Gravitational stress-induced changes in the phosphoproteom of Arabidopsis thaliana cell cultures

NASA Astrophysics Data System (ADS)

Hampp, Ruediger; Hausmann, Niklas; Neef, Maren; Schuetz, Wolfgang; Madlung, Johannes; Fladerer, Claudia; Nordheim, Alfred; Costa, Alex; Barjaktarovic, Zarko

Callus cell cultures of Arabidopsis thaliana respond to changes in gravitational field strengths by changes in protein expression. Using ESI-MS/MS for proteins with differential abundance after separation by 2D-PAGE, 28 spots which changed reproducibly and significantly (P¡0.05) in amount after 2h of hypergravity (18 up-, 10 down-regulated) could be identified. The corre-sponding proteins were largely involved in stress responses, including detoxification of reactive oxygen species (ROS; Barjaktaroviá et al., J. Exptl. Bot. 58:4357 (2007)). In the present study, c we extended these investigations to phosphorylated proteins. For this purpose, callus cell cul-tures of Arabidopsis thaliana were exposed to hypergravity (8 g) and simulated weightlessness (random positioning; RP) for up to 30 min, a period of time which yielded most reliable data. First changes, however, were visible as early as 10 min after start of treatment. Out of the protein spots altered in phosphorylation, we were able to identify 24 from those responding to random positioning and 12 which responded to 8 g. The respective proteins are involved in scavenging and detoxification of ROS (32Most recent data obtained from parabolic flights indicate that exposure times to g of as little as 20 s are sufficient to alter the phosphorylation of proteins pattern. This is accompanied by changes in the cellular Ca2+ and H2O2 contents.

Model-Based Analysis of Arabidopsis Leaf Epidermal Cells Reveals Distinct Division and Expansion Patterns for Pavement and Guard Cells1[W][OA

PubMed Central

Asl, Leila Kheibarshekan; Dhondt, Stijn; Boudolf, Véronique; Beemster, Gerrit T.S.; Beeckman, Tom; Inzé, Dirk; Govaerts, Willy; De Veylder, Lieven

2011-01-01

To efficiently capture sunlight for photosynthesis, leaves typically develop into a flat and thin structure. This development is driven by cell division and expansion, but the individual contribution of these processes is currently unknown, mainly because of the experimental difficulties to disentangle them in a developing organ, due to their tight interconnection. To circumvent this problem, we built a mathematic model that describes the possible division patterns and expansion rates for individual epidermal cells. This model was used to fit experimental data on cell numbers and sizes obtained over time intervals of 1 d throughout the development of the first leaf pair of Arabidopsis (Arabidopsis thaliana). The parameters were obtained by a derivative-free optimization method that minimizes the differences between the predicted and experimentally observed cell size distributions. The model allowed us to calculate probabilities for a cell to divide into guard or pavement cells, the maximum size at which it can divide, and its average cell division and expansion rates at each point during the leaf developmental process. Surprisingly, average cell cycle duration remained constant throughout leaf development, whereas no evidence for a maximum cell size threshold for cell division of pavement cells was found. Furthermore, the model predicted that neighboring cells of different sizes within the epidermis expand at distinctly different relative rates, which could be verified by direct observations. We conclude that cell division seems to occur independently from the status of cell expansion, whereas the cell cycle might act as a timer rather than as a size-regulated machinery. PMID:21693673

A proteomics dissection of Arabidopsis thaliana vacuoles isolated from cell culture

PubMed Central

Jaquinod, Michel; Villiers, Florent; Kieffer-Jaquinod, Sylvie; Hugouvieux, Véronique; Bruley, Christophe; Garin, Jérôme; Bourguignon, Jacques

2007-01-01

To better understand the mechanisms governing cellular traffic, storage of various metabolites and their ultimate degradation, Arabidopsis thaliana vacuoles proteomes were established. To this aim, a procedure was developed to prepare highly purified vacuoles from protoplasts isolated from Arabidopsis cell cultures using Ficoll density gradients. Based on the specific activity of the vacuolar marker α-mannosidase, the enrichment factor of the vacuoles was estimated at approximately 42 fold with an average yield of 2.1%. Absence of significant contamination by other cellular compartments was validated by western blot using antibodies raised against specific markers of chloroplasts, mitochondria, plasma membrane and endoplasmic reticulum. Based on these results, vacuole preparations showed the necessary degree of purity for proteomic study. Therefore, a proteomic approach was developed in order to identify the protein components present in both the membrane and soluble fractions of the Arabidopsis cell vacuoles. This approach includes: (i) a mild oxidation step leading to the transformation of cysteine residues into cysteic acid and methionine to methionine sulfoxide, (ii) an in-solution proteolytic digestion of very hydrophobic proteins, (iii) a pre-fractionation of proteins by short migration on SDS-PAGE followed by analysis by liquid chromatography coupled to tandem mass spectrometry. This procedure allowed the identification of more than 650 proteins, 2/3 of which copurify with the membrane hydrophobic fraction and 1/3 with the soluble fraction. Among the 416 proteins identified from the membrane fraction, 195 were considered integral membrane proteins based on the presence of one or more predicted transmembrane domains, and 110 transporters and related proteins were identified (91 putative transporters and 19 proteins related to the V-ATPase pump). With regard to function, about 20% of the proteins identified were previously known to be associated with vacuolar

Brassinosteroid Regulates Seed Size and Shape in Arabidopsis1[W][OPEN

PubMed Central

Jiang, Wen-Bo; Huang, Hui-Ya; Hu, Yu-Wei; Zhu, Sheng-Wei; Wang, Zhi-Yong; Lin, Wen-Hui

2013-01-01

Seed development is important for agriculture productivity. We demonstrate that brassinosteroid (BR) plays crucial roles in determining the size, mass, and shape of Arabidopsis (Arabidopsis thaliana) seeds. The seeds of the BR-deficient mutant de-etiolated2 (det2) are smaller and less elongated than those of wild-type plants due to a decreased seed cavity, reduced endosperm volume, and integument cell length. The det2 mutant also showed delay in embryo development, with reduction in both the size and number of embryo cells. Pollination of det2 flowers with wild-type pollen yielded seeds of normal size but still shortened shape, indicating that the BR produced by the zygotic embryo and endosperm is sufficient for increasing seed volume but not for seed elongation, which apparently requires BR produced from maternal tissues. BR activates expression of SHORT HYPOCOTYL UNDER BLUE1, MINISEED3, and HAIKU2, which are known positive regulators of seed size, but represses APETALA2 and AUXIN RESPONSE FACTOR2, which are negative regulators of seed size. These genes are bound in vivo by the BR-activated transcription factor BRASSINAZOLE-RESISTANT1 (BZR1), and they are known to influence specific processes of integument, endosperm, and embryo development. Our results demonstrate that BR regulates seed size and seed shape by transcriptionally modulating specific seed developmental pathways. PMID:23771896

FAMA Is an Essential Component for the Differentiation of Two Distinct Cell Types, Myrosin Cells and Guard Cells, in Arabidopsis[W

PubMed Central

Shirakawa, Makoto; Ueda, Haruko; Nagano, Atsushi J.; Shimada, Tomoo; Kohchi, Takayuki; Hara-Nishimura, Ikuko

2014-01-01

Brassicales plants, including Arabidopsis thaliana, have an ingenious two-compartment defense system, which sequesters myrosinase from the substrate glucosinolate and produces a toxic compound when cells are damaged by herbivores. Myrosinase is stored in vacuoles of idioblast myrosin cells. The molecular mechanism that regulates myrosin cell development remains elusive. Here, we identify the basic helix-loop-helix transcription factor FAMA as an essential component for myrosin cell development along Arabidopsis leaf veins. FAMA is known as a regulator of stomatal development. We detected FAMA expression in myrosin cell precursors in leaf primordia in addition to stomatal lineage cells. FAMA deficiency caused defects in myrosin cell development and in the biosynthesis of myrosinases THIOGLUCOSIDE GLUCOHYDROLASE1 (TGG1) and TGG2. Conversely, ectopic FAMA expression conferred myrosin cell characteristics to hypocotyl and root cells, both of which normally lack myrosin cells. The FAMA interactors ICE1/SCREAM and its closest paralog SCREAM2/ICE2 were essential for myrosin cell development. DNA microarray analysis identified 32 candidate genes involved in myrosin cell development under the control of FAMA. This study provides a common regulatory pathway that determines two distinct cell types in leaves: epidermal guard cells and inner-tissue myrosin cells. PMID:25304202

Arabidopsis NUCLEOSTEMIN-LIKE 1 (NSN1) regulates cell cycling potentially by cooperating with nucleosome assembly protein AtNAP1;1.

PubMed

Wang, Zhen; Wang, Xiaomin; Xie, Bo; Hong, Zonglie; Yang, Qingchuan

2018-06-01

In mammals, nucleostemin (NS), a nucleolar GTPase, is involved in stem cell proliferation, embryogenesis and ribosome biogenesis. Arabidopsis NUCLEOSTEMIN-LIKE 1 (NSN1) has previously been shown to be essential for plant growth and development. However, the role of NSN1 in cell proliferation is largely unknown. Using nsn1, a loss-of-function mutant of Arabidopsis NSN1, we investigated the function of NSN1 in plant cell proliferation and cell cycle regulation. Morphologically, nsn1 exhibited developmental defects in both leaves and roots, producing severely reduced vegetative organs with a much smaller number of cells than those in the wild type. Dynamic analysis of leaf and root growth revealed a lower cell proliferation rate and slower cell division in nsn1. Consistently, the transcriptional levels of key cell  cycle genes, including those regulating the transition of G1-S and G2-M, were reduced drastically in nsn1. The introduction of CYCLIN B1::GUS into nsn1 resulted in confined expression of GUS in both the leaf primordia and root meristem, indicating that cell proliferation was hampered by the mutation of NSN1. Upon subjection to treatment with bleomycin and methyl methanesulfonate (MMS), nsn1 plants exhibited hypersensitivity to the genotoxic agents. In the nucleus, NSN1 interacted with nucleosome assembly protein1 (AtNAP1;1), a highly conserved histone chaperone functioning in cell proliferation. Notably, the N-terminal conserved domains of Arabidopsis NSN1 were critical for the physical interaction. As a conserved homolog of mammalian nucleostemin, Arabidopsis NSN1 plays pivotal roles in embryogenesis and ribosome biogenesis. In this study, NSN1 was found to function as a positive regulator in cell cycle progression. The interaction between NSN1 and histone chaperone AtNAP1;1, and the high resemblance in sensitivity to genotoxics between nsn1 and atnap1;1 imply the indispensability of the two nuclear proteins for cell cycle regulation

RNA-Seq of Arabidopsis Pollen Uncovers Novel Transcription and Alternative Splicing1[C][W][OA

PubMed Central

Loraine, Ann E.; McCormick, Sheila; Estrada, April; Patel, Ketan; Qin, Peng

2013-01-01

Pollen grains of Arabidopsis (Arabidopsis thaliana) contain two haploid sperm cells enclosed in a haploid vegetative cell. Upon germination, the vegetative cell extrudes a pollen tube that carries the sperm to an ovule for fertilization. Knowing the identity, relative abundance, and splicing patterns of pollen transcripts will improve our understanding of pollen and allow investigation of tissue-specific splicing in plants. Most Arabidopsis pollen transcriptome studies have used the ATH1 microarray, which does not assay splice variants and lacks specific probe sets for many genes. To investigate the pollen transcriptome, we performed high-throughput sequencing (RNA-Seq) of Arabidopsis pollen and seedlings for comparison. Gene expression was more diverse in seedling, and genes involved in cell wall biogenesis were highly expressed in pollen. RNA-Seq detected at least 4,172 protein-coding genes expressed in pollen, including 289 assayed only by nonspecific probe sets. Additional exons and previously unannotated 5′ and 3′ untranslated regions for pollen-expressed genes were revealed. We detected regions in the genome not previously annotated as expressed; 14 were tested and 12 were confirmed by polymerase chain reaction. Gapped read alignments revealed 1,908 high-confidence new splicing events supported by 10 or more spliced read alignments. Alternative splicing patterns in pollen and seedling were highly correlated. For most alternatively spliced genes, the ratio of variants in pollen and seedling was similar, except for some encoding proteins involved in RNA splicing. This study highlights the robustness of splicing patterns in plants and the importance of ongoing annotation and visualization of RNA-Seq data using interactive tools such as Integrated Genome Browser. PMID:23590974

Salicylic Acid–Independent ENHANCED DISEASE SUSCEPTIBILITY1 Signaling in Arabidopsis Immunity and Cell Death Is Regulated by the Monooxygenase FMO1 and the Nudix Hydrolase NUDT7[W

PubMed Central

Bartsch, Michael; Gobbato, Enrico; Bednarek, Pawel; Debey, Svenja; Schultze, Joachim L.; Bautor, Jaqueline; Parker, Jane E.

2006-01-01

Arabidopsis thaliana ENHANCED DISEASE SUSCEPTIBILITY1 (EDS1) controls defense activation and programmed cell death conditioned by intracellular Toll-related immune receptors that recognize specific pathogen effectors. EDS1 is also needed for basal resistance to invasive pathogens by restricting the progression of disease. In both responses, EDS1, assisted by its interacting partner, PHYTOALEXIN-DEFICIENT4 (PAD4), regulates accumulation of the phenolic defense molecule salicylic acid (SA) and other as yet unidentified signal intermediates. An Arabidopsis whole genome microarray experiment was designed to identify genes whose expression depends on EDS1 and PAD4, irrespective of local SA accumulation, and potential candidates of an SA-independent branch of EDS1 defense were found. We define two new immune regulators through analysis of corresponding Arabidopsis loss-of-function insertion mutants. FLAVIN-DEPENDENT MONOOXYGENASE1 (FMO1) positively regulates the EDS1 pathway, and one member (NUDT7) of a family of cytosolic Nudix hydrolases exerts negative control of EDS1 signaling. Analysis of fmo1 and nudt7 mutants alone or in combination with sid2-1, a mutation that severely depletes pathogen-induced SA production, points to SA-independent functions of FMO1 and NUDT7 in EDS1-conditioned disease resistance and cell death. We find instead that SA antagonizes initiation of cell death and stunting of growth in nudt7 mutants. PMID:16531493

Gene expression analysis of WRKY transcription factors in Arabidopsis thaliana cell cultures during a parabolic flight

NASA Astrophysics Data System (ADS)

Babbick, Maren; Barjaktarović, Žarko; Hampp, Ruediger

Plants sense gravity by specialized cells (statocytes) and adjust growth and development accordingly. It has, however, also been shown that plant cells which are not part of specialized tissues are also able to sense gravitational forces. Therefore we used undifferentiated, homogeneous cell cultures of Arabidopsis thaliana (cv. Columbia) in order to identify early alterations in gene expression as a response to altered gravitational field strengths. In this contribution we report on cell cultures exposed to parabolic flights (approximately 20 sec of microgravity). For this short-term exposure study, we specifically checked for genes at the beginning of signal transduction chains, such as those coding for transcription factors (TFs). TFs are small proteins that regulate expression of their target genes by binding to specific promoter sequences. Our main focus were members of the so-called WRKY TF family. WRKY TFs are known to be involved in various physiological processes like senescence and pathogen defense. By quantifying transcriptional changes of these genes by real-time RT-PCR, we wanted to find out, how gene expression is affected by both hyperand microgravity conditions during a parabolic flight. For this purpose Arabidopsis thaliana callus cultures were metabolically quenched by the injection of RNAlater at the end of the microgravity-phase of each parabola. The data we present will show how fast changes in amounts of transcripts will occur, and to what degree the expression profiles are comparable with data obtained from exposures to hypergravity and simulated microgravity.

The Organization Pattern of Root Border-Like Cells of Arabidopsis Is Dependent on Cell Wall Homogalacturonan12[C][W

PubMed Central

Durand, Caroline; Vicré-Gibouin, Maïté; Follet-Gueye, Marie Laure; Duponchel, Ludovic; Moreau, Myriam; Lerouge, Patrice; Driouich, Azeddine

2009-01-01

Border-like cells are released by Arabidopsis (Arabidopsis thaliana) root tips as organized layers of several cells that remain attached to each other rather than completely detached from each other, as is usually observed in border cells of many species. Unlike border cells, cell attachment between border-like cells is maintained after their release into the external environment. To investigate the role of cell wall polysaccharides in the attachment and organization of border-like cells, we have examined their release in several well-characterized mutants defective in the biosynthesis of xyloglucan, cellulose, or pectin. Our data show that among all mutants examined, only quasimodo mutants (qua1-1 and qua2-1), which have been characterized as producing less homogalacturonan, had an altered border-like cell phenotype as compared with the wild type. Border-like cells in both lines were released as isolated cells separated from each other, with the phenotype being much more pronounced in qua1-1 than in qua2-1. Further analysis of border-like cells in the qua1-1 mutant using immunocytochemistry and a set of anti-cell wall polysaccharide antibodies showed that the loss of the wild-type phenotype was accompanied by (1) a reduction in homogalacturonan-JIM5 epitope in the cell wall of border-like cells, confirmed by Fourier transform infrared microspectrometry, and (2) the secretion of an abundant mucilage that is enriched in xylogalacturonan and arabinogalactan-protein epitopes, in which the cells are trapped in the vicinity of the root tip. PMID:19448034

Golgi Traffic and Integrity Depend on N-Myristoyl Transferase-1 in Arabidopsis[W

PubMed Central

Renna, Luciana; Stefano, Giovanni; Majeran, Wojciech; Micalella, Chiara; Meinnel, Thierry; Giglione, Carmela; Brandizzi, Federica

2013-01-01

N-myristoylation is a crucial irreversible eukaryotic lipid modification allowing a key subset of proteins to be targeted at the periphery of specific membrane compartments. Eukaryotes have conserved N-myristoylation enzymes, involving one or two N-myristoyltransferases (NMT1 and NMT2), among which NMT1 is the major enzyme. In the postembryonic developmental stages, defects in NMT1 lead to aberrant cell polarity, flower differentiation, fruit maturation, and innate immunity; however, no specific NMT1 target responsible for such deficiencies has hitherto been identified. Using a confocal microscopy forward genetics screen for the identification of Arabidopsis thaliana secretory mutants, we isolated STINGY, a recessive mutant with defective Golgi traffic and integrity. We mapped STINGY to a substitution at position 160 of Arabidopsis NMT1 (NMT1A160T). In vitro kinetic studies with purified NMT1A160T enzyme revealed a significant reduction in its activity due to a remarkable decrease in affinity for both myristoyl-CoA and peptide substrates. We show here that this recessive mutation is responsible for the alteration of Golgi traffic and integrity by predominantly affecting the Golgi membrane/cytosol partitioning of ADP-ribosylation factor proteins. Our results provide important functional insight into N-myristoylation in plants by ascribing postembryonic functions of Arabidopsis NMT1 that involve regulation of the functional and morphological integrity of the plant endomembranes. PMID:23673980

Strigolactones Suppress Adventitious Rooting in Arabidopsis and Pea1[C][W][OA

PubMed Central

Rasmussen, Amanda; Mason, Michael Glenn; De Cuyper, Carolien; Brewer, Philip B.; Herold, Silvia; Agusti, Javier; Geelen, Danny; Greb, Thomas; Goormachtig, Sofie; Beeckman, Tom; Beveridge, Christine Anne

2012-01-01

Adventitious root formation is essential for the propagation of many commercially important plant species and involves the formation of roots from nonroot tissues such as stems or leaves. Here, we demonstrate that the plant hormone strigolactone suppresses adventitious root formation in Arabidopsis (Arabidopsis thaliana) and pea (Pisum sativum). Strigolactone-deficient and response mutants of both species have enhanced adventitious rooting. CYCLIN B1 expression, an early marker for the initiation of adventitious root primordia in Arabidopsis, is enhanced in more axillary growth2 (max2), a strigolactone response mutant, suggesting that strigolactones restrain the number of adventitious roots by inhibiting the very first formative divisions of the founder cells. Strigolactones and cytokinins appear to act independently to suppress adventitious rooting, as cytokinin mutants are strigolactone responsive and strigolactone mutants are cytokinin responsive. In contrast, the interaction between the strigolactone and auxin signaling pathways in regulating adventitious rooting appears to be more complex. Strigolactone can at least partially revert the stimulatory effect of auxin on adventitious rooting, and auxin can further increase the number of adventitious roots in max mutants. We present a model depicting the interaction of strigolactones, cytokinins, and auxin in regulating adventitious root formation. PMID:22323776

Apoplastic Alkalinization Is Instrumental for the Inhibition of Cell Elongation in the Arabidopsis Root by the Ethylene Precursor 1-Aminocyclopropane-1-Carboxylic Acid1[W][OA

PubMed Central

Staal, Marten; De Cnodder, Tinne; Simon, Damien; Vandenbussche, Filip; Van Der Straeten, Dominique; Verbelen, Jean-Pierre; Elzenga, Theo; Vissenberg, Kris

2011-01-01

In Arabidopsis (Arabidopsis thaliana; Columbia-0) roots, the so-called zone of cell elongation comprises two clearly different domains: the transition zone, a postmeristematic region (approximately 200–450 μm proximal of the root tip) with a low rate of elongation, and a fast elongation zone, the adjacent proximal region (450 μm away from the root tip up to the first root hair) with a high rate of elongation. In this study, the surface pH was measured in both zones using the microelectrode ion flux estimation technique. The surface pH is highest in the apical part of the transition zone and is lowest at the basal part of the fast elongation zone. Fast cell elongation is inhibited within minutes by the ethylene precursor 1-aminocyclopropane-1-carboxylic acid; concomitantly, apoplastic alkalinization occurs in the affected root zone. Fusicoccin, an activator of the plasma membrane H+-ATPase, can partially rescue this inhibition of cell elongation, whereas the inhibitor N,N′-dicyclohexylcarbodiimide does not further reduce the maximal cell length. Microelectrode ion flux estimation experiments with auxin mutants lead to the final conclusion that control of the activity state of plasma membrane H+-ATPases is one of the mechanisms by which ethylene, via auxin, affects the final cell length in the root. PMID:21282405

Chloroplast Dysfunction Causes Multiple Defects in Cell Cycle Progression in the Arabidopsis crumpled leaf Mutant1[C][W

PubMed Central

Hudik, Elodie; Yoshioka, Yasushi; Domenichini, Séverine; Bourge, Mickaël; Soubigout-Taconnat, Ludivine; Mazubert, Christelle; Yi, Dalong; Bujaldon, Sandrine; Hayashi, Hiroyuki; De Veylder, Lieven; Bergounioux, Catherine; Benhamed, Moussa; Raynaud, Cécile

2014-01-01

The majority of research on cell cycle regulation is focused on the nuclear events that govern the replication and segregation of the genome between the two daughter cells. However, eukaryotic cells contain several compartmentalized organelles with specialized functions, and coordination among these organelles is required for proper cell cycle progression, as evidenced by the isolation of several mutants in which both organelle function and overall plant development were affected. To investigate how chloroplast dysfunction affects the cell cycle, we analyzed the crumpled leaf (crl) mutant of Arabidopsis (Arabidopsis thaliana), which is deficient for a chloroplastic protein and displays particularly severe developmental defects. In the crl mutant, we reveal that cell cycle regulation is altered drastically and that meristematic cells prematurely enter differentiation, leading to reduced plant stature and early endoreduplication in the leaves. This response is due to the repression of several key cell cycle regulators as well as constitutive activation of stress-response genes, among them the cell cycle inhibitor SIAMESE-RELATED5. One unique feature of the crl mutant is that it produces aplastidic cells in several organs, including the root tip. By investigating the consequence of the absence of plastids on cell cycle progression, we showed that nuclear DNA replication occurs in aplastidic cells in the root tip, which opens future research prospects regarding the dialogue between plastids and the nucleus during cell cycle regulation in higher plants. PMID:25037213

Flavonols Accumulate Asymmetrically and Affect Auxin Transport in Arabidopsis1[C][W][OA

PubMed Central

Kuhn, Benjamin M.; Geisler, Markus; Bigler, Laurent; Ringli, Christoph

2011-01-01

Flavonoids represent a class of secondary metabolites with diverse functions in plants including ultraviolet protection, pathogen defense, and interspecies communication. They are also known as modulators of signaling processes in plant and animal systems and therefore are considered to have beneficial effects as nutraceuticals. The rol1-2 (for repressor of lrx1) mutation of Arabidopsis (Arabidopsis thaliana) induces aberrant accumulation of flavonols and a cell-growth phenotype in the shoot. The hyponastic cotyledons, aberrant shape of pavement cells, and deformed trichomes in rol1-2 mutants are suppressed by blocking flavonoid biosynthesis, suggesting that the altered flavonol accumulation in these plants induces the shoot phenotype. Indeed, the identification of several transparent testa, myb, and fls1 (for flavonol synthase1) alleles in a rol1-2 suppressor screen provides genetic evidence that flavonols interfere with shoot development in rol1-2 seedlings. The increased accumulation of auxin in rol1-2 seedlings appears to be caused by a flavonol-induced modification of auxin transport. Quantification of auxin export from mesophyll protoplasts revealed that naphthalene-1-acetic acid but not indole-3-acetic acid transport is affected by the rol1-2 mutation. Inhibition of flavonol biosynthesis in rol1-2 fls1-3 restores naphthalene-1-acetic acid transport to wild-type levels, indicating a very specific mode of action of flavonols on the auxin transport machinery. PMID:21502189

Intracellular Calcium Decreases Upon Hyper Gravity-Treatment of Arabidopsis Thaliana Cell Cultures

NASA Astrophysics Data System (ADS)

Neef, Maren; Denn, Tamara; Ecke, Margret; Hampp, Rüdiger

2016-06-01

Cell cultures of Arabidopsis thaliana ( A. t.) respond to changes in the gravitational field strength with fluctuations of the amount of cytosolic calcium (Ca2+). In parabolic flight experiments, where hyper- and μg phases follow each other, μg clearly increased Ca2+, while hyper-g caused a slight reduction. Since the latter observation had not been reported before, we studied this effect in more detail. Using a special centrifuge for heavy items (ZARM, Bremen, Germany), we determined the hyper-g-dependent intracellular Ca2+ level with transgenic cell lines expressing the Ca2+ sensor, cameleon. This sensor exhibits a shift in fluorescence from 480 to 530 nm in response to Ca2+ binding. The data show a drop in the intracellular Ca2+ concentration with a threshold gravity of around 3 g. This is above hypergravity levels achieved during parabolic flights (1.8 g). The use of mutants with different sub-cellular targets of cameleon expression (nucleus, tonoplast, plasma membrane) gave the same results, i.e. Ca2+ is obviously exported from several intracellular compartments.

Identification of nuclear target proteins for S-nitrosylation in pathogen-treated Arabidopsis thaliana cell cultures.

PubMed

Chaki, Mounira; Shekariesfahlan, Azam; Ageeva, Alexandra; Mengel, Alexander; von Toerne, Christine; Durner, Jörg; Lindermayr, Christian

2015-09-01

Nitric oxide (NO) is a significant signalling molecule involved in the regulation of many different physiological processes in plants. One of the most imperative regulatory modes of action of NO is protein S-nitrosylation--the covalent attachment of an NO group to the sulfur atom of cysteine residues. In this study, we focus on S-nitrosylation of Arabidopsis nuclear proteins after pathogen infection. After treatment of Arabidopsis suspension cell cultures with pathogens, nuclear proteins were extracted and treated with the S-nitrosylating agent S-nitrosoglutathione (GSNO). A biotin switch assay was performed and biotin-labelled proteins were purified by neutravidin affinity chromatography and identified by mass spectrometry. A total of 135 proteins were identified, whereas nuclear localization has been described for 122 proteins of them. 117 of these proteins contain at least one cysteine residue. Most of the S-nitrosylated candidates were involved in protein and RNA metabolism, stress response, and cell organization and division. Interestingly, two plant-specific histone deacetylases were identified suggesting that nitric oxide regulated epigenetic processes in plants. In sum, this work provides a new collection of targets for protein S-nitrosylation in Arabidopsis and gives insight into the regulatory function of NO in the nucleus during plant defense response. Moreover, our data extend the knowledge on the regulatory function of NO in events located in the nucleus. Copyright © 2015 Elsevier Ireland Ltd. All rights reserved.

Arabidopsis N-MYC DOWNREGULATED-LIKE1, a Positive Regulator of Auxin Transport in a G Protein–Mediated Pathway[W

PubMed Central

Mudgil, Yashwanti; Uhrig, Joachm F.; Zhou, Jiping; Temple, Brenda; Jiang, Kun; Jones, Alan M.

2009-01-01

Root architecture results from coordinated cell division and expansion in spatially distinct cells of the root and is established and maintained by gradients of auxin and nutrients such as sugars. Auxin is transported acropetally through the root within the central stele and then, upon reaching the root apex, auxin is transported basipetally through the outer cortical and epidermal cells. The two Gβγ dimers of the Arabidopsis thaliana heterotrimeric G protein complex are differentially localized to the central and cortical tissues of the Arabidopsis roots. A null mutation in either the single β (AGB1) or the two γ (AGG1 and AGG2) subunits confers phenotypes that disrupt the proper architecture of Arabidopsis roots and are consistent with altered auxin transport. Here, we describe an evolutionarily conserved interaction between AGB1/AGG dimers and a protein designated N-MYC DOWNREGULATED-LIKE1 (NDL1). The Arabidopsis genome encodes two homologs of NDL1 (NDL2 and NDL3), which also interact with AGB1/AGG1 and AGB1/AGG2 dimers. We show that NDL proteins act in a signaling pathway that modulates root auxin transport and auxin gradients in part by affecting the levels of at least two auxin transport facilitators. Reduction of NDL family gene expression and overexpression of NDL1 alter root architecture, auxin transport, and auxin maxima. AGB1, auxin, and sugars are required for NDL1 protein stability in regions of the root where auxin gradients are established; thus, the signaling mechanism contains feedback loops. PMID:19948787

Chloroplast and reactive oxygen species involvement in apoptotic-like programmed cell death in Arabidopsis suspension cultures

PubMed Central

Doyle, Siamsa M.; Diamond, Mark; McCabe, Paul F.

2010-01-01

Chloroplasts produce reactive oxygen species (ROS) during cellular stress. ROS are known to act as regulators of programmed cell death (PCD) in plant and animal cells, so it is possible that chloroplasts have a role in regulating PCD in green tissue. Arabidopsis thaliana cell suspension cultures are model systems in which to test this, as here it is shown that their cells contain well-developed, functional chloroplasts when grown in the light, but not when grown in the dark. Heat treatment at 55 °C induced apoptotic-like (AL)-PCD in the cultures, but light-grown cultures responded with significantly less AL-PCD than dark-grown cultures. Chloroplast-free light-grown cultures were established using norflurazon, spectinomycin, and lincomycin and these cultures responded to heat treatment with increased AL-PCD, demonstrating that chloroplasts affect AL-PCD induction in light-grown cultures. Antioxidant treatment of light-grown cultures also resulted in increased AL-PCD induction, suggesting that chloroplast-produced ROS may be involved in AL-PCD regulation. Cycloheximide treatment of light-grown cultures prolonged cell viability and attenuated AL-PCD induction; however, this effect was less pronounced in dark-grown cultures, and did not occur in antioxidant-treated light-grown cultures. This suggests that a complex interplay between light, chloroplasts, ROS, and nuclear protein synthesis occurs during plant AL-PCD. The results of this study highlight the importance of taking into account the time-point at which cells are observed and whether the cells are light-grown and chloroplast-containing or not, for any study on plant AL-PCD, as it appears that chloroplasts can play a significant role in AL-PCD regulation. PMID:19933317

Rice Blast Fungus (Magnaporthe oryzae) Infects Arabidopsis via a Mechanism Distinct from That Required for the Infection of Rice1[W][OA

PubMed Central

Park, Ju-Young; Jin, Jianming; Lee, Yin-Won; Kang, Seogchan; Lee, Yong-Hwan

2009-01-01

Magnaporthe oryzae is a hemibiotrophic fungal pathogen that causes rice (Oryza sativa) blast. Although M. oryzae as a whole infects a wide variety of monocotyledonous hosts, no dicotyledonous plant has been reported as a host. We found that two rice pathogenic strains of M. oryzae, KJ201 and 70-15, interacted differentially with 16 ecotypes of Arabidopsis (Arabidopsis thaliana). Strain KJ201 infected all ecotypes with varying degrees of virulence, whereas strain 70-15 caused no symptoms in certain ecotypes. In highly susceptible ecotypes, small chlorotic lesions appeared on infected leaves within 3 d after inoculation and subsequently expanded across the affected leaves. The fungus produced spores in susceptible ecotypes but not in resistant ecotypes. Fungal cultures recovered from necrotic lesions caused the same symptoms in healthy plants, satisfying Koch's postulates. Histochemical analyses showed that infection by the fungus caused an accumulation of reactive oxygen species and eventual cell death. Similar to the infection process in rice, the fungus differentiated to form appressorium and directly penetrated the leaf surface in Arabidopsis. However, the pathogenic mechanism in Arabidopsis appears distinct from that in rice; three fungal genes essential for pathogenicity in rice played only limited roles in causing disease symptoms in Arabidopsis, and the fungus seems to colonize Arabidopsis as a necrotroph through the secretion of phytotoxic compounds, including 9,12-octadecadienoic acid. Expression of PR-1 and PDF1.2 was induced in response to infection by the fungus, suggesting the activation of salicylic acid- and jasmonic acid/ethylene-dependent signaling pathways. However, the roles of these signaling pathways in defense against M. oryzae remain unclear. In combination with the wealth of genetic and genomic resources available for M. oryzae, this newly established pathosystem allows comparison of the molecular and cellular mechanisms underlying

Arabidopsis VASCULAR-RELATED UNKNOWN PROTEIN1 Regulates Xylem Development and Growth by a Conserved Mechanism That Modulates Hormone Signaling1[W][OPEN

PubMed Central

Grienenberger, Etienne; Douglas, Carl J.

2014-01-01

Despite a strict conservation of the vascular tissues in vascular plants (tracheophytes), our understanding of the genetic basis underlying the differentiation of secondary cell wall-containing cells in the xylem of tracheophytes is still far from complete. Using coexpression analysis and phylogenetic conservation across sequenced tracheophyte genomes, we identified a number of Arabidopsis (Arabidopsis thaliana) genes of unknown function whose expression is correlated with secondary cell wall deposition. Among these, the Arabidopsis VASCULAR-RELATED UNKNOWN PROTEIN1 (VUP1) gene encodes a predicted protein of 24 kD with no annotated functional domains but containing domains that are highly conserved in tracheophytes. Here, we show that the VUP1 expression pattern, determined by promoter-β-glucuronidase reporter gene expression, is associated with vascular tissues, while vup1 loss-of-function mutants exhibit collapsed morphology of xylem vessel cells. Constitutive overexpression of VUP1 caused dramatic and pleiotropic developmental defects, including severe dwarfism, dark green leaves, reduced apical dominance, and altered photomorphogenesis, resembling brassinosteroid-deficient mutants. Constitutive overexpression of VUP homologs from multiple tracheophyte species induced similar defects. Whole-genome transcriptome analysis revealed that overexpression of VUP1 represses the expression of many brassinosteroid- and auxin-responsive genes. Additionally, deletion constructs and site-directed mutagenesis were used to identify critical domains and amino acids required for VUP1 function. Altogether, our data suggest a conserved role for VUP1 in regulating secondary wall formation during vascular development by tissue- or cell-specific modulation of hormone signaling pathways. PMID:24567189

Disruption of Abscisic Acid Signaling Constitutively Activates Arabidopsis Resistance to the Necrotrophic Fungus Plectosphaerella cucumerina1[W

PubMed Central

Sánchez-Vallet, Andrea; López, Gemma; Ramos, Brisa; Delgado-Cerezo, Magdalena; Riviere, Marie-Pierre; Llorente, Francisco; Fernández, Paula Virginia; Miedes, Eva; Estevez, José Manuel; Grant, Murray; Molina, Antonio

2012-01-01

Plant resistance to necrotrophic fungi is regulated by a complex set of signaling pathways that includes those mediated by the hormones salicylic acid (SA), ethylene (ET), jasmonic acid (JA), and abscisic acid (ABA). The role of ABA in plant resistance remains controversial, as positive and negative regulatory functions have been described depending on the plant-pathogen interaction analyzed. Here, we show that ABA signaling negatively regulates Arabidopsis (Arabidopsis thaliana) resistance to the necrotrophic fungus Plectosphaerella cucumerina. Arabidopsis plants impaired in ABA biosynthesis, such as the aba1-6 mutant, or in ABA signaling, like the quadruple pyr/pyl mutant (pyr1pyl1pyl2pyl4), were more resistant to P. cucumerina than wild-type plants. In contrast, the hab1-1abi1-2abi2-2 mutant impaired in three phosphatases that negatively regulate ABA signaling displayed an enhanced susceptibility phenotype to this fungus. Comparative transcriptomic analyses of aba1-6 and wild-type plants revealed that the ABA pathway negatively regulates defense genes, many of which are controlled by the SA, JA, or ET pathway. In line with these data, we found that aba1-6 resistance to P. cucumerina was partially compromised when the SA, JA, or ET pathway was disrupted in this mutant. Additionally, in the aba1-6 plants, some genes encoding cell wall-related proteins were misregulated. Fourier transform infrared spectroscopy and biochemical analyses of cell walls from aba1-6 and wild-type plants revealed significant differences in their Fourier transform infrared spectratypes and uronic acid and cellulose contents. All these data suggest that ABA signaling has a complex function in Arabidopsis basal resistance, negatively regulating SA/JA/ET-mediated resistance to necrotrophic fungi. PMID:23037505

Light-Induced Acclimation of the Arabidopsis chlorina1 Mutant to Singlet Oxygen[C][W

PubMed Central

Ramel, Fanny; Ksas, Brigitte; Akkari, Elsy; Mialoundama, Alexis S.; Monnet, Fabien; Krieger-Liszkay, Anja; Ravanat, Jean-Luc; Mueller, Martin J.; Bouvier, Florence; Havaux, Michel

2013-01-01

Singlet oxygen (1O2) is a reactive oxygen species that can function as a stress signal in plant leaves leading to programmed cell death. In microalgae, 1O2-induced transcriptomic changes result in acclimation to 1O2. Here, using a chlorophyll b–less Arabidopsis thaliana mutant (chlorina1 [ch1]), we show that this phenomenon can also occur in vascular plants. The ch1 mutant is highly photosensitive due to a selective increase in the release of 1O2 by photosystem II. Under photooxidative stress conditions, the gene expression profile of ch1 mutant leaves very much resembled the gene responses to 1O2 reported in the Arabidopsis mutant flu. Preexposure of ch1 plants to moderately elevated light intensities eliminated photooxidative damage without suppressing 1O2 formation, indicating acclimation to 1O2. Substantial differences in gene expression were observed between acclimation and high-light stress: A number of transcription factors were selectively induced by acclimation, and contrasting effects were observed for the jasmonate pathway. Jasmonate biosynthesis was strongly induced in ch1 mutant plants under high-light stress and was noticeably repressed under acclimation conditions, suggesting the involvement of this hormone in 1O2-induced cell death. This was confirmed by the decreased tolerance to photooxidative damage of jasmonate-treated ch1 plants and by the increased tolerance of the jasmonate-deficient mutant delayed-dehiscence2. PMID:23590883

B1-Phytoprostanes Trigger Plant Defense and Detoxification Responses1[w

PubMed Central

Loeffler, Christiane; Berger, Susanne; Guy, Alexandre; Durand, Thierry; Bringmann, Gerhard; Dreyer, Michael; von Rad, Uta; Durner, Jörg; Mueller, Martin J.

2005-01-01

Phytoprostanes are prostaglandin/jasmonate-like products of nonenzymatic lipid peroxidation that not only occur ubiquitously in healthy plants but also increase in response to oxidative stress. In this work, we show that the two naturally occurring B1-phytoprostanes (PPB1) regioisomers I and II (each comprising two enantiomers) are short-lived stress metabolites that display a broad spectrum of biological activities. Gene expression analysis of Arabidopsis (Arabidopsis thaliana) cell cultures treated with PPB1-I or -II revealed that both regioisomers triggered a massive detoxification and defense response. Interestingly, expression of several glutathione S-transferases, glycosyl transferases, and putative ATP-binding cassette transporters was found to be increased by one or both PPB1 regioisomers, and hence, may enhance the plant's capacity to inactivate and sequester reactive products of lipid peroxidation. Moreover, pretreatment of tobacco (Nicotiana tabacum) suspension cells with PPB1 considerably prevented cell death caused by severe CuSO4 poisoning. Several Arabidopsis genes induced by PPB1, such as those coding for adenylylsulfate reductase, tryptophan synthase β-chain, and PAD3 pointed to an activation of the camalexin biosynthesis pathway that indeed led to the accumulation of camalexin in PPB1 treated leaves of Arabidopsis. Stimulation of secondary metabolism appears to be a common plant reaction in response to PPB1. In three different plant species, PPB1-II induced a concentration dependent accumulation of phytoalexins that was comparable to that induced by methyl jasmonate. PPB1-I was much weaker active or almost inactive. No differences were found between the enantiomers of each regioisomer. Thus, results suggest that PPB1 represent stress signals that improve plants capacity to cope better with a variety of stresses. PMID:15618427

Arabidopsis EDS1 connects pathogen effector recognition to cell compartment-specific immune responses.

PubMed

Heidrich, Katharina; Wirthmueller, Lennart; Tasset, Céline; Pouzet, Cécile; Deslandes, Laurent; Parker, Jane E

2011-12-09

Pathogen effectors are intercepted by plant intracellular nucleotide binding-leucine-rich repeat (NB-LRR) receptors. However, processes linking receptor activation to downstream defenses remain obscure. Nucleo-cytoplasmic basal resistance regulator EDS1 (ENHANCED DISEASE SUSCEPTIBILITY1) is indispensible for immunity mediated by TIR (Toll-interleukin-1 receptor)-NB-LRR receptors. We show that Arabidopsis EDS1 molecularly connects TIR-NB-LRR disease resistance protein RPS4 recognition of bacterial effector AvrRps4 to defense pathways. RPS4-EDS1 and AvrRps4-EDS1 complexes are detected inside nuclei of living tobacco cells after transient coexpression and in Arabidopsis soluble leaf extracts after resistance activation. Forced AvrRps4 localization to the host cytoplasm or nucleus reveals cell compartment-specific RPS4-EDS1 defense branches. Although nuclear processes restrict bacterial growth, programmed cell death and transcriptional resistance reinforcement require nucleo-cytoplasmic coordination. Thus, EDS1 behaves as an effector target and activated TIR-NB-LRR signal transducer for defenses across cell compartments.

Oxidative DNA Damage Bypass in Arabidopsis thaliana Requires DNA Polymerase λ and Proliferating Cell Nuclear Antigen 2[W

PubMed Central

Amoroso, Alessandra; Concia, Lorenzo; Maggio, Caterina; Raynaud, Cécile; Bergounioux, Catherine; Crespan, Emmanuele; Cella, Rino; Maga, Giovanni

2011-01-01

The oxidized base 7,8-oxoguanine (8-oxo-G) is the most common DNA lesion generated by reactive oxygen species. This lesion is highly mutagenic due to the frequent misincorporation of A opposite 8-oxo-G during DNA replication. In mammalian cells, the DNA polymerase (pol) family X enzyme DNA pol λ catalyzes the correct incorporation of C opposite 8-oxo-G, together with the auxiliary factor proliferating cell nuclear antigen (PCNA). Here, we show that Arabidopsis thaliana DNA pol λ, the only member of the X family in plants, is as efficient in performing error-free translesion synthesis past 8-oxo-G as its mammalian homolog. Arabidopsis, in contrast with animal cells, possesses two genes for PCNA. Using in vitro and in vivo approaches, we observed that PCNA2, but not PCNA1, physically interacts with DNA pol λ, enhancing its fidelity and efficiency in translesion synthesis. The levels of DNA pol λ in transgenic plantlets characterized by overexpression or silencing of Arabidopsis POLL correlate with the ability of cell extracts to perform error-free translesion synthesis. The important role of DNA pol λ is corroborated by the observation that the promoter of POLL is activated by UV and that both overexpressing and silenced plants show altered growth phenotypes. PMID:21325140

The anisotropy1 D604N Mutation in the Arabidopsis Cellulose Synthase1 Catalytic Domain Reduces Cell Wall Crystallinity and the Velocity of Cellulose Synthase Complexes1[W][OA

PubMed Central

Fujita, Miki; Himmelspach, Regina; Ward, Juliet; Whittington, Angela; Hasenbein, Nortrud; Liu, Christine; Truong, Thy T.; Galway, Moira E.; Mansfield, Shawn D.; Hocart, Charles H.; Wasteneys, Geoffrey O.

2013-01-01

Multiple cellulose synthase (CesA) subunits assemble into plasma membrane complexes responsible for cellulose production. In the Arabidopsis (Arabidopsis thaliana) model system, we identified a novel D604N missense mutation, designated anisotropy1 (any1), in the essential primary cell wall CesA1. Most previously identified CesA1 mutants show severe constitutive or conditional phenotypes such as embryo lethality or arrest of cellulose production but any1 plants are viable and produce seeds, thus permitting the study of CesA1 function. The dwarf mutants have reduced anisotropic growth of roots, aerial organs, and trichomes. Interestingly, cellulose microfibrils were disordered only in the epidermal cells of the any1 inflorescence stem, whereas they were transverse to the growth axis in other tissues of the stem and in all elongated cell types of roots and dark-grown hypocotyls. Overall cellulose content was not altered but both cell wall crystallinity and the velocity of cellulose synthase complexes were reduced in any1. We crossed any1 with the temperature-sensitive radial swelling1-1 (rsw1-1) CesA1 mutant and observed partial complementation of the any1 phenotype in the transheterozygotes at rsw1-1’s permissive temperature (21°C) and full complementation by any1 of the conditional rsw1-1 root swelling phenotype at the restrictive temperature (29°C). In rsw1-1 homozygotes at restrictive temperature, a striking dissociation of cellulose synthase complexes from the plasma membrane was accompanied by greatly diminished motility of intracellular cellulose synthase-containing compartments. Neither phenomenon was observed in the any1 rsw1-1 transheterozygotes, suggesting that the proteins encoded by the any1 allele replace those encoded by rsw1-1 at restrictive temperature. PMID:23532584

A Novel Approach to Dissect the Abscission Process in Arabidopsis1[C][W][OA

PubMed Central

González-Carranza, Zinnia Haydee; Shahid, Ahmad Ali; Zhang, Li; Liu, Yang; Ninsuwan, Unchalee; Roberts, Jeremy Alan

2012-01-01

Abscission is the consequence of a specialized layer of cells undergoing a complex series of molecular and biochemical events. Analysis of the specific molecular changes associated with abscission is hampered by contamination from neighboring nonseparating tissues. Moreover, studies of abscission frequently involve the examination of events that take place in isolated segments of tissue exposed to nonphysiological concentrations of ethylene or indole-3-acetic acid for protracted periods (more than 24 h) of time. To resolve these problems, we have adopted the use of a transgenic line of Arabidopsis (Arabidopsis thaliana) where the promoter of an abscission-specific polygalacturonase gene (At2g41850/ARABIDOPSIS DEHISCENCE ZONE POLYGALACTURONASE2) has been fused to a green fluorescent protein reporter. RNA was extracted from green fluorescent protein-tagged cells, released from abscising floral organs, and used to generate a complementary DNA library. This library was used to probe a microarray, and a population of abscission-related transcripts was studied in detail. Seven novel abscission-related genes were identified, four of which encode proteins of unknown function. Reverse transcription-polymerase chain reaction analyses and promoter fusions to the β-glucuronidase reporter gene confirmed the expression of these genes in the abscission zone and revealed other places of expression during seedling development. Three of these genes were studied further by crossing reporter lines to the abscission mutants inflorescence deficient in abscission (ida) and blade-on-petiole1 (bop1)/bop2 and an IDA-overexpressing line. Phenotypic analysis of an At3g14380 transfer DNA insertion line indicates that this gene plays a functional role in floral organ shedding. This strategy has enabled us to uncover new genes involved in abscission, and their possible contribution to the process is discussed. PMID:22992509

Quantifying the Labeling and the Levels of Plant Cell Wall Precursors Using Ion Chromatography Tandem Mass Spectrometry1[W][OA

PubMed Central

Alonso, Ana P.; Piasecki, Rebecca J.; Wang, Yan; LaClair, Russell W.; Shachar-Hill, Yair

2010-01-01

The biosynthesis of cell wall polymers involves enormous fluxes through central metabolism that are not fully delineated and whose regulation is poorly understood. We have established and validated a liquid chromatography tandem mass spectrometry method using multiple reaction monitoring mode to separate and quantify the levels of plant cell wall precursors. Target analytes were identified by their parent/daughter ions and retention times. The method allows the quantification of precursors at low picomole quantities with linear responses up to the nanomole quantity range. When applying the technique to Arabidopsis (Arabidopsis thaliana) T87 cell cultures, 16 hexose-phosphates (hexose-Ps) and nucleotide-sugars (NDP-sugars) involved in cell wall biosynthesis were separately quantified. Using hexose-P and NDP-sugar standards, we have shown that hot water extraction allows good recovery of the target metabolites (over 86%). This method is applicable to quantifying the levels of hexose-Ps and NDP-sugars in different plant tissues, such as Arabidopsis T87 cells in culture and fenugreek (Trigonella foenum-graecum) endosperm tissue, showing higher levels of galacto-mannan precursors in fenugreek endosperm. In Arabidopsis cells incubated with [U-13CFru]sucrose, the method was used to track the labeling pattern in cell wall precursors. As the fragmentation of hexose-Ps and NDP-sugars results in high yields of [PO3]−/or [H2PO4]− ions, mass isotopomers can be quantified directly from the intensity of selected tandem mass spectrometry transitions. The ability to directly measure 13C labeling in cell wall precursors makes possible metabolic flux analysis of cell wall biosynthesis based on dynamic labeling experiments. PMID:20442274

A Role for the TOC Complex in Arabidopsis Root Gravitropism1[W][OA

PubMed Central

Stanga, John P.; Boonsirichai, Kanokporn; Sedbrook, John C.; Otegui, Marisa S.; Masson, Patrick H.

2009-01-01

Arabidopsis (Arabidopsis thaliana) roots perceive gravity and reorient their growth accordingly. Starch-dense amyloplasts within the columella cells of the root cap are important for gravitropism, and starchless mutants such as pgm1 display an attenuated response to gravistimulation. The altered response to gravity1 (arg1) mutant is known to be involved with the early phases of gravity signal transduction. arg1 responds slowly to gravistimulation and is in a genetically distinct pathway from pgm1, as pgm1 mutants enhance the gravitropic defect of arg1. arg1 seeds were mutagenized with ethylmethane sulfonate to identify new mutants that enhance the gravitropic defect of arg1. Two modifier of arg1 mutants (mar1 and mar2) grow in random directions only when arg1 is present, do not affect phototropism, and respond like the wild type to application of phytohormones. Both have mutations affecting different components of the Translocon of Outer Membrane of Chloroplasts (TOC) complex. mar1 possesses a mutation in the TOC75-III gene; mar2 possesses a mutation in the TOC132 gene. Overexpression of TOC132 rescues the random growth phenotype of mar2 arg1 roots. Root cap amyloplasts in mar2 arg1 appear ultrastructurally normal. They saltate like the wild type and sediment at wild-type rates upon gravistimulation. These data point to a role for the plastidic TOC complex in gravity signal transduction within the statocytes. PMID:19211693

Molecular Characterization of Organelle-Type Nudix Hydrolases in Arabidopsis1[W

PubMed Central

Ogawa, Takahisa; Yoshimura, Kazuya; Miyake, Hiroe; Ishikawa, Kazuya; Ito, Daisuke; Tanabe, Noriaki; Shigeoka, Shigeru

2008-01-01

Nudix (for nucleoside diphosphates linked to some moiety X) hydrolases act to hydrolyze ribonucleoside and deoxyribonucleoside triphosphates, nucleotide sugars, coenzymes, or dinucleoside polyphosphates. Arabidopsis (Arabidopsis thaliana) contains 27 genes encoding Nudix hydrolase homologues (AtNUDX1 to -27) with a predicted distribution in the cytosol, mitochondria, and chloroplasts. Previously, cytosolic Nudix hydrolases (AtNUDX1 to -11 and -25) were characterized. Here, we conducted a characterization of organelle-type AtNUDX proteins (AtNUDX12 to -24, -26, and -27). AtNUDX14 showed pyrophosphohydrolase activity toward both ADP-ribose and ADP-glucose, although its Km value was approximately 100-fold lower for ADP-ribose (13.0 ± 0.7 μm) than for ADP-glucose (1,235 ± 65 μm). AtNUDX15 hydrolyzed not only reduced coenzyme A (118.7 ± 3.4 μm) but also a wide range of its derivatives. AtNUDX19 showed pyrophosphohydrolase activity toward both NADH (335.3 ± 5.4 μm) and NADPH (36.9 ± 3.5 μm). AtNUDX23 had flavin adenine dinucleotide pyrophosphohydrolase activity (9.1 ± 0.9 μm). Both AtNUDX26 and AtNUDX27 hydrolyzed diadenosine polyphosphates (n = 4–5). A confocal microscopic analysis using a green fluorescent protein fusion protein showed that AtNUDX15 is distributed in mitochondria and AtNUDX14 -19, -23, -26, and -27 are distributed in chloroplasts. These AtNUDX mRNAs were detected ubiquitously in various Arabidopsis tissues. The T-DNA insertion mutants of AtNUDX13, -14, -15, -19, -20, -21, -25, -26, and -27 did not exhibit any phenotypical differences under normal growth conditions. These results suggest that Nudix hydrolases in Arabidopsis control a variety of metabolites and are pertinent to a wide range of physiological processes. PMID:18815383

Arabidopsis Response Regulator1 and Arabidopsis Histidine Phosphotransfer Protein2 (AHP2), AHP3, and AHP5 Function in Cold Signaling1[W][OA

PubMed Central

Jeon, Jin; Kim, Jungmook

2013-01-01

The Arabidopsis (Arabidopsis thaliana) two-component signaling system, which is composed of sensor histidine kinases, histidine phosphotransfer proteins, and response regulators, mediates the cytokinin response and various other plant responses. We have previously shown that ARABIDOPSIS HISTIDINE KINASE2 (AHK2), AHK3, and cold-inducible type A ARABIDOPSIS RESPONSE REGULATORS (ARRs) play roles in cold signaling. However, the roles of type B ARRs and ARABIDOPSIS HISTIDINE PHOSPHOTRANSFER PROTEINS (AHPs) have not been investigated in cold signaling. Here, we show that ARR1 and AHP2, AHP3, and AHP5 play positive roles in the cold-inducible expression of type A ARRs. arr1 mutants showed greatly reduced cold-responsive expression of type A ARRs compared with the wild type, whereas ARR1-overexpressing Arabidopsis exhibited the hypersensitive cold response of type A ARRs as well as enhanced freezing tolerance with cytokinin, suggesting that ARR1 functions as a positive factor of cold signaling. Transgenic Arabidopsis expressing ARR1ΔDDK:GR lacking the amino-terminal receiver domain showed wild-type expression levels of type A ARRs in response to cold, indicating that the signal receiver domain of ARR1 might be important for cold-responsive expression of type A ARRs. ahp2 ahp3 ahp5 triple mutations greatly reduced type A ARR expression in response to cold, whereas the single or double ahp mutants displayed wild-type levels of ARR expression, suggesting that AHP2, AHP3, and AHP5 are redundantly involved in cold signaling. Taken together, these results suggest that ARR1 mediates cold signal via AHP2, AHP3, or AHP5 from AHK2 and AHK3 to express type A ARRs. We further identified a cold transcriptome affected by ahk2 ahk3 mutations by microarray analysis, revealing a new cold-responsive gene network regulated downstream of AHK2 and AHK3. PMID:23124324

Progressive Transverse Microtubule Array Organization in Hormone-Induced Arabidopsis Hypocotyl Cells[W

PubMed Central

Vineyard, Laura; Elliott, Andrew; Dhingra, Sonia; Lucas, Jessica R.; Shaw, Sidney L.

