Chloroplast proteome response to drought stress and recovery in tomato (Solanum lycopersicum L.).

PubMed

Tamburino, Rachele; Vitale, Monica; Ruggiero, Alessandra; Sassi, Mauro; Sannino, Lorenza; Arena, Simona; Costa, Antonello; Batelli, Giorgia; Zambrano, Nicola; Scaloni, Andrea; Grillo, Stefania; Scotti, Nunzia

2017-02-10

Drought is a major constraint for plant growth and crop productivity that is receiving an increased attention due to global climate changes. Chloroplasts act as environmental sensors, however, only partial information is available on stress-induced mechanisms within plastids. Here, we investigated the chloroplast response to a severe drought treatment and a subsequent recovery cycle in tomato through physiological, metabolite and proteomic analyses. Under stress conditions, tomato plants showed stunted growth, and elevated levels of proline, abscisic acid (ABA) and late embryogenesis abundant gene transcript. Proteomics revealed that water deficit deeply affects chloroplast protein repertoire (31 differentially represented components), mainly involving energy-related functional species. Following the rewatering cycle, physiological parameters and metabolite levels indicated a recovery of tomato plant functions, while proteomics revealed a still ongoing adjustment of the chloroplast protein repertoire, which was even wider than during the drought phase (54 components differentially represented). Changes in gene expression of candidate genes and accumulation of ABA suggested the activation under stress of a specific chloroplast-to-nucleus (retrograde) signaling pathway and interconnection with the ABA-dependent network. Our results give an original overview on the role of chloroplast as enviromental sensor by both coordinating the expression of nuclear-encoded plastid-localised proteins and mediating plant stress response. Although our data suggest the activation of a specific retrograde signaling pathway and interconnection with ABA signaling network in tomato, the involvement and fine regulation of such pathway need to be further investigated through the development and characterization of ad hoc designed plant mutants.

Chloroplast and nuclear photorelocation movements

PubMed Central

WADA, Masamitsu

2016-01-01

Chloroplasts move toward weak light to increase photosynthetic efficiency, and migrate away from strong light to protect chloroplasts from photodamage and eventual cell death. These chloroplast behaviors were first observed more than 100 years ago, but the underlying mechanism has only recently been identified. Ideal plant materials, such as fern gametophytes for photobiological and cell biological approaches, and Arabidopsis thaliana for genetic analyses, have been used along with sophisticated methods, such as partial cell irradiation and time-lapse video recording under infrared light to study chloroplast movement. These studies have revealed precise chloroplast behavior, and identified photoreceptors, other relevant protein components, and novel actin filament structures required for chloroplast movement. In this review, our findings regarding chloroplast and nuclear movements are described. PMID:27840388

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

Two distinct redox cascades cooperatively regulate chloroplast functions and sustain plant viability.

PubMed

Yoshida, Keisuke; Hisabori, Toru

2016-07-05

The thiol-based redox regulation system is believed to adjust chloroplast functions in response to changes in light environments. A redox cascade via the ferredoxin-thioredoxin reductase (FTR)/thioredoxin (Trx) pathway has been traditionally considered to serve as a transmitter of light signals to target enzymes. However, emerging data indicate that chloroplasts have a complex redox network composed of diverse redox-mediator proteins and target enzymes. Despite extensive research addressing this system, two fundamental questions are still unresolved: How are redox pathways orchestrated within chloroplasts, and why are chloroplasts endowed with a complicated redox network? In this report, we show that NADPH-Trx reductase C (NTRC) is a key redox-mediator protein responsible for regulatory functions distinct from those of the classically known FTR/Trx system. Target screening and subsequent biochemical assays indicated that NTRC and the Trx family differentially recognize their target proteins. In addition, we found that NTRC is an electron donor to Trx-z, which is a key regulator of gene expression in chloroplasts. We further demonstrate that cooperative control of chloroplast functions via the FTR/Trx and NTRC pathways is essential for plant viability. Arabidopsis double mutants impaired in FTR and NTRC expression displayed lethal phenotypes under autotrophic growth conditions. This severe growth phenotype was related to a drastic loss of photosynthetic performance. These combined results provide an expanded map of the chloroplast redox network and its biological functions.

Two distinct redox cascades cooperatively regulate chloroplast functions and sustain plant viability

PubMed Central

Yoshida, Keisuke; Hisabori, Toru

2016-01-01

The thiol-based redox regulation system is believed to adjust chloroplast functions in response to changes in light environments. A redox cascade via the ferredoxin-thioredoxin reductase (FTR)/thioredoxin (Trx) pathway has been traditionally considered to serve as a transmitter of light signals to target enzymes. However, emerging data indicate that chloroplasts have a complex redox network composed of diverse redox-mediator proteins and target enzymes. Despite extensive research addressing this system, two fundamental questions are still unresolved: How are redox pathways orchestrated within chloroplasts, and why are chloroplasts endowed with a complicated redox network? In this report, we show that NADPH-Trx reductase C (NTRC) is a key redox-mediator protein responsible for regulatory functions distinct from those of the classically known FTR/Trx system. Target screening and subsequent biochemical assays indicated that NTRC and the Trx family differentially recognize their target proteins. In addition, we found that NTRC is an electron donor to Trx-z, which is a key regulator of gene expression in chloroplasts. We further demonstrate that cooperative control of chloroplast functions via the FTR/Trx and NTRC pathways is essential for plant viability. Arabidopsis double mutants impaired in FTR and NTRC expression displayed lethal phenotypes under autotrophic growth conditions. This severe growth phenotype was related to a drastic loss of photosynthetic performance. These combined results provide an expanded map of the chloroplast redox network and its biological functions. PMID:27335455

Analyses of charophyte chloroplast genomes help characterize the ancestral chloroplast genome of land plants.

PubMed

Civaň, Peter; Foster, Peter G; Embley, Martin T; Séneca, Ana; Cox, Cymon J

2014-04-01

Despite the significance of the relationships between embryophytes and their charophyte algal ancestors in deciphering the origin and evolutionary success of land plants, few chloroplast genomes of the charophyte algae have been reconstructed to date. Here, we present new data for three chloroplast genomes of the freshwater charophytes Klebsormidium flaccidum (Klebsormidiophyceae), Mesotaenium endlicherianum (Zygnematophyceae), and Roya anglica (Zygnematophyceae). The chloroplast genome of Klebsormidium has a quadripartite organization with exceptionally large inverted repeat (IR) regions and, uniquely among streptophytes, has lost the rrn5 and rrn4.5 genes from the ribosomal RNA (rRNA) gene cluster operon. The chloroplast genome of Roya differs from other zygnematophycean chloroplasts, including the newly sequenced Mesotaenium, by having a quadripartite structure that is typical of other streptophytes. On the basis of the improbability of the novel gain of IR regions, we infer that the quadripartite structure has likely been lost independently in at least three zygnematophycean lineages, although the absence of the usual rRNA operonic synteny in the IR regions of Roya may indicate their de novo origin. Significantly, all zygnematophycean chloroplast genomes have undergone substantial genomic rearrangement, which may be the result of ancient retroelement activity evidenced by the presence of integrase-like and reverse transcriptase-like elements in the Roya chloroplast genome. Our results corroborate the close phylogenetic relationship between Zygnematophyceae and land plants and identify 89 protein-coding genes and 22 introns present in the chloroplast genome at the time of the evolutionary transition of plants to land, all of which can be found in the chloroplast genomes of extant charophytes.

Analyses of Charophyte Chloroplast Genomes Help Characterize the Ancestral Chloroplast Genome of Land Plants

PubMed Central

Civáň, Peter; Foster, Peter G.; Embley, Martin T.; Séneca, Ana; Cox, Cymon J.

2014-01-01

Despite the significance of the relationships between embryophytes and their charophyte algal ancestors in deciphering the origin and evolutionary success of land plants, few chloroplast genomes of the charophyte algae have been reconstructed to date. Here, we present new data for three chloroplast genomes of the freshwater charophytes Klebsormidium flaccidum (Klebsormidiophyceae), Mesotaenium endlicherianum (Zygnematophyceae), and Roya anglica (Zygnematophyceae). The chloroplast genome of Klebsormidium has a quadripartite organization with exceptionally large inverted repeat (IR) regions and, uniquely among streptophytes, has lost the rrn5 and rrn4.5 genes from the ribosomal RNA (rRNA) gene cluster operon. The chloroplast genome of Roya differs from other zygnematophycean chloroplasts, including the newly sequenced Mesotaenium, by having a quadripartite structure that is typical of other streptophytes. On the basis of the improbability of the novel gain of IR regions, we infer that the quadripartite structure has likely been lost independently in at least three zygnematophycean lineages, although the absence of the usual rRNA operonic synteny in the IR regions of Roya may indicate their de novo origin. Significantly, all zygnematophycean chloroplast genomes have undergone substantial genomic rearrangement, which may be the result of ancient retroelement activity evidenced by the presence of integrase-like and reverse transcriptase-like elements in the Roya chloroplast genome. Our results corroborate the close phylogenetic relationship between Zygnematophyceae and land plants and identify 89 protein-coding genes and 22 introns present in the chloroplast genome at the time of the evolutionary transition of plants to land, all of which can be found in the chloroplast genomes of extant charophytes. PMID:24682153

Salinity inhibits post transcriptional processing of chloroplast 16S rRNA in shoot cultures of jojoba (Simmondsia chinesis).

PubMed

Mizrahi-Aviv, Ela; Mills, David; Benzioni, Aliza; Bar-Zvi, Dudy

2005-03-01

Chloroplast metabolism is rapidly affected by salt stress. Photosynthesis is one of the first processes known to be affected by salinity. Here, we report that salinity inhibits chloroplast post-transcriptional RNA processing. A differentially expressed 680-bp cDNA, containing the 3' sequence of 16S rRNA, transcribed intergenic spacer, exon 1 and intron of tRNA(Ile), was isolated by differential display reverse transcriptase PCR from salt-grown jojoba (Simmondsia chinesis) shoot cultures. Northern blot analysis indicated that although most rRNA appears to be fully processed, partially processed chloroplast 16S rRNA accumulates in salt-grown cultures. Thus, salinity appears to decrease the processing of the rrn transcript. The possible effect of this decreased processing on physiological processes is, as yet, unknown.

Salt-stress-responsive chloroplast proteins in Brassica juncea genotypes with contrasting salt tolerance and their quantitative PCR analysis.

PubMed

Yousuf, Peerzada Yasir; Ahmad, Altaf; Aref, Ibrahim M; Ozturk, Munir; Hemant; Ganie, Arshid Hussain; Iqbal, Muhammad

2016-11-01

Brassica juncea is mainly cultivated in the arid and semi-arid regions of India where its production is significantly affected by soil salinity. Adequate knowledge of the mechanisms underlying the salt tolerance at sub-cellular levels must aid in developing the salt-tolerant plants. A proper functioning of chloroplasts under salinity conditions is highly desirable to maintain crop productivity. The adaptive molecular mechanisms offered by plants at the chloroplast level to cope with salinity stress must be a prime target in developing the salt-tolerant plants. In the present study, we have analyzed differential expression of chloroplast proteins in two Brassica juncea genotypes, Pusa Agrani (salt-sensitive) and CS-54 (salt-tolerant), under the effect of sodium chloride. The chloroplast proteins were isolated and resolved using 2DE, which facilitated identification and quantification of 12 proteins that differed in expression in the salt-tolerant and salt-sensitive genotypes. The identified proteins were related to a variety of chloroplast-associated molecular processes, including oxygen-evolving process, PS I and PS II functioning, Calvin cycle and redox homeostasis. Expression analysis of genes encoding differentially expressed proteins through real time PCR supported our findings with proteomic analysis. The study indicates that modulating the expression of chloroplast proteins associated with stabilization of photosystems and oxidative defence plays imperative roles in adaptation to salt stress.

The chloroplast min system functions differentially in two specific nongreen plastids in Arabidopsis thaliana.

PubMed

Wang, Peng; Zhang, Jie; Su, Jianbin; Wang, Peng; Liu, Jun; Liu, Bing; Feng, Dongru; Wang, Jinfa; Wang, Hongbin

2013-01-01

The nongreen plastids, such as etioplasts, chromoplasts, etc., as well as chloroplasts, are all derived from proplastids in the meristem. To date, the Min system members in plants have been identified as regulators of FtsZ-ring placement, which are essential for the symmetrical division of chloroplasts. However, the regulation of FtsZ-ring placement in nongreen plastids is poorly understood. In this study, we investigated the division site placement of nongreen plastids by examining the etioplasts as representative in Arabidopsis Min system mutants. Surprisingly, the shape and number of etioplasts in cotyledons of arc3, arc11 and mcd1 mutants were similar to that observed in wild-type plants, whereas arc12 and parc6 mutants exhibited enlarged etioplasts that were reduced in number. In order to examine nongreen plastids in true leaves, we silenced the ALB3 gene in these Min system mutant backgrounds to produce immature chloroplasts without the thylakoidal network using virus induced gene silencing (VIGS). Interestingly, consistent with our observations in etioplasts, enlarged and fewer nongreen plastids were only detected in leaves of parc6 (VIGS-ALB3) and arc12 (VIGS-ALB3) plants. Further, the FtsZ-ring assembled properly at the midpoint in nongreen plastids of arc3, arc11 and mcd1 (VIGS-ALB3) plants, but organized into multiple rings in parc6 (VIGS-ALB3) and presented fragmented filaments in arc12 (VIGS-ALB3) plants, suggesting that division site placement in nongreen plastids requires fewer components of the plant Min system. Taken together, these results suggest that division site placement in nongreen plastids is different from that in chloroplasts.

The Chloroplast Min System Functions Differentially in Two Specific Nongreen Plastids in Arabidopsis thaliana

PubMed Central

Wang, Peng; Zhang, Jie; Su, Jianbin; Wang, Peng; Liu, Jun; Liu, Bing; Feng, Dongru; Wang, Jinfa; Wang, Hongbin

2013-01-01

The nongreen plastids, such as etioplasts, chromoplasts, etc., as well as chloroplasts, are all derived from proplastids in the meristem. To date, the Min system members in plants have been identified as regulators of FtsZ-ring placement, which are essential for the symmetrical division of chloroplasts. However, the regulation of FtsZ-ring placement in nongreen plastids is poorly understood. In this study, we investigated the division site placement of nongreen plastids by examining the etioplasts as representative in Arabidopsis Min system mutants. Surprisingly, the shape and number of etioplasts in cotyledons of arc3, arc11 and mcd1 mutants were similar to that observed in wild-type plants, whereas arc12 and parc6 mutants exhibited enlarged etioplasts that were reduced in number. In order to examine nongreen plastids in true leaves, we silenced the ALB3 gene in these Min system mutant backgrounds to produce immature chloroplasts without the thylakoidal network using virus induced gene silencing (VIGS). Interestingly, consistent with our observations in etioplasts, enlarged and fewer nongreen plastids were only detected in leaves of parc6 (VIGS-ALB3) and arc12 (VIGS-ALB3) plants. Further, the FtsZ-ring assembled properly at the midpoint in nongreen plastids of arc3, arc11 and mcd1 (VIGS-ALB3) plants, but organized into multiple rings in parc6 (VIGS-ALB3) and presented fragmented filaments in arc12 (VIGS-ALB3) plants, suggesting that division site placement in nongreen plastids requires fewer components of the plant Min system. Taken together, these results suggest that division site placement in nongreen plastids is different from that in chloroplasts. PMID:23936263

Transient foreign gene expression in chloroplasts of cultured tobacco cells after biolistic delivery of chloroplast vectors.

PubMed Central

Daniell, H; Vivekananda, J; Nielsen, B L; Ye, G N; Tewari, K K; Sanford, J C

1990-01-01

Expression of chloramphenicol acetyltransferase (cat) by suitable vectors in chloroplasts of cultured tobacco cells, delivered by high-velocity microprojectiles, is reported here. Several chloroplast expression vectors containing bacterial cat genes, placed under the control of either psbA promoter region from pea (pHD series) or rbcL promoter region from maize (pAC series) have been used in this study. In addition, chloroplast expression vectors containing replicon fragments from pea, tobacco, or maize chloroplast DNA have also been tested for efficiency and duration of cat expression in chloroplasts of tobacco cells. Cultured NT1 tobacco cells collected on filter papers were bombarded with tungsten particles coated with pUC118 (negative control), 35S-CAT (nuclear expression vector), pHD312 (repliconless chloroplast expression vector), and pHD407, pACp18, and pACp19 (chloroplast expression vectors with replicon). Sonic extracts of cells bombarded with pUC118 showed no detectable cat activity in the autoradiograms. Nuclear expression of cat reached two-thirds of the maximal 48 hr after bombardment and the maximal at 72 hr. Cells bombarded with chloroplast expression vectors showed a low level of expression until 48 hr of incubation. A dramatic increase in the expression of cat was observed 24 hr after the addition of fresh medium to cultured cells in samples bombarded with pHD407; the repliconless vector pHD312 showed about 50% of this maximal activity. The expression of nuclear cat and the repliconless chloroplast vector decreased after 72 hr, but a high level of chloroplast cat expression was maintained in cells bombarded with pHD407. Organelle-specific expression of cat in appropriate compartments was checked by introducing various plasmid constructions into tobacco protoplasts by electroporation. Although the nuclear expression vector 35S-CAT showed expression of cat, no activity was observed with any chloroplast vectors. Images PMID:2404285

Transient foreign gene expression in chloroplasts of cultured tobacco cells after biolistic delivery of chloroplast vectors.

PubMed

Daniell, H; Vivekananda, J; Nielsen, B L; Ye, G N; Tewari, K K; Sanford, J C

1990-01-01

Expression of chloramphenicol acetyltransferase (cat) by suitable vectors in chloroplasts of cultured tobacco cells, delivered by high-velocity microprojectiles, is reported here. Several chloroplast expression vectors containing bacterial cat genes, placed under the control of either psbA promoter region from pea (pHD series) or rbcL promoter region from maize (pAC series) have been used in this study. In addition, chloroplast expression vectors containing replicon fragments from pea, tobacco, or maize chloroplast DNA have also been tested for efficiency and duration of cat expression in chloroplasts of tobacco cells. Cultured NT1 tobacco cells collected on filter papers were bombarded with tungsten particles coated with pUC118 (negative control), 35S-CAT (nuclear expression vector), pHD312 (repliconless chloroplast expression vector), and pHD407, pACp18, and pACp19 (chloroplast expression vectors with replicon). Sonic extracts of cells bombarded with pUC118 showed no detectable cat activity in the autoradiograms. Nuclear expression of cat reached two-thirds of the maximal 48 hr after bombardment and the maximal at 72 hr. Cells bombarded with chloroplast expression vectors showed a low level of expression until 48 hr of incubation. A dramatic increase in the expression of cat was observed 24 hr after the addition of fresh medium to cultured cells in samples bombarded with pHD407; the repliconless vector pHD312 showed about 50% of this maximal activity. The expression of nuclear cat and the repliconless chloroplast vector decreased after 72 hr, but a high level of chloroplast cat expression was maintained in cells bombarded with pHD407. Organelle-specific expression of cat in appropriate compartments was checked by introducing various plasmid constructions into tobacco protoplasts by electroporation. Although the nuclear expression vector 35S-CAT showed expression of cat, no activity was observed with any chloroplast vectors.

Mitochondrion-to-Chloroplast DNA Transfers and Intragenomic Proliferation of Chloroplast Group II Introns in Gloeotilopsis Green Algae (Ulotrichales, Ulvophyceae)

PubMed Central

Turmel, Monique; Otis, Christian; Lemieux, Claude

2016-01-01

Abstract To probe organelle genome evolution in the Ulvales/Ulotrichales clade, the newly sequenced chloroplast and mitochondrial genomes of Gloeotilopsis planctonica and Gloeotilopsis sarcinoidea (Ulotrichales) were compared with those of Pseudendoclonium akinetum (Ulotrichales) and of the few other green algae previously sampled in the Ulvophyceae. At 105,236 bp, the G. planctonica mitochondrial DNA (mtDNA) is the largest mitochondrial genome reported so far among chlorophytes, whereas the 221,431-bp G. planctonica and 262,888-bp G. sarcinoidea chloroplast DNAs (cpDNAs) are the largest chloroplast genomes analyzed among the Ulvophyceae. Gains of non-coding sequences largely account for the expansion of these genomes. Both Gloeotilopsis cpDNAs lack the inverted repeat (IR) typically found in green plants, indicating that two independent IR losses occurred in the Ulvales/Ulotrichales. Our comparison of the Pseudendoclonium and Gloeotilopsis cpDNAs offered clues regarding the mechanism of IR loss in the Ulotrichales, suggesting that internal sequences from the rDNA operon were differentially lost from the two original IR copies during this process. Our analyses also unveiled a number of genetic novelties. Short mtDNA fragments were discovered in two distinct regions of the G. sarcinoidea cpDNA, providing the first evidence for intracellular inter-organelle gene migration in green algae. We identified for the first time in green algal organelles, group II introns with LAGLIDADG ORFs as well as group II introns inserted into untranslated gene regions. We discovered many group II introns occupying sites not previously documented for the chloroplast genome and demonstrated that a number of them arose by intragenomic proliferation, most likely through retrohoming. PMID:27503298

Mutational Dynamics of Aroid Chloroplast Genomes

PubMed Central

Ahmed, Ibrar; Biggs, Patrick J.; Matthews, Peter J.; Collins, Lesley J.; Hendy, Michael D.; Lockhart, Peter J.

2012-01-01

A characteristic feature of eukaryote and prokaryote genomes is the co-occurrence of nucleotide substitution and insertion/deletion (indel) mutations. Although similar observations have also been made for chloroplast DNA, genome-wide associations have not been reported. We determined the chloroplast genome sequences for two morphotypes of taro (Colocasia esculenta; family Araceae) and compared these with four publicly available aroid chloroplast genomes. Here, we report the extent of genome-wide association between direct and inverted repeats, indels, and substitutions in these aroid chloroplast genomes. We suggest that alternative but not mutually exclusive hypotheses explain the mutational dynamics of chloroplast genome evolution. PMID:23204304

Chloroplast precursor proteins compete to form early import intermediates in isolated pea chloroplasts.

PubMed

Row, P E; Gray, J C

2001-01-01

In order to ascertain whether there is one site for the import of precursor proteins into chloroplasts or whether different precursor proteins are imported via different import machineries, chloroplasts were incubated with large quantities of the precursor of the 33 kDa subunit of the oxygen-evolving complex (pOE33) or the precursor of the light-harvesting chlorophyll a/b-binding protein (pLHCP) and tested for their ability to import a wide range of other chloroplast precursor proteins. Both pOE33 and pLHCP competed for import into chloroplasts with precursors of the stromally-targeted small subunit of Rubisco (pSSu), ferredoxin NADP(+) reductase (pFNR) and porphobilinogen deaminase; the thylakoid membrane proteins LHCP and the Rieske iron-sulphur protein (pRieske protein); ferrochelatase and the gamma subunit of the ATP synthase (which are both associated with the thylakoid membrane); the thylakoid lumenal protein plastocyanin and the phosphate translocator, an integral membrane protein of the inner envelope. The concentrations of pOE33 or pLHCP required to cause half-maximal inhibition of import ranged between 0.2 and 4.9 microM. These results indicate that all of these proteins are imported into the chloroplast by a common import machinery. Incubation of chloroplasts with pOE33 inhibited the formation of early import intermediates of pSSu, pFNR and pRieske protein.

REDUCED CHLOROPLAST COVERAGE genes from Arabidopsis thaliana help to establish the size of the chloroplast compartment

DOE PAGES

Larkin, Robert M.; Stefano, Giovanni; Ruckle, Michael E.; ...

2016-02-09

Eukaryotic cells require mechanisms to establish the proportion of cellular volume devoted to particular organelles. These mechanisms are poorly understood. From a screen for plastid-to-nucleus signaling mutants in Arabidopsis thaliana, we cloned a mutant allele of a gene that encodes a protein of unknown function that is homologous to two other Arabidopsis genes of unknown function and Arabidopsis. In contrast to FRIENDLY, these three homologs of FRIENDLY are found only in photosynthetic organisms. Based on these data, we proposed that FRIENDLY expanded into a small gene family to help regulate the energy metabolism of cells that contain both mitochondria andmore » chloroplasts. Indeed, we found that knocking out these genes caused a number of chloroplast phenotypes, including a reduction in the proportion of cellular volume devoted to chloroplasts to 50% of wild type. Thus, we refer to these genes as REDUCED CHLOROPLAST COVERAGE (REC). The size of the chloroplast compartment was reduced most in rec1 mutants. The REC1 protein accumulated in the cytosol and the nucleus. REC1 was excluded from the nucleus when plants were treated with amitrole, which inhibits cell expansion and chloroplast function. Finally, we conclude that REC1 is an extraplastidic protein that helps to establish the size of the chloroplast compartment, and that signals derived from cell expansion or chloroplasts may regulate REC1.« less

Chloroplast-derived enzyme cocktails hydrolyse lignocellulosic biomass and release fermentable sugars

PubMed Central

Verma, Dheeraj; Kanagaraj, Anderson; Jin, Shuangxia; Singh, Nameirakpam D.; Kolattukudy, Pappachan E; Daniell, Henry

2009-01-01

Summary It is widely recognized that biofuel production from lignocellulosic materials is limited by inadequate technology to efficiently and economically release fermentable sugars from the complex multi-polymeric raw materials. Therefore, endoglucanases, exoglucanase, pectate lyases, cutinase, swollenin, xylanase, acetyl xylan esterase, beta glucosidase and lipase genes from bacteria or fungi were expressed in E. coli or tobacco chloroplasts. A PCR based method was used to clone genes without introns from Trichoderma reesei genomic DNA. Homoplasmic transplastomic lines showed normal phenotype and were fertile. Based on observed expression levels, up to 49, 64 and 10,751 million units of pectate lyases or endoglucanase can be produced annually, per acre of tobacco. Plant production cost of endoglucanase is 3,100-fold and pectate lyase is 1,057 or 1,480 fold lower than the same recombinant enzymes sold commercially, produced via fermentation. Chloroplast-derived enzymes had higher temperature stability and wider pH optima than enzymes expressed in E. coli. Plant crude-extracts showed higher enzyme activity than E. coli with increasing protein concentration, demonstrating their direct utility without purification. Addition of E. coli extracts to the chloroplast-derived enzymes significantly decreased their activity. Chloroplast-derived crude-extract enzyme cocktails yielded more (up to 3,625%) glucose from filter paper, pine wood or citrus peel than commercial cocktails. Furthermore, pectate lyase transplastomic plants showed enhanced resistance to Erwina soft rot. This is the first report of using plant-derived enzyme cocktails for production of fermentable sugars from lignocellulosic biomass. Limitations of higher cost and lower production capacity of fermentation systems are addressed by chloroplast-derived enzyme cocktails. PMID:20070870

Differential changes in photosynthetic capacity, 77 K chlorophyll fluorescence and chloroplast ultrastructure between Zn-efficient and Zn-inefficient rice genotypes (Oryza sativa) under low zinc stress.

PubMed

Chen, Wenrong; Yang, Xiaoe; He, Zhenli; Feng, Ying; Hu, Fenghong

2008-01-01

The relationship of zinc (Zn) efficiency in rice to differential tolerance of photosynthetic capacity and chloroplast function to low Zn stress was studied using Zn-efficient (IR8192) and Zn-inefficient (Erjiufeng) rice genotypes (Oryza sativa L.). Zinc deficiency caused extensive declines in leaf chlorophyll (Chl) content, ratios of chl a:b, Pn, Fv/Fm and Fv/Fo, indicating that the intrinsic quantum efficiency of the photosystem II (PSII) units was damaged. A greater decline was observed in the inefficient genotype (Erjiufeng) than the efficient genotype (IR8192). The 77 K chl fluorescence emission spectrum revealed that Zn deficiency blocked energy spillover from PSII to PSI and more excitation energy was distributed to PSII in IR8192 than Erjiufeng. The spectrum of Zn-deficient Erjiufeng was completely disordered, implying that the photosynthetic centers were seriously damaged. Electron microscopy showed that Zn deficiency caused a severe damage to the fine structure of chloroplasts, but IR8192 had a better preserved chloroplast ultrastructure as compared with Erjiufeng. These differences may result from the higher levels of the antioxidant enzyme activities and lower oxidant stress level in IR8192. These results indicate that Zn deficiency decreases leaf photosynthetic capacity primarily by reducing the number of PSII units per unit leaf area, and also reducing the photochemical capacity of the remaining PSII units. Therefore, the maintenance of more efficient photochemical capacity under low Zn stress is a key factor for the high Zn efficiency in rice, which may result from less antioxidant damage caused by low Zn to the chloroplast ultrastructure.

Genome Sequences of Populus tremula Chloroplast and Mitochondrion: Implications for Holistic Poplar Breeding

PubMed Central

Mader, Malte; Le Paslier, Marie-Christine; Bounon, Rémi; Berard, Aurélie; Vettori, Cristina; Schroeder, Hilke; Leplé, Jean-Charles; Fladung, Matthias

2016-01-01

Complete Populus genome sequences are available for the nucleus (P. trichocarpa; section Tacamahaca) and for chloroplasts (seven species), but not for mitochondria. Here, we provide the complete genome sequences of the chloroplast and the mitochondrion for the clones P. tremula W52 and P. tremula x P. alba 717-1B4 (section Populus). The organization of the chloroplast genomes of both Populus clones is described. A phylogenetic tree constructed from all available complete chloroplast DNA sequences of Populus was not congruent with the assignment of the related species to different Populus sections. In total, 3,024 variable nucleotide positions were identified among all compared Populus chloroplast DNA sequences. The 5-prime part of the LSC from trnH to atpA showed the highest frequency of variations. The variable positions included 163 positions with SNPs allowing for differentiating the two clones with P. tremula chloroplast genomes (W52, 717-1B4) from the other seven Populus individuals. These potential P. tremula-specific SNPs were displayed as a whole-plastome barcode on the P. tremula W52 chloroplast DNA sequence. Three of these SNPs and one InDel in the trnH-psbA linker were successfully validated by Sanger sequencing in an extended set of Populus individuals. The complete mitochondrial genome sequence of P. tremula is the first in the family of Salicaceae. The mitochondrial genomes of the two clones are 783,442 bp (W52) and 783,513 bp (717-1B4) in size, structurally very similar and organized as single circles. DNA sequence regions with high similarity to the W52 chloroplast sequence account for about 2% of the W52 mitochondrial genome. The mean SNP frequency was found to be nearly six fold higher in the chloroplast than in the mitochondrial genome when comparing 717-1B4 with W52. The availability of the genomic information of all three DNA-containing cell organelles will allow a holistic approach in poplar molecular breeding in the future. PMID:26800039

Mitochondrion-to-Chloroplast DNA Transfers and Intragenomic Proliferation of Chloroplast Group II Introns in Gloeotilopsis Green Algae (Ulotrichales, Ulvophyceae).

PubMed

Turmel, Monique; Otis, Christian; Lemieux, Claude

2016-09-19

To probe organelle genome evolution in the Ulvales/Ulotrichales clade, the newly sequenced chloroplast and mitochondrial genomes of Gloeotilopsis planctonica and Gloeotilopsis sarcinoidea (Ulotrichales) were compared with those of Pseudendoclonium akinetum (Ulotrichales) and of the few other green algae previously sampled in the Ulvophyceae. At 105,236 bp, the G planctonica mitochondrial DNA (mtDNA) is the largest mitochondrial genome reported so far among chlorophytes, whereas the 221,431-bp G planctonica and 262,888-bp G sarcinoidea chloroplast DNAs (cpDNAs) are the largest chloroplast genomes analyzed among the Ulvophyceae. Gains of non-coding sequences largely account for the expansion of these genomes. Both Gloeotilopsis cpDNAs lack the inverted repeat (IR) typically found in green plants, indicating that two independent IR losses occurred in the Ulvales/Ulotrichales. Our comparison of the Pseudendoclonium and Gloeotilopsis cpDNAs offered clues regarding the mechanism of IR loss in the Ulotrichales, suggesting that internal sequences from the rDNA operon were differentially lost from the two original IR copies during this process. Our analyses also unveiled a number of genetic novelties. Short mtDNA fragments were discovered in two distinct regions of the G sarcinoidea cpDNA, providing the first evidence for intracellular inter-organelle gene migration in green algae. We identified for the first time in green algal organelles, group II introns with LAGLIDADG ORFs as well as group II introns inserted into untranslated gene regions. We discovered many group II introns occupying sites not previously documented for the chloroplast genome and demonstrated that a number of them arose by intragenomic proliferation, most likely through retrohoming. © The Author 2016. Published by Oxford University Press on behalf of the Society for Molecular Biology and Evolution.

Phosphoinositides play differential roles in regulating phototropin1- and phototropin2-mediated chloroplast movements in Arabidopsis.

PubMed

Aggarwal, Chhavi; Labuz, Justyna; Gabryś, Halina

2013-01-01

Phototropins are UVA/blue-light receptors involved in controlling the light-dependent physiological responses which serve to optimize the photosynthetic activity of plants and promote growth. The phototropin-induced phosphoinositide (PI) metabolism has been shown to be essential for stomatal opening and phototropism. However, the role of PIs in phototropin-induced chloroplast movements remains poorly understood. The aim of this work is to determine which PI species are involved in the control of chloroplast movements in Arabidopsis and the nature of their involvement. We present the effects of the inactivation of phospholipase C (PLC), PI3-kinase (PI3K) and PI4-kinase (PI4K) on chloroplast relocations in Arabidopsis. The inhibition of the phosphatidylinositol 4,5-bisphospahte [PI(4,5)P2]-PLC pathway, using neomycin and U73122, suppressed the phot2-mediated chloroplast accumulation and avoidance responses, without affecting movement responses controlled by phot1. On the other hand, PI3K and PI4K activities are more restricted to phot1- and phot2-induced weak-light responses. The inactivation of PI3K and PI4K by wortmannin and LY294002 severely affected the weak blue-light-activated accumulation response but had little effect on the strong blue-light-activated avoidance response. The inhibitory effect observed with PI metabolism inhibitors is, at least partly, due to a disturbance in Ca(2+) ((c)) signaling. Using the transgenic aequorin system, we show that the application of these inhibitors suppresses the blue-light-induced transient Ca(2+) ((c)) rise. These results demonstrate the importance of PIs in chloroplast movements, with the PI(4,5)P2-PLC pathway involved in phot2 signaling while PI3K and PI4K are required for the phot1- and phot2-induced accumulation response. Our results suggest that these PIs modulate cytosolic Ca(2+) signaling during movements.

A family of selfish minicircular chromosomes with jumbled chloroplast gene fragments from a dinoflagellate.

PubMed

Zhang, Z; Cavalier-Smith, T; Green, B R

2001-08-01

Chloroplast genes of several dinoflagellate species are located on unigenic DNA minicircular chromosomes. We have now completely sequenced five aberrant minicircular chromosomes from the dinoflagellate Heterocapsa triquetra. These probably nonfunctional DNA circles lack complete genes, with each being composed of several short fragments of two or three different chloroplast genes and a common conserved region with a tripartite 9G-9A-9G core like the putative replicon origin of functional single-gene circular chloroplast chromosomes. Their sequences imply that all five circles evolved by differential deletions and duplications from common ancestral circles bearing fragments of four genes: psbA, psbC, 16S rRNA, and 23S rRNA. It appears that recombination between separate unigenic chromosomes initially gave intermediate heterodimers, which were subsequently stabilized by deletions that included part or all of one putative replicon origin. We suggest that homologous recombination at the 9G-9A-9G core regions produced a psbA/psbC heterodimer which generated two distinct chimeric circles by differential deletions and duplications. A 23S/16S rRNA heterodimer more likely formed by illegitimate recombination between 16S and 23S rRNA genes. Homologous recombination between the 9G-9A-9G core regions of both heterodimers and additional differential deletions and duplications could then have yielded the other three circles. Near identity of the gene fragments and 9G-9A-9G cores, despite diverging adjacent regions, may be maintained by gene conversion. The conserved organization of the 9G-9A-9G cores alone favors the idea that they are replicon origins and suggests that they may enable the aberrant minicircles to parasitize the chloroplast's replication machinery as selfish circles.

Chloroplast Phosphofructokinase

PubMed Central

Kelly, Grahame J.; Latzko, Erwin

1977-01-01

Chloroplast phosphofructokinase from spinach (Spinacia oleracea L.) was purified approximately 40-fold by a combination of fractionations with ammonium sulfate and acetone followed by chromatography on DEAE-Sephadex A-50. Positive cooperative kinetics was observed for the interaction between the enzyme and the substrate fructose 6-phosphate. The optimum pH shifted from 7.7 toward 7.0 as the fructose 6-phosphate concentration was taken below 0.5 mm. The second substrate was MgATP2− (Michaelis constant 30 μm). Free ATP inhibited the enzyme. Chloroplast phosphofructokinase was sensitive to inhibition by low concentration of phosphoenolpyruvate and glycolate 2-phosphate (especially at higher pH); these compounds inhibited in a positively cooperative fashion. Inhibitions by glycerate 2-phosphate (and probably glycerate 3-phosphate), citrate, and inorganic phosphate were also recorded; however, inorganic phosphate effectively relieved the inhibitions by phosphoenolpyruvate and glycolate 2-phosphate. These regulatory properties are considered to complement those of ADP-glucose pyrophosphorylase and fructosebisphosphatase in the regulation of chloroplast starch metabolism. PMID:16660079

The wheat chloroplastic proteome.

PubMed

Kamal, Abu Hena Mostafa; Cho, Kun; Choi, Jong-Soon; Bae, Kwang-Hee; Komatsu, Setsuko; Uozumi, Nobuyuki; Woo, Sun Hee

2013-11-20

With the availability of plant genome sequencing, analysis of plant proteins with mass spectrometry has become promising and admired. Determining the proteome of a cell is still a challenging assignment, which is convoluted by proteome dynamics and convolution. Chloroplast is fastidious curiosity for plant biologists due to their intricate biochemical pathways for indispensable metabolite functions. In this review, an overview on proteomic studies conducted in wheat with a special focus on subcellular proteomics of chloroplast, salt and water stress. In recent years, we and other groups have attempted to understand the photosynthesis in wheat and abiotic stress under salt imposed and water deficit during vegetative stage. Those studies provide interesting results leading to better understanding of the photosynthesis and identifying the stress-responsive proteins. Indeed, recent studies aimed at resolving the photosynthesis pathway in wheat. Proteomic analysis combining two complementary approaches such as 2-DE and shotgun methods couple to high through put mass spectrometry (LTQ-FTICR and MALDI-TOF/TOF) in order to better understand the responsible proteins in photosynthesis and abiotic stress (salt and water) in wheat chloroplast will be focused. In this review we discussed the identification of the most abundant protein in wheat chloroplast and stress-responsive under salt and water stress in chloroplast of wheat seedlings, thus providing the proteomic view of the events during the development of this seedling under stress conditions. Chloroplast is fastidious curiosity for plant biologists due to their intricate biochemical pathways for indispensable metabolite functions. An overview on proteomic studies conducted in wheat with a special focus on subcellular proteomics of chloroplast, salt and water stress. We have attempted to understand the photosynthesis in wheat and abiotic stress under salt imposed and water deficit during seedling stage. Those studies

The Chloroplast Genome of Symplocarpus renifolius: A Comparison of Chloroplast Genome Structure in Araceae.

PubMed

Choi, Kyoung Su; Park, Kyu Tae; Park, SeonJoo

2017-11-16

Symplocarpus renifolius is a member of Araceae family that is extraordinarily diverse in appearance. Previous studies on chloroplast genomes in Araceae were focused on duckweeds (Lemnoideae) and root crops ( Colocasia , commonly known as taro). Here, we determined the chloroplast genome of Symplocarpus renifolius and compared the factors, such as genes and inverted repeat (IR) junctions and performed phylogenetic analysis using other Araceae species. The chloroplast genome of S. renifolius is 158,521 bp and includes 113 genes. A comparison among the Araceae chloroplast genomes showed that infA in Lemna , Spirodela , Wolffiella , Wolffia , Dieffenbachia and Colocasia has been lost or has become a pseudogene and has only been retained in Symplocarpus . In the Araceae chloroplast DNA (cpDNA), psbZ is retained. However, psbZ duplication occurred in Wolffia species and tandem repeats were noted around the duplication regions. A comparison of the IR junction in Araceae species revealed the presence of ycf1 and rps15 in the small single copy region, whereas duckweed species contained ycf1 and rps15 in the IR region. The phylogenetic analyses of the chloroplast genomes revealed that Symplocarpus are a basal group and are sister to the other Araceae species. Consequently, infA deletion or pseudogene events in Araceae occurred after the divergence of Symplocarpus and aquatic plants (duckweeds) in Araceae and duplication events of rps15 and ycf1 occurred in the IR region.

The Chloroplast Genome of Symplocarpus renifolius: A Comparison of Chloroplast Genome Structure in Araceae

PubMed Central

Park, Kyu Tae

2017-01-01

Symplocarpus renifolius is a member of Araceae family that is extraordinarily diverse in appearance. Previous studies on chloroplast genomes in Araceae were focused on duckweeds (Lemnoideae) and root crops (Colocasia, commonly known as taro). Here, we determined the chloroplast genome of Symplocarpus renifolius and compared the factors, such as genes and inverted repeat (IR) junctions and performed phylogenetic analysis using other Araceae species. The chloroplast genome of S. renifolius is 158,521 bp and includes 113 genes. A comparison among the Araceae chloroplast genomes showed that infA in Lemna, Spirodela, Wolffiella, Wolffia, Dieffenbachia and Colocasia has been lost or has become a pseudogene and has only been retained in Symplocarpus. In the Araceae chloroplast DNA (cpDNA), psbZ is retained. However, psbZ duplication occurred in Wolffia species and tandem repeats were noted around the duplication regions. A comparison of the IR junction in Araceae species revealed the presence of ycf1 and rps15 in the small single copy region, whereas duckweed species contained ycf1 and rps15 in the IR region. The phylogenetic analyses of the chloroplast genomes revealed that Symplocarpus are a basal group and are sister to the other Araceae species. Consequently, infA deletion or pseudogene events in Araceae occurred after the divergence of Symplocarpus and aquatic plants (duckweeds) in Araceae and duplication events of rps15 and ycf1 occurred in the IR region. PMID:29144427

Tobacco mosaic virus RNA enters chloroplasts in vivo

PubMed Central

Schoelz, James E.; Zaitlin, Milton

1989-01-01

Several lines of evidence are presented to allow us to conclude that tobacco mosaic virus (TMV) RNA enters the chloroplast in vivo. Chloroplasts were prepared from either directly inoculated or systemically infected leaves of tobacco plants inoculated with one of several strains of the virus and from uninfected control plants. Intact chloroplasts were isolated on Percoll gradients and treated with pancreatic RNase and thermolysin to destroy potential TMV virions and RNA on the outside or bound to their surfaces. Northern blot analysis of RNA extracted from these chloroplasts demonstrated that full-length TMV RNA was present within the chloroplasts prepared from both directly inoculated and systemically invaded leaves. Only genomic length, but not subgenomic length, RNA was found in the chloroplast extracts, indicating a selectivity of the transport of the viral RNA into the chloroplast. A temperature-sensitive TMV mutant (Ts 38), in which no virions are formed at 35°C, was used to demonstrate that at that restrictive temperature viral RNA is detected in the chloroplast, indicating that free viral RNA can enter the chloroplast rather than intact virions. To our knowledge, the transport of a foreign RNA species into chloroplasts has not been reported previously. Images PMID:16578844

Plastid Transcriptomics and Translatomics of Tomato Fruit Development and Chloroplast-to-Chromoplast Differentiation: Chromoplast Gene Expression Largely Serves the Production of a Single Protein[W][OA

PubMed Central

Kahlau, Sabine; Bock, Ralph

2008-01-01

Plastid genes are expressed at high levels in photosynthetically active chloroplasts but are generally believed to be drastically downregulated in nongreen plastids. The genome-wide changes in the expression patterns of plastid genes during the development of nongreen plastid types as well as the contributions of transcriptional versus translational regulation are largely unknown. We report here a systematic transcriptomics and translatomics analysis of the tomato (Solanum lycopersicum) plastid genome during fruit development and chloroplast-to-chromoplast conversion. At the level of RNA accumulation, most but not all plastid genes are strongly downregulated in fruits compared with leaves. By contrast, chloroplast-to-chromoplast differentiation during fruit ripening is surprisingly not accompanied by large changes in plastid RNA accumulation. However, most plastid genes are translationally downregulated during chromoplast development. Both transcriptional and translational downregulation are more pronounced for photosynthesis-related genes than for genes involved in gene expression, indicating that some low-level plastid gene expression must be sustained in chromoplasts. High-level expression during chromoplast development identifies accD, the only plastid-encoded gene involved in fatty acid biosynthesis, as the target gene for which gene expression activity in chromoplasts is maintained. In addition, we have determined the developmental patterns of plastid RNA polymerase activities, intron splicing, and RNA editing and report specific developmental changes in the splicing and editing patterns of plastid transcripts. PMID:18441214

The Chloroplast Genome of Pellia endiviifolia: Gene Content, RNA-Editing Pattern, and the Origin of Chloroplast Editing

PubMed Central

Grosche, Christopher; Funk, Helena T.; Maier, Uwe G.; Zauner, Stefan

2012-01-01

RNA editing is a post-transcriptional process that can act upon transcripts from mitochondrial, nuclear, and chloroplast genomes. In chloroplasts, single-nucleotide conversions in mRNAs via RNA editing occur at different frequencies across the plant kingdom. These range from several hundred edited sites in some mosses and ferns to lower frequencies in seed plants and the complete lack of RNA editing in the liverwort Marchantia polymorpha. Here, we report the sequence and edited sites of the chloroplast genome from the liverwort Pellia endiviifolia. The type and frequency of chloroplast RNA editing display a pattern highly similar to that in seed plants. Analyses of the C to U conversions and the genomic context in which the editing sites are embedded provide evidence in favor of the hypothesis that chloroplast RNA editing evolved to compensate mutations in the first land plants. PMID:23221608

Chloroplast Galactolipids: The Link Between Photosynthesis, Chloroplast Shape, Jasmonates, Phosphate Starvation and Freezing Tolerance.

PubMed

Li, Hsou-Min; Yu, Chun-Wei

2018-06-01

Monogalactosyldiacylglycerol (MGDG) and digalactosyldiacylglycerol (DGDG) together constitute approximately 80% of chloroplast lipids. Apart from facilitating the photosynthesis light reaction in the thylakoid membrane, these two lipids are important for maintaining chloroplast morphology and for plant survival under abiotic stresses such as phosphate starvation and freezing. Recently it was shown that severe growth retardation phenotypes of the DGDG-deficient mutant dgd1 were due to jasmonate overproduction, linking MGDG and DGDG homeostasis with phytohormone production and suggesting MGDG as a major substrate for jasmonate biosynthesis. Induction of jasmonate synthesis and jasmonic acid (JA) signaling was also observed under conditions of phosphate starvation. We hypothesize that when DGDG is recruited to substitute for phospholipids in extraplastidic membranes during phosphate deficiency, the altered MGDG to DGDG ratio in the chloroplast envelope triggers the conversion of galactolipids into jasmonates. The conversion may contribute to rebalancing the MGDG to DGDG ratio rapidly to maintain chloroplast shape, and jasmonate production can reduce the growth rate and enhance predator deterrence. We also hypothesize that other conditions, such as suppression of dgd1 phenotypes by trigalactosyldiacylglycerol (tgd) mutations, may all be linked to altered jasmonate production, indicating that caution should be exercised when interpreting phenotypes caused by conditions that may alter the MGDG to DGDG ratio at the chloroplast envelope.

Redox-shuttling between chloroplast and cytosol: integration of intra-chloroplast and extra-chloroplast metabolism.

PubMed

Taniguchi, Mitsutaka; Miyake, Hiroshi

2012-06-01

Reducing equivalents produced in the chloroplast are essential for many key cellular metabolic enzyme reactions. Two redox shuttle systems transfer reductant out of the chloroplast; these systems consist of metabolite transporters, coupled with stromal and cytosolic dehydrogenase isozymes. The transporters function in the redox shuttle and also operate as key enzymes in carbon/nitrogen metabolism. To maintain adequate levels of reductant and proper metabolic balance, the shuttle systems are finely controlled. Also, in the leaves of C(4) plants, cell-specific division of carbon and nitrogen assimilation includes cell-specific localization of the redox shuttle systems. The redox shuttle systems are tightly linked to cellular metabolic pathways and are essential for maintaining metabolic balance between energy and reducing equivalents. Copyright © 2012 Elsevier Ltd. All rights reserved.

Flow Cytometry of Spinach Chloroplasts 1

PubMed Central

Schröder, Wolfgang P.; Petit, Patrice X.

