinase (MAPK) cascades are central signaling pathways activated in plants after sensing internal developmental and external stress cues. Knowledge about the downstream substrate proteins of MAPKs is still limited in plants. We screened Arabidopsis WRKY transcription factors as potential targets downstream of MAPKs, and concentrated on characterizing WRKY46 as a substrate of the MAPK, MPK3. Mass spectrometry revealed in vitro phosphorylation of WRKY46 at amino acid position S168 by MPK3. However, mutagenesis studies showed that a second phosphosite, S250, can also be phosphorylated. Elicitation with pathogen-associated molecular patterns (PAMPs), such as the bacterial flagellin-derived flg22 peptide led to in vivo destabilization of WRKY46 in Arabidopsis protoplasts. Mutation of either phosphorylation site reduced the PAMP-induced degradation of WRKY46. Furthermore, the protein for the double phosphosite mutant is expressed at higher levels compared to wild-type proteins or single phosphosite mutants. In line with its nuclear localization and predicted function as a transcriptional activator, overexpression of WRKY46 in protoplasts raised basal plant defense as reflected by the increase in promoter activity of the PAMP-responsive gene, NHL10, in a MAPK-dependent manner. Thus, MAPK-mediated regulation of WRKY46 is a mechanism to control plant defense. PMID:26870073

  20. The 73 kD Subunit of the Cleavage and Polyadenylation Specificity Factor (CPSF) Complex Affects Reproductive Development in Arabidopsis

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Cleavage and polyadenylation specificity factor (CPSF) is an important multi-subunit component of the mRNA 3’-end processing apparatus in eukaryotes. We have identified the Arabidopsis CPSF complex that involves five protein subunits named AtCPSF160, AtCPSF100, AtCPSF73-I, AtCPSF73-II and AtCPSF30....

  1. The ULTRAPETALA1 trxG factor contributes to patterning the Arabidopsis adaxial-abaxial leaf polarity axis

    Technology Transfer Automated Retrieval System (TEKTRAN)

    The SAND domain protein ULTRAPETALA1 (ULT1) functions as a trithorax group factor that regulates a variety of developmental processes in Arabidopsis. We have recently shown that ULT1 regulates developmental patterning in the gynoecia and leaves. ULT1 acts together with the KANADI1 (KAN1) transcripti...

  2. Chemical genetic discovery of PARP targets reveals a role for PARP-1 in transcription elongation.

    PubMed

    Gibson, Bryan A; Zhang, Yajie; Jiang, Hong; Hussey, Kristine M; Shrimp, Jonathan H; Lin, Hening; Schwede, Frank; Yu, Yonghao; Kraus, W Lee

    2016-07-01

    Poly[adenosine diphosphate (ADP)-ribose] polymerases (PARPs) are a family of enzymes that modulate diverse biological processes through covalent transfer of ADP-ribose from the oxidized form of nicotinamide adenine dinucleotide (NAD(+)) onto substrate proteins. Here we report a robust NAD(+) analog-sensitive approach for PARPs, which allows PARP-specific ADP-ribosylation of substrates that is suitable for subsequent copper-catalyzed azide-alkyne cycloaddition reactions. Using this approach, we mapped hundreds of sites of ADP-ribosylation for PARPs 1, 2, and 3 across the proteome, as well as thousands of PARP-1-mediated ADP-ribosylation sites across the genome. We found that PARP-1 ADP-ribosylates and inhibits negative elongation factor (NELF), a protein complex that regulates promoter-proximal pausing by RNA polymerase II (Pol II). Depletion or inhibition of PARP-1 or mutation of the ADP-ribosylation sites on NELF-E promotes Pol II pausing, providing a clear functional link between PARP-1, ADP-ribosylation, and NELF. This analog-sensitive approach should be broadly applicable across the PARP family and has the potential to illuminate the ADP-ribosylated proteome and the molecular mechanisms used by individual PARPs to mediate their responses to cellular signals. PMID:27256882

  3. Differential control of seed primary dormancy in Arabidopsis ecotypes by the transcription factor SPATULA.

    PubMed

    Vaistij, Fabián E; Gan, Yinbo; Penfield, Steven; Gilday, Alison D; Dave, Anuja; He, Zhesi; Josse, Eve-Marie; Choi, Giltsu; Halliday, Karen J; Graham, Ian A

    2013-06-25

    Freshly matured seeds exhibit primary dormancy, which prevents germination until environmental conditions are favorable. The establishment of dormancy occurs during seed development and involves both genetic and environmental factors that impact on the ratio of two antagonistic phytohormones: abscisic acid (ABA), which promotes dormancy, and gibberellic acid, which promotes germination. Although our understanding of dormancy breakage in mature seeds is well advanced, relatively little is known about the mechanisms involved in establishing dormancy during seed maturation. We previously showed that the SPATULA (SPT) transcription factor plays a key role in regulating seed germination. Here we investigate its role during seed development and find that, surprisingly, it has opposite roles in setting dormancy in Landsberg erecta and Columbia Arabidopsis ecotypes. We also find that SPT regulates expression of five transcription factor encoding genes: ABA-INSENSITIVE4 (ABI4) and ABI5, which mediate ABA signaling; REPRESSOR-OF-GA (RGA) and RGA-LIKE3 involved in gibberellic acid signaling; and MOTHER-OF-FT-AND-TFL1 (MFT) that we show here promotes Arabidopsis seed dormancy. Although ABI4, RGA, and MFT are repressed by SPT, ABI5 and RGL3 are induced. Furthermore, we show that RGA, MFT, and ABI5 are direct targets of SPT in vivo. We present a model in which SPT drives two antagonistic "dormancy-repressing" and "dormancy-promoting" routes that operate simultaneously in freshly matured seeds. Each of these routes has different impacts and this in turn explains the opposite effect of SPT on seed dormancy of the two ecotypes analyzed here. PMID:23754415

  4. Chrysanthemum transcription factor CmLBD1 direct lateral root formation in Arabidopsis thaliana.

    PubMed

    Zhu, Lu; Zheng, Chen; Liu, Ruixia; Song, Aiping; Zhang, Zhaohe; Xin, Jingjing; Jiang, Jiafu; Chen, Sumei; Zhang, Fei; Fang, Weimin; Chen, Fadi

    2016-01-01

    The plant-specific LATERAL ORGAN BOUNDARIES DOMAIN (LBD) genes are important regulators of growth and development. Here, a chrysanthemum class I LBD transcription factor gene, designated CmLBD1, was isolated and its function verified. CmLBD1 was transcribed in both the root and stem, but not in the leaf. The gene responded to auxin and was shown to participate in the process of adventitious root primordium formation. Its heterologous expression in Arabidopsis thaliana increased the number of lateral roots formed. When provided with exogenous auxin, lateral root emergence was promoted. CmLBD1 expression also favored callus formation from A. thaliana root explants in the absence of exogenously supplied phytohormones. In planta, CmLBD1 probably acts as a positive regulator of the response to auxin fluctuations and connects auxin signaling with lateral root formation. PMID:26819087

  5. An Arabidopsis Transcriptional Regulatory Map Reveals Distinct Functional and Evolutionary Features of Novel Transcription Factors.

    PubMed

    Jin, Jinpu; He, Kun; Tang, Xing; Li, Zhe; Lv, Le; Zhao, Yi; Luo, Jingchu; Gao, Ge

    2015-07-01

    Transcription factors (TFs) play key roles in both development and stress responses. By integrating into and rewiring original systems, novel TFs contribute significantly to the evolution of transcriptional regulatory networks. Here, we report a high-confidence transcriptional regulatory map covering 388 TFs from 47 families in Arabidopsis. Systematic analysis of this map revealed the architectural heterogeneity of developmental and stress response subnetworks and identified three types of novel network motifs that are absent from unicellular organisms and essential for multicellular development. Moreover, TFs of novel families that emerged during plant landing present higher binding specificities and are preferentially wired into developmental processes and these novel network motifs. Further unveiled connection between the binding specificity and wiring preference of TFs explains the wiring preferences of novel-family TFs. These results reveal distinct functional and evolutionary features of novel TFs, suggesting a plausible mechanism for their contribution to the evolution of multicellular organisms. PMID:25750178

  6. Chrysanthemum transcription factor CmLBD1 direct lateral root formation in Arabidopsis thaliana

    PubMed Central

    Zhu, Lu; Zheng, Chen; Liu, Ruixia; Song, Aiping; Zhang, Zhaohe; Xin, Jingjing; Jiang, Jiafu; Chen, Sumei; Zhang, Fei; Fang, Weimin; Chen, Fadi

    2016-01-01

    The plant-specific LATERAL ORGAN BOUNDARIES DOMAIN (LBD) genes are important regulators of growth and development. Here, a chrysanthemum class I LBD transcription factor gene, designated CmLBD1, was isolated and its function verified. CmLBD1 was transcribed in both the root and stem, but not in the leaf. The gene responded to auxin and was shown to participate in the process of adventitious root primordium formation. Its heterologous expression in Arabidopsis thaliana increased the number of lateral roots formed. When provided with exogenous auxin, lateral root emergence was promoted. CmLBD1 expression also favored callus formation from A. thaliana root explants in the absence of exogenously supplied phytohormones. In planta, CmLBD1 probably acts as a positive regulator of the response to auxin fluctuations and connects auxin signaling with lateral root formation. PMID:26819087

  7. A subgroup of MYB transcription factor genes undergoes highly conserved alternative splicing in Arabidopsis and rice.

    PubMed

    Li, Jigang; Li, Xiaojuan; Guo, Lei; Lu, Feng; Feng, Xiaojie; He, Kun; Wei, Liping; Chen, Zhangliang; Qu, Li-Jia; Gu, Hongya

    2006-01-01

    MYB transcription factor genes play important roles in many developmental processes and in various defence responses of plants. Two Arabidopsis R2R3-type MYB genes, AtMYB59 and AtMYB48, were found to undergo similar alternative splicing. Both genes have four distinctively spliced transcripts that encode either MYB-related proteins or R2R3-MYB proteins. An extensive BLAST search of the GenBank database resulted in finding and cloning two rice homologues, both of which were also found to share a similar alternative splicing pattern. In a semi-quantitative study, the expression of one splice variant of AtMYB59 was found to be differentially regulated in treatments with different phytohormones and stresses. GFP fusion protein analysis revealed that both of the two predicted nuclear localization signals (NLSs) in the R3 domain are required for localizing to the nucleus. Promoter-GUS analysis in transgenic plants showed that 5'-UTR is sufficient for the translation initiation of type 3 transcripts (encoding R2R3-MYB proteins), but not for type 2 transcripts (encoding MYB-related proteins). Moreover, a new type of non-canonical intron, with the same nucleotide repeats at the 5' and 3' splice sites, was identified. Thirty-eight Arabidopsis and rice genes were found to have this type of non-canonical intron, most of which undergo alternative splicing. These data suggest that this subgroup of transcription factor genes may be involved in multiple biological processes and may be transcriptionally regulated by alternative splicing. PMID:16531467

  8. Connecting RNA Processing to Abiotic Environmental Response in Arabidopsis: the role of a polyadenylation factor

    NASA Astrophysics Data System (ADS)

    Li, Q. Q.; Xu, R.; Hunt, A. G.; Falcone, D. L.

    Plants are constantly challenged by numerous environmental stresses both biotic and abiotic It is clear that plants have evolved to counter these stresses using all but limited means We recently discovered the potential role of a messenger RNA processing factor namely the Arabidopsis cleavage and polyadenylation specificity factor 30 kDa subunit AtCPSF30 when a mutant deficient in this factor displayed altered responses to an array of abiotic stresses This AtCPSF30 mutant named oxt6 exhibited an elevated tolerance to oxidative stress Microarray experiments of oxt6 and its complemented lines revealed an altered gene expression profile among which were antioxidative defense genes Interestingly the same gene encoding AtCPSF30 can also be transcribed into a large transcript that codes for a potential splicing factor Both protein products have a domain for RNA binding and a calmodulin binding domain activities of which have been confirmed by biochemical assays Surprisingly binding of AtCPSF30 to calmodulin inhibits the RNA-binding activity of the protein Mutational analysis shows that a small part of the protein is responsible for calmodulin binding and point mutations in this region abolished both RNA binding activity and the inhibition of this activity by calmodulin Analyses of the potential splicing factor are on going and the results will be presented The interesting possibilities for both the interplay between splicing and polyadenylation and the regulation of these processes by stimuli that act through

  9. Activation of immobilized, biotinylated choleragen AI protein by a 19-kilodalton guanine nucleotide-binding protein.

    PubMed

    Noda, M; Tsai, S C; Adamik, R; Bobak, D A; Moss, J; Vaughan, M

    1989-09-19

    Cholera toxin catalyzes the ADP-ribosylation that results in activation of the stimulatory guanine nucleotide-binding protein of the adenylyl cyclase system, known as Gs. The toxin also ADP-ribosylates other proteins and simple guanidino compounds and auto-ADP-ribosylates its AI protein (CTA1). All of the ADP-ribosyltransferase activities of CTAI are enhanced by 19-21-kDa guanine nucleotide-binding proteins known as ADP-ribosylation factors, or ARFs. CTAI contains a single cysteine located near the carboxy terminus. CTAI was immobilized through this cysteine by reaction with iodoacetyl-N-biotinyl-hexylenediamine and binding of the resulting biotinylated protein to avidin-agarose. Immobilized CTAI catalyzed the ARF-stimulated ADP-ribosylation of agmatine. The reaction was enhanced by detergents and phospholipid, but the fold stimulation by purified sARF-II from bovine brain was considerably less than that observed with free CTA. ADP-ribosylation of Gsa by immobilized CTAI, which was somewhat enhanced by sARF-II, was much less than predicted on the basis of the NAD:agmatine ADP-ribosyltransferase activity. Immobilized CTAI catalyzed its own auto-ADP-ribosylation as well as the ADP-ribosylation of the immobilized avidin and CTA2, with relatively little stimulation by sARF-II. ADP-ribosylation of CTA2 by free CTAI is minimal. These observations are consistent with the conclusion that the cysteine near the carboxy terminus of the toxin is not critical for ADP-ribosyltransferase activity or for its regulation by sARF-II. Biotinylation and immobilization of the toxin through this cysteine may, however, limit accessibility to Gsa or SARF-II, or perhaps otherwise reduce interaction with these proteins whether as substrates or activator. PMID:2514798

  10. Transcription Factor ATAF1 in Arabidopsis Promotes Senescence by Direct Regulation of Key Chloroplast Maintenance and Senescence Transcriptional Cascades.

    PubMed

    Garapati, Prashanth; Xue, Gang-Ping; Munné-Bosch, Sergi; Balazadeh, Salma

    2015-07-01

    Senescence represents a fundamental process of late leaf development. Transcription factors (TFs) play an important role for expression reprogramming during senescence; however, the gene regulatory networks through which they exert their functions, and their physiological integration, are still largely unknown. Here, we identify the Arabidopsis (Arabidopsis thaliana) abscisic acid (ABA)- and hydrogen peroxide-activated TF Arabidopsis thaliana activating factor1 (ATAF1) as a novel upstream regulator of senescence. ATAF1 executes its physiological role by affecting both key chloroplast maintenance and senescence-promoting TFs, namely GOLDEN2-LIKE1 (GLK1) and ORESARA1 (Arabidopsis NAC092), respectively. Notably, while ATAF1 activates ORESARA1, it represses GLK1 expression by directly binding to their promoters, thereby generating a transcriptional output that shifts the physiological balance toward the progression of senescence. We furthermore demonstrate a key role of ATAF1 for ABA- and hydrogen peroxide-induced senescence, in accordance with a direct regulatory effect on ABA homeostasis genes, including nine-CIS-epoxycarotenoid dioxygenase3 involved in ABA biosynthesis and ABC transporter G family member40, encoding an ABA transport protein. Thus, ATAF1 serves as a core transcriptional activator of senescence by coupling stress-related signaling with photosynthesis- and senescence-related transcriptional cascades. PMID:25953103

  11. Membrane-associated transcription factor peptidase, site-2 protease, antagonizes ABA signaling in Arabidopsis.

    PubMed

    Zhou, Shun-Fan; Sun, Le; Valdés, Ana Elisa; Engström, Peter; Song, Ze-Ting; Lu, Sun-Jie; Liu, Jian-Xiang

    2015-10-01

    Abscisic acid plays important roles in maintaining seed dormancy while gibberellins (GA) and other phytohormones antagonize ABA to promote germination. However, how ABA signaling is desensitized during the transition from dormancy to germination is still poorly understood. We functionally characterized the role of membrane-associated transcription factor peptidase, site-2 protease (S2P), in ABA signaling during seed germination in Arabidopsis. Genetic analysis showed that loss-of-function of S2P conferred high ABA sensitivity during seed germination, and expression of the activated form of membrane-associated transcription factor bZIP17, in which the transmembrane domain and endoplasmic reticulum (ER) lumen-facing C-terminus were deleted, in the S2P mutant rescued its ABA-sensitive phenotype. MYC and green fluorescent protein (GFP)-tagged bZIP17 were processed and translocated from the ER to the nucleus in response to ABA treatment. Furthermore, genes encoding negative regulators of ABA signaling, such as the transcription factor ATHB7 and its target genes HAB1, HAB2, HAI1 and AHG3, were up-regulated in seeds of the wild-type upon ABA treatment; this up-regulation was impaired in seeds of S2P mutants. Our results suggest that S2P desensitizes ABA signaling during seed germination through regulating the activation of the membrane-associated transcription factor bZIP17 and therefore controlling the expression level of genes encoding negative regulators of ABA signaling. PMID:25919792

  12. The Arabidopsis Transcription Factor MYB77 Modulates Auxin Signal Transduction[W

    PubMed Central

    Shin, Ryoung; Burch, Adrien Y.; Huppert, Kari A.; Tiwari, Shiv B.; Murphy, Angus S.; Guilfoyle, Tom J.; Schachtman, Daniel P.

    2007-01-01

    Auxin is a key plant hormone that regulates plant development, apical dominance, and growth-related tropisms, such as phototropism and gravitropism. In this study, we report a new Arabidopsis thaliana transcription factor, MYB77, that is involved in auxin response. In MYB77 knockout plants, we found that auxin-responsive gene expression was greatly attenuated. Lateral root density in the MYB77 knockout was lower than the wild type at low concentrations of indole-3-acetic acid (IAA) and also under low nutrient conditions. MYB77 interacts with auxin response factors (ARFs) in vitro through the C terminus (domains III and IV) of ARFs and the activation domain of MYB77. A synergistic genetic interaction was demonstrated between MYB77 and ARF7 that resulted in a strong reduction in lateral root numbers. Experiments with protoplasts confirmed that the coexpression of MYB77 and an ARF C terminus enhance reporter gene expression. R2R3 MYB transcription factors have not been previously implicated in regulating the expression of auxin-inducible genes. Also it was previously unknown that ARFs interact with proteins other than those in the Aux/IAA family via conserved domains. The interaction between MYB77 and ARFs defines a new type of combinatorial transcriptional control in plants. This newly defined transcription factor interaction is part of the plant cells' repertoire for modulating response to auxin, thereby controlling lateral root growth and development under changing environmental conditions. PMID:17675404

  13. The cytokinin response factors modulate root and shoot growth and promote leaf senescence in Arabidopsis.

    PubMed

    Raines, Tracy; Shanks, Carly; Cheng, Chia-Yi; McPherson, Duncan; Argueso, Cristiana T; Kim, Hyo J; Franco-Zorrilla, José M; López-Vidriero, Irene; Solano, Roberto; Vaňková, Radomíra; Schaller, G Eric; Kieber, Joseph J

    2016-01-01

    The cytokinin response factors (CRFs) are a group of related AP2/ERF transcription factors that are transcriptionally induced by cytokinin. Here we explore the role of the CRFs in Arabidopsis thaliana growth and development by analyzing lines with decreased and increased CRF function. While single crf mutations have no appreciable phenotypes, disruption of multiple CRFs results in larger rosettes, delayed leaf senescence, a smaller root apical meristem (RAM), reduced primary and lateral root growth, and, in etiolated seedlings, shorter hypocotyls. In contrast, overexpression of CRFs generally results in the opposite phenotypes. The crf1,2,5,6 quadruple mutant is embryo lethal, indicating that CRF function is essential for embryo development. Disruption of the CRFs results in partially insensitivity to cytokinin in a root elongation assay and affects the basal expression of a significant number of cytokinin-regulated genes, including the type-A ARRs, although it does not impair the cytokinin induction of the type-A ARRs. Genes encoding homeobox transcription factors are mis-expressed in the crf1,3,5,6 mutant, including STIMPY/WOX9 that is required for root and shoot apical meristem maintenance roots and which has previously been linked to cytokinin. These results indicate that the CRF transcription factors play important roles in multiple aspects of plant growth and development, in part through a complex interaction with cytokinin signaling. PMID:26662515

  14. TOPP4 Regulates the Stability of PHYTOCHROME INTERACTING FACTOR5 during Photomorphogenesis in Arabidopsis.

    PubMed

    Yue, Jing; Qin, Qianqian; Meng, Siyuan; Jing, Huiting; Gou, Xiaoping; Li, Jia; Hou, Suiwen

    2016-03-01

    In plants, photoreceptors transfer light signals to phytochrome-interacting factors (PIFs), inducing the rapid phosphorylation and degradation of PIFs to promote photomorphogenesis. However, the phosphatase responsible for PIF dephosphorylation remains unknown. In this study, we identified a type 1 protein phosphatase, TOPP4, that is essential for PIF5 protein stability in Arabidopsis (Arabidopsis thaliana). Compared with the wild type, the dominant-negative mutant, topp4-1, displayed reduced hypocotyl length and larger apical hook and cotyledon opening angle under red light. Overexpression of topp4-1 in the wild type led to defects that were similar to those in the topp4-1 mutant. Red light induced phytochrome B (phyB)-dependent TOPP4 expression in hypocotyls. The topp4-1 mutation weakened the closed cotyledon angle of phyB-9 and phyA-211 phyB-9, while overexpression of TOPP4 significantly repressed the short hypocotyls of phyB-green fluorescent protein seedlings, indicating that TOPP4 and phyB function in an antagonistic way during photomorphogenesis. Protein interaction assays and phosphorylation studies demonstrate that TOPP4 interacts directly with PIF5 and dephosphorylates it. Furthermore, TOPP4 inhibits the red light-induced ubiquitination and degradation of PIF5. These findings demonstrate that dephosphorylation of PIF5 by TOPP4 inhibits its ubiquitin-mediated degradation during photomorphogenesis. These data outline a novel phytochrome signaling mechanism by which TOPP4-mediated dephosphorylation of PIF5 attenuates phytochrome-dependent light responses. PMID:26704640

  15. The Miscanthus NAC transcription factor MlNAC9 enhances abiotic stress tolerance in transgenic Arabidopsis.

    PubMed

    Zhao, Xun; Yang, Xuanwen; Pei, Shengqiang; He, Guo; Wang, Xiaoyu; Tang, Qi; Jia, Chunlin; Lu, Ying; Hu, Ruibo; Zhou, Gongke

    2016-07-15

    NAC (NAM, ATAF1/2, and CUC2) transcription factors are known to play important roles in responses to abiotic stresses in plants. Currently, little information regarding the functional roles of NAC genes in stress tolerance is available in Miscanthus lutarioriparius, a promising bioenergy plant for cellulosic ethanol production. In this study, we carried out the functional characterization of MlNAC9 in abiotic stresses. MlNAC9 was shown to act as a nuclear localized transcription activator with the activation domain in its C-terminus. The overexpression of MlNAC9 in Arabidopsis conferred hypersensitivity to abscisic acid (ABA) at seed germination and root elongation stages. In addition, the overexpression of MlNAC9 led to increased seed germination rate and root growth under salt (NaCl) treatment. Meanwhile, the transgenic Arabidopsis overexpressing MlNAC9 showed enhanced tolerance to drought and cold stresses. The expression of stress-responsive marker genes was significantly increased in MlNAC9 overexpression lines compared to that of WT under ABA, drought, salt, and cold stresses. Correspondingly, the activities of antioxidant enzymes superoxide dismutase (SOD), peroxidase (POD), and catalase (CAT) were significantly increased and the malondialdehyde (MDA) content was lower accumulated in MlNAC9 overexpression lines under drought and salt treatments. These results indicated that the overexpression of MlNAC9 improved the tolerance to abiotic stresses via an ABA-dependent pathway, and the enhanced tolerance of transgenic plants was mainly attributed to the increased expression of stress-responsive genes and the enhanced scavenging capability of reactive oxygen species (ROS). PMID:27085481

  16. Arabidopsis NAC transcription factor JUB1 regulates GA/BR metabolism and signalling.

    PubMed

    Shahnejat-Bushehri, Sara; Tarkowska, Danuse; Sakuraba, Yasuhito; Balazadeh, Salma

    2016-01-01

    Gibberellins (GAs) and brassinosteroids (BRs) are important phytohormones that control plant development and responses to environmental cues by involving DELLA proteins and BRASSINAZOLE-RESISTANT1 (BZR1) respectively as key transcription factors. Here, we reveal a new role for JUNGBRUNNEN1 (JUB1) as a transcriptional regulator of GA/BR signalling in Arabidopsis thaliana. JUB1 directly represses the hormone biosynthesis genes GA3ox1 and DWARF4 (DWF4), leading to reduced levels of GAs and BRs and typical GA/BR deficiency phenotypes exhibiting short hypocotyls, dwarfism, late flowering and male sterility. JUB1 also directly represses PHYTOCHROME INTERACTING FACTOR4 (PIF4), a transcription factor connecting hormonal and environmental stimuli. On the other hand, JUB1 activates the DELLA genes GA INSENSITIVE (GAI) and RGA-LIKE 1 (RGL1). In addition, BZR1 and PIF4 act as direct transcriptional repressors upstream of JUB1, establishing a negative feedback loop. Thus, JUB1 forms the core of a robust regulatory module that triggers DELLA accumulation, thereby restricting cell elongation while concomitantly enhancing stress tolerance. PMID:27249348

  17. A Dof transcription factor, SCAP1, is essential for the development of functional stomata in Arabidopsis.

    PubMed

    Negi, Juntaro; Moriwaki, Kosuke; Konishi, Mineko; Yokoyama, Ryusuke; Nakano, Toshiaki; Kusumi, Kensuke; Hashimoto-Sugimoto, Mimi; Schroeder, Julian I; Nishitani, Kazuhiko; Yanagisawa, Shuichi; Iba, Koh

    2013-03-18

    Stomata are highly specialized organs that consist of pairs of guard cells and regulate gas and water vapor exchange in plants [1-3]. Although early stages of guard cell differentiation have been described [4-10] and were interpreted in analogy to processes of cell type differentiation in animals [11], the downstream development of functional stomatal guard cells remains poorly understood. We have isolated an Arabidopsis mutant, stomatal carpenter 1 (scap1), that develops irregularly shaped guard cells and lacks the ability to control stomatal aperture, including CO2-induced stomatal closing and light-induced stomatal opening. SCAP1 was identified as a plant-specific Dof-type transcription factor expressed in maturing guard cells, but not in guard mother cells. SCAP1 regulates the expression of genes encoding key elements of stomatal functioning and morphogenesis, such as K(+) channel protein, MYB60 transcription factor, and pectin methylesterase. Consequently, ion homeostasis was disturbed in scap1 guard cells, and esterification of extracellular pectins was impaired so that the cell walls lining the pores did not mature normally. We conclude that SCAP1 regulates essential processes of stomatal guard cell maturation and functions as a key transcription factor regulating the final stages of guard cell differentiation. PMID:23453954

  18. AUXIN RESPONSE FACTOR7 Restores the Expression of Auxin-Responsive Genes in Mutant Arabidopsis Leaf Mesophyll ProtoplastsW⃞

    PubMed Central

    Wang, Shucai; Tiwari, Shiv B.; Hagen, Gretchen; Guilfoyle, Tom J.

    2005-01-01

    AUXIN RESPONSE FACTOR7 (ARF7) is one of five ARF transcriptional activators in Arabidopsis thaliana that is proposed to regulate auxin-responsive expression of genes containing TGTCTC auxin response elements in their promoters. An Arabidopsis mutant (nonphototropic hypocotyl4-1 [nph4-1]) that is a null for ARF7 showed strongly reduced expression of integrated auxin-responsive reporter genes and natural genes that were monitored in Arabidopsis leaf mesophyll protoplasts. Expression of the reporter and natural genes was restored in an auxin-dependent manner when protoplasts were transfected with a 35S:ARF7 effector gene, encoding a full-length ARF7 protein. Transfection of effector genes encoding other ARF activators restored auxin-responsive gene expression to varying degrees, but less than that observed with the ARF7 effector gene. Arabidopsis lines that were null for ARF6, ARF8, or ARF19 were not defective in expression of the reporter and natural auxin response genes assayed in mesophyll protoplasts, suggesting that ARF7 plays a major role in regulating expression of a subset of auxin response genes in leaf mesophyll cells. Auxin-responsive gene expression was induced in wild-type protoplasts and restored in nph4-1 protoplasts only with auxin and not with other hormones, including brassinolide. In the presence of auxin, however, brassinolide modestly enhanced auxin-responsive gene expression. PMID:15923351

  19. Expression of Aberrant Forms of AUXIN RESPONSE FACTOR8 Stimulates Parthenocarpy in Arabidopsis and Tomato1[W][OA

    PubMed Central

    Goetz, Marc; Hooper, Lauren C.; Johnson, Susan D.; Rodrigues, Julio Carlyle Macedo; Vivian-Smith, Adam; Koltunow, Anna M.

    2007-01-01

    Fruit initiation in Arabidopsis (Arabidopsis thaliana) is generally repressed until fertilization occurs. However, mutations in AUXIN RESPONSE FACTOR8 (ARF8) uncouple fruit initiation from fertilization, resulting in the formation of seedless, parthenocarpic fruit. Here we induced parthenocarpy in wild-type Arabidopsis by introducing either the mutant genomic (g) Atarf8-4 sequence or gAtARF8:β-glucuronidase translational fusion constructs by plant transformation. Silencing of endogenous AtARF8 transcription was not observed, indicating that the introduced, aberrant ARF8 transcripts were compromising the function of endogenous ARF8 and/or associated factors involved in suppressing fruit initiation. To analyze the role of ARF8 in tomato (Solanum lycopersicum) we initially emasculated 23 tomato cultivars to test for background parthenocarpy. Surprisingly, all had a predisposition to initiate fertilization-independent fruit growth. Expression of gAtarf8-4 in transgenic tomato (‘Monalbo’) resulted in a significant increase in the number and size of parthenocarpic fruit. Isolation of tomato ARF8 cDNA indicated significant sequence conservation with AtARF8. SlARF8 may therefore control tomato fruit initiation in a similar manner as AtARF8 does in Arabidopsis. Two SlARF8 cDNAs differing in size by 5 bp were found, both arising from the same gene. The smaller cDNA is a splice variant and is also present in Arabidopsis. We propose that low endogenous levels of the splice variant products might interfere with efficient formation/function of a complex repressing fruit initiation, thereby providing an explanation for the observed ovary expansion in tomato and also Arabidopsis after emasculation. Increasing the levels of aberrant Atarf8-4 transcripts may further destabilize formation/function of the complex in a dosage-dependent manner enhancing tomato parthenocarpic fruit initiation frequency and size and mimicking the parthenocarpic dehiscent silique phenotype found in

  20. Jasmonate regulates the inducer of cbf expression-C-repeat binding factor/DRE binding factor1 cascade and freezing tolerance in Arabidopsis.

    PubMed

    Hu, Yanru; Jiang, Liqun; Wang, Fang; Yu, Diqiu

    2013-08-01

    The inducer of cbf expression (ICE)-C-repeat binding factor/DRE binding factor1 (CBF/DREB1) transcriptional pathway plays a critical role in modulating cold stress responses in Arabidopsis thaliana. Dissecting crucial upstream regulatory signals or components of the ICE-CBF/DREB1 cascade will enhance our understanding of plant cold-tolerance mechanisms. Here, we show that jasmonate positively regulates plant responses to freezing stress in Arabidopsis. Exogenous application of jasmonate significantly enhanced plant freezing tolerance with or without cold acclimation. By contrast, blocking endogenous jasmonate biosynthesis and signaling rendered plants hypersensitive to freezing stress. Consistent with the positive role of jasmonate in freezing stress, production of endogenous jasmonate was triggered by cold treatment. In addition, cold induction of genes acting in the CBF/DREB1 signaling pathway was upregulated by jasmonate. Further investigation revealed that several jasmonate ZIM-domain (JAZ) proteins, the repressors of jasmonate signaling, physically interact with ICE1 and ICE2 transcription factors. JAZ1 and JAZ4 repress the transcriptional function of ICE1, thereby attenuating the expression of its regulon. Consistent with this, overexpression of JAZ1 or JAZ4 represses freezing stress responses of Arabidopsis. Taken together, our study provides evidence that jasmonate functions as a critical upstream signal of the ICE-CBF/DREB1 pathway to positively regulate Arabidopsis freezing tolerance. PMID:23933884

  1. Jasmonate Regulates the INDUCER OF CBF EXPRESSION–C-REPEAT BINDING FACTOR/DRE BINDING FACTOR1 Cascade and Freezing Tolerance in Arabidopsis[W

    PubMed Central

    Hu, Yanru; Jiang, Liqun; Wang, Fang; Yu, Diqiu

    2013-01-01

    The INDUCER OF CBF EXPRESSION (ICE)–C-REPEAT BINDING FACTOR/DRE BINDING FACTOR1 (CBF/DREB1) transcriptional pathway plays a critical role in modulating cold stress responses in Arabidopsis thaliana. Dissecting crucial upstream regulatory signals or components of the ICE-CBF/DREB1 cascade will enhance our understanding of plant cold-tolerance mechanisms. Here, we show that jasmonate positively regulates plant responses to freezing stress in Arabidopsis. Exogenous application of jasmonate significantly enhanced plant freezing tolerance with or without cold acclimation. By contrast, blocking endogenous jasmonate biosynthesis and signaling rendered plants hypersensitive to freezing stress. Consistent with the positive role of jasmonate in freezing stress, production of endogenous jasmonate was triggered by cold treatment. In addition, cold induction of genes acting in the CBF/DREB1 signaling pathway was upregulated by jasmonate. Further investigation revealed that several JASMONATE ZIM-DOMAIN (JAZ) proteins, the repressors of jasmonate signaling, physically interact with ICE1 and ICE2 transcription factors. JAZ1 and JAZ4 repress the transcriptional function of ICE1, thereby attenuating the expression of its regulon. Consistent with this, overexpression of JAZ1 or JAZ4 represses freezing stress responses of Arabidopsis. Taken together, our study provides evidence that jasmonate functions as a critical upstream signal of the ICE-CBF/DREB1 pathway to positively regulate Arabidopsis freezing tolerance. PMID:23933884

  2. Auxin response factors mediate Arabidopsis organ asymmetry via modulation of KANADI activity.

    PubMed

    Pekker, Irena; Alvarez, John Paul; Eshed, Yuval

    2005-11-01

    Members of the KANADI gene family in Arabidopsis thaliana regulate abaxial identity and laminar growth of lateral organs. Promoter APETALA3-mediated ectopic expression of KANADI restricts petal expansion and was used in a genetic screen for factors involved in KANADI-mediated signaling. Through this screen, mutations in ETTIN (ETT; also known as Auxin Response Factor3 [ARF3]) were isolated as second site suppressors and found to ameliorate ectopic KANADI activity throughout the plant as well. Mutant phenotypes of ett are restricted to flowers; however, double mutants with a closely related gene ARF4 exhibit transformation of abaxial tissues into adaxial ones in all aerial parts, resembling mutations in KANADI. Accordingly, the common RNA expression domain of both ARFs was found to be on the abaxial side of all lateral organs. Truncated, negatively acting gene products of strong ett alleles map to an ARF-specific, N-terminal domain of ETT. Such gene products strongly enhance abaxial tissue loss only when ARF activities are compromised. As KANADI is not required for either ETT or ARF4 transcription, and their overexpression cannot rescue kanadi mutants, cooperative activity is implied. ARF proteins are pivotal in mediating auxin responses; thus, we present a model linking transient local auxin gradients and gradual partitioning of lateral organs along the abaxial/adaxial axis. PMID:16199616

  3. Identification of three LRR-RKs involved in perception of root meristem growth factor in Arabidopsis.

    PubMed

    Shinohara, Hidefumi; Mori, Ayaka; Yasue, Naoko; Sumida, Kumiko; Matsubayashi, Yoshikatsu

    2016-04-01

    A peptide hormone, root meristem growth factor (RGF), regulates root meristem development through the PLETHORA (PLT) stem cell transcription factor pathway, but it remains to be uncovered how extracellular RGF signals are transduced to the nucleus. Here we identified, using a combination of a custom-made receptor kinase (RK) expression library and exhaustive photoaffinity labeling, three leucine-rich repeat RKs (LRR-RKs) that directly interact with RGF peptides in Arabidopsis These three LRR-RKs, which we named RGFR1, RGFR2, and RGFR3, are expressed in root tissues including the proximal meristem, the elongation zone, and the differentiation zone. The triple rgfr mutant was insensitive to externally applied RGF peptide and displayed a short root phenotype accompanied by a considerable decrease in meristematic cell number. In addition, PLT1 and PLT2 protein gradients, observed as a gradual gradient decreasing toward the elongation zone from the stem cell area in wild type, steeply declined at the root tip in the triple mutant. Because RGF peptides have been shown to create a diffusion-based concentration gradient extending from the stem cell area, our results strongly suggest that RGFRs mediate the transformation of an RGF peptide gradient into a PLT protein gradient in the proximal meristem, thereby acting as key regulators of root meristem development. PMID:27001831

  4. Expression of the Arabidopsis Sigma Factor SIG5 Is Photoreceptor and Photosynthesis Controlled

    PubMed Central

    Mellenthin, Marina; Ellersiek, Ulrike; Börger, Anna; Baier, Margarete

    2014-01-01

    Two collections of Arabidopsis GAL4 enhancer trap lines were screened for light-intensity dependent reporter gene activation. Line N9313 was isolated for its strong light-intensity regulation. The T-DNA element trapped distant enhancers of the SIG5 promoter, which drives expression of a sigma factor involved in regulation of chloroplast genes for photosystem II core proteins. The T-DNA insertion 715 bp upstream of the transcription initiation site splits the promoter in a distal and proximal part. Both parts are sensitive to blue and red light and depend on photosynthetic electron transport activity between photosystem II and the plastoquinone pool. The mainblue-light sensitivity is localized within a 196-bp sequence (–887 to –691 bp) in the proximal promoter region It is preferentially CRY1 and PHYB controlled. Type-I and type-II phytochromes mediate red-light sensitivity via various promoter elements spread over the proximal and distal upstream region. This work characterizes SIG5 as an anterograde control factor of chloroplast gene expression, which is controlled by chloroplast signals in a retrograde manner. PMID:27135509

  5. The Myb-domain protein ULTRAPETALA1 INTERACTING FACTOR 1 controls floral meristem activities in Arabidopsis.

    PubMed

    Moreau, Fanny; Thévenon, Emmanuel; Blanvillain, Robert; Lopez-Vidriero, Irene; Franco-Zorrilla, Jose Manuel; Dumas, Renaud; Parcy, François; Morel, Patrice; Trehin, Christophe; Carles, Cristel C

    2016-04-01

    Higher plants continuously and iteratively produce new above-ground organs in the form of leaves, stems and flowers. These organs arise from shoot apical meristems whose homeostasis depends on coordination between self-renewal of stem cells and their differentiation into organ founder cells. This coordination is stringently controlled by the central transcription factor WUSCHEL (WUS), which is both necessary and sufficient for stem cell specification in Arabidopsis thaliana ULTRAPETALA1 (ULT1) was previously identified as a plant-specific, negative regulator of WUS expression. However, molecular mechanisms underlying this regulation remain unknown. ULT1 protein contains a SAND putative DNA-binding domain and a B-box, previously proposed as a protein interaction domain in eukaryotes. Here, we characterise a novel partner of ULT1, named ULT1 INTERACTING FACTOR 1 (UIF1), which contains a Myb domain and an EAR motif. UIF1 and ULT1 function in the same pathway for regulation of organ number in the flower. Moreover, UIF1 displays DNA-binding activity and specifically binds to WUS regulatory elements. We thus provide genetic and molecular evidence that UIF1 and ULT1 work together in floral meristem homeostasis, probably by direct repression of WUS expression. PMID:26903506

  6. Ethylene Response Factor 6 Is a Regulator of Reactive Oxygen Species Signaling in Arabidopsis

    PubMed Central

    Sewelam, Nasser; Kazan, Kemal; Thomas-Hall, Skye R.; Kidd, Brendan N.; Manners, John M.; Schenk, Peer M.

    2013-01-01

    Reactive oxygen species (ROS) are produced in plant cells in response to diverse biotic and abiotic stresses as well as during normal growth and development. Although a large number of transcription factor (TF) genes are up- or down-regulated by ROS, currently very little is known about the functions of these TFs during oxidative stress. In this work, we examined the role of ERF6 (ETHYLENE RESPONSE FACTOR6), an AP2/ERF domain-containing TF, during oxidative stress responses in Arabidopsis. Mutant analyses showed that NADPH oxidase (RbohD) and calcium signaling are required for ROS-responsive expression of ERF6. erf6 insertion mutant plants showed reduced growth and increased H2O2 and anthocyanin levels. Expression analyses of selected ROS-responsive genes during oxidative stress identified several differentially expressed genes in the erf6 mutant. In particular, a number of ROS responsive genes, such as ZAT12, HSFs, WRKYs, MAPKs, RBOHs, DHAR1, APX4, and CAT1 were more strongly induced by H2O2 in erf6 plants than in wild-type. In contrast, MDAR3, CAT3, VTC2 and EX1 showed reduced expression levels in the erf6 mutant. Taken together, our results indicate that ERF6 plays an important role as a positive antioxidant regulator during plant growth and in response to biotic and abiotic stresses. PMID:23940555

  7. AthaMap-assisted transcription factor target gene identification in Arabidopsis thaliana.

    PubMed

    Bülow, Lorenz; Brill, Yuri; Hehl, Reinhard

    2010-01-01

    The AthaMap database generates a map of potential transcription factor binding sites (TFBS) and small RNA target sites in the Arabidopsis thaliana genome. The database contains sites for 115 different transcription factors (TFs). TFBS were identified with positional weight matrices (PWMs) or with single binding sites. With the new web tool 'Gene Identification', it is possible to identify potential target genes for selected TFs. For these analyses, the user can define a region of interest of up to 6000 bp in all annotated genes. For TFBS determined with PWMs, the search can be restricted to high-quality TFBS. The results are displayed in tables that identify the gene, position of the TFBS and, if applicable, individual score of the TFBS. In addition, data files can be downloaded that harbour positional information of TFBS of all TFs in a region between -2000 and +2000 bp relative to the transcription or translation start site. Also, data content of AthaMap was increased and the database was updated to the TAIR8 genome release. Database URL: http://www.athamap.de/gene_ident.php. PMID:21177332

  8. AthaMap-assisted transcription factor target gene identification in Arabidopsis thaliana

    PubMed Central

    Bülow, Lorenz; Brill, Yuri; Hehl, Reinhard

    2010-01-01

    The AthaMap database generates a map of potential transcription factor binding sites (TFBS) and small RNA target sites in the Arabidopsis thaliana genome. The database contains sites for 115 different transcription factors (TFs). TFBS were identified with positional weight matrices (PWMs) or with single binding sites. With the new web tool ‘Gene Identification’, it is possible to identify potential target genes for selected TFs. For these analyses, the user can define a region of interest of up to 6000 bp in all annotated genes. For TFBS determined with PWMs, the search can be restricted to high-quality TFBS. The results are displayed in tables that identify the gene, position of the TFBS and, if applicable, individual score of the TFBS. In addition, data files can be downloaded that harbour positional information of TFBS of all TFs in a region between −2000 and +2000 bp relative to the transcription or translation start site. Also, data content of AthaMap was increased and the database was updated to the TAIR8 genome release. Database URL: http://www.athamap.de/gene_ident.php PMID:21177332

  9. Screen Identifying Arabidopsis Transcription Factors Involved in the Response to 9-Lipoxygenase-Derived Oxylipins

    PubMed Central

    Walper, Elisabeth; Weiste, Christoph; Mueller, Martin J.; Hamberg, Mats; Dröge-Laser, Wolfgang

    2016-01-01

    13-Lipoxygenase-derived oxylipins, such as jasmonates act as potent signaling molecules in plants. Although experimental evidence supports the impact of oxylipins generated by the 9-Lipoxygenase (9-LOX) pathway in root development and pathogen defense, their signaling function in plants remains largely elusive. Based on the root growth inhibiting properties of the 9-LOX-oxylipin 9-HOT (9-hydroxy-10,12,15-octadecatrienoic acid), we established a screening approach aiming at identifying transcription factors (TFs) involved in signaling and/or metabolism of this oxylipin. Making use of the AtTORF-Ex (Arabidopsis thaliana Transcription Factor Open Reading Frame Expression) collection of plant lines overexpressing TF genes, we screened for those TFs which restore root growth on 9-HOT. Out of 6,000 lines, eight TFs were recovered at least three times and were therefore selected for detailed analysis. Overexpression of the basic leucine Zipper (bZIP) TF TGA5 and its target, the monoxygenase CYP81D11 reduced the effect of added 9-HOT, presumably due to activation of a detoxification pathway. The highly related ETHYLENE RESPONSE FACTORs ERF106 and ERF107 induce a broad detoxification response towards 9-LOX-oxylipins and xenobiotic compounds. From a set of 18 related group S-bZIP factors isolated in the screen, bZIP11 is known to participate in auxin-mediated root growth and may connect oxylipins to root meristem function. The TF candidates isolated in this screen provide starting points for further attempts to dissect putative signaling pathways involving 9-LOX-derived oxylipins. PMID:27073862

  10. Roles of Arabidopsis WRKY3 and WRKY4 Transcription Factors in Plant Responses to Pathogens

    PubMed Central

    Lai, Zhibing; Vinod, KM; Zheng, Zuyu; Fan, Baofang; Chen, Zhixiang

    2008-01-01

    Background Plant WRKY DNA-binding transcription factors are involved in plant responses to biotic and abiotic responses. It has been previously shown that Arabidopsis WRKY3 and WRKY4, which encode two structurally similar WRKY transcription factors, are induced by pathogen infection and salicylic acid (SA). However, the role of the two WRKY transcription factors in plant disease resistance has not been directly analyzed. Results Both WRKY3 and WRKY4 are nuclear-localized and specifically recognize the TTGACC W-box sequences in vitro. Expression of WRKY3 and WRKY4 was induced rapidly by stress conditions generated by liquid infiltration or spraying. Stress-induced expression of WRKY4 was further elevated by pathogen infection and SA treatment. To determine directly their role in plant disease resistance, we have isolated T-DNA insertion mutants and generated transgenic overexpression lines for WRKY3 and WRKY4. Both the loss-of-function mutants and transgenic overexpression lines were examined for responses to the biotrophic bacterial pathogen Pseudomonas syringae and the necrotrophic fungal pathogen Botrytis cinerea. The wrky3 and wrky4 single and double mutants exhibited more severe disease symptoms and support higher fungal growth than wild-type plants after Botrytis infection. Although disruption of WRKY3 and WRKY4 did not have a major effect on plant response to P. syringae, overexpression of WRKY4 greatly enhanced plant susceptibility to the bacterial pathogen and suppressed pathogen-induced PR1 gene expression. Conclusion The nuclear localization and sequence-specific DNA-binding activity support that WRKY3 and WRKY4 function as transcription factors. Functional analysis based on T-DNA insertion mutants and transgenic overexpression lines indicates that WRKY3 and WRKY4 have a positive role in plant resistance to necrotrophic pathogens and WRKY4 has a negative effect on plant resistance to biotrophic pathogens. PMID:18570649

  11. The GATA Family of Transcription Factors in Arabidopsis and Rice1

    PubMed Central

    Reyes, José C.; Muro-Pastor, M. Isabel; Florencio, Francisco J.

    2004-01-01

    GATA transcription factors are a group of DNA binding proteins broadly distributed in eukaryotes. The GATA factors DNA binding domain is a class IV zinc finger motif in the form CX2CX17–20CX2C followed by a basic region. In plants, GATA DNA motifs have been implicated in light-dependent and nitrate-dependent control of transcription. Herein, we show that the Arabidopsis and the rice (Oryza sativa) genomes present 29 and 28 loci, respectively, that encode for putative GATA factors. A phylogenetic analysis of the 57 GATA factors encoding genes, as well as the study of their intron-exon structure, indicates the existence of seven subfamilies of GATA genes. Some of these subfamilies are represented in both species but others are exclusive for one of them. In addition to the GATA zinc finger motif, polypeptides of the different subfamilies are characterized by the presence of additional domains such as an acidic domain, a CCT (CONSTANS, CO-like, and TOC1) domain, or a transposase-like domain also found in FAR1 and FHY3. Subfamily VI comprises genes that encode putative bi-zinc finger polypeptides, also found in metazoan and fungi, and a tri-zinc finger protein which has not been previously reported in eukaryotes. The phylogeny of the GATA zinc finger motif, excluding flanking regions, evidenced the existence of four classes of GATA zinc fingers, three of them containing 18 residues in the zinc finger loop and one containing a 20-residue loop. Our results support multiple models of evolution of the GATA gene family in plants including gene duplication and exon shuffling. PMID:15084732

  12. Screen Identifying Arabidopsis Transcription Factors Involved in the Response to 9-Lipoxygenase-Derived Oxylipins.

    PubMed

    Walper, Elisabeth; Weiste, Christoph; Mueller, Martin J; Hamberg, Mats; Dröge-Laser, Wolfgang

    2016-01-01

    13-Lipoxygenase-derived oxylipins, such as jasmonates act as potent signaling molecules in plants. Although experimental evidence supports the impact of oxylipins generated by the 9-Lipoxygenase (9-LOX) pathway in root development and pathogen defense, their signaling function in plants remains largely elusive. Based on the root growth inhibiting properties of the 9-LOX-oxylipin 9-HOT (9-hydroxy-10,12,15-octadecatrienoic acid), we established a screening approach aiming at identifying transcription factors (TFs) involved in signaling and/or metabolism of this oxylipin. Making use of the AtTORF-Ex (Arabidopsis thaliana Transcription Factor Open Reading Frame Expression) collection of plant lines overexpressing TF genes, we screened for those TFs which restore root growth on 9-HOT. Out of 6,000 lines, eight TFs were recovered at least three times and were therefore selected for detailed analysis. Overexpression of the basic leucine Zipper (bZIP) TF TGA5 and its target, the monoxygenase CYP81D11 reduced the effect of added 9-HOT, presumably due to activation of a detoxification pathway. The highly related ETHYLENE RESPONSE FACTORs ERF106 and ERF107 induce a broad detoxification response towards 9-LOX-oxylipins and xenobiotic compounds. From a set of 18 related group S-bZIP factors isolated in the screen, bZIP11 is known to participate in auxin-mediated root growth and may connect oxylipins to root meristem function. The TF candidates isolated in this screen provide starting points for further attempts to dissect putative signaling pathways involving 9-LOX-derived oxylipins. PMID:27073862

  13. Distinctive interactions of the Arabidopsis homolog of the 30 kD subunit of the cleavage and polyadenylation specificity factor (AtCPSF30) with other polyadenylation factor subunits

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Background: The Arabidopsis ortholog of the 30 kD subunit of the mammalian Cleavage and Polyadenylation Specificity Factor (AtCPSF30) is an RNA-binding endonuclease that is associated with other Arabidopsis CPSF subunits (orthologs of the 160, 100, and 73 kD subunits of CPSF). In order to better u...

  14. Phytochrome-interacting factors PIF4 and PIF5 negatively regulate anthocyanin biosynthesis under red light in Arabidopsis seedlings.