2013-01-01

The acentriolar cortical microtubule arrays in dark-grown hypocotyl cells organize into a transverse coaligned pattern that is critical for axial plant growth. In light-grown Arabidopsis thaliana seedlings, the cortical array on the outer (periclinal) cell face creates a variety of array patterns with a significant bias (>3:1) for microtubules polymerizing edge-ward and into the side (anticlinal) faces of the cell. To study the mechanisms required for creating the transverse coalignment, we developed a dual-hormone protocol that synchronously induces ∼80% of the light-grown hypocotyl cells to form transverse arrays over a 2-h period. Repatterning occurred in two phases, beginning with an initial 30 to 40% decrease in polymerizing plus ends prior to visible changes in the array pattern. Transverse organization initiated at the cell’s midzone by 45 min after induction and progressed bidirectionally toward the apical and basal ends of the cell. Reorganization corrected the edge-ward bias in polymerization and proceeded without transiting through an obligate intermediate pattern. Quantitative comparisons of uninduced and induced microtubule arrays showed a limited deconstruction of the initial periclinal array followed by a progressive array reorganization to transverse coordinated between the anticlinal and periclinal cell faces. PMID:23444330

Physical Interaction of Floral Organs Controls Petal Morphogenesis in Arabidopsis1[W][OA

PubMed Central

Takeda, Seiji; Iwasaki, Akira; Matsumoto, Noritaka; Uemura, Tomohiro; Tatematsu, Kiyoshi; Okada, Kiyotaka

2013-01-01

Flowering plants bear beautiful flowers to attract pollinators. Petals are the most variable organs in flowering plants, with their color, fragrance, and shape. In Arabidopsis (Arabidopsis thaliana), petal primordia arise at a similar time to stamen primordia and elongate at later stages through the narrow space between anthers and sepals. Although many of the genes involved in regulating petal identity and primordia growth are known, the molecular mechanism for the later elongation process remains unknown. We found a mutant, folded petals1 (fop1), in which normal petal development is inhibited during their growth through the narrow space between sepals and anthers, resulting in formation of folded petals at maturation. During elongation, the fop1 petals contact the sepal surface at several sites. The conical-shaped petal epidermal cells are flattened in the fop1 mutant, as if they had been pressed from the top. Surgical or genetic removal of sepals in young buds restores the regular growth of petals, suggesting that narrow space within a bud is the cause of petal folding in the fop1 mutant. FOP1 encodes a member of the bifunctional wax ester synthase/diacylglycerol acyltransferase family, WSD11, which is expressed in elongating petals and localized to the plasma membrane. These results suggest that the FOP1/WSD11 products synthesized in the petal epidermis may act as a lubricant, enabling uninhibited growth of the petals as they extend between the sepals and the anthers. PMID:23314942

Arabidopsis MALE STERILITY1 Encodes a PHD-Type Transcription Factor and Regulates Pollen and Tapetum Development[W

PubMed Central

Ito, Takuya; Nagata, Noriko; Yoshiba, Yoshu; Ohme-Takagi, Masaru; Ma, Hong; Shinozaki, Kazuo

2007-01-01

The Arabidopsis thaliana MALE STERILITY1 (MS1) gene encodes a nuclear protein with Leu zipper–like and PHD-finger motifs and is important for postmeiotic pollen development. Here, we examined MS1 function using both cell biological and molecular biological approaches. We introduced a fusion construct of MS1 and a transcriptional repression domain (MS1-SRDX) into wild-type Arabidopsis, and the transgenic plants showed a semisterile phenotype similar to that of ms1. Since the repression domain can convert various kinds of transcriptional activators to dominant repressors, this suggested that MS1 functioned as a transcriptional activator. The Leu zipper–like region and the PHD motif were required for the MS1 function. Phenotypic analysis of the ms1 mutant and the MS1-SRDX transgenic Arabidopsis indicated that MS1 was involved in formation of pollen exine and pollen cytosolic components as well as tapetum development. Next, we searched for MS1 downstream genes by analyzing publicly available microarray data and identified 95 genes affected by MS1. Using a transgenic ms1 plant showing dexamethasone-inducible recovery of fertility, we further examined whether these genes were immediately downstream of MS1. From these results, we discuss a role of MS1 in pollen and tapetum development and the conservation of MS1 function in flowering plants. PMID:18032630

A Diffusible Signal from Arbuscular Mycorrhizal Fungi Elicits a Transient Cytosolic Calcium Elevation in Host Plant Cells1[W

PubMed Central

Navazio, Lorella; Moscatiello, Roberto; Genre, Andrea; Novero, Mara; Baldan, Barbara; Bonfante, Paola; Mariani, Paola

2007-01-01

The implication of calcium as intracellular messenger in the arbuscular mycorrhizal (AM) symbiosis has not yet been directly demonstrated, although often envisaged. We used soybean (Glycine max) cell cultures stably expressing the bioluminescent Ca2+ indicator aequorin to detect intracellular Ca2+ changes in response to the culture medium of spores of Gigaspora margarita germinating in the absence of the plant partner. Rapid and transient elevations in cytosolic free Ca2+ were recorded, indicating that diffusible molecules released by the mycorrhizal fungus are perceived by host plant cells through a Ca2+-mediated signaling. Similar responses were also triggered by two Glomus isolates. The fungal molecules active in generating the Ca2+ transient were constitutively released in the medium, and the induced Ca2+ signature was not modified by the coculture of germinating spores with plant cells. Even ungerminated spores were able to generate the signaling molecules, as proven when the germination was blocked by a low temperature. The fungal molecules were found to be stable to heat treatment, of small molecular mass (<3 kD), and, on the basis of extraction with an organic solvent, partially lipophilic. Evidence for the specificity of such an early fungal signal to the AM symbiosis is suggested by the lack of a Ca2+ response in cultured cells of the nonhost plant Arabidopsis (Arabidopsis thaliana) and by the up-regulation in soybean cells of genes related to Medicago truncatula DMI1, DMI2, and DMI3 and considered essential for the establishment of the AM symbiosis. PMID:17142489

Isolation of plasmodesmata from Arabidopsis suspension culture cells.

PubMed

Grison, Magali S; Fernandez-Calvino, Lourdes; Mongrand, Sébastien; Bayer, Emmanuelle M F

2015-01-01

Due to their position firmly anchored within the plant cell wall, plasmodesmata (PD) are notoriously difficult to isolate from plant tissue. Yet, getting access to isolated PD represents the most straightforward strategy for the identification of their molecular components. Proteomic and lipidomic analyses of such PD fractions have provided and will continue to provide critical information on the functional and structural elements that define these membranous nano-pores. Here, we describe a two-step simple purification procedure that allows isolation of pure PD-derived membranes from Arabidopsis suspension cells. The first step of this procedure consists in isolating cell wall fragments containing intact PD while free of contamination from other cellular compartments. The second step relies on an enzymatic degradation of the wall matrix and the subsequent release of "free" PD. Isolated PD membranes provide a suitable starting material for the analysis of PD-associated proteins and lipids.

Systematic Analysis of Arabidopsis Organelles and a Protein Localization Database for Facilitating Fluorescent Tagging of Full-Length Arabidopsis Proteins1[W

PubMed Central

Li, Shijun; Ehrhardt, David W.; Rhee, Seung Y.

2006-01-01

Cells are organized into a complex network of subcellular compartments that are specialized for various biological functions. Subcellular location is an important attribute of protein function. To facilitate systematic elucidation of protein subcellular location, we analyzed experimentally verified protein localization data of 1,300 Arabidopsis (Arabidopsis thaliana) proteins. The 1,300 experimentally verified proteins are distributed among 40 different compartments, with most of the proteins localized to four compartments: mitochondria (36%), nucleus (28%), plastid (17%), and cytosol (13.3%). About 19% of the proteins are found in multiple compartments, in which a high proportion (36.4%) is localized to both cytosol and nucleus. Characterization of the overrepresented Gene Ontology molecular functions and biological processes suggests that the Golgi apparatus and peroxisome may play more diverse functions but are involved in more specialized processes than other compartments. To support systematic empirical determination of protein subcellular localization using a technology called fluorescent tagging of full-length proteins, we developed a database and Web application to provide preselected green fluorescent protein insertion position and primer sequences for all Arabidopsis proteins to study their subcellular localization and to store experimentally verified protein localization images, videos, and their annotations of proteins generated using the fluorescent tagging of full-length proteins technology. The database can be searched, browsed, and downloaded using a Web browser at http://aztec.stanford.edu/gfp/. The software can also be downloaded from the same Web site for local installation. PMID:16617091

Zinc tolerance and accumulation in stable cell suspension cultures and in vitro regenerated plants of the emerging model plant Arabidopsis halleri (Brassicaceae).

PubMed

Vera-Estrella, Rosario; Miranda-Vergara, Maria Cristina; Barkla, Bronwyn J

2009-03-01

Arabidopsis halleri is increasingly employed as a model plant for studying heavy metal hyperaccumulation. With the aim of providing valuable tools for studies on cellular physiology and molecular biology of metal tolerance and transport, this study reports the development of successful and highly efficient methods for the in vitro regeneration of A. halleri plants and production of stable cell suspension lines. Plants were regenerated from leaf explants of A. halleri via a three-step procedure: callus induction, somatic embryogenesis and shoot development. Efficiency of callus proliferation and regeneration depended on the initial callus induction media and was optimal in the presence of 1 mg L(-1) 2,4-dichlorophenoxyacetic acid, and 0.05 mg L(-1) benzylaminopurine. Subsequent shoot and root regeneration from callus initiated under these conditions reached levels of 100% efficiency. High friability of the callus supported the development of cell suspension cultures with minimal cellular aggregates. Characterization of regenerated plants and cell cultures determined that they maintained not only the zinc tolerance and requirement of the whole plant but also the ability to accumulate zinc; with plants accumulating up to 50.0 micromoles zinc g(-1) FW, and cell suspension cultures 30.9 micromoles zinc g(-1) DW. Together this work will provide the experimental basis for furthering our knowledge of A. halleri as a model heavy metal hyperaccumulating plant.

Dissecting Arabidopsis Gβ Signal Transduction on the Protein Surface1[W][OA

PubMed Central

Jiang, Kun; Frick-Cheng, Arwen; Trusov, Yuri; Delgado-Cerezo, Magdalena; Rosenthal, David M.; Lorek, Justine; Panstruga, Ralph; Booker, Fitzgerald L.; Botella, José Ramón; Molina, Antonio; Ort, Donald R.; Jones, Alan M.

2012-01-01

The heterotrimeric G-protein complex provides signal amplification and target specificity. The Arabidopsis (Arabidopsis thaliana) Gβ-subunit of this complex (AGB1) interacts with and modulates the activity of target cytoplasmic proteins. This specificity resides in the structure of the interface between AGB1 and its targets. Important surface residues of AGB1, which were deduced from a comparative evolutionary approach, were mutated to dissect AGB1-dependent physiological functions. Analysis of the capacity of these mutants to complement well-established phenotypes of Gβ-null mutants revealed AGB1 residues critical for specific AGB1-mediated biological processes, including growth architecture, pathogen resistance, stomata-mediated leaf-air gas exchange, and possibly photosynthesis. These findings provide promising new avenues to direct the finely tuned engineering of crop yield and traits. PMID:22570469

Ethylene Upregulates Auxin Biosynthesis in Arabidopsis Seedlings to Enhance Inhibition of Root Cell Elongation[W

PubMed Central

Swarup, Ranjan; Perry, Paula; Hagenbeek, Dik; Van Der Straeten, Dominique; Beemster, Gerrit T.S.; Sandberg, Göran; Bhalerao, Rishikesh; Ljung, Karin; Bennett, Malcolm J.

2007-01-01

Ethylene represents an important regulatory signal for root development. Genetic studies in Arabidopsis thaliana have demonstrated that ethylene inhibition of root growth involves another hormone signal, auxin. This study investigated why auxin was required by ethylene to regulate root growth. We initially observed that ethylene positively controls auxin biosynthesis in the root apex. We subsequently demonstrated that ethylene-regulated root growth is dependent on (1) the transport of auxin from the root apex via the lateral root cap and (2) auxin responses occurring in multiple elongation zone tissues. Detailed growth studies revealed that the ability of the ethylene precursor 1-aminocyclopropane-1-carboxylic acid to inhibit root cell elongation was significantly enhanced in the presence of auxin. We conclude that by upregulating auxin biosynthesis, ethylene facilitates its ability to inhibit root cell expansion. PMID:17630275

Dual Roles of Reactive Oxygen Species and NADPH Oxidase RBOHD in an Arabidopsis-Alternaria Pathosystem1[W

PubMed Central

Pogány, Miklós; von Rad, Uta; Grün, Sebastian; Dongó, Anita; Pintye, Alexandra; Simoneau, Philippe; Bahnweg, Günther; Kiss, Levente; Barna, Balázs; Durner, Jörg

2009-01-01

Arabidopsis (Arabidopsis thaliana) NADPH oxidases have been reported to suppress the spread of pathogen- and salicylic acid-induced cell death. Here, we present dual roles of RBOHD (for respiratory burst oxidase homolog D) in an Arabidopsis-Alternaria pathosystem, suggesting either initiation or prevention of cell death dependent on the distance from pathogen attack. Our data demonstrate that a rbohD knockout mutant exhibits increased spread of cell death at the macroscopic level upon inoculation with the fungus Alternaria brassicicola. However, the cellular patterns of reactive oxygen species accumulation and cell death are fundamentally different in the AtrbohD mutant compared with the wild type. Functional RBOHD causes marked extracellular hydrogen peroxide accumulation as well as cell death in distinct, single cells of A. brassicicola-infected wild-type plants. This single cell response is missing in the AtrbohD mutant, where infection triggers spreading-type necrosis preceded by less distinct chloroplastic hydrogen peroxide accumulation in large clusters of cells. While the salicylic acid analog benzothiadiazole induces the action of RBOHD and the development of cell death in infected tissues, the ethylene inhibitor aminoethoxyvinylglycine inhibits cell death, indicating that both salicylic acid and ethylene positively regulate RBOHD and cell death. Moreover, A. brassicicola-infected AtrbohD plants hyperaccumulate ethylene and free salicylic acid compared with the wild type, suggesting negative feedback regulation of salicylic acid and ethylene by RBOHD. We propose that functional RBOHD triggers death in cells that are damaged by fungal infection but simultaneously inhibits death in neighboring cells through the suppression of free salicylic acid and ethylene levels. PMID:19726575

Target of Rapamycin Regulates Development and Ribosomal RNA Expression through Kinase Domain in Arabidopsis1[W][OA

PubMed Central

Ren, Maozhi; Qiu, Shuqing; Venglat, Prakash; Xiang, Daoquan; Feng, Li; Selvaraj, Gopalan; Datla, Raju

2011-01-01

Target of rapamycin (TOR) is a central regulator of cell growth, cell death, nutrition, starvation, hormone, and stress responses in diverse eukaryotes. However, very little is known about TOR signaling and the associated functional domains in plants. We have taken a genetic approach to dissect TOR functions in Arabidopsis (Arabidopsis thaliana) and report here that the kinase domain is essential for the role of TOR in embryogenesis and 45S rRNA expression. Twelve new T-DNA insertion mutants, spanning 14.2 kb of TOR-encoding genomic region, have been characterized. Nine of these share expression of defective kinase domain and embryo arrest at 16 to 32 cell stage. However, three T-DNA insertion lines affecting FATC domain displayed normal embryo development, indicating that FATC domain was dispensable in Arabidopsis. Genetic complementation showed that the TOR kinase domain alone in tor-10/tor-10 mutant background can rescue early embryo lethality and restore normal development. Overexpression of full-length TOR or kinase domain in Arabidopsis displayed developmental abnormalities in meristem, leaf, root, stem, flowering time, and senescence. We further show that TOR, especially the kinase domain, plays a role in ribosome biogenesis by activating 45S rRNA production. Of the six putative nuclear localization sequences in the kinase domain, nuclear localization sequence 6 was identified to confer TOR nuclear targeting in transient expression assays. Chromatin immunoprecipitation studies revealed that the HEAT repeat domain binds to 45S rRNA promoter and the 5′ external transcribed spacer elements motif. Together, these results show that TOR controls the embryogenesis, postembryonic development, and 45S rRNA production through its kinase domain in Arabidopsis. PMID:21266656

Apyrase (Nucleoside Triphosphate-Diphosphohydrolase) and Extracellular Nucleotides Regulate Cotton Fiber Elongation in Cultured Ovules1[W][OA

PubMed Central

Clark, Greg; Torres, Jonathan; Finlayson, Scott; Guan, Xueying; Handley, Craig; Lee, Jinsuk; Kays, Julia E.; Chen, Z. Jeffery; Roux, Stanley J.

2010-01-01

Ectoapyrase enzymes remove the terminal phosphate from extracellular nucleoside tri- and diphosphates. In Arabidopsis (Arabidopsis thaliana), two ectoapyrases, AtAPY1 and AtAPY2, have been implicated as key modulators of growth. In fibers of cotton (Gossypium hirsutum), transcript levels for GhAPY1 and GhAPY2, two closely related ectoapyrases that have high sequence similarity to AtAPY1 and AtAPY2, are up-regulated when fibers enter their rapid growth phase. In an ovule culture system, fibers release ATP as they grow, and when their ectoapyrase activity is blocked by the addition of polyclonal anti-apyrase antibodies or by two different small molecule inhibitors, the medium ATP level rises and fiber growth is suppressed. High concentrations of the poorly hydrolyzable nucleotides ATPγS and ADPβS applied to the medium inhibit fiber growth, and low concentrations of them stimulate growth, but treatment with adenosine 5′-O-thiomonophosphate causes no change in the growth rate. Both the inhibition and stimulation of growth by applied nucleotides can be blocked by an antagonist that blocks purinoceptors in animal cells, and by adenosine. Treatment of cotton ovule cultures with ATPγS induces increased levels of ethylene, and two ethylene antagonists, aminovinylglycine and silver nitrate, block both the growth stimulatory and growth inhibitory effects of applied nucleotides. In addition, the ethylene precursor, 1-aminocyclopropane-1-carboxylic acid, lowers the concentration of nucleotide needed to promote fiber growth. These data indicate that ectoapyrases and extracellular nucleotides play a significant role in regulating cotton fiber growth and that ethylene is a likely downstream component of the signaling pathway. PMID:20018604

Adenosine Kinase Modulates Root Gravitropism and Cap Morphogenesis in Arabidopsis1[W][OA

PubMed Central

Young, Li-Sen; Harrison, Benjamin R.; U.M., Narayana Murthy; Moffatt, Barbara A.; Gilroy, Simon; Masson, Patrick H.

2006-01-01

Adenosine kinase (ADK) is a key enzyme that regulates intra- and extracellular levels of adenosine, thereby modulating methyltransferase reactions, production of polyamines and secondary compounds, and cell signaling in animals. Unfortunately, little is known about ADK's contribution to the regulation of plant growth and development. Here, we show that ADK is a modulator of root cap morphogenesis and gravitropism. Upon gravistimulation, soluble ADK levels and activity increase in the root tip. Mutation in one of two Arabidopsis (Arabidopsis thaliana) ADK genes, ADK1, results in cap morphogenesis defects, along with alterations in root sensitivity to gravistimulation and slower kinetics of root gravitropic curvature. The kinetics defect can be partially rescued by adding spermine to the growth medium, whereas the defects in cap morphogenesis and gravitropic sensitivity cannot. The root morphogenesis and gravitropism defects of adk1-1 are accompanied by altered expression of the PIN3 auxin efflux facilitator in the cap and decreased expression of the auxin-responsive DR5-GUS reporter. Furthermore, PIN3 fails to relocalize to the bottom membrane of statocytes upon gravistimulation. Consequently, adk1-1 roots cannot develop a lateral auxin gradient across the cap, necessary for the curvature response. Interestingly, adk1-1 does not affect gravity-induced cytoplasmic alkalinization of the root statocytes, suggesting either that ADK1 functions between cytoplasmic alkalinization and PIN3 relocalization in a linear pathway or that the pH and PIN3-relocalization responses to gravistimulation belong to distinct branches of the pathway. Our data are consistent with a role for ADK and the S-adenosyl-l-methionine pathway in the control of root gravitropism and cap morphogenesis. PMID:16891550

Ethylene Inhibits Cell Proliferation of the Arabidopsis Root Meristem1[OPEN

PubMed Central

Street, Ian H.; Aman, Sitwat; Zubo, Yan; Ramzan, Aleena; Wang, Xiaomin; Shakeel, Samina N.; Kieber, Joseph J.; Schaller, G. Eric

2015-01-01

The root system of plants plays a critical role in plant growth and survival, with root growth being dependent on both cell proliferation and cell elongation. Multiple phytohormones interact to control root growth, including ethylene, which is primarily known for its role in controlling root cell elongation. We find that ethylene also negatively regulates cell proliferation at the root meristem of Arabidopsis (Arabidopsis thaliana). Genetic analysis indicates that the inhibition of cell proliferation involves two pathways operating downstream of the ethylene receptors. The major pathway is the canonical ethylene signal transduction pathway that incorporates CONSTITUTIVE TRIPLE RESPONSE1, ETHYLENE INSENSITIVE2, and the ETHYLENE INSENSITIVE3 family of transcription factors. The secondary pathway is a phosphorelay based on genetic analysis of receptor histidine kinase activity and mutants involving the type B response regulators. Analysis of ethylene-dependent gene expression and genetic analysis supports SHORT HYPOCOTYL2, a repressor of auxin signaling, as one mediator of the ethylene response and furthermore, indicates that SHORT HYPOCOTYL2 is a point of convergence for both ethylene and cytokinin in negatively regulating cell proliferation. Additional analysis indicates that ethylene signaling contributes but is not required for cytokinin to inhibit activity of the root meristem. These results identify key elements, along with points of cross talk with cytokinin and auxin, by which ethylene negatively regulates cell proliferation at the root apical meristem. PMID:26149574

The Cysteine Protease CEP1, a Key Executor Involved in Tapetal Programmed Cell Death, Regulates Pollen Development in Arabidopsis[W][OPEN

PubMed Central

Zhang, Dandan; Liu, Di; Lv, Xiaomeng; Wang, Ying; Xun, Zhili; Liu, Zhixiong; Li, Fenglan; Lu, Hai

2014-01-01

Tapetal programmed cell death (PCD) is a prerequisite for pollen grain development in angiosperms, and cysteine proteases are the most ubiquitous hydrolases involved in plant PCD. We identified a papain-like cysteine protease, CEP1, which is involved in tapetal PCD and pollen development in Arabidopsis thaliana. CEP1 is expressed specifically in the tapetum from stages 5 to 11 of anther development. The CEP1 protein first appears as a proenzyme in precursor protease vesicles and is then transported to the vacuole and transformed into the mature enzyme before rupture of the vacuole. cep1 mutants exhibited aborted tapetal PCD and decreased pollen fertility with abnormal pollen exine. A transcriptomic analysis revealed that 872 genes showed significantly altered expression in the cep1 mutants, and most of them are important for tapetal cell wall organization, tapetal secretory structure formation, and pollen development. CEP1 overexpression caused premature tapetal PCD and pollen infertility. ELISA and quantitative RT-PCR analyses confirmed that the CEP1 expression level showed a strong relationship to the degree of tapetal PCD and pollen fertility. Our results reveal that CEP1 is a crucial executor during tapetal PCD and that proper CEP1 expression is necessary for timely degeneration of tapetal cells and functional pollen formation. PMID:25035401

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

Arabidopsis Nitrate Transporter NRT1.9 Is Important in Phloem Nitrate Transport[W][OA

PubMed Central

Wang, Ya-Yun; Tsay, Yi-Fang

2011-01-01

This study of the Arabidopsis thaliana nitrate transporter NRT1.9 reveals an important function for a NRT1 family member in phloem nitrate transport. Functional analysis in Xenopus laevis oocytes showed that NRT1.9 is a low-affinity nitrate transporter. Green fluorescent protein and β-glucuronidase reporter analyses indicated that NRT1.9 is a plasma membrane transporter expressed in the companion cells of root phloem. In nrt1.9 mutants, nitrate content in root phloem exudates was decreased, and downward nitrate transport was reduced, suggesting that NRT1.9 may facilitate loading of nitrate into the root phloem and enhance downward nitrate transport in roots. Under high nitrate conditions, the nrt1.9 mutant showed enhanced root-to-shoot nitrate transport and plant growth. We conclude that phloem nitrate transport is facilitated by expression of NRT1.9 in root companion cells. In addition, enhanced root-to-shoot xylem transport of nitrate in nrt1.9 mutants points to a negative correlation between xylem and phloem nitrate transport. PMID:21571952

Endosome-Associated CRT1 Functions Early in Resistance Gene–Mediated Defense Signaling in Arabidopsis and Tobacco[W

PubMed Central

Kang, Hong-Gu; Oh, Chang-Sik; Sato, Masanao; Katagiri, Fumiaki; Glazebrook, Jane; Takahashi, Hideki; Kachroo, Pradeep; Martin, Gregory B.; Klessig, Daniel F.

2010-01-01

Resistance gene–mediated immunity confers protection against pathogen infection in a wide range of plants. A genetic screen for Arabidopsis thaliana mutants compromised for recognition of turnip crinkle virus previously identified CRT1, a member of the GHKL ATPase/kinase superfamily. Here, we demonstrate that CRT1 interacts with various resistance proteins from different structural classes, and this interaction is disrupted when these resistance proteins are activated. The Arabidopsis mutant crt1-2 crh1-1, which lacks CRT1 and its closest homolog, displayed compromised resistance to avirulent Pseudomonas syringae and Hyaloperonospora arabidopsidis. Additionally, resistance-associated hypersensitive cell death was suppressed in Nicotiana benthamiana silenced for expression of CRT1 homolog(s). Thus, CRT1 appears to be a general factor for resistance gene–mediated immunity. Since elevation of cytosolic calcium triggered by avirulent P. syringae was compromised in crt1-2 crh1-1 plants, but cell death triggered by Nt MEK2DD was unaffected in CRT1-silenced N. benthamiana, CRT1 likely functions at an early step in this pathway. Genome-wide transcriptome analysis led to identification of CRT1-Associated genes, many of which are associated with transport processes, responses to (a)biotic stress, and the endomembrane system. Confocal microscopy and subcellular fractionation revealed that CRT1 localizes to endosome-like vesicles, suggesting a key process in resistance protein activation/signaling occurs in this subcellular compartment. PMID:20332379

Genetic Resources for Maize Cell Wall Biology1[C][W][OA

PubMed Central

Penning, Bryan W.; Hunter, Charles T.; Tayengwa, Reuben; Eveland, Andrea L.; Dugard, Christopher K.; Olek, Anna T.; Vermerris, Wilfred; Koch, Karen E.; McCarty, Donald R.; Davis, Mark F.; Thomas, Steven R.; McCann, Maureen C.; Carpita, Nicholas C.

2009-01-01

Grass species represent a major source of food, feed, and fiber crops and potential feedstocks for biofuel production. Most of the biomass is contributed by cell walls that are distinct in composition from all other flowering plants. Identifying cell wall-related genes and their functions underpins a fundamental understanding of growth and development in these species. Toward this goal, we are building a knowledge base of the maize (Zea mays) genes involved in cell wall biology, their expression profiles, and the phenotypic consequences of mutation. Over 750 maize genes were annotated and assembled into gene families predicted to function in cell wall biogenesis. Comparative genomics of maize, rice (Oryza sativa), and Arabidopsis (Arabidopsis thaliana) sequences reveal differences in gene family structure between grass species and a reference eudicot species. Analysis of transcript profile data for cell wall genes in developing maize ovaries revealed that expression within families differed by up to 100-fold. When transcriptional analyses of developing ovaries before pollination from Arabidopsis, rice, and maize were contrasted, distinct sets of cell wall genes were expressed in grasses. These differences in gene family structure and expression between Arabidopsis and the grasses underscore the requirement for a grass-specific genetic model for functional analyses. A UniformMu population proved to be an important resource in both forward- and reverse-genetics approaches to identify hundreds of mutants in cell wall genes. A forward screen of field-grown lines by near-infrared spectroscopic screen of mature leaves yielded several dozen lines with heritable spectroscopic phenotypes. Pyrolysis-molecular beam mass spectrometry confirmed that several nir mutants had altered carbohydrate-lignin compositions. PMID:19926802

Singlet oxygen triggers chloroplast rupture and cell death in the zeaxanthin epoxidase defective mutant aba1 of Arabidopsis thaliana under high light stress.

PubMed

Sánchez-Corrionero, Álvaro; Sánchez-Vicente, Inmaculada; González-Pérez, Sergio; Corrales, Ascensión; Krieger-Liszkay, Anja; Lorenzo, Óscar; Arellano, Juan B

2017-09-01

The two Arabidopsis thaliana mutants, aba1 and max4, were previously identified as sharing a number of co-regulated genes with both the flu mutant and Arabidopsis cell suspension cultures exposed to high light (HL). On this basis, we investigated whether aba1 and max4 were generating high amounts of singlet oxygen ( 1 O 2 ) and activating 1 O 2 -mediated cell death. Thylakoids of aba1 produced twice as much 1 O 2 as thylakoids of max4 and wild type (WT) plants when illuminated with strong red light. 1 O 2 was measured using the spin probe 2,2,6,6-tetramethyl-4-piperidone hydrochloride. 77-K chlorophyll fluorescence emission spectra of thylakoids revealed lower aggregation of the light harvesting complex II in aba1. This was rationalized as a loss of connectivity between photosystem II (PSII) units and as the main cause for the high yield of 1 O 2 generation in aba1. Up-regulation of the 1 O 2 responsive gene AAA-ATPase was only observed with statistical significant in aba1 under HL. Two early jasmonate (JA)-responsive genes, JAZ1 and JAZ5, encoding for two repressor proteins involved in the negative feedback regulation of JA signalling, were not up-regulated to the WT plant levels. Chloroplast aggregation followed by chloroplast rupture and eventual cell death was observed by confocal imaging of the fluorescence emission of leaf cells of transgenic aba1 plants expressing the chimeric fusion protein SSU-GFP. Cell death was not associated with direct 1 O 2 cytotoxicity in aba1, but rather with a delayed stress response. In contrast, max4 did not show evidence of 1 O 2 -mediated cell death. In conclusion, aba1 may serve as an alternative model to other 1 O 2 -overproducing mutants of Arabidopsis for investigating 1 O 2 -mediated cell death. Copyright © 2017 Elsevier GmbH. All rights reserved.

AtFXG1, an Arabidopsis Gene Encoding α-l-Fucosidase Active against Fucosylated Xyloglucan Oligosaccharides1

PubMed Central

de la Torre, Francisco; Sampedro, Javier; Zarra, Ignacio; Revilla, Gloria

2002-01-01

An α-l-fucosidase (EC 3.2.1.51) able to release the t-fucosyl residue from the side chain of xyloglucan oligosaccharides has been detected in the leaves of Arabidopsis plants. Moreover, an α-l-fucosidase with similar substrate specificity was purified from cabbage (Brassica oleracea) leaves to render a single band on SDS-PAGE. Two peptide sequences were obtained from this protein band, and they were used to identify an Arabidopsis gene coding for an α-fucosidase that we propose to call AtFXG1. In addition, an Arabidopsis gene with homology with known α-l-fucosidases has been also found, and we proposed to name it as AtFUC1. Both AtFXG1 and ATFUC1 were heterologously expressed in Pichia pastoris cells and the α-l-fucosidase activities secreted to the culture medium. The α-l-fucosidase encoded by AtFXG1 was active against the oligosaccharides from xyloglucan XXFG as well as against 2′-fucosyl-lactitol but not against p-nitrophenyl-α-l-fucopyranoside. However, the AtFUC1 heterologously expressed was active only against 2′-fucosyl-lactitol. Thus, the former must be related to xyloglucan metabolism. PMID:11788770

Loss-of-Function Mutation of REDUCED WALL ACETYLATION2 in Arabidopsis Leads to Reduced Cell Wall Acetylation and Increased Resistance to Botrytis cinerea1[W][OA

PubMed Central

Manabe, Yuzuki; Nafisi, Majse; Verhertbruggen, Yves; Orfila, Caroline; Gille, Sascha; Rautengarten, Carsten; Cherk, Candice; Marcus, Susan E.; Somerville, Shauna; Pauly, Markus; Knox, J. Paul; Sakuragi, Yumiko; Scheller, Henrik Vibe

2011-01-01

Nearly all polysaccharides in plant cell walls are O-acetylated, including the various pectic polysaccharides and the hemicelluloses xylan, mannan, and xyloglucan. However, the enzymes involved in the polysaccharide acetylation have not been identified. While the role of polysaccharide acetylation in vivo is unclear, it is known to reduce biofuel yield from lignocellulosic biomass by the inhibition of microorganisms used for fermentation. We have analyzed four Arabidopsis (Arabidopsis thaliana) homologs of the protein Cas1p known to be involved in polysaccharide O-acetylation in Cryptococcus neoformans. Loss-of-function mutants in one of the genes, designated REDUCED WALL ACETYLATION2 (RWA2), had decreased levels of acetylated cell wall polymers. Cell wall material isolated from mutant leaves and treated with alkali released about 20% lower amounts of acetic acid when compared with the wild type. The same level of acetate deficiency was found in several pectic polymers and in xyloglucan. Thus, the rwa2 mutations affect different polymers to the same extent. There were no obvious morphological or growth differences observed between the wild type and rwa2 mutants. However, both alleles of rwa2 displayed increased tolerance toward the necrotrophic fungal pathogen Botrytis cinerea. PMID:21212300

RanGAP1 is a continuous marker of the Arabidopsis cell division plane

PubMed Central

Xu, Xianfeng Morgan; Zhao, Qiao; Rodrigo-Peiris, Thushani; Brkljacic, Jelena; He, Chao Sylvia; Müller, Sabine; Meier, Iris

2008-01-01

In higher plants, the plane of cell division is faithfully predicted by the preprophase band (PPB). The PPB, a cortical ring of microtubules and F-actin, disassembles upon nuclear-envelope breakdown. During cytokinesis, the expanding cell plate fuses with the plasma membrane at the cortical division site, the site of the former PPB. The nature of the “molecular memory” that is left behind by the PPB and is proposed to guide the cell plate to the cortical division site is unknown. RanGAP is the GTPase activating protein of the small GTPase Ran, which provides spatial information for nucleocytoplasmic transport and various mitotic processes in animals. Here, we show that, in dividing root cells, Arabidopsis RanGAP1 concentrates at the PPB and remains associated with the cortical division site during mitosis and cytokinesis, requiring its N-terminal targeting domain. In a fass/ton2 mutant, which affects PPB formation, RanGAP1 recruitment to the PPB site is lost, while its PPB retention is microtubule-independent. RanGAP1 persistence at the cortical division site, but not its initial accumulation at the PPB requires the 2 cytokinesis-regulating kinesins POK1 and POK2. Depletion of RanGAP by inducible RNAi leads to oblique cell walls and cell-wall stubs in root cell files, consistent with cytokinesis defects. We propose that Arabidopsis RanGAP, a continuous positive protein marker of the plant division plane, has a role in spatial signaling during plant cell division. PMID:19011093

Lack of α-Xylosidase Activity in Arabidopsis Alters Xyloglucan Composition and Results in Growth Defects1[W][OA

PubMed Central

Sampedro, Javier; Pardo, Brenda; Gianzo, Cristina; Guitián, Esteban; Revilla, Gloria; Zarra, Ignacio

2010-01-01

Xyloglucan is the main hemicellulose in the primary cell walls of most seed plants and is thought to play a role in regulating the separation of cellulose microfibrils during growth. Xylose side chains block the degradation of the backbone, and α-xylosidase activity is necessary to remove them. Two Arabidopsis (Arabidopsis thaliana) mutant lines with insertions in the α-xylosidase gene AtXYL1 were characterized in this work. Both lines showed a reduction to undetectable levels of α-xylosidase activity against xyloglucan oligosaccharides. This reduction resulted in the accumulation of XXXG and XXLG in the liquid growth medium of Atxyl1 seedlings. The presence of XXLG suggests that it is a poor substrate for xyloglucan β-galactosidase. In addition, the polymeric xyloglucan of Atxyl1 lines was found to be enriched in XXLG subunits, with a concomitant decrease in XXFG and XLFG. This change can be explained by extensive exoglycosidase activity at the nonreducing ends of xyloglucan chains. These enzymes could thus have a larger role than previously thought in the metabolism of xyloglucan. Finally, Atxyl1 lines showed a reduced ability to control the anisotropic growth pattern of different organs, pointing to the importance of xyloglucan in this process. The promoter of AtXYL1 was shown to direct expression to many different organs and cell types undergoing cell wall modifications, including trichomes, vasculature, stomata, and elongating anther filaments. PMID:20801759

Suberin-Associated Fatty Alcohols in Arabidopsis: Distributions in Roots and Contributions to Seed Coat Barrier Properties1[W

PubMed Central

Vishwanath, Sollapura J.; Kosma, Dylan K.; Pulsifer, Ian P.; Scandola, Sabine; Pascal, Stéphanie; Joubès, Jérôme; Dittrich-Domergue, Franziska; Lessire, René; Rowland, Owen; Domergue, Frédéric

2013-01-01

Suberin is found in a variety of tissues, such as root endoderms and periderms, storage tuber periderms, tree cork layer, and seed coats. It acts as a hydrophobic barrier to control the movement of water, gases, and solutes as well as an antimicrobial barrier. Suberin consists of polymerized phenolics, glycerol, and a variety of fatty acid derivatives, including primary fatty alcohols. We have conducted an in-depth analysis of the distribution of the C18:0 to C22:0 fatty alcohols in Arabidopsis (Arabidopsis thaliana) roots and found that only 20% are part of the root suberin polymer, together representing about 5% of its aliphatic monomer composition, while the remaining 80% are found in the nonpolymeric (soluble) fraction. Down-regulation of Arabidopsis FATTY ACYL REDUCTASE1 (FAR1), FAR4, and FAR5, which collectively produce the fatty alcohols found in suberin, reduced their levels by 70% to 80% in (1) the polymeric and nonpolymeric fractions from roots of tissue culture-grown plants, (2) the suberin-associated root waxes from 7-week-old soil-grown plants, and (3) the seed coat suberin polymer. By contrast, the other main monomers of suberin were not altered, indicating that reduced levels of fatty alcohols did not influence the suberin polymerization process. Nevertheless, the 75% reduction in total fatty alcohol and diol loads in the seed coat resulted in increased permeability to tetrazolium salts and a higher sensitivity to abscisic acid. These results suggest that fatty alcohols and diols play an important role in determining the functional properties of the seed coat suberin barrier. PMID:24019425

Hypocotyl Directional Growth in Arabidopsis: A Complex Trait1[W][OA

PubMed Central

Gupta, Aditi; Singh, Manjul; Jones, Alan M.; Laxmi, Ashverya

2012-01-01

The growth direction of the Arabidopsis (Arabidopsis thaliana) etiolated-seedling hypocotyl is a complex trait that is controlled by extrinsic signals such as gravity and touch as well as intrinsic signals such as hormones (brassinosteroid [BR], auxin, cytokinin, ethylene) and nutrient status (glucose [Glc], sucrose). We used a genetic approach to identify the signaling elements and their relationship underlying hypocotyl growth direction. BR randomizes etiolated-seedling growth by inhibiting negative gravitropism of the hypocotyls via modulating auxin homeostasis for which we designate as reset, not to be confused with the gravity set point angle. Cytokinin signaling antagonizes this BR reset of gravity sensing and/or tropism by affecting ethylene biosynthesis/signaling. Glc also antagonizes BR reset but acts independently of cytokinin and ethylene signaling pathways via inhibiting BR-regulated gene expression quantitatively and spatially, by altering protein degradation, and by antagonizing BR-induced changes in microtubule organization and cell patterning associated with hypocotyl agravitropism. This BR reset is reduced in the presence of the microtubule organization inhibitor oryzalin, suggesting a central role for cytoskeleton reorganization. A unifying and hierarchical model of Glc and hormone signaling interplay is proposed. The biological significance of BR-mediated changes in hypocotyl graviresponse lies in the fact that BR signaling sensitizes the dark-grown seedling hypocotyl to the presence of obstacles, overriding gravitropism, to enable efficient circumnavigation through soil. PMID:22689891

Expression of genomic AtCYCD2;1 in Arabidopsis induces cell division at smaller cell sizes: implications for the control of plant growth.

PubMed

Qi, Ruhu; John, Peter Crook Lloyd

2007-07-01

The Arabidopsis (Arabidopsis thaliana) CYCD2;1 gene introduced in genomic form increased cell formation in the Arabidopsis root apex and leaf, while generating full-length mRNA, raised CDK/CYCLIN enzyme activity, reduced G1-phase duration, and reduced size of cells at S phase and division. Other cell cycle genes, CDKA;1, CYCLIN B;1, and the cDNA form of CYCD2;1 that produced an aberrantly spliced mRNA, produced smaller or zero increases in CDK/CYCLIN activity and did not increase the number of cells formed. Plants with a homozygous single insert of genomic CYCD2;1 grew with normal morphology and without accelerated growth of root or shoot, not providing evidence that cell formation or CYCLIN D2 controls growth of postembryonic vegetative tissues. At the root apex, cells progressed normally from meristem to elongation, but their smaller size enclosed less growth and a 40% reduction in final size of epidermal and cortical cells was seen. Smaller elongated cell size inhibited endoreduplication, indicating a cell size requirement. Leaf cells were also smaller and more numerous during proliferation and epidermal pavement and palisade cells attained 59% and 69% of controls, whereas laminas reached normal size. Autonomous control of expansion was therefore not evident in abundant cell types that formed tissues of root or leaf. Cell size was reduced by a greater number formed in a tissue prior to cell and tissue expansion. Initiation and termination of expansion did not correlate with cell dimension or number and may be determined by tissue-wide signals acting across cellular boundaries.

The Transcription Factor WIN1/SHN1 Regulates Cutin Biosynthesis in Arabidopsis thaliana[W

PubMed Central

Kannangara, Rubini; Branigan, Caroline; Liu, Yan; Penfield, Teresa; Rao, Vijaya; Mouille, Grégory; Höfte, Herman; Pauly, Markus; Riechmann, José Luis; Broun, Pierre

2007-01-01

The composition and permeability of the cuticle has a large influence on its ability to protect the plant against various forms of biotic and abiotic stress. WAX INDUCER1 (WIN1) and related transcription factors have recently been shown to trigger wax production, enhance drought tolerance, and modulate cuticular permeability when overexpressed in Arabidopsis thaliana. We found that WIN1 influences the composition of cutin, a polyester that forms the backbone of the cuticle. WIN1 overexpression induces compositional changes and an overall increase in cutin production in vegetative and reproductive organs, while its downregulation has the opposite effect. Changes in cutin composition are preceded by the rapid and coordinated induction of several genes known or likely to be involved in cutin biosynthesis. This transcriptional response is followed after a delay by the induction of genes associated with wax biosynthesis, suggesting that the regulation of cutin and wax production by WIN1 is a two-step process. We demonstrate that at least one of the cutin pathway genes, which encodes long-chain acyl-CoA synthetase LACS2, is likely to be directly targeted by WIN1. Overall, our results suggest that WIN1 modulates cuticle permeability in Arabidopsis by regulating genes encoding cutin pathway enzymes. PMID:17449808

Methylation of Gibberellins by Arabidopsis GAMT1 and GAMT2[W

PubMed Central

Varbanova, Marina; Yamaguchi, Shinjiro; Yang, Yue; McKelvey, Katherine; Hanada, Atsushi; Borochov, Roy; Yu, Fei; Jikumaru, Yusuke; Ross, Jeannine; Cortes, Diego; Ma, Choong Je; Noel, Joseph P.; Mander, Lew; Shulaev, Vladimir; Kamiya, Yuji; Rodermel, Steve; Weiss, David; Pichersky, Eran

2007-01-01

Arabidopsis thaliana GAMT1 and GAMT2 encode enzymes that catalyze formation of the methyl esters of gibberellins (GAs). Ectopic expression of GAMT1 or GAMT2 in Arabidopsis, tobacco (Nicotiana tabacum), and petunia (Petunia hybrida) resulted in plants with GA deficiency and typical GA deficiency phenotypes, such as dwarfism and reduced fertility. GAMT1 and GAMT2 are both expressed mainly in whole siliques (including seeds), with peak transcript levels from the middle until the end of silique development. Within whole siliques, GAMT2 was previously shown to be expressed mostly in developing seeds, and we show here that GAMT1 expression is also localized mostly to seed, suggesting a role in seed development. Siliques of null single GAMT1 and GAMT2 mutants accumulated high levels of various GAs, with particularly high levels of GA1 in the double mutant. Methylated GAs were not detected in wild-type siliques, suggesting that methylation of GAs by GAMT1 and GAMT2 serves to deactivate GAs and initiate their degradation as the seeds mature. Seeds of homozygous GAMT1 and GAMT2 null mutants showed reduced inhibition of germination, compared with the wild type, when placed on plates containing the GA biosynthesis inhibitor ancymidol, with the double mutant showing the least inhibition. These results suggest that the mature mutant seeds contained higher levels of active GAs than wild-type seeds. PMID:17220201

Increasing cellular level of phosphatidic acid enhances FGF-1 production in long term-cultured rat astrocytes.

PubMed

Nagayasu, Yuko; Morita, Shin-Ya; Hayashi, Hideki; Miura, Yutaka; Yokoyama, Kazuki; Michikawa, Makoto; Ito, Jin-Ichi

2014-05-14

We found in a previous study that both mRNA expression and release of fibroblast growth factor 1 (FGF-1) are greater in rat astrocytes that are long term-cultured for one month (W/M cells) than in the cells cultured for one week (W/W cells). However, FGF-1 does not enhance phosphorylation of Akt, MEK, and ERK in W/M cells, while it does in W/W cells. In this work we studied the mechanism to cause these differences between W/W and W/M cells in culture. As it is known that long term culture generates oxidative stress, we characterized the stresses which W/M cells undergo in comparison with W/W cells. The levels of superoxide dismutase 1 (SOD1) and mitochondrial Bax were higher in W/M cells than in W/W cells. W/M cells recovered their ability to respond to FGF-1 to enhance phosphorylation of Akt, MEK, and ERK in the presence of antioxidants. Oxidative stress induced by hydrogen peroxide (H2O2) had no effect on mRNA expression of FGF-1 in W/W cells, although H2O2 enhances release of FGF-1 from W/W cells without inducing apoptosis. The influence of cell density was studied on mRNA expression of FGF-1 and cellular response to FGF-1, as an increasing cell density is observed in W/M cells. The increasing cell density enhanced mRNA expression of FGF-1 in W/W cells without suppression of responses to FGF-1. The decrease in cell density lowered the FGF-1 mRNA expression in W/M cells without recovery of the response to FGF-1 to enhance phosphorylation of Akt, MEK, and ERK. These findings suggest that oxidative stress attenuate sensitivity to FGF-1 and higher cell density may enhance FGF-1 expression in W/M cells. In addition, we found that the cellular level of phosphatidic acid (PA) increased in H2O2-treated W/W and W/M cells and decreased by the treatment with antioxidants, and that PA enhances the mRNA expression of FGF-1 in the W/W cells. These findings suggest that the increasing PA production may enhance FGF-1 expression to protect astrocytes against oxidative stress

Asymmetric leaves1 mediates leaf patterning and stem cell function in Arabidopsis.

PubMed

Byrne, M E; Barley, R; Curtis, M; Arroyo, J M; Dunham, M; Hudson, A; Martienssen, R A

Meristem function in plants requires both the maintenance of stem cells and the specification of founder cells from which lateral organs arise. Lateral organs are patterned along proximodistal, dorsoventral and mediolateral axes. Here we show that the Arabidopsis mutant asymmetric leaves1 (as1) disrupts this process. AS1 encodes a myb domain protein, closely related to PHANTASTICA in Antirrhinum and ROUGH SHEATH2 in maize, both of which negatively regulate knotted-class homeobox genes. AS1 negatively regulates the homeobox genes KNAT1 and KNAT2 and is, in turn, negatively regulated by the meristematic homeobox gene SHOOT MERISTEMLESS. This genetic pathway defines a mechanism for differentiating between stem cells and organ founder cells within the shoot apical meristem and demonstrates that genes expressed in organ primordia interact with meristematic genes to regulate shoot morphogenesis.

Profilin-Dependent Nucleation and Assembly of Actin Filaments Controls Cell Elongation in Arabidopsis1[OPEN

PubMed Central

Cao, Lingyan; Blanchoin, Laurent; Staiger, Christopher J.

2016-01-01

Actin filaments in plant cells are incredibly dynamic; they undergo incessant remodeling and assembly or disassembly within seconds. These dynamic events are choreographed by a plethora of actin-binding proteins, but the exact mechanisms are poorly understood. Here, we dissect the contribution of Arabidopsis (Arabidopsis thaliana) PROFILIN1 (PRF1), a conserved actin monomer-binding protein, to actin organization and single filament dynamics during axial cell expansion of living epidermal cells. We found that reduced PRF1 levels enhanced cell and organ growth. Surprisingly, we observed that the overall frequency of nucleation events in prf1 mutants was dramatically decreased and that a subpopulation of actin filaments that assemble at high rates was reduced. To test whether profilin cooperates with plant formin proteins to execute actin nucleation and rapid filament elongation in cells, we used a pharmacological approach. Here, we used Small Molecule Inhibitor of Formin FH2 (SMIFH2), after validating its mode of action on a plant formin in vitro, and observed a reduced nucleation frequency of actin filaments in live cells. Treatment of wild-type epidermal cells with SMIFH2 mimicked the phenotype of prf1 mutants, and the nucleation frequency in prf1-2 mutant was completely insensitive to these treatments. Our data provide compelling evidence that PRF1 coordinates the stochastic dynamic properties of actin filaments by modulating formin-mediated actin nucleation and assembly during plant cell expansion. PMID:26574597

A Genome-Wide Functional Investigation into the Roles of Receptor-Like Proteins in Arabidopsis1[W][OA

PubMed Central

Wang, Guodong; Ellendorff, Ursula; Kemp, Ben; Mansfield, John W.; Forsyth, Alec; Mitchell, Kathy; Bastas, Kubilay; Liu, Chun-Ming; Woods-Tör, Alison; Zipfel, Cyril; de Wit, Pierre J.G.M.; Jones, Jonathan D.G.; Tör, Mahmut; Thomma, Bart P.H.J.

2008-01-01

Receptor-like proteins (RLPs) are cell surface receptors that typically consist of an extracellular leucine-rich repeat domain, a transmembrane domain, and a short cytoplasmatic tail. In several plant species, RLPs have been found to play a role in disease resistance, such as the tomato (Solanum lycopersicum) Cf and Ve proteins and the apple (Malus domestica) HcrVf2 protein that mediate resistance against the fungal pathogens Cladosporium fulvum, Verticillium spp., and Venturia inaequalis, respectively. In addition, RLPs play a role in plant development; Arabidopsis (Arabidopsis thaliana) TOO MANY MOUTHS (TMM) regulates stomatal distribution, while Arabidopsis CLAVATA2 (CLV2) and its functional maize (Zea mays) ortholog FASCINATED EAR2 regulate meristem maintenance. In total, 57 RLP genes have been identified in the Arabidopsis genome and a genome-wide collection of T-DNA insertion lines was assembled. This collection was functionally analyzed with respect to plant growth and development and sensitivity to various stress responses, including susceptibility toward pathogens. A number of novel developmental phenotypes were revealed for our CLV2 and TMM insertion mutants. In addition, one AtRLP gene was found to mediate abscisic acid sensitivity and another AtRLP gene was found to influence nonhost resistance toward Pseudomonas syringae pv phaseolicola. This genome-wide collection of Arabidopsis RLP gene T-DNA insertion mutants provides a tool for future investigations into the biological roles of RLPs. PMID:18434605

A Galacturonic Acid–Containing Xyloglucan Is Involved in Arabidopsis Root Hair Tip Growth[W

PubMed Central

Peña, Maria J.; Kong, Yingzhen; York, William S.; O’Neill, Malcolm A.

2012-01-01

Root hairs provide a model system to study plant cell growth, yet little is known about the polysaccharide compositions of their walls or the role of these polysaccharides in wall expansion. We report that Arabidopsis thaliana root hair walls contain a previously unidentified xyloglucan that is composed of both neutral and galacturonic acid–containing subunits, the latter containing the β-d-galactosyluronic acid-(1→2)-α-d-xylosyl-(1→ and/or α-l-fucosyl-(1→2)-β-d-galactosyluronic acid-(1→2)-α-d-xylosyl-(1→) side chains. Arabidopsis mutants lacking root hairs have no acidic xyloglucan. A loss-of-function mutation in At1g63450, a root hair–specific gene encoding a family GT47 glycosyltransferase, results in the synthesis of xyloglucan that lacks galacturonic acid. The root hairs of this mutant are shorter than those of the wild type. This mutant phenotype and the absence of galacturonic acid in the root xyloglucan are complemented by At1g63450. The leaf and stem cell walls of wild-type Arabidopsis contain no acidic xyloglucan. However, overexpression of At1g63450 led to the synthesis of galacturonic acid–containing xyloglucan in these tissues. We propose that At1g63450 encodes XYLOGLUCAN-SPECIFIC GALACTURONOSYLTRANSFERASE1, which catalyzes the formation of the galactosyluronic acid-(1→2)-α-d-xylopyranosyl linkage and that the acidic xyloglucan is present only in root hair cell walls. The role of the acidic xyloglucan in root hair tip growth is discussed. PMID:23175743

Arabidopsis SHR and SCR transcription factors and AUX1 auxin influx carrier control the switch between adventitious rooting and xylogenesis in planta and in in vitro cultured thin cell layers.

PubMed

Della Rovere, F; Fattorini, L; D'Angeli, S; Veloccia, A; Del Duca, S; Cai, G; Falasca, G; Altamura, M M

2015-03-01

Adventitious roots (ARs) are essential for vegetative propagation. The Arabidopsis thaliana transcription factors SHORT ROOT (SHR) and SCARECROW (SCR) affect primary/lateral root development, but their involvement in AR formation is uncertain. LAX3 and AUX1 auxin influx carriers contribute to primary/lateral root development. LAX3 expression is regulated by SHR, and LAX3 contributes to AR tip auxin maximum. In contrast, AUX1 involvement in AR development is unknown. Xylogenesis is induced by auxin plus cytokinin as is AR formation, but the genes involved are largely unknown. Stem thin cell layers (TCLs) form ARs and undergo xylogenesis under the same auxin plus cytokinin input. The aim of this research was to investigate SHR, SCR, AUX1 and LAX3 involvement in AR formation and xylogenesis in intact hypocotyls and stem TCLs in arabidopsis. Hypocotyls of scr-1, shr-1, lax3, aux1-21 and lax3/aux1-21 Arabidopsis thaliana null mutant seedlings grown with or without auxin plus cytokinin were examined histologically, as were stem TCLs cultured with auxin plus cytokinin. SCR and AUX1 expression was monitored using pSCR::GFP and AUX1::GUS lines, and LAX3 expression and auxin localization during xylogenesis were monitored by using LAX3::GUS and DR5::GUS lines. AR formation was inhibited in all mutants, except lax3. SCR was expressed in pericycle anticlinally derived AR-forming cells of intact hypocotyls, and in cell clumps forming AR meristemoids of TCLs. The apex was anomalous in shr and scr ARs. In all mutant hypocotyls, the pericycle divided periclinally to produce xylogenesis. Xylary element maturation was favoured by auxin plus cytokinin in shr and aux1-21. Xylogenesis was enhanced in TCLs, and in aux1-21 and shr in particular. AUX1 was expressed before LAX3, i.e. in the early derivatives leading to either ARs or xylogenesis. AR formation and xylogenesis are developmental programmes that are inversely related, but they involve fine-tuning by the same proteins, namely SHR

The Plastidial Glyceraldehyde-3-Phosphate Dehydrogenase Is Critical for Viable Pollen Development in Arabidopsis1[W

PubMed Central

Muñoz-Bertomeu, Jesús; Cascales-Miñana, Borja; Irles-Segura, Asunción; Mateu, Isabel; Nunes-Nesi, Adriano; Fernie, Alisdair R.; Segura, Juan; Ros, Roc

2010-01-01

Plant metabolism is highly coordinated with development. However, an understanding of the whole picture of metabolism and its interactions with plant development is scarce. In this work, we show that the deficiency in the plastidial glycolytic glyceraldehyde-3-phosphate dehydrogenase (GAPCp) leads to male sterility in Arabidopsis (Arabidopsis thaliana). Pollen from homozygous gapcp double mutant plants (gapcp1gapcp2) displayed shrunken and collapsed forms and were unable to germinate when cultured in vitro. The pollen alterations observed in gapcp1gapcp2 were attributed to a disorganized tapetum layer. Accordingly, the expression of several of the genes involved in tapetum development was down-regulated in gapcp1gapcp2. The fertility of gapcp1gapcp2 was rescued by transforming this mutant with a construct carrying the GAPCp1 cDNA under the control of its native promoter (pGAPCp1::GAPCp1c). However, the GAPCp1 or GAPCp2 cDNA under the control of the 35S promoter (p35S::GAPCp), which is poorly expressed in the tapetum, did not complement the mutant fertility. Mutant GAPCp isoforms deficient in the catalytic activity of the enzyme were unable to complement the sterile phenotype of gapcp1gapcp2, thus confirming that both the expression and catalytic activity of GAPCp in anthers are necessary for mature pollen development. A metabolomic study in flower buds indicated that the most important difference between the sterile (gapcp1gapcp2, gapcp1gapcp2-p35S::GAPCp) and the fertile (wild-type plants, gapcp1gapcp2-pGAPCp1::GAPCp1c) lines was the increase in the signaling molecule trehalose. This work corroborates the importance of plastidial glycolysis in plant metabolism and provides evidence for the crucial role of GAPCps in pollen development. It additionally brings new insights into the complex interactions between metabolism and development. PMID:20107025

Mutation of the Inducible ARABIDOPSIS THALIANA CYTOCHROME P450 REDUCTASE2 Alters Lignin Composition and Improves Saccharification1[W][OPEN

PubMed Central

Sundin, Lisa; Vanholme, Ruben; Geerinck, Jan; Goeminne, Geert; Höfer, René; Kim, Hoon; Ralph, John; Boerjan, Wout

2014-01-01

ARABIDOPSIS THALIANA CYTOCHROME P450 REDUCTASE1 (ATR1) and ATR2 provide electrons from NADPH to a large number of CYTOCHROME P450 (CYP450) enzymes in Arabidopsis (Arabidopsis thaliana). Whereas ATR1 is constitutively expressed, the expression of ATR2 appears to be induced during lignin biosynthesis and upon stresses. Therefore, ATR2 was hypothesized to be preferentially involved in providing electrons to the three CYP450s involved in lignin biosynthesis: CINNAMATE 4-HYDROXYLASE (C4H), p-COUMARATE 3-HYDROXYLASE1 (C3H1), and FERULATE 5-HYDROXYLASE1 (F5H1). Here, we show that the atr2 mutation resulted in a 6% reduction in total lignin amount in the main inflorescence stem and a compositional shift of the remaining lignin to a 10-fold higher fraction of p-hydroxyphenyl units at the expense of syringyl units. Phenolic profiling revealed shifts in lignin-related phenolic metabolites, in particular with the substrates of C4H, C3H1 and F5H1 accumulating in atr2 mutants. Glucosinolate and flavonol glycoside biosynthesis, both of which also rely on CYP450 activities, appeared less affected. The cellulose in the atr2 inflorescence stems was more susceptible to enzymatic hydrolysis after alkaline pretreatment, making ATR2 a potential target for engineering plant cell walls for biofuel production. PMID:25315601

Simulated microgravity, Mars gravity, and 2g hypergravity affect cell cycle regulation, ribosome biogenesis, and epigenetics in Arabidopsis cell cultures.