1992-01-01

Intact spinach (Spinacia oleracea) chloroplasts, thylakoid membranes, and inside-out or right-side-out thylakoid vesicles have been characterized by flow cytometry with respect to forward angle light scatter, right angle light scatter, and chlorophyll fluorescence. Analysis of intact chloroplasts with respect to forward light scatter and the chlorophyll fluorescence parameter revealed the presence of truly “intact” and “disrupted” chloroplasts. The forward light scatter parameter, normally considered to reflect object size, was instead found to reflect the particle density. One essential advantage of flow cytometry is that additional parameters such as Ricinus communis agglutinin (linked to fluorescein isothiocyanate) fluorescence can be determined through logical conditions placed on bit-maps, amounting to an analytical purification procedure. In the present case, chloroplast subpopulations with fully preserved envelopes, thylakoid membrane, and inside-out or right-side-out thylakoid membranes vesicles can be distinguished. Flow cytometry is also a useful tool to address the question of availability of glycosyl moities on the membrane surfaces if one keeps in mind that organelle-to-organelle interactions could be partially mediated through a recognition process. A high specific binding of R. communis agglutinin and peanut lectin to the chloroplast envelope was detected. This showed that galactose residues were exposed and accessible to specific lectins on the chloroplast surface. No exposed glucose, fucose, or mannose residues could be detected by the appropriate lectins. Ricin binding to the intact chloroplasts caused a strong aggregation. Disruption of these aggregates by resuspension or during passage in the flow cytometer induced partial breakage of the chloroplasts. Only minor binding of R. communis agglutinin and peanut lectin to the purified thylakoid membranes was detected; the binding was found to be low for both inside-out and right

Genome-wide investigation reveals high evolutionary rates in annual model plants.

PubMed

Yue, Jia-Xing; Li, Jinpeng; Wang, Dan; Araki, Hitoshi; Tian, Dacheng; Yang, Sihai

2010-11-09

Rates of molecular evolution vary widely among species. While significant deviations from molecular clock have been found in many taxa, effects of life histories on molecular evolution are not fully understood. In plants, annual/perennial life history traits have long been suspected to influence the evolutionary rates at the molecular level. To date, however, the number of genes investigated on this subject is limited and the conclusions are mixed. To evaluate the possible heterogeneity in evolutionary rates between annual and perennial plants at the genomic level, we investigated 85 nuclear housekeeping genes, 10 non-housekeeping families, and 34 chloroplast genes using the genomic data from model plants including Arabidopsis thaliana and Medicago truncatula for annuals and grape (Vitis vinifera) and popular (Populus trichocarpa) for perennials. According to the cross-comparisons among the four species, 74-82% of the nuclear genes and 71-97% of the chloroplast genes suggested higher rates of molecular evolution in the two annuals than those in the two perennials. The significant heterogeneity in evolutionary rate between annuals and perennials was consistently found both in nonsynonymous sites and synonymous sites. While a linear correlation of evolutionary rates in orthologous genes between species was observed in nonsynonymous sites, the correlation was weak or invisible in synonymous sites. This tendency was clearer in nuclear genes than in chloroplast genes, in which the overall evolutionary rate was small. The slope of the regression line was consistently lower than unity, further confirming the higher evolutionary rate in annuals at the genomic level. The higher evolutionary rate in annuals than in perennials appears to be a universal phenomenon both in nuclear and chloroplast genomes in the four dicot model plants we investigated. Therefore, such heterogeneity in evolutionary rate should result from factors that have genome-wide influence, most likely those

Rangewide Genetic Variation in Coast Redwood Populations at a Chloroplast Microsatellite Locus

Treesearch

Chris Brinegar

2012-01-01

Old growth and second growth populations of coast redwood (Sequoia sempervirens) were sampled at 10 locations throughout its range and analyzed at a highly variable chloroplast microsatellite locus. Very low FST values indicated that there was no significant genetic differentiation between adjacent old growth and second growth populations at each location. Genetic...

Multiplexed fragaria chloroplast genome sequencing

Treesearch

W. Njuguna; A. Liston; R. Cronn; N.V. Bassil

2010-01-01

A method to sequence multiple chloroplast genomes using ultra high throughput sequencing technologies was recently described. Complete chloroplast genome sequences can resolve phylogenetic relationships at low taxonomic levels and identify informative point mutations and indels. The objective of this research was to sequence multiple Fragaria...

Chloroplast evolution, structure and functions

PubMed Central

Jensen, Poul Erik

2014-01-01

In this review, we consider a selection of recent advances in chloroplast biology. These include new findings concerning chloroplast evolution, such as the identification of Chlamydiae as a third partner in primary endosymbiosis, a second instance of primary endosymbiosis represented by the chromatophores found in amoebae of the genus Paulinella, and a new explanation for the longevity of captured chloroplasts (kleptoplasts) in sacoglossan sea slugs. The controversy surrounding the three-dimensional structure of grana, its recent resolution by tomographic analyses, and the role of the CURVATURE THYLAKOID1 (CURT1) proteins in supporting grana formation are also discussed. We also present an updated inventory of photosynthetic proteins and the factors involved in the assembly of thylakoid multiprotein complexes, and evaluate findings that reveal that cyclic electron flow involves NADPH dehydrogenase (NDH)- and PGRL1/PGR5-dependent pathways, both of which receive electrons from ferredoxin. Other topics covered in this review include new protein components of nucleoids, an updated inventory of the chloroplast proteome, new enzymes in chlorophyll biosynthesis and new candidate messengers in retrograde signaling. Finally, we discuss the first successful synthetic biology approaches that resulted in chloroplasts in which electrons from the photosynthetic light reactions are fed to enzymes derived from secondary metabolism. PMID:24991417

Phylogenomic Analysis and Dynamic Evolution of Chloroplast Genomes in Salicaceae

PubMed Central

Huang, Yuan; Wang, Jun; Yang, Yongping; Fan, Chuanzhu; Chen, Jiahui

2017-01-01

Chloroplast genomes of plants are highly conserved in both gene order and gene content. Analysis of the whole chloroplast genome is known to provide much more informative DNA sites and thus generates high resolution for plant phylogenies. Here, we report the complete chloroplast genomes of three Salix species in family Salicaceae. Phylogeny of Salicaceae inferred from complete chloroplast genomes is generally consistent with previous studies but resolved with higher statistical support. Incongruences of phylogeny, however, are observed in genus Populus, which most likely results from homoplasy. By comparing three Salix chloroplast genomes with the published chloroplast genomes of other Salicaceae species, we demonstrate that the synteny and length of chloroplast genomes in Salicaceae are highly conserved but experienced dynamic evolution among species. We identify seven positively selected chloroplast genes in Salicaceae, which might be related to the adaptive evolution of Salicaceae species. Comparative chloroplast genome analysis within the family also indicates that some chloroplast genes are lost or became pseudogenes, infer that the chloroplast genes horizontally transferred to the nucleus genome. Based on the complete nucleus genome sequences from two Salicaceae species, we remarkably identify that the entire chloroplast genome is indeed transferred and integrated to the nucleus genome in the individual of the reference genome of P. trichocarpa at least once. This observation, along with presence of the large nuclear plastid DNA (NUPTs) and NUPTs-containing multiple chloroplast genes in their original order in the chloroplast genome, favors the DNA-mediated hypothesis of organelle to nucleus DNA transfer. Overall, the phylogenomic analysis using chloroplast complete genomes clearly elucidates the phylogeny of Salicaceae. The identification of positively selected chloroplast genes and dynamic chloroplast-to-nucleus gene transfers in Salicaceae provide

Transcriptome and proteomic analyses reveal multiple differences associated with chloroplast development in the spaceflight-induced wheat albino mutant mta.

PubMed

Shi, Kui; Gu, Jiayu; Guo, Huijun; Zhao, Linshu; Xie, Yongdun; Xiong, Hongchun; Li, Junhui; Zhao, Shirong; Song, Xiyun; Liu, Luxiang

2017-01-01

Chloroplast development is an integral part of plant survival and growth, and occurs in parallel with chlorophyll biosynthesis. However, little is known about the mechanisms underlying chloroplast development in hexaploid wheat. Here, we obtained a spaceflight-induced wheat albino mutant mta. Chloroplast ultra-structural observation showed that chloroplasts of mta exhibit abnormal morphology and distribution compared to wild type. Photosynthetic pigments content was also significantly decreased in mta. Transcriptome and chloroplast proteome profiling of mta and wild type were done to identify differentially expressed genes (DEGs) and proteins (DEPs), respectively. In total 4,588 DEGs including 1,980 up- and 2,608 down-regulated, and 48 chloroplast DEPs including 15 up- and 33 down-regulated were identified in mta. Classification of DEGs revealed that most were involved in chloroplast development, chlorophyll biosynthesis, or photosynthesis. Besides, transcription factors such as PIF3, GLK and MYB which might participate in those pathways were also identified. The correlation analysis between DEGs and DEPs revealed that the transcript-to-protein in abundance was functioned into photosynthesis and chloroplast relevant groups. Real time qPCR analysis validated that the expression level of genes encoding photosynthetic proteins was significantly decreased in mta. Together, our results suggest that the molecular mechanism for albino leaf color formation in mta is a thoroughly regulated and complicated process. The combined analysis of transcriptome and proteome afford comprehensive information for further research on chloroplast development mechanism in wheat. And spaceflight provides a potential means for mutagenesis in crop breeding.

Chloroplast genomes: diversity, evolution, and applications in genetic engineering

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

Daniell, Henry; Lin, Choun -Sea; Yu, Ming

Chloroplasts play a crucial role in sustaining life on earth. The availability of over 800 sequenced chloroplast genomes from a variety of land plants has enhanced our understanding of chloroplast biology, intracellular gene transfer, conservation, diversity, and the genetic basis by which chloroplast transgenes can be engineered to enhance plant agronomic traits or to produce high-value agricultural or biomedical products. In this review, we discuss the impact of chloroplast genome sequences on understanding the origins of economically important cultivated species and changes that have taken place during domestication. Here, we also discuss the potential biotechnological applications of chloroplast genomes.

Chloroplast genomes: diversity, evolution, and applications in genetic engineering

DOE PAGES

Daniell, Henry; Lin, Choun -Sea; Yu, Ming; ...

2016-06-23

Chloroplasts play a crucial role in sustaining life on earth. The availability of over 800 sequenced chloroplast genomes from a variety of land plants has enhanced our understanding of chloroplast biology, intracellular gene transfer, conservation, diversity, and the genetic basis by which chloroplast transgenes can be engineered to enhance plant agronomic traits or to produce high-value agricultural or biomedical products. In this review, we discuss the impact of chloroplast genome sequences on understanding the origins of economically important cultivated species and changes that have taken place during domestication. Here, we also discuss the potential biotechnological applications of chloroplast genomes.

Complete chloroplast genome sequences of Solanum commersonii and its application to chloroplast genotype in somatic hybrids with Solanum tuberosum.

PubMed

Cho, Kwang-Soo; Cheon, Kyeong-Sik; Hong, Su-Young; Cho, Ji-Hong; Im, Ju-Seong; Mekapogu, Manjulatha; Yu, Yei-Soo; Park, Tae-Ho

2016-10-01

Chloroplast genome of Solanum commersonii and S olanum tuberosum were completely sequenced, and Indel markers were successfully applied to distinguish chlorotypes demonstrating the chloroplast genome was randomly distributed during protoplast fusion. Somatic hybridization has been widely employed for the introgression of resistance to several diseases from wild Solanum species to overcome sexual barriers in potato breeding. Solanum commersonii is a major resource used as a parent line in somatic hybridization to improve bacterial wilt resistance in interspecies transfer to cultivated potato (S. tuberosum). Here, we sequenced the complete chloroplast genomes of Lz3.2 (S. commersonii) and S. tuberosum (PT56), which were used to develop fusion products, then compared them with those of five members of the Solanaceae family, S. tuberosum, Capsicum annum, S. lycopersicum, S. bulbocastanum and S. nigrum and Coffea arabica as an out-group. We then developed Indel markers for application in chloroplast genotyping. The complete chloroplast genome of Lz3.2 is composed of 155,525 bp, which is larger than the PT56 genome with 155,296 bp. Gene content, order and orientation of the S. commersonii chloroplast genome were highly conserved with those of other Solanaceae species, and the phylogenetic tree revealed that S. commersonii is located within the same node of S. tuberosum. However, sequence alignment revealed nine Indels between S. commersonii and S. tuberosum in their chloroplast genomes, allowing two Indel markers to be developed. The markers could distinguish the two species and were successfully applied to chloroplast genotyping (chlorotype) in somatic hybrids and their progenies. The results obtained in this study confirmed the random distribution of the chloroplast genome during protoplast fusion and its maternal inheritance and can be applied to select proper plastid genotypes in potato breeding program.

Seamless editing of the chloroplast genome in plants.

PubMed

Martin Avila, Elena; Gisby, Martin F; Day, Anil

2016-07-29

Gene editing technologies enable the precise insertion of favourable mutations and performance enhancing trait genes into chromosomes whilst excluding all excess DNA from modified genomes. The technology gives rise to a new class of biotech crops which is likely to have widespread applications in agriculture. Despite progress in the nucleus, the seamless insertions of point mutations and non-selectable foreign genes into the organelle genomes of crops have not been described. The chloroplast genome is an attractive target to improve photosynthesis and crop performance. Current chloroplast genome engineering technologies for introducing point mutations into native chloroplast genes leave DNA scars, such as the target sites for recombination enzymes. Seamless editing methods to modify chloroplast genes need to address reversal of site-directed point mutations by template mediated repair with the vast excess of wild type chloroplast genomes that are present early in the transformation process. Using tobacco, we developed an efficient two-step method to edit a chloroplast gene by replacing the wild type sequence with a transient intermediate. This was resolved to the final edited gene by recombination between imperfect direct repeats. Six out of 11 transplastomic plants isolated contained the desired intermediate and at the second step this was resolved to the edited chloroplast gene in five of six plants tested. Maintenance of a single base deletion mutation in an imperfect direct repeat of the native chloroplast rbcL gene showed the limited influence of biased repair back to the wild type sequence. The deletion caused a frameshift, which replaced the five C-terminal amino acids of the Rubisco large subunit with 16 alternative residues resulting in a ~30-fold reduction in its accumulation. We monitored the process in vivo by engineering an overlapping gusA gene downstream of the edited rbcL gene. Translational coupling between the overlapping rbcL and gusA genes

The chloroplast ATP synthase features the characteristic redox regulation machinery.

PubMed

Hisabori, Toru; Sunamura, Ei-Ichiro; Kim, Yusung; Konno, Hiroki

2013-11-20

Regulation of the activity of the chloroplast ATP synthase is largely accomplished by the chloroplast thioredoxin system, the main redox regulation system in chloroplasts, which is directly coupled to the photosynthetic reaction. We review the current understanding of the redox regulation system of the chloroplast ATP synthase. The thioredoxin-targeted portion of the ATP synthase consists of two cysteines located on the central axis subunit γ. The redox state of these two cysteines is under the influence of chloroplast thioredoxin, which directly controls rotation during catalysis by inducing a conformational change in this subunit. The molecular mechanism of redox regulation of the chloroplast ATP synthase has recently been determined. Regulation of the activity of the chloroplast ATP synthase is critical in driving efficiency into the ATP synthesis reaction in chloroplasts. The molecular architecture of the chloroplast ATP synthase, which confers redox regulatory properties requires further investigation, in light of the molecular structure of the enzyme complex as well as the physiological significance of the regulation system.

The complete chloroplast genome of Aconitum chiisanense Nakai (Ranunculaceae).

PubMed

Lim, Chae Eun; Kim, Goon-Bo; Baek, Seunghoon; Han, Su-Min; Yu, Hee-Ju; Mun, Jeong-Hwan

2017-01-01

We determined the complete chloroplast DNA sequence of Aconitum chiisanense Nakai, a rare Aconitum species endemic to Korea. The chloroplast genome is 155 934 bp in length and contains 4 rRNA, 30 tRNA, and 78 protein-coding genes. Phylogenetic analysis revealed that the chloroplast genome of A. chiisanense is closely related to that of A. barbatum var. puberulum. Sequence comparison with other Ranunculaceae chloroplasts identified a unique deletion in the rps16 gene of A. chiisanense chloroplast DNA that can serve as a molecular marker for species identification.

The Chloroplast ATP Synthase Features the Characteristic Redox Regulation Machinery

PubMed Central

Sunamura, Ei-Ichiro; Kim, Yusung; Konno, Hiroki

2013-01-01

Abstract Significance: Regulation of the activity of the chloroplast ATP synthase is largely accomplished by the chloroplast thioredoxin system, the main redox regulation system in chloroplasts, which is directly coupled to the photosynthetic reaction. We review the current understanding of the redox regulation system of the chloroplast ATP synthase. Recent Advances: The thioredoxin-targeted portion of the ATP synthase consists of two cysteines located on the central axis subunit γ. The redox state of these two cysteines is under the influence of chloroplast thioredoxin, which directly controls rotation during catalysis by inducing a conformational change in this subunit. The molecular mechanism of redox regulation of the chloroplast ATP synthase has recently been determined. Critical Issues: Regulation of the activity of the chloroplast ATP synthase is critical in driving efficiency into the ATP synthesis reaction in chloroplasts. Future Directions: The molecular architecture of the chloroplast ATP synthase, which confers redox regulatory properties requires further investigation, in light of the molecular structure of the enzyme complex as well as the physiological significance of the regulation system. Antioxid. Redox Signal. 19, 1846–1854. PMID:23145525

Looking for a substituent of spinach (Spinacia oleracea) chloroplasts

NASA Astrophysics Data System (ADS)

Chang, Ying Ping; Yeoh, Loo Yew; Chee, Swee Yong; Lim, Tuck Meng

2017-04-01

Spinach's chloroplasts electron transport features are often adapted to build biofuel cells or biosensors for environment conservation. This approach may raise food security issues. The present study aimed to test on in vitro functional activity of chloroplasts from selected underutilized leaves of: Pandan (Pandanus amaryllifolius), oil palm (Elaeis guineensis) and water lettuce (Pistia stratiotes) in comparison with spinach (Spinacia oleracea). The leaves' electrical conductivity was measured to evaluate the initial cell permeability. We applied Hill's reaction to determine the photoreduction capacity of the chloroplasts. Initial electrical conductivity of leaves ranged from 11.5 to 18.5 µs/cm/g followed the order of water lettucechloroplasts. Chloroplasts of oil palm frond and water lettuce showed low photoreduction rate of 14 to 22%. On the other hand, the chloroplasts of both spinach and pandan leaves exerted an initial photoreduction rate which was above 90%. The photoreduction rate of these chloroplasts remained to above 60% even after 30 day-storage at -20°C. In comparison with spinach, pandan leaves' chloroplasts possessed similar in vitro functional activity and storage stability at 4°C and -20°C. This warrants further investigation on chloroplasts of pandan leaves for higher-value applications.

Export of carbon from chloroplasts at night

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

Schleucher, J.; Vanderveer, P.J.; Sharkey, T.D.

Hexose export from chloroplasts at night has been inferred in previous studies of mutant and transgenic plants. The authors have tested whether hexose export is the normal route of carbon export from chloroplasts at night. The authors used nuclear magnetic resonance to distinguish glucose (Glc) made from hexose export and Glc made from triose export. Glc synthesized in vitro from fructose-6-phosphate in the presence of deuterium-labeled water had deuterium incorporated at C-2, whereas synthesis from triose phosphates caused C-2 through C-5 to become deuterated. In both tomato (Lycopersicon esculentum L.) and bean (phaseolus vulgaris L.), Glc from sucrose made atmore » night in the presence of deuterium-enriched water was deuterated only in the C-2 position, indicating that >75% of carbon is exported as hexoses at night. In darkness the phosphate in the cytosol was 28 mM, whereas that in the chloroplasts was 5 mW, but hexose phosphates were 10-fold higher in the cytosol than in the chloroplasts. Therefore, hexose phosphates would not move out of chloroplasts without the input of energy. The authors conclude that most carbon leaves chloroplasts at night as Glc, maltose, or higher maltodextrins under normal conditions.« less

Divergent copies of the large inverted repeat in the chloroplast genomes of ulvophycean green algae.

PubMed

Turmel, Monique; Otis, Christian; Lemieux, Claude

2017-04-20

The chloroplast genomes of many algae and almost all land plants carry two identical copies of a large inverted repeat (IR) sequence that can pair for flip-flop recombination and undergo expansion/contraction. Although the IR has been lost multiple times during the evolution of the green algae, the underlying mechanisms are still largely unknown. A recent comparison of IR-lacking and IR-containing chloroplast genomes of chlorophytes from the Ulvophyceae (Ulotrichales) suggested that differential elimination of genes from the IR copies might lead to IR loss. To gain deeper insights into the evolutionary history of the chloroplast genome in the Ulvophyceae, we analyzed the genomes of Ignatius tetrasporus and Pseudocharacium americanum (Ignatiales, an order not previously sampled), Dangemannia microcystis (Oltmannsiellopsidales), Pseudoneochloris marina (Ulvales) and also Chamaetrichon capsulatum and Trichosarcina mucosa (Ulotrichales). Our comparison of these six chloroplast genomes with those previously reported for nine ulvophyceans revealed unsuspected variability. All newly examined genomes feature an IR, but remarkably, the copies of the IR present in the Ignatiales, Pseudoneochloris, and Chamaetrichon diverge in sequence, with the tRNA genes from the rRNA operon missing in one IR copy. The implications of this unprecedented finding for the mechanism of IR loss and flip-flop recombination are discussed.

DNA Gyrase Is Involved in Chloroplast Nucleoid Partitioning

PubMed Central

Cho, Hye Sun; Lee, Sang Sook; Kim, Kwang Dong; Hwang, Inhwan; Lim, Jong-Seok; Park, Youn-Il; Pai, Hyun-Sook

2004-01-01

DNA gyrase, which catalyzes topological transformation of DNA, plays an essential role in replication and transcription in prokaryotes. Virus-induced gene silencing of NbGyrA or NbGyrB, which putatively encode DNA gyrase subunits A and B, respectively, resulted in leaf yellowing phenotypes in Nicotiana benthamiana. NbGyrA and NbGyrB complemented the gyrA and gyrB temperature-sensitive mutations of Escherichia coli, respectively, which indicates that the plant and bacterial subunits are functionally similar. NbGyrA and NbGyrB were targeted to both chloroplasts and mitochondria, and depletion of these subunits affected both organelles by reducing chloroplast numbers and inducing morphological and physiological abnormalities in both organelles. Flow cytometry analysis revealed that the average DNA content in the affected chloroplasts and mitochondria was significantly higher than in the control organelles. Furthermore, 4′,6-diamidino-2-phenylindole staining revealed that the abnormal chloroplasts contained one or a few large nucleoids instead of multiple small nucleoids dispersed throughout the stroma. Pulse-field gel electrophoresis analyses of chloroplasts demonstrated that the sizes and/or structure of the DNA molecules in the abnormal chloroplast nucleoids are highly aberrant. Based on these results, we propose that DNA gyrase plays a critical role in chloroplast nucleoid partitioning by regulating DNA topology. PMID:15367714

Effects and mechanism of acid rain on plant chloroplast ATP synthase.

PubMed

Sun, Jingwen; Hu, Huiqing; Li, Yueli; Wang, Lihong; Zhou, Qing; Huang, Xiaohua

2016-09-01

Acid rain can directly or indirectly affect plant physiological functions, especially photosynthesis. The enzyme ATP synthase is the key in photosynthetic energy conversion, and thus, it affects plant photosynthesis. To clarify the mechanism by which acid rain affects photosynthesis, we studied the effects of acid rain on plant growth, photosynthesis, chloroplast ATP synthase activity and gene expression, chloroplast ultrastructure, intracellular H(+) level, and water content of rice seedlings. Acid rain at pH 4.5 remained the chloroplast structure unchanged but increased the expression of six chloroplast ATP synthase subunits, promoted chloroplast ATP synthase activity, and increased photosynthesis and plant growth. Acid rain at pH 4.0 or less decreased leaf water content, destroyed chloroplast structure, inhibited the expression of six chloroplast ATP synthase subunits, decreased chloroplast ATP synthase activity, and reduced photosynthesis and plant growth. In conclusion, acid rain affected the chloroplast ultrastructure, chloroplast ATPase transcription and activity, and P n by changing the acidity in the cells, and thus influencing the plant growth and development. Finally, the effects of simulated acid rain on the test indices were found to be dose-dependent.

Mechanism of protein import across the chloroplast envelope.

PubMed

Chen, K; Chen, X; Schnell, D J

2000-01-01

The development and maintenance of chloroplasts relies on the contribution of protein subunits from both plastid and nuclear genomes. Most chloroplast proteins are encoded by nuclear genes and are post-translationally imported into the organelle across the double membrane of the chloroplast envelope. Protein import into the chloroplast consists of two essential elements: the specific recognition of the targeting signals (transit sequences) of cytoplasmic preproteins by receptors at the outer envelope membrane and the subsequent translocation of preproteins simultaneously across the double membrane of the envelope. These processes are mediated via the co-ordinate action of protein translocon complexes in the outer (Toc apparatus) and inner (Tic apparatus) envelope membranes.

Chloroplast Biogenesis: Control of Plastid Development, Protein Import, Division and Inheritance

PubMed Central

Sakamoto, Wataru; Miyagishima, Shin-ya; Jarvis, Paul

2008-01-01

The chloroplast is a multi-copy cellular organelle that not only performs photosynthesis but also synthesizes amino acids, lipids and phytohormones. The plastid also responds to environmental stimuli such as gravitropism. Biogenesis of chloroplasts is initiated from proplastids in shoot meristems, and involves a series of important events. In the last decade, considerable progress has been made towards understanding various aspects of chloroplast biogenesis at the molecular level, via studies in model systems such as Arabidopsis. This review focuses on two important aspects of chloroplast biogenesis, synthesis/assembly and division/transmission. Chloroplasts originated through endosymbiosis from an ancestor of extant cyanobacteria, and thus contain their own genomes. DNA in chloroplasts is organized into complexes with proteins, and these are called nucleoids. The synthesis of chloroplast proteins is regulated at various steps. However, a majority of proteins are synthesized in the cytosol, and their proper import into chloroplast compartments is a prerequisite for chloroplast development. Fundamental aspects of plastid gene expression/regulation and chloroplast protein transport are described, together with recent proteome analyses of the organelle. Chloroplasts are not de novo synthesized, but instead are propagated from pre-existing plastids. In addition, plastids are transmitted from generation to generation with a unique mode of inheritance. Our current knowledge on the division machinery and the inheritance of plastids is described. PMID:22303235

Redirecting the Cyanobacterial Bicarbonate Transporters BicA and SbtA to the Chloroplast Envelope: Soluble and Membrane Cargos Need Different Chloroplast Targeting Signals in Plants

PubMed Central

Rolland, Vivien; Badger, Murray R.; Price, G. Dean

2016-01-01

Most major crops used for human consumption are C3 plants, which yields are limited by photosynthetic inefficiency. To circumvent this, it has been proposed to implement the cyanobacterial CO2-concentrating mechanism (CCM), principally consisting of bicarbonate transporters and carboxysomes, into plant chloroplasts. As it is currently not possible to recover homoplasmic transplastomic monocots, foreign genes must be introduced in these plants via nuclear transformation. Consequently, it is paramount to ensure that resulting proteins reach the appropriate sub-cellular compartment, which for cyanobacterial transporters BicA and SbtA, is the chloroplast inner-envelope membrane (IEM). At present, targeting signals to redirect large transmembrane proteins from non-chloroplastic organisms to plant chloroplast envelopes are unknown. The goal of this study was to identify such signals, using agrobacteria-mediated transient expression and confocal microscopy to determine the sub-cellular localization of ∼37 GFP-tagged chimeras. Initially, fragments of chloroplast proteins known to target soluble cargos to the stroma were tested for their ability to redirect BicA, but they proved ineffective. Next, different N-terminal regions from Arabidopsis IEM transporters were tested. We demonstrated that the N-terminus of AtHP59, AtPLGG1 or AtNTT1 (92–115 amino acids), containing a cleavable chloroplast transit peptide (cTP) and a membrane protein leader (MPL), was sufficient to redirect BicA or SbtA to the chloroplast envelope. This constitutes the first evidence that nuclear-encoded transmembrane proteins from non-chloroplastic organisms can be targeted to the envelope of plant chloroplasts; a finding which represents an important advance in chloroplast engineering by opening up the door to further manipulation of the chloroplastic envelope. PMID:26973659

Chloroplast Osmotic Adjustment and Water Stress Effects on Photosynthesis 1

PubMed Central

Gupta, Ashima Sen; Berkowitz, Gerald A.

1988-01-01

Previous studies have suggested that chloroplast stromal volume reduction may mediate the inhibition of photosynthesis under water stress. In this study, the effects of spinach (Spinacia oleracea, var `Winter Bloomsdale') plant water deficits on chloroplast photosynthetic capacity, solute concentrations in chloroplasts, and chloroplast volume were studied. In situ (gas exchange) and in vitro measurements indicated that chloroplast photosynthetic capacity was maintained during initial leaf water potential (Ψw) and relative water content (RWC) decline. During the latter part of the stress period, photosynthesis dropped precipitously. Chloroplast stromal volume apparently remained constant during the initial period of decline in RWC, but as leaf Ψw reached −1.2 megapascals, stromal volume began to decline. The apparent maintenance of stromal volume over the initial RWC decline during a stress cycle suggested that chloroplasts are capable of osmotic adjustment in response to leaf water deficits. This hypothesis was confirmed by measuring chloroplast solute levels, which increased during stress. The results of these experiments suggest that stromal volume reduction in situ may be associated with loss of photosynthetic capacity and that one mechanism of photosynthetic acclimation to low Ψw may involve stromal volume maintenance. PMID:16666266

Engineered Chloroplast Genome just got Smarter

PubMed Central

Jin, Shuangxia; Daniell, Henry

2015-01-01

Chloroplasts are known to sustain life on earth by providing food, fuel and oxygen through the process of photosynthesis. However, the chloroplast genome has also been smartly engineered to confer valuable agronomic traits and/or serve as bioreactors for production of industrial enzymes, biopharmaceuticals, bio-products or vaccines. The recent breakthrough in hyper-expression of biopharmaceuticals in edible leaves has facilitated the advancement to clinical studies by major pharmaceutical companies. This review critically evaluates progress in developing new tools to enhance or simplify expression of targeted genes in chloroplasts. These tools hold the promise to further the development of novel fuels and products, enhance the photosynthetic process, and increase our understanding of retrograde signaling and cellular processes. PMID:26440432

Chloroplast-to-nucleus communication

PubMed Central

Chan, Kai Xun; Crisp, Peter Alexander; Estavillo, Gonzalo Martin

2010-01-01

In order for plant cells to function efficiently under different environmental conditions, chloroplastic processes have to be tightly regulated by the nucleus. It is widely believed that there is inter-organelle communication from the chloroplast to the nucleus, called retrograde signaling. Although some pathways of communication have been identified, the actual signals that move between the two cellular compartments are largely unknown. This review provides an overview of retrograde signaling including its importance to the cell, candidate signals, recent advances and current experimental systems. In addition, we highlight the potential of using drought stress as a model for studying retrograde signaling. PMID:21512326

[Identification of medicinal plant Dendrobium based on the chloroplast psbK-psbI intergenic spacer].

PubMed

Yao, Hui; Yang, Pei; Zhou, Hong; Ma, Shuang-jiao; Song, Jing-yuan; Chen, Shi-lin

2015-06-01

In this paper, the chloroplast psbK-psbI intergenic spacers of 18 species of Dendrobium and their adulterants were amplified and sequenced, and then the sequence characteristics were analyzed. The sequence lengths of chloroplast psbK-psbI regions of Dendrobium ranged from 474 to 513 bp and the GC contents were 25.4%-27.6%. The variable sites were 71 while the informative sites were 46. The inter-specific genetic distances calculated by Kimura 2-parameter (K2P) of Dendrobium were 0.006 1-0.058 1, with an average of 0.028 4. The K2P genetic distances between Dendrobium species and Bulbophyllum odoratissimum were 0.093 2-0.120 4. The NJ tree showed that the Dendrobium species can be easily differentiated from each other and 6 samples of the inspected Dendrobium species were identified successfully through sequencing the psbK-psbI intergenic spacer. Therefore, the chloroplast psbK-psbI intergenic spacer can be used as a candidate marker to identify Dendrobium species and its adulterants.

Chloroplast targeting of FtsHprotease is essential for chloroplast development and thylakoid stability at elevated temperatures in plants

USDA-ARS?s Scientific Manuscript database

AtFtsH11 is a chloroplast and mitochondria dual targeted metalloprotease, identified as essential for Arabidopsis plant to survive at moderate high temperatures at all developmental stages. Our study showed that FtsH11 plays critical roles in both the early stages of chloroplast biogenesis and main...

Ferns, mosses and liverworts as model systems for light-mediated chloroplast movements.

PubMed

Suetsugu, Noriyuki; Higa, Takeshi; Wada, Masamitsu

2017-11-01

Light-induced chloroplast movement is found in most plant species, including algae and land plants. In land plants with multiple small chloroplasts, under weak light conditions, the chloroplasts move towards the light and accumulate on the periclinal cell walls to efficiently perceive light for photosynthesis (the accumulation response). Under strong light conditions, chloroplasts escape from light to avoid photodamage (the avoidance response). In most plant species, blue light induces chloroplast movement, and phototropin receptor kinases are the blue light receptors. Molecular mechanisms for photoreceptors, signal transduction and chloroplast motility systems are being studied using the model plant Arabidopsis thaliana. However, to further understand the molecular mechanisms and evolutionary history of chloroplast movement in green plants, analyses using other plant systems are required. Here, we review recent works on chloroplast movement in green algae, liverwort, mosses and ferns that provide new insights on chloroplast movement. © 2016 John Wiley & Sons Ltd.

Mollusc-algal chloroplast endosymbiosis. Photosynthesis, thylakoid protein maintenance, and chloroplast gene expression continue for many months in the absence of the algal nucleus.

PubMed

Green, B J; Li, W Y; Manhart, J R; Fox, T C; Summer, E J; Kennedy, R A; Pierce, S K; Rumpho, M E

2000-09-01

Early in its life cycle, the marine mollusc Elysia chlorotica Gould forms an intracellular endosymbiotic association with chloroplasts of the chromophytic alga Vaucheria litorea C. Agardh. As a result, the dark green sea slug can be sustained in culture solely by photoautotrophic CO(2) fixation for at least 9 months if provided with only light and a source of CO(2). Here we demonstrate that the sea slug symbiont chloroplasts maintain photosynthetic oxygen evolution and electron transport activity through photosystems I and II for several months in the absence of any external algal food supply. This activity is correlated to the maintenance of functional levels of chloroplast-encoded photosystem proteins, due in part at least to de novo protein synthesis of chloroplast proteins in the sea slug. Levels of at least one putative algal nuclear encoded protein, a light-harvesting complex protein homolog, were also maintained throughout the 9-month culture period. The chloroplast genome of V. litorea was found to be 119.1 kb, similar to that of other chromophytic algae. Southern analysis and polymerase chain reaction did not detect an algal nuclear genome in the slug, in agreement with earlier microscopic observations. Therefore, the maintenance of photosynthetic activity in the captured chloroplasts is regulated solely by the algal chloroplast and animal nuclear genomes.

Remodeling of chloroplast proteome under salinity affects salt tolerance of Festuca arundinacea.

PubMed

Pawłowicz, Izabela; Waśkiewicz, Agnieszka; Perlikowski, Dawid; Rapacz, Marcin; Ratajczak, Dominika; Kosmala, Arkadiusz

2018-06-07

Acclimation of photosynthetic apparatus to variable environmental conditions is an important component of tolerance to dehydration stresses, including salinity. The present study deals with the research on alterations in chloroplast proteome of the forage grasses. Based on chlorophyll fluorescence parameters, two genotypes of a model grass species-Festuca arundinacea with distinct levels of salinity tolerance: low salt tolerant (LST) and high salt tolerant (HST), were selected. Next, two-dimensional electrophoresis and mass spectrometry were applied under both control and salt stress conditions to identify proteins accumulated differentially between these two genotypes. The physiological analysis revealed that under NaCl treatment the studied plants differed in photosystem II activity, water content, and ion accumulation. The differentially accumulated proteins included ATPase B, ATP synthase, ribulose-1,5-bisphosphate carboxylase large and small subunits, cytochrome b6-f complex iron-sulfur subunit, oxygen-evolving enhancer proteins (OEE), OEE1 and OEE2, plastidic fructose-bisphosphate aldolase (pFBA), and lipocalin. A higher level of lipocalin, potentially involved in prevention of lipid peroxidation under stress, was also observed in the HST genotype. Our physiological and proteomic results performed for the first time on the species of forage grasses clearly showed that chloroplast metabolism adjustment could be a crucial factor in developing salinity tolerance.

Global RNA association with the transcriptionally active chromosome of chloroplasts.

PubMed

Lehniger, Marie-Kristin; Finster, Sabrina; Melonek, Joanna; Oetke, Svenja; Krupinska, Karin; Schmitz-Linneweber, Christian

2017-10-01

Processed chloroplast RNAs are co-enriched with preparations of the chloroplast transcriptionally active chromosome. Chloroplast genomes are organized as a polyploid DNA-protein structure called the nucleoid. Transcriptionally active chloroplast DNA together with tightly bound protein factors can be purified by gel filtration as a functional entity called the transcriptionally active chromosome (TAC). Previous proteomics analyses of nucleoids and of TACs demonstrated a considerable overlap in protein composition including RNA binding proteins. Therefore the RNA content of TAC preparations from Nicotiana tabacum was determined using whole genome tiling arrays. A large number of chloroplast RNAs was found to be associated with the TAC. The pattern of RNAs attached to the TAC consists of RNAs produced by different chloroplast RNA polymerases and differs from the pattern of RNA found in input controls. An analysis of RNA splicing and RNA editing of selected RNA species demonstrated that TAC-associated RNAs are processed to a similar extent as the RNA in input controls. Thus, TAC fractions contain a specific subset of the processed chloroplast transcriptome.

The evolution of blue-greens and the origins of chloroplasts

NASA Technical Reports Server (NTRS)

Schwartz, R. M.; Dayhoff, M. O.

1981-01-01

All of the available molecular data support the theory that the chloroplasts of eukaryote cells were originally free-living blue-greens. Of great interest is what the relationships are between contemporary types of blue-greens and eukaryote chloroplasts and whether the chloroplasts of the various eukaryotes are the result of one or more than one symbiosis. By combining information from phylogenetic trees based on cytochrome c6 and 2Fe-2S ferredoxin sequences, it is shown that the chloroplasts of a number of eukaryote algae as well as the protist Euglena are polyphyletic; the chloroplasts of green algae and the higher plants may be the result of a single symbiosis.

The rice nuclear gene, VIRESCENT 2, is essential for chloroplast development and encodes a novel type of guanylate kinase targeted to plastids and mitochondria.

PubMed

Sugimoto, Hiroki; Kusumi, Kensuke; Noguchi, Ko; Yano, Masahiro; Yoshimura, Atsushi; Iba, Koh

2007-11-01

Guanylate kinase (GK) is a critical enzyme in guanine nucleotide metabolism pathways, catalyzing the phosphorylation of (d)GMP to (d)GDP. Here we show that a novel gene, VIRESCENT 2 (V2), encodes a new type of GK (designated pt/mtGK) that is localized in plastids and mitochondria. We initially identified the V2 gene by positional cloning of the rice v2 mutant. The v2 mutant is temperature-sensitive and develops chlorotic leaves at restrictive temperatures. The v2 mutation causes inhibition of chloroplast differentiation; in particular, it disrupts the chloroplast translation machinery during early leaf development [Sugimoto et al. (2004)Plant Cell Physiol. 45, 985]. In the bacterial and animal species studied to date, GK is localized in the cytoplasm and participates in maintenance of the guanine nucleotide pools required for many fundamental cellular processes. Phenotypic analysis of rice seedlings with RNAi knockdown of cytosolic GK (designated cGK) showed that cGK is indispensable for the growth and development of plants, but not for chloroplast development. Thus, rice has two types of GK, as does Arabidopsis, suggesting that higher plants have two types of GK. Our results suggest that, of the two types of GK, only pt/mtGK is essential for chloroplast differentiation.

RNA-stabilization factors in chloroplasts of vascular plants.

PubMed

Manavski, Nikolay; Schmid, Lisa-Marie; Meurer, Jörg

2018-04-13

In contrast to the cyanobacterial ancestor, chloroplast gene expression is predominantly governed on the post-transcriptional level such as modifications of the RNA sequence, decay rates, exo- and endonucleolytic processing as well as translational events. The concerted function of numerous chloroplast RNA-binding proteins plays a fundamental and often essential role in all these processes but our understanding of their impact in regulation of RNA degradation is only at the beginning. Moreover, metabolic processes and post-translational modifications are thought to affect the function of RNA protectors. These protectors contain a variety of different RNA-recognition motifs, which often appear as multiple repeats. They are required for normal plant growth and development as well as diverse stress responses and acclimation processes. Interestingly, most of the protectors are plant specific which reflects a fast-evolving RNA metabolism in chloroplasts congruent with the diverging RNA targets. Here, we mainly focused on the characteristics of known chloroplast RNA-binding proteins that protect exonuclease-sensitive sites in chloroplasts of vascular plants. © 2018 The Author(s).

Entire Photodamaged Chloroplasts Are Transported to the Central Vacuole by Autophagy[OPEN

PubMed Central

2017-01-01

Turnover of dysfunctional organelles is vital to maintain homeostasis in eukaryotic cells. As photosynthetic organelles, plant chloroplasts can suffer sunlight-induced damage. However, the process for turnover of entire damaged chloroplasts remains unclear. Here, we demonstrate that autophagy is responsible for the elimination of sunlight-damaged, collapsed chloroplasts in Arabidopsis thaliana. We found that vacuolar transport of entire chloroplasts, termed chlorophagy, was induced by UV-B damage to the chloroplast apparatus. This transport did not occur in autophagy-defective atg mutants, which exhibited UV-B-sensitive phenotypes and accumulated collapsed chloroplasts. Use of a fluorescent protein marker of the autophagosomal membrane allowed us to image autophagosome-mediated transport of entire chloroplasts to the central vacuole. In contrast to sugar starvation, which preferentially induced distinct type of chloroplast-targeted autophagy that transports a part of stroma via the Rubisco-containing body (RCB) pathway, photooxidative damage induced chlorophagy without prior activation of RCB production. We further showed that chlorophagy is induced by chloroplast damage caused by either artificial visible light or natural sunlight. Thus, this report establishes that an autophagic process eliminates entire chloroplasts in response to light-induced damage. PMID:28123106

Ubiquitin facilitates a quality-control pathway that removes damaged chloroplasts

DOE PAGES

Woodson, Jesse D.; Joens, Matthew S.; Sinson, Andrew B.; ...

2015-10-23

Energy production by chloroplasts and mitochondria causes constant oxidative damage. A functioning photosynthetic cell requires quality-control mechanisms to turn over and degrade chloroplasts damaged by reactive oxygen species (ROS). Here in this study, we generated a conditionally lethal Arabidopsis mutant that accumulated excess protoporphyrin IX in the chloroplast and produced singlet oxygen. Damaged chloroplasts were subsequently ubiquitinated and selectively degraded. A genetic screen identified the plant U-box 4 (PUB4) E3 ubiquitin ligase as being necessary for this process. pub4-6 mutants had defects in stress adaptation and longevity. As a result, we have identified a signal that leads to the targetedmore » removal of ROS-overproducing chloroplasts.« less

Ubiquitin facilitates a quality-control pathway that removes damaged chloroplasts

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

Woodson, Jesse D.; Joens, Matthew S.; Sinson, Andrew B.

Energy production by chloroplasts and mitochondria causes constant oxidative damage. A functioning photosynthetic cell requires quality-control mechanisms to turn over and degrade chloroplasts damaged by reactive oxygen species (ROS). Here in this study, we generated a conditionally lethal Arabidopsis mutant that accumulated excess protoporphyrin IX in the chloroplast and produced singlet oxygen. Damaged chloroplasts were subsequently ubiquitinated and selectively degraded. A genetic screen identified the plant U-box 4 (PUB4) E3 ubiquitin ligase as being necessary for this process. pub4-6 mutants had defects in stress adaptation and longevity. As a result, we have identified a signal that leads to the targetedmore » removal of ROS-overproducing chloroplasts.« less

Assessing the Ability of Chloroplast and Nuclear DNA Gene Markers to Verify the Geographic Origin of Jatoba (Hymenaea courbaril L.) Timber.

PubMed

Chaves, Camila L; Degen, Bernd; Pakull, Birte; Mader, Malte; Honorio, Euridice; Ruas, Paulo; Tysklind, Niklas; Sebbenn, Alexandre M

2018-06-27

Deforestation-reinforced by illegal logging-is a serious problem in many tropical regions and causes pervasive environmental and economic damage. Existing laws that intend to reduce illegal logging need efficient, fraud resistant control methods. We developed a genetic reference database for Jatoba (Hymenaea courbaril), an important, high value timber species from the Neotropics. The data set can be used for controls on declarations of wood origin. Samples from 308 Hymenaea trees from 12 locations in Brazil, Bolivia, Peru, and French Guiana have been collected and genotyped on 10 nuclear microsatellites (nSSRs), 13 chloroplast SNPs (cpSNP), and 1 chloroplast indel marker. The chloroplast gene markers have been developed using Illumina DNA sequencing. Bayesian cluster analysis divided the individuals based on the nSSRs into 8 genetic groups. Using self-assignment tests, the power of the genetic reference database to judge on declarations on the location has been tested for 3 different assignment methods. We observed a strong genetic differentiation among locations leading to high and reliable self-assignment rates for the locations between 50% to 100% (average of 88%). Although all 3 assignment methods came up with similar mean self-assignment rates, there were differences for some locations linked to the level of genetic diversity, differentiation, and heterozygosity. Our results show that the nuclear and chloroplast gene markers are effective to be used for a genetic certification system and can provide national and international authorities with a robust tool to confirm legality of timber.

Differential responses of double petal and multi petal jasmine to shading: I. Photosynthetic characteristics and chloroplast ultrastructure.

PubMed

Deng, Yanming; Li, Congcong; Shao, Qingsong; Ye, Xiaoqing; She, Jianming

2012-06-01

A double petal (DP) and a multi petal (MP) type jasmine (Jasminum sambac Ait.) growth and flowering was known largely affected by different levels of irradiance. Here, our objective was to determine the effects of shade on photosynthesis related characteristics and chloroplast ultrastructure of these two types. In both types, net photosynthetic rate (Pn), stomatal conductance (g(s)) and transpiration rate increased with decreasing irradiance from 100% to 20%, while both maximum and variable fluorescence showed a steady increase, and photochemical and nonphotochemical quenching indexes declined. At each conducted time, chlorophyll a, b and carotenoids contents in DP type shaded leaves increased whereas those in MP type decreased at 5% irradiance (considered as extreme shade). The maximum photochemical efficiency of photosystem II of DP plants showed subtle changes but that of MP plants declined by shading thereafter 21 days of treatment. Observation of chloroplast ultrastructure showed its best development in the leaves of DP and MP types mostly from 50% to 20% irradiance (considered as weak and moderate shade, respectively). At each shade treatment, Pn, g(s) and water use efficiency of DP-jasmine were always higher than those of MP-jasmine, thus the shade tolerance ability of the former was higher than that of the latter. The results showed that full sunlight and 5% natural irradiance caused photoinhibition and light deficiency of jasmine plants respectively, and modulating chloroplast development by the more numbers of thylakoids and grana to contain more photosynthetic pigments is an important shade tolerance mechanism of DP type. Copyright © 2012 Elsevier Masson SAS. All rights reserved.

Two Effects of Electrical Fields on Chloroplasts 1

PubMed Central

Arnold, William A.; Azzi, Jim R.

1977-01-01

An electrical field across a suspension of Chenopodium chloroplasts stimulates the emission of delayed light during the time the field is on. This stimulation can be used to calculate the distance over which the electron moves in the untrapping process that gives the delayed light. An electrical field applied at the time of illumination gives a polarization to the suspension of chloroplasts that lasts for some seconds. This polarization is a new way to study delayed light and fluorescence from chloroplasts. Images PMID:16660112

Arabidopsis VARIEGATED 3 encodes a chloroplast-targeted, zinc-finger protein required for chloroplast and palisade cell development.

PubMed

Naested, Henrik; Holm, Agnethe; Jenkins, Tom; Nielsen, H Bjørn; Harris, Cassandra A; Beale, Michael H; Andersen, Mathias; Mant, Alexandra; Scheller, Henrik; Camara, Bilal; Mattsson, Ole; Mundy, John

2004-09-15

The stable, recessive Arabidopsis variegated 3 (var3) mutant exhibits a variegated phenotype due to somatic areas lacking or containing developmentally retarded chloroplasts and greatly reduced numbers of palisade cells. The VAR3 gene, isolated by transposon tagging, encodes the 85.9 kDa VAR3 protein containing novel repeats and zinc fingers described as protein interaction domains. VAR3 interacts specifically in yeast and in vitro with NCED4, a putative polyene chain or carotenoid dioxygenase, and both VAR3 and NCED4 accumulate in the chloroplast stroma. Metabolic profiling demonstrates that pigment profiles are qualitatively similar in wild type and var3, although var3 accumulates lower levels of chlorophylls and carotenoids. These results indicate that VAR3 is a part of a protein complex required for normal chloroplast and palisade cell development.

Phylogenic study of Lemnoideae (duckweeds) through complete chloroplast genomes for eight accessions.