    PubMed

    Liu, Zhongjuan; Zhang, Yongqiang; Wang, Jianfeng; Li, Ping; Zhao, Chengzhou; Chen, Yadi; Bi, Yurong

    2015-09-01

    Light is an important environmental factor inducing anthocyanin accumulation in plants. Phytochrome-interacting factors (PIFs) have been shown to be a family of bHLH transcription factors involved in light signaling in Arabidopsis. Red light effectively increased anthocyanin accumulation in wild-type Col-0, whereas the effects were enhanced in pif4 and pif5 mutants but impaired in overexpression lines PIF4OX and PIF5OX, indicating that PIF4 and PIF5 are both negative regulators for red light-induced anthocyanin accumulation. Consistently, transcript levels of several genes involved in anthocyanin biosynthesis and regulatory pathway, including CHS, F3'H, DFR, LDOX, PAP1 and TT8, were significantly enhanced in mutants pif4 and pif5 but decreased in PIF4OX and PIF5OX compared to in Col-0, indicating that PIF4 and PIF5 are transcriptional repressor of these gene. Transient expression assays revealed that PIF4 and PIF5 could repress red light-induced promoter activities of F3'H and DFR in Arabidopsis protoplasts. Furthermore, chromatin immunoprecipitation-quantitative PCR (ChIP-qPCR) test and electrophoretic mobility shift assay (EMSA) showed that PIF5 could directly bind to G-box motifs present in the promoter of DFR. Taken together, these results suggest that PIF4 and PIF5 negatively regulate red light-induced anthocyanin accumulation through transcriptional repression of the anthocyanin biosynthetic genes in Arabidopsis. PMID:26259175

  15. Splicing factor SR34b mutation reduces cadmium tolerance in Arabidopsis by regulating iron-regulated transporter 1 gene

    SciTech Connect

    Zhang, Wentao; Du, Bojing; Liu, Di; Qi, Xiaoting

    2014-12-12

    Highlights: • Arabidopsis splicing factor SR34b gene is cadmium-inducible. • SR34b T-DNA insertion mutant is sensitive to cadmium due to high cadmium uptake. • SR34b is a regulator of cadmium transporter IRT1 at the posttranscription level. • These results highlight the roles of splicing factors in cadmium tolerance of plant. - Abstract: Serine/arginine-rich (SR) proteins are important splicing factors. However, the biological functions of plant SR proteins remain unclear especially in abiotic stresses. Cadmium (Cd) is a non-essential element that negatively affects plant growth and development. In this study, we provided clear evidence for SR gene involved in Cd tolerance in planta. Systemic expression analysis of 17 Arabidopsis SR genes revealed that SR34b is the only SR gene upregulated by Cd, suggesting its potential roles in Arabidopsis Cd tolerance. Consistent with this, a SR34b T-DNA insertion mutant (sr34b) was moderately sensitive to Cd, which had higher Cd{sup 2+} uptake rate and accumulated Cd in greater amounts than wild-type. This was due to the altered expression of iron-regulated transporter 1 (IRT1) gene in sr34b mutant. Under normal growth conditions, IRT1 mRNAs highly accumulated in sr34b mutant, which was a result of increased stability of IRT1 mRNA. Under Cd stress, however, sr34b mutant plants had a splicing defect in IRT1 gene, thus reducing the IRT1 mRNA accumulation. Despite of this, sr34b mutant plants still constitutively expressed IRT1 proteins under Cd stress, thereby resulting in Cd stress-sensitive phenotype. We therefore propose the essential roles of SR34b in posttranscriptional regulation of IRT1 expression and identify it as a regulator of Arabidopsis Cd tolerance.

  16. A "Whirly" transcription factor is required for salicylic acid-dependent disease resistance in Arabidopsis.

    PubMed

    Desveaux, Darrell; Subramaniam, Rajagopal; Després, Charles; Mess, Jean-Nicholas; Lévesque, Caroline; Fobert, Pierre R; Dangl, Jeffery L; Brisson, Normand

    2004-02-01

    Transcriptional reprogramming is critical for plant disease resistance responses; its global control is not well understood. Salicylic acid (SA) can induce plant defense gene expression and a long-lasting disease resistance state called systemic acquired resistance (SAR). Plant-specific "Whirly" DNA binding proteins were previously implicated in defense gene regulation. We demonstrate that the potato StWhy1 protein is a transcriptional activator of genes containing the PBF2 binding PB promoter element. DNA binding activity of AtWhy1, the Arabidopsis StWhy1 ortholog, is induced by SA and is required for both SA-dependent disease resistance and SA-induced expression of an SAR response gene. AtWhy1 is required for both full basal and specific disease resistance responses. The transcription factor-associated protein NPR1 is also required for SAR. Surprisingly, AtWhy1 activation by SA is NPR1 independent, suggesting that AtWhy1 works in conjunction with NPR1 to transduce the SA signal. Our analysis of AtWhy1 adds a critical component to the SA-dependent plant disease resistance response. PMID:14960277

  17. Quiescent center initiation in the Arabidopsis lateral root primordia is dependent on the SCARECROW transcription factor.

    PubMed

    Goh, Tatsuaki; Toyokura, Koichi; Wells, Darren M; Swarup, Kamal; Yamamoto, Mayuko; Mimura, Tetsuro; Weijers, Dolf; Fukaki, Hidehiro; Laplaze, Laurent; Bennett, Malcolm J; Guyomarc'h, Soazig

    2016-09-15

    Lateral root formation is an important determinant of root system architecture. In Arabidopsis, lateral roots originate from pericycle cells, which undergo a program of morphogenesis to generate a new lateral root meristem. Despite its importance for root meristem organization, the onset of quiescent center (QC) formation during lateral root morphogenesis remains unclear. Here, we used live 3D confocal imaging to monitor cell organization and identity acquisition during lateral root development. Our dynamic observations revealed an early morphogenesis phase and a late meristem formation phase as proposed in the bi-phasic growth model. Establishment of lateral root QCs coincided with this developmental phase transition. QC precursor cells originated from the outer layer of stage II lateral root primordia, within which the SCARECROW (SCR) transcription factor was specifically expressed. Disrupting SCR function abolished periclinal divisions in this lateral root primordia cell layer and perturbed the formation of QC precursor cells. We conclude that de novo QC establishment in lateral root primordia operates via SCR-mediated formative cell division and coincides with the developmental phase transition. PMID:27510971

  18. The cytochrome c oxidase biogenesis factor AtCOX17 modulates stress responses in Arabidopsis.

    PubMed

    Garcia, Lucila; Welchen, Elina; Gey, Uta; Arce, Agustín L; Steinebrunner, Iris; Gonzalez, Daniel H

    2016-03-01

    COX17 is a soluble protein from the mitochondrial intermembrane space that participates in the transfer of copper for cytochrome c oxidase (COX) assembly in eukaryotic organisms. In this work, we studied the function of both Arabidopsis thaliana AtCOX17 genes using plants with altered expression levels of these genes. Silencing of AtCOX17-1 in a cox17-2 knockout background generates plants with smaller rosettes and decreased expression of genes involved in the response of plants to different stress conditions, including several genes that are induced by mitochondrial dysfunctions. Silencing of either of the AtCOX17 genes does not affect plant development or COX activity but causes a decrease in the response of genes to salt stress. In addition, these plants contain higher reactive oxygen and lipid peroxidation levels after irrigation with high NaCl concentrations and are less sensitive to abscisic acid. In agreement with a role of AtCOX17 in stress and abscisic acid responses, both AtCOX17 genes are induced by several stress conditions, abscisic acid and mutation of the transcription factor ABI4. The results indicate that AtCOX17 is required for optimal expression of a group of stress-responsive genes, probably as a component of signalling pathways that link stress conditions to gene expression responses. PMID:26436309

  19. AUXIN RESPONSE FACTOR8 is a negative regulator of fruit initiation in Arabidopsis.

    PubMed

    Goetz, Marc; Vivian-Smith, Adam; Johnson, Susan D; Koltunow, Anna M

    2006-08-01

    Fruit and seed formation in plants is normally initiated after pollination and fertilization, and, in the absence of fertilization, flowers senesce. In the Arabidopsis thaliana mutant fruit without fertilization, a mutation in AUXIN RESPONSE FACTOR8 (ARF8) results in the uncoupling of fruit development from pollination and fertilization and gives rise to seedless (parthenocarpic) fruit. Parthenocarpy was confirmed in two additional recessive alleles and was caused by mutations within the coding region of ARF8. Genetic experiments indicate that ARF8 acts as an inhibitor to stop further carpel development in the absence of fertilization and the generation of signals required to initiate fruit and seed development. Expression of ARF8 was found to be regulated at multiple levels, and transcriptional autoregulation of ARF8 was observed. Analysis of plants transformed with a transcriptional P(ARF8):beta-glucuronidase (GUS) construct or a translational ARF8:GUS fusion construct displayed distinct developmental regulation of the reporter in floral tissues involved in pollination and fertilization and in the carpel wall. After fertilization, the level of GUS activity declined in the developing seed, while in unfertilized ovules that are destined to senesce, ARF8:GUS expression spread throughout the ovule. This is consistent with a proposed role for ARF8 in restricting signal transduction processes in ovules and growth in pistils until the fruit initiation cue. PMID:16829592

  20. SHINE transcription factors act redundantly to pattern the archetypal surface of Arabidopsis flower organs.

    PubMed

    Shi, Jian Xin; Malitsky, Sergey; De Oliveira, Sheron; Branigan, Caroline; Franke, Rochus B; Schreiber, Lukas; Aharoni, Asaph

    2011-05-01

    Floral organs display tremendous variation in their exterior that is essential for organogenesis and the interaction with the environment. This diversity in surface characteristics is largely dependent on the composition and structure of their coating cuticular layer. To date, mechanisms of flower organ initiation and identity have been studied extensively, while little is known regarding the regulation of flower organs surface formation, cuticle composition, and its developmental significance. Using a synthetic microRNA approach to simultaneously silence the three SHINE (SHN) clade members, we revealed that these transcription factors act redundantly to shape the surface and morphology of Arabidopsis flowers. It appears that SHNs regulate floral organs' epidermal cell elongation and decoration with nanoridges, particularly in petals. Reduced activity of SHN transcription factors results in floral organs' fusion and earlier abscission that is accompanied by a decrease in cutin load and modified cell wall properties. SHN transcription factors possess target genes within four cutin- and suberin-associated protein families including, CYP86A cytochrome P450s, fatty acyl-CoA reductases, GSDL-motif lipases, and BODYGUARD1-like proteins. The results suggest that alongside controlling cuticular lipids metabolism, SHNs act to modify the epidermis cell wall through altering pectin metabolism and structural proteins. We also provide evidence that surface formation in petals and other floral organs during their growth and elongation or in abscission and dehiscence through SHNs is partially mediated by gibberellin and the DELLA signaling cascade. This study therefore demonstrates the need for a defined composition and structure of the cuticle and cell wall in order to form the archetypal features of floral organs surfaces and control their cell-to-cell separation processes. Furthermore, it will promote future investigation into the relation between the regulation of organ

  1. Arabidopsis WRKY2 transcription factor mediates seed germination and postgermination arrest of development by abscisic acid

    PubMed Central

    Jiang, Wenbo; Yu, Diqiu

    2009-01-01

    Background Plant WRKY DNA-binding transcription factors are key regulators in certain developmental programs. A number of studies have suggested that WRKY genes may mediate seed germination and postgermination growth. However, it is unclear whether WRKY genes mediate ABA-dependent seed germination and postgermination growth arrest. Results To determine directly the role of Arabidopsis WRKY2 transcription factor during ABA-dependent seed germination and postgermination growth arrest, we isolated T-DNA insertion mutants. Two independent T-DNA insertion mutants for WRKY2 were hypersensitive to ABA responses only during seed germination and postgermination early growth. wrky2 mutants displayed delayed or decreased expression of ABI5 and ABI3, but increased or prolonged expression of Em1 and Em6. wrky2 mutants and wild type showed similar levels of expression for miR159 and its target genes MYB33 and MYB101. Analysis of WRKY2 expression level in ABA-insensitive and ABA-deficient mutants abi5-1, abi3-1, aba2-3 and aba3-1 further indicated that ABA-induced WRKY2 accumulation during germination and postgermination early growth requires ABI5, ABI3, ABA2 and ABA3. Conclusion ABA hypersensitivity of the wrky2 mutants during seed germination and postgermination early seedling establishment is attributable to elevated mRNA levels of ABI5, ABI3 and ABI5-induced Em1 and Em6 in the mutants. WRKY2-mediated ABA responses are independent of miR159 and its target genes MYB33 and MYB101. ABI5, ABI3, ABA2 and ABA3 are important regulators of the transcripts of WRKY2 by ABA treatment. Our results suggest that WRKY2 transcription factor mediates seed germination and postgermination developmental arrest by ABA. PMID:19622176

  2. Pyocyanin, a virulence factor produced by Pseudomonas aeruginosa, alters root development through reactive oxygen species and ethylene signaling in Arabidopsis.

    PubMed

    Ortiz-Castro, Randy; Pelagio-Flores, Ramón; Méndez-Bravo, Alfonso; Ruiz-Herrera, León Francisco; Campos-García, Jesús; López-Bucio, José

    2014-04-01

    Pyocyanin acts as a virulence factor in Pseudomonas aeruginosa, a plant and animal pathogen. In this study, we evaluated the effect of pyocyanin on growth and development of Arabidopsis seedlings. Root inoculation with P. aeruginosa PAO1 strain inhibited primary root growth in wild-type (WT) Arabidopsis seedlings. In contrast, single lasI- and double rhlI-/lasI- mutants of P. aeruginosa defective in pyocyanin production showed decreased root growth inhibition concomitant with an increased phytostimulation. Treatment with pyocyanin modulates root system architecture, inhibiting primary root growth and promoting lateral root and root hair formation without affecting meristem viability or causing cell death. These effects correlated with altered proportions of hydrogen peroxide and superoxide in root tips and with an inhibition of cell division and elongation. Mutant analyses showed that pyocyanin modulation of root growth was likely independent of auxin, cytokinin, and abscisic acid but required ethylene signaling because the Arabidopsis etr1-1, ein2-1, and ein3-1 ethylene-related mutants were less sensitive to pyocyanin-induced root stoppage and reactive oxygen species (ROS) distribution. Our findings suggest that pyocyanin is an important factor modulating the interplay between ROS production and root system architecture by an ethylene-dependent signaling. PMID:24224532

  3. TaNAC2, a NAC-type wheat transcription factor conferring enhanced multiple abiotic stress tolerances in Arabidopsis

    PubMed Central

    Mao, Xinguo; Zhang, Hongying; Qian, Xueya; Li, Ang; Zhao, Guangyao; Jing, Ruilian

    2012-01-01

    Environmental stresses such as drought, salinity, and cold are major factors that significantly limit agricultural productivity. NAC transcription factors play essential roles in response to various abiotic stresses. However, the paucity of wheat NAC members functionally characterized to date does not match the importance of this plant as a world staple crop. Here, the function of TaNAC2 was characterized in Arabidopsis thaliana. A fragment of TaNAC2 was obtained from suppression subtractive cDNA libraries of wheat treated with polyethylene glycol, and its full-length cDNA was obtained by searching a full-length wheat cDNA library. Gene expression profiles indicated that TaNAC2 was involved in response to drought, salt, cold, and abscisic acid treatment. To test its function, transgenic Arabidopsis lines overexpressing TaNAC2–GFP controlled by the cauliflower mosaic virus 35S promoter were generated. Overexpression of TaNAC2 resulted in enhanced tolerances to drought, salt, and freezing stresses in Arabidopsis, which were simultaneously demonstrated by enhanced expression of abiotic stress-response genes and several physiological indices. Therefore, TaNAC2 has potential for utilization in transgenic breeding to improve abiotic stress tolerances in crops. PMID:22330896

  4. Extensive Modulation of the Transcription Factor Transcriptome during Somatic Embryogenesis in Arabidopsis thaliana

    PubMed Central

    Gliwicka, Marta; Nowak, Katarzyna; Balazadeh, Salma; Mueller-Roeber, Bernd; Gaj, Malgorzata D.

    2013-01-01

    Molecular mechanisms controlling plant totipotency are largely unknown and studies on somatic embryogenesis (SE), the process through which already differentiated cells reverse their developmental program and become embryogenic, provide a unique means for deciphering molecular mechanisms controlling developmental plasticity of somatic cells. Among various factors essential for embryogenic transition of somatic cells transcription factors (TFs), crucial regulators of genetic programs, are believed to play a central role. Herein, we used quantitative real-time polymerase chain reaction (qRT-PCR) to identify TF genes affected during SE induced by in vitro culture in Arabidopsis thaliana. Expression profiles of 1,880 TFs were evaluated in the highly embryogenic Col-0 accession and the non-embryogenic tanmei/emb2757 mutant. Our study revealed 729 TFs whose expression changes during the 10-days incubation period of SE; 141 TFs displayed distinct differences in expression patterns in embryogenic versus non-embryogenic cultures. The embryo-induction stage of SE occurring during the first 5 days of culture was associated with a robust and dramatic change of the TF transcriptome characterized by the drastic up-regulation of the expression of a great majority (over 80%) of the TFs active during embryogenic culture. In contrast to SE induction, the advanced stage of embryo formation showed attenuation and stabilization of transcript levels of many TFs. In total, 519 of the SE-modulated TFs were functionally annotated and transcripts related with plant development, phytohormones and stress responses were found to be most abundant. The involvement of selected TFs in SE was verified using T-DNA insertion lines and a significantly reduced embryogenic response was found for the majority of them. This study provides comprehensive data focused on the expression of TF genes during SE and suggests directions for further research on functional genomics of SE. PMID:23874927

  5. Template-based structure prediction and classification of transcription factors in Arabidopsis thaliana

    PubMed Central

    Lu, Tao; Yang, Yuedong; Yao, Bo; Liu, Song; Zhou, Yaoqi; Zhang, Chi

    2012-01-01

    Transcription factors (TFs) play important roles in plants. However, there is no systematic study of their structures and functions of most TFs in plants. Here, we performed template-based structure prediction for all TFs in Arabidopsis thaliana, with their full-length sequences as well as C-terminal and N-terminal regions. A total of 2918 model structures were obtained with a high confidence score. We find that TF families employ only a smaller number of templates for DNA-binding domains (DBD) but a diverse number of templates for transcription regulatory domains (TRD). Although TF families are classified according to DBD, their sizes have a significant correlation with the number of unique non-DNA-binding templates employed in the family (Pearson correlation coefficient of 0.74). That is, the size of TF family is related to its functional diversity. Network analysis reveals new connections between TF families based on shared TRD or DBD templates; 81% TF families share DBD and 67% share TRD templates. Two large fully connected family clusters in this network are observed along with 69 island families. In addition, 25 genes with unknown functions are found to be DNA-binding and/or TF factors according to predicted structures. This work provides a global view of the classification of TFs based on their DBD or TRD templates, and hence, a deeper understanding of DNA-binding and regulatory functions from structural perspective. All structural models of TFs are deposited in the online database for public usage at http://sysbio.unl.edu/AthTF. PMID:22549903

  6. Role of Transcription Factor HAT1 in Modulating Arabidopsis thaliana Response to Cucumber mosaic virus.

    PubMed

    Zou, Li-Juan; Deng, Xing-Guang; Han, Xue-Ying; Tan, Wen-Rong; Zhu, Li-Jun; Xi, De-Hui; Zhang, Da-Wei; Lin, Hong-Hui

    2016-09-01

    Arabidopsis thaliana homeodomain-leucine zipper protein 1 (HAT1) belongs to the homeodomain-leucine zipper (HD-Zip) family class II that plays important roles in plant growth and development as a transcription factor. To elucidate further the role of HD-Zip II transcription factors in plant defense, the A. thaliana hat1, hat1hat3 and hat1hat2hat3 mutants and HAT1 overexpression plants (HAT1OX) were challenged with Cucumber mosaic virus (CMV). HAT1OX displayed more susceptibility, while loss-of-function mutants of HAT1 exhibited less susceptibility to CMV infection. HAT1 and its close homologs HAT2 and HAT3 function redundantly, as the triple mutant hat1hat2hat3 displayed increased virus resistance compared with the hat1 and hat1hat3 mutants. Furthermore, the induction of the antioxidant system (the activities and expression of enzymatic antioxidants) and the expression of defense-associated genes were down-regulated in HAT1OX but up-regulated in hat1hat2hat3 when compared with Col-0 after CMV infection. Further evidence showed that the involvement of HAT1 in the anti-CMV defense response might be dependent on salicylic acid (SA) but not jasmonic acid (JA). The SA level or expression of SA synthesis-related genes was decreased in HAT1OX but increased in hat1hat2hat3 compared with Col-0 after CMV infection, but there were little difference in JA level or JA synthesis-related gene expression among HAT1OX or defective plants. In addition, HAT1 expression is dependent on SA accumulation. Taken together, our study indicated that HAT1 negatively regulates plant defense responses to CMV. PMID:27328697

  7. A Recently Evolved Isoform of the Transcription Factor BES1 Promotes Brassinosteroid Signaling and Development in Arabidopsis thaliana

    PubMed Central

    Zhang, Chi; Wang, Xuelu

    2015-01-01

    Brassinosteroids (BRs) are essential steroid hormones that regulate plant growth and development. The transcription factor BRI1-EMS-SUPPRESSOR1 (BES1) regulates the expression of thousands of target genes in response to BRs. Here, we report an Arabidopsis thaliana-specific long isoform of BES1, BES1-L, which has stronger activity in promoting BR signaling than the canonical and widely used short BES1-S. The BES1-L isoform contains an additional N-terminal bipartite nuclear localization signal, which strongly promotes its nuclear localization. BES1-L also promotes the nuclear localization of BES1-S and BRASSINAZOLE-RESISTANT1 via dimerization. The transcription of BES1-L and BES1-S is differentially regulated by BRs due to the presence of G-box element in the BES1-S promoter. Moreover, BES1-L uniquely exists in the majority of A. thaliana ecotypes, but not in other species, even its Brassicaceae relatives, including Arabidopsis lyrata. The phenotypes of the BES1-L overexpression lines and plants with truncated BES1-L indicate that BES1-L is a more important isoform of BES1 in Arabidopsis and may have contributed to the evolution and expansion of A. thaliana. PMID:25649439

  8. The transcript elongation factor SPT4/SPT5 is involved in auxin-related gene expression in Arabidopsis

    PubMed Central

    Dürr, Julius; Lolas, Ihab B.; Sørensen, Brian B.; Schubert, Veit; Houben, Andreas; Melzer, Michael; Deutzmann, Rainer; Grasser, Marion; Grasser, Klaus D.

    2014-01-01

    The heterodimeric complex SPT4/SPT5 is a transcript elongation factor (TEF) that directly interacts with RNA polymerase II (RNAPII) to regulate messenger RNA synthesis in the chromatin context. We provide biochemical evidence that in Arabidopsis, SPT4 occurs in a complex with SPT5, demonstrating that the SPT4/SPT5 complex is conserved in plants. Each subunit is encoded by two genes SPT4-1/2 and SPT5-1/2. A mutant affected in the tissue-specifically expressed SPT5-1 is viable, whereas inactivation of the generally expressed SPT5-2 is homozygous lethal. RNAi-mediated downregulation of SPT4 decreases cell proliferation and causes growth reduction and developmental defects. These plants display especially auxin signalling phenotypes. Consistently, auxin-related genes, most strikingly AUX/IAA genes, are downregulated in SPT4–RNAi plants that exhibit an enhanced auxin response. In Arabidopsis nuclei, SPT5 clearly localizes to the transcriptionally active euchromatin, and essentially co-localizes with transcribing RNAPII. Typical for TEFs, SPT5 is found over the entire transcription unit of RNAPII-transcribed genes. In SPT4–RNAi plants, elevated levels of RNAPII and SPT5 are detected within transcribed regions (including those of downregulated genes), indicating transcript elongation defects in these plants. Therefore, SPT4/SPT5 acts as a TEF in Arabidopsis, regulating transcription during the elongation stage with particular impact on the expression of certain auxin-related genes. PMID:24497194

  9. The gene family encoding the Arabidopsis thaliana translation elongation factor EF-1 alpha: molecular cloning, characterization and expression.

    PubMed

    Axelos, M; Bardet, C; Liboz, T; Le Van Thai, A; Curie, C; Lescure, B

    1989-10-01

    The gene family encoding the Arabidopsis thaliana translation elongation factor (EF-1 alpha) was analysed. This family contains four genes (A1-A4) organized in a similar manner in different varieties of Arabidopsis. Based upon both their physical separation and a comparison of their sequences, it is suggested that the A4 gene and the A1, A2, and A3 genes constitute two distinct subfamilies within the genome. By introducing chimaeric gene constructs into Arabidopsis cells, we showed that the A1 gene promoter mediates a transient expression about twofold higher than that obtained using the CaMV 35 S promoter. This expression depends on a 348 bp DNA fragment extending from -982 to -634 bp upstream of the initiation codon. This element contains a characteristic telomeric sequence (AACCCTAA) which is also found in the promoters of the A2 and A4 genes as well as in the promoters of the Drosophila EF-1 alpha F1 gene and of several highly expressed plant genes. PMID:2615757

  10. AUXIN RESPONSE FACTOR17 Is Essential for Pollen Wall Pattern Formation in Arabidopsis1[C][W][OA

    PubMed Central

    Yang, Jun; Tian, Lei; Sun, Ming-Xi; Huang, Xue-Yong; Zhu, Jun; Guan, Yue-Feng; Jia, Qi-Shi; Yang, Zhong-Nan

    2013-01-01

    In angiosperms, pollen wall pattern formation is determined by primexine deposition on the microspores. Here, we show that AUXIN RESPONSE FACTOR17 (ARF17) is essential for primexine formation and pollen development in Arabidopsis (Arabidopsis thaliana). The arf17 mutant exhibited a male-sterile phenotype with normal vegetative growth. ARF17 was expressed in microsporocytes and microgametophytes from meiosis to the bicellular microspore stage. Transmission electron microscopy analysis showed that primexine was absent in the arf17 mutant, which leads to pollen wall-patterning defects and pollen degradation. Callose deposition was also significantly reduced in the arf17 mutant, and the expression of CALLOSE SYNTHASE5 (CalS5), the major gene for callose biosynthesis, was approximately 10% that of the wild type. Chromatin immunoprecipitation and electrophoretic mobility shift assays showed that ARF17 can directly bind to the CalS5 promoter. As indicated by the expression of DR5-driven green fluorescent protein, which is an synthetic auxin response reporter, auxin signaling appeared to be specifically impaired in arf17 anthers. Taken together, our results suggest that ARF17 is essential for pollen wall patterning in Arabidopsis by modulating primexine formation at least partially through direct regulation of CalS5 gene expression. PMID:23580594

  11. Arabidopsis VQ MOTIF-CONTAINING PROTEIN29 represses seedling deetiolation by interacting with PHYTOCHROME-INTERACTING FACTOR1.

    PubMed

    Li, Yunliang; Jing, Yanjun; Li, Junjiao; Xu, Gang; Lin, Rongcheng

    2014-04-01

    Seedling deetiolation, a critical process in early plant development, is regulated by an intricate transcriptional network. Here, we identified VQ MOTIF-CONTAINING PROTEIN29 (VQ29) as a novel regulator of the photomorphogenic response in Arabidopsis (Arabidopsis thaliana). We showed that 29 of the 34 VQ proteins present in Arabidopsis exhibit transcriptional activity in plant cells and that mutations in the VQ motif affect the transcriptional activity of VQ29. We then functionally characterized VQ29 and showed that the hypocotyl growth of plants overexpressing VQ29 is hyposensitive to far-red and low-intensity white light, whereas a vq29 loss-of-function mutant exhibits decreased hypocotyl elongation under a low intensity of far-red or white light. Consistent with this, VQ29 expression is repressed by light in a phytochrome-dependent manner. Intriguingly, our yeast (Saccharomyces cerevisiae) two-hybrid, bimolecular fluorescence complementation, and coimmunoprecipitation assays showed that VQ29 physically interacts with PHYTOCHROME-INTERACTING FACTOR1 (PIF1). We then showed that VQ29 and PIF1 directly bind to the promoter of a cell elongation-related gene, XYLOGLUCAN ENDOTRANSGLYCOSYLASE7, and coactivate its expression. Furthermore, the vq29 pif1 double mutant has shorter hypocotyls than either of the corresponding single mutants. Therefore, our study reveals that VQ29 is a negative transcriptional regulator of light-mediated inhibition of hypocotyl elongation that likely promotes the transcriptional activity of PIF1 during early seedling development. PMID:24569844

  12. Genome-Wide Analysis of Basic/Helix-Loop-Helix Transcription Factor Family in Rice and Arabidopsis1[W

    PubMed Central

    Li, Xiaoxing; Duan, Xuepeng; Jiang, Haixiong; Sun, Yujin; Tang, Yuanping; Yuan, Zheng; Guo, Jingkang; Liang, Wanqi; Chen, Liang; Yin, Jingyuan; Ma, Hong; Wang, Jian; Zhang, Dabing

    2006-01-01

    The basic/helix-loop-helix (bHLH) transcription factors and their homologs form a large family in plant and animal genomes. They are known to play important roles in the specification of tissue types in animals. On the other hand, few plant bHLH proteins have been studied functionally. Recent completion of whole genome sequences of model plants Arabidopsis (Arabidopsis thaliana) and rice (Oryza sativa) allows genome-wide analysis and comparison of the bHLH family in flowering plants. We have identified 167 bHLH genes in the rice genome, and their phylogenetic analysis indicates that they form well-supported clades, which are defined as subfamilies. In addition, sequence analysis of potential DNA-binding activity, the sequence motifs outside the bHLH domain, and the conservation of intron/exon structural patterns further support the evolutionary relationships among these proteins. The genome distribution of rice bHLH genes strongly supports the hypothesis that genome-wide and tandem duplication contributed to the expansion of the bHLH gene family, consistent with the birth-and-death theory of gene family evolution. Bioinformatics analysis suggests that rice bHLH proteins can potentially participate in a variety of combinatorial interactions, endowing them with the capacity to regulate a multitude of transcriptional programs. In addition, similar expression patterns suggest functional conservation between some rice bHLH genes and their close Arabidopsis homologs. PMID:16896230

  13. The sunflower transcription factor HaHB11 improves yield, biomass and tolerance to flooding in transgenic Arabidopsis plants.

    PubMed

    Cabello, Julieta V; Giacomelli, Jorge I; Piattoni, Claudia V; Iglesias, Alberto A; Chan, Raquel L

    2016-03-20

    HaHB11 is a member of the sunflower homeodomain-leucine zipper I subfamily of transcription factors. The analysis of a sunflower microarray hybridized with RNA from HaHB11-transformed leaf-disks indicated the regulation of many genes encoding enzymes from glycolisis and fermentative pathways. A 1300bp promoter sequence, fused to the GUS reporter gene, was used to transform Arabidopsis plants showing an induction of expression after flooding treatments, concurrently with HaHB11 regulation by submergence in sunflower. Arabidopsis transgenic plants expressing HaHB11 under the control of the CaMV 35S promoter and its own promoter were obtained and these plants exhibited significant increases in rosette and stem biomass. All the lines produced more seeds than controls and particularly, those of high expression level doubled seeds yield. Transgenic plants also showed tolerance to flooding stress, both to submergence and waterlogging. Carbohydrates contents were higher in the transgenics compared to wild type and decreased less after submergence treatments. Finally, transcript levels of selected genes involved in glycolisis and fermentative pathways as well as the corresponding enzymatic activities were assessed both, in sunflower and transgenic Arabidopsis plants, before and after submergence. Altogether, the present work leads us to propose HaHB11 as a biotechnological tool to improve crops yield, biomass and flooding tolerance. PMID:26876611

  14. Peroxidation due to cryoprotectant treatment is a vital factor for cell survival in Arabidopsis cryopreservation

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Cryopreservation is a safe and cost-effective tool for the long-term storage of plant germplasm, but damage to plant tissues and death of plants sometimes occurs. Determining the causes of this damage is vital to improving plant recovery from cryopreservation. When Arabidopsis germinating seeds were...

  15. Xanthomonas campestris overcomes Arabidopsis stomatal innate immunity through a DSF cell-to-cell signal-regulated virulence factor.

    PubMed

    Gudesblat, Gustavo E; Torres, Pablo S; Vojnov, Adrián A

    2009-02-01

    Pathogen-induced stomatal closure is part of the plant innate immune response. Phytopathogens using stomata as a way of entry into the leaf must avoid the stomatal response of the host. In this article, we describe a factor secreted by the bacterial phytopathogen Xanthomonas campestris pv campestris (Xcc) capable of interfering with stomatal closure induced by bacteria or abscisic acid (ABA). We found that living Xcc, as well as ethyl acetate extracts from Xcc culture supernatants, are capable of reverting stomatal closure induced by bacteria, lipopolysaccharide, or ABA. Xcc ethyl acetate extracts also complemented the infectivity of Pseudomonas syringae pv tomato (Pst) mutants deficient in the production of the coronatine toxin, which is required to overcome stomatal defense. By contrast, the rpfF and rpfC mutant strains of Xcc, which are unable to respectively synthesize or perceive a diffusible molecule involved in bacterial cell-to-cell signaling, were incapable of reverting stomatal closure, indicating that suppression of stomatal response by Xcc requires an intact rpf/diffusible signal factor system. In addition, we found that guard cell-specific Arabidopsis (Arabidopsis thaliana) Mitogen-Activated Protein Kinase3 (MPK3) antisense mutants were unresponsive to bacteria or lipopolysaccharide in promotion of stomatal closure, and also more sensitive to Pst coronatine-deficient mutants, showing that MPK3 is required for stomatal immune response. Additionally, we found that, unlike in wild-type Arabidopsis, ABA-induced stomatal closure in MPK3 antisense mutants is not affected by Xcc or by extracts from Xcc culture supernatants, suggesting that the Xcc factor might target some signaling component in the same pathway as MPK3. PMID:19091877

  16. Down regulation of virulence factors of Pseudomonas aeruginosa by salicylic acid attenuates its virulence on Arabidopsis thaliana and Caenorhabditis elegans.

    PubMed

    Prithiviraj, B; Bais, H P; Weir, T; Suresh, B; Najarro, E H; Dayakar, B V; Schweizer, H P; Vivanco, J M

    2005-09-01

    Salicylic acid (SA) is a phenolic metabolite produced by plants and is known to play an important role in several physiological processes, such as the induction of plant defense responses against pathogen attack. Here, using the Arabidopsis thaliana-Pseudomonas aeruginosa pathosystem, we provide evidence that SA acts directly on the pathogen, down regulating fitness and virulence factor production of the bacteria. Pseudomonas aeruginosa PA14 showed reduced attachment and biofilm formation on the roots of the Arabidopsis mutants lox2 and cpr5-2, which produce elevated amounts of SA, as well as on wild-type Arabidopsis plants primed with exogenous SA, a treatment known to enhance endogenous SA concentration. Salicylic acid at a concentration that did not inhibit PA14 growth was sufficient to significantly affect the ability of the bacteria to attach and form biofilm communities on abiotic surfaces. Furthermore, SA down regulated three known virulence factors of PA14: pyocyanin, protease, and elastase. Interestingly, P. aeruginosa produced more pyocyanin when infiltrated into leaves of the Arabidopsis transgenic line NahG, which accumulates less SA than wild-type plants. This finding suggests that endogenous SA plays a role in down regulating the synthesis and secretion of pyocyanin in vivo. To further test if SA directly affects the virulence of P. aeruginosa, we used the Caenorhabditis elegans-P. aeruginosa infection model. The addition of SA to P. aeruginosa lawns significantly diminished the bacterium's ability to kill the worms, without affecting the accumulation of bacteria inside the nematodes' guts, suggesting that SA negatively affects factors that influence the virulence of P. aeruginosa. We employed microarray technology to identify SA target genes. These analyses showed that SA treatment affected expression of 331 genes. It selectively repressed transcription of exoproteins and other virulence factors, while it had no effect on expression of housekeeping

  17. Modulation of NADH Levels by Arabidopsis Nudix Hydrolases, AtNUDX6 and 7, and the Respective Proteins Themselves Play Distinct Roles in the Regulation of Various Cellular Responses Involved in Biotic/Abiotic Stresses.

    PubMed

    Ogawa, Takahisa; Muramoto, Kohei; Takada, Risa; Nakagawa, Shouya; Shigeoka, Shigeru; Yoshimura, Kazuya

    2016-06-01

    Arabidopsis Nudix hydrolases, AtNUDX6 and 7, exhibit pyrophosphohydrolase activities toward NADH and contribute to the modulation of various defense responses, such as the poly(ADP-ribosyl)ation (PAR) reaction and salicylic acid (SA)-induced Nonexpresser of Pathogenesis-Related genes 1 (NPR1)-dependent defense pathway, against biotic and abiotic stresses. However, the mechanisms by which these enzymes regulate such cellular responses remain unclear. To clarify the functional role(s) of AtNUDX6 and 7 and NADH metabolism, we examined the effects of the transient expression of the active and inactive forms of AtNUDX6 and 7 under the control of an estrogen (ES)-inducible system on various stress responses. The transient expression of active AtNUDX6 and 7 proteins suppressed NADH levels and induced PAR activity, whereas that of their inactive forms did not, indicating the involvement of NADH metabolism in the regulation of the PAR reaction. A transcriptome analysis using KO-nudx6, KO-nudx7 and double KO-nudx6/7 plants, in which intracellular NADH levels increased, identified genes (NADH-responsive genes, NRGs) whose expression levels positively and negatively correlated with NADH levels. Many NRGs did not overlap with the genes whose expression was reported to be responsive to various types of oxidants and reductants, suggesting a novel role for intracellular NADH levels as a redox signaling cue. The active and inactive AtNUDX6 proteins induced the expression of thioredoxin-h5, the activator of NPR1 and SA-induced NPR1-dependent defense genes, while the active and inactive AtNUDX7 proteins suppressed the accumulation of SA and subsequent gene expression, indicating that AtNUDX6 and 7 proteins themselves play distinct roles in stress responses. PMID:27095738

  18. Arabidopsis Small Rubber Particle Protein Homolog SRPs Play Dual Roles as Positive Factors for Tissue Growth and Development and in Drought Stress Responses.

    PubMed

    Kim, Eun Yu; Park, Ki Youl; Seo, Young Sam; Kim, Woo Taek

    2016-04-01

    Lipid droplets (LDs) act as repositories for fatty acids and sterols, which are used for various cellular processes such as energy production and membrane and hormone synthesis. LD-associated proteins play important roles in seed development and germination, but their functions in postgermination growth are not well understood. Arabidopsis (Arabidopsis thaliana) contains three SRP homologs (SRP1, SRP2, and SRP3) that share sequence identities with small rubber particle proteins of the rubber tree (Hevea brasiliensis). In this report, the possible cellular roles of SRPs in postgermination growth and the drought tolerance response were investigated. Arabidopsis SRPs appeared to be LD-associated proteins and displayed polymerization properties in vivo and in vitro. SRP-overexpressing transgenic Arabidopsis plants (35S:SRP1, 35S:SRP2, and 35S:SRP3) exhibited higher vegetative and reproductive growth and markedly better tolerance to drought stress than wild-type Arabidopsis. In addition, constitutive over-expression of SRPs resulted in increased numbers of large LDs in postgermination seedlings. In contrast, single (srp1, 35S:SRP2-RNAi, and srp3) and triple (35S:SRP2-RNAi/srp1srp3) loss-of-function mutant lines exhibited the opposite phenotypes. Our results suggest that Arabidopsis SRPs play dual roles as positive factors in postgermination growth and the drought stress tolerance response. The possible relationships between LD-associated proteins and the drought stress response are discussed. PMID:26903535

  19. Arabidopsis Small Rubber Particle Protein Homolog SRPs Play Dual Roles as Positive Factors for Tissue Growth and Development and in Drought Stress Responses1[OPEN

    PubMed Central

    Kim, Eun Yu; Park, Ki Youl; Seo, Young Sam; Kim, Woo Taek

    2016-01-01

    Lipid droplets (LDs) act as repositories for fatty acids and sterols, which are used for various cellular processes such as energy production and membrane and hormone synthesis. LD-associated proteins play important roles in seed development and germination, but their functions in postgermination growth are not well understood. Arabidopsis (Arabidopsis thaliana) contains three SRP homologs (SRP1, SRP2, and SRP3) that share sequence identities with small rubber particle proteins of the rubber tree (Hevea brasiliensis). In this report, the possible cellular roles of SRPs in postgermination growth and the drought tolerance response were investigated. Arabidopsis SRPs appeared to be LD-associated proteins and displayed polymerization properties in vivo and in vitro. SRP-overexpressing transgenic Arabidopsis plants (35S:SRP1, 35S:SRP2, and 35S:SRP3) exhibited higher vegetative and reproductive growth and markedly better tolerance to drought stress than wild-type Arabidopsis. In addition, constitutive over-expression of SRPs resulted in increased numbers of large LDs in postgermination seedlings. In contrast, single (srp1, 35S:SRP2-RNAi, and srp3) and triple (35S:SRP2-RNAi/srp1srp3) loss-of-function mutant lines exhibited the opposite phenotypes. Our results suggest that Arabidopsis SRPs play dual roles as positive factors in postgermination growth and the drought stress tolerance response. The possible relationships between LD-associated proteins and the drought stress response are discussed. PMID:26903535

  20. Leaf Variegation of Thylakoid Formation1 Is Suppressed by Mutations of Specific σ-Factors in Arabidopsis1[OPEN

    PubMed Central

    Zhu, Ying; Wu, Wenjuan

    2015-01-01

    Thylakoid Formation1 (THF1) has been shown to play roles in chloroplast development, resistance to excessive light, and chlorophyll degradation in Arabidopsis (Arabidopsis thaliana). To elucidate mechanisms underlying THF1-regulated chloroplast development, we mutagenized thf1 seeds with ethyl methanesulfonate and screened second-site recessive mutations that suppress its leaf variegation phenotype. Here, we characterized a unique suppressor line, 42-6, which displays a leaf virescent phenotype. Map-based cloning and genetic complementation results showed that thf1 variegation was suppressed by a mutation in σ-FACTOR6 (SIG6), which is a plastid transcription factor specifically controlling gene expression through the plastid-encoded RNA polymerase. Northern-blot analysis revealed that plastid gene expression was down-regulated in not only 42-6 and sig6 but also, thf1 at the early stage of chloroplast development. Interestingly, mutations in SIG2 but not in other σ-factors also suppressed thf1 leaf variegation. Furthermore, we found that leaf variegation of thf1 and var2 could be suppressed by several virescent mutations, including yellow seedling1, brz-insensitive-pale green2, and nitric oxide-associated protein1, indicating that virescent mutations suppress leaf variegation. Taken together, our results provide unique insights into thf1-mediated leaf variegation, which might be triggered by defects in plastid gene transcription. PMID:25999408

  1. Ectopic expression of a grapevine transcription factor VvWRKY11 contributes to osmotic stress tolerance in Arabidopsis.

    PubMed

    Liu, Huaying; Yang, Wenlong; Liu, Dongcheng; Han, Yuepeng; Zhang, Aimin; Li, Shaohua

    2011-01-01

    Plant WRKY transcriptional factors play an important role in response to biotic and abiotic stresses. In this study, a WRKY transcription factor was isolated from grapevine. This transcription factor showed 66% and 58% identity at the DNA and amino acid sequence levels, respectively, with Arabidopsis AtWRKY11 genes, and was therefore designated VvWRKY11. Phylogenetic analysis and structure comparison indicated that VvWRKY11 protein belongs to group IIc. The VvWRKY11 protein was shown to be located in the nucleus based on green fluorescent protein analysis. Yeast one-hybrid analysis further indicated that VvWRKY11 protein binds specifically to the W-box element. The expression profile of VvWRKY11 in response to treatment with phytohormone salicylic acid or pathogen Plasmopara viticola is rapid and transient. Transgenic Arabidopsis seedlings overexpressing VvWRKY11 showed higher tolerance to water stress induced by mannitol than wild-type plants. These results clearly demonstrated that the VvWRKY11 gene is involved in the response to dehydration stress. In addition, the role of VvWRKY11 protein in regulating the expression of two stress response genes, AtRD29A and AtRD29B, is also discussed. PMID:20354906

  2. Zinc-Finger Transcription Factor ZAT6 Positively Regulates Cadmium Tolerance through the Glutathione-Dependent Pathway in Arabidopsis.

    PubMed

    Chen, Jian; Yang, Libo; Yan, Xingxing; Liu, Yunlei; Wang, Ren; Fan, Tingting; Ren, Yongbing; Tang, Xiaofeng; Xiao, Fangming; Liu, Yongsheng; Cao, Shuqing

    2016-05-01

    Cadmium (Cd) is an environmental pollutant with high toxicity to animals and plants. It has been established that the glutathione (GSH)-dependent phytochelatin (PC) synthesis pathway is one of the most important mechanisms contributing to Cd accumulation and tolerance in plants. However, the transcription factors involved in regulating GSH-dependent PC synthesis pathway remain largely unknown. Here, we identified an Arabidopsis (Arabidopsis thaliana) Cd-resistant mutant xcd2-D (XVE system-induced cadmium-tolerance2) using a forward genetics approach. The mutant gene underlying xcd2-D mutation was revealed to encode a known zinc-finger transcription factor, ZAT6. Transgenic plants overexpressing ZAT6 showed significant increase of Cd tolerance, whereas loss of function of ZAT6 led to decreased Cd tolerance. Increased Cd accumulation and tolerance in ZAT6-overexpressing lines was GSH dependent and associated with Cd-activated synthesis of PC, which was correlated with coordinated activation of PC-synthesis related gene expression. By contrast, loss of function of ZAT6 reduced Cd accumulation and tolerance, which was accompanied by abolished PC synthesis and gene expression. Further analysis revealed that ZAT6 positively regulates the transcription of GSH1, GSH2, PCS1, and PCS2, but ZAT6 is capable of specifically binding to GSH1 promoter in vivo. Consistently, overexpression of GSH1 has been shown to restore Cd sensitivity in the zat6-1 mutant, suggesting that GSH1 is a key target of ZAT6. Taken together, our data provide evidence that ZAT6 coordinately activates PC synthesis-related gene expression and directly targets GSH1 to positively regulate Cd accumulation and tolerance in Arabidopsis. PMID:26983992

  3. LLM-Domain Containing B-GATA Factors Control Different Aspects of Cytokinin-Regulated Development in Arabidopsis thaliana.

    PubMed

    Ranftl, Quirin L; Bastakis, Emmanouil; Klermund, Carina; Schwechheimer, Claus

    2016-04-01

    Leu-Leu-Met (LLM)-domain B-GATAs are a subfamily of the 30-membered GATA transcription factor family from Arabidopsis. Only two of the six Arabidopsis LLM-domain B-GATAs, i.e. GATA, NITRATE-INDUCIBLE, CARBON METABOLISM-INVOLVED (GNC) and its paralog GNC-LIKE/CYTOKININ-RESPONSIVE GATA FACTOR1 (GNL), have already been analyzed with regard to their biological function. Together, GNC and GNL control germination, greening, flowering time, and senescence downstream from auxin, cytokinin (CK), gibberellin (GA), and light signaling. Whereas overexpression and complementation analyses suggest a redundant biochemical function between GNC and GNL, nothing is known about the biological role of the four other LLM-domain B-GATAs, GATA15, GATA16, GATA17, and GATA17L (GATA17-LIKE), based on loss-of-function mutant phenotypes. Here, we examine insertion mutants of the six Arabidopsis B-GATA genes and reveal the role of these genes in the control of greening, hypocotyl elongation, phyllotaxy, floral organ initiation, accessory meristem formation, flowering time, and senescence. Several of these phenotypes had previously not been described for the gnc and gnl mutants or were enhanced in the more complex mutants when compared to gnc gnl mutants. Some of the respective responses may be mediated by CK signaling, which activates the expression of all six GATA genes. CK-induced gene expression is partially compromised in LLM-domain B-GATA mutants, suggesting that B-GATA genes play a role in CK responses. We furthermore provide evidence for a transcriptional cross regulation between these GATAs that may, in at least some cases, be at the basis of their apparent functional redundancy. PMID:26829982

  4. The ETHYLENE RESPONSE FACTORs ERF6 and ERF11 Antagonistically Regulate Mannitol-Induced Growth Inhibition in Arabidopsis1[OPEN

    PubMed Central

    Dubois, Marieke; Van den Broeck, Lisa; Claeys, Hannes; Van Vlierberghe, Kaatje; Matsui, Minami; Inzé, Dirk

    2015-01-01

    Leaf growth is a tightly regulated and complex process, which responds in a dynamic manner to changing environmental conditions, but the mechanisms that reduce growth under adverse conditions are rather poorly understood. We previously identified a growth inhibitory pathway regulating leaf growth upon exposure to a low concentration of mannitol and characterized the ETHYLENE RESPONSE FACTOR (ERF)/APETALA2 transcription factor ERF6 as a central activator of both leaf growth inhibition and induction of stress tolerance genes. Here, we describe the role of the transcriptional repressor ERF11 in relation to the ERF6-mediated stress response in Arabidopsis (Arabidopsis thaliana). Using inducible overexpression lines, we show that ERF6 induces the expression of ERF11. ERF11 in turn molecularly counteracts the action of ERF6 and represses at least some of the ERF6-induced genes by directly competing for the target gene promoters. As a phenotypical consequence of the ERF6-ERF11 antagonism, the extreme dwarfism caused by ERF6 overexpression is suppressed by overexpression of ERF11. Together, our data demonstrate that dynamic mechanisms exist to fine-tune the stress response and that ERF11 counteracts ERF6 to maintain a balance between plant growth and stress defense. PMID:25995327

  5. A Member of the Arabidopsis Mitochondrial Transcription Termination Factor Family Is Required for Maturation of Chloroplast Transfer RNAIle(GAU).

    PubMed

    Romani, Isidora; Manavski, Nikolay; Morosetti, Arianna; Tadini, Luca; Maier, Swetlana; Kühn, Kristina; Ruwe, Hannes; Schmitz-Linneweber, Christian; Wanner, Gerhard; Leister, Dario; Kleine, Tatjana

    2015-09-01

    Plastid gene expression is crucial for organelle function, but the factors that control it are still largely unclear. Members of the so-called mitochondrial transcription termination factor (mTERF) family are found in metazoans and plants and regulate organellar gene expression at different levels. Arabidopsis (Arabidopsis thaliana) mTERF6 is localized in chloroplasts and mitochondria, and its knockout perturbs plastid development and results in seedling lethality. In the leaky mterf6-1 mutant, a defect in photosynthesis is associated with reduced levels of photosystem subunits, although corresponding messenger RNA levels are unaffected, whereas translational capacity and maturation of chloroplast ribosomal RNAs (rRNAs) are perturbed in mterf6-1 mutants. Bacterial one-hybrid screening, electrophoretic mobility shift assays, and coimmunoprecipitation experiments reveal a specific interaction between mTERF6 and an RNA sequence in the chloroplast isoleucine transfer RNA gene (trnI.2) located in the rRNA operon. In vitro, recombinant mTERF6 bound to its plastid DNA target site can terminate transcription. At present, it is unclear whether disturbed rRNA maturation is a primary or secondary defect. However, it is clear that mTERF6 is required for the maturation of trnI.2. This points to an additional function of mTERFs. PMID:26152711

  6. The Arabidopsis Transcription Factor LUH/MUM1 Is Required for Extrusion of Seed Coat Mucilage1[W][OA

    PubMed Central

    Huang, Jun; DeBowles, Danisha; Esfandiari, Elahe; Dean, Gillian; Carpita, Nicholas C.; Haughn, George W.