PubMed

Kamal, Khaled Y; Herranz, Raúl; van Loon, Jack J W A; Medina, F Javier

2018-04-23

Gravity is the only component of Earth environment that remained constant throughout the entire process of biological evolution. However, it is still unclear how gravity affects plant growth and development. In this study, an in vitro cell culture of Arabidopsis thaliana was exposed to different altered gravity conditions, namely simulated reduced gravity (simulated microgravity, simulated Mars gravity) and hypergravity (2g), to study changes in cell proliferation, cell growth, and epigenetics. The effects after 3, 14, and 24-hours of exposure were evaluated. The most relevant alterations were found in the 24-hour treatment, being more significant for simulated reduced gravity than hypergravity. Cell proliferation and growth were uncoupled under simulated reduced gravity, similarly, as found in meristematic cells from seedlings grown in real or simulated microgravity. The distribution of cell cycle phases was changed, as well as the levels and gene transcription of the tested cell cycle regulators. Ribosome biogenesis was decreased, according to levels and gene transcription of nucleolar proteins and the number of inactive nucleoli. Furthermore, we found alterations in the epigenetic modifications of chromatin. These results show that altered gravity effects include a serious disturbance of cell proliferation and growth, which are cellular functions essential for normal plant development.

The Role of Oxophytodienoate Reductases in the Detoxification of the Explosive 2,4,6-Trinitrotoluene by Arabidopsis1[W][OA

PubMed Central

Beynon, Emily R.; Symons, Zoe C.; Jackson, Rosamond G.; Lorenz, Astrid; Rylott, Elizabeth L.; Bruce, Neil C.

2009-01-01

The explosive 2,4,6-trinitrotoluene (TNT) is a significant environmental pollutant that is both toxic and recalcitrant to degradation. Phytoremediation is being increasingly proposed as a viable alternative to conventional remediation technologies to clean up explosives-contaminated sites. Despite the potential of this technology, relatively little is known about the innate enzymology of TNT detoxification in plants. To further elucidate this, we used microarray analysis to identify Arabidopsis (Arabidopsis thaliana) genes up-regulated by exposure to TNT and found that the expression of oxophytodienoate reductases (OPRs) increased in response to TNT. The OPRs share similarity with the Old Yellow Enzyme family, bacterial members of which have been shown to transform explosives. The three predominantly expressed forms, OPR1, OPR2, and OPR3, were recombinantly expressed and affinity purified. Subsequent biochemical characterization revealed that all three OPRs are able to transform TNT to yield nitro-reduced TNT derivatives, with OPR1 additionally producing the aromatic ring-reduced products hydride and dihydride Meisenheimer complexes. Arabidopsis plants overexpressing OPR1 removed TNT more quickly from liquid culture, produced increased levels of transformation products, and maintained higher fresh weight biomasses than wild-type plants. In contrast, OPR1,2 RNA interference lines removed less TNT, produced fewer transformation products, and had lower biomasses. When grown on solid medium, two of the three OPR1 lines and all of the OPR2-overexpressing lines exhibited significantly enhanced tolerance to TNT. These data suggest that, in concert with other detoxification mechanisms, OPRs play a physiological role in xenobiotic detoxification. PMID:19605548

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

DOE Office of Scientific and Technical Information (OSTI.GOV)

Aggarwal, Pooja; Padmanabhan, Bhavna; Bhat, Abhay

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, theirmore » 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.« less

Isolation and Proteomic Characterization of the Arabidopsis Golgi Defines Functional and Novel Components Involved in Plant Cell Wall Biosynthesis1[W][OA

PubMed Central

Parsons, Harriet T.; Christiansen, Katy; Knierim, Bernhard; Carroll, Andrew; Ito, Jun; Batth, Tanveer S.; Smith-Moritz, Andreia M.; Morrison, Stephanie; McInerney, Peter; Hadi, Masood Z.; Auer, Manfred; Mukhopadhyay, Aindrila; Petzold, Christopher J.; Scheller, Henrik V.; Loqué, Dominique; Heazlewood, Joshua L.

2012-01-01

The plant Golgi plays a pivotal role in the biosynthesis of cell wall matrix polysaccharides, protein glycosylation, and vesicle trafficking. Golgi-localized proteins have become prospective targets for reengineering cell wall biosynthetic pathways for the efficient production of biofuels from plant cell walls. However, proteomic characterization of the Golgi has so far been limited, owing to the technical challenges inherent in Golgi purification. In this study, a combination of density centrifugation and surface charge separation techniques have allowed the reproducible isolation of Golgi membranes from Arabidopsis (Arabidopsis thaliana) at sufficiently high purity levels for in-depth proteomic analysis. Quantitative proteomic analysis, immunoblotting, enzyme activity assays, and electron microscopy all confirm high purity levels. A composition analysis indicated that approximately 19% of proteins were likely derived from contaminating compartments and ribosomes. The localization of 13 newly assigned proteins to the Golgi using transient fluorescent markers further validated the proteome. A collection of 371 proteins consistently identified in all replicates has been proposed to represent the Golgi proteome, marking an appreciable advancement in numbers of Golgi-localized proteins. A significant proportion of proteins likely involved in matrix polysaccharide biosynthesis were identified. The potential within this proteome for advances in understanding Golgi processes has been demonstrated by the identification and functional characterization of the first plant Golgi-resident nucleoside diphosphatase, using a yeast complementation assay. Overall, these data show key proteins involved in primary cell wall synthesis and include a mixture of well-characterized and unknown proteins whose biological roles and importance as targets for future research can now be realized. PMID:22430844

In Vivo Function of Tryptophans in the Arabidopsis UV-B Photoreceptor UVR8[W

PubMed Central

O’Hara, Andrew; Jenkins, Gareth I.

2012-01-01

Arabidopsis thaliana UV RESISTANCE LOCUS8 (UVR8) is a photoreceptor specifically for UV-B light that initiates photomorphogenic responses in plants. UV-B exposure causes rapid conversion of UVR8 from dimer to monomer, accumulation in the nucleus, and interaction with CONSTITUTIVELY PHOTOMORPHOGENIC1 (COP1), which functions with UVR8 in UV-B responses. Studies in yeast and with purified UVR8 implicate several tryptophan amino acids in UV-B photoreception. However, their roles in UV-B responses in plants, and the functional significance of all 14 UVR8 tryptophans, are not known. Here we report the functions of the UVR8 tryptophans in vivo. Three tryptophans in the β-propeller core are important in maintaining structural stability and function of UVR8. However, mutation of three other core tryptophans and four at the dimeric interface has no apparent effect on function in vivo. Mutation of three tryptophans implicated in UV-B photoreception, W233, W285, and W337, impairs photomorphogenic responses to different extents. W285 is essential for UVR8 function in plants, whereas W233 is important but not essential for function, and W337 has a lesser role. Ala mutants of these tryptophans appear monomeric and constitutively bind COP1 in plants, but their responses indicate that monomer formation and COP1 binding are not sufficient for UVR8 function. PMID:23012433

Intact Arabidopsis RPB1 functions in stem cell niches maintenance and cell cycling control.

PubMed

Zhang, Qian-Qian; Li, Ying; Fu, Zhao-Ying; Liu, Xun-Biao; Yuan, Kai; Fang, Ying; Liu, Yan; Li, Gang; Zhang, Xian-Sheng; Chong, Kang; Ge, Lei

2018-05-12

Plant meristem activity depends on accurate execution of transcriptional networks required for establishing optimum functioning of stem cell niches. An Arabidopsis mutant card1-1 (constitutive auxin response with DR5:GFP) that encodes a truncated RPB1 (RNA Polymerase II's largest subunit) with shortened C-terminal domain (CTD) was identified. Phosphorylation of the CTD repeats of RPB1 is coupled to transcription in eukaryotes. Here we uncover that the truncated CTD of RPB1 disturbed cell cycling and enlarged the size of shoot and root meristem. The defects in patterning of root stem cell niche in card1-1 indicates that intact CTD of RPB1 is necessary for fine-tuning the specific expression of genes responsible for cell-fate determination. The gene-edited plants with different CTD length of RPB1, created by CRISPR-CAS9 technology, confirmed that both the full length and the DK-rich tail of RPB1's CTD play roles in the accurate transcription of CYCB1;1 encoding a cell-cycle marker protein in root meristem and hence participate in maintaining root meristem size. Our experiment proves that the intact RPB1 CTD is necessary for stem cell niche maintenance, which is mediated by transcriptional regulation of cell cycling genes. © 2018 The Authors The Plant Journal © 2018 John Wiley & Sons Ltd.

A 3D cell culture system: separation distance between INS-1 cell and endothelial cell monolayers co-cultured in fibrin influences INS-1 cells insulin secretion.

PubMed

Sabra, Georges; Vermette, Patrick

2013-02-01

The aim of this study was to develop an in vitro cell culture system allowing studying the effect of separation distance between monolayers of rat insulinoma cells (INS-1) and human umbilical vein endothelial cells (HUVEC) co-cultured in fibrin over INS-1 cell insulin secretion. For this purpose, a three-dimensional (3D) cell culture chamber was designed, built using micro-fabrication techniques and validated. The co-culture was successfully carried out and the effect on INS-1 cell insulin secretion was investigated. After 48 and 72 h, INS-1 cells co-cultured with HUVEC separated by a distance of 100 µm revealed enhanced insulin secretion compared to INS-1 cells cultured alone or co-cultured with HUVEC monolayers separated by a distance of 200 µm. These results illustrate the importance of the separation distance between two cell niches for cell culture design and the possibility to further enhance the endocrine function of beta cells when this factor is considered. Copyright © 2012 Wiley Periodicals, Inc.

Leaf Oil Body Functions as a Subcellular Factory for the Production of a Phytoalexin in Arabidopsis1[W

PubMed Central

Shimada, Takashi L.; Takano, Yoshitaka; Shimada, Tomoo; Fujiwara, Masayuki; Fukao, Yoichiro; Mori, Masashi; Okazaki, Yozo; Saito, Kazuki; Sasaki, Ryosuke; Aoki, Koh; Hara-Nishimura, Ikuko

2014-01-01

Oil bodies are intracellular structures present in the seed and leaf cells of many land plants. Seed oil bodies are known to function as storage compartments for lipids. However, the physiological function of leaf oil bodies is unknown. Here, we show that leaf oil bodies function as subcellular factories for the production of a stable phytoalexin in response to fungal infection and senescence. Proteomic analysis of oil bodies prepared from Arabidopsis (Arabidopsis thaliana) leaves identified caleosin (CLO3) and α-dioxygenase (α-DOX1). Both CLO3 and α-DOX1 were localized on the surface of oil bodies. Infection with the pathogenic fungus Colletotrichum higginsianum promoted the formation of CLO3- and α-DOX1-positive oil bodies in perilesional areas surrounding the site of infection. α-DOX1 catalyzes the reaction from α-linolenic acid (a major fatty acid component of oil bodies) to an unstable compound, 2-hydroperoxy-octadecatrienoic acid (2-HPOT). Intriguingly, a combination of α-DOX1 and CLO3 produced a stable compound, 2-hydroxy-octadecatrienoic acid (2-HOT), from α-linolenic acid. This suggests that the colocalization of α-DOX1 and CLO3 on oil bodies might prevent the degradation of unstable 2-HPOT by efficiently converting 2-HPOT into the stable compound 2-HOT. We found that 2-HOT had antifungal activity against members of the genus Colletotrichum and that infection with C. higginsianum induced 2-HOT production. These results defined 2-HOT as an Arabidopsis phytoalexin. This study provides, to our knowledge, the first evidence that leaf oil bodies produce a phytoalexin under a pathological condition, which suggests a new mechanism of plant defense. PMID:24214535

Elevated Early Callose Deposition Results in Complete Penetration Resistance to Powdery Mildew in Arabidopsis1[C][W][OA

PubMed Central

Ellinger, Dorothea; Naumann, Marcel; Falter, Christian; Zwikowics, Claudia; Jamrow, Torsten; Manisseri, Chithra; Somerville, Shauna C.; Voigt, Christian A.

2013-01-01

A common response by plants to fungal attack is deposition of callose, a (1,3)-β-glucan polymer, in the form of cell wall thickenings called papillae, at site of wall penetration. While it has been generally believed that the papillae provide a structural barrier to slow fungal penetration, this idea has been challenged in recent studies of Arabidopsis (Arabidopsis thaliana), where fungal resistance was found to be independent of callose deposition. To the contrary, we show that callose can strongly support penetration resistance when deposited in elevated amounts at early time points of infection. We generated transgenic Arabidopsis lines that express POWDERY MILDEW RESISTANT4 (PMR4), which encodes a stress-induced callose synthase, under the control of the constitutive 35S promoter. In these lines, we detected callose synthase activity that was four times higher than that in wild-type plants 6 h post inoculation with the virulent powdery mildew Golovinomyces cichoracearum. The callose synthase activity was correlated with enlarged callose deposits and the focal accumulation of green fluorescent protein-tagged PMR4 at sites of attempted fungal penetration. We observed similar results from infection studies with the nonadapted powdery mildew Blumeria graminis f. sp. hordei. Haustoria formation was prevented in resistant transgenic lines during both types of powdery mildew infection, and neither the salicylic acid-dependent nor jasmonate-dependent pathways were induced. We present a schematic model that highlights the differences in callose deposition between the resistant transgenic lines and the susceptible wild-type plants during compatible and incompatible interactions between Arabidopsis and powdery mildew. PMID:23335625

Mapping methyl jasmonate-mediated transcriptional reprogramming of metabolism and cell cycle progression in cultured Arabidopsis cells

PubMed Central

Pauwels, Laurens; Morreel, Kris; De Witte, Emilie; Lammertyn, Freya; Van Montagu, Marc; Boerjan, Wout; Inzé, Dirk; Goossens, Alain

2008-01-01

Jasmonates (JAs) are plant-specific signaling molecules that steer a diverse set of physiological and developmental processes. Pathogen attack and wounding inflicted by herbivores induce the biosynthesis of these hormones, triggering defense responses both locally and systemically. We report on alterations in the transcriptome of a fast-dividing cell culture of the model plant Arabidopsis thaliana after exogenous application of methyl JA (MeJA). Early MeJA response genes encoded the JA biosynthesis pathway proteins and key regulators of MeJA responses, including most JA ZIM domain proteins and MYC2, together with transcriptional regulators with potential, but yet unknown, functions in MeJA signaling. In a second transcriptional wave, MeJA reprogrammed cellular metabolism and cell cycle progression. Up-regulation of the monolignol biosynthesis gene set resulted in an increased production of monolignols and oligolignols, the building blocks of lignin. Simultaneously, MeJA repressed activation of M-phase genes, arresting the cell cycle in G2. MeJA-responsive transcription factors were screened for their involvement in early signaling events, in particular the regulation of JA biosynthesis. Parallel screens based on yeast one-hybrid and transient transactivation assays identified both positive (MYC2 and the AP2/ERF factor ORA47) and negative (the C2H2 Zn finger proteins STZ/ZAT10 and AZF2) regulators, revealing a complex control of the JA autoregulatory loop and possibly other MeJA-mediated downstream processes. PMID:18216250

Plasticity of the Arabidopsis Root System under Nutrient Deficiencies1[C][W][OPEN

PubMed Central

Gruber, Benjamin D.; Giehl, Ricardo F.H.; Friedel, Swetlana; von Wirén, Nicolaus

2013-01-01

Plant roots show a particularly high variation in their morphological response to different nutrient deficiencies. Although such changes often determine the nutrient efficiency or stress tolerance of plants, it is surprising that a comprehensive and comparative analysis of root morphological responses to different nutrient deficiencies has not yet been conducted. Since one reason for this is an inherent difficulty in obtaining nutrient-deficient conditions in agar culture, we first identified conditions appropriate for producing nutrient-deficient plants on agar plates. Based on a careful selection of agar specifically for each nutrient being considered, we grew Arabidopsis (Arabidopsis thaliana) plants at four levels of deficiency for 12 nutrients and quantified seven root traits. In combination with measurements of biomass and elemental concentrations, we observed that the nutritional status and type of nutrient determined the extent and type of changes in root system architecture (RSA). The independent regulation of individual root traits further pointed to a differential sensitivity of root tissues to nutrient limitations. To capture the variation in RSA under different nutrient supplies, we used principal component analysis and developed a root plasticity chart representing the overall modulations in RSA under a given treatment. This systematic comparison of RSA responses to nutrient deficiencies provides a comprehensive view of the overall changes in root plasticity induced by the deficiency of single nutrients and provides a solid basis for the identification of nutrient-sensitive steps in the root developmental program. PMID:23852440

SUPPRESSOR OF MORE AXILLARY GROWTH2 1 Controls Seed Germination and Seedling Development in Arabidopsis1[W][OPEN

PubMed Central

Stanga, John P.; Smith, Steven M.; Briggs, Winslow R.; Nelson, David C.

2013-01-01

Abiotic chemical signals discovered in smoke that are known as karrikins (KARs) and the endogenous hormone strigolactone (SL) control plant growth through a shared MORE AXILLARY GROWTH2 (MAX2)-dependent pathway. A SL biosynthetic pathway and candidate KAR/SL receptors have been characterized, but signaling downstream of MAX2 is poorly defined. A screen for genetic suppressors of the enhanced seed dormancy phenotype of max2 in Arabidopsis (Arabidopsis thaliana) led to identification of a suppressor of max2 1 (smax1) mutant. smax1 restores the seed germination and seedling photomorphogenesis phenotypes of max2 but does not affect the lateral root formation, axillary shoot growth, or senescence phenotypes of max2. Expression of three transcriptional markers of KAR/SL signaling, D14-LIKE2, KAR-UP F-BOX1, and INDOLE-3-ACETIC ACID INDUCIBLE1, is rescued in smax1 max2 seedlings. SMAX1 is a member of an eight-gene family in Arabidopsis that has weak similarity to HEAT SHOCK PROTEIN 101, which encodes a caseinolytic peptidase B chaperonin required for thermotolerance. SMAX1 and the SMAX1-like (SMXL) homologs are differentially expressed in Arabidopsis tissues. SMAX1 transcripts are most abundant in dry seed, consistent with its function in seed germination control. Several SMXL genes are up-regulated in seedlings treated with the synthetic SL GR24. SMAX1 and SMXL2 transcripts are reduced in max2 seedlings, which could indicate negative feedback regulation by KAR/SL signaling. smax1 seed and seedling growth mimics the wild type treated with KAR/SL, but smax1 seedlings are still responsive to 2H-furo[2,3-c]pyran-2-one (KAR2) or GR24. We conclude that SMAX1 is an important component of KAR/SL signaling during seed germination and seedling growth but is not necessary for all MAX2-dependent responses. We hypothesize that one or more SMXL proteins may also act downstream of MAX2 to control the diverse developmental responses to KARs and SLs. PMID:23893171

The Arabidopsis Halophytic Relative Thellungiella halophila Tolerates Nitrogen-Limiting Conditions by Maintaining Growth, Nitrogen Uptake, and Assimilation1[W][OA

PubMed Central

Kant, Surya; Bi, Yong-Mei; Weretilnyk, Elizabeth; Barak, Simon; Rothstein, Steven J.

2008-01-01

A comprehensive knowledge of mechanisms regulating nitrogen (N) use efficiency is required to reduce excessive input of N fertilizers while maintaining acceptable crop yields under limited N supply. Studying plant species that are naturally adapted to low N conditions could facilitate the identification of novel regulatory genes conferring better N use efficiency. Here, we show that Thellungiella halophila, a halophytic relative of Arabidopsis (Arabidopsis thaliana), grows better than Arabidopsis under moderate (1 mm nitrate) and severe (0.4 mm nitrate) N-limiting conditions. Thellungiella exhibited a lower carbon to N ratio than Arabidopsis under N limitation, which was due to Thellungiella plants possessing higher N content, total amino acids, total soluble protein, and lower starch content compared with Arabidopsis. Furthermore, Thellungiella had higher amounts of several metabolites, such as soluble sugars and organic acids, under N-sufficient conditions (4 mm nitrate). Nitrate reductase activity and NR2 gene expression in Thellungiella displayed less of a reduction in response to N limitation than in Arabidopsis. Thellungiella shoot GS1 expression was more induced by low N than in Arabidopsis, while in roots, Thellungiella GS2 expression was maintained under N limitation but was decreased in Arabidopsis. Up-regulation of NRT2.1 and NRT3.1 expression was higher and repression of NRT1.1 was lower in Thellungiella roots under N-limiting conditions compared with Arabidopsis. Differential transporter gene expression was correlated with higher nitrate influx in Thellungiella at low 15NO3− supply. Taken together, our results suggest that Thellungiella is tolerant to N-limited conditions and could act as a model system to unravel the mechanisms for low N tolerance. PMID:18467466

Chlorophyll b Reductase Plays an Essential Role in Maturation and Storability of Arabidopsis Seeds1[W

PubMed Central

Nakajima, Saori; Ito, Hisashi; Tanaka, Ryouichi; Tanaka, Ayumi

2012-01-01

Although seeds are a sink organ, chlorophyll synthesis and degradation occurs during embryogenesis and in a manner similar to that observed in photosynthetic leaves. Some mutants retain chlorophyll after seed maturation, and they are disturbed in seed storability. To elucidate the effects of chlorophyll retention on the seed storability of Arabidopsis (Arabidopsis thaliana), we examined the non-yellow coloring1 (nyc1)/nyc1-like (nol) mutants that do not degrade chlorophyll properly. Approximately 10 times more chlorophyll was retained in the dry seeds of the nyc1/nol mutant than in the wild-type seeds. The germination rates rapidly decreased during storage, with most of the mutant seeds failing to germinate after storage for 23 months, whereas 75% of the wild-type seeds germinated after 42 months. These results indicate that chlorophyll retention in the seeds affects seed longevity. Electron microscopic studies indicated that many small oil bodies appeared in the embryonic cotyledons of the nyc1/nol mutant; this finding indicates that the retention of chlorophyll affects the development of organelles in embryonic cells. A sequence analysis of the NYC1 promoter identified a potential abscisic acid (ABA)-responsive element. An electrophoretic mobility shift assay confirmed the binding of an ABA-responsive transcriptional factor to the NYC1 promoter DNA fragment, thus suggesting that NYC1 expression is regulated by ABA. Furthermore, NYC1 expression was repressed in the ABA-insensitive mutants during embryogenesis. These data indicate that chlorophyll degradation is induced by ABA during seed maturation to produce storable seeds. PMID:22751379

Arabidopsis PECTIN METHYLESTERASEs Contribute to Immunity against Pseudomonas syringae1[C][W][OPEN

PubMed Central

Bethke, Gerit; Grundman, Rachael E.; Sreekanta, Suma; Truman, William; Katagiri, Fumiaki; Glazebrook, Jane

2014-01-01

Pectins, major components of dicot cell walls, are synthesized in a heavily methylesterified form in the Golgi and are partially deesterified by pectin methylesterases (PMEs) upon export to the cell wall. PME activity is important for the virulence of the necrotrophic fungal pathogen Botrytis cinerea. Here, the roles of Arabidopsis PMEs in pattern-triggered immunity and immune responses to the necrotrophic fungus Alternaria brassicicola and the bacterial hemibiotroph Pseudomonas syringae pv maculicola ES4326 (Pma ES4326) were studied. Plant PME activity increased during pattern-triggered immunity and after inoculation with either pathogen. The increase of PME activity in response to pathogen treatment was concomitant with a decrease in pectin methylesterification. The pathogen-induced PME activity did not require salicylic acid or ethylene signaling, but was dependent on jasmonic acid signaling. In the case of induction by A. brassicicola, the ethylene response factor, but not the MYC2 branch of jasmonic acid signaling, contributed to induction of PME activity, whereas in the case of induction by Pma ES4326, both branches contributed. There are 66 PME genes in Arabidopsis, suggesting extensive genetic redundancy. Nevertheless, selected pme single, double, triple and quadruple mutants allowed significantly more growth of Pma ES4326 than wild-type plants, indicating a role of PMEs in resistance to this pathogen. No decreases in total PME activity were detected in these pme mutants, suggesting that the determinant of immunity is not total PME activity; rather, it is some specific effect of PMEs such as changes in the pattern of pectin methylesterification. PMID:24367018

Sporophytic and gametophytic functions of the cell cycle-associated Mob1 gene in Arabidopsis thaliana L.

PubMed

Galla, Giulio; Zenoni, Sara; Marconi, Gianpiero; Marino, Giada; Botton, Alessandro; Pinosa, Francesco; Citterio, Sandra; Ruperti, Benedetto; Palme, Klaus; Albertini, Emidio; Pezzotti, Mario; Mau, Martin; Sharbel, Timothy F; De Storme, Nico; Geelen, Danny; Barcaccia, Gianni

2011-09-15

Mob1 genes are primarily involved in the cell cycle progression and mitosis exit in yeasts and animals. The function of a Mob1-like gene (At5g45550) from Arabidopsis thaliana was investigated using RNAi and immunological staining. AtMob1-like RNAi silenced lines showed a reduced radial expansion of the inflorescence stem and a reduced elongation zone of the primary root. Morphological features of plant organs were accompanied by a reduction in cell size. The fertility of AtMob1-like RNAi silenced lines was very low as seed production was strongly reduced. About 2% of the progeny of AtMob1-like RNAi silenced plants were tetraploid. The female and male sporogenesis was affected differentially. The ovules developed irregularly and one third of the megaspores and embryo sacs degenerated prematurely. Up to 20% of the ovules produced binucleated megaspores that failed to develop further, being their degeneration likely accompanied with a delayed programmed cell death. The anthers produced about 30% of aborted pollen grains, showing also a strong variation in their size. Together, the results show that Arabidopsis MOB1-like is required to regulate cell expansion and cell division, presumably by affecting the mitotic as well as the meiotic cell cycle. Copyright © 2011 Elsevier B.V. All rights reserved.

Broad-Spectrum Suppression of Innate Immunity Is Required for Colonization of Arabidopsis Roots by the Fungus Piriformospora indica1[C][W

PubMed Central

Jacobs, Sophie; Zechmann, Bernd; Molitor, Alexandra; Trujillo, Marco; Petutschnig, Elena; Lipka, Volker; Kogel, Karl-Heinz; Schäfer, Patrick

2011-01-01

Piriformospora indica is a root-colonizing basidiomycete that confers a wide range of beneficial traits to its host. The fungus shows a biotrophic growth phase in Arabidopsis (Arabidopsis thaliana) roots followed by a cell death-associated colonization phase, a colonization strategy that, to our knowledge, has not yet been reported for this plant. P. indica has evolved an extraordinary capacity for plant root colonization. Its broad host spectrum encompasses gymnosperms and monocotyledonous as well as dicotyledonous angiosperms, which suggests that it has an effective mechanism(s) for bypassing or suppressing host immunity. The results of our work argue that P. indica is confronted with a functional root immune system. Moreover, the fungus does not evade detection but rather suppresses immunity triggered by various microbe-associated molecular patterns. This ability to suppress host immunity is compromised in the jasmonate mutants jasmonate insensitive1-1 and jasmonate resistant1-1. A quintuple-DELLA mutant displaying constitutive gibberellin (GA) responses and the GA biosynthesis mutant ga1-6 (for GA requiring 1) showed higher and lower degrees of colonization, respectively, in the cell death-associated stage, suggesting that P. indica recruits GA signaling to help establish proapoptotic root cell colonization. Our study demonstrates that mutualists, like pathogens, are confronted with an effective innate immune system in roots and that colonization success essentially depends on the evolution of strategies for immunosuppression. PMID:21474434

Proper selection of 1 g controls in simulated microgravity research as illustrated with clinorotated plant cell suspension cultures

NASA Astrophysics Data System (ADS)

Kamal, Khaled Y.; Hemmersbach, Ruth; Medina, F. Javier; Herranz, Raúl

2015-04-01

Understanding the physical and biological effects of the absence of gravity is necessary to conduct operations on space environments. It has been previously shown that the microgravity environment induces the dissociation of cell proliferation from cell growth in young seedling root meristems, but this source material is limited to few cells in each row of meristematic layers. Plant cell cultures, composed by a large and homogeneous population of proliferating cells, are an ideal model to study the effects of altered gravity on cellular mechanisms regulating cell proliferation and associated cell growth. Cell suspension cultures of Arabidopsis thaliana cell line (MM2d) were exposed to 2D-clinorotation in a pipette clinostat for 3.5 or 14 h, respectively, and were then processed either by quick freezing, to be used in flow cytometry, or by chemical fixation, for microscopy techniques. After long-term clinorotation, the proportion of cells in G1 phase was increased and the nucleolus area, as revealed by immunofluorescence staining with anti-nucleolin, was decreased. Despite the compatibility of these results with those obtained in real microgravity on seedling meristems, we provide a technical discussion in the context of clinorotation and proper 1 g controls with respect to suspension cultures. Standard 1 g procedure of sustaining the cell suspension is achieved by continuously shaking. Thus, we compare the mechanical forces acting on cells in clinorotated samples, in a control static sample and in the standard 1 g conditions of suspension cultures in order to define the conditions of a complete and reliable experiment in simulated microgravity with corresponding 1 g controls.

Sugar suppresses cell death caused by disruption of fumarylacetoacetate hydrolase in Arabidopsis.

PubMed

Zhi, Tiantian; Zhou, Zhou; Huang, Yi; Han, Chengyun; Liu, Yan; Zhu, Qi; Ren, Chunmei

2016-09-01

Sugar negatively regulates cell death resulting from the loss of fumarylacetoacetate hydrolase that catalyzes the last step in the Tyr degradation pathway in Arabidopsis . Fumarylacetoacetate hydrolase (FAH) hydrolyzes fumarylacetoacetate to fumarate and acetoacetate, the final step in the tyrosine (Tyr) degradation pathway that is essential to animals. Previously, we first found that the Tyr degradation pathway plays an important role in plants. Mutation of the SSCD1 gene encoding FAH in Arabidopsis leads to spontaneous cell death under short-day conditions. In this study, we presented that the lethal phenotype of the short-day sensitive cell death1 (sscd1) seedlings was suppressed by sugars including sucrose, glucose, fructose, and maltose in a dose-dependent manner. Real-time quantitative PCR (RT-qPCR) analysis showed the expression of Tyr degradation pathway genes homogentisate dioxygenase and maleylacetoacetate isomerase, and sucrose-processing genes cell-wall invertase 1 and alkaline/neutral invertase G, was up-regulated in the sscd1 mutant, however, this up-regulation could be repressed by sugar. In addition, a high concentration of sugar attenuated cell death of Arabidopsis wild-type seedlings caused by treatment with exogenous succinylacetone, an abnormal metabolite resulting from the loss of FAH in the Tyr degradation pathway. These results indicated that (1) sugar could suppress cell death in sscd1, which might be because sugar supply enhances the resistance of Arabidopsis seedlings to toxic effects of succinylacetone and reduces the accumulation of Tyr degradation intermediates, resulting in suppression of cell death; and (2) sucrose-processing genes cell-wall invertase 1 and alkaline/neutral invertase G might be involved in the cell death in sscd1. Our work provides insights into the relationship between sugar and sscd1-mediated cell death, and contributes to elucidation of the regulation of cell death resulting from the loss of FAH in plants.

WVD2 and WDL1 modulate helical organ growth and anisotropic cell expansion in Arabidopsis

NASA Technical Reports Server (NTRS)

Yuen, Christen Y L.; Pearlman, Rebecca S.; Silo-Suh, Laura; Hilson, Pierre; Carroll, Kathleen L.; Masson, Patrick H.

2003-01-01

Wild-type Arabidopsis roots develop a wavy pattern of growth on tilted agar surfaces. For many Arabidopsis ecotypes, roots also grow askew on such surfaces, typically slanting to the right of the gravity vector. We identified a mutant, wvd2-1, that displays suppressed root waving and leftward root slanting under these conditions. These phenotypes arise from transcriptional activation of the novel WAVE-DAMPENED2 (WVD2) gene by the cauliflower mosaic virus 35S promoter in mutant plants. Seedlings overexpressing WVD2 exhibit constitutive right-handed helical growth in both roots and etiolated hypocotyls, whereas the petioles of WVD2-overexpressing rosette leaves exhibit left-handed twisting. Moreover, the anisotropic expansion of cells is impaired, resulting in the formation of shorter and stockier organs. In roots, the phenotype is accompanied by a change in the arrangement of cortical microtubules within peripheral cap cells and cells at the basal end of the elongation zone. WVD2 transcripts are detectable by reverse transcriptase-polymerase chain reaction in multiple organs of wild-type plants. Its predicted gene product contains a conserved region named "KLEEK," which is found only in plant proteins. The Arabidopsis genome possesses seven other genes predicted to encode KLEEK-containing products. Overexpression of one of these genes, WVD2-LIKE 1, which encodes a protein with regions of similarity to WVD2 extending beyond the KLEEK domain, results in phenotypes that are highly similar to wvd2-1. Silencing of WVD2 and its paralogs results in enhanced root skewing in the wild-type direction. Our observations suggest that at least two members of this gene family may modulate both rotational polarity and anisotropic cell expansion during organ growth.

A γ-Glutamyl Transpeptidase-Independent Pathway of Glutathione Catabolism to Glutamate via 5-Oxoproline in Arabidopsis1[W][OA

PubMed Central

Ohkama-Ohtsu, Naoko; Oikawa, Akira; Zhao, Ping; Xiang, Chengbin; Saito, Kazuki; Oliver, David J.

2008-01-01

The degradation pathway of glutathione (GSH) in plants is not well understood. In mammals, GSH is predominantly metabolized through the γ-glutamyl cycle, where GSH is degraded by the sequential reaction of γ-glutamyl transpeptidase (GGT), γ-glutamyl cyclotransferase, and 5-oxoprolinase to yield glutamate (Glu) and dipeptides that are subject to peptidase action. In this study, we examined if GSH is degraded through the same pathway in Arabidopsis (Arabidopsis thaliana) as occurs in mammals. In Arabidopsis, the oxoprolinase knockout mutants (oxp1-1 and oxp1-2) accumulate more 5-oxoproline (5OP) and less Glu than wild-type plants, suggesting substantial metabolite flux though 5OP and that 5OP is a major contributor to Glu steady-state levels. In the ggt1-1/ggt4-1/oxp1-1 triple mutant with no GGT activity in any organs except young siliques, the 5OP concentration in leaves was not different from that in oxp1-1, suggesting that GGTs are not major contributors to 5OP production in Arabidopsis. 5OP formation strongly tracked the level of GSH in Arabidopsis plants, suggesting that GSH is the precursor of 5OP in a GGT-independent reaction. Kinetics analysis suggests that γ-glutamyl cyclotransferase is the major source of GSH degradation and 5OP formation in Arabidopsis. This discovery led us to propose a new pathway for GSH turnover in plants where GSH is converted to 5OP and then to Glu by the combined action of γ-glutamyl cyclotransferase and 5-oxoprolinase in the cytoplasm. PMID:18768907

AUXIN BINDING PROTEIN1 Links Cell Wall Remodeling, Auxin Signaling, and Cell Expansion in Arabidopsis[W

PubMed Central

Paque, Sébastien; Mouille, Grégory; Grandont, Laurie; Alabadí, David; Gaertner, Cyril; Goyallon, Arnaud; Muller, Philippe; Primard-Brisset, Catherine; Sormani, Rodnay; Blázquez, Miguel A.; Perrot-Rechenmann, Catherine

2014-01-01

Cell expansion is an increase in cell size and thus plays an essential role in plant growth and development. Phytohormones and the primary plant cell wall play major roles in the complex process of cell expansion. In shoot tissues, cell expansion requires the auxin receptor AUXIN BINDING PROTEIN1 (ABP1), but the mechanism by which ABP1 affects expansion remains unknown. We analyzed the effect of functional inactivation of ABP1 on transcriptomic changes in dark-grown hypocotyls and investigated the consequences of gene expression on cell wall composition and cell expansion. Molecular and genetic evidence indicates that ABP1 affects the expression of a broad range of cell wall–related genes, especially cell wall remodeling genes, mainly via an SCFTIR/AFB-dependent pathway. ABP1 also functions in the modulation of hemicellulose xyloglucan structure. Furthermore, fucosidase-mediated defucosylation of xyloglucan, but not biosynthesis of nonfucosylated xyloglucan, rescued dark-grown hypocotyl lengthening of ABP1 knockdown seedlings. In muro remodeling of xyloglucan side chains via an ABP1-dependent pathway appears to be of critical importance for temporal and spatial control of cell expansion. PMID:24424095

Reduced Carbohydrate Availability Enhances the Susceptibility of Arabidopsis toward Colletotrichum higginsianum1[W][OA

PubMed Central

Engelsdorf, Timo; Horst, Robin J.; Pröls, Reinhard; Pröschel, Marlene; Dietz, Franziska; Hückelhoven, Ralph; Voll, Lars M.

2013-01-01

Colletotrichum higginsianum is a hemibiotrophic ascomycete fungus that is adapted to Arabidopsis (Arabidopsis thaliana). After breaching the host surface, the fungus establishes an initial biotrophic phase in the penetrated epidermis cell, before necrotrophic growth is initiated upon further host colonization. We observed that partitioning of major leaf carbohydrates was shifted in favor of sucrose and at the expense of starch during necrotrophic fungal growth. Arabidopsis mutants with impaired starch turnover were more susceptible toward C. higginsianum infection, exhibiting a strong negative correlation between diurnal carbohydrate accumulation and fungal proliferation for the tested genotypes. By altering the length of the light phase and employing additional genotypes impaired in nocturnal carbon mobilization, we revealed that reduced availability of carbon enhances susceptibility in the investigated pathosystem. Systematic starvation experiments resulted in two important findings. First, we showed that carbohydrate supply by the host is dispensable during biotrophic growth of C. higginsianum, while carbon deficiency was most harmful to the host during the necrotrophic colonization phase. Compared with the wild type, the increases in the total salicylic acid pool and camalexin accumulation were reduced in starch-free mutants at late interaction stages, while an increased ratio of free to total salicylic acid did not convey elevated pathogenesis-related gene expression in starch-free mutants. These observations suggest that reduced carbon availability dampens induced defense responses. In contrast, starch-free mutants were more resistant toward the fungal biotroph Erysiphe cruciferarum, indicating that reduced carbohydrate availability influences susceptibility differently in the interaction with the investigated hemibiotrophic and biotrophic fungal pathogens. PMID:23487433

Real-time Recording of Cytosolic Calcium Levels in Arabidopsis thaliana Cell Cultures during Parabolic Flights

NASA Astrophysics Data System (ADS)

Neef, Maren; Ecke, Margret; Hampp, Rüdiger

2015-07-01

In plants, like in other organisms, calcium (Ca2+) is an important second messenger which participates in the conversion of environmental signals into molecular responses. There is increasing evidence, that sensing of changes in gravitation or reorientation of tissues is an example for such signaling cascades in which Ca2+ is involved. In order to determine g-dependent changes in the cytosolic calcium (Ca^{2+}_{ {cyt}}) concentration of plant cells, semisolid transgenic callus cell cultures of Arabidopsis thaliana (A.t.), expressing the calcium sensor YC3.6 (cameleon), were exposed to g-forces between 1.8 g and μ g during parabolic flights. Using such cells, intracellular calcium transients can be monitored by FRET in vivo and in real-time. Interestingly we observed a slight decrease of the Ca^{2+}_{ {cyt}} level during the hypergravity phases of a parabola but a significant increase of the Ca^{2+}_{ {cyt}} concentration during microgravity. Application of known Ca2+ inhibitors and antagonists yielded the following effects: nifedipine (Ca2+ channel blocker) showed no effect, whereas LaCl3, GdCl3 (both inhibitors of uptake at the plasma membrane), DPI (inhibitor of NADP oxidase), and DMSO (solvent) diminished the gravity-alteration-related Ca^{2+}_{ {cyt}} response. EGTA (binding of Ca2+) and eosin yellow (inhibitor of a plasma membrane-located Ca2+ pump) suppressed the respective Ca^{2+}_{ {cyt}} changes entirely. We thus conclude that the significant increase in Ca^{2+}_{ {cyt}} under microgravity is largely due to extracellular Ca2+ sources.

Analyses of a Gravistimulation-Specific Ca2+ Signature in Arabidopsis using Parabolic Flights1[W][OPEN

PubMed Central

Toyota, Masatsugu; Furuichi, Takuya; Sokabe, Masahiro; Tatsumi, Hitoshi

2013-01-01

Gravity is a critical environmental factor affecting the morphology and functions of organisms on the Earth. Plants sense changes in the gravity vector (gravistimulation) and regulate their growth direction accordingly. In Arabidopsis (Arabidopsis thaliana) seedlings, gravistimulation, achieved by rotating the specimens under the ambient 1g of the Earth, is known to induce a biphasic (transient and sustained) increase in cytoplasmic calcium concentration ([Ca2+]c). However, the [Ca2+]c increase genuinely caused by gravistimulation has not been identified because gravistimulation is generally accompanied by rotation of specimens on the ground (1g), adding an additional mechanical signal to the treatment. Here, we demonstrate a gravistimulation-specific Ca2+ response in Arabidopsis seedlings by separating rotation from gravistimulation by using the microgravity (less than 10−4g) conditions provided by parabolic flights. Gravistimulation without rotating the specimen caused a sustained [Ca2+]c increase, which corresponds closely to the second sustained [Ca2+]c increase observed in ground experiments. The [Ca2+]c increases were analyzed under a variety of gravity intensities (e.g. 0.5g, 1.5g, or 2g) combined with rapid switching between hypergravity and microgravity, demonstrating that Arabidopsis seedlings possess a very rapid gravity-sensing mechanism linearly transducing a wide range of gravitational changes (0.5g–2g) into Ca2+ signals on a subsecond time scale. PMID:23835410

AtFXG1, an Arabidopsis gene encoding alpha-L-fucosidase active against fucosylated xyloglucan oligosaccharides.

PubMed

de La Torre, Francisco; Sampedro, Javier; Zarra, Ignacio; Revilla, Gloria

2002-01-01

An alpha-L-fucosidase (EC 3.2.1.51) able to release the t-fucosyl residue from the side chain of xyloglucan oligosaccharides has been detected in the leaves of Arabidopsis plants. Moreover, an alpha-L-fucosidase with similar substrate specificity was purified from cabbage (Brassica oleracea) leaves to render a single band on SDS-PAGE. Two peptide sequences were obtained from this protein band, and they were used to identify an Arabidopsis gene coding for an alpha-fucosidase that we propose to call AtFXG1. In addition, an Arabidopsis gene with homology with known alpha-L-fucosidases has been also found, and we proposed to name it as AtFUC1. Both AtFXG1 and ATFUC1 were heterologously expressed in Pichia pastoris cells and the alpha-L-fucosidase activities secreted to the culture medium. The alpha-L-fucosidase encoded by AtFXG1 was active against the oligosaccharides from xyloglucan XXFG as well as against 2'-fucosyl-lactitol but not against p-nitrophenyl-alpha-L-fucopyranoside. However, the AtFUC1 heterologously expressed was active only against 2'-fucosyl-lactitol. Thus, the former must be related to xyloglucan metabolism.

VIP1 is very important/interesting protein 1 regulating touch responses of Arabidopsis.

PubMed

Tsugama, Daisuke; Liu, Shenkui; Takano, Tetsuo

2016-06-02

VIP1 (VIRE2-INTERACTING PROTEIN 1) is a bZIP transcription factor in Arabidopsis thaliana. VIP1 and its close homologs (i.e., Arabidopsis group I bZIP proteins) are present in the cytoplasm under steady conditions, but are transiently localized to the nucleus when cells are exposed to hypo-osmotic conditions, which mimic mechanical stimuli such as touch. Recently we have reported that overexpression of a repression domain-fused form of VIP1 represses the expression of some touch-responsive genes, changes structures and/or local auxin responses of the root cap cells, and enhances the touch-induced root waving. This raises the possibility that VIP1 suppresses touch-induced responses. VIP1 should be useful to further characterize touch responses of plants. Here we discuss 2 seemingly interesting perspectives about VIP1: (1) What factors are involved in regulating the nuclear localization of VIP1?; (2) What can be done to further characterize the physiological functions of VIP1 and other Arabidopsis group I bZIP proteins?

The Sterol Methyltransferases SMT1, SMT2, and SMT3 Influence Arabidopsis Development through Nonbrassinosteroid Products1[W][OA

PubMed Central

Carland, Francine; Fujioka, Shozo; Nelson, Timothy

2010-01-01

Plant sterols are structural components of cell membranes that provide rigidity, permeability, and regional identity to membranes. Sterols are also the precursors to the brassinosteroid signaling molecules. Evidence is accumulating that specific sterols have roles in pattern formation during development. COTYLEDON VASCULAR PATTERNING1 (CVP1) encodes C-24 STEROL METHYLTRANSFERASE2 (SMT2), one of three SMTs in Arabidopsis (Arabidopsis thaliana). SMT2 and SMT3, which also encodes a C-24 SMT, catalyze the reaction that distinguishes the synthesis of structural sterols from signaling brassinosteroid derivatives and are highly regulated. The deficiency of SMT2 in the cvp1 mutant results in moderate developmental defects, including aberrant cotyledon vein patterning, serrated floral organs, and reduced stature, but plants are viable, suggesting that SMT3 activity can substitute for the loss of SMT2. To test the distinct developmental roles of SMT2 and SMT3, we identified a transcript null smt3 mutant. Although smt3 single mutants appear wild type, cvp1 smt3 double mutants show enhanced defects relative to cvp1 mutants, such as discontinuous cotyledon vein pattern, and produce novel phenotypes, including defective root growth, loss of apical dominance, sterility, and homeotic floral transformations. These phenotypes are correlated with major alterations in the profiles of specific sterols but without significant alterations to brassinosteroid profiles. The alterations to sterol profiles in cvp1 mutants affect auxin response, demonstrated by weak auxin insensitivity, enhanced axr1 auxin resistance, ectopically expressed DR5:β-glucuronidase in developing embryos, and defective response to auxin-inhibited PIN2-green fluorescent protein endocytosis. We discuss the developmental roles of sterols implied by these results. PMID:20421456

A Class I ADP-Ribosylation Factor GTPase-Activating Protein Is Critical for Maintaining Directional Root Hair Growth in Arabidopsis1[W][OA

PubMed Central

Yoo, Cheol-Min; Wen, Jiangqi; Motes, Christy M.; Sparks, J. Alan; Blancaflor, Elison B.

2008-01-01

Membrane trafficking and cytoskeletal dynamics are important cellular processes that drive tip growth in root hairs. These processes interact with a multitude of signaling pathways that allow for the efficient transfer of information to specify the direction in which tip growth occurs. Here, we show that AGD1, a class I ADP ribosylation factor GTPase-activating protein, is important for maintaining straight growth in Arabidopsis (Arabidopsis thaliana) root hairs, since mutations in the AGD1 gene resulted in wavy root hair growth. Live cell imaging of growing agd1 root hairs revealed bundles of endoplasmic microtubules and actin filaments extending into the extreme tip. The wavy phenotype and pattern of cytoskeletal distribution in root hairs of agd1 partially resembled that of mutants in an armadillo repeat-containing kinesin (ARK1). Root hairs of double agd1 ark1 mutants were more severely deformed compared with single mutants. Organelle trafficking as revealed by a fluorescent Golgi marker was slightly inhibited, and Golgi stacks frequently protruded into the extreme root hair apex of agd1 mutants. Transient expression of green fluorescent protein-AGD1 in tobacco (Nicotiana tabacum) epidermal cells labeled punctate bodies that partially colocalized with the endocytic marker FM4-64, while ARK1-yellow fluorescent protein associated with microtubules. Brefeldin A rescued the phenotype of agd1, indicating that the altered activity of an AGD1-dependent ADP ribosylation factor contributes to the defective growth, organelle trafficking, and cytoskeletal organization of agd1 root hairs. We propose that AGD1, a regulator of membrane trafficking, and ARK1, a microtubule motor, are components of converging signaling pathways that affect cytoskeletal organization to specify growth orientation in Arabidopsis root hairs. PMID:18539780

iTRAQ Analysis Reveals Mechanisms of Growth Defects Due to Excess Zinc in Arabidopsis1[W][OA

PubMed Central

Fukao, Yoichiro; Ferjani, Ali; Tomioka, Rie; Nagasaki, Nahoko; Kurata, Rie; Nishimori, Yuka; Fujiwara, Masayuki; Maeshima, Masayoshi

2011-01-01

The micronutrient zinc is essential for all living organisms, but it is toxic at high concentrations. Here, to understand the effects of excess zinc on plant cells, we performed an iTRAQ (for isobaric tags for relative and absolute quantification)-based quantitative proteomics approach to analyze microsomal proteins from Arabidopsis (Arabidopsis thaliana) roots. Our approach was sensitive enough to identify 521 proteins, including several membrane proteins. Among them, IRT1, an iron and zinc transporter, and FRO2, a ferric-chelate reductase, increased greatly in response to excess zinc. The expression of these two genes has been previously reported to increase under iron-deficient conditions. Indeed, the concentration of iron was significantly decreased in roots and shoots under excess zinc. Also, seven subunits of the vacuolar H+-ATPase (V-ATPase), a proton pump on the tonoplast and endosome, were identified, and three of them decreased significantly in response to excess zinc. In addition, excess zinc in the wild type decreased V-ATPase activity and length of roots and cells to levels comparable to those of the untreated de-etiolated3-1 mutant, which bears a mutation in V-ATPase subunit C. Interestingly, excess zinc led to the formation of branched and abnormally shaped root hairs, a phenotype that correlates with decreased levels of proteins of several root hair-defective mutants. Our results point out mechanisms of growth defects caused by excess zinc in which cross talk between iron and zinc homeostasis and V-ATPase activity might play a central role. PMID:21325567

Dissecting Phosphite-Induced Priming in Arabidopsis Infected with Hyaloperonospora arabidopsidis1[W

PubMed Central

Massoud, Kamal; Barchietto, Thierry; Le Rudulier, Thomas; Pallandre, Laurane; Didierlaurent, Laure; Garmier, Marie; Ambard-Bretteville, Françoise; Seng, Jean-Marc; Saindrenan, Patrick

2012-01-01

Phosphite (Phi), a phloem-mobile oxyanion of phosphorous acid (H3PO3), protects plants against diseases caused by oomycetes. Its mode of action is unclear, as evidence indicates both direct antibiotic effects on pathogens as well as inhibition through enhanced plant defense responses, and its target(s) in the plants is unknown. Here, we demonstrate that the biotrophic oomycete Hyaloperonospora arabidopsidis (Hpa) exhibits an unusual biphasic dose-dependent response to Phi after inoculation of Arabidopsis (Arabidopsis thaliana), with characteristics of indirect activity at low doses (10 mm or less) and direct inhibition at high doses (50 mm or greater). The effect of low doses of Phi on Hpa infection was nullified in salicylic acid (SA)-defective plants (sid2-1, NahG) and in a mutant impaired in SA signaling (npr1-1). Compromised jasmonate (jar1-1) and ethylene (ein2-1) signaling or abscisic acid (aba1-5) biosynthesis, reactive oxygen generation (atrbohD), or accumulation of the phytoalexins camalexin (pad3-1) and scopoletin (f6′h1-1) did not affect Phi activity. Low doses of Phi primed the accumulation of SA and Pathogenesis-Related protein1 transcripts and mobilized two essential components of basal resistance, Enhanced Disease Susceptibility1 and Phytoalexin Deficient4, following pathogen challenge. Compared with inoculated, Phi-untreated plants, the gene expression, accumulation, and phosphorylation of the mitogen-activated protein kinase MPK4, a negative regulator of SA-dependent defenses, were reduced in plants treated with low doses of Phi. We propose that Phi negatively regulates MPK4, thus priming SA-dependent defense responses following Hpa infection. PMID:22408091

Multigene Engineering of Triacylglycerol Metabolism Boosts Seed Oil Content in Arabidopsis1[W][OPEN

PubMed Central

van Erp, Harrie; Kelly, Amélie A.; Menard, Guillaume; Eastmond, Peter J.

2014-01-01

Increasing the yield of oilseed crops is an important objective for biotechnologists. A number of individual genes involved in triacylglycerol metabolism have previously been reported to enhance the oil content of seeds when their expression is altered. However, it has yet to be established whether specific combinations of these genes can be used to achieve an additive effect and whether this leads to enhanced yield. Using Arabidopsis (Arabidopsis thaliana) as an experimental system, we show that seed-specific overexpression of WRINKLED1 (a transcriptional regulator of glycolysis and fatty acid synthesis) and DIACYLGLYCEROL ACYLTRANSFERASE1 (a triacylglycerol biosynthetic enzyme) combined with suppression of the triacylglycerol lipase SUGAR-DEPENDENT1 results in a higher percentage seed oil content and greater seed mass than manipulation of each gene individually. Analysis of total seed yield per plant suggests that, despite a reduction in seed number, the total yield of oil is also increased. PMID:24696520

Nuclear Trapping Controls the Position-Dependent Localization of CAPRICE in the Root Epidermis of Arabidopsis1[C][W

PubMed Central

Kang, Yeon Hee; Song, Sang-Kee; Schiefelbein, John; Lee, Myeong Min

2013-01-01

Cell fate determination and differentiation are central processes in the development of multicellular organisms, and the Arabidopsis (Arabidopsis thaliana) root epidermis provides a model system to study the molecular basis of these processes. A lateral inhibition mechanism mediated by an R3 single-repeat MYB protein, CAPRICE (CPC), has been proposed to explain the specification of the two types of root epidermal cells (hair cells and nonhair cells). However, it is not clear how CPC acts preferentially in the H-position cells, rather than the N-position cells, where its gene is expressed. To explore this issue, we examined the effect of misexpressed CPC on cell fate specification and CPC localization in the root epidermis. We show that CPC is able to move readily within the root epidermis when its expression level is high and that CPC can induce the hair cell fate in a cell-autonomous manner. We provide evidence that CPC is capable of moving from the stele tissue in the center of the root to the outermost epidermal layer, where it can induce the hair cell fate. In addition, we show that CPC protein accumulates primarily in the nuclei of H-position cells in the early meristematic region, and this localization requires the H-cell-expressed ENHANCER OF GLABRA3 (EGL3) basic helix-loop-helix transcription factor. These results suggest that cell-cell movement of CPC occurs readily within the meristematic region of the root and that EGL3 preferentially traps the CPC protein in the H-position cells of the epidermis. PMID:23832626

Mutation of the Membrane-Associated M1 Protease APM1 Results in Distinct Embryonic and Seedling Developmental Defects in Arabidopsis[C][W

PubMed Central

Peer, Wendy Ann; Hosein, Fazeeda N.; Bandyopadhyay, Anindita; Makam, Srinivas N.; Otegui, Marisa S.; Lee, Gil-Je; Blakeslee, Joshua J.; Cheng, Yan; Titapiwatanakun, Boosaree; Yakubov, Bahktiyor; Bangari, Bharat; Murphy, Angus S.