PubMed

Ding, Yanqiang; Fang, Yang; Guo, Ling; Li, Zhidan; He, Kaize; Zhao, Yun; Zhao, Hai

2017-01-01

Phylogenetic relationship within different genera of Lemnoideae, a kind of small aquatic monocotyledonous plants, was not well resolved, using either morphological characters or traditional markers. Given that rich genetic information in chloroplast genome makes them particularly useful for phylogenetic studies, we used chloroplast genomes to clarify the phylogeny within Lemnoideae. DNAs were sequenced with next-generation sequencing. The duckweeds chloroplast genomes were indirectly filtered from the total DNA data, or directly obtained from chloroplast DNA data. To test the reliability of assembling the chloroplast genome based on the filtration of the total DNA, two methods were used to assemble the chloroplast genome of Landoltia punctata strain ZH0202. A phylogenetic tree was built on the basis of the whole chloroplast genome sequences using MrBayes v.3.2.6 and PhyML 3.0. Eight complete duckweeds chloroplast genomes were assembled, with lengths ranging from 165,775 bp to 171,152 bp, and each contains 80 protein-coding sequences, four rRNAs, 30 tRNAs and two pseudogenes. The identity of L. punctata strain ZH0202 chloroplast genomes assembled through two methods was 100%, and their sequences and lengths were completely identical. The chloroplast genome comparison demonstrated that the differences in chloroplast genome sizes among the Lemnoideae primarily resulted from variation in non-coding regions, especially from repeat sequence variation. The phylogenetic analysis demonstrated that the different genera of Lemnoideae are derived from each other in the following order: Spirodela , Landoltia , Lemna , Wolffiella , and Wolffia . This study demonstrates potential of whole chloroplast genome DNA as an effective option for phylogenetic studies of Lemnoideae. It also showed the possibility of using chloroplast DNA data to elucidate those phylogenies which were not yet solved well by traditional methods even in plants other than duckweeds.

Phylogenic study of Lemnoideae (duckweeds) through complete chloroplast genomes for eight accessions

PubMed Central

Ding, Yanqiang; Fang, Yang; Guo, Ling; Li, Zhidan; He, Kaize

2017-01-01

Background Phylogenetic relationship within different genera of Lemnoideae, a kind of small aquatic monocotyledonous plants, was not well resolved, using either morphological characters or traditional markers. Given that rich genetic information in chloroplast genome makes them particularly useful for phylogenetic studies, we used chloroplast genomes to clarify the phylogeny within Lemnoideae. Methods DNAs were sequenced with next-generation sequencing. The duckweeds chloroplast genomes were indirectly filtered from the total DNA data, or directly obtained from chloroplast DNA data. To test the reliability of assembling the chloroplast genome based on the filtration of the total DNA, two methods were used to assemble the chloroplast genome of Landoltia punctata strain ZH0202. A phylogenetic tree was built on the basis of the whole chloroplast genome sequences using MrBayes v.3.2.6 and PhyML 3.0. Results Eight complete duckweeds chloroplast genomes were assembled, with lengths ranging from 165,775 bp to 171,152 bp, and each contains 80 protein-coding sequences, four rRNAs, 30 tRNAs and two pseudogenes. The identity of L. punctata strain ZH0202 chloroplast genomes assembled through two methods was 100%, and their sequences and lengths were completely identical. The chloroplast genome comparison demonstrated that the differences in chloroplast genome sizes among the Lemnoideae primarily resulted from variation in non-coding regions, especially from repeat sequence variation. The phylogenetic analysis demonstrated that the different genera of Lemnoideae are derived from each other in the following order: Spirodela, Landoltia, Lemna, Wolffiella, and Wolffia. Discussion This study demonstrates potential of whole chloroplast genome DNA as an effective option for phylogenetic studies of Lemnoideae. It also showed the possibility of using chloroplast DNA data to elucidate those phylogenies which were not yet solved well by traditional methods even in plants other than

Mollusc-Algal Chloroplast Endosymbiosis. Photosynthesis, Thylakoid Protein Maintenance, and Chloroplast Gene Expression Continue for Many Months in the Absence of the Algal Nucleus1

PubMed Central

Green, Brian J.; Li, Wei-Ye; Manhart, James R.; Fox, Theodore C.; Summer, Elizabeth J.; Kennedy, Robert A.; Pierce, Sidney K.; Rumpho, Mary E.

2000-01-01

Early in its life cycle, the marine mollusc Elysia chlorotica Gould forms an intracellular endosymbiotic association with chloroplasts of the chromophytic alga Vaucheria litorea C. Agardh. As a result, the dark green sea slug can be sustained in culture solely by photoautotrophic CO2 fixation for at least 9 months if provided with only light and a source of CO2. Here we demonstrate that the sea slug symbiont chloroplasts maintain photosynthetic oxygen evolution and electron transport activity through photosystems I and II for several months in the absence of any external algal food supply. This activity is correlated to the maintenance of functional levels of chloroplast-encoded photosystem proteins, due in part at least to de novo protein synthesis of chloroplast proteins in the sea slug. Levels of at least one putative algal nuclear encoded protein, a light-harvesting complex protein homolog, were also maintained throughout the 9-month culture period. The chloroplast genome of V. litorea was found to be 119.1 kb, similar to that of other chromophytic algae. Southern analysis and polymerase chain reaction did not detect an algal nuclear genome in the slug, in agreement with earlier microscopic observations. Therefore, the maintenance of photosynthetic activity in the captured chloroplasts is regulated solely by the algal chloroplast and animal nuclear genomes. PMID:10982447

A new type of subchloroplast fragments isolated from pea chloroplasts in the presence of digitonin.

PubMed

Kochubey, S M; Bondarenko, O Yu; Shevchenko, V V

2007-09-01

Heavy fragments were isolated from pea chloroplasts using digitonin treatment and differential centrifugation. The particles were characterized by a significantly lowered chlorophyll a/b ratio, contents of photosystem I (PS I) proteins and ATPase, as well as of amount of P700. The content of photosystem II (PS II) proteins decreased insignificantly, whereas that of proteins of the light-harvesting complex II did not change. The absorption and low-temperature fluorescence spectra were indicative of a decreased content of PS I. Electron microscopy of ultrathin sections of heavy fragment preparations identified them as grana with reduced content of thylakoids. The diameter of these particles was practically the same as within chloroplasts. Comparison of various characteristics of the fragments and chloroplasts from which the fragments were isolated allowed us to define a high degree of preservation of marginal regions in thylakoids present in the heavy fragment particles. Analysis of the results shows that the procedure of fragmentation produces grana with high extent of thylakoid integrity. The phenomenon of reduction of the thylakoid content in grana, occurring as our heavy fragments, is considered in the frame of our previous hypothesis concerning the peculiarities of grana organization in the transversal direction.

Use of the chloroplast gene ycf1 for the genetic differentiation of pine nuts obtained from consumers experiencing dysgeusia.

PubMed

Handy, Sara M; Parks, Matthew B; Deeds, Jonathan R; Liston, Aaron; de Jager, Lowri S; Luccioli, Stefano; Kwegyir-Afful, Ernest; Fardin-Kia, Ali R; Begley, Timothy H; Rader, Jeanne I; Diachenko, Gregory W

2011-10-26

Pine nuts are a part of traditional cooking in many parts of the world and have seen a significant increase in availability/use in the United States over the past 10 years. The U.S. Food and Drug Administration (US FDA) field offices received 411 complaints from U.S. consumers over the past three years regarding taste disturbances following the consumption of pine nuts. Using analysis of fatty acids by gas chromatography with flame ionization detection, previous reports have implicated nuts from Pinus armandii (Armand Pine) as the causative species for similar taste disturbances. This method was found to provide insufficient species resolution to link FDA consumer complaint samples to a single species of pine, particularly when samples contained species mixtures of pine nuts. Here we describe a DNA based method for differentiating pine nut samples using the ycf1 chloroplast gene. Although the exact cause of pine nut associated dysgeusia is still not known, we found that 15 of 15 samples from consumer complaints contained at least some Pinus armandii, confirming the apparent association of this species with taste disturbances.

The complete chloroplast genome of the Dendrobium strongylanthum (Orchidaceae: Epidendroideae).

PubMed

Li, Jing; Chen, Chen; Wang, Zhe-Zhi

2016-07-01

Complete chloroplast genome sequence is very useful for studying the phylogenetic and evolution of species. In this study, the complete chloroplast genome of Dendrobium strongylanthum was constructed from whole-genome Illumina sequencing data. The chloroplast genome is 153 058 bp in length with 37.6% GC content and consists of two inverted repeats (IRs) of 26 316 bp. The IR regions are separated by large single-copy region (LSC, 85 836 bp) and small single-copy (SSC, 14 590 bp) region. A total of 130 chloroplast genes were successfully annotated, including 84 protein coding genes, 38 tRNA genes, and eight rRNA genes. Phylogenetic analyses showed that the chloroplast genome of Dendrobium strongylanthum is related to that of the Dendrobium officinal.

Pb-Induced Avoidance-Like Chloroplast Movements in Fronds of Lemna trisulca L.

PubMed Central

Samardakiewicz, Sławomir; Krzeszowiec-Jeleń, Weronika; Bednarski, Waldemar; Jankowski, Artur; Suski, Szymon; Gabryś, Halina; Woźny, Adam

2015-01-01

Lead ions are particularly dangerous to the photosynthetic apparatus, but little is known about the effects of trace metals, including Pb, on regulation of chloroplast redistribution. In this study a new effect of lead on chloroplast distribution patterns and movements was demonstrated in mesophyll cells of a small-sized aquatic angiosperm Lemna trisulca L. (star duckweed). An analysis of confocal microscopy images of L. trisulca fronds treated with lead (15 μM Pb2+, 24 h) in darkness or in weak white light revealed an enhanced accumulation of chloroplasts in the profile position along the anticlinal cell walls, in comparison to untreated plants. The rearrangement of chloroplasts in their response to lead ions in darkness was similar to the avoidance response of chloroplasts in plants treated with strong white light. Transmission electron microscopy X-ray microanalysis showed that intracellular chloroplast arrangement was independent of the location of Pb deposits, suggesting that lead causes redistribution of chloroplasts, which looks like a light-induced avoidance response, but is not a real avoidance response to the metal. Furthermore, a similar redistribution of chloroplasts in L. trisulca cells in darkness was observed also under the influence of exogenously applied hydrogen peroxide (H2O2). In addition, we detected an enhanced accumulation of endogenous H2O2 after treatment of plants with lead. Interestingly, H2O2-specific scavenger catalase partly abolished the Pb-induced chloroplast response. These results suggest that H2O2 can be involved in the avoidance-like movement of chloroplasts induced by lead. Analysis of photometric measurements revealed also strong inhibition (but not complete) of blue-light-induced chloroplast movements by lead. This inhibition may result from disturbances in the actin cytoskeleton, as we observed fragmentation and disappearance of actin filaments around chloroplasts. Results of this study show that the mechanisms of the toxic

Chloroplast redox homeostasis is essential for lateral root formation in Arabidopsis.

PubMed

Ferrández, Julia; González, Maricruz; Cejudo, Francisco Javier

2012-09-01

Redox regulation based on dithiol-disulphide interchange is an essential component of the control of chloroplast metabolism. In contrast to heterotrophic organisms, and non-photosynthetic plant tissues, chloroplast redox regulation relies on ferredoxin (Fd) reduced by the photosynthetic electron transport chain, thus being highly dependent on light. The finding of the NADPH-dependent thioredoxin reductase C (NTRC), a chloroplast-localized NTR with a joint thioredoxin domain, showed that NADPH is also used as source of reducing power for chloroplast redox homeostasis. Recently we have found that NTRC is also in plastids of non-photosynthetic tissues. Because these non-green plastids lack photochemical reactions, their redox homeostasis depends exclusively on NADPH produced from sugars and, thus, NTRC may play an essential role maintaining the redox homeostasis in these plastids. The fact that redox regulation occurs in any type of plastids raises the possibility that the functions of chloroplasts and non-green plastids, such as amyloplasts, are integrated to harmonize the growth of the different organs of the plant. To address this question, we generated Arabidopsis plants the redox homeostasis of which is recovered exclusively in chloroplasts, by leaf-specific expression of NTRC in the ntrc mutant, or exclusively in amyloplasts, by root-specific expression of NTRC. The analysis of these plants suggests that chloroplasts exert a pivotal role on plant growth, as expected because chloroplasts constitute the major source of nutrients and energy, derived from photosynthesis, for growth of heterotrophic tissues. However, NTRC deficiency causes impairment of auxin synthesis and lateral root formation. Interestingly, recovery of redox homeostasis of chloroplasts, but not of amyloplasts, was sufficient to restore wild type levels of lateral roots, showing the important signaling function of chloroplasts for the development of heterotrophic organs.

PDV2 has a dosage effect on chloroplast division in Arabidopsis.

PubMed

Chang, Ning; Sun, Qingqing; Li, Yiqiong; Mu, Yajuan; Hu, Jinglei; Feng, Yue; Liu, Xiaomin; Gao, Hongbo

2017-03-01

PDV2 has a dosage effect on chloroplast division in Arabidopsis thaliana , but this effect may vary in different plants. Chloroplasts have to be divided as plants grow to maintain an optimized number in the cell. Chloroplasts are divided by protein complexes across the double membranes from the stroma side to the cytosolic side. PDV2 is a chloroplast division protein on the chloroplast outer membrane. It recruits the dynamin-related GTPase ARC5 to the division site. The C-terminus of PDV2 and the C-terminus of ARC6 interact in the intermembrane space, which is important for the localization of PDV2. Previously, it was shown that overexpression of PDV2 can increase the division of chloroplasts in Arabidopsis and moss, so the authors concluded that PDV2 determines the rate of chloroplast division in land plants. PDV2 was also shown to inhibit the GTPase activity of ARC5 by in vitro experiment. These results look to be contradictory. Here, we identified a null allele of PDV2 in Arabidopsis and studied plants with different levels of PDV2. Our results suggested that the chloroplast division phenotype in Arabidopsis is sensitive to the level of PDV2, while this is not the case for ARC6. The level of PDV2 protein is reduced sharply in fast-growing leaves, while the level of ARC6 is not. The levels of PDV2 and ARC6 in several other plant species at different developmental stages were also investigated. The results indicated that their expression pattern varies in different species. Thus, PDV2 is an important positive factor of chloroplast division with an apparent dosage effect in Arabidopsis, but this effect for different chloroplast division proteins in different plants may vary.

Chloroplast Translation: Structural and Functional Organization, Operational Control, and Regulation[OPEN

PubMed Central

2018-01-01

Chloroplast translation is essential for cellular viability and plant development. Its positioning at the intersection of organellar RNA and protein metabolism makes it a unique point for the regulation of gene expression in response to internal and external cues. Recently obtained high-resolution structures of plastid ribosomes, the development of approaches allowing genome-wide analyses of chloroplast translation (i.e., ribosome profiling), and the discovery of RNA binding proteins involved in the control of translational activity have greatly increased our understanding of the chloroplast translation process and its regulation. In this review, we provide an overview of the current knowledge of the chloroplast translation machinery, its structure, organization, and function. In addition, we summarize the techniques that are currently available to study chloroplast translation and describe how translational activity is controlled and which cis-elements and trans-factors are involved. Finally, we discuss how translational control contributes to the regulation of chloroplast gene expression in response to developmental, environmental, and physiological cues. We also illustrate the commonalities and the differences between the chloroplast and bacterial translation machineries and the mechanisms of protein biosynthesis in these two prokaryotic systems. PMID:29610211

Mergers and acquisitions: malaria and the great chloroplast heist.

PubMed

McFadden, G I

2000-01-01

The origin of the relict chloroplast recently identified in malarial parasites has been mysterious. Several new papers suggest that the parasites obtained their chloroplasts in an ancient endosymbiotic event that also created some major algal groups.

Chloroplast-cytoplasmic interrelations involved in chloroplast development in Chlamydomonas reinhardi y-1: effect of selective depletion of chloroplast translates

PubMed Central

1980-01-01

Chlamydomonas reinhardi y-1 cells grown in the dark in the presence of chloramphenicol (CD cells) are depleted of photosynthetic membranes and 70S translates. These cells were found to be unable to synthesize chlorophyll in the light until chloroplast protein synthesis was resumed. On the other hand, CD cells acquired the capacity to partially green in the presence of cycloheximide. This greening was characterized by the development of photosynthetic activity, as demonstrated by light- dependent oxygen evolution of whole cells and by measurements of ribulose-1,5-bisphosphate carboxylase and fluorescence kinetics. The chlorophyll synthesized de novo during greening in the absence of 80S ribosomal activity was organized in chlorophyll-protein complexes, as ascertained by low-temperature fluorescence-emission spectra. The morphology of these cells appeared to be normal. A model has been proposed as a working hypothesis, which could account for the phenomena described above and previously reported data pertaining to chloroplast development. PMID:7419587

Mergers and acquisitions: malaria and the great chloroplast heist

PubMed Central

McFadden, Geoffrey I

2000-01-01

The origin of the relict chloroplast recently identified in malarial parasites has been mysterious. Several new papers suggest that the parasites obtained their chloroplasts in an ancient endosymbiotic event that also created some major algal groups. PMID:11178253

Multiple feedbacks between chloroplast and whole plant in the context of plant adaptation and acclimation to the environment

PubMed Central

Demmig-Adams, Barbara; Stewart, Jared J.; Adams, William W.

2014-01-01

This review focuses on feedback pathways that serve to match plant energy acquisition with plant energy utilization, and thereby aid in the optimization of chloroplast and whole-plant function in a given environment. First, the role of source–sink signalling in adjusting photosynthetic capacity (light harvesting, photochemistry and carbon fixation) to meet whole-plant carbohydrate demand is briefly reviewed. Contrasting overall outcomes, i.e. increased plant growth versus plant growth arrest, are described and related to respective contrasting environments that either do or do not present opportunities for plant growth. Next, new insights into chloroplast-generated oxidative signals, and their modulation by specific components of the chloroplast's photoprotective network, are reviewed with respect to their ability to block foliar phloem-loading complexes, and, thereby, affect both plant growth and plant biotic defences. Lastly, carbon export capacity is described as a newly identified tuning point that has been subjected to the evolution of differential responses in plant varieties (ecotypes) and species from different geographical origins with contrasting environmental challenges. PMID:24591724

Chloroplast overexpression of rice caffeic acid O-methyltransferase increases melatonin production in chloroplasts via the 5-methoxytryptamine pathway in transgenic rice plants.

PubMed

Choi, Geun-Hee; Lee, Hyoung Yool; Back, Kyoungwhan

2017-08-01

Recent analyses of the enzymatic features of various melatonin biosynthetic genes from bacteria, animals, and plants have led to the hypothesis that melatonin could be synthesized via the 5-methoxytryptamine (5-MT) pathway. 5-MT is known to be synthesized in vitro from serotonin by the enzymatic action of O-methyltransferases, including N-acetylserotonin methyltransferase (ASMT) and caffeic acid O-methyltransferase (COMT), leading to melatonin synthesis by the subsequent enzymatic reaction with serotonin N-acetyltransferase (SNAT). Here, we show that 5-MT was produced and served as a precursor for melatonin synthesis in plants. When rice seedlings were challenged with senescence treatment, 5-MT levels and melatonin production were increased in transgenic rice seedlings overexpressing the rice COMT in chloroplasts, while no such increases were observed in wild-type or transgenic seedlings overexpressing the rice COMT in the cytosol, suggesting a 5-MT transport limitation from the cytosol to chloroplasts. In contrast, cadmium treatment led to results different from those in senescence. The enhanced melatonin production was not observed in the chloroplast COMT lines relative over the cytosol COMT lines although 5-MT levels were equally induced in all genotypes upon cadmium treatment. The transgenic seedlings with enhanced melatonin in their chloroplasts exhibited improved seedling growth vs the wild type under continuous light conditions. This is the first report describing enhanced melatonin production in chloroplasts via the 5-MT pathway with the ectopic overexpression of COMT in chloroplasts in plants. © 2017 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.

The evolutionary fate of the chloroplast and nuclear rps16 genes as revealed through the sequencing and comparative analyses of four novel legume chloroplast genomes from Lupinus

PubMed Central

Keller, J.; Rousseau-Gueutin, M.; Martin, G.E.; Morice, J.; Boutte, J.; Coissac, E.; Ourari, M.; Aïnouche, M.; Salmon, A.; Cabello-Hurtado, F.

2017-01-01

Abstract The Fabaceae family is considered as a model system for understanding chloroplast genome evolution due to the presence of extensive structural rearrangements, gene losses and localized hypermutable regions. Here, we provide sequences of four chloroplast genomes from the Lupinus genus, belonging to the underinvestigated Genistoid clade. Notably, we found in Lupinus species the functional loss of the essential rps16 gene, which was most likely replaced by the nuclear rps16 gene that encodes chloroplast and mitochondrion targeted RPS16 proteins. To study the evolutionary fate of the rps16 gene, we explored all available plant chloroplast, mitochondrial and nuclear genomes. Whereas no plant mitochondrial genomes carry an rps16 gene, many plants still have a functional nuclear and chloroplast rps16 gene. Ka/Ks ratios revealed that both chloroplast and nuclear rps16 copies were under purifying selection. However, due to the dual targeting of the nuclear rps16 gene product and the absence of a mitochondrial copy, the chloroplast gene may be lost. We also performed comparative analyses of lupine plastomes (SNPs, indels and repeat elements), identified the most variable regions and examined their phylogenetic utility. The markers identified here will help to reveal the evolutionary history of lupines, Genistoids and closely related clades. PMID:28338826

Isolation of Intact Chloroplasts from Euglena gracilis by Zonal Centrifugation 1

PubMed Central

Vasconcelos, Aurea; Pollack, Marilyn; Mendiola, Leticia R.; Hoffmann, H.-P.; Brown, D. H.; Price, C. A.

1971-01-01

Chloroplasts were separated from Euglena gracilis by zonal centrifugation at low speed in density gradients of Ficoll or dextran. The chloroplasts were intact by the criteria of ultrastructure and their content of ribulose diphosphate carboxylase and soluble protein. The chloroplasts also contained ribosomes and ribosomal RNA uncontaminated by the corresponding cytoplasmic particles. Images PMID:16657599

The TOC complex: preprotein gateway to the chloroplast.

PubMed

Andrès, Charles; Agne, Birgit; Kessler, Felix

2010-06-01

Photosynthetic eukaryotes strongly depend on chloroplast metabolic pathways. Most if not all involve nuclear encoded proteins. These are synthesized as cytosolic preproteins with N-terminal, cleavable targeting sequences (transit peptide). Preproteins are imported by a major pathway composed of two proteins complexes: TOC and TIC (Translocon of the Outer and Inner membranes of the Chloroplasts, respectively). These selectively recognize the preproteins and facilitate their transport across the chloroplast envelope. The TOC core complex consists of three types of components, each belonging to a small family: Toc34, Toc75 and Toc159. Toc34 and Toc159 isoforms represent a subfamily of the GTPase superfamily. The members of the Toc34 and Toc159 subfamily act as GTP-dependent receptors at the chloroplast surface and distinct members of each occur in defined, substrate-specific TOC complexes. Toc75, a member of the Omp85 family, is conserved from prokaryotes and functions as the unique protein-conducting channel at the outer membrane. In this review we will describe the current state of knowledge regarding the composition and function of the TOC complex.

Isolation and Characterization of Chloroplast DNA from the Duckweed Spirodela oligorrhiza

PubMed Central

van Ee, Jan H.; Veld, Willem A. Man In'T; Planta, Rudi J.

1980-01-01

Chloroplast DNA of the duckweed Spirodela oligorrhiza, isolated by CsCl gradient centrifugation, was characterized by its buoyant density, guanine + cytosine content, melting behavior, circularity, and contour length. In all these characteristics, chloroplast DNA of S. oligorrhiza is similar to the chloroplast genomes of other higher plants, except that it has a significantly larger size. Images PMID:16661479

Highly effective sequencing whole chloroplast genomes of angiosperms by nine novel universal primer pairs.

PubMed

Yang, Jun-Bo; Li, De-Zhu; Li, Hong-Tao

2014-09-01

Chloroplast genomes supply indispensable information that helps improve the phylogenetic resolution and even as organelle-scale barcodes. Next-generation sequencing technologies have helped promote sequencing of complete chloroplast genomes, but compared with the number of angiosperms, relatively few chloroplast genomes have been sequenced. There are two major reasons for the paucity of completely sequenced chloroplast genomes: (i) massive amounts of fresh leaves are needed for chloroplast sequencing and (ii) there are considerable gaps in the sequenced chloroplast genomes of many plants because of the difficulty of isolating high-quality chloroplast DNA, preventing complete chloroplast genomes from being assembled. To overcome these obstacles, all known angiosperm chloroplast genomes available to date were analysed, and then we designed nine universal primer pairs corresponding to the highly conserved regions. Using these primers, angiosperm whole chloroplast genomes can be amplified using long-range PCR and sequenced using next-generation sequencing methods. The primers showed high universality, which was tested using 24 species representing major clades of angiosperms. To validate the functionality of the primers, eight species representing major groups of angiosperms, that is, early-diverging angiosperms, magnoliids, monocots, Saxifragales, fabids, malvids and asterids, were sequenced and assembled their complete chloroplast genomes. In our trials, only 100 mg of fresh leaves was used. The results show that the universal primer set provided an easy, effective and feasible approach for sequencing whole chloroplast genomes in angiosperms. The designed universal primer pairs provide a possibility to accelerate genome-scale data acquisition and will therefore magnify the phylogenetic resolution and species identification in angiosperms. © 2014 John Wiley & Sons Ltd.

Chloroplasts do not have a polarity for light-induced accumulation movement.

PubMed

Tsuboi, Hidenori; Yamashita, Hiroko; Wada, Masamitsu

2009-01-01

Chloroplast photorelocation movement in green plants is generally mediated by blue light. However, in cryptogam plants, including ferns, mosses, and algae, both red light and blue light are effective. Although the photoreceptors required for this phenomenon have been identified, the mechanisms underlying this movement response are not yet known. In order to analyze this response in more detail, chloroplast movement was induced in dark-adapted Adiantum capillus-veneris gametophyte cells by partial cell irradiation with a microbeam of red and/or blue light. In each case, chloroplasts were found to move toward the microbeam-irradiated area. A second microbeam was also applied to the cell at a separate location before the chloroplasts had reached the destination of the first microbeam. Under these conditions, chloroplasts were found to change their direction of movement without turning and move toward the second microbeam-irradiated area after a lag time of a few minutes. These findings indicate that chloroplasts can move in any direction and do not exhibit a polarity for chloroplast accumulation movement. This phenomenon was analyzed in detail in Adiantum and subsequently confirmed in Arabidopsis thaliana palisade cells. Interestingly, the lag time for direction change toward the second microbeam in Adiantum was longer in the red light than in the blue light. However, the reason for this discrepancy is not yet understood.

The evolutionary fate of the chloroplast and nuclear rps16 genes as revealed through the sequencing and comparative analyses of four novel legume chloroplast genomes from Lupinus.

PubMed

Keller, J; Rousseau-Gueutin, M; Martin, G E; Morice, J; Boutte, J; Coissac, E; Ourari, M; Aïnouche, M; Salmon, A; Cabello-Hurtado, F; Aïnouche, A

2017-08-01

The Fabaceae family is considered as a model system for understanding chloroplast genome evolution due to the presence of extensive structural rearrangements, gene losses and localized hypermutable regions. Here, we provide sequences of four chloroplast genomes from the Lupinus genus, belonging to the underinvestigated Genistoid clade. Notably, we found in Lupinus species the functional loss of the essential rps16 gene, which was most likely replaced by the nuclear rps16 gene that encodes chloroplast and mitochondrion targeted RPS16 proteins. To study the evolutionary fate of the rps16 gene, we explored all available plant chloroplast, mitochondrial and nuclear genomes. Whereas no plant mitochondrial genomes carry an rps16 gene, many plants still have a functional nuclear and chloroplast rps16 gene. Ka/Ks ratios revealed that both chloroplast and nuclear rps16 copies were under purifying selection. However, due to the dual targeting of the nuclear rps16 gene product and the absence of a mitochondrial copy, the chloroplast gene may be lost. We also performed comparative analyses of lupine plastomes (SNPs, indels and repeat elements), identified the most variable regions and examined their phylogenetic utility. The markers identified here will help to reveal the evolutionary history of lupines, Genistoids and closely related clades. © The Author 2017. Published by Oxford University Press on behalf of Kazusa DNA Research Institute.

Arabidopsis chloroplast chaperonin 10 is a calmodulin-binding protein

NASA Technical Reports Server (NTRS)

Yang, T.; Poovaiah, B. W.

2000-01-01

Calcium regulates diverse cellular activities in plants through the action of calmodulin (CaM). By using (35)S-labeled CaM to screen an Arabidopsis seedling cDNA expression library, a cDNA designated as AtCh-CPN10 (Arabidopsis thaliana chloroplast chaperonin 10) was cloned. Chloroplast CPN10, a nuclear-encoded protein, is a functional homolog of E. coli GroES. It is believed that CPN60 and CPN10 are involved in the assembly of Rubisco, a key enzyme involved in the photosynthetic pathway. Northern analysis revealed that AtCh-CPN10 is highly expressed in green tissues. The recombinant AtCh-CPN10 binds to CaM in a calcium-dependent manner. Deletion mutants revealed that there is only one CaM-binding site in the last 31 amino acids of the AtCh-CPN10 at the C-terminal end. The CaM-binding region in AtCh-CPN10 has higher homology to other chloroplast CPN10s in comparison to GroES and mitochondrial CPN10s, suggesting that CaM may only bind to chloroplast CPN10s. Furthermore, the results also suggest that the calcium/CaM messenger system is involved in regulating Rubisco assembly in the chloroplast, thereby influencing photosynthesis. Copyright 2000 Academic Press.

Expression of non-toxic mutant of Escherichia coli heat-labile enterotoxin in tobacco chloroplasts.

PubMed

Kang, Tae-Jin; Han, So-Chon; Kim, Mi-Young; Kim, Young-Sook; Yang, Moon-Sik

2004-11-01

Chloroplast transformation systems offer unique advantages in biotechnology, including high level of foreign gene expression, maternal inheritance, and polycistronic expression. We studied chloroplast expression of LTK63 (change Ser-->Lys at position 63 in the A subunit) which is the mutant of Escherichia coli heat-labile toxin. LTK63 is devoid of any toxic activity, but still retains its mucosal adjuvanticity. The LTK63 was cloned into chloroplast targeting vector and transformed to tobacco chloroplasts by particle bombardment. PCR and Southern blot analyses confirmed stable homologous recombination of the LTK63 gene into the chloroplast genome. The amount of LTK63 protein detected in tobacco chloroplasts was approximately 3.7% of the total soluble protein. The GM1-ganglioside binding assay confirmed that chloroplast-synthesized LTB of LTK63 binds to the intestinal membrane GM1-ganglioside receptor. Thus, the expression of LTK63 in chloroplasts provides a potential route toward the development of a plant-based edible vaccine for high expression system and environmentally friendly approach.

Computer modeling of electron and proton transport in chloroplasts.

PubMed

Tikhonov, Alexander N; Vershubskii, Alexey V

2014-07-01

Photosynthesis is one of the most important biological processes in biosphere, which provides production of organic substances from atmospheric CO2 and water at expense of solar energy. In this review, we contemplate computer models of oxygenic photosynthesis in the context of feedback regulation of photosynthetic electron transport in chloroplasts, the energy-transducing organelles of the plant cell. We start with a brief overview of electron and proton transport processes in chloroplasts coupled to ATP synthesis and consider basic regulatory mechanisms of oxygenic photosynthesis. General approaches to computer simulation of photosynthetic processes are considered, including the random walk models of plastoquinone diffusion in thylakoid membranes and deterministic approach to modeling electron transport in chloroplasts based on the mass action law. Then we focus on a kinetic model of oxygenic photosynthesis that includes key stages of the linear electron transport, alternative pathways of electron transfer around photosystem I (PSI), transmembrane proton transport and ATP synthesis in chloroplasts. This model includes different regulatory processes: pH-dependent control of the intersystem electron transport, down-regulation of photosystem II (PSII) activity (non-photochemical quenching), the light-induced activation of the Bassham-Benson-Calvin (BBC) cycle. The model correctly describes pH-dependent feedback control of electron transport in chloroplasts and adequately reproduces a variety of experimental data on induction events observed under different experimental conditions in intact chloroplasts (variations of CO2 and O2 concentrations in atmosphere), including a complex kinetics of P700 (primary electron donor in PSI) photooxidation, CO2 consumption in the BBC cycle, and photorespiration. Finally, we describe diffusion-controlled photosynthetic processes in chloroplasts within the framework of the model that takes into account complex architecture of

Sonication-based isolation and enrichment of Chlorella protothecoides chloroplasts for illumina genome sequencing

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

Angelova, Angelina; Park, Sang-Hycuk; Kyndt, John

2013-09-01

With the increasing world demand for biofuel, a number of oleaginous algal species are being considered as renewable sources of oil. Chlorella protothecoides Krüger synthesizes triacylglycerols (TAGs) as storage compounds that can be converted into renewable fuel utilizing an anabolic pathway that is poorly understood. The paucity of algal chloroplast genome sequences has been an important constraint to chloroplast transformation and for studying gene expression in TAGs pathways. In this study, the intact chloroplasts were released from algal cells using sonication followed by sucrose gradient centrifugation, resulting in a 2.36-fold enrichment of chloroplasts from C. protothecoides, based on qPCR analysis.more » The C. protothecoides chloroplast genome (cpDNA) was determined using the Illumina HiSeq 2000 sequencing platform and found to be 84,576 Kb in size (8.57 Kb) in size, with a GC content of 30.8 %. This is the first report of an optimized protocol that uses a sonication step, followed by sucrose gradient centrifugation, to release and enrich intact chloroplasts from a microalga (C. prototheocoides) of sufficient quality to permit chloroplast genome sequencing with high coverage, while minimizing nuclear genome contamination. The approach is expected to guide chloroplast isolation from other oleaginous algal species for a variety of uses that benefit from enrichment of chloroplasts, ranging from biochemical analysis to genomics studies.« less

Characterization of the snowy cotyledon 1 mutant of Arabidopsis thaliana: the impact of chloroplast elongation factor G on chloroplast development and plant vitality.

PubMed

Albrecht, Verónica; Ingenfeld, Anke; Apel, Klaus

2006-03-01

During seedling development chloroplast formation marks the transition from heterotrophic to autotrophic growth. The development and activity of chloroplasts may differ in cotyledons that initially serve as a storage organ and true leaves whose primary function is photosynthesis. A genetic screen was used for the identification of genes that affect selectively chloroplast function in cotyledons of Arabidopsis thaliana. Several mutants exhibiting pale cotyledons and green true leaves were isolated and dubbed snowy cotyledon (sco). One of the mutants, sco1, was characterized in more detail. The mutated gene was identified using map-based cloning. The mutant contains a point mutation in a gene encoding the chloroplast elongation factor G, leading to an amino acid exchange within the predicted 70S ribosome-binding domain. The mutation results in a delay in the onset of germination. At this early developmental stage embryos still contain undifferentiated proplastids, whose proper function seems necessary for seed germination. In light-grown sco1 seedlings the greening of cotyledons is severely impaired, whereas the following true leaves develop normally as in wild-type plants. Despite this apparent similarity of chloroplast development in true leaves of mutant and wild-type plants various aspects of mature plant development are also affected by the sco1 mutation such as the onset of flowering, the growth rate, and seed production. The onset of senescence in the mutant and the wild-type plants occurs, however, at the same time, suggesting that in the mutant this particular developmental step does not seem to suffer from reduced protein translation efficiency in chloroplasts.

Unique localization of the plastid-specific ribosomal proteins in the chloroplast ribosome small subunit provides mechanistic insights into the chloroplastic translation

PubMed Central

Ahmed, Tofayel; Shi, Jian

2017-01-01

Abstract Chloroplastic translation is mediated by a bacterial-type 70S chloroplast ribosome. During the evolution, chloroplast ribosomes have acquired five plastid-specific ribosomal proteins or PSRPs (cS22, cS23, bTHXc, cL37 and cL38) which have been suggested to play important regulatory roles in translation. However, their exact locations on the chloroplast ribosome remain elusive due to lack of a high-resolution structure, hindering our progress to understand their possible roles. Here we present a cryo-EM structure of the 70S chloroplast ribosome from spinach resolved to 3.4 Å and focus our discussion mainly on the architecture of the 30S small subunit (SSU) which is resolved to 3.7 Å. cS22 localizes at the SSU foot where it seems to compensate for the deletions in 16S rRNA. The mRNA exit site is highly remodeled due to the presence of cS23 suggesting an alternative mode of translation initiation. bTHXc is positioned at the SSU head and appears to stabilize the intersubunit bridge B1b during thermal fluctuations. The translation factor plastid pY binds to the SSU on the intersubunit side and interacts with the conserved nucleotide bases involved in decoding. Most of the intersubunit bridges are conserved compared to the bacteria, except for a new bridge involving uL2c and bS6c. PMID:28582576

Uncovering the Protein Lysine and Arginine Methylation Network in Arabidopsis Chloroplasts

PubMed Central

Mininno, Morgane; Brugière, Sabine; Gilgen, Annabelle; Ma, Sheng; Mazzoleni, Meryl; Gigarel, Océane; Martin-Laffon, Jacqueline; Ferro, Myriam; Ravanel, Stéphane

2014-01-01

Post-translational modification of proteins by the addition of methyl groups to the side chains of Lys and Arg residues is proposed to play important roles in many cellular processes. In plants, identification of non-histone methylproteins at a cellular or subcellular scale is still missing. To gain insights into the extent of this modification in chloroplasts we used a bioinformatics approach to identify protein methyltransferases targeted to plastids and set up a workflow to specifically identify Lys and Arg methylated proteins from proteomic data used to produce the Arabidopsis chloroplast proteome. With this approach we could identify 31 high-confidence Lys and Arg methylation sites from 23 chloroplastic proteins, of which only two were previously known to be methylated. These methylproteins are split between the stroma, thylakoids and envelope sub-compartments. They belong to essential metabolic processes, including photosynthesis, and to the chloroplast biogenesis and maintenance machinery (translation, protein import, division). Also, the in silico identification of nine protein methyltransferases that are known or predicted to be targeted to plastids provided a foundation to build the enzymes/substrates relationships that govern methylation in chloroplasts. Thereby, using in vitro methylation assays with chloroplast stroma as a source of methyltransferases we confirmed the methylation sites of two targets, plastid ribosomal protein L11 and the β-subunit of ATP synthase. Furthermore, a biochemical screening of recombinant chloroplastic protein Lys methyltransferases allowed us to identify the enzymes involved in the modification of these substrates. The present study provides a useful resource to build the methyltransferases/methylproteins network and to elucidate the role of protein methylation in chloroplast biology. PMID:24748391

Perturbations of carotenoid and tetrapyrrole biosynthetic pathways result in differential alterations in chloroplast function and plastid signaling.

PubMed

Park, Joon-Heum; Jung, Sunyo

2017-01-22

In this study, we used the biosynthetic inhibitors of carotenoid and tetrapyrrole biosynthetic pathways, norflurazon (NF) and oxyfluorfen (OF), as tools to gain insight into mechanisms of photooxidation in rice plants. NF resulted in bleaching symptom on leaves of the treated plants, whereas OF treatment developed a fast symptom of an apparent necrotic phenotype. Both plants exhibited decreases in photosynthetic efficiency, as indicated by F v /F m . NF caused severe disruption in thylakoid membranes, whereas OF-treated plants exhibited disruption of chloroplast envelope and plasma membrane. Levels of Lhca and Lhcb proteins in photosystem I (PSI) and PSII were reduced by photooxidative stress in NF- and OF-treated plants, with a greater decrease in NF plants. The down-regulation of nuclear-encoded photosynthesis genes Lhcb and rbcS was also found in both NF- and OF-treated plants, whereas plastid-encoded photosynthetic genes including RbcL, PsaC, and PsbD accumulated normally in NF plants but decreased drastically in OF plants. This proposes that the plastids in NF plants retain their potential to develop thylakoid membranes and that photobleaching is mainly controlled by nuclear genes. Distinct photooxidation patterns between NF- and OF-treated plants developed differential signaling, which might enable the plant to coordinate the expression of photosynthetic genes from the nuclear and plastidic genomes. Copyright © 2016 Elsevier Inc. All rights reserved.

Protein targeting and integration signal for the chloroplastic outer envelope membrane.

PubMed Central

Li, H M; Chen, L J

1996-01-01

Most proteins in chloroplasts are encoded by the nuclear genome and synthesized in the cytosol. With the exception of most quter envelope membrane proteins, nuclear-encoded chloroplastic proteins are synthesized with N-terminal extensions that contain the chloroplast targeting information of these proteins. Most outer membrane proteins, however, are synthesized without extensions in the cytosol. Therefore, it is not clear where the chloroplastic outer membrane targeting information resides within these polypeptides. We have analyzed a chloroplastic outer membrane protein, OEP14 (outer envelope membrane protein of 14 kD, previously named OM14), and localized its outer membrane targeting and integration signal to the first 30 amino acids of the protein. This signal consists of a positively charged N-terminal portion followed by a hydrophobic core, bearing resemblance to the signal peptides of proteins targeted to the endoplasmic reticulum. However, a chimeric protein containing this signal fused to a passenger protein did not integrate into the endoplasmic reticulum membrane. Furthermore, membrane topology analysis indicated that the signal inserts into the chloroplastic outer membrane in an orientation opposite to that predicted by the "positive inside" rule. PMID:8953775

Rust fungal effectors mimic host transit peptides to translocate into chloroplasts.

PubMed

Petre, Benjamin; Lorrain, Cécile; Saunders, Diane G O; Win, Joe; Sklenar, Jan; Duplessis, Sébastien; Kamoun, Sophien

2016-04-01

Parasite effector proteins target various host cell compartments to alter host processes and promote infection. How effectors cross membrane-rich interfaces to reach these compartments is a major question in effector biology. Growing evidence suggests that effectors use molecular mimicry to subvert host cell machinery for protein sorting. We recently identified chloroplast-targeted protein 1 (CTP1), a candidate effector from the poplar leaf rust fungus Melampsora larici-populina that carries a predicted transit peptide and accumulates in chloroplasts and mitochondria. Here, we show that the CTP1 transit peptide is necessary and sufficient for accumulation in the stroma of chloroplasts. CTP1 is part of a Melampsora-specific family of polymorphic secreted proteins. Two members of that family, CTP2 and CTP3, also translocate in chloroplasts in an N-terminal signal-dependent manner. CTP1, CTP2 and CTP3 are cleaved when they accumulate in chloroplasts, while they remain intact when they do not translocate into chloroplasts. Our findings reveal that fungi have evolved effector proteins that mimic plant-specific sorting signals to traffic within plant cells. © 2015 John Wiley & Sons Ltd.

Analysis of Protein Interactions at Native Chloroplast Membranes by Ellipsometry

PubMed Central

Kriechbaumer, Verena; Nabok, Alexei; Mustafa, Mohd K.; Al-Ammar, Rukaiah; Tsargorodskaya, Anna; Smith, David P.; Abell, Ben M.

2012-01-01

Membrane bound receptors play vital roles in cell signaling, and are the target for many drugs, yet their interactions with ligands are difficult to study by conventional techniques due to the technical difficulty of monitoring these interactions in lipid environments. In particular, the ability to analyse the behaviour of membrane proteins in their native membrane environment is limited. Here, we have developed a quantitative approach to detect specific interactions between low-abundance chaperone receptors within native chloroplast membranes and their soluble chaperone partners. Langmuir-Schaefer film deposition was used to deposit native chloroplasts onto gold-coated glass slides, and interactions between the molecular chaperones Hsp70 and Hsp90 and their receptors in the chloroplast membranes were detected and quantified by total internal reflection ellipsometry (TIRE). We show that native chloroplast membranes deposited on gold-coated glass slides using Langmuir-Schaefer films retain functional receptors capable of binding chaperones with high specificity and affinity. Taking into account the low chaperone receptor abundance in native membranes, these binding properties are consistent with data generated using soluble forms of the chloroplast chaperone receptors, OEP61 and Toc64. Therefore, we conclude that chloroplasts have the capacity to selectively bind chaperones, consistent with the notion that chaperones play an important role in protein targeting to chloroplasts. Importantly, this method of monitoring by TIRE does not require any protein labelling. This novel combination of techniques should be applicable to a wide variety of membranes and membrane protein receptors, thus presenting the opportunity to quantify protein interactions involved in fundamental cellular processes, and to screen for drugs that target membrane proteins. PMID:22479632

The demise of chloroplast DNA in Arabidopsis.

PubMed

Rowan, Beth A; Oldenburg, Delene J; Bendich, Arnold J

2004-09-01

Although it might be expected that chloroplast DNA (cpDNA) would be stably maintained in mature leaves, we report the surprising observation that cpDNA levels decline during plastid development in Arabidopsis thaliana (Col.) until most of the leaves contain little or no DNA long before the onset of senescence. We measured the cpDNA content in developing cotyledons, rosette leaves, and cauline leaves. The amount of cpDNA per chloroplast decreases as the chloroplasts develop, reaching undetectable levels in mature leaves. In young cauline leaves, most individual molecules of cpDNA are found in complex, branched forms. In expanded cauline leaves, cpDNA is present in smaller branched forms only at the base of the leaf and is virtually absent in the distal part of the leaf. We conclude that photosynthetic activity may persist long after the demise of the cpDNA. Copyright 2004 Springer-Verlag

Dissipation of the Proton Electrochemical Potential in Intact and Lysed Chloroplasts 1

PubMed Central

Nishio, John N.; Whitmarsh, John

1991-01-01

Effective ionophore:chlorophyll ratios were determined for various ionophores that decrease the electrical potential across thylakoid membranes in intact and hypo-osmotically lysed chloroplasts isolated from spinach (Spinacia oleracea). The efficacy of gramicidin D, valinomycin, carbonylcyanide m-chlorophenylhydrazone, and dicyclohexano-18-crown-6 in collapsing the electrical potential was determined spectrophotometrically by the decay half-time of the absorbance change at 518 nanometers induced by a saturating, single turnover flash. The results show that the effectiveness of the ionophores in collapsing the electrical potential in intact and lysed chloroplasts depends on the amount of ionophore-accessible membrane in the assay medium. Only gramicidin exhibited a significant difference in efficacy between intact and lysed chloroplasts. The ratio of gramicidin to chlorophyll required to collapse the electrical potential was more than 50 times higher in intact chloroplasts than in lysed chloroplasts. The efficacy of carbonylcyanide m-chlorophenylhydrazone was significantly reduced in the presence of bovine serum albumin. The other ionophores tested maintained their potency in the presence of bovine serum albumin. Valinomycin was the most effective ionophore tested for collapsing the electrical potential in intact chloroplasts, whereas gramicidin was the most potent ionophore in lysed chloroplasts. The significance of the ionophore:chlorophyll ratios required to collapse the electrical potential is discussed with regard to bioenergetic studies, especially those that examine the contribution of the transmembrane electrochemical potential to protein transport into chloroplasts. PMID:16668015

Promotion of chloroplast proliferation upon enhanced post-mitotic cell expansion in leaves.

PubMed

Kawade, Kensuke; Horiguchi, Gorou; Ishikawa, Naoko; Hirai, Masami Yokota; Tsukaya, Hirokazu

2013-09-28

Leaves are determinate organs; hence, precise control of cell proliferation and post-mitotic cell expansion is essential for their growth. A defect in cell proliferation often triggers enhanced post-mitotic cell expansion in leaves. This phenomenon is referred to as 'compensation'. Several lines of evidence from studies on compensation have shown that cell proliferation and post-mitotic cell expansion are coordinately regulated during leaf development. Therefore, compensation has attracted much attention to the mechanisms for leaf growth. However, our understanding of compensation at the subcellular level remains limited because studies of compensation have focused mainly on cellular-level phenotypes. Proper leaf growth requires quantitative control of subcellular components in association with cellular-level changes. To gain insight into the subcellular aspect of compensation, we investigated the well-known relationship between cell area and chloroplast number per cell in compensation-exhibiting lines, and asked whether chloroplast proliferation is modulated in response to the induction of compensation. We first established a convenient and reliable method for observation of chloroplasts in situ. Using this method, we analyzed Arabidopsis thaliana mutants fugu5 and angustifolia3 (an3), and a transgenic line KIP-RELATED PROTEIN2 overexpressor (KRP2 OE), which are known to exhibit typical features of compensation. We here showed that chloroplast number per cell increased in the subepidermal palisade tissue of these lines. We analyzed tetraploidized wild type, fugu5, an3 and KRP2 OE, and found that cell area itself, but not nuclear ploidy, is a key parameter that determines the activity of chloroplast proliferation. In particular, in the case of an3, we uncovered that promotion of chloroplast proliferation depends on the enhanced post-mitotic cell expansion. The expression levels of chloroplast proliferation-related genes are similar to or lower than that in the wild

Promotion of chloroplast proliferation upon enhanced post-mitotic cell expansion in leaves

PubMed Central

2013-01-01

Background Leaves are determinate organs; hence, precise control of cell proliferation and post-mitotic cell expansion is essential for their growth. A defect in cell proliferation often triggers enhanced post-mitotic cell expansion in leaves. This phenomenon is referred to as ‘compensation’. Several lines of evidence from studies on compensation have shown that cell proliferation and post-mitotic cell expansion are coordinately regulated during leaf development. Therefore, compensation has attracted much attention to the mechanisms for leaf growth. However, our understanding of compensation at the subcellular level remains limited because studies of compensation have focused mainly on cellular-level phenotypes. Proper leaf growth requires quantitative control of subcellular components in association with cellular-level changes. To gain insight into the subcellular aspect of compensation, we investigated the well-known relationship between cell area and chloroplast number per cell in compensation-exhibiting lines, and asked whether chloroplast proliferation is modulated in response to the induction of compensation. Results We first established a convenient and reliable method for observation of chloroplasts in situ. Using this method, we analyzed Arabidopsis thaliana mutants fugu5 and angustifolia3 (an3), and a transgenic line KIP-RELATED PROTEIN2 overexpressor (KRP2 OE), which are known to exhibit typical features of compensation. We here showed that chloroplast number per cell increased in the subepidermal palisade tissue of these lines. We analyzed tetraploidized wild type, fugu5, an3 and KRP2 OE, and found that cell area itself, but not nuclear ploidy, is a key parameter that determines the activity of chloroplast proliferation. In particular, in the case of an3, we uncovered that promotion of chloroplast proliferation depends on the enhanced post-mitotic cell expansion. The expression levels of chloroplast proliferation-related genes are similar to or

Chloroplast avoidance movement is not functional in plants grown under strong sunlight.