    2011-01-01

    During differentiation, the Arabidopsis (Arabidopsis thaliana) seed coat epidermal cells secrete mucilage composed primarily of rhamnogalacturonan I that is extruded from the seed coat upon imbibition. The mucilage of the mucilage modified1 (mum1) mutant contains rhamnogalacturonan I that is more highly branched and lacks the ability to be extruded when exposed to water. Our cloning of the MUM1 gene shows that it encodes a putative transcription factor, LEUNIG_HOMOLOG (LUH). Cellular localization and transcriptional assay results suggest that LUH/MUM1 is a nucleus-localized transcriptional activator. LUH/MUM1 is expressed in all the tissues examined, including the seed coat. Quantitative reverse transcription-polymerase chain reaction data suggest that LUH/MUM1 is expressed throughout seed coat development, reaching peak expression late in differentiation. LUH1/MUM1 expression in plants homozygous for mutations in several genes encoding regulators of seed coat mucilage was unchanged. Thus, LUH/MUM1 expression appears to be independent of other transcription factors known to regulate aspects of seed coat mucilage biology. The expression in the luh/mum1 mutant of three genes encoding enzymes needed for mucilage extrusion, MUM2, SUBSILIN PROTEASE1.7, and β-XYLOSIDASE1, was reduced relative to that of the wild type. Overexpression of MUM2 could partially rescue the mum1 phenotype. These data suggest that LUH/MUM1 is a positive regulator of all three genes. PMID:21518777

  7. The Trihelix Transcription Factor GTL1 Regulates Ploidy-Dependent Cell Growth in the Arabidopsis Trichome[W][OA

    PubMed Central

    Breuer, Christian; Kawamura, Ayako; Ichikawa, Takanari; Tominaga-Wada, Rumi; Wada, Takuji; Kondou, Youichi; Muto, Shu; Matsui, Minami; Sugimoto, Keiko

    2009-01-01

    Leaf trichomes in Arabidopsis thaliana develop through several distinct cellular processes, such as patterning, differentiation, and growth. Although recent studies have identified several key transcription factors as regulating early patterning and differentiation steps, it is still largely unknown how these regulatory proteins mediate subsequent trichome development, which is accompanied by rapid cell growth and branching. Here, we report a novel trichome mutation in Arabidopsis, which in contrast with previously identified mutants, increases trichome cell size without altering its overall patterning or branching. We show that the corresponding gene encodes a GT-2-LIKE1 (GTL1) protein, a member of the trihelix transcription factor family. GTL1 is present within the nucleus during the postbranching stages of trichome development, and its loss of function leads to an increase in the nuclear DNA content only in trichomes that have completed branching. Our data further demonstrate that the gtl1 mutation modifies the expression of several cell cycle genes and partially rescues the ploidy defects in the cyclin-dependent kinase inhibitor mutant siamese. Taken together, this study provides the genetic evidence for the requirement of transcriptional regulation in the repression of ploidy-dependent plant cell growth as well as for an involvement of GTL trihelix proteins in this regulation. PMID:19717615

  8. CYTOKININ RESPONSE FACTOR2 (CRF2) and CRF3 Regulate Lateral Root Development in Response to Cold Stress in Arabidopsis.

    PubMed

    Jeon, Jin; Cho, Chuloh; Lee, Mi Rha; Van Binh, Nguyen; Kim, Jungmook

    2016-08-01

    Lateral roots (LRs) are a major determinant of the root system architecture in plants, and developmental plasticity of LR formation is critical for the survival of plants in changing environmental conditions. In Arabidopsis thaliana, genetic pathways have been identified that regulate LR branching in response to numerous environmental cues, including some nutrients, salt, and gravity. However, it is not known how genetic components are involved in the LR adaptation response to cold. Here, we demonstrate that CYTOKININ RESPONSE FACTOR2 (CRF2) and CRF3, encoding APETALA2 transcription factors, play an important role in regulating Arabidopsis LR initiation under cold stress. Analysis of LR developmental kinetics demonstrated that both CRF2 and CRF3 regulate LR initiation. crf2 and crf3 single mutants exhibited decreased LR initiation under cold stress compared with the wild type, and the crf2 crf3 double mutants showed additively decreased LR densities compared with the single mutants. Conversely, CRF2 or CRF3 overexpression caused increased LR densities. CRF2 was induced by cold via a subset of the cytokinin two-component signaling (TCS) pathway, whereas CRF3 was upregulated by cold via TCS-independent pathways. Our results suggest that CRF2 and CRF3 respond to cold via TCS-dependent and TCS-independent pathways and control LR initiation and development, contributing to LR adaptation to cold stress. PMID:27432872

  9. The TCP4 transcription factor of Arabidopsis blocks cell division in yeast at G1 {yields} S transition

    SciTech Connect

    Aggarwal, Pooja; Padmanabhan, Bhavna; Bhat, Abhay; Sarvepalli, Kavitha; Sadhale, Parag P.; Nath, Utpal

    2011-07-01

    Highlights: {yields} TCP4 is a class II TCP transcription factor, that represses cell division in Arabidopsis. {yields} TCP4 expression in yeast retards cell division by blocking G1 {yields} S transition. {yields} Genome-wide expression studies and Western analysis reveals stabilization of cell cycle inhibitor Sic1, as possible mechanism. -- Abstract: The TCP transcription factors control important aspects of plant development. Members of class I TCP proteins promote cell cycle by regulating genes directly involved in cell proliferation. In contrast, members of class II TCP proteins repress cell division. While it has been postulated that class II proteins induce differentiation signal, their exact role on cell cycle has not been studied. Here, we report that TCP4, a class II TCP protein from Arabidopsis that repress cell proliferation in developing leaves, inhibits cell division by blocking G1 {yields} S transition in budding yeast. Cells expressing TCP4 protein with increased transcriptional activity fail to progress beyond G1 phase. By analyzing global transcriptional status of these cells, we show that expression of a number of cell cycle genes is altered. The possible mechanism of G1 {yields} S arrest is discussed.

  10. Brefeldin A-inhibited guanine exchange factor 2 regulates filamin A phosphorylation and neuronal migration.

    PubMed

    Zhang, Jingping; Neal, Jason; Lian, Gewei; Shi, Bingxing; Ferland, Russell J; Sheen, Volney

    2012-09-01

    Periventricular heterotopia (PH) is a human malformation of cortical development associated with gene mutations in ADP-ribosylation factor guanine exchange factor 2 (ARFGEF2 encodes for Big2 protein) and Filamin A (FLNA). PH is thought to derive from neuroependymal disruption, but the extent to which neuronal migration contributes to this phenotype is unknown. Here, we show that Arfgef2 null mice develop PH and exhibit impaired neural migration with increased protein expression for both FlnA and phosphoFlnA at Ser2152. Big2 physically interacts with FlnA and overexpression of phosphomimetic Ser2512 FLNA impairs neuronal migration. FlnA phosphorylation directs FlnA localization toward the cell cytoplasm, diminishes its binding affinity to actin skeleton, and alters the number and size of paxillin focal adhesions. Collectively, our results demonstrate a molecular mechanism whereby Big2 inhibition promotes phosphoFlnA (Ser2152) expression, and increased phosphoFlnA impairs its actin binding affinity and the distribution of focal adhesions, thereby disrupting cell intrinsic neuronal migration. PMID:22956851

  11. Ultrastructural Localization of Endogenous Exchange Factor for ARF6 in Adrenocortical Cells In Situ of Mice

    PubMed Central

    Chomphoo, Surang; Mothong, Wilaiwan; Sawatpanich, Tarinee; Kanla, Pipatphong; Sakagami, Hiroyuki; Kondo, Hisatake; Hipkaeo, Wiphawi

    2016-01-01

    EFA6 (exchange factor for ARF6) activates Arf6 (ADP ribosylation factor 6) by exchanging ADP to ATP, and the resulting activated form of Arf6 is involved in the membrane dynamics and actin re-organization of cells. The present study was attempted to localize EFA6 type D (EFA6D) in mouse adrenocortical cells in situ whose steroid hormone secretion is generally considered not to depend on the vesicle-involved regulatory mechanism. In immunoblotting, an immunoreactive band with the same size as brain EFA6D was detected in homogenates of adrenal cortical tissues almost free of adrenal capsules and medulla. In immuno-light microscopy, EFA6D-immunoreactivity was positive in adrenocortical cells and it was often distinct along the plasmalemma, especially along portions of the cell columns facing the interstitium. In immuno-electron microscopy, the gold-labeling was more dense in the peripheral intracellular domains than the central domain of the immunopositive cells. The labeling was deposited on the plasma membranes in a discontinuous pattern and in cytoplasmic domains rich in filaments. It was also associated with some, but not all, of pleiomorphic vesicles and coated pits/vesicles. No labeling was seen in association with lipid droplets or smooth endoplasmic reticulum. The present finding is in support of the importance of EFA6D for activation of Arf6 in adrenocortical cells. PMID:27462133

  12. Ultrastructural Localization of Endogenous Exchange Factor for ARF6 in Adrenocortical Cells In Situ of Mice.

    PubMed

    Chomphoo, Surang; Mothong, Wilaiwan; Sawatpanich, Tarinee; Kanla, Pipatphong; Sakagami, Hiroyuki; Kondo, Hisatake; Hipkaeo, Wiphawi

    2016-06-28

    EFA6 (exchange factor for ARF6) activates Arf6 (ADP ribosylation factor 6) by exchanging ADP to ATP, and the resulting activated form of Arf6 is involved in the membrane dynamics and actin re-organization of cells. The present study was attempted to localize EFA6 type D (EFA6D) in mouse adrenocortical cells in situ whose steroid hormone secretion is generally considered not to depend on the vesicle-involved regulatory mechanism. In immunoblotting, an immunoreactive band with the same size as brain EFA6D was detected in homogenates of adrenal cortical tissues almost free of adrenal capsules and medulla. In immuno-light microscopy, EFA6D-immunoreactivity was positive in adrenocortical cells and it was often distinct along the plasmalemma, especially along portions of the cell columns facing the interstitium. In immuno-electron microscopy, the gold-labeling was more dense in the peripheral intracellular domains than the central domain of the immunopositive cells. The labeling was deposited on the plasma membranes in a discontinuous pattern and in cytoplasmic domains rich in filaments. It was also associated with some, but not all, of pleiomorphic vesicles and coated pits/vesicles. No labeling was seen in association with lipid droplets or smooth endoplasmic reticulum. The present finding is in support of the importance of EFA6D for activation of Arf6 in adrenocortical cells. PMID:27462133

  13. Overexpression of Heat Shock Factor Gene HsfA3 Increases Galactinol Levels and Oxidative Stress Tolerance in Arabidopsis.

    PubMed

    Song, Chieun; Chung, Woo Sik; Lim, Chae Oh

    2016-06-30

    Heat shock factors (Hsfs) are central regulators of abiotic stress responses, especially heat stress responses, in plants. In the current study, we characterized the activity of the Hsf gene HsfA3 in Arabidopsis under oxidative stress conditions. HsfA3 transcription in seedlings was induced by reactive oxygen species (ROS), exogenous hydrogen peroxide (H2O2), and an endogenous H2O2 propagator, 2,5-dibromo-3-methyl-6-isopropyl-p-benzoquinone (DBMIB). HsfA3-overexpressing transgenic plants exhibited increased oxidative stress tolerance compared to untransformed wild-type plants (WT), as revealed by changes in fresh weight, chlorophyll fluorescence, and ion leakage under light conditions. The expression of several genes encoding galactinol synthase (GolS), a key enzyme in the biosynthesis of raffinose family oligosaccharides (RFOs), which function as antioxidants in plant cells, was induced in HsfA3 overexpressors. In addition, galactinol levels were higher in HsfA3 overexpressors than in WT under unstressed conditions. In transient transactivation assays using Arabidopsis leaf protoplasts, HsfA3 activated the transcription of a reporter gene driven by the GolS1 or GolS2 promoter. Electrophoretic mobility shift assays showed that GolS1 and GolS2 are directly regulated by HsfA3. Taken together, these findings provide evidence that GolS1 and GolS2 are directly regulated by HsfA3 and that GolS enzymes play an important role in improving oxidative stress tolerance by increasing galactinol biosynthesis in Arabidopsis. PMID:27109422

  14. Overexpression of Heat Shock Factor Gene HsfA3 Increases Galactinol Levels and Oxidative Stress Tolerance in Arabidopsis

    PubMed Central

    Song, Chieun; Chung, Woo Sik; Lim, Chae Oh

    2016-01-01

    Heat shock factors (Hsfs) are central regulators of abiotic stress responses, especially heat stress responses, in plants. In the current study, we characterized the activity of the Hsf gene HsfA3 in Arabidopsis under oxidative stress conditions. HsfA3 transcription in seedlings was induced by reactive oxygen species (ROS), exogenous hydrogen peroxide (H2O2), and an endogenous H2O2 propagator, 2,5-dibromo-3-methyl-6-isopropyl-p-benzoquinone (DBMIB). HsfA3-overexpressing transgenic plants exhibited increased oxidative stress tolerance compared to untransformed wild-type plants (WT), as revealed by changes in fresh weight, chlorophyll fluorescence, and ion leakage under light conditions. The expression of several genes encoding galactinol synthase (GolS), a key enzyme in the biosynthesis of raffinose family oligosaccharides (RFOs), which function as antioxidants in plant cells, was induced in HsfA3 overexpressors. In addition, galactinol levels were higher in HsfA3 overexpressors than in WT under unstressed conditions. In transient transactivation assays using Arabidopsis leaf protoplasts, HsfA3 activated the transcription of a reporter gene driven by the GolS1 or GolS2 promoter. Electrophoretic mobility shift assays showed that GolS1 and GolS2 are directly regulated by HsfA3. Taken together, these findings provide evidence that GolS1 and GolS2 are directly regulated by HsfA3 and that GolS enzymes play an important role in improving oxidative stress tolerance by increasing galactinol biosynthesis in Arabidopsis. PMID:27109422

  15. The Arabidopsis SR45 Splicing Factor, a Negative Regulator of Sugar Signaling, Modulates SNF1-Related Protein Kinase 1 Stability.

    PubMed

    Carvalho, Raquel F; Szakonyi, Dóra; Simpson, Craig G; Barbosa, Inês C R; Brown, John W S; Baena-González, Elena; Duque, Paula

    2016-08-01

    The ability to sense and respond to sugar signals allows plants to cope with environmental and metabolic changes by adjusting growth and development accordingly. We previously reported that the SR45 splicing factor negatively regulates glucose signaling during early seedling development in Arabidopsis thaliana Here, we show that under glucose-fed conditions, the Arabidopsis sr45-1 loss-of-function mutant contains higher amounts of the energy-sensing SNF1-Related Protein Kinase 1 (SnRK1) despite unaffected SnRK1 transcript levels. In agreement, marker genes for SnRK1 activity are upregulated in sr45-1 plants, and the glucose hypersensitivity of sr45-1 is attenuated by disruption of the SnRK1 gene. Using a high-resolution RT-PCR panel, we found that the sr45-1 mutation broadly targets alternative splicing in vivo, including that of the SR45 pre-mRNA itself. Importantly, the enhanced SnRK1 levels in sr45-1 are suppressed by a proteasome inhibitor, indicating that SR45 promotes targeting of the SnRK1 protein for proteasomal destruction. Finally, we demonstrate that SR45 regulates alternative splicing of the Arabidopsis 5PTase13 gene, which encodes an inositol polyphosphate 5-phosphatase previously shown to interact with and regulate the stability of SnRK1 in vitro, thus providing a mechanistic link between SR45 function and the modulation of degradation of the SnRK1 energy sensor in response to sugars. PMID:27436712

  16. AtSIG6, a plastid sigma factor from Arabidopsis, reveals functional impact of cpCK2 phosphorylation

    PubMed Central

    Schweer, Jennifer; Türkeri, Hacer; Link, Brigitte; Link, Gerhard

    2010-01-01

    Plastids contain sigma factors, i.e. gene-regulatory proteins for promoter binding and transcription initiation. Despite the physical and functional similarity shared with their prokaryotic counterparts, the plant sigma factors have distinguishing features: most notably the existence of a variable extra sequence comprising their N-terminal portions. This distinct architecture is reflected by functional differences, including phosphorylation control by organellar protein kinase(s) closely related to nucleocytosolic, rather than bacterial-type, enzymes. In particular, cpCK2, a nuclear-coded plastid-targeted casein kinase 2, has been implicated as a key component in plant sigma factor phosphorylation and transcriptional regulation (Eur. J. Biochem. 269, 2002, 3329; Planta, 219, 2004, 298). Although this notion is based mainly on biochemical evidence and in vitro systems, the recent availability of Arabidopsis sigma knock-out lines for complementation by intact and mutant sigma cDNAs has opened up new strategies for the study of transcription regulatory mechanisms in vivo. Using Arabidopsis sigma factor 6 (AtSIG6) as a paradigm, we present data suggesting that: (i) this factor is a substrate for regulatory phosphorylation by cpCK2 both in vitro and in vivo; (ii) cpCK2 phosphorylation of SIG6 occurs at multiple sites, which can widely differ in their effect on the visual and/or molecular phenotype; (iii) in vivo usage of the perhaps most critical cpCK2 site defined by Ser174 requires (pre-)phosphorylation at the n + 3 serine residue Ser177, pointing to ‘pathfinder’ kinase activity capable of generating a functional cpCK2 substrate site. PMID:20088902

  17. Generation of serrated and wavy petals by inhibition of the activity of TCP transcription factors in Arabidopsis thaliana

    PubMed Central

    Ohme-Takagi, Masaru; Sato, Fumihiko

    2011-01-01

    The final shape of shoot lateral organs, namely, leaves and flowers, is determined by coordinated growth after the initiation of primordia from shoot meristems in seed plants. This coordination is achieved by the complex action of many transcription factors, which include the TEOSINTE BRANCHED1, CYCLOIDEA and PCF (TCP) family. We have recently reported that CINCINNATA-like (CIN-like) TCP genes act dose-dependently to regulate the flat and smooth morphology of leaves in Arabidopsis thaliana. In contrast, the roles of CIN-like TCP genes in flower development are poorly understood. In this report, using multiple tcp mutants and transgenic plants in which the activity of CIN-like TCP transcription factors is dominantly inhibited, we found that these TCPs regulate the smooth and flat morphology of petals. Based on these findings, we discuss a possible strategy to generate a fringed morphology in floricultural plants. PMID:21455021

  18. Identification of an Arabidopsis transmembrane bZIP transcription factor involved in the endoplasmic reticulum stress response

    SciTech Connect

    Tajima, Hiromi; Iwata, Yuji; Iwano, Megumi; Takayama, Seiji; Koizumi, Nozomu

    2008-09-19

    Among 75 bZIP transcription factors identified in Arabidopsis, 3 (AtbZIP17, AtbZIP28, and AtbZIP49) possess a putative transmembrane domain (TMD) in addition to AtbZIP60, which was characterized previously. In the present study, cDNAs of AtbZIP17 and AtbZIP28 were isolated. Truncated forms of AtbZIP17 and AtbZIP28 lacking the C-terminal domain including TMD were examined as putative active forms. One of them, AtbZIP28{delta}C, activated BiP1 and BiP3 promoters through the cis-elements P-UPRE and ERSE responsible for the ER stress response. Subsequently, a fusion protein of green fluorescent protein (GFP) and AtbZIP28 was expressed in Arabidopsis cultured cells. Under non-stress conditions, GFP fluorescence localization almost overlapped with an ER marker; however, tunicamycin and dithiothreitol treatment clearly increased GFP fluorescence in the nucleus suggesting that the N-terminal fragment of AtbZIP28 translocates to the nucleus in response to ER stress.

  19. Arabidopsis FIMBRIN5, an Actin Bundling Factor, Is Required for Pollen Germination and Pollen Tube Growth[W

    PubMed Central

    Wu, Youjun; Yan, Jin; Zhang, Ruihui; Qu, Xiaolu; Ren, Sulin; Chen, Naizhi; Huang, Shanjin

    2010-01-01

    Actin cables in pollen tubes serve as molecular tracks for cytoplasmic streaming and organelle movement and are formed by actin bundling factors like villins and fimbrins. However, the precise mechanisms by which actin cables are generated and maintained remain largely unknown. Fimbrins comprise a family of five members in Arabidopsis thaliana. Here, we characterized a fimbrin isoform, Arabidopsis FIMBRIN5 (FIM5). Our results show that FIM5 is required for the organization of actin cytoskeleton in pollen grains and pollen tubes, and FIM5 loss-of-function associates with a delay of pollen germination and inhibition of pollen tube growth. FIM5 decorates actin filaments throughout pollen grains and tubes. Actin filaments become redistributed in fim5 pollen grains and disorganized in fim5 pollen tubes. Specifically, actin cables protrude into the extreme tips, and their longitudinal arrangement is disrupted in the shank of fim5 pollen tubes. Consequently, the pattern and velocity of cytoplasmic streaming were altered in fim5 pollen tubes. Additionally, loss of FIM5 function rendered pollen germination and tube growth hypersensitive to the actin-depolymerizing drug latrunculin B. In vitro biochemical analyses indicated that FIM5 exhibits actin bundling activity and stabilizes actin filaments. Thus, we propose that FIM5 regulates actin dynamics and organization during pollen germination and tube growth via stabilizing actin filaments and organizing them into higher-order structures. PMID:21098731

  20. [Nucleotide polymorphism in the drought induced transcription factor CBF4 region of Arabidopsis thaliana and its molecular evolution analyses].

    PubMed

    Hao, Gang-Ping; Wu, Zhong-Yi; Cao, Ming-Qing; Pelletier, Georges; Brunel, Dominique; Huang, Cong-Lin; Yang, Qing

    2004-12-01

    Intraspecific nucleotide polymorphism in the drought induced transcription factor CBF4 region of Arabidopsis thaliana was analyzed with 17 core accessions growing in different ecoclimate. High density of single nucleotide polymorphism (SNP) and insertion/deletion (Indel) were found, on average 1 SNP per 35.8 bp and 1 Indel per 143 bp. Nucleotide polymorphism in non-coding region was three times higher than that in coding region. In coding region of CBF4, SNP frequency is one SNP per 96.4 bp, one nonsynonymous mutation was detected from 25 av, 203 av and 244 av accessions, which is the 205th site amino acid variation: gly <--> val caused by the 1034th site (corresponding to 19,696 site nucleotide of GenBank No. AB015478 as 1) nucleotide variation: G <--> T. Statistical result of nucleotide diversity showed that linkage disequilibrium (LD) existed in large-scale region of CBF4 and recombination event was also detected in 5' non-coding region. Identical to the results of other genes of Arabidopsis, different regions of the gene were seemingly under different selective pressures. Balancing selection resulted in high nucleotide diversity in 3' non-coding region, and the neutral mutation hypothesis can explain the DNA polymorphism in coding region, whereas, nature positive selection in the population affected nucleotide variation in 5' non-coding region of gene. PMID:15633649

  1. Co-localisation studies of Arabidopsis SR splicing factors reveal different types of speckles in plant cell nuclei

    SciTech Connect

    Lorkovic, Zdravko J.; Barta, Andrea

    2008-10-15

    SR proteins are multidomain splicing factors which are important for spliceosome assembly and for regulation of alternative splicing. In mammalian nuclei these proteins localise to speckles from where they are recruited to transcription sites. By using fluorescent protein fusion technology and different experimental approaches it has been shown that Arabidopsis SR proteins, in addition to diffuse nucleoplasmic staining, localise into an irregular nucleoplasmic network resembling speckles in mammalian cells. As Arabidopsis SR proteins fall into seven conserved sub-families we investigated co-localisation of members of the different sub-families in transiently transformed tobacco protoplast. Here we demonstrate the new finding that members of different SR protein sub-families localise into distinct populations of nuclear speckles with no, partial or complete co-localisation. This is particularly interesting as we also show that these proteins do interact in a yeast two-hybrid assay as well as in pull-down and in co-immunopreciptiation assays. Our data raise the interesting possibility that SR proteins are partitioned into distinct populations of nuclear speckles to allow a more specific recruitment to the transcription/pre-mRNA processing sites of particular genes depending on cell type and developmental stage.

  2. The Arabidopsis Transcription Factor MYB112 Promotes Anthocyanin Formation during Salinity and under High Light Stress1[OPEN

    PubMed Central

    Lotkowska, Magda E.; Tohge, Takayuki; Fernie, Alisdair R.; Xue, Gang-Ping; Balazadeh, Salma; Mueller-Roeber, Bernd

    2015-01-01

    MYB transcription factors (TFs) are important regulators of flavonoid biosynthesis in plants. Here, we report MYB112 as a formerly unknown regulator of anthocyanin accumulation in Arabidopsis (Arabidopsis thaliana). Expression profiling after chemically induced overexpression of MYB112 identified 28 up- and 28 down-regulated genes 5 h after inducer treatment, including MYB7 and MYB32, which are both induced. In addition, upon extended induction, MYB112 also positively affects the expression of PRODUCTION OF ANTHOCYANIN PIGMENT1, a key TF of anthocyanin biosynthesis, but acts negatively toward MYB12 and MYB111, which both control flavonol biosynthesis. MYB112 binds to an 8-bp DNA fragment containing the core sequence (A/T/G)(A/C)CC(A/T)(A/G/T)(A/C)(T/C). By electrophoretic mobility shift assay and chromatin immunoprecipitation coupled to quantitative polymerase chain reaction, we show that MYB112 binds in vitro and in vivo to MYB7 and MYB32 promoters, revealing them as direct downstream target genes. We further show that MYB112 expression is up-regulated by salinity and high light stress, environmental parameters that both require the MYB112 TF for anthocyanin accumulation under these stresses. In contrast to several other MYB TFs affecting anthocyanin biosynthesis, MYB112 expression is not controlled by nitrogen limitation or an excess of carbon. Thus, MYB112 constitutes a regulator that promotes anthocyanin accumulation under abiotic stress conditions. PMID:26378103

  3. The Small Ethylene Response Factor ERF96 is Involved in the Regulation of the Abscisic Acid Response in Arabidopsis

    DOE PAGESBeta

    Wang, Xiaoping; Liu, Shanda; Tian, Hainan; Wang, Shucai; Chen, Jin-Gui

    2015-11-26

    We report that ethylene regulates many aspects of plant growth and development including seed germination, leaf senescence, and fruit ripening, and of plant responses to environmental stimuli including both biotic and abiotic stresses. Ethylene response factors (ERFs) are plant-specific transcription factors and are a subfamily of the AP2 (APETALA2)/ERF transcription factor family. The function of many members in this large gene family remains largely unknown. ERF96, a member of the Group IX ERF family transcription factors, has recently been shown to be a transcriptional activator that is involved in plant defense response in Arabidopsis. Here we provide evidence that ERF96more » is a positive regulator of abscisic acid (ABA) responses. Bioinformatics analysis indicated that there are a total four small ERFs in Arabidopsis including ERF95, ERF96, ERF97, and ERF98, and that ERF96 forms a cluster with ERF95 and ERF97. By using quantitative RT-PCR, we found that ERF96 is expressed in all tissues and organs examined except roots, with relatively high expression in flowers and seeds. Results from the protoplast transfection assay indicated that the EDLL motif-containing C-terminal domain is responsible for ERF96’s transcriptional activity. Although loss-of-function mutant of ERF96 was morphologically similar to wild type plants, transgenic plants overexpressing ERF96 had smaller rosette size and were delayed in flowering time. In ABA sensitivity assays, we found that ERF96 overexpression plants were hypersensitive to ABA in terms of ABA inhibition of seed germination, early seedling development and root elongation. Consistent with these observations, elevated transcript levels of some ABA-responsive genes including RD29A, ABI5, ABF3, ABF4, P5CS, and COR15A were observed in the transgenic plants in the presence of ABA. However, in the absence of ABA treatment, the transcript levels of these ABA-responsive genes remained largely unchanged. Our experiments also showed

  4. The Small Ethylene Response Factor ERF96 is Involved in the Regulation of the Abscisic Acid Response in Arabidopsis

    PubMed Central

    Wang, Xiaoping; Liu, Shanda; Tian, Hainan; Wang, Shucai; Chen, Jin-Gui

    2015-01-01

    Ethylene regulates many aspects of plant growth and development including seed germination, leaf senescence, and fruit ripening, and of plant responses to environmental stimuli including both biotic and abiotic stresses. Ethylene response factors (ERFs) are plant-specific transcription factors and are a subfamily of the AP2 (APETALA2)/ERF transcription factor family. The function of many members in this large gene family remains largely unknown. ERF96, a member of the Group IX ERF family transcription factors, has recently been shown to be a transcriptional activator that is involved in plant defense response in Arabidopsis. Here we provide evidence that ERF96 is a positive regulator of abscisic acid (ABA) responses. Bioinformatics analysis indicated that there are a total four small ERFs in Arabidopsis including ERF95, ERF96, ERF97, and ERF98, and that ERF96 forms a cluster with ERF95 and ERF97. By using quantitative RT-PCR, we found that ERF96 is expressed in all tissues and organs examined except roots, with relatively high expression in flowers and seeds. Results from the protoplast transfection assay indicated that the EDLL motif-containing C-terminal domain is responsible for ERF96’s transcriptional activity. Although loss-of-function mutant of ERF96 was morphologically similar to wild type plants, transgenic plants overexpressing ERF96 had smaller rosette size and were delayed in flowering time. In ABA sensitivity assays, we found that ERF96 overexpression plants were hypersensitive to ABA in terms of ABA inhibition of seed germination, early seedling development and root elongation. Consistent with these observations, elevated transcript levels of some ABA-responsive genes including RD29A, ABI5, ABF3, ABF4, P5CS, and COR15A were observed in the transgenic plants in the presence of ABA. However, in the absence of ABA treatment, the transcript levels of these ABA-responsive genes remained largely unchanged. Our experiments also showed that water loss in ERF

  5. The Small Ethylene Response Factor ERF96 is Involved in the Regulation of the Abscisic Acid Response in Arabidopsis

    DOE PAGESBeta

    Wang, Xiaoping; Liu, Shanda; Tian, Hainan; Wang, Shucai; Chen, Jin-Gui

    2015-11-26

    Ethylene regulates many aspects of plant growth and development including seed germination, leaf senescence, and fruit ripening, and of plant responses to environmental stimuli including both biotic and abiotic stresses. Ethylene response factors (ERFs) are plant-specific transcription factors and are a subfamily of the AP2 (APETALA2)/ERF transcription factor family. The function of many members in this large gene family remains largely unknown. ERF96, a member of the Group IX ERF family transcription factors, has recently been shown to be a transcriptional activator that is involved in plant defense response in Arabidopsis. In this article, we provide evidence that ERF96 ismore » a positive regulator of abscisic acid (ABA) responses. Bioinformatics analysis indicated that there are a total four small ERFs in Arabidopsis including ERF95, ERF96, ERF97, and ERF98, and that ERF96 forms a cluster with ERF95 and ERF97. By using quantitative RT-PCR, we found that ERF96 is expressed in all tissues and organs examined except roots, with relatively high expression in flowers and seeds. Results from the protoplast transfection assay indicated that the EDLL motif-containing C-terminal domain is responsible for ERF96’s transcriptional activity. Although loss-of-function mutant of ERF96 was morphologically similar to wild type plants, transgenic plants overexpressing ERF96 had smaller rosette size and were delayed in flowering time. In ABA sensitivity assays, we found that ERF96 overexpression plants were hypersensitive to ABA in terms of ABA inhibition of seed germination, early seedling development and root elongation. Consistent with these observations, elevated transcript levels of some ABA-responsive genes including RD29A, ABI5, ABF3, ABF4, P5CS, and COR15A were observed in the transgenic plants in the presence of ABA. However, in the absence of ABA treatment, the transcript levels of these ABA-responsive genes remained largely unchanged. Our experiments also showed

  6. The Small Ethylene Response Factor ERF96 is Involved in the Regulation of the Abscisic Acid Response in Arabidopsis

    SciTech Connect

    Wang, Xiaoping; Liu, Shanda; Tian, Hainan; Wang, Shucai; Chen, Jin-Gui

    2015-11-26

    Ethylene regulates many aspects of plant growth and development including seed germination, leaf senescence, and fruit ripening, and of plant responses to environmental stimuli including both biotic and abiotic stresses. Ethylene response factors (ERFs) are plant-specific transcription factors and are a subfamily of the AP2 (APETALA2)/ERF transcription factor family. The function of many members in this large gene family remains largely unknown. ERF96, a member of the Group IX ERF family transcription factors, has recently been shown to be a transcriptional activator that is involved in plant defense response in Arabidopsis. In this article, we provide evidence that ERF96 is a positive regulator of abscisic acid (ABA) responses. Bioinformatics analysis indicated that there are a total four small ERFs in Arabidopsis including ERF95, ERF96, ERF97, and ERF98, and that ERF96 forms a cluster with ERF95 and ERF97. By using quantitative RT-PCR, we found that ERF96 is expressed in all tissues and organs examined except roots, with relatively high expression in flowers and seeds. Results from the protoplast transfection assay indicated that the EDLL motif-containing C-terminal domain is responsible for ERF96’s transcriptional activity. Although loss-of-function mutant of ERF96 was morphologically similar to wild type plants, transgenic plants overexpressing ERF96 had smaller rosette size and were delayed in flowering time. In ABA sensitivity assays, we found that ERF96 overexpression plants were hypersensitive to ABA in terms of ABA inhibition of seed germination, early seedling development and root elongation. Consistent with these observations, elevated transcript levels of some ABA-responsive genes including RD29A, ABI5, ABF3, ABF4, P5CS, and COR15A were observed in the transgenic plants in the presence of ABA. However, in the absence of ABA treatment, the transcript levels of these ABA-responsive genes remained largely unchanged. Our experiments also showed that water

  7. The Small Ethylene Response Factor ERF96 is Involved in the Regulation of the Abscisic Acid Response in Arabidopsis

    SciTech Connect

    Wang, Xiaoping; Liu, Shanda; Tian, Hainan; Wang, Shucai; Chen, Jin-Gui

    2015-11-26

    We report that ethylene regulates many aspects of plant growth and development including seed germination, leaf senescence, and fruit ripening, and of plant responses to environmental stimuli including both biotic and abiotic stresses. Ethylene response factors (ERFs) are plant-specific transcription factors and are a subfamily of the AP2 (APETALA2)/ERF transcription factor family. The function of many members in this large gene family remains largely unknown. ERF96, a member of the Group IX ERF family transcription factors, has recently been shown to be a transcriptional activator that is involved in plant defense response in Arabidopsis. Here we provide evidence that ERF96 is a positive regulator of abscisic acid (ABA) responses. Bioinformatics analysis indicated that there are a total four small ERFs in Arabidopsis including ERF95, ERF96, ERF97, and ERF98, and that ERF96 forms a cluster with ERF95 and ERF97. By using quantitative RT-PCR, we found that ERF96 is expressed in all tissues and organs examined except roots, with relatively high expression in flowers and seeds. Results from the protoplast transfection assay indicated that the EDLL motif-containing C-terminal domain is responsible for ERF96’s transcriptional activity. Although loss-of-function mutant of ERF96 was morphologically similar to wild type plants, transgenic plants overexpressing ERF96 had smaller rosette size and were delayed in flowering time. In ABA sensitivity assays, we found that ERF96 overexpression plants were hypersensitive to ABA in terms of ABA inhibition of seed germination, early seedling development and root elongation. Consistent with these observations, elevated transcript levels of some ABA-responsive genes including RD29A, ABI5, ABF3, ABF4, P5CS, and COR15A were observed in the transgenic plants in the presence of ABA. However, in the absence of ABA treatment, the transcript levels of these ABA-responsive genes remained largely unchanged. Our experiments also showed that

  8. Proteolytic elimination of N-myristoyl modifications by the Shigella virulence factor IpaJ

    PubMed Central

    Burnaevskiy, Nikolay; Fox, Thomas G.; Plymire, Daniel A.; Ertelt, James M.; Weigele, Bethany A.; Selyunin, Andrey S.; Way, Sing Sing; Patrie, Steven M.; Alto, Neal M.

    2013-01-01

    Protein N-myristoylation is a 14-carbon fatty-acid modification that is conserved across eukaryotic species and occurs on nearly 1% of the cellular proteome1,2. The ability of the myristoyl group to facilitate dynamic protein–protein and protein–membrane interactions (known as the myristoyl switch) makes it an essential feature of many signal transduction systems3. Thus pathogenic strategies that facilitate protein demyristoylation would markedly alter the signalling landscape of infected host cells. Here we describe an irreversible mechanism of protein demyristoylation catalysed by invasion plasmid antigen J (IpaJ), a previously uncharacterized Shigella flexneri type III effector protein with cysteine protease activity. A yeast genetic screen for IpaJ substrates identified ADP-ribosylation factor (ARF)1p and ARF2p, small molecular mass GTPases that regulate cargo transport through the Golgi apparatus4. Mass spectrometry showed that IpaJ cleaved the peptide bond between N-myristoylated glycine-2 and asparagine-3 of human ARF1, thereby providing a new mechanism for host secretory inhibition by a bacterial pathogen5,6. We further demonstrate that IpaJ cleaves an array of N-myristoylated proteins involved in cellular growth, signal transduction, autophagasome maturation and organelle function. Taken together, these findings show a previously unrecognized pathogenic mechanism for the site-specific elimination of N-myristoyl protein modification. PMID:23535599

  9. Modulation of transmembrane signalling in HL-60 granulocytes by tumour necrosis factor-alpha.

    PubMed Central

    McLeish, K R; Klein, J B; Schepers, T; Sonnenfeld, G

    1991-01-01

    Differentiated HL-60 granulocytes were used to study the mechanism by which tumour necrosis factor-alpha (TNF) enhances responses to N-formyl-methionyl-leucylphenylalanine (FMLP). Cultivation of differentiated HL-60 cells with 100 units of TNF/ml for 24 h resulted in a 3-fold increase in superoxide release and 4-fold increase in prostaglandin E2 production on stimulation with 1 microM-FMLP. On the other hand, cultivation with TNF failed to increase phorbol diester stimulation of superoxide release. Formyl-peptide-receptor expression determined on isolated membranes from cells cultivated with TNF (TNF-M) was increased by 50% compared with membranes from control cells (NM). Similarly, FMLP binding to intact HL-60 cells was increased by cultivation with TNF. Guanine-nucleotide-binding proteins (G-protein) levels were not different between TNF-M and NM, as determined by pertussis-toxin-catalysed ADP-ribosylation and by immunoblotting with antisera recognizing alpha i2 subunit. Binding of guanosine 5'-[gamma-thio]triphosphate and GTP hydrolysis stimulated by FMLP were enhanced by about 50% in TNF-M. The efficiency of G-protein activation by formyl-peptide receptors did not differ between TNF-M and NM. TNF regulates expression of formyl-peptide receptors independently of G-protein levels. The regulation of receptor expression is one mechanism by which TNF enhances cell responses to formylated peptides. Images Fig. 4. PMID:1659380

  10. Arabidopsis HD-Zip II transcription factors control apical embryo development and meristem function.

    PubMed

    Turchi, Luana; Carabelli, Monica; Ruzza, Valentino; Possenti, Marco; Sassi, Massimiliano; Peñalosa, Andrés; Sessa, Giovanna; Salvi, Sergio; Forte, Valentina; Morelli, Giorgio; Ruberti, Ida

    2013-05-01

    The Arabidopsis genome encodes ten Homeodomain-Leucine zipper (HD-Zip) II proteins. ARABIDOPSIS THALIANA HOMEOBOX 2 (ATHB2), HOMEOBOX ARABIDOPSIS THALIANA 1 (HAT1), HAT2, HAT3 and ATHB4 are regulated by changes in the red/far red light ratio that induce shade avoidance in most of the angiosperms. Here, we show that progressive loss of HAT3, ATHB4 and ATHB2 activity causes developmental defects from embryogenesis onwards in white light. Cotyledon development and number are altered in hat3 athb4 embryos, and these defects correlate with changes in auxin distribution and response. athb2 gain-of-function mutation and ATHB2 expression driven by its promoter in hat3 athb4 result in significant attenuation of phenotypes, thus demonstrating that ATHB2 is functionally redundant to HAT3 and ATHB4. In analogy to loss-of-function mutations in HD-Zip III genes, loss of HAT3 and ATHB4 results in organ polarity defects, whereas triple hat3 athb4 athb2 mutants develop one or two radialized cotyledons and lack an active shoot apical meristem (SAM). Consistent with overlapping expression pattern of HD-Zip II and HD-Zip III gene family members, bilateral symmetry and SAM defects are enhanced when hat3 athb4 is combined with mutations in PHABULOSA (PHB), PHAVOLUTA (PHV) or REVOLUTA (REV). Finally, we show that ATHB2 is part of a complex regulatory circuit directly involving both HD-Zip II and HD-Zip III proteins. Taken together, our study provides evidence that a genetic system consisting of HD-Zip II and HD-Zip III genes cooperates in establishing bilateral symmetry and patterning along the adaxial-abaxial axis in the embryo as well as in controlling SAM activity. PMID:23578926

  11. Expanding functions of GIT Arf GTPase-activating proteins, PIX Rho guanine nucleotide exchange factors and GIT-PIX complexes.

    PubMed

    Zhou, Wu; Li, Xiaobo; Premont, Richard T

    2016-05-15

    The GIT proteins, GIT1 and GIT2, are GTPase-activating proteins (inactivators) for the ADP-ribosylation factor (Arf) small GTP-binding proteins, and function to limit the activity of Arf proteins. The PIX proteins, α-PIX and β-PIX (also known as ARHGEF6 and ARHGEF7, respectively), are guanine nucleotide exchange factors (activators) for the Rho family small GTP-binding protein family members Rac1 and Cdc42. Through their multi-domain structures, GIT and PIX proteins can also function as signaling scaffolds by binding to numerous protein partners. Importantly, the constitutive association of GIT and PIX proteins into oligomeric GIT-PIX complexes allows these two proteins to function together as subunits of a larger structure that coordinates two distinct small GTP-binding protein pathways and serves as multivalent scaffold for the partners of both constituent subunits. Studies have revealed the involvement of GIT and PIX proteins, and of the GIT-PIX complex, in numerous fundamental cellular processes through a wide variety of mechanisms, pathways and signaling partners. In this Commentary, we discuss recent findings in key physiological systems that exemplify current understanding of the function of this important regulatory complex. Further, we draw attention to gaps in crucial information that remain to be filled to allow a better understanding of the many roles of the GIT-PIX complex in health and disease. PMID:27182061

  12. Genome-wide CRISPR screen reveals novel host factors required for Staphylococcus aureus α-hemolysin-mediated toxicity

    PubMed Central

    Virreira Winter, Sebastian; Zychlinsky, Arturo; Bardoel, Bart W.

    2016-01-01

    Staphylococcus aureus causes a wide variety of infections and antibiotic resistant strains are a major problem in hospitals. One of the best studied virulence factors of S. aureus is the pore-forming toxin alpha hemolysin (αHL) whose mechanism of action is incompletely understood. We performed a genome-wide loss-of-function screen using CRISPR/Cas9 technology to identify host targets required for αHL susceptibility in human myeloid cells. We found gRNAs for ten genes enriched after intoxication with αHL and focused on the top five hits. Besides a disintegrin and metalloproteinase domain-containing protein 10 (ADAM10), the host receptor for αHL, we identified three proteins, Sys1 golgi trafficking protein (SYS1), ADP-ribosylation factor 1 (ARFRP1), and tetraspanin-14 (TSPAN14) which regulate the presentation of ADAM10 on the plasma membrane post-translationally. Interestingly, we also showed that cells lacking sphingomyelin synthase 1 (SGMS1) resist αHL intoxication, but have only a slightly reduced ADAM10 surface expression. SGMS1 regulates lipid raft formation, suggesting that αHL requires these membrane microdomains for attachment and cytotoxicity. PMID:27066838

  13. A Conserved Peptide Pattern from a Widespread Microbial Virulence Factor Triggers Pattern-Induced Immunity in Arabidopsis

    PubMed Central

    Oome, Stan; Raaymakers, Tom M.; Van den Ackerveken, Guido; Nürnberger, Thorsten

    2014-01-01

    Microbe- or host damage-derived patterns mediate activation of pattern-triggered immunity (PTI) in plants. Microbial virulence factor (effector)-triggered immunity (ETI) constitutes a second layer of plant protection against microbial attack. Various necrosis and ethylene-inducing peptide 1 (Nep1)-like proteins (NLPs) produced by bacterial, oomycete and fungal microbes are phytotoxic virulence factors that exert immunogenic activities through phytotoxin-induced host cell damage. We here show that multiple cytotoxic NLPs also carry a pattern of 20 amino acid residues (nlp20) that triggers immunity-associated plant defenses and immunity to microbial infection in Arabidopsis thaliana and related plant species with similar characteristics as the prototype pattern, bacterial flagellin. Characteristic differences in flagellin and nlp20 plant responses exist however, as nlp20s fail to trigger extracellular alkalinization in Arabidopsis cell suspensions and seedling growth inhibition. Immunogenic nlp20 peptide motifs are frequently found in bacterial, oomycete and fungal NLPs. Such an unusually broad taxonomic distribution within three phylogenetic kingdoms is unprecedented among microbe-derived triggers of immune responses in either metazoans or plants. Our findings suggest that cytotoxic NLPs carrying immunogenic nlp20 motifs trigger PTI in two ways as typical patterns and by inflicting host cell damage. We further propose that conserved structures within a microbial virulence factor might have driven the emergence of a plant pattern recognition system mediating PTI. As this is reminiscent of the evolution of immune receptors mediating ETI, our findings support the idea that there is a continuum between PTI and ETI. PMID:25375108

  14. ULTRAPETALA trxG Genes Interact with KANADI Transcription Factor Genes to Regulate Arabidopsis Gynoecium Patterning[C][W][OPEN

    PubMed Central

    Monfared, Mona M.; Shemyakina, Elena A.; Fletcher, Jennifer C.

    2014-01-01

    Organ formation relies upon precise patterns of gene expression that are under tight spatial and temporal regulation. Transcription patterns are specified by several cellular processes during development, including chromatin remodeling, but little is known about how chromatin-remodeling factors contribute to plant organogenesis. We demonstrate that the trithorax group (trxG) gene ULTRAPETALA1 (ULT1) and the GARP transcription factor gene KANADI1 (KAN1) organize the Arabidopsis thaliana gynoecium along two distinct polarity axes. We show that ULT1 activity is required for the kan1 adaxialized polarity defect, indicating that ULT1 and KAN1 act oppositely to regulate the adaxial-abaxial axis. Conversely, ULT1 and KAN1 together establish apical-basal polarity by promoting basal cell fate in the gynoecium, restricting the expression domain of the basic helix-loop-helix transcription factor gene SPATULA. Finally, we show that ult alleles display dose-dependent genetic interactions with kan alleles and that ULT and KAN proteins can associate physically. Our findings identify a dual role for plant trxG factors in organ patterning, with ULT1 and KAN1 acting antagonistically to pattern the adaxial-abaxial polarity axis but jointly to pattern the apical-basal axis. Our data indicate that the ULT proteins function to link chromatin-remodeling factors with DNA binding transcription factors to regulate target gene expression. PMID:25381352

  15. Repression of AS2 by WOX family transcription factors is required for leaf development in Medicago and Arabidopsis

    PubMed Central

    Zhang, Fei; Tadege, Million

    2015-01-01

    WOX transcription factors are key regulators of meristematic activity in plants. The Medicago WOX gene, STF, functions in maintenance of leaf marginal meristem, analogous to the function of WUS in the shoot apical meristem. Both STF and WUS directly repress AS2 expression in their respective domains. Ectopic expression of AS2 with WUS promoter leads to a narrow leaf phenotype and other phenotypes similar to the wus mutant. We also found that a wox1 prs wus triple mutant produces much narrower leaf blades than the wox1 prs double mutant, indicating that WUS genetically interacts with WOX1 and PRS in Arabidopsis leaf blade development. Our data points to a general requirement for AS2 repression in meristematic regions to allow cell proliferation. PMID:25807065

  16. Repression of AS2 by WOX family transcription factors is required for leaf development in Medicago and Arabidopsis.

    PubMed

    Zhang, Fei; Tadege, Million

    2015-01-01

    WOX transcription factors are key regulators of meristematic activity in plants. The Medicago WOX gene, STF, functions in maintenance of leaf marginal meristem, analogous to the function of WUS in the shoot apical meristem. Both STF and WUS directly repress AS2 expression in their respective domains. Ectopic expression of AS2 with WUS promoter leads to a narrow leaf phenotype and other phenotypes similar to the wus mutant. We also found that a wox1 prs wus triple mutant produces much narrower leaf blades than the wox1 prs double mutant, indicating that WUS genetically interacts with WOX1 and PRS in Arabidopsis leaf blade development. Our data points to a general requirement for AS2 repression in meristematic regions to allow cell proliferation. PMID:25807065

  17. Ectopic expression of R3 MYB transcription factor gene OsTCL1 in Arabidopsis, but not rice, affects trichome and root hair formation.