2009-01-01

Aminopeptidase M1 (APM1), a single copy gene in Arabidopsis thaliana, encodes a metallopeptidase originally identified via its affinity for, and hydrolysis of, the auxin transport inhibitor 1-naphthylphthalamic acid (NPA). Mutations in this gene result in haploinsufficiency. Loss-of-function mutants show irregular, uncoordinated cell divisions throughout embryogenesis, affecting the shape and number of cotyledons and the hypophysis, and is seedling lethal at 5 d after germination due to root growth arrest. Quiescent center and cell cycle markers show no signals in apm1-1 knockdown mutants, and the ground tissue specifiers SHORTROOT and SCARECROW are misexpressed or mislocalized. apm1 mutants have multiple, fused cotyledons and hypocotyls with enlarged epidermal cells with cell adhesion defects. apm1 alleles show defects in gravitropism and auxin transport. Gravistimulation decreases APM1 expression in auxin-accumulating root epidermal cells, and auxin treatment increases expression in the stele. On sucrose gradients, APM1 occurs in unique light membrane fractions. APM1 localizes at the margins of Golgi cisternae, plasma membrane, select multivesicular bodies, tonoplast, dense intravacuolar bodies, and maturing metaxylem cells. APM1 associates with brefeldin A–sensitive endomembrane structures and the plasma membrane in cortical and epidermal cells. The auxin-related phenotypes and mislocalization of auxin efflux proteins in apm1 are consistent with biochemical interactions between APM1 and NPA. PMID:19531600

Xyloglucan Deficiency Disrupts Microtubule Stability and Cellulose Biosynthesis in Arabidopsis, Altering Cell Growth and Morphogenesis1[OPEN

PubMed Central

Xiao, Chaowen; Zhang, Tian; Zheng, Yunzhen

2016-01-01

Xyloglucan constitutes most of the hemicellulose in eudicot primary cell walls and functions in cell wall structure and mechanics. Although Arabidopsis (Arabidopsis thaliana) xxt1 xxt2 mutants lacking detectable xyloglucan are viable, they display growth defects that are suggestive of alterations in wall integrity. To probe the mechanisms underlying these defects, we analyzed cellulose arrangement, microtubule patterning and dynamics, microtubule- and wall-integrity-related gene expression, and cellulose biosynthesis in xxt1 xxt2 plants. We found that cellulose is highly aligned in xxt1 xxt2 cell walls, that its three-dimensional distribution is altered, and that microtubule patterning and stability are aberrant in etiolated xxt1 xxt2 hypocotyls. We also found that the expression levels of microtubule-associated genes, such as MAP70-5 and CLASP, and receptor genes, such as HERK1 and WAK1, were changed in xxt1 xxt2 plants and that cellulose synthase motility is reduced in xxt1 xxt2 cells, corresponding with a reduction in cellulose content. Our results indicate that loss of xyloglucan affects both the stability of the microtubule cytoskeleton and the production and patterning of cellulose in primary cell walls. These findings establish, to our knowledge, new links between wall integrity, cytoskeletal dynamics, and wall synthesis in the regulation of plant morphogenesis. PMID:26527657

Investigating the Molecular Mechanism of TSO1 Function in Arabidopsis cell division and meristem development

DOE Office of Scientific and Technical Information (OSTI.GOV)

Zhongchi Liu

2004-10-01

Unlike animals, plants are constantly exposed to environmental mutagens including ultraviolet light and reactive oxygen species. Further, plant cells are totipotent with highly plastic developmental programs. An understanding of molecular mechanisms underlying the ability of plants to monitor and repair its DNA and to eliminate damaged cells are of great importance. Previously we have identified two genes, TSO1 and TSO2, from a flowering plant Arabidopsis thaliana. Mutations in these two genes cause callus-like flowers, fasciated shoot apical meristems, and abnormal cell division, indicating that TSO1 and TSO2 may encode important cell cycle regulators. Previous funding from DOE led to themore » molecular cloning of TSO1, which was shown to encode a novel nuclear protein with two CXC domains suspected to bind DNA. This DOE grant has allowed us to characterize and isolate TSO2 that encodes the small subunit of the ribonucleotide reductase (RNR). RNR comprises two large subunits (R1) an d two small subunits (R2), catalyzes a rate-limiting step in the production of deoxyribonucleotides needed for DNA replication and repair. Previous studies in yeast and mammals indicated that defective RNR often led to cell cycle arrest, growth retardation and p53-dependent apoptosis while abnormally elevated RNR activities led to higher mutation rates. Subsequently, we identified two additional R2 genes, R2A and R2B in the Arabidopsis genome. Using reverse genetics, mutations in R2A and R2B were isolated, and double and triple mutants among the three R2 genes (TSO2, R2A and R2B) were constructed and analyzed. We showed that Arabidopsis tso2 mutants, with reduced dNTP levels, were more sensitive to UV-C. While r2a or r2b single mutants did not exhibit any phenotypes, tso2 r2b double mutants were embryonic lethal and tso2 r2a double mutants were seedling lethal indicating redundant functions among the three R2 genes. Furthermore, tso2 r2a double mutants exhibited increased DNA dam

Changes in Gene Expression of Arabidopsis Thaliana Cell Cultures Upon Exposure to Real and Simulated Partial- g Forces

NASA Astrophysics Data System (ADS)

Fengler, Svenja; Spirer, Ina; Neef, Maren; Ecke, Margret; Hauslage, Jens; Hampp, Rüdiger

2016-06-01

Cell cultures of the plant model organism Arabidopsis thaliana were exposed to partial- g forces during parabolic flight and clinostat experiments (0.16 g, 0.38 g and 0.5 g were tested). In order to investigate gravity-dependent alterations in gene expression, samples were metabolically quenched by the fixative RNA later Ⓡ to stabilize nucleic acids and used for whole-genome microarray analysis. An attempt to identify the potential threshold acceleration for the gravity-dependent response showed that the smaller the experienced g-force, the greater was the susceptibility of the cell cultures. Compared to short-term μ g during a parabolic flight, the number of differentially expressed genes under partial- g was lower. In addition, the effect on the alteration of amounts of transcripts decreased during partial- g parabolic flight due to the sequence of the different parabolas (0.38 g, 0.16 g and μ g). A time-dependent analysis under simulated 0.5 g indicates that adaptation occurs within minutes. Differentially expressed genes (at least 2-fold up- or down-regulated in expression) under real flight conditions were to some extent identical with those affected by clinorotation. The highest number of homologuous genes was detected within seconds of exposure to 0.38 g (both flight and clinorotation). To a considerable part, these genes deal with cell wall properties. Additionally, responses specific for clinorotation were observed.

Arabidopsis WRKY45 transcription factor activates PHOSPHATE TRANSPORTER1;1 expression in response to phosphate starvation.

PubMed

Wang, Hui; Xu, Qian; Kong, You-Han; Chen, Yun; Duan, Jun-Ye; Wu, Wei-Hua; Chen, Yi-Fang

2014-04-01

The WRKY transcription factor family has more than 70 members in the Arabidopsis (Arabidopsis thaliana) genome, and some of them are involved in plant responses to biotic and abiotic stresses. This study evaluated the role of WRKY45 in regulating phosphate (Pi) uptake in Arabidopsis. WRKY45 was localized in the nucleus and mainly expressed in roots. During Pi starvation, WRKY45 expression was markedly induced, typically in roots. WRKY45 overexpression in Arabidopsis increased Pi content and uptake, while RNA interference suppression of WRKY45 decreased Pi content and uptake. Furthermore, the WRKY45-overexpressing lines were more sensitive to arsenate, the analog of Pi, compared with wild-type seedlings. These results indicate that WRKY45 positively regulates Arabidopsis Pi uptake. Quantitative real-time polymerase chain reaction and β-glucuronidase staining assays showed that PHOSPHATE TRANSPORTER1;1 (PHT1;1) expression was enhanced in the WRKY45-overexpressing lines and slightly repressed in the WRKY45 RNA interference line. Chromatin immunoprecipitation and electrophoretic mobility shift assay results indicated that WRKY45 can bind to two W-boxes within the PHT1;1 promoter, confirming the role of WRKY45 in directly up-regulating PHT1;1 expression. The pht1;1 mutant showed decreased Pi content and uptake, and overexpression of PHT1;1 resulted in enhanced Pi content and uptake. Furthermore, the PHT1;1-overexpressing line was much more sensitive to arsenate than WRKY45-overexpressing and wild-type seedlings, indicating that PHT1;1 overexpression can enhance Arabidopsis Pi uptake. Moreover, the enhanced Pi uptake and the increased arsenate sensitivity of the WRKY45-overexpressing line was impaired by pht1;1 (35S:WRKY45-18::pht1;1), demonstrating an epistatic genetic regulation between WRKY45 and PHT1;1. Together, our results demonstrate that WRKY45 is involved in Arabidopsis response to Pi starvation by direct up-regulation of PHT1;1 expression.

Utilisation of water-in-oil-water (W1/O/W2) double emulsion in a set-type yogurt model for the delivery of probiotic Lactobacillus paracasei.

PubMed

El Kadri, Hani; Lalou, Sofia; Mantzouridou, FaniTh; Gkatzionis, Konstantinos

2018-05-01

W 1 /O/W 2 emulsion in set-type yogurt has the potential to segregate probiotics in order to avoid interference with the starter culture as well as protection against harsh processing and digestion conditions. Lactobacillus paracasei subsp. paracasei DC 412 probiotic cells in milk-based W 1 /O/W 2 emulsions were incorporated in yogurt, in addition to starter cultures Lactobacillus bulgaricus and Streptococcus thermophilus, and the effect on the fermentation, bacterial growth kinetics, physicochemical properties, and structural characteristics was investigated. Stability of W 1 /O/W 2 was monitored with optical microscopy and cryo-SEM and localisation of encapsulated L. paracasei in yogurt was monitored using fluorescent microscopy. During fermentation, starter culture was not affected by introduction of L. paracasei and/or W 1 /O/W 2 emulsion. The viability of L. paracasei encapsulated in W 1 /O/W 2 emulsion was enhanced during storage and after exposure to simulated gastrointestinal conditions. L. paracasei remained within the inner W 1 phase till the end of the storage period (28 days at 4 °C). Moreover, W 1 /O/W 2 emulsion altered physicochemical and textural properties; however, these were within acceptable range. These results demonstrate the capability of W 1 /O/W 2 emulsion to be utilised for probiotic fortification of yogurt to increase functionality without interfering with starter culture and fermentation. Copyright © 2018 Elsevier Ltd. All rights reserved.

LYK4, a Lysin Motif Receptor-Like Kinase, Is Important for Chitin Signaling and Plant Innate Immunity in Arabidopsis1[C][W][OA

PubMed Central

Wan, Jinrong; Tanaka, Kiwamu; Zhang, Xue-Cheng; Son, Geon Hui; Brechenmacher, Laurent; Nguyen, Tran Hong Nha; Stacey, Gary

2012-01-01

Chitin is commonly found in fungal cell walls and is one of the well-studied microbe/pathogen-associated molecular patterns. Previous studies showed that lysin motif (LysM)-containing proteins are essential for plant recognition of chitin, leading to the activation of plant innate immunity. In Arabidopsis (Arabidopsis thaliana), the LYK1/CERK1 (for LysM-containing receptor-like kinase1/chitin elicitor receptor kinase1) was shown to be essential for chitin recognition, whereas in rice (Oryza sativa), the LysM-containing protein, CEBiP (for chitin elicitor-binding protein), was shown to be involved in chitin recognition. Unlike LYK1/CERK1, CEBiP lacks an intracellular kinase domain. Arabidopsis possesses three CEBiP-like genes. Our data show that mutations in these genes, either singly or in combination, did not compromise the response to chitin treatment. Arabidopsis also contains five LYK genes. Analysis of mutations in LYK2, -3, -4, or -5 showed that LYK4 is also involved in chitin signaling. The lyk4 mutants showed reduced induction of chitin-responsive genes and diminished chitin-induced cytosolic calcium elevation as well as enhanced susceptibility to both the bacterial pathogen Pseudomonas syringae pv tomato DC3000 and the fungal pathogen Alternaria brassicicola, although these phenotypes were not as dramatic as that seen in the lyk1/cerk1 mutants. Similar to LYK1/CERK1, the LYK4 protein was also localized to the plasma membrane. Therefore, LYK4 may play a role in the chitin recognition receptor complex to assist chitin signal transduction and plant innate immunity. PMID:22744984

Pectin Biosynthesis: GALS1 in Arabidopsis thaliana Is a β-1,4-Galactan β-1,4-Galactosyltransferase[C][W][OA

PubMed Central

Liwanag, April Jennifer Madrid; Ebert, Berit; Verhertbruggen, Yves; Rennie, Emilie A.; Rautengarten, Carsten; Oikawa, Ai; Andersen, Mathias C.F.; Clausen, Mads H.; Scheller, Henrik Vibe

2012-01-01

β-1,4-Galactans are abundant polysaccharides in plant cell walls, which are generally found as side chains of rhamnogalacturonan I. Rhamnogalacturonan I is a major component of pectin with a backbone of alternating rhamnose and galacturonic acid residues and side chains that include α-1,5-arabinans, β-1,4-galactans, and arabinogalactans. Many enzymes are required to synthesize pectin, but few have been identified. Pectin is most abundant in primary walls of expanding cells, but β-1,4-galactan is relatively abundant in secondary walls, especially in tension wood that forms in response to mechanical stress. We investigated enzymes in glycosyltransferase family GT92, which has three members in Arabidopsis thaliana, which we designated GALACTAN SYNTHASE1, (GALS1), GALS2 and GALS3. Loss-of-function mutants in the corresponding genes had a decreased β-1,4-galactan content, and overexpression of GALS1 resulted in plants with 50% higher β-1,4-galactan content. The plants did not have an obvious growth phenotype. Heterologously expressed and affinity-purified GALS1 could transfer Gal residues from UDP-Gal onto β-1,4-galactopentaose. GALS1 specifically formed β-1,4-galactosyl linkages and could add successive β-1,4-galactosyl residues to the acceptor. These observations confirm the identity of the GT92 enzyme as β-1,4-galactan synthase. The identification of this enzyme could provide an important tool for engineering plants with improved bioenergy properties. PMID:23243126

Monitoring Meso-Scale Ordering of Cellulose in Intact Plant Cell Walls Using Sum Frequency Generation Spectroscopy1[C][W][OPEN

PubMed Central

Park, Yong Bum; Lee, Christopher M.; Koo, Bon-Wook; Park, Sunkyu; Cosgrove, Daniel J.; Kim, Seong H.

2013-01-01

Sum frequency generation (SFG) vibration spectroscopy can selectively detect crystalline cellulose without spectral interference from cell wall matrix components. Here, we show that the cellulose SFG spectrum is sensitive to cellulose microfibril alignment and packing within the cell wall. SFG intensity at 2,944 cm−1 correlated well with crystalline cellulose contents of various regions of the Arabidopsis (Arabidopsis thaliana) inflorescence, while changes in the 3,320/2,944 cm−1 intensity ratio suggest subtle changes in cellulose ordering as tissues mature. SFG analysis of two cellulose synthase mutants (irx1/cesa8 and irx3/cesa7) indicates a reduction in cellulose content without evidence of altered cellulose structure. In primary cell walls of Arabidopsis, cellulose exhibited a characteristic SFG peak at 2,920 and 3,320 cm−1, whereas in secondary cell walls, it had peaks at 2,944 and 3,320 cm−1. Starch (amylose) gave an SFG peak at 2,904 cm−1 (CH methine) whose intensity increased with light exposure prior to harvest. Selective removal of matrix polysaccharides from primary cell walls by acid hydrolysis resulted in an SFG spectrum resembling that of secondary wall cellulose. Our results show that SFG spectroscopy is sensitive to the ordering of cellulose microfibrils in plant cell walls at the meso scale (nm to μm) that is important for cell wall architecture but cannot be probed by other spectroscopic or diffraction techniques. PMID:23995148

Gain-of-function mutation of AtDICE1, encoding a putative endoplasmic reticulum-localized membrane protein, causes defects in anisotropic cell elongation by disturbing cell wall integrity in Arabidopsis.

PubMed

Le, Phi-Yen; Jeon, Hyung-Woo; Kim, Min-Ha; Park, Eung-Jun; Lee, Hyoshin; Hwang, Indeok; Han, Kyung-Hwan; Ko, Jae-Heung

2018-04-05

Anisotropic cell elongation depends on cell wall relaxation and cellulose microfibril arrangement. The aim of this study was to characterize the molecular function of AtDICE1 encoding a novel transmembrane protein involved in anisotropic cell elongation in Arabidopsis. Phenotypic characterizations of transgenic Arabidopsis plants mis-regulating AtDICE1 expression with different pharmacological treatments were made, and biochemical, cell biological and transcriptome analyses were performed. Upregulation of AtDICE1 in Arabidopsis (35S::AtDICE1) resulted in severe dwarfism, probably caused by defects in anisotropic cell elongation. Epidermal cell swelling was evident in all tissues, and abnormal secondary wall thickenings were observed in pith cells of stems. These phenotypes were reproduced not only by inducible expression of AtDICE1 but also by overexpression of its poplar homologue in Arabidopsis. RNA interference suppression lines of AtDICE1 resulted in no observable phenotypic changes. Interestingly, wild-type plants treated with isoxaben, a cellulose biosynthesis inhibitor, phenocopied the 35S::AtDICE1 plants, suggesting that cellulose biosynthesis was compromised in the 35S::AtDICE1 plants. Indeed, disturbed cortical microtubule arrangements in 35S::AtDICE1/GFP-TuA6 plants were observed, and the cellulose content was significantly reduced in 35S::AtDICE1 plants. A promoter::GUS analysis showed that AtDICE1 is mainly expressed in vascular tissue, and transient expression of GFP:AtDICE1 in tobacco suggests that AtDICE1 is probably localized in the endoplasmic reticulum (ER). In addition, the external N-terminal conserved domain of AtDICE1 was found to be necessary for AtDICE1 function. Whole transcriptome analyses of 35S::AtDICE1 revealed that many genes involved in cell wall modification and stress/defence responses were mis-regulated. AtDICE1, a novel ER-localized transmembrane protein, may contribute to anisotropic cell elongation in the formation of vascular

Turbidostat Culture of Saccharomyces cerevisiae W303-1A under Selective Pressure Elicited by Ethanol Selects for Mutations in SSD1 and UTH1

PubMed Central

Avrahami-Moyal, Liat; Engelberg, David; Wenger, Jared. W.; Sherlock, Gavin; Braun, Sergei

2012-01-01

We investigated the genetic causes of ethanol tolerance by characterizing mutations selected in Saccharomyces cerevisiae W303-1A under the selective pressure of ethanol. W303-1A was subjected to three rounds of turbidostat, in medium supplemented with increasing amounts of ethanol. By the end of selection, the growth rate of the culture has increased from 0.029 h-1 to 0.32 h-1. Unlike the progenitor strain, all yeast cells isolated from this population were able to form colonies on medium supplemented with 7% ethanol within six days, our definition of ethanol tolerance. Several clones selected from all three stages of selection were able to form dense colonies within two days on solid medium supplemented with 9% ethanol. We sequenced the whole genomes of 6 clones and identified mutations responsible for ethanol tolerance. Thirteen additional clones were tested for the presence of similar mutations. In 15 out of 19 tolerant clones the stop-codon in ssd1-d was replaced with an aminoacid-encoding codon. Three other clones contained one of two mutations in UTH1, and one clone did not contain mutations in either SSD1 or UTH1. We showed that the mutations in SSD1 and UTH1 increased tolerance of the cell wall to zymolyase and conclude that stability of the cell wall is a major factor in increased tolerance to ethanol. PMID:22443114

Genetic Analysis of Growth-Regulator-Induced Parthenocarpy in Arabidopsis1

PubMed Central

Vivian-Smith, Adam; Koltunow, Anna M.

1999-01-01

In Arabidopsis, seedless silique development or parthenocarpy can be induced by the application of various plant growth regulators (PGRs) to unfertilized pistils. Ecotype-specific responses were observed in the Arabidopsis ecotypes Columbia and Landsberg relative to the type of PGR and level applied. The parthenocarpic response was greatest in ecotype Landsberg, and comparisons of fruit growth and morphology were studied primarily in this ecotype. Gibberellic acid application (10 μmol pistil−1) caused development similar to that in pollinated pistils, while benzyladenine (1 μmol pistil−1) and naphthylacetic acid (10 μmol pistil−1) treatment produced shorter siliques. Naphthylacetic acid primarily modified mesocarp cell expansion. Arabidopsis mutants were employed to examine potential dependencies on gibberellin biosynthesis (ga1-3, ga4-1, and ga5-1) and perception (spy-4 and gai) during parthenocarpic silique development. Emasculated spy-4 pistils were neither obviously parthenocarpic nor deficient in PGR perception. By contrast, emasculated gai mutants did not produce parthenocarpic siliques following gibberellic acid application, but silique development occurred following pollination or application of auxin and cytokinin. Pollinated gai siliques had decreased cell numbers and morphologically resembled auxin-induced parthenocarpic siliques. This shows that a number of independent and possibly redundant pathways can direct hormone-induced parthenocarpy, and that endogenous gibberellins play a role in regulating cell expansion and promoting cell division in carpels. PMID:10517835

Arabidopsis REGULATOR OF AXILLARY MERISTEMS1 controls a leaf axil stem cell niche and modulates vegetative development.

PubMed

Keller, Thomas; Abbott, Jessica; Moritz, Thomas; Doerner, Peter

2006-03-01

Shoot branching is a major determinant of variation in plant stature. Branches, which form secondary growth axes, originate from stem cells activated in leaf axils. The initial steps by which axillary meristems (AMs) are specified and their stem cells organized are still poorly understood. We identified gain- and loss-of-function alleles at the Arabidopsis thaliana REGULATOR OF AXILLARY MERISTEMS1 (RAX1) locus. RAX1 is encoded by the Myb-like transcription factor MYB37 and is an Arabidopsis homolog of the tomato (Solanum lycopersicum) Blind gene. RAX1 is transiently expressed in a small central domain within the boundary zone separating shoot apical meristem and leaf primordia early in leaf primordium development. RAX1 genetically interacts with CUP-SHAPED COTYLEDON (CUC) genes and is required for the expression of CUC2 in the RAX1 expression domain, suggesting that RAX1 acts through CUC2. We propose that RAX1 functions to positionally specify a stem cell niche for AM formation. RAX1 also affects the timing of developmental phase transitions by negatively regulating gibberellic acid levels in the shoot apex. RAX1 thus defines a novel activity that links the specification of AM formation with the modulation of the rate of progression through developmental phases.

KIRA1 and ORESARA1 terminate flower receptivity by promoting cell death in the stigma of Arabidopsis.

PubMed

Gao, Zhen; Daneva, Anna; Salanenka, Yuliya; Van Durme, Matthias; Huysmans, Marlies; Lin, Zongcheng; De Winter, Freya; Vanneste, Steffen; Karimi, Mansour; Van de Velde, Jan; Vandepoele, Klaas; Van de Walle, Davy; Dewettinck, Koen; Lambrecht, Bart N; Nowack, Moritz K

2018-05-28

Flowers have a species-specific functional life span that determines the time window in which pollination, fertilization and seed set can occur. The stigma tissue plays a key role in flower receptivity by intercepting pollen and initiating pollen tube growth toward the ovary. In this article, we show that a developmentally controlled cell death programme terminates the functional life span of stigma cells in Arabidopsis. We identified the leaf senescence regulator ORESARA1 (also known as ANAC092) and the previously uncharacterized KIRA1 (also known as ANAC074) as partially redundant transcription factors that modulate stigma longevity by controlling the expression of programmed cell death-associated genes. KIRA1 expression is sufficient to induce cell death and terminate floral receptivity, whereas lack of both KIRA1 and ORESARA1 substantially increases stigma life span. Surprisingly, the extension of stigma longevity is accompanied by only a moderate extension of flower receptivity, suggesting that additional processes participate in the control of the flower's receptive life span.

Genome-Wide Identification and Testing of Superior Reference Genes for Transcript Normalization in Arabidopsis1[w

PubMed Central

Czechowski, Tomasz; Stitt, Mark; Altmann, Thomas; Udvardi, Michael K.; Scheible, Wolf-Rüdiger

2005-01-01

Gene transcripts with invariant abundance during development and in the face of environmental stimuli are essential reference points for accurate gene expression analyses, such as RNA gel-blot analysis or quantitative reverse transcription-polymerase chain reaction (PCR). An exceptionally large set of data from Affymetrix ATH1 whole-genome GeneChip studies provided the means to identify a new generation of reference genes with very stable expression levels in the model plant species Arabidopsis (Arabidopsis thaliana). Hundreds of Arabidopsis genes were found that outperform traditional reference genes in terms of expression stability throughout development and under a range of environmental conditions. Most of these were expressed at much lower levels than traditional reference genes, making them very suitable for normalization of gene expression over a wide range of transcript levels. Specific and efficient primers were developed for 22 genes and tested on a diverse set of 20 cDNA samples. Quantitative reverse transcription-PCR confirmed superior expression stability and lower absolute expression levels for many of these genes, including genes encoding a protein phosphatase 2A subunit, a coatomer subunit, and an ubiquitin-conjugating enzyme. The developed PCR primers or hybridization probes for the novel reference genes will enable better normalization and quantification of transcript levels in Arabidopsis in the future. PMID:16166256

CER4 Encodes an Alcohol-Forming Fatty Acyl-Coenzyme A Reductase Involved in Cuticular Wax Production in Arabidopsis1[W

PubMed Central

Rowland, Owen; Zheng, Huanquan; Hepworth, Shelley R.; Lam, Patricia; Jetter, Reinhard; Kunst, Ljerka

2006-01-01

A waxy cuticle that serves as a protective barrier against uncontrolled water loss and environmental damage coats the aerial surfaces of land plants. It is composed of a cutin polymer matrix and waxes. Cuticular waxes are complex mixtures of very-long-chain fatty acids and their derivatives. We report here the molecular cloning and characterization of CER4, a wax biosynthetic gene from Arabidopsis (Arabidopsis thaliana). Arabidopsis cer4 mutants exhibit major decreases in stem primary alcohols and wax esters, and slightly elevated levels of aldehydes, alkanes, secondary alcohols, and ketones. This phenotype suggested that CER4 encoded an alcohol-forming fatty acyl-coenzyme A reductase (FAR). We identified eight FAR-like genes in Arabidopsis that are highly related to an alcohol-forming FAR expressed in seeds of jojoba (Simmondsia chinensis). Molecular characterization of CER4 alleles and genomic complementation revealed that one of these eight genes, At4g33790, encoded the FAR required for cuticular wax production. Expression of CER4 cDNA in yeast (Saccharomyces cerevisiae) resulted in the accumulation of C24:0 and C26:0 primary alcohols. Fully functional green fluorescent protein-tagged CER4 protein was localized to the endoplasmic reticulum in yeast cells by confocal microscopy. Analysis of gene expression by reverse transcription-PCR indicated that CER4 was expressed in leaves, stems, flowers, siliques, and roots. Expression of a β-glucuronidase reporter gene driven by the CER4 promoter in transgenic plants was detected in epidermal cells of leaves and stems, consistent with a dedicated role for CER4 in cuticular wax biosynthesis. CER4 was also expressed in all cell types in the elongation zone of young roots. These data indicate that CER4 is an alcohol-forming FAR that has specificity for very-long-chain fatty acids and is responsible for the synthesis of primary alcohols in the epidermal cells of aerial tissues and in roots. PMID:16980563

Biochemical and immunocytological characterizations of Arabidopsis pollen tube cell wall.

PubMed

Dardelle, Flavien; Lehner, Arnaud; Ramdani, Yasmina; Bardor, Muriel; Lerouge, Patrice; Driouich, Azeddine; Mollet, Jean-Claude

2010-08-01

During plant sexual reproduction, pollen germination and tube growth require development under tight spatial and temporal control for the proper delivery of the sperm cells to the ovules. Pollen tubes are fast growing tip-polarized cells able to perceive multiple guiding signals emitted by the female organ. Adhesion of pollen tubes via cell wall molecules may be part of the battery of signals. In order to study these processes, we investigated the cell wall characteristics of in vitro-grown Arabidopsis (Arabidopsis thaliana) pollen tubes using a combination of immunocytochemical and biochemical techniques. Results showed a well-defined localization of cell wall epitopes. Low esterified homogalacturonan epitopes were found mostly in the pollen tube wall back from the tip. Xyloglucan and arabinan from rhamnogalacturonan I epitopes were detected along the entire tube within the two wall layers and the outer wall layer, respectively. In contrast, highly esterified homogalacturonan and arabinogalactan protein epitopes were found associated predominantly with the tip region. Chemical analysis of the pollen tube cell wall revealed an important content of arabinosyl residues (43%) originating mostly from (1-->5)-alpha-L-arabinan, the side chains of rhamnogalacturonan I. Finally, matrix-assisted laser desorption ionization time-of-flight mass spectrometry analysis of endo-glucanase-sensitive xyloglucan showed mass spectra with two dominant oligosaccharides (XLXG/XXLG and XXFG), both being mono O-acetylated, and accounting for over 68% of the total ion signals. These findings demonstrate that the Arabidopsis pollen tube wall has its own characteristics compared with other cell types in the Arabidopsis sporophyte. These structural features are discussed in terms of pollen tube cell wall biosynthesis and growth dynamics.

The Pepper Extracellular Xyloglucan-Specific Endo-β-1,4-Glucanase Inhibitor Protein Gene, CaXEGIP1, Is Required for Plant Cell Death and Defense Responses1[C][W][OA

PubMed Central

Choi, Hyong Woo; Kim, Nak Hyun; Lee, Yeon Kyeong; Hwang, Byung Kook

2013-01-01

Plants produce various proteinaceous inhibitors to protect themselves against microbial pathogen attack. A xyloglucan-specific endo-β-1,4-glucanase inhibitor1 gene, CaXEGIP1, was isolated and functionally characterized in pepper (Capsicum annuum) plants. CaXEGIP1 was rapidly and strongly induced in pepper leaves infected with avirulent Xanthomonas campestris pv vesicatoria, and purified CaXEGIP1 protein significantly inhibited the hydrolytic activity of the glycoside hydrolase74 family xyloglucan-specific endo-β-1,4-glucanase from Clostridium thermocellum. Soluble-modified green fluorescent protein-tagged CaXEGIP1 proteins were mainly localized to the apoplast of onion (Allium cepa) epidermal cells. Agrobacterium tumefaciens-mediated overexpression of CaXEGIP1 triggered pathogen-independent, spontaneous cell death in pepper and Nicotiana benthamiana leaves. CaXEGIP1 silencing in pepper conferred enhanced susceptibility to virulent and avirulent X. campestris pv vesicatoria, accompanied by a compromised hypersensitive response and lowered expression of defense-related genes. Overexpression of dexamethasone:CaXEGIP1 in Arabidopsis (Arabidopsis thaliana) enhanced resistance to Hyaloperonospora arabidopsidis infection. Comparative histochemical and proteomic analyses revealed that CaXEGIP1 overexpression induced a spontaneous cell death response and also increased the expression of some defense-related proteins in transgenic Arabidopsis leaves. This response was also accompanied by cell wall thickening and darkening. Together, these results suggest that pathogen-inducible CaXEGIP1 positively regulates cell death-mediated defense responses in plants. PMID:23093361

ARABIDOPSIS THALIANA HOMEOBOX25 Uncovers a Role for Gibberellins in Seed Longevity1[C][W

PubMed Central

Bueso, Eduardo; Muñoz-Bertomeu, Jesús; Campos, Francisco; Brunaud, Veronique; Martínez, Liliam; Sayas, Enric; Ballester, Patricia; Yenush, Lynne; Serrano, Ramón

2014-01-01

Seed longevity is crucial for agriculture and plant genetic diversity, but it is limited by cellular damage during storage. Seeds are protected against aging by cellular defenses and by structures such as the seed coat. We have screened an activation-tagging mutant collection of Arabidopsis (Arabidopsis thaliana) and selected four dominant mutants with improved seed longevity (isl1-1D to isl4-1D) under both natural and accelerated aging conditions. In the isl1-1D mutant, characterized in this work, overexpression of the transcription factor ARABIDOPSIS THALIANA HOMEOBOX25 (ATHB25; At5g65410) increases the expression of GIBBERELLIC ACID3-OXIDASE2, encoding a gibberellin (GA) biosynthetic enzyme, and the levels of GA1 and GA4 are higher (3.2- and 1.4-fold, respectively) in the mutant than in the wild type. The morphological and seed longevity phenotypes of the athb25-1D mutant were recapitulated in transgenic plants with moderate (4- to 6-fold) overexpression of ATHB25. Simultaneous knockdown of ATHB25, ATHB22, and ATHB31 expression decreases seed longevity, as does loss of ATHB25 and ATHB22 function in a double mutant line. Seeds from wild-type plants treated with GA and from a quintuple DELLA mutant (with constitutive GA signaling) are more tolerant to aging, providing additional evidence for a role of GA in seed longevity. A correlation was observed in several genotypes between seed longevity and mucilage formation at the seed surface, suggesting that GA may act by reinforcing the seed coat. This mechanism was supported by the observation of a maternal effect in reciprocal crosses between the wild type and the athb25-1D mutant. PMID:24335333

Polarized localization and borate-dependent degradation of the Arabidopsis borate transporter BOR1 in tobacco BY-2 cells

PubMed Central

Matsuoka, Ken

2013-01-01

In Arabidopsis the borate transporter BOR1, which is located in the plasma membrane, is degraded in the presence of excess boron by an endocytosis-mediated mechanism. A similar mechanism was suggested in rice as excess boron decreased rice borate transporter levels, although in this case whether the decrease was dependent on an increase in degradation or a decrease in protein synthesis was not elucidated. To address whether the borate-dependent degradation mechanism is conserved among plant cells, we analyzed the fate of GFP-tagged BOR1 (BOR1-GFP) in transformed tobacco BY-2 cells. Cells expressing BOR1-GFP displayed GFP fluorescence at the plasma membrane, especially at the membrane between two attached cells. The plasma membrane signal was abolished when cells were incubated in medium with a high concentration of borate (3 to 5 mM). This decrease in BOR1-GFP signal was mediated by a specific degradation of the protein after internalization by endocytosis from the plasma membrane. Pharmacological analysis indicated that the decrease in BOR1-GFP largely depends on the increase in degradation rate and that the degradation was mediated by a tyrosine-motif and the actin cytoskeleton. Tyr mutants of BOR1-GFP, which has been shown to inhibit borate-dependent degradation in Arabidopsis root cells, did not show borate-dependent endocytosis in tobacco BY-2 cells. These findings indicate that the borate-dependent degradation machinery of the borate transporter is conserved among plant species. PMID:24715955

Polarized localization and borate-dependent degradation of the Arabidopsis borate transporter BOR1 in tobacco BY-2 cells.

PubMed

Yamauchi, Noboru; Gosho, Tadashi; Asatuma, Satoru; Toyooka, Kiminori; Fujiwara, Toru; Matsuoka, Ken

2013-01-01

In Arabidopsis the borate transporter BOR1, which is located in the plasma membrane, is degraded in the presence of excess boron by an endocytosis-mediated mechanism. A similar mechanism was suggested in rice as excess boron decreased rice borate transporter levels, although in this case whether the decrease was dependent on an increase in degradation or a decrease in protein synthesis was not elucidated. To address whether the borate-dependent degradation mechanism is conserved among plant cells, we analyzed the fate of GFP-tagged BOR1 (BOR1-GFP) in transformed tobacco BY-2 cells. Cells expressing BOR1-GFP displayed GFP fluorescence at the plasma membrane, especially at the membrane between two attached cells. The plasma membrane signal was abolished when cells were incubated in medium with a high concentration of borate (3 to 5 mM). This decrease in BOR1-GFP signal was mediated by a specific degradation of the protein after internalization by endocytosis from the plasma membrane. Pharmacological analysis indicated that the decrease in BOR1-GFP largely depends on the increase in degradation rate and that the degradation was mediated by a tyrosine-motif and the actin cytoskeleton. Tyr mutants of BOR1-GFP, which has been shown to inhibit borate-dependent degradation in Arabidopsis root cells, did not show borate-dependent endocytosis in tobacco BY-2 cells. These findings indicate that the borate-dependent degradation machinery of the borate transporter is conserved among plant species.

Arabidopsis Class I and Class II TCP Transcription Factors Regulate Jasmonic Acid Metabolism and Leaf Development Antagonistically1[C][W

PubMed Central

Danisman, Selahattin; van der Wal, Froukje; Dhondt, Stijn; Waites, Richard; de Folter, Stefan; Bimbo, Andrea; van Dijk, Aalt DJ; Muino, Jose M.; Cutri, Lucas; Dornelas, Marcelo C.; Angenent, Gerco C.; Immink, Richard G.H.

2012-01-01

TEOSINTE BRANCHED1/CYCLOIDEA/PROLIFERATING CELL FACTOR1 (TCP) transcription factors control developmental processes in plants. The 24 TCP transcription factors encoded in the Arabidopsis (Arabidopsis thaliana) genome are divided into two classes, class I and class II TCPs, which are proposed to act antagonistically. We performed a detailed phenotypic analysis of the class I tcp20 mutant, showing an increase in leaf pavement cell sizes in 10-d-old seedlings. Subsequently, a glucocorticoid receptor induction assay was performed, aiming to identify potential target genes of the TCP20 protein during leaf development. The LIPOXYGENASE2 (LOX2) and class I TCP9 genes were identified as TCP20 targets, and binding of TCP20 to their regulatory sequences could be confirmed by chromatin immunoprecipitation analyses. LOX2 encodes for a jasmonate biosynthesis gene, which is also targeted by class II TCP proteins that are under the control of the microRNA JAGGED AND WAVY (JAW), although in an antagonistic manner. Mutation of TCP9, the second identified TCP20 target, resulted in increased pavement cell sizes during early leaf developmental stages. Analysis of senescence in the single tcp9 and tcp20 mutants and the tcp9tcp20 double mutants showed an earlier onset of this process in comparison with wild-type control plants in the double mutant only. Both the cell size and senescence phenotypes are opposite to the known class II TCP mutant phenotype in JAW plants. Altogether, these results point to an antagonistic function of class I and class II TCP proteins in the control of leaf development via the jasmonate signaling pathway. PMID:22718775

Arabidopsis annexin1 mediates the radical-activated plasma membrane Ca²+- and K+-permeable conductance in root cells.

PubMed

Laohavisit, Anuphon; Shang, Zhonglin; Rubio, Lourdes; Cuin, Tracey A; Véry, Anne-Aliénor; Wang, Aihua; Mortimer, Jennifer C; Macpherson, Neil; Coxon, Katy M; Battey, Nicholas H; Brownlee, Colin; Park, Ohkmae K; Sentenac, Hervé; Shabala, Sergey; Webb, Alex A R; Davies, Julia M

2012-04-01

Plant cell growth and stress signaling require Ca²⁺ influx through plasma membrane transport proteins that are regulated by reactive oxygen species. In root cell growth, adaptation to salinity stress, and stomatal closure, such proteins operate downstream of the plasma membrane NADPH oxidases that produce extracellular superoxide anion, a reactive oxygen species that is readily converted to extracellular hydrogen peroxide and hydroxyl radicals, OH•. In root cells, extracellular OH• activates a plasma membrane Ca²⁺-permeable conductance that permits Ca²⁺ influx. In Arabidopsis thaliana, distribution of this conductance resembles that of annexin1 (ANN1). Annexins are membrane binding proteins that can form Ca²⁺-permeable conductances in vitro. Here, the Arabidopsis loss-of-function mutant for annexin1 (Atann1) was found to lack the root hair and epidermal OH•-activated Ca²⁺- and K⁺-permeable conductance. This manifests in both impaired root cell growth and ability to elevate root cell cytosolic free Ca²⁺ in response to OH•. An OH•-activated Ca²⁺ conductance is reconstituted by recombinant ANN1 in planar lipid bilayers. ANN1 therefore presents as a novel Ca²⁺-permeable transporter providing a molecular link between reactive oxygen species and cytosolic Ca²⁺ in plants.

ZmXTH1, a new xyloglucan endotransglucosylase/hydrolase in maize, affects cell wall structure and composition in Arabidopsis thaliana.

PubMed

Genovesi, Valeria; Fornalé, Silvia; Fry, Stephen C; Ruel, Katia; Ferrer, Pau; Encina, Antonio; Sonbol, Fathi-Mohamed; Bosch, Josep; Puigdomènech, Pere; Rigau, Joan; Caparrós-Ruiz, David

2008-01-01

Xyloglucan endotransglucosylase/hydrolases (XTHs; EC 2.4.1.207 and/or EC 3.2.1.151) are enzymes involved in the modification of cell wall structure by cleaving and, often, also re-joining xyloglucan molecules in primary plant cell walls. Using a pool of antibodies raised against an enriched cell wall protein fraction, a new XTH cDNA in maize, ZmXTH1, has been isolated from a cDNA expression library obtained from the elongation zone of the maize root. The predicted protein has a putative N-terminal signal peptide and possesses the typical domains of this enzyme family, such as a catalytic domain that is homologous to that of Bacillus macerans beta-glucanase, a putative N-glycosylation motif, and four cysteine residues in the central and C terminal regions of the ZmXTH1 protein. Phylogenetic analysis of ZmXTH1 reveals that it belongs to subgroup 4, so far only reported from Poaceae monocot species. ZmXTH1 has been expressed in Pichia pastoris (a methylotrophic yeast) and the recombinant enzyme showed xyloglucan endotransglucosylase but not xyloglucan endohydrolase activity, representing the first enzyme belonging to subgroup 4 characterized in maize so far. Expression data indicate that ZmXTH1 is expressed in elongating tissues, modulated by culture conditions, and induced by gibberellins. Transient expression assays in onion cells reveal that ZmXTH1 is directed to the cell wall, although weakly bound. Finally, Arabidopsis thaliana plants expressing ZmXTH1 show slightly increased xyloglucan endohydrolase activity and alterations in the cell wall structure and composition.

Protein Repair l-Isoaspartyl Methyltransferase1 Is Involved in Both Seed Longevity and Germination Vigor in Arabidopsis[W

PubMed Central

Ogé, Laurent; Bourdais, Gildas; Bove, Jérôme; Collet, Boris; Godin, Béatrice; Granier, Fabienne; Boutin, Jean-Pierre; Job, Dominique; Jullien, Marc; Grappin, Philippe

2008-01-01

The formation of abnormal amino acid residues is a major source of spontaneous age-related protein damage in cells. The protein l-isoaspartyl methyltransferase (PIMT) combats protein misfolding resulting from l-isoaspartyl formation by catalyzing the conversion of abnormal l-isoaspartyl residues to their normal l-aspartyl forms. In this way, the PIMT repair enzyme system contributes to longevity and survival in bacterial and animal kingdoms. Despite the discovery of PIMT activity in plants two decades ago, the role of this enzyme during plant stress adaptation and in seed longevity remains undefined. In this work, we have isolated Arabidopsis thaliana lines exhibiting altered expression of PIMT1, one of the two genes encoding the PIMT enzyme in Arabidopsis. PIMT1 overaccumulation reduced the accumulation of l-isoaspartyl residues in seed proteins and increased both seed longevity and germination vigor. Conversely, reduced PIMT1 accumulation was associated with an increase in the accumulation of l-isoaspartyl residues in the proteome of freshly harvested dry mature seeds, thus leading to heightened sensitivity to aging treatments and loss of seed vigor under stressful germination conditions. These data implicate PIMT1 as a major endogenous factor that limits abnormal l-isoaspartyl accumulation in seed proteins, thereby improving seed traits such as longevity and vigor. The PIMT repair pathway likely works in concert with other anti-aging pathways to actively eliminate deleterious protein products, thus enabling successful seedling establishment and strengthening plant proliferation in natural environments. PMID:19011119

LC-MS metabolic profiling of Arabidopsis thaliana plant leaves and cell cultures: optimization of pre-LC-MS procedure parameters.

PubMed

t'Kindt, Ruben; De Veylder, Lieven; Storme, Michael; Deforce, Dieter; Van Bocxlaer, Jan

2008-08-01

This study treats the optimization of methods for homogenizing Arabidopsis thaliana plant leaves as well as cell cultures, and extracting their metabolites for metabolomics analysis by conventional liquid chromatography electrospray ionization mass spectrometry (LC-ESI/MS). Absolute recovery, process efficiency and procedure repeatability have been compared between different pre-LC-MS homogenization/extraction procedures through the use of samples fortified before extraction with a range of representative metabolites. Hereby, the magnitude of the matrix effect observed in the ensuing LC-MS based metabolomics analysis was evaluated. Based on relative recovery and repeatability of key metabolites, comprehensiveness of extraction (number of m/z-retention time pairs) and clean-up potential of the approach (minimum matrix effects), the most appropriate sample pre-treatment was adopted. It combines liquid nitrogen homogenization for plant leaves with thermomixer based extraction using MeOH/H(2)O 80/20. As such, an efficient and highly reproducible LC-MS plant metabolomics set-up is achieved, as illustrated by the obtained results for both LC-MS (8.88%+/-5.16 versus 7.05%+/-4.45) and technical variability (12.53%+/-11.21 versus 9.31%+/-6.65) data in a comparative investigation of A. thaliana plant leaves and cell cultures, respectively.

Natural Variation of Root Hydraulics in Arabidopsis Grown in Normal and Salt-Stressed Conditions1[C][W

PubMed Central

Sutka, Moira; Li, Guowei; Boudet, Julie; Boursiac, Yann; Doumas, Patrick; Maurel, Christophe

2011-01-01

To gain insights into the natural variation of root hydraulics and its molecular components, genotypic differences related to root water transport and plasma membrane intrinsic protein (PIP) aquaporin expression were investigated in 13 natural accessions of Arabidopsis (Arabidopsis thaliana). The hydraulic conductivity of excised root systems (Lpr) showed a 2-fold variation among accessions. The contribution of aquaporins to water uptake was characterized using as inhibitors mercury, propionic acid, and azide. The aquaporin-dependent and -independent paths of water transport made variable contributions to the total hydraulic conductivity in the different accessions. The distinct suberization patterns observed among accessions were not correlated with their root hydraulic properties. Real-time reverse transcription-polymerase chain reaction revealed, by contrast, a positive overall correlation between Lpr and certain highly expressed PIP transcripts. Root hydraulic responses to salt stress were characterized in a subset of five accessions (Bulhary-1, Catania-1, Columbia-0, Dijon-M, and Monte-Tosso-0 [Mr-0]). Lpr was down-regulated in all accessions except Mr-0. In Mr-0 and Catania-1, cortical cell hydraulic conductivity was unresponsive to salt, whereas it was down-regulated in the three other accessions. By contrast, the five accessions showed qualitatively similar aquaporin transcriptional profiles in response to salt. The overall work provides clues on how hydraulic regulation allows plant adaptation to salt stress. It also shows that a wide range of root hydraulic profiles, as previously reported in various species, can be observed in a single model species. This work paves the way for a quantitative genetics analysis of root hydraulics. PMID:21212301

LINE-1 Cultured Cell Retrotransposition Assay

PubMed Central

Kopera, Huira C.; Larson, Peter A.; Moldovan, John B.; Richardson, Sandra R.; Liu, Ying; Moran, John V.

2016-01-01

Summary The Long INterspersed Element-1 (LINE-1 or L1) retrotransposition assay has facilitated the discovery and characterization of active (i.e., retrotransposition-competent) LINE-1 sequences from mammalian genomes. In this assay, an engineered LINE-1 containing a retrotransposition reporter cassette is transiently transfected into a cultured cell line. Expression of the reporter cassette, which occurs only after a successful round of retrotransposition, allows the detection and quantification of the LINE-1 retrotransposition efficiency. This assay has yielded insight into the mechanism of LINE-1 retrotransposition. It also has provided a greater understanding of how the cell regulates LINE-1 retrotransposition and how LINE-1 retrotransposition impacts the structure of mammalian genomes. Below, we provide a brief introduction to LINE-1 biology and then detail how the LINE-1 retrotransposition assay is performed in cultured mammalian cells. PMID:26895052

Transepithelial resistance and claudin expression in trout RTgill-W1 cell line: effects of osmoregulatory hormones.

PubMed

Trubitt, Rebecca T; Rabeneck, D Brett; Bujak, Joanna K; Bossus, Maryline C; Madsen, Steffen S; Tipsmark, Christian K

2015-04-01

In the present study, we examined the trout gill cell line RTgill-W1 as a possible tool for in vitro investigation of epithelial gill function in fish. After seeding in transwells, transepithelial resistance (TER) increased until reaching a plateau after 1-2 days (20-80Ω⋅cm(2)), which was then maintained for more than 6 days. Tetrabromocinnamic acid, a known stimulator of TER via casein kinase II inhibition, elevated TER in the cell line to 125% of control values after 2 and 6h. Treatment with ethylenediaminetetraacetic acid induced a decrease in TER to <15% of pre-treatment level. Cortisol elevated TER after 12-72 h in a concentration-dependent manner, and this increase was antagonized by growth hormone (Gh). The effects of three osmoregulatory hormones, Gh, prolactin, and cortisol, on the mRNA expression of three tight junction proteins were examined: claudin-10e (Cldn-10e), Cldn-30, and zonula occludens-1 (Zo-1). The expression of cldn-10e was stimulated by all three hormones but with the strongest effect of Gh (50-fold). cldn-30 expression was stimulated especially by cortisol (20-fold) and also by Gh (4-fold). Finally, zo-1 was unresponsive to hormone treatment. Western blot analysis detected Cldn-10e and Cldn-30 immunoreactive proteins of expected molecular weight in samples from rainbow trout gills but not from RTgill-W1 cultures, possibly due to low expression levels. Collectively, these results show that the RTgill-W1 cell layers have tight junctions between cells, are sensitive to hormone treatments, and may provide a useful model for in vitro study of some in vivo gill phenomena. Copyright © 2014 Elsevier Inc. All rights reserved.

MYB46 Modulates Disease Susceptibility to Botrytis cinerea in Arabidopsis12[W

PubMed Central

Ramírez, Vicente; Agorio, Astrid; Coego, Alberto; García-Andrade, Javier; Hernández, M. José; Balaguer, Begoña; Ouwerkerk, Pieter B.F.; Zarra, Ignacio; Vera, Pablo

2011-01-01

In this study, we show that the Arabidopsis (Arabidopsis thaliana) transcription factor MYB46, previously described to regulate secondary cell wall biosynthesis in the vascular tissue of the stem, is pivotal for mediating disease susceptibility to the fungal pathogen Botrytis cinerea. We identified MYB46 by its ability to bind to a new cis-element located in the 5′ promoter region of the pathogen-induced Ep5C gene, which encodes a type III cell wall-bound peroxidase. We present genetic and molecular evidence indicating that MYB46 modulates the magnitude of Ep5C gene induction following pathogenic insults. Moreover, we demonstrate that different myb46 knockdown mutant plants exhibit increased disease resistance to B. cinerea, a phenotype that is accompanied by selective transcriptional reprogramming of a set of genes encoding cell wall proteins and enzymes, of which extracellular type III peroxidases are conspicuous. In essence, our results substantiate that defense-related signaling pathways and cell wall integrity are interconnected and that MYB46 likely functions as a disease susceptibility modulator to B. cinerea through the integration of cell wall remodeling and downstream activation of secondary lines of defense. PMID:21282403

Interactions between the S-Domain Receptor Kinases and AtPUB-ARM E3 Ubiquitin Ligases Suggest a Conserved Signaling Pathway in Arabidopsis1[W][OA

PubMed Central

Samuel, Marcus A.; Mudgil, Yashwanti; Salt, Jennifer N.; Delmas, Frédéric; Ramachandran, Shaliny; Chilelli, Andrea; Goring, Daphne R.

2008-01-01

The Arabidopsis (Arabidopsis thaliana) genome encompasses multiple receptor kinase families with highly variable extracellular domains. Despite their large numbers, the various ligands and the downstream interacting partners for these kinases have been deciphered only for a few members. One such member, the S-receptor kinase, is known to mediate the self-incompatibility (SI) response in Brassica. S-receptor kinase has been shown to interact and phosphorylate a U-box/ARM-repeat-containing E3 ligase, ARC1, which, in turn, acts as a positive regulator of the SI response. In an effort to identify conserved signaling pathways in Arabidopsis, we performed yeast two-hybrid analyses of various S-domain receptor kinase family members with representative Arabidopsis plant U-box/ARM-repeat (AtPUB-ARM) E3 ligases. The kinase domains from S-domain receptor kinases were found to interact with ARM-repeat domains from AtPUB-ARM proteins. These kinase domains, along with M-locus protein kinase, a positive regulator of SI response, were also able to phosphorylate the ARM-repeat domains in in vitro phosphorylation assays. Subcellular localization patterns were investigated using transient expression assays in tobacco (Nicotiana tabacum) BY-2 cells and changes were detected in the presence of interacting kinases. Finally, potential links to the involvement of these interacting modules to the hormone abscisic acid (ABA) were investigated. Interestingly, AtPUB9 displayed redistribution to the plasma membrane of BY-2 cells when either treated with ABA or coexpressed with the active kinase domain of ARK1. As well, T-DNA insertion mutants for ARK1 and AtPUB9 lines were altered in their ABA sensitivity during germination and acted at or upstream of ABI3, indicating potential involvement of these proteins in ABA responses. PMID:18552232

The MYB23 Gene Provides a Positive Feedback Loop for Cell Fate Specification in the Arabidopsis Root Epidermis[C][W

PubMed Central

Kang, Yeon Hee; Kirik, Victor; Hulskamp, Martin; Nam, Kyoung Hee; Hagely, Katherine; Lee, Myeong Min; Schiefelbein, John

2009-01-01

The specification of cell fates during development requires precise regulatory mechanisms to ensure robust cell type patterns. Theoretical models of pattern formation suggest that a combination of negative and positive feedback mechanisms are necessary for efficient specification of distinct fates in a field of differentiating cells. Here, we examine the role of the R2R3-MYB transcription factor gene, AtMYB23 (MYB23), in the establishment of the root epidermal cell type pattern in Arabidopsis thaliana. MYB23 is closely related to, and is positively regulated by, the WEREWOLF (WER) MYB gene during root epidermis development. Furthermore, MYB23 is able to substitute for the function of WER and to induce its own expression when controlled by WER regulatory sequences. We also show that the MYB23 protein binds to its own promoter, suggesting a MYB23 positive feedback loop. The localization of MYB23 transcripts and MYB23-green fluorescent protein (GFP) fusion protein, as well as the effect of a chimeric MYB23-SRDX repressor construct, links MYB23 function to the developing non-hair cell type. Using mutational analyses, we find that MYB23 is necessary for precise establishment of the root epidermal pattern, particularly under conditions that compromise the cell specification process. These results suggest that MYB23 participates in a positive feedback loop to reinforce cell fate decisions and ensure robust establishment of the cell type pattern in the Arabidopsis root epidermis. PMID:19395683

Pinoresinol reductase 1 impacts lignin distribution during secondary cell wall biosynthesis in Arabidopsis

DOE PAGES

Zhao, Qiao; Zeng, Yining; Yin, Yanbin; ...