PubMed

Higa, Takeshi; Wada, Masamitsu

2016-04-01

Chloroplast movement in nine climbing plant species was investigated. It is thought that chloroplasts generally escape from strong light to avoid photodamage but accumulate towards weak light to perform photosynthesis effectively. Unexpectedly, however, the leaves of climbing plants grown under strong sunlight showed very low or no chloroplast photorelocation responses to either weak or strong blue light when detected by red light transmittance through leaves. Direct observations of Cayratia japonica leaves, for example, revealed that the average number of chloroplasts in upper periclinal walls of palisade tissue cells was only 1.2 after weak blue-light irradiation and almost all of the chloroplasts remained at the anticlinal wall, the state of chloroplast avoidance response. The leaves grown under strong light have thin and columnar palisade tissue cells comparing with the leaves grown under low light. Depending on our analyses and our schematic model, the thinner cells in a unit leaf area have a wider total plasma membrane area, such that more chloroplasts can exist on the plasma membrane in the thinner cells than in the thicker cells in a unit leaf-area basis. The same strategy might be used in other plant leaves grown under direct sunlight. © 2015 John Wiley & Sons Ltd.

Dated tribe-wide whole chloroplast genome phylogeny indicates recurrent hybridizations within Triticeae.

PubMed

Bernhardt, Nadine; Brassac, Jonathan; Kilian, Benjamin; Blattner, Frank R

2017-06-16

Triticeae, the tribe of wheat grasses, harbours the cereals barley, rye and wheat and their wild relatives. Although economically important, relationships within the tribe are still not understood. We analysed the phylogeny of chloroplast lineages among nearly all monogenomic Triticeae taxa and polyploid wheat species aiming at a deeper understanding of the tribe's evolution. We used on- and off-target reads of a target-enrichment experiment followed by Illumina sequencing. The read data was used to assemble the plastid locus ndhF for 194 individuals and the whole chloroplast genome for 183 individuals, representing 53 Triticeae species and 15 genera. We conducted Bayesian and multispecies coalescent analyses to infer relationships and estimate divergence times of the taxa. We present the most comprehensive dated Triticeae chloroplast phylogeny and review previous hypotheses in the framework of our results. Monophyly of Triticeae chloroplasts could not be confirmed, as either Bromus or Psathyrostachys captured a chloroplast from a lineage closely related to a Bromus-Triticeae ancestor. The most recent common ancestor of Triticeae occurred approximately between ten and 19 million years ago. The comparison of the chloroplast phylogeny with available nuclear data in several cases revealed incongruences indicating past hybridizations. Recent events of chloroplast capture were detected as individuals grouped apart from con-specific accessions in otherwise monopyhletic groups.

The complete chloroplast genome of common walnut (Juglans regia)

Treesearch

Yiheng ​Hu; Keith E. Woeste; Meng Dang; Tao Zhou; Xiaojia Feng; Guifang Zhao; Zhanlin Liu; Zhonghu Li; Peng Zhao

2016-01-01

Common walnut (Juglans regia L.) is cultivated in temperate regions worldwide for its wood and nuts. The complete chloroplast genome of J. regia was sequenced using the Illumina MiSeq platform. This is the first complete chloroplast sequence for the Juglandaceae, a family that includes numerous species of economic importance....

Eukaryotic Hsp70 chaperones in the intermembrane space of chloroplasts.

PubMed

Bionda, Tihana; Gross, Lucia E; Becker, Thomas; Papasotiriou, Dimitrios G; Leisegang, Matthias S; Karas, Michael; Schleiff, Enrico

2016-03-01

Multiple eukaryotic Hsp70 typically localized in the cytoplasm are also distributed to the intermembrane space of chloroplasts and might thereby represent the missing link in energizing protein translocation. Protein translocation into organelles is a central cellular process that is tightly regulated. It depends on signals within the preprotein and on molecular machines catalyzing the process. Molecular chaperones participate in transport and translocation of preproteins into organelles to control folding and to provide energy for the individual steps. While most of the processes are explored and the components are identified, the transfer of preproteins into and across the intermembrane space of chloroplasts is not yet understood. The existence of an energy source in this compartment is discussed, because the required transit peptide length for successful translocation into chloroplasts is shorter than that found for mitochondria where energy is provided exclusively by matrix chaperones. Furthermore, a cytosolic-type Hsp70 homologue was proposed as component of the chloroplast translocon in the intermembrane space energizing the initial translocation. The molecular identity of such intermembrane space localized Hsp70 remained unknown, which led to a controversy concerning its existence. We identified multiple cytosolic Hsp70s by mass spectrometry on isolated, thermolysin-treated Medicago sativa chloroplasts. The localization of these Hsp70s of M. sativa or Arabidopsis thaliana in the intermembrane space was confirmed by a self-assembly GFP-based in vivo system. The localization of cytosolic Hsp70s in the stroma of chloroplasts or different mitochondrial compartments could not be observed. Similarly, we could not identify any cytosolic Hsp90 in the intermembrane space of chloroplast. With respect to our results we discuss the possible targeting and function of the Hsp70 found in the intermembrane space.

A DIRECT LIGHT EFFECT ON MAINTAINING PHOTOSYNTHETIC ACTIVITY OF NITELLA CHLOROPLASTS

PubMed Central

Craig, I. W.; Gibor, A.

1970-01-01

The chloroplasts of internodal cells of Nitella are fixed to a stationary layer of cytoplasm whereas the nuclei and most of the cytoplasm stream along the longitudinal axis. Isolated internodal cells were maintained for several days with half the cell kept in the dark, the other half kept under continuous light. Photosynthetic activity of the cells was checked by placing the cell evenly illuminated in a 14CO2 atmosphere. Chloroplasts of the previously dark half of the cell were found to fix only half as much CO2 as the chloroplasts which were continuously illuminated. These results are discussed in relation to the possible direct effect of light on biosynthetic reactions of mature chloroplasts. PMID:5411077

The Chloroplast atpA Gene Cluster in Chlamydomonas reinhardtii1

PubMed Central

Drapier, Dominique; Suzuki, Hideki; Levy, Haim; Rimbault, Blandine; Kindle, Karen L.; Stern, David B.; Wollman, Francis-André

1998-01-01

Most chloroplast genes in vascular plants are organized into polycistronic transcription units, which generate a complex pattern of mono-, di-, and polycistronic transcripts. In contrast, most Chlamydomonas reinhardtii chloroplast transcripts characterized to date have been monocistronic. This paper describes the atpA gene cluster in the C. reinhardtii chloroplast genome, which includes the atpA, psbI, cemA, and atpH genes, encoding the α-subunit of the coupling-factor-1 (CF1) ATP synthase, a small photosystem II polypeptide, a chloroplast envelope membrane protein, and subunit III of the CF0 ATP synthase, respectively. We show that promoters precede the atpA, psbI, and atpH genes, but not the cemA gene, and that cemA mRNA is present only as part of di-, tri-, or tetracistronic transcripts. Deletions introduced into the gene cluster reveal, first, that CF1-α can be translated from di- or polycistronic transcripts, and, second, that substantial reductions in mRNA quantity have minimal effects on protein synthesis rates. We suggest that posttranscriptional mRNA processing is common in C. reinhardtii chloroplasts, permitting the expression of multiple genes from a single promoter. PMID:9625716

Chloroplast behaviour and interactions with other organelles in Arabidopsis thaliana pavement cells.

PubMed

Barton, Kiah A; Wozny, Michael R; Mathur, Neeta; Jaipargas, Erica-Ashley; Mathur, Jaideep

2018-01-29

Chloroplasts are a characteristic feature of green plants. Mesophyll cells possess the majority of chloroplasts and it is widely believed that, with the exception of guard cells, the epidermal layer in most higher plants does not contain chloroplasts. However, recent observations on Arabidopsis thaliana have shown a population of chloroplasts in pavement cells that are smaller than mesophyll chloroplasts and have a high stroma to grana ratio. Here, using stable transgenic lines expressing fluorescent proteins targeted to the plastid stroma, plasma membrane, endoplasmic reticulum, tonoplast, nucleus, mitochondria, peroxisomes, F-actin and microtubules, we characterize the spatiotemporal relationships between the pavement cell chloroplasts (PCCs) and their subcellular environment. Observations on the PCCs suggest a source-sink relationship between the epidermal and the mesophyll layers, and experiments with the Arabidopsis mutants glabra2 ( gl2 ) and immutans ( im ), which show altered epidermal plastid development, underscored their developmental plasticity. Our findings lay down the foundation for further investigations aimed at understanding the precise role and contributions of PCCs in plant interactions with the environment. © 2018. Published by The Company of Biologists Ltd.

Identification of the triazine receptor protein as a chloroplast gene product

PubMed Central

Steinback, Katherine E.; McIntosh, Lee; Bogorad, Lawrence; Arntzen, Charles J.

1981-01-01

The triazine herbicides inhibit photosynthesis by blocking electron transport at the second stable electron acceptor of photosystem II. This electron transport component of chloroplast thylakoid membranes is a protein-plastoquinone complex termed “B.” The polypeptide that is believed to be a component of the B complex has recently been identified as a 32- to 34-kilo-dalton polypeptide by using a photoaffinity labeling probe, azido-[14C]atrazine. A 34-kilodalton polypeptide of pea chloroplasts rapidly incorporates [35S]methionine in vivo and is also a rapidly labeled product of chloroplast-directed protein synthesis. Trypsin treatment of membranes tagged with azido-[14C]atrazine, [35S]methionine in vivo, or [35S]methionine in isolated intact chloroplasts results in identical, sequential alterations of the 34-kilo-dalton polypeptide to species of 32, then 18 and 16 kilodaltons. From the identical pattern of susceptibility to trypsin we conclude that the rapidly synthesized 34-kilodalton polypeptide that is a product of chloroplast-directed protein synthesis is identical to the triazine herbicide-binding protein of photosystem II. Chloroplasts of both triazine-susceptible and triazine-resistant biotypes of Amaranthus hybridus synthesize the 34-kilodalton polypeptide, but that of the resistant biotype does not bind the herbicide. Images PMID:16593133

Chloroplast microsatellite primers for cacao (Theobroma cacao) and other Malvaceae.

PubMed

Yang, Ji Y; Motilal, Lambert A; Dempewolf, Hannes; Maharaj, Kamaldeo; Cronk, Q C B

2011-12-01

Chloroplast microsatellites were developed in Theobroma cacao to examine the genetic diversity of cacao cultivars in Trinidad and Tobago. Nine polymorphic microsatellites were designed from the chloroplast genomes of two T. cacao accessions. These microsatellites were tested in 95 hybrid accessions from Trinidad and Tobago. An average of 2.9 alleles per locus was found. These chloroplast microsatellites, particularly the highly polymorphic pentameric repeat, were useful in assessing genetic variation in T. cacao. In addition, these markers should also prove to be useful for population genetic studies in other species of Malvaceae.

Constitutive Transcription and Stable RNA Accumulation in Plastids during the Conversion of Chloroplasts to Chromoplasts in Ripening Tomato Fruits 1

PubMed Central

Marano, María Rosa; Carrillo, Néstor

1992-01-01

The size distribution of plastid transcripts during chromoplast differentiation in ripening tomato (Lycopersicon esculentum L.) fruits was determined using northern blot analysis. Hybridization of total cellular RNA from leaves and fruits with several tobacco chloroplast DNA probes showed distinct transcript patterns in chloroplasts and chromoplasts. We also compared transcriptional rates by probing immobilized DNA fragments of small size (representing about 85% of the plastid genome) with run-on transcripts from tomato plastids. The relative rates of transcription of the various DNA regions were very similar in chloro- and chromoplasts. Parallel determination of the steady-state levels of plastid RNA showed no strict correlation between synthesis rate and RNA accumulation. Differences in the relative abundance of transcripts between chloro- and chromoplasts were not very pronounced and were limited to a small number of genes. The results indicate that the conversion of chloroplasts to chromoplasts at the onset of tomato fruit ripening proceeds with no important variations in the relative transcription rates and with only moderate changes in the relative stability of plastid-encoded transcripts. Images Figure 1 Figure 4 PMID:16653091

DNA-based differentiation of the Ecuadorian cocoa types CCN-51 and Arriba based on sequence differences in the chloroplast genome.

PubMed

Herrmann, Luise; Haase, Ilka; Blauhut, Maike; Barz, Nadine; Fischer, Markus

2014-12-17

Two cocoa types, Arriba and CCN-51, are being cultivated in Ecuador. With regard to the unique aroma, Arriba is considered a fine cocoa type, while CCN-51 is a bulk cocoa because of its weaker aroma. Because it is being assumed that Arriba is mixed with CCN-51, there is an interest in the analytical differentiation of the two types. Two methods to identify CCN-51 adulterations in Arriba cocoa were developed on the basis of differences in the chloroplast DNA. On the one hand, a different repeat of the sequence TAAAG in the inverted repeat region results in a different length of amplicons for the two cocoa types, which can be detected by agarose gel electrophoresis, capillary gel electrophoresis, and denaturing high-performance liquid chromatography. On the other hand, single nucleotide polymorphisms (SNPs) between the CCN-51 and Arriba sequences represent restriction sites, which can be used for restriction fragment length polymorphism analysis. A semi-quantitative analysis based on these SNPs is feasible. A method for an exact quantitation based on these results is not realizable. These sequence variations were confirmed for a comprehensive cultivar collection of Arriba and CCN-51, for both bean and leaf samples.

Population Genetic Structure and Phylogeography of Camellia flavida (Theaceae) Based on Chloroplast and Nuclear DNA Sequences

PubMed Central

Wei, Su-Juan; Lu, Yong-Bin; Ye, Quan-Qing; Tang, Shao-Qing

2017-01-01

Camellia flavida is an endangered species of yellow camellia growing in limestone mountains in southwest China. The current classification of C. flavida into two varieties, var. flavida and var. patens, is controversial. We conducted a genetic analysis of C. flavida to determine its taxonomic structure. A total of 188 individual plants from 20 populations across the entire distribution range in southwest China were analyzed using two DNA fragments: a chloroplast DNA fragment from the small single copy region and a single-copy nuclear gene called phenylalanine ammonia-lyase (PAL). Sequences from both chloroplast and nuclear DNA were highly diverse; with high levels of genetic differentiation and restricted gene flow. This result can be attributed to the high habitat heterogeneity in limestone karst, which isolates C. flavida populations from each other. Our nuclear DNA results demonstrate that there are three differentiated groups within C. flavida: var. flavida 1, var. flavida 2, and var. patens. These genetic groupings are consistent with the morphological characteristics of the plants. We suggest that the samples included in this study constitute three taxa and the var. flavida 2 group is the genuine C. flavida. The three groups should be recognized as three management units for conservation concerns. PMID:28579991

Structure-Function Analysis of Chloroplast Proteins via Random Mutagenesis Using Error-Prone PCR.

PubMed

Dumas, Louis; Zito, Francesca; Auroy, Pascaline; Johnson, Xenie; Peltier, Gilles; Alric, Jean

2018-06-01

Site-directed mutagenesis of chloroplast genes was developed three decades ago and has greatly advanced the field of photosynthesis research. Here, we describe a new approach for generating random chloroplast gene mutants that combines error-prone polymerase chain reaction of a gene of interest with chloroplast complementation of the knockout Chlamydomonas reinhardtii mutant. As a proof of concept, we targeted a 300-bp sequence of the petD gene that encodes subunit IV of the thylakoid membrane-bound cytochrome b 6 f complex. By sequencing chloroplast transformants, we revealed 149 mutations in the 300-bp target petD sequence that resulted in 92 amino acid substitutions in the 100-residue target subunit IV sequence. Our results show that this method is suited to the study of highly hydrophobic, multisubunit, and chloroplast-encoded proteins containing cofactors such as hemes, iron-sulfur clusters, and chlorophyll pigments. Moreover, we show that mutant screening and sequencing can be used to study photosynthetic mechanisms or to probe the mutational robustness of chloroplast-encoded proteins, and we propose that this method is a valuable tool for the directed evolution of enzymes in the chloroplast. © 2018 American Society of Plant Biologists. All rights reserved.

Localization of Carbamoylphosphate Synthetase and Aspartate Carbamoyltransferase in Chloroplasts

PubMed Central

Shibata, Hitoshi; Ochiai, Hideo; Sawa, Yoshihiro; Miyoshi, Shoji

1986-01-01

The localization of carbamoylphosphate synthetase (CPSase) and aspartate carbamoyltransferase (ACTase), the first two enzymes of the pyrimidine biosynthetic pathway, in chloroplasts was investigated. In dark-grown radish (Raphanus sativus) seedlings, light induced a prominent increase in CPSase activity, but had little effect on ACTase activity. Both enzymes were found in chloroplasts isolated from radish cotyledons and leaves of spinach (Spinacia oleracea), soybean (Glycine max), and corn (Zea mays). The higher activity of ACTase relative to CPSase is discussed in relation to the instability of carbamoylphosphate, the product of the CPSase, and to the control of pyrimidine synthesis. Based on these results, the function of CPSase and ACTase in chloroplasts is discussed. PMID:16664566

An Ancient Bacterial Signaling Pathway Regulates Chloroplast Function to Influence Growth and Development in Arabidopsis.

PubMed

Sugliani, Matteo; Abdelkefi, Hela; Ke, Hang; Bouveret, Emmanuelle; Robaglia, Christophe; Caffarri, Stefano; Field, Ben

2016-03-01

The chloroplast originated from the endosymbiosis of an ancient photosynthetic bacterium by a eukaryotic cell. Remarkably, the chloroplast has retained elements of a bacterial stress response pathway that is mediated by the signaling nucleotides guanosine penta- and tetraphosphate (ppGpp). However, an understanding of the mechanism and outcomes of ppGpp signaling in the photosynthetic eukaryotes has remained elusive. Using the model plant Arabidopsis thaliana, we show that ppGpp is a potent regulator of chloroplast gene expression in vivo that directly reduces the quantity of chloroplast transcripts and chloroplast-encoded proteins. We then go on to demonstrate that the antagonistic functions of different plant RelA SpoT homologs together modulate ppGpp levels to regulate chloroplast function and show that they are required for optimal plant growth, chloroplast volume, and chloroplast breakdown during dark-induced and developmental senescence. Therefore, our results show that ppGpp signaling is not only linked to stress responses in plants but is also an important mediator of cooperation between the chloroplast and the nucleocytoplasmic compartment during plant growth and development. © 2016 American Society of Plant Biologists. All rights reserved.

Chloroplast and mitochondrial DNA are paternally inherited in Sequoia sempervirens D. Don Endl

PubMed Central

Neale, David B.; Marshall, Kimberly A.; Sederoff, Ronald R.

1989-01-01

Restriction fragment length polymorphisms in controlled crosses were used to infer the mode of inheritance of chloroplast DNA and mitochondrial DNA in coast redwood (Sequoia sempervirens D. Don Endl.). Chloroplast DNA was paternally inherited, as is true for all other conifers studied thus far. Surprisingly, a restriction fragment length polymorphism detected by a mitochondrial probe was paternally inherited as well. This polymorphism could not be detected in hybridizations with chloroplast probes covering the entire chloroplast genome, thus providing evidence that the mitochondrial probe had not hybridized to chloroplast DNA on the blot. We conclude that mitochondrial DNA is paternally inherited in coast redwood. To our knowledge, paternal inheritance of mitochondrial DNA in sexual crosses of a multicellular eukaryotic organism has not been previously reported. Images PMID:16594091

Chloroplast incorporation and long-term photosynthetic performance through the life cycle in laboratory cultures of Elysia timida (Sacoglossa, Heterobranchia)

PubMed Central

2014-01-01

Introduction The Mediterranean sacoglossan Elysia timida is one of the few sea slug species with the ability to sequester chloroplasts from its food algae and to subsequently store them in a functional state in the digestive gland cells for more than a month, during which time the plastids retain high photosynthetic activity (= long-term retention). Adult E. timida have been described to feed on the unicellular alga Acetabularia acetabulum in their natural environment. The suitability of E. timida as a laboratory model culture system including its food source was studied. Results In contrast to the literature reporting that juvenile E. timida feed on Cladophora dalmatica first, and later on switch to the adult diet A. acetabulum, the juveniles in this study fed directly on A. acetabulum (young, non-calcified stalks); they did not feed on the various Cladophora spp. (collected from the sea or laboratory culture) offered. This could possibly hint to cryptic speciation with no clear morphological differences, but incipient ecological differentiation. Transmission electron microscopy of chloroplasts from A. acetabulum after initial intake by juvenile E. timida showed different states of degradation — in conglomerations or singularly — and fragments of phagosome membranes, but differed from kleptoplast images of C. dalmatica in juvenile E. timida from the literature. Based on the finding that the whole life cycle of E. timida can be completed with A. acetabulum as the sole food source, a laboratory culture system was established. An experiment with PAM-fluorometry showed that cultured E. timida are also able to store chloroplasts in long-term retention from Acetabularia peniculus, which stems from the Indo-Pacific and is not abundant in the natural environment of E. timida. Variations between three experiment groups indicated potential influences of temperature on photosynthetic capacities. Conclusions E. timida is a viable laboratory model system to study

Comparison of intraspecific, interspecific and intergeneric chloroplast diversity in Cycads

PubMed Central

Jiang, Guo-Feng; Hinsinger, Damien Daniel; Strijk, Joeri Sergej

2016-01-01

Cycads are among the most threatened plant species. Increasing the availability of genomic information by adding whole chloroplast data is a fundamental step in supporting phylogenetic studies and conservation efforts. Here, we assemble a dataset encompassing three taxonomic levels in cycads, including ten genera, three species in the genus Cycas and two individuals of C. debaoensis. Repeated sequences, SSRs and variations of the chloroplast were analyzed at the intraspecific, interspecific and intergeneric scale, and using our sequence data, we reconstruct a phylogenomic tree for cycads. The chloroplast was 162,094 bp in length, with 133 genes annotated, including 87 protein-coding, 37 tRNA and 8 rRNA genes. We found 7 repeated sequences and 39 SSRs. Seven loci showed promising levels of variations for application in DNA-barcoding. The chloroplast phylogeny confirmed the division of Cycadales in two suborders, each of them being monophyletic, revealing a contradiction with the current family circumscription and its evolution. Finally, 10 intraspecific SNPs were found. Our results showed that despite the extremely restricted distribution range of C. debaoensis, using complete chloroplast data is useful not only in intraspecific studies, but also to improve our understanding of cycad evolution and in defining conservation strategies for this emblematic group. PMID:27558458

Ion and metabolite transport in the chloroplast of algae: lessons from land plants.

PubMed

Marchand, Justine; Heydarizadeh, Parisa; Schoefs, Benoît; Spetea, Cornelia

2018-06-01

Chloroplasts are endosymbiotic organelles and play crucial roles in energy supply and metabolism of eukaryotic photosynthetic organisms (algae and land plants). They harbor channels and transporters in the envelope and thylakoid membranes, mediating the exchange of ions and metabolites with the cytosol and the chloroplast stroma and between the different chloroplast subcompartments. In secondarily evolved algae, three or four envelope membranes surround the chloroplast, making more complex the exchange of ions and metabolites. Despite the importance of transport proteins for the optimal functioning of the chloroplast in algae, and that many land plant homologues have been predicted, experimental evidence and molecular characterization are missing in most cases. Here, we provide an overview of the current knowledge about ion and metabolite transport in the chloroplast from algae. The main aspects reviewed are localization and activity of the transport proteins from algae and/or of homologues from other organisms including land plants. Most chloroplast transporters were identified in the green alga Chlamydomonas reinhardtii, reside in the envelope and participate in carbon acquisition and metabolism. Only a few identified algal transporters are located in the thylakoid membrane and play role in ion transport. The presence of genes for putative transporters in green algae, red algae, diatoms, glaucophytes and cryptophytes is discussed, and roles in the chloroplast are suggested. A deep knowledge in this field is required because algae represent a potential source of biomass and valuable metabolites for industry, medicine and agriculture.

Differentiation of Populus species using chloroplast single nucleotide polymorphism (SNP) markers--essential for comprehensible and reliable poplar breeding.

PubMed

Schroeder, H; Hoeltken, A M; Fladung, M

2012-03-01

Within the genus Populus several species belonging to different sections are cross-compatible. Hence, high numbers of interspecies hybrids occur naturally and, additionally, have been artificially produced in huge breeding programmes during the last 100 years. Therefore, determination of a single poplar species, used for the production of 'multi-species hybrids' is often difficult, and represents a great challenge for the use of molecular markers in species identification. Within this study, over 20 chloroplast regions, both intergenic spacers and coding regions, have been tested for their ability to differentiate different poplar species using 23 already published barcoding primer combinations and 17 newly designed primer combinations. About half of the published barcoding primers yielded amplification products, whereas the new primers designed on the basis of the total sequenced cpDNA genome of Populus trichocarpa Torr. & Gray yielded much higher amplification success. Intergenic spacers were found to be more variable than coding regions within the genus Populus. The highest discrimination power of Populus species was found in the combination of two intergenic spacers (trnG-psbK, psbK-psbl) and the coding region rpoC. In barcoding projects, the coding regions matK and rbcL are often recommended, but within the genus Populus they only show moderate variability and are not efficient in species discrimination. © 2011 German Botanical Society and The Royal Botanical Society of the Netherlands.

Chloroplast Growth and Replication in Germinating Spinach Cotyledons following Massive γ-Irradiation of the Seed

PubMed Central

Rose, Ray; Possingham, John

1976-01-01

Spinach seeds (Spinacia oleracea L.) given massive doses of γ-irradiation (500 krad) germinate and form a seedling with two green cotyledons and a radicle, but develop no further. Irradiated cotyledons show no increase in cell number or total DNA over a 7-day period in the light, while in control cotyledons there is a small increase in cell number and large increases in total DNA and chloroplast number. The chloroplasts of irradiated cotyledons are delayed in their division, become greatly enlarged and contain large amounts of starch. The whole population of chloroplasts subsequently undergoes a wave of division. The daughter chloroplasts show normal thylakoid development, but have some abnormal structural features caused by the radiation stress. Information on the effect of X-irradiation, ultraviolet irradiation, and 5-fluorodeoxyuridine on chloroplast replication and on chloroplast and nuclear DNA synthesis was obtained from cultured spinach leaf discs. It appears that chloroplast replication is more resistant to ionizing radiation than cell division and can proceed in the absence of nuclear DNA synthesis and greatly reduced chloroplast DNA synthesis. Images PMID:16659421

A chloroplast-targeted cabbage DEAD-box RNA helicase BrRH22 confers abiotic stress tolerance to transgenic Arabidopsis plants by affecting translation of chloroplast transcripts.

PubMed

Nawaz, Ghazala; Lee, Kwanuk; Park, Su Jung; Kim, Yeon-Ok; Kang, Hunseung

2018-06-01

Although the roles of many DEAD-box RNA helicases (RHs) have been determined in the nucleus as well as in cytoplasm during stress responses, the importance of chloroplast-targeted DEAD-box RHs in stress response remains largely unknown. In this study, we determined the function of BrRH22, a chloroplast-targeted DEAD-box RH in cabbage (Brassica rapa), in abiotic stress responses. The expression of BrRH22 was markedly increased by drought, heat, salt, or cold stress and by ABA treatment, but was largely decreased by UV stress. Expression of BrRH22 in Arabidopsis enhanced germination and plantlet growth under high salinity or drought stress. BrRH22-expressing plants displayed a higher cotyledon greening and better plantlet growth upon ABA treatment due to decreases in the levels of ABI3, ABI4, and ABI5. Further, BrRH22 affected translation of several chloroplast transcripts under stress. Notably, BrRH22 had RNA chaperone function. These results altogether suggest that chloroplast-transported BrRH22 contributes positively to the response of transgenic Arabidopsis to abiotic stress by affecting translation of chloroplast genes via its RNA chaperone activity. Copyright © 2018 Elsevier Masson SAS. All rights reserved.

Evolution of Chloroplast Transcript Processing in Plasmodium and Its Chromerid Algal Relatives

PubMed Central

Dorrell, Richard G.; Drew, James; Nisbet, R. Ellen R.; Howe, Christopher J.

2014-01-01

It is well understood that apicomplexan parasites, such as the malaria pathogen Plasmodium, are descended from free-living algae, and maintain a vestigial chloroplast that has secondarily lost all genes of photosynthetic function. Recently, two fully photosynthetic relatives of parasitic apicomplexans have been identified, the ‘chromerid’ algae Chromera velia and Vitrella brassicaformis, which retain photosynthesis genes within their chloroplasts. Elucidating the processes governing gene expression in chromerid chloroplasts might provide valuable insights into the origins of parasitism in the apicomplexans. We have characterised chloroplast transcript processing pathways in C. velia, V. brassicaformis and P. falciparum with a focus on the addition of an unusual, 3′ poly(U) tail. We demonstrate that poly(U) tails in chromerids are preferentially added to transcripts that encode proteins that are directly involved in photosynthetic electron transfer, over transcripts for proteins that are not involved in photosynthesis. To our knowledge, this represents the first chloroplast transcript processing pathway to be associated with a particular functional category of genes. In contrast, Plasmodium chloroplast transcripts are not polyuridylylated. We additionally present evidence that poly(U) tail addition in chromerids is involved in the alternative processing of polycistronic precursors covering multiple photosynthesis genes, and appears to be associated with high levels of transcript abundance. We propose that changes to the chloroplast transcript processing machinery were an important step in the loss of photosynthesis in ancestors of parasitic apicomplexans. PMID:24453981

Chloroplast two-component systems: evolution of the link between photosynthesis and gene expression

PubMed Central

Puthiyaveetil, Sujith; Allen, John F.

2009-01-01

Two-component signal transduction, consisting of sensor kinases and response regulators, is the predominant signalling mechanism in bacteria. This signalling system originated in prokaryotes and has spread throughout the eukaryotic domain of life through endosymbiotic, lateral gene transfer from the bacterial ancestors and early evolutionary precursors of eukaryotic, cytoplasmic, bioenergetic organelles—chloroplasts and mitochondria. Until recently, it was thought that two-component systems inherited from an ancestral cyanobacterial symbiont are no longer present in chloroplasts. Recent research now shows that two-component systems have survived in chloroplasts as products of both chloroplast and nuclear genes. Comparative genomic analysis of photosynthetic eukaryotes shows a lineage-specific distribution of chloroplast two-component systems. The components and the systems they comprise have homologues in extant cyanobacterial lineages, indicating their ancient cyanobacterial origin. Sequence and functional characteristics of chloroplast two-component systems point to their fundamental role in linking photosynthesis with gene expression. We propose that two-component systems provide a coupling between photosynthesis and gene expression that serves to retain genes in chloroplasts, thus providing the basis of cytoplasmic, non-Mendelian inheritance of plastid-associated characters. We discuss the role of this coupling in the chronobiology of cells and in the dialogue between nuclear and cytoplasmic genetic systems. PMID:19324807

Low-molecular-weight (4.5S) ribonucleic acid in higher-plant chloroplast ribosomes.

PubMed Central

Whitfeld, P R; Leaver, C J; Bottomley, W; Atchison, B

1978-01-01

A species of RNA that migrates on 10% (w/v) polyacrylamide gels between 5S and 4S RNA was detected in spinach chloroplasts. This RNA (referred to as 4.5 S RNA) was present in amounts equimolar to the 5S RNA and its molecular weight was estimated to be approx. 33 000. Fractionation of the chloroplast components showed that the 4.5S RNA was associated with the 50 S ribosomal subunit and that it could be removed by washing the ribosomes with a buffer containing 0.01 M-EDTA and 0.5 M-KCl. It did not appear to be a cleavage product of the labile 23 S RNA of spinach chloroplast ribosomes. When 125I-labelled 4.5 S RNA was hybridized to fragments of spinach chloroplast DNA produced by SmaI restriction endonuclease, a single fragment (mol.wt. 1.15 times 10(6)) became labelled. The same DNA fragment also hybridized to chloroplast 5 S RNA and part of the 23 S RNA. It was concluded that the coding sequence for 4.5 S RNA was part of, or immediately adjacent to, the rRNA-gene region in chloroplast DNA . A comparable RNA species was observed in chloroplasts of tobacco and pea leaves. Images Fig. 8. PMID:743229

Chloroplast two-component systems: evolution of the link between photosynthesis and gene expression.

PubMed

Puthiyaveetil, Sujith; Allen, John F

2009-06-22

Two-component signal transduction, consisting of sensor kinases and response regulators, is the predominant signalling mechanism in bacteria. This signalling system originated in prokaryotes and has spread throughout the eukaryotic domain of life through endosymbiotic, lateral gene transfer from the bacterial ancestors and early evolutionary precursors of eukaryotic, cytoplasmic, bioenergetic organelles-chloroplasts and mitochondria. Until recently, it was thought that two-component systems inherited from an ancestral cyanobacterial symbiont are no longer present in chloroplasts. Recent research now shows that two-component systems have survived in chloroplasts as products of both chloroplast and nuclear genes. Comparative genomic analysis of photosynthetic eukaryotes shows a lineage-specific distribution of chloroplast two-component systems. The components and the systems they comprise have homologues in extant cyanobacterial lineages, indicating their ancient cyanobacterial origin. Sequence and functional characteristics of chloroplast two-component systems point to their fundamental role in linking photosynthesis with gene expression. We propose that two-component systems provide a coupling between photosynthesis and gene expression that serves to retain genes in chloroplasts, thus providing the basis of cytoplasmic, non-Mendelian inheritance of plastid-associated characters. We discuss the role of this coupling in the chronobiology of cells and in the dialogue between nuclear and cytoplasmic genetic systems.

Breakthrough in chloroplast genetic engineering of agronomically important crops

PubMed Central

Daniell, Henry; Kumar, Shashi; Dufourmantel, Nathalie

2012-01-01

Chloroplast genetic engineering offers several unique advantages, including high-level transgene expression, multi-gene engineering in a single transformation event and transgene containment by maternal inheritance, as well as a lack of gene silencing, position and pleiotropic effects and undesirable foreign DNA. More than 40 transgenes have been stably integrated and expressed using the tobacco chloroplast genome to confer desired agronomic traits or express high levels of vaccine antigens and biopharmaceuticals. Despite such significant progress, this technology has not been extended to major crops. However, highly efficient soybean, carrot and cotton plastid transformation has recently been accomplished through somatic embryogenesis using species-specific chloroplast vectors. This review focuses on recent exciting developments in this field and offers directions for further research and development. PMID:15866001

An Ancient Bacterial Signaling Pathway Regulates Chloroplast Function to Influence Growth and Development in Arabidopsis[OPEN

PubMed Central

Sugliani, Matteo; Ke, Hang; Bouveret, Emmanuelle; Robaglia, Christophe; Caffarri, Stefano

2016-01-01

The chloroplast originated from the endosymbiosis of an ancient photosynthetic bacterium by a eukaryotic cell. Remarkably, the chloroplast has retained elements of a bacterial stress response pathway that is mediated by the signaling nucleotides guanosine penta- and tetraphosphate (ppGpp). However, an understanding of the mechanism and outcomes of ppGpp signaling in the photosynthetic eukaryotes has remained elusive. Using the model plant Arabidopsis thaliana, we show that ppGpp is a potent regulator of chloroplast gene expression in vivo that directly reduces the quantity of chloroplast transcripts and chloroplast-encoded proteins. We then go on to demonstrate that the antagonistic functions of different plant RelA SpoT homologs together modulate ppGpp levels to regulate chloroplast function and show that they are required for optimal plant growth, chloroplast volume, and chloroplast breakdown during dark-induced and developmental senescence. Therefore, our results show that ppGpp signaling is not only linked to stress responses in plants but is also an important mediator of cooperation between the chloroplast and the nucleocytoplasmic compartment during plant growth and development. PMID:26908759

Maternal inheritance of the chloroplast genome in Eucalyptus globulus and interspecific hybrids.

PubMed

Mckinnon, A E; Vaillancourt, R E; Tilyard, P A; Potts, B M

2001-10-01

The utility of chloroplast DNA (cpDNA) in Eucalyptus, either as a molecular marker for genetic studies or as a potential vehicle for genetic manipulation, is based on knowledge of its mode of inheritance. Chloroplast inheritance in angiosperms can vary among and within species, and anomalous inheritance has been reported in some interspecific-hybrid combinations. In Eucalyptus, abnormalities of pollen-tube growth occur in a number of interspecific-hybrid combinations, and this might increase the likelihood of anomalous chloroplast transmission. We used a rapid PCR technique to determine chloroplast heritability in 425 progeny of Eucalyptus, comprising 194 progeny of the premier pulpwood species E. globulus and 231 interspecific hybrids between E. globulus and E. nitens (F1, F2, and backcrosses). At this sampling intensity, no pollen-mediated transmission of cpDNA was found in any of the 40 families tested. The results are discussed with reference to chloroplast engineering and the use of cpDNA as a seed-specific marker in phylogeographic studies of Eucalyptus.

SChloro: directing Viridiplantae proteins to six chloroplastic sub-compartments.

PubMed

Savojardo, Castrense; Martelli, Pier Luigi; Fariselli, Piero; Casadio, Rita

2017-02-01

Chloroplasts are organelles found in plants and involved in several important cell processes. Similarly to other compartments in the cell, chloroplasts have an internal structure comprising several sub-compartments, where different proteins are targeted to perform their functions. Given the relation between protein function and localization, the availability of effective computational tools to predict protein sub-organelle localizations is crucial for large-scale functional studies. In this paper we present SChloro, a novel machine-learning approach to predict protein sub-chloroplastic localization, based on targeting signal detection and membrane protein information. The proposed approach performs multi-label predictions discriminating six chloroplastic sub-compartments that include inner membrane, outer membrane, stroma, thylakoid lumen, plastoglobule and thylakoid membrane. In comparative benchmarks, the proposed method outperforms current state-of-the-art methods in both single- and multi-compartment predictions, with an overall multi-label accuracy of 74%. The results demonstrate the relevance of the approach that is eligible as a good candidate for integration into more general large-scale annotation pipelines of protein subcellular localization. The method is available as web server at http://schloro.biocomp.unibo.it gigi@biocomp.unibo.it.

AtPAP2 modulates the import of the small subunit of Rubisco into chloroplasts.

PubMed

Zhang, Renshan; Guan, Xiaoqian; Law, Yee-Song; Sun, Feng; Chen, Shuai; Wong, Kam Bo; Lim, Boon Leong

2016-10-02

Arabidopsis thaliana purple acid phosphatase 2 (AtPAP2) is the only phosphatase that is dual-targeted to both chloroplasts and mitochondria. Like Toc33/34 of the TOC and Tom 20 of the TOM, AtPAP2 is anchored to the outer membranes of chloroplasts and mitochondria via a hydrophobic C-terminal motif. AtPAP2 on the mitochondria was previously shown to recognize the presequences of several nuclear-encoded mitochondrial proteins and modulate the import of pMORF3 into the mitochondria. Here we show that AtPAP2 binds to the small subunit of Rubisco (pSSU) and that chloroplast import experiments demonstrated that pSSU was imported less efficiently into pap2 chloroplasts than into wild-type chloroplasts. We propose that AtPAP2 is an outer membrane-bound phosphatase receptor that facilitates the import of selected proteins into chloroplasts.

The Complete Chloroplast Genome of Banana (Musa acuminata, Zingiberales): Insight into Plastid Monocotyledon Evolution

PubMed Central

Martin, Guillaume; Baurens, Franc-Christophe; Cardi, Céline; Aury, Jean-Marc; D’Hont, Angélique

2013-01-01

Background Banana (genus Musa) is a crop of major economic importance worldwide. It is a monocotyledonous member of the Zingiberales, a sister group of the widely studied Poales. Most cultivated bananas are natural Musa inter-(sub-)specific triploid hybrids. A Musa acuminata reference nuclear genome sequence was recently produced based on sequencing of genomic DNA enriched in nucleus. Methodology/Principal Findings The Musa acuminata chloroplast genome was assembled with chloroplast reads extracted from whole-genome-shotgun sequence data. The Musa chloroplast genome is a circular molecule of 169,972 bp with a quadripartite structure containing two single copy regions, a Large Single Copy region (LSC, 88,338 bp) and a Small Single Copy region (SSC, 10,768 bp) separated by Inverted Repeat regions (IRs, 35,433 bp). Two forms of the chloroplast genome relative to the orientation of SSC versus LSC were found. The Musa chloroplast genome shows an extreme IR expansion at the IR/SSC boundary relative to the most common structures found in angiosperms. This expansion consists of the integration of three additional complete genes (rps15, ndhH and ycf1) and part of the ndhA gene. No such expansion has been observed in monocots so far. Simple Sequence Repeats were identified in the Musa chloroplast genome and a new set of Musa chloroplastic markers was designed. Conclusion The complete sequence of M. acuminata ssp malaccensis chloroplast we reported here is the first one for the Zingiberales order. As such it provides new insight in the evolution of the chloroplast of monocotyledons. In particular, it reinforces that IR/SSC expansion has occurred independently several times within monocotyledons. The discovery of new polymorphic markers within Musa chloroplast opens new perspectives to better understand the origin of cultivated triploid bananas. PMID:23840670

The complete chloroplast genome of banana (Musa acuminata, Zingiberales): insight into plastid monocotyledon evolution.

PubMed

Martin, Guillaume; Baurens, Franc-Christophe; Cardi, Céline; Aury, Jean-Marc; D'Hont, Angélique

2013-01-01

Banana (genus Musa) is a crop of major economic importance worldwide. It is a monocotyledonous member of the Zingiberales, a sister group of the widely studied Poales. Most cultivated bananas are natural Musa inter-(sub-)specific triploid hybrids. A Musa acuminata reference nuclear genome sequence was recently produced based on sequencing of genomic DNA enriched in nucleus. The Musa acuminata chloroplast genome was assembled with chloroplast reads extracted from whole-genome-shotgun sequence data. The Musa chloroplast genome is a circular molecule of 169,972 bp with a quadripartite structure containing two single copy regions, a Large Single Copy region (LSC, 88,338 bp) and a Small Single Copy region (SSC, 10,768 bp) separated by Inverted Repeat regions (IRs, 35,433 bp). Two forms of the chloroplast genome relative to the orientation of SSC versus LSC were found. The Musa chloroplast genome shows an extreme IR expansion at the IR/SSC boundary relative to the most common structures found in angiosperms. This expansion consists of the integration of three additional complete genes (rps15, ndhH and ycf1) and part of the ndhA gene. No such expansion has been observed in monocots so far. Simple Sequence Repeats were identified in the Musa chloroplast genome and a new set of Musa chloroplastic markers was designed. The complete sequence of M. acuminata ssp malaccensis chloroplast we reported here is the first one for the Zingiberales order. As such it provides new insight in the evolution of the chloroplast of monocotyledons. In particular, it reinforces that IR/SSC expansion has occurred independently several times within monocotyledons. The discovery of new polymorphic markers within Musa chloroplast opens new perspectives to better understand the origin of cultivated triploid bananas.

Glucose respiration in the intact chloroplast of Chlamydomonas reinhardtii

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

Changguo Chen; Gibbs, M.

1991-01-01

Chloroplastic respiration was monitored by measuring {sup 14}CO{sub 2} from {sup 14}C glucose in the darkened Chlamydomonas reinhardtii F-60 chloroplast, The patterns of {sup 14}CO{sub 2} evolution from labeled glucose in the absence and presence of the inhibitors iodoacetamide, glycolate-2-phosphate, and phosphoenolypyruvate were those expected from the oxidative pentose phosphate cycle and glycolysis. The K{sub m} for glucose was 56 micromolar and for MgATP was 200 micromolar. Release of {sup 14}CO{sub 2} was inhibited by phloretin and inorganic phosphate. Comparing the inhibition of CO{sub 2} evolution generated by pH 7.5 with respect to pH 8.2 (optimum) in chloroplasts given C-1,more » C-2, and C-6 labeled glucose indicated that a suboptimum pH affects the recycling of the pentose phosphate intermediates to a greater extent than CO{sub 2} evolution from C-1 of glucose. Respiratory inhibition by pH 7.5 in the darkened chloroplast was alleviated by NH{sub 4}Cl and KCl (stromal alkalating agents), iodoacetamide (an inhibitor of glyceraldehyde 3-phosphate dehydrogenase), or phosphoenolypyruvate (an inhibitor of phosphofructokinase). It is concluded that the site which primarily mediates respiration in the darkened Chlamydomonas chloroplast is the fructose-1,6-bisphosphatase/phosphofructokinase junction. The respiratory pathways described here can account for the total oxidation of a hexose to Co{sub 2} and for interactions between carbohydrate metabolism and the oxyhydrogen reaction in algal cells adapted to a hydrogen metabolism.« less

A High-Resolution Gene Map of the Chloroplast Genome of the Red Alga Porphyra purpurea.

PubMed Central

Reith, M; Munholland, J

1993-01-01

Extensive DNA sequencing of the chloroplast genome of the red alga Porphyra purpurea has resulted in the detection of more than 125 genes. Fifty-eight (approximately 46%) of these genes are not found on the chloroplast genomes of land plants. These include genes encoding 17 photosynthetic proteins, three tRNAs, and nine ribosomal proteins. In addition, nine genes encoding proteins related to biosynthetic functions, six genes encoding proteins involved in gene expression, and at least five genes encoding miscellaneous proteins are among those not known to be located on land plant chloroplast genomes. The increased coding capacity of the P. purpurea chloroplast genome, along with other characteristics such as the absence of introns and the conservation of ancestral operons, demonstrate the primitive nature of the P. purpurea chloroplast genome. In addition, evidence for a monophyletic origin of chloroplasts is suggested by the identification of two groups of genes that are clustered in chloroplast genomes but not in cyanobacteria. PMID:12271072

Light-stimulated Production of a Chloroplast-localized System for Protein Synthesis in Euglena gracilis1

PubMed Central

Reger, Bonnie J.; Smillie, R. M.; Fuller, R. C.

1972-01-01

Chloroplasts and proplastids isolated respectively from autotrophic and dark-adapted cells of Euglena gracilis strain Z incorporated 14C-l-leucine into protein. In each case the incorporation was inhibited by chloramphenicol (50% inhibition at about 5 μg/ml for chloroplasts and 30 μg/ml for proplastids), but not appreciably by cycloheximide at concentrations up to 200 μg/ml. Chloroplasts from autotrophic cells incorporated leucine into protein at rates of about 10 pg leucine per mg RNA in one minute, but isolated proplastids were only 5 to 10% as active. When dark-adapted cells were illuminated there was little increase in the activity of the chloroplast fraction during the first 12 hr. Between 12 and 24 hr, when there was a rapid increase in the rate of synthesis of chlorophyll, the capacity of the chloroplast fraction for protein synthesis increased markedly. Suppression of the formation of a chloroplast-localized system for protein synthesis by treating the cells with chloramphenicol and the lack of such an effect with cycloheximide suggests that certain of the proteins which form part of a functional chloroplast system for protein synthesis are themselves synthesized within the chloroplasts. PMID:16658126

A simple low-cost microcontroller-based photometric instrument for monitoring chloroplast movement.

PubMed

Berg, Robert; Königer, Martina; Schjeide, Brit-Maren; Dikmak, George; Kohler, Susan; Harris, Gary C

2006-03-01

A new microcontroller-based photometric instrument for monitoring blue light dependent changes in leaf transmission (chloroplast movement) was developed based on a modification of the double-beam technique developed by Walzcak and Gabrys [(1980) Photosynthetica 14: 65-72]. A blue and red bicolor light emitting diode (LED) provided both a variable intensity blue actinic light and a low intensity red measuring beam. A phototransistor detected the intensity of the transmitted measuring light. An inexpensive microcontroller independently and precisely controlled the light emission of the bicolor LED. A typical measurement event involved turning off the blue actinic light for 100 mus to create a narrow temporal window for turning on and measuring the transmittance of the red light. The microcontroller was programmed using LogoChip Logo (http://www.wellesley.edu/Physics/Rberg/logochip/) to record fluence rate response curves. Laser scanning confocal microscopy was utilized to correlate the changes in leaf transmission with intercellular chloroplast position. In the dark, the chloroplasts in the spongy mesophyll exhibited no evident asymmetries in their distribution, however, in the palisade layer the cell surface in contact with the overlying epidermis was devoid of chloroplasts. The low light dependent decrease in leaf transmittance in dark acclimated leaves was correlated with the movement of chloroplasts within the palisade layer into the regions previously devoid of chloroplasts. Changes in leaf transmittance were evident within one minute following the onset of illumination. Minimal leaf transmittance was correlated with chloroplasts having retreated from cell surfaces perpendicular to the incident light (avoidance reaction) in both spongy and palisade layers.

The complete chloroplast genome of two Brassica species, Brassica nigra and B. Oleracea.

PubMed

Seol, Young-Joo; Kim, Kyunghee; Kang, Sang-Ho; Perumal, Sampath; Lee, Jonghoon; Kim, Chang-Kug

2017-03-01

The two Brassica species, Brassica nigra and Brassica oleracea, are important agronomic crops. The chloroplast genome sequences were generated by de novo assembly using whole genome next-generation sequences. The chloroplast genomes of B. nigra and B. oleracea were 153 633 bp and 153 366 bp in size, respectively, and showed conserved typical chloroplast structure. The both chloroplast genomes contained a total of 114 genes including 80 protein-coding genes, 30 tRNA genes, and 4 rRNA genes. Phylogenetic analysis revealed that B. oleracea is closely related to B. rapa and B. napus but B. nigra is more diverse than the neighbor species Raphanus sativus.