    PubMed

    Zheng, Kaijie; Tian, Hainan; Hu, Qingnan; Guo, Hongyan; Yang, Li; Cai, Ling; Wang, Xutong; Liu, Bao; Wang, Shucai

    2016-01-01

    In Arabidopsis, a MYB-bHLH-WD40 (MBW) transcriptional activator complex activates the homeodomain protein gene GLABRA2 (GL2), leading to the promotion of trichome formation and inhibition of root hair formation. The same MBW complex also activates single-repeat R3 MYB genes. R3 MYBs in turn, play a negative feedback role by competing with R2R3 MYB proteins for binding bHLH proteins, thus blocking the formation of the MBW complex. By BLASTing the rice (Oryza sativa) protein database using the entire amino acid sequence of Arabidopsis R3 MYB transcription factor TRICHOMELESS1 (TCL1), we found that there are two genes in rice genome encoding R3 MYB transcription factors, namely Oryza sativa TRICHOMELESS1 (OsTCL1) and OsTCL2. Expressing OsTCL1 in Arabidopsis inhibited trichome formation and promoted root hair formation, and OsTCL1 interacted with GL3 when tested in Arabidopsis protoplasts. Consistent with these observations, expression levels of GL2, R2R3 MYB transcription factor gene GLABRA1 (GL1) and several R3 MYB genes were greatly reduced, indicating that OsTCL1 is functional R3 MYB. However, trichome and root hair formation in transgenic rice plants overexpressing OsTCL1 remained largely unchanged, and elevated expression of OsGL2 was observed in the transgenic rice plants, indicating that rice may use different mechanisms to regulate trichome formation. PMID:26758286

  18. Ectopic expression of R3 MYB transcription factor gene OsTCL1 in Arabidopsis, but not rice, affects trichome and root hair formation

    PubMed Central

    Zheng, Kaijie; Tian, Hainan; Hu, Qingnan; Guo, Hongyan; Yang, Li; Cai, Ling; Wang, Xutong; Liu, Bao; Wang, Shucai

    2016-01-01

    In Arabidopsis, a MYB-bHLH-WD40 (MBW) transcriptional activator complex activates the homeodomain protein gene GLABRA2 (GL2), leading to the promotion of trichome formation and inhibition of root hair formation. The same MBW complex also activates single-repeat R3 MYB genes. R3 MYBs in turn, play a negative feedback role by competing with R2R3 MYB proteins for binding bHLH proteins, thus blocking the formation of the MBW complex. By BLASTing the rice (Oryza sativa) protein database using the entire amino acid sequence of Arabidopsis R3 MYB transcription factor TRICHOMELESS1 (TCL1), we found that there are two genes in rice genome encoding R3 MYB transcription factors, namely Oryza sativa TRICHOMELESS1 (OsTCL1) and OsTCL2. Expressing OsTCL1 in Arabidopsis inhibited trichome formation and promoted root hair formation, and OsTCL1 interacted with GL3 when tested in Arabidopsis protoplasts. Consistent with these observations, expression levels of GL2, R2R3 MYB transcription factor gene GLABRA1 (GL1) and several R3 MYB genes were greatly reduced, indicating that OsTCL1 is functional R3 MYB. However, trichome and root hair formation in transgenic rice plants overexpressing OsTCL1 remained largely unchanged, and elevated expression of OsGL2 was observed in the transgenic rice plants, indicating that rice may use different mechanisms to regulate trichome formation. PMID:26758286

  19. Arf guanine nucleotide-exchange factors BIG1 and BIG2 regulate nonmuscle myosin IIA activity by anchoring myosin phosphatase complex

    PubMed Central

    Le, Kang; Li, Chun-Chun; Ye, Guan; Moss, Joel; Vaughan, Martha

    2013-01-01

    Brefeldin A-inhibited guanine nucleotide-exchange factors BIG1 and BIG2 activate, through their Sec7 domains, ADP ribosylation factors (Arfs) by accelerating the replacement of Arf-bound GDP with GTP for initiation of vesicular transport or activation of specific enzymes that modify important phospholipids. They are also implicated in regulation of cell polarization and actin dynamics for directed migration. Reciprocal coimmunoprecipitation of endogenous HeLa cell BIG1 and BIG2 with myosin IIA was demonstrably independent of Arf guanine nucleotide-exchange factor activity, because effects of BIG1 and BIG2 depletion were reversed by overexpression of the cognate BIG molecule C-terminal sequence that follows the Arf activation site. Selective depletion of BIG1 or BIG2 enhanced specific phosphorylation of myosin regulatory light chain (T18/S19) and F-actin content, which impaired cell migration in Transwell assays. Our data are clear evidence of these newly recognized functions for BIG1 and BIG2 in transduction or integration of mechanical signals from integrin adhesions and myosin IIA-dependent actin dynamics. Thus, by anchoring or scaffolding the assembly, organization, and efficient operation of multimolecular myosin phosphatase complexes that include myosin IIA, protein phosphatase 1δ, and myosin phosphatase-targeting subunit 1, BIG1 and BIG2 serve to integrate diverse biophysical and biochemical events in cells. PMID:23918382

  20. Structural and Functional Analysis of VQ Motif-Containing Proteins in Arabidopsis as Interacting Proteins of WRKY Transcription Factors1[W][OA

    PubMed Central

    Cheng, Yuan; Zhou, Yuan; Yang, Yan; Chi, Ying-Jun; Zhou, Jie; Chen, Jian-Ye; Wang, Fei; Fan, Baofang; Shi, Kai; Zhou, Yan-Hong; Yu, Jing-Quan; Chen, Zhixiang

    2012-01-01

    WRKY transcription factors are encoded by a large gene superfamily with a broad range of roles in plants. Recently, several groups have reported that proteins containing a short VQ (FxxxVQxLTG) motif interact with WRKY proteins. We have recently discovered that two VQ proteins from Arabidopsis (Arabidopsis thaliana), SIGMA FACTOR-INTERACTING PROTEIN1 and SIGMA FACTOR-INTERACTING PROTEIN2, act as coactivators of WRKY33 in plant defense by specifically recognizing the C-terminal WRKY domain and stimulating the DNA-binding activity of WRKY33. In this study, we have analyzed the entire family of 34 structurally divergent VQ proteins from Arabidopsis. Yeast (Saccharomyces cerevisiae) two-hybrid assays showed that Arabidopsis VQ proteins interacted specifically with the C-terminal WRKY domains of group I and the sole WRKY domains of group IIc WRKY proteins. Using site-directed mutagenesis, we identified structural features of these two closely related groups of WRKY domains that are critical for interaction with VQ proteins. Quantitative reverse transcription polymerase chain reaction revealed that expression of a majority of Arabidopsis VQ genes was responsive to pathogen infection and salicylic acid treatment. Functional analysis using both knockout mutants and overexpression lines revealed strong phenotypes in growth, development, and susceptibility to pathogen infection. Altered phenotypes were substantially enhanced through cooverexpression of genes encoding interacting VQ and WRKY proteins. These findings indicate that VQ proteins play an important role in plant growth, development, and response to environmental conditions, most likely by acting as cofactors of group I and IIc WRKY transcription factors. PMID:22535423

  1. Pertussis toxin treatment does not block inhibition by atrial natriuretic factor of aldosterone secretion in cultured bovine zona glomerulosa cells

    SciTech Connect

    De Lean, A.; Cantin, M.

    1986-03-05

    The authors have previously reported that atrial natriuretic factor (ANF) potently inhibits PGE or forskolin-stimulation aldosterone secretion in bovine zona glomerulosa (ZG) by acting through specific high affinity receptors. In order to evaluate the functional role of the regulatory protein N/sub i/ and the inhibition of adenylate cyclase activity (AC) in ZG, the authors have studied the effect of treatment with PT on inhibition by ANF of aldosterone production. Primary cultures of ZG were treated for 18 hours in serum-free F12 medium with (0-100 ng/ml PT). No effect of PT pretreatment was observed either on basal, PGE-stimulated or ANF-inhibited levels of steroidogenesis. When membranes prepared from control ZG were ADP-ribosylated with (/sup 32/P) NAD in the presence of PT, two toxin-specific bands with 39 Kd and 41 Kd were documented on SDS gel. Cell pretreatment with as low as 1 ng/ml drastically reduced further labelling of these two bands while higher doses completely abolished them. Since PT treatment covalently modifies completely the toxin substrate without altering ANF inhibition of adrenal steroidogenesis, the authors conclude that N/sub i/ is not involved in the mode of action of ANF on aldosterone production.

  2. Mutational Evidence for the Critical Role of CBF Transcription Factors in Cold Acclimation in Arabidopsis.

    PubMed

    Zhao, Chunzhao; Zhang, Zhengjing; Xie, Shaojun; Si, Tong; Li, Yuanya; Zhu, Jian-Kang

    2016-08-01

    The three tandemly arranged CBF genes, CBF1, CBF2, and CBF3, are involved in cold acclimation. Due to the lack of stable loss-of-function Arabidopsis (Arabidopsis thaliana) mutants deficient in all three CBF genes, it is still unclear whether the CBF genes are essential for freezing tolerance and whether they may have other functions besides cold acclimation. In this study, we used the CRISPR/Cas9 system to generate cbf single, double, and triple mutants. Compared to the wild type, the cbf triple mutants are extremely sensitive to freezing after cold acclimation, demonstrating that the three CBF genes are essential for cold acclimation. Our results show that the three CBF genes also contribute to basal freezing tolerance. Unexpectedly, we found that the cbf triple mutants are defective in seedling development and salt stress tolerance. Transcript profiling revealed that the CBF genes regulate 414 cold-responsive (COR) genes, of which 346 are CBF-activated genes and 68 are CBF-repressed genes. The analysis suggested that CBF proteins are extensively involved in the regulation of carbohydrate and lipid metabolism, cell wall modification, and gene transcription. Interestingly, like the triple mutants, cbf2 cbf3 double mutants are more sensitive to freezing after cold acclimation compared to the wild type, but cbf1 cbf3 double mutants are more resistant, suggesting that CBF2 is more important than CBF1 and CBF3 in cold acclimation-dependent freezing tolerance. Our results not only demonstrate that the three CBF genes together are required for cold acclimation and freezing tolerance, but also reveal that they are important for salt tolerance and seedling development. PMID:27252305

  3. Misregulation of AUXIN RESPONSE FACTOR 8 underlies the developmental abnormalities caused by three distinct viral silencing suppressors in Arabidopsis.

    PubMed

    Jay, Florence; Wang, Yu; Yu, Agnès; Taconnat, Ludivine; Pelletier, Sandra; Colot, Vincent; Renou, Jean-Pierre; Voinnet, Olivier

    2011-05-01

    In Arabidopsis, micro (mi)RNAs and trans-acting (ta-si)RNAs synthesized directly or indirectly through the DICER-LIKE-1 (DCL1) ribonuclease have roles in patterning and hormonal responses, while DCL2,3,4-dependent small-interfering (si)RNAs are mainly involved in silencing of transposable elements and antiviral defense. Viral suppressors of RNA silencing (VSRs) produced by phytoviruses to counter plant defense may perturb plant developmental programs because of the collision of their inhibitory effects with the regulatory action of endogenous miRNAs and ta-siRNAs. This could explain the similar developmental aberrations displayed by Arabidopsis miRNA/ta-siRNA pathway mutants, including dcl1, and by some VSR-expressing plants. Nonetheless, the molecular bases for these morphological aberrations have remained mysterious, and their contribution to viral disease symptoms/virulence unexplored. The extent of VSR inhibitory actions to other types of endogenous small RNAs remains also unclear. Here, we present an in-depth analysis of transgenic Arabidopsis expressing constitutively HcPro, P19 and P15, three unrelated VSRs. We show that VSR expression has comparable, yet modest effects on known miRNA and ta-siRNA target RNA levels, similar to those observed using an hypomorphic dcl1 mutation. However, by combining results of transcriptome studies with deep-sequencing data from immuno-precipitated small RNAs, additional, novel endogenous targets of miRNA and ta-siRNA were identified, unraveling an unsuspected complexity in the origin and scope-of-action of these molecules. Other stringent analyses pinpointed misregulation of the miR167 target AUXIN RESPONSE FACTOR 8 (ARF8) as a major cause for the developmental aberrations exhibited by VSR transgenic plants, but also for the phenotypes induced during normal viral infection caused by the HcPro-encoding Turnip mosaic virus (TuMV). Neither RNA silencing, its suppression by VSRs, nor the virulence/accumulation of TuMV was

  4. The N-Terminus of the Floral Arabidopsis TGA Transcription Factor PERIANTHIA Mediates Redox-Sensitive DNA-Binding

    PubMed Central

    Gutsche, Nora; Zachgo, Sabine

    2016-01-01

    The Arabidopsis TGA transcription factor (TF) PERIANTHIA (PAN) regulates the formation of the floral organ primordia as revealed by the pan mutant forming an abnormal pentamerous arrangement of the outer three floral whorls. The Arabidopsis TGA bZIP TF family comprises 10 members, of which PAN and TGA9/10 control flower developmental processes and TGA1/2/5/6 participate in stress-responses. For the TGA1 protein it was shown that several cysteines can be redox-dependently modified. TGA proteins interact in the nucleus with land plant-specific glutaredoxins, which may alter their activities posttranslationally. Here, we investigated the DNA-binding of PAN to the AAGAAT motif under different redox-conditions. The AAGAAT motif is localized in the second intron of the floral homeotic regulator AGAMOUS (AG), which controls stamen and carpel development as well as floral determinacy. Whereas PAN protein binds to this regulatory cis-element under reducing conditions, the interaction is strongly reduced under oxidizing conditions in EMSA studies. The redox-sensitive DNA-binding is mediated via a special PAN N-terminus, which is not present in other Arabidopsis TGA TFs and comprises five cysteines. Two N-terminal PAN cysteines, Cys68 and Cys87, were shown to form a disulfide bridge and Cys340, localized in a C-terminal putative transactivation domain, can be S-glutathionylated. Comparative land plant analyses revealed that the AAGAAT motif exists in asterid and rosid plant species. TGA TFs with N-terminal extensions of variable length were identified in all analyzed seed plants. However, a PAN-like N-terminus exists only in the rosids and exclusively Brassicaceae homologs comprise four to five of the PAN N-terminal cysteines. Redox-dependent modifications of TGA cysteines are known to regulate the activity of stress-related TGA TFs. Here, we show that the N-terminal PAN cysteines participate in a redox-dependent control of the PAN interaction with a highly conserved

  5. Transcription Factor ATAF1 in Arabidopsis Promotes Senescence by Direct Regulation of Key Chloroplast Maintenance and Senescence Transcriptional Cascades1[OPEN

    PubMed Central

    Garapati, Prashanth; Xue, Gang-Ping

    2015-01-01

    Senescence represents a fundamental process of late leaf development. Transcription factors (TFs) play an important role for expression reprogramming during senescence; however, the gene regulatory networks through which they exert their functions, and their physiological integration, are still largely unknown. Here, we identify the Arabidopsis (Arabidopsis thaliana) abscisic acid (ABA)- and hydrogen peroxide-activated TF Arabidopsis thaliana ACTIVATING FACTOR1 (ATAF1) as a novel upstream regulator of senescence. ATAF1 executes its physiological role by affecting both key chloroplast maintenance and senescence-promoting TFs, namely GOLDEN2-LIKE1 (GLK1) and ORESARA1 (ARABIDOPSIS NAC092), respectively. Notably, while ATAF1 activates ORESARA1, it represses GLK1 expression by directly binding to their promoters, thereby generating a transcriptional output that shifts the physiological balance toward the progression of senescence. We furthermore demonstrate a key role of ATAF1 for ABA- and hydrogen peroxide-induced senescence, in accordance with a direct regulatory effect on ABA homeostasis genes, including NINE-CIS-EPOXYCAROTENOID DIOXYGENASE3 involved in ABA biosynthesis and ABC TRANSPORTER G FAMILY MEMBER40, encoding an ABA transport protein. Thus, ATAF1 serves as a core transcriptional activator of senescence by coupling stress-related signaling with photosynthesis- and senescence-related transcriptional cascades. PMID:25953103

  6. Cell-to-Cell Movement of Two Interacting AT-Hook Factors in Arabidopsis Root Vascular Tissue Patterning[W

    PubMed Central

    Zhou, Jing; Wang, Xu; Lee, Jung-Youn; Lee, Ji-Young

    2013-01-01

    The xylem and phloem, major conducting and supporting tissues in vascular plants, are established by cell division and cell-type specification in the procambium/cambium. The organization of the xylem, phloem, and procambium/cambium is tightly controlled. However, the underlying regulatory mechanisms remain largely unknown. In this study, we report the discovery of two transcription factors, AT-HOOK MOTIF NUCLEAR LOCALIZED PROTEIN 3 (AHL3) and AHL4, which regulate vascular tissue boundaries in Arabidopsis thaliana roots. In either of the knockout mutants of AHL3 and AHL4, encoding closely related AT-hook transcription factors, a misspecification of tissue boundaries between the xylem and procambium occurred and ectopic xylem developed in the procambium domain. In plants, specific types of transcription factors can serve as direct intercellular signals by moving from one cell to another, playing crucial roles in tissue patterning. Adding to this paradigm, AHL4 moves actively from the procambium to xylem in the root meristem to regulate the tissue boundaries. When the intercellular movement of AHL4 was impaired, AHL4 could not complement the xylem phenotype in the ahl4. Furthermore, AHL4 revealed unique characteristics in that it interacts with AHL3 in vivo and that this interaction facilitates their intercellular trafficking. Taken together, this study uncovered a novel mechanism in vascular tissue patterning that requires the intercellular trafficking of two interacting transcription factors. PMID:23335615

  7. Arabidopsis Ensemble Reverse-Engineered Gene Regulatory Network Discloses Interconnected Transcription Factors in Oxidative Stress[W

    PubMed Central

    Vermeirssen, Vanessa; De Clercq, Inge; Van Parys, Thomas; Van Breusegem, Frank; Van de Peer, Yves

    2014-01-01

    The abiotic stress response in plants is complex and tightly controlled by gene regulation. We present an abiotic stress gene regulatory network of 200,014 interactions for 11,938 target genes by integrating four complementary reverse-engineering solutions through average rank aggregation on an Arabidopsis thaliana microarray expression compendium. This ensemble performed the most robustly in benchmarking and greatly expands upon the availability of interactions currently reported. Besides recovering 1182 known regulatory interactions, cis-regulatory motifs and coherent functionalities of target genes corresponded with the predicted transcription factors. We provide a valuable resource of 572 abiotic stress modules of coregulated genes with functional and regulatory information, from which we deduced functional relationships for 1966 uncharacterized genes and many regulators. Using gain- and loss-of-function mutants of seven transcription factors grown under control and salt stress conditions, we experimentally validated 141 out of 271 predictions (52% precision) for 102 selected genes and mapped 148 additional transcription factor-gene regulatory interactions (49% recall). We identified an intricate core oxidative stress regulatory network where NAC13, NAC053, ERF6, WRKY6, and NAC032 transcription factors interconnect and function in detoxification. Our work shows that ensemble reverse-engineering can generate robust biological hypotheses of gene regulation in a multicellular eukaryote that can be tested by medium-throughput experimental validation. PMID:25549671

  8. Hydrogen sulfide is a novel potential virulence factor of Mycoplasma pneumoniae: characterization of the unusual cysteine desulfurase/desulfhydrase HapE.

    PubMed

    Großhennig, Stephanie; Ischebeck, Till; Gibhardt, Johannes; Busse, Julia; Feussner, Ivo; Stülke, Jörg

    2016-04-01

    Mycoplasma pneumoniae is a human pathogen causing atypical pneumonia with a minimalized and highly streamlined genome. So far, hydrogen peroxide production, cytadherence, and the ADP-ribosylating CARDS toxin have been identified as pathogenicity determinants. We have studied haemolysis caused by M. pneumoniae, and discovered that hydrogen peroxide is responsible for the oxidation of heme, but not for lysis of erythrocytes. This feature could be attributed to hydrogen sulfide, a compound that has previously not been identified as virulence factor in lung pathogens. Indeed, we observed hydrogen sulfide production by M. pneumoniae. The search for a hydrogen sulfide-producing enzyme identified HapE, a protein with similarity to cysteine desulfurases. In contrast to typical cysteine desulfurases, HapE is a bifunctional enzyme: it has both the cysteine desulfurase activity to produce alanine and the cysteine desulfhydrase activity to produce pyruvate and hydrogen sulfide. Experiments with purified HapE showed that the enzymatic activity of the protein is responsible for haemolysis, demonstrating that HapE is a novel potential virulence factor of M. pneumoniae. PMID:26711628

  9. Overexpression of the poplar NF-YB7 transcription factor confers drought tolerance and improves water-use efficiency in Arabidopsis.

    PubMed

    Han, Xiao; Tang, Sha; An, Yi; Zheng, Dong-Chao; Xia, Xin-Li; Yin, Wei-Lun

    2013-11-01

    Water deficit is a serious environmental factor limiting the growth and productivity of plants worldwide. Improvement of drought tolerance and efficient water use are significant strategies to overcome this dilemma. In this study, a drought-responsive transcription factor, nuclear factor Y subunit B 7 (PdNF-YB7), induced by osmotic stress (PEG6000) and abscisic acid, was isolated from fast-growing poplar clone NE-19 [Populus nigra × (Populus deltoides × Populus nigra)]. Ectopic overexpression of PdNF-YB7 (oxPdB7) in Arabidopsis enhanced drought tolerance and whole-plant and instantaneous leaf water-use efficiency (WUE, the ratio of biomass produced to water consumed). Overexpressing lines had an increase in germination rate and root length and decrease in water loss and displayed higher photosynthetic rate, instantaneous leaf WUE, and leaf water potential to exhibit enhanced drought tolerance under water scarcity. Additionally, overexpression of PdNF-YB7 in Arabidopsis improved whole-plant WUE by increasing carbon assimilation and reducing transpiration with water abundance. These drought-tolerant, higher WUE transgenic Arabidopsis had earlier seedling establishment and higher biomass than controls under normal and drought conditions. In contrast, Arabidopsis mutant nf-yb3 was more sensitive to drought stress with lower WUE. However, complementation analysis indicated that complementary lines (nf-yb3/PdB7) had almost the same drought response and WUE as wild-type Col-0. Taken together, these results suggest that PdNF-YB7 positively confers drought tolerance and improves WUE in Arabidopsis; thus it could potentially be used in breeding drought-tolerant plants with increased production even under water deficiency. PMID:24006421

  10. The TRANSPLANTA collection of Arabidopsis lines: a resource for functional analysis of transcription factors based on their conditional overexpression.

    PubMed

    Coego, Alberto; Brizuela, Esther; Castillejo, Pablo; Ruíz, Sandra; Koncz, Csaba; del Pozo, Juan C; Piñeiro, Manuel; Jarillo, José A; Paz-Ares, Javier; León, José

    2014-03-01

    Transcription factors (TFs) are key regulators of gene expression in all organisms. In eukaryotes, TFs are often represented by functionally redundant members of large gene families. Overexpression might prove a means to unveil the biological functions of redundant TFs; however, constitutive overexpression of TFs frequently causes severe developmental defects, preventing their functional characterization. Conditional overexpression strategies help to overcome this problem. Here, we report on the TRANSPLANTA collection of Arabidopsis lines, each expressing one of 949 TFs under the control of a β-estradiol-inducible promoter. Thus far, 1636 independent homozygous lines, representing an average of 2.6 lines for every TF, have been produced for the inducible expression of 634 TFs. Along with a GUS-GFP reporter, randomly selected TRANSPLANTA lines were tested and confirmed for conditional transgene expression upon β-estradiol treatment. As a proof of concept for the exploitation of this resource, β-estradiol-induced proliferation of root hairs, dark-induced senescence, anthocyanin accumulation and dwarfism were observed in lines conditionally expressing full-length cDNAs encoding RHD6, WRKY22, MYB123/TT2 and MYB26, respectively, in agreement with previously reported phenotypes conferred by these TFs. Further screening performed with other TRANSPLANTA lines allowed the identification of TFs involved in different plant biological processes, illustrating that the collection is a powerful resource for the functional characterization of TFs. For instance, ANAC058 and a TINY/AP2 TF were identified as modulators of ABA-mediated germination potential, and RAP2.10/DEAR4 was identified as a regulator of cell death in the hypocotyl-root transition zone. Seeds of TRANSPLANTA lines have been deposited at the Nottingham Arabidopsis Stock Centre for further distribution. PMID:24456507

  11. The Elucidation of the Interactome of 16 Arabidopsis bZIP Factors Reveals Three Independent Functional Networks

    PubMed Central

    Llorca, Carles Marco; Berendzen, Kenneth Wayne; Malik, Waqas Ahmed; Mahn, Stefan; Piepho, Hans-Peter; Zentgraf, Ulrike

    2015-01-01

    The function of the bZIP transcription factors is strictly dependent on their ability to dimerize. Heterodimerization has proven to be highly specific and is postulated to operate as a combinatorial mechanism allowing the generation of a large variety of dimers with unique qualities by specifically combining a small set of monomers; an assumption that has not yet been tested systematically. Here, the interaction pattern and the transactivation properties of 16 Arabidopsis thaliana bZIPs are examined in transiently transformed Arabidopsis protoplasts to deliver a perspective on the relationship between bZIP dimerization and function. An interaction matrix of bZIPs belonging to the C, G, H, and S1 bZIP groups was resolved by Bimolecular Fluorescent Complementation (BiFC) coupled to quantitative flow cytometric analysis, while an extensive GUS reporter gene assay was carried out to determine the effect of different bZIP pairs on the expression of four different known bZIP-targeted promoters. Statistical data treatment and complementary bioinformatic analysis were performed to substantiate the biological findings. According to these results, the 16 bZIPs interact in three isolated networks, within which their members dimerize non-specifically and exhibit a significant level of functional redundancy. A coherent explanation for these results is supported by in silico analysis of differences in the length, structure and composition of their leucine zippers and appears to explain their dimerization specificity and dynamics observed in vivo quite well. A model in which the bZIP networks act as functional units is proposed. PMID:26452049

  12. A single-repeat R3-MYB transcription factor MYBC1 negatively regulates freezing tolerance in Arabidopsis

    SciTech Connect

    Zhai, Hong; Bai, Xi; Zhu, Yanming; Li, Yong; Cai, Hua; Ji, Wei; Ji, Zuojun; Liu, Xiaofei; Liu, Xin; Li, Jing

    2010-04-16

    We had previously identified the MYBC1 gene, which encodes a single-repeat R3-MYB protein, as a putative osmotic responding gene; however, no R3-MYB transcription factor has been reported to regulate osmotic stress tolerance. Thus, we sought to elucidate the function of MYBC1 in response to osmotic stresses. Real-time RT-PCR analysis indicated that MYBC1 expression responded to cold, dehydration, salinity and exogenous ABA at the transcript level. mybc1 mutants exhibited an increased tolerance to freezing stress, whereas 35S::MYBC1 transgenic plants exhibited decreased cold tolerance. Transcript levels of some cold-responsive genes, including CBF/DREB genes, KIN1, ADC1, ADC2 and ZAT12, though, were not altered in the mybc1 mutants or the 35S::MYBC1 transgenic plants in response to cold stress, as compared to the wild type. Microarray analysis results that are publically available were investigated and found transcript level of MYBC1 was not altered by overexpression of CBF1, CBF2, and CBF3, suggesting that MYBC1 is not down regulated by these CBF family members. Together, these results suggested that MYBC1is capable of negatively regulating the freezing tolerance of Arabidopsis in the CBF-independent pathway. In transgenic Arabidopsis carrying an MYBC1 promoter driven {beta}-glucuronidase (GUS) construct, GUS activity was observed in all tissues and was relatively stronger in the vascular tissues. Fused MYBC1 and GFP protein revealed that MYBC1 was localized exclusively in the nuclear compartment.

  13. Eukaryotic release factor 1-2 affects Arabidopsis responses to glucose and phytohormones during germination and early seedling development

    PubMed Central

    Zhou, Xiangjun; Cooke, Peter; Li, Li

    2010-01-01

    Germination and early seedling development are coordinately regulated by glucose and phytohormones such as ABA, GA, and ethylene. However, the molecules that affect plant responses to glucose and phytohormones remain to be fully elucidated. Eukaryotic release factor 1 (eRF1) is responsible for the recognition of the stop codons in mRNAs during protein synthesis. Accumulating evidence indicates that eRF1 functions in other processes in addition to translation termination. The physiological role of eRF1-2, a member of the eRF1 family, in Arabidopsis was examined here. The eRF1-2 gene was found to be specifically induced by glucose. Arabidopsis plants overexpressing eRF1-2 were hypersensitive to glucose during germination and early seedling development. Such hypersensitivity to glucose was accompanied by a dramatic reduction of the expression of glucose-regulated genes, chlorophyll a/b binding protein and plastocyanin. The hypersensitive response was not due to the enhanced accumulation of ABA. In addition, the eRF1-2 overexpressing plants showed increased sensitivity to paclobutrazol, an inhibitor of GA biosynthesis, and exogenous GA restored their normal growth. By contrast, the loss-of-function erf1-2 mutant exhibited resistance to paclobutrazol, suggesting that eRF1-2 may exert a negative effect on the GA signalling pathway. Collectively, these data provide evidence in support of a novel role of eRF1-2 in affecting glucose and phytohormone responses in modulating plant growth and development. PMID:19939886

  14. Novel NAC Transcription Factor TaNAC67 Confers Enhanced Multi-Abiotic Stress Tolerances in Arabidopsis

    PubMed Central

    Mao, Xinguo; Chen, Shuangshuang; Li, Ang; Zhai, Chaochao; Jing, Ruilian

    2014-01-01

    Abiotic stresses are major environmental factors that affect agricultural productivity worldwide. NAC transcription factors play pivotal roles in abiotic stress signaling in plants. As a staple crop, wheat production is severely constrained by abiotic stresses whereas only a few NAC transcription factors have been characterized functionally. To promote the application of NAC genes in wheat improvement by biotechnology, a novel NAC gene designated TaNAC67 was characterized in common wheat. To determine its role, transgenic Arabidopsis overexpressing TaNAC67-GFP controlled by the CaMV-35S promoter was generated and subjected to various abiotic stresses for morphological and physiological assays. Gene expression showed that TaNAC67 was involved in response to drought, salt, cold and ABA treatments. Localization assays revealed that TaNAC67 localized in the nucleus. Morphological analysis indicated the transgenics had enhanced tolerances to drought, salt and freezing stresses, simultaneously supported by enhanced expression of multiple abiotic stress responsive genes and improved physiological traits, including strengthened cell membrane stability, retention of higher chlorophyll contents and Na+ efflux rates, improved photosynthetic potential, and enhanced water retention capability. Overexpression of TaNAC67 resulted in pronounced enhanced tolerances to drought, salt and freezing stresses, therefore it has potential for utilization in transgenic breeding to improve abiotic stress tolerance in crops. PMID:24427285

  15. Phosphorylation of a WRKY Transcription Factor by MAPKs Is Required for Pollen Development and Function in Arabidopsis

    PubMed Central

    Guan, Yuefeng; Meng, Xiangzong; Khanna, Reshma; LaMontagne, Erica; Liu, Yidong; Zhang, Shuqun

    2014-01-01

    Plant male gametogenesis involves complex and dynamic changes in gene expression. At present, little is known about the transcription factors involved in this process and how their activities are regulated. Here, we show that a pollen-specific transcription factor, WRKY34, and its close homolog, WRKY2, are required for male gametogenesis in Arabidopsis thaliana. When overexpressed using LAT52, a strong pollen-specific promoter, epitope-tagged WRKY34 is temporally phosphorylated by MPK3 and MPK6, two mitogen-activated protein kinases (MAPKs, or MPKs), at early stages in pollen development. During pollen maturation, WRKY34 is dephosphorylated and degraded. Native promoter-driven WRKY34-YFP fusion also follows the same expression pattern at the protein level. WRKY34 functions redundantly with WRKY2 in pollen development, germination, and pollen tube growth. Loss of MPK3/MPK6 phosphorylation sites in WRKY34 compromises the function of WRKY34 in vivo. Epistasis interaction analysis confirmed that MPK6 belongs to the same genetic pathway of WRKY34 and WRKY2. Our study demonstrates the importance of temporal post-translational regulation of WRKY transcription factors in the control of developmental phase transitions in plants. PMID:24830428

  16. An Arabidopsis F-box protein acts as a transcriptional co-factor to regulate floral development.

    PubMed

    Chae, Eunyoung; Tan, Queenie K-G; Hill, Theresa A; Irish, Vivian F

    2008-04-01

    Plants flower in response to both environmental and endogenous signals. The Arabidopsis LEAFY (LFY) transcription factor is crucial in integrating these signals, and acts in part by activating the expression of multiple floral homeotic genes. LFY-dependent activation of the homeotic APETALA3 (AP3) gene requires the activity of UNUSUAL FLORAL ORGANS (UFO), an F-box component of an SCF ubiquitin ligase, yet how this regulation is effected has remained unclear. Here, we show that UFO physically interacts with LFY both in vitro and in vivo, and this interaction is necessary to recruit UFO to the AP3 promoter. Furthermore, a transcriptional repressor domain fused to UFO reduces endogenous LFY activity in plants, supporting the idea that UFO acts as part of a transcriptional complex at the AP3 promoter. Moreover, chemical or genetic disruption of proteasome activity compromises LFY-dependent AP3 activation, indicating that protein degradation is required to promote LFY activity. These results define an unexpected role for an F-box protein in functioning as a DNA-associated transcriptional co-factor in regulating floral homeotic gene expression. These results suggest a novel mechanism for promoting flower development via protein degradation and concomitant activation of the LFY transcription factor. This mechanism may be widely conserved, as homologs of UFO and LFY have been identified in a wide array of plant species. PMID:18287201

  17. Structural Basis for the Oligomerization of the MADS Domain Transcription Factor SEPALLATA3 in Arabidopsis[W

    PubMed Central

    Puranik, Sriharsha; Acajjaoui, Samira; Conn, Simon; Costa, Luca; Conn, Vanessa; Vial, Anthony; Marcellin, Romain; Melzer, Rainer; Brown, Elizabeth; Hart, Darren; Theißen, Günter; Silva, Catarina S.; Parcy, François; Dumas, Renaud; Nanao, Max; Zubieta, Chloe

    2014-01-01

    In plants, MADS domain transcription factors act as central regulators of diverse developmental pathways. In Arabidopsis thaliana, one of the most central members of this family is SEPALLATA3 (SEP3), which is involved in many aspects of plant reproduction, including floral meristem and floral organ development. SEP3 has been shown to form homo and heterooligomeric complexes with other MADS domain transcription factors through its intervening (I) and keratin-like (K) domains. SEP3 function depends on its ability to form specific protein-protein complexes; however, the atomic level determinants of oligomerization are poorly understood. Here, we report the 2.5-Å crystal structure of a small portion of the intervening and the complete keratin-like domain of SEP3. The domains form two amphipathic alpha helices separated by a rigid kink, which prevents intramolecular association and presents separate dimerization and tetramerization interfaces comprising predominantly hydrophobic patches. Mutations to the tetramerization interface demonstrate the importance of highly conserved hydrophobic residues for tetramer stability. Atomic force microscopy was used to show SEP3-DNA interactions and the role of oligomerization in DNA binding and conformation. Based on these data, the oligomerization patterns of the larger family of MADS domain transcription factors can be predicted and manipulated based on the primary sequence. PMID:25228343

  18. Enhancement of choleragen ADP-ribosyltransferase activities by guanyl nucleotides and a 19-kDa membrane protein.

    PubMed Central

    Tsai, S C; Noda, M; Adamik, R; Moss, J; Vaughan, M

    1987-01-01

    Choleragen activates adenylate cyclase by catalyzing, in the presence of NAD, the ADP-ribosylation of Gs alpha, the stimulatory guanyl nucleotide-binding protein of the cyclase system. Kahn and Gilman [Kahn, R. A. & Gilman, A. G. (1986) J. Biol. Chem. 261, 7906-7911] identified another guanyl nucleotide-binding protein termed ADP-ribosylation factor (ARF) that stimulated this reaction. It was proposed that the toxin substrate is an ARF-Gs alpha complex and that ARF may have a physiological role in regulation of Gs alpha activity. We have found that purified ARF from bovine brain enhances not only the ADP-ribosylation of Gs alpha but also Gs alpha-independent choleragen-catalyzed reactions. These are (i) ADP-ribosylation of agmatine, a low molecular weight guanidino compound; (ii) ADP-ribosylation of several proteins unrelated to Gs alpha; and (iii) auto-ADP-ribosylation of the toxin A1 peptide. These reactions, as well as the ADP-ribosylation of ARF itself, were stimulated by GTP or stable GTP analogues such as guanyl-5'-yl imido-beta gamma-diphosphate and guanosine 5'-O-[gamma-thio]triphosphate; GDP and guanosine 5'-O-[beta-thio]diphosphate were inactive. These observations are consistent with the conclusion that ARF interacts directly with the A subunit of choleragen in a GTP-dependent fashion thereby enhancing catalytic activity manifest as transfer of ADP-ribose to Gs alpha and other proteins, to the toxin A1 peptide, or to agmatine. It is tempting to speculate that ARF may be involved in regulating one or another of the ADP-ribosyltransferases found in animal cells. Images PMID:3110784

  19. CFL1, a WW Domain Protein, Regulates Cuticle Development by Modulating the Function of HDG1, a Class IV Homeodomain Transcription Factor, in Rice and Arabidopsis[W

    PubMed Central

    Wu, Renhong; Li, Shibai; He, Shan; Waßmann, Friedrich; Yu, Caihong; Qin, Genji; Schreiber, Lukas; Qu, Li-Jia; Gu, Hongya

    2011-01-01

    Plants have a chemically heterogeneous lipophilic layer, the cuticle, which protects them from biotic and abiotic stresses. The mechanisms that regulate cuticle development are poorly understood. We identified a rice (Oryza sativa) dominant curly leaf mutant, curly flag leaf1 (cfl1), and cloned CFL1, which encodes a WW domain protein. We overexpressed both rice and Arabidopsis CFL1 in Arabidopsis thaliana; these transgenic plants showed severely impaired cuticle development, similar to that in cfl1 rice. Reduced expression of At CFL1 resulted in reinforcement of cuticle structure. At CFL1 was predominantly expressed in specialized epidermal cells and in regions where dehiscence and abscission occur. Biochemical evidence showed that At CFL1 interacts with HDG1, a class IV homeodomain-leucine zipper transcription factor. Suppression of HDG1 function resulted in similar defective cuticle phenotypes in wild-type Arabidopsis but much alleviated phenotypes in At cfl1-1 mutants. The expression of two cuticle development-associated genes, BDG and FDH, was downregulated in At CFL1 overexpressor and HDG1 suppression plants. HDG1 binds to the cis-element L1 box, which exists in the regulatory regions of BDG and FDH. Our results suggest that rice and Arabidopsis CFL1 negatively regulate cuticle development by affecting the function of HDG1, which regulates the downstream genes BDG and FDH. PMID:21954461

  20. Tracking transcription factor mobility and interaction in Arabidopsis roots with fluorescence correlation spectroscopy

    PubMed Central

    Clark, Natalie M; Hinde, Elizabeth; Winter, Cara M; Fisher, Adam P; Crosti, Giuseppe; Blilou, Ikram; Gratton, Enrico; Benfey, Philip N; Sozzani, Rosangela

    2016-01-01

    To understand complex regulatory processes in multicellular organisms, it is critical to be able to quantitatively analyze protein movement and protein-protein interactions in time and space. During Arabidopsis development, the intercellular movement of SHORTROOT (SHR) and subsequent interaction with its downstream target SCARECROW (SCR) control root patterning and cell fate specification. However, quantitative information about the spatio-temporal dynamics of SHR movement and SHR-SCR interaction is currently unavailable. Here, we quantify parameters including SHR mobility, oligomeric state, and association with SCR using a combination of Fluorescent Correlation Spectroscopy (FCS) techniques. We then incorporate these parameters into a mathematical model of SHR and SCR, which shows that SHR reaches a steady state in minutes, while SCR and the SHR-SCR complex reach a steady-state between 18 and 24 hr. Our model reveals the timing of SHR and SCR dynamics and allows us to understand how protein movement and protein-protein stoichiometry contribute to development. DOI: http://dx.doi.org/10.7554/eLife.14770.001 PMID:27288545

  1. Arabidopsis cotyledon-specific chloroplast biogenesis factor CYO1 is a protein disulfide isomerase.

    PubMed

    Shimada, Hiroshi; Mochizuki, Mariko; Ogura, Kan; Froehlich, John E; Osteryoung, Katherine W; Shirano, Yumiko; Shibata, Daisuke; Masuda, Shinji; Mori, Kazuki; Takamiya, Ken-Ichiro

    2007-10-01

    Chloroplast development in cotyledons differs in a number of ways from that in true leaves, but the cotyledon-specific program of chloroplast biogenesis has not been clarified. The cyo1 mutant in Arabidopsis thaliana has albino cotyledons but normal green true leaves. Chloroplasts develop abnormally in cyo1 mutant plants grown in the light, but etioplasts are normal in mutants grown in the dark. We isolated CYO1 by T-DNA tagging and verified that the mutant allele was responsible for the albino cotyledon phenotype by complementation. CYO1 has a C(4)-type zinc finger domain similar to that of Escherichia coli DnaJ. CYO1 is expressed mainly in young plants under light conditions, and the CYO1 protein localizes to the thylakoid membrane in chloroplasts. Transcription of nuclear photosynthetic genes is generally unaffected by the cyo1 mutation, but the level of photosynthetic proteins is decreased in cyo1 mutants. Recombinant CYO1 accelerates disulfide bond reduction in the model substrate insulin and renatures RNase A, indicating that CYO1 has protein disulfide isomerase activity. These results suggest that CYO1 has a chaperone-like activity required for thylakoid biogenesis in cotyledons. PMID:17921316

  2. Identification and Roles of Photosystem II Assembly, Stability, and Repair Factors in Arabidopsis.

    PubMed

    Lu, Yan

    2016-01-01

    Photosystem II (PSII) is a multi-component pigment-protein complex that is responsible for water splitting, oxygen evolution, and plastoquinone reduction. Components of PSII can be classified into core proteins, low-molecular-mass proteins, extrinsic oxygen-evolving complex (OEC) proteins, and light-harvesting complex II proteins. In addition to these PSII subunits, more than 60 auxiliary proteins, enzymes, or components of thylakoid protein trafficking/targeting systems have been discovered to be directly or indirectly involved in de novo assembly and/or the repair and reassembly cycle of PSII. For example, components of thylakoid-protein-targeting complexes and the chloroplast-vesicle-transport system were found to deliver PSII subunits to thylakoid membranes. Various auxiliary proteins, such as PsbP-like (Psb stands for PSII) and light-harvesting complex-like proteins, atypical short-chain dehydrogenase/reductase family proteins, and tetratricopeptide repeat proteins, were discovered to assist the de novo assembly and stability of PSII and the repair and reassembly cycle of PSII. Furthermore, a series of enzymes were discovered to catalyze important enzymatic steps, such as C-terminal processing of the D1 protein, thiol/disulfide-modulation, peptidylprolyl isomerization, phosphorylation and dephosphorylation of PSII core and antenna proteins, and degradation of photodamaged PSII proteins. This review focuses on the current knowledge of the identities and molecular functions of different types of proteins that influence the assembly, stability, and repair of PSII in the higher plant Arabidopsis thaliana. PMID:26909098

  3. Ligand Recognition by the TPR Domain of the Import Factor Toc64 from Arabidopsis thaliana

    PubMed Central

    Panigrahi, Rashmi; Adina-Zada, Abdussalam; Whelan, James; Vrielink, Alice

    2013-01-01

    The specific targeting of protein to organelles is achieved by targeting signals being recognised by their cognate receptors. Cytosolic chaperones, bound to precursor proteins, are recognized by specific receptors of the import machinery enabling transport into the specific organelle. The aim of this study was to gain greater insight into the mode of recognition of the C-termini of Hsp70 and Hsp90 chaperones by the Tetratricopeptide Repeat (TPR) domain of the chloroplast import receptor Toc64 from Arabidopsis thaliana (At). The monomeric TPR domain binds with 1∶1 stoichiometry in similar micromolar affinity to both Hsp70 and Hsp90 as determined by isothermal titration calorimetry (ITC). Mutations of the terminal EEVD motif caused a profound decrease in affinity. Additionally, this study considered the contributions of residues upstream as alanine scanning experiments of these residues showed reduced binding affinity. Molecular dynamics simulations of the TPR domain helices upon peptide binding predicted that two helices within the TPR domain move backwards, exposing the cradle surface for interaction with the peptide. Our findings from ITC and molecular dynamics studies suggest that AtToc64_TPR does not discriminate between C-termini peptides of Hsp70 and Hsp90. PMID:24391770

  4. Tracking transcription factor mobility and interaction in Arabidopsis roots with fluorescence correlation spectroscopy.

    PubMed

    Clark, Natalie M; Hinde, Elizabeth; Winter, Cara M; Fisher, Adam P; Crosti, Giuseppe; Blilou, Ikram; Gratton, Enrico; Benfey, Philip N; Sozzani, Rosangela

    2016-01-01

    To understand complex regulatory processes in multicellular organisms, it is critical to be able to quantitatively analyze protein movement and protein-protein interactions in time and space. During Arabidopsis development, the intercellular movement of SHORTROOT (SHR) and subsequent interaction with its downstream target SCARECROW (SCR) control root patterning and cell fate specification. However, quantitative information about the spatio-temporal dynamics of SHR movement and SHR-SCR interaction is currently unavailable. Here, we quantify parameters including SHR mobility, oligomeric state, and association with SCR using a combination of Fluorescent Correlation Spectroscopy (FCS) techniques. We then incorporate these parameters into a mathematical model of SHR and SCR, which shows that SHR reaches a steady state in minutes, while SCR and the SHR-SCR complex reach a steady-state between 18 and 24 hr. Our model reveals the timing of SHR and SCR dynamics and allows us to understand how protein movement and protein-protein stoichiometry contribute to development. PMID:27288545

  5. Identification and Roles of Photosystem II Assembly, Stability, and Repair Factors in Arabidopsis

    PubMed Central

    Lu, Yan

    2016-01-01

    Photosystem II (PSII) is a multi-component pigment-protein complex that is responsible for water splitting, oxygen evolution, and plastoquinone reduction. Components of PSII can be classified into core proteins, low-molecular-mass proteins, extrinsic oxygen-evolving complex (OEC) proteins, and light-harvesting complex II proteins. In addition to these PSII subunits, more than 60 auxiliary proteins, enzymes, or components of thylakoid protein trafficking/targeting systems have been discovered to be directly or indirectly involved in de novo assembly and/or the repair and reassembly cycle of PSII. For example, components of thylakoid-protein-targeting complexes and the chloroplast-vesicle-transport system were found to deliver PSII subunits to thylakoid membranes. Various auxiliary proteins, such as PsbP-like (Psb stands for PSII) and light-harvesting complex-like proteins, atypical short-chain dehydrogenase/reductase family proteins, and tetratricopeptide repeat proteins, were discovered to assist the de novo assembly and stability of PSII and the repair and reassembly cycle of PSII. Furthermore, a series of enzymes were discovered to catalyze important enzymatic steps, such as C-terminal processing of the D1 protein, thiol/disulfide-modulation, peptidylprolyl isomerization, phosphorylation and dephosphorylation of PSII core and antenna proteins, and degradation of photodamaged PSII proteins. This review focuses on the current knowledge of the identities and molecular functions of different types of proteins that influence the assembly, stability, and repair of PSII in the higher plant Arabidopsis thaliana. PMID:26909098

  6. A NAC transcription factor and SNI1 cooperatively suppress basal pathogen resistance in Arabidopsis thaliana

    PubMed Central

    Kim, Ho Soo; Park, Hyeong Cheol; Kim, Kyung Eun; Jung, Mi Soon; Han, Hay Ju; Kim, Sun Ho; Kwon, Young Sang; Bahk, Sunghwa; An, Jonguk; Bae, Dong Won; Yun, Dae-Jin; Kwak, Sang-Soo; Chung, Woo Sik

    2012-01-01

    Transcriptional repression of pathogen defense-related genes is essential for plant growth and development. Several proteins are known to be involved in the transcriptional regulation of plant defense responses. However, mechanisms by which expression of defense-related genes are regulated by repressor proteins are poorly characterized. Here, we describe the in planta function of CBNAC, a calmodulin-regulated NAC transcriptional repressor in Arabidopsis. A T-DNA insertional mutant (cbnac1) displayed enhanced resistance to a virulent strain of the bacterial pathogen Pseudomonas syringae DC3000 (PstDC3000), whereas resistance was reduced in transgenic CBNAC overexpression lines. The observed changes in disease resistance were correlated with alterations in pathogenesis-related protein 1 (PR1) gene expression. CBNAC bound directly to the PR1 promoter. SNI1 (suppressor of nonexpressor of PR genes1, inducible 1) was identified as a CBNAC-binding protein. Basal resistance to PstDC3000 and derepression of PR1 expression was greater in the cbnac1 sni1 double mutant than in either cbnac1 or sni1 mutants. SNI1 enhanced binding of CBNAC to its cognate PR1 promoter element. CBNAC and SNI1 are hypothesized to work as repressor proteins in the cooperative suppression of plant basal defense. PMID:22826500

  7. Molecular cloning of the black tiger shrimp (Penaeus monodon) elongation factor 2 (EF-2): sequence analysis and its expression on the ovarian maturation stage.

    PubMed

    Qiu, Lihua; Jiang, Shigui; Zhou, Falin; Zhang, Dianchang; Huang, Jianhua; Guo, Yihui

    2008-09-01

    The techniques of homology cloning and anchored PCR were used to clone the elongation factor 2 (EF-2) gene from black tiger shrimp (Penaeus monodon). The full length cDNA of black tiger shrimp EF-2 (btsEF-2) contained a 5' untranslated region (UTR) of 73 bp, an ORF of 2541 bp encoding a polypeptide of 846 amino acids with an estimated molecular mass of 95 kDa, and a 3( UTR of 112 bp. The searches for protein sequence similarities with BLAST analysis indicated that the deduced amino acid sequence of btsEF-2 was homological to the EF-2 of other species and even the mammalians. The conserved signature sequence of EF-2 gene family, GTPase effector domain and ADP-ribosylation domain were found in the btsEF-2 deduced amino acid sequence. The temporal expressions of gene in the different ovarian stages were measured by real time PCR. The mRNA expressions of the gene were constitutively expressed in ovary and different during the maturation stages. The result indicated that EF-2 gene was constitutively expressed and could play a critical role in the ovarian maturation stage. PMID:17629788

  8. β-Amylase–Like Proteins Function as Transcription Factors in Arabidopsis, Controlling Shoot Growth and Development[C][W][OA

    PubMed Central

    Reinhold, Heike; Soyk, Sebastian; Šimková, Klára; Hostettler, Carmen; Marafino, John; Mainiero, Samantha; Vaughan, Cara K.; Monroe, Jonathan D.; Zeeman, Samuel C.

    2011-01-01

    Plants contain β-amylase–like proteins (BAMs; enzymes usually associated with starch breakdown) present in the nucleus rather than targeted to the chloroplast. They possess BRASSINAZOLE RESISTANT1 (BZR1)-type DNA binding domains—also found in transcription factors mediating brassinosteroid (BR) responses. The two Arabidopsis thaliana BZR1-BAM proteins (BAM7 and BAM8) bind a cis-regulatory element that both contains a G box and resembles a BR-responsive element. In protoplast transactivation assays, these BZR1-BAMs activate gene expression. Structural modeling suggests that the BAM domain’s glucan binding cleft is intact, but the recombinant proteins are at least 1000 times less active than chloroplastic β-amylases. Deregulation of BZR1-BAMs (the bam7bam8 double mutant and BAM8-overexpressing plants) causes altered leaf growth and development. Of the genes upregulated in plants overexpressing BAM8 and downregulated in bam7bam8 plants, many carry the cis-regulatory element in their promoters. Many genes that respond to BRs are inversely regulated by BZR1-BAMs. We propose a role for BZR1-BAMs in controlling plant growth and development through crosstalk with BR signaling. Furthermore, we speculate that BZR1-BAMs may transmit metabolic signals by binding a ligand in their BAM domain, although diurnal changes in the concentration of maltose, a candidate ligand produced by chloroplastic β-amylases, do not influence their transcription factor function. PMID:21487098

  9. ZmNAC55, a maize stress-responsive NAC transcription factor, confers drought resistance in transgenic Arabidopsis.

    PubMed

    Mao, Hude; Yu, Lijuan; Han, Ran; Li, Zhanjie; Liu, Hui

    2016-08-01

    Abiotic stress has been shown to significantly limit the growth and productivity of crops. NAC transcription factors play essential roles in response to various abiotic stresses. However, only little information regarding stress-related NAC genes is available in maize. Here, we cloned a maize NAC transcription factor ZmNAC55 and identified its function in drought stress. Transient expression and transactivation assay demonstrated that ZmNAC55 was localized in the nucleus and had transactivation activity. Expression analysis of ZmNAC55 in maize showed that this gene was induced by drought, high salinity and cold stresses and by abscisic acid (ABA). Promoter analysis demonstrated that multiple stress-related cis-acting elements exist in promoter region of ZmNAC55. Overexpression of ZmNAC55 in Arabidopsis led to hypersensitivity to ABA at the germination stage, but enhanced drought resistence compared to wild-type seedlings. Transcriptome analysis identified a number of differentially expressed genes between 35S::ZmNAC55 transgenic and wild-type plants, and many of which are involved in stress response, including twelve qRT-PCR confirmed well-known drought-responsive genes. These results highlight the important role of ZmNAC55 in positive regulates of drought resistence, and may have potential applications in transgenic breeding of drought-tolerant crops. PMID:27085597

  10. The NAC transcription factor ANAC046 is a positive regulator of chlorophyll degradation and senescence in Arabidopsis leaves

    PubMed Central

    Oda-Yamamizo, Chihiro; Mitsuda, Nobutaka; Sakamoto, Shingo; Ogawa, Daisuke; Ohme-Takagi, Masaru; Ohmiya, Akemi

    2016-01-01

    Chlorophyll (Chl) degradation occurs during leaf senescence, embryo degreening, bud breaking, and fruit ripening. The Chl catabolic pathway has been intensively studied and nearly all the enzymes involved are identified and characterized; however, the molecular regulatory mechanisms of this pathway are largely unknown. In this study, we performed yeast one-hybrid screening using a transcription factor cDNA library to search for factors controlling the expression of Chl catabolic genes. We identified ANAC046 as a common regulator that directly binds to the promoter regions of NON-YELLOW COLORING1, STAY-GREEN1 (SGR1), SGR2, and PHEOPHORBIDE a OXYGENASE. Transgenic plants overexpressing ANAC046 exhibited an early-senescence phenotype and a lower Chl content in comparison with the wild-type plants, whereas loss-of-function mutants exhibited a delayed-senescence phenotype and a higher Chl content. Microarray analysis of ANAC046 transgenic plants showed that not only Chl catabolic genes but also senescence-associated genes were positively regulated by ANAC046. We conclude that ANAC046 is a positive regulator of Arabidopsis leaf senescence and exerts its effect by controlling the expression of Chl catabolic genes and senescence-associated genes. PMID:27021284

  11. Orchestration of the Floral Transition and Floral Development in Arabidopsis by the Bifunctional Transcription Factor APETALA2[W][OA

    PubMed Central

    Yant, Levi; Mathieu, Johannes; Dinh, Thanh Theresa; Ott, Felix; Lanz, Christa; Wollmann, Heike; Chen, Xuemei; Schmid, Markus

    2010-01-01

    The Arabidopsis thaliana transcription factor APETALA2 (AP2) has numerous functions, including roles in seed development, stem cell maintenance, and specification of floral organ identity. To understand the relationship between these different roles, we mapped direct targets of AP2 on a genome-wide scale in two tissue types. We find that AP2 binds to thousands of loci in the developing flower, many of which exhibit AP2-dependent transcription. Opposing, logical effects are evident in AP2 binding to two microRNA genes that influence AP2 expression, with AP2 positively regulating miR156 and negatively regulating miR172, forming a complex direct feedback loop, which also included all but one of the AP2-like miR172 target clade members. We compare the genome-wide direct target repertoire of AP2 with that of SCHLAFMÜTZE, a closely related transcription factor that also represses the transition to flowering. We detect clear similarities and important differences in the direct target repertoires that are also tissue specific. Finally, using an inducible expression system, we demonstrate that AP2 has dual molecular roles. It functions as both a transcriptional activator and repressor, directly inducing the expression of the floral repressor AGAMOUS-LIKE15 and directly repressing the transcription of floral activators like SUPPRESSOR OF OVEREXPRESSION OF CONSTANS1. PMID:20675573

  12. LLM-Domain B-GATA Transcription Factors Promote Stomatal Development Downstream of Light Signaling Pathways in Arabidopsis thaliana Hypocotyls.