2014-08-05

In this paper, pinoresinol reductase (PrR) catalyzes the conversion of the lignan (-)-pinoresinol to (-)-lariciresinol in Arabidopsis thaliana, where it is encoded by two genes, PrR1 and PrR2, that appear to act redundantly. PrR1 is highly expressed in lignified inflorescence stem tissue, whereas PrR2 expression is barely detectable in stems. Co-expression analysis has indicated that PrR1 is co-expressed with many characterized genes involved in secondary cell wall biosynthesis, whereas PrR2 expression clusters with a different set of genes. The promoter of the PrR1 gene is regulated by the secondary cell wall related transcription factors SND1 and MYB46. The loss-of-function mutantmore » of PrR1 shows, in addition to elevated levels of pinoresinol, significantly decreased lignin content and a slightly altered lignin structure with lower abundance of cinnamyl alcohol end groups. Stimulated Raman scattering (SRS) microscopy analysis indicated that the lignin content of the prr1-1 loss-of-function mutant is similar to that of wild-type plants in xylem cells, which exhibit a normal phenotype, but is reduced in the fiber cells. Finally, together, these data suggest an association of the lignan biosynthetic enzyme encoded by PrR1 with secondary cell wall biosynthesis in fiber cells.« less

Distinct Cell Wall Architectures in Seed Endosperms in Representatives of the Brassicaceae and Solanaceae1[C][W][OA

PubMed Central

Lee, Kieran J.D.; Dekkers, Bas J.W.; Steinbrecher, Tina; Walsh, Cherie T.; Bacic, Antony; Bentsink, Leónie; Leubner-Metzger, Gerhard; Knox, J. Paul

2012-01-01

In some species, a crucial role has been demonstrated for the seed endosperm during germination. The endosperm has been shown to integrate environmental cues with hormonal networks that underpin dormancy and seed germination, a process that involves the action of cell wall remodeling enzymes (CWREs). Here, we examine the cell wall architectures of the endosperms of two related Brassicaceae, Arabidopsis (Arabidopsis thaliana) and the close relative Lepidium (Lepidium sativum), and that of the Solanaceous species, tobacco (Nicotiana tabacum). The Brassicaceae species have a similar cell wall architecture that is rich in pectic homogalacturonan, arabinan, and xyloglucan. Distinctive features of the tobacco endosperm that are absent in the Brassicaceae representatives are major tissue asymmetries in cell wall structural components that reflect the future site of radicle emergence and abundant heteromannan. Cell wall architecture of the micropylar endosperm of tobacco seeds has structural components similar to those seen in Arabidopsis and Lepidium endosperms. In situ and biomechanical analyses were used to study changes in endosperms during seed germination and suggest a role for mannan degradation in tobacco. In the case of the Brassicaceae representatives, the structurally homogeneous cell walls of the endosperm can be acted on by spatially regulated CWRE expression. Genetic manipulations of cell wall components present in the Arabidopsis seed endosperm demonstrate the impact of cell wall architectural changes on germination kinetics. PMID:22961130

Uridine-Ribohydrolase Is a Key Regulator in the Uridine Degradation Pathway of Arabidopsis[W

PubMed Central

Jung, Benjamin; Flörchinger, Martin; Kunz, Hans-Henning; Traub, Michaela; Wartenberg, Ruth; Jeblick, Wolfgang; Neuhaus, H. Ekkehard; Möhlmann, Torsten

2009-01-01

Nucleoside degradation and salvage are important metabolic pathways but hardly understood in plants. Recent work on human pathogenic protozoans like Leishmania and Trypanosoma substantiates an essential function of nucleosidase activity. Plant nucleosidases are related to those from protozoans and connect the pathways of nucleoside degradation and salvage. Here, we describe the cloning of such an enzyme from Arabidopsis thaliana, Uridine-Ribohydrolase 1 (URH1) and the characterization by complementation of a yeast mutant. Furthermore, URH1 was synthesized as a recombinant protein in Escherichia coli. The pure recombinant protein exhibited highest hydrolase activity for uridine, followed by inosine and adenosine, the corresponding Km values were 0.8, 1.4, and 0.7 mM, respectively. In addition, URH1 was able to cleave the cytokinin derivative isopentenyladenine-riboside. Promoter β-glucuronidase fusion studies revealed that URH1 is mainly transcribed in the vascular cells of roots and in root tips, guard cells, and pollen. Mutants expressing the Arabidopsis enzyme or the homolog from rice (Oryza sativa) exhibit resistance toward toxic fluorouridine, fluorouracil, and fluoroorotic acid, providing clear evidence for a pivotal function of URH1 as regulative in pyrimidine degradation. Moreover, mutants with increased and decreased nucleosidase activity are delayed in germination, indicating that this enzyme activity must be well balanced in the early phase of plant development. PMID:19293370

TORNADO1 regulates root epidermal patterning through the WEREWOLF pathway in Arabidopsis thaliana.

PubMed

Kwak, Su-Hwan; Song, Sang-Kee; Lee, Myeong Min; Schiefelbein, John

2015-01-01

Cell fate in the root epidermis of Arabidopsis thaliana is determined in a position-dependent manner. SCRAMBLED (SCM), an atypical leucine-rich repeat receptor-like kinase, mediates this positional regulation via its effect on WEREWOLF (WER) expression, and subsequently, its downstream transcription factor, GLABRA2 (GL2), which are required for nonhair cell development. Previously, TORNADO1 (TRN1), a plant-specific protein with a leucine-rich repeat ribonuclease inhibitor-like domain, was shown to be required for proper epidermal patterning in Arabidopsis roots. In this work, we analyzed the possible involvement of TRN1 in the known root epidermal gene network. We discovered that the trn1 mutant caused the ectopic expression of WER and the randomized expression of GL2 and EGL3. This suggests that TRN1 regulates the position-dependent cell fate determination by affecting WER expression in Arabidopsis root epidermis. Additionally, the distinct phenotypes of the aerial parts of the trn1-t and scm-2 mutant suggest that TRN1 and SCM might have different functions in the development of aerial parts.

TORNADO1 regulates root epidermal patterning through the WEREWOLF pathway in Arabidopsis thaliana

PubMed Central

Kwak, Su-Hwan; Song, Sang-Kee; Lee, Myeong Min; Schiefelbein, John

2015-01-01

Cell fate in the root epidermis of Arabidopsis thaliana is determined in a position-dependent manner. SCRAMBLED (SCM), an atypical leucine-rich repeat receptor-like kinase, mediates this positional regulation via its effect on WEREWOLF (WER) expression, and subsequently, its downstream transcription factor, GLABRA2 (GL2), which are required for nonhair cell development. Previously, TORNADO1 (TRN1), a plant-specific protein with a leucine-rich repeat ribonuclease inhibitor-like domain, was shown to be required for proper epidermal patterning in Arabidopsis roots. In this work, we analyzed the possible involvement of TRN1 in the known root epidermal gene network. We discovered that the trn1 mutant caused the ectopic expression of WER and the randomized expression of GL2 and EGL3. This suggests that TRN1 regulates the position-dependent cell fate determination by affecting WER expression in Arabidopsis root epidermis. Additionally, the distinct phenotypes of the aerial parts of the trn1-t and scm-2 mutant suggest that TRN1 and SCM might have different functions in the development of aerial parts. PMID:26451798

Chlorophyll Degradation: The Tocopherol Biosynthesis-Related Phytol Hydrolase in Arabidopsis Seeds Is Still Missing1[C][W][OPEN

PubMed Central

Zhang, Wei; Liu, Tianqi; Ren, Guodong; Hörtensteiner, Stefan; Zhou, Yongming; Cahoon, Edgar B.; Zhang, Chunyu

2014-01-01

Phytyl diphosphate (PDP) is the prenyl precursor for tocopherol biosynthesis. Based on recent genetic evidence, PDP is supplied to the tocopherol biosynthetic pathway primarily by chlorophyll degradation and sequential phytol phosphorylation. Three enzymes of Arabidopsis (Arabidopsis thaliana) are known to be capable of removing the phytol chain from chlorophyll in vitro: chlorophyllase1 (CLH1), CLH2, and pheophytin pheophorbide hydrolase (PPH), which specifically hydrolyzes pheophytin. While PPH, but not chlorophyllases, is required for in vivo chlorophyll breakdown during Arabidopsis leaf senescence, little is known about the involvement of these phytol-releasing enzymes in tocopherol biosynthesis. To explore the origin of PDP for tocopherol synthesis, seed tocopherol concentrations were determined in Arabidopsis lines engineered for seed-specific overexpression of PPH and in single and multiple mutants in the three genes encoding known dephytylating enzymes. Except for modestly increasing tocopherol content observed in the PPH overexpressor, none of the remaining lines exhibited significantly reduced tocopherol concentrations, suggesting that the known chlorophyll-derived phytol-releasing enzymes do not play major roles in tocopherol biosynthesis. Tocopherol content of seeds from double mutants in NONYELLOWING1 (NYE1) and NYE2, regulators of chlorophyll degradation, had modest reduction compared with wild-type seeds, although mature seeds of the double mutant retained significantly higher chlorophyll levels. These findings suggest that NYEs may play limited roles in regulating an unknown tocopherol biosynthesis-related phytol hydrolase. Meanwhile, seeds of wild-type over-expressing NYE1 had lower tocopherol levels, suggesting that phytol derived from NYE1-dependent chlorophyll degradation probably doesn’t enter tocopherol biosynthesis. Potential routes of chlorophyll degradation are discussed in relation to tocopherol biosynthesis. PMID:25059706

CPK3-phosphorylated RhoGDI1 is essential in the development of Arabidopsis seedlings and leaf epidermal cells

PubMed Central

Wu, Yan

2013-01-01

The regulation of Rho of plants (ROP) in morphogenesis of leaf epidermal cells has been well studied, but the roles concerning regulators of ROPs such as RhoGDIs are poorly understood. This study reports that AtRhoGDI1 (GDI1) acts as a versatile regulator to modulate development of seedlings and leaf pavement cells. In mutant gdi1, leaf pavement cells showed shorter lobes in comparison with those in wild type. In GDI1-14 seedlings (GDI1-overexpression line) the growth of lobes in pavement cells was severely suppressed and the development of seedlings was altered. These results indicate that GDI1 plays an essential role in morphogenesis of epidermal pavement cells through modulating the ROP signalling pathways. The interaction between GDI1 and ROP2 or ROP6 was detected in the leaf pavement cells using FRET analysis. Dominant negative, not constitutively active, DN-rop6 could weaken the effect caused by overexpression of GDI1; because the pleiotropic phenotype of GDI1-14 plants was eliminated in the hybrid line GDI1-14 DN-rop6. GDI1 could be phosphorylated by CPK3. Three conserved Ser/Thr residues in GDI1 were determined as targeted amino acids for CPK3. Overexpression of GDI1(3D), not GDI1(3A), could rescue the abnormal growth phenotypes of gdi1-1 seedlings, demonstrating the impact of GDI1 phosphorylation in the development of Arabidopsis. In summary, these results suggest that GDI1 regulation in morphogenesis of seedlings and leaf pavement cells could be undergone through modulating the ROP signalling pathways and the phosphorylation of GDI1 by CPK3 was required for the developmental modulation in Arabidopsis. PMID:23846874

AtPDCD5 Plays a Role in Programmed Cell Death after UV-B Exposure in Arabidopsis1[OPEN

PubMed Central

Falcone Ferreyra, María Lorena; D’Andrea, Lucio; AbdElgawad, Hamada

2016-01-01

DNA damage responses have evolved to sense and react to DNA damage; the induction of DNA repair mechanisms can lead to genomic restoration or, if the damaged DNA cannot be adequately repaired, to the execution of a cell death program. In this work, we investigated the role of an Arabidopsis (Arabidopsis thaliana) protein, AtPDCD5, which is highly similar to the human PDCD5 protein; it is induced by ultraviolet (UV)-B radiation and participates in programmed cell death in the UV-B DNA damage response. Transgenic plants expressing AtPDCD5 fused to GREEN FLUORESCENT PROTEIN indicate that AtPDCD5 is localized both in the nucleus and the cytosol. By use of pdcd5 mutants, we here demonstrate that these plants have an altered antioxidant metabolism and accumulate higher levels of DNA damage after UV-B exposure, similar to levels in ham1ham2 RNA interference transgenic lines with decreased expression of acetyltransferases from the MYST family. By coimmunoprecipitation and pull-down assays, we provide evidence that AtPDCD5 interacts with HAM proteins, suggesting that both proteins participate in the same pathway of DNA damage responses. Plants overexpressing AtPDCD5 show less DNA damage but more cell death in root tips upon UV-B exposure. Finally, we here show that AtPDCD5 also participates in age-induced programmed cell death. Together, the data presented here demonstrate that AtPDCD5 plays an important role during DNA damage responses induced by UV-B radiation in Arabidopsis and also participates in programmed cell death programs. PMID:26884483

Three Arabidopsis Fatty Acyl-Coenzyme A Reductases, FAR1, FAR4, and FAR5, Generate Primary Fatty Alcohols Associated with Suberin Deposition1[C][W][OA

PubMed Central

Domergue, Frédéric; Vishwanath, Sollapura J.; Joubès, Jérôme; Ono, Jasmine; Lee, Jennifer A.; Bourdon, Matthieu; Alhattab, Reem; Lowe, Christine; Pascal, Stéphanie; Lessire, René; Rowland, Owen

2010-01-01

Suberin is a protective hydrophobic barrier consisting of phenolics, glycerol, and a variety of fatty acid derivatives, including C18:0-C22:0 primary fatty alcohols. An eight-member gene family encoding alcohol-forming fatty acyl-coenzyme A reductases (FARs) has been identified in Arabidopsis (Arabidopsis thaliana). Promoter-driven expression of the β-glucuronidase reporter gene indicated that three of these genes, FAR1(At5g22500), FAR4(At3g44540), and FAR5(At3g44550), are expressed in root endodermal cells. The three genes were transcriptionally induced by wounding and salt stress. These patterns of gene expression coincide with known sites of suberin deposition. We then characterized a set of mutants with T-DNA insertions in FAR1, FAR4, or FAR5 and found that the suberin compositions of roots and seed coats were modified in each far mutant. Specifically, C18:0-OH was reduced in far5-1, C20:0-OH was reduced in far4-1, and C22:0-OH was reduced in far1-1. We also analyzed the composition of polymer-bound lipids of leaves before and after wounding and found that the basal levels of C18:0-C22:0 primary alcohols in wild-type leaves were increased by wounding. In contrast, C18:0-OH and C22:0-OH were not increased by wounding in far5-1 and far1-1 mutants, respectively. Heterologous expression of FAR1, FAR4, and FAR5 in yeast confirmed that they are indeed active alcohol-forming FARs with distinct, but overlapping, chain length specificities ranging from C18:0 to C24:0. Altogether, these results indicate that Arabidopsis FAR1, FAR4, and FAR5 generate the fatty alcohols found in root, seed coat, and wound-induced leaf tissue. PMID:20571114

Plastidial Glycolytic Glyceraldehyde-3-Phosphate Dehydrogenase Is an Important Determinant in the Carbon and Nitrogen Metabolism of Heterotrophic Cells in Arabidopsis1

PubMed Central

Anoman, Armand D.; Muñoz-Bertomeu, Jesús; Rosa-Téllez, Sara; Flores-Tornero, María; Serrano, Ramón; Bueso, Eduardo; Fernie, Alisdair R.; Segura, Juan; Ros, Roc

2015-01-01

This study functionally characterizes the Arabidopsis (Arabidopsis thaliana) plastidial glycolytic isoforms of glyceraldehyde-3-phosphate dehydrogenase (GAPCp) in photosynthetic and heterotrophic cells. We expressed the enzyme in gapcp double mutants (gapcp1gapcp2) under the control of photosynthetic (Rubisco small subunit RBCS2B [RBCS]) or heterotrophic (phosphate transporter PHT1.2 [PHT]) cell-specific promoters. Expression of GAPCp1 under the control of RBCS in gapcp1gapcp2 had no significant effect on the metabolite profile or growth in the aerial part (AP). GAPCp1 expression under the control of the PHT promoter clearly affected Arabidopsis development by increasing the number of lateral roots and having a major effect on AP growth and metabolite profile. Our results indicate that GAPCp1 is not functionally important in photosynthetic cells but plays a fundamental role in roots and in heterotrophic cells of the AP. Specifically, GAPCp activity may be required in root meristems and the root cap for normal primary root growth. Transcriptomic and metabolomic analyses indicate that the lack of GAPCp activity affects nitrogen and carbon metabolism as well as mineral nutrition and that glycerate and glutamine are the main metabolites responding to GAPCp activity. Thus, GAPCp could be an important metabolic connector of glycolysis with other pathways, such as the phosphorylated pathway of serine biosynthesis, the ammonium assimilation pathway, or the metabolism of γ-aminobutyrate, which in turn affect plant development. PMID:26134167

Arabidopsis CPR5 regulates ethylene signaling via molecular association with the ETR1 receptor.

PubMed

Wang, Feifei; Wang, Lijuan; Qiao, Longfei; Chen, Jiacai; Pappa, Maria Belen; Pei, Haixia; Zhang, Tao; Chang, Caren; Dong, Chun-Hai

2017-11-01

The plant hormone ethylene plays various functions in plant growth, development and response to environmental stress. Ethylene is perceived by membrane-bound ethylene receptors, and among the homologous receptors in Arabidopsis, the ETR1 ethylene receptor plays a major role. The present study provides evidence demonstrating that Arabidopsis CPR5 functions as a novel ETR1 receptor-interacting protein in regulating ethylene response and signaling. Yeast split ubiquitin assays and bi-fluorescence complementation studies in plant cells indicated that CPR5 directly interacts with the ETR1 receptor. Genetic analyses indicated that mutant alleles of cpr5 can suppress ethylene insensitivity in both etr1-1 and etr1-2, but not in other dominant ethylene receptor mutants. Overexpression of Arabidopsis CPR5 either in transgenic Arabidopsis plants, or ectopically in tobacco, significantly enhanced ethylene sensitivity. These findings indicate that CPR5 plays a critical role in regulating ethylene signaling. CPR5 is localized to endomembrane structures and the nucleus, and is involved in various regulatory pathways, including pathogenesis, leaf senescence, and spontaneous cell death. This study provides evidence for a novel regulatory function played by CPR5 in the ethylene receptor signaling pathway in Arabidopsis. © 2017 Institute of Botany, Chinese Academy of Sciences.

Arabidopsis Deficient in Cutin Ferulate Encodes a Transferase Required for Feruloylation of ω-Hydroxy Fatty Acids in Cutin Polyester1[W][OA

PubMed Central

Rautengarten, Carsten; Ebert, Berit; Ouellet, Mario; Nafisi, Majse; Baidoo, Edward E.K.; Benke, Peter; Stranne, Maria; Mukhopadhyay, Aindrila; Keasling, Jay D.; Sakuragi, Yumiko; Scheller, Henrik Vibe

2012-01-01

The cuticle is a complex aliphatic polymeric layer connected to the cell wall and covers surfaces of all aerial plant organs. The cuticle prevents nonstomatal water loss, regulates gas exchange, and acts as a barrier against pathogen infection. The cuticle is synthesized by epidermal cells and predominantly consists of an aliphatic polymer matrix (cutin) and intracuticular and epicuticular waxes. Cutin monomers are primarily C16 and C18 unsubstituted, ω-hydroxy, and α,ω-dicarboxylic fatty acids. Phenolics such as ferulate and p-coumarate esters also contribute to a minor extent to the cutin polymer. Here, we present the characterization of a novel acyl-coenzyme A (CoA)-dependent acyl-transferase that is encoded by a gene designated Deficient in Cutin Ferulate (DCF). The DCF protein is responsible for the feruloylation of ω-hydroxy fatty acids incorporated into the cutin polymer of aerial Arabidopsis (Arabidopsis thaliana) organs. The enzyme specifically transfers hydroxycinnamic acids using ω-hydroxy fatty acids as acyl acceptors and hydroxycinnamoyl-CoAs, preferentially feruloyl-CoA and sinapoyl-CoA, as acyl donors in vitro. Arabidopsis mutant lines carrying DCF loss-of-function alleles are devoid of rosette leaf cutin ferulate and exhibit a 50% reduction in ferulic acid content in stem insoluble residues. DCF is specifically expressed in the epidermis throughout all green Arabidopsis organs. The DCF protein localizes to the cytosol, suggesting that the feruloylation of cutin monomers takes place in the cytoplasm. PMID:22158675

Plastids Are Major Regulators of Light Signaling in Arabidopsis1[W][OA

PubMed Central

Ruckle, Michael E.; Burgoon, Lyle D.; Lawrence, Lauren A.; Sinkler, Christopher A.; Larkin, Robert M.

2012-01-01

We previously provided evidence that plastid signaling regulates the downstream components of a light signaling network and that this signal integration coordinates chloroplast biogenesis with both the light environment and development by regulating gene expression. We tested these ideas by analyzing light- and plastid-regulated transcriptomes in Arabidopsis (Arabidopsis thaliana). We found that the enrichment of Gene Ontology terms in these transcriptomes is consistent with the integration of light and plastid signaling (1) down-regulating photosynthesis and inducing both repair and stress tolerance in dysfunctional chloroplasts and (2) helping coordinate processes such as growth, the circadian rhythm, and stress responses with the degree of chloroplast function. We then tested whether factors that contribute to this signal integration are also regulated by light and plastid signals by characterizing T-DNA insertion alleles of genes that are regulated by light and plastid signaling and that encode proteins that are annotated as contributing to signaling, transcription, or no known function. We found that a high proportion of these mutant alleles induce chloroplast biogenesis during deetiolation. We quantified the expression of four photosynthesis-related genes in seven of these enhanced deetiolation (end) mutants and found that photosynthesis-related gene expression is attenuated. This attenuation is particularly striking for Photosystem II subunit S expression. We conclude that the integration of light and plastid signaling regulates a number of END genes that help optimize chloroplast function and that at least some END genes affect photosynthesis-related gene expression. PMID:22383539

MGOUN1 encodes an Arabidopsis type IB DNA topoisomerase required in stem cell regulation and to maintain developmentally regulated gene silencing.

PubMed

Graf, Philipp; Dolzblasz, Alicja; Würschum, Tobias; Lenhard, Michael; Pfreundt, Ulrike; Laux, Thomas

2010-03-01

Maintenance of stem cells in the Arabidopsis thaliana shoot meristem is regulated by signals from the underlying cells of the organizing center, provided through the transcription factor WUSCHEL (WUS). Here, we report the isolation of several independent mutants of MGOUN1 (MGO1) as genetic suppressors of ectopic WUS activity and enhancers of stem cell defects in hypomorphic wus alleles. mgo1 mutants have previously been reported to result in a delayed progression of meristem cells into differentiating organ primordia (Laufs et al., 1998). Genetic analyses indicate that MGO1 functions together with WUS in stem cell maintenance at all stages of shoot and floral meristems. Synergistic interactions of mgo1 with several chromatin mutants suggest that MGO1 affects gene expression together with chromatin remodeling pathways. In addition, the expression states of developmentally regulated genes are randomly switched in mgo1 in a mitotically inheritable way, indicating that MGO1 stabilizes epigenetic states against stochastically occurring changes. Positional cloning revealed that MGO1 encodes a putative type IB topoisomerase, which in animals and yeast has been shown to be required for regulation of DNA coiling during transcription and replication. The specific developmental defects in mgo1 mutants link topoisomerase IB function in Arabidopsis to stable propagation of developmentally regulated gene expression.

Arabidopsis thaliana Somatic Embryogenesis Receptor Kinase 1 protein is present in sporophytic and gametophytic cells and undergoes endocytosis.

PubMed

Kwaaitaal, M A C J; de Vries, S C; Russinova, E

2005-10-01

Arabidopsis thaliana plants expressing AtSERK1 fused to yellow-fluorescent protein were generated. Fluorescence was detected predominantly at the cell periphery, most likely the plasma membrane, of cells in ovules, embryo sacs, anthers, and embryos and in seedlings. The AtSERK1 protein was detected in diverse cell types including the epidermis and the vascular bundles. In some cells, fluorescent receptors were seen in small vesicle-like compartments. After application of the fungal toxin Brefeldin A, the fluorescent receptors were rapidly internalized in the root meristem and root vascular tissue. We conclude that the AtSERK1 receptor functions in a common signalling pathway employed in both sporophytic and gametophytic cells.

Toward Stable Genetic Engineering of Human O-Glycosylation in Plants1[C][W][OA

PubMed Central

Yang, Zhang; Bennett, Eric P.; Jørgensen, Bodil; Drew, Damian P.; Arigi, Emma; Mandel, Ulla; Ulvskov, Peter; Levery, Steven B.; Clausen, Henrik; Petersen, Bent L.

2012-01-01

Glycosylation is the most abundant and complex posttranslational modification to be considered for recombinant production of therapeutic proteins. Mucin-type (N-acetylgalactosamine [GalNAc]-type) O-glycosylation is found in eumetazoan cells but absent in plants and yeast, making these cell types an obvious choice for de novo engineering of this O-glycosylation pathway. We previously showed that transient implementation of O-glycosylation capacity in plants requires introduction of the synthesis of the donor substrate UDP-GalNAc and one or more polypeptide GalNAc-transferases for incorporating GalNAc residues into proteins. Here, we have stably engineered O-glycosylation capacity in two plant cell systems, soil-grown Arabidopsis (Arabidopsis thaliana) and tobacco (Nicotiana tabacum) Bright Yellow-2 suspension culture cells. Efficient GalNAc O-glycosylation of two stably coexpressed substrate O-glycoproteins was obtained, but a high degree of proline hydroxylation and hydroxyproline-linked arabinosides, on a mucin (MUC1)-derived substrate, was also observed. Addition of the prolyl 4-hydroxylase inhibitor 2,2-dipyridyl, however, effectively suppressed proline hydroxylation and arabinosylation of MUC1 in Bright Yellow-2 cells. In summary, stably engineered mammalian type O-glycosylation was established in transgenic plants, demonstrating that plants may serve as host cells for the production of recombinant O-glycoproteins. However, the present stable implementation further strengthens the notion that elimination of endogenous posttranslational modifications may be needed for the production of protein therapeutics. PMID:22791304

Fluctuations of the transcription factor ATML1 generate the pattern of giant cells in the Arabidopsis sepal

PubMed Central

Meyer, Heather M; Teles, José; Formosa-Jordan, Pau; Refahi, Yassin; San-Bento, Rita; Ingram, Gwyneth; Jönsson, Henrik; Locke, James C W; Roeder, Adrienne H K

2017-01-01

Multicellular development produces patterns of specialized cell types. Yet, it is often unclear how individual cells within a field of identical cells initiate the patterning process. Using live imaging, quantitative image analyses and modeling, we show that during Arabidopsis thaliana sepal development, fluctuations in the concentration of the transcription factor ATML1 pattern a field of identical epidermal cells to differentiate into giant cells interspersed between smaller cells. We find that ATML1 is expressed in all epidermal cells. However, its level fluctuates in each of these cells. If ATML1 levels surpass a threshold during the G2 phase of the cell cycle, the cell will likely enter a state of endoreduplication and become giant. Otherwise, the cell divides. Our results demonstrate a fluctuation-driven patterning mechanism for how cell fate decisions can be initiated through a random yet tightly regulated process. DOI: http://dx.doi.org/10.7554/eLife.19131.001 PMID:28145865

Dynamic cell culture system (7-IML-1)

NASA Technical Reports Server (NTRS)

Cogoli, Augusto

1992-01-01

This experiment is one of the Biorack experiments being flown on the International Microgravity Laboratory 1 (MIL-1) mission as part of an investigation studying cell proliferation and performance in space. One of the objectives of this investigation is to assess the potential benefits of bioprocessing in space with the ultimate goal of developing a bioreactor for continuous cell cultures in space. This experiment will test the operation of an automated culture chamber that was designed for use in a Bioreactor in space. The device to be tested is called the Dynamic Cell Culture System (DCCS). It is a simple device in which media are renewed or chemicals are injected automatically, by means of osmotic pumps. This experiment uses four Type I/O experiment containers. One DCCS unit, which contains a culture chamber with renewal of medium and a second chamber without a medium supply fits in each container. Two DCCS units are maintained under zero gravity conditions during the on-orbit period. The other two units are maintained under 1 gh conditions in a 1 g centrifuge. The schedule for incubator transfer is given.

Gibberellic Acid-Stimulated Arabidopsis6 Serves as an Integrator of Gibberellin, Abscisic Acid, and Glucose Signaling during Seed Germination in Arabidopsis1[OPEN

PubMed Central

Zhong, Chunmei; Xu, Hao; Ye, Siting; Wang, Shiyi; Li, Lingfei; Zhang, Shengchun; Wang, Xiaojing

2015-01-01

The DELLA protein REPRESSOR OF ga1-3-LIKE2 (RGL2) plays an important role in seed germination under different conditions through a number of transcription factors. However, the functions of the structural genes associated with RGL2-regulated germination are less defined. Here, we report the role of an Arabidopsis (Arabidopsis thaliana) cell wall-localized protein, Gibberellic Acid-Stimulated Arabidopsis6 (AtGASA6), in functionally linking RGL2 and a cell wall loosening expansin protein (Arabidopsis expansin A1 [AtEXPA1]), resulting in the control of embryonic axis elongation and seed germination. AtGASA6-overexpressing seeds showed precocious germination, whereas transfer DNA and RNA interference mutant seeds displayed delayed seed germination under abscisic acid, paclobutrazol, and glucose (Glc) stress conditions. The differences in germination rates resulted from corresponding variation in cell elongation in the hypocotyl-radicle transition region of the embryonic axis. AtGASA6 was down-regulated by RGL2, GLUCOSE INSENSITIVE2, and ABSCISIC ACID-INSENSITIVE5 genes, and loss of AtGASA6 expression in the gasa6 mutant reversed the insensitivity shown by the rgl2 mutant to paclobutrazol and the gin2 mutant to Glc-induced stress, suggesting that it is involved in regulating both the gibberellin and Glc signaling pathways. Furthermore, it was found that the promotion of seed germination and length of embryonic axis by AtGASA6 resulted from a promotion of cell elongation at the embryonic axis mediated by AtEXPA1. Taken together, the data indicate that AtGASA6 links RGL2 and AtEXPA1 functions and plays a role as an integrator of gibberellin, abscisic acid, and Glc signaling, resulting in the regulation of seed germination through a promotion of cell elongation. PMID:26400990

Identification and Characterization of Arabidopsis Seed Coat Mucilage Proteins1[OPEN

PubMed Central

Tsai, Allen Yi-Lun; Kunieda, Tadashi; Rogalski, Jason; Foster, Leonard J.; Ellis, Brian E.

2017-01-01

Plant cell wall proteins are important regulators of cell wall architecture and function. However, because cell wall proteins are difficult to extract and analyze, they are generally poorly understood. Here, we describe the identification and characterization of proteins integral to the Arabidopsis (Arabidopsis thaliana) seed coat mucilage, a specialized layer of the extracellular matrix composed of plant cell wall carbohydrates that is used as a model for cell wall research. The proteins identified in mucilage include those previously identified by genetic analysis, and several mucilage proteins are reduced in mucilage-deficient mutant seeds, suggesting that these proteins are genuinely associated with the mucilage. Arabidopsis mucilage has both nonadherent and adherent layers. Both layers have similar protein profiles except for proteins involved in lipid metabolism, which are present exclusively in the adherent mucilage. The most abundant mucilage proteins include a family of proteins named TESTA ABUNDANT1 (TBA1) to TBA3; a less abundant fourth homolog was named TBA-LIKE (TBAL). TBA and TBAL transcripts and promoter activities were detected in developing seed coats, and their expression requires seed coat differentiation regulators. TBA proteins are secreted to the mucilage pocket during differentiation. Although reverse genetics failed to identify a function for TBAs/TBAL, the TBA promoters are highly expressed and cell type specific and so should be very useful tools for targeting proteins to the seed coat epidermis. Altogether, these results highlight the mucilage proteome as a model for cell walls in general, as it shares similarities with other cell wall proteomes while also containing mucilage-specific features. PMID:28003327

Functional Analysis of Cellulose and Xyloglucan in the Walls of Stomatal Guard Cells of Arabidopsis1[OPEN

PubMed Central

Rui, Yue; Anderson, Charles T.

2016-01-01

Stomatal guard cells are pairs of specialized epidermal cells that control water and CO2 exchange between the plant and the environment. To fulfill the functions of stomatal opening and closure that are driven by changes in turgor pressure, guard cell walls must be both strong and flexible, but how the structure and dynamics of guard cell walls enable stomatal function remains poorly understood. To address this question, we applied cell biological and genetic analyses to investigate guard cell walls and their relationship to stomatal function in Arabidopsis (Arabidopsis thaliana). Using live-cell spinning disk confocal microscopy, we measured the motility of cellulose synthase (CESA)-containing complexes labeled by green fluorescent protein (GFP)-CESA3 and observed a reduced proportion of GFP-CESA3 particles colocalizing with microtubules upon stomatal closure. Imaging cellulose organization in guard cells revealed a relatively uniform distribution of cellulose in the open state and a more fibrillar pattern in the closed state, indicating that cellulose microfibrils undergo dynamic reorganization during stomatal movements. In cesa3je5 mutants defective in cellulose synthesis and xxt1 xxt2 mutants lacking the hemicellulose xyloglucan, stomatal apertures, changes in guard cell length, and cellulose reorganization were aberrant during fusicoccin-induced stomatal opening or abscisic acid-induced stomatal closure, indicating that sufficient cellulose and xyloglucan are required for normal guard cell dynamics. Together, these results provide new insights into how guard cell walls allow stomata to function as responsive mediators of gas exchange at the plant surface. PMID:26729799

Arabidopsis FH1 Formin Affects Cotyledon Pavement Cell Shape by Modulating Cytoskeleton Dynamics.

PubMed

Rosero, Amparo; Oulehlová, Denisa; Stillerová, Lenka; Schiebertová, Petra; Grunt, Michal; Žárský, Viktor; Cvrčková, Fatima

2016-03-01

Plant cell morphogenesis involves concerted rearrangements of microtubules and actin microfilaments. We previously reported that FH1, the main Arabidopsis thaliana housekeeping Class I membrane-anchored formin, contributes to actin dynamics and microtubule stability in rhizodermis cells. Here we examine the effects of mutations affecting FH1 (At3g25500) on cell morphogenesis and above-ground organ development in seedlings, as well as on cytoskeletal organization and dynamics, using a combination of confocal and variable angle epifluorescence microscopy with a pharmacological approach. Homozygous fh1 mutants exhibited cotyledon epinasty and had larger cotyledon pavement cells with more pronounced lobes than the wild type. The pavement cell shape alterations were enhanced by expression of the fluorescent microtubule marker GFP-microtubule-associated protein 4 (MAP4). Mutant cotyledon pavement cells exhibited reduced density and increased stability of microfilament bundles, as well as enhanced dynamics of microtubules. Analogous results were also obtained upon treatments with the formin inhibitor SMIFH2 (small molecule inhibitor of formin homology 2 domains). Pavement cell shape in wild-type (wt) and fh1 plants in some situations exhibited a differential response towards anti-cytoskeletal drugs, especially the microtubule disruptor oryzalin. Our observations indicate that FH1 participates in the control of microtubule dynamics, possibly via its effects on actin, subsequently influencing cell morphogenesis and macroscopic organ development. © The Author 2016. Published by Oxford University Press on behalf of Japanese Society of Plant Physiologists. All rights reserved. For permissions, please email: journals.permissions@oup.com.

A Plant Small Polypeptide Is a Novel Component of DNA-Binding Protein Phosphatase 1-Mediated Resistance to Plum pox virus in Arabidopsis1[C][W

PubMed Central

Castelló, María José; Carrasco, Jose Luis; Navarrete-Gómez, Marisa; Daniel, Jacques; Granot, David; Vera, Pablo

2011-01-01

DNA-binding protein phosphatases (DBPs) have been identified as a novel class of plant-specific regulatory factors playing a role in plant-virus interactions. NtDBP1 from tobacco (Nicotiana tabacum) was shown to participate in transcriptional regulation of gene expression in response to virus infection in compatible interactions, and AtDBP1, its closest relative in the model plant Arabidopsis (Arabidopsis thaliana), has recently been found to mediate susceptibility to potyvirus, one of the most speciose taxa of plant viruses. Here, we report on the identification of a novel family of highly conserved small polypeptides that interact with DBP1 proteins both in tobacco and Arabidopsis, which we have designated DBP-interacting protein 2 (DIP2). The interaction of AtDIP2 with AtDBP1 was demonstrated in vivo by bimolecular fluorescence complementation, and AtDIP2 was shown to functionally interfere with AtDBP1 in yeast. Furthermore, reducing AtDIP2 gene expression leads to increased susceptibility to the potyvirus Plum pox virus and to a lesser extent also to Turnip mosaic virus, whereas overexpression results in enhanced resistance. Therefore, we describe a novel family of conserved small polypeptides in plants and identify AtDIP2 as a novel host factor contributing to resistance to potyvirus in Arabidopsis. PMID:22021419

POLYGALACTURONASE INVOLVED IN EXPANSION1 functions in cell elongation and flower development in Arabidopsis.

PubMed

Xiao, Chaowen; Somerville, Chris; Anderson, Charles T

2014-03-01

Pectins are acidic carbohydrates that comprise a significant fraction of the primary walls of eudicotyledonous plant cells. They influence wall porosity and extensibility, thus controlling cell and organ growth during plant development. The regulated degradation of pectins is required for many cell separation events in plants, but the role of pectin degradation in cell expansion is poorly defined. Using an activation tag screen designed to isolate genes involved in wall expansion, we identified a gene encoding a putative polygalacturonase that, when overexpressed, resulted in enhanced hypocotyl elongation in etiolated Arabidopsis thaliana seedlings. We named this gene POLYGALACTURONASE INVOLVED IN EXPANSION1 (PGX1). Plants lacking PGX1 display reduced hypocotyl elongation that is complemented by transgenic PGX1 expression. PGX1 is expressed in expanding tissues throughout development, including seedlings, roots, leaves, and flowers. PGX1-GFP (green fluorescent protein) localizes to the apoplast, and heterologously expressed PGX1 displays in vitro polygalacturonase activity, supporting a function for this protein in apoplastic pectin degradation. Plants either overexpressing or lacking PGX1 display alterations in total polygalacturonase activity, pectin molecular mass, and wall composition and also display higher proportions of flowers with extra petals, suggesting PGX1's involvement in floral organ patterning. These results reveal new roles for polygalacturonases in plant development.

Atkinesin-13A Modulates Cell-Wall Synthesis and Cell Expansion in Arabidopsis thaliana via the THESEUS1 Pathway

PubMed Central

Fujikura, Ushio; Elsaesser, Lore; Breuninger, Holger; Sánchez-Rodríguez, Clara; Ivakov, Alexander; Laux, Thomas; Findlay, Kim; Persson, Staffan; Lenhard, Michael

2014-01-01

Growth of plant organs relies on cell proliferation and expansion. While an increasingly detailed picture about the control of cell proliferation is emerging, our knowledge about the control of cell expansion remains more limited. We demonstrate here that the internal-motor kinesin AtKINESIN-13A (AtKIN13A) limits cell expansion and cell size in Arabidopsis thaliana, with loss-of-function atkin13a mutants forming larger petals with larger cells. The homolog, AtKINESIN-13B, also affects cell expansion and double mutants display growth, gametophytic and early embryonic defects, indicating a redundant role of the two genes. AtKIN13A is known to depolymerize microtubules and influence Golgi motility and distribution. Consistent with this function, AtKIN13A interacts genetically with ANGUSTIFOLIA, encoding a regulator of Golgi dynamics. Reduced AtKIN13A activity alters cell wall structure as assessed by Fourier-transformed infrared-spectroscopy and triggers signalling via the THESEUS1-dependent cell-wall integrity pathway, which in turn promotes the excess cell expansion in the atkin13a mutant. Thus, our results indicate that the intracellular activity of AtKIN13A regulates cell expansion and wall architecture via THESEUS1, providing a compelling case of interplay between cell wall integrity sensing and expansion. PMID:25232944

The MADS-box XAANTAL1 increases proliferation at the Arabidopsis root stem-cell niche and participates in transition to differentiation by regulating cell-cycle components

PubMed Central

García-Cruz, Karla V.; García-Ponce, Berenice; Garay-Arroyo, Adriana; Sanchez, María De La Paz; Ugartechea-Chirino, Yamel; Desvoyes, Bénédicte; Pacheco-Escobedo, Mario A.; Tapia-López, Rosalinda; Ransom-Rodríguez, Ivan; Gutierrez, Crisanto; Alvarez-Buylla, Elena R.

2016-01-01

Background Morphogenesis depends on the concerted modulation of cell proliferation and differentiation. Such modulation is dynamically adjusted in response to various external and internal signals via complex transcriptional regulatory networks that mediate between such signals and regulation of cell-cycle and cellular responses (proliferation, growth, differentiation). In plants, which are sessile, the proliferation/differentiation balance is plastically adjusted during their life cycle and transcriptional networks are important in this process. MADS-box genes are key developmental regulators in eukaryotes, but their role in cell proliferation and differentiation modulation in plants remains poorly studied. Methods We characterize the XAL1 loss-of-function xal1-2 allele and overexpression lines using quantitative cellular and cytometry analyses to explore its role in cell cycle, proliferation, stem-cell patterning and transition to differentiation. We used quantitative PCR and cellular markers to explore if XAL1 regulates cell-cycle components and PLETHORA1 (PLT1) gene expression, as well as confocal microscopy to analyse stem-cell niche organization. Key Results We previously showed that XAANTAL1 (XAL1/AGL12) is necessary for Arabidopsis root development as a promoter of cell proliferation in the root apical meristem. Here, we demonstrate that XAL1 positively regulates the expression of PLT1 and important components of the cell cycle: CYCD3;1, CYCA2;3, CYCB1;1, CDKB1;1 and CDT1a. In addition, we show that xal1-2 mutant plants have a premature transition to differentiation with root hairs appearing closer to the root tip, while endoreplication in these plants is partially compromised. Coincidently, the final size of cortex cells in the mutant is shorter than wild-type cells. Finally, XAL1 overexpression-lines corroborate that this transcription factor is able to promote cell proliferation at the stem-cell niche. Conclusion XAL1 seems to be an important component

The organization pattern of root border-like cells of Arabidopsis is dependent on cell wall homogalacturonan.

PubMed

Durand, Caroline; Vicré-Gibouin, Maïté; Follet-Gueye, Marie Laure; Duponchel, Ludovic; Moreau, Myriam; Lerouge, Patrice; Driouich, Azeddine

2009-07-01

Border-like cells are released by Arabidopsis (Arabidopsis thaliana) root tips as organized layers of several cells that remain attached to each other rather than completely detached from each other, as is usually observed in border cells of many species. Unlike border cells, cell attachment between border-like cells is maintained after their release into the external environment. To investigate the role of cell wall polysaccharides in the attachment and organization of border-like cells, we have examined their release in several well-characterized mutants defective in the biosynthesis of xyloglucan, cellulose, or pectin. Our data show that among all mutants examined, only quasimodo mutants (qua1-1 and qua2-1), which have been characterized as producing less homogalacturonan, had an altered border-like cell phenotype as compared with the wild type. Border-like cells in both lines were released as isolated cells separated from each other, with the phenotype being much more pronounced in qua1-1 than in qua2-1. Further analysis of border-like cells in the qua1-1 mutant using immunocytochemistry and a set of anti-cell wall polysaccharide antibodies showed that the loss of the wild-type phenotype was accompanied by (1) a reduction in homogalacturonan-JIM5 epitope in the cell wall of border-like cells, confirmed by Fourier transform infrared microspectrometry, and (2) the secretion of an abundant mucilage that is enriched in xylogalacturonan and arabinogalactan-protein epitopes, in which the cells are trapped in the vicinity of the root tip.

ARABIDOPSIS HOMOLOG of TRITHORAX1 (ATX1) is required for cell production, patterning, and morphogenesis in root development

PubMed Central

Napsucialy-Mendivil, Selene; Alvarez-Venegas, Raúl; Shishkova, Svetlana; Dubrovsky, Joseph G.

2014-01-01

ARABIDOPSIS HOMOLOG of TRITHORAX1 (ATX1/SDG27), a known regulator of flower development, encodes a H3K4histone methyltransferase that maintains a number of genes in an active state. In this study, the role of ATX1 in root development was evaluated. The loss-of-function mutant atx1-1 was impaired in primary root growth. The data suggest that ATX1 controls root growth by regulating cell cycle duration, cell production, and the transition from cell proliferation in the root apical meristem (RAM) to cell elongation. In atx1-1, the quiescent centre (QC) cells were irregular in shape and more expanded than those of the wild type. This feature, together with the atypical distribution of T-divisions, the presence of oblique divisions, and the abnormal cell patterning in the RAM, suggests a lack of coordination between cell division and cell growth in the mutant. The expression domain of QC-specific markers was expanded both in the primary RAM and in the developing lateral root primordia of atx1-1 plants. These abnormalities were independent of auxin-response gradients. ATX1 was also found to be required for lateral root initiation, morphogenesis, and emergence. The time from lateral root initiation to emergence was significantly extended in the atx1-1 mutant. Overall, these data suggest that ATX1 is involved in the timing of root development, stem cell niche maintenance, and cell patterning during primary and lateral root development. Thus, ATX1 emerges as an important player in root system architecture. PMID:25205583

Roles of BOR2, a Boron Exporter, in Cross Linking of Rhamnogalacturonan II and Root Elongation under Boron Limitation in Arabidopsis1[W

PubMed Central

Miwa, Kyoko; Wakuta, Shinji; Takada, Shigeki; Ide, Koji; Takano, Junpei; Naito, Satoshi; Omori, Hiroyuki; Matsunaga, Toshiro; Fujiwara, Toru

2013-01-01

Boron (B) is required for cross linking of the pectic polysaccharide rhamnogalacturonan II (RG-II) and is consequently essential for the maintenance of cell wall structure. Arabidopsis (Arabidopsis thaliana) BOR1 is an efflux B transporter for xylem loading of B. Here, we describe the roles of BOR2, the most similar paralog of BOR1. BOR2 encodes an efflux B transporter localized in plasma membrane and is strongly expressed in lateral root caps and epidermis of elongation zones of roots. Transfer DNA insertion of BOR2 reduced root elongation by 68%, whereas the mutation in BOR1 reduced it by 32% under low B availability (0.1 µm), but the reduction in shoot growth was not as obvious as that in the BOR1 mutant. A double mutant of BOR1 and BOR2 exhibited much more severe growth defects in both roots and shoots under B-limited conditions than the corresponding single mutants. All single and double mutants grew normally under B-sufficient conditions. These results suggest that both BOR1 and BOR2 are required under B limitation and that their roles are, at least in part, different. The total B concentrations in roots of BOR2 mutants were not significantly different from those in wild-type plants, but the proportion of cross-linked RG-II was reduced under low B availability. Such a reduction in RG-II cross linking was not evident in roots of the BOR1 mutant. Thus, we propose that under B-limited conditions, transport of boric acid/borate by BOR2 from symplast to apoplast is required for effective cross linking of RG-II in cell wall and root cell elongation. PMID:24114060

Arabidopsis mitochondrial protein slow embryo development1 is essential for embryo development

DOE Office of Scientific and Technical Information (OSTI.GOV)

Ju, Yan; Liu, Chunying; Lu, Wenwen

The plant seeds formation are crucial parts in reproductive process in seed plants as well as food source for humans. Proper embryo development ensure viable seed formation. Here, we showed an Arabidopsis T-DNA insertion mutant slow embryo development1 (sed1) which exhibited retarded embryogenesis, led to aborted seeds. Embryo without SED1 developed slower compared to normal one and could be recognized at early globular stage by its white appearance. In later development stage, storage accumulated poorly with less protein and lipid body production. In vitro culture did not rescue albino embryo. SED1 encoded a protein targeted to mitochondria. Transmission electron microscopic analysismore » revealed that mitochondria developed abnormally, and more strikingly plastid failed to construct grana in time in sed1/sed1 embryo. These data indicated that SED1 is indispensable for embryogenesis in Arabidopsis, and the mitochondria may be involved in the regulation of many aspects of seed development. -- Highlights: •Arabidopsis SED1 is essential for embryo development. •The sed1 embryo accumulates less storage and has abnormal ultrastructure. •SED1 localizes to the mitochondrion.« less

Integration of Auxin and Salt Signals by the NAC Transcription Factor NTM2 during Seed Germination in Arabidopsis1[W

PubMed Central

Park, Jungmin; Kim, Youn-Sung; Kim, Sang-Gyu; Jung, Jae-Hoon; Woo, Je-Chang; Park, Chung-Mo

2011-01-01

Seed germination is regulated through elaborately interacting signaling networks that integrate diverse environmental cues into hormonal signaling pathways. Roles of gibberellic acid and abscisic acid in germination have been studied extensively using Arabidopsis (Arabidopsis thaliana) mutants having alterations in seed germination. Auxin has also been implicated in seed germination. However, how auxin influences germination is largely unknown. Here, we demonstrate that auxin is linked via the IAA30 gene with a salt signaling cascade mediated by the NAM-ATAF1/2-CUC2 transcription factor NTM2/Arabidopsis NAC domain-containing protein 69 (for NAC with Transmembrane Motif1) during seed germination. Germination of the NTM2-deficient ntm2-1 mutant seeds exhibited enhanced resistance to high salinity. However, the salt resistance disappeared in the ntm2-1 mutant overexpressing the IAA30 gene, which was induced by salt in a NTM2-dependent manner. Auxin exhibited no discernible effects on germination under normal growth conditions. Under high salinity, however, whereas exogenous application of auxin further suppressed the germination of control seeds, the auxin effects were reduced in the ntm2-1 mutant. Consistent with the inhibitory effects of auxin on germination, germination of YUCCA 3-overexpressing plants containing elevated levels of active auxin was more severely influenced by salt. These observations indicate that auxin delays seed germination under high salinity through cross talk with the NTM2-mediated salt signaling in Arabidopsis. PMID:21450938

Cameleon calcium indicator reports cytoplasmic calcium dynamics in Arabidopsis guard cells

NASA Technical Reports Server (NTRS)

Allen, G. J.; Kwak, J. M.; Chu, S. P.; Llopis, J.; Tsien, R. Y.; Harper, J. F.; Schroeder, J. I.; Evans, M. L. (Principal Investigator)

1999-01-01

Cytoplasmic free calcium ([Ca2+]cyt) acts as a stimulus-induced second messenger in plant cells and multiple signal transduction pathways regulate [Ca2+]cyt in stomatal guard cells. Measuring [Ca2+]cyt in guard cells has previously required loading of calcium-sensitive dyes using invasive and technically difficult micro-injection techniques. To circumvent these problems, we have constitutively expressed the pH-independent, green fluorescent protein-based calcium indicator yellow cameleon 2.1 in Arabidopsis thaliana (Miyawaki et al. 1999; Proc. Natl. Acad. Sci. USA 96, 2135-2140). This yellow cameleon calcium indicator was expressed in guard cells and accumulated predominantly in the cytoplasm. Fluorescence ratio imaging of yellow cameleon 2.1 allowed time-dependent measurements of [Ca2+]cyt in Arabidopsis guard cells. Application of extracellular calcium or the hormone abscisic acid (ABA) induced repetitive [Ca2+]cyt transients in guard cells. [Ca2+]cyt changes could be semi-quantitatively determined following correction of the calibration procedure for chloroplast autofluorescence. Extracellular calcium induced repetitive [Ca2+]cyt transients with peak values of up to approximately 1.5 microM, whereas ABA-induced [Ca2+]cyt transients had peak values up to approximately 0.6 microM. These values are similar to stimulus-induced [Ca2+]cyt changes previously reported in plant cells using ratiometric dyes or aequorin. In some guard cells perfused with low extracellular KCl concentrations, spontaneous calcium transients were observed. As yellow cameleon 2.1 was expressed in all guard cells, [Ca2+]cyt was measured independently in the two guard cells of single stomates for the first time. ABA-induced, calcium-induced or spontaneous [Ca2+]cyt increases were not necessarily synchronized in the two guard cells. Overall, these data demonstrate that that GFP-based cameleon calcium indicators are suitable to measure [Ca2+]cyt changes in guard cells and enable the pattern of [Ca

Synthesis of Hydroxylated Sterols in Transgenic Arabidopsis Plants Alters Growth and Steroid Metabolism1[C][W][OA

PubMed Central

Beste, Lisa; Nahar, Nurun; Dalman, Kerstin; Fujioka, Shozo; Jonsson, Lisbeth; Dutta, Paresh C.; Sitbon, Folke

2011-01-01

To explore mechanisms in plant sterol homeostasis, we have here increased the turnover of sterols in Arabidopsis (Arabidopsis thaliana) and potato (Solanum tuberosum) plants by overexpressing four mouse cDNA encoding cholesterol hydroxylases (CHs), hydroxylating cholesterol at the C-7, C-24, C-25, or C-27 positions. Compared to the wild type, the four types of Arabidopsis transformant showed varying degrees of phenotypic alteration, the strongest one being in CH25 lines, which were dark-green dwarfs resembling brassinosteroid-related mutants. Gas chromatography-mass spectrometry analysis of extracts from wild-type Arabidopsis plants revealed trace levels of α and β forms of 7-hydroxycholesterol, 7-hydroxycampesterol, and 7-hydroxysitosterol. The expected hydroxycholesterol metabolites in CH7-, CH24-, and CH25 transformants were identified and quantified using gas chromatography-mass spectrometry. Additional hydroxysterol forms were also observed, particularly in CH25 plants. In CH24 and CH25 lines, but not in CH7 ones, the presence of hydroxysterols was correlated with a considerable alteration of the sterol profile and an increased sterol methyltransferase activity in microsomes. Moreover, CH25 lines contained clearly reduced levels of brassinosteroids, and displayed an enhanced drought tolerance. Equivalent transformations of potato plants with the CH25 construct increased hydroxysterol levels, but without the concomitant alteration of growth and sterol profiles observed in Arabidopsis. The results suggest that an increased hydroxylation of cholesterol and/or other sterols in Arabidopsis triggers compensatory processes, acting to maintain sterols at adequate levels. PMID:21746809

Partial Shotgun Sequencing of the Boechera stricta Genome Reveals Extensive Microsynteny and Promoter Conservation with Arabidopsis1[W

PubMed Central

Windsor, Aaron J.; Schranz, M. Eric; Formanová, Nataša; Gebauer-Jung, Steffi; Bishop, John G.; Schnabelrauch, Domenica; Kroymann, Juergen; Mitchell-Olds, Thomas

2006-01-01

Comparative genomics provides insight into the evolutionary dynamics that shape discrete sequences as well as whole genomes. To advance comparative genomics within the Brassicaceae, we have end sequenced 23,136 medium-sized insert clones from Boechera stricta, a wild relative of Arabidopsis (Arabidopsis thaliana). A significant proportion of these sequences, 18,797, are nonredundant and display highly significant similarity (BLASTn e-value ≤ 10−30) to low copy number Arabidopsis genomic regions, including more than 9,000 annotated coding sequences. We have used this dataset to identify orthologous gene pairs in the two species and to perform a global comparison of DNA regions 5′ to annotated coding regions. On average, the 500 nucleotides upstream to coding sequences display 71.4% identity between the two species. In a similar analysis, 61.4% identity was observed between 5′ noncoding sequences of Brassica oleracea and Arabidopsis, indicating that regulatory regions are not as diverged among these lineages as previously anticipated. By mapping the B. stricta end sequences onto the Arabidopsis genome, we have identified nearly 2,000 conserved blocks of microsynteny (bracketing 26% of the Arabidopsis genome). A comparison of fully sequenced B. stricta inserts to their homologous Arabidopsis genomic regions indicates that indel polymorphisms >5 kb contribute substantially to the genome size difference observed between the two species. Further, we demonstrate that microsynteny inferred from end-sequence data can be applied to the rapid identification and cloning of genomic regions of interest from nonmodel species. These results suggest that among diploid relatives of Arabidopsis, small- to medium-scale shotgun sequencing approaches can provide rapid and cost-effective benefits to evolutionary and/or functional comparative genomic frameworks. PMID:16607030

Abnormal Glycosphingolipid Mannosylation Triggers Salicylic Acid–Mediated Responses in Arabidopsis[W][OA

PubMed Central

Mortimer, Jenny C.; Yu, Xiaolan; Albrecht, Sandra; Sicilia, Francesca; Huichalaf, Mariela; Ampuero, Diego; Michaelson, Louise V.; Murphy, Alex M.; Matsunaga, Toshiro; Kurz, Samantha; Stephens, Elaine; Baldwin, Timothy C.; Ishii, Tadashi; Napier, Johnathan A.; Weber, Andreas P.M.; Handford, Michael G.; Dupree, Paul

2013-01-01

The Arabidopsis thaliana protein GOLGI-LOCALIZED NUCLEOTIDE SUGAR TRANSPORTER (GONST1) has been previously identified as a GDP-d-mannose transporter. It has been hypothesized that GONST1 provides precursors for the synthesis of cell wall polysaccharides, such as glucomannan. Here, we show that in vitro GONST1 can transport all four plant GDP-sugars. However, gonst1 mutants have no reduction in glucomannan quantity and show no detectable alterations in other cell wall polysaccharides. By contrast, we show that a class of glycosylated sphingolipids (glycosylinositol phosphoceramides [GIPCs]) contains Man and that this mannosylation is affected in gonst1. GONST1 therefore is a Golgi GDP-sugar transporter that specifically supplies GDP-Man to the Golgi lumen for GIPC synthesis. gonst1 plants have a dwarfed phenotype and a constitutive hypersensitive response with elevated salicylic acid levels. This suggests an unexpected role for GIPC sugar decorations in sphingolipid function and plant defense signaling. Additionally, we discuss these data in the context of substrate channeling within the Golgi. PMID:23695979

Lifting DELLA Repression of Arabidopsis Seed Germination by Nonproteolytic Gibberellin Signaling1[C][W][OPEN

PubMed Central

Ariizumi, Tohru; Hauvermale, Amber L.; Nelson, Sven K.; Hanada, Atsushi; Yamaguchi, Shinjiro; Steber, Camille M.