Short-term effects of salt exposure on the maize chloroplast protein pattern.

PubMed

Zörb, Christian; Herbst, Ramona; Forreiter, Christoph; Schubert, Sven

2009-09-01

It is of fundamental importance to understand the physiological differences leading to salt resistance and to get access to the molecular mechanisms underlying this physiological response. The aim of this work was to investigate the effects of short-term salt exposure on the proteome of maize chloroplasts in the initial phase of salt stress (up to 4 h). It could be shown that sodium ions accumulate quickly and excessively in chloroplasts in the initial phase of moderate salt stress. A change in the chloroplast protein pattern was observed without a change in water potential of the leaves. 2-DE revealed that 12 salt-responsive chloroplast proteins increased while eight chloroplast proteins decreased. Some of the maize chloroplast proteins such as CF1e and a Ca(2+)-sensing receptor show a rather transient response for the first 4 h of salt exposure. The enhanced abundance of the ferredoxin NADPH reductase, the 23 kDa polypeptide of the photosystem II, and the FtsH-like protein might reflect mechanism to attenuate the detrimental effects of Na(+) on the photosynthetic machinery. The observed transient increase and subsequent decrease of selected proteins may exhibit a counterbalancing effect of target proteins in this context. Intriguingly, several subunits of the CF1-CF0 complex are unequally affected, whereas others do not respond at all.

The Unicellular Green Alga Chlamydomonas reinhardtii as an Experimental System to Study Chloroplast RNA Metabolism

NASA Astrophysics Data System (ADS)

Nickelsen, J.; Kück, U.

Chloroplasts are typical organelles of photoautotrophic eukaryotic cells which drive a variety of functions, including photosynthesis. For many years the unicellular green alga Chlamydomonas reinhardtii has served as an experimental organism for studying photosynthetic processes. The recent development of molecular tools for this organism together with efficient methods of genetic analysis and the availability of many photosynthesis mutants has now made this alga a powerful model system for the analysis of chloroplast biogenesis. For example, techniques have been developed to transfer recombinant DNA into both the nuclear and the chloroplast genome. This allows both complementation tests and analyses of gene functions in vivo. Moreover, site-specific DNA recombinations in the chloroplast allow targeted gene disruption experiments which enable a "reverse genetics" to be performed. The potential of the algal system for the study of chloroplast biogenesis is illustrated in this review by the description of regulatory systems of gene expression involved in organelle biogenesis. One example concerns the regulation of trans-splicing of chloroplast mRNAs, a process which is controlled by both multiple nuclear- and chloroplast-encoded factors. The second example involves the stabilization of chloroplast mRNAs. The available data lead us predict distinct RNA elements, which interact with trans-acting factors to protect the RNA against nucleolytic attacks.

Chloroplast Dynamics and Photosynthetic Efficiency: Final Technical Report

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

Hanson, Maureen

This project investigated the mechanism by which chloroplasts position themselves to maximize solar energy utilization, to enhance gas exchange, to minimize environmental stress, and to promote efficient exchange of metabolites with other compartments within the plant cell. Chloroplasts move within leaf cells to optimize light levels, moving toward levels of light useful for photosynthesis while moving away from excess light. Plastids sometimes extend their reach by sending out projections (stromules) that can connect anchor chloroplasts in position within the cell or provide close contacts with plasma membrane, mitochondria, peroxisomes, endoplasmic reticulum, and the nucleus. The intracellular location of chloroplasts inmore » relation to other organelles with which they share biosynthetic pathways, such as peroxisomes and mitochondria in photorespiration, affects metabolite flow. This work contributed to the knowledge of the mechanisms of organelle movement and anchoring in specific locations in plant cells and how proteins traffic within the cell. We identified two domains on 12 of the 13 Arabidopsis myosins that were similar to the vacuole-binding (V) domain characterized in yeast and to the DIL domain characterized in yeast and mouse as required for secretory vesicle or melanosome movement, respectively. Because all of the Arabidopsis regions with homology to the V domain contain the amino acid sequence PAL, we refer to this region as the Arabidopsis PAL domain. We have used the yeast Myo2p tail structural information to model the 12 myosin XI tail domains containing the homologous PAL and DIL domains. Eight YFP::DIL domain fusions labeled peroxisomes; none labeled mitochondria or chloroplasts. Six myosin XI Vacuole domains labeled mitochondria and seven labeled Golgi bodies. The Arabidopsis myosin XI-F PAL domain and the homologous myosin XI-F PAL domain from N. benthamiana labels chloroplasts and stromules in N. benthamiana leaves. Using an Arabidopsis

Choline oxidation by intact spinach chloroplasts. [Spinacia oleracea L

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

Weigel, P.; Lerma, C.; Hanson, A.D.

1988-01-01

Plants synthesize betaine by a two-step oxidation of choline (choline ..-->.. betaine aldehyde ..-->.. betaine). Protoplast-derived chloroplasts of spinach (Spinacia oleracea L.) carry out both reactions, more rapidly in light than in darkness. We investigated the light-stimulated oxidation of choline, using spinach chloroplasts isolated directly from leaves. The rates of choline oxidation obtained (dark and light rates: 10-50 and 100-300 nanomoles per hour per milligram chlorophyll, respectively) were approximately 20-fold higher than for protoplast-derived chloroplasts. Betaine aldehyde was the main product. Choline oxidation in darkness and light was suppressed by hypoxia. Neither uncouplers not the Calvin cycle inhibitor glyceraldehyde greatlymore » affected choline oxidation in the light, and maximal choline oxidation was attained far below light saturation of CO/sub 2/ fixation. The light stimulation of choline oxidation was abolished by the PSII inhibitors DCMU and dibromothymoquinone, and was partially restored by adding reduced diaminodurene, an electron donor to PSI. Both methyl viologen and phenazine methosulfate prevented choline oxidation. Adding dihydroxyacetone phosphate, which can generate NADPH in organello, doubled the dark rate of choline oxidation. These results indicate that choline oxidation in chloroplasts requires oxygen, and reducing power generated from PSI. Enzymic reactions consistent with these requirements are discussed.« less

The complete chloroplast DNA sequence of the green alga Nephroselmis olivacea: Insights into the architecture of ancestral chloroplast genomes

PubMed Central

Turmel, Monique; Otis, Christian; Lemieux, Claude

1999-01-01

Green plants seem to form two sister lineages: Chlorophyta, comprising the green algal classes Prasinophyceae, Ulvophyceae, Trebouxiophyceae, and Chlorophyceae, and Streptophyta, comprising the Charophyceae and land plants. We have determined the complete chloroplast DNA (cpDNA) sequence (200,799 bp) of Nephroselmis olivacea, a member of the class (Prasinophyceae) thought to include descendants of the earliest-diverging green algae. The 127 genes identified in this genome represent the largest gene repertoire among the green algal and land plant cpDNAs completely sequenced to date. Of the Nephroselmis genes, 2 (ycf81 and ftsI, a gene involved in peptidoglycan synthesis) have not been identified in any previously investigated cpDNA; 5 genes [ftsW, rnE, ycf62, rnpB, and trnS(cga)] have been found only in cpDNAs of nongreen algae; and 10 others (ndh genes) have been described only in land plant cpDNAs. Nephroselmis and land plant cpDNAs share the same quadripartite structure—which is characterized by the presence of a large rRNA-encoding inverted repeat and two unequal single-copy regions—and very similar sets of genes in corresponding genomic regions. Given that our phylogenetic analyses place Nephroselmis within the Chlorophyta, these structural characteristics were most likely present in the cpDNA of the common ancestor of chlorophytes and streptophytes. Comparative analyses of chloroplast genomes indicate that the typical quadripartite architecture and gene-partitioning pattern of land plant cpDNAs are ancient features that may have been derived from the genome of the cyanobacterial progenitor of chloroplasts. Our phylogenetic data also offer insight into the chlorophyte ancestor of euglenophyte chloroplasts. PMID:10468594

Transcriptome analysis of ectopic chloroplast development in green curd cauliflower (Brassica oleracea L. var. botrytis)

USDA-ARS?s Scientific Manuscript database

Chloroplasts are the green plastids where photosynthesis takes place. The biogenesis of chloroplasts requires the coordinate expression of both nuclear and chloroplast genes and is regulated by developmental and environmental signals. Despite extensive studies of this process, the genetic basis and ...

Chloroplast parameters differ in wild type and transgenic poplars overexpressing gsh1 in the cytosol.

PubMed

Ivanova, L A; Ronzhina, D A; Ivanov, L A; Stroukova, L V; Peuke, A D; Rennenberg, H

2009-07-01

Poplar mutants overexpressing the bacterial genes gsh1 or gsh2 encoding the enzymes of glutathione biosynthesis are among the best-characterised transgenic plants. However, this characterisation originates exclusively from laboratory studies, and the performance of these mutants under field conditions is largely unknown. Here, we report a field experiment in which the wild-type poplar hybrid Populus tremula x P. alba and a transgenic line overexpressing the bacterial gene gsh1 encoding gamma-glutamylcysteine synthetase in the cytosol were grown for 3 years at a relatively clean (control) field site and a field site contaminated with heavy metals. Aboveground biomass accumulation was slightly smaller in transgenic compared to wild-type plants; soil contamination significantly decreased biomass accumulation in both wild-type and transgenic plants by more than 40%. Chloroplasts parameters, i.e., maximal diameter, projection area and perimeter, surface area and volume, surface/volume ratio and a two-dimensional form coefficient, were found to depend on plant type, leaf tissue and soil contamination. The greatest differences between wild and transgenic poplars were observed at the control site. Under these conditions, chloroplast sizes in palisade tissue of transgenic poplar significantly exceeded those of the wild type. In contrast to the wild type, palisade chloroplast volume exceeded that of spongy chloroplasts in transgenic poplars at both field sites. Chlorophyll content per chloroplast was the same in wild and transgenic poplars. Apparently, the increase in chloroplast volume was not connected to changes in the photosynthetic centres. Chloroplasts of transgenic poplar at the control site were more elongated in palisade cells and close to spherical in spongy mesophyll chloroplasts. At the contaminated site, palisade and spongy cell chloroplasts of leaves from transgenic trees and the wild type were the same shape. Transgenic poplars also had a smaller chloroplast

Verdant: automated annotation, alignment and phylogenetic analysis of whole chloroplast genomes.

PubMed

McKain, Michael R; Hartsock, Ryan H; Wohl, Molly M; Kellogg, Elizabeth A

2017-01-01

Chloroplast genomes are now produced in the hundreds for angiosperm phylogenetics projects, but current methods for annotation, alignment and tree estimation still require some manual intervention reducing throughput and increasing analysis time for large chloroplast systematics projects. Verdant is a web-based software suite and database built to take advantage a novel annotation program, annoBTD. Using annoBTD, Verdant provides accurate annotation of chloroplast genomes without manual intervention. Subsequent alignment and tree estimation can incorporate newly annotated and publically available plastomes and can accommodate a large number of taxa. Verdant sharply reduces the time required for analysis of assembled chloroplast genomes and removes the need for pipelines and software on personal hardware. Verdant is available at: http://verdant.iplantcollaborative.org/plastidDB/ It is implemented in PHP, Perl, MySQL, Javascript, HTML and CSS with all major browsers supported. mrmckain@gmail.comSupplementary information: Supplementary data are available at Bioinformatics online. © The Author 2016. Published by Oxford University Press.

Interaction of Chloroplasts with Inhibitors

PubMed Central

Ridley, Stuart M.

1977-01-01

A primary symptom of diuron (DCMU) phytotoxicity in plants is the destruction of chlorophyll. To study this process in vitro, chloroplasts from pea leaves (Pisum sativum L.) have been incubated in the light with DCMU for periods of up to 34 hours. The sequence of photodestruction of chlorophylls and carotenoids has been followed to try and establish the nature of the chloroplast protection mechanisms that are destroyed by DCMU. β-Carotene decays most rapidly, followed by chlorophyll a and xanthophylls which are destroyed in a constant ratio, followed finally by chlorophyll b. Bypassing the DCMU block in the electron transport system with an artificial electron donor provides complete protection against chlorophyll and carotenoid photodestruction. The same protection by this electron donor system is afforded to stroma-free lamellae from which soluble reductants have been removed so that NADPH formation, which has been proposed as an essential part of a protective xanthophyll cycle, is not possible. Both this and the simultaneous loss of chlorophyll a and xanthophylls tend to preclude the breakdown of a xanthophyll cycle from the possible protective mechanisms inhibited or destroyed by DCMU. Cofactors of cyclic electron transport also protect against DCMU-induced photodestruction of pigments. Their concentration dependence for this protection appears to reflect their various abilities to catalyze cyclic photophosphorylation. The extent to which the chlorophylls are destroyed in the major pigment-protein complexes from chloroplasts illuminated with and without DCMU has been measured. In the absence of DCMU, the light-harvesting chlorophyll a/b protein complex is destroyed most rapidly. In the presence of DCMU, the losses of chlorophyll a from the photosystem I P700-chlorophyll a protein and the chlorophyll a/b complex are about the same. Chlorophyll losses are matched by simultaneous losses of the protein moieties; spectral analyses show that the remaining

Effect of Flooding on Starch Accumulation in Chloroplasts of Sunflower (Helianthus annuus L.) 1

PubMed Central

Wample, Robert L.; Davis, Ronald W.

1983-01-01

Chloroplasts in leaves of sunflower (Helianthus annuus L. cv hybrid 894) whose roots were flooded for 4 days showed an increase in the level of starch in chloroplasts when examined with the electron microscope. Starch determination showed significantly higher levels in leaves of flooded plants. Chloroplast and mitochondrial structure seemed otherwise normal. Images Fig. 1 Fig. 2 PMID:16663176

Chloroplast chlB gene is required for light-independent chlorophyll accumulation in Chlamydomonas reinhardtii.

PubMed

Liu, X Q; Xu, H; Huang, C

1993-10-01

Light-independent chlorophyll synthesis occurs in some algae, lower plants, and gymnosperms, but not in angiosperms. We have identified a new chloroplast gene, chlB, that is required for the light-independent accumulation of chlorophyll in the green alga Chlamydomonas reinhardtii. The chlB gene was cloned, sequenced, and then disrupted by performing particle gun-mediated chloroplast transformation. The resulting homoplasmic mutant was unable to accumulate chlorophyll in the dark and thus exhibited a 'yellow-in-the-dark' phenotype. The chlB gene encodes a polypeptide of 688 amino acid residues, and is distinct from two previously characterized chloroplast genes (chlN and chlL) also required for light-independent chlorophyll accumulation in C. reinhardtii. Three unidentified open reading frames in chloroplast genomes of liverwort, black pine, and Chlamydomonas moewusii were also identified as chlB genes, based on their striking sequence similarities to the C. reinhardtii chlB gene. A chlB-like gene is absent in chloroplast genomes of tobacco and rice, consistent with the lack of light-independent chlorophyll synthesis in these plants. Polypeptides encoded by the chloroplast chlB genes also show significant sequence similarities with the bchB gene product of Rhodobacter capsulatus. Comparisons among the chloroplast chlB and the bacterial bchB gene products revealed five highly conserved sequence areas that are interspersed by four stretches of highly variable and probably insertional sequences.

Comparative chromatography of chloroplast pigment

NASA Technical Reports Server (NTRS)

Grandolfo, M.; Sherma, J.; Strain, H. H.

1969-01-01

Methods for isolation of low concentration pigments of the cocklebur species are described. The methods entail two step chromatography so that the different sorption properties of the various pigments in varying column parameters can be utilized. Columnar and thin layer methods are compared. Many conditions influence separability of the chloroplasts.

Transcriptome analysis of ectopic chloroplast development in green curd cauliflower (Brassica oleracea L. var. botrytis).

PubMed

Zhou, Xiangjun; Fei, Zhangjun; Thannhauser, Theodore W; Li, Li

2011-11-23

Chloroplasts are the green plastids where photosynthesis takes place. The biogenesis of chloroplasts requires the coordinate expression of both nuclear and chloroplast genes and is regulated by developmental and environmental signals. Despite extensive studies of this process, the genetic basis and the regulatory control of chloroplast biogenesis and development remain to be elucidated. Green cauliflower mutant causes ectopic development of chloroplasts in the curd tissue of the plant, turning the otherwise white curd green. To investigate the transcriptional control of chloroplast development, we compared gene expression between green and white curds using the RNA-seq approach. Deep sequencing produced over 15 million reads with lengths of 86 base pairs from each cDNA library. A total of 7,155 genes were found to exhibit at least 3-fold changes in expression between green and white curds. These included light-regulated genes, genes encoding chloroplast constituents, and genes involved in chlorophyll biosynthesis. Moreover, we discovered that the cauliflower ELONGATED HYPOCOTYL5 (BoHY5) was expressed higher in green curds than white curds and that 2616 HY5-targeted genes, including 1600 up-regulated genes and 1016 down-regulated genes, were differently expressed in green in comparison to white curd tissue. All these 1600 up-regulated genes were HY5-targeted genes in the light. The genome-wide profiling of gene expression by RNA-seq in green curds led to the identification of large numbers of genes associated with chloroplast development, and suggested the role of regulatory genes in the high hierarchy of light signaling pathways in mediating the ectopic chloroplast development in the green curd cauliflower mutant.

Transcriptome analysis of ectopic chloroplast development in green curd cauliflower (Brassica oleracea L. var. botrytis)

PubMed Central

2011-01-01

Background Chloroplasts are the green plastids where photosynthesis takes place. The biogenesis of chloroplasts requires the coordinate expression of both nuclear and chloroplast genes and is regulated by developmental and environmental signals. Despite extensive studies of this process, the genetic basis and the regulatory control of chloroplast biogenesis and development remain to be elucidated. Results Green cauliflower mutant causes ectopic development of chloroplasts in the curd tissue of the plant, turning the otherwise white curd green. To investigate the transcriptional control of chloroplast development, we compared gene expression between green and white curds using the RNA-seq approach. Deep sequencing produced over 15 million reads with lengths of 86 base pairs from each cDNA library. A total of 7,155 genes were found to exhibit at least 3-fold changes in expression between green and white curds. These included light-regulated genes, genes encoding chloroplast constituents, and genes involved in chlorophyll biosynthesis. Moreover, we discovered that the cauliflower ELONGATED HYPOCOTYL5 (BoHY5) was expressed higher in green curds than white curds and that 2616 HY5-targeted genes, including 1600 up-regulated genes and 1016 down-regulated genes, were differently expressed in green in comparison to white curd tissue. All these 1600 up-regulated genes were HY5-targeted genes in the light. Conclusions The genome-wide profiling of gene expression by RNA-seq in green curds led to the identification of large numbers of genes associated with chloroplast development, and suggested the role of regulatory genes in the high hierarchy of light signaling pathways in mediating the ectopic chloroplast development in the green curd cauliflower mutant. PMID:22112144

Unbiased estimation of chloroplast number in mesophyll cells: advantage of a genuine three-dimensional approach

PubMed Central

Kubínová, Zuzana

2014-01-01

Chloroplast number per cell is a frequently examined quantitative anatomical parameter, often estimated by counting chloroplast profiles in two-dimensional (2D) sections of mesophyll cells. However, a mesophyll cell is a three-dimensional (3D) structure and this has to be taken into account when quantifying its internal structure. We compared 2D and 3D approaches to chloroplast counting from different points of view: (i) in practical measurements of mesophyll cells of Norway spruce needles, (ii) in a 3D model of a mesophyll cell with chloroplasts, and (iii) using a theoretical analysis. We applied, for the first time, the stereological method of an optical disector based on counting chloroplasts in stacks of spruce needle optical cross-sections acquired by confocal laser-scanning microscopy. This estimate was compared with counting chloroplast profiles in 2D sections from the same stacks of sections. Comparing practical measurements of mesophyll cells, calculations performed in a 3D model of a cell with chloroplasts as well as a theoretical analysis showed that the 2D approach yielded biased results, while the underestimation could be up to 10-fold. We proved that the frequently used method for counting chloroplasts in a mesophyll cell by counting their profiles in 2D sections did not give correct results. We concluded that the present disector method can be efficiently used for unbiased estimation of chloroplast number per mesophyll cell. This should be the method of choice, especially in coniferous needles and leaves with mesophyll cells with lignified cell walls where maceration methods are difficult or impossible to use. PMID:24336344

Conflict amongst chloroplast DNA sequences obscures the phylogeny of a group of Asplenium ferns.

PubMed

Shepherd, Lara D; Holland, Barbara R; Perrie, Leon R

2008-07-01

A previous study of the relationships amongst three subgroups of the Austral Asplenium ferns found conflicting signal between the two chloroplast loci investigated. Because organelle genomes like those of chloroplasts and mitochondria are thought to be non-recombining, with a single evolutionary history, we sequenced four additional chloroplast loci with the expectation that this would resolve these relationships. Instead, the conflict was only magnified. Although tree-building analyses favoured one of the three possible trees, one of the alternative trees actually had one more supporting site (six versus five) and received greater support in spectral and neighbor-net analyses. Simulations suggested that chance alone was unlikely to produce strong support for two of the possible trees and none for the third. Likelihood permutation tests indicated that the concatenated chloroplast sequence data appeared to have experienced recombination. However, recombination between the chloroplast genomes of different species would be highly atypical, and corollary supporting observations, like chloroplast heteroplasmy, are lacking. Wider taxon sampling clarified the composition of the Austral group, but the conflicting signal meant analyses (e.g., morphological evolution, biogeographic) conditional on a well-supported phylogeny could not be performed.

Native architecture of the Chlamydomonas chloroplast revealed by in situ cryo-electron tomography

PubMed Central

Engel, Benjamin D; Schaffer, Miroslava; Kuhn Cuellar, Luis; Villa, Elizabeth; Plitzko, Jürgen M; Baumeister, Wolfgang

2015-01-01

Chloroplast function is orchestrated by the organelle's intricate architecture. By combining cryo-focused ion beam milling of vitreous Chlamydomonas cells with cryo-electron tomography, we acquired three-dimensional structures of the chloroplast in its native state within the cell. Chloroplast envelope inner membrane invaginations were frequently found in close association with thylakoid tips, and the tips of multiple thylakoid stacks converged at dynamic sites on the chloroplast envelope, implicating lipid transport in thylakoid biogenesis. Subtomogram averaging and nearest neighbor analysis revealed that RuBisCO complexes were hexagonally packed within the pyrenoid, with ∼15 nm between their centers. Thylakoid stacks and the pyrenoid were connected by cylindrical pyrenoid tubules, physically bridging the sites of light-dependent photosynthesis and light-independent carbon fixation. Multiple parallel minitubules were bundled within each pyrenoid tubule, possibly serving as conduits for the targeted one-dimensional diffusion of small molecules such as ATP and sugars between the chloroplast stroma and the pyrenoid matrix. DOI: http://dx.doi.org/10.7554/eLife.04889.001 PMID:25584625

The complete chloroplast genomes of two Wisteria species, W. floribunda and W. sinensis (Fabaceae).

PubMed

Kim, Na-Rae; Kim, Kyunghee; Lee, Sang-Choon; Lee, Jung-Hoon; Cho, Seong-Hyun; Yu, Yeisoo; Kim, Young-Dong; Yang, Tae-Jin

2016-11-01

Wisteria floribunda and Wisteria sinensis are ornamental woody vines in the Fabaceae. The complete chloroplast genome sequences of the two species were generated by de novo assembly using whole genome next generation sequences. The chloroplast genomes of W. floribunda and W. sinensis were 130 960 bp and 130 561 bp long, respectively, and showed inverted repeat (IR)-lacking structures as those reported in IRLC in the Fabaceae. The chloroplast genomes of both species contained same number of protein-coding sequences (77), tRNA genes (30), and rRNA genes (4). The phylogenetic analysis with the reported chloroplast genomes confirmed close taxonomical relationship of W. floribunda and W. sinensis.

Towards an understanding of wheat chloroplasts: a methodical investigation of thylakoid proteome.

PubMed

Kamal, Abu Hena Mostafa; Cho, Kun; Komatsu, Setsuko; Uozumi, Nobuyuki; Choi, Jong-Soon; Woo, Sun Hee

2012-05-01

We utilized Percoll density gradient centrifugation to isolate and fractionate chloroplasts of Korean winter wheat cultivar cv. Kumgang (Triticum aestivum L.). The resulting protein fractions were separated by one dimensional polyacrylamide gel electrophoresis (1D-PAGE) coupled with LTQ-FTICR mass spectrometry. This enabled us to detect and identify 767 unique proteins. Our findings represent the most comprehensive exploration of a proteome to date. Based on annotation information from the UniProtKB/Swiss-Prot database and our analyses via WoLF PSORT and PSORT, these proteins are localized in the chloroplast (607 proteins), chloroplast stroma (145), thylakoid membrane (342), lumens (163), and integral membranes (166). In all, 67% were confirmed as chloroplast thylakoid proteins. Although nearly complete protein coverage (89% proteins) has been accomplished for the key chloroplast pathways in wheat, such as for photosynthesis, many other proteins are involved in regulating carbon metabolism. The identified proteins were assigned to 103 functional categories according to a classification system developed by the iProClass database and provided through Protein Information Resources. Those functions include electron transport, energy, cellular organization and biogenesis, transport, stress responses, and other metabolic processes. Whereas most of these proteins are associated with known complexes and metabolic pathways, about 13% of the proteins have unknown functions. The chloroplast proteome contains many proteins that are localized to the thylakoids but as yet have no known function. We propose that some of these familiar proteins participate in the photosynthetic pathway. Thus, our new and comprehensive protein profile may provide clues for better understanding that photosynthetic process in wheat.

Phenotypic, histological and proteomic analyses reveal multiple differences associated with chloroplast development in yellow and variegated variants from Camellia sinensis

PubMed Central

Ma, Chengying; Cao, Junxi; Li, Jianke; Zhou, Bo; Tang, Jinchi; Miao, Aiqing

2016-01-01

Leaf colour variation is observed in several plants. We obtained two types of branches with yellow and variegated leaves from Camellia sinensis. To reveal the mechanisms that underlie the leaf colour variations, combined morphological, histological, ionomic and proteomic analyses were performed using leaves from abnormal branches (variants) and normal branches (CKs). The measurement of the CIE-Lab coordinates showed that the brightness and yellowness of the variants were more intense than the CKs. When chloroplast profiles were analysed, HY1 (branch with yellow leaves) and HY2 (branch with variegated leaves) displayed abnormal chloroplast structures and a reduced number and size compared with the CKs, indicating that the abnormal chloroplast development might be tightly linked to the leaf colour variations. Moreover, the concentration of elemental minerals was different between the variants and the CKs. Furthermore, DEPs (differentially expressed proteins) were identified in the variants and the CKs by a quantitative proteomics analysis using the label-free approach. The DEPs were significantly involved in photosynthesis and included PSI, PSII, cytochrome b6/f complex, photosynthetic electron transport, LHC and F-type ATPase. Our results suggested that a decrease in the abundance of photosynthetic proteins might be associated with the changes of leaf colours in tea plants. PMID:27633059

Copper-Deficiency in Brassica napus Induces Copper Remobilization, Molybdenum Accumulation and Modification of the Expression of Chloroplastic Proteins

PubMed Central

Billard, Vincent; Ourry, Alain; Maillard, Anne; Garnica, Maria; Coquet, Laurent; Jouenne, Thierry; Cruz, Florence; Garcia-Mina, José-Maria; Yvin, Jean-Claude; Etienne, Philippe

2014-01-01

During the last 40 years, crop breeding has strongly increased yields but has had adverse effects on the content of micronutrients, such as Fe, Mg, Zn and Cu, in edible products despite their sufficient supply in most soils. This suggests that micronutrient remobilization to edible tissues has been negatively selected. As a consequence, the aim of this work was to quantify the remobilization of Cu in leaves of Brassica napus L. during Cu deficiency and to identify the main metabolic processes that were affected so that improvements can be achieved in the future. While Cu deficiency reduced oilseed rape growth by less than 19% compared to control plants, Cu content in old leaves decreased by 61.4%, thus demonstrating a remobilization process between leaves. Cu deficiency also triggered an increase in Cu transporter expression in roots (COPT2) and leaves (HMA1), and more surprisingly, the induction of the MOT1 gene encoding a molybdenum transporter associated with a strong increase in molybdenum (Mo) uptake. Proteomic analysis of leaves revealed 33 proteins differentially regulated by Cu deficiency, among which more than half were located in chloroplasts. Eleven differentially expressed proteins are known to require Cu for their synthesis and/or activity. Enzymes that were located directly upstream or downstream of Cu-dependent enzymes were also differentially expressed. The overall results are then discussed in relation to remobilization of Cu, the interaction between Mo and Cu that occurs through the synthesis pathway of Mo cofactor, and finally their putative regulation within the Calvin cycle and the chloroplastic electron transport chain. PMID:25333918

Mutations Altering Chloroplast Ribosome Phenotype in Chlamydomonas, II. A New Mendelian Mutation*

PubMed Central

Boynton, John E.; Gillham, Nicholas W.; Burkholder, Barbara

1970-01-01

A new mutation of Chlamydomonas reinhardi, cr-1, is characterized. The mutation exhibits Mendelian inheritance and affects the sedimentation velocity and formation of intact chloroplast ribosomes. The mutant grows reasonably well when supplied with sodium acetate as a carbon source, but poorly when forced to grow photosynthetically using carbon dioxide. Since the mutant cr-1 accumulates large subunits of the chloroplast ribosome, we postulate that it is blocked in the formation of the small subunit. A tentative model explaining the behavior of the several mutants in Chlamydomonas now known to have altered chloroplast ribosomal phenotypes is presented. Images PMID:16591885

Palisade cell shape affects the light-induced chloroplast movements and leaf photosynthesis.

PubMed

Gotoh, Eiji; Suetsugu, Noriyuki; Higa, Takeshi; Matsushita, Tomonao; Tsukaya, Hirokazu; Wada, Masamitsu

2018-01-24

Leaf photosynthesis is regulated by multiple factors that help the plant to adapt to fluctuating light conditions. Leaves of sun-light-grown plants are thicker and contain more columnar palisade cells than those of shade-grown plants. Light-induced chloroplast movements are also essential for efficient leaf photosynthesis and facilitate efficient light utilization in leaf cells. Previous studies have demonstrated that leaves of most of the sun-grown plants exhibited no or very weak chloroplast movements and could accomplish efficient photosynthesis under strong light. To examine the relationship between palisade cell shape, chloroplast movement and distribution, and leaf photosynthesis, we used an Arabidopsis thaliana mutant, angustifolia (an), which has thick leaves that contain columnar palisade cells similar to those in the sun-grown plants. In the highly columnar cells of an mutant leaves, chloroplast movements were restricted. Nevertheless, under white light condition (at 120 µmol m -2 s -1 ), the an mutant plants showed higher chlorophyll content per unit leaf area and, thus, higher light absorption by the leaves than the wild type, which resulted in enhanced photosynthesis per unit leaf area. Our findings indicate that coordinated regulation of leaf cell shape and chloroplast movement according to the light conditions is pivotal for efficient leaf photosynthesis.

Genetic manipulation of isoprene emissions in poplar plants remodels the chloroplast proteome.

PubMed

Velikova, Violeta; Ghirardo, Andrea; Vanzo, Elisa; Merl, Juliane; Hauck, Stefanie M; Schnitzler, Jörg-Peter

2014-04-04

Biogenic isoprene (2-methyl-1,3-butadiene) improves the integrity and functionality of thylakoid membranes and scavenges reactive oxygen species (ROS) in plant tissue under stress conditions. On the basis of available physiological studies, we hypothesized that the suppression of isoprene production in the poplar plant by genetic engineering would cause changes in the chloroplast protein pattern, which in turn would compensate for changes in chloroplast functionality and overall plant performance under abiotic stress. To test this hypothesis, we used a stable isotope-coded protein-labeling technique in conjunction with polyacrylamide gel electrophoresis and liquid chromatography tandem mass spectrometry. We analyzed quantitative and qualitative changes in the chloroplast proteome of isoprene-emitting and non isoprene-emitting poplars. Here we demonstrate that suppression of isoprene synthase by RNA interference resulted in decreased levels of chloroplast proteins involved in photosynthesis and increased levels of histones, ribosomal proteins, and proteins related to metabolism. Overall, our results show that the absence of isoprene triggers a rearrangement of the chloroplast protein profile to minimize the negative stress effects resulting from the absence of isoprene. The present data strongly support the idea that isoprene improves/stabilizes thylakoid membrane structure and interferes with the production of ROS.

Translation efficiencies of synonymous codons are not always correlated with codon usage in tobacco chloroplasts.

PubMed

Nakamura, Masayuki; Sugiura, Masahiro

2007-01-01

Codon usage in chloroplasts is different from that in prokaryotic and eukaryotic nuclear genomes. However, no experimental approach has been made to analyse the translation efficiency of individual codons in chloroplasts. We devised an in vitro assay for translation efficiencies using synthetic mRNAs, and measured the translation efficiencies of five synonymous codon groups in tobacco chloroplasts. Among four alanine codons (GCN, where N is U, C, A or G), GCU was the most efficient for translation, whereas the chloroplast genome lacks tRNA genes corresponding to GCU. Phenylalanine and tyrosine are each encoded by two codons (UUU/C and UAU/C, respectively). Phenylalanine UUC and tyrosine UAC were translated more than twice as efficiently than UUU and UAU, respectively, contrary to their codon usage, whereas translation efficiencies of synonymous codons for alanine, aspartic acid and asparagine were parallel to their codon usage. These observations indicate that translation efficiencies of individual codons are not always correlated with codon usage in vitro in chloroplasts. This raises an important issue for foreign gene expression in chloroplasts.

Mesophyll cells of C4 plants have fewer chloroplasts than those of closely related C3 plants.

PubMed

Stata, Matt; Sage, Tammy L; Rennie, Troy D; Khoshravesh, Roxana; Sultmanis, Stefanie; Khaikin, Yannay; Ludwig, Martha; Sage, Rowan F

2014-11-01

The evolution of C(4) photosynthesis from C(3) ancestors eliminates ribulose bisphosphate carboxylation in the mesophyll (M) cell chloroplast while activating phosphoenolpyruvate (PEP) carboxylation in the cytosol. These changes may lead to fewer chloroplasts and different chloroplast positioning within M cells. To evaluate these possibilities, we compared chloroplast number, size and position in M cells of closely related C(3), C(3) -C(4) intermediate and C(4) species from 12 lineages of C(4) evolution. All C(3) species had more chloroplasts per M cell area than their C(4) relatives in high-light growth conditions. C(3) species also had higher chloroplast coverage of the M cell periphery than C(4) species, particularly opposite intercellular air spaces. In M cells from 10 of the 12 C(4) lineages, a greater fraction of the chloroplast envelope was pulled away from the plasmalemma in the C(4) species than their C(3) relatives. C(3) -C(4) intermediate species generally exhibited similar patterns as their C(3) relatives. We interpret these results to reflect adaptive shifts that facilitate efficient C(4) function by enhancing diffusive access to the site of primary carbon fixation in the cytosol. Fewer chloroplasts in C(4) M cells would also reduce shading of the bundle sheath chloroplasts, which also generate energy required by C(4) photosynthesis. © 2014 John Wiley & Sons Ltd.

Chloroplast-derived vaccine antigens and biopharmaceuticals: expression, folding, assembly and functionality.

PubMed

Chebolu, S; Daniell, H

2009-01-01

Chloroplast genetic engineering offers several advantages, including high levels of transgene expression, transgene containment via maternal inheritance, and multi-gene expression in a single transformation event. Oral delivery is facilitated by hyperexpression of vaccine antigens against cholera, tetanus, anthrax, plague, or canine parvovirus (4%-31% of total soluble protein, TSP) in transgenic chloroplasts (leaves) or non-green plastids (carrots, tomato) as well as the availability of antibiotic free selectable markers or the ability to excise selectable marker genes. Hyperexpression of several therapeutic proteins, including human serum albumin (11.1% TSP), somatotropin (7% TSP), interferon-alpha (19% TSP), interferon-gamma (6% TSP), and antimicrobial peptide (21.5% TSP), facilitates efficient and economic purification. Also, the presence of chaperones and enzymes in chloroplasts facilitates assembly of complex multisubunit proteins and correct folding of human blood proteins with proper disulfide bonds. Functionality of chloroplast-derived vaccine antigens and therapeutic proteins has been demonstrated by several assays, including the macrophage lysis assay, GM1-ganglioside binding assay, protection of HeLA cells or human lung carcinoma cells against encephalomyocarditis virus, systemic immune response, protection against pathogen challenge, and growth or inhibition of cell cultures. Purification of human proinsulin has been achieved using novel purification strategies (inverse temperature transition property) that do not require expensive column chromatography techniques. Thus, transgenic chloroplasts are ideal bio-reactors for production of functional human and animal therapeutic proteins in an environmentally friendly manner.

Chloroplast-Derived Vaccine Antigens and Biopharmaceuticals: Expression, Folding, Assembly and Functionality

PubMed Central

Chebolu, S.; Daniell, H.

2009-01-01

Chloroplast genetic engineering offers several advantages, including high levels of transgene expression, transgene containment via maternal inheritance, and multi-gene expression in a single transformation event. Oral delivery is facilitated by hyperexpression of vaccine antigens against cholera, tetanus, anthrax, plague, or canine parvovirus (4%–31% of total soluble protein, TSP) in transgenic chloroplasts (leaves) or non-green plastids (carrots, tomato) as well as the availability of antibiotic free selectable markers or the ability to excise selectable marker genes. Hyperexpression of several therapeutic proteins, including human serum albumin (11.1% TSP), somatotropin (7% TSP), interferon-alpha (19% TSP), interferon-gamma (6% TSP), and antimicrobial peptide (21.5% TSP), facilitates efficient and economic purification. Also, the presence of chaperones and enzymes in chloroplasts facilitates assembly of complex multisubunit proteins and correct folding of human blood proteins with proper disulfide bonds. Functionality of chloroplast-derived vaccine antigens and therapeutic proteins has been demonstrated by several assays, including the macrophage lysis assay, GM1-ganglioside binding assay, protection of HeLA cells or human lung carcinoma cells against encephalomyocarditis virus, systemic immune response, protection against pathogen challenge, and growth or inhibition of cell cultures. Purification of human proinsulin has been achieved using novel purification strategies (inverse temperature transition property) that do not require expensive column chromatography techniques. Thus, transgenic chloroplasts are ideal bioreactors for production of functional human and animal therapeutic proteins in an environmentally friendly manner. PMID:19401820

Integration of light and circadian signals that regulate chloroplast transcription by a nuclear-encoded sigma factor.

PubMed

Belbin, Fiona E; Noordally, Zeenat B; Wetherill, Sarah J; Atkins, Kelly A; Franklin, Keara A; Dodd, Antony N

2017-01-01

We investigated the signalling pathways that regulate chloroplast transcription in response to environmental signals. One mechanism controlling plastid transcription involves nuclear-encoded sigma subunits of plastid-encoded plastid RNA polymerase. Transcripts encoding the sigma factor SIG5 are regulated by light and the circadian clock. However, the extent to which a chloroplast target of SIG5 is regulated by light-induced changes in SIG5 expression is unknown. Moreover, the photoreceptor signalling pathways underlying the circadian regulation of chloroplast transcription by SIG5 are unidentified. We monitored the regulation of chloroplast transcription in photoreceptor and sigma factor mutants under controlled light regimes in Arabidopsis thaliana. We established that a chloroplast transcriptional response to light intensity was mediated by SIG5; a chloroplast transcriptional response to the relative proportions of red and far red light was regulated by SIG5 through phytochrome and photosynthetic signals; and the circadian regulation of chloroplast transcription by SIG5 was predominantly dependent on blue light and cryptochrome. Our experiments reveal the extensive integration of signals concerning the light environment by a single sigma factor to regulate chloroplast transcription. This may originate from an evolutionarily ancient mechanism that protects photosynthetic bacteria from high light stress, which subsequently became integrated with higher plant phototransduction networks. © 2016 The Authors. New Phytologist © 2016 New Phytologist Trust.

Light, genotype, and abscisic acid affect chloroplast positioning in guard cells of Arabidopsis thaliana leaves in distinct ways.

PubMed

Königer, Martina; Jessen, Brita; Yang, Rui; Sittler, Dorothea; Harris, Gary C

2010-09-01

The goal of this study was to investigate the effects of light intensity, genotype, and various chemical treatments on chloroplast movement in guard cells of Arabidopsis thaliana leaves. After treatment at various light intensities (dark, low, and high light), leaf discs were fixed with glutaraldehyde, and imaged using confocal laser microscopy. Each chloroplast was assigned a horizontal (close to pore, center, or epidermal side) and vertical (outer, middle, inner) position. White light had a distinct effect on chloroplast positioning, most notably under high light (HL) when chloroplasts on the upper leaf surface of wild-type (WT) moved from epidermal and center positions toward the pore. This was not the case for phot1-5/phot2-1 or phot2-1 plants, thus phototropins are essential for chloroplast positioning in guard cells. In npq1-2 mutants, fewer chloroplasts moved to the pore position under HL than in WT plants, indicating that white light can affect chloroplast positioning also in a zeaxanthin-dependent way. Cytochalasin B inhibited the movement of chloroplasts to the pore under HL, while oryzalin did not, supporting the idea that actin plays a role in the movement. The movement along actin cables is dependent on CHUP1 since chloroplast positioning in chup1 was significantly altered. Abscisic acid (ABA) caused most chloroplasts in WT and phot1-5/phot2-1 to be localized in the center, middle part of the guard cells irrespective of light treatment. This indicates that not only light but also water stress influences chloroplast positioning.

Chloroplast heterogeneity and historical admixture within the genus Malus.

PubMed

Volk, Gayle M; Henk, Adam D; Baldo, Angela; Fazio, Gennaro; Chao, C Thomas; Richards, Christopher M

2015-07-01

• The genus Malus represents a unique and complex evolutionary context in which to study domestication. Several Malus species have provided novel alleles and traits to the cultivars. The extent of admixture among wild Malus species has not been well described, due in part to limited sampling of individuals within a taxon.• Four chloroplast regions (1681 bp total) were sequenced and aligned for 412 Malus individuals from 30 species. Phylogenetic relationships were reconstructed using maximum parsimony. The distribution of chloroplast haplotypes among species was examined using statistical parsimony, phylogenetic trees, and a median-joining network.• Chloroplast haplotypes are shared among species within Malus. Three major haplotype-sharing networks were identified. One includes species native to China, Western North America, as well as Malus domestica Borkh, and its four primary progenitor species: M. sieversii (Ledeb.) M. Roem., M. orientalis Uglitzk., M. sylvestris (L.) Mill., and M. prunifolia (Willd.) Borkh; another includes five Chinese Malus species, and a third includes the three Malus species native to Eastern North America.• Chloroplast haplotypes found in M. domestica belong to a single, highly admixed network. Haplotypes shared between the domesticated apple and its progenitors may reflect historical introgression or the retention of ancestral polymorphisms. Multiple individuals should be sampled within Malus species to reveal haplotype heterogeneity, if complex maternal contributions to named species are to be recognized. © 2015 Botanical Society of America, Inc.

Desaturation of oleoyl groups in envelope membranes from spinach chloroplasts.

PubMed Central

Schmidt, H; Heinz, E

1990-01-01

Envelope membranes isolated from chloroplasts of spinach (Spinacia oleracea) desaturate oleoyl groups in monogalactosyl diacylglycerol to linoleoyl groups. The desaturation requires NADPH in combination with ferredoxin and is not restricted to monogalactosyl diacylglycerol, since it is also observed in biosynthetic intermediates as, for example, in phosphatidic acid. This indicates a certain degree of unspecificity of the oleate desaturase in isolated envelope membranes. Lipid desaturation is another important function of chloroplast envelopes. PMID:11607123

Insights from the complete chloroplast genome into the evolution of Sesamum indicum L.

PubMed

Zhang, Haiyang; Li, Chun; Miao, Hongmei; Xiong, Songjin

2013-01-01

Sesame (Sesamum indicum L.) is one of the oldest oilseed crops. In order to investigate the evolutionary characters according to the Sesame Genome Project, apart from sequencing its nuclear genome, we sequenced the complete chloroplast genome of S. indicum cv. Yuzhi 11 (white seeded) using Illumina and 454 sequencing. Comparisons of chloroplast genomes between S. indicum and the 18 other higher plants were then analyzed. The chloroplast genome of cv. Yuzhi 11 contains 153,338 bp and a total of 114 unique genes (KC569603). The number of chloroplast genes in sesame is the same as that in Nicotiana tabacum, Vitis vinifera and Platanus occidentalis. The variation in the length of the large single-copy (LSC) regions and inverted repeats (IR) in sesame compared to 18 other higher plant species was the main contributor to size variation in the cp genome in these species. The 77 functional chloroplast genes, except for ycf1 and ycf2, were highly conserved. The deletion of the cp ycf1 gene sequence in cp genomes may be due either to its transfer to the nuclear genome, as has occurred in sesame, or direct deletion, as has occurred in Panax ginseng and Cucumis sativus. The sesame ycf2 gene is only 5,721 bp in length and has lost about 1,179 bp. Nucleotides 1-585 of ycf2 when queried in BLAST had hits in the sesame draft genome. Five repeats (R10, R12, R13, R14 and R17) were unique to the sesame chloroplast genome. We also found that IR contraction/expansion in the cp genome alters its rate of evolution. Chloroplast genes and repeats display the signature of convergent evolution in sesame and other species. These findings provide a foundation for further investigation of cp genome evolution in Sesamum and other higher plants.

Identification and Characterization of a Chloroplast-Targeted Obg GTPase in Dendrobium officinale.

PubMed

Chen, Ji; Deng, Feng; Deng, Mengsheng; Han, Jincheng; Chen, Jianbin; Wang, Li; Yan, Shen; Tong, Kai; Liu, Fan; Tian, Mengliang

2016-12-01

Bacterial homologous chloroplast-targeted Obg GTPases (ObgCs) belong to the plant-typical Obg group, which is involved in diverse physiological processes during chloroplast development. However, the evolutionarily conserved function of ObgC in plants remains elusive and requires further investigation. In this study, we identified DoObgC from an epiphytic plant Dendrobium officinale and demonstrated the characteristics of DoObgC. Sequence analysis indicated that DoObgC is highly conserved with other plant ObgCs, which contain the chloroplast transit peptide (cTP), Obg fold, G domain, and OCT regions. The C terminus of DoObgC lacking the chloroplast-targeting cTP region, DoObgC Δ1-160 , showed strong similarity to ObgE and other bacterial Obgs. Overexpression of DoObgC Δ1-160 in Escherichia coli caused slow cell growth and an increased number of elongated cells. This phenotype was consistent with the phenotype of cells overexpressing ObgE. Furthermore, the expression of recombinant DoObgC Δ1-160 enhanced the cell persistence of E. coli to streptomycin. Results of transient expression assays revealed that DoObgC was localized to chloroplasts. Moreover, we demonstrated that DoObgC could rescue the embryotic lethal phenotype of the Arabidopsis obgc-t mutant, suggesting that DoObgC is a functional homolog to Arabidopsis AtObgC in D. officinale. Gene expression profiles showed that DoObgC was expressed in leaf-specific and light-dependent patterns and that DoObgC responded to wounding treatments. Our previous and present studies reveal that ObgC has an evolutionarily conserved role in ribosome biogenesis to adapt chloroplast development to the environment.

Tic40, a membrane-anchored co-chaperone homolog in the chloroplast protein translocon

PubMed Central

Chou, Ming-Lun; Fitzpatrick, Lynda M.; Tu, Shuh-Long; Budziszewski, Gregory; Potter-Lewis, Sharon; Akita, Mitsuru; Levin, Joshua Z.; Keegstra, Kenneth; Li, Hsou-min

2003-01-01

The function of Tic40 during chloroplast protein import was investigated. Tic40 is an inner envelope membrane protein with a large hydrophilic domain located in the stroma. Arabidopsis null mutants of the atTic40 gene were very pale green and grew slowly but were not seedling lethal. Isolated mutant chloroplasts imported precursor proteins at a lower rate than wild-type chloroplasts. Mutant chloroplasts were normal in allowing binding of precursor proteins. However, during subsequent translocation across the inner membrane, fewer precursors were translocated and more precursors were released from the mutant chloroplasts. Cross-linking experiments demonstrated that Tic40 was part of the translocon complex and functioned at the same stage of import as Tic110 and Hsp93, a member of the Hsp100 family of molecular chaperones. Tertiary structure prediction and immunological studies indicated that the C-terminal portion of Tic40 contains a TPR domain followed by a domain with sequence similarity to co-chaperones Sti1p/Hop and Hip. We propose that Tic40 functions as a co-chaperone in the stromal chaperone complex that facilitates protein translocation across the inner membrane. PMID:12805212

Stable chloroplast transformation of immature scutella and inflorescences in wheat (Triticum aestivum L.).