    PubMed

    Klermund, Carina; Ranftl, Quirin L; Diener, Julia; Bastakis, Emmanouil; Richter, René; Schwechheimer, Claus

    2016-03-01

    Stomata are pores that regulate the gas and water exchange between the environment and aboveground plant tissues, including hypocotyls, leaves, and stems. Here, we show that mutants of Arabidopsis thaliana LLM-domain B-GATA genes are defective in stomata formation in hypocotyls. Conversely, stomata formation is strongly promoted by overexpression of various LLM-domain B-class GATA genes, most strikingly in hypocotyls but also in cotyledons. Genetic analyses indicate that these B-GATAs act upstream of the stomata formation regulators SPEECHLESS(SPCH), MUTE, and SCREAM/SCREAM2 and downstream or independent of the patterning regulators TOO MANY MOUTHS and STOMATAL DENSITY AND DISTRIBUTION1 The effects of the GATAs on stomata formation are light dependent but can be induced in dark-grown seedlings by red, far-red, or blue light treatments. PHYTOCHROME INTERACTING FACTOR(PIF) mutants form stomata in the dark, and in this genetic background, GATA expression is sufficient to induce stomata formation in the dark. Since the expression of the LLM-domain B-GATAs GNC(GATA, NITRATE-INDUCIBLE, CARBON METABOLISM-INVOLVED) and GNC-LIKE/CYTOKININ-RESPONSIVE GATA FACTOR1 as well as that of SPCH is red light induced but the induction of SPCH is compromised in a GATA gene mutant background, we hypothesize that PIF- and light-regulated stomata formation in hypocotyls is critically dependent on LLM-domain B-GATA genes. PMID:26917680

  13. Arabidopsis DE-ETIOLATED1 Represses Photomorphogenesis by Positively Regulating Phytochrome-Interacting Factors in the Dark[C][W

    PubMed Central

    Dong, Jie; Tang, Dafang; Gao, Zhaoxu; Yu, Renbo; Li, Kunlun; He, Hang; Terzaghi, William; Deng, Xing Wang

    2014-01-01

    Arabidopsis thaliana seedlings undergo photomorphogenic development even in darkness when the function of DE-ETIOLATED1 (DET1), a repressor of photomorphogenesis, is disrupted. However, the mechanism by which DET1 represses photomorphogenesis remains unclear. Our results indicate that DET1 directly interacts with a group of transcription factors known as the phytochrome-interacting factors (PIFs). Furthermore, our results suggest that DET1 positively regulates PIF protein levels primarily by stabilizing PIF proteins in the dark. Genetic analysis showed that each pif single mutant could enhance the det1-1 phenotype, and ectopic expression of each PIF in det1-1 partially suppressed the det1-1 phenotype, based on hypocotyl elongation and cotyledon opening angles observed in darkness. Genomic analysis also revealed that DET1 may modulate the expression of light-regulated genes to mediate photomorphogenesis partially through PIFs. The observed interaction and regulation between DET1 and PIFs not only reveal how DET1 represses photomorphogenesis, but also suggest a possible mechanism by which two groups of photomorphogenic repressors, CONSTITUTIVE PHOTOMORPHOGENESIS/DET/FUSCA and PIFs, work in concert to repress photomorphogenesis in darkness. PMID:25248553

  14. AtERF71/HRE2 transcription factor mediates osmotic stress response as well as hypoxia response in Arabidopsis.

    PubMed

    Park, Hee-Yeon; Seok, Hye-Yeon; Woo, Dong-Hyuk; Lee, Sun-Young; Tarte, Vaishali N; Lee, Eun-Hye; Lee, Choon-Hwan; Moon, Yong-Hwan

    2011-10-14

    Various transcription factors are involved in the response to environmental stresses in plants. In this study, we characterized AtERF71/HRE2, a member of the Arabidopsis AP2/ERF family, as an important regulator of the osmotic and hypoxic stress responses in plants. Transcript level of AtERF71/HRE2 was highly increased by anoxia, NaCl, mannitol, ABA, and MV treatments. aterf71/hre2 loss-of-function mutants displayed higher sensitivity to osmotic stress such as high salt and mannitol, accumulating higher levels of ROS under high salt treatment. In contrast, AtERF71/HRE2-overexpressing transgenic plants showed tolerance to salt and mannitol as well as flooding and MV stresses, exhibiting lower levels of ROS under high salt treatment. AtERF71/HRE2 protein was localized in the nucleus, and the C-terminal region of AtERF71/HRE2 was required for transcription activation activity. Taken together, our results suggest that AtERF71/HRE2 might function as a transcription factor involved in the response to osmotic stress as well as hypoxia. PMID:21946064

  15. Salt-induced transcription factor MYB74 is regulated by the RNA-directed DNA methylation pathway in Arabidopsis

    PubMed Central

    Xu, Rui; Wang, Yuhan; Zheng, Hao; Lu, Wei; Wu, Changai; Huang, Jinguang; Yan, Kang; Yang, Guodong; Zheng, Chengchao

    2015-01-01

    Salt stress is one of the major abiotic stresses in agriculture worldwide that causes crop failure by interfering with the profile of gene expression and cell metabolism. Transcription factors and RNA-directed DNA methylation (RdDM) play an important role in the regulation of gene activation under abiotic stress in plants. This work characterized AtMYB74, a member of the R2R3-MYB gene family, which is transcriptionally regulated mainly by RdDM as a response in salt stress in Arabidopsis. Bisulphite sequencing indicated that 24-nt siRNAs target a region approximately 500bp upstream of the transcription initiation site of AtMYB74, which is heavily methylated. Levels of DNA methylation in this region were significantly reduced in wild type plants under salt stress, whereas no changes were found in RdDM mutants. Northern blot and quantitative real-time reverse transcription PCR analysis showed that the accumulation of 24-nt siRNAs was decreased in WT plants under salt stress. Further promoter deletion analysis revealed that the siRNA target region is essential for maintaining AtMYB74 expression patterns. In addition, transgenic plants overexpressing AtMYB74 displayed hypersensitivity to NaCl during seed germination. These results suggest that changes in the levels of the five 24-nt siRNAs regulate the AtMYB74 transcription factor via RdDM in response to salt stress. PMID:26139822

  16. Overexpression of a NF-YB3 transcription factor from Picea wilsonii confers tolerance to salinity and drought stress in transformed Arabidopsis thaliana.

    PubMed

    Zhang, Tong; Zhang, Dun; Liu, Yajing; Luo, Chaobing; Zhou, Yanni; Zhang, Lingyun

    2015-09-01

    Nuclear factor Y (NF-Y) is a highly conserved transcription factor comprising NF-YA, NF-YB and NF-YC subunits. To date, the roles of NF-Y subunit in plant still remain elusive. In this study, a subunit NF-YB (PwNF-YB3), was isolated from Picea wilsonii Mast. and its role was studied. PwNF-YB3 transcript was detected in all vegetative and reproductive tissues with higher levels in stem and root and was greatly induced by salinity, heat and PEG but not by cold and ABA treatment. Over-expression of PwNF-YB3 in Arabidopsis showed a significant acceleration in the onset of flowering and resulted in more vigorous seed germination and significant tolerance for seedlings under salinity, drought and osmotic stress compared with wild type plants. Transcription levels of salinity-responsive gene (SOS3) and drought-induced gene (CDPK1) were substantially higher in transgenic Arabidopsis than in wild-type plants. Importantly, CBF pathway markers (COR15B, KIN1, LEA76), but not ABA pathway markers CBF4, were greatly induced under condition of drought. The nuclear localization showed that NF-YB3 acted as a transcription factor. Taken together, the data provide evidence that PwNF-YB3 positively confers significant tolerance to salt, osmotic and drought stress in transformed Arabidopsis plants probably through modulating gene regulation in CBF-dependent pathway. PMID:26093308

  17. Overexpression of a citrus basic helix-loop-helix transcription factor (CubHLH1), which is homologous to Arabidopsis activation-tagged bri1 suppressor 1 interacting factor genes, modulates carotenoid metabolism in transgenic tomato.

    PubMed

    Endo, Tomoko; Fujii, Hiroshi; Sugiyama, Aiko; Nakano, Michiharu; Nakajima, Naoko; Ikoma, Yoshinori; Omura, Mitsuo; Shimada, Takehiko

    2016-02-01

    To explore the transcription factors associated with carotenoid metabolism in citrus fruit, one transcription factor (CubHLH1) was selected through microarray screening in Satsuma mandarin (Citrus unshiu Marc.) fruit, which was treated with exogenous ethylene or gibberellin (GA), accelerating or retarding carotenoid accumulation in peel, respectively. The amino acid sequence of CubHLH1 has homology to Arabidopsis activation-tagged bri1 suppressor 1 (ATBS1) interacting factor (AIF), which is functionally characterized as a negative regulator of the brassinolide (BR) signalling pathway. Yeast two-hybrid analysis revealed that protein for CubHLH1 could interact with Arabidopsis and tomato ATBS1. Overexpression of CubHLH1 caused a dwarf phenotype in transgenic tomato (Solanum lycopersicum L.), suggesting that CubHLH1 has a similar function to Arabidopsis AIF. In the transgenic tomato fruit at ripening stage, the lycopene content was reduced along with the changes in carotenoid biosynthetic gene expression. The abscisic acid (ABA) content of all the transgenic tomato fruit was higher than that of the wild type. These results implied that CubHLH1 is considered to have a similar function to Arabidopsis AIFs and might be directly involved in carotenoid metabolism in mature citrus fruit. PMID:26795149

  18. MTR4, a putative RNA helicase and exosome co-factor, is required for proper rRNA biogenesis and development in Arabidopsis thaliana.

    PubMed

    Lange, Heike; Sement, François M; Gagliardi, Dominique

    2011-10-01

    The exosome is a conserved protein complex that is responsible for essential 3'→5' RNA degradation in both the nucleus and the cytosol. It is composed of a nine-subunit core complex to which co-factors confer both RNA substrate recognition and ribonucleolytic activities. Very few exosome co-factors have been identified in plants. Here, we have characterized a putative RNA helicase, AtMTR4, that is involved in the degradation of several nucleolar exosome substrates in Arabidopsis thaliana. We show that AtMTR4, rather than its closely related protein HEN2, is required for proper rRNA biogenesis in Arabidopsis. AtMTR4 is mostly localized in the nucleolus, a subcellular compartmentalization that is shared with another exosome co-factor, RRP6L2. AtMTR4 and RRP6L2 cooperate in several steps of rRNA maturation and surveillance, such as processing the 5.8S rRNA and removal of rRNA maturation by-products. Interestingly, degradation of the Arabidopsis 5' external transcribed spacer (5' ETS) requires cooperation of both the 5'→3' and 3'→5' exoribonucleolytic pathways. Accumulating AtMTR4 targets give rise to illegitimate small RNAs; however, these do not affect rRNA metabolism or contribute to the phenotype of mtr4 mutants. Plants lacking AtMTR4 are viable but show several developmental defects, including aberrant vein patterning and pointed first leaves. The mtr4 phenotype resembles that of several ribosomal protein and nucleolin mutants, and may be explained by delayed ribosome biogenesis, as we observed a reduced rate of rRNA accumulation in mtr4 mutants. Taken together, these data link AtMTR4 with rRNA biogenesis and development in Arabidopsis. PMID:21682783

  19. Prediction of Regulatory Interactions from Genome Sequences Using a Biophysical Model for the Arabidopsis LEAFY Transcription Factor[C][W

    PubMed Central

    Moyroud, Edwige; Minguet, Eugenio Gómez; Ott, Felix; Yant, Levi; Posé, David; Monniaux, Marie; Blanchet, Sandrine; Bastien, Olivier; Thévenon, Emmanuel; Weigel, Detlef; Schmid, Markus; Parcy, François

    2011-01-01

    Despite great advances in sequencing technologies, generating functional information for nonmodel organisms remains a challenge. One solution lies in an improved ability to predict genetic circuits based on primary DNA sequence in combination with detailed knowledge of regulatory proteins that have been characterized in model species. Here, we focus on the LEAFY (LFY) transcription factor, a conserved master regulator of floral development. Starting with biochemical and structural information, we built a biophysical model describing LFY DNA binding specificity in vitro that accurately predicts in vivo LFY binding sites in the Arabidopsis thaliana genome. Applying the model to other plant species, we could follow the evolution of the regulatory relationship between LFY and the AGAMOUS (AG) subfamily of MADS box genes and show that this link predates the divergence between monocots and eudicots. Remarkably, our model succeeds in detecting the connection between LFY and AG homologs despite extensive variation in binding sites. This demonstrates that the cis-element fluidity recently observed in animals also exists in plants, but the challenges it poses can be overcome with predictions grounded in a biophysical model. Therefore, our work opens new avenues to deduce the structure of regulatory networks from mere inspection of genomic sequences. PMID:21515819

  20. Arabidopsis IRE1 catalyses unconventional splicing of bZIP60 mRNA to produce the active transcription factor

    PubMed Central

    Nagashima, Yukihiro; Mishiba, Kei-ichiro; Suzuki, Eiji; Shimada, Yukihisa; Iwata, Yuji; Koizumi, Nozomu

    2011-01-01

    IRE1 plays an essential role in the endoplasmic reticulum (ER) stress response in yeast and mammals. We found that a double mutant of Arabidopsis IRE1A and IRE1B (ire1a/ire1b) is more sensitive to the ER stress inducer tunicamycin than the wild-type. Transcriptome analysis revealed that genes whose induction was reduced in ire1a/ire1b largely overlapped those in the bzip60 mutant. We observed that the active form of bZIP60 protein detected in the wild-type was missing in ire1a/ire1b. We further demonstrated that bZIP60 mRNA is spliced by ER stress, removing 23 ribonucleotides and therefore causing a frameshift that replaces the C-terminal region of bZIP60 including the transmembrane domain (TMD) with a shorter region without a TMD. This splicing was detected in ire1a and ire1b single mutants, but not in the ire1a/ire1b double mutant. We conclude that IRE1A and IRE1B catalyse unconventional splicing of bZIP60 mRNA to produce the active transcription factor. PMID:22355548

  1. The MADS transcription factor XAL2/AGL14 modulates auxin transport during Arabidopsis root development by regulating PIN expression

    PubMed Central

    Garay-Arroyo, Adriana; Ortiz-Moreno, Enrique; de la Paz Sánchez, María; Murphy, Angus S; García-Ponce, Berenice; Marsch-Martínez, Nayelli; de Folter, Stefan; Corvera-Poiré, Adriana; Jaimes-Miranda, Fabiola; Pacheco-Escobedo, Mario A; Dubrovsky, Joseph G; Pelaz, Soraya; Álvarez-Buylla, Elena R

    2013-01-01

    Elucidating molecular links between cell-fate regulatory networks and dynamic patterning modules is a key for understanding development. Auxin is important for plant patterning, particularly in roots, where it establishes positional information for cell-fate decisions. PIN genes encode plasma membrane proteins that serve as auxin efflux transporters; mutations in members of this gene family exhibit smaller roots with altered root meristems and stem-cell patterning. Direct regulators of PIN transcription have remained elusive. Here, we establish that a MADS-box gene (XAANTAL2, XAL2/AGL14) controls auxin transport via PIN transcriptional regulation during Arabidopsis root development; mutations in this gene exhibit altered stem-cell patterning, root meristem size, and root growth. XAL2 is necessary for normal shootward and rootward auxin transport, as well as for maintaining normal auxin distribution within the root. Furthermore, this MADS-domain transcription factor upregulates PIN1 and PIN4 by direct binding to regulatory regions and it is required for PIN4-dependent auxin response. In turn, XAL2 expression is regulated by auxin levels thus establishing a positive feedback loop between auxin levels and PIN regulation that is likely to be important for robust root patterning. PMID:24121311

  2. The MADS domain factors AGL15 and AGL18 act redundantly as repressors of the floral transition in Arabidopsis.

    PubMed

    Adamczyk, Benjamin J; Lehti-Shiu, Melissa D; Fernandez, Donna E

    2007-06-01

    The developmental roles of AGL15 and AGL18, members of the AGL15-like clade of MADS domain regulatory factors, have not been defined previously. Analysis of transgenic Arabidopsis plants showed that overexpression of AGL18 produces the same phenotypic changes as overexpression of AGL15, and the two genes have partially overlapping expression patterns. Functional redundancy was confirmed through analysis of loss-of-function mutants. agl15 agl18 double mutants, but not single mutants, flower early under non-inductive conditions, indicating that AGL15 and AGL18 act in a redundant fashion as repressors of the floral transition. Further genetic analyses and expression studies were used to examine the relationship between AGL15 and AGL18 activity and other regulators of the floral transition. AGL15 and AGL18 act upstream of the floral integrator FT, and a combination of agl15 and agl18 mutations partially suppresses defects in the photoperiod pathway. agl15 agl18 mutations show an additive relationship with mutations in genes encoding other MADS domain floral repressors, and further acceleration of flowering is seen in triple and quadruple mutants under both inductive and non-inductive conditions. Thus, flowering time is determined by the additive effect of multiple MADS domain floral repressors, with important contributions from AGL15 and AGL18. PMID:17521410

  3. Regulation of Arabidopsis thaliana plasma membrane glucose-responsive regulator (AtPGR) expression by A. thaliana storekeeper-like transcription factor, AtSTKL, modulates glucose response in Arabidopsis.

    PubMed

    Chung, Moon-Soo; Lee, Sungbeom; Min, Ji-Hee; Huang, Ping; Ju, Hyun-Woo; Kim, Cheol Soo

    2016-07-01

    Biochemical, genetic, physiological, and molecular research in plants has demonstrated a central role of glucose (Glc) in the control of plant growth, metabolism, and development, and has revealed networks that integrate light, stresses, nutrients, and hormone signaling. Previous studies have reported that AtPGR protein as potential candidates for Glc signaling protein. In the present study, we characterized transcription factors that bind to the upstream region of the AtPGR gene isolated using the yeast one-hybrid screening with an Arabidopsis cDNA library. One of the selected genes (AtSTKL) appeared to confer elevated sensitivity to Glc response. Overexpression of AtSTKLs (AtSTKL1 and AtSTKL2) increased the sensitivity to Glc during the post-germination stages. In contrast, atstkl1 and atstkl2 antisense lines displayed reduced sensitivity to high Glc concentration during the early seedling stage. Furthermore, we showed that the two AtSTKLs bind to the 5'-GCCT-3' element of the upstream promoter region of the AtPGR gene in vitro and repress the beta-glucuronidase (GUS) activity in AtPGR promoter-GUS (P999-GUS) transgenic plants. Green fluorescent protein (GFP)-tagged AtSTKLs were localized in the nuclei of transgenic Arabidopsis cells. Collectively, these results suggest that AtSTKL1 and AtSTKL2 function both as repressors of AtPGR transcription and as novel transcription factors in the Glc signaling pathway. PMID:27031427

  4. Mechanism of cholera toxin activation by a guanine nucleotide-dependent 19 kDa protein.

    PubMed

    Noda, M; Tsai, S C; Adamik, R; Moss, J; Vaughan, M

    1990-05-16

    Cholera toxin causes the devastating diarrheal syndrome characteristic of cholera by catalyzing the ADP-ribosylation of Gs alpha, a GTP-binding regulatory protein, resulting in activation of adenylyl cyclase. ADP-ribosylation of Gs alpha is enhanced by 19 kDa guanine nucleotide-binding proteins known as ADP-ribosylation factors or ARFs. We investigated the effects of agents known to alter toxin-catalyzed activation of adenylyl cyclase on the stimulation of toxin- and toxin subunit-catalyzed ADP-ribosylation of Gs alpha and other substrates by an ADP-ribosylation factor purified from a soluble fraction of bovine brain (sARF II). In the presence of GTP, sARF II enhanced activity of both the toxin catalytic unit and a reduced and alkylated fragment ('A1'), as a result of an increase in substrate affinity with no significant effects on Vmax. Activation of toxin was independent of Gs alpha and was stimulated 4-fold by sodium dodecyl sulfate, but abolished by Triton X-100. sARF II therefore serves as a direct allosteric activator of the A1 protein and may thus amplify the pathological effects of cholera toxin. PMID:2112955

  5. The Solanum lycopersicum Zinc Finger2 Cysteine-2/Histidine-2 Repressor-Like Transcription Factor Regulates Development and Tolerance to Salinity in Tomato and Arabidopsis1[W

    PubMed Central

    Hichri, Imène; Muhovski, Yordan; Žižková, Eva; Dobrev, Petre I.; Franco-Zorrilla, Jose Manuel; Solano, Roberto; Lopez-Vidriero, Irene; Motyka, Vaclav; Lutts, Stanley

    2014-01-01

    The zinc finger superfamily includes transcription factors that regulate multiple aspects of plant development and were recently shown to regulate abiotic stress tolerance. Cultivated tomato (Solanum lycopersicum Zinc Finger2 [SIZF2]) is a cysteine-2/histidine-2-type zinc finger transcription factor bearing an ERF-associated amphiphilic repression domain and binding to the ACGTCAGTG sequence containing two AGT core motifs. SlZF2 is ubiquitously expressed during plant development, and is rapidly induced by sodium chloride, drought, and potassium chloride treatments. Its ectopic expression in Arabidopsis (Arabidopsis thaliana) and tomato impaired development and influenced leaf and flower shape, while causing a general stress visible by anthocyanin and malonyldialdehyde accumulation. SlZF2 enhanced salt sensitivity in Arabidopsis, whereas SlZF2 delayed senescence and improved tomato salt tolerance, particularly by maintaining photosynthesis and increasing polyamine biosynthesis, in salt-treated hydroponic cultures (125 mm sodium chloride, 20 d). SlZF2 may be involved in abscisic acid (ABA) biosynthesis/signaling, because SlZF2 is rapidly induced by ABA treatment and 35S::SlZF2 tomatoes accumulate more ABA than wild-type plants. Transcriptome analysis of 35S::SlZF2 revealed that SlZF2 both increased and reduced expression of a comparable number of genes involved in various physiological processes such as photosynthesis, polyamine biosynthesis, and hormone (notably ABA) biosynthesis/signaling. Involvement of these different metabolic pathways in salt stress tolerance is discussed. PMID:24567191

  6. The nuclear localization of the Arabidopsis transcription factor TIP is blocked by its interaction with the coat protein of Turnip crinkle virus

    SciTech Connect

    Ren Tao; Qu Feng; Morris, T. Jack . E-mail: jmorris@unlnotes.unl.edu

    2005-01-20

    We have previously reported that TIP, an Arabidopsis protein, interacts with the coat protein (CP) of Turnip crinkle virus (TCV) in yeast cells and that this interaction correlated with the resistance response in the TCV-resistant Arabidopsis ecotype Dijon-17. TIP was also able to activate transcription of reporter genes in yeast cells, suggesting that it is likely a transcription factor. We have now verified the physical interaction between TIP and TCV CP in vitro and showed that CP mutants unable to interact with TIP in yeast cells bind TIP with much lower affinity in vitro. Secondly, we have performed gel shift experiments demonstrating that TIP does not bind to DNA in a sequence-specific manner. The subcellular localization of TIP was also investigated by transiently expressing green fluorescence protein (GFP)-tagged TIP in Nicotiana benthamiana plant cells, which showed that GFP-tagged TIP localizes primarily to nuclei. Significantly, co-expression of TCVCP and GFP-TIP prevented the nuclear localization of TIP. Together, these results suggest that TIP might be a transcription factor involved in regulating the defense response of Arabidopsis to TCV and that its normal role is compromised by interaction with the invading viral CP.

  7. Arabidopsis GARP transcriptional activators interact with the Pro-rich activation domain shared by G-box-binding bZIP factors.

    PubMed

    Tamai, Hiroki; Iwabuchi, Masaki; Meshi, Tetsuo

    2002-01-01

    The Pro-rich regions, found in a subset of plant bZIP transcription factors, including G-box-binding factors (GBFs) of Arabidopsis thaliana, are thought to be deeply involved in transcriptional regulation. However, the molecular mechanisms of the Pro-rich region-mediated transcriptional regulation are still largely unknown. Here we report evidence showing that two closely related Arabidopsis proteins, designated GPRI1 and GPRI2, containing a GARP DNA-binding domain, are likely partners of one or more GBFs. The results of yeast two-hybrid assays and in vitro binding assays indicated that GPRI1 can interact with the Pro-rich regions of GBF1 and GBF3. GPRI2 interacted with the Pro-rich region of GBF1. GPRI1 and GPRI2 transactivated transcription in yeast. In GPRI1 the region responsible for this activation was mapped in the N-terminal third of the protein. Transient assays showed that in Arabidopsis cells not only the N-terminal but also the C-terminal regions of GPRI1 can function as a separable activation domain. GPRI1 and GPRI2 may function in some promoters in concert with a GBF through interaction with its Pro-rich region to enhance the transcriptional level of the corresponding genes. PMID:11828027

  8. mRNA biogenesis-related helicase eIF4AIII from Arabidopsis thaliana is an important factor for abiotic stress adaptation.

    PubMed

    Pascuan, Cecilia; Frare, Romina; Alleva, Karina; Ayub, Nicolás Daniel; Soto, Gabriela

    2016-05-01

    Similar to other plant species, Arabidopsis has a huge repertoire of predicted helicases, including the eIF4AIII factor, a putative component of the exon junction complex related to mRNA biogenesis. In this article, we integrated evolutionary and functional approaches to have a better understanding of eIF4AIII function in plants. Phylogenetic analysis showed that the mRNA biogenesis-related helicase eIF4AIII is the ortholog of the stress-related helicases PDH45 from Pisum sativum and MH1 from Medicago sativa, suggesting evolutionary and probably functional equivalences between mRNA biogenesis and stress-related plant helicases. Molecular and genetic analyses confirmed the relevance of eIF4AIII during abiotic stress adaptation in Arabidopsis. Therefore, in addition to its function in mRNA biogenesis, eIF4AIII can play a role in abiotic stress adaptation. PMID:26883227

  9. Nuclear activity of ROXY1, a glutaredoxin interacting with TGA factors, is required for petal development in Arabidopsis thaliana.

    PubMed

    Li, Shutian; Lauri, Andrea; Ziemann, Mark; Busch, Andrea; Bhave, Mrinal; Zachgo, Sabine

    2009-02-01

    Glutaredoxins (GRXs) have thus far been associated mainly with redox-regulated processes participating in stress responses. However, ROXY1, encoding a GRX, has recently been shown to regulate petal primorida initiation and further petal morphogenesis in Arabidopsis thaliana. ROXY1 belongs to a land plant-specific class of GRXs that has a CC-type active site motif, which deviates from ubiquitously occurring CPYC and CGFS GRXs. Expression studies of yellow fluorescent protein-ROXY1 fusion genes driven by the cauliflower mosaic virus 35S promoter reveal a nucleocytoplasmic distribution of ROXY1. We demonstrate that nuclear localization of ROXY1 is indispensable and thus crucial for its activity in flower development. Yeast two-hybrid screens identified TGA transcription factors as interacting proteins, which was confirmed by bimolecular fluorescence complementation experiments showing their nuclear interaction in planta. Overlapping expression patterns of ROXY1 and TGA genes during flower development demonstrate that ROXY1/TGA protein interactions can occur in vivo and support their biological relevance in petal development. Deletion analysis of ROXY1 demonstrates the importance of the C terminus for its functionality and for mediating ROXY1/TGA protein interactions. Phenotypic analysis of the roxy1-2 pan double mutant and an engineered chimeric repressor mutant from PERIANTHIA (PAN), a floral TGA gene, supports a dual role of ROXY1 in petal development. Together, our results show that the ROXY1 protein functions in the nucleus, likely by modifying PAN posttranslationally and thereby regulating its activity in petal primordia initiation. Additionally, ROXY1 affects later petal morphogenesis, probably by modulating other TGA factors that might act redundantly during differentiation of second whorl organs. PMID:19218396

  10. Improvement of enzymatic saccharification yield in Arabidopsis thaliana by ectopic expression of the rice SUB1A-1 transcription factor

    PubMed Central

    Núñez-López, Lizeth; Aguirre-Cruz, Andrés

    2015-01-01

    Saccharification of polysaccharides releases monosaccharides that can be used by ethanol-producing microorganisms in biofuel production. To improve plant biomass as a raw material for saccharification, factors controlling the accumulation and structure of carbohydrates must be identified. Rice SUB1A-1 is a transcription factor that represses the turnover of starch and postpones energy-consuming growth processes under submergence stress. Arabidopsis was employed to test if heterologous expression of SUB1A-1 or SUB1C-1 (a related gene) can be used to improve saccharification. Cellulolytic and amylolytic enzymatic treatments confirmed that SUB1A-1 transgenics had better saccharification yield than wild-type (Col-0), mainly from accumulated starch. This improved saccharification yield was developmentally controlled; when compared to Col-0, young transgenic vegetative plants yielded 200–300% more glucose, adult vegetative plants yielded 40–90% more glucose and plants in reproductive stage had no difference in yield. We measured photosynthetic parameters, starch granule microstructure, and transcript abundance of genes involved in starch degradation (SEX4, GWD1), juvenile transition (SPL3-5) and meristematic identity (FUL, SOC1) but found no differences to Col-0, indicating that starch accumulation may be controlled by down-regulation of CONSTANS and FLOWERING LOCUS T by SUB1A-1 as previously reported. SUB1A-1 transgenics also offered less resistance to deformation than wild-type concomitant to up-regulation of AtEXP2 expansin and BGL2 glucan-1,3,-beta-glucosidase. We conclude that heterologous SUB1A-1 expression can improve saccharification yield and softness, two traits needed in bioethanol production. PMID:25780769

  11. Computational Study of the Binding Mechanism of Actin-Depolymerizing Factor 1 with Actin in Arabidopsis thaliana

    PubMed Central

    Wang, Xue; Dong, Chun-Hai; Yang, Jian Ming; Yao, Xiao Jun

    2016-01-01

    Actin is a highly conserved protein. It plays important roles in cellular function and exists either in the monomeric (G-actin) or polymeric form (F-actin). Members of the actin-depolymerizing factor (ADF)/cofilin protein family bind to both G-actin and F-actin and play vital roles in actin dynamics by manipulating the rates of filament polymerization and depolymerization. It has been reported that the S6D and R98A/K100A mutants of actin-depolymerizing factor 1 (ADF1) in Arabidopsis thaliana decreased the binding affinity of ADF for the actin monomer. To investigate the binding mechanism and dynamic behavior of the ADF1–actin complex, we constructed a homology model of the AtADF1–actin complex based on the crystal structure of AtADF1 and the twinfilin C-terminal ADF-H domain in a complex with a mouse actin monomer. The model was then refined for subsequent molecular dynamics simulations. Increased binding energy of the mutated system was observed using the Molecular Mechanics Generalized Born Surface Area and Poisson–Boltzmann Surface Area (MM-GB/PBSA) methods. To determine the residues that make decisive contributions to the ADF1 actin-binding affinity, per-residue decomposition and computational alanine scanning analyses were performed, which provided more detailed information on the binding mechanism. Root-mean-square fluctuation and principal component analyses confirmed that the S6D and R98A/K100A mutants induced an increased conformational flexibility. The comprehensive molecular insight gained from this study is of great importance for understanding the binding mechanism of ADF1 and G-actin. PMID:27414648

  12. Eukaryotic Translation Initiation Factor eIFiso4G Is Required to Regulate Violaxanthin De-epoxidase Expression in Arabidopsis*

    PubMed Central

    Chen, Zhong; Jolley, Blair; Caldwell, Christian; Gallie, Daniel R.

    2014-01-01

    The eukaryotic translation initiation factor (eIF) 4G is a scaffold protein that organizes the assembly of those initiation factors needed to recruit the 40 S ribosomal subunit to an mRNA. Plants, like many eukaryotes, express two eIF4G isoforms. eIFiso4G, one of the isoforms specific to plants, is unique among eukaryotic eIF4G proteins in that it is highly divergent and unusually small in size, raising the possibility of functional specialization. In this study, the role of eIFiso4G in plant growth was investigated using null mutants for the eIF4G isoforms in Arabidopsis. eIFiso4G loss of function mutants exhibited smaller cell, leaf, plant size, and biomass accumulation that correlated with its reduced photosynthetic activity, phenotypes not observed with the eIF4G loss of function mutant. Although no change in photorespiration or dark respiration was observed in the eIFiso4G loss of function mutant, a reduction in chlorophyll levels and an increase in the level of nonphotochemical quenching were observed. An increase in xanthophyll cycle activity and the generation of reactive oxygen species contributed to the qE and qI components of nonphotochemical quenching, respectively. An increase in the transcript and protein levels of violaxanthin de-epoxidase in the eIFiso4G loss of function mutant and an increase in its xanthophyll de-epoxidation state correlated with the higher qE associated with loss of eIFiso4G expression. These observations indicate that eIFiso4G expression is required to regulate violaxanthin de-epoxidase expression and to support photosynthetic activity. PMID:24706761

  13. A wheat salinity-induced WRKY transcription factor TaWRKY93 confers multiple abiotic stress tolerance in Arabidopsis thaliana.

    PubMed

    Qin, Yuxiang; Tian, Yanchen; Liu, Xiuzhi

    2015-08-21

    Wheat is an important crop in the world. But most of the cultivars are salt sensitive, and often adversely affected by salt stress. WRKY transcription factors play a major role in plant responses to salt stress, but the effective salinity regulatory WRKYs identified in bread wheat are limited and the mechanism of salt stress tolerance is also not well explored. Here, we identified a salt (NaCl) induced class II WRKY transcription factor TaWRKY93. Its transcript level was strongly induced by salt (NaCl) and exogenous abscisic acid (ABA). Over-expression of TaWRKY93 in Arabidopsis thaliana enhanced salt (NaCl), drought, low temperature and osmotic (mannitol) stress tolerance, mainly demonstrated by transgenic plants forming longer primary roots or more lateral roots on MS plates supplemented with NaCl and mannitol individually, higher survival rate under drought and low temperature stress. Further, transgenic plants maintained a more proline content, higher relative water content and less electrolyte leakage than the wild type plants. The transcript abundance of a series of abiotic stress-related genes was up-regulated in the TaWRKY93 transgenic plants. In summary, TaWRKY93 is a new positive regulator of abiotic stress, it may increase salinity, drought and low temperature stress tolerance through enhancing osmotic adjustment, maintaining membrane stability and increasing transcription of stress related genes, and contribute to the superior agricultural traits of SR3 through promoting root development. It can be used as a candidate gene for wheat transgenic engineering breeding against abiotic stress. PMID:26106823

  14. Computational Study of the Binding Mechanism of Actin-Depolymerizing Factor 1 with Actin in Arabidopsis thaliana.

    PubMed

    Du, Juan; Wang, Xue; Dong, Chun-Hai; Yang, Jian Ming; Yao, Xiao Jun

    2016-01-01

    Actin is a highly conserved protein. It plays important roles in cellular function and exists either in the monomeric (G-actin) or polymeric form (F-actin). Members of the actin-depolymerizing factor (ADF)/cofilin protein family bind to both G-actin and F-actin and play vital roles in actin dynamics by manipulating the rates of filament polymerization and depolymerization. It has been reported that the S6D and R98A/K100A mutants of actin-depolymerizing factor 1 (ADF1) in Arabidopsis thaliana decreased the binding affinity of ADF for the actin monomer. To investigate the binding mechanism and dynamic behavior of the ADF1-actin complex, we constructed a homology model of the AtADF1-actin complex based on the crystal structure of AtADF1 and the twinfilin C-terminal ADF-H domain in a complex with a mouse actin monomer. The model was then refined for subsequent molecular dynamics simulations. Increased binding energy of the mutated system was observed using the Molecular Mechanics Generalized Born Surface Area and Poisson-Boltzmann Surface Area (MM-GB/PBSA) methods. To determine the residues that make decisive contributions to the ADF1 actin-binding affinity, per-residue decomposition and computational alanine scanning analyses were performed, which provided more detailed information on the binding mechanism. Root-mean-square fluctuation and principal component analyses confirmed that the S6D and R98A/K100A mutants induced an increased conformational flexibility. The comprehensive molecular insight gained from this study is of great importance for understanding the binding mechanism of ADF1 and G-actin. PMID:27414648

  15. Transcription factors WRKY70 and WRKY11 served as regulators in rhizobacterium Bacillus cereus AR156-induced systemic resistance to Pseudomonas syringae pv. tomato DC3000 in Arabidopsis.

    PubMed

    Jiang, Chun-Hao; Huang, Zi-Yang; Xie, Ping; Gu, Chun; Li, Ke; Wang, Da-Chen; Yu, Yi-Yang; Fan, Zhi-Hang; Wang, Chun-Juan; Wang, Yun-Peng; Guo, Ya-Hui; Guo, Jian-Hua

    2016-01-01

    The activation of both the SA and JA/ETsignalling pathways may lead to more efficient general and broad resistance to Pst DC3000 by non-pathogenic rhizobacteria. However, the mechanisms that govern this simultaneous activation are unclear. Using Arabidopsis as a model system, two transcription factors, WRKY11 and WRKY70, were identified as important regulators involved in Induced Systemic Resistance (ISR) triggered by Bacillus cereus AR156. The results revealed that AR156 treatment significantly stimulated the transcription of WRKY70, but suppressed that of WRKY11 in Arabidopsis leaves. Furthermore, they were shown to be required for AR156 enhancing the activation of cellular defence responses and the transcription level of the plant defence response gene. Overexpression of the two transcription factors in Arabidopsis also showed that they were essential for AR156 to elicit ISR. AR156-triggered ISR was completely abolished in the double mutant of the two transcription factors, but still partially retained in the single mutants, indicating that the regulation of the two transcription factors depend on two different pathways. The target genes of the two transcription factors and epistasis analysis suggested that WRKY11 regulated AR156-triggered ISR through activating the JA signalling pathway, and WRKY70 regulated the ISR through activating the SA signalling pathway. In addition, both WRKY11 and WRKY70 modulated AR156-triggered ISR in a NPR1-dependent manner. In conclusion, WRKY11 and WRKY70 played an important role in regulating the signalling transduction pathways involved in AR156-triggered ISR. This study is the first to illustrate the mechanism by which a single rhizobacterium elicits ISR by simultaneously activating both the SA and JA/ET signalling pathways. PMID:26433201

  16. miR396-targeted AtGRF transcription factors are required for coordination of cell division and differentiation during leaf development in Arabidopsis

    PubMed Central

    Wang, Li; Gu, Xiaolu; Xu, Deyang; Wang, Wei; Wang, Hua; Zeng, Minhuan; Chang, Zhaoyang; Huang, Hai; Cui, Xiaofeng

    2011-01-01

    In plants, cell proliferation and polarized cell differentiation along the adaxial–abaxial axis in the primordium is critical for leaf morphogenesis, while the temporal–spatial relationships between these two processes remain largely unexplored. Here, it is reported that microRNA396 (miR396)-targeted Arabidopsis growth-regulating factors (AtGRFs) are required for leaf adaxial–abaxial polarity in Arabidopsis. Reduction of the expression of AtGRF genes by transgenic miR396 overexpression in leaf polarity mutants asymmetric leaves1 (as1) and as2 resulted in plants with enhanced leaf adaxial–abaxial defects, as a consequence of reduced cell proliferation. Moreover, transgenic miR396 overexpression markedly decreased the cell division activity and the expression of cell cycle-related genes, but resulted in an increased percentage of leaf cells with a higher ploidy level, indicating that miR396 negatively regulates cell proliferation by controlling entry into the mitotic cell cycle. miR396 is mainly expressed in the leaf cells arrested for cell division, coinciding with its roles in cell cycle regulation. These results together suggest that cell division activity mediated by miR396-targeted AtGRFs is important for polarized cell differentiation along the adaxial–abaxial axis during leaf morphogenesis in Arabidopsis. PMID:21036927

  17. Regulation of secondary cell wall biosynthesis by poplar R2R3 MYB transcription factor PtrMYB152 in Arabidopsis

    SciTech Connect

    Wang, Shucai; Li, Eryang; Porth, Ilga; Chen, Jin-Gui; Mansfield, Shawn D.; Douglas, Carl

    2014-05-23

    Poplar has 192 annotated R2R3 MYB genes, of which only three have been shown to play a role in the regulation of secondary cell wall formation. Here we report the characterization of PtrMYB152, a poplar homolog of the Arabidopsis R2R3 MYB transcription factor AtMYB43, in the regulation of secondary cell wall biosynthesis. The expression of PtrMYB152 in secondary xylem is about 18 times of that in phloem. When expressed in Arabidopsis under the control of either 35S or PtrCesA8 promoters, PtrMYB152 increased secondary cell wall thickness, which is likely caused by increased lignification. Accordingly, elevated expression of genes encoding sets of enzymes in secondary wall biosynthesis were observed in transgenic plants expressing PtrMYB152. Arabidopsis protoplast transfection assays suggested that PtrMYB152 functions as a transcriptional activator. Taken together, our results suggest that PtrMYB152 may be part of a regulatory network activating expression of discrete sets of secondary cell wall biosynthesis genes.

  18. Basic helix-loop-helix transcription factor from wild rice (OrbHLH2) improves tolerance to salt- and osmotic stress in Arabidopsis.

    PubMed

    Zhou, Jing; Li, Fei; Wang, Jin-Lan; Ma, Yun; Chong, Kang; Xu, Yun-yuan

    2009-08-15

    Salt stress adversely affects plant growth and development. Some plants reduce the damage of high-salt stress by expressing a series of salt-responsive genes. Studies of the molecular mechanism of the salt-stress response have focused on the characterization of components involved in signal perception and transduction. In the present work, we cloned and characterized a basic helix-loop-helix (bHLH) encoding gene, OrbHLH2, from wild rice (Oryza rufipogon), which encodes a homologue protein of ICE1 in Arabidopsis. OrbHLH2 protein localized in the nucleus. Overexpression of OrbHLH2 in Arabidopsis conferred increased tolerance to salt and osmotic stress, and the stress-responsive genes DREB1A/CBF3, RD29A, COR15A and KIN1 were upregulated in transgenic plants. Abscisic acid (ABA) treatment showed a similar effect on the seed germination or transcriptional expression of stress-responsive genes in both wild type and OrbHLH2-overexpressed plants, which implies that OrbHLH2 does not depend on ABA in responding to salt stress. OrbHLH2 may function as a transcription factor and positively regulate salt-stress signals independent of ABA in Arabidopsis, which provides some useful data for improving salt tolerance in crops. PMID:19324458

  19. MYB-related transcription factors function as regulators of the circadian clock and anthocyanin biosynthesis in Arabidopsis

    PubMed Central

    Nguyen, Nguyen Hoai; Lee, Hojoung

    2016-01-01

    ABSTRACT In Arabidopsis, the MYB (myeloblastosis) gene family contains more than 190 members, which play a number of roles in plant growth and development. Based on their protein structure, this gene family was divided into several subclasses, including the MYB-related class. Currently, an MYB-related gene designated as MYB-like Domain (AtMYBD) has been shown to function as a positive regulator of anthocyanin biosynthesis in Arabidopsis. This gene was found to belong to the CCA1-like (circadian clock-associated 1) group, which represents several genes that are master regulators of the circadian clocks of plants. Here, we speculate that AtMYBD is able to regulate anthocyanin biosynthesis in Arabidopsis thaliana in a circadian clock-related manner. PMID:26905954

  20. Interaction of Arabidopsis Trihelix-Domain Transcription Factors VFP3 and VFP5 with Agrobacterium Virulence Protein VirF

    PubMed Central

    García-Cano, Elena; Magori, Shimpei; Sun, Qi; Ding, Zehong; Lazarowitz, Sondra G.; Citovsky, Vitaly

    2015-01-01

    Agrobacterium is a natural genetic engineer of plants that exports several virulence proteins into host cells in order to take advantage of the cell machinery to facilitate transformation and support bacterial growth. One of these effectors is the F-box protein VirF, which presumably uses the host ubiquitin/proteasome system (UPS) to uncoat the packaging proteins from the invading bacterial T-DNA. By analogy to several other bacterial effectors, VirF most likely has several functions in the host cell and, therefore, several interacting partners among host proteins. Here we identify one such interactor, an Arabidopsis trihelix-domain transcription factor VFP3, and further show that its very close homolog VFP5 also interacted with VirF. Interestingly, interactions of VirF with either VFP3 or VFP5 did not activate the host UPS, suggesting that VirF might play other UPS-independent roles in bacterial infection. To better understand the potential scope of VFP3 function, we used RNAi to reduce expression of the VFP3 gene. Transcriptome profiling of these VFP3-silenced plants using high-throughput cDNA sequencing (RNA-seq) revealed that VFP3 substantially affected plant gene expression; specifically, 1,118 genes representing approximately 5% of all expressed genes were significantly either up- or down-regulated in the VFP3 RNAi line compared to wild-type Col-0 plants. Among the 507 up-regulated genes were genes implicated in the regulation of transcription, protein degradation, calcium signaling, and hormone metabolism, whereas the 611 down-regulated genes included those involved in redox regulation, light reactions of photosynthesis, and metabolism of lipids, amino acids, and cell wall. Overall, this pattern of changes in gene expression is characteristic of plants under stress. Thus, VFP3 likely plays an important role in controlling plant homeostasis. PMID:26571494

  1. Heat Stress- and Heat Shock Transcription Factor-Dependent Expression and Activity of Ascorbate Peroxidase in Arabidopsis1

    PubMed Central

    Panchuk, Irina I.; Volkov, Roman A.; Schöffl, Friedrich

    2002-01-01

    To find evidence for a connection between heat stress response, oxidative stress, and common stress tolerance, we studied the effects of elevated growth temperatures and heat stress on the activity and expression of ascorbate peroxidase (APX). We compared wild-type Arabidopsis with transgenic plants overexpressing heat shock transcription factor 3 (HSF3), which synthesize heat shock proteins and are improved in basal thermotolerance. Following heat stress, APX activity was positively affected in transgenic plants and correlated with a new thermostable isoform, APXS. This enzyme was present in addition to thermolabile cytosolic APX1, the prevalent isoform in unstressed cells. In HSF3-transgenic plants, APXS activity was detectable at normal temperature and persisted after severe heat stress at 44°C. In nontransgenic plants, APXS was undetectable at normal temperature, but could be induced by moderate heat stress. The mRNA expression profiles of known and three new Apx genes were determined using real-time PCR. Apx1 and Apx2 genes encoding cytosolic APX were heat stress and HSF dependently expressed, but only the representations of Apx2 mRNA met the criteria that suggest identity between APXS and APX2: not expressed at normal temperature in wild type, strong induction by heat stress, and HSF3-dependent expression in transgenic plants. Our data suggest that Apx2 is a novel heat shock gene and that the enzymatic activity of APX2/APXS is required to compensate heat stress-dependent decline of APX1 activity in the cytosol. The functional roles of modulations of APX expression and the interdependence of heat stress and oxidative stress response and signaling mechanisms are discussed. PMID:12068123

  2. Characterization of MADS-domain transcription factor complexes in Arabidopsis flower development

    PubMed Central

    Smaczniak, Cezary; Immink, Richard G. H.; Muiño, Jose M.; Blanvillain, Robert; Busscher, Marco; Busscher-Lange, Jacqueline; Dinh, Q. D. (Peter); Liu, Shujing; Westphal, Adrie H.; Boeren, Sjef; Parcy, François; Xu, Lin; Carles, Cristel C.; Angenent, Gerco C.; Kaufmann, Kerstin

    2012-01-01

    Floral organs are specified by the combinatorial action of MADS-domain transcription factors, yet the mechanisms by which MADS-domain proteins activate or repress the expression of their target genes and the nature of their cofactors are still largely unknown. Here, we show using affinity purification and mass spectrometry that five major floral homeotic MADS-domain proteins (AP1, AP3, PI, AG, and SEP3) interact in floral tissues as proposed in the “floral quartet” model. In vitro studies confirmed a flexible composition of MADS-domain protein complexes depending on relative protein concentrations and DNA sequence. In situ bimolecular fluorescent complementation assays demonstrate that MADS-domain proteins interact during meristematic stages of flower development. By applying a targeted proteomics approach we were able to establish a MADS-domain protein interactome that strongly supports a mechanistic link between MADS-domain proteins and chromatin remodeling factors. Furthermore, members of other transcription factor families were identified as interaction partners of floral MADS-domain proteins suggesting various specific combinatorial modes of action. PMID:22238427

  3. HEMERA Couples the Proteolysis and Transcriptional Activity of PHYTOCHROME INTERACTING FACTORs in Arabidopsis Photomorphogenesis

    PubMed Central

    Qiu, Yongjian; Li, Meina; Pasoreck, Elise K.; Long, Lingyun; Shi, Yiting; Galvão, Rafaelo M.; Chou, Conrad L.; Wang, He; Sun, Amanda Y.; Zhang, Yiyin C.; Jiang, Anna; Chen, Meng

    2015-01-01

    Phytochromes (phys) are red and far-red photoreceptors that control plant development and growth by promoting the proteolysis of a family of antagonistically acting basic helix-loop-helix transcription factors, the PHYTOCHROME-INTERACTING FACTORs (PIFs). We have previously shown that the degradation of PIF1 and PIF3 requires HEMERA (HMR). However, the biochemical function of HMR and the mechanism by which it mediates PIF degradation remain unclear. Here, we provide genetic evidence that HMR acts upstream of PIFs in regulating hypocotyl growth. Surprisingly, genome-wide analysis of HMR- and PIF-dependent genes reveals that HMR is also required for the transactivation of a subset of PIF direct-target genes. We show that HMR interacts with all PIFs. The HMR-PIF interaction is mediated mainly by HMR’s N-terminal half and PIFs’ conserved active-phytochrome B binding motif. In addition, HMR possesses an acidic nine-amino-acid transcriptional activation domain (9aaTAD) and a loss-of-function mutation in this 9aaTAD impairs the expression of PIF target genes and the destruction of PIF1 and PIF3. Together, these in vivo results support a regulatory mechanism for PIFs in which HMR is a transcriptional coactivator binding directly to PIFs and the 9aaTAD of HMR couples the degradation of PIF1 and PIF3 with the transactivation of PIF target genes. PMID:25944101

  4. Arabidopsis MYC Transcription Factors Are the Target of Hormonal Salicylic Acid/Jasmonic Acid Cross Talk in Response to Pieris brassicae Egg Extract.