2013-01-01

DELLA repression of Arabidopsis (Arabidopsis thaliana) seed germination can be lifted either through DELLA proteolysis by the ubiquitin-proteasome pathway or through proteolysis-independent gibberellin (GA) hormone signaling. GA binding to the GIBBERELLIN-INSENSITIVE DWARF1 (GID1) GA receptors stimulates GID1-GA-DELLA complex formation, which in turn triggers DELLA protein ubiquitination and proteolysis via the SCFSLY1 E3 ubiquitin ligase and 26S proteasome. Although DELLA cannot be destroyed in the sleepy1-2 (sly1-2) F-box mutant, long dry after-ripening and GID1 overexpression can relieve the strong sly1-2 seed dormancy phenotype. It appears that sly1-2 seed dormancy results from abscisic acid (ABA) signaling downstream of DELLA, since dormant sly1-2 seeds accumulate high levels of ABA hormone and loss of ABA sensitivity rescues sly1-2 seed germination. DELLA positively regulates the expression of XERICO, an inducer of ABA biosynthesis. GID1b overexpression rescues sly1-2 germination through proteolysis-independent DELLA down-regulation associated with increased expression of GA-inducible genes and decreased ABA accumulation, apparently as a result of decreased XERICO messenger RNA levels. Higher levels of GID1 overexpression are associated with more efficient sly1 germination and increased GID1-GA-DELLA complex formation, suggesting that GID1 down-regulates DELLA through protein binding. After-ripening results in increased GA accumulation and GID1a-dependent GA signaling, suggesting that after-ripening triggers GA-stimulated GID1-GA-DELLA protein complex formation, which in turn blocks DELLA transcriptional activation of the XERICO inhibitor of seed germination. PMID:23818171

Combining Enhanced Root and Shoot Growth Reveals Cross Talk between Pathways That Control Plant Organ Size in Arabidopsis1[C][W][OA

PubMed Central

Vercruyssen, Liesbeth; Gonzalez, Nathalie; Werner, Tomáš; Schmülling, Thomas; Inzé, Dirk

2011-01-01

Functionally distinct Arabidopsis (Arabidopsis thaliana) genes that positively affect root or shoot growth when ectopically expressed were combined to explore the feasibility of enhanced biomass production. Enhanced root growth resulting from cytokinin deficiency was obtained by overexpressing CYTOKININ OXIDASE/DEHYDROGENASE3 (CKX3) under the control of the root-specific PYK10 promoter. Plants harboring the PYK10-CKX3 construct were crossed with four different transgenic lines showing enhanced leaf growth. For all combinations, the phenotypic traits of the individual lines could be combined, resulting in an overall growth increase. Unexpectedly, three out of four combinations had more than additive effects. Both leaf and root growth were synergistically enhanced in plants ectopically expressing CKX3 and BRASSINOSTEROID INSENSITIVE1, indicating cross talk between cytokinins and brassinosteroids. In agreement, treatment of PYK10-CKX3 plants with brassinolide resulted in a dramatic increase in lateral root growth that could not be observed in wild-type plants. Coexpression of CKX3 and the GROWTH-REGULATING FACTOR5 (GRF5) antagonized the effects of GRF5 overexpression, revealing an interplay between cytokinins and GRF5 during leaf cell proliferation. The combined overexpression of CKX3 and GIBBERELLIN 20-OXIDASE1 led to a synergistic increase in leaf growth, suggesting an antagonistic growth control by cytokinins and gibberellins. Only additive effects on root and shoot growth were visible in plants ectopically expressing both CKX3 and ARABIDOPSIS VACUOLAR PYROPHOSPHATASE1, hinting at an independent action mode. Our results show new interactions and contribute to the molecular and physiological understanding of biomass production at the whole plant level. PMID:21205622

Coupling of a 2.5 kW steam reformer with a 1 kW el PEM fuel cell

NASA Astrophysics Data System (ADS)

Mathiak, J.; Heinzel, A.; Roes, J.; Kalk, Th.; Kraus, H.; Brandt, H.

The University of Duisburg-Essen has developed a compact multi-fuel steam reformer suitable for natural gas, propane and butane. This steam reformer was combined with a polymer electrolyte membrane fuel cell (PEM FC) and a system test of the process chain was performed. The fuel processor comprises a prereformer step, a primary reformer, water gas shift reactors, a steam generator, internal heat exchangers in order to achieve an optimised heat integration and an external burner for heat supply as well as a preferential oxidation step (PROX) as CO purification. The fuel processor is designed to deliver a thermal hydrogen power output from 500 W to 2.5 kW. The PEM fuel cell stack provides about 1 kW electrical power. In the following paper experimental results of measurements of the single components PEM fuel cell and fuel processor as well as results of the coupling of both to form a process chain are presented.

Shoot Na+ Exclusion and Increased Salinity Tolerance Engineered by Cell Type–Specific Alteration of Na+ Transport in Arabidopsis[W][OA

PubMed Central

Møller, Inge S.; Gilliham, Matthew; Jha, Deepa; Mayo, Gwenda M.; Roy, Stuart J.; Coates, Juliet C.; Haseloff, Jim; Tester, Mark

2009-01-01

Soil salinity affects large areas of cultivated land, causing significant reductions in crop yield globally. The Na+ toxicity of many crop plants is correlated with overaccumulation of Na+ in the shoot. We have previously suggested that the engineering of Na+ exclusion from the shoot could be achieved through an alteration of plasma membrane Na+ transport processes in the root, if these alterations were cell type specific. Here, it is shown that expression of the Na+ transporter HKT1;1 in the mature root stele of Arabidopsis thaliana decreases Na+ accumulation in the shoot by 37 to 64%. The expression of HKT1;1 specifically in the mature root stele is achieved using an enhancer trap expression system for specific and strong overexpression. The effect in the shoot is caused by the increased influx, mediated by HKT1;1, of Na+ into stelar root cells, which is demonstrated in planta and leads to a reduction of root-to-shoot transfer of Na+. Plants with reduced shoot Na+ also have increased salinity tolerance. By contrast, plants constitutively expressing HKT1;1 driven by the cauliflower mosaic virus 35S promoter accumulated high shoot Na+ and grew poorly. Our results demonstrate that the modification of a specific Na+ transport process in specific cell types can reduce shoot Na+ accumulation, an important component of salinity tolerance of many higher plants. PMID:19584143

The TCP4 transcription factor of Arabidopsis blocks cell division in yeast at G1→S transition.

PubMed

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

2011-07-01

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→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→S arrest is discussed. Copyright © 2011 Elsevier Inc. All rights reserved.

Proteomics and Functional Analyses of Pepper Abscisic Acid–Responsive 1 (ABR1), Which Is Involved in Cell Death and Defense Signaling[C][W

PubMed Central

Choi, Du Seok; Hwang, Byung Kook

2011-01-01

Abscisic acid (ABA) is a key regulator of plant growth and development, as well as plant defense responses. A high-throughput in planta proteome screen identified the pepper (Capsicum annuum) GRAM (for glucosyltransferases, Rab-like GTPase activators, and myotubularins) domain-containing ABA-RESPONSIVE1 (ABR1), which is highly induced by infection with avirulent Xanthomonas campestris pv vesicatoria and also by treatment with ABA. The GRAM domain is essential for the cell death response and for the nuclear localization of ABR1. ABR1 is required for priming cell death and reactive oxygen species production, as well as ABA-salicylic acid (SA) antagonism. Silencing of ABR1 significantly compromised the hypersensitive response but enhanced bacterial pathogen growth and ABA levels in pepper. High levels of ABA in ABR1-silenced plants antagonized the SA levels induced by pathogen infection. Heterologous transgenic expression of ABR1 in Arabidopsis thaliana conferred enhanced resistance to Pseudomonas syringae pv tomato and Hyaloperonospora arabidopsidis infection. The susceptibility of the Arabidopsis ABR1 putative ortholog mutant, abr1, to these pathogens also supports the involvement of ABR1 in disease resistance. Together, these results reveal ABR1 as a novel negative regulator of ABA signaling and suggest that the nuclear ABR1 pool is essential for the cell death induction associated with ABA-SA antagonism. PMID:21335377

Repression of Growth Regulating Factors by the MicroRNA396 Inhibits Cell Proliferation by UV-B Radiation in Arabidopsis Leaves[C][W

PubMed Central

Casadevall, Romina; Rodriguez, Ramiro E.; Debernardi, Juan M.; Palatnik, Javier F.; Casati, Paula

2013-01-01

Because of their sessile lifestyle, plants are continuously exposed to solar UV-B radiation. Inhibition of leaf growth is one of the most consistent responses of plants upon exposure to UV-B radiation. In this work, we investigated the role of GROWTH-REGULATING FACTORs (GRFs) and of microRNA miR396 in UV-B–mediated inhibition of leaf growth in Arabidopsis thaliana plants. We demonstrate that miRNA396 is upregulated by UV-B radiation in proliferating tissues and that this induction is correlated with a decrease in GRF1, GRF2, and GRF3 transcripts. Induction of miR396 results in inhibition of cell proliferation, and this outcome is independent of the UV-B photoreceptor UV resistance locus 8, as well as ATM AND RAD3–RELATED and the mitogen-activated protein kinase MPK6, but is dependent on MPK3. Transgenic plants expressing an artificial target mimic directed against miR396 (MIM396) with a decrease in the endogenous microRNA activity or plants expressing miR396-resistant copies of several GRFs are less sensitive to this inhibition. Consequently, at intensities that can induce DNA damage in Arabidopsis plants, UV-B radiation limits leaf growth by inhibiting cell division in proliferating tissues, a process mediated by miR396 and GRFs. PMID:24076976

Karrikins Discovered in Smoke Trigger Arabidopsis Seed Germination by a Mechanism Requiring Gibberellic Acid Synthesis and Light1[W][OA

PubMed Central

Nelson, David C.; Riseborough, Julie-Anne; Flematti, Gavin R.; Stevens, Jason; Ghisalberti, Emilio L.; Dixon, Kingsley W.; Smith, Steven M.

2009-01-01

Discovery of the primary seed germination stimulant in smoke, 3-methyl-2H-furo[2,3-c]pyran-2-one (KAR1), has resulted in identification of a family of structurally related plant growth regulators, karrikins. KAR1 acts as a key germination trigger for many species from fire-prone, Mediterranean climates, but a molecular mechanism for this response remains unknown. We demonstrate that Arabidopsis (Arabidopsis thaliana), an ephemeral of the temperate northern hemisphere that has never, to our knowledge, been reported to be responsive to fire or smoke, rapidly and sensitively perceives karrikins. Thus, these signaling molecules may have greater significance among angiosperms than previously realized. Karrikins can trigger germination of primary dormant Arabidopsis seeds far more effectively than known phytohormones or the structurally related strigolactone GR-24. Natural variation and depth of seed dormancy affect the degree of KAR1 stimulation. Analysis of phytohormone mutant germination reveals suppression of KAR1 responses by abscisic acid and a requirement for gibberellin (GA) synthesis. The reduced germination of sleepy1 mutants is partially recovered by KAR1, which suggests that germination enhancement by karrikin is only partly DELLA dependent. While KAR1 has little effect on sensitivity to exogenous GA, it enhances expression of the GA biosynthetic genes GA3ox1 and GA3ox2 during seed imbibition. Neither abscisic acid nor GA levels in seed are appreciably affected by KAR1 treatment prior to radicle emergence, despite marked differences in germination outcome. KAR1 stimulation of Arabidopsis germination is light-dependent and reversible by far-red exposure, although limited induction of GA3ox1 still occurs in the dark. The observed requirements for light and GA biosynthesis provide the first insights into the karrikin mode of action. PMID:19074625

Functional analysis of the theobroma cacao NPR1 gene in arabidopsis

PubMed Central

2010-01-01

Background The Arabidopsis thaliana NPR1 gene encodes a transcription coactivator (NPR1) that plays a major role in the mechanisms regulating plant defense response. After pathogen infection and in response to salicylic acid (SA) accumulation, NPR1 translocates from the cytoplasm into the nucleus where it interacts with other transcription factors resulting in increased expression of over 2000 plant defense genes contributing to a pathogen resistance response. Results A putative Theobroma cacao NPR1 cDNA was isolated by RT-PCR using degenerate primers based on homologous sequences from Brassica, Arabidopsis and Carica papaya. The cDNA was used to isolate a genomic clone from Theobroma cacao containing a putative TcNPR1 gene. DNA sequencing revealed the presence of a 4.5 kb coding region containing three introns and encoding a polypeptide of 591 amino acids. The predicted TcNPR1 protein shares 55% identity and 78% similarity to Arabidopsis NPR1, and contains each of the highly conserved functional domains indicative of this class of transcription factors (BTB/POZ and ankyrin repeat protein-protein interaction domains and a nuclear localization sequence (NLS)). To functionally define the TcNPR1 gene, we transferred TcNPR1 into an Arabidopsis npr1 mutant that is highly susceptible to infection by the plant pathogen Pseudomonas syringae pv. tomato DC3000. Driven by the constitutive CaMV35S promoter, the cacao TcNPR1 gene partially complemented the npr1 mutation in transgenic Arabidopsis plants, resulting in 100 fold less bacterial growth in a leaf infection assay. Upon induction with SA, TcNPR1 was shown to translocate into the nucleus of leaf and root cells in a manner identical to Arabidopsis NPR1. Cacao NPR1 was also capable of participating in SA-JA signaling crosstalk, as evidenced by the suppression of JA responsive gene expression in TcNPR1 overexpressing transgenic plants. Conclusion Our data indicate that the TcNPR1 is a functional ortholog of Arabidopsis NPR1

Functional analysis of the Theobroma cacao NPR1 gene in Arabidopsis.

PubMed

Shi, Zi; Maximova, Siela N; Liu, Yi; Verica, Joseph; Guiltinan, Mark J

2010-11-15

The Arabidopsis thaliana NPR1 gene encodes a transcription coactivator (NPR1) that plays a major role in the mechanisms regulating plant defense response. After pathogen infection and in response to salicylic acid (SA) accumulation, NPR1 translocates from the cytoplasm into the nucleus where it interacts with other transcription factors resulting in increased expression of over 2000 plant defense genes contributing to a pathogen resistance response. A putative Theobroma cacao NPR1 cDNA was isolated by RT-PCR using degenerate primers based on homologous sequences from Brassica, Arabidopsis and Carica papaya. The cDNA was used to isolate a genomic clone from Theobroma cacao containing a putative TcNPR1 gene. DNA sequencing revealed the presence of a 4.5 kb coding region containing three introns and encoding a polypeptide of 591 amino acids. The predicted TcNPR1 protein shares 55% identity and 78% similarity to Arabidopsis NPR1, and contains each of the highly conserved functional domains indicative of this class of transcription factors (BTB/POZ and ankyrin repeat protein-protein interaction domains and a nuclear localization sequence (NLS)). To functionally define the TcNPR1 gene, we transferred TcNPR1 into an Arabidopsis npr1 mutant that is highly susceptible to infection by the plant pathogen Pseudomonas syringae pv. tomato DC3000. Driven by the constitutive CaMV35S promoter, the cacao TcNPR1 gene partially complemented the npr1 mutation in transgenic Arabidopsis plants, resulting in 100 fold less bacterial growth in a leaf infection assay. Upon induction with SA, TcNPR1 was shown to translocate into the nucleus of leaf and root cells in a manner identical to Arabidopsis NPR1. Cacao NPR1 was also capable of participating in SA-JA signaling crosstalk, as evidenced by the suppression of JA responsive gene expression in TcNPR1 overexpressing transgenic plants. Our data indicate that the TcNPR1 is a functional ortholog of Arabidopsis NPR1, and is likely to play a

Integrated Metabolite and Transcript Profiling Identify a Biosynthetic Mechanism for Hispidol in Medicago truncatula Cell Cultures1[C][W][OA

PubMed Central

Farag, Mohamed A.; Deavours, Bettina E.; de Fátima, Ângelo; Naoumkina, Marina; Dixon, Richard A.; Sumner, Lloyd W.

2009-01-01

Metabolic profiling of elicited barrel medic (Medicago truncatula) cell cultures using high-performance liquid chromatography coupled to photodiode and mass spectrometry detection revealed the accumulation of the aurone hispidol (6-hydroxy-2-[(4-hydroxyphenyl)methylidene]-1-benzofuran-3-one) as a major response to yeast elicitor. Parallel, large-scale transcriptome profiling indicated that three peroxidases, MtPRX1, MtPRX2, and MtPRX3, were coordinately induced with the accumulation of hispidol. MtPRX1 and MtPRX2 exhibited aurone synthase activity based upon in vitro substrate specificity and product profiles of recombinant proteins expressed in Escherichia coli. Hispidol possessed significant antifungal activity relative to other M. truncatula phenylpropanoids tested but has not been reported in this species before and was not found in differentiated roots in which high levels of the peroxidase transcripts accumulated. We propose that hispidol is formed in cell cultures by metabolic spillover when the pool of its precursor, isoliquiritigenin, builds up as a result of an imbalance between the upstream and downstream segments of the phenylpropanoid pathway, reflecting the plasticity of plant secondary metabolism. The results illustrate that integration of metabolomics and transcriptomics in genetically reprogrammed plant cell cultures is a powerful approach for the discovery of novel bioactive secondary metabolites and the mechanisms underlying their generation. PMID:19571306

Arabidopsis ETR1 and ERS1 Differentially Repress the Ethylene Response in Combination with Other Ethylene Receptor Genes1[W

PubMed Central

Liu, Qian; Wen, Chi-Kuang

2012-01-01

The ethylene response is negatively regulated by a family of five ethylene receptor genes in Arabidopsis (Arabidopsis thaliana). The five members of the ethylene receptor family can physically interact and form complexes, which implies that cooperativity for signaling may exist among the receptors. The ethylene receptor gene mutations etr1-1(C65Y)(for ethylene response1-1), ers1-1(I62P) (for ethylene response sensor1-1), and ers1C65Y are dominant, and each confers ethylene insensitivity. In this study, the repression of the ethylene response by these dominant mutant receptor genes was examined in receptor-defective mutants to investigate the functional significance of receptor cooperativity in ethylene signaling. We showed that etr1-1(C65Y), but not ers1-1(I62P), substantially repressed various ethylene responses independent of other receptor genes. In contrast, wild-type receptor genes differentially supported the repression of ethylene responses by ers1-1(I62P); ETR1 and ETHYLENE INSENSITIVE4 (EIN4) supported ers1-1(I62P) functions to a greater extent than did ERS2, ETR2, and ERS1. The lack of both ETR1 and EIN4 almost abolished the repression of ethylene responses by ers1C65Y, which implied that ETR1 and EIN4 have synergistic effects on ers1C65Y functions. Our data indicated that a dominant ethylene-insensitive receptor differentially repressed ethylene responses when coupled with a wild-type ethylene receptor, which supported the hypothesis that the formation of a variety of receptor complexes may facilitate differential receptor signal output, by which ethylene responses can be repressed to different extents. We hypothesize that plants can respond to a broad ethylene concentration range and exhibit tissue-specific ethylene responsiveness with differential cooperation of the multiple ethylene receptors. PMID:22227969

Propidium Iodide Competes with Ca2+ to Label Pectin in Pollen Tubes and Arabidopsis Root Hairs1[W][OA

PubMed Central

Rounds, Caleb M.; Lubeck, Eric; Hepler, Peter K.; Winship, Lawrence J.

2011-01-01

We have used propidium iodide (PI) to investigate the dynamic properties of the primary cell wall at the apex of Arabidopsis (Arabidopsis thaliana) root hairs and pollen tubes and in lily (Lilium formosanum) pollen tubes. Our results show that in root hairs, as in pollen tubes, oscillatory peaks in PI fluorescence precede growth rate oscillations. Pectin forms the primary component of the cell wall at the tip of both root hairs and pollen tubes. Given the electronic structure of PI, we investigated whether PI binds to pectins in a manner analogous to Ca2+ binding. We first show that Ca2+ is able to abrogate PI growth inhibition in a dose-dependent manner. PI fluorescence itself also relies directly on the amount of Ca2+ in the growth solution. Exogenous pectin methyl esterase treatment of pollen tubes, which demethoxylates pectins, freeing more Ca2+-binding sites, leads to a dramatic increase in PI fluorescence. Treatment with pectinase leads to a corresponding decrease in fluorescence. These results are consistent with the hypothesis that PI binds to demethoxylated pectins. Unlike other pectin stains, PI at low yet useful concentration is vital and specifically does not alter the tip-focused Ca2+ gradient or growth oscillations. These data suggest that pectin secretion at the apex of tip-growing plant cells plays a critical role in regulating growth, and PI represents an excellent tool for examining the role of pectin and of Ca2+ in tip growth. PMID:21768649

Sodium Fluxes through Nonselective Cation Channels in the Plasma Membrane of Protoplasts from Arabidopsis Roots1

PubMed Central

Demidchik, Vadim; Tester, Mark

2002-01-01

The aim of the present work was to characterize Na+ currents through nonselective cation channels (NSCCs) in protoplasts derived from root cells of Arabidopsis. The procedure of the protoplast isolation was modified to increase the stability of Arabidopsis root protoplasts in low external Ca2+ by digesting tissue in elevated Ca2+. Experiments in whole-cell and outside-out modes were carried out. We found that Na+ currents in Arabidopsis root protoplasts were mediated by cation channels that were insensitive to externally applied tetraethylammonium+ and verapamil, had no time-dependent activation (permanently opened or completely activated within 1–2 ms), were voltage independent, and were weakly selective for monovalent cations. The selectivity sequence was as follows: K+ (1.49) > NH4+ (1.24) > Rb+ (1.15) ≈ Cs+ (1.10) ≈ Na+ (1.00) > Li+ (0.73) > tetraethylammonium+ (0.47). Arabidopsis root NSCCs were blocked by H+ (pK ≈ 6.0), Ca2+ (K1/2 ≈ 0.1 mm), Ba2+, Zn2+, La3+, Gd3+, quinine, and the His modifier diethylpyrocarbonate. They were insensitive to most organic blockers (nifedipine, verapamil, flufenamate, and amiloride) and to the SH-group modifier p-chloromercuriphenyl sulfonic acid. Voltage-insensitive, Ca2+-sensitive single channels were also resolved. Properties of Arabidopsis root NSCCs are discussed and compared with characteristics of similar conductances studied previously in plants and animals. It is suggested that NSCCs present a distinct group of plant ion channels, mediating toxic Na+ influx to the cell and probably having other important roles in physiological processes of plants. PMID:11842142

MEDIATOR25 Acts as an Integrative Hub for the Regulation of Jasmonate-Responsive Gene Expression in Arabidopsis1[C][W

PubMed Central

Çevik, Volkan; Kidd, Brendan N.; Zhang, Peijun; Hill, Claire; Kiddle, Steve; Denby, Katherine J.; Holub, Eric B.; Cahill, David M.; Manners, John M.; Schenk, Peer M.; Beynon, Jim; Kazan, Kemal

2012-01-01

The PHYTOCHROME AND FLOWERING TIME1 gene encoding the MEDIATOR25 (MED25) subunit of the eukaryotic Mediator complex is a positive regulator of jasmonate (JA)-responsive gene expression in Arabidopsis (Arabidopsis thaliana). Based on the function of the Mediator complex as a bridge between DNA-bound transcriptional activators and the RNA polymerase II complex, MED25 has been hypothesized to function in association with transcriptional regulators of the JA pathway. However, it is currently not known mechanistically how MED25 functions to regulate JA-responsive gene expression. In this study, we show that MED25 physically interacts with several key transcriptional regulators of the JA signaling pathway, including the APETALA2 (AP2)/ETHYLENE RESPONSE FACTOR (ERF) transcription factors OCTADECANOID-RESPONSIVE ARABIDOPSIS AP2/ERF59 and ERF1 as well as the master regulator MYC2. Physical interaction detected between MED25 and four group IX AP2/ERF transcription factors was shown to require the activator interaction domain of MED25 as well as the recently discovered Conserved Motif IX-1/EDLL transcription activation motif of MED25-interacting AP2/ERFs. Using transcriptional activation experiments, we also show that OCTADECANOID-RESPONSIVE ARABIDOPSIS AP2/ERF59- and ERF1-dependent activation of PLANT DEFENSIN1.2 as well as MYC2-dependent activation of VEGETATIVE STORAGE PROTEIN1 requires a functional MED25. In addition, MED25 is required for MYC2-dependent repression of pathogen defense genes. These results suggest an important role for MED25 as an integrative hub within the Mediator complex during the regulation of JA-associated gene expression. PMID:22822211

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

Arabidopsis Enhanced Drought Tolerance1/HOMEODOMAIN GLABROUS11 Confers Drought Tolerance in Transgenic Rice without Yield Penalty1[W][OA

PubMed Central

Yu, Linhui; Chen, Xi; Wang, Zhen; Wang, Shimei; Wang, Yuping; Zhu, Qisheng; Li, Shigui; Xiang, Chengbin

2013-01-01

Enhancing drought tolerance without yield decrease has been a great challenge in crop improvement. Here, we report the Arabidopsis (Arabidopsis thaliana) homodomain-leucine zipper transcription factor Enhanced Drought Tolerance/HOMEODOMAIN GLABROUS11 (EDT1/HDG11) was able to confer drought tolerance and increase grain yield in transgenic rice (Oryza sativa) plants. The improved drought tolerance was associated with a more extensive root system, reduced stomatal density, and higher water use efficiency. The transgenic rice plants also had higher levels of abscisic acid, proline, soluble sugar, and reactive oxygen species-scavenging enzyme activities during stress treatments. The increased grain yield of the transgenic rice was contributed by improved seed setting, larger panicle, and more tillers as well as increased photosynthetic capacity. Digital gene expression analysis indicated that AtEDT1/HDG11 had a significant influence on gene expression profile in rice, which was consistent with the observed phenotypes of transgenic rice plants. Our study shows that AtEDT1/HDG11 can improve both stress tolerance and grain yield in rice, demonstrating the efficacy of AtEDT1/HDG11 in crop improvement. PMID:23735506

The MADS-box XAANTAL1 increases proliferation at the Arabidopsis root stem-cell niche and participates in transition to differentiation by regulating cell-cycle components.

PubMed

García-Cruz, Karla V; García-Ponce, Berenice; Garay-Arroyo, Adriana; Sanchez, María De La Paz; Ugartechea-Chirino, Yamel; Desvoyes, Bénédicte; Pacheco-Escobedo, Mario A; Tapia-López, Rosalinda; Ransom-Rodríguez, Ivan; Gutierrez, Crisanto; Alvarez-Buylla, Elena R

2016-07-29

Morphogenesis depends on the concerted modulation of cell proliferation and differentiation. Such modulation is dynamically adjusted in response to various external and internal signals via complex transcriptional regulatory networks that mediate between such signals and regulation of cell-cycle and cellular responses (proliferation, growth, differentiation). In plants, which are sessile, the proliferation/differentiation balance is plastically adjusted during their life cycle and transcriptional networks are important in this process. MADS-box genes are key developmental regulators in eukaryotes, but their role in cell proliferation and differentiation modulation in plants remains poorly studied. We characterize the XAL1 loss-of-function xal1-2 allele and overexpression lines using quantitative cellular and cytometry analyses to explore its role in cell cycle, proliferation, stem-cell patterning and transition to differentiation. We used quantitative PCR and cellular markers to explore if XAL1 regulates cell-cycle components and PLETHORA1 (PLT1) gene expression, as well as confocal microscopy to analyse stem-cell niche organization. We previously showed that XAANTAL1 (XAL1/AGL12) is necessary for Arabidopsis root development as a promoter of cell proliferation in the root apical meristem. Here, we demonstrate that XAL1 positively regulates the expression of PLT1 and important components of the cell cycle: CYCD3;1, CYCA2;3, CYCB1;1, CDKB1;1 and CDT1a In addition, we show that xal1-2 mutant plants have a premature transition to differentiation with root hairs appearing closer to the root tip, while endoreplication in these plants is partially compromised. Coincidently, the final size of cortex cells in the mutant is shorter than wild-type cells. Finally, XAL1 overexpression-lines corroborate that this transcription factor is able to promote cell proliferation at the stem-cell niche. XAL1 seems to be an important component of the networks that modulate cell

Arabidopsis LEAFY COTYLEDON1 controls cell fate determination during post-embryonic development

PubMed Central

Huang, Mingkun; Hu, Yilong; Liu, Xu; Li, Yuge; Hou, Xingliang

2015-01-01

Arabidopsis LEAFY COTYLEDON1 (LEC1) transcription factor is a master regulator that shapes plant embryo development and post-embryonic seedling establishment. Loss-of-function of LEC1 alters the cotyledon identity, causing the formation of ectopic trichomes, which does not occur in wild-type seedlings, implying that LEC1 might regulate embryonic cell fate determination during post-embryonic development. To test this hypothesis, we compared the expression of trichome development-related genes between the wild-type and the lec1 mutant. We observed that transcripts of GLABROUS1 (GL1), GL2, and GL3, genes encoding the positive regulators in trichome development, were significantly upregulated, while the TRICHOMELESS1 (TCL2), ENHANCER OF TRY AND CPC1 (ETC1), and ETC2 genes, encoding the negative regulators in trichome development, were downregulated in the lec1 mutant. Furthermore, overexpression of LEC1 activated the expressions of TCL2, CAPPICE (CPC), and ETC1, resulting in production of cotyledonary leaves with no or fewer trichomes during vegetative development. In addition, we demonstrated that LEC1 interacts with TCL2 in yeast and in vitro. A genetic experiment showed that loss-of-function of GL2 rescued the ectopic trichome formation in the lec1 mutant. These findings strongly support that LEC1 regulates trichome development, providing direct evidence for the role of LEC1 in cell fate determination during post-embryonic development. PMID:26579186

Light-Dark Changes in Cytosolic Nitrate Pools Depend on Nitrate Reductase Activity in Arabidopsis Leaf Cells1[w

PubMed Central

Cookson, Sarah J.; Williams, Lorraine E.; Miller, Anthony J.

2005-01-01

Several different cellular processes determine the size of the metabolically available nitrate pool in the cytoplasm. These processes include not only ion fluxes across the plasma membrane and tonoplast but also assimilation by the activity of nitrate reductase (NR). In roots, the maintenance of cytosolic nitrate activity during periods of nitrate starvation and resupply (M. van der Leij, S.J. Smith, A.J. Miller [1998] Planta 205: 64–72; R.-G. Zhen, H.-W. Koyro, R.A. Leigh, A.D. Tomos, A.J. Miller [1991] Planta 185: 356–361) suggests that this pool is regulated. Under nitrate-replete conditions vacuolar nitrate is a membrane-bound store that can release nitrate to the cytoplasm; after depletion of cytosolic nitrate, tonoplast transporters would serve to restore this pool. To study the role of assimilation, specifically the activity of NR in regulating the size of the cytosolic nitrate pool, we have compared wild-type and mutant plants. In leaf mesophyll cells, light-to-dark transitions increase cytosolic nitrate activity (1.5–2.8 mm), and these changes were reversed by dark-to-light transitions. Such changes were not observed in nia1nia2 NR-deficient plants indicating that this change in cytosolic nitrate activity was dependent on the presence of functional NR. Furthermore, in the dark, the steady-state cytosolic nitrate activities were not statistically different between the two types of plant, indicating that NR has little role in determining resting levels of nitrate. Epidermal cells of both wild type and NR mutants had cytosolic nitrate activities that were not significantly different from mesophyll cells in the dark and were unaltered by dark-to-light transitions. We propose that the NR-dependent changes in cytosolic nitrate provide a cellular mechanism for the diurnal changes in vacuolar nitrate storage, and the results are discussed in terms of the possible signaling role of cytosolic nitrate. PMID:15908593

Arabidopsis synaptotagmin 1 is required for the maintenance of plasma membrane integrity and cell viability.

PubMed

Schapire, Arnaldo L; Voigt, Boris; Jasik, Jan; Rosado, Abel; Lopez-Cobollo, Rosa; Menzel, Diedrik; Salinas, Julio; Mancuso, Stefano; Valpuesta, Victoriano; Baluska, Frantisek; Botella, Miguel A

2008-12-01

Plasma membrane repair in animal cells uses synaptotagmin 7, a Ca(2+)-activated membrane fusion protein that mediates delivery of intracellular membranes to wound sites by a mechanism resembling neuronal Ca(2+)-regulated exocytosis. Here, we show that loss of function of the homologous Arabidopsis thaliana Synaptotagmin 1 protein (SYT1) reduces the viability of cells as a consequence of a decrease in the integrity of the plasma membrane. This reduced integrity is enhanced in the syt1-2 null mutant in conditions of osmotic stress likely caused by a defective plasma membrane repair. Consistent with a role in plasma membrane repair, SYT1 is ubiquitously expressed, is located at the plasma membrane, and shares all domains characteristic of animal synaptotagmins (i.e., an N terminus-transmembrane domain and a cytoplasmic region containing two C2 domains with phospholipid binding activities). Our analyses support that membrane trafficking mediated by SYT1 is important for plasma membrane integrity and plant fitness.

ACR4, a putative receptor kinase gene of Arabidopsis thaliana, that is expressed in the outer cell layers of embryos and plants, is involved in proper embryogenesis.

PubMed

Tanaka, Hirokazu; Watanabe, Masaru; Watanabe, Daisuke; Tanaka, Toshihiro; Machida, Chiyoko; Machida, Yasunori

2002-04-01

The surfaces of higher plants are characterized by epidermis, which usually consists of a single layer of cells. The epidermis is derived from the outer cell layer of the embryo or protoderm, which arises as a result of periclinal cell division. After seed germination, most of the epidermal cells of the aerial parts of plants are derived from the outer cell layer of the shoot apical meristem (the L1 layer). Thus, knowledge of how the protoderm and/or L1 layer is established is fundamental to understanding the morphogenesis of higher plants. Here, we report the isolation of a gene encoding an Arabidopsis homologue (ACR4) of the maize putative receptor kinase CRINKLY4 (CR4), which is involved in epidermal differentiation. The domain organization of the predicted amino acid sequence of ACR4 is essentially identical to that of CR4. ACR4-GFP fusion protein localized to the cell surface when expressed in tobacco cell (BY-2) culture. ACR4 transcripts were detected in all the organs of the Arabidopsis plant. In developing embryos and shoot apices, ACR4 transcripts accumulated in protoderm and epidermis at relatively higher levels than in the inner tissues. Over-expression of antisense ACR4 in Arabidopsis plants resulted in malformation of embryos to varying degrees. These results suggest that ACR4 is, at a minimum, involved in the normal morphogenesis of embryos, most likely through properly differentiating protoderm cells.

Arabidopsis SYT1 maintains stability of cortical endoplasmic reticulum networks and VAP27-1-enriched endoplasmic reticulum-plasma membrane contact sites.

PubMed

Siao, Wei; Wang, Pengwei; Voigt, Boris; Hussey, Patrick J; Baluska, Frantisek

2016-11-01

Arabidopsis synaptotagmin 1 (SYT1) is localized on the endoplasmic reticulum-plasma membrane (ER-PM) contact sites in leaf and root cells. The ER-PM localization of Arabidopsis SYT1 resembles that of the extended synaptotagmins (E-SYTs) in animal cells. In mammals, E-SYTs have been shown to regulate calcium signaling, lipid transfer, and endocytosis. Arabidopsis SYT1 was reported to be essential for maintaining cell integrity and virus movement. This study provides detailed insight into the subcellular localization of SYT1 and VAP27-1, another ER-PM-tethering protein. SYT1 and VAP27-1 were shown to be localized on distinct ER-PM contact sites. The VAP27-1-enriched ER-PM contact sites (V-EPCSs) were always in contact with the SYT1-enriched ER-PM contact sites (S-EPCSs). The V-EPCSs still existed in the leaf epidermal cells of the SYT1 null mutant; however, they were less stable than those in the wild type. The polygonal networks of cortical ER disassembled and the mobility of VAP27-1 protein on the ER-PM contact sites increased in leaf cells of the SYT1 null mutant. These results suggest that SYT1 is responsible for stabilizing the ER network and V-EPCSs. © The Author 2016. Published by Oxford University Press on behalf of the Society for Experimental Biology.

β-Amylase1 and β-Amylase3 Are Plastidic Starch Hydrolases in Arabidopsis That Seem to Be Adapted for Different Thermal, pH, and Stress Conditions1[W][OPEN

PubMed Central

Monroe, Jonathan D.; Storm, Amanda R.; Badley, Elizabeth M.; Lehman, Michael D.; Platt, Samantha M.; Saunders, Lauren K.; Schmitz, Jonathan M.; Torres, Catherine E.

2014-01-01

Starch degradation in chloroplasts requires β-amylase (BAM) activity, which is encoded by a multigene family. Of nine Arabidopsis (Arabidopsis thaliana) BAM genes, six encode plastidic enzymes, but only four of these are catalytically active. In vegetative plants, BAM1 acts during the day in guard cells, whereas BAM3 is the dominant activity in mesophyll cells at night. Plastidic BAMs have been difficult to assay in leaf extracts, in part because of a cytosolic activity encoded by BAM5. We generated a series of double mutants lacking BAM5 and each of the active plastidic enzymes (BAM1, BAM2, BAM3, and BAM6) and found that most of the plastidic activity in 5-week-old plants was encoded by BAM1 and BAM3. Both of these activities were relatively constant during the day and the night. Analysis of leaf extracts from double mutants and purified BAM1 and BAM3 proteins revealed that these proteins have distinct properties. Using soluble starch as the substrate, BAM1 and BAM3 had optimum activity at pH 6.0 to 6.5, but at high pH, BAM1 was more active than BAM3, consistent with its known daytime role in the guard cell stroma. The optimum temperature for BAM1, which is transcriptionally induced by heat stress, was about 10°C higher than that of BAM3, which is transcriptionally induced by cold stress. The amino acid composition of BAM1 and BAM3 orthologs reflected differences that are consistent with known adaptations of proteins from heat- and cold-adapted organisms, suggesting that these day- and night-active enzymes have undergone thermal adaptation. PMID:25293962

High-Throughput Cryopreservation of Plant Cell Cultures for Functional Genomics

PubMed Central

Ogawa, Yoichi; Sakurai, Nozomu; Oikawa, Akira; Kai, Kosuke; Morishita, Yoshihiko; Mori, Kumiko; Moriya, Kanami; Fujii, Fumiko; Aoki, Koh; Suzuki, Hideyuki; Ohta, Daisaku; Saito, Kazuki; Shibata, Daisuke

2012-01-01

Suspension-cultured cell lines from plant species are useful for genetic engineering. However, maintenance of these lines is laborious, involves routine subculturing and hampers wider use of transgenic lines, especially when many lines are required for a high-throughput functional genomics application. Cryopreservation of these lines may reduce the need for subculturing. Here, we established a simple protocol for cryopreservation of cell lines from five commonly used plant species, Arabidopsis thaliana, Daucus carota, Lotus japonicus, Nicotiana tabacum and Oryza sativa. The LSP solution (2 M glycerol, 0.4 M sucrose and 86.9 mM proline) protected cells from damage during freezing and was only mildly toxic to cells kept at room temperature for at least 2 h. More than 100 samples were processed for freezing simultaneously. Initially, we determined the conditions for cryopreservation using a programmable freezer; we then developed a modified simple protocol that did not require a programmable freezer. In the simple protocol, a thick expanded polystyrene (EPS) container containing the vials with the cell–LSP solution mixtures was kept at −30°C for 6 h to cool the cells slowly (pre-freezing); samples from the EPS containers were then plunged into liquid nitrogen before long-term storage. Transgenic Arabidopsis cells were subjected to cryopreservation, thawed and then re-grown in culture; transcriptome and metabolome analyses indicated that there was no significant difference in gene expression or metabolism between cryopreserved cells and control cells. The simplicity of the protocol will accelerate the pace of research in functional plant genomics. PMID:22437846

Mutations in MYB3R1 and MYB3R4 Cause Pleiotropic Developmental Defects and Preferential Down-Regulation of Multiple G2/M-Specific Genes in Arabidopsis1[C][W

PubMed Central

Haga, Nozomi; Kobayashi, Kosuke; Suzuki, Takamasa; Maeo, Kenichiro; Kubo, Minoru; Ohtani, Misato; Mitsuda, Nobutaka; Demura, Taku; Nakamura, Kenzo; Jürgens, Gerd; Ito, Masaki

2011-01-01

R1R2R3-Myb proteins represent an evolutionarily conserved class of Myb family proteins important for cell cycle regulation and differentiation in eukaryotic cells. In plants, this class of Myb proteins are believed to regulate the transcription of G2/M phase-specific genes by binding to common cis-elements, called mitosis-specific activator (MSA) elements. In Arabidopsis (Arabidopsis thaliana), MYB3R1 and MYB3R4 act as transcriptional activators and positively regulate cytokinesis by activating the transcription of KNOLLE, which encodes a cytokinesis-specific syntaxin. Here, we show that the double mutation myb3r1 myb3r4 causes pleiotropic developmental defects, some of which are due to deficiency of KNOLLE whereas other are not, suggesting that multiple target genes are involved. Consistently, microarray analysis of the double mutant revealed altered expression of many genes, among which G2/M-specific genes showed significant overrepresentation of the MSA motif and a strong tendency to be down-regulated by the double mutation. Our results demonstrate, on a genome-wide level, the importance of the MYB3R-MSA pathway for regulating G2/M-specific transcription. In addition, MYB3R1 and MYB3R4 may have diverse roles during plant development by regulating G2/M-specific genes with various functions as well as genes possibly unrelated to the cell cycle. PMID:21862669

Genome-Wide Analysis of the Core DNA Replication Machinery in the Higher Plants Arabidopsis and Rice1[W][OA

PubMed Central

Shultz, Randall W.; Tatineni, Vinaya M.; Hanley-Bowdoin, Linda; Thompson, William F.

2007-01-01

Core DNA replication proteins mediate the initiation, elongation, and Okazaki fragment maturation functions of DNA replication. Although this process is generally conserved in eukaryotes, important differences in the molecular architecture of the DNA replication machine and the function of individual subunits have been reported in various model systems. We have combined genome-wide bioinformatic analyses of Arabidopsis (Arabidopsis thaliana) and rice (Oryza sativa) with published experimental data to provide a comprehensive view of the core DNA replication machinery in plants. Many components identified in this analysis have not been studied previously in plant systems, including the GINS (go ichi ni san) complex (PSF1, PSF2, PSF3, and SLD5), MCM8, MCM9, MCM10, NOC3, POLA2, POLA3, POLA4, POLD3, POLD4, and RNASEH2. Our results indicate that the core DNA replication machinery from plants is more similar to vertebrates than single-celled yeasts (Saccharomyces cerevisiae), suggesting that animal models may be more relevant to plant systems. However, we also uncovered some important differences between plants and vertebrate machinery. For example, we did not identify geminin or RNASEH1 genes in plants. Our analyses also indicate that plants may be unique among eukaryotes in that they have multiple copies of numerous core DNA replication genes. This finding raises the question of whether specialized functions have evolved in some cases. This analysis establishes that the core DNA replication machinery is highly conserved across plant species and displays many features in common with other eukaryotes and some characteristics that are unique to plants. PMID:17556508

Visualizing the Genetic Landscape of Arabidopsis Seed Performance1[W][OA

PubMed Central

Joosen, Ronny Viktor Louis; Arends, Danny; Willems, Leo Albert Jan; Ligterink, Wilco; Jansen, Ritsert C.; Hilhorst, Henk W.M.

2012-01-01

Perfect timing of germination is required to encounter optimal conditions for plant survival and is the result of a complex interaction between molecular processes, seed characteristics, and environmental cues. To detangle these processes, we made use of natural genetic variation present in an Arabidopsis (Arabidopsis thaliana) Bayreuth × Shahdara recombinant inbred line population. For a detailed analysis of the germination response, we characterized rate, uniformity, and maximum germination and discuss the added value of such precise measurements. The effects of after-ripening, stratification, and controlled deterioration as well as the effects of salt, mannitol, heat, cold, and abscisic acid (ABA) with and without cold stratification were analyzed for these germination characteristics. Seed morphology (size and length) of both dry and imbibed seeds was quantified by using image analysis. For the overwhelming amount of data produced in this study, we developed new approaches to perform and visualize high-throughput quantitative trait locus (QTL) analysis. We show correlation of trait data, (shared) QTL positions, and epistatic interactions. The detection of similar loci for different stresses indicates that, often, the molecular processes regulating environmental responses converge into similar pathways. Seven major QTL hotspots were confirmed using a heterogeneous inbred family approach. QTLs colocating with previously reported QTLs and well-characterized mutants are discussed. A new connection between dormancy, ABA, and a cripple mucilage formation due to a naturally occurring mutation in the MUCILAGE-MODIFIED2 gene is proposed, and this is an interesting lead for further research on the regulatory role of ABA in mucilage production and its multiple effects on germination parameters. PMID:22158761

Arabidopsis dynamin-related protein 1A polymers bind, but do not tubulate, liposomes

DOE Office of Scientific and Technical Information (OSTI.GOV)

Backues, Steven K.; Bednarek, Sebastian Y., E-mail: sybednar@wisc.edu

2010-03-19

The Arabidopsis dynamin-related protein 1A (AtDRP1A) is involved in endocytosis and cell plate maturation in Arabidopsis. Unlike dynamin, AtDRP1A does not have any recognized membrane binding or protein-protein interaction domains. We report that GTPase active AtDRP1A purified from Escherichia coli as a fusion to maltose binding protein forms homopolymers visible by negative staining electron microscopy. These polymers interact with protein-free liposomes whose lipid composition mimics that of the inner leaflet of the Arabidopsis plasma membrane, suggesting that lipid-binding may play a role in AtDRP1A function. However, AtDRP1A polymers do not appear to assemble and disassemble in a dynamic fashion andmore » do not have the ability to tubulate liposomes in vitro, suggesting that additional factors or modifications are necessary for AtDRP1A's in vivo function.« less

Gravitropism of Arabidopsis thaliana Roots Requires the Polarization of PIN2 toward the Root Tip in Meristematic Cortical Cells[C][W

PubMed Central

Rahman, Abidur; Takahashi, Maho; Shibasaki, Kyohei; Wu, Shuang; Inaba, Takehito; Tsurumi, Seiji; Baskin, Tobias I.

2010-01-01

In the root, the transport of auxin from the tip to the elongation zone, referred to here as shootward, governs gravitropic bending. Shootward polar auxin transport, and hence gravitropism, depends on the polar deployment of the PIN-FORMED auxin efflux carrier PIN2. In Arabidopsis thaliana, PIN2 has the expected shootward localization in epidermis and lateral root cap; however, this carrier is localized toward the root tip (rootward) in cortical cells of the meristem, a deployment whose function is enigmatic. We use pharmacological and genetic tools to cause a shootward relocation of PIN2 in meristematic cortical cells without detectably altering PIN2 polarization in other cell types or PIN1 polarization. This relocation of cortical PIN2 was negatively regulated by the membrane trafficking factor GNOM and by the regulatory A1 subunit of type 2-A protein phosphatase (PP2AA1) but did not require the PINOID protein kinase. When GNOM was inhibited, PINOID abundance increased and PP2AA1 was partially immobilized, indicating both proteins are subject to GNOM-dependent regulation. Shootward PIN2 specifically in the cortex was accompanied by enhanced shootward polar auxin transport and by diminished gravitropism. These results demonstrate that auxin flow in the root cortex is important for optimal gravitropic response. PMID:20562236

PIC1, an Ancient Permease in Arabidopsis Chloroplasts, Mediates Iron Transport[W

PubMed Central

Duy, Daniela; Wanner, Gerhard; Meda, Anderson R.; von Wirén, Nicolaus; Soll, Jürgen; Philippar, Katrin

2007-01-01

In chloroplasts, the transition metals iron and copper play an essential role in photosynthetic electron transport and act as cofactors for superoxide dismutases. Iron is essential for chlorophyll biosynthesis, and ferritin clusters in plastids store iron during germination, development, and iron stress. Thus, plastidic homeostasis of transition metals, in particular of iron, is crucial for chloroplast as well as plant development. However, very little is known about iron uptake by chloroplasts. Arabidopsis thaliana PERMEASE IN CHLOROPLASTS1 (PIC1), identified in a screen for metal transporters in plastids, contains four predicted α-helices, is targeted to the inner envelope, and displays homology with cyanobacterial permease-like proteins. Knockout mutants of PIC1 grew only heterotrophically and were characterized by a chlorotic and dwarfish phenotype reminiscent of iron-deficient plants. Ultrastructural analysis of plastids revealed severely impaired chloroplast development and a striking increase in ferritin clusters. Besides upregulation of ferritin, pic1 mutants showed differential regulation of genes and proteins related to iron stress or transport, photosynthesis, and Fe-S cluster biogenesis. Furthermore, PIC1 and its cyanobacterial homolog mediated iron accumulation in an iron uptake–defective yeast mutant. These observations suggest that PIC1 functions in iron transport across the inner envelope of chloroplasts and hence in cellular metal homeostasis. PMID:17337631

PAUSED Encodes the Arabidopsis Exportin-t Ortholog1

PubMed Central

Hunter, Christine A.; Aukerman, Milo J.; Sun, Hui; Fokina, Maria; Poethig, R. Scott

2003-01-01

Los1p/exportin-t (XPOT) mediates the nuclear export of tRNAs in yeast and mammals. The requirements for this transport pathway are unclear, however, because los1 mutations do not affect yeast growth, and the phenotype of XPOT mutations in mammals is unknown. Here, we show that PAUSED (PSD) is the Arabidopsis ortholog of LOS1/XPOT and is capable of rescuing the tRNA export defect of los1 in Brewer's yeast (Saccharomyces cerevisiae), suggesting that its function has been conserved. Putative null alleles of PSD disrupt the initiation of the shoot apical meristem and delay leaf initiation after germination, the emergence of the radicle and lateral roots, and the transition to flowering. Plants doubly mutant for psd and hasty, the Arabidopsis ortholog of exportin 5, are viable but have a more severe phenotype than either single mutant. These results suggest that PSD plays a role in tRNA export in Arabidopsis, but that at least one—and perhaps several—additional tRNA export pathways also exist. The PSD transcript is broadly expressed during development and is alternatively spliced in the 3′-untranslated region. No temporal or spatial difference in the abundance of different splice forms was observed. We propose that the mutant phenotype of psd reflects defects in developmental events and cell/tissue types that require elevated levels of protein synthesis and are therefore acutely sensitive to a reduction in tRNA export. PMID:12913168

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

Jasmonate Controls Leaf Growth by Repressing Cell Proliferation and the Onset of Endoreduplication while Maintaining a Potential Stand-By Mode1[W][OA

PubMed Central

Noir, Sandra; Bömer, Moritz; Takahashi, Naoki; Ishida, Takashi; Tsui, Tjir-Li; Balbi, Virginia; Shanahan, Hugh; Sugimoto, Keiko; Devoto, Alessandra

2013-01-01

Phytohormones regulate plant growth from cell division to organ development. Jasmonates (JAs) are signaling molecules that have been implicated in stress-induced responses. However, they have also been shown to inhibit plant growth, but the mechanisms are not well understood. The effects of methyl jasmonate (MeJA) on leaf growth regulation were investigated in Arabidopsis (Arabidopsis thaliana) mutants altered in JA synthesis and perception, allene oxide synthase and coi1-16B (for coronatine insensitive1), respectively. We show that MeJA inhibits leaf growth through the JA receptor COI1 by reducing both cell number and size. Further investigations using flow cytometry analyses allowed us to evaluate ploidy levels and to monitor cell cycle progression in leaves and cotyledons of Arabidopsis and/or Nicotiana benthamiana at different stages of development. Additionally, a novel global transcription profiling analysis involving continuous treatment with MeJA was carried out to identify the molecular players whose expression is regulated during leaf development by this hormone and COI1. The results of these studies revealed that MeJA delays the switch from the mitotic cell cycle to the endoreduplication cycle, which accompanies cell expansion, in a COI1-dependent manner and inhibits the mitotic cycle itself, arresting cells in G1 phase prior to the S-phase transition. Significantly, we show that MeJA activates critical regulators of endoreduplication and affects the expression of key determinants of DNA replication. Our discoveries also suggest that MeJA may contribute to the maintenance of a cellular “stand-by mode” by keeping the expression of ribosomal genes at an elevated level. Finally, we propose a novel model for MeJA-regulated COI1-dependent leaf growth inhibition. PMID:23439917

Rapid Reversion from Monomer to Dimer Regenerates the Ultraviolet-B Photoreceptor UV RESISTANCE LOCUS8 in Intact Arabidopsis Plants1[W][OA

PubMed Central

Heilmann, Monika; Jenkins, Gareth I.

2013-01-01

Arabidopsis (Arabidopsis thaliana) UV RESISTANCE LOCUS8 (UVR8) is a photoreceptor that specifically mediates photomorphogenic responses to ultraviolet (UV)-B in plants. UV-B photoreception induces the conversion of the UVR8 dimer into a monomer that interacts with the CONSTITUTIVELY PHOTOMORPHOGENIC1 (COP1) protein to regulate gene expression. However, it is not known how the dimeric photoreceptor is regenerated in plants. Here, we show, by using inhibitors of protein synthesis and degradation via the proteasome, that the UVR8 dimer is not regenerated by rapid de novo synthesis following destruction of the monomer. Rather, regeneration occurs by reversion from the monomer to the dimer. However, regeneration of dimeric UVR8 in darkness following UV-B exposure occurs much more rapidly in vivo than in vitro with illuminated plant extracts or purified UVR8, indicating that rapid regeneration requires intact cells. Rapid dimer regeneration in vivo requires protein synthesis, the presence of a carboxyl-terminal 27-amino acid region of UVR8, and the presence of COP1, which is known to interact with the carboxyl-terminal region. However, none of these factors can account fully for the difference in regeneration kinetics in vivo and in vitro, indicating that additional proteins or processes are involved in UVR8 dimer regeneration in vivo. PMID:23129206

Analysis of cell division patterns in the Arabidopsis shoot apical meristem

DOE PAGES

Shapiro, Bruce E.; Tobin, Cory; Mjolsness, Eric; ...

2015-03-30

The stereotypic pattern of cell shapes in the Arabidopsis shoot apical meristem (SAM) suggests that strict rules govern the placement of new walls during cell division. When a cell in the SAM divides, a new wall is built that connects existing walls and divides the cytoplasm of the daughter cells. Because features that are determined by the placement of new walls such as cell size, shape, and number of neighbors are highly regular, rules must exist for maintaining such order. Here in this paper we present a quantitative model of these rules that incorporates different observed features of cell division.more » Each feature is incorporated into a "potential function" that contributes a single term to a total analog of potential energy. New cell walls are predicted to occur at locations where the potential function is minimized. Quantitative terms that represent the well-known historical rules of plant cell division, such as those given by Hofmeister, Errera, and Sachs are developed and evaluated against observed cell divisions in the epidermal layer (L1) of Arabidopsis thaliana SAM. The method is general enough to allow additional terms for nongeometric properties such as internal concentration gradients and mechanical tensile forces.« less

Myosins XI-K, XI-1, and XI-2 are required for development of pavement cells, trichomes, and stigmatic papillae in Arabidopsis

PubMed Central

2012-01-01

Background The positioning and dynamics of vesicles and organelles, and thus the growth of plant cells, is mediated by the acto-myosin system. In Arabidopsis there are 13 class XI myosins which mediate vesicle and organelle transport in different cell types. So far the involvement of five class XI myosins in cell expansion during the shoot and root development has been shown, three of which, XI-1, XI-2, and XI-K, are essential for organelle transport. Results Simultaneous depletion of Arabidopsis class XI myosins XI-K, XI-1, and XI-2 in double and triple mutant plants affected the growth of several types of epidermal cells. The size and shape of trichomes, leaf pavement cells and the elongation of the stigmatic papillae of double and triple mutant plants were affected to different extent. Reduced cell size led to significant size reduction of shoot organs in the case of triple mutant, affecting bolt formation, flowering time and fertility. Phenotype analysis revealed that the reduced fertility of triple mutant plants was caused by delayed or insufficient development of pistils. Conclusions We conclude that the class XI myosins XI-K, XI-1 and XI-2 have partially redundant roles in the growth of shoot epidermis. Myosin XI-K plays more important role whereas myosins XI-1 and XI-2 have minor roles in the determination of size and shape of epidermal cells, because the absence of these two myosins is compensated by XI-K. Co-operation between myosins XI-K and XI-2 appears to play an important role in these processes. PMID:22672737

Functional Analysis of SPINDLY in Gibberellin Signaling in Arabidopsis1[C][W][OA

PubMed Central

Silverstone, Aron L.; Tseng, Tong-Seung; Swain, Stephen M.; Dill, Alyssa; Jeong, Sun Yong; Olszewski, Neil E.; Sun, Tai-ping

2007-01-01

The Arabidopsis (Arabidopsis thaliana) SPINDLY (SPY) protein negatively regulates the gibberellin (GA) signaling pathway. SPY is an O-linked N-acetylglucosamine (GlcNAc) transferase (OGT) with a protein-protein interaction domain consisting of 10 tetratricopeptide repeats (TPR). OGTs add a GlcNAc monosaccharide to serine/threonine residues of nuclear and cytosolic proteins. Determination of the molecular defects in 14 new spy alleles reveals that these mutations cluster in three TPRs and the C-terminal catalytic region. Phenotypic characterization of 12 spy alleles indicates that TPRs 6, 8, and 9 and the catalytic domain are crucial for GA-regulated stem elongation, floral induction, and fertility. TPRs 8 and 9 and the catalytic region are also important for modulating trichome morphology and inflorescence phyllotaxy. Consistent with a role for SPY in embryo development, several alleles affect seedling cotyledon number. These results suggest that three of the TPRs and the OGT activity in SPY are required for its function in GA signal transduction. We also examined the effect of spy mutations on another negative regulator of GA signaling, REPRESSOR OF ga1-3 (RGA). The DELLA motif in RGA is essential for GA-induced proteolysis of RGA, and deletion of this motif (as in rga-Δ17) causes a GA-insensitive dwarf phenotype. Here, we demonstrate that spy partially suppresses the rga-Δ17 phenotype but does not reduce rga-Δ17 or RGA protein levels or alter RGA nuclear localization. We propose that SPY may function as a negative regulator of GA response by increasing the activity of RGA, and presumably other DELLA proteins, by GlcNAc modification. PMID:17142481

Exposure of cultured astroglial and microglial brain cells to 900 MHz microwave radiation.