PubMed

Cui, Cuiju; Song, Fei; Tan, Yi; Zhou, Xuan; Zhao, Wen; Ma, Fengyun; Liu, Yunyi; Hussain, Javeed; Wang, Yuesheng; Yang, Guangxiao; He, Guangyuan

2011-04-01

Chloroplast transformation in wheat was achieved by bombardment of scutella from immature embryos and immature inflorescences, respectively. A wheat chloroplast site-specific expression vector, pBAGNRK, was constructed by placing an expression cassette containing neomycin phosphotransferase II (nptII) and green fluorescent protein (gfp) as selection and reporter genes, respectively, in the intergenic spacer between atpB and rbcL of wheat chloroplast genome. Integration of gfp gene in the plastome was identified by polymerase chain reaction (PCR) analysis and Southern blotting using gfp gene as a probe. Expression of GFP protein was examined by western blot. Three positive transformants were obtained and the Southern blot of partial fragment of atpB and rbcL (targeting site) probes verified that one of them was homoplasmic. Stable expression of GFP fluorescence was confirmed by confocal microscopy in the leaf tissues from T(1) progeny seedlings. PCR analysis of gfp gene also confirmed the inheritance of transgene in the T(1) progeny. These results strengthen the feasibility of wheat chloroplast transformation and also give a novel method for the introduction of important agronomic traits in wheat through chloroplast transformation.

Inferring Phylogenetic Relationships of Indian Citron (Citrus medica L.) based on rbcL and matK Sequences of Chloroplast DNA.

PubMed

Uchoi, Ajit; Malik, Surendra Kumar; Choudhary, Ravish; Kumar, Susheel; Rohini, M R; Pal, Digvender; Ercisli, Sezai; Chaudhury, Rekha

2016-06-01

Phylogenetic relationships of Indian Citron (Citrus medica L.) with other important Citrus species have been inferred through sequence analyses of rbcL and matK gene region of chloroplast DNA. The study was based on 23 accessions of Citrus genotypes representing 15 taxa of Indian Citrus, collected from wild, semi-wild, and domesticated stocks. The phylogeny was inferred using the maximum parsimony (MP) and neighbor-joining (NJ) methods. Both MP and NJ trees separated all the 23 accessions of Citrus into five distinct clusters. The chloroplast DNA (cpDNA) analysis based on rbcL and matK sequence data carried out in Indian taxa of Citrus was useful in differentiating all the true species and species/varieties of probable hybrid origin in distinct clusters or groups. Sequence analysis based on rbcL and matK gene provided unambiguous identification and disposition of true species like C. maxima, C. medica, C. reticulata, and related hybrids/cultivars. The separation of C. maxima, C. medica, and C. reticulata in distinct clusters or sub-clusters supports their distinctiveness as the basic species of edible Citrus. However, the cpDNA sequence analysis of rbcL and matK gene could not find any clear cut differentiation between subgenera Citrus and Papeda as proposed in Swingle's system of classification.

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

Multiplex sequencing of plant chloroplast genomes using Solexa sequencing-by-synthesis technology

Treesearch

Richard Cronn; Aaron Liston; Matthew Parks; David S. Gernandt; Rongkun Shen; Todd Mockler

2008-01-01

Organellar DNA sequences are widely used in evolutionary and population genetic studies; however, the conservative nature of chloroplast gene and genome evolution often limits phylogenetic resolution and statistical power. To gain maximal access to the historical record contained within chloroplast genomes, we have adapted multiplex sequencing-by-synthesis (MSBS) to...

Chloroplast-encoded serotonin N-acetyltransferase in the red alga Pyropia yezoensis: gene transition to the nucleus from chloroplasts.

PubMed

Byeon, Yeong; Yool Lee, Hyoung; Choi, Dong-Woog; Back, Kyoungwhan

2015-02-01

Melatonin biosynthesis involves the N-acetylation of arylalkylamines such as serotonin, which is catalysed by serotonin N-acetyltransferase (SNAT), the penultimate enzyme of melatonin biosynthesis in both animals and plants. Here, we report the functional characterization of a putative N-acetyltransferase gene in the chloroplast genome of the alga laver (Pyropia yezoensis, formerly known as Porphyra yezoensis) with homology to the rice SNAT gene. To confirm that the putative Pyropia yezoensis SNAT (PySNAT) gene encodes an SNAT, we cloned the full-length chloroplastidic PySNAT gene by PCR and purified the recombinant PySNAT protein from Escherichia coli. PySNAT was 174 aa and had 50% amino acid identity with cyanobacteria SNAT. Purified recombinant PySNAT showed a peak activity at 55 °C with a K m of 467 µM and V max of 28 nmol min-1 mg(-1) of protein. Unlike other plant SNATs, PySNAT localized to the cytoplasm due to a lack of N-terminal chloroplast transit peptides. Melatonin was present at 0.16ng g(-1) of fresh mass but increased during heat stress. Phylogenetic analysis of the sequence suggested that PySNAT has evolved from the cyanobacteria SNAT gene via endosymbiotic gene transfer. Additionally, the chloroplast transit peptides of plant SNATs were acquired 1500 million years ago, concurrent with the appearance of green algae. © The Author 2014. Published by Oxford University Press on behalf of the Society for Experimental Biology.

Cryo-EM structure of the large subunit of the spinach chloroplast ribosome

PubMed Central

Ahmed, Tofayel; Yin, Zhan; Bhushan, Shashi

2016-01-01

Protein synthesis in the chloroplast is mediated by the chloroplast ribosome (chloro-ribosome). Overall architecture of the chloro-ribosome is considerably similar to the Escherichia coli (E. coli) ribosome but certain differences are evident. The chloro-ribosome proteins are generally larger because of the presence of chloroplast-specific extensions in their N- and C-termini. The chloro-ribosome harbours six plastid-specific ribosomal proteins (PSRPs); four in the small subunit and two in the large subunit. Deletions and insertions occur throughout the rRNA sequence of the chloro-ribosome (except for the conserved peptidyl transferase center region) but the overall length of the rRNAs do not change significantly, compared to the E. coli. Although, recent advancements in cryo-electron microscopy (cryo-EM) have provided detailed high-resolution structures of ribosomes from many different sources, a high-resolution structure of the chloro-ribosome is still lacking. Here, we present a cryo-EM structure of the large subunit of the chloro-ribosome from spinach (Spinacia oleracea) at an average resolution of 3.5 Å. High-resolution map enabled us to localize and model chloro-ribosome proteins, chloroplast-specific protein extensions, two PSRPs (PSRP5 and 6) and three rRNA molecules present in the chloro-ribosome. Although comparable to E. coli, the polypeptide tunnel and the tunnel exit site show chloroplast-specific features. PMID:27762343

Sequence evidence for the symbiotic origins of chloroplasts and mitochondria

NASA Technical Reports Server (NTRS)

George, D. G.; Hunt, L. T.; Dayhoff, M. O.

1983-01-01

The origin of mitochondria and chloroplasts is investigated on the basis of prokaryotic and early-eukaryotic evolutionary trees derived from protein and nucleic-acid sequences by the method of Dayhoff (1979). Trees for bacterial ferrodoxins, 5S ribosomal RNA, c-type cytochromes, the lipid-binding subunit of ATPase, and dihydrofolate reductase are presented and discussed. Good agreement among the trees is found, and it is argued that the mitochondria and chloroplasts evolved by multiple symbiotic events.

Different effects of eubacterial and eukaryotic DNA topoisomerase II inhibitors on chloroplasts ofEuglena gracilis

NASA Astrophysics Data System (ADS)

Krajčovič, Juraj; Ebringer, Libor

1990-03-01

Inhibitors of eubacterial and eukaryotic DNA topoisomerases type II exhibited different effects on chloroplasts of the flagellateEuglena gracilis. Antibacterial agents (cinoxacin, nalidixic and oxolinic acids, ciprofloxacin, enoxacin, norfloxacin and ofloxacin) from the group of quinolones and coumarins (coumermycin A1, clorobiocin and novobiocin) — all inhibitors of prokaryotic DNA topoisomerase II — were very potent eliminators of chloroplasts fromE. gracilis. In contrast, antitumor drugs (adriamycin, etoposide, teniposide and mitoxantrone) — antagonists of the eukaryotic counterpart — did not affect these semiautonomous photosynthetic organelles. These findings point out again the close evolutionary relationships between eubacteria and chloroplasts and are in agreement with the hypothesis of an endosymbiotic origin of chloroplasts.

Chloroplast microsatellites reveal population genetic diversity in red pine, Pinus resinosa Ait

Treesearch

Craig S. Echt; L.L. DeVerno; M. Anzidei; G.G. Vendramin

1998-01-01

Variation in paternally inherited chloroplast microsatellite (cpSSR) DNA was used to study population genetic structure in red pine (Pinus resinosa Ait.), a species characterized by morphological uniformity, no allozyme variation, and limited RAPD variation. Using nine cpSSR loci, a total of 23 chloroplast haplotypes and 25 cpSSR alleles were were...

Phylogeographical patterns of an alpine plant, Rhodiola dumulosa (Crassulaceae), inferred from chloroplast DNA sequences.

PubMed

Hou, Yan; Lou, Anru

2014-01-01

The phylogeographical patterns of Rhodiola dumulosa, an alpine plant species restrictedly growing in the crevices of rock piles, were investigated based on 4 fragments of the chloroplast genome. To cover the full distribution of R. dumulosa in China, 19 populations from 3 major disjunct distribution areas (northern, central, and northwestern China) were sampled. A total of 5881bp (after alignment) of chloroplast DNA (cpDNA) from 100 individuals were sequenced. The combined cpDNA data set yielded 36 haplotypes. The total genetic diversity of R. dumulosa was remarkably high (H(T) = 0.981). The interpopulation genetic differentiation was significantly large (F(ST) = 0.8537, P < 0.001), possibly due to the long-term isolation of the natural populations. N(ST) was significantly larger than G(ST) (P < 0.001), indicating the presence of phylogeographical structure among the R. dumulosa populations. We propose 2 migration steps to explain the current distribution of R. dumulosa in China. First, this species migrated from refugia in the Qinghai-Tibetan Plateau to northern areas via the intervening highlands when temperatures increased; second, the highland populations migrated toward the mountaintops when temperatures increased further because R. dumulosa is adapted to cold environments. During the second migration step, the common ancestral haplotypes may have been gradually lost.

Enzymic synthesis of γ-coniceine in Conium maculatum chloroplasts and mitochondria.

PubMed

Roberts, M F

1981-08-01

Further studies of the transaminase responsible for the first committed step in alkaloid formation in Conium maculatum have shown the L-alanine: 5-ketooctanal transaminase to occur in both the mitochondria and chloroplast. Experiments suggest that these enzymes are the isoenzymes Transaminase A and B respectively previously isolated by the author. It is suggested that the chloroplast enzyme is normally responsible for alkaloid production.

Mesophyll Chloroplast Investment in C3, C4 and C2 Species of the Genus Flaveria.

PubMed

Stata, Matt; Sage, Tammy L; Hoffmann, Natalie; Covshoff, Sarah; Ka-Shu Wong, Gane; Sage, Rowan F

2016-05-01

The mesophyll (M) cells of C4 plants contain fewer chloroplasts than observed in related C3 plants; however, it is uncertain where along the evolutionary transition from C3 to C4 that the reduction in M chloroplast number occurs. Using 18 species in the genus Flaveria, which contains C3, C4 and a range of C3-C4 intermediate species, we examined changes in chloroplast number and size per M cell, and positioning of chloroplasts relative to the M cell periphery. Chloroplast number and coverage of the M cell periphery declined in proportion to increasing strength of C4 metabolism in Flaveria, while chloroplast size increased with increasing C4 cycle strength. These changes increase cytosolic exposure to the cell periphery which could enhance diffusion of inorganic carbon to phosphenolpyruvate carboxylase (PEPC), a cytosolic enzyme. Analysis of the transcriptome from juvenile leaves of nine Flaveria species showed that the transcript abundance of four genes involved in plastid biogenesis-FtsZ1, FtsZ2, DRP5B and PARC6-was negatively correlated with variation in C4 cycle strength and positively correlated with M chloroplast number per planar cell area. Chloroplast size was negatively correlated with abundance of FtsZ1, FtsZ2 and PARC6 transcripts. These results indicate that natural selection targeted the proteins of the contractile ring assembly to effect the reduction in chloroplast numbers in the M cells of C4 Flaveria species. If so, efforts to engineer the C4 pathway into C3 plants might evaluate whether inducing transcriptome changes similar to those observed in Flaveria could reduce M chloroplast numbers, and thus introduce a trait that appears essential for efficient C4 function. © 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.

Chloroplastic biosynthesis of melatonin and its involvement in protection of plants from salt stress

PubMed Central

Zheng, Xiaodong; Tan, Dun X.; Allan, Andrew C.; Zuo, Bixiao; Zhao, Yu; Reiter, Russel J.; Wang, Lin; Wang, Zhi; Guo, Yan; Zhou, Jingzhe; Shan, Dongqian; Li, Qingtian; Han, Zhenhai; Kong, Jin

2017-01-01

Within the chloroplasts reactive oxygen species (ROS) are generated during photosynthesis and stressful conditions. Excessive ROS damages chloroplasts and reduces photosynthesis if not properly detoxified. In this current study, we document that chloroplasts produce melatonin, a recently-discovered plant antioxidant molecule. When N-acetylserotonin, a substrate for melatonin synthesis, was fed to purified chloroplasts, they produced melatonin in a dose-response manner. To further confirm this function of chloroplasts, the terminal enzyme for melatonin synthesis, N-acetylserotonin-O-methyltransferase (ASMT), was cloned from apple rootstock, Malus zumi. The in vivo fluorescence observations and Western blots confirmed MzASMT9 was localized in the chloroplasts. A study of enzyme kinetics revealed that the Km and Vmax of the purified recombinant MzASMT9 protein for melatonin synthesis were 500 μM and 12 pmol/min·mg protein, respectively. Arabidopsis ectopically-expressing MzASMT9 possessed improved melatonin level. Importantly, the MzASMT9 gene was found to be upregulated by high light intensity and salt stress. Increased melatonin due to the highly-expressed MzASMT9 resulted in Arabidopsis lines with enhanced salt tolerance than wild type plants, as indicated by reduced ROS, lowered lipid peroxidation and enhanced photosynthesis. These findings have agricultural applications for the genetic enhancement of melatonin-enriched plants for increasing crop production under a variety of unfavorable environmental conditions. PMID:28145449

Effects of catalase on chloroplast arrangement in Opuntia streptacantha chlorenchyma cells under salt stress.

PubMed

Arias-Moreno, Diana Marcela; Jiménez-Bremont, Juan Francisco; Maruri-López, Israel; Delgado-Sánchez, Pablo

2017-08-17

In arid and semiarid regions, low precipitation rates lead to soil salinity problems, which may limit plant establishment, growth, and survival. Herein, we investigated the NaCl stress effect on chlorophyll fluorescence, photosynthetic-pigments, movement and chloroplasts ultrastructure in chlorenchyma cells of Opuntia streptacantha cladodes. Cladodes segments were exposed to salt stress at 0, 100, 200, and 300 mM NaCl for 8, 16, and 24 h. The results showed that salt stress reduced chlorophyll content, F v /F m , ΦPSII, and qP values. Under the highest salt stress treatments, the chloroplasts were densely clumped toward the cell center and thylakoid membranes were notably affected. We analyzed the effect of exogenous catalase in salt-stressed cladode segments during 8, 16, and 24 h. The catalase application to salt-stressed cladodes counteracted the NaCl adverse effects, increasing the chlorophyll fluorescence parameters, photosynthetic-pigments, and avoided chloroplast clustering. Our results indicate that salt stress triggered the chloroplast clumping and affected the photosynthesis in O. streptacantha chlorenchyma cells. The exogenous catalase reverted the H 2 O 2 accumulation and clustering of chloroplast, which led to an improvement of the photosynthetic efficiency. These data suggest that H 2 O 2 detoxification by catalase is important to protect the chloroplast, thus conserving the photosynthetic activity in O. streptacantha under stress.

Expression and Chloroplast Targeting of Cholesterol Oxidase in Transgenic Tobacco Plants

PubMed Central

Corbin, David R.; Grebenok, Robert J.; Ohnmeiss, Thomas E.; Greenplate, John T.; Purcell, John P.

2001-01-01

Cholesterol oxidase represents a novel type of insecticidal protein with potent activity against the cotton boll weevil (Anthonomus grandis grandis Boheman). We transformed tobacco (Nicotiana tabacum) plants with the cholesterol oxidase choM gene and expressed cytosolic and chloroplast-targeted versions of the ChoM protein. Transgenic leaf tissues expressing cholesterol oxidase exerted insecticidal activity against boll weevil larvae. Our results indicate that cholesterol oxidase can metabolize phytosterols in vivo when produced cytosolically or when targeted to chloroplasts. The transgenic plants exhibiting cytosolic expression accumulated low levels of saturated sterols known as stanols, and displayed severe developmental aberrations. In contrast, the transgenic plants expressing chloroplast-targeted cholesterol oxidase maintained a greater accumulation of stanols, and appeared phenotypically and developmentally normal. These results are discussed within the context of plant sterol distribution and metabolism. PMID:11457962

The SAL-PAP Chloroplast Retrograde Pathway Contributes to Plant Immunity by Regulating Glucosinolate Pathway and Phytohormone Signaling.

PubMed

Ishiga, Yasuhiro; Watanabe, Mutsumi; Ishiga, Takako; Tohge, Takayuki; Matsuura, Takakazu; Ikeda, Yoko; Hoefgen, Rainer; Fernie, Alisdair R; Mysore, Kirankumar S

2017-10-01

Chloroplasts have a crucial role in plant immunity against pathogens. Increasing evidence suggests that phytopathogens target chloroplast homeostasis as a pathogenicity mechanism. In order to regulate the performance of chloroplasts under stress conditions, chloroplasts produce retrograde signals to alter nuclear gene expression. Many signals for the chloroplast retrograde pathway have been identified, including chlorophyll intermediates, reactive oxygen species, and metabolic retrograde signals. Although there is a reasonably good understanding of chloroplast retrograde signaling in plant immunity, some signals are not well-understood. In order to understand the role of chloroplast retrograde signaling in plant immunity, we investigated Arabidopsis chloroplast retrograde signaling mutants in response to pathogen inoculation. sal1 mutants (fry1-2 and alx8) responsible for the SAL1-PAP retrograde signaling pathway showed enhanced disease symptoms not only to the hemibiotrophic pathogen Pseudomonas syringae pv. tomato DC3000 but, also, to the necrotrophic pathogen Pectobacterium carotovorum subsp. carotovorum EC1. Glucosinolate profiles demonstrated the reduced accumulation of aliphatic glucosinolates in the fry1-2 and alx8 mutants compared with the wild-type Col-0 in response to DC3000 infection. In addition, quantification of multiple phytohormones and analyses of their gene expression profiles revealed that both the salicylic acid (SA)- and jasmonic acid (JA)-mediated signaling pathways were down-regulated in the fry1-2 and alx8 mutants. These results suggest that the SAL1-PAP chloroplast retrograde pathway is involved in plant immunity by regulating the SA- and JA-mediated signaling pathways.

Regulation of chloroplast biogenesis: the immutans mutant of Arabidopsis

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

Rodermel, Steven

The immutans (im) variegation mutant of Arabidopsis is an ideal model to gain insight into factors that control chloroplast biogenesis. im defines the gene for PTOX, a plastoquinol terminal oxidase that participates in control of thylakoid redox. Here, we report that the im defect can be suppressed during the late stages of plant development by gigantea (gi2), which defines the gene for GIGANTEA (GI), a central component of the circadian clock that plays a poorly-understood role in diverse plant developmental processes. imgi2 mutants are late-flowering and display other well-known phenotypes associated with gi2, such as starch accumulation and resistance tomore » oxidative stress. We show that the restoration of chloroplast biogenesis in imgi2 is caused by a developmental-specific de-repression of cytokinin signaling that involves crosstalk with signaling pathways mediated by gibberellin (GA) and SPINDLY (SPY), a GA response inhibitor. Suppression of the plastid defect in imgi2 is likely caused by a relaxation of excitation pressures in developing plastids by factors contributed by gi2, including enhanced rates of photosynthesis and increased resistance to oxidative stress. Interestingly, the suppression phenotype of imgi can be mimicked by crossing im with the starch accumulation mutant, sex1, perhaps because sex1 utilizes pathways similar to gi. We conclude that our studies provide a direct genetic linkage between GIGANTEA and chloroplast biogenesis, and we construct a model of interactions between signaling pathways mediated by gi, GA, SPY, cytokinins, and sex1 that are required for chloroplast biogenesis.« less

Induction of Hexose-Phosphate Translocator Activity in Spinach Chloroplasts.

PubMed Central

Quick, W. P.; Scheibe, R.; Neuhaus, H. E.

1995-01-01

Many environmental and experimental conditions lead to accumulation of carbohydrates in photosynthetic tissues. This situation is typically associated with major changes in the mRNA and protein complement of the cell, including metabolic repression of photosynthetic gene expression, which can be induced by feeding carbohydrates directly to leaves. In this study we examined the carbohydrate transport properties of chloroplasts isolated from spinach (Spinacia oleracea L.) leaves fed with glucose for several days. These chloroplasts contain large quantities of starch, can perform photosynthetic 3-phosphoglycerate reduction, and surprisingly also have the ability to perform starch synthesis from exogenous glucose-6-phosphate (Glc-6-P) both in the light and in darkness, similarly to heterotrophic plastids. Glucose-1-phosphate does not act as an exogenous precursor for starch synthesis. Light, ATP, and 3-phosphoglyceric acid stimulate Glc-6-P-dependent starch synthesis. Short-term uptake experiments indicate that a novel Glc-6-P-translocator capacity is present in the envelope membrane, exhibiting an apparent Km of 0.54 mM and a Vmax of 2.9 [mu]mol Glc-6-P mg-1 chlorophyll h-1. Similar results were obtained with chloroplasts isolated from glucose-fed potato leaves and from water-stressed spinach leaves. The generally held view that sugar phosphates transported by chloroplasts are confined to triose phosphates is not supported by these results. A physiological role for a Glc-6-P translocator in green plastids is presented with reference to the source/sink function of the leaf. PMID:12228584

The complete chloroplast genome sequence of tung tree (Vernicia fordii): Organization and phylogenetic relationships with other angiosperms

USDA-ARS?s Scientific Manuscript database

Tung tree (Vernicia fordii) is an economically important plant widely cultivated for industrial oil production in China. To better understand the molecular basis of tung tree chloroplasts, we sequenced and characterized the complete chloroplast genome. The chloroplast genome was 161,524 bp in length...

Pea amyloplast DNA is qualitatively similar to pea chloroplast DNA

NASA Technical Reports Server (NTRS)

Gaynor, J. J.

1984-01-01

Amyloplast DNA (apDNA), when subjected to digestion with restriction endonucleases, yields patterns nearly identical to that of DNA from mature pea chloroplasts (ctDNA). Southern transfers of apDNA and ctDNA, probed with the large subunit (LS) gene of ribulose-1,5-bisphosphate carboxylase (Rubisco), shows hybridization to the expected restriction fragments for both apDNA and ctDNA. However, Northern transfers of total RNA from chloroplasts and amyloplasts, probed again with the LS gene of Rubisco, shows that no detectable LS meggage is found in amyloplasts although LS expression in mature chloroplasts is high. Likewise, two dimensional polyacrylamide gel electrophoresis of etiolated gravisensitive pea tissue shows that both large and small subunits of Rubisco are conspicuously absent; however, in greening tissue these two constitute the major soluble proteins. These findings suggest that although the informational content of these two organelle types is equivalent, gene expression is quite different and is presumably under nuclear control.

The KAC family of kinesin-like proteins is essential for the association of chloroplasts with the plasma membrane in land plants.

PubMed

Suetsugu, Noriyuki; Sato, Yoshikatsu; Tsuboi, Hidenori; Kasahara, Masahiro; Imaizumi, Takato; Kagawa, Takatoshi; Hiwatashi, Yuji; Hasebe, Mitsuyasu; Wada, Masamitsu

2012-11-01

Chloroplasts require association with the plasma membrane for movement in response to light and for appropriate positioning within the cell to capture photosynthetic light efficiently. In Arabidopsis, CHLOROPLAST UNUSUAL POSITIONING 1 (CHUP1), KINESIN-LIKE PROTEIN FOR ACTIN-BASED CHLOROPLAST MOVEMENT 1 (KAC1) and KAC2 are required for both the proper movement of chloroplasts and the association of chloroplasts with the plasma membrane, through the reorganization of short actin filaments located on the periphery of the chloroplasts. Here, we show that KAC and CHUP1 orthologs (AcKAC1, AcCHUP1A and AcCHUP1B, and PpKAC1 and PpKAC2) play important roles in chloroplast positioning in the fern Adiantum capillus-veneris and the moss Physcomitrella patens. The knockdown of AcKAC1 and two AcCHUP1 genes induced the aggregation of chloroplasts around the nucleus. Analyses of A. capillus-veneris mutants containing perinuclear-aggregated chloroplasts confirmed that AcKAC1 is required for chloroplast-plasma membrane association. In addition, P. patens lines in which two KAC genes had been knocked out showed an aggregated chloroplast phenotype similar to that of the fern kac1 mutants. These results indicate that chloroplast positioning and movement are mediated through the activities of KAC and CHUP1 proteins, which are conserved in land plants.

Protein methylation in pea chloroplasts. [Pisum sativum

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

Niemi, K.J.; Adler, J.; Selman, B.R.

1990-07-01

The methylation of chloroplast proteins has been investigated by incubating intact pea (Pisum sativum) chloroplasts with ({sup 3}H-methyl)-S-adenosylmethionine. Incubation in the light increases the amount of methylation in both the thylakoid and stromal fractions. Numerous thylakoid proteins serve as substrates for the methyltransfer reactions. Three of these thylakoid proteins are methylated to a significantly greater extent in the light than in the dark. The primary stromal polypeptide methylated is the large subunit of ribulose bisphosphate carboxylase/oxygenase. One other stromal polypeptide is also methylated much more in the light than in the dark. Two distinct types of protein methylation occur. Onemore » methylinkage is stable to basic conditions whereas a second type is base labile. The base-stable linkage is indicative of N-methylation of amino acid residues while base-lability is suggestive of carboxymethylation of amino acid residues. Labeling in the light increases the percentage of methylation that is base labile in the thylakoid fraction while no difference is observed in the amount of base-labile methylations in light-labeled and dark-labeled stromal proteins. Also suggestive of carboxymethylation is the detection of volatile ({sup 3}H)methyl radioactivity which increases during the labeling period and is greater in chloroplasts labeled in the light as opposed to being labeled in the dark; this implies in vivo turnover of the ({sup 3}H)methyl group.« less

Designing specific chloroplast markers for black walnut from a set of universal primers

Treesearch

Erin Victory; Rodney L. Robichaud; Keith Woeste

2003-01-01

Chloroplasts are a valuable source of genetic information because their sequence is highly conserved, they undergo little or no recombination, and they are uniparentally inherited. Chloroplast polymorphisms are powerful genetic tools for identifying matrilineal family groups, studying gene flow from seed versus pollen movement, reconstructing phylogeographic...

An optimized transit peptide for effective targeting of diverse foreign proteins into chloroplasts in rice.

PubMed

Shen, Bo-Ran; Zhu, Cheng-Hua; Yao, Zhen; Cui, Li-Li; Zhang, Jian-Jun; Yang, Cheng-Wei; He, Zheng-Hui; Peng, Xin-Xiang

2017-04-11

Various chloroplast transit peptides (CTP) have been used to successfully target some foreign proteins into chloroplasts, but for other proteins these same CTPs have reduced localization efficiencies or fail completely. The underlying cause of the failures remains an open question, and more effective CTPs are needed. In this study, we initially observed that two E.coli enzymes, EcTSR and EcGCL, failed to be targeted into rice chloroplasts by the commonly-used rice rbcS transit peptide (rCTP) and were subsequently degraded. Further analyses revealed that the N-terminal unfolded region of cargo proteins is critical for their localization capability, and that a length of about 20 amino acids is required to attain the maximum localization efficiency. We considered that the unfolded region may alleviate the steric hindrance produced by the cargo protein, by functioning as a spacer to which cytosolic translocators can bind. Based on this inference, an optimized CTP, named RC2, was constructed. Analyses showed that RC2 can more effectively target diverse proteins, including EcTSR and EcGCL, into rice chloroplasts. Collectively, our results provide further insight into the mechanism of CTP-mediated chloroplastic localization, and more importantly, RC2 can be widely applied in future chloroplastic metabolic engineering, particularly for crop plants.

Enzyme-Triggered Defined Protein Nanoarrays: Efficient Light-Harvesting Systems to Mimic Chloroplasts.

PubMed

Zhao, Linlu; Zou, Haoyang; Zhang, Hao; Sun, Hongcheng; Wang, Tingting; Pan, Tiezheng; Li, Xiumei; Bai, Yushi; Qiao, Shanpeng; Luo, Quan; Xu, Jiayun; Hou, Chunxi; Liu, Junqiu

2017-01-24

The elegance and efficiency by which chloroplasts harvest solar energy and conduct energy transfer have been a source of inspiration for chemists to mimic such process. However, precise manipulation to obtain orderly arranged antenna chromophores in constructing artificial chloroplast mimics was a great challenge, especially from the structural similarity and bioaffinity standpoints. Here we reported a design strategy that combined covalent and noncovalent interactions to prepare a protein-based light-harvesting system to mimic chloroplasts. Cricoid stable protein one (SP1) was utilized as a building block model. Under enzyme-triggered covalent protein assembly, mutant SP1 with tyrosine (Tyr) residues at the designated sites can couple together to form nanostructures. Through controlling the Tyr sites on the protein surface, we can manipulate the assembly orientation to respectively generate 1D nanotubes and 2D nanosheets. The excellent stability endowed the self-assembled protein architectures with promising applications. We further integrated quantum dots (QDs) possessing optical and electronic properties with the 2D nanosheets to fabricate chloroplast mimics. By attaching different sized QDs as donor and acceptor chromophores to the negatively charged surface of SP1-based protein nanosheets via electrostatic interactions, we successfully developed an artificial light-harvesting system. The assembled protein nanosheets structurally resembled the natural thylakoids, and the QDs can achieve pronounced FRET phenomenon just like the chlorophylls. Therefore, the coassembled system was meaningful to explore the photosynthetic process in vitro, as it was designed to mimic the natural chloroplast.

Characterisation and changes in the antioxidant system of chloroplasts and chromoplasts isolated from green and mature pepper fruits.

PubMed

Martí, M C; Camejo, D; Olmos, E; Sandalio, L M; Fernández-García, N; Jiménez, A; Sevilla, F

2009-07-01

Purification and characterisation of pepper (Capsicum annuum L) chloroplasts and chromoplasts isolated from commercial green, red and yellow mature fruits were undertaken. Induction of the synthesis of several antioxidants in organelles isolated from mature fruits was found. The ultrastructure of organelles and the presence and activity of SOD isozymes and enzymes involved in the ASC-GSH cycle, together with the non-enzymatic antioxidant content and some oxidative parameters, were analysed. It was found that lipids, rather than proteins, seem to be a target for oxidation in the chromoplasts. The ascorbate and glutathione contents were elicited during differentiation of chloroplasts into chromoplasts in both red and yellow fruits. The activity of SOD and of components of the ASC-GSH cycle was up-regulated, suggesting that these enzymes may play a role in the protection of plastids and could act as modulators of signal molecules such as O(2) ( -) and H(2)O(2) during fruit maturation. The presence of an Mn-SOD in chromoplasts isolated from yellow pepper fruits was also investigated in terms of structural and antioxidant differences between the two cultivars.

Purification and characterization of O-acetylserine (thiol) lyase from spinach chloroplasts.

PubMed

Droux, M; Martin, J; Sajus, P; Douce, R

1992-06-01

O-Acetylserine (thiol) lyase, the last enzyme in the cysteine biosynthetic pathway, was purified to homogeneity from spinach leaf chloroplasts. The enzyme has a molecular mass of 68,000 and consists of two identical subunits of Mr 35,000. The absorption spectrum obtained at pH 7.5 exhibited a peak at 407 nm due to pyridoxal phosphate, and addition of O-acetylserine induced a considerable modification of the spectrum. The pyridoxal phosphate content was found to be 1.1 per subunit of 35,000, and the chromophore was displaced from the enzyme by O-acetylserine, leading to a progressive inactivation of the holoenzyme. Upon gel filtration chromatography on Superdex 200, part of the chloroplastic O-acetylserine (thiol) lyase eluted in association with serine acetyltransferase at a position corresponding to a molecular mass of 310,000 (such a complex called cysteine synthase has been characterized in bacteria). The activity of O-acetylserine (thiol) lyase was optimum between pH 7.5 and 8.5. The apparent Km for O-acetylserine was 1.3 mM and for sulfide was 0.25 mM. The calculated activation energy was 12.6 kcal/mol at 10 mM O-acetylserine. The overall amino-acid composition of spinach chloroplast O-acetylserine (thiol) lyase was different than that determined for the same enzyme (cytosolic?) obtained from a crude extract of spinach leaves. A polyclonal antibody prepared against the chloroplastic O-acetylserine (thiol) lyase exhibited a very low cross-reactivity with a preparation of mitochondrial matrix and cytosolic proteins suggesting that the chloroplastic isoform was distinct from the mitochondrial and cytosolic counterparts.

Production of biopharmaceuticals and vaccines in plants via the chloroplast genome.

PubMed

Daniell, Henry

2006-10-01

Transgenic plants offer many advantages, including low cost of production (by elimination of fermenters), storage and transportation; heat stability; and absence of human pathogens. When therapeutic proteins are orally delivered, plant cells protect antigens in the stomach through bioencapsulation and eliminate the need for expensive purification and sterile injections, in addition to development of both systemic and mucosal immunity. Chloroplast genetic engineering offers several advantages, including high levels of transgene expression, transgene containment via maternal inheritance and multi-gene expression in a single transformation event. Hyper-expression of vaccine antigens against cholera, tetanus, anthrax, plague or canine parvovirus (4-31% of total soluble protein, tsp) in transgenic chloroplasts (leaves) or non-green plastids (carrots, tomato), as well as the availability of antibiotic-free selectable markers or the ability to excise selectable marker genes, facilitate oral delivery. Hyper-expression of several therapeutic proteins, including human serum albumin (11.1% tsp), somatotropin (7% tsp), interferon-gamma (6% tsp), anti-microbial peptide (21.5% tsp), facilitates efficient and economic purification. Also, the presence of chaperones and enzymes in chloroplasts facilitate assembly of complex multi-subunit proteins and correct folding of human blood proteins with proper disulfide bonds. Functionality of chloroplast-derived vaccine antigens and therapeutic proteins has been demonstrated by several assays, including the macrophage lysis assay, GM1-ganglioside binding assay, protection of HeLa cells or human lung carcinoma cells against encephalomyocarditis virus, systemic immune response, protection against pathogen challenge, and growth or inhibition of cell cultures. Thus, transgenic chloroplasts are ideal bioreactors for production of functional human and animal therapeutic proteins in an environmentally friendly manner.

The Cytoskeleton and the Peroxisomal-Targeted SNOWY COTYLEDON3 Protein Are Required for Chloroplast Development in Arabidopsis[W

PubMed Central

Albrecht, Verónica; Šimková, Klára; Carrie, Chris; Delannoy, Etienne; Giraud, Estelle; Whelan, Jim; Small, Ian David; Apel, Klaus; Badger, Murray R.; Pogson, Barry James

2010-01-01

Here, we describe the snowy cotyledon3 (sco3-1) mutation, which impairs chloroplast and etioplast development in Arabidopsis thaliana seedlings. SCO3 is a member of a largely uncharacterized protein family unique to the plant kingdom. The sco3-1 mutation alters chloroplast morphology and development, reduces chlorophyll accumulation, impairs thylakoid formation and photosynthesis in seedlings, and results in photoinhibition under extreme CO2 concentrations in mature leaves. There are no readily apparent changes to chloroplast biology, such as transcription or assembly that explain the disruption to chloroplast biogenesis. Indeed, SCO3 is actually targeted to another organelle, specifically to the periphery of peroxisomes. However, impaired chloroplast development cannot be attributed to perturbed peroxisomal metabolic processes involving germination, fatty acid β-oxidation or photorespiration, though there are so far undescribed changes in low and high CO2 sensitivity in seedlings and young true leaves. Many of the chloroplasts are bilobed, and some have persistent membranous extensions that encircle other cellular components. Significantly, there are changes to the cytoskeleton in sco3-1, and microtubule inhibitors have similar effects on chloroplast biogenesis as sco3-1 does. The localization of SCO3 to the periphery of the peroxisomes was shown to be dependent on a functional microtubule cytoskeleton. Therefore, the microtubule and peroxisome-associated SCO3 protein is required for chloroplast development, and sco3-1, along with microtubule inhibitors, demonstrates an unexpected role for the cytoskeleton and peroxisomes in chloroplast biogenesis. PMID:20978221

Robust expression of a bioactive mammalian protein in chlamydomonas chloroplast

DOEpatents

Mayfield, Stephen P.

2010-03-16

Methods and compositions are disclosed to engineer chloroplast comprising heterologous mammalian genes via a direct replacement of chloroplast Photosystem II (PSII) reaction center protein coding regions to achieve expression of recombinant protein above 5% of total protein. When algae is used, algal expressed protein is produced predominantly as a soluble protein where the functional activity of the peptide is intact. As the host algae is edible, production of biologics in this organism for oral delivery or proteins/peptides, especially gut active proteins, without purification is disclosed.

Robust expression of a bioactive mammalian protein in Chlamydomonas chloroplast

DOEpatents

Mayfield, Stephen P

2015-01-13

Methods and compositions are disclosed to engineer chloroplast comprising heterologous mammalian genes via a direct replacement of chloroplast Photosystem II (PSII) reaction center protein coding regions to achieve expression of recombinant protein above 5% of total protein. When algae is used, algal expressed protein is produced predominantly as a soluble protein where the functional activity of the peptide is intact. As the host algae is edible, production of biologics in this organism for oral delivery of proteins/peptides, especially gut active proteins, without purification is disclosed.

Circadian oscillations of cytosolic and chloroplastic free calcium in plants

NASA Technical Reports Server (NTRS)

Johnson, C. H.; Knight, M. R.; Kondo, T.; Masson, P.; Sedbrook, J.; Haley, A.; Trewavas, A.

1995-01-01

Tobacco and Arabidopsis plants, expressing a transgene for the calcium-sensitive luminescent protein apoaequorin, revealed circadian oscillations in free cytosolic calcium that can be phase-shifted by light-dark signals. When apoaequorin was targeted to the chloroplast, circadian chloroplast calcium rhythms were likewise observed after transfer of the seedlings to constant darkness. Circadian oscillations in free calcium concentrations can be expected to control many calcium-dependent enzymes and processes accounting for circadian outputs. Regulation of calcium flux is therefore fundamental to the organization of circadian systems.

The complete chloroplast genome of a medicinal plant Epimedium koreanum Nakai (Berberidaceae).

PubMed

Lee, Jung-Hoon; Kim, Kyunghee; Kim, Na-Rae; Lee, Sang-Choon; Yang, Tae-Jin; Kim, Young-Dong

2016-11-01

Epimedium koreanum is a perennial medicinal plant distributed in Eastern Asia. The complete chloroplast genome sequences of E. koreanum was obtained by de novo assembly using whole genome next-generation sequences. The chloroplast genome of E. koreanum was 157 218 bp in length and separated into four distinct regions such as large single copy region (89 600 bp), small single copy region (17 222 bp) and a pair of inverted repeat regions (25 198 bp). The genome contained a total of 112 genes including 78 protein-coding genes, 30 tRNA genes, and 4 rRNA genes. Phylogenetic analysis with the reported chloroplast genomes revealed that E. koreanum is most closely related to Berberis bealei, a traditional medicinal plant in the Berberidaceae family.

Characterization of polymorphic chloroplast microsatellites in Prunus species and maternal lineages in peach genotypes

USDA-ARS?s Scientific Manuscript database

Several available Prunus chloroplast genomes have not been exploited to develop polymorphic chloroplast microsatellites that could be useful in Prunus maternal lineage and phylogenetic analysis. In this study, using available bioinformatics tools, 80, 75, and 78 microsatellites were identified from ...

Combined Analysis of the Chloroplast Genome and Transcriptome of the Antarctic Vascular Plant Deschampsia antarctica Desv

PubMed Central

Lee, Jungeun; Kang, Yoonjee; Shin, Seung Chul; Park, Hyun; Lee, Hyoungseok

2014-01-01

Background Antarctic hairgrass (Deschampsia antarctica Desv.) is the only natural grass species in the maritime Antarctic. It has been researched as an important ecological marker and as an extremophile plant for studies on stress tolerance. Despite its importance, little genomic information is available for D. antarctica. Here, we report the complete chloroplast genome, transcriptome profiles of the coding/noncoding genes, and the posttranscriptional processing by RNA editing in the chloroplast system. Results The complete chloroplast genome of D. antarctica is 135,362 bp in length with a typical quadripartite structure, including the large (LSC: 79,881 bp) and small (SSC: 12,519 bp) single-copy regions, separated by a pair of identical inverted repeats (IR: 21,481 bp). It contains 114 unique genes, including 81 unique protein-coding genes, 29 tRNA genes, and 4 rRNA genes. Sequence divergence analysis with other plastomes from the BEP clade of the grass family suggests a sister relationship between D. antarctica, Festuca arundinacea and Lolium perenne of the Poeae tribe, based on the whole plastome. In addition, we conducted high-resolution mapping of the chloroplast-derived transcripts. Thus, we created an expression profile for 81 protein-coding genes and identified ndhC, psbJ, rps19, psaJ, and psbA as the most highly expressed chloroplast genes. Small RNA-seq analysis identified 27 small noncoding RNAs of chloroplast origin that were preferentially located near the 5′- or 3′-ends of genes. We also found >30 RNA-editing sites in the D. antarctica chloroplast genome, with a dominance of C-to-U conversions. Conclusions We assembled and characterized the complete chloroplast genome sequence of D. antarctica and investigated the features of the plastid transcriptome. These data may contribute to a better understanding of the evolution of D. antarctica within the Poaceae family for use in molecular phylogenetic studies and may also help researchers understand the

Combined analysis of the chloroplast genome and transcriptome of the Antarctic vascular plant Deschampsia antarctica Desv.

PubMed

Lee, Jungeun; Kang, Yoonjee; Shin, Seung Chul; Park, Hyun; Lee, Hyoungseok

2014-01-01

Antarctic hairgrass (Deschampsia antarctica Desv.) is the only natural grass species in the maritime Antarctic. It has been researched as an important ecological marker and as an extremophile plant for studies on stress tolerance. Despite its importance, little genomic information is available for D. antarctica. Here, we report the complete chloroplast genome, transcriptome profiles of the coding/noncoding genes, and the posttranscriptional processing by RNA editing in the chloroplast system. The complete chloroplast genome of D. antarctica is 135,362 bp in length with a typical quadripartite structure, including the large (LSC: 79,881 bp) and small (SSC: 12,519 bp) single-copy regions, separated by a pair of identical inverted repeats (IR: 21,481 bp). It contains 114 unique genes, including 81 unique protein-coding genes, 29 tRNA genes, and 4 rRNA genes. Sequence divergence analysis with other plastomes from the BEP clade of the grass family suggests a sister relationship between D. antarctica, Festuca arundinacea and Lolium perenne of the Poeae tribe, based on the whole plastome. In addition, we conducted high-resolution mapping of the chloroplast-derived transcripts. Thus, we created an expression profile for 81 protein-coding genes and identified ndhC, psbJ, rps19, psaJ, and psbA as the most highly expressed chloroplast genes. Small RNA-seq analysis identified 27 small noncoding RNAs of chloroplast origin that were preferentially located near the 5'- or 3'-ends of genes. We also found >30 RNA-editing sites in the D. antarctica chloroplast genome, with a dominance of C-to-U conversions. We assembled and characterized the complete chloroplast genome sequence of D. antarctica and investigated the features of the plastid transcriptome. These data may contribute to a better understanding of the evolution of D. antarctica within the Poaceae family for use in molecular phylogenetic studies and may also help researchers understand the characteristics of the chloroplast

Evolutionary, Molecular and Genetic Analyses of Tic22 Homologues in Arabidopsis thaliana Chloroplasts

PubMed Central

Kasmati, Ali Reza; Patel, Ramesh; Ling, Qihua; Karim, Sazzad; Aronsson, Henrik; Jarvis, Paul

2013-01-01

The Tic22 protein was previously identified in pea as a putative component of the chloroplast protein import apparatus. It is a peripheral protein of the inner envelope membrane, residing in the intermembrane space. In Arabidopsis, there are two Tic22 homologues, termed atTic22-III and atTic22-IV, both of which are predicted to localize in chloroplasts. These two proteins defined clades that are conserved in all land plants, which appear to have evolved at a similar rates since their separation >400 million years ago, suggesting functional conservation. The atTIC22-IV gene was expressed several-fold more highly than atTIC22-III, but the genes exhibited similar expression profiles and were expressed throughout development. Knockout mutants lacking atTic22-IV were visibly normal, whereas those lacking atTic22-III exhibited moderate chlorosis. Double mutants lacking both isoforms were more strongly chlorotic, particularly during early development, but were viable and fertile. Double-mutant chloroplasts were small and under-developed relative to those in wild type, and displayed inefficient import of precursor proteins. The data indicate that the two Tic22 isoforms act redundantly in chloroplast protein import, and that their function is non-essential but nonetheless required for normal chloroplast biogenesis, particularly during early plant development. PMID:23675512

Photophysiology of kleptoplasts: photosynthetic use of light by chloroplasts living in animal cells.

PubMed

Serôdio, João; Cruz, Sónia; Cartaxana, Paulo; Calado, Ricardo

2014-04-19

Kleptoplasty is a remarkable type of photosynthetic association, resulting from the maintenance of functional chloroplasts--the 'kleptoplasts'--in the tissues of a non-photosynthetic host. It represents a biologically unique condition for chloroplast and photosynthesis functioning, occurring in different phylogenetic lineages, namely dinoflagellates, ciliates, foraminiferans and, most interestingly, a single taxon of metazoans, the sacoglossan sea slugs. In the case of sea slugs, chloroplasts from macroalgae are often maintained as intracellular organelles in cells of these marine gastropods, structurally intact and photosynthetically competent for extended periods of time. Kleptoplasty has long attracted interest owing to the longevity of functional kleptoplasts in the absence of the original algal nucleus and the limited number of proteins encoded by the chloroplast genome. This review updates the state-of-the-art on kleptoplast photophysiology, focusing on the comparative analysis of the responses to light of the chloroplasts when in their original, macroalgal cells, and when sequestered in animal cells and functioning as kleptoplasts. It covers fundamental but ecologically relevant aspects of kleptoplast light responses, such as the occurrence of photoacclimation in hospite, operation of photoprotective processes and susceptibility to photoinhibition. Emphasis is given to host-mediated processes unique to kleptoplastic associations, reviewing current hypotheses on behavioural photoprotection and host-mediated enhancement of photosynthetic performance, and identifying current gaps in sacoglossan kleptoplast photophysiology research.

Purification of intact chloroplasts from marine plant Posidonia oceanica suitable for organelle proteomics.

PubMed

Piro, Amalia; Serra, Ilia Anna; Spadafora, Antonia; Cardilio, Monica; Bianco, Linda; Perrotta, Gaetano; Santos, Rui; Mazzuca, Silvia

2015-12-01

Posidonia oceanica is a marine angiosperm, or seagrass, adapted to grow to the underwater life from shallow waters to 50 m depth. This raises questions of how their photosynthesis adapted to the attenuation of light through the water column and leads to the assumption that biochemistry and metabolism of the chloroplast are the basis of adaptive capacity. In the present study, we described a protocol that was adapted from those optimized for terrestrial plants, to extract chloroplasts from as minimal tissue as possible. We obtained the best balance between tissue amount/intact chloroplasts yield using one leaf from one plant. After isopynic separations, the chloroplasts purity and integrity were evaluated by biochemical assay and using a proteomic approach. Chloroplast proteins were extracted from highly purified organelles and resolved by 1DE SDS-PAGE. Proteins were sequenced by nLC-ESI-IT-MS/MS of 1DE gel bands and identified against NCBInr green plant databases, Dr. Zompo database for seagrasses in a local customized dataset. The curated localization of proteins in sub-plastidial compartments (i.e. envelope, stroma and thylakoids) was retrieved in the AT_CHLORO database. This purification protocol and the validation of compartment markers may serve as basis for sub-cellular proteomics in P. oceanica and other seagrasses. © 2015 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Identification of a maize nucleic acid-binding protein (NBP) belonging to a family of nuclear-encoded chloroplast proteins.

PubMed Central

Cook, W B; Walker, J C

1992-01-01

A cDNA encoding a nuclear-encoded chloroplast nucleic acid-binding protein (NBP) has been isolated from maize. Identified as an in vitro DNA-binding activity, NBP belongs to a family of nuclear-encoded chloroplast proteins which share a common domain structure and are thought to be involved in posttranscriptional regulation of chloroplast gene expression. NBP contains an N-terminal chloroplast transit peptide, a highly acidic domain and a pair of ribonucleoprotein consensus sequence domains. NBP is expressed in a light-dependent, organ-specific manner which is consistent with its involvement in chloroplast biogenesis. The relationship of NBP to the other members of this protein family and their possible regulatory functions are discussed. Images PMID:1346929

Nuclear, chloroplast, and mitochondrial data of a US cannabis DNA database.