    PubMed

    Schmiesing, André; Emonet, Aurélia; Gouhier-Darimont, Caroline; Reymond, Philippe

    2016-04-01

    Arabidopsis (Arabidopsis thaliana) plants recognize insect eggs and activate the salicylic acid (SA) pathway. As a consequence, expression of defense genes regulated by the jasmonic acid (JA) pathway is suppressed and larval performance is enhanced. Cross talk between defense signaling pathways is common in plant-pathogen interactions, but the molecular mechanism mediating this phenomenon is poorly understood. Here, we demonstrate that egg-induced SA/JA antagonism works independently of the APETALA2/ETHYLENE RESPONSE FACTOR (AP2/ERF) transcription factor ORA59, which controls the ERF branch of the JA pathway. In addition, treatment with egg extract did not enhance expression or stability of JASMONATE ZIM-domain transcriptional repressors, and SA/JA cross talk did not involve JASMONATE ASSOCIATED MYC2-LIKEs, which are negative regulators of the JA pathway. Investigating the stability of MYC2, MYC3, and MYC4, three basic helix-loop-helix transcription factors that additively control jasmonate-related defense responses, we found that egg extract treatment strongly diminished MYC protein levels in an SA-dependent manner. Furthermore, we identified WRKY75 as a novel and essential factor controlling SA/JA cross talk. These data indicate that insect eggs target the MYC branch of the JA pathway and uncover an unexpected modulation of SA/JA antagonism depending on the biological context in which the SA pathway is activated. PMID:26884488

  5. Arabidopsis MYC Transcription Factors Are the Target of Hormonal Salicylic Acid/Jasmonic Acid Cross Talk in Response to Pieris brassicae Egg Extract1[OPEN

    PubMed Central

    Schmiesing, André; Gouhier-Darimont, Caroline

    2016-01-01

    Arabidopsis (Arabidopsis thaliana) plants recognize insect eggs and activate the salicylic acid (SA) pathway. As a consequence, expression of defense genes regulated by the jasmonic acid (JA) pathway is suppressed and larval performance is enhanced. Cross talk between defense signaling pathways is common in plant-pathogen interactions, but the molecular mechanism mediating this phenomenon is poorly understood. Here, we demonstrate that egg-induced SA/JA antagonism works independently of the APETALA2/ETHYLENE RESPONSE FACTOR (AP2/ERF) transcription factor ORA59, which controls the ERF branch of the JA pathway. In addition, treatment with egg extract did not enhance expression or stability of JASMONATE ZIM-domain transcriptional repressors, and SA/JA cross talk did not involve JASMONATE ASSOCIATED MYC2-LIKEs, which are negative regulators of the JA pathway. Investigating the stability of MYC2, MYC3, and MYC4, three basic helix-loop-helix transcription factors that additively control jasmonate-related defense responses, we found that egg extract treatment strongly diminished MYC protein levels in an SA-dependent manner. Furthermore, we identified WRKY75 as a novel and essential factor controlling SA/JA cross talk. These data indicate that insect eggs target the MYC branch of the JA pathway and uncover an unexpected modulation of SA/JA antagonism depending on the biological context in which the SA pathway is activated. PMID:26884488

  6. WRKY6 Transcription Factor Restricts Arsenate Uptake and Transposon Activation in Arabidopsis[W

    PubMed Central

    Castrillo, Gabriel; Sánchez-Bermejo, Eduardo; de Lorenzo, Laura; Crevillén, Pedro; Fraile-Escanciano, Ana; TC, Mohan; Mouriz, Alfonso; Catarecha, Pablo; Sobrino-Plata, Juan; Olsson, Sanna; Leo del Puerto, Yolanda; Mateos, Isabel; Rojo, Enrique; Hernández, Luis E.; Jarillo, Jose A.; Piñeiro, Manuel; Paz-Ares, Javier; Leyva, Antonio

    2013-01-01

    Stress constantly challenges plant adaptation to the environment. Of all stress types, arsenic was a major threat during the early evolution of plants. The most prevalent chemical form of arsenic is arsenate, whose similarity to phosphate renders it easily incorporated into cells via the phosphate transporters. Here, we found that arsenate stress provokes a notable transposon burst in plants, in coordination with arsenate/phosphate transporter repression, which immediately restricts arsenate uptake. This repression was accompanied by delocalization of the phosphate transporter from the plasma membrane. When arsenate was removed, the system rapidly restored transcriptional expression and membrane localization of the transporter. We identify WRKY6 as an arsenate-responsive transcription factor that mediates arsenate/phosphate transporter gene expression and restricts arsenate-induced transposon activation. Plants therefore have a dual WRKY-dependent signaling mechanism that modulates arsenate uptake and transposon expression, providing a coordinated strategy for arsenate tolerance and transposon gene silencing. PMID:23922208

  7. The Arabidopsis thaliana TCP transcription factors: A broadening horizon beyond development

    PubMed Central

    Li, Shutian

    2015-01-01

    The TCP family of transcription factors is named after the first 4 characterized members, namely TEOSINTE BRANCHED1 (TB1) from maize (Zea mays), CYCLOIDEA (CYC) from snapdragon (Antirrhinum majus), as well as PROLIFERATING CELL NUCLEAR ANTIGEN FACTOR1 (PCF1) and PCF2 from rice (Oryza sativa). Phylogenic analysis of this plant-specific protein family unveils a conserved bHLH-containing DNA-binding motif known as the TCP domain. In accordance with the structure of this shared domain, TCP proteins are grouped into class I (TCP-P) and class II (TCP-C), which are suggested to antagonistically modulate plant growth and development via competitively binding similar cis-regulatory modules called site II elements. Over the last decades, TCPs across the plant kingdom have been demonstrated to control a plethora of plant processes. Notably, TCPs also regulate plant development and defense responses via stimulating the biosynthetic pathways of bioactive metabolites, such as brassinosteroid (BR), jasmonic acid (JA) and flavonoids. Besides, mutagenesis analysis coupled with biochemical experiments identifies several crucial amino acids located within the TCP domain, which confer the redox sensitivity of class I TCPs and determine the distinct DNA-binding properties of TCPs. In this review, developmental functions of TCPs in various biological pathways are briefly described with an emphasis on their involvement in the synthesis of bioactive substances. Furthermore, novel biochemical aspects of TCPs with respect to redox regulation and DNA-binding preferences are elaborated. In addition, the unexpected participation of TCPs in effector-triggered immunity (ETI) and defense against insects indicates that the widely recognized developmental regulators are capable of fine-tuning defense signaling and thereby enable plants to evade deleterious developmental phenotypes. Altogether, these recent impressive breakthroughs remarkably advance our understanding as to how TCPs integrate

  8. Arabidopsis Sigma Factor Binding Proteins Are Activators of the WRKY33 Transcription Factor in Plant Defense[W

    PubMed Central

    Lai, Zhibing; Li, Ying; Wang, Fei; Cheng, Yuan; Fan, Baofang; Yu, Jing-Quan; Chen, Zhixiang

    2011-01-01

    Necrotrophic pathogens are important plant pathogens that cause many devastating plant diseases. Despite their impact, our understanding of the plant defense response to necrotrophic pathogens is limited. The WRKY33 transcription factor is important for plant resistance to necrotrophic pathogens; therefore, elucidation of its functions will enhance our understanding of plant immunity to necrotrophic pathogens. Here, we report the identification of two WRKY33-interacting proteins, nuclear-encoded SIGMA FACTOR BINDING PROTEIN1 (SIB1) and SIB2, which also interact with plastid-encoded plastid RNA polymerase SIGMA FACTOR1. Both SIB1 and SIB2 contain an N-terminal chloroplast targeting signal and a putative nuclear localization signal, suggesting that they are dual targeted. Bimolecular fluorescence complementation indicates that WRKY33 interacts with SIBs in the nucleus of plant cells. Both SIB1 and SIB2 contain a short VQ motif that is important for interaction with WRKY33. The two VQ motif–containing proteins recognize the C-terminal WRKY domain and stimulate the DNA binding activity of WRKY33. Like WRKY33, both SIB1 and SIB2 are rapidly and strongly induced by the necrotrophic pathogen Botrytis cinerea. Resistance to B. cinerea is compromised in the sib1 and sib2 mutants but enhanced in SIB1-overexpressing transgenic plants. These results suggest that dual-targeted SIB1 and SIB2 function as activators of WRKY33 in plant defense against necrotrophic pathogens. PMID:21990940

  9. Xanthomonas campestris Overcomes Arabidopsis Stomatal Innate Immunity through a DSF Cell-to-Cell Signal-Regulated Virulence Factor1[OA

    PubMed Central

    Gudesblat, Gustavo E.; Torres, Pablo S.; Vojnov, Adrián A.

    2009-01-01

    Pathogen-induced stomatal closure is part of the plant innate immune response. Phytopathogens using stomata as a way of entry into the leaf must avoid the stomatal response of the host. In this article, we describe a factor secreted by the bacterial phytopathogen Xanthomonas campestris pv campestris (Xcc) capable of interfering with stomatal closure induced by bacteria or abscisic acid (ABA). We found that living Xcc, as well as ethyl acetate extracts from Xcc culture supernatants, are capable of reverting stomatal closure induced by bacteria, lipopolysaccharide, or ABA. Xcc ethyl acetate extracts also complemented the infectivity of Pseudomonas syringae pv tomato (Pst) mutants deficient in the production of the coronatine toxin, which is required to overcome stomatal defense. By contrast, the rpfF and rpfC mutant strains of Xcc, which are unable to respectively synthesize or perceive a diffusible molecule involved in bacterial cell-to-cell signaling, were incapable of reverting stomatal closure, indicating that suppression of stomatal response by Xcc requires an intact rpf/diffusible signal factor system. In addition, we found that guard cell-specific Arabidopsis (Arabidopsis thaliana) Mitogen-Activated Protein Kinase3 (MPK3) antisense mutants were unresponsive to bacteria or lipopolysaccharide in promotion of stomatal closure, and also more sensitive to Pst coronatine-deficient mutants, showing that MPK3 is required for stomatal immune response. Additionally, we found that, unlike in wild-type Arabidopsis, ABA-induced stomatal closure in MPK3 antisense mutants is not affected by Xcc or by extracts from Xcc culture supernatants, suggesting that the Xcc factor might target some signaling component in the same pathway as MPK3. PMID:19091877

  10. Putative cold acclimation pathways in Arabidopsis thaliana identified by a combined analysis of mRNA co-expression patterns, promoter motifs and transcription factors

    PubMed Central

    Chawade, Aakash; Bräutigam, Marcus; Lindlöf, Angelica; Olsson, Olof; Olsson, Björn

    2007-01-01

    Background With the advent of microarray technology, it has become feasible to identify virtually all genes in an organism that are induced by developmental or environmental changes. However, relying solely on gene expression data may be of limited value if the aim is to infer the underlying genetic networks. Development of computational methods to combine microarray data with other information sources is therefore necessary. Here we describe one such method. Results By means of our method, previously published Arabidopsis microarray data from cold acclimated plants at six different time points, promoter motif sequence data extracted from ~24,000 Arabidopsis promoters and known transcription factor binding sites were combined to construct a putative genetic regulatory interaction network. The inferred network includes both previously characterised and hitherto un-described regulatory interactions between transcription factor (TF) genes and genes that encode other TFs or other proteins. Part of the obtained transcription factor regulatory network is presented here. More detailed information is available in the additional files. Conclusion The rule-based method described here can be used to infer genetic networks by combining data from microarrays, promoter sequences and known promoter binding sites. This method should in principle be applicable to any biological system. We tested the method on the cold acclimation process in Arabidopsis and could identify a more complex putative genetic regulatory network than previously described. However, it should be noted that information on specific binding sites for individual TFs were in most cases not available. Thus, gene targets for the entire TF gene families were predicted. In addition, the networks were built solely by a bioinformatics approach and experimental verifications will be necessary for their final validation. On the other hand, since our method highlights putative novel interactions, more directed experiments

  11. Transcription factor WRKY46 regulates osmotic stress responses and stomatal movement independently in Arabidopsis.

    PubMed

    Ding, Zhong Jie; Yan, Jing Ying; Xu, Xiao Yan; Yu, Di Qiu; Li, Gui Xin; Zhang, Shu Qun; Zheng, Shao Jian

    2014-07-01

    Drought and salt stress severely inhibit plant growth and development; however, the regulatory mechanisms of plants in response to these stresses are not fully understood. Here we report that the expression of a WRKY transcription factor WRKY46 is rapidly induced by drought, salt and oxidative stresses. T-DNA insertion of WRKY46 leads to more sensitivity to drought and salt stress, whereas overexpression of WRKY46 (OV46) results in hypersensitivity in soil-grown plants, with a higher water loss rate, but with increased tolerance on the sealed agar plates. Stomatal closing in the OV46 line is insensitive to ABA because of a reduced accumulation of reactive oxygen species (ROS) in the guard cells. We further find that WRKY46 is expressed in guard cells, where its expression is not affected by dehydration, and is involved in light-dependent stomatal opening. Microarray analysis reveals that WRKY46 regulates a set of genes involved in cellular osmoprotection and redox homeostasis under dehydration stress, which is confirmed by ROS and malondialdehyde (MDA) levels in stressed seedlings. Moreover, WRKY46 modulates light-dependent starch metabolism in guard cells via regulating QUA-QUINE STARCH (QQS) gene expression. Taken together, we demonstrate that WRKY46 plays dual roles in regulating plant responses to drought and salt stress and light-dependent stomatal opening in guard cells. PMID:24773321

  12. LLM-Domain Containing B-GATA Factors Control Different Aspects of Cytokinin-Regulated Development in Arabidopsis thaliana1[OPEN

    PubMed Central

    Ranftl, Quirin L.; Bastakis, Emmanouil; Klermund, Carina

    2016-01-01

    Leu-Leu-Met (LLM)-domain B-GATAs are a subfamily of the 30-membered GATA transcription factor family from Arabidopsis. Only two of the six Arabidopsis LLM-domain B-GATAs, i.e. GATA, NITRATE-INDUCIBLE, CARBON METABOLISM-INVOLVED (GNC) and its paralog GNC-LIKE/CYTOKININ-RESPONSIVE GATA FACTOR1 (GNL), have already been analyzed with regard to their biological function. Together, GNC and GNL control germination, greening, flowering time, and senescence downstream from auxin, cytokinin (CK), gibberellin (GA), and light signaling. Whereas overexpression and complementation analyses suggest a redundant biochemical function between GNC and GNL, nothing is known about the biological role of the four other LLM-domain B-GATAs, GATA15, GATA16, GATA17, and GATA17L (GATA17-LIKE), based on loss-of-function mutant phenotypes. Here, we examine insertion mutants of the six Arabidopsis B-GATA genes and reveal the role of these genes in the control of greening, hypocotyl elongation, phyllotaxy, floral organ initiation, accessory meristem formation, flowering time, and senescence. Several of these phenotypes had previously not been described for the gnc and gnl mutants or were enhanced in the more complex mutants when compared to gnc gnl mutants. Some of the respective responses may be mediated by CK signaling, which activates the expression of all six GATA genes. CK-induced gene expression is partially compromised in LLM-domain B-GATA mutants, suggesting that B-GATA genes play a role in CK responses. We furthermore provide evidence for a transcriptional cross regulation between these GATAs that may, in at least some cases, be at the basis of their apparent functional redundancy. PMID:26829982

  13. Zinc-Finger Transcription Factor ZAT6 Positively Regulates Cadmium Tolerance through the Glutathione-Dependent Pathway in Arabidopsis1[OPEN

    PubMed Central

    Chen, Jian; Yan, Xingxing; Liu, Yunlei; Wang, Ren; Fan, Tingting; Ren, Yongbing; Tang, Xiaofeng; Xiao, Fangming

    2016-01-01

    Cadmium (Cd) is an environmental pollutant with high toxicity to animals and plants. It has been established that the glutathione (GSH)-dependent phytochelatin (PC) synthesis pathway is one of the most important mechanisms contributing to Cd accumulation and tolerance in plants. However, the transcription factors involved in regulating GSH-dependent PC synthesis pathway remain largely unknown. Here, we identified an Arabidopsis (Arabidopsis thaliana) Cd-resistant mutant xcd2-D (XVE system-induced cadmium-tolerance2) using a forward genetics approach. The mutant gene underlying xcd2-D mutation was revealed to encode a known zinc-finger transcription factor, ZAT6. Transgenic plants overexpressing ZAT6 showed significant increase of Cd tolerance, whereas loss of function of ZAT6 led to decreased Cd tolerance. Increased Cd accumulation and tolerance in ZAT6-overexpressing lines was GSH dependent and associated with Cd-activated synthesis of PC, which was correlated with coordinated activation of PC-synthesis related gene expression. By contrast, loss of function of ZAT6 reduced Cd accumulation and tolerance, which was accompanied by abolished PC synthesis and gene expression. Further analysis revealed that ZAT6 positively regulates the transcription of GSH1, GSH2, PCS1, and PCS2, but ZAT6 is capable of specifically binding to GSH1 promoter in vivo. Consistently, overexpression of GSH1 has been shown to restore Cd sensitivity in the zat6-1 mutant, suggesting that GSH1 is a key target of ZAT6. Taken together, our data provide evidence that ZAT6 coordinately activates PC synthesis-related gene expression and directly targets GSH1 to positively regulate Cd accumulation and tolerance in Arabidopsis. PMID:26983992

  14. Identification of Arabidopsis Cyclase-associated Protein 1 as the First Nucleotide Exchange Factor for Plant Actin

    PubMed Central

    Chaudhry, Faisal; Guérin, Christophe; von Witsch, Matthias

    2007-01-01

    The actin cytoskeleton powers organelle movements, orchestrates responses to abiotic stresses, and generates an amazing array of cell shapes. Underpinning these diverse functions of the actin cytoskeleton are several dozen accessory proteins that coordinate actin filament dynamics and construct higher-order assemblies. Many actin-binding proteins from the plant kingdom have been characterized and their function is often surprisingly distinct from mammalian and fungal counterparts. The adenylyl cyclase-associated protein (CAP) has recently been shown to be an important regulator of actin dynamics in vivo and in vitro. The disruption of actin organization in cap mutant plants indicates defects in actin dynamics or the regulated assembly and disassembly of actin subunits into filaments. Current models for actin dynamics maintain that actin-depolymerizing factor (ADF)/cofilin removes ADP–actin subunits from filament ends and that profilin recharges these monomers with ATP by enhancing nucleotide exchange and delivery of subunits onto filament barbed ends. Plant profilins, however, lack the essential ability to stimulate nucleotide exchange on actin, suggesting that there might be a missing link yet to be discovered from plants. Here, we show that Arabidopsis thaliana CAP1 (AtCAP1) is an abundant cytoplasmic protein; it is present at a 1:3 M ratio with total actin in suspension cells. AtCAP1 has equivalent affinities for ADP– and ATP–monomeric actin (Kd ∼ 1.3 μM). Binding of AtCAP1 to ATP–actin monomers inhibits polymerization, consistent with AtCAP1 being an actin sequestering protein. However, we demonstrate that AtCAP1 is the first plant protein to increase the rate of nucleotide exchange on actin. Even in the presence of ADF/cofilin, AtCAP1 can recharge actin monomers and presumably provide a polymerizable pool of subunits to profilin for addition onto filament ends. In turnover assays, plant profilin, ADF, and CAP act cooperatively to promote flux of

  15. Effects of botulinum toxin type D on secretion of tumor necrosis factor from human monocytes

    SciTech Connect

    Imamura, K.; Spriggs, D.; Ohno, T.; Kufe, D.

    1989-05-01

    Botulinum toxins are potent neurotoxins which block the release of neurotransmitters. The effects of these toxins on hematopoietic cells, however, are unknown. Monocytes secrete a variety of polypeptide growth factors, including tumor necrosis factor (TNF). In the study reported here, the effects of botulinum toxin type D on the secretion of TNF from human monocytes were examined. The results demonstrate that biotulinum toxin type D inhibits the release of TNF from monocytes activated by lipopolysaccharide (LPS) but not by 12-O-tetradecanoylphorbol-13-acetate. Botulinum toxin type D had no detectable effect on intracellular TNF levels in LPS-treated monocytes, indicating that the effects of this toxin involve the secretory process. This inhibitory effect of botulinum toxin type D on TNF secretion from LPS-treated monocytes was partially reversed by treatment with 12-O-tetradecanoylphorbol-13-acetate or introduction of guanosine 5'-(/gamma/-thio)t-riphosphate into these cells. The results demonstrate that TNF secretion is regulated by at least two distinct guanine nucleotide-binding proteins, one responsible for the activation of phospholiphase C and another which acts as a substrate for botulinum toxin type D. ADP-ribosylation of monocyte membranes by botulinum toxin type D demonstrated the presence of three substrates with M/sub r/s of 45,000, 21,000, and 17,000. While the role of these substrates in exocytosis is unknown, the results suggest that the M/sub r/ 21,000 substrate is involved in a process other than TNF secretion.

  16. Arabidopsis histidine-containing phosphotransfer factor 4 (AHP4) negatively regulates secondary wall thickening of the anther endothecium during flowering.

    PubMed

    Jung, Kwang Wook; Oh, Seung-Ick; Kim, Yun Young; Yoo, Kyoung Shin; Cui, Mei Hua; Shin, Jeong Sheop

    2008-04-30

    Cytokinins are essential hormones in plant development. Arabidopsis histidine-containing phosphotransfer proteins (AHPs) are mediators in a multistep phosphorelay pathway for cytokinin signaling. The exact role of AHP4 has not been elucidated. In this study, we demonstrated young flower-specific expression of AHP4, and compared AHP4-overexpressing (Ox) trangenic Arabidopsis lines and an ahp4 knock-out line. AHP4-Ox plants had reduced fertility due to a lack of secondary cell wall thickening in the anther endothecium and inhibition of IRREGURAR XYLEMs (IRXs) expression in young flowers. Conversely, ahp4 anthers had more lignified anther walls than the wild type, and increased IRXs expression. Our study indicates that AHP4 negatively regulates thickening of the secondary cell wall of the anther endothecium, and provides new insight into the role of cytokinins in formation of secondary cell walls via the action of AHP4. PMID:18413999

  17. AtDOF5.4/OBP4, a DOF Transcription Factor Gene that Negatively Regulates Cell Cycle Progression and Cell Expansion in Arabidopsis thaliana

    PubMed Central

    Xu, Peipei; Chen, Haiying; Ying, Lu; Cai, Weiming

    2016-01-01

    In contrast to animals, plant development involves continuous organ formation, which requires strict regulation of cell proliferation. The core cell cycle machinery is conserved across plants and animals, but plants have developed new mechanisms that precisely regulate cell proliferation in response to internal and external stimuli. Here, we report that the DOF transcription factor OBP4 negatively regulates cell proliferation and expansion. OBP4 is a nuclear protein. Constitutive and inducible overexpression of OBP4 reduced the cell size and number, resulting in dwarf plants. Inducible overexpression of OBP4 in Arabidopsis also promoted early endocycle onset and inhibited cell expansion, while inducible overexpression of OBP4 fused to the VP16 activation domain in Arabidopsis delayed endocycle onset and promoted plant growth. Furthermore, gene expression analysis showed that cell cycle regulators and cell wall expansion factors were largely down-regulated in the OBP4 overexpression lines. Short-term inducible analysis coupled with in vivo ChIP assays indicated that OBP4 targets the CyclinB1;1, CDKB1;1 and XTH genes. These results strongly suggest that OBP4 is a negative regulator of cell cycle progression and cell growth. These findings increase our understanding of the transcriptional regulation of the cell cycle in plants. PMID:27297966

  18. The Arabidopsis Transcription Factor NAC016 Promotes Drought Stress Responses by Repressing AREB1 Transcription through a Trifurcate Feed-Forward Regulatory Loop Involving NAP[OPEN

    PubMed Central

    Sakuraba, Yasuhito; Kim, Ye-Sol; Han, Su-Hyun; Lee, Byoung-Doo; Paek, Nam-Chon

    2015-01-01

    Drought and other abiotic stresses negatively affect plant growth and development and thus reduce productivity. The plant-specific NAM/ATAF1/2/CUC2 (NAC) transcription factors have important roles in abiotic stress-responsive signaling. Here, we show that Arabidopsis thaliana NAC016 is involved in drought stress responses; nac016 mutants have high drought tolerance, and NAC016-overexpressing (NAC016-OX) plants have low drought tolerance. Using genome-wide gene expression microarray analysis and MEME motif searches, we identified the NAC016-specific binding motif (NAC16BM), GATTGGAT[AT]CA, in the promoters of genes downregulated in nac016-1 mutants. The NAC16BM sequence does not contain the core NAC binding motif CACG (or its reverse complement CGTG). NAC016 directly binds to the NAC16BM in the promoter of ABSCISIC ACID-RESPONSIVE ELEMENT BINDING PROTEIN1 (AREB1), which encodes a central transcription factor in the stress-responsive abscisic acid signaling pathway and represses AREB1 transcription. We found that knockout mutants of the NAC016 target gene NAC-LIKE, ACTIVATED BY AP3/PI (NAP) also exhibited strong drought tolerance; moreover, NAP binds to the AREB1 promoter and suppresses AREB1 transcription. Taking these results together, we propose that a trifurcate feed-forward pathway involving NAC016, NAP, and AREB1 functions in the drought stress response, in addition to affecting leaf senescence in Arabidopsis. PMID:26059204

  19. Ectopic Overexpression of SsCBF1, a CRT/DRE-Binding Factor from the Nightshade Plant Solanum lycopersicoides, Confers Freezing and Salt Tolerance in Transgenic Arabidopsis

    PubMed Central

    Zhang, Lili; Li, Zhenjun; Li, Jingfu; Wang, Aoxue

    2013-01-01

    The C-repeat (CRT)/dehydration-responsive element (DRE) binding factor (CBF/DREB1) transcription factors play a key role in cold response. However, the detailed roles of many plant CBFs are far from fully understood. A CBF gene (SsCBF1) was isolated from the cold-hardy plant Solanum lycopersicoides. A subcellular localization study using GFP fusion protein indicated that SsCBF1 is localized in the nucleus. We delimited the SsCBF1 transcriptional activation domain to the C-terminal segment comprising amino acid residues 193–228 (SsCBF1193–228). The expression of SsCBF1 could be dramatically induced by cold, drought and high salinity. Transactivation assays in tobacco leaves revealed that SsCBF1 could specifically bind to the CRT cis-elements in vivo to activate the expression of downstream reporter genes. The ectopic overexpression of SsCBF1 conferred increased freezing and high-salinity tolerance and late flowering phenotype to transgenic Arabidopsis. RNA-sequencing data exhibited that a set of cold and salt stress responsive genes were up-regulated in transgenic Arabidopsis. Our results suggest that SsCBF1 behaves as a typical CBF to contribute to plant freezing tolerance. Increased resistance to high-salinity and late flowering phenotype derived from SsCBF1 OE lines lend more credence to the hypothesis that plant CBFs participate in diverse physiological and biochemical processes related to adverse conditions. PMID:23755095

  20. The Membrane-Bound NAC Transcription Factor ANAC013 Functions in Mitochondrial Retrograde Regulation of the Oxidative Stress Response in Arabidopsis[C][W

    PubMed Central

    De Clercq, Inge; Vermeirssen, Vanessa; Van Aken, Olivier; Vandepoele, Klaas; Murcha, Monika W.; Law, Simon R.; Inzé, Annelies; Ng, Sophia; Ivanova, Aneta; Rombaut, Debbie; van de Cotte, Brigitte; Jaspers, Pinja; Van de Peer, Yves; Kangasjärvi, Jaakko; Whelan, James; Van Breusegem, Frank

    2013-01-01

    Upon disturbance of their function by stress, mitochondria can signal to the nucleus to steer the expression of responsive genes. This mitochondria-to-nucleus communication is often referred to as mitochondrial retrograde regulation (MRR). Although reactive oxygen species and calcium are likely candidate signaling molecules for MRR, the protein signaling components in plants remain largely unknown. Through meta-analysis of transcriptome data, we detected a set of genes that are common and robust targets of MRR and used them as a bait to identify its transcriptional regulators. In the upstream regions of these mitochondrial dysfunction stimulon (MDS) genes, we found a cis-regulatory element, the mitochondrial dysfunction motif (MDM), which is necessary and sufficient for gene expression under various mitochondrial perturbation conditions. Yeast one-hybrid analysis and electrophoretic mobility shift assays revealed that the transmembrane domain–containing NO APICAL MERISTEM/ARABIDOPSIS TRANSCRIPTION ACTIVATION FACTOR/CUP-SHAPED COTYLEDON transcription factors (ANAC013, ANAC016, ANAC017, ANAC053, and ANAC078) bound to the MDM cis-regulatory element. We demonstrate that ANAC013 mediates MRR-induced expression of the MDS genes by direct interaction with the MDM cis-regulatory element and triggers increased oxidative stress tolerance. In conclusion, we characterized ANAC013 as a regulator of MRR upon stress in Arabidopsis thaliana. PMID:24045019

  1. Arabidopsis WRKY6 Transcription Factor Acts as a Positive Regulator of Abscisic Acid Signaling during Seed Germination and Early Seedling Development.

    PubMed

    Huang, Yun; Feng, Cui-Zhu; Ye, Qing; Wu, Wei-Hua; Chen, Yi-Fang

    2016-02-01

    The phytohormone abscisic acid (ABA) plays important roles during seed germination and early seedling development. Here, we characterized the function of the Arabidopsis WRKY6 transcription factor in ABA signaling. The transcript of WRKY6 was repressed during seed germination and early seedling development, and induced by exogenous ABA. The wrky6-1 and wrky6-2 mutants were ABA insensitive, whereas WRKY6-overexpressing lines showed ABA-hypersensitive phenotypes during seed germination and early seedling development. The expression of RAV1 was suppressed in the WRKY6-overexpressing lines and elevated in the wrky6 mutants, and the expression of ABI3, ABI4, and ABI5, which was directly down-regulated by RAV1, was enhanced in the WRKY6-overexpressing lines and repressed in the wrky6 mutants. Electrophoretic mobility shift and chromatin immunoprecipitation assays showed that WRKY6 could bind to the RAV1 promoter in vitro and in vivo. Overexpression of RAV1 in WRKY6-overexpressing lines abolished their ABA-hypersensitive phenotypes, and the rav1 wrky6-2 double mutant showed an ABA-hypersensitive phenotype, similar to rav1 mutant. Together, the results demonstrated that the Arabidopsis WRKY6 transcription factor played important roles in ABA signaling by directly down-regulating RAV1 expression. PMID:26829043

  2. Arabidopsis WRKY6 Transcription Factor Acts as a Positive Regulator of Abscisic Acid Signaling during Seed Germination and Early Seedling Development

    PubMed Central

    Wu, Wei-Hua; Chen, Yi-Fang

    2016-01-01

    The phytohormone abscisic acid (ABA) plays important roles during seed germination and early seedling development. Here, we characterized the function of the Arabidopsis WRKY6 transcription factor in ABA signaling. The transcript of WRKY6 was repressed during seed germination and early seedling development, and induced by exogenous ABA. The wrky6-1 and wrky6-2 mutants were ABA insensitive, whereas WRKY6-overexpressing lines showed ABA-hypersensitive phenotypes during seed germination and early seedling development. The expression of RAV1 was suppressed in the WRKY6-overexpressing lines and elevated in the wrky6 mutants, and the expression of ABI3, ABI4, and ABI5, which was directly down-regulated by RAV1, was enhanced in the WRKY6-overexpressing lines and repressed in the wrky6 mutants. Electrophoretic mobility shift and chromatin immunoprecipitation assays showed that WRKY6 could bind to the RAV1 promoter in vitro and in vivo. Overexpression of RAV1 in WRKY6-overexpressing lines abolished their ABA-hypersensitive phenotypes, and the rav1 wrky6-2 double mutant showed an ABA-hypersensitive phenotype, similar to rav1 mutant. Together, the results demonstrated that the Arabidopsis WRKY6 transcription factor played important roles in ABA signaling by directly down-regulating RAV1 expression. PMID:26829043

  3. AtDOF5.4/OBP4, a DOF Transcription Factor Gene that Negatively Regulates Cell Cycle Progression and Cell Expansion in Arabidopsis thaliana.

    PubMed

    Xu, Peipei; Chen, Haiying; Ying, Lu; Cai, Weiming

    2016-01-01

    In contrast to animals, plant development involves continuous organ formation, which requires strict regulation of cell proliferation. The core cell cycle machinery is conserved across plants and animals, but plants have developed new mechanisms that precisely regulate cell proliferation in response to internal and external stimuli. Here, we report that the DOF transcription factor OBP4 negatively regulates cell proliferation and expansion. OBP4 is a nuclear protein. Constitutive and inducible overexpression of OBP4 reduced the cell size and number, resulting in dwarf plants. Inducible overexpression of OBP4 in Arabidopsis also promoted early endocycle onset and inhibited cell expansion, while inducible overexpression of OBP4 fused to the VP16 activation domain in Arabidopsis delayed endocycle onset and promoted plant growth. Furthermore, gene expression analysis showed that cell cycle regulators and cell wall expansion factors were largely down-regulated in the OBP4 overexpression lines. Short-term inducible analysis coupled with in vivo ChIP assays indicated that OBP4 targets the CyclinB1;1, CDKB1;1 and XTH genes. These results strongly suggest that OBP4 is a negative regulator of cell cycle progression and cell growth. These findings increase our understanding of the transcriptional regulation of the cell cycle in plants. PMID:27297966

  4. An eukaryotic translation initiation factor, AteIF5A-2, affects cadmium accumulation and sensitivity in Arabidopsis.

    PubMed

    Xu, Xiao-Yan; Ding, Zhong-Jie; Chen, Lei; Yan, Jin-Ying; Li, Gui-Xin; Zheng, Shao-Jian

    2015-10-01

    Cadmium (Cd) is one of the most toxic elements and can be accumulated in plants easily; meanwhile, eIF5A is a highly conserved protein in all eukaryotic organisms. The present work tried to investigate whether eIF5A is involved in Cd accumulation and sensitivity in Arabidopsis (Arabidopsis thaliana L.) by comparing the wild-type Columbia-0 (Col-0) with a knockdown mutant of AteIF5A-2, fbr12-3 under Cd stress conditions. The results showed that the mutant fbr12-3 accumulated more Cd in roots and shoots and had significantly lower chlorophyll content, shorter root length, and smaller biomass, suggesting that downregulation of AteIF5A-2 makes the mutant more Cd sensitive. Real-time polymerase chain reaction revealed that the expressions of metal transporters involved in Cd uptake and translocation including IRT1, ZIP1, AtNramp3, and AtHMA4 were significantly increased but the expressions of PCS1 and PCS2 related to Cd detoxification were decreased notably in fbr12-3 compared with Col-0. As a result, an increase in MDA and H2 O2 content but decrease in root trolox, glutathione and proline content under Cd stress was observed, indicating that a severer oxidative stress occurs in the mutant. All these results demonstrated for the first time that AteIF5A influences Cd sensitivity by affecting Cd uptake, accumulation, and detoxification in Arabidopsis. PMID:25559189

  5. Stability of the rhizosphere and endophytic bacterial communities associated with Arabidopsis thaliana (L.) Heynh under impact of cosmic factors

    NASA Astrophysics Data System (ADS)

    Kordium, V. A.; Adamchuk-Chala, N. I.; Moshinec, H. V.

    The orbital experiment will involve a growing of Arabidopsis plant seed to seed in the presence of a plant probiotic bacteria consortium introduced into the system The purpose of experiment is to characterize microbial community associated with Arabidopsis thaliana and determine how consortium of introduced bacteria along with the endemic plant-associated bacteria influences the plant development reproductive system and seed formation in spaceflight conditions The first study will be an examination of the survival of model bacteria in on the inoculated plant The second complex study is to examine the plant traits in particular the ultrastructure of root statocytes in order to determine whether the plant development proceeds normally under microgravity conditions on background of introduced bacteria and to assess the structural changes occurring in the cotyledons generative organs and seeds The third set of observations will concern studies of the structure of microbial community associated with Arabidopsis plants with traditional and molecular tools The fourth part of the work will be an examination of mobile genetic elements that can play a role in adaptation of bacteria to the spaceflight conditions however they may affect the stability of bacterial endo- and rhizosphere communities The final part of the proposal initiates the study of possible risk of the bacterial consortium use for a plant inoculation in spaceflight conditions An evaluation of this risk will be performed via examination of expression of the Klebsiella

  6. ZmSOC1, a MADS-box transcription factor from Zea mays, promotes flowering in Arabidopsis.

    PubMed

    Zhao, Suzhou; Luo, Yanzhong; Zhang, Zhanlu; Xu, Miaoyun; Wang, Weibu; Zhao, Yangmin; Zhang, Lan; Fan, Yunliu; Wang, Lei

    2014-01-01

    Zea mays is an economically important crop, but its molecular mechanism of flowering remains largely uncharacterized. The gene, SUPPRESSOR OF OVEREXPRESSION OF CONSTANS 1 (SOC1), integrates multiple flowering signals to regulate floral transition in Arabidopsis. In this study, ZmSOC1 was isolated from Zea mays. Sequence alignment and phylogenetic analysis demonstrated that the ZmSOC1 protein contained a highly conserved MADS domain and a typical SOC1 motif. ZmSOC1 protein was localized in the nucleus in protoplasts and showed no transcriptional activation activity in yeast cells. ZmSOC1 was highly expressed in maize reproductive organs, including filaments, ear and endosperm, but expression was very low in embryos; on the other hand, the abiotic stresses could repress ZmSOC1 expression. Overexpression of ZmSOC1 resulted in early flowering in Arabidopsis through increasing the expression of AtLFY and AtAP1. Overall, these results suggest that ZmSOC1 is a flowering promoter in Arabidopsis. PMID:25372944

  7. A Myb-related transcription factor is involved in the phytochrome regulation of an Arabidopsis Lhcb gene.

    PubMed Central

    Wang, Z Y; Kenigsbuch, D; Sun, L; Harel, E; Ong, M S; Tobin, E M

    1997-01-01

    We have isolated the gene for a protein designated CCA1. This protein can bind to a region of the promoter of an Arabidopsis light-harvesting chlorophyll a/b protein gene, Lhcb1*3, which is necessary for its regulation by phytochrome. The CCA1 protein interacted with two imperfect repeats in the Lhcb1*3 promoter, AAA/cAATCT, a sequence that is conserved in Lhcb genes. A region near the N terminus of CCA1, which has some homology to the repeated sequence found in the DNA binding domain of Myb proteins, is required for binding to the Lhcb1*3 promoter. Lines of transgenic Arabidopsis plants expressing antisense RNA for CCA1 showed reduced phytochrome induction of the endogenous Lhcb1*3 gene, whereas expression of another phytochrome-regulated gene, rbcS-1A, which encodes the small subunit of ribulose-1,5-bisphosphate carboxylase/oxygenase, was not affected. Thus, the CCA1 protein acts as a specific activator of Lhcb1*3 transcription in response to brief red illumination. The expression of CCA1 RNA was itself transiently increased when etiolated seedlings were transferred to light. We conclude that the CCA1 protein is a key element in the functioning of the phytochrome signal transduction pathway leading to increased transcription of this Lhcb gene in Arabidopsis. PMID:9144958

  8. ZmSOC1, an MADS-Box Transcription Factor from Zea mays, Promotes Flowering in Arabidopsis

    PubMed Central

    Zhao, Suzhou; Luo, Yanzhong; Zhang, Zhanlu; Xu, Miaoyun; Wang, Weibu; Zhao, Yangmin; Zhang, Lan; Fan, Yunliu; Wang, Lei

    2014-01-01

    Zea mays is an economically important crop, but its molecular mechanism of flowering remains largely uncharacterized. The gene, SUPPRESSOR OF OVEREXPRESSION OF CONSTANS 1 (SOC1), integrates multiple flowering signals to regulate floral transition in Arabidopsis. In this study, ZmSOC1 was isolated from Zea mays. Sequence alignment and phylogenetic analysis demonstrated that the ZmSOC1 protein contained a highly conserved MADS domain and a typical SOC1 motif. ZmSOC1 protein was localized in the nucleus in protoplasts and showed no transcriptional activation activity in yeast cells. ZmSOC1 was highly expressed in maize reproductive organs, including filaments, ear and endosperm, but expression was very low in embryos; on the other hand, the abiotic stresses could repress ZmSOC1 expression. Overexpression of ZmSOC1 resulted in early flowering in Arabidopsis through increasing the expression of AtLFY and AtAP1. Overall, these results suggest that ZmSOC1 is a flowering promoter in Arabidopsis. PMID:25372944

  9. Filamin A regulates neuronal migration through brefeldin A-inhibited guanine exchange factor 2-dependent Arf1 activation.

    PubMed

    Zhang, Jingping; Neal, Jason; Lian, Gewei; Hu, Jianjun; Lu, Jie; Sheen, Volney

    2013-10-01

    Periventricular heterotopias is a malformation of cortical development, characterized by ectopic neuronal nodules around ventricle lining and caused by an initial migration defect during early brain development. Human mutations in the Filamin A (FLNA) and ADP-ribosylation factor guanine exchange factor 2 [ARFGEF2; encoding brefeldin-A-inhibited guanine exchange factor-2 (BIG2)] genes give rise to this disorder. Previously, we have reported that Big2 inhibition impairs neuronal migration and binds to FlnA, and its loss promotes FlnA phosphorylation. FlnA phosphorylation dictates FlnA-actin binding affinity and consequently alters focal adhesion size and number to effect neuronal migration. Here we show that FlnA loss similarly impairs migration, reciprocally enhances Big2 expression, but also alters Big2 subcellular localization in both null and conditional FlnA mice. FlnA phosphorylation promotes relocalization of Big2 from the Golgi toward the lipid ruffles, thereby activating Big2-dependent Arf1 at the cell membrane. Loss of FlnA phosphorylation or Big2 function impairs Arf1-dependent vesicle trafficking at the periphery, and Arf1 is required for maintenance of cell-cell junction connectivity and focal adhesion assembly. Loss of Arf1 activity disrupts neuronal migration and cell adhesion. Collectively, these studies demonstrate a potential mechanism whereby coordinated interactions between actin (through FlnA) and vesicle trafficking (through Big2-Arf) direct the assembly and disassembly of membrane protein complexes required for neuronal migration and neuroependymal integrity. PMID:24089482

  10. The Transcription Factor AtDOF4.7 Is Involved in Ethylene- and IDA-Mediated Organ Abscission in Arabidopsis

    PubMed Central

    Wang, Gao-Qi; Wei, Peng-Cheng; Tan, Feng; Yu, Man; Zhang, Xiao-Yan; Chen, Qi-Jun; Wang, Xue-Chen

    2016-01-01

    Organ abscission is an important plant developmental process that occurs in response to environmental stress or pathogens. In Arabidopsis, ligand signals, such as ethylene or INFLORESCENCE DEFICIENT IN ABSCISSION (IDA), can regulate organ abscission. Previously, we reported that overexpression of AtDOF4.7, a transcription factor gene, directly suppresses the expression of the abscission-related gene ARABIDOPSIS DEHISCENCE ZONE POLYGALACTURONASE 2 (ADPG2), resulting in a deficiency of floral organ abscission. However, the relationship between AtDOF4.7 and abscission pathways still needs to be investigated. In this study, we showed that ethylene regulates the expression of AtDOF4.7, and the peptide ligand, IDA negatively regulates AtDOF4.7 at the transcriptional level. Genetic evidence indicates that AtDOF4.7 and IDA are involved in a common pathway, and a MAPK cascade can phosphorylate AtDOF4.7 in vitro. Further in vivo data suggest that AtDOF4.7 protein levels may be regulated by this phosphorylation. Collectively, our results indicate that ethylene regulates AtDOF4.7 that is involved in the IDA-mediated floral organ abscission pathway. PMID:27379143

  11. A Shoot-Specific Hypoxic Response of Arabidopsis Sheds Light on the Role of the Phosphate-Responsive Transcription Factor PHOSPHATE STARVATION RESPONSE11[W

    PubMed Central

    Klecker, Maria; Gasch, Philipp; Peisker, Helga; Dörmann, Peter; Schlicke, Hagen; Grimm, Bernhard; Mustroph, Angelika

    2014-01-01

    Plant responses to biotic and abiotic stresses are often very specific, but signal transduction pathways can partially or completely overlap. Here, we demonstrate that in Arabidopsis (Arabidopsis thaliana), the transcriptional responses to phosphate starvation and oxygen deficiency stress comprise a set of commonly induced genes. While the phosphate deficiency response is systemic, under oxygen deficiency, most of the commonly induced genes are found only in illuminated shoots. This jointly induced response to the two stresses is under control of the transcription factor PHOSPHATE STARVATION RESPONSE1 (PHR1), but not of the oxygen-sensing N-end rule pathway, and includes genes encoding proteins for the synthesis of galactolipids, which replace phospholipids in plant membranes under phosphate starvation. Despite the induction of galactolipid synthesis genes, total galactolipid content and plant survival are not severely affected by the up-regulation of galactolipid gene expression in illuminated leaves during hypoxia. However, changes in galactolipid molecular species composition point to an adaptation of lipid fluxes through the endoplasmic reticulum and chloroplast pathways during hypoxia. PHR1-mediated signaling of phosphate deprivation was also light dependent. Because a photoreceptor-mediated PHR1 activation was not detectable under hypoxia, our data suggest that a chloroplast-derived retrograde signal, potentially arising from metabolic changes, regulates PHR1 activity under both oxygen and phosphate deficiency. PMID:24753539

  12. STA1, an Arabidopsis pre-mRNA processing factor 6 homolog, is a new player involved in miRNA biogenesis

    PubMed Central

    Ben Chaabane, Samir; Liu, Renyi; Chinnusamy, Viswanathan; Kwon, Yerim; Park, Joo-hyuk; Kim, Seo Yeon; Zhu, Jian-Kang; Yang, Seong Wook; Lee, Byeong-ha

    2013-01-01

    MicroRNAs (miRNAs) are small regulatory RNAs that have important regulatory roles in numerous developmental and metabolic processes in most eukaryotes. In Arabidopsis, DICER-LIKE1 (DCL1), HYPONASTIC LEAVES 1, SERRATE, HUA ENHANCER1 and HASTY are involved in processing of primary miRNAs (pri-miRNAs) to yield precursor miRNAs (pre-miRNAs) and eventually miRNAs. In addition to these components, mRNA cap-binding proteins, CBP80/ABA HYPERSENSITIVE1 and CBP20, also participate in miRNA biogenesis. Here, we show that STABILIZED1 (STA1), an Arabidopsis pre-mRNA processing factor 6 homolog, is also involved in the biogenesis of miRNAs. Similar to other miRNA biogenesis-defective mutants, sta1-1 accumulated significantly lower levels of mature miRNAs and concurrently higher levels of pri-miRNAs than wild type. The dramatic reductions of mature miRNAs were associated with the accumulation of their target gene transcripts and developmental defects. Furthermore, sta1-1 impaired splicing of intron containing pri-miRNAs and decreased transcript levels of DCL1. These results suggest that STA1 is involved in miRNA biogenesis directly by functioning in pri-miRNA splicing and indirectly by modulating the DCL1 transcript level. PMID:23268445

  13. The Arabidopsis transcription factor IIB-related protein BRP4 is involved in the regulation of mitotic cell-cycle progression during male gametogenesis

    PubMed Central

    Hu, Yuxin

    2014-01-01

    Male gametogenesis in angiosperms involves two rounds of mitosis that are essential for the generation of two sperm cells to achieve double fertilization, a distinct event in the sexual reproduction of flowering plants. Precise regulation of mitosis during male gametogenesis is critically important for the establishment of the male germline. However, the molecular mechanisms underlying mitotic division during male gametophyte development have not been characterized fully. Here, we report that the Arabidopsis transcription initiation factor TFIIB-related protein BRP4 is involved in the regulation of mitotic cell-cycle progression during male gametogenesis. BRP4 was expressed predominately in developing male gametophytes. Knockdown expression of BRP4 by a native promoter-driven RNA interference construct in Arabidopsis resulted in arrest of the mitotic progression of male gametophytes, leading to a defect in pollen development. Moreover, we showed that the level of expression of a gene encoding a subunit of the origin recognition complex, ORC6, was decreased in BRP4 knockdown plants, and that the ORC6 knockdown transgenic plants phenocopied the male gametophyte defect observed in BRP4 knockdown plants, suggesting that ORC6 acts downstream of BRP4 to mediate male mitotic progression. Taken together, our results reveal that BRP4 plays an important role in the regulation of mitotic cell-cycle progression during male gametogenesis. PMID:24723406

  14. The Transcription Factor AtDOF4.7 Is Involved in Ethylene- and IDA-Mediated Organ Abscission in Arabidopsis.

    PubMed

    Wang, Gao-Qi; Wei, Peng-Cheng; Tan, Feng; Yu, Man; Zhang, Xiao-Yan; Chen, Qi-Jun; Wang, Xue-Chen

    2016-01-01

    Organ abscission is an important plant developmental process that occurs in response to environmental stress or pathogens. In Arabidopsis, ligand signals, such as ethylene or INFLORESCENCE DEFICIENT IN ABSCISSION (IDA), can regulate organ abscission. Previously, we reported that overexpression of AtDOF4.7, a transcription factor gene, directly suppresses the expression of the abscission-related gene ARABIDOPSIS DEHISCENCE ZONE POLYGALACTURONASE 2 (ADPG2), resulting in a deficiency of floral organ abscission. However, the relationship between AtDOF4.7 and abscission pathways still needs to be investigated. In this study, we showed that ethylene regulates the expression of AtDOF4.7, and the peptide ligand, IDA negatively regulates AtDOF4.7 at the transcriptional level. Genetic evidence indicates that AtDOF4.7 and IDA are involved in a common pathway, and a MAPK cascade can phosphorylate AtDOF4.7 in vitro. Further in vivo data suggest that AtDOF4.7 protein levels may be regulated by this phosphorylation. Collectively, our results indicate that ethylene regulates AtDOF4.7 that is involved in the IDA-mediated floral organ abscission pathway. PMID:27379143

  15. A Generic Tool for Transcription Factor Target Gene Discovery in Arabidopsis Cell Suspension Cultures Based on Tandem Chromatin Affinity Purification1[W][OPEN

    PubMed Central

    Verkest, Aurine; Abeel, Thomas; Heyndrickx, Ken S.; Van Leene, Jelle; Lanz, Christa; Van De Slijke, Eveline; De Winne, Nancy; Eeckhout, Dominique; Persiau, Geert; Van Breusegem, Frank; Inzé, Dirk; Vandepoele, Klaas; De Jaeger, Geert

    2014-01-01

    Genome-wide identification of transcription factor (TF) binding sites is pivotal to our understanding of gene expression regulation. Although much progress has been made in the determination of potential binding regions of proteins by chromatin immunoprecipitation, this method has some inherent limitations regarding DNA enrichment efficiency and antibody necessity. Here, we report an alternative strategy for assaying in vivo TF-DNA binding in Arabidopsis (Arabidopsis thaliana) cells by tandem chromatin affinity purification (TChAP). Evaluation of TChAP using the E2Fa TF and comparison with traditional chromatin immunoprecipitation and single chromatin affinity purification illustrates the suitability of TChAP and provides a resource for exploring the E2Fa transcriptional network. Integration with transcriptome, cis-regulatory element, functional enrichment, and coexpression network analyses demonstrates the quality of the E2Fa TChAP sequencing data and validates the identification of new direct E2Fa targets. TChAP enhances both TF target mapping throughput, by circumventing issues related to antibody availability, and output, by improving DNA enrichment efficiency. PMID:24453163

  16. The Beet Cyst Nematode Heterodera schachtii Modulates the Expression of WRKY Transcription Factors in Syncytia to Favour Its Development in Arabidopsis Roots

    PubMed Central

    Ali, Muhammad Amjad; Wieczorek, Krzysztof; Kreil, David P.; Bohlmann, Holger

    2014-01-01

    Cyst nematodes invade the roots of their host plants as second stage juveniles and induce a syncytium which is the only source of nutrients throughout their life. A recent transcriptome analysis of syncytia induced by the beet cyst nematode Heterodera schachtii in Arabidopsis roots has shown that thousands of genes are up-regulated or down-regulated in syncytia as compared to root segments from uninfected plants. Among the down-regulated genes are many which code for WRKY transcription factors. Arabidopsis contains 66 WRKY genes with 59 represented by the ATH1 GeneChip. Of these, 28 were significantly down-regulated and 6 up-regulated in syncytia as compared to control root segments. We have studied here the down-regulated genes WRKY6, WRKY11, WRKY17 and WRKY33 in detail. We confirmed the down-regulation in syncytia with promoter::GUS lines. Using various overexpression lines and mutants it was shown that the down-regulation of these WRKY genes is important for nematode development, probably through interfering with plant defense reactions. In case of WRKY33, this might involve the production of the phytoalexin camalexin. PMID:25033038

  17. Arabidopsis Actin Depolymerizing Factor4 Modulates the Stochastic Dynamic Behavior of Actin Filaments in the Cortical Array of Epidermal Cells[C][W

    PubMed Central

    Henty, Jessica L.; Bledsoe, Samuel W.; Khurana, Parul; Meagher, Richard B.; Day, Brad; Blanchoin, Laurent; Staiger, Christopher J.