PubMed

Thorlin, Thorleif; Rouquette, Jean-Michel; Hamnerius, Yngve; Hansson, Elisabeth; Persson, Mikael; Björklund, Ulrika; Rosengren, Lars; Rönnbäck, Lars; Persson, Mikael

2006-08-01

The rapid rise in the use of mobile communications has raised concerns about health issues related to low-level microwave radiation. The head and brain are usually the most exposed targets in mobile phone users. In the brain, two types of glial cells, the astroglial and the microglial cells, are interesting in the context of biological effects from microwave exposure. These cells are widely distributed in the brain and are directly involved in the response to brain damage as well as in the development of brain cancer. The aim of the present study was to investigate whether 900 MHz radiation could affect these two different glial cell types in culture by studying markers for damage-related processes in the cells. Primary cultures enriched in astroglial cells were exposed to 900 MHz microwave radiation in a temperature-controlled exposure system at specific absorption rates (SARs) of 3 W/kg GSM modulated wave (mw) for 4, 8 and 24 h or 27 W/kg continuous wave (cw) for 24 h, and the release into the extracellular medium of the two pro-inflammatory cytokines interleukin 6 (Il6) and tumor necrosis factor-alpha (Tnfa) was analyzed. In addition, levels of the astroglial cell-specific reactive marker glial fibrillary acidic protein (Gfap), whose expression dynamics is different from that of cytokines, were measured in astroglial cultures and in astroglial cell-conditioned cell culture medium at SARs of 27 and 54 W/kg (cw) for 4 or 24 h. No significant differences could be detected for any of the parameters studied at any time and for any of the radiation characteristics. Total protein levels remained constant during the experiments. Microglial cell cultures were exposed to 900 MHz radiation at an SAR of 3 W/kg (mw) for 8 h, and I16, Tnfa, total protein and the microglial reactivity marker ED-1 (a macrophage activation antigen) were measured. No significant differences were found. The morphology of the cultured astroglial cells and microglia was studied and appeared to be

The Lateral Organ Boundaries Domain Transcription Factor LBD20 Functions in Fusarium Wilt Susceptibility and Jasmonate Signaling in Arabidopsis1[W

PubMed Central

Thatcher, Louise F.; Powell, Jonathan J.; Aitken, Elizabeth A.B.; Kazan, Kemal; Manners, John M.

2012-01-01

The LATERAL ORGAN BOUNDARIES (LOB) DOMAIN (LBD) gene family encodes plant-specific transcriptional regulators functioning in organ development. In a screen of Arabidopsis (Arabidopsis thaliana) sequence-indexed transferred DNA insertion mutants, we found disruption of the LOB DOMAIN-CONTAINING PROTEIN20 (LBD20) gene led to increased resistance to the root-infecting vascular wilt pathogen Fusarium oxysporum. In wild-type plants, LBD20 transcripts were barely detectable in leaves but abundant in roots, where they were further induced after F. oxysporum inoculation or methyl jasmonate treatment. Induction of LBD20 expression in roots was abolished in coronatine insensitive1 (coi1) and myc2 (allelic to jasmonate insensitive1) mutants, suggesting LBD20 may function in jasmonate (JA) signaling. Consistent with this, expression of the JA-regulated THIONIN2.1 (Thi2.1) and VEGETATIVE STORAGE PROTEIN2 (VSP2) genes were up-regulated in shoots of lbd20 following treatment of roots with F. oxysporum or methyl jasmonate. However, PLANT DEFENSIN1.2 expression was unaltered, indicating a repressor role for LBD20 in a branch of the JA-signaling pathway. Plants overexpressing LBD20 (LBD20-OX) had reduced Thi2.1 and VSP2 expression. There was a significant correlation between increased LBD20 expression in the LBD20-OX lines with both Thi2.1 and VSP2 repression, and reduced survival following F. oxysporum infection. Chlorosis resulting from application of F. oxysporum culture filtrate was also reduced in lbd20 leaves relative to the wild type. Taken together, LBD20 is a F. oxysporum susceptibility gene that appears to regulate components of JA signaling downstream of COI1 and MYC2 that are required for full elicitation of F. oxysporum- and JA-dependent responses. To our knowledge, this is the first demonstration of a role for a LBD gene family member in either biotic stress or JA signaling. PMID:22786889

Exogenous Cellulase Switches Cell Interdigitation to Cell Elongation in an RIC1-dependent Manner in Arabidopsis thaliana Cotyledon Pavement Cells.

PubMed

Higaki, Takumi; Takigawa-Imamura, Hisako; Akita, Kae; Kutsuna, Natsumaro; Kobayashi, Ryo; Hasezawa, Seiichiro; Miura, Takashi

2017-01-01

Pavement cells in cotyledons and true leaves exhibit a jigsaw puzzle-like morphology in most dicotyledonous plants. Among the molecular mechanisms mediating cell morphogenesis, two antagonistic Rho-like GTPases regulate local cell outgrowth via cytoskeletal rearrangements. Analyses of several cell wall-related mutants suggest the importance of cell wall mechanics in the formation of interdigitated patterns. However, how these factors are integrated is unknown. In this study, we observed that the application of exogenous cellulase to hydroponically grown Arabidopsis thaliana cotyledons switched the interdigitation of pavement cells to the production of smoothly elongated cells. The cellulase-induced inhibition of cell interdigitation was not observed in a RIC1 knockout mutant. This gene encodes a Rho-like GTPase-interacting protein important for localized cell growth suppression via microtubule bundling on concave cell interfaces. Additionally, to characterize pavement cell morphologies, we developed a mathematical model that considers the balance between cell and cell wall growth, restricted global cell growth orientation, and regulation of local cell outgrowth mediated by a Rho-like GTPase-cytoskeleton system. Our computational simulations fully support our experimental observations, and suggest that interdigitated patterns form because of mechanical buckling in the absence of Rho-like GTPase-dependent regulation of local cell outgrowth. Our model clarifies the cell wall mechanics influencing pavement cell morphogenesis. © The Author 2016. Published by Oxford University Press on behalf of Japanese Society of Plant Physiologists. All rights reserved. For permissions, please email: journals.permissions@oup.com.

METACASPASE9 modulates autophagy to confine cell death to the target cells during Arabidopsis vascular xylem differentiation

PubMed Central

Escamez, Sacha; André, Domenique; Zhang, Bo; Bollhöner, Benjamin; Pesquet, Edouard; Tuominen, Hannele

2016-01-01

ABSTRACT We uncovered that the level of autophagy in plant cells undergoing programmed cell death determines the fate of the surrounding cells. Our approach consisted of using Arabidopsis thaliana cell cultures capable of differentiating into two different cell types: vascular tracheary elements (TEs) that undergo programmed cell death (PCD) and protoplast autolysis, and parenchymatic non-TEs that remain alive. The TE cell type displayed higher levels of autophagy when expression of the TE-specific METACASPASE9 (MC9) was reduced using RNAi (MC9-RNAi). Misregulation of autophagy in the MC9-RNAi TEs coincided with ectopic death of the non-TEs, implying the existence of an autophagy-dependent intercellular signalling from within the TEs towards the non-TEs. Viability of the non-TEs was restored when AUTOPHAGY2 (ATG2) was downregulated specifically in MC9-RNAi TEs, demonstrating the importance of autophagy in the spatial confinement of cell death. Our results suggest that other eukaryotic cells undergoing PCD might also need to tightly regulate their level of autophagy to avoid detrimental consequences for the surrounding cells. PMID:26740571

Cytosolic Calcium, hydrogen peroxide, and related gene expression and protein modulation in Arabidopsis thaliana cell cultures respond immediately to altered gravitation: Parabolic flight data

NASA Astrophysics Data System (ADS)

Hampp, Ruediger; Hausmann, Niklas; Neef, Maren; Fengler, Svenja

Callus cell cultures of Arabidopsis thaliana (cv. Columbia) were exposed to parabolic flights in order to assess molecular short-term responses to altered gravity fields. Using transgenic cell lines, hydrogen peroxide and cytosolic Ca2+ were continuously monitored. In parallel, the metabolism of samples was chemically quenched (RNAlater, Ambion, for RNA; acid/base for NADPH, NADP) at typical stages of a parabola (1g before pull up; end of pull up (1.8 g), end of microgravity (µg, 20 sec), and end of pull out (1.8 g)). Cells exhibited an increase of both Ca2+ and hydrogen peroxide with the onset of µg, and a decline thereafter. This behaviour was accompanied by a decrease of the NADPH/NADP redox ratio, indicating a Ca2+-dependent activation of a NADPH oxidase. Microarray analyses revealed concomitant expression profiles. At the end of the microgravity phase, 396 transcripts were specifically up-, while 485 were down-regulated. Up-regulation was dominated by Ca2+- and ROS(reactive oxygen species)-related gene products. The same material was also used for the analysis of phosphopeptides by 2D SDS PAGE. Relevant spots were identified by liquid chromatography-MS. With the exception of a chaperone (HSP 70-3), hypergravity (1.8 g) and microgravity modified different sets of proteins. These are partly involved in primary metabolism (glycolysis, gluconeogenesis, citrate cycle) and detoxification of reactive oxygen species. Taken together, these data show that both gene expression and protein modulation jointly respond within seconds to alterations in the gravity field, with a focus on metabolic adaptation, signalling and control of ROS.

Target of Rapamycin Signaling Regulates Metabolism, Growth, and Life Span in Arabidopsis[W][OA

PubMed Central

Ren, Maozhi; Venglat, Prakash; Qiu, Shuqing; Feng, Li; Cao, Yongguo; Wang, Edwin; Xiang, Daoquan; Wang, Jinghe; Alexander, Danny; Chalivendra, Subbaiah; Logan, David; Mattoo, Autar; Selvaraj, Gopalan; Datla, Raju

2012-01-01

Target of Rapamycin (TOR) is a major nutrition and energy sensor that regulates growth and life span in yeast and animals. In plants, growth and life span are intertwined not only with nutrient acquisition from the soil and nutrition generation via photosynthesis but also with their unique modes of development and differentiation. How TOR functions in these processes has not yet been determined. To gain further insights, rapamycin-sensitive transgenic Arabidopsis thaliana lines (BP12) expressing yeast FK506 Binding Protein12 were developed. Inhibition of TOR in BP12 plants by rapamycin resulted in slower overall root, leaf, and shoot growth and development leading to poor nutrient uptake and light energy utilization. Experimental limitation of nutrient availability and light energy supply in wild-type Arabidopsis produced phenotypes observed with TOR knockdown plants, indicating a link between TOR signaling and nutrition/light energy status. Genetic and physiological studies together with RNA sequencing and metabolite analysis of TOR-suppressed lines revealed that TOR regulates development and life span in Arabidopsis by restructuring cell growth, carbon and nitrogen metabolism, gene expression, and rRNA and protein synthesis. Gain- and loss-of-function Ribosomal Protein S6 (RPS6) mutants additionally show that TOR function involves RPS6-mediated nutrition and light-dependent growth and life span in Arabidopsis. PMID:23275579

Transformation of Arabidopsis with a Brassica SLG/SRK region and ARC1 gene is not sufficient to transfer the self-incompatibility phenotype.

PubMed

Bi, Y M; Brugière, N; Cui, Y; Goring, D R; Rothstein, S J

2000-05-01

Self-incompatibility (SI) promotes outbreeding in flowering plants, and in Brassica SI is genetically controlled by the S locus. Self-incompatible Brassica and self-fertile Arabidopsis belong to the same crucifer family. In addition, a comparative analysis reveals a high degree of microsynteny between the B. campestris S locus and its homologous region in Arabidopsis--with the notable exception that the Brassica SI genes, SLG and SRK, are missing. Brassica ARC1 encodes a component of the SRK signal transduction pathway leading to self-pollen rejection, and no closely related ARC1 homolog has been identified in Arabidopsis. The purpose of the research reported here was to introduce Brassica SI components into Arabidopsis in an attempt to compensate for the missing genes and to investigate whether the SI phenotype can be transferred. Inserts of approximately 40 kb from the fosmid clones F20 and F22, which span the B. napus W1 SLG-SRK region, were cloned into the plant transformation vector pBIBAC2. Transgenic plants were generated that expressed the Brassica SI genes in the flower buds. In addition, the endogenous, SLG-like, gene AtS1 was not co-suppressed by the Brassica SLG transgene. No SI phenotype was observed among the T1 BIBAC2-F20 and BIBAC2-F22 transgenic plants. When the ARC1 gene was transformed into BIBAC2-F20 or BIBAC2-F22 plants, the resulting BIBAC2-F20-ARC1 and BIBAC2-F22-ARC1 plants still set seeds normally, and no rejection response was observed when self-incompatible B. napus W1 pollen was placed on BIBAC2-F20-ARC1 or BIBAC2-F22-ARC1 Arabidopsis stigmas. Taken together, our results suggest that complementing Arabidopsis genome with Brassica SLG, SRK and ARC1 genes is unlikely to be sufficient to transfer the SI phenotype.

Involvement of the VEP1 gene in vascular strand development in Arabidopsis thaliana.

PubMed

Jun, Ji Hyung; Ha, Chan Man; Nam, Hong Gil

2002-03-01

A dominant mutant line characterized by abnormal leaf venation pattern was isolated from a transgenic Arabidopsis plant pool that was generated with Agrobacterium culture harboring an Arabidopsis antisense cDNA library. In the mutant line, the phenotype was due to antisense suppression of a gene we named VEP1 (Vein Patterning). The predicted amino acid sequence of the gene contained a motif related to the mammalian death domain that is found in the apoptotic machinery. Reduced expression of the VEP1 gene resulted in the reduced complexity of the venation pattern of the cotyledons and foliar leaves, which was mainly due to the reduced number of the minor veins and their incomplete connection. The analysis of mutant embryos indicated that the phenotype was originated, at least in part, from a defect in the procambium patterning. In the mutant, the stem and root were thinner than those in wild type. This phenotype was associated with reduced vascular development. The promoter activity of the VEP1 gene was detected preferentially in the vascular regions. We propose that the death domain-containing protein VEP1 functions as a positive element required for vascular strand development in Arabidopsis thaliana.

Determination of L1 retrotransposition kinetics in cultured cells

PubMed Central

Ostertag, Eric M.; Luning Prak, Eline T.; DeBerardinis, Ralph J.; Moran, John V.; Kazazian, Haig H.

2000-01-01

L1 retrotransposons are autonomous retroelements that are active in the human and mouse genomes. Previously, we developed a cultured cell assay that uses a neomycin phosphotransferase (neo) retrotransposition cassette to determine relative retrotransposition frequencies among various L1 elements. Here, we describe a new retrotransposition assay that uses an enhanced green fluorescent protein (EGFP) retrotransposition cassette to determine retrotransposition kinetics in cultured cells. We show that retrotransposition is not detected in cultured cells during the first 48 h post-transfection, but then proceeds at a continuous high rate for at least 16 days. We also determine the relative retrotransposition rates of two similar human L1 retrotransposons, L1RP and L1.3. L1RP retrotransposed in the EGFP assay at a rate of ~0.5% of transfected cells/day, ~3-fold higher than the rate measured for L1.3. We conclude that the new assay detects near real time retrotransposition in a single cell and is sufficiently sensitive to differentiate retrotransposition rates among similar L1 elements. The EGFP assay exhibits improved speed and accuracy compared to the previous assay when used to determine relative retrotransposition frequencies. Furthermore, the EGFP cassette has an expanded range of experimental applications. PMID:10684937

Chloroplast 2010: A Database for Large-Scale Phenotypic Screening of Arabidopsis Mutants1[W][OA

PubMed Central

Lu, Yan; Savage, Linda J.; Larson, Matthew D.; Wilkerson, Curtis G.; Last, Robert L.

2011-01-01

Large-scale phenotypic screening presents challenges and opportunities not encountered in typical forward or reverse genetics projects. We describe a modular database and laboratory information management system that was implemented in support of the Chloroplast 2010 Project, an Arabidopsis (Arabidopsis thaliana) reverse genetics phenotypic screen of more than 5,000 mutants (http://bioinfo.bch.msu.edu/2010_LIMS; www.plastid.msu.edu). The software and laboratory work environment were designed to minimize operator error and detect systematic process errors. The database uses Ruby on Rails and Flash technologies to present complex quantitative and qualitative data and pedigree information in a flexible user interface. Examples are presented where the database was used to find opportunities for process changes that improved data quality. We also describe the use of the data-analysis tools to discover mutants defective in enzymes of leucine catabolism (heteromeric mitochondrial 3-methylcrotonyl-coenzyme A carboxylase [At1g03090 and At4g34030] and putative hydroxymethylglutaryl-coenzyme A lyase [At2g26800]) based upon a syndrome of pleiotropic seed amino acid phenotypes that resembles previously described isovaleryl coenzyme A dehydrogenase (At3g45300) mutants. In vitro assay results support the computational annotation of At2g26800 as hydroxymethylglutaryl-coenzyme A lyase. PMID:21224340

Involvement of the Electrophilic Isothiocyanate Sulforaphane in Arabidopsis Local Defense Responses1

PubMed Central

Andersson, Mats X.; Nilsson, Anders K.; Johansson, Oskar N.; Boztaş, Gülin; Adolfsson, Lisa E.; Pinosa, Francesco; Petit, Christel Garcia; Aronsson, Henrik; Mackey, David; Tör, Mahmut; Hamberg, Mats; Ellerström, Mats

2015-01-01

Plants defend themselves against microbial pathogens through a range of highly sophisticated and integrated molecular systems. Recognition of pathogen-secreted effector proteins often triggers the hypersensitive response (HR), a complex multicellular defense reaction where programmed cell death of cells surrounding the primary site of infection is a prominent feature. Even though the HR was described almost a century ago, cell-to-cell factors acting at the local level generating the full defense reaction have remained obscure. In this study, we sought to identify diffusible molecules produced during the HR that could induce cell death in naive tissue. We found that 4-methylsulfinylbutyl isothiocyanate (sulforaphane) is released by Arabidopsis (Arabidopsis thaliana) leaf tissue undergoing the HR and that this compound induces cell death as well as primes defense in naive tissue. Two different mutants impaired in the pathogen-induced accumulation of sulforaphane displayed attenuated programmed cell death upon bacterial and oomycete effector recognition as well as decreased resistance to several isolates of the plant pathogen Hyaloperonospora arabidopsidis. Treatment with sulforaphane provided protection against a virulent H. arabidopsidis isolate. Glucosinolate breakdown products are recognized as antifeeding compounds toward insects and recently also as intracellular signaling and bacteriostatic molecules in Arabidopsis. The data presented here indicate that these compounds also trigger local defense responses in Arabidopsis tissue. PMID:25371552

Statolith Sedimentation Kinetics and Force Transduction to the Cortical Endoplasmic Reticulum in Gravity-Sensing Arabidopsis Columella Cells[W][OA

PubMed Central

Leitz, Guenther; Kang, Byung-Ho; Schoenwaelder, Monica E.A.; Staehelin, L. Andrew

2009-01-01

The starch statolith hypothesis of gravity sensing in plants postulates that the sedimentation of statoliths in specialized statocytes (columella cells) provides the means for converting the gravitational potential energy into a biochemical signal. We have analyzed the sedimentation kinetics of statoliths in the central S2 columella cells of Arabidopsis thaliana. The statoliths can form compact aggregates with gap sizes between statoliths approaching <30 nm. Significant intra-aggregate sliding motions of individual statoliths suggest a contribution of hydrodynamic forces to the motion of statoliths. The reorientation of the columella cells accelerates the statoliths toward the central cytoplasm within <1 s of reorientation. During the subsequent sedimentation phase, the statoliths tend to move at a distance to the cortical endoplasmic reticulum (ER) boundary and interact only transiently with the ER. Statoliths moved by laser tweezers against the ER boundary experience an elastic lift force upon release from the optical trap. High-resolution electron tomography analysis of statolith-to-ER contact sites indicate that the weight of statoliths is sufficient to locally deform the ER membranes that can potentially activate mechanosensitive ion channels. We suggest that in root columella cells, the transduction of the kinetic energy of sedimenting statoliths into a biochemical signal involves a combination of statolith-driven motion of the cytosol, statolith-induced deformation of the ER membranes, and a rapid release of kinetic energy from the ER during reorientation to activate mechanosensitive sites within the central columella cells. PMID:19276442

The Cotton Transcription Factor TCP14 Functions in Auxin-Mediated Epidermal Cell Differentiation and Elongation1[C][W

PubMed Central

Wang, Miao-Ying; Zhao, Pi-Ming; Cheng, Huan-Qing; Han, Li-Bo; Wu, Xiao-Min; Gao, Peng; Wang, Hai-Yun; Yang, Chun-Lin; Zhong, Nai-Qin; Zuo, Jian-Ru; Xia, Gui-Xian

2013-01-01

Plant-specific TEOSINTE-BRANCHED1/CYCLOIDEA/PCF (TCP) transcription factors play crucial roles in development, but their functional mechanisms remain largely unknown. Here, we characterized the cellular functions of the class I TCP transcription factor GhTCP14 from upland cotton (Gossypium hirsutum). GhTCP14 is expressed predominantly in fiber cells, especially at the initiation and elongation stages of development, and its expression increased in response to exogenous auxin. Induced heterologous overexpression of GhTCP14 in Arabidopsis (Arabidopsis thaliana) enhanced initiation and elongation of trichomes and root hairs. In addition, root gravitropism was severely affected, similar to mutant of the auxin efflux carrier PIN-FORMED2 (PIN2) gene. Examination of auxin distribution in GhTCP14-expressing Arabidopsis by observation of auxin-responsive reporters revealed substantial alterations in auxin distribution in sepal trichomes and root cortical regions. Consistent with these changes, expression of the auxin uptake carrier AUXIN1 (AUX1) was up-regulated and PIN2 expression was down-regulated in the GhTCP14-expressing plants. The association of GhTCP14 with auxin responses was also evidenced by the enhanced expression of auxin response gene IAA3, a gene in the AUXIN/INDOLE-3-ACETIC ACID (Aux/IAA) family. Electrophoretic mobility shift assays showed that GhTCP14 bound the promoters of PIN2, IAA3, and AUX1, and transactivation assays indicated that GhTCP14 had transcription activation activity. Taken together, these results demonstrate that GhTCP14 is a dual-function transcription factor able to positively or negatively regulate expression of auxin response and transporter genes, thus potentially acting as a crucial regulator in auxin-mediated differentiation and elongation of cotton fiber cells. PMID:23715527

Comparative interactomics: analysis of arabidopsis 14-3-3 complexes reveals highly conserved 14-3-3 interactions between humans and plants.

PubMed

Paul, Anna-Lisa; Liu, Li; McClung, Scott; Laughner, Beth; Chen, Sixue; Ferl, Robert J

2009-04-01

As a first step in the broad characterization of plant 14-3-3 multiprotein complexes in vivo, stringent and specific antibody affinity purification was used to capture 14-3-3s together with their interacting proteins from extracts of Arabidopsis cell suspension cultures. Approximately 120 proteins were identified as potential in vivo 14-3-3 interacting proteins by mass spectrometry of the recovered complexes. Comparison of the proteins in this data set with the 14-3-3 interacting proteins from a similar study in human embryonic kidney cell cultures revealed eight interacting proteins that likely represent reasonably abundant, fundamental 14-3-3 interaction complexes that are highly conserved across all eukaryotes. The Arabidopsis 14-3-3 interaction data set was also compared to a yeast in vivo 14-3-3 interaction data set. Four 14-3-3 interacting proteins are conserved in yeast, humans, and Arabidopsis. Comparisons of the data sets based on biochemical function revealed many additional similarities in the human and Arabidopsis data sets that represent conserved functional interactions, while also leaving many proteins uniquely identified in either Arabidopsis or human cells. In particular, the Arabidopsis interaction data set is enriched for proteins involved in metabolism.

Plastid Ontogeny during Petal Development in Arabidopsis1

PubMed Central

Pyke, Kevin A.; Page, Anton M.

1998-01-01

Imaging of chlorophyll autofluorescence by confocal microscopy in intact whole petals of Arabidopsis thaliana has been used to analyze chloroplast development and redifferentiation during petal development. Young petals dissected from unopened buds contained green chloroplasts throughout their structure, but as the upper part of the petal lamina developed and expanded, plastids lost their chlorophyll and redifferentiated into leukoplasts, resulting in a white petal blade. Normal green chloroplasts remained in the stalk of the mature petal. In epidermal cells the chloroplasts were normal and green, in stark contrast with leaf epidermal cell plastids. In addition, the majority of these chloroplasts had dumbbell shapes, typical of dividing chloroplasts, and we suggest that the rapid expansion of petal epidermal cells may be a trigger for the initiation of chloroplast division. In petals of the Arabidopsis plastid division mutant arc6, the conversion of chloroplasts into leukoplasts was unaffected in spite of the greatly enlarged size and reduced number of arc6 chloroplasts in cells in the petal base, resulting in few enlarged leukoplasts in cells from the white lamina of arc6 petals. PMID:9489024

Comparative Genomics and Reverse Genetics Analysis Reveal Indispensable Functions of the Serine Acetyltransferase Gene Family in Arabidopsis[W][OA

PubMed Central

Watanabe, Mutsumi; Mochida, Keiichi; Kato, Tomohiko; Tabata, Satoshi; Yoshimoto, Naoko; Noji, Masaaki; Saito, Kazuki

2008-01-01

Ser acetyltransferase (SERAT), which catalyzes O-acetyl-Ser (OAS) formation, plays a key role in sulfur assimilation and Cys synthesis. Despite several studies on SERATs from various plant species, the in vivo function of multiple SERAT genes in plant cells remains unaddressed. Comparative genomics studies with the five genes of the SERAT gene family in Arabidopsis thaliana indicated that all three Arabidopsis SERAT subfamilies are conserved across five plant species with available genome sequences. Single and multiple knockout mutants of all Arabidopsis SERAT gene family members were analyzed. All five quadruple mutants with a single gene survived, with three mutants showing dwarfism. However, the quintuple mutant lacking all SERAT genes was embryo-lethal. Thus, all five isoforms show functional redundancy in vivo. The developmental and compartment-specific roles of each SERAT isoform were also demonstrated. Mitochondrial SERAT2;2 plays a predominant role in cellular OAS formation, while plastidic SERAT2;1 contributes less to OAS formation and subsequent Cys synthesis. Three cytosolic isoforms, SERAT1;1, SERAT3;1, and SERAT3;2, may play a major role during seed development. Thus, the evolutionally conserved SERAT gene family is essential in cellular processes, and the substrates and products of SERAT must be exchangeable between the cytosol and organelles. PMID:18776059

An Early Nodulin-Like Protein Accumulates in the Sieve Element Plasma Membrane of Arabidopsis1[OA

PubMed Central

Khan, Junaid A.; Wang, Qi; Sjölund, Richard D.; Schulz, Alexander; Thompson, Gary A.

2007-01-01

Membrane proteins within the sieve element-companion cell complex have essential roles in the physiological functioning of the phloem. The monoclonal antibody line RS6, selected from hybridomas raised against sieve elements isolated from California shield leaf (Streptanthus tortuosus; Brassicaceae) tissue cultures, recognizes an antigen in the Arabidopsis (Arabidopsis thaliana) ecotype Columbia that is associated specifically with the plasma membrane of sieve elements, but not companion cells, and accumulates at the earliest stages of sieve element differentiation. The identity of the RS6 antigen was revealed by reverse transcription-PCR of Arabidopsis leaf RNA using degenerate primers to be an early nodulin (ENOD)-like protein that is encoded by the expressed gene At3g20570. Arabidopsis ENOD-like proteins are encoded by a multigene family composed of several types of structurally related phytocyanins that have a similar overall domain structure of an amino-terminal signal peptide, plastocyanin-like copper-binding domain, proline/serine-rich domain, and carboxy-terminal hydrophobic domain. The amino- and carboxy-terminal domains of the 21.5-kD sieve element-specific ENOD are posttranslationally cleaved from the precursor protein, resulting in a mature peptide of approximately 15 kD that is attached to the sieve element plasma membrane via a carboxy-terminal glycosylphosphatidylinositol membrane anchor. Many of the Arabidopsis ENOD-like proteins accumulate in gametophytic tissues, whereas in both floral and vegetative tissues, the sieve element-specific ENOD is expressed only within the phloem. Members of the ENOD subfamily of the cupredoxin superfamily do not appear to bind copper and have unknown functions. Phenotypic analysis of homozygous T-DNA insertion mutants for the gene At3g20570 shows minimal alteration in vegetative growth but a significant reduction in the overall reproductive potential. PMID:17293437

Arabidopsis Formin3 Directs the Formation of Actin Cables and Polarized Growth in Pollen Tubes[W

PubMed Central

Ye, Jianrong; Zheng, Yiyan; Yan, An; Chen, Naizhi; Wang, Zhangkui; Huang, Shanjin; Yang, Zhenbiao

2009-01-01

Cytoplasmic actin cables are the most prominent actin structures in plant cells, but the molecular mechanism underlying their formation is unknown. The function of these actin cables, which are proposed to modulate cytoplasmic streaming and intracellular movement of many organelles in plants, has not been studied by genetic means. Here, we show that Arabidopsis thaliana formin3 (AFH3) is an actin nucleation factor responsible for the formation of longitudinal actin cables in pollen tubes. The Arabidopsis AFH3 gene encodes a 785–amino acid polypeptide, which contains a formin homology 1 (FH1) and a FH2 domain. In vitro analysis revealed that the AFH3 FH1FH2 domains interact with the barbed end of actin filaments and have actin nucleation activity in the presence of G-actin or G actin-profilin. Overexpression of AFH3 in tobacco (Nicotiana tabacum) pollen tubes induced excessive actin cables, which extended into the tubes' apices. Specific downregulation of AFH3 eliminated actin cables in Arabidopsis pollen tubes and reduced the level of actin polymers in pollen grains. This led to the disruption of the reverse fountain streaming pattern in pollen tubes, confirming a role for actin cables in the regulation of cytoplasmic streaming. Furthermore, these tubes became wide and short and swelled at their tips, suggesting that actin cables may regulate growth polarity in pollen tubes. Thus, AFH3 regulates the formation of actin cables, which are important for cytoplasmic streaming and polarized growth in pollen tubes. PMID:20023198

Arabidopsis and Brachypodium distachyon Transgenic Plants Expressing Aspergillus nidulans Acetylesterases Have Decreased Degree of Polysaccharide Acetylation and Increased Resistance to Pathogens1[C][W][OA

PubMed Central

Pogorelko, Gennady; Lionetti, Vincenzo; Fursova, Oksana; Sundaram, Raman M.; Qi, Mingsheng; Whitham, Steven A.; Bogdanove, Adam J.; Bellincampi, Daniela; Zabotina, Olga A.

2013-01-01

The plant cell wall has many significant structural and physiological roles, but the contributions of the various components to these roles remain unclear. Modification of cell wall properties can affect key agronomic traits such as disease resistance and plant growth. The plant cell wall is composed of diverse polysaccharides often decorated with methyl, acetyl, and feruloyl groups linked to the sugar subunits. In this study, we examined the effect of perturbing cell wall acetylation by making transgenic Arabidopsis (Arabidopsis thaliana) and Brachypodium (Brachypodium distachyon) plants expressing hemicellulose- and pectin-specific fungal acetylesterases. All transgenic plants carried highly expressed active Aspergillus nidulans acetylesterases localized to the apoplast and had significant reduction of cell wall acetylation compared with wild-type plants. Partial deacetylation of polysaccharides caused compensatory up-regulation of three known acetyltransferases and increased polysaccharide accessibility to glycosyl hydrolases. Transgenic plants showed increased resistance to the fungal pathogens Botrytis cinerea and Bipolaris sorokiniana but not to the bacterial pathogens Pseudomonas syringae and Xanthomonas oryzae. These results demonstrate a role, in both monocot and dicot plants, of hemicellulose and pectin acetylation in plant defense against fungal pathogens. PMID:23463782

Expression of Arabidopsis Hexokinase in Citrus Guard Cells Controls Stomatal Aperture and Reduces Transpiration.

PubMed

Lugassi, Nitsan; Kelly, Gilor; Fidel, Lena; Yaniv, Yossi; Attia, Ziv; Levi, Asher; Alchanatis, Victor; Moshelion, Menachem; Raveh, Eran; Carmi, Nir; Granot, David

2015-01-01

Hexokinase (HXK) is a sugar-phosphorylating enzyme involved in sugar-sensing. It has recently been shown that HXK in guard cells mediates stomatal closure and coordinates photosynthesis with transpiration in the annual species tomato and Arabidopsis. To examine the role of HXK in the control of the stomatal movement of perennial plants, we generated citrus plants that express Arabidopsis HXK1 (AtHXK1) under KST1, a guard cell-specific promoter. The expression of KST1 in the guard cells of citrus plants has been verified using GFP as a reporter gene. The expression of AtHXK1 in the guard cells of citrus reduced stomatal conductance and transpiration with no negative effect on the rate of photosynthesis, leading to increased water-use efficiency. The effects of light intensity and humidity on stomatal behavior were examined in rooted leaves of the citrus plants. The optimal intensity of photosynthetically active radiation and lower humidity enhanced stomatal closure of AtHXK1-expressing leaves, supporting the role of sugar in the regulation of citrus stomata. These results suggest that HXK coordinates photosynthesis and transpiration and stimulates stomatal closure not only in annual species, but also in perennial species.

Expression of Arabidopsis Hexokinase in Citrus Guard Cells Controls Stomatal Aperture and Reduces Transpiration

PubMed Central

Lugassi, Nitsan; Kelly, Gilor; Fidel, Lena; Yaniv, Yossi; Attia, Ziv; Levi, Asher; Alchanatis, Victor; Moshelion, Menachem; Raveh, Eran; Carmi, Nir; Granot, David

2015-01-01

Hexokinase (HXK) is a sugar-phosphorylating enzyme involved in sugar-sensing. It has recently been shown that HXK in guard cells mediates stomatal closure and coordinates photosynthesis with transpiration in the annual species tomato and Arabidopsis. To examine the role of HXK in the control of the stomatal movement of perennial plants, we generated citrus plants that express Arabidopsis HXK1 (AtHXK1) under KST1, a guard cell-specific promoter. The expression of KST1 in the guard cells of citrus plants has been verified using GFP as a reporter gene. The expression of AtHXK1 in the guard cells of citrus reduced stomatal conductance and transpiration with no negative effect on the rate of photosynthesis, leading to increased water-use efficiency. The effects of light intensity and humidity on stomatal behavior were examined in rooted leaves of the citrus plants. The optimal intensity of photosynthetically active radiation and lower humidity enhanced stomatal closure of AtHXK1-expressing leaves, supporting the role of sugar in the regulation of citrus stomata. These results suggest that HXK coordinates photosynthesis and transpiration and stimulates stomatal closure not only in annual species, but also in perennial species. PMID:26734024

Phytoplasma Effector SAP54 Induces Indeterminate Leaf-Like Flower Development in Arabidopsis Plants1[C][W][OA

PubMed Central

MacLean, Allyson M.; Sugio, Akiko; Makarova, Olga V.; Findlay, Kim C.; Grieve, Victoria M.; Tóth, Réka; Nicolaisen, Mogens; Hogenhout, Saskia A.

2011-01-01

Phytoplasmas are insect-transmitted bacterial plant pathogens that cause considerable damage to a diverse range of agricultural crops globally. Symptoms induced in infected plants suggest that these phytopathogens may modulate developmental processes within the plant host. We report herein that Aster Yellows phytoplasma strain Witches’ Broom (AY-WB) readily infects the model plant Arabidopsis (Arabidopsis thaliana) ecotype Columbia, inducing symptoms that are characteristic of phytoplasma infection, such as the production of green leaf-like flowers (virescence and phyllody) and increased formation of stems and branches (witches’ broom). We found that the majority of genes encoding secreted AY-WB proteins (SAPs), which are candidate effector proteins, are expressed in Arabidopsis and the AY-WB insect vector Macrosteles quadrilineatus (Hemiptera; Cicadellidae). To identify which of these effector proteins induce symptoms of phyllody and virescence, we individually expressed the effector genes in Arabidopsis. From this screen, we have identified a novel AY-WB effector protein, SAP54, that alters floral development, resulting in the production of leaf-like flowers that are similar to those produced by plants infected with this phytoplasma. This study offers novel insight into the effector profile of an insect-transmitted plant pathogen and reports to our knowledge the first example of a microbial pathogen effector protein that targets flower development in a host. PMID:21849514

G Protein–Coupled Receptor-Type G Proteins Are Required for Light-Dependent Seedling Growth and Fertility in Arabidopsis[W

PubMed Central

Jaffé, Felix W.; Freschet, Gian-Enrico C.; Valdes, Billy M.; Runions, John; Terry, Matthew J.; Williams, Lorraine E.

2012-01-01

G protein–coupled receptor-type G proteins (GTGs) are highly conserved membrane proteins in plants, animals, and fungi that have eight to nine predicted transmembrane domains. They have been classified as G protein–coupled receptor-type G proteins that function as abscisic acid (ABA) receptors in Arabidopsis thaliana. We cloned Arabidopsis GTG1 and GTG2 and isolated new T-DNA insertion alleles of GTG1 and GTG2 in both Wassilewskija and Columbia backgrounds. These gtg1 gtg2 double mutants show defects in fertility, hypocotyl and root growth, and responses to light and sugars. Histological studies of shoot tissue reveal cellular distortions that are particularly evident in the epidermal layer. Stable expression of GTG1pro:GTG1-GFP (for green fluorescent protein) in Arabidopsis and transient expression in tobacco (Nicotiana tabacum) indicate that GTG1 is localized primarily to Golgi bodies and to the endoplasmic reticulum. Microarray analysis comparing gene expression profiles in the wild type and double mutant revealed differences in expression of genes important for cell wall function, hormone response, and amino acid metabolism. The double mutants isolated here respond normally to ABA in seed germination assays, root growth inhibition, and gene expression analysis. These results are inconsistent with their proposed role as ABA receptors but demonstrate that GTGs are fundamentally important for plant growth and development. PMID:23001037

The Ubiquitin Receptor DA1 Interacts with the E3 Ubiquitin Ligase DA2 to Regulate Seed and Organ Size in Arabidopsis[C][W

PubMed Central

Xia, Tian; Li, Na; Dumenil, Jack; Li, Jie; Kamenski, Andrei; Bevan, Michael W.; Gao, Fan; Li, Yunhai

2013-01-01

Seed size in higher plants is determined by the coordinated growth of the embryo, endosperm, and maternal tissue. Several factors that act maternally to regulate seed size have been identified, such as AUXIN RESPONSE FACTOR2, APETALA2, KLUH, and DA1, but the genetic and molecular mechanisms of these factors in seed size control are almost totally unknown. We previously demonstrated that the ubiquitin receptor DA1 acts synergistically with the E3 ubiquitin ligase ENHANCER1 OF DA1 (EOD1)/BIG BROTHER to regulate the final size of seeds in Arabidopsis thaliana. Here, we describe another RING-type protein with E3 ubiquitin ligase activity, encoded by DA2, which regulates seed size by restricting cell proliferation in the maternal integuments of developing seeds. The da2-1 mutant forms large seeds, while overexpression of DA2 decreases seed size of wild-type plants. Overexpression of rice (Oryza sativa) GRAIN WIDTH AND WEIGHT2, a homolog of DA2, restricts seed growth in Arabidopsis. Genetic analyses show that DA2 functions synergistically with DA1 to regulate seed size, but does so independently of EOD1. Further results reveal that DA2 interacts physically with DA1 in vitro and in vivo. Therefore, our findings define the genetic and molecular mechanisms of three ubiquitin-related proteins DA1, DA2, and EOD1 in seed size control and indicate that they are promising targets for crop improvement. PMID:24045020

Intracellular Localization of Arabidopsis Sulfurtransferases1

PubMed Central

Bauer, Michael; Dietrich, Christof; Nowak, Katharina; Sierralta, Walter D.; Papenbrock, Jutta

2004-01-01

Sulfurtransferases (Str) comprise a group of enzymes widely distributed in archaea, eubacteria, and eukaryota which catalyze the transfer of a sulfur atom from suitable sulfur donors to nucleophilic sulfur acceptors. In all organisms analyzed to date, small gene families encoding Str proteins have been identified. The gene products were localized to different compartments of the cells. Our interest concerns the localization of Str proteins encoded in the nuclear genome of Arabidopsis. Computer-based prediction methods revealed localization in different compartments of the cell for six putative AtStrs. Several methods were used to determine the localization of the AtStr proteins experimentally. For AtStr1, a mitochondrial localization was demonstrated by immunodetection in the proteome of isolated mitochondria resolved by one- and two-dimensional gel electrophoresis and subsequent blotting. The respective mature AtStr1 protein was identified by mass spectrometry sequencing. The same result was obtained by transient expression of fusion constructs with the green fluorescent protein in Arabidopsis protoplasts, whereas AtStr2 was exclusively localized to the cytoplasm by this method. Three members of the single-domain AtStr were localized in the chloroplasts as demonstrated by transient expression of green fluorescent protein fusions in protoplasts and stomata, whereas the single-domain AtStr18 was shown to be cytoplasmic. The remarkable subcellular distribution of AtStr15 was additionally analyzed by transmission electron immunomicroscopy using a monospecific antibody against green fluorescent protein, indicating an attachment to the thylakoid membrane. The knowledge of the intracellular localization of the members of this multiprotein family will help elucidate their specific functions in the organism. PMID:15181206

The anisotropy1 D604N mutation in the Arabidopsis cellulose synthase1 catalytic domain reduces cell wall crystallinity and the velocity of cellulose synthase complexes.

PubMed

Fujita, Miki; Himmelspach, Regina; Ward, Juliet; Whittington, Angela; Hasenbein, Nortrud; Liu, Christine; Truong, Thy T; Galway, Moira E; Mansfield, Shawn D; Hocart, Charles H; Wasteneys, Geoffrey O

2013-05-01

Multiple cellulose synthase (CesA) subunits assemble into plasma membrane complexes responsible for cellulose production. In the Arabidopsis (Arabidopsis thaliana) model system, we identified a novel D604N missense mutation, designated anisotropy1 (any1), in the essential primary cell wall CesA1. Most previously identified CesA1 mutants show severe constitutive or conditional phenotypes such as embryo lethality or arrest of cellulose production but any1 plants are viable and produce seeds, thus permitting the study of CesA1 function. The dwarf mutants have reduced anisotropic growth of roots, aerial organs, and trichomes. Interestingly, cellulose microfibrils were disordered only in the epidermal cells of the any1 inflorescence stem, whereas they were transverse to the growth axis in other tissues of the stem and in all elongated cell types of roots and dark-grown hypocotyls. Overall cellulose content was not altered but both cell wall crystallinity and the velocity of cellulose synthase complexes were reduced in any1. We crossed any1 with the temperature-sensitive radial swelling1-1 (rsw1-1) CesA1 mutant and observed partial complementation of the any1 phenotype in the transheterozygotes at rsw1-1's permissive temperature (21°C) and full complementation by any1 of the conditional rsw1-1 root swelling phenotype at the restrictive temperature (29°C). In rsw1-1 homozygotes at restrictive temperature, a striking dissociation of cellulose synthase complexes from the plasma membrane was accompanied by greatly diminished motility of intracellular cellulose synthase-containing compartments. Neither phenomenon was observed in the any1 rsw1-1 transheterozygotes, suggesting that the proteins encoded by the any1 allele replace those encoded by rsw1-1 at restrictive temperature.

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

A pharmacological study of Arabidopsis cell fusion between the persistent synergid and endosperm.

PubMed

Motomura, Kazuki; Kawashima, Tomokazu; Berger, Frédéric; Kinoshita, Tetsu; Higashiyama, Tetsuya; Maruyama, Daisuke

2018-01-29

Cell fusion is a pivotal process in fertilization and multinucleate cell formation. A plant cell is ubiquitously surrounded by a hard cell wall, and very few cell fusions have been observed except for gamete fusions. We recently reported that the fertilized central cell (the endosperm) absorbs the persistent synergid, a highly differentiated cell necessary for pollen tube attraction. The synergid-endosperm fusion (SE fusion) appears to eliminate the persistent synergid from fertilized ovule in Arabidopsis thaliana Here, we analyzed the effects of various inhibitors on SE fusion in an in vitro culture system. Different from other cell fusions, neither disruption of actin polymerization nor protein secretion impaired SE fusion. However, transcriptional and translational inhibitors decreased the SE fusion success rate and also inhibited endosperm division. Failures of SE fusion and endosperm nuclear proliferation were also induced by roscovitine, an inhibitor of cyclin-dependent kinases (CDK). These data indicate unique aspects of SE fusion such as independence of filamentous actin support and the importance of CDK-mediated mitotic control. © 2018. Published by The Company of Biologists Ltd.

A Major Facilitator Superfamily Transporter Plays a Dual Role in Polar Auxin Transport and Drought Stress Tolerance in Arabidopsis[W

PubMed Central

Remy, Estelle; Cabrito, Tânia R.; Baster, Pawel; Batista, Rita A.; Teixeira, Miguel C.; Friml, Jiri; Sá-Correia, Isabel; Duque, Paula

2013-01-01

Many key aspects of plant development are regulated by the polarized transport of the phytohormone auxin. Cellular auxin efflux, the rate-limiting step in this process, has been shown to rely on the coordinated action of PIN-formed (PIN) and B-type ATP binding cassette (ABCB) carriers. Here, we report that polar auxin transport in the Arabidopsis thaliana root also requires the action of a Major Facilitator Superfamily (MFS) transporter, Zinc-Induced Facilitator-Like 1 (ZIFL1). Sequencing, promoter-reporter, and fluorescent protein fusion experiments indicate that the full-length ZIFL1.1 protein and a truncated splice isoform, ZIFL1.3, localize to the tonoplast of root cells and the plasma membrane of leaf stomatal guard cells, respectively. Using reverse genetics, we show that the ZIFL1.1 transporter regulates various root auxin-related processes, while the ZIFL1.3 isoform mediates drought tolerance by regulating stomatal closure. Auxin transport and immunolocalization assays demonstrate that ZIFL1.1 indirectly modulates cellular auxin efflux during shootward auxin transport at the root tip, likely by regulating plasma membrane PIN2 abundance. Finally, heterologous expression in yeast revealed that ZIFL1.1 and ZIFL1.3 share H+-coupled K+ transport activity. Thus, by determining the subcellular and tissue distribution of two isoforms, alternative splicing dictates a dual function for the ZIFL1 transporter. We propose that this MFS carrier regulates stomatal movements and polar auxin transport by modulating potassium and proton fluxes in Arabidopsis cells. PMID:23524662

Transcriptional Regulation of Arabidopsis MIR168a and ARGONAUTE1 Homeostasis in Abscisic Acid and Abiotic Stress Responses1[W

PubMed Central

Li, Wei; Cui, Xiao; Meng, Zhaolu; Huang, Xiahe; Xie, Qi; Wu, Heng; Jin, Hailing; Zhang, Dabing; Liang, Wanqi

2012-01-01

The accumulation of a number of small RNAs in plants is affected by abscisic acid (ABA) and abiotic stresses, but the underlying mechanisms are poorly understood. The miR168-mediated feedback regulatory loop regulates ARGONAUTE1 (AGO1) homeostasis, which is crucial for gene expression modulation and plant development. Here, we reveal a transcriptional regulatory mechanism by which MIR168 controls AGO1 homeostasis during ABA treatment and abiotic stress responses in Arabidopsis (Arabidopsis thaliana). Plants overexpressing MIR168a and the AGO1 loss-of-function mutant ago1-27 display ABA hypersensitivity and drought tolerance, while the mir168a-2 mutant shows ABA hyposensitivity and drought hypersensitivity. Both the precursor and mature miR168 were induced under ABA and several abiotic stress treatments, but no obvious decrease for the target of miR168, AGO1, was shown under the same conditions. However, promoter activity analysis indicated that AGO1 transcription activity was increased under ABA and drought treatments, suggesting that transcriptional elevation of MIR168a is required for maintaining a stable AGO1 transcript level during the stress response. Furthermore, we showed both in vitro and in vivo that the transcription of MIR168a is directly regulated by four abscisic acid-responsive element (ABRE) binding factors, which bind to the ABRE cis-element within the MIR168a promoter. This ABRE motif is also found in the promoter of MIR168a homologs in diverse plant species. Our findings suggest that transcriptional regulation of miR168 and posttranscriptional control of AGO1 homeostasis may play an important and conserved role in stress response and signal transduction in plants. PMID:22247272

The Arabidopsis Rho of Plants GTPase AtROP6 Functions in Developmental and Pathogen Response Pathways1[C][W][OA

PubMed Central

Poraty-Gavra, Limor; Zimmermann, Philip; Haigis, Sabine; Bednarek, Paweł; Hazak, Ora; Stelmakh, Oksana Rogovoy; Sadot, Einat; Schulze-Lefert, Paul; Gruissem, Wilhelm; Yalovsky, Shaul

2013-01-01

How plants coordinate developmental processes and environmental stress responses is a pressing question. Here, we show that Arabidopsis (Arabidopsis thaliana) Rho of Plants6 (AtROP6) integrates developmental and pathogen response signaling. AtROP6 expression is induced by auxin and detected in the root meristem, lateral root initials, and leaf hydathodes. Plants expressing a dominant negative AtROP6 (rop6DN) under the regulation of its endogenous promoter are small and have multiple inflorescence stems, twisted leaves, deformed leaf epidermis pavement cells, and differentially organized cytoskeleton. Microarray analyses of rop6DN plants revealed that major changes in gene expression are associated with constitutive salicylic acid (SA)-mediated defense responses. In agreement, their free and total SA levels resembled those of wild-type plants inoculated with a virulent powdery mildew pathogen. The constitutive SA-associated response in rop6DN was suppressed in mutant backgrounds defective in SA signaling (nonexpresser of PR genes1 [npr1]) or biosynthesis (salicylic acid induction deficient2 [sid2]). However, the rop6DN npr1 and rop6DN sid2 double mutants retained the aberrant developmental phenotypes, indicating that the constitutive SA response can be uncoupled from ROP function(s) in development. rop6DN plants exhibited enhanced preinvasive defense responses to a host-adapted virulent powdery mildew fungus but were impaired in preinvasive defenses upon inoculation with a nonadapted powdery mildew. The host-adapted powdery mildew had a reduced reproductive fitness on rop6DN plants, which was retained in mutant backgrounds defective in SA biosynthesis or signaling. Our findings indicate that both the morphological aberrations and altered sensitivity to powdery mildews of rop6DN plants result from perturbations that are independent from the SA-associated response. These perturbations uncouple SA-dependent defense signaling from disease resistance execution. PMID

Spatially and temporally restricted expression of PtrMYB021 regulates secondary cell wall formation in Arabidopsis

DOE PAGES

Wang, Wei; Li, Eryang; Porth, Ilga; ...

2016-02-02

Among the R2R3 MYB transcription factors that involve in the regulation of secondary cell wall formation in Arabidopsis, MYB46 alone is sufficient to induce the entire secondary cell wall biosynthesis program. PtrMYB021, the poplar homolog of MYB46, has been reported to regulate secondary cell wall formation when expressed in Arabidopsis. We report here that spatially and temporally restricted expression of PtrMYB021 is critical for its function in regulating secondary cell wall formation. By using quantitative RT-PCR, we found that PtrMYB021 was expressed primarily in xylem tissues. When expressed in Arabidopsis under the control of PtrCesA8, but not the 35S promoter,more » PtrMYB021 increased secondary cell wall thickness, which is likely caused by increased lignification as well as changes in cell wall carbohydrate composition. Consistent with this, elevated expression of lignin and cellulose biosynthetic genes were observed in the transgenic plants. Finally, when expressed in Arabidopsis protoplasts as fusion proteins to the Gal4 DNA binding domain, PtrMYB021 activated the reporter gene Gal4-GUS. In summary, our results suggest that PtrMYB021 is a transcriptional activator, and spatially and temporally restricted expression of PtrMYB021 in Arabidopsis regulates secondary cell wall formation by activating a subset of secondary cell wall biosynthesis genes.« less

Spatially and temporally restricted expression of PtrMYB021 regulates secondary cell wall formation in Arabidopsis

DOE Office of Scientific and Technical Information (OSTI.GOV)

Wang, Wei; Li, Eryang; Porth, Ilga

Among the R2R3 MYB transcription factors that involve in the regulation of secondary cell wall formation in Arabidopsis, MYB46 alone is sufficient to induce the entire secondary cell wall biosynthesis program. PtrMYB021, the poplar homolog of MYB46, has been reported to regulate secondary cell wall formation when expressed in Arabidopsis. We report here that spatially and temporally restricted expression of PtrMYB021 is critical for its function in regulating secondary cell wall formation. By using quantitative RT-PCR, we found that PtrMYB021 was expressed primarily in xylem tissues. When expressed in Arabidopsis under the control of PtrCesA8, but not the 35S promoter,more » PtrMYB021 increased secondary cell wall thickness, which is likely caused by increased lignification as well as changes in cell wall carbohydrate composition. Consistent with this, elevated expression of lignin and cellulose biosynthetic genes were observed in the transgenic plants. Finally, when expressed in Arabidopsis protoplasts as fusion proteins to the Gal4 DNA binding domain, PtrMYB021 activated the reporter gene Gal4-GUS. In summary, our results suggest that PtrMYB021 is a transcriptional activator, and spatially and temporally restricted expression of PtrMYB021 in Arabidopsis regulates secondary cell wall formation by activating a subset of secondary cell wall biosynthesis genes.« less

A Common Position-Dependent Mechanism Controls Cell-Type Patterning and GLABRA2 Regulation in the Root and Hypocotyl Epidermis of Arabidopsis1

PubMed Central

Hung, Chen-Yi; Lin, Yan; Zhang, Meng; Pollock, Susan; David Marks, M.; Schiefelbein, John

1998-01-01

A position-dependent pattern of epidermal cell types is produced during root development in Arabidopsis thaliana. This pattern is reflected in the expression pattern of GLABRA2 (GL2), a homeobox gene that regulates cell differentiation in the root epidermis. GL2 promoter::GUS fusions were used to show that the TTG gene, a regulator of root epidermis development, is necessary for maximal GL2 activity but is not required for the pattern of GL2 expression. Furthermore, GL2-promoter activity is influenced by expression of the myc-like maize R gene (35S::R) in Arabidopsis but is not affected by gl2 mutations. A position-dependent pattern of cell differentiation and GL2-promoter activity was also discovered in the hypocotyl epidermis that was analogous to the pattern in the root. Non-GL2-expressing cell files in the hypocotyl epidermis located outside anticlinal cortical cell walls exhibit reduced cell length and form stomata. Like the root, the hypocotyl GL2 activity was shown to be influenced by ttg and 35S::R but not by gl2. The parallel pattern of cell differentiation in the root and hypocotyl indicates that TTG and GL2 participate in a common position-dependent mechanism to control cell-type patterning throughout the apical-basal axis of the Arabidopsis seedling. PMID:9576776

Arabidopsis GRI is involved in the regulation of cell death induced by extracellular ROS.

PubMed

Wrzaczek, Michael; Brosché, Mikael; Kollist, Hannes; Kangasjärvi, Jaakko

2009-03-31

Reactive oxygen species (ROS) have important functions in plant stress responses and development. In plants, ozone and pathogen infection induce an extracellular oxidative burst that is involved in the regulation of cell death. However, very little is known about how plants can perceive ROS and regulate the initiation and the containment of cell death. We have identified an Arabidopsis thaliana protein, GRIM REAPER (GRI), that is involved in the regulation of cell death induced by extracellular ROS. Plants with an insertion in GRI display an ozone-sensitive phenotype. GRI is an Arabidopsis ortholog of the tobacco flower-specific Stig1 gene. The GRI protein appears to be processed in leaves with a release of an N-terminal fragment of the protein. Infiltration of the N-terminal fragment of the GRI protein into leaves caused cell death in a superoxide- and salicylic acid-dependent manner. Analysis of the extracellular GRI protein yields information on how plants can initiate ROS-induced cell death during stress response and development.

The Arabidopsis transcription factor AtTCP15 regulates endoreduplication by modulating expression of key cell-cycle genes.