PubMed

Houston, Rachel; Birck, Matthew; LaRue, Bobby; Hughes-Stamm, Sheree; Gangitano, David

2018-05-01

As Cannabis sativa (marijuana) is a controlled substance in many parts of the world, the ability to track biogeographical origin of cannabis could provide law enforcement with investigative leads regarding its trade and distribution. Population substructure and inbreeding may cause cannabis plants to become more genetically related. This genetic relatedness can be helpful for intelligence purposes. Analysis of autosomal, chloroplast, and mitochondrial DNA allows for not only prediction of biogeographical origin of a plant but also discrimination between individual plants. A previously validated, 13-autosomal STR multiplex was used to genotype 510 samples. Samples were analyzed from four different sites: 21 seizures at the US-Mexico border, Northeastern Brazil, hemp seeds purchased in the US, and the Araucania area of Chile. In addition, a previously reported multi-loci system was modified and optimized to genotype five chloroplast and two mitochondrial markers. For this purpose, two methods were designed: a homopolymeric STR pentaplex and a SNP triplex with one chloroplast (Cscp001) marker shared by both methods for quality control. For successful mitochondrial and chloroplast typing, a novel real-time PCR quantitation method was developed and validated to accurately estimate the quantity of the chloroplast DNA (cpDNA) using a synthetic DNA standard. Moreover, a sequenced allelic ladder was also designed for accurate genotyping of the homopolymeric STR pentaplex. For autosomal typing, 356 unique profiles were generated from the 425 samples that yielded full STR profiles and 25 identical genotypes within seizures were observed. Phylogenetic analysis and case-to-case pairwise comparisons of 21 seizures at the US-Mexico border, using the Fixation Index (F ST ) as genetic distance, revealed the genetic association of nine seizures that formed a reference population. For mitochondrial and chloroplast typing, subsampling was performed, and 134 samples were genotyped

High-throughput m6A-seq reveals RNA m6A methylation patterns in the chloroplast and mitochondria transcriptomes of Arabidopsis thaliana.

PubMed

Wang, Zegang; Tang, Kai; Zhang, Dayong; Wan, Yizhen; Wen, Yan; Lu, Quanyou; Wang, Lei

2017-01-01

This study is the first to comprehensively characterize m6A patterns in the Arabidopsis chloroplast and mitochondria transcriptomes based on our open accessible data deposited in NCBI's Gene Expression Omnibus with GEO Series accession number of GSE72706. Over 86% of the transcripts were methylated by m6A in the two organelles. Over 550 and 350 m6A sites were mapped, with ~5.6 to ~5.8 and ~4.6 to ~4.9 m6A sites per transcript, to the chloroplast and mitochondria genome, respectively. The overall m6A methylation extent in the two organelles was greatly higher than that in the nucleus. The m6A motif sequences in the transcriptome of two organelles were similar to the nuclear motifs, suggesting that selection of the m6A motifs for RNA methylation was conserved between the nucleus and organelle transcriptomes. The m6A patterns of rRNAs and tRNAs in the organelle were similar to those in the nucleus. However, the m6A patterns in coding RNAs were distinct between the nucleus and the organelle, suggesting that that regulation of the m6A methylation patterns may be different between the nuclei and the organelles. The extensively methylated transcripts in the two organelles were mainly associated with rRNA, ribosomal proteins, photosystem reaction proteins, tRNA, NADH dehydrogenase and redox. On average, 64% and 79% of the transcripts in the two organelles showed differential m6A methylation across three organs of the leaves, flowers and roots. The m6A methylation extent in the chloroplast was higher than that in the mitochondria. This study provides deep insights into the m6A methylation topology and differentiation in the plant organelle transcriptomes.

Two Isoforms of Dihydroxyacetone Phosphate Reductase from the Chloroplasts of Dunaliella tertiolecta.

PubMed

Gee, R.; Goyal, A.; Byerrum, R. U.; Tolbert, N. E.

1993-09-01

Three isoforms of dihydroxyacetone phosphate reductase in extracts from Dunaliella tertiolecta have been separated by a diethylaminoethyl cellulose column chromatography with a shallow NaCl gradient. The chloroplasts contained the two major isoforms, and the third, minor form was in the cytosol. The isoforms are unstable in the absence of glycerol and they are cold labile, but they may be partially reactivated at 35[deg]C. The first chloroplast form to elute from the DEAE cellulose column was the major form when the cells were grown on high NaCl and it has been referred to as the form for glycerol production for osmoregulation or "osmoregulator form." The second form increased in specific activity when inorganic phosphate was increased in the growth media to stimulate growth, and it has been given the designation for the form for glyceride synthesis, "glyceride form." The osmoregulator form was stimulated by NaCl added to the enzyme assay, but not by reduced Escherichia coli thioredoxin. The glyceride form had properties similar to the enzyme in leaf chloroplast, such as inhibition by NaCl and by fatty acyl-coenzyme A derivatives and some stimulation by dithiothreitol, uridine diphosphate galactose, cyti-dine diphosphate dipalmatoyl diglyceride, and reduced E. coli thioredoxin. Thus, Dunaliella chloroplasts have a salt-stimulated osmoregulatory form of dihydroxyacetone phosphate reductase, which seems to have a role in glycerol production, and an isoform, which may be involved in glyceride synthesis and which has properties similar to the enzyme in chloroplasts of higher plants.

Two Isoforms of Dihydroxyacetone Phosphate Reductase from the Chloroplasts of Dunaliella tertiolecta.

PubMed Central

Gee, R.; Goyal, A.; Byerrum, R. U.; Tolbert, N. E.

1993-01-01

Three isoforms of dihydroxyacetone phosphate reductase in extracts from Dunaliella tertiolecta have been separated by a diethylaminoethyl cellulose column chromatography with a shallow NaCl gradient. The chloroplasts contained the two major isoforms, and the third, minor form was in the cytosol. The isoforms are unstable in the absence of glycerol and they are cold labile, but they may be partially reactivated at 35[deg]C. The first chloroplast form to elute from the DEAE cellulose column was the major form when the cells were grown on high NaCl and it has been referred to as the form for glycerol production for osmoregulation or "osmoregulator form." The second form increased in specific activity when inorganic phosphate was increased in the growth media to stimulate growth, and it has been given the designation for the form for glyceride synthesis, "glyceride form." The osmoregulator form was stimulated by NaCl added to the enzyme assay, but not by reduced Escherichia coli thioredoxin. The glyceride form had properties similar to the enzyme in leaf chloroplast, such as inhibition by NaCl and by fatty acyl-coenzyme A derivatives and some stimulation by dithiothreitol, uridine diphosphate galactose, cyti-dine diphosphate dipalmatoyl diglyceride, and reduced E. coli thioredoxin. Thus, Dunaliella chloroplasts have a salt-stimulated osmoregulatory form of dihydroxyacetone phosphate reductase, which seems to have a role in glycerol production, and an isoform, which may be involved in glyceride synthesis and which has properties similar to the enzyme in chloroplasts of higher plants. PMID:12231930

Release of proteins from intact chloroplasts induced by reactive oxygen species during biotic and abiotic stress.

PubMed

Kwon, Kwang-Chul; Verma, Dheeraj; Jin, Shuangxia; Singh, Nameirakpam D; Daniell, Henry

2013-01-01

Plastids sustain life on this planet by providing food, feed, essential biomolecules and oxygen. Such diverse metabolic and biosynthetic functions require efficient communication between plastids and the nucleus. However, specific factors, especially large molecules, released from plastids that regulate nuclear genes have not yet been fully elucidated. When tobacco and lettuce transplastomic plants expressing GFP within chloroplasts, were challenged with Erwinia carotovora (biotic stress) or paraquat (abiotic stress), GFP was released into the cytoplasm. During this process GFP moves gradually towards the envelope, creating a central red zone of chlorophyll fluorescence. GFP was then gradually released from intact chloroplasts into the cytoplasm with an intact vacuole and no other visible cellular damage. Different stages of GFP release were observed inside the same cell with a few chloroplasts completely releasing GFP with detection of only red chlorophyll fluorescence or with no reduction in GFP fluorescence or transitional steps between these two phases. Time lapse imaging by confocal microscopy clearly identified sequence of these events. Intactness of chloroplasts during this process was evident from chlorophyll fluorescence emanated from thylakoid membranes and in vivo Chla fluorescence measurements (maximum quantum yield of photosystem II) made before or after infection with pathogens to evaluate their photosynthetic competence. Hydrogen peroxide and superoxide anion serve as signal molecules for generation of reactive oxygen species and Tiron, scavenger of superoxide anion, blocked release of GFP from chloroplasts. Significant increase in ion leakage in the presence of paraquat and light suggests changes in the chloroplast envelope to facilitate protein release. Release of GFP-RC101 (an antimicrobial peptide), which was triggered by Erwinia infection, ceased after conferring protection, further confirming this export phenomenon. These results suggest a

Release of Proteins from Intact Chloroplasts Induced by Reactive Oxygen Species during Biotic and Abiotic Stress

PubMed Central

Singh, Nameirakpam D.; Daniell, Henry

2013-01-01

Plastids sustain life on this planet by providing food, feed, essential biomolecules and oxygen. Such diverse metabolic and biosynthetic functions require efficient communication between plastids and the nucleus. However, specific factors, especially large molecules, released from plastids that regulate nuclear genes have not yet been fully elucidated. When tobacco and lettuce transplastomic plants expressing GFP within chloroplasts, were challenged with Erwinia carotovora (biotic stress) or paraquat (abiotic stress), GFP was released into the cytoplasm. During this process GFP moves gradually towards the envelope, creating a central red zone of chlorophyll fluorescence. GFP was then gradually released from intact chloroplasts into the cytoplasm with an intact vacuole and no other visible cellular damage. Different stages of GFP release were observed inside the same cell with a few chloroplasts completely releasing GFP with detection of only red chlorophyll fluorescence or with no reduction in GFP fluorescence or transitional steps between these two phases. Time lapse imaging by confocal microscopy clearly identified sequence of these events. Intactness of chloroplasts during this process was evident from chlorophyll fluorescence emanated from thylakoid membranes and in vivo Chla fluorescence measurements (maximum quantum yield of photosystem II) made before or after infection with pathogens to evaluate their photosynthetic competence. Hydrogen peroxide and superoxide anion serve as signal molecules for generation of reactive oxygen species and Tiron, scavenger of superoxide anion, blocked release of GFP from chloroplasts. Significant increase in ion leakage in the presence of paraquat and light suggests changes in the chloroplast envelope to facilitate protein release. Release of GFP-RC101 (an antimicrobial peptide), which was triggered by Erwinia infection, ceased after conferring protection, further confirming this export phenomenon. These results suggest a

Pyrosequencing of the northern red oak (Quercus rubra L.) chloroplast genome reveals high quality polymorphisms for population management

Treesearch

Lisa W. Alexander; Keith E. Woeste

2014-01-01

Given the low intraspecific chloroplast diversity detected in northern red oak (Quercus rubra L.), more powerful genetic tools are necessary to accurately characterize Q. rubra chloroplast diversity and structure. We report the sequencing, assembly, and annotation of the chloroplast genome of northern red oak via pyrosequencing and...

More introgression with less gene flow: chloroplast vs. mitochondrial DNA in the Picea asperata complex in China, and comparison with other Conifers.

PubMed

Du, Fang K; Petit, Rémy J; Liu, Jian Quan

2009-04-01

Recent work has suggested that rates of introgression should be inversely related to levels of gene flow because introgressed populations cannot be 'rescued' by intraspecific gene flow if it is too low. Mitochondrial and chloroplast DNA (mtDNA and cpDNA) experience very different levels of gene flow in conifers due to their contrasted maternal and paternal modes of transmission, hence the prediction that mtDNA should introgress more readily than cpDNA in this group. Here, we use sequence data from both mtDNA and cpDNA to test this hypothesis in a group of closely related spruces species, the Picea asperata complex from China. Nine mitochondrial and nine chloroplast haplotypes were recovered from 459 individuals in 46 natural populations belonging to five species of the Picea asperata complex. Low variation was found in the two mtDNA introns along with a high level of differentiation among populations (G(ST) = 0.90). In contrast, we detected higher variation and lower differentiation among populations at cpDNA markers (G(ST) = 0.56), a trend shared by most conifer species studied so far. We found that cpDNA variation, although far from being fully diagnostic, is more species-specific than mtDNA variation: four groups of populations were identified using cpDNA markers, all of them related to species or groups of species, whereas for mtDNA, geographical variation prevails over species differentiation. The literature suggests that mtDNA haplotypes are often shared among related conifer species, whereas cpDNA haplotypes are more species-specific. Hence, increased intraspecific gene flow appears to decrease differentiation within species but not among species.

Phaseolin expression in tobacco chloroplast reveals an autoregulatory mechanism in heterologous protein translation.

PubMed

De Marchis, Francesca; Bellucci, Michele; Pompa, Andrea

2016-02-01

Plastid DNA engineering is a well-established research area of plant biotechnology, and plastid transgenes often give high expression levels. However, it is still almost impossible to predict the accumulation rate of heterologous protein in transplastomic plants, and there are many cases of unsuccessful transgene expression. Chloroplasts regulate their proteome at the post-transcriptional level, mainly through translation control. One of the mechanisms to modulate the translation has been described in plant chloroplasts for the chloroplast-encoded subunits of multiprotein complexes, and the autoregulation of the translation initiation of these subunits depends on the availability of their assembly partners [control by epistasy of synthesis (CES)]. In Chlamydomonas reinhardtii, autoregulation of endogenous proteins recruited in the assembly of functional complexes has also been reported. In this study, we revealed a self-regulation mechanism triggered by the accumulation of a soluble recombinant protein, phaseolin, in the stroma of chloroplast-transformed tobacco plants. Immunoblotting experiments showed that phaseolin could avoid this self-regulation mechanism when targeted to the thylakoids in transplastomic plants. To inhibit the thylakoid-targeted phaseolin translation as well, this protein was expressed in the presence of a nuclear version of the phaseolin gene with a transit peptide. Pulse-chase and polysome analysis revealed that phaseolin mRNA translation on plastid ribosomes was repressed due to the accumulation in the stroma of the same soluble polypeptide imported from the cytosol. We suggest that translation autoregulation in chloroplast is not limited to heteromeric protein subunits but also involves at least some of the foreign soluble recombinant proteins, leading to the inhibition of plastome-encoded transgene expression in chloroplast. © 2015 Society for Experimental Biology, Association of Applied Biologists and John Wiley & Sons Ltd.

Enhanced green fluorescent protein (egfp) gene expression in Tetraselmis subcordiformis chloroplast with endogenous regulators.

PubMed

Cui, Yulin; Zhao, Jialin; Hou, Shichang; Qin, Song

2016-05-01

On the basis of fundamental genetic transformation technologies, the goal of this study was to optimize Tetraselmis subcordiformis chloroplast transformation through the use of endogenous regulators. The genes rrn16S, rbcL, psbA, and psbC are commonly highly expressed in chloroplasts, and the regulators of these genes are often used in chloroplast transformation. For lack of a known chloroplast genome sequence, the genome-walking method was used here to obtain full sequences of T. subcordiformis endogenous regulators. The resulting regulators, including three promoters, two terminators, and a ribosome combination sequence, were inserted into the previously constructed plasmid pPSC-R, with the egfp gene included as a reporter gene, and five chloroplast expression vectors prepared. These vectors were successfully transformed into T. subcordiformis by particle bombardment and the efficiency of each vector tested by assessing EGFP fluorescence via microscopy. The results showed that these vectors exhibited higher efficiency than the former vector pPSC-G carrying exogenous regulators, and the vector pRFA with Prrn, psbA-5'RE, and TpsbA showed the highest efficiency. This research provides a set of effective endogenous regulators for T. subcordiformis and will facilitate future fundamental studies of this alga.

The DCL gene of tomato is required for chloroplast development and palisade cell morphogenesis in leaves.

PubMed

Keddie, J S; Carroll, B; Jones, J D; Gruissem, W

1996-08-15

The defective chloroplasts and leaves-mutable (dcl-m) mutation of tomato was identified in a Ds mutagenesis screen. This unstable mutation affects both chloroplast development and palisade cell morphogenesis in leaves. Mutant plants are clonally variegated as a result of somatic excision of Ds and have albino leaves with green sectors. Leaf midribs and stems are light green with sectors of dark green tissue but fruit and petals are wild-type in appearance. Within dark green sectors of dcl-m leaves, palisade cells are normal, whereas in albino areas of dcl-m leaves, palisade cells do not expand to become their characteristic columnar shape. The development of chloroplasts from proplastids in albino areas is apparently blocked at an early stage. DCL was cloned using Ds as a tag and encodes a novel protein of approximately 25 kDa, containing a chloroplast transit peptide and an acidic alpha-helical region. DCL protein was imported into chloroplasts in vitro and processed to a mature form. Because of the ubiquitous expression of DCL and the proplastid-like appearance of dcl-affected plastids, the DCL protein may regulate a basic and universal function of the plastid. The novel dcl-m phenotype suggests that chloroplast development is required for correct palisade cell morphogenesis during leaf development.

The novel chloroplast outer membrane kinase KOC1 is a required component of the plastid protein import machinery.

PubMed

Zufferey, Mónica; Montandon, Cyrille; Douet, Véronique; Demarsy, Emilie; Agne, Birgit; Baginsky, Sacha; Kessler, Felix

2017-04-28

The biogenesis and maintenance of cell organelles such as mitochondria and chloroplasts require the import of many proteins from the cytosol, a process that is controlled by phosphorylation. In the case of chloroplasts, the import of hundreds of different proteins depends on translocons at the outer and inner chloroplast membrane (TOC and TIC, respectively) complexes. The essential protein TOC159 functions thereby as an import receptor. It has an N-terminal acidic (A-) domain that extends into the cytosol, controls receptor specificity, and is highly phosphorylated in vivo However, kinases that phosphorylate the TOC159 A-domain to enable protein import have remained elusive. Here, using co-purification with TOC159 from Arabidopsis , we discovered a novel component of the chloroplast import machinery, the regulatory kinase at the outer chloroplast membrane 1 (KOC1). We found that KOC1 is an integral membrane protein facing the cytosol and stably associates with TOC. Moreover, KOC1 phosphorylated the A-domain of TOC159 in vitro , and in mutant koc1 chloroplasts, preprotein import efficiency was diminished. koc1 Arabidopsis seedlings had reduced survival rates after transfer from the dark to the light in which protein import into plastids is required to rapidly complete chloroplast biogenesis. In summary, our data indicate that KOC1 is a functional component of the TOC machinery that phosphorylates import receptors, supports preprotein import, and contributes to efficient chloroplast biogenesis. © 2017 by The American Society for Biochemistry and Molecular Biology, Inc.

The novel chloroplast outer membrane kinase KOC1 is a required component of the plastid protein import machinery

PubMed Central

Zufferey, Mónica; Montandon, Cyrille; Douet, Véronique; Demarsy, Emilie; Agne, Birgit; Baginsky, Sacha; Kessler, Felix

2017-01-01

The biogenesis and maintenance of cell organelles such as mitochondria and chloroplasts require the import of many proteins from the cytosol, a process that is controlled by phosphorylation. In the case of chloroplasts, the import of hundreds of different proteins depends on translocons at the outer and inner chloroplast membrane (TOC and TIC, respectively) complexes. The essential protein TOC159 functions thereby as an import receptor. It has an N-terminal acidic (A-) domain that extends into the cytosol, controls receptor specificity, and is highly phosphorylated in vivo. However, kinases that phosphorylate the TOC159 A-domain to enable protein import have remained elusive. Here, using co-purification with TOC159 from Arabidopsis, we discovered a novel component of the chloroplast import machinery, the regulatory kinase at the outer chloroplast membrane 1 (KOC1). We found that KOC1 is an integral membrane protein facing the cytosol and stably associates with TOC. Moreover, KOC1 phosphorylated the A-domain of TOC159 in vitro, and in mutant koc1 chloroplasts, preprotein import efficiency was diminished. koc1 Arabidopsis seedlings had reduced survival rates after transfer from the dark to the light in which protein import into plastids is required to rapidly complete chloroplast biogenesis. In summary, our data indicate that KOC1 is a functional component of the TOC machinery that phosphorylates import receptors, supports preprotein import, and contributes to efficient chloroplast biogenesis. PMID:28283569

Transport of Phosphoenolpyruvate by Chloroplasts from Mesembryanthemum crystallinum L. Exhibiting Crassulacean Acid Metabolism 1

PubMed Central

Neuhaus, H. Ekkehard; Holtum, Joseph A. M.; Latzko, Erwin

1988-01-01

Chloroplasts from CAM-Mesembryanthemum crystallinum can transport phosphoenolpyruvate (PEP) across the envelope. The initial velocities of PEP uptake in the dark at 4°C exhibited saturation kinetics with increasing external PEP concentration. PEP uptake had a Vmax of 6.46 (±0.05) micromoles per milligram chlorophyll per hour and an apparent Kmpep of 0.148 (±0.004) millimolar. The uptake was competitively inhibited by Pi (apparent Ki = 0.19 millimolar), by glycerate 3-phosphate (apparent Ki = 0.13 millimolar), and by dihydroxyacetone phosphate, but malate and pyruvate were without effect. The chloroplasts were able to synthesize PEP when presented with pyruvate. PEP synthesis was light dependent. The prolonged synthesis and export of PEP from the chloroplasts required the presence of Pi or glycerate 3-phosphate in the external medium. It is suggested that the transport of pyruvate and PEP across the chloroplasts envelope is required during the gluconeogenic conversion of carbon from malate to storage carbohydrate in the light. PMID:16666128

CLA1, a novel gene required for chloroplast development, is highly conserved in evolution.

PubMed

Mandel, M A; Feldmann, K A; Herrera-Estrella, L; Rocha-Sosa, M; León, P

1996-05-01

An albino mutant designated cla1-1 (for "cloroplastos alterados', or "altered chloroplasts') has been isolated from a T-DNA-generated library of Arabidopsis thaliana. In cla1-1 plants, chloroplast development is arrested at an early stage. cla1-1 plants behave like wild-type in their capacity to etiolate and produce anthocyanins indicating that the light signal transduction pathway seems to be unaffected. Genetic and molecular analyses show that the disruption of a single gene, CLA1, by the T-DNA insertion is responsible for the mutant phenotype. RNA expression patterns indicate that CLA1 is positively regulated by light and that it has different effects on the steady-state RNA levels of some nuclear- and chloroplast-encoded photosynthetic genes. Although the specific function of the CLA1 gene is still unknown, it encodes a novel protein conserved in evolution between photosynthetic bacteria and plants which is essential for chloroplast development in Arabidopsis.

Comparative analysis of complete chloroplast genome sequence and inversion variation in Lasthenia burkei (Madieae, Asteraceae).

PubMed

Walker, Joseph F; Zanis, Michael J; Emery, Nancy C

2014-04-01

Complete chloroplast genome studies can help resolve relationships among large, complex plant lineages such as Asteraceae. We present the first whole plastome from the Madieae tribe and compare its sequence variation to other chloroplast genomes in Asteraceae. We used high throughput sequencing to obtain the Lasthenia burkei chloroplast genome. We compared sequence structure and rates of molecular evolution in the small single copy (SSC), large single copy (LSC), and inverted repeat (IR) regions to those for eight Asteraceae accessions and one Solanaceae accession. The chloroplast sequence of L. burkei is 150 746 bp and contains 81 unique protein coding genes and 4 coding ribosomal RNA sequences. We identified three major inversions in the L. burkei chloroplast, all of which have been found in other Asteraceae lineages, and a previously unreported inversion in Lactuca sativa. Regions flanking inversions contained tRNA sequences, but did not have particularly high G + C content. Substitution rates varied among the SSC, LSC, and IR regions, and rates of evolution within each region varied among species. Some observed differences in rates of molecular evolution may be explained by the relative proportion of coding to noncoding sequence within regions. Rates of molecular evolution vary substantially within and among chloroplast genomes, and major inversion events may be promoted by the presence of tRNAs. Collectively, these results provide insight into different mechanisms that may promote intramolecular recombination and the inversion of large genomic regions in the plastome.

Is chloroplast import of photosynthesis proteins facilitated by an actin-TOC-TIC-VIPP1 complex?

PubMed

Jouhet, Juliette; Gray, John C

2009-10-01

Actin filaments are major components of the cytoskeleton that interact with chloroplast envelope membranes to allow chloroplast positioning and movement, stromule mobility and gravitropism perception. We recently reported that Toc159, a component of the TOC complex of the chloroplast protein import apparatus, interacts directly with actin. The interaction of Toc159 and actin was identified by co-immunoprecipitation and co-sedimentation experiments with detergent-solubilised pea chloroplast envelope membranes. In addition, many of the components of the TOC-TIC protein import apparatus and VIPP1 (vesicle-inducing protein in plastids 1) were identified by mass spectroscopy in the material co-immunoprecipitated with antibodies to actin. Toc159 is the receptor for the import of photosynthesis proteins and VIPP1 is involved in thylakoid membrane formation by inducing vesicle formation from the chloroplast inner envelope membrane, suggesting we may have identified an actin-TOC-TIC-VIPP1 complex that may provide a means of channeling cytosolic preproteins to the thylakoid membrane. The interaction of Toc159 with actin may facilitate exchange between the putative soluble and membrane forms of Toc159 and promote the interaction of cytosolic preproteins with the TOC complex.

High-throughput sequencing of three Lemnoideae (duckweeds) chloroplast genomes from total DNA.

PubMed

Wang, Wenqin; Messing, Joachim

2011-01-01

Chloroplast genomes provide a wealth of information for evolutionary and population genetic studies. Chloroplasts play a particularly important role in the adaption for aquatic plants because they float on water and their major surface is exposed continuously to sunlight. The subfamily of Lemnoideae represents such a collection of aquatic species that because of photosynthesis represents one of the fastest growing plant species on earth. We sequenced the chloroplast genomes from three different genera of Lemnoideae, Spirodela polyrhiza, Wolffiella lingulata and Wolffia australiana by high-throughput DNA sequencing of genomic DNA using the SOLiD platform. Unfractionated total DNA contains high copies of plastid DNA so that sequences from the nucleus and mitochondria can easily be filtered computationally. Remaining sequence reads were assembled into contiguous sequences (contigs) using SOLiD software tools. Contigs were mapped to a reference genome of Lemna minor and gaps, selected by PCR, were sequenced on the ABI3730xl platform. This combinatorial approach yielded whole genomic contiguous sequences in a cost-effective manner. Over 1,000-time coverage of chloroplast from total DNA were reached by the SOLiD platform in a single spot on a quadrant slide without purification. Comparative analysis indicated that the chloroplast genome was conserved in gene number and organization with respect to the reference genome of L. minor. However, higher nucleotide substitution, abundant deletions and insertions occurred in non-coding regions of these genomes, indicating a greater genomic dynamics than expected from the comparison of other related species in the Pooideae. Noticeably, there was no transition bias over transversion in Lemnoideae. The data should have immediate applications in evolutionary biology and plant taxonomy with increased resolution and statistical power.

Biparental chloroplast inheritance leads to rescue from cytonuclear incompatibility.

PubMed

Barnard-Kubow, Karen B; McCoy, Morgan A; Galloway, Laura F

2017-02-01

Although organelle inheritance is predominantly maternal across animals and plants, biparental chloroplast inheritance has arisen multiple times in the angiosperms. Biparental inheritance has the potential to impact the evolutionary dynamics of cytonuclear incompatibility, interactions between nuclear and organelle genomes that are proposed to be among the earliest types of genetic incompatibility to arise in speciation. We examine the interplay between biparental inheritance and cytonuclear incompatibility in Campanulastrum americanum, a plant species exhibiting both traits. We first determine patterns of chloroplast inheritance in genetically similar and divergent crosses, and then associate inheritance with hybrid survival across multiple generations. There is substantial biparental inheritance in C. americanum. The frequency of biparental inheritance is greater in divergent crosses and in the presence of cytonuclear incompatibility. Biparental inheritance helps to mitigate cytonuclear incompatibility, leading to increased fitness of F 1 hybrids and recovery in the F 2 generation. This study demonstrates the potential for biparental chloroplast inheritance to rescue cytonuclear compatibility, reducing cytonuclear incompatibility's contribution to reproductive isolation and potentially slowing speciation. The efficacy of rescue depended upon the strength of incompatibility, with a greater persistence of weak incompatibilities in later generations. These findings suggest that incompatible plastids may lead to selection for biparental inheritance. © 2016 The Authors. New Phytologist © 2016 New Phytologist Trust.

Method of producing metallized chloroplasts and use thereof in the photochemical production of hydrogen and oxygen

DOEpatents

Greenbaum, Elias

1987-01-01

The invention is primarily a metallized chloroplast composition for use in a photosynthetic reaction. A catalytic metal is precipitated on a chloroplast membrane at the location where a catalyzed reduction reaction occurs. This metallized chloroplast is stabilized by depositing it on a support medium such as fiber so that it can be easily handled. A possible application of this invention is the splitting of water to form hydrogen and oxygen that can be used as a renewable energy source.

Effects of temperature and light on the formation of chloroplast protrusions in leaf mesophyll cells of high alpine plants.

PubMed

Buchner, Othmar; Holzinger, Andreas; Lütz, Cornelius

2007-11-01

Chloroplasts of many alpine plants have the ability to form marked, stroma-filled protrusions that do not contain thylakoids. Effects of temperature and light intensity on the frequency of chloroplasts with such protrusions in leaf mesophyll cells of nine different alpine plant species (Carex curvula All., Leontodon helveticus Merat., Oxyria digyna (L.) Hill., Poa alpina L. ssp. vivipara, Polygonum viviparum L., Ranunculus glacialis L., Ranunculus alpestris L., Silene acaulis L. and Soldanella pusilla Baumg.) covering seven different families were studied. Leaves were exposed to either darkness and a stepwise increase in temperature (10-38 degrees C) or to different light intensities (500 and 2000 micromol photons m(-2) s(-1)) and a constant temperature of 10 or 30 degrees C in a special temperature-regulated chamber. A chloroplast protrusions index characterising the relative proportion of chloroplasts with protrusions was defined. Seven of the nine species showed a significant increase in chloroplast protrusions when temperature was elevated to over 20 degrees C. In contrast, the light level did not generally affect the abundance of chloroplasts with protrusions. Chloroplast protrusions lead to a dynamic enlargement of the chloroplast surface area. They do not appear to be directly connected to a distinct photosystem II (PSII) (F(v)/F(m)) status and thus seem to be involved in secondary, not primary, photosynthetic processes.

Separation of Chloroplast Pigments Using Reverse Phase Chromatography.

ERIC Educational Resources Information Center

Reese, R. Neil

1997-01-01

Presents a protocol that uses reverse phase chromatography for the separation of chloroplast pigments. Provides a simple and relatively safe procedure for use in teaching laboratories. Discusses pigment extraction, chromatography, results, and advantages of the process. (JRH)

Formation and Change of Chloroplast-Located Plant Metabolites in Response to Light Conditions.

PubMed

Chen, Yiyong; Zhou, Bo; Li, Jianlong; Tang, Hao; Tang, Jinchi; Yang, Ziyin

2018-02-26

Photosynthesis is the central energy conversion process for plant metabolism and occurs within mature chloroplasts. Chloroplasts are also the site of various metabolic reactions involving amino acids, lipids, starch, and sulfur, as well as where the production of some hormones takes place. Light is one of the most important environmental factors, acting as an essential energy source for plants, but also as an external signal influencing their growth and development. Plants experience large fluctuations in the intensity and spectral quality of light, and many attempts have been made to improve or modify plant metabolites by treating them with different light qualities (artificial lighting) or intensities. In this review, we discuss how changes in light intensity and wavelength affect the formation of chloroplast-located metabolites in plants.

Nano-scale characterization of the dynamics of the chloroplast Toc translocon.

PubMed

Reddick, L Evan; Chotewutmontri, Prakitchai; Crenshaw, Will; Dave, Ashita; Vaughn, Michael; Bruce, Barry D

2008-01-01

Translocons are macromolecular nano-scale machines that facilitate the selective translocation of proteins across membranes. Although common in function, different translocons have evolved diverse molecular mechanisms for protein translocation. Subcellular organelles of endosymbiotic origin such as the chloroplast and mitochondria had to evolve/acquire translocons capable of importing proteins whose genes were transferred to the host genome. These gene products are expressed on cytosolic ribosomes as precursor proteins and targeted back to the organelle by an N-terminal extension called the transit peptide or presequence. In chloroplasts the transit peptide is specifically recognized by the Translocon of the Outer Chloroplast membrane (Toc) which is composed of receptor GTPases that potentially function as gate-like switches, where GTP binding and hydrolysis somehow facilitate preprotein binding and translocation. Compared to other translocons, the dynamics of the Toc translocon are probably more complex and certainly less understood. We have developed biochemical/biophysical, imaging, and computational techniques to probe the dynamics of the Toc translocon at the nanoscale. In this chapter we provide detailed protocols for kinetic and binding analysis of precursor interactions in organeller, measurement of the activity and nucleotide binding of the Toc GTPases, native electrophoretic analysis of the assembly/organization of the Toc complex, visualization of the distribution and mobility of Toc apparatus on the surface of chloroplasts, and conclude with the identification and molecular modeling Toc75 POTRA domains. With these new methodologies we discuss future directions of the field.

Comparative chloroplast genomics and phylogenetics of Fagopyrum esculentum ssp. ancestrale – A wild ancestor of cultivated buckwheat

PubMed Central

Logacheva, Maria D; Samigullin, Tahir H; Dhingra, Amit; Penin, Aleksey A

2008-01-01

Background Chloroplast genome sequences are extremely informative about species-interrelationships owing to its non-meiotic and often uniparental inheritance over generations. The subject of our study, Fagopyrum esculentum, is a member of the family Polygonaceae belonging to the order Caryophyllales. An uncertainty remains regarding the affinity of Caryophyllales and the asterids that could be due to undersampling of the taxa. With that background, having access to the complete chloroplast genome sequence for Fagopyrum becomes quite pertinent. Results We report the complete chloroplast genome sequence of a wild ancestor of cultivated buckwheat, Fagopyrum esculentum ssp. ancestrale. The sequence was rapidly determined using a previously described approach that utilized a PCR-based method and employed universal primers, designed on the scaffold of multiple sequence alignment of chloroplast genomes. The gene content and order in buckwheat chloroplast genome is similar to Spinacia oleracea. However, some unique structural differences exist: the presence of an intron in the rpl2 gene, a frameshift mutation in the rpl23 gene and extension of the inverted repeat region to include the ycf1 gene. Phylogenetic analysis of 61 protein-coding gene sequences from 44 complete plastid genomes provided strong support for the sister relationships of Caryophyllales (including Polygonaceae) to asterids. Further, our analysis also provided support for Amborella as sister to all other angiosperms, but interestingly, in the bayesian phylogeny inference based on first two codon positions Amborella united with Nymphaeales. Conclusion Comparative genomics analyses revealed that the Fagopyrum chloroplast genome harbors the characteristic gene content and organization as has been described for several other chloroplast genomes. However, it has some unique structural features distinct from previously reported complete chloroplast genome sequences. Phylogenetic analysis of the dataset

AT_CHLORO, a comprehensive chloroplast proteome database with subplastidial localization and curated information on envelope proteins.

PubMed

Ferro, Myriam; Brugière, Sabine; Salvi, Daniel; Seigneurin-Berny, Daphné; Court, Magali; Moyet, Lucas; Ramus, Claire; Miras, Stéphane; Mellal, Mourad; Le Gall, Sophie; Kieffer-Jaquinod, Sylvie; Bruley, Christophe; Garin, Jérôme; Joyard, Jacques; Masselon, Christophe; Rolland, Norbert

2010-06-01

Recent advances in the proteomics field have allowed a series of high throughput experiments to be conducted on chloroplast samples, and the data are available in several public databases. However, the accurate localization of many chloroplast proteins often remains hypothetical. This is especially true for envelope proteins. We went a step further into the knowledge of the chloroplast proteome by focusing, in the same set of experiments, on the localization of proteins in the stroma, the thylakoids, and envelope membranes. LC-MS/MS-based analyses first allowed building the AT_CHLORO database (http://www.grenoble.prabi.fr/protehome/grenoble-plant-proteomics/), a comprehensive repertoire of the 1323 proteins, identified by 10,654 unique peptide sequences, present in highly purified chloroplasts and their subfractions prepared from Arabidopsis thaliana leaves. This database also provides extensive proteomics information (peptide sequences and molecular weight, chromatographic retention times, MS/MS spectra, and spectral count) for a unique chloroplast protein accurate mass and time tag database gathering identified peptides with their respective and precise analytical coordinates, molecular weight, and retention time. We assessed the partitioning of each protein in the three chloroplast compartments by using a semiquantitative proteomics approach (spectral count). These data together with an in-depth investigation of the literature were compiled to provide accurate subplastidial localization of previously known and newly identified proteins. A unique knowledge base containing extensive information on the proteins identified in envelope fractions was thus obtained, allowing new insights into this membrane system to be revealed. Altogether, the data we obtained provide unexpected information about plastidial or subplastidial localization of some proteins that were not suspected to be associated to this membrane system. The spectral counting-based strategy was further

Complete chloroplast genome of Tetragonia tetragonioides: Molecular phylogenetic relationships and evolution in Caryophyllales.

PubMed

Choi, Kyoung Su; Kwak, Myounghai; Lee, Byoungyoon; Park, SeonJoo

2018-01-01

The chloroplast genome of Tetragonia tetragonioides (Aizoaceae; Caryophyllales) was sequenced to provide information for studies on phylogeny and evolution within Caryophyllales. The chloroplast genome of Tetragonia tetragonioides is 149,506 bp in length and includes a pair of inverted repeats (IRs) of 24,769 bp that separate a large single copy (LSC) region of 82,780 bp and a small single copy (SSC) region of 17,188 bp. Comparative analysis of the chloroplast genome showed that Caryphyllales species have lost many genes. In particular, the rpl2 intron and infA gene were not found in T. tetragonioides, and core Caryophyllales lack the rpl2 intron. Phylogenetic analyses were conducted using 55 genes in 16 complete chloroplast genomes. Caryophyllales was found to divide into two clades; core Caryophyllales and noncore Caryophyllales. The genus Tetragonia is closely related to Mesembryanthemum. Comparisons of the synonymous (Ks), nonsynonymous (Ka), and Ka/Ks substitution rates revealed that nonsynonymous substitution rates were lower than synonymous substitution rates and that Ka/Ks rates were less than 1. The findings of the present study suggest that most genes are a purified selection.

Arginine Decarboxylase Is Localized in Chloroplasts.

PubMed Central

Borrell, A.; Culianez-Macia, F. A.; Altabella, T.; Besford, R. T.; Flores, D.; Tiburcio, A. F.

1995-01-01

Plants, unlike animals, can use either ornithine decarboxylase or arginine decarboxylase (ADC) to produce the polyamine precursor putrescine. Lack of knowledge of the exact cellular and subcellular location of these enzymes has been one of the main obstacles to our understanding of the biological role of polyamines in plants. We have generated polyclonal antibodies to oat (Avena sativa L.) ADC to study the spatial distribution and subcellular localization of ADC protein in different oat tissues. By immunoblotting and immunocytochemistry, we show that ADC is organ specific. By cell fractionation and immunoblotting, we show that ADC is localized in chloroplasts associated with the thylakoid membrane. The results also show that increased levels of ADC protein are correlated with high levels of ADC activity and putrescine in osmotically stressed oat leaves. A model of compartmentalization for the arginine pathway and putrescine biosynthesis in active photosynthetic tissues has been proposed. In the context of endosymbiote-driven metabolic evolution in plants, the location of ADC in the chloroplast compartment may have major evolutionary significance, since it explains (a) why plants can use two alternative pathways for putrescine biosynthesis and (b) why animals do not possess ADC. PMID:12228631

The First Complete Chloroplast Genome Sequences in Actinidiaceae: Genome Structure and Comparative Analysis.

PubMed

Yao, Xiaohong; Tang, Ping; Li, Zuozhou; Li, Dawei; Liu, Yifei; Huang, Hongwen

2015-01-01

Actinidia chinensis is an important economic plant belonging to the basal lineage of the asterids. Availability of a complete Actinidia chloroplast genome sequence is crucial to understanding phylogenetic relationships among major lineages of angiosperms and facilitates kiwifruit genetic improvement. We report here the complete nucleotide sequences of the chloroplast genomes for Actinidia chinensis and A. chinensis var deliciosa obtained through de novo assembly of Illumina paired-end reads produced by total DNA sequencing. The total genome size ranges from 155,446 to 157,557 bp, with an inverted repeat (IR) of 24,013 to 24,391 bp, a large single copy region (LSC) of 87,984 to 88,337 bp and a small single copy region (SSC) of 20,332 to 20,336 bp. The genome encodes 113 different genes, including 79 unique protein-coding genes, 30 tRNA genes and 4 ribosomal RNA genes, with 16 duplicated in the inverted repeats, and a tRNA gene (trnfM-CAU) duplicated once in the LSC region. Comparisons of IR boundaries among four asterid species showed that IR/LSC borders were extended into the 5' portion of the psbA gene and IR contraction occurred in Actinidia. The clap gene has been lost from the chloroplast genome in Actinidia, and may have been transferred to the nucleus during chloroplast evolution. Twenty-seven polymorphic simple sequence repeat (SSR) loci were identified in the Actinidia chloroplast genome. Maximum parsimony analyses of a 72-gene, 16 taxa angiosperm dataset strongly support the placement of Actinidiaceae in Ericales within the basal asterids.

High-Throughput Sequencing of Three Lemnoideae (Duckweeds) Chloroplast Genomes from Total DNA

PubMed Central

Wang, Wenqin; Messing, Joachim

2011-01-01

Background Chloroplast genomes provide a wealth of information for evolutionary and population genetic studies. Chloroplasts play a particularly important role in the adaption for aquatic plants because they float on water and their major surface is exposed continuously to sunlight. The subfamily of Lemnoideae represents such a collection of aquatic species that because of photosynthesis represents one of the fastest growing plant species on earth. Methods We sequenced the chloroplast genomes from three different genera of Lemnoideae, Spirodela polyrhiza, Wolffiella lingulata and Wolffia australiana by high-throughput DNA sequencing of genomic DNA using the SOLiD platform. Unfractionated total DNA contains high copies of plastid DNA so that sequences from the nucleus and mitochondria can easily be filtered computationally. Remaining sequence reads were assembled into contiguous sequences (contigs) using SOLiD software tools. Contigs were mapped to a reference genome of Lemna minor and gaps, selected by PCR, were sequenced on the ABI3730xl platform. Conclusions This combinatorial approach yielded whole genomic contiguous sequences in a cost-effective manner. Over 1,000-time coverage of chloroplast from total DNA were reached by the SOLiD platform in a single spot on a quadrant slide without purification. Comparative analysis indicated that the chloroplast genome was conserved in gene number and organization with respect to the reference genome of L. minor. However, higher nucleotide substitution, abundant deletions and insertions occurred in non-coding regions of these genomes, indicating a greater genomic dynamics than expected from the comparison of other related species in the Pooideae. Noticeably, there was no transition bias over transversion in Lemnoideae. The data should have immediate applications in evolutionary biology and plant taxonomy with increased resolution and statistical power. PMID:21931804

Melatonin biosynthesis in plants: multiple pathways catalyze tryptophan to melatonin in the cytoplasm or chloroplasts.

PubMed

Back, Kyoungwhan; Tan, Dun-Xian; Reiter, Russel J

2016-11-01

Melatonin is an animal hormone as well as a signaling molecule in plants. It was first identified in plants in 1995, and almost all enzymes responsible for melatonin biosynthesis had already been characterized in these species. Melatonin biosynthesis from tryptophan requires four-step reactions. However, six genes, that is, TDC, TPH, T5H, SNAT, ASMT, and COMT, have been implicated in the synthesis of melatonin in plants, suggesting the presence of multiple pathways. Two major pathways have been proposed based on the enzyme kinetics: One is the tryptophan/tryptamine/serotonin/N-acetylserotonin/melatonin pathway, which may occur under normal growth conditions; the other is the tryptophan/tryptamine/serotonin/5-methoxytryptamine/melatonin pathway, which may occur when plants produce large amounts of serotonin, for example, upon senescence. The melatonin biosynthetic capacity associated with conversion of tryptophan to serotonin is much higher than that associated with conversion of serotonin to melatonin, which yields a low level of melatonin synthesis in plants. Many melatonin intermediates are produced in various subcellular compartments, such as the cytoplasm, endoplasmic reticulum, and chloroplasts, which either facilitates or impedes the subsequent enzymatic steps. Depending on the pathways, the final subcellular sites of melatonin synthesis vary at either the cytoplasm or chloroplasts, which may differentially affect the mode of action of melatonin in plants. © 2016 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.

Chloroplast microsatellites reveal colonization and metapopulation dynamics in the Canary Island pine

PubMed Central

Navascués, Miguel; Vaxevanidou, Zafeiro; González-Martínez, Santiago C; Climent, José; Gil, Luis; Emerson, Brent C

2006-01-01

Chloroplast microsatellites are becoming increasingly popular markers for population genetic studies in plants, but there has been little focus on their potential for demographic inference. In this work the utility of chloroplast microsatellites for the study of population expansions was explored. First, we investigated the power of mismatch distribution analysis and the FS test with coalescent simulations of different demographic scenarios. We then applied those methods to empirical data obtained for the Canary Island pine (Pinus canariensis). The results of the simulations showed that chloroplast microsatellites are sensitive to sudden population growth. The power of the FS test and accuracy of demographic parameter estimates, such as the time of expansion, were reduced proportionally to the level of homoplasy within the data. The analysis of Canary Island pine chloroplast microsatellite data indicated population expansions for almost all sample localities. Demographic expansions at the island level can be explained by the colonisation of the archipelago by the pine, while population expansions of different ages in different localities within an island appear to be the result of local extinctions and recolonisation dynamics. Comparable mitochondrial DNA sequence data from a parasite of P. canariensis, the weevil Brachyderes rugatus, supports this scenario, suggesting a key role for volcanism in the evolution of pine forest communities in the Canary Islands. PMID:16911194

Construction of a restriction map and gene map of the lettuce chloroplast small single-copy region using Southern cross-hybridization.

PubMed

Mitchelson, K R

1996-01-01

The small single-copy region (SSCR) of the chloroplast genome of many higher plants typically contain ndh genes encoding proteins that share homology with subunits of the respiratory-chain reduced nicotinamide adenine dinucleotide (NADH) dehydrogenase complex of mitochondria. A map of the lettuce chloroplast SSCR has been determined by Southern cross-hybridization, taking advantage of the high degree of homology between a tobacco small single-copy fragment and a corresponding lettuce chloroplast fragment. The gene order of the SSCR of lettuce and tobacco chloroplasts is similar. The cross-hybridization method can rapidly create a primary gene map of unknown chloroplast fragments, thus providing detailed information of the localization and arrangement of genes and conserved open reading frame regions.

Complete Chloroplast Genome Sequences of Mongolia Medicine Artemisia frigida and Phylogenetic Relationships with Other Plants

PubMed Central

Liu, Yue; Huo, Naxin; Dong, Lingli; Wang, Yi; Zhang, Shuixian; Young, Hugh A.; Feng, Xiaoxiao; Gu, Yong Qiang

2013-01-01

Background Artemisia frigida Willd. is an important Mongolian traditional medicinal plant with pharmacological functions of stanch and detumescence. However, there is little sequence and genomic information available for Artemisia frigida, which makes phylogenetic identification, evolutionary studies, and genetic improvement of its value very difficult. We report the complete chloroplast genome sequence of Artemisia frigida based on 454 pyrosequencing. Methodology/Principal Findings The complete chloroplast genome of Artemisia frigida is 151,076 bp including a large single copy (LSC) region of 82,740 bp, a small single copy (SSC) region of 18,394 bp and a pair of inverted repeats (IRs) of 24,971 bp. The genome contains 114 unique genes and 18 duplicated genes. The chloroplast genome of Artemisia frigida contains a small 3.4 kb inversion within a large 23 kb inversion in the LSC region, a unique feature in Asteraceae. The gene order in the SSC region of Artemisia frigida is inverted compared with the other 6 Asteraceae species with the chloroplast genomes sequenced. This inversion is likely caused by an intramolecular recombination event only occurred in Artemisia frigida. The existence of rich SSR loci in the Artemisia frigida chloroplast genome provides a rare opportunity to study population genetics of this Mongolian medicinal plant. Phylogenetic analysis demonstrates a sister relationship between Artemisia frigida and four other species in Asteraceae, including Ageratina adenophora, Helianthus annuus, Guizotia abyssinica and Lactuca sativa, based on 61 protein-coding sequences. Furthermore, Artemisia frigida was placed in the tribe Anthemideae in the subfamily Asteroideae (Asteraceae) based on ndhF and trnL-F sequence comparisons. Conclusion The chloroplast genome sequence of Artemisia frigida was assembled and analyzed in this study, representing the first plastid genome sequenced in the Anthemideae tribe. This complete chloroplast genome sequence will be

The complete chloroplast genome of Cinnamomum camphora and its comparison with related Lauraceae species.

PubMed

Chen, Caihui; Zheng, Yongjie; Liu, Sian; Zhong, Yongda; Wu, Yanfang; Li, Jiang; Xu, Li-An; Xu, Meng

2017-01-01

Cinnamomum camphora , a member of the Lauraceae family, is a valuable aromatic and timber tree that is indigenous to the south of China and Japan. All parts of Cinnamomum camphora have secretory cells containing different volatile chemical compounds that are utilized as herbal medicines and essential oils. Here, we reported the complete sequencing of the chloroplast genome of Cinnamomum camphora using illumina technology. The chloroplast genome of Cinnamomum camphora is 152,570 bp in length and characterized by a relatively conserved quadripartite structure containing a large single copy region of 93,705 bp, a small single copy region of 19,093 bp and two inverted repeat (IR) regions of 19,886 bp. Overall, the genome contained 123 coding regions, of which 15 were repeated in the IR regions. An analysis of chloroplast sequence divergence revealed that the small single copy region was highly variable among the different genera in the Lauraceae family. A total of 40 repeat structures and 83 simple sequence repeats were detected in both the coding and non-coding regions. A phylogenetic analysis indicated that Calycanthus is most closely related to Lauraceae , both being members of Laurales , which forms a sister group to Magnoliids . The complete sequence of the chloroplast of Cinnamomum camphora will aid in in-depth taxonomical studies of the Lauraceae family in the future. The genetic sequence information will also have valuable applications for chloroplast genetic engineering.

Fine-scale mergers of chloroplast and mitochondrial genes create functional, transcompartmentally chimeric mitochondrial genes.

PubMed

Hao, Weilong; Palmer, Jeffrey D

2009-09-29

The mitochondrial genomes of flowering plants possess a promiscuous proclivity for taking up sequences from the chloroplast genome. All characterized chloroplast integrants exist apart from native mitochondrial genes, and only a few, involving chloroplast tRNA genes that have functionally supplanted their mitochondrial counterparts, appear to be of functional consequence. We developed a novel computational approach to search for homologous recombination (gene conversion) in a large number of sequences and applied it to 22 mitochondrial and chloroplast gene pairs, which last shared common ancestry some 2 billion years ago. We found evidence of recurrent conversion of short patches of mitochondrial genes by chloroplast homologs during angiosperm evolution, but no evidence of gene conversion in the opposite direction. All 9 putative conversion events involve the atp1/atpA gene encoding the alpha subunit of ATP synthase, which is unusually well conserved between the 2 organelles and the only shared gene that is widely sequenced across plant mitochondria. Moreover, all conversions were limited to the 2 regions of greatest nucleotide and amino acid conservation of atp1/atpA. These observations probably reflect constraints operating on both the occurrence and fixation of recombination between ancient homologs. These findings indicate that recombination between anciently related sequences is more frequent than previously appreciated and creates functional mitochondrial genes of chimeric origin. These results also have implications for the widespread use of mitochondrial atp1 in phylogeny reconstruction.

Reference-free comparative genomics of 174 chloroplasts.

PubMed

Kua, Chai-Shian; Ruan, Jue; Harting, John; Ye, Cheng-Xi; Helmus, Matthew R; Yu, Jun; Cannon, Charles H

2012-01-01

Direct analysis of unassembled genomic data could greatly increase the power of short read DNA sequencing technologies and allow comparative genomics of organisms without a completed reference available. Here, we compare 174 chloroplasts by analyzing the taxanomic distribution of short kmers across genomes [1]. We then assemble de novo contigs centered on informative variation. The localized de novo contigs can be separated into two major classes: tip = unique to a single genome and group = shared by a subset of genomes. Prior to assembly, we found that ~18% of the chloroplast was duplicated in the inverted repeat (IR) region across a four-fold difference in genome sizes, from a highly reduced parasitic orchid [2] to a massive algal chloroplast [3], including gnetophytes [4] and cycads [5]. The conservation of this ratio between single copy and duplicated sequence was basal among green plants, independent of photosynthesis and mechanism of genome size change, and different in gymnosperms and lower plants. Major lineages in the angiosperm clade differed in the pattern of shared kmers and de novo contigs. For example, parasitic plants demonstrated an expected accelerated overall rate of evolution, while the hemi-parasitic genomes contained a great deal more novel sequence than holo-parasitic plants, suggesting different mechanisms at different stages of genomic contraction. Additionally, the legumes are diverging more quickly and in different ways than other major families. Small duplicated fragments of the rrn23 genes were deeply conserved among seed plants, including among several species without the IR regions, indicating a crucial functional role of this duplication. Localized de novo assembly of informative kmers greatly reduces the complexity of large comparative analyses by confining the analysis to a small partition of data and genomes relevant to the specific question, allowing direct analysis of next-gen sequence data from previously unstudied genomes and

The complete chloroplast DNA sequence of the green alga Oltmannsiellopsis viridis reveals a distinctive quadripartite architecture in the chloroplast genome of early diverging ulvophytes

PubMed Central

Pombert, Jean-François; Lemieux, Claude; Turmel, Monique

2006-01-01

Background The phylum Chlorophyta contains the majority of the green algae and is divided into four classes. The basal position of the Prasinophyceae has been well documented, but the divergence order of the Ulvophyceae, Trebouxiophyceae and Chlorophyceae is currently debated. The four complete chloroplast DNA (cpDNA) sequences presently available for representatives of these classes have revealed extensive variability in overall structure, gene content, intron composition and gene order. The chloroplast genome of Pseudendoclonium (Ulvophyceae), in particular, is characterized by an atypical quadripartite architecture that deviates from the ancestral type by a large inverted repeat (IR) featuring an inverted rRNA operon and a small single-copy (SSC) region containing 14 genes normally found in the large single-copy (LSC) region. To gain insights into the nature of the events that led to the reorganization of the chloroplast genome in the Ulvophyceae, we have determined the complete cpDNA sequence of Oltmannsiellopsis viridis, a representative of a distinct, early diverging lineage. Results The 151,933 bp IR-containing genome of Oltmannsiellopsis differs considerably from Pseudendoclonium and other chlorophyte cpDNAs in intron content and gene order, but shares close similarities with its ulvophyte homologue at the levels of quadripartite architecture, gene content and gene density. Oltmannsiellopsis cpDNA encodes 105 genes, contains five group I introns, and features many short dispersed repeats. As in Pseudendoclonium cpDNA, the rRNA genes in the IR are transcribed toward the single copy region featuring the genes typically found in the ancestral LSC region, and the opposite single copy region harbours genes characteristic of both the ancestral SSC and LSC regions. The 52 genes that were transferred from the ancestral LSC to SSC region include 12 of those observed in Pseudendoclonium cpDNA. Surprisingly, the overall gene organization of Oltmannsiellopsis cp

Variability of chloroplast DNA and nuclear ribosomal DNA in cassava (Manihot esculenta Crantz) and its wild relatives.

PubMed

Fregene, M A; Vargas, J; Ikea, J; Angel, F; Tohme, J; Asiedu, R A; Akoroda, M O; Roca, W M

1994-11-01

Chloroplast DNA (cp) and nuclear ribosomal DNA (rDNA) variation was investigated in 45 accessions of cultivated and wild Manihot species. Ten independent mutations, 8 point mutations and 2 length mutations were identified, using eight restriction enzymes and 12 heterologous cpDNA probes from mungbean. Restriction fragment length polymorphism analysis defined nine distinct chloroplast types, three of which were found among the cultivated accessions and six among the wild species. Cladistic analysis of the cpDNA data using parsimony yielded a hypothetical phylogeny of lineages among the cpDNAs of cassava and its wild relatives that is congruent with morphological evolutionary differentiation in the genus. The results of our survey of cpDNA, together with rDNA restriction site change at the intergenic spacer region and rDNA repeat unit length variation (using rDNA cloned fragments from taro as probe), suggest that cassava might have arisen from the domestication of wild tuberous accessions of some Manihot species, followed by intensive selection. M. esculenta subspp flabellifolia is probably a wild progenitor. Introgressive hybridization with wild forms and pressures to adapt to the widely varying climates and topography in which cassava is found might have enhanced the crop's present day variability.

Photoinduction of cyclosis-mediated interactions between distant chloroplasts.

PubMed

Bulychev, Alexander A; Komarova, Anna V

2015-01-01

Communications between chloroplasts and other organelles based on the exchange of metabolites, including redox active substances, are recognized as a part of intracellular regulation, chlororespiration, and defense against oxidative stress. Similar communications may operate between spatially distant chloroplasts in large cells where photosynthetic and respiratory activities are distributed unevenly under fluctuating patterned illumination. Microfluorometry of chlorophyll fluorescence in vivo in internodal cells of the alga Chara corallina revealed that a 30-s pulse of localized light induces a transient increase (~25%) in F' fluorescence of remote cell parts exposed to dim background light at a 1.5-mm distance on the downstream side from the illuminated spot in the plane of unilateral cytoplasmic streaming but has no effect on F' at equal distance on the upstream side. An abrupt arrest of cytoplasmic streaming for about 30s by triggering the action potential extended either the ascending or descending fronts of the F' fluorescence response, depending on the exact moment of streaming cessation. The response of F' fluorescence to localized illumination of a distant cell region was absent in dark-adapted internodes, when the localized light was applied within the first minute after switching on continuous background illumination of the whole cell, but it appeared in full after longer exposures to continuous background light. These results and the elimination of the F' response by methyl viologen known to redirect electron transport pathways beyond photosystem I indicate the importance of photosynthetic induction and the stromal redox state for long-distance communications of chloroplasts in vivo. Copyright © 2015 Elsevier B.V. All rights reserved.

Distribution of Metabolites between Chloroplast and Cytoplasm during the Induction Phase of Photosynthesis in Leaf Protoplasts 1

PubMed Central

Robinson, Simon P.; Walker, David A.

1980-01-01

A method for rapid separation of the chloroplast and cytoplasmic fractions from isolated leaf protoplasts of wheat and spinach has been used to determine the distribution of 14C-labeled products during photosynthesis. In the dark, CO2 fixation was only 1 to 2% of that in the light and the products were mainly in the cytoplasmic fraction suggesting fixation by phosphoenolpyruvate carboxylase. Label appeared rapidly in the chloroplast fraction following illumination but the amount leveled off after 4 to 5 minutes reflecting the buildup of intermediates to steady state levels. There was only a slight lag before label appeared in the cytoplasmic fraction and it continued to increase at a constant rate reflecting synthesis of neutral products. In the light, the percentage of label in the chloroplast fraction decreased rapidly in the first minute of illumination and was only 10 to 20% in the steady-state. It is suggested that the chloroplast phosphate transporter promotes a rapid transfer of sugar phosphates from the chloroplast to the cytoplasm, even during the induction phase of photosynthesis. PMID:16661305

Mode of inheritance and evidence for cistron heterogeneity of chloroplast 16S ribosomal RNA genes in Nicotiana.

PubMed

Vacek, A T; Bourque, D P

1980-09-01

Oligonucleotide maps (fingerprints) of T1 RNase digests of 125I-labeled 16 S chloroplast rRNA of Nicotiana tabacum and N. gossei revealed the presence of T1 oligonucleotide fragment 100 in the 16 S rRNA of N. gossei while N. tabacum 16 S rRNA had a unique T1 oligonucleotide (fragment 101) as well as some fragment 100. From the positions in the fingerprints and from fingerprints of secondary enzymatic digestion of the fragments, we conclude that fragments 100 and 101 are similar in sequence and size, but fragment 100 probably contains an extra uracil residue. This difference is shown to be maternally inherited, thus confirming the location of 16 S chloroplast rRNA genes on chloroplast DNA and ruling out the possibility of genetically active chloroplast rRNA genes in the nucleus. The presence of both fragments 100 and 101 in N. tabacum may indicate sequence heterogeneity between the two cistrons for 16 S chloroplast rRNA. These results demonstrate the feasibility of determining the inheritance of organelle genes by genetic analysis of their primary transcripts.

Photoregulation of fructose and glucose respiration in the intact chloroplasts of Chlamydomonas reinhardtii F-60 and spinach

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

Singh, K.K.; Changguo Chen; Gibbs, M.

1993-04-01

The photoregulation of chloroplastic respiration was studied by monitoring in darkness and in light the release of [sup 14]CO[sub 2] from whole chloroplasts of Chlamydomonas reinhardtii F-60 and spinach (Spinacia oleracea L.) supplied externally with [[sup 14]C]glucose and [[sup 14]C]fructose, respectively. CO[sub 2] release was inhibited more than 90% in both chloroplasts by a light intensity of 4 W m[sup [minus]2]. Oxidants, oxaloacetate in Chlamydomonas, nitrite in spinach, and phenazine methosulfate in both chloroplasts, reversed the inhibition. The onset of the photoinhibitory effect on CO[sub 2] release was relatively rapid compared to the restoration of CO[sub 2] release following illumination.more » In both darkened chloroplasts, dithiothreitol inhibited release. Of the four enzymes (fructokinase, phosphoglucose isomerase, glucose-6-P dehydrogenase, and gluconate-6-P dehydrogenase) in the pathway catalyzing the release of CO[sub 2] from fructose, only glucose-6-P dehydrogenase was deactivated by light and by dithiothreitol. 33 refs., 3 figs., 4 tabs.« less

Identification of the 64 kilodalton chloroplast stromal phosphoprotein as phosphoglucomutase. [Pisum sativum

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

Salvucci, M.E.; Drake, R.R.; Broadbent, K.P.

1990-05-01

Phosphorylation of the 64 kilodalton stromal phosphoprotein by incubation of pea (Pisum sativum) chloroplast extracts with ({gamma}-{sup 32}P)ATP decreased in the presence of Glc-6-P and Glc-1,6-P{sub 2}, but was stimulated by glucose. Two-dimensional gel electrophoresis following incubation of intact chloroplasts and stromal extracts with ({gamma}-{sup 32}P)ATP, or incubation of stromal extracts and partially purified phosphoglucomutase (EC 2.7.5.1) with ({sup 32}P)Glc-1-P showed that the identical 64 kilodalton polypeptide was labeled. A 62 kilodalton polypeptide was phosphorylated by incubation of tobacco (Nicotiana sylvestris) stromal extracts with either ({gamma}-{sup 32}P)ATP or ({sup 32}P)Glc-1-P. In contrast, an analogous polypeptide was not phosphorylated in extractsmore » from a tobacco mutant deficient in plastid phosphoglucomutase activity. The results indicate that the 64 (or 62) kilodalton chloroplast stromal phosphoprotein is phosphoglucomutase.« less

Analysis of Arabidopsis Accessions Hypersensitive to a Loss of Chloroplast Translation1[OPEN

PubMed Central

Parker, Nicole; Wang, Yixing; Meinke, David

2016-01-01

Natural accessions of Arabidopsis (Arabidopsis thaliana) differ in their ability to tolerate a loss of chloroplast translation. These differences can be attributed in part to variation in a duplicated nuclear gene (ACC2) that targets homomeric acetyl-coenzyme A carboxylase (ACCase) to plastids. This functional redundancy allows limited fatty acid biosynthesis to occur in the absence of heteromeric ACCase, which is encoded in part by the plastid genome. In the presence of functional ACC2, tolerant alleles of several nuclear genes, not yet identified, enhance the growth of seedlings and embryos disrupted in chloroplast translation. ACC2 knockout mutants, by contrast, are hypersensitive. Here we describe an expanded search for hypersensitive accessions of Arabidopsis, evaluate whether all of these accessions are defective in ACC2, and characterize genotype-to-phenotype relationships for homomeric ACCase variants identified among 855 accessions with sequenced genomes. Null alleles with ACC2 nonsense mutations, frameshift mutations, small deletions, genomic rearrangements, and defects in RNA splicing are included among the most sensitive accessions examined. By contrast, most missense mutations affecting highly conserved residues failed to eliminate ACC2 function. Several accessions were identified where sensitivity could not be attributed to a defect in either ACC2 or Tic20-IV, the chloroplast membrane channel required for ACC2 uptake. Overall, these results underscore the central role of ACC2 in mediating Arabidopsis response to a loss of chloroplast translation, highlight future applications of this system to analyzing chloroplast protein import, and provide valuable insights into the mutational landscape of an important metabolic enzyme that is highly conserved throughout eukaryotes. PMID:27707889

Stable megadalton TOC-TIC supercomplexes as major mediators of protein import into chloroplasts.

PubMed

Chen, Lih-Jen; Li, Hsou-Min

2017-10-01

Preproteins are believed to be imported into chloroplasts through membrane contact sites where the translocon complexes of the outer (TOC) and inner (TIC) envelope membranes are assembled together. However, a single TOC-TIC supercomplex containing preproteins undergoing active import has not yet been directly observed. We optimized the blue native polyacrylamide gel electrophoresis (PAGE) (BN-PAGE) system to detect and resolve megadalton (MD)-sized complexes. Using this optimized system, the outer-membrane channel Toc75 from pea chloroplasts was found in at least two complexes: the 880-kD TOC complex and a previously undetected 1-MD complex. Two-dimensional BN-PAGE immunoblots further showed that Toc75, Toc159, Toc34, Tic20, Tic56 and Tic110 were all located in the 880-kD to 1.3-MD region. During active preprotein import, preproteins were transported mostly through the 1-MD complex and a smaller amount of preproteins was also detected in a complex of 1.25 MD. Antibody-shift assays showed that the 1-MD complex is a TOC-TIC supercomplex containing at least Toc75, Toc159, Toc34 and Tic110. Results from crosslinking and import with Arabidopsis chloroplasts suggest that the 1.25-MD complex is also a supercomplex. Our data provide direct evidence supporting that chloroplast preproteins are imported through TOC-TIC supercomplexes, and also provide the first size estimation of these supercomplexes. Furthermore, unlike in mitochondria where translocon supercomplexes are only transiently assembled during preprotein import, in chloroplasts at least some of the supercomplexes are preassembled stable structures. © 2017 The Authors The Plant Journal published by John Wiley & Sons Ltd and Society for Experimental Biology.

Transformation of Chloroplast Ribosomal RNA Genes in Chlamydomonas: Molecular and Genetic Characterization of Integration Events

PubMed Central

Newman, S. M.; Boynton, J. E.; Gillham, N. W.; Randolph-Anderson, B. L.; Johnson, A. M.; Harris, E. H.

1990-01-01

Transformation of chloroplast ribosomal RNA (rRNA) genes in Chlamydomonas has been achieved by the biolistic process using cloned chloroplast DNA fragments carrying mutations that confer antibiotic resistance. The sites of exchange employed during the integration of the donor DNA into the recipient genome have been localized using a combination of antibiotic resistance mutations in the 16S and 23S rRNA genes and restriction fragment length polymorphisms that flank these genes. Complete or nearly complete replacement of a region of the chloroplast genome in the recipient cell by the corresponding sequence from the donor plasmid was the most common integration event. Exchange events between the homologous donor and recipient sequences occurred preferentially near the vector:insert junctions. Insertion of the donor rRNA genes and flanking sequences into one inverted repeat of the recipient genome was followed by intramolecular copy correction so that both copies of the inverted repeat acquired identical sequences. Increased frequencies of rRNA gene transformants were achieved by reducing the copy number of the chloroplast genome in the recipient cells and by decreasing the heterology between donor and recipient DNA sequences flanking the selectable markers. In addition to producing bona fide chloroplast rRNA transformants, the biolistic process induced mutants resistant to low levels of streptomycin, typical of nuclear mutations in Chlamydomonas. PMID:1981764

Recognition of RNA Editing Sites Is Directed by Unique Proteins in Chloroplasts: Biochemical Identification of cis-Acting Elements and trans-Acting Factors Involved in RNA Editing in Tobacco and Pea Chloroplasts

PubMed Central

Miyamoto, Tetsuya; Obokata, Junichi; Sugiura, Masahiro

2002-01-01

RNA editing in higher-plant chloroplasts involves C-to-U conversions at specific sites. Although in vivo analyses have been performed, little is known about the biochemical aspects of chloroplast editing reactions. Here we improved our original in vitro system and devised a procedure for preparing active chloroplast extracts not only from tobacco plants but also from pea plants. Using our tobacco in vitro system, cis-acting elements were defined for psbE and petB mRNAs. Distinct proteins were found to bind specifically to each cis-element, a 56-kDa protein to the psbE site and a 70-kDa species to the petB site. Pea chloroplasts lack the corresponding editing site in psbE since T is already present in the DNA. Parallel in vitro analyses with tobacco and pea extracts revealed that the pea plant has no editing activity for psbE mRNAs and lacks the 56-kDa protein, whereas petB mRNAs are edited and the 70-kDa protein is also present. Therefore, coevolution of an editing site and its cognate trans-factor was demonstrated biochemically in psbE mRNA editing between tobacco and pea plants. PMID:12215530

Complete chloroplast genome of Trachelium caeruleum: extensiverearrangements are associated with repeats and tRNAs

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

Haberle, Rosemarie C.; Fourcade, Matthew L.; Boore, Jeffrey L.

2006-01-09

Chloroplast genome structure, gene order and content arehighly conserved in land plants. We sequenced the complete chloroplastgenome sequence of Trachelium caeruleum (Campanulaceae) a member of anangiosperm family known for highly rearranged chloroplast genomes. Thetotal genome size is 162,321 bp with an IR of 27,273 bp, LSC of 100,113bp and SSC of 7,661 bp. The genome encodes 115 unique genes, with 19duplicated in the IR, a tRNA (trnI-CAU) duplicated once in the LSC and aprotein coding gene (psbJ) duplicated twice, for a total of 137 genes.Four genes (ycf15, rpl23, infA and accD) are truncated and likelynonfunctional; three others (clpP, ycf1 andmore » ycf2) are so highly divergedthat they may now be pseudogenes. The most conspicuous feature of theTrachelium genome is the presence of eighteen internally unrearrangedblocks of genes that have been inverted or relocated within the genome,relative to the typical gene order of most angiosperm chloroplastgenomes. Recombination between repeats or tRNAs has been suggested as twomeans of chloroplast genome rearrangements. We compared the relativenumber of repeats in Trachelium to eight other angiosperm chloroplastgenomes, and evaluated the location of repeats and tRNAs in relation torearrangements. Trachelium has the highest number and largest repeats,which are concentrated near inversion endpoints or other rearrangements.tRNAs occur at many but not all inversion endpoints. There is likely nosingle mechanism responsible for the remarkable number of alterations inthis genome, but both repeats and tRNAs are clearly associated with theserearrangements. Land plant chloroplast genomes are highly conserved instructure, gene order and content. The chloroplast genomes of ferns, thegymnosperm Ginkgo, and most angiosperms are nearly collinear, reflectingthe gene order in lineages that diverged from lycopsids and the ancestralchloroplast gene order over 350 million years ago (Raubeson and Jansen,1992). Although earlier mapping

Differential Contribution of Endoplasmic Reticulum and Chloroplast ω-3 Fatty Acid Desaturase Genes to the Linolenic Acid Content of Olive (Olea europaea) Fruit.

PubMed

Hernández, M Luisa; Sicardo, M Dolores; Martínez-Rivas, José M

2016-01-01

Linolenic acid is a polyunsaturated fatty acid present in plant lipids, which plays key roles in plant metabolism as a structural component of storage and membrane lipids, and as a precursor of signaling molecules. The synthesis of linolenic acid is catalyzed by two different ω-3 fatty acid desaturases, which correspond to microsomal- (FAD3) and chloroplast- (FAD7 and FAD8) localized enzymes. We have investigated the specific contribution of each enzyme to the linolenic acid content in olive fruit. With that aim, we isolated two different cDNA clones encoding two ω-3 fatty acid desaturases from olive (Olea europaea cv. Picual). Sequence analysis indicates that they code for microsomal (OepFAD3B) and chloroplast (OepFAD7-2) ω-3 fatty acid desaturase enzymes, different from the previously characterized OekFAD3A and OekFAD7-1 genes. Functional expression in yeast of the corresponding OepFAD3A and OepFAD3B cDNAs confirmed that they encode microsomal ω-3 fatty acid desaturases. The linolenic acid content and transcript levels of olive FAD3 and FAD7 genes were measured in different tissues of Picual and Arbequina cultivars, including mesocarp and seed during development and ripening of olive fruit. Gene expression and lipid analysis indicate that FAD3A is the gene mainly responsible for the linolenic acid present in the seed, while FAD7-1 and FAD7-2 contribute mostly to the linolenic acid present in the mesocarp and, therefore, in the olive oil. These results also indicate the relevance of lipid trafficking between the endoplasmic reticulum and chloroplast in determining the linolenic acid content of membrane and storage lipids in oil-accumulating photosynthetic tissues. © The Author 2015. Published by Oxford University Press on behalf of Japanese Society of Plant Physiologists. All rights reserved. For permissions, please email: journals.permissions@oup.com.

ChloroSeq, an optimized chloroplast RNA-Seq bioinformatic pipeline, reveals remodeling of the organellar transcriptome under heat stress

DOE PAGES

Castandet, Benoît; Hotto, Amber M.; Strickler, Susan R.; ...

2016-07-06

Although RNA-Seq has revolutionized transcript analysis, organellar transcriptomes are rarely assessed even when present in published datasets. Here, we describe the development and application of a rapid and convenient method, ChloroSeq, to delineate qualitative and quantitative features of chloroplast RNA metabolism from strand-specific RNA-Seq datasets, including processing, editing, splicing, and relative transcript abundance. The use of a single experiment to analyze systematically chloroplast transcript maturation and abundance is of particular interest due to frequent pleiotropic effects observed in mutants that affect chloroplast gene expression and/or photosynthesis. To illustrate its utility, ChloroSeq was applied to published RNA-Seq datasets derived from Arabidopsismore » thaliana grown under control and abiotic stress conditions, where the organellar transcriptome had not been examined. The most appreciable effects were found for heat stress, which induces a global reduction in splicing and editing efficiency, and leads to increased abundance of chloroplast transcripts, including genic, intergenic, and antisense transcripts. Moreover, by concomitantly analyzing nuclear transcripts that encode chloroplast gene expression regulators from the same libraries, we demonstrate the possibility of achieving a holistic understanding of the nucleus-organelle system. In conclusion, ChloroSeq thus represents a unique method for streamlining RNA-Seq data interpretation of the chloroplast transcriptome and its regulators.« less

ChloroSeq, an optimized chloroplast RNA-Seq bioinformatic pipeline, reveals remodeling of the organellar transcriptome under heat stress

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

Castandet, Benoît; Hotto, Amber M.; Strickler, Susan R.

Although RNA-Seq has revolutionized transcript analysis, organellar transcriptomes are rarely assessed even when present in published datasets. Here, we describe the development and application of a rapid and convenient method, ChloroSeq, to delineate qualitative and quantitative features of chloroplast RNA metabolism from strand-specific RNA-Seq datasets, including processing, editing, splicing, and relative transcript abundance. The use of a single experiment to analyze systematically chloroplast transcript maturation and abundance is of particular interest due to frequent pleiotropic effects observed in mutants that affect chloroplast gene expression and/or photosynthesis. To illustrate its utility, ChloroSeq was applied to published RNA-Seq datasets derived from Arabidopsismore » thaliana grown under control and abiotic stress conditions, where the organellar transcriptome had not been examined. The most appreciable effects were found for heat stress, which induces a global reduction in splicing and editing efficiency, and leads to increased abundance of chloroplast transcripts, including genic, intergenic, and antisense transcripts. Moreover, by concomitantly analyzing nuclear transcripts that encode chloroplast gene expression regulators from the same libraries, we demonstrate the possibility of achieving a holistic understanding of the nucleus-organelle system. In conclusion, ChloroSeq thus represents a unique method for streamlining RNA-Seq data interpretation of the chloroplast transcriptome and its regulators.« less

Chloroplast and nuclear microsatellite analysis of Aegilops cylindrica.

PubMed

Gandhi, Harish T; Vales, M Isabel; Watson, Christy J W; Mallory-Smith, Carol A; Mori, Naoki; Rehman, Maqsood; Zemetra, Robert S; Riera-Lizarazu, Oscar

2005-08-01

Aegilops cylindrica Host (2n = 4x = 28, genome CCDD) is an allotetraploid formed by hybridization between the diploid species Ae. tauschii Coss. (2n = 2x = 14, genome DD) and Ae. markgrafii (Greuter) Hammer (2n = 2x = 14, genome CC). Previous research has shown that Ae. tauschii contributed its cytoplasm to Ae. cylindrica. However, our analysis with chloroplast microsatellite markers showed that 1 of the 36 Ae. cylindrica accessions studied, TK 116 (PI 486249), had a plastome derived from Ae. markgrafii rather than Ae. tauschii. Thus, Ae. markgrafii has also contributed its cytoplasm to Ae. cylindrica. Our analysis of chloroplast and nuclear microsatellite markers also suggests that D-type plastome and the D genome in Ae. cylindrica were closely related to, and were probably derived from, the tauschii gene pool of Ae. tauschii. A determination of the likely source of the C genome and the C-type plastome in Ae. cylindrica was not possible.

RPT2/NCH1 subfamily of NPH3-like proteins is essential for the chloroplast accumulation response in land plants.

PubMed

Suetsugu, Noriyuki; Takemiya, Atsushi; Kong, Sam-Geun; Higa, Takeshi; Komatsu, Aino; Shimazaki, Ken-Ichiro; Kohchi, Takayuki; Wada, Masamitsu

2016-09-13

In green plants, the blue light receptor kinase phototropin mediates various photomovements and developmental responses, such as phototropism, chloroplast photorelocation movements (accumulation and avoidance), stomatal opening, and leaf flattening, which facilitate photosynthesis. In Arabidopsis, two phototropins (phot1 and phot2) redundantly mediate these responses. Two phototropin-interacting proteins, NONPHOTOTROPIC HYPOCOTYL 3 (NPH3) and ROOT PHOTOTROPISM 2 (RPT2), which belong to the NPH3/RPT2-like (NRL) family of BTB (broad complex, tramtrack, and bric à brac) domain proteins, mediate phototropism and leaf flattening. However, the roles of NRL proteins in chloroplast photorelocation movement remain to be determined. Here, we show that another phototropin-interacting NRL protein, NRL PROTEIN FOR CHLOROPLAST MOVEMENT 1 (NCH1), and RPT2 redundantly mediate the chloroplast accumulation response but not the avoidance response. NPH3, RPT2, and NCH1 are not involved in the chloroplast avoidance response or stomatal opening. In the liverwort Marchantia polymorpha, the NCH1 ortholog, MpNCH1, is essential for the chloroplast accumulation response but not the avoidance response, indicating that the regulation of the phototropin-mediated chloroplast accumulation response by RPT2/NCH1 is conserved in land plants. Thus, the NRL protein combination could determine the specificity of diverse phototropin-mediated responses.

The chloroplast division mutant caa33 of Arabidopsis thaliana reveals a crucial impact of chloroplast homeostasis on stress acclimation and retrograde plastid-to-nucleus signaling

PubMed Central

Šimková, Klára; Kim, Chanhong; Gacek, Katarzyna; Baruah, Aiswarya; Laloi, Christophe; Apel, Klaus

2011-01-01

SUMMARY Retrograde plastid-to-nucleus signaling tightly controls and coordinates nuclear and plastid gene expression that is required for plastid biogenesis and chloroplast activities. As chloroplasts act as sensors of environmental changes, plastid-derived signaling also modulates stress responses of plants by transferring stress-related signals and altering nuclear gene expression. Various mutant screens have been undertaken to identify constituents of plastid signaling pathways. Almost all mutations identified in these screens have in common that they target plastid-specific but not extra-plastidic functions. They have been suggested to define either genuine constituents of retrograde signaling pathways or components required for the synthesis of plastid signals. Here we report the characterization of the caa33 (constitutive activator of AAA-ATPase) mutant, which reveals another way of how mutations that affect plastid functions may modulate retrograde plastid signaling. caa33 disturbs a plastid-specific function by impeding plastid division thereby perturbing plastid homeostasis. This results in pre-conditioning plants by activating the expression of stress genes, enhancing pathogen resistance and attenuating the plant’s capacity to respond to plastid signals. Our study reveals an intimate link between chloroplast activity and the plant’s susceptibility to stress and emphasizes the need to consider the possible impact of pre-conditioning on retrograde plastid-to-nucleus signaling. PMID:22014227

Abiotic stresses affect differently the intron splicing and expression of chloroplast genes in coffee plants (Coffea arabica) and rice (Oryza sativa).

PubMed

Nguyen Dinh, Sy; Sai, Than Zaw Tun; Nawaz, Ghazala; Lee, Kwanuk; Kang, Hunseung

2016-08-20

Despite the increasing understanding of the regulation of chloroplast gene expression in plants, the importance of intron splicing and processing of chloroplast RNA transcripts under stress conditions is largely unknown. Here, to understand how abiotic stresses affect the intron splicing and expression patterns of chloroplast genes in dicots and monocots, we carried out a comprehensive analysis of the intron splicing and expression patterns of chloroplast genes in the coffee plant (Coffea arabica) as a dicot and rice (Oryza sativa) as a monocot under abiotic stresses, including drought, cold, or combined drought and heat stresses. The photosynthetic activity of both coffee plants and rice seedlings was significantly reduced under all stress conditions tested. Analysis of the transcript levels of chloroplast genes revealed that the splicing of tRNAs and mRNAs in coffee plants and rice seedlings were significantly affected by abiotic stresses. Notably, abiotic stresses affected differently the splicing of chloroplast tRNAs and mRNAs in coffee plants and rice seedlings. The transcript levels of most chloroplast genes were markedly downregulated in both coffee plants and rice seedlings upon stress treatment. Taken together, these results suggest that coffee and rice plants respond to abiotic stresses via regulating the intron splicing and expression of different sets of chloroplast genes. Copyright © 2016 Elsevier GmbH. All rights reserved.

Chloroplast Ultrastructure of the Alga Phaeocystis antarctica Karsten: A New Structural Model Using Electron Tomography

NASA Technical Reports Server (NTRS)

Moisan, Tiffany A.; Ellisman, M. H.; Sosinsky, G. E.; Gerlach, John C. (Technical Monitor)

2001-01-01

Understanding the light-harvesting properties of algae and higher plants are a fundamental topic in photosynthesis research. Using thick sections obtained from fixed and embedded cultures of colonial P antarctica, we calculate tomographic reconstructions of individual chloroplasts under light-limiting and saturating conditions for net photosynthesis. Our goal is to gain an understanding of the continuity of thylakoid membranes and understand the spatial relationship between the pyrenoid, the starch containing organelle, and thylakoid membranes. We found that Phaeocystis showed considerable morphological and physiological flexibility in response to environmental light levels. We found that the thylakoids generally run parallel to the chloroplast membrane with many junctures and bifurcations, many of which are in contact with the chloroplast membrane itself. The considerable flexibility in the. thylakoid membranes allows for the accommodation of the pyrenoid structure. The arrangement of the thylakoids within these structures resemble those found in new structures of mitochondria cristae. We present a new structural model for algal chloroplasts which greatly revises current concepts of thylakoid membrane structure in relation to photoacclimation.

Tic20 forms a channel independent of Tic110 in chloroplasts

PubMed Central

2011-01-01

Background The Tic complex (Translocon at the inner envelope membrane of chloroplasts) mediates the translocation of nuclear encoded chloroplast proteins across the inner envelope membrane. Tic110 forms one prominent protein translocation channel. Additionally, Tic20, another subunit of the complex, was proposed to form a protein import channel - either together with or independent of Tic110. However, no experimental evidence for Tic20 channel activity has been provided so far. Results We performed a comprehensive biochemical and electrophysiological study to characterize Tic20 in more detail and to gain a deeper insight into its potential role in protein import into chloroplasts. Firstly, we compared transcript and protein levels of Tic20 and Tic110 in both Pisum sativum and Arabidopsis thaliana. We found the Tic20 protein to be generally less abundant, which was particularly pronounced in Arabidopsis. Secondly, we demonstrated that Tic20 forms a complex larger than 700 kilodalton in the inner envelope membrane, which is clearly separate from Tic110, migrating as a dimer at about 250 kilodalton. Thirdly, we defined the topology of Tic20 in the inner envelope, and found its N- and C-termini to be oriented towards the stromal side. Finally, we successfully reconstituted overexpressed and purified full-length Tic20 into liposomes. Using these Tic20-proteoliposomes, we could demonstrate for the first time that Tic20 can independently form a cation selective channel in vitro. Conclusions The presented data provide first biochemical evidence to the notion that Tic20 can act as a channel protein within the chloroplast import translocon complex. However, the very low abundance of Tic20 in the inner envelope membranes indicates that it cannot form a major protein translocation channel. Furthermore, the independent complex formation of Tic20 and Tic110 argues against a joint channel formation. Thus, based on the observed channel activity of Tic20 in proteoliposomes, we

A genomic approach for isolating chloroplast microsatellite markers for Pachyptera kerere (Bignoniaceae)1

PubMed Central

Francisco, Jessica N. C.; Nazareno, Alison G.; Lohmann, Lúcia G.

2016-01-01

Premise of the study: In this study, we developed chloroplast microsatellite markers (cpSSRs) for Pachyptera kerere (Bignoniaceae) to investigate the population structure and genetic diversity of this species. Methods and Results: We used Illumina HiSeq data to reconstruct the chloroplast genome of P. kerere by a combination of de novo and reference-guided assembly. We then used the chloroplast genome to develop a set of cpSSRs from intergenic regions. Overall, 24 primer pairs were designed, 21 of which amplified successfully and were polymorphic, presenting three to nine alleles per locus. The unbiased haploid diversity per locus varied from 0.207 (Pac28) to 0.817 (Pac04). All but one locus amplified for all other taxa of Pachyptera. Conclusions: The markers reported here will serve as a basis for studies to assess the genetic structure and phylogeographic history of Pachyptera. PMID:27672522

Combined effects of simulated acid rain and lanthanum chloride on chloroplast structure and functional elements in rice.

PubMed

Hu, Huiqing; Wang, Lihong; Zhou, Qing; Huang, Xiaohua

2016-05-01

Acid rain and rare earth element (REE) pollution exist simultaneously in many agricultural regions. However, how REE pollution and acid rain affect plant growth in combination remains largely unknown. In this study, the combined effects of simulated acid rain and lanthanum chloride (LaCl3) on chloroplast morphology, chloroplast ultrastructure, functional element contents, chlorophyll content, and the net photosynthetic rate (P n) in rice (Oryza sativa) were investigated by simulating acid rain and rare earth pollution. Under the combined treatment of simulated acid rain at pH 4.5 and 0.08 mM LaCl3, the chloroplast membrane was smooth, proteins on this membrane were uniform, chloroplast structure was integrated, and the thylakoids were orderly arranged, and simulated acid rain and LaCl3 exhibited a mild antagonistic effect; the Mg, Ca, Mn contents, the chlorophyll content, and the P n increased under this combined treatment, with a synergistic effect of simulated acid rain and LaCl3. Under other combined treatments of simulated acid rain and LaCl3, the chloroplast membrane surface was uneven, a clear "hole" was observed on the surface of chloroplasts, and the thylakoids were dissolved and loose; and the P n and contents of functional elements (P, Mg, K, Ca, Mn, Fe, Ni, Cu, Zn and Mo) and chlorophyll decreased. Under these combined treatments, simulated acid rain and LaCl3 exhibited a synergistic effect. Based on the above results, a model of the combined effects of simulated acid rain and LaCl3 on plant photosynthesis was established in order to reveal the combined effects on plant photosynthesis, especially on the photosynthetic organelle-chloroplast. Our results would provide some references for further understanding the mechanism of the combined effects of simulated acid rain and LaCl3 on plant photosynthesis.

Pea chloroplast DnaJ-J8 and Toc12 are encoded by the same gene and localized in the stroma.

PubMed

Chiu, Chi-Chou; Chen, Lih-Jen; Li, Hsou-min

2010-11-01

Toc12 is a novel J domain-containing protein identified in pea (Pisum sativum) chloroplasts. It was shown to be an integral outer membrane protein localizing in the intermembrane space of the chloroplast envelope. Furthermore, Toc12 was shown to associate with an intermembrane space Hsp70, suggesting that Toc12 is important for protein translocation across the chloroplast envelope. Toc12 shares a high degree of sequence similarity with Arabidopsis (Arabidopsis thaliana) DnaJ-J8, which has been suggested to be a soluble protein of the chloroplast stroma. Here, we isolated genes encoding DnaJ-J8 from pea and found that Toc12 is a truncated clone of one of the pea DnaJ-J8s. Protein import analyses indicate that Toc12 and DnaJ-J8s possess a cleavable transit peptide and are localized in the stroma. Arabidopsis mutants with T-DNA insertions in the DnaJ-J8 gene show no defect in chloroplast protein import. Implications of these results in the energetics and mechanisms of chloroplast protein import are discussed.

Temporal programming of chloroplast and cytoplasmic ribosomal RNA transcription in the synchronous cell cycle of Chlamydomonas reinhardtii

PubMed Central

1977-01-01

Approximately 90% of the Chlamydomonas reinhardtii chloroplast and cytoplasmic rRNAs was transcribed in the nuclear G1 phase, which occurred during the light period under an alternating light-dark synchronization regime of 12 h each. The remaining 10% of chloroplast and cytoplasmic rRNAs was transcribed from its respective DNAs in the dark period, in the midst of an apparent turnover of a transcription appeared to be prokaryotic in sophistication. The transcription was not interrupted during chloroplast DNA synthesis which occurred during the light period. However, transcription of the nuclear DNA was repressed severely during the nuclear S phase in the dark period. The patterns of incorporation of 32P into chloroplast and cytoplasmic rRNA species in the cell cycle were similar to those of the actual rRNA synthesis as measured optically. However, the quantity of 32P incorporation per unit amount of rRNA synthesized varied considerably during the cell cycle, increasing in all rRNA's during the dark period. 32P incorporation data obtained from continuous and pulse 32P-labeling experiments also revealed a turnover of a small amount of both cytoplasmic and chloroplast rRNAs at the end of the S phase. The 32P incorporation into cytoplasmic and chloroplast rRNAs was well matched temporally with the 32P incorporation into their corresponding ribosomes, indicating that the newly synthesized rRNA molecules are utilized without delay throughout the cell cycle in the assembly of ribosomes. PMID:833204

An Auxilin-Like J-Domain Protein, JAC1, Regulates Phototropin-Mediated Chloroplast Movement in Arabidopsis1[w

PubMed Central

Suetsugu, Noriyuki; Kagawa, Takatoshi; Wada, Masamitsu

2005-01-01

The ambient-light conditions mediate chloroplast relocation in plant cells. Under the low-light conditions, chloroplasts accumulate in the light (accumulation response), while under the high-light conditions, they avoid the light (avoidance response). In Arabidopsis (Arabidopsis thaliana), the accumulation response is mediated by two blue-light receptors, termed phototropins (phot1 and phot2) that act redundantly, and the avoidance response is mediated by phot2 alone. A mutant, J-domain protein required for chloroplast accumulation response 1 (jac1), lacks the accumulation response under weak blue light but shows a normal avoidance response under strong blue light. In dark-adapted wild-type cells, chloroplasts accumulate on the bottom of cells. Both the jac1 and phot2 mutants are defective in this chloroplast movement in darkness. Positional cloning of JAC1 reveals that this gene encodes a J-domain protein, resembling clathrin-uncoating factor auxilin at its C terminus. The amounts of JAC1 transcripts and JAC1 proteins are not regulated by light and by phototropins. A green fluorescent protein-JAC1 fusion protein showed a similar localization pattern to green fluorescent protein alone in a transient expression assay using Arabidopsis mesophyll cells and onion (Allium cepa) epidermal cells, suggesting that the JAC1 protein may be a soluble cytosolic protein. Together, these results suggest that JAC1 is an essential component of phototropin-mediated chloroplast movement. PMID:16113208

Polyglycine Acts as a Rejection Signal for Protein Transport at the Chloroplast Envelope.

PubMed

Endow, Joshua K; Rocha, Agostinho Gomes; Baldwin, Amy J; Roston, Rebecca L; Yamaguchi, Toshio; Kamikubo, Hironari; Inoue, Kentaro

2016-01-01

PolyGly is present in many proteins in various organisms. One example is found in a transmembrane β-barrel protein, translocon at the outer-envelope-membrane of chloroplasts 75 (Toc75). Toc75 requires its N-terminal extension (t75) for proper localization. t75 comprises signals for chloroplast import (n75) and envelope sorting (c75) in tandem. n75 and c75 are removed by stromal processing peptidase and plastidic type I signal peptidase 1, respectively. PolyGly is present within c75 and its deletion or substitution causes mistargeting of Toc75 to the stroma. Here we have examined the properties of polyGly-dependent protein targeting using two soluble passenger proteins, the mature portion of the small subunit of ribulose-1,5-bisphosphate carboxylase/oxygenase (mSS) and enhanced green fluorescent protein (EGFP). Both t75-mSS and t75-EGFP were imported into isolated chloroplasts and their n75 removed. Resultant c75-mSS was associated with the envelope at the intermembrane space, whereas c75-EGFP was partially exposed outside the envelope. Deletion of polyGly or substitution of tri-Ala for the critical tri-Gly segment within polyGly caused each passenger to be targeted to the stroma. Transient expression of t75-EGFP in Nicotiana benthamiana resulted in accumulation of c75-EGFP exposed at the surface of the chloroplast, but the majority of the EGFP passenger was found free in the cytosol with most of its c75 attachment removed. Results of circular dichroism analyses suggest that polyGly within c75 may form an extended conformation, which is disrupted by tri-Ala substitution. These data suggest that polyGly is distinct from a canonical stop-transfer sequence and acts as a rejection signal at the chloroplast inner envelope.

Polyglycine Acts as a Rejection Signal for Protein Transport at the Chloroplast Envelope

DOE PAGES

Endow, Joshua K.; Rocha, Agostinho Gomes; Baldwin, Amy J.; ...

2016-12-09

PolyGly is present in many proteins in various organisms. One example is found in a transmembrane β-barrel protein, translocon at the outer-envelope-membrane of chloroplasts 75 (Toc75). Toc75 requires its N-terminal extension (t75) for proper localization. t75 comprises signals for chloroplast import (n75) and envelope sorting (c75) in tandem. n75 and c75 are removed by stromal processing peptidase and plastidic type I signal peptidase 1, respectively. PolyGly is present within c75 and its deletion or substitution causes mistargeting of Toc75 to the stroma. Here in this study we have examined the properties of polyGly-dependent protein targeting using two soluble passenger proteins,more » the mature portion of the small subunit of ribulose-1,5-bisphosphate carboxylase/oxygenase (mSS) and enhanced green fluorescent protein (EGFP). Both t75-mSS and t75-EGFP were imported into isolated chloroplasts and their n75 removed. Resultant c75-mSS was associated with the envelope at the intermembrane space, whereas c75-EGFP was partially exposed outside the envelope. Deletion of polyGly or substitution of tri-Ala for the critical tri-Gly segment within polyGly caused each passenger to be targeted to the stroma. Transient expression of t75-EGFP in Nicotiana benthamiana resulted in accumulation of c75-EGFP exposed at the surface of the chloroplast, but the majority of the EGFP passenger was found free in the cytosol with most of its c75 attachment removed. Results of circular dichroism analyses suggest that polyGly within c75 may form an extended conformation, which is disrupted by tri-Ala substitution. These data suggest that polyGly is distinct from a canonical stop-transfer sequence and acts as a rejection signal at the chloroplast inner envelope.« less

Light Affects the Chloroplast Ultrastructure and Post-Storage Photosynthetic Performance of Watermelon (Citrullus lanatus) Plug Seedlings

PubMed Central

Duan, Qingqing; Jiang, Wu; Ding, Ming; Lin, Ye; Huang, Danfeng

2014-01-01

Watermelon [Citrullus lanatus (Thunb.) Matsum. and Nakai] plug seedlings were stored at 15°C in the light at a photosynthetic photon flux density of 15 µmol·m−2·s−1 or in darkness for 6 days, to evaluate their chloroplast ultrastructure, and associated photosynthetic characteristics. Storage in the dark caused swelling, disordered granal arrangement, and starch grain disappearance in the chloroplasts. In contrast, the chloroplasts stored in the light were relatively normal. As a result, the light-stored seedlings had a significantly higher chlorophyll content, Fv/Fm, and Pn than did dark-stored seedlings. Regardless of whether the seedlings were stored in light or darkness, the Gs and Ls of the seedlings significantly decreased, while the Ci obviously increased when the Pn decreased after 6 days of storage. This result suggests that the decreased Pn is not solely a stomatal effect, as the effects on the chloroplasts contributed to this photosynthetic inhibition. Six days after transplanting, seedlings that were stored in the light or darkness for 2 or 4 days showed complete recovery of chloroplast ultrastructure, chlorophyll content, Fv/Fm, Gs and Pn. When the storage period increased to 6 days, the dark-stored seedlings had a significantly lower Fv/Fm and Pn than the light-stored and control seedlings 6 days after transplanting, which was mainly ascribed to incomplete recovery of chloroplast ultrastructure. Furthermore, the light-stored seedlings exhibited a significantly higher shoot dry weight during storage and a higher percentage dry weight increase after transplanting than the dark-stored seedlings. These effects were enhanced by prolonged storage (4 to 6 days). This study demonstrated that dim light during storage is beneficial for maintaining chloroplast ultrastructure as well as photosynthetic efficiency in watermelon seedlings, thus contributing to the rapid recovery of post-storage photosynthetic performance, which ensures the transplant quality

Chloroplast DNA variation of northern red oak

Treesearch

Jeanne Romero-Severson; Preston Aldrich; Yi Feng; Weilin Sun; Charles Michler

2003-01-01

Chloroplast DNA (cpDNA) variation was examined in 48 northern red oaks at 14 sites representing contrasting glacial histories and age structures within the state of Indiana in the United States. PCR-RFLP of three intergenic regions revealed five haplotypes. Haplotype I was common to seven sites and was the most frequent (17 trees). Haplotype II was common to five sites...

Chloroplast genes transferred to the nuclear plant genome have adjusted to nuclear base composition and codon usage.

PubMed Central

Oliver, J L; Marín, A; Martínez-Zapater, J M

1990-01-01

During plant evolution, some plastid genes have been moved to the nuclear genome. These transferred genes are now correctly expressed in the nucleus, their products being transported into the chloroplast. We compared the base compositions, the distributions of some dinucleotides and codon usages of transferred, nuclear and chloroplast genes in two dicots and two monocots plant species. Our results indicate that transferred genes have adjusted to nuclear base composition and codon usage, being now more similar to the nuclear genes than to the chloroplast ones in every species analyzed. PMID:2308