    2011-01-01

    Actin filament arrays are constantly remodeled as the needs of cells change as well as during responses to biotic and abiotic stimuli. Previous studies demonstrate that many single actin filaments in the cortical array of living Arabidopsis thaliana epidermal cells undergo stochastic dynamics, a combination of rapid growth balanced by disassembly from prolific severing activity. Filament turnover and dynamics are well understood from in vitro biochemical analyses and simple reconstituted systems. However, the identification in living cells of the molecular players involved in controlling actin dynamics awaits the use of model systems, especially ones where the power of genetics can be combined with imaging of individual actin filaments at high spatial and temporal resolution. Here, we test the hypothesis that actin depolymerizing factor (ADF)/cofilin contributes to stochastic filament severing and facilitates actin turnover. A knockout mutant for Arabidopsis ADF4 has longer hypocotyls and epidermal cells when compared with wild-type seedlings. This correlates with a change in actin filament architecture; cytoskeletal arrays in adf4 cells are significantly more bundled and less dense than in wild-type cells. Several parameters of single actin filament turnover are also altered. Notably, adf4 mutant cells have a 2.5-fold reduced severing frequency as well as significantly increased actin filament lengths and lifetimes. Thus, we provide evidence that ADF4 contributes to the stochastic dynamic turnover of actin filaments in plant cells. PMID:22010035

  18. RNA Processing Factor 7 and Polynucleotide Phosphorylase Are Necessary for Processing and Stability of nad2 mRNA in Arabidopsis Mitochondria

    PubMed Central

    Stoll, Birgit; Zendler, Daniel; Binder, Stefan

    2014-01-01

    Post-transcriptional maturation of plant mitochondrial transcripts requires several steps. Among these, the generation of mature 5′ ends is still one of the most enigmatic processes. Toward a characterization of proteins involved in 5′ processing of mitochondrial transcripts in Arabidopsis (Arabidopsis thaliana), we now analyzed 5′ maturation of nad2 transcripts. Based on natural genetic variation affecting 5′ ends of nad2 transcripts in ecotype Can-0 and complementation studies we now identified RNA processing factor 7, which takes part in the generation of the 5′ terminus of the mature nad2 mRNA. RPF7 is a relatively short regular P-class pentatricopeptide repeat protein comprising seven canonical P repeats and a single short S repeat. The corresponding allele in Can-0 encodes a truncated version of this protein lacking two C-terminal repeats, which are essential for the function of RPF7. Furthermore we established transgenic plants expressing artifical microRNAs targeting the mitochondrial polynucleotide phosphorylase (PNPase), which results in substantial reduction of the PNPase mRNA levels and strong knockdown of this gene. Detailed quantitative studies of 5′ and 3′ extended nad2 precursor RNAs in these knockdown plants as well as in the rpf7–1 knockout mutant suggest that 5′ processing contributes to the stability of mitochondrial transcripts in plants. PMID:25181358

  19. Gladiolus hybridus ABSCISIC ACID INSENSITIVE 5 (GhABI5) is an important transcription factor in ABA signaling that can enhance Gladiolus corm dormancy and Arabidopsis seed dormancy

    PubMed Central

    Wu, Jian; Seng, Shanshan; Sui, Juanjuan; Vonapartis, Eliana; Luo, Xian; Gong, Benhe; Liu, Chen; Wu, Chenyu; Liu, Chao; Zhang, Fengqin; He, Junna; Yi, Mingfang

    2015-01-01

    The phytohormone abscisic acid (ABA) regulates plant development and is crucial for abiotic stress response. In this study, cold storage contributes to reducing endogenous ABA content, resulting in dormancy breaking of Gladiolus. The ABA inhibitor fluridone also promotes germination, suggesting that ABA is an important hormone that regulates corm dormancy. Here, we report the identification and functional characterization of the Gladiolus ABI5 homolog (GhABI5), which is a basic leucine zipper motif transcriptional factor (TF). GhABI5 is expressed in dormant vegetative organs (corm, cormel, and stolon) as well as in reproductive organs (stamen), and it is up-regulated by ABA or drought. Complementation analysis reveals that GhABI5 rescues the ABA insensitivity of abi5-3 during seed germination and induces the expression of downstream ABA response genes in Arabidopsis thaliana (EM1, EM6, and RD29B). Down-regulation of GhABI5 in dormant cormels via virus induced gene silence promotes sprouting and reduces the expression of downstream genes (GhLEA and GhRD29B). The results of this study reveal that GhABI5 regulates bud dormancy (vegetative organ) in Gladiolus in addition to its well-studied function in Arabidopsis seeds (reproductive organ). PMID:26579187

  20. Dysfunction of Chromatin Assembly Factor 1 Induces Shortening of Telomeres and Loss of 45S rDNA in Arabidopsis thaliana[W][OA

    PubMed Central

    Mozgová, Iva; Mokroš, Petr; Fajkus, Jiří

    2010-01-01

    Chromatin Assembly Factor 1 (CAF1) is a three-subunit H3/H4 histone chaperone responsible for replication-dependent nucleosome assembly. It is composed of CAC 1-3 in yeast; p155, p60, and p48 in humans; and FASCIATA1 (FAS1), FAS2, and MULTICOPY SUPPRESSOR OF IRA1 in Arabidopsis thaliana. We report that disruption of CAF1 function by fas mutations in Arabidopsis results in telomere shortening and loss of 45S rDNA, while other repetitive sequences (5S rDNA, centromeric 180-bp repeat, CACTA, and Athila) are unaffected. Substantial telomere shortening occurs immediately after the loss of functional CAF1 and slows down at telomeres shortened to median lengths around 1 to 1.5 kb. The 45S rDNA loss is progressive, leaving 10 to 15% of the original number of repeats in the 5th generation of mutants affecting CAF1, but the level of the 45S rRNA transcripts is not altered in these mutants. Increasing severity of the fas phenotype is accompanied by accumulation of anaphase bridges, reduced viability, and plant sterility. Our results show that appropriate replication-dependent chromatin assembly is specifically required for stable maintenance of telomeres and 45S rDNA. PMID:20699390

  1. Overexpression of soybean R2R3-MYB transcription factor, GmMYB12B2, and tolerance to UV radiation and salt stress in transgenic Arabidopsis.

    PubMed

    Li, X W; Wang, Y; Yan, F; Li, J W; Zhao, Y; Zhao, X; Zhai, Y; Wang, Q Y

    2016-01-01

    MYB, v-myb avian myeloblastosis viral oncogene homolog, proteins play central roles in plant stress response. Previously, we identified a novel R2R3-MYB transcription factor, GmMYB12B2, which affected the expression levels of some key enzyme genes involved in flavonoid biosynthesis in transgenic Arabidopsis. In the present study, we analyzed the expression levels of GmMYB12B2 under salt, low temperature, drought, abscisic acid (ABA), and ultraviolet (UV) radiation treatments in soybean using semi-quantitative reverse transcription polymerase chain reaction. The expression of GmMYB12B2 was drastically induced by UV irradiation and salt treatment, but no response was detected under low temperature, drought, and ABA stresses. A detailed characterization of the GmMYB12B2 overexpression lines revealed that GmMYB12B2 might be involved in response of plants to UV radiation and salt stresses. Transgenic Arabidopsis lines constitutively expressing GmMYB12B2 showed an increased tolerance to salt and UV radiation treatment compared with wild-type plants. The expression levels of certain salt stress-responsive genes, such as DREB2A and RD17, were found to be elevated in the transgenic plants. These results indicate that GmMYB12B2 acts as a regulator in the plant stress response. PMID:27323089

  2. A MYB transcription factor regulates very-long-chain fatty acid biosynthesis for activation of the hypersensitive cell death response in Arabidopsis.

    PubMed

    Raffaele, Sylvain; Vailleau, Fabienne; Léger, Amandine; Joubès, Jérôme; Miersch, Otto; Huard, Carine; Blée, Elisabeth; Mongrand, Sébastien; Domergue, Frédéric; Roby, Dominique

    2008-03-01

    Plant immune responses to pathogen attack include the hypersensitive response (HR), a form of programmed cell death occurring at invasion sites. We previously reported on Arabidopsis thaliana MYB30, a transcription factor that acts as a positive regulator of a cell death pathway conditioning the HR. Here, we show by microarray analyses of Arabidopsis plants misexpressing MYB30 that the genes encoding the four enzymes forming the acyl-coA elongase complex are putative MYB30 targets. The acyl-coA elongase complex synthesizes very-long-chain fatty acids (VLCFAs), and the accumulation of extracellular VLCFA-derived metabolites (leaf epidermal wax components) was affected in MYB30 knockout mutant and overexpressing lines. In the same lines, a lipid extraction procedure allowing high recovery of sphingolipids revealed changes in VLCFA contents that were amplified in response to inoculation. Finally, the exacerbated HR phenotype of MYB30-overexpressing lines was altered by the loss of function of the acyl-ACP thioesterase FATB, which causes severe defects in the supply of fatty acids for VLCFA biosynthesis. Based on these findings, we propose a model in which MYB30 modulates HR via VLCFAs by themselves, or VLCFA derivatives, as cell death messengers in plants. PMID:18326828

  3. Control of root hair development in Arabidopsis thaliana by an endoplasmic reticulum anchored member of the R2R3-MYB transcription factor family.

    PubMed

    Slabaugh, Erin; Held, Michael; Brandizzi, Federica

    2011-08-01

    The evolution of roots and root hairs was a crucial innovation that contributed to the adaptation of plants to a terrestrial environment. Initiation of root hairs involves transcriptional cues that in part determine cell patterning of the root epidermis. Once root hair initiation has occurred, elongation of the root hair takes place. Although many genes have been identified as being involved in root hair development, many contributors remain uncharacterized. In this study we report on the involvement of a member (here dubbed maMYB) of the plant-specific R2R3-MYB family of transcription factors in root hair elongation in Arabidopsis. We show that maMYB is associated with the endoplasmic reticulum membrane with the transcription factor domain exposed to the cytosol, suggesting that it may function as a membrane-tethered transcription factor. We demonstrate that a truncated form of maMYB (maMYB⁸⁴⁻³⁰⁹), which contains the R2R3-MYB transcription factor domain, is localized and retained in the nucleus, where it regulates gene expression. Silencing of maMyb resulted in plants with significantly shorter root hairs but similar root hair density compared with wild type, implying a role of the protein in root hair elongation. 2,4-D (2,4-dichlorophenoxyacetic acid), an exogenous auxin analog that promotes root hair elongation, rescued the short root hair phenotype and maMyb mRNA was induced in the presence of 2,4-D and IAA (indole-3-acetic acid). These results indicate a functional role of maMYB, which is integrated with auxin, in root hair elongation in Arabidopsis. PMID:21477080

  4. Modeling the Arabidopsis seed shape by a cardioid: efficacy of the adjustment with a scale change with factor equal to the Golden Ratio and analysis of seed shape in ethylene mutants.

    PubMed

    Cervantes, Emilio; Javier Martín, José; Ardanuy, Ramón; de Diego, Juana G; Tocino, Angel

    2010-03-15

    A new model for the description of Arabidopsis seed shape based on the comparison of the outline of its longitudinal section with a transformed cardioid is presented. The transformation consists of scaling the horizontal axis by a factor equal to the Golden Ratio. The elongated cardioid approximates the shape of the Arabidopsis seed with more accuracy than other figures. The length to width ratio in wild-type Columbia Arabidopsis dry seeds is close to the Golden Ratio and decreases over the course of imbibition. Dry seeds of etr1-1 mutants presented a reduced length to width ratio. Application of the new model based on the cardioid allows for comparison of shape between wild-type and mutant genotypes, revealing other general alterations in the seeds in ethylene signaling pathway mutants (etr1-1). PMID:19880215

  5. Nuclear Function of Subclass I Actin-Depolymerizing Factor Contributes to Susceptibility in Arabidopsis to an Adapted Powdery Mildew Fungus1[OPEN

    PubMed Central

    Inada, Noriko; Higaki, Takumi; Hasezawa, Seiichiro

    2016-01-01

    Actin-depolymerizing factors (ADFs) are conserved proteins that function in regulating the structure and dynamics of actin microfilaments in eukaryotes. In this study, we present evidence that Arabidopsis (Arabidopsis thaliana) subclass I ADFs, particularly ADF4, functions as a susceptibility factor for an adapted powdery mildew fungus. The null mutant of ADF4 significantly increased resistance against the adapted powdery mildew fungus Golovinomyces orontii. The degree of resistance was further enhanced in transgenic plants in which the expression of all subclass I ADFs (i.e. ADF1–ADF4) was suppressed. Microscopic observations revealed that the enhanced resistance of adf4 and ADF1-4 knockdown plants (ADF1-4Ri) was associated with the accumulation of hydrogen peroxide and cell death specific to G. orontii-infected cells. The increased resistance and accumulation of hydrogen peroxide in ADF1-4Ri were suppressed by the introduction of mutations in the salicylic acid- and jasmonic acid-signaling pathways but not by a mutation in the ethylene-signaling pathway. Quantification by microscopic images detected an increase in the level of actin microfilament bundling in ADF1-4Ri but not in adf4 at early G. orontii infection time points. Interestingly, complementation analysis revealed that nuclear localization of ADF4 was crucial for susceptibility to G. orontii. Based on its G. orontii-infected-cell-specific phenotype, we suggest that subclass I ADFs are susceptibility factors that function in a direct interaction between the host plant and the powdery mildew fungus. PMID:26747284

  6. The Arabidopsis GAGA-Binding Factor BASIC PENTACYSTEINE6 Recruits the POLYCOMB-REPRESSIVE COMPLEX1 Component LIKE HETEROCHROMATIN PROTEIN1 to GAGA DNA Motifs1

    PubMed Central

    Hecker, Andreas; Brand, Luise H.; Peter, Sébastien; Simoncello, Nathalie; Kilian, Joachim; Gaudin, Valérie

    2015-01-01

    Polycomb-repressive complexes (PRCs) play key roles in development by repressing a large number of genes involved in various functions. Much, however, remains to be discovered about PRC-silencing mechanisms as well as their targeting to specific genomic regions. Besides other mechanisms, GAGA-binding factors in animals can guide PRC members in a sequence-specific manner to Polycomb-responsive DNA elements. Here, we show that the Arabidopsis (Arabidopsis thaliana) GAGA-motif binding factor protein BASIC PENTACYSTEINE6 (BPC6) interacts with LIKE HETEROCHROMATIN PROTEIN1 (LHP1), a PRC1 component, and associates with VERNALIZATION2 (VRN2), a PRC2 component, in vivo. By using a modified DNA-protein interaction enzyme-linked immunosorbant assay, we could show that BPC6 was required and sufficient to recruit LHP1 to GAGA motif-containing DNA probes in vitro. We also found that LHP1 interacts with VRN2 and, therefore, can function as a possible scaffold between BPC6 and VRN2. The lhp1-4 bpc4 bpc6 triple mutant displayed a pleiotropic phenotype, extreme dwarfism and early flowering, which disclosed synergistic functions of LHP1 and group II plant BPC members. Transcriptome analyses supported this synergy and suggested a possible function in the concerted repression of homeotic genes, probably through histone H3 lysine-27 trimethylation. Hence, our findings suggest striking similarities between animal and plant GAGA-binding factors in the recruitment of PRC1 and PRC2 components to Polycomb-responsive DNA element-like GAGA motifs, which must have evolved through convergent evolution. PMID:26025051

  7. The cytohesin guanosine exchange factors (GEFs) are required to promote HGF-mediated renal recovery after acute kidney injury (AKI) in mice

    PubMed Central

    Reviriego-Mendoza, Marta M; Santy, Lorraine C

    2015-01-01

    The lack of current treatment and preventable measures for acute kidney injury (AKI) in hospitalized patients results in an increased mortality rate of up to 80% and elevated health costs. Additionally, if not properly repaired, those who survive AKI may develop fibrosis and long-term kidney damage. The molecular aspects of kidney injury and repair are still uncertain. Hepatocyte growth factor (HGF) promotes recovery of the injured kidney by inducing survival and migration of tubular epithelial cells to repopulate bare tubule areas. HGF-stimulated kidney epithelial cell migration requires the activation of ADP-ribosylation factor 6 (Arf6) and Rac1 via the cytohesin family of Arf-guanine-nucleotide exchange factors (GEFs), in vitro. We used an ischemia and reperfusion injury (IRI) mouse model to analyze the effects of modulating this signaling pathway on kidney recovery. We treated IRI mice with either HGF, the cytohesin inhibitor SecinH3, or a combination of both. As previously reported, HGF treatment promoted rapid improvement of kidney function as evidenced by creatinine (Cre) and blood urea nitrogen (BUN) levels. In contrast, simultaneous treatment with SecinH3 and HGF blocks the ability of HGF to promote kidney recovery. Immunohistochemistry showed that HGF treatment promoted recovery of tubule structure, and had enhanced levels of active, GTP-bound Arf6 and GTP-Rac1. SecinH3 treatment, however, caused a dramatic decrease in GTP-Arf6 and GTP-Rac1 levels when compared to kidney sections from HGF-treated IRI mice. Additionally, SecinH3 counteracted the renal reparative effects of HGF. Our results support the conclusion that cytohesin function is required for HGF-stimulated renal IRI repair. PMID:26116550

  8. MIXTA-Like Transcription Factors and WAX INDUCER1/SHINE1 Coordinately Regulate Cuticle Development in Arabidopsis and Torenia fournieri[C][W

    PubMed Central

    Oshima, Yoshimi; Shikata, Masahito; Koyama, Tomotsugu; Ohtsubo, Norihiro; Mitsuda, Nobutaka; Ohme-Takagi, Masaru

    2013-01-01

    The waxy plant cuticle protects cells from dehydration, repels pathogen attack, and prevents organ fusion during development. The transcription factor WAX INDUCER1/SHINE1 (WIN1/SHN1) regulates the biosynthesis of waxy substances in Arabidopsis thaliana. Here, we show that the MIXTA-like MYB transcription factors MYB106 and MYB16, which regulate epidermal cell morphology, also regulate cuticle development coordinately with WIN1/SHN1 in Arabidopsis and Torenia fournieri. Expression of a MYB106 chimeric repressor fusion (35S:MYB106-SRDX) and knockout/down of MYB106 and MYB16 induced cuticle deficiencies characterized by organ adhesion and reduction of epicuticular wax crystals and cutin nanoridges. A similar organ fusion phenotype was produced by expression of a WIN1/SHN1 chimeric repressor. Conversely, the dominant active form of MYB106 (35S:MYB106-VP16) induced ectopic production of cutin nanoridges and increased expression of WIN1/SHN1 and wax biosynthetic genes. Microarray experiments revealed that MYB106 and WIN1/SHN1 regulate similar sets of genes, predominantly those involved in wax and cutin biosynthesis. Furthermore, WIN1/SHN1 expression was induced by MYB106-VP16 and repressed by MYB106-SRDX. These results indicate that the regulatory cascade of MIXTA-like proteins and WIN1/SHN1 coordinately regulate cutin biosynthesis and wax accumulation. This study reveals an additional key aspect of MIXTA-like protein function and suggests a unique relationship between cuticle development and epidermal cell differentiation. PMID:23709630

  9. Protein intrinsic disorder in Arabidopsis NAC transcription factors: transcriptional activation by ANAC013 and ANAC046 and their interactions with RCD1.

    PubMed

    O'Shea, Charlotte; Kryger, Mikael; Stender, Emil G P; Kragelund, Birthe B; Willemoës, Martin; Skriver, Karen

    2015-01-15

    Protein ID (intrinsic disorder) plays a significant, yet relatively unexplored role in transcription factors (TFs). In the present paper, analysis of the transcription regulatory domains (TRDs) of six phylogenetically representative, plant-specific NAC [no apical meristem, ATAF (Arabidopsis transcription activation factor), cup-shaped cotyledon] TFs shows that the domains are present in similar average pre-molten or molten globule-like states, but have different patterns of order/disorder and MoRFs (molecular recognition features). ANAC046 (Arabidopsis NAC 046) was selected for further studies because of its simple MoRF pattern and its ability to interact with RCD1 (radical-induced cell death 1). Experiments in yeast and thermodynamic characterization suggest that its single MoRF region is sufficient for both transcriptional activation and interaction with RCD1. The remainder of the large regulatory domain is unlikely to contribute to the interaction, since the domain and truncations thereof have similar affinities for RCD1, which are also similar for ANAC013-RCD1 interactions. However, different enthalpic and entropic contributions to binding were revealed for ANAC046 and ANAC013, suggestive of differences in binding mechanisms. Although substitution of both hydrophobic and acidic residues of the ANAC046 MoRF region abolished binding, substitution of other residues, even with α-helix-breaking proline, was less disruptive. Together, the biophysical analyses suggest that RCD1-ANAC046 complex formation does not involve folding-upon-binding, but rather fuzziness or an unknown structure in ANAC046. We suggest that the ANAC046 regulatory domain functions as an entropic chain with a terminal hot spot interacting with RCD1. RCD1, a cellular hub, may be able to interact with many different TFs by exploiting their ID-based flexibility, as demonstrated for its interactions with ANAC046 and ANAC013. PMID:25348421

  10. A Dual-Function Transcription Factor, AtYY1, Is a Novel Negative Regulator of the Arabidopsis ABA Response Network.

    PubMed

    Li, Tian; Wu, Xiu-Yun; Li, Hui; Song, Jian-Hui; Liu, Jin-Yuan

    2016-05-01

    Abscisic acid (ABA) plays crucial roles in plant growth and development, as well as in response to various environmental stresses. To date, many regulatory genes involved in the ABA response network have been identified; however, their roles have remained to be fully elucidated. In this study, we identified AtYY1, an Arabidopsis homolog of the mammalian C2H2 zinc-finger transcription factor Yin Yang 1 (YY1), as a novel negative regulator of the ABA response. AtYY1 is a dual-function transcription factor with both repression and activation domains. The expression of AtYY1 was induced by ABA and stress conditions including high salt and dehydration. The yy1 mutant was more sensitive to ABA and NaCl than the wild-type, while overexpressing AtYY1 plants were less sensitive. AtYY1 loss also enhanced ABA-induced stomatal closing and drought resistance. Moreover, AtYY1 can bind the ABA REPRESSOR1 (ABR1) promoter and directly upregulate ABR1 expression, as well as negatively regulate ABA- and salt-responsive gene expression. Additional analysis indicated that ABA INSENSITIVE4 (ABI4) might positively regulate AtYY1 expression and that ABR1 can antagonize this regulation. Our findings provide direct evidence that AtYY1 is a novel negative regulator of the ABA response network and that the ABI4-AtYY1-ABR1 regulatory pathway may fine-tune ABA-responsive gene expression in Arabidopsis. PMID:26961720

  11. Molecular Evidence for Functional Divergence and Decay of a Transcription Factor Derived from Whole-Genome Duplication in Arabidopsis thaliana1[OPEN

    PubMed Central

    Lehti-Shiu, Melissa D.; Uygun, Sahra; Moghe, Gaurav D.; Panchy, Nicholas; Fang, Liang; Hufnagel, David E.; Jasicki, Hannah L.; Feig, Michael; Shiu, Shin-Han

    2015-01-01

    Functional divergence between duplicate transcription factors (TFs) has been linked to critical events in the evolution of land plants and can result from changes in patterns of expression, binding site divergence, and/or interactions with other proteins. Although plant TFs tend to be retained post polyploidization, many are lost within tens to hundreds of million years. Thus, it can be hypothesized that some TFs in plant genomes are in the process of becoming pseudogenes. Here, we use a pair of salt tolerance-conferring transcription factors, DWARF AND DELAYED FLOWERING1 (DDF1) and DDF2, that duplicated through paleopolyploidy 50 to 65 million years ago, as examples to illustrate potential mechanisms leading to duplicate retention and loss. We found that the expression patterns of Arabidopsis thaliana (At)DDF1 and AtDDF2 have diverged in a highly asymmetric manner, and AtDDF2 has lost most inferred ancestral stress responses. Consistent with promoter disablement, the AtDDF2 promoter has fewer predicted cis-elements and a methylated repetitive element. Through comparisons of AtDDF1, AtDDF2, and their Arabidopsis lyrata orthologs, we identified significant differences in binding affinities and binding site preference. In particular, an AtDDF2-specific substitution within the DNA-binding domain significantly reduces binding affinity. Cross-species analyses indicate that both AtDDF1 and AtDDF2 are under selective constraint, but among A. thaliana accessions, AtDDF2 has a higher level of nonsynonymous nucleotide diversity compared with AtDDF1. This may be the result of selection in different environments or may point toward the possibility of ongoing functional decay despite retention for millions of years after gene duplication. PMID:26103993

  12. Identification of Insulin-Like Growth Factor-I Receptor (IGF-IR) Gene Promoter-Binding Proteins in Estrogen Receptor (ER)-Positive and ER-Depleted Breast Cancer Cells

    PubMed Central

    Sarfstein, Rive; Belfiore, Antonino; Werner, Haim

    2010-01-01

    The insulin-like growth factor I receptor (IGF-IR) has been implicated in the etiology of breast cancer. Overexpression of the IGF-IR gene is a typical feature of most primary breast cancers, whereas low IGF-IR levels are seen at advanced stages. Hence, evaluation of IGF-IR levels might be important for assessing prognosis. In the present study, we employed a proteomic approach based on DNA affinity chromatography followed either by mass spectroscopy (MS) or Western blot analysis to identify transcription factors that may associate with the IGF-IR promoter in estrogen receptor (ER)-positive and ER-depleted breast cancer cells. A biotinylated IGF-IR promoter fragment was bound to streptavidin magnetic beads and incubated with nuclear extracts of breast cancer cells. IGF-IR promoter-binding proteins were eluted with high salt and analyzed by MS and Western blots. Among the proteins that were found to bind to the IGF-IR promoter we identified zinc finger transcription factors Sp1 and KLF6, ER-α, p53, c-jun, and poly (ADP-ribosylation) polymerase. Furthermore, chromatin immune-precipitation (ChIP) analysis confirmed the direct in vivo binding of some of these transcription factors to IGF-IR promoter DNA. The functional relevance of binding data was assessed by cotransfection experiments with specific expression vectors along with an IGF-IR promoter reporter. In summary, we identified nuclear proteins that are potentially responsible for the differential expression of the IGF-IR gene in ER-positive and ER-depleted breast cancer cells. PMID:24281069

  13. Genome-wide classification and evolutionary analysis of the bHLH family of transcription factors in Arabidopsis, poplar, rice, moss, and algae.

    PubMed

    Carretero-Paulet, Lorenzo; Galstyan, Anahit; Roig-Villanova, Irma; Martínez-García, Jaime F; Bilbao-Castro, Jose R; Robertson, David L

    2010-07-01

    Basic helix-loop-helix proteins (bHLHs) are found throughout the three eukaryotic kingdoms and constitute one of the largest families of transcription factors. A growing number of bHLH proteins have been functionally characterized in plants. However, some of these have not been previously classified. We present here an updated and comprehensive classification of the bHLHs encoded by the whole sequenced genomes of Arabidopsis (Arabidopsis thaliana), Populus trichocarpa, Oryza sativa, Physcomitrella patens, and five algae species. We define a plant bHLH consensus motif, which allowed the identification of novel highly diverged atypical bHLHs. Using yeast two-hybrid assays, we confirm that (1) a highly diverged bHLH has retained protein interaction activity and (2) the two most conserved positions in the consensus play an essential role in dimerization. Phylogenetic analysis permitted classification of the 638 bHLH genes identified into 32 subfamilies. Evolutionary and functional relationships within subfamilies are supported by intron patterns, predicted DNA-binding motifs, and the architecture of conserved protein motifs. Our analyses reveal the origin and evolutionary diversification of plant bHLHs through differential expansions, domain shuffling, and extensive sequence divergence. At the functional level, this would translate into different subfamilies evolving specific DNA-binding and protein interaction activities as well as differential transcriptional regulatory roles. Our results suggest a role for bHLH proteins in generating plant phenotypic diversity and provide a solid framework for further investigations into the role carried out in the transcriptional regulation of key growth and developmental processes. PMID:20472752

  14. The Arabidopsis transcription factor BRASSINOSTEROID INSENSITIVE1-ETHYL METHANESULFONATE-SUPPRESSOR1 is a direct substrate of MITOGEN-ACTIVATED PROTEIN KINASE6 and regulates immunity.

    PubMed

    Kang, Sining; Yang, Fan; Li, Lin; Chen, Huamin; Chen, She; Zhang, Jie

    2015-03-01

    Pathogen-associated molecular patterns (PAMPs) are recognized by plant pattern recognition receptors to activate PAMP-triggered immunity (PTI). Mitogen-activated protein kinases (MAPKs), as well as other cytoplasmic kinases, integrate upstream immune signals and, in turn, dissect PTI signaling via different substrates to regulate defense responses. However, only a few direct substrates of these signaling kinases have been identified. Here, we show that PAMP perception enhances phosphorylation of BRASSINOSTEROID INSENSITIVE1-ETHYL METHANESULFONATE-SUPPRESSOR1 (BES1), a transcription factor involved in brassinosteroid (BR) signaling pathway, through pathogen-induced MAPKs in Arabidopsis (Arabidopsis thaliana). BES1 interacts with MITOGEN-ACTIVATED PROTEIN KINASE6 (MPK6) and is phosphorylated by MPK6. bes1 loss-of-function mutants display compromised resistance to bacterial pathogen Pseudomonas syringae pv tomato DC3000. BES1 S286A/S137A double mutation (BES1(SSAA)) impairs PAMP-induced phosphorylation and fails to restore bacterial resistance in bes1 mutant, indicating a positive role of BES1 phosphorylation in plant immunity. BES1 is phosphorylated by glycogen synthase kinase3 (GSK3)-like kinase BR-insensitive2 (BIN2), a negative regulator of BR signaling. BR perception inhibits BIN2 activity, allowing dephosphorylation of BES1 to regulate plant development. However, BES1(SSAA) does not affect BR-mediated plant growth, suggesting differential residue requirements for the modulation of BES1 phosphorylation in PTI and BR signaling. Our study identifies BES1 as a unique direct substrate of MPK6 in PTI signaling. This finding reveals MAPK-mediated BES1 phosphorylation as another BES1 modulation mechanism in plant cell signaling, in addition to GSK3-like kinase-mediated BES1 phosphorylation and F box protein-mediated BES1 degradation. PMID:25609555

  15. Protein Disulfide Isomerase-2 of Arabidopsis Mediates Protein Folding and Localizes to Both the Secretory Pathway and Nucleus, Where It Interacts with Maternal Effect Embryo Arrest Factor

    PubMed Central

    Cho, Eun Ju; Yuen, Christen Y.L.; Kang, Byung-Ho; Ondzighi, Christine A.; Staehelin, L. Andrew; Christopher, David A.

    2011-01-01

    Protein disulfide isomerase (PDI) is a thiodisulfide oxidoreductase that catalyzes the formation, reduction and rearrangement of disulfide bonds in proteins of eukaryotes. The classical PDI has a signal peptide, two CXXCcontaining thioredoxin catalytic sites (a,a′), two noncatalytic thioredoxin fold domains (b,b′), an acidic domain (c) and a C-terminal endoplasmic reticulum (ER) retention signal. Although PDI resides in the ER where it mediates the folding of nascent polypeptides of the secretory pathway, we recently showed that PDI5 of Arabidopsis thaliana chaperones and inhibits cysteine proteases during trafficking to vacuoles prior to programmed cell death of the endothelium in developing seeds. Here we describe Arabidopsis PDI2, which shares a primary structure similar to that of classical PDI. Recombinant PDI2 is imported into ER-derived microsomes and complements the E. coli protein- folding mutant, dsbA. PDI2 interacted with proteins in both the ER and nucleus, including ER-resident protein folding chaperone, BiP1, and nuclear embryo transcription factor, MEE8. The PDI2-MEE8 interaction was confirmed to occur in vitro and in vivo. Transient expression of PDI2- GFP fusions in mesophyll protoplasts resulted in labeling of the ER, nucleus and vacuole. PDI2 is expressed in multiple tissues, with relatively high expression in seeds and root tips. Immunoelectron microscopy with GFP- and PDI2-specific antisera on transgenic seeds (PDI2-GFP) and wild type roots demonstrated that PDI2 was found in the secretory pathway (ER, Golgi, vacuole, cell wall) and the nuclei. Our results indicate that PDI2 mediates protein folding in the ER and has new functional roles in the nucleus. PMID:21909944

  16. An R2R3-type transcription factor gene AtMYB59 regulates root growth and cell cycle progression in Arabidopsis.

    PubMed

    Mu, Rui-Ling; Cao, Yang-Rong; Liu, Yun-Feng; Lei, Gang; Zou, Hong-Feng; Liao, Yong; Wang, Hui-Wen; Zhang, Wan-Ke; Ma, Biao; Du, Ji-Zhou; Yuan, Ming; Zhang, Jin-Song; Chen, Shou-Yi

    2009-11-01

    MYB proteins play important roles in eukaryotic organisms. In plants, the R1R2R3-type MYB proteins function in cell cycle control. However, whether the R2R3-type MYB protein is also involved in the cell division process remains unknown. Here, we report that an R2R3-type transcription factor gene, AtMYB59, is involved in the regulation of cell cycle progression and root growth. The AtMYB59 protein is localized in the nuclei of onion epidermal cells and has transactivation activity. Expression of AtMYB59 in yeast cells suppresses cell proliferation, and the transformants have more nuclei and higher aneuploid DNA content with longer cells. Mutation in the conserved domain of AtMYB59 abolishes its effects on yeast cell growth. In synchronized Arabidopsis cell suspensions, the AtMYB59 gene is specifically expressed in the S phase during cell cycle progression. Expression and promoter-GUS analysis reveals that the AtMYB59 gene is abundantly expressed in roots. Transgenic plants overexpressing AtMYB59 have shorter roots compared with wild-type plants (Arabidopsis accession Col-0), and around half of the mitotic cells in root tips are at metaphase. Conversely, the null mutant myb59-1 has longer roots and fewer mitotic cells at metaphase than Col, suggesting that AtMYB59 may inhibit root growth by extending the metaphase of mitotic cells. AtMYB59 regulates many downstream genes, including the CYCB1;1 gene, probably through binding to MYB-responsive elements. These results support a role for AtMYB59 in cell cycle regulation and plant root growth. PMID:19581938

  17. RGF1 INSENSITIVE 1 to 5, a group of LRR receptor-like kinases, are essential for the perception of root meristem growth factor 1 in Arabidopsis thaliana.

    PubMed

    Ou, Yang; Lu, Xiaoting; Zi, Quaner; Xun, Qingqing; Zhang, Jingjie; Wu, Yujun; Shi, Hongyong; Wei, Zhuoyun; Zhao, Baolin; Zhang, Xiaoyue; He, Kai; Gou, Xiaoping; Li, Chuanyou; Li, Jia

    2016-06-01

    RGF1, a secreted peptide hormone, plays key roles in root meristem development in Arabidopsis. Previous studies indicated that a functional RGF1 needs to be sulfated at a tyrosine residue by a tyrosylprotein sulfotransferase and that RGF1 regulates the root meristem activity mainly via two downstream transcription factors, PLETHORA 1 (PLT1) and PLT2. How extracellular RGF1 is perceived by a plant cell, however, is unclear. Using genetic approaches, we discovered a clade of leucine-rich repeat receptor-like kinases, designated as RGF1 INSENSITIVE 1 (RGI1) to RGI5, serving as receptors of RGF1. Two independent rgi1 rgi2 rgi3 rgi4 rgi5 quintuple mutants display a consistent short primary root phenotype with a small size of meristem. An rgi1 rgi2 rgi3 rgi4 quadruple mutant shows a significantly reduced sensitivity to RGF1, and the quintuple mutant is completely insensitive to RGF1. The expression of PLT1 and PLT2 is almost undetectable in the quintuple mutant. Ectopic expression of PLT2 driven by an RGI2 promoter in the quintuple mutant greatly rescued its root meristem defects. One of the RGIs, RGI1, was subsequently analyzed biochemically in detail. In vitro dot blotting and pull-down analyses indicated that RGI1 can physically interact with RGF1. Exogenous application of RGF1 can quickly and simultaneously induce the phosphorylation and ubiquitination of RGI1, indicating that RGI1 can perceive and transduce the RGF1 peptide signal. Yet, the activated RGI1 is likely turned over rapidly. These results demonstrate that RGIs, acting as the receptors of RGF1, play essential roles in RGF1-PLT-mediated root meristem development in Arabidopsis thaliana. PMID:27229312

  18. Genome-Wide Classification and Evolutionary Analysis of the bHLH Family of Transcription Factors in Arabidopsis, Poplar, Rice, Moss, and Algae1[W

    PubMed Central

    Carretero-Paulet, Lorenzo; Galstyan, Anahit; Roig-Villanova, Irma; Martínez-García, Jaime F.; Bilbao-Castro, Jose R.; Robertson, David L.

    2010-01-01

    Basic helix-loop-helix proteins (bHLHs) are found throughout the three eukaryotic kingdoms and constitute one of the largest families of transcription factors. A growing number of bHLH proteins have been functionally characterized in plants. However, some of these have not been previously classified. We present here an updated and comprehensive classification of the bHLHs encoded by the whole sequenced genomes of Arabidopsis (Arabidopsis thaliana), Populus trichocarpa, Oryza sativa, Physcomitrella patens, and five algae species. We define a plant bHLH consensus motif, which allowed the identification of novel highly diverged atypical bHLHs. Using yeast two-hybrid assays, we confirm that (1) a highly diverged bHLH has retained protein interaction activity and (2) the two most conserved positions in the consensus play an essential role in dimerization. Phylogenetic analysis permitted classification of the 638 bHLH genes identified into 32 subfamilies. Evolutionary and functional relationships within subfamilies are supported by intron patterns, predicted DNA-binding motifs, and the architecture of conserved protein motifs. Our analyses reveal the origin and evolutionary diversification of plant bHLHs through differential expansions, domain shuffling, and extensive sequence divergence. At the functional level, this would translate into different subfamilies evolving specific DNA-binding and protein interaction activities as well as differential transcriptional regulatory roles. Our results suggest a role for bHLH proteins in generating plant phenotypic diversity and provide a solid framework for further investigations into the role carried out in the transcriptional regulation of key growth and developmental processes. PMID:20472752

  19. Phosphorylation and Dephosphorylation of the Presequence of Precursor MULTIPLE ORGANELLAR RNA EDITING FACTOR3 during Import into Mitochondria from Arabidopsis1[OPEN

    PubMed Central

    Law, Yee-Song; Zhang, Renshan; Guan, Xiaoqian; Cheng, Shifeng; Sun, Feng; Duncan, Owen; Murcha, Monika W.; Whelan, James; Lim, Boon Leong

    2015-01-01

    The nucleus-encoded mitochondria-targeted proteins, multiple organellar RNA editing factors (MORF3, MORF5, and MORF6), interact with Arabidopsis (Arabidopsis thaliana) PURPLE ACID PHOSPHATASE2 (AtPAP2) located on the chloroplast and mitochondrial outer membranes in a presequence-dependent manner. Phosphorylation of the presequence of the precursor MORF3 (pMORF3) by endogenous kinases in wheat germ translation lysate, leaf extracts, or STY kinases, but not in rabbit reticulocyte translation lysate, resulted in the inhibition of protein import into mitochondria. This inhibition of import could be overcome by altering threonine/serine residues to alanine on the presequence, thus preventing phosphorylation. Phosphorylated pMORF3, but not the phosphorylation-deficient pMORF3, can form a complex with 14-3-3 proteins and HEAT SHOCK PROTEIN70. The phosphorylation-deficient mutant of pMORF3 also displayed faster rates of import when translated in wheat germ lysates. Mitochondria isolated from plants with altered amounts of AtPAP2 displayed altered protein import kinetics. The import rate of pMORF3 synthesized in wheat germ translation lysate into pap2 mitochondria was slower than that into wild-type mitochondria, and this rate disparity was not seen for pMORF3 synthesized in rabbit reticulocyte translation lysate, the latter translation lysate largely deficient in kinase activity. Taken together, these results support a role for the phosphorylation and dephosphorylation of pMORF3 during the import into plant mitochondria. These results suggest that kinases, possibly STY kinases, and AtPAP2 are involved in the import of protein into both mitochondria and chloroplasts and provide a mechanism by which the import of proteins into both organelles may be coordinated. PMID:26304849

  20. Identification and isoprenylation of plant GTP-binding proteins.

    PubMed

    Biermann, B; Randall, S K; Crowell, D N

    1996-08-01

    To identify isoprenylated plant GTP-binding proteins, Arabidopsis thaliana and Nicotiana tabacum cDNA expression libraries were screened for cDNA-encoded proteins capable of binding [32P]GTP in vitro. ATGB2, an Arabidopsis homologue of the GTP-binding protein Rab2, was found to bind GTP in vitro and to be a substrate for a geranylgeranyl:protein transferase (GGTase) present in plant extracts. The carboxyl terminus of this protein contains a -GCCG sequence, which has not previously been shown to be recognized by any prenyl:protein transferase (PTase), but which most closely resembles that isoprenylated by the type II GGTase (-XXCC, -XCXC, or -CCXX). In vitro geranylgeranylation of an Arabidopsis Rab1 protein containing a carboxyl-terminal-CCGQ sequence confirmed the presence of a type II GGTase-like activity in plant extracts. Several other proteins were also identified by in vitro GTP binding, including Arabidopsis and tobacco homologues of Rab11, ARF (ADP-ribosylation factor) and Sar proteins, as well as a novel 22 kDa Arabidopsis protein (ATG81). This 22 kDa protein had consensus GTP-binding motifs and bound GTP with high specificity, but its structure was not closely related to that of any known GTP-binding protein (it most resembled proteins within the ARF/Sar and G protein alpha-subunit superfamilies). PMID:8843944

  1. Poly(ADP-ribose) Polymerase 1 Interacts with Nuclear Respiratory Factor 1 (NRF-1) and Plays a Role in NRF-1 Transcriptional Regulation*S⃞

    PubMed Central

    Hossain, Mohammad B.; Ji, Ping; Anish, Ramakrishnan; Jacobson, Raymond H.; Takada, Shinako

    2009-01-01

    Nuclear respiratory factor 1 (NRF-1) is one of the key transcriptional activators for nuclear-coded genes involved in mitochondrial biogenesis and function as well as for many housekeeping genes. A transcriptional co-activator PGC-1 and its related family member PRC have previously been shown to interact with NRF-1 and co-activate NRF-1. We show here that NRF-1 can also directly interact with poly(ADP-ribose) polymerase 1 (PARP-1) and co-purify the PARP-1·DNA-PK·Ku80·Ku70·topoisomerase IIβ-containing protein complex. Our in vitro binding experiments show that DNA-binding/dimerization domain of NRF-1 and the N-terminal half of PARP-1, which contains two Zinc fingers and the auto-modification domain, are responsible for the interaction, and that this interaction occurs with or without PARP-1 poly(ADP-ribosyl)ation (PARylation). DNA-bound NRF-1 can form a complex with PARP-1, suggesting that NRF-1 can recruit the PARP-1·DNA-PK·Ku80·Ku70·topoisomerase IIβ-containing protein complex to the promoter. PARP-1 can also PARylate the DNA-binding domain of NRF-1 and negatively regulate NRF-1·PARP-1 interaction. Transient transfection and chromatin immunoprecipitation experiments suggest that PARP-1 plays a role during transcriptional activation by NRF-1. Our finding identifies a new aspect of transcriptional regulation used by NRF-1. PMID:19181665

  2. Molecular Control of the Amount, Subcellular Location and Activity State of Translation Elongation Factor 2 (eEF-2) in Neurons Experiencing Stress

    PubMed Central

    Argüelles-Castilla, Sandro; Camandola, Simonetta; Hutchison, Emmette R.; Cutler, Roy G.; Ayala, Antonio; Mattson, Mark P.

    2013-01-01

    Eukaryotic elongation factor 2 (eEF-2) is an important regulator of the protein translation machinery wherein it controls the movement of the ribosome along the mRNA. The activity of eEF-2 is regulated by changes in cellular energy status and nutrient availability, and posttranslational modifications such as phosphorylation and mono-ADP-ribosylation. However, the mechanisms regulating protein translation under conditions of cellular stress in neurons are unknown. Here we show that when rat hippocampal neurons experience oxidative stress (lipid peroxidation induced by exposure to cumene hydroperoxide; CH), eEF-2 is hyperphosphorylated and ribosylated resulting in reduced translational activity. The degradation of eEF-2 requires calpain proteolytic activity and is accompanied by accumulation of eEF-2 in the nuclear compartment. The subcellular localization of both native and phosphorylated forms of eEF-2 is influenced by CRM1 and 14.3.3, respectively. In hippocampal neurons p53 interacts with non-phosphorylated (active) eEF-2, but not with its phosphorylated form. The p53 – eEF-2 complexes are present in cytoplasm and nucleus, and their abundance increases when neurons experience oxidative stress. The nuclear localization of active eEF-2 depends upon its interaction with p53, as cells lacking p53 contain less active eEF-2 in the nuclear compartment. Overexpression of eEF-2 in hippocampal neurons results in increased nuclear levels of eEF-2, and decreased cell death following exposure to CH. Our results reveal novel molecular mechanisms controlling the differential subcellular localization and activity state of eEF-2 that may influence the survival status of neurons during periods of elevated oxidative stress. PMID:23542375

  3. The activation process of Arabidopsis thaliana A1 gene encoding the translation elongation factor EF-1 alpha is conserved among angiosperms.

    PubMed

    Curie, C; Liboz, T; Montané, M H; Rouan, D; Axelos, M; Lescure, B

    1992-04-01

    In Arabidopsis thaliana, the activation process of the A1 EF-1 alpha gene depends on several elements. Using the GUS reporter gene, transient expression experiments have shown that mutations of upstream cis-acting elements of the A1 promoter, or the deletion of an intron located within the 5' non-coding region, similarly affect expression in dicot or monocot protoplasts. The results reported here strongly suggest that this 5' intron is properly spliced in Zea mays. We show that two trans-acting factors, specifically interacting with an upstream activating sequence (the TEF 1 box), are present in nuclear extracts prepared from A. thaliana, Brassica rapa, Nicotiana tabacum and Z. mays. In addition, a DNA sequence homologous to the TEF 1 box, found at approximately the same location within a Lycopersicon esculentum EF-1 alpha promoter, interacts with the same trans-acting factors. Homologies found between the A. thaliana and L. esculentum TEF 1 box sequences have allowed us to define mutations of this upstream element which affect the interaction with the corresponding trans-acting factors. These results support the notion that the activation processes of A. thaliana EF-1 alpha genes have been conserved among angiosperms and provide interesting data on the functional structure of the TEF 1 box. PMID:1600144

  4. Progressive Regulation of Sesquiterpene Biosynthesis in Arabidopsis and Patchouli (Pogostemon cablin) by the miR156-Targeted SPL Transcription Factors.

    PubMed

    Yu, Zong-Xia; Wang, Ling-Jian; Zhao, Bo; Shan, Chun-Min; Zhang, Yu-Hua; Chen, Dong-Fang; Chen, Xiao-Ya

    2014-10-29

    Plant metabolites vary at different stages of life cycle. Although it is well documented that environmental factors stimulate biosynthesis of secondary metabolites, the regulation by endogenous developmental cues remains poorly understood. The microRNA156 (miR156)-tageted SQUAMOSA PROMOTER BINDING PROTEIN-LIKE (SPL) factors function as a major age cue in regulating developmental phase transition and flowering. We show here that the miR156-targeted SPL transcription factor plays an important role in the spatiotemporal regulation of sesquiterpene biosynthesis. In Arabidopsis thaliana, the miR156-SPL module regulates the formation of (E)-β-caryophyllene in flowering stage through modulating expression of the sesquiterpene synthase gene TPS21. We demonstrated that SPL9 directly binds to TPS21 promoter and activates its expression. In the perennial fragrant herb Pogostemon cablin, the accumulation of "patchouli oil", largely composed of sesquiterpenes dominated by (-)-patchoulol, is also age-regulated, and the SPL promotes biosynthesis of sesquiterpenes in elder plants by up-regulating patchoulol synthase (PatPTS) gene expression. As miR156-SPLs are highly conserved in plants, our finding not only uncovers a molecular link between developmental timing and sesquiterpene production, but also suggests a new strategy to engineer plant for accelerated growth with enhanced production of terpenoids. PMID:25355059

  5. Progressive regulation of sesquiterpene biosynthesis in Arabidopsis and Patchouli (Pogostemon cablin) by the miR156-targeted SPL transcription factors.

    PubMed

    Yu, Zong-Xia; Wang, Ling-Jian; Zhao, Bo; Shan, Chun-Min; Zhang, Yu-Hua; Chen, Dong-Fang; Chen, Xiao-Ya

    2015-01-01

    Plant metabolites vary at different stages of their life cycle. Although it is well documented that environmental factors stimulate biosynthesis of secondary metabolites, the regulation by endogenous developmental cues remains poorly understood. The microRNA156 (miR156)-targeted squamosa promoter binding protein-like (SPL) factors function as a major age cue in regulating developmental phase transition and flowering. We show here that the miR156-targeted SPL transcription factor plays an important role in the spatiotemporal regulation of sesquiterpene biosynthesis. In Arabidopsis thaliana, the miR156-SPL module regulates the formation of (E)-β-caryophyllene in the flowering stage through modulating expression of the sesquiterpene synthase gene TPS21. We demonstrated that SPL9 directly binds to TPS21 promoter and activates its expression. In the perennial fragrant herb Pogostemon cablin, the accumulation of patchouli oil, largely composed of sesquiterpenes dominated by (-)-patchoulol, is also age-regulated, and the SPL promotes biosynthesis of sesquiterpenes in elder plants by upregulating patchoulol synthase (PatPTS) gene expression. As miR156-SPLs are highly conserved in plants, our finding not only uncovers a molecular link between developmental timing and sesquiterpene production but also suggests a new strategy to engineer plants for accelerated growth with enhanced production of terpenoids. PMID:25578275

  6. Renal vasoconstriction by vasopressin V1a receptors is modulated by nitric oxide, prostanoids, and superoxide but not the ADP ribosyl cyclase CD38

    PubMed Central

    Kopple, Tayler E.; Arendshorst, William J.

    2014-01-01

    Renal blood flow (RBF) responses to arginine vasopressin (AVP) were tested in anesthetized wild-type (WT) and CD38−/− mice that lack the major calcium-mobilizing second messenger cyclic ADP ribose. AVP (3–25 ng) injected intravenously produced dose-dependent decreases in RBF, reaching a maximum of 25 ± 2% below basal RBF in WT and 27 ± 2% in CD38−/− mice with 25 ng of AVP. Renal vascular resistance (RVR) increased 75 ± 6% and 78 ± 6% in WT and CD38−/− mice. Inhibition of nitric oxide (NO) synthase with nitro-l-arginine methyl ester (l-NAME) increased the maximum RVR response to AVP to 308 ± 76% in WT and 388 ± 81% in CD38−/− (P < 0.001 for both). Cyclooxygenase inhibition with indomethacin increased the maximum RVR response to 125 ± 15% in WT and 120 ± 14% in CD38−/− mice (P < 0.001, <0.05). Superoxide suppression with tempol inhibited the maximum RVR response to AVP by 38% in both strains (P < 0.005) but was ineffective when administered after l-NAME. The rate of RBF recovery (relaxation) after AVP was slowed by l-NAME and indomethacin (P < 0.001, <0.005) but was unchanged by tempol. All vascular responses to AVP were abolished by an AVP V1a receptor antagonist. A V2 receptor agonist or antagonist had no effect on AVP-induced renal vasoconstriction. Taken together, the results indicate that renal vasoconstriction by AVP in the mouse is strongly buffered by vasodilatory actions of NO and prostanoids. The vasoconstriction depends on V1a receptor activation without involvement of CD38 or concomitant vasodilatation by V2 receptors. The role of superoxide is to enhance the contractile response to AVP, most likely by reducing the availability of NO rather than directly stimulating intracellular contraction signaling pathways. PMID:24623148

  7. Single molecule detection of PARP1 and PARP2 interaction with DNA strand breaks and their poly(ADP-ribosyl)ation using high-resolution AFM imaging.

    PubMed

    Sukhanova, Maria V; Abrakhi, Sanae; Joshi, Vandana; Pastre, David; Kutuzov, Mikhail M; Anarbaev, Rashid O; Curmi, Patrick A; Hamon, Loic; Lavrik, Olga I

    2016-04-01

    PARP1 and PARP2 are implicated in the synthesis of poly(ADP-ribose) (PAR) after detection of DNA damage. The specificity of PARP1 and PARP2 interaction with long DNA fragments containing single- and/or double-strand breaks (SSBs and DSBs) have been studied using atomic force microscopy (AFM) imaging in combination with biochemical approaches. Our data show that PARP1 localizes mainly on DNA breaks and exhibits a slight preference for nicks over DSBs, although the protein has a moderately high affinity for undamaged DNA. In contrast to PARP1, PARP2 is mainly detected at a single DNA nick site, exhibiting a low level of binding to undamaged DNA and DSBs. The enhancement of binding affinity of PARP2 for DNA containing a single nick was also observed using fluorescence titration. AFM studies reveal that activation of both PARPs leads to the synthesis of highly branched PAR whose size depends strongly on the presence of SSBs and DSBs for PARP1 and of SSBs for PARP2. The initial affinity between the PARP1, PARP2 and the DNA damaged site appears to influence both the size of the PAR synthesized and the time of residence of PARylated PARP1 and PARP2 on DNA damages. PMID:26673720

  8. The Poly(ADP-ribose) Polymerase Enzyme Tankyrase Antagonizes Activity of the β-Catenin Destruction Complex through ADP-ribosylation of Axin and APC2.

    PubMed

    Croy, Heather E; Fuller, Caitlyn N; Giannotti, Jemma; Robinson, Paige; Foley, Andrew V A; Yamulla, Robert J; Cosgriff, Sean; Greaves, Bradford D; von Kleeck, Ryan A; An, Hyun Hyung; Powers, Catherine M; Tran, Julie K; Tocker, Aaron M; Jacob, Kimberly D; Davis, Beckley K; Roberts, David M

    2016-06-10

    Most colon cancer cases are initiated by truncating mutations in the tumor suppressor, adenomatous polyposis coli (APC). APC is a critical negative regulator of the Wnt signaling pathway that participates in a multi-protein "destruction complex" to target the key effector protein β-catenin for ubiquitin-mediated proteolysis. Prior work has established that the poly(ADP-ribose) polymerase (PARP) enzyme Tankyrase (TNKS) antagonizes destruction complex activity by promoting degradation of the scaffold protein Axin, and recent work suggests that TNKS inhibition is a promising cancer therapy. We performed a yeast two-hybrid (Y2H) screen and uncovered TNKS as a putative binding partner of Drosophila APC2, suggesting that TNKS may play multiple roles in destruction complex regulation. We find that TNKS binds a C-terminal RPQPSG motif in Drosophila APC2, and that this motif is conserved in human APC2, but not human APC1. In addition, we find that APC2 can recruit TNKS into the β-catenin destruction complex, placing the APC2/TNKS interaction at the correct intracellular location to regulate β-catenin proteolysis. We further show that TNKS directly PARylates both Drosophila Axin and APC2, but that PARylation does not globally regulate APC2 protein levels as it does for Axin. Moreover, TNKS inhibition in colon cancer cells decreases β-catenin signaling, which we find cannot be explained solely through Axin stabilization. Instead, our findings suggest that TNKS regulates destruction complex activity at the level of both Axin and APC2, providing further mechanistic insight into TNKS inhibition as a potential Wnt pathway cancer therapy. PMID:27068743

  9. Single molecule detection of PARP1 and PARP2 interaction with DNA strand breaks and their poly(ADP-ribosyl)ation using high-resolution AFM imaging

    PubMed Central

    Sukhanova, Maria V.; Abrakhi, Sanae; Joshi, Vandana; Pastre, David; Kutuzov, Mikhail M.; Anarbaev, Rashid O.; Curmi, Patrick A.; Hamon, Loic; Lavrik, Olga I.

    2016-01-01

    PARP1 and PARP2 are implicated in the synthesis of poly(ADP-ribose) (PAR) after detection of DNA damage. The specificity of PARP1 and PARP2 interaction with long DNA fragments containing single- and/or double-strand breaks (SSBs and DSBs) have been studied using atomic force microscopy (AFM) imaging in combination with biochemical approaches. Our data show that PARP1 localizes mainly on DNA breaks and exhibits a slight preference for nicks over DSBs, although the protein has a moderately high affinity for undamaged DNA. In contrast to PARP1, PARP2 is mainly detected at a single DNA nick site, exhibiting a low level of binding to undamaged DNA and DSBs. The enhancement of binding affinity of PARP2 for DNA containing a single nick was also observed using fluorescence titration. AFM studies reveal that activation of both PARPs leads to the synthesis of highly branched PAR whose size depends strongly on the presence of SSBs and DSBs for PARP1 and of SSBs for PARP2. The initial affinity between the PARP1, PARP2 and the DNA damaged site appears to influence both the size of the PAR synthesized and the time of residence of PARylated PARP1 and PARP2 on DNA damages. PMID:26673720

  10. Increased poly(ADP-ribosyl)ation in skeletal muscle tissue of pediatric patients with severe burn injury: prevention by propranolol treatment

    PubMed Central

    Oláh, Gábor; Finnerty, Celeste; Sbrana, Elena; Elijah, Itoro; Gerö, Domokos; Herndon, David; Szabó, Csaba

    2011-01-01

    Summary Activation of the nuclear enzyme poly (ADP-ribose) polymerase (PARP) has been shown to promote cellular energetic collapse and cellular necrosis in various forms of critical illness. Most of the evidence implicating the PARP pathway in disease processes is derived from preclinical studies. With respect to PARP and burns, studies in rodent and large animal models of burn injury have demonstrated the activation of PARP in various tissues and the beneficial effect of its pharmacological inhibition. The aim of the current study was to measure the activation of PARP in human skeletal muscle biopsies at various stages of severe pediatric burn injury and to identify the cell types where this activation may occur. Another aim of the study was to test the effect of propranolol (an effective treatment of patients with burns), on the activation of PARP in skeletal muscle biopsies. PARP activation was measured by Western blotting for its product, poly(ADP-ribose) (PAR). The localization of PARP activation was determined by PAR immunohistochemistry. The results showed that PARP becomes activated in the skeletal muscle tissue after burns, with the peak of the activation occurring in the middle stage of the disease (13–18 days after burns). Even at the late stage of the disease (69–369 days post-burn) an elevated degree of PARP activation persisted in some of the patients. Immunohistochemical studies localized the staining of PAR primarily to vascular endothelial cells, and occasionally to resident mononuclear cells. There was a marked suppression of PARP activation in the skeletal muscle biopsies of patients who received propranolol treatment. We conclude that human burn injury is associated with the activation of PARP. We hypothesize that this response may contribute to the inflammatory responses and cell dysfunction in burns. Some of the clinical benefit of propranolol in burns may be related to its inhibitory effect on PARP activation. PMID:21368715

  11. Inhibition of potentially lethal radiation damage repair in normal and neoplastic human cells by 3-aminobenzamide: an inhibitor of poly(ADP-ribosylation)

    SciTech Connect

    Thraves, P.J.; Mossman, K.L.; Frazier, D.T.; Dritschilo, A.

    1986-08-01

    The effect of 3-aminobenzamide (3AB), an inhibitor of poly(ADP-ribose) synthetase, on potentially lethal damage repair (PLDR) was investigated in normal human fibroblasts and four human tumor cell lines from tumors with varying degrees of radiocurability. The tumor lines selected were: Ewing's sarcoma, a bone tumor considered radiocurable and, human lung adenocarcinoma, osteosarcoma, and melanoma, three tumors considered nonradiocurable. PLDR was measured by comparing cell survival when cells were irradiated in a density-inhibited state and replated at appropriate cell numbers at specified times following irradiation to cell survival when cells were replated immediately following irradiation. 3AB was added to cultures 2 hr prior to irradiation and removed at the time of replating. Different test radiation doses were used for the various cell lines to obtain equivalent levels of cell survival. In the absence of inhibitor, PLDR was similar in all cell lines tested. In the presence of 8 mM 3AB, differential inhibition of PLDR was observed. PLDR was almost completely inhibited in Ewing's sarcoma cells and partially inhibited in normal fibroblast cells and osteosarcoma cells. No inhibition of PLDR was observed in the lung adenocarcinoma or melanoma cells. Except for the osteosarcoma cells, inhibition of PLDR by 3AB correlated well with radiocurability.

  12. The transcription factor PHR1 regulates lipid remodeling and triacylglycerol accumulation in Arabidopsis thaliana during phosphorus starvation.

    PubMed

    Pant, Bikram Datt; Burgos, Asdrubal; Pant, Pooja; Cuadros-Inostroza, Alvaro; Willmitzer, Lothar; Scheible, Wolf-Rüdiger

    2015-04-01

    Lipid remodeling is one of the most dramatic metabolic responses to phosphorus (P) starvation. It consists of the degradation of phospholipids to release the phosphate needed by the cell and the accumulation of glycolipids to replace phospholipids in the membranes. It is shown that PHR1, a well-described transcriptional regulator of P starvation of the MYB family, largely controls this response. Glycerolipid composition and the expression of most lipid-remodeling gene transcripts analysed were altered in the phr1 mutant under phosphate starvation in comparison to wild-type plants. In addition to these results, the lipidomic characterization of wild-type plants showed two novel features of the lipid response to P starvation for Arabidopsis. Triacylglycerol (TAG) accumulates dramatically under P starvation (by as much as ~20-fold in shoots and ~13-fold in roots), a response known to occur in green algae but hardly known in plants. Surprisingly, there was an increase in phosphatidylglycerol (PG) in P-starved roots, a response that may be adaptive as it was suppressed in the phr1 mutant. PMID:25680792

  13. Arabidopsis thaliana gonidialess A/Zuotin related factors (GlsA/ZRF) are essential for maintenance of meristem integrity.

    PubMed

    Guzmán-López, José Alfredo; Abraham-Juárez, María Jazmín; Lozano-Sotomayor, Paulina; de Folter, Stefan; Simpson, June

    2016-05-01

    Observation of a differential expression pattern, including strong expression in meristematic tissue of an Agave tequilana GlsA/ZRF ortholog suggested an important role for this gene during bulbil formation and developmental changes in this species. In order to better understand this role, the two GlsA/ZFR orthologs present in the genome of Arabidopsis thaliana were functionally characterized by analyzing expression patterns, double mutant phenotypes, promoter-GUS fusions and expression of hormone related or meristem marker genes. Patterns of expression for A. thaliana show that GlsA/ZFR genes are strongly expressed in SAMs and RAMs in mature plants and developing embryos and double mutants showed multiple changes in morphology related to both SAM and RAM tissues. Typical double mutants showed stunted growth of aerial and root tissue, formation of multiple ectopic meristems and effects on cotyledons, leaves and flowers. The KNOX genes STM and BP were overexpressed in double mutants whereas CLV3, WUSCHEL and AS1 were repressed and lack of AtGlsA expression was also associated with changes in localization of auxin and cytokinin. These results suggest that GlsA/ZFR is an essential component of the machinery that maintains the integrity of SAM and RAM tissue and underline the potential to identify new genes or gene functions based on observations in non-model plants. PMID:26826012

  14. Negative Regulation of Anthocynanin Biosynthesis in Arabidopsis by a miR156-Targeted SPL Transcription Factor

    SciTech Connect

    Gou, J.Y.; Liu, C.; Felippes, F. F.; Weigel, D.; Wang, J.-W.

    2011-04-01

    Flavonoids are synthesized through an important metabolic pathway that leads to the production of diverse secondary metabolites, including anthocyanins, flavonols, flavones, and proanthocyanidins. Anthocyanins and flavonols are derived from Phe and share common precursors, dihydroflavonols, which are substrates for both flavonol synthase and dihydroflavonol 4-reductase. In the stems of Arabidopsis thaliana, anthocyanins accumulate in an acropetal manner, with the highest level at the junction between rosette and stem. We show here that this accumulation pattern is under the regulation of miR156-targeted SQUAMOSA PROMOTER BINDING PROTEIN-LIKE (SPL) genes, which are deeply conserved and known to have important roles in regulating phase change and flowering. Increased miR156 activity promotes accumulation of anthocyanins, whereas reduced miR156 activity results in high levels of flavonols. We further provide evidence that at least one of the miR156 targets, SPL9, negatively regulates anthocyanin accumulation by directly preventing expression of anthocyanin biosynthetic genes through destabilization of a MYB-bHLH-WD40 transcriptional activation complex. Our results reveal a direct link between the transition to flowering and secondary metabolism and provide a potential target for manipulation of anthocyanin and flavonol content in plants.

  15. The Alfin-like homeodomain finger protein AL5 suppresses multiple negative factors to confer abiotic stress tolerance in Arabidopsis.

    PubMed

    Wei, Wei; Zhang, Yu-Qin; Tao, Jian-Jun; Chen, Hao-Wei; Li, Qing-Tian; Zhang, Wan-Ke; Ma, Biao; Lin, Qing; Zhang, Jin-Song; Chen, Shou-Yi

    2015-03-01

    Plant homeodomain (PHD) finger proteins affect processes of growth and development by changing transcription and reading epigenetic histone modifications, but their functions in abiotic stress responses remain largely unclear. Here we characterized seven Arabidopsis thaliana Alfin1-like PHD finger proteins (ALs) in terms of the responses to abiotic stresses. ALs localized to the nucleus and repressed transcription. Except AL6, all the ALs bound to G-rich elements. Mutations of the amino acids at positions 34 and 35 in AL6 caused loss of ability to bind to G-rich elements. Expression of the AL genes responded differentially to osmotic stress, salt, cold and abscisic acid treatments. AL5-over-expressing plants showed higher tolerance to salt, drought and freezing stress than Col-0. Consistently, al5 mutants showed reduced stress tolerance. We used ChIP-Seq assays to identify eight direct targets of AL5, and found that AL5 binds to the promoter regions of these genes. Knockout mutants of five of these target genes exhibited varying tolerances to stresses. These results indicate that AL5 inhibits multiple signaling pathways to confer stress tolerance. Our study sheds light on mechanisms of AL5-mediated signaling in abiotic stress responses, and provides tools for improvement of stress tolerance in crop plants. PMID:25619813

  16. Subunit-selective N-Methyl-d-aspartate (NMDA) Receptor Signaling through Brefeldin A-resistant Arf Guanine Nucleotide Exchange Factors BRAG1 and BRAG2 during Synapse Maturation.

    PubMed

    Elagabani, Mohammad Nael; Briševac, Dušica; Kintscher, Michael; Pohle, Jörg; Köhr, Georg; Schmitz, Dietmar; Kornau, Hans-Christian

    2016-04-22

    The maturation of glutamatergic synapses in the CNS is regulated by NMDA receptors (NMDARs) that gradually change from a GluN2B- to a GluN2A-dominated subunit composition during postnatal development. Here we show that NMDARs control the activity of the small GTPase ADP-ribosylation factor 6 (Arf6) by consecutively recruiting two related brefeldin A-resistant Arf guanine nucleotide exchange factors, BRAG1 and BRAG2, in a GluN2 subunit-dependent manner. In young cortical cultures, GluN2B and BRAG1 tonically activated Arf6. In mature cultures, Arf6 was activated through GluN2A and BRAG2 upon NMDA treatment, whereas the tonic Arf6 activation was not detectable any longer. This shift in Arf6 regulation and the associated drop in Arf6 activity were reversed by a knockdown of BRAG2. Given their sequential recruitment during development, we examined whether BRAG1 and BRAG2 influence synaptic currents in hippocampal CA1 pyramidal neurons using patch clamp recordings in acute slices from mice at different ages. The number of AMPA receptor (AMPAR) miniature events was reduced by depletion of BRAG1 but not by depletion of BRAG2 during the first 2 weeks after birth. In contrast, depletion of BRAG2 during postnatal weeks 4 and 5 reduced the number of AMPAR miniature events and compromised the quantal sizes of both AMPAR and NMDAR currents evoked at Schaffer collateral synapses. We conclude that both Arf6 activation through GluN2B-BRAG1 during early development and the transition from BRAG1- to BRAG2-dependent Arf6 signaling induced by the GluN2 subunit switch are critical for the development of mature glutamatergic synapses. PMID:26884337

  17. Function search in a large transcription factor gene family in Arabidopsis: assessing the potential of reverse genetics to identify insertional mutations in R2R3 MYB genes.

    PubMed Central

    Meissner, R C; Jin, H; Cominelli, E; Denekamp, M; Fuertes, A; Greco, R; Kranz, H D; Penfield, S; Petroni, K; Urzainqui, A; Martin, C; Paz-Ares, J; Smeekens, S; Tonelli, C; Weisshaar, B; Baumann, E; Klimyuk, V; Marillonnet, S; Patel, K; Speulman, E; Tissier, A F; Bouchez, D; Jones, J J; Pereira, A; Wisman, E

    1999-01-01

    More than 92 genes encoding MYB transcription factors of the R2R3 class have been described in Arabidopsis. The functions of a few members of this large gene family have been described, indicating important roles for R2R3 MYB transcription factors in the regulation of secondary metabolism, cell shape, and disease resistance, and in responses to growth regulators and stresses. For the majority of the genes in this family, however, little functional information is available. As the first step to characterizing these genes functionally, the sequences of >90 family members, and the map positions and expression profiles of >60 members, have been determined previously. An important second step in the functional analysis of the MYB family, through a process of reverse genetics that entails the isolation of insertion mutants, is described here. For this purpose, a variety of gene disruption resources has been used, including T-DNA-insertion populations and three distinct populations that harbor transposon insertions. We report the isolation of 47 insertions into 36 distinct MYB genes by screening a total of 73 genes. These defined insertion lines will provide the foundation for subsequent detailed functional analyses for the assignment of specific functions to individual members of the R2R3 MYB gene family. PMID:10521515

  18. Arabidopsis GROWTH-REGULATING FACTOR7 Functions as a Transcriptional Repressor of Abscisic Acid– and Osmotic Stress–Responsive Genes, Including DREB2A[W

    PubMed Central

    Kim, June-Sik; Mizoi, Junya; Kidokoro, Satoshi; Maruyama, Kyonoshin; Nakajima, Jun; Nakashima, Kazuo; Mitsuda, Nobutaka; Takiguchi, Yuko; Ohme-Takagi, Masaru; Kondou, Youichi; Yoshizumi, Takeshi; Matsui, Minami; Shinozaki, Kazuo; Yamaguchi-Shinozaki, Kazuko

    2012-01-01

    Arabidopsis thaliana DEHYDRATION-RESPONSIVE ELEMENT BINDING PROTEIN2A (DREB2A) functions as a transcriptional activator that increases tolerance to osmotic and heat stresses; however, its expression also leads to growth retardation and reduced reproduction. To avoid these adverse effects, the expression of DREB2A is predicted to be tightly regulated. We identified a short promoter region of DREB2A that represses its expression under nonstress conditions. Yeast one-hybrid screening for interacting factors identified GROWTH-REGULATING FACTOR7 (GRF7). GRF7 bound to the DREB2A promoter and repressed its expression. In both artificial miRNA-silenced lines and a T-DNA insertion line of GRF7, DREB2A transcription was increased compared with the wild type under nonstress conditions. A previously undiscovered cis-element, GRF7-targeting cis-element (TGTCAGG), was identified as a target sequence of GRF7 in the short promoter region of DREB2A via electrophoretic mobility shift assays. Microarray analysis of GRF7 knockout plants showed that a large number of the upregulated genes in the mutant plants were also responsive to osmotic stress and/or abscisic acid. These results suggest that GRF7 functions as a repressor of a broad range of osmotic stress–responsive genes to prevent growth inhibition under normal conditions. PMID:22942381

  19. The rolB gene activates secondary metabolism in Arabidopsis calli via selective activation of genes encoding MYB and bHLH transcription factors.

    PubMed

    Bulgakov, Victor P; Veremeichik, Galina N; Grigorchuk, Valeria P; Rybin, Viacheslav G; Shkryl, Yuri N

    2016-05-01

    It is known that the rolB gene of Agrobacterium rhizogenes increases the production of secondary metabolites in transformed plant cells, but its mechanism of action remains unclear. In this report, we demonstrate that rolB expression in Arabidopsis thaliana calli led to the activation of most genes encoding secondary metabolism-specific MYB and bHLH transcription factors (TFs), such as MYB11, MYB12, MYB28, MYB76, MYB34, MYB51, MYB122, TT2 and TT8. Accordingly, a higher transcript abundance of main biosynthetic genes related to these factors was detected. The rolB-transformed calli produced 3-fold higher levels of indolic glucosinolates (GSs) compared with normal calli but did not produce secondary metabolites from other groups. Enhanced accumulation of indolic GSs was caused by activation of MYB34, MYB51 and MYB122, and the absence of aliphatic GSs in transformed calli was caused by the inability of rolB to induce MYB29. The inability of rolB-calli to produce flavonoids was caused by the lack of MYB111 expression, induced by the rolB-mediated conversion of MYB expression from cotyledon-specific to root-specific patterns. The high specificity of rolB on secondary metabolism-specific TFs was demonstrated for the first time. PMID:26913794

  20. The Heterologous Expression of the Chrysanthemum R2R3-MYB Transcription Factor CmMYB1 Alters Lignin Composition and Represses Flavonoid Synthesis in Arabidopsis thaliana

    PubMed Central

    Chen, Sumei; Jiang, Jiafu; Gu, Chunsun; Zhou, Guoqin; Chen, Yu; Song, Aiping; Chen, Fadi

    2013-01-01

    Plant R2R3-MYB transcription factor genes are widely distributed in higher plants and play important roles in the regulation of many secondary metabolites at the transcriptional level. In this study, a chrysanthemum subgroup 4 R2R3-MYB transcription factor gene, designated CmMYB1, was isolated through screening chrysanthemum EST (expressed sequence tag) libraries and using rapid application of cDNA ends (RACE) methods and functionally characterized. CmMYB1 is expressed in the root, stem, leaf and flowers, but most strongly in the stem and most weakly in the root. Its heterologous expression in Arabidopsis thaliana reduced the lignin content and altered the lignin composition. The heterologous expression also repressed the flavonoids content in A. thaliana. Together, these results suggested that CmMYB1 is a negative regulator of genes involved in the lignin pathway and flavonoid pathway, it may be a promising gene for controlling lignin and flavonoids profiles in plants. PMID:23840353

  1. Blue light alters miR167 expression and microRNA-targeted auxin response factor genes in Arabidopsis thaliana plants.

    PubMed

    Pashkovskiy, Pavel P; Kartashov, Alexander V; Zlobin, Ilya E; Pogosyan, Sergei I; Kuznetsov, Vladimir V

    2016-07-01

    The effect of blue LED (450 nm) on the photomorphogenesis of Arabidopsis thaliana Col-0 plants and the transcript levels of several genes, including miRNAs, photoreceptors and auxin response factors (ARF) was investigated. It was observed that blue light accelerated the generative development, reduced the rosette leaf number, significantly reduced the leaf area, dry biomass and led to the disruption of conductive tissue formation. The blue LED differentially influenced the transcript levels of several phytochromes (PHY a, b, c, d, and e), cryptochromes (CRY 1 and 2) and phototropins (PHOT 1 and 2). At the same time, the blue LED significantly increased miR167 expression compared to a fluorescent lamp or white LEDs. This increase likely resulted in the enhanced transcription of the auxin response factor genes ARF4 and ARF8, which are regulated by this miRNA. These findings support the hypothesis that the effects of blue light on A. thaliana are mediated by auxin signalling pathway involving miRNA-dependent regulation of ARF gene expression. PMID:27031426

  2. Stress induced and nuclear localized HIPP26 from Arabidopsis thaliana interacts via its heavy metal associated domain with the drought stress related zinc finger transcription factor ATHB29.

    PubMed

    Barth, Olaf; Vogt, Sebastian; Uhlemann, Ria; Zschiesche, Wiebke; Humbeck, Klaus

    2009-01-01

    HIPP26 from Arabidopsis thaliana belongs to a novel class of plant proteins, characterized by a heavy metal associated domain and an additional isoprenylation motif. It is induced during cold, salt and drought stress. The nuclear localization of HIPP26, predicted by a NLS motif, could be confirmed in onion epidermal cells overexpressing GFP-HIPP26. Experiments with modified HIPP26 indicate that the isoprenylation plays a role in the spatial distribution in the nucleus. Using promoter-GUS constructs, a tissue specific expression pattern of HIPP26 could be shown, with high expression in the vascular tissue. By a yeast-two-hybrid approach a strong interaction of HIPP26 with the zinc finger homeodomain transcription factor ATHB29, which is known to play a role in dehydration stress response could be detected. This was confirmed by GST pull-down assays. When using a modified HIPP26 lacking the two central cysteines of the heavy metal associated domain, ATHB29 was not bound in the GST pull-down assay, indicating that this structure is necessary for the interaction. Further yeast-two-hybrid analyses testing interaction of different members of the HIPP family with related zinc finger transcription factors revealed a specific interaction of ATHB29 with several HIPP proteins. A functional relationship between HIPP26 and ATHB29 is also indicated by experiments with mutants of HIPP26 showing altered expression levels of such genes known to be regulated by ATHB29. PMID:18974936

  3. Overexpression of the MYB37 transcription factor enhances abscisic acid sensitivity, and improves both drought tolerance and seed productivity in Arabidopsis thaliana.

    PubMed

    Yu, Yong-Tao; Wu, Zhen; Lu, Kai; Bi, Chao; Liang, Shan; Wang, Xiao-Fang; Zhang, Da-Peng

    2016-02-01

    Although a lot of genes have been revealed to participate in abscisic acid (ABA) signaling, many of the additional components involved in ABA signaling remain to be discovered. Here we report that overexpression of MYB37, a R2R3 MYB subgroup 14 transcription factor in Arabidopsis thaliana, confers hypersensitive phenotypes to exogenous ABA in all the major ABA responses, including ABA-induced inhibition of seed germination, cotyledon greening and early seedling growth, and ABA-induced stomatal closure and inhibition of stomatal opening. Interestingly and importantly, MYB37-overexpression improves plant tolerance to drought, enhances growth of mature plants and seed productivity, thought it delays flowering, which suggests that this gene may be used for improving crop adaptability to drought environment and productivity. However, a myb37-1 knockout mutant displays wild-type ABA responses most likely due to a functional redundancy of the multiple MYB members. Real-time PCR analysis shows that upregulation of the MYB37 expression changes expression of a subset of ABA-responsive genes. Together, these findings suggest that the MYB37 transcription factor plays an important, positive role in plant response to ABA and drought stress, and meanwhile, it plays a positive role in the regulation of seed production. PMID:26646286

  4. Arabidopsis Basic Helix-Loop-Helix Transcription Factors MYC2, MYC3, and MYC4 Regulate Glucosinolate Biosynthesis, Insect Performance, and Feeding Behavior[W][OPEN

    PubMed Central

    Schweizer, Fabian; Fernández-Calvo, Patricia; Zander, Mark; Diez-Diaz, Monica; Fonseca, Sandra; Glauser, Gaétan; Lewsey, Mathew G.; Ecker, Joseph R.; Solano, Roberto; Reymond, Philippe

    2013-01-01

    Arabidopsis thaliana plants fend off insect attack by constitutive and inducible production of toxic metabolites, such as glucosinolates (GSs). A triple mutant lacking MYC2, MYC3, and MYC4, three basic helix-loop-helix transcription factors that are known to additively control jasmonate-related defense responses, was shown to have a highly reduced expression of GS biosynthesis genes. The myc2 myc3 myc4 (myc234) triple mutant was almost completely devoid of GS and was extremely susceptible to the generalist herbivore Spodoptera littoralis. On the contrary, the specialist Pieris brassicae was unaffected by the presence of GS and preferred to feed on wild-type plants. In addition, lack of GS in myc234 drastically modified S. littoralis feeding behavior. Surprisingly, the expression of MYB factors known to regulate GS biosynthesis genes was not altered in myc234, suggesting that MYC2/MYC3/MYC4 are necessary for direct transcriptional activation of GS biosynthesis genes. To support this, chromatin immunoprecipitation analysis showed that MYC2 binds directly to the promoter of several GS biosynthesis genes in vivo. Furthermore, yeast two-hybrid and pull-down experiments indicated that MYC2/MYC3/MYC4 interact directly with GS-related MYBs. This specific MYC–MYB interaction plays a crucial role in the regulation of defense secondary metabolite production and underlines the importance of GS in shaping plant interactions with adapted and nonadapted herbivores. PMID:23943862

  5. Fast Retrograde Signaling in Response to High Light Involves Metabolite Export, MITOGEN-ACTIVATED PROTEIN KINASE6, and AP2/ERF Transcription Factors in Arabidopsis[C][W

    PubMed Central

    Vogel, Marc Oliver; Moore, Marten; König, Katharina; Pecher, Pascal; Alsharafa, Khalid; Lee, Justin; Dietz, Karl-Josef

    2014-01-01

    Regulation of the expression of nuclear genes encoding chloroplast proteins allows for metabolic adjustment in response to changing environmental conditions. This regulation is linked to retrograde signals that transmit information on the metabolic state of the chloroplast to the nucleus. Transcripts of several APETALA2/ETHYLENE RESPONSE FACTOR transcription factors (AP2/ERF-TFs) were found to respond within 10 min after transfer of low-light-acclimated Arabidopsis thaliana plants to high light. Initiation of this transcriptional response was completed within 1 min after transfer to high light. The fast responses of four AP2/ERF genes, ERF6, RRTF1, ERF104, and ERF105, were entirely deregulated in triose phosphate/phosphate translocator (tpt) mutants. Similarly, activation of MITOGEN-ACTIVATED PROTEIN KINASE6 (MPK6) was upregulated after 1 min in the wild type but not in the tpt mutant. Based on this, together with altered transcript regulation in mpk6 and erf6 mutants, a retrograde signal transmission model is proposed starting with metabolite export through the triose phosphate/phosphate translocator with subsequent MPK6 activation leading to initiation of AP2/ERF-TF gene expression and other downstream gene targets. The results show that operational retrograde signaling in response to high light involves a metabolite-linked pathway in addition to previously described redox and hormonal pathways. PMID:24668746

  6. AtTGA4, a bZIP transcription factor, confers drought resistance by enhancing nitrate transport and assimilation in Arabidopsis thaliana.

    PubMed

    Zhong, Li; Chen, Dandan; Min, Donghong; Li, Weiwei; Xu, Zhaoshi; Zhou, Yongbin; Li, Liancheng; Chen, Ming; Ma, Youzhi

    2015-02-13

    To cope with environmental stress caused by global climate change and excessive nitrogen application, it is important to improve water and nitrogen use efficiencies in crop plants. It has been reported that higher nitrogen uptake could alleviate the damaging impact of drought stress. However, there is scant evidence to explain how nitrogen uptake affects drought resistance. In this study we observed that bZIP transcription factor AtTGA4 (TGACG motif-binding factor 4) was induced by both drought and low nitrogen stresses, and that overexpression of AtTGA4 simultaneously improved drought resistance and reduced nitrogen starvation in Arabidopsis. Following drought stress there were higher nitrogen and proline contents in transgenic AtTGA4 plants than in wild type controls, and activity of the key enzyme nitrite reductase (NIR) involved in nitrate assimilation processes was also higher. Expressions of the high-affinity nitrate transporter genes NRT2.1 and NRT2.2 and nitrate reductase genes NIA1 and NIA2 in transgenic plants were all higher than in wild type indicating that higher levels of nitrate transport and assimilation activity contributed to enhanced drought resistance of AtTGA4 transgenic plants. Thus genetic transformation with AtTGA4 may provide a new approach to simultaneously improve crop tolerance to drought and low nitrogen stresses. PMID:25596127

  7. WRKY8 transcription factor functions in the TMV-cg defense response by mediating both abscisic acid and ethylene signaling in Arabidopsis

    PubMed Central

    Chen, Ligang; Zhang, Liping; Li, Daibo; Wang, Fang; Yu, Diqiu

    2013-01-01

    WRKY transcription factors are key players in the plant immune response, but less is known about their involvement in antiviral defense than about their roles in defense against bacterial or fungi pathogens. Here, we report that Arabidopsis thaliana WRKY DNA-binding protein 8 (WRKY8) has a role in mediating the long-distance movement of crucifer-infecting tobacco mosaic virus (TMV-cg). The expression of WRKY8 was inhibited by TMV-cg infection, and mutation of WRKY8 accelerated the accumulation of TMV-cg in systemically infected leaves. Quantitative RT-PCR analysis showed that the expression of ABA insensitive 4 (ABI4) was reduced and the expression of 1-aminocyclopropane-1-carboxylic acid synthase 6 (ACS6) and ethylene response factor 104 (ERF104) was enhanced in the systemically infected leaves of wrky8. Immunoprecipitation assays demonstrated that WRKY8 could bind selectively to putative W-boxes of the ABI4, ACS6, and ERF104 promoters. Furthermore, TMV-cg infection enhanced WRKY8 binding to the ABI4 promoter but reduced the binding of WRKY8 to the ACS6 and ERF104 promoters, indicating that regulation of ABI4, ACS6, and ERF104 by WRKY8 is at least partially dependent on TMV-cg. Exogenous applications of abscisic acid (ABA) reduced the systemic accumulation of TMV-cg. Mutations in ABA deficient 1, ABA deficient 2, ABA deficient 3, or abi4 accelerated systemic TMV-cg accumulation. In contrast, exogenous application of aminocyclopropane-1-carboxylic acid enhanced the systemic accumulation of TMV-cg, but mutations in acs6, erf104, or an octuple acs mutant inhibited systemic TMV-cg accumulation. Our results demonstrate that WRKY8 is involved in the defense response against TMV-cg through the direct regulation of the expression of ABI4, ACS6, and ERF104 and may mediate the crosstalk between ABA and ethylene signaling during the TMV-cg–Arabidopsis interaction. PMID:23650359

  8. The Rose (Rosa hybrida) NAC Transcription Factor 3 Gene, RhNAC3, Involved in ABA Signaling Pathway Both in Rose and Arabidopsis

    PubMed Central

    Lü, Peitao; Liu, Jitao; Gao, Junping; Zhang, Changqing

    2014-01-01

    Plant transcription factors involved in stress responses are generally classified by their involvement in either the abscisic acid (ABA)-dependent or the ABA-independent regulatory pathways. A stress-associated NAC gene from rose (Rosa hybrida), RhNAC3, was previously found to increase dehydration tolerance in both rose and Arabidopsis. However, the regulatory mechanism involved in RhNAC3 action is still not fully understood. In this study, we isolated and analyzed the upstream regulatory sequence of RhNAC3 and found many stress-related cis-elements to be present in the promoter, with five ABA-responsive element (ABRE) motifs being of particular interest. Characterization of Arabidopsis thaliana plants transformed with the putative RhNAC3 promoter sequence fused to the β-glucuronidase (GUS) reporter gene revealed that RhNAC3 is expressed at high basal levels in leaf guard cells and in vascular tissues. Moreover, the ABRE motifs in the RhNAC3 promoter were observed to have a cumulative effect on the transcriptional activity of this gene both in the presence and absence of exogenous ABA. Overexpression of RhNAC3 in A. thaliana resulted in ABA hypersensitivity during seed germination and promoted leaf closure after ABA or drought treatments. Additionally, the expression of 11 ABA-responsive genes was induced to a greater degree by dehydration in the transgenic plants overexpressing RhNAC3 than control lines transformed with the vector alone. Further analysis revealed that all these genes contain NAC binding cis-elements in their promoter regions, and RhNAC3 was found to partially bind to these putative NAC recognition sites. We further found that of 219 A. thaliana genes previously shown by microarray analysis to be regulated by heterologous overexpression RhNAC3, 85 are responsive to ABA. In rose, the expression of genes downstream of the ABA-signaling pathways was also repressed in RhNAC3-silenced petals. Taken together, we propose that the rose RhNAC3 protein

  9. A Ploidy-Sensitive Mechanism Regulates Aperture Formation on the Arabidopsis Pollen Surface and Guides Localization of the Aperture Factor INP1.

    PubMed

    Reeder, Sarah H; Lee, Byung Ha; Fox, Ronald; Dobritsa, Anna A

    2016-05-01

    Pollen presents a powerful model for studying mechanisms of precise formation and deposition of extracellular structures. Deposition of the pollen wall exine leads to the generation of species-specific patterns on pollen surface. In most species, exine does not develop uniformly across the pollen surface, resulting in the formation of apertures-openings in the exine that are species-specific in number, morphology and location. A long time ago, it was proposed that number and positions of apertures might be determined by the geometry of tetrads of microspores-the precursors of pollen grains arising via meiotic cytokinesis, and by the number of last-contact points between sister microspores. We have tested this model by characterizing Arabidopsis mutants with ectopic apertures and/or abnormal geometry of meiotic products. Here we demonstrate that contact points per se do not act as aperture number determinants and that a correct geometric conformation of a tetrad is neither necessary nor sufficient to generate a correct number of apertures. A mechanism sensitive to pollen ploidy, however, is very important for aperture number and positions and for guiding the aperture factor INP1 to future aperture sites. In the mutants with ectopic apertures, the number and positions of INP1 localization sites change depending on ploidy or ploidy-related cell size and not on INP1 levels, suggesting that sites for aperture formation are specified before INP1 is brought to them. PMID:27177036

  10. Myrosin Idioblast Cell Fate and Development Are Regulated by the Arabidopsis Transcription Factor FAMA, the Auxin Pathway, and Vesicular Trafficking[W

    PubMed Central

    Li, Meng; Sack, Fred D.

    2014-01-01

    Crucifer shoots harbor a glucosinolate-myrosinase system that defends against insect predation. Arabidopsis thaliana myrosinase (thioglucoside glucohydrolase [TGG]) accumulates in stomata and in myrosin idioblasts (MIs). This work reports that the basic helix-loop-helix transcription factor FAMA that is key to stomatal development is also expressed in MIs. The loss of FAMA function abolishes MI fate as well as the expression of the myrosinase genes TGG1 and TGG2. MI cells have previously been reported to be located in the phloem. Instead, we found that MIs arise from the ground meristem rather than provascular tissues and thus are not homologous with phloem. Moreover, MI patterning and morphogenesis are abnormal when the function of the ARF-GEF gene GNOM is lost as well as when auxin efflux and vesicular trafficking are chemically disrupted. Stomata and MI cells constitute part of a wider system that reduces plant predation, the so-called “mustard oil bomb,” in which vacuole breakage in cells harboring myrosinase and glucosinolate yields a brew toxic to many animals, especially insects. This identification of the gene that confers the fate of MIs, as well as stomata, might facilitate the development of strategies for engineering crops to mitigate predation. PMID:25304201

  11. A R2R3-MYB transcription factor that is specifically expressed in cotton (Gossypium hirsutum) fibers affects secondary cell wall biosynthesis and deposition in transgenic Arabidopsis.

    PubMed

    Sun, Xiang; Gong, Si-Ying; Nie, Xiao-Ying; Li, Yang; Li, Wen; Huang, Geng-Qing; Li, Xue-Bao

    2015-07-01

    Secondary cell wall (SCW) is an important industrial raw material for pulping, papermaking, construction, lumbering, textiles and potentially for biofuel production. The process of SCW thickening of cotton fibers lays down the cellulose that will constitute the bulk (up to 96%) of the fiber at maturity. In this study, a gene encoding a MYB-domain protein was identified in cotton (Gossypium hirsutum) and designated as GhMYBL1. Quantitative real-time polymerase chain reaction (RT-PCR) analysis revealed that GhMYBL1 was specifically expressed in cotton fibers at the stage of secondary wall deposition. Further analysis indicated that this protein is a R2R3-MYB transcription factor, and is targeted to the cell nucleus. Overexpression of GhMYBL1 in Arabidopsis affected the formation of SCW in the stem xylem of the transgenic plants. The enhanced SCW thickening also occurred in the interfascicular fibers, xylary fibers and vessels of the GhMYBL1-overexpression transgenic plants. The expression of secondary wall-associated genes, such as CesA4, CesA7, CesA8, PAL1, F5H and 4CL1, were upregulated, and consequently, cellulose and lignin biosynthesis were enhanced in the GhMYBL1 transgenic plants. These data suggested that GhMYBL1 may participate in modulating the process of secondary wall biosynthesis and deposition of cotton fibers. PMID:25534543

  12. Enhancement of chlorogenic acid production in hairy roots of Platycodon grandiflorum by over-expression of an Arabidopsis thaliana transcription factor AtPAP1.

    PubMed

    Tuan, Pham Anh; Kwon, Do Yeon; Lee, Sanghyun; Arasu, Mariadhas Valan; Al-Dhabi, Naif Abdullah; Park, Nam Il; Park, Sang Un

    2014-01-01

    To improve the production of chlorogenic acid (CGA) in hairy roots of Platycodon grandiflorum, we induced over-expression of Arabidopsis thaliana transcription factor production of anthocyanin pigment (AtPAP1) using an Agrobacterium rhizogenes-mediated transformation system. Twelve hairy root lines showing over-expression of AtPAP1 were generated. In order to investigate the regulation of AtPAP1 on the activities of CGA biosynthetic genes, the expression levels of seven P. grandiflorum CGA biosynthetic genes were analyzed in the hairy root line that had the greatest accumulation of AtPAP1 transcript, OxPAP1-1. The introduction of AtPAP1 increased the mRNA levels of all examined CGA biosynthetic genes and resulted in a 900% up-regulation of CGA accumulation in OxPAP1-1 hairy roots relative to controls. This suggests that P. grandiflorum hairy roots that over-express the AtPAP1 gene are a potential alternative source of roots for the production of CGA. PMID:25153629

  13. IRT1 DEGRADATION FACTOR1, a RING E3 Ubiquitin Ligase, Regulates the Degradation of IRON-REGULATED TRANSPORTER1 in Arabidopsis[C][W][OPEN

    PubMed Central

    Shin, Lung-Jiun; Lo, Jing-Chi; Chen, Guan-Hong; Callis, Judy; Fu, Hongyong; Yeh, Kuo-Chen

    2013-01-01

    Fe is an essential micronutrient for plant growth and development; plants have developed sophisticated strategies to acquire ferric Fe from the soil. Nongraminaceous plants acquire Fe by a reduction-based mechanism, and graminaceous plants use a chelation-based mechanism. In Arabidopsis thaliana, which uses the reduction-based method, IRON-REGULATED TRANSPORTER1 (IRT1) functions as the most important transporter for ferrous Fe uptake. Rapid and constitutive degradation of IRT1 allows plants to quickly respond to changing conditions to maintain Fe homeostasis. IRT1 degradation involves ubiquitination. To identify the specific E3 ubiquitin ligases involved in IRT1 degradation, we screened a set of insertional mutants in RING-type E3 ligases and identified a mutant that showed delayed degradation of IRT1 and loss of IRT1-ubiquitin complexes. The corresponding gene was designated IRT1 DEGRADATION FACTOR1 (IDF1). Evidence of direct interaction between IDF1 and IRT1 in the plasma membrane supported the role of IDF1 in IRT1 degradation. IRT1 accumulation was reduced when coexpressed with IDF1 in yeast or Xenopus laevis oocytes. IDF1 function was RING domain dependent. The idf1 mutants showed increased tolerance to Fe deficiency, resulting from increased IRT1 levels. This evidence indicates that IDF1 directly regulates IRT1 degradation through its RING-type E3 ligase activity. PMID:23995086

  14. Actin-Depolymerizing Factor2-Mediated Actin Dynamics Are Essential for Root-Knot Nematode Infection of Arabidopsis[C][W

    PubMed Central

    Clément, Mathilde; Ketelaar, Tijs; Rodiuc, Natalia; Banora, Mohamed Youssef; Smertenko, Andrei; Engler, Gilbert; Abad, Pierre; Hussey, Patrick J.; de Almeida Engler, Janice

    2009-01-01

    Reorganization of the actin and microtubule networks is known to occur in targeted vascular parenchymal root cells upon infection with the nematode Meloidogyne incognita. Here, we show that actin-depolymerizing factor (ADF) is upregulated in the giant feeding cells of Arabidopsis thaliana that develop upon nematode infection and that knockdown of a specific ADF isotype inhibits nematode proliferation. Analysis of the levels of transcript and the localization of seven ADF genes shows that five are upregulated in galls that result from the infection and that ADF2 expression is particularly increased between 14 and 21 d after nematode inoculation. Further analysis of ADF2 function in inducible RNA interference lines designed to knock down ADF2 expression reveals that this protein is required for normal cell growth and plant development. The net effect of decreased levels of ADF2 is F-actin stabilization in cells, resulting from decreased F-actin turnover. In nematode-infected plants with reduced levels of ADF2, the galls containing the giant feeding cells and growing nematodes do not develop due to the arrest in growth of the giant multinucleate feeding cells, which in turn is due to an aberrant actin network. PMID:19794115

  15. Induction of Dormancy in Arabidopsis Summer Annuals Requires Parallel Regulation of DOG1 and Hormone Metabolism by Low Temperature and CBF Transcription Factors[W][OA

    PubMed Central

    Kendall, Sarah L.; Hellwege, Anja; Marriot, Poppy; Whalley, Celina; Graham, Ian A.; Penfield, Steven

    2011-01-01

    Summer annuals overwinter as seeds in the soil seed bank. This is facilitated by a cold-induced increase in dormancy during seed maturation followed by a switch to a state during seed imbibition in which cold instead promotes germination. Here, we show that the seed maturation transcriptome in Arabidopsis thaliana is highly temperature sensitive and reveal that low temperature during seed maturation induces several genes associated with dormancy, including DELAY OF GERMINATION1 (DOG1), and influences gibberellin and abscisic acid levels in mature seeds. Mutants lacking DOG1, or with altered gibberellin or abscisic acid synthesis or signaling, in turn show reduced ability to enter the deeply dormant states in response to low seed maturation temperatures. In addition, we find that DOG1 promotes gibberellin catabolism during maturation. We show that C-REPEAT BINDING FACTORS (CBFs) are necessary for regulation of dormancy and of GA2OX6 and DOG1 expression caused by low temperatures. However, the temperature sensitivity of CBF transcription is markedly reduced in seeds and is absent in imbibed seeds. Our data demonstrate that inhibition of CBF expression is likely a critical feature allowing cold to promote rather than inhibit germination and support a model in which CBFs act in parallel to a low-temperature signaling pathway in the regulation of dormancy. PMID:21803937

  16. Arabidopsis Cuticular Wax Biosynthesis Is Negatively Regulated by the DEWAX Gene Encoding an AP2/ERF-Type Transcription Factor[W][OPEN

    PubMed Central

    Go, Young Sam; Kim, Hyojin; Kim, Hae Jin; Suh, Mi Chung

    2014-01-01

    The aerial parts of plants are protected from desiccation and other stress by surface cuticular waxes. The total cuticular wax loads and the expression of wax biosynthetic genes are significantly downregulated in Arabidopsis thaliana under dark conditions. We isolated Decrease Wax Biosynthesis (DEWAX), which encodes an AP2/ERF-type transcription factor that is preferentially expressed in the epidermis and induced by darkness. Disruption of DEWAX leads to an increase in total leaf and stem wax loads, and the excess wax phenotype of dewax was restored to wild type levels in complementation lines. Moreover, overexpression of DEWAX resulted in a reduction in total wax loads in leaves and stems compared with the wild type and altered the ultrastructure of cuticular layers. DEWAX negatively regulates the expression of alkane-forming enzyme, long-chain acyl-CoA synthetase, ATP citrate lyase A subunit, enoyl-CoA reductase, and fatty acyl-CoA reductase, and chromatin immunoprecipitation analysis suggested that DEWAX directly interacts with the promoters of wax biosynthesis genes. Cuticular wax biosynthesis is negatively regulated twice a day by the expression of DEWAX, throughout the night and at stomata closing. Significantly higher levels (10- to 100-fold) of DEWAX transcripts were found in leaves than in stems, suggesting that DEWAX-mediated transcriptional repression may be an additional mechanism contributing to the different total wax loads in leaves and stems. PMID:24692420

  17. A Ploidy-Sensitive Mechanism Regulates Aperture Formation on the Arabidopsis Pollen Surface and Guides Localization of the Aperture Factor INP1

    PubMed Central

    Reeder, Sarah H.; Lee, Byung Ha; Fox, Ronald; Dobritsa, Anna A.

    2016-01-01

    Pollen presents a powerful model for studying mechanisms of precise formation and deposition of extracellular structures. Deposition of the pollen wall exine leads to the generation of species-specific patterns on pollen surface. In most species, exine does not develop uniformly across the pollen surface, resulting in the formation of apertures–openings in the exine that are species-specific in number, morphology and location. A long time ago, it was proposed that number and positions of apertures might be determined by the geometry of tetrads of microspores–the precursors of pollen grains arising via meiotic cytokinesis, and by the number of last-contact points between sister microspores. We have tested this model by characterizing Arabidopsis mutants with ectopic apertures and/or abnormal geometry of meiotic products. Here we demonstrate that contact points per se do not act as aperture number determinants and that a correct geometric conformation of a tetrad is neither necessary nor sufficient to generate a correct number of apertures. A mechanism sensitive to pollen ploidy, however, is very important for aperture number and positions and for guiding the aperture factor INP1 to future aperture sites. In the mutants with ectopic apertures, the number and positions of INP1 localization sites change depending on ploidy or ploidy-related cell size and not on INP1 levels, suggesting that sites for aperture formation are specified before INP1 is brought to them. PMID:27177036

  18. Identification of a novel type of WRKY transcription factor binding site in elicitor-responsive cis-sequences from Arabidopsis thaliana.

    PubMed

    Machens, Fabian; Becker, Marlies; Umrath, Felix; Hehl, Reinhard

    2014-03-01

    Using a combination of bioinformatics and synthetic promoters, novel elicitor-responsive cis-sequences were discovered in promoters of pathogen-upregulated genes from Arabidopsis thaliana. One group of functional sequences contains the conserved core sequence GACTTTT. This core sequence and adjacent nucleotides are essential for elicitor-responsive gene expression in a parsley protoplast system. By yeast one-hybrid screening, WRKY70 was selected with a cis-sequence harbouring the core sequence GACTTTT but no known WRKY binding site (W-box). Transactivation experiments, mutation analyses, and electrophoretic mobility shift assays demonstrate that the sequence CGACTTTT is the binding site for WRKY70 in the investigated cis-sequence and is required for WRKY70-activated gene expression. Using several cis-sequences in transactivation experiments and binding studies, the CGACTTTT sequence can be extended to propose YGACTTTT as WRKY70 binding site. This binding site, designated WT-box, is enriched in promoters of genes upregulated in a WRKY70 overexpress