PubMed

Li, Zi-Yu; Li, Bin; Dong, Ai-Wu

2012-01-01

Plant cells frequently undergo endoreduplication, a modified cell cycle in which genome is repeatedly replicated without cytokinesis. As the key step to achieve final size and function for cells, endoreduplication is prevalent during plant development. However, mechanisms to control the balance between endoreduplication and mitotic cell division are still poorly understood. Here, we show that the Arabidopsis TCP (CINCINNATA-like TEOSINTE BRANCHED1-CYCLOIDEA-PCF)-family transcription factor gene AtTCP15 is expressed in trichomes, as well as in rapidly dividing and vascular tissues. Expression of AtTCP15SRDX, AtTCP15 fused with a SRDX repressor domain, induces extra endoreduplication in trichomes and cotyledon cells in transgenic Arabidopsis. On the contrary, overexpression of AtTCP15 suppresses endoreduplication in trichomes and other examined cells. Misregulation of AtTCP15 affects the expression of several important genes involved in cell-cycle regulation. AtTCP15 protein binds directly to the promoter regions of CYCA2;3 and RETINOBLASTOMA-RELATED (RBR) genes, which play key roles in endoreduplication. Taken together, AtTCP15 plays an important role in regulating endoreduplication during Arabidopsis development.

The Arabidopsis domain of unknown function 1218 (DUF1218) containing proteins, MODIFYING WALL LIGNIN-1 and 2 (At1g31720/MWL-1 and At4g19370/MWL-2) function redundantly to alter secondary cell wall lignin content

DOE Office of Scientific and Technical Information (OSTI.GOV)

Mewalal, Ritesh; Mizrachi, Eshchar; Coetzee, Berdine

DUF1218 is a land plant-specific innovation and has previously been shown to be associated with cell wall biology, vasculature patterning and abiotic/biotic stress response. The Arabidopsis genome encodes 15 members, two of which (At1g31720 and At4g27435) are preferentially expressed in the secondary cell wall depositing inflorescence stems. To further our understanding of the roles of DUF1218-containing proteins in secondary cell wall biology, we functionally characterized At1g31720 (herein referred to as MODIFYING WALL LIGNIN-1 or MWL-1). Since related gene family members may contribute to functional redundancy, we also characterized At4g19370 ( MWL-2), the most closely related gene to MWL-1 in themore » protein family. Subcellular localization revealed that both Arabidopsis proteins are targeted to the cell periphery. The single T-DNA knockout lines, mwl-1 and mwl-2, and independent overexpression lines showed no significant differences in plant growth or changes in total lignin content relative to wild-type (WT) control plants. However, the double homozygous mutant, mwl-1/ mwl-2, had smaller rosettes with a significant decrease in rosette fresh weight and stem height relative to the WT control at four weeks and six weeks, respectively. Moreover, mwl-1/ mwl-2 showed a significant reduction in total lignin content (by ca. 11% relative to WT) and an increase in syringyl/guaiacyl (S/G) monomer ratio relative to the control plants. Lastly, our study has identified two additional members of the DUF1218 family in Arabidopsis as novel contributors to secondary cell wall biology, specifically lignin biosynthesis, and these proteins appear to function redundantly.« less

The Arabidopsis domain of unknown function 1218 (DUF1218) containing proteins, MODIFYING WALL LIGNIN-1 and 2 (At1g31720/MWL-1 and At4g19370/MWL-2) function redundantly to alter secondary cell wall lignin content

DOE PAGES

Mewalal, Ritesh; Mizrachi, Eshchar; Coetzee, Berdine; ...

2016-03-01

DUF1218 is a land plant-specific innovation and has previously been shown to be associated with cell wall biology, vasculature patterning and abiotic/biotic stress response. The Arabidopsis genome encodes 15 members, two of which (At1g31720 and At4g27435) are preferentially expressed in the secondary cell wall depositing inflorescence stems. To further our understanding of the roles of DUF1218-containing proteins in secondary cell wall biology, we functionally characterized At1g31720 (herein referred to as MODIFYING WALL LIGNIN-1 or MWL-1). Since related gene family members may contribute to functional redundancy, we also characterized At4g19370 ( MWL-2), the most closely related gene to MWL-1 in themore » protein family. Subcellular localization revealed that both Arabidopsis proteins are targeted to the cell periphery. The single T-DNA knockout lines, mwl-1 and mwl-2, and independent overexpression lines showed no significant differences in plant growth or changes in total lignin content relative to wild-type (WT) control plants. However, the double homozygous mutant, mwl-1/ mwl-2, had smaller rosettes with a significant decrease in rosette fresh weight and stem height relative to the WT control at four weeks and six weeks, respectively. Moreover, mwl-1/ mwl-2 showed a significant reduction in total lignin content (by ca. 11% relative to WT) and an increase in syringyl/guaiacyl (S/G) monomer ratio relative to the control plants. Lastly, our study has identified two additional members of the DUF1218 family in Arabidopsis as novel contributors to secondary cell wall biology, specifically lignin biosynthesis, and these proteins appear to function redundantly.« less

Nuclear Phytochrome A Signaling Promotes Phototropism in Arabidopsis[W][OA

PubMed Central

Kami, Chitose; Hersch, Micha; Trevisan, Martine; Genoud, Thierry; Hiltbrunner, Andreas; Bergmann, Sven; Fankhauser, Christian

2012-01-01

Phototropin photoreceptors (phot1 and phot2 in Arabidopsis thaliana) enable responses to directional light cues (e.g., positive phototropism in the hypocotyl). In Arabidopsis, phot1 is essential for phototropism in response to low light, a response that is also modulated by phytochrome A (phyA), representing a classical example of photoreceptor coaction. The molecular mechanisms underlying promotion of phototropism by phyA remain unclear. Most phyA responses require nuclear accumulation of the photoreceptor, but interestingly, it has been proposed that cytosolic phyA promotes phototropism. By comparing the kinetics of phototropism in seedlings with different subcellular localizations of phyA, we show that nuclear phyA accelerates the phototropic response, whereas in the fhy1 fhl mutant, in which phyA remains in the cytosol, phototropic bending is slower than in the wild type. Consistent with this data, we find that transcription factors needed for full phyA responses are needed for normal phototropism. Moreover, we show that phyA is the primary photoreceptor promoting the expression of phototropism regulators in low light (e.g., PHYTOCHROME KINASE SUBSTRATE1 [PKS1] and ROOT PHOTO TROPISM2 [RPT2]). Although phyA remains cytosolic in fhy1 fhl, induction of PKS1 and RPT2 expression still occurs in fhy1 fhl, indicating that a low level of nuclear phyA signaling is still present in fhy1 fhl. PMID:22374392

Transport of Indole-3-Butyric Acid and Indole-3-Acetic Acid in Arabidopsis Hypocotyls Using Stable Isotope Labeling1[C][W][OA

PubMed Central

Liu, Xing; Barkawi, Lana; Gardner, Gary; Cohen, Jerry D.

2012-01-01

The polar transport of the natural auxins indole-3-butyric acid (IBA) and indole-3-acetic acid (IAA) has been described in Arabidopsis (Arabidopsis thaliana) hypocotyls using radioactive tracers. Because radioactive assays alone cannot distinguish IBA from its metabolites, the detected transport from applied [3H]IBA may have resulted from the transport of IBA metabolites, including IAA. To test this hypothesis, we used a mass spectrometry-based method to quantify the transport of IBA in Arabidopsis hypocotyls by following the movement of [13C1]IBA and the [13C1]IAA derived from [13C1]IBA. We also assayed [13C6]IAA transport in a parallel control experiment. We found that the amount of transported [13C1]IBA was dramatically lower than [13C6]IAA, and the IBA transport was not reduced by the auxin transport inhibitor N-1-naphthylphthalamic acid. Significant amounts of the applied [13C1]IBA were converted to [13C1]IAA during transport, but [13C1]IBA transport was independent of IBA-to-IAA conversion. We also found that most of the [13C1]IBA was converted to ester-linked [13C1]IBA at the apical end of hypocotyls, and ester-linked [13C1]IBA was also found in the basal end at a level higher than free [13C1]IBA. In contrast, most of the [13C6]IAA was converted to amide-linked [13C6]IAA at the apical end of hypocotyls, but very little conjugated [13C6]IAA was found in the basal end. Our results demonstrate that the polar transport of IBA is much lower than IAA in Arabidopsis hypocotyls, and the transport mechanism is distinct from IAA transport. These experiments also establish a method for quantifying the movement of small molecules in plants using stable isotope labeling. PMID:22323783

Analysis of Cell Division and Elongation Underlying the Developmental Acceleration of Root Growth in Arabidopsis thaliana1

PubMed Central

Beemster, Gerrit T.S.; Baskin, Tobias I.

1998-01-01

To investigate the relation between cell division and expansion in the regulation of organ growth rate, we used Arabidopsis thaliana primary roots grown vertically at 20°C with an elongation rate that increased steadily during the first 14 d after germination. We measured spatial profiles of longitudinal velocity and cell length and calculated parameters of cell expansion and division, including rates of local cell production (cells mm−1 h−1) and cell division (cells cell−1 h−1). Data were obtained for the root cortex and also for the two types of epidermal cell, trichoblasts and atrichoblasts. Accelerating root elongation was caused by an increasingly longer growth zone, while maximal strain rates remained unchanged. The enlargement of the growth zone and, hence, the accelerating root elongation rate, were accompanied by a nearly proportionally increased cell production. This increased production was caused by increasingly numerous dividing cells, whereas their rates of division remained approximately constant. Additionally, the spatial profile of cell division rate was essentially constant. The meristem was longer than generally assumed, extending well into the region where cells elongated rapidly. In the two epidermal cell types, meristem length and cell division rate were both very similar to that of cortical cells, and differences in cell length between the two epidermal cell types originated at the apex of the meristem. These results highlight the importance of controlling the number of dividing cells, both to generate tissues with different cell lengths and to regulate the rate of organ enlargement. PMID:9536070

Expression of transcription factors after short-term exposure of Arabidopsis thaliana cell cultures to hypergravity and simulated microgravity (2-D/3-D clinorotation, magnetic levitation)

NASA Astrophysics Data System (ADS)

Babbick, M.; Dijkstra, C.; Larkin, O. J.; Anthony, P.; Davey, M. R.; Power, J. B.; Lowe, K. C.; Cogoli-Greuter, M.; Hampp, R.

Gravity is an important environmental factor that controls plant growth and development. Studies have shown that the perception of gravity is not only a property of specialized cells, but can also be performed by undifferentiated cultured cells. In this investigation, callus of Arabidopsis thaliana cv. Columbia was used to investigate the initial steps of gravity-related signalling cascades, through altered expression of transcription factors (TFs). TFs are families of small proteins that regulate gene expression by binding to specific promoter sequences. Based on microarray studies, members of the gene families WRKY, MADS-box, MYB, and AP2/EREBP were selected for investigation, as well as members of signalling chains, namely IAA 19 and phosphoinositol-4-kinase. Using qRT-PCR, transcripts were quantified within a period of 30 min in response to hypergravity (8 g), clinorotation [2-D clinostat and 3-D random positioning machine (RPM)] and magnetic levitation (ML). The data indicated that (1) changes in gravity induced stress-related signalling, and (2) exposure in the RPM induced changes in gene expression which resemble those of magnetic levitation. Two dimensional clinorotation resulted in responses similar to those caused by hypergravity. It is suggested that RPM and ML are preferable to simulate microgravity than clinorotation.

Cell-specific vacuolar calcium storage mediated by "CAX1" regulates apoplastic calcium concentration, gas exchange, and plant productivity in "Arabidopsis"

USDA-ARS?s Scientific Manuscript database

The physiological role and mechanism of nutrient storage within vacuoles of specific cell types is poorly understood. Transcript profiles from "Arabidopsis thaliana" leaf cells differing in calcium concentration ([Ca], epidermis <10 mM versus mesophyll >60 mM) were compared using a microarray screen...

Lipase Activity in Insect Oral Secretions Mediates Defense Responses in Arabidopsis1[C][W][OA

PubMed Central

Schäfer, Martin; Fischer, Christine; Meldau, Stefan; Seebald, Eileen; Oelmüller, Ralf; Baldwin, Ian T.

2011-01-01

How plants perceive herbivory is not yet well understood. We investigated early responses of the model plant Arabidopsis (Arabidopsis thaliana) to attack from the generalist grasshopper herbivore, Schistocerca gregaria (Caelifera). When compared with wounding alone, S. gregaria attack and the application of grasshopper oral secretions (GS) to puncture wounds elicited a rapid accumulation of various oxylipins, including 13-hydroperoxy octadecatrienoic acid, 12-oxo-phytodienoic acid (OPDA), jasmonic acid, and jasmonic acid-isoleucine. Additionally, GS increased cytosolic calcium levels, mitogen-activated protein kinase (MPK3 and MPK6) activity, and ethylene emission but not the accumulation of hydrogen peroxide. Although GS contain caeliferin A16:0, a putative elicitor of caeliferan herbivores, treatment with pure, synthetic caeliferin A16:0 did not induce any of the observed responses. With mutant plants, we demonstrate that the observed changes in oxylipin levels are independent of MPK3 and MPK6 activity but that MPK6 is important for the GS-induced ethylene release. Biochemical and pharmacological analyses revealed that the lipase activity of GS plays a central role in the GS-induced accumulation of oxylipins, especially OPDA, which could be fully mimicked by treating puncture wounds only with a lipase from Rhizopus arrhizus. GS elicitation increased the levels of OPDA-responsive transcripts. Because the oral secretions of most insects used to study herbivory-induced responses in Arabidopsis rapidly elicit similar accumulations of OPDA, we suggest that lipids containing OPDA (arabidopsides) play an important role in the activation of herbivory-induced responses. PMID:21546453

Overexpression of Arabidopsis Plasmodesmata Germin-Like Proteins Disrupts Root Growth and Development[C][W

PubMed Central

Ham, Byung-Kook; Li, Gang; Kang, Byung-Ho; Zeng, Fanchang; Lucas, William J.

2012-01-01

In plants, a population of non-cell-autonomous proteins (NCAPs), including numerous transcription factors, move cell to cell through plasmodesmata (PD). In many cases, the intercellular trafficking of these NCAPs is regulated by their interaction with specific PD components. To gain further insight into the functions of this NCAP pathway, coimmunoprecipitation experiments were performed on a tobacco (Nicotiana tabacum) plasmodesmal-enriched cell wall protein preparation using as bait the NCAP, pumpkin (Cucurbita maxima) PHLOEM PROTEIN16 (Cm-PP16). A Cm-PP16 interaction partner, Nt-PLASMODESMAL GERMIN-LIKE PROTEIN1 (Nt-PDGLP1) was identified and shown to be a PD-located component. Arabidopsis thaliana putative orthologs, PDGLP1 and PDGLP2, were identified; expression studies indicated that, postgermination, these proteins were preferentially expressed in the root system. The PDGLP1 signal peptide was shown to function in localization to the PD by a novel mechanism involving the endoplasmic reticulum-Golgi secretory pathway. Overexpression of various tagged versions altered root meristem function, leading to reduced primary root but enhanced lateral root growth. This effect on root growth was corrected with an inability of these chimeric proteins to form stable PD-localized complexes. PDGLP1 and PDGLP2 appear to be involved in regulating primary root growth by controlling phloem-mediated allocation of resources between the primary and lateral root meristems. PMID:22960910

Twin plants from supernumerary egg cells in Arabidopsis.

PubMed

Kong, Jixiang; Lau, Steffen; Jürgens, Gerd

2015-01-19

Sexual reproduction of flowering plants is distinguished by double fertilization—the two sperm cells delivered by a pollen tube fuse with the two gametic cells of the female gametophyte, the egg and the central cell—inside the ovule to give rise to the embryo and the nutritive endosperm, respectively. The pollen tube is attracted by nongametic synergid cells, and how these two cells of the female gametophyte are specified is currently unclear. Here, we show that ALTERED MERISTEM PROGRAM 1 (AMP1), encoding a protein associated with the endoplasmic reticulum, is required for synergid cell fate during Arabidopsis female gametophyte development. Loss of AMP1 function leads to supernumerary egg cells at the expense of synergids, enabling the generation of dizygotic twins. However, if twin embryos are formed, endosperm formation is prevented, eventually resulting in ovule abortion. The latter can be overcome by the delivery of supernumerary sperm cells in tetraspore (tes) pollen, enabling the formation of twin plants. Thus, both primary and supernumerary egg cells are fully functional in amp1 mutant plants. Sporophytic AMP1 expression is sufficient to prevent cell-fate change of synergids, indicating that one or more AMP1-dependent mobile signals from outside the female gametophyte can contribute to its patterning, in addition to the previously reported lateral inhibition between gametophytic cells. Our results provide insight into the mechanism of synergid fate specification and emphasize the importance of specifying only one egg cell within the female gametophyte to ensure central-cell fertilization by the second sperm cell. Copyright © 2015 Elsevier Ltd. All rights reserved.

The gravitropism defective 2 Mutants of Arabidopsis Are Deficient in a Protein Implicated in Endocytosis in Caenorhabditis elegans1[w

PubMed Central

Silady, Rebecca A.; Kato, Takehide; Lukowitz, Wolfgang; Sieber, Patrick; Tasaka, Masao; Somerville, Chris R.

2004-01-01

The gravitropism defective 2 (grv2) mutants of Arabidopsis show reduced shoot phototropism and gravitropism. Amyloplasts in the shoot endodermal cells of grv2 do not sediment to the same degree as in wild type. The GRV2 gene encodes a 277-kD polypeptide that is 42% similar to the Caenorhabditis elegans RME-8 protein, which is required for endocytosis. We hypothesize that a defect in endocytosis may affect both the initial gravity sensing via amyloplasts sedimentation and the subsequent more general tropic growth response. PMID:15466218

A simple microfluidic device for live cell imaging of Arabidopsis cotyledons, leaves, and seedlings.

PubMed

Vang, Shia; Seitz, Kati; Krysan, Patrick J

2018-06-01

One of the challenges of performing live-cell imaging in plants is establishing a system for securing the sample during imaging that allows for the rapid addition of treatments. Here we report how a commercially available device called a HybriWell ™ can be repurposed to create an imaging chamber suitable for Arabidopsis seedlings, cotyledons and leaves. Liquid in the imaging chamber can be rapidly exchanged to introduce chemical treatments via microfluidic passive pumping. When used in conjunction with fluorescent biosensors, this system can facilitate live-cell imaging studies of signal transduction pathways triggered by different treatments. As a demonstration, we show how the HybriWell can be used to monitor flg22-induced calcium transients using the R-GECO1 calcium indicator in detached Arabidopsis leaves.

Cell fate in the Arabidopsis root epidermis is determined by competition between WEREWOLF and CAPRICE.

PubMed

Song, Sang-Kee; Ryu, Kook Hui; Kang, Yeon Hee; Song, Jae Hyo; Cho, Young-Hee; Yoo, Sang-Dong; Schiefelbein, John; Lee, Myeong Min

2011-11-01

The root hair and nonhair cells in the Arabidopsis (Arabidopsis thaliana) root epidermis are specified by a suite of transcriptional regulators. Two of these are WEREWOLF (WER) and CAPRICE (CPC), which encode MYB transcription factors that are required for promoting the nonhair cell fate and the hair cell fate, respectively. However, the precise function and relationship between these transcriptional regulators have not been fully defined experimentally. Here, we examine these issues by misexpressing the WER gene using the GAL4-upstream activation sequence transactivation system. We find that WER overexpression in the Arabidopsis root tip is sufficient to cause epidermal cells to adopt the nonhair cell fate through direct induction of GLABRA2 (GL2) gene expression. We also show that GLABRA3 (GL3) and ENHANCER OF GLABRA3 (EGL3), two closely related bHLH proteins, are required for the action of the overexpressed WER and that WER interacts with these bHLHs in plant cells. Furthermore, we find that CPC suppresses the WER overexpression phenotype quantitatively. These results show that WER acts together with GL3/EGL3 to induce GL2 expression and that WER and CPC compete with one another to define cell fates in the Arabidopsis root epidermis.

NIP1;2 is a plasma membrane-localized transporter mediating aluminum uptake, translocation, and tolerance in Arabidopsis

PubMed Central

Wang, Yuqi; Li, Ruihong; Li, Demou; Jia, Xiaomin; Zhou, Dangwei; Li, Jianyong; Lyi, Sangbom M.; Hou, Siyu; Huang, Yulan

2017-01-01

Members of the aquaporin (AQP) family have been suggested to transport aluminum (Al) in plants; however, the Al form transported by AQPs and the roles of AQPs in Al tolerance remain elusive. Here we report that NIP1;2, a plasma membrane-localized member of the Arabidopsis nodulin 26-like intrinsic protein (NIP) subfamily of the AQP family, facilitates Al-malate transport from the root cell wall into the root symplasm, with subsequent Al xylem loading and root-to-shoot translocation, which are critical steps in an internal Al tolerance mechanism in Arabidopsis. We found that NIP1;2 transcripts are expressed mainly in the root tips, and that this expression is enhanced by Al but not by other metal stresses. Mutations in NIP1;2 lead to hyperaccumulation of toxic Al3+ in the root cell wall, inhibition of root-to-shoot Al translocation, and a significant reduction in Al tolerance. NIP1;2 facilitates the transport of Al-malate, but not Al3+ ions, in both yeast and Arabidopsis. We demonstrate that the formation of the Al-malate complex in the root tip apoplast is a prerequisite for NIP1;2-mediated Al removal from the root cell wall, and that this requires a functional root malate exudation system mediated by the Al-activated malate transporter, ALMT1. Taken together, these findings reveal a critical linkage between the previously identified Al exclusion mechanism based on root malate release and an internal Al tolerance mechanism identified here through the coordinated function of NIP1;2 and ALMT1, which is required for Al removal from the root cell wall, root-to-shoot Al translocation, and overall Al tolerance in Arabidopsis. PMID:28439024

Arabidopsis N-MYC DOWNREGULATED-LIKE1, a positive regulator of auxin transport in a G protein-mediated pathway.

PubMed

Mudgil, Yashwanti; Uhrig, Joachm F; Zhou, Jiping; Temple, Brenda; Jiang, Kun; Jones, Alan M

2009-11-01

Root architecture results from coordinated cell division and expansion in spatially distinct cells of the root and is established and maintained by gradients of auxin and nutrients such as sugars. Auxin is transported acropetally through the root within the central stele and then, upon reaching the root apex, auxin is transported basipetally through the outer cortical and epidermal cells. The two Gbetagamma dimers of the Arabidopsis thaliana heterotrimeric G protein complex are differentially localized to the central and cortical tissues of the Arabidopsis roots. A null mutation in either the single beta (AGB1) or the two gamma (AGG1 and AGG2) subunits confers phenotypes that disrupt the proper architecture of Arabidopsis roots and are consistent with altered auxin transport. Here, we describe an evolutionarily conserved interaction between AGB1/AGG dimers and a protein designated N-MYC DOWNREGULATED-LIKE1 (NDL1). The Arabidopsis genome encodes two homologs of NDL1 (NDL2 and NDL3), which also interact with AGB1/AGG1 and AGB1/AGG2 dimers. We show that NDL proteins act in a signaling pathway that modulates root auxin transport and auxin gradients in part by affecting the levels of at least two auxin transport facilitators. Reduction of NDL family gene expression and overexpression of NDL1 alter root architecture, auxin transport, and auxin maxima. AGB1, auxin, and sugars are required for NDL1 protein stability in regions of the root where auxin gradients are established; thus, the signaling mechanism contains feedback loops.

FUSCA3 activates triacylglycerol accumulation in Arabidopsis seedlings and tobacco BY2 cells

DOE PAGES

Zhang, Meng; Cao, Xia; Jia, Qingli; ...

2016-06-11

Triacylglycerol (TAG) is the main storage lipid in plant seeds and the major form of plant oil used for food and, increasingly, for industrial and biofuel applications. Several transcription factors, including FUSCA3 (At3 g26790, FUS3), are associated with embryo maturation and oil biosynthesis in seeds. However, the ability of FUS3 to increase TAG biosynthesis in other tissues has not been quantitatively examined. Here, we evaluated the ability of FUS3 to activate TAG accumulation in non-seed tissues. Overexpression of FUS3 driven by an estradiol-inducible promoter increased oil contents in Arabidopsis seedlings up to 6% of dry weight; more than 50-fold overmore » controls. Eicosenoic acid, a characteristic fatty acid of Arabidopsis seed oil, accumulated to over 20% of fatty acids in cotyledons and leaves. These large increases depended on added sucrose, although without sucrose TAG increased three- to four-fold. Inducing the expression of FUS3 in tobacco BY2 cells also increased TAG accumulation, and co-expression of FUS3 and diacylglycerol acyltransferase 1 (DGAT1) further increased TAG levels to 4% of dry weight. BY2 cell growth was not altered by FUS3 expression, although Arabidopsis seedling development was impaired, consistent with the ability of FUS3 to induce embryo characteristics in non-seed tissues. Microarrays of Arabidopsis seedlings revealed that FUS3 overexpression increased the expression of a higher proportion of genes involved in TAG biosynthesis than genes involved in fatty acid biosynthesis or other lipid pathways. Together these results provide additional insights into FUS3 functions in TAG metabolism and suggest complementary strategies for engineering vegetative oil accumulation.« less

Phosphatidylinositol (4,5)Bisphosphate Inhibits K+-Efflux Channel Activity in NT1 Tobacco Cultured Cells1[W][OA

PubMed Central

Ma, Xiaohong; Shor, Oded; Diminshtein, Sofia; Yu, Ling; Im, Yang Ju; Perera, Imara; Lomax, Aaron; Boss, Wendy F.; Moran, Nava

2009-01-01

In the animal world, the regulation of ion channels by phosphoinositides (PIs) has been investigated extensively, demonstrating a wide range of channels controlled by phosphatidylinositol (4,5)bisphosphate (PtdInsP2). To understand PI regulation of plant ion channels, we examined the in planta effect of PtdInsP2 on the K+-efflux channel of tobacco (Nicotiana tabacum), NtORK (outward-rectifying K channel). We applied a patch clamp in the whole-cell configuration (with fixed “cytosolic” Ca2+ concentration and pH) to protoplasts isolated from cultured tobacco cells with genetically manipulated plasma membrane levels of PtdInsP2 and cellular inositol (1,4,5)trisphosphate: “Low PIs” had depressed levels of these PIs, and “High PIs” had elevated levels relative to controls. In all of these cells, K channel activity, reflected in the net, steady-state outward K+ currents (IK), was inversely related to the plasma membrane PtdInsP2 level. Consistent with this, short-term manipulations decreasing PtdInsP2 levels in the High PIs, such as pretreatment with the phytohormone abscisic acid (25 μm) or neutralizing the bath solution from pH 5.6 to pH 7, increased IK (i.e. NtORK activity). Moreover, increasing PtdInsP2 levels in controls or in abscisic acid-treated high-PI cells, using the specific PI-phospholipase C inhibitor U73122 (2.5–4 μm), decreased NtORK activity. In all cases, IK decreases stemmed largely from decreased maximum attainable NtORK channel conductance and partly from shifted voltage dependence of channel gating to more positive potentials, making it more difficult to activate the channels. These results are consistent with NtORK inhibition by the negatively charged PtdInsP2 in the internal plasma membrane leaflet. Such effects are likely to underlie PI signaling in intact plant cells. PMID:19052153

FLYING SAUCER1 Is a Transmembrane RING E3 Ubiquitin Ligase That Regulates the Degree of Pectin Methylesterification in Arabidopsis Seed Mucilage[W

PubMed Central

Voiniciuc, Cătălin; Dean, Gillian H.; Griffiths, Jonathan S.; Kirchsteiger, Kerstin; Hwang, Yeen Ting; Gillett, Alan; Dow, Graham; Western, Tamara L.; Estelle, Mark; Haughn, George W.

2013-01-01

Pectins are complex polysaccharides that form the gel matrix of the primary cell wall and are abundant in the middle lamella that holds plant cells together. Their degree of methylesterification (DM) impacts wall strength and cell adhesion since unesterified pectin regions can cross-link via Ca2+ ions to form stronger gels. Here, we characterize flying saucer1 (fly1), a novel Arabidopsis thaliana seed coat mutant, which displays primary wall detachment, reduced mucilage extrusion, and increased mucilage adherence. These defects appear to result from a lower DM in mucilage and are enhanced by the addition of Ca2+ or completely rescued using alkaline Ca2+ chelators. FLY1 encodes a transmembrane protein with a RING-H2 domain that has in vitro E3 ubiquitin ligase activity. FLY1 is orthologous to TRANSMEMBRANE UBIQUITIN LIGASE1, a Golgi-localized E3 ligase involved in the quality control of membrane proteins in yeast. However, FLY1–yellow fluorescent protein (YFP) fusions are localized in punctae that are predominantly distinct from the Golgi and the trans-Golgi network/early endosome in the seed coat epidermis. Wortmannin treatment, which induces the fusion of late endosomes in plants, resulted in enlarged FLY1-YFP bodies. We propose that FLY1 regulates the DM of pectin in mucilage, potentially by recycling pectin methylesterase enzymes in the endomembrane system of seed coat epidermal cells. PMID:23482858

Nanog1 in NTERA-2 and Recombinant NanogP8 from Somatic Cancer Cells Adopt Multiple Protein Conformations and Migrate at Multiple M.W Species

PubMed Central

Liu, Bigang; Badeaux, Mark D.; Choy, Grace; Chandra, Dhyan; Shen, Irvin; Jeter, Collene R.; Rycaj, Kiera; Lee, Chia-Fang; Person, Maria D.; Liu, Can; Chen, Yueping; Shen, Jianjun; Jung, Sung Yun; Qin, Jun; Tang, Dean G.

2014-01-01

Human Nanog1 is a 305-amino acid (aa) homeodomain-containing transcription factor critical for the pluripotency of embryonic stem (ES) and embryonal carcinoma (EC) cells. Somatic cancer cells predominantly express a retrogene homolog of Nanog1 called NanogP8, which is ∼99% similar to Nanog at the aa level. Although the predicted M.W of Nanog1/NanogP8 is ∼35 kD, both have been reported to migrate, on Western blotting (WB), at apparent molecular masses of 29–80 kD. Whether all these reported protein bands represent authentic Nanog proteins is unclear. Furthermore, detailed biochemical studies on Nanog1/NanogpP8 have been lacking. By combining WB using 8 anti-Nanog1 antibodies, immunoprecipitation, mass spectrometry, and studies using recombinant proteins, here we provide direct evidence that the Nanog1 protein in NTERA-2 EC cells exists as multiple M.W species from ∼22 kD to 100 kD with a major 42 kD band detectable on WB. We then demonstrate that recombinant NanogP8 (rNanogP8) proteins made in bacteria using cDNAs from multiple cancer cells also migrate, on denaturing SDS-PAGE, at ∼28 kD to 180 kD. Interestingly, different anti-Nanog1 antibodies exhibit differential reactivity towards rNanogP8 proteins, which can spontaneously form high M.W protein species. Finally, we show that most long-term cultured cancer cell lines seem to express very low levels of or different endogenous NanogP8 protein that cannot be readily detected by immunoprecipitation. Altogether, the current study reveals unique biochemical properties of Nanog1 in EC cells and NanogP8 in somatic cancer cells. PMID:24598770

Arabidopsis thaliana RALF1 opposes brassinosteroid effects on root cell elongation and lateral root formation

PubMed Central

Moura, Daniel S.

2014-01-01

Rapid alkalinization factor (RALF) is a peptide signal that plays a basic role in cell biology and most likely regulates cell expansion. In this study, transgenic Arabidopsis thaliana lines with high and low levels of AtRALF1 transcripts were used to investigate this peptide’s mechanism of action. Overexpression of the root-specific isoform AtRALF1 resulted in reduced cell size. Conversely, AtRALF1 silencing increased root length by increasing the size of root cells. AtRALF1-silenced plants also showed an increase in the number of lateral roots, whereas AtRALF1 overexpression produced the opposite effect. In addition, four AtRALF1-inducible genes were identified: two genes encoding proline-rich proteins (AtPRP1 and AtPRP3), one encoding a hydroxyproline-rich glycoprotein (AtHRPG2), and one encoding a xyloglucan endotransglucosylase (TCH4). These genes were expressed in roots and involved in cell-wall rearrangement, and their induction was concentration dependent. Furthermore, AtRALF1-overexpressing plants were less sensitive to exogenous brassinolide (BL); upon BL treatment, the plants showed no increase in root length and a compromised increase in hypocotyl elongation. In addition, the treatment had no effect on the number of emerged lateral roots. AtRALF1 also induces two brassinosteroid (BR)-downregulated genes involved in the BR biosynthetic pathway: the cytochrome P450 monooxygenases CONSTITUTIVE PHOTOMORPHISM AND DWARFISM (CPD) and DWARF4 (DWF4). Simultaneous treatment with both AtRALF1 and BL caused a reduction in AtRALF1-inducible gene expression levels, suggesting that these signals may compete for components shared by both pathways. Taken together, these results indicate an opposing effect of AtRALF1 and BL, and suggest that RALF’s mechanism of action could be to interfere with the BR signalling pathway. PMID:24620000

Interplay between Heat Shock Proteins HSP101 and HSA32 Prolongs Heat Acclimation Memory Posttranscriptionally in Arabidopsis1[W][OA

PubMed Central

Wu, Ting-ying; Juan, Yu-ting; Hsu, Yang-hsin; Wu, Sze-hsien; Liao, Hsiu-ting; Fung, Raymond W.M.; Charng, Yee-yung

2013-01-01

Heat acclimation improves the tolerance of organisms to severe heat stress. Our previous work showed that in Arabidopsis (Arabidopsis thaliana), the “memory” of heat acclimation treatment decayed faster in the absence of the heat-stress-associated 32-kD protein HSA32, a heat-induced protein predominantly found in plants. The HSA32 null mutant attains normal short-term acquired thermotolerance but is defective in long-term acquired thermotolerance. To further explore this phenomenon, we isolated Arabidopsis defective in long-term acquired thermotolerance (dlt) mutants using a forward genetic screen. Two recessive missense alleles, dlt1-1 and dlt1-2, encode the molecular chaperone heat shock protein101 (HSP101). Results of immunoblot analyses suggest that HSP101 enhances the translation of HSA32 during recovery after heat treatment, and in turn, HSA32 retards the decay of HSP101. The dlt1-1 mutation has little effect on HSP101 chaperone activity and thermotolerance function but compromises the regulation of HSA32. In contrast, dlt1-2 impairs the chaperone activity and thermotolerance function of HSP101 but not the regulation of HSA32. These results suggest that HSP101 has a dual function, which could be decoupled by the mutations. Pulse-chase analysis showed that HSP101 degraded faster in the absence of HSA32. The autophagic proteolysis inhibitor E-64d, but not the proteasome inhibitor MG132, inhibited the degradation of HSP101. Ectopic expression of HSA32 confirmed its effect on the decay of HSP101 at the posttranscriptional level and showed that HSA32 was not sufficient to confer long-term acquired thermotolerance when the HSP101 level was low. Taken together, we propose that a positive feedback loop between HSP101 and HSA32 at the protein level is a novel mechanism for prolonging the memory of heat acclimation. PMID:23439916

Near-IR laser-triggered target cell collection using a carbon nanotube-based cell-cultured substrate.

PubMed

Sada, Takao; Fujigaya, Tsuyohiko; Niidome, Yasuro; Nakazawa, Kohji; Nakashima, Naotoshi

2011-06-28

Unique near-IR optical properties of single-walled carbon nanotube (SWNTs) are of interest in many biological applications. Here we describe the selective cell detachment and collection from an SWNT-coated cell-culture dish triggered by near-IR pulse laser irradiation. First, HeLa cells were cultured on an SWNT-coated dish prepared by a spraying of an aqueous SWNT dispersion on a glass dish. The SWNT-coated dish was found to show a good cell adhesion behavior as well as a cellular proliferation rate similar to a conventional glass dish. We discovered, by near-IR pulse laser irradiation (at the laser power over 25 mW) to the cell under optical microscopic observation, a quick single-cell detachment from the SWNT-coated surface. Shockwave generation from the irradiated SWNTs is expected to play an important role for the cell detachment. Moreover, we have succeeded in catapulting the target single cell from the cultured medium when the depth of the medium was below 150 μm and the laser power was stronger than 40 mW. The captured cell maintained its original shape. The retention of the genetic information of the cell was confirmed by the polymerase chain reaction (PCR) technique. A target single-cell collection from a culture medium under optical microscopic observation is significant in wide fields of single-cell studies in biological areas.

Patatin-Related Phospholipase pPLAIIIβ-Induced Changes in Lipid Metabolism Alter Cellulose Content and Cell Elongation in Arabidopsis[C][W

PubMed Central

Li, Maoyin; Bahn, Sung Chul; Guo, Liang; Musgrave, William; Berg, Howard; Welti, Ruth; Wang, Xuemin

2011-01-01

The release of fatty acids from membrane lipids has been implicated in various plant processes, and the patatin-related phospholipases (pPLAs) constitute a major enzyme family that catalyzes fatty acid release. The Arabidopsis thaliana pPLA family has 10 members that are classified into three groups. Group 3 pPLAIII has four members but lacks the canonical lipase/esterase consensus catalytic sequences, and their enzymatic activity and cellular functions have not been delineated. Here, we show that pPLAIIIβ hydrolyzes phospholipids and galactolipids and additionally has acyl-CoA thioesterase activity. Alterations of pPLAIIIβ result in changes in lipid levels and composition. pPLAIIIβ-KO plants have longer leaves, petioles, hypocotyls, primary roots, and root hairs than wild-type plants, whereas pPLAIIIβ-OE plants exhibit the opposite phenotype. In addition, pPLAIIIβ-OE plants have significantly lower cellulose content and mechanical strength than wild-type plants. Root growth of pPLAIIIβ-KO plants is less sensitive to treatment with free fatty acids, the enzymatic products of pPLAIIIβ, than wild-type plants; root growth of pPLAIIIβ-OE plants is more sensitive. These data suggest that alteration of pPLAIIIβ expression and the resulting lipid changes alter cellulose content and cell elongation in Arabidopsis. PMID:21447788

The Arabidopsis E3 Ubiquitin Ligase HOS1 Negatively Regulates CONSTANS Abundance in the Photoperiodic Control of Flowering[W

PubMed Central

Lazaro, Ana; Valverde, Federico; Piñeiro, Manuel; Jarillo, Jose A.

2012-01-01

The Arabidopsis thaliana early in short days6 (esd6) mutant was isolated in a screen for mutations that accelerate flowering time. Among other developmental alterations, esd6 displays early flowering in both long- and short-day conditions. Fine mapping of the mutation showed that the esd6 phenotype is caused by a lesion in the HIGH EXPRESSION OF OSMOTICALLY RESPONSIVE GENES1 (HOS1) locus, which encodes a RING finger–containing E3 ubiquitin ligase. The esd6/hos1 mutation causes decreased FLOWERING LOCUS C expression and requires CONSTANS (CO) protein for its early flowering phenotype under long days. Moreover, CO and HOS1 physically interact in vitro and in planta, and HOS1 regulates CO abundance, particularly during the daylight period. Accordingly, hos1 causes a shift in the regular long-day pattern of expression of FLOWERING LOCUS T (FT) transcript, starting to rise 4 h after dawn in the mutant. In addition, HOS1 interacts synergistically with CONSTITUTIVE PHOTOMORPHOGENIC1, another regulator of CO protein stability, in the regulation of flowering time. Taken together, these results indicate that HOS1 is involved in the control of CO abundance, ensuring that CO activation of FT occurs only when the light period reaches a certain length and preventing precocious flowering in Arabidopsis. PMID:22408073

Building bridges: formin1 of Arabidopsis forms a connection between the cell wall and the actin cytoskeleton.

PubMed

Martinière, Alexandre; Gayral, Philippe; Hawes, Chris; Runions, John

2011-04-01

Actin microfilament (MF) organization and remodelling is critical to cell function. The formin family of actin binding proteins are involved in nucleating MFs in Arabidopsis thaliana. They all contain formin homology domains in the intracellular, C-terminal half of the protein that interacts with MFs. Formins in class I are usually targeted to the plasma membrane and this is true of Formin1 (AtFH1) of A. thaliana. In this study, we have investigated the extracellular domain of AtFH1 and we demonstrate that AtFH1 forms a bridge from the actin cytoskeleton, across the plasma membrane and is anchored within the cell wall. AtFH1 has a large, extracellular domain that is maintained by purifying selection and that contains four conserved regions, one of which is responsible for immobilising the protein. Protein anchoring within the cell wall is reduced in constructs that express truncations of the extracellular domain and in experiments in protoplasts without primary cell walls. The 18 amino acid proline-rich extracellular domain that is responsible for AtFH1 anchoring has homology with cell-wall extensins. We also have shown that anchoring of AtFH1 in the cell wall promotes actin bundling within the cell and that overexpression of AtFH1 has an inhibitory effect on organelle actin-dependant dynamics. Thus, the AtFH1 bridge provides stable anchor points for the actin cytoskeleton and is probably a crucial component of the signalling response and actin-remodelling mechanisms. © 2011 The Authors. The Plant Journal © 2011 Blackwell Publishing Ltd.

Reassessment of an Arabidopsis cell wall invertase inhibitor AtCIF1 reveals its role in seed germination and early seedling growth.

PubMed

Su, Tao; Wolf, Sebastian; Han, Mei; Zhao, Hongbo; Wei, Hongbin; Greiner, Steffen; Rausch, Thomas

2016-01-01

In higher plants, cell wall invertase (CWI) and vacuolar invertase (VI) are recognized as essential players in sugar metabolism and sugar signaling, thereby affecting source-sink interactions, plant development and responses to environmental cues. CWI and VI expression levels are transcriptionally controlled; however, both enzymes are also subject to posttranslational control by invertase inhibitor proteins. The physiological significances of inhibitor proteins during seed germination and early seedling development are not yet fully understood. Here, we demonstrate that the inhibitor isoform AtCIF1 impacted on seed germination and early seedling growth in Arabidopsis. The primary target of AtCIF1 was shown to be localized to the apoplast after expressing an AtCIF1 YFP-fusion construct in tobacco epidermis and transgenic Arabidopsis root. The analysis of expression patterns showed that AtCWI1 was co-expressed spatiotemporally with AtCIF1 within the early germinating seeds. Seed germination was observed to be accelerated independently of exogenous abscisic acid (ABA) in the AtCIF1 loss-of-function mutant cif1-1. This effect coincided with a drastic increase of CWI activity in cif1-1 mutant seeds by 24 h after the onset of germination, both in vitro and in planta. Accordingly, quantification of sugar content showed that hexose levels were significantly boosted in germinating seeds of the cif1-1 mutant. Further investigation of AtCIF1 overexpressors in Arabidopsis revealed a markedly suppressed CWI activity as well as delayed seed germination. Thus, we conclude that the posttranslational modulation of CWI activity by AtCIF1 helps to orchestrate seed germination and early seedling growth via fine-tuning sucrose hydrolysis and, possibly, sugar signaling.

Serendipitous solution to the problem of culturing Arabidopsis plants in sealed containers for spaceflights of long duration

NASA Technical Reports Server (NTRS)

Brown, A. H.; Odowd, P.; Loercher, L.; Kuniewicz, R.; Dahl, A. O.

1979-01-01

The Arabidopsis thaliana plant species is tested to determine how a higher plant will develop from seed to maturity when deprived of all gravitational information that it might use to control its growth. Experimental results show that Arabidopsis seedlings can develop to maturity by means of a light-dependent but CO2-independent metabolism that feeds on organic compounds derived from the culture medium. This process is identified as photoassimilation. The ability of a higher plant to nourish itself by photoassimilation and thereby to survive in a heremetically sealed chamber of small dimensions is more than a biochemical curiosity. It allows the botanical investigator to design a culture system convenient for space-flight applications, which ensures isolation of each test plant from the gaseous environment of the spacecraft.

The vacuolar transport of aleurain-GFP and 2S albumin-GFP fusions is mediated by the same pre-vacuolar compartments in tobacco BY-2 and Arabidopsis suspension cultured cells.

PubMed

Miao, Yansong; Li, Kwun Yee; Li, Hong-Ye; Yao, Xiaoqiang; Jiang, Liwen

2008-12-01

Soluble proteins reach vacuoles because they contain vacuolar sorting determinants (VSDs) that are recognized by vacuolar sorting receptor (VSR) proteins. Pre-vacuolar compartments (PVCs), defined by VSRs and GFP-VSR reporters in tobacco BY-2 cells, are membrane-bound intermediate organelles that mediate protein traffic from the Golgi apparatus to the vacuole in plant cells. Multiple pathways have been demonstrated to be responsible for vacuolar transport of lytic enzymes and storage proteins to the lytic vacuole (LV) and the protein storage vacuole (PSV), respectively. However, the nature of PVCs for LV and PSV pathways remains unclear. Here, we used two fluorescent reporters, aleurain-GFP and 2S albumin-GFP, that represent traffic of lytic enzymes and storage proteins to LV and PSV, respectively, to study the PVC-mediated transport pathways via transient expression in suspension cultured cells. We demonstrated that the vacuolar transport of aleurain-GFP and 2S albumin-GFP was mediated by the same PVC populations in both tobacco BY-2 and Arabidopsis suspension cultured cells. These PVCs were defined by the seven GFP-AtVSR reporters. In wortmannin-treated cells, the vacuolated PVCs contained the mRFP-AtVSR reporter in their limiting membranes, whereas the soluble aleurain-GFP or 2S albumin-GFP remained in the lumen of the PVCs, indicating a possible in vivo relationship between receptor and cargo within PVCs.

Diuretics Prime Plant Immunity in Arabidopsis thaliana

PubMed Central

Noutoshi, Yoshiteru; Ikeda, Mika; Shirasu, Ken

2012-01-01

Plant activators are agrochemicals that activate the plant immune system, thereby enhancing disease resistance. Due to their prophylactic and durable effects on a wide spectrum of diseases, plant activators can provide synergistic crop protection when used in combination with traditional pest controls. Although plant activators have achieved great success in wet-rice farming practices in Asia, their use is still limited. To isolate novel plant activators applicable to other crops, we screened a chemical library using a method that can selectively identify immune-priming compounds. Here, we report the isolation and characterization of three diuretics, bumetanide, bendroflumethiazide and clopamide, as immune-priming compounds. These drugs upregulate the immunity-related cell death of Arabidopsis suspension-cultured cells induced with an avirulent strain of Pseudomonas syringae pv. tomato in a concentration-dependent manner. The application of these compounds to Arabidopsis plants confers disease resistance to not only the avirulent but also a virulent strain of the pathogen. Unlike salicylic acid, an endogenous phytohormone that governs disease resistance in response to biotrophic pathogens, the three diuretic compounds analyzed here do not induce PR1 or inhibit plant growth, showing potential as lead compounds in a practical application. PMID:23144763

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

Arabidopsis GRI is involved in the regulation of cell death induced by extracellular ROS

PubMed Central

Wrzaczek, Michael; Brosché, Mikael; Kollist, Hannes; Kangasjärvi, Jaakko

2009-01-01

Reactive oxygen species (ROS) have important functions in plant stress responses and development. In plants, ozone and pathogen infection induce an extracellular oxidative burst that is involved in the regulation of cell death. However, very little is known about how plants can perceive ROS and regulate the initiation and the containment of cell death. We have identified an Arabidopsis thaliana protein, GRIM REAPER (GRI), that is involved in the regulation of cell death induced by extracellular ROS. Plants with an insertion in GRI display an ozone-sensitive phenotype. GRI is an Arabidopsis ortholog of the tobacco flower-specific Stig1 gene. The GRI protein appears to be processed in leaves with a release of an N-terminal fragment of the protein. Infiltration of the N-terminal fragment of the GRI protein into leaves caused cell death in a superoxide- and salicylic acid-dependent manner. Analysis of the extracellular GRI protein yields information on how plants can initiate ROS-induced cell death during stress response and development. PMID:19279211

Golgi- and Trans-Golgi Network-Mediated Vesicle Trafficking Is Required for Wax Secretion from Epidermal Cells1[W][OPEN

PubMed Central

McFarlane, Heather E.; Watanabe, Yoichiro; Yang, Weili; Huang, Yan; Ohlrogge, John; Samuels, A. Lacey

2014-01-01

Lipid secretion from epidermal cells to the plant surface is essential to create the protective plant cuticle. Cuticular waxes are unusual secretory products, consisting of a variety of highly hydrophobic compounds including saturated very-long-chain alkanes, ketones, and alcohols. These compounds are synthesized in the endoplasmic reticulum (ER) but must be trafficked to the plasma membrane for export by ATP-binding cassette transporters. To test the hypothesis that wax components are trafficked via the endomembrane system and packaged in Golgi-derived secretory vesicles, Arabidopsis (Arabidopsis thaliana) stem wax secretion was assayed in a series of vesicle-trafficking mutants, including gnom like1-1 (gnl1-1), transport particle protein subunit120-4, and echidna (ech). Wax secretion was dependent upon GNL1 and ECH. Independent of secretion phenotypes, mutants with altered ER morphology also had decreased wax biosynthesis phenotypes, implying that the biosynthetic capacity of the ER is closely related to its structure. These results provide genetic evidence that wax export requires GNL1- and ECH-dependent endomembrane vesicle trafficking to deliver cargo to plasma membrane-localized ATP-binding cassette transporters. PMID:24468625

Pectin Biosynthesis Is Critical for Cell Wall Integrity and Immunity in Arabidopsis thaliana

PubMed Central

Bethke, Gerit; Thao, Amanda; Xiong, Guangyan; Hatsugai, Noriyuki; Katagiri, Fumiaki; Pauly, Markus

2016-01-01

Plant cell walls are important barriers against microbial pathogens. Cell walls of Arabidopsis thaliana leaves contain three major types of polysaccharides: cellulose, various hemicelluloses, and pectins. UDP-d-galacturonic acid, the key building block of pectins, is produced from the precursor UDP-d-glucuronic acid by the action of glucuronate 4-epimerases (GAEs). Pseudomonas syringae pv maculicola ES4326 (Pma ES4326) repressed expression of GAE1 and GAE6 in Arabidopsis, and immunity to Pma ES4326 was compromised in gae6 and gae1 gae6 mutant plants. These plants had brittle leaves and cell walls of leaves had less galacturonic acid. Resistance to specific Botrytis cinerea isolates was also compromised in gae1 gae6 double mutant plants. Although oligogalacturonide (OG)-induced immune signaling was unaltered in gae1 gae6 mutant plants, immune signaling induced by a commercial pectinase, macerozyme, was reduced. Macerozyme treatment or infection with B. cinerea released less soluble uronic acid, likely reflecting fewer OGs, from gae1 gae6 cell walls than from wild-type Col-0. Although both OGs and macerozyme-induced immunity to B. cinerea in Col-0, only OGs also induced immunity in gae1 gae6. Pectin is thus an important contributor to plant immunity, and this is due at least in part to the induction of immune responses by soluble pectin, likely OGs, that are released during plant-pathogen interactions. PMID:26813622

Xyloglucan Deficiency Disrupts Microtubule Stability and Cellulose Biosynthesis in Arabidopsis, Altering Cell Growth and Morphogenesis

DOE PAGES

Xiao, Chaowen; Zhang, Tian; Zheng, Yunzhen; ...

2015-11-02

Here, xyloglucan constitutes most of the hemicellulose in eudicot primary cell walls and functions in cell wall structure and mechanics. Although Arabidopsis ( Arabidopsis thaliana) xxt1 xxt2 mutants lacking detectable xyloglucan are viable, they display growth defects that are suggestive of alterations in wall integrity. To probe the mechanisms underlying these defects, we analyzed cellulose arrangement, microtubule patterning and dynamics, microtubule- and wall-integrity-related gene expression, and cellulose biosynthesis in xxt1 xxt2 plants. We found that cellulose is highly aligned in xxt1 xxt2 cell walls, that its three-dimensional distribution is altered, and that microtubule patterning and stability are aberrant in etiolatedmore » xxt1 xxt2 hypocotyls. We also found that the expression levels of microtubule-associated genes, such as MAP70-5 and CLASP, and receptor genes, such as HERK1 and WAK1, were changed in xxt1 xxt2 plants and that cellulose synthase motility is reduced in xxt1 xxt2 cells, corresponding with a reduction in cellulose content. Our results indicate that loss of xyloglucan affects both the stability of the microtubule cytoskeleton and the production and patterning of cellulose in primary cell walls. These findings establish, to our knowledge, new links between wall integrity, cytoskeletal dynamics, and wall synthesis in the regulation of plant morphogenesis.« less

Xyloglucan Deficiency Disrupts Microtubule Stability and Cellulose Biosynthesis in Arabidopsis, Altering Cell Growth and Morphogenesis.

PubMed

Xiao, Chaowen; Zhang, Tian; Zheng, Yunzhen; Cosgrove, Daniel J; Anderson, Charles T

2016-01-01

Xyloglucan constitutes most of the hemicellulose in eudicot primary cell walls and functions in cell wall structure and mechanics. Although Arabidopsis (Arabidopsis thaliana) xxt1 xxt2 mutants lacking detectable xyloglucan are viable, they display growth defects that are suggestive of alterations in wall integrity. To probe the mechanisms underlying these defects, we analyzed cellulose arrangement, microtubule patterning and dynamics, microtubule- and wall-integrity-related gene expression, and cellulose biosynthesis in xxt1 xxt2 plants. We found that cellulose is highly aligned in xxt1 xxt2 cell walls, that its three-dimensional distribution is altered, and that microtubule patterning and stability are aberrant in etiolated xxt1 xxt2 hypocotyls. We also found that the expression levels of microtubule-associated genes, such as MAP70-5 and CLASP, and receptor genes, such as HERK1 and WAK1, were changed in xxt1 xxt2 plants and that cellulose synthase motility is reduced in xxt1 xxt2 cells, corresponding with a reduction in cellulose content. Our results indicate that loss of xyloglucan affects both the stability of the microtubule cytoskeleton and the production and patterning of cellulose in primary cell walls. These findings establish, to our knowledge, new links between wall integrity, cytoskeletal dynamics, and wall synthesis in the regulation of plant morphogenesis. © 2016 American Society of Plant Biologists. All Rights Reserved.

Comparative Cytotoxicity Study of Silver Nanoparticles (AgNPs) in a Variety of Rainbow Trout Cell Lines (RTL-W1, RTH-149, RTG-2) and Primary Hepatocytes

PubMed Central

Connolly, Mona; Fernandez-Cruz, Maria-Luisa; Quesada-Garcia, Alba; Alte, Luis; Segner, Helmut; Navas, Jose M.

2015-01-01

Among all classes of nanomaterials, silver nanoparticles (AgNPs) have potentially an important ecotoxicological impact, especially in freshwater environments. Fish are particularly susceptible to the toxic effects of silver ions and, with knowledge gaps regarding the contribution of dissolution and unique particle effects to AgNP toxicity, they represent a group of vulnerable organisms. Using cell lines (RTL-W1, RTH-149, RTG-2) and primary hepatocytes of rainbow trout (Oncorhynchus mykiss) as in vitro test systems, we assessed the cytotoxicity of the representative AgNP, NM-300K, and AgNO3 as an Ag+ ion source. Lack of AgNP interference with the cytotoxicity assays (AlamarBlue, CFDA-AM, NRU assay) and their simultaneous application point to the compatibility and usefulness of such a battery of assays. The RTH-149 and RTL-W1 liver cell lines exhibited similar sensitivity as primary hepatocytes towards AgNP toxicity. Leibovitz’s L-15 culture medium composition (high amino acid content) had an important influence on the behaviour and toxicity of AgNPs towards the RTL-W1 cell line. The obtained results demonstrate that, with careful consideration, such an in vitro approach can provide valuable toxicological data to be used in an integrated testing strategy for NM-300K risk assessment. PMID:26006119

The Basic Helix-Loop-Helix Transcription Factor MYC2 Directly Represses PLETHORA Expression during Jasmonate-Mediated Modulation of the Root Stem Cell Niche in Arabidopsis[W][OA

PubMed Central

Chen, Qian; Sun, Jiaqiang; Zhai, Qingzhe; Zhou, Wenkun; Qi, Linlin; Xu, Li; Wang, Bao; Chen, Rong; Jiang, Hongling; Qi, Jing; Li, Xugang; Palme, Klaus; Li, Chuanyou

2011-01-01

The root stem cell niche, which in the Arabidopsis thaliana root meristem is an area of four mitotically inactive quiescent cells (QCs) and the surrounding mitotically active stem cells, is critical for root development and growth. We report here that during jasmonate-induced inhibition of primary root growth, jasmonate reduces root meristem activity and leads to irregular QC division and columella stem cell differentiation. Consistently, jasmonate reduces the expression levels of the AP2-domain transcription factors PLETHORA1 (PLT1) and PLT2, which form a developmentally instructive protein gradient and mediate auxin-induced regulation of stem cell niche maintenance. Not surprisingly, the effects of jasmonate on root stem cell niche maintenance and PLT expression require the functioning of MYC2/JASMONATE INSENSITIVE1, a basic helix-loop-helix transcription factor that involves versatile aspects of jasmonate-regulated gene expression. Gel shift and chromatin immunoprecipitation experiments reveal that MYC2 directly binds the promoters of PLT1 and PLT2 and represses their expression. We propose that MYC2-mediated repression of PLT expression integrates jasmonate action into the auxin pathway in regulating root meristem activity and stem cell niche maintenance. This study illustrates a molecular framework for jasmonate-induced inhibition of root growth through interaction with the growth regulator auxin. PMID:21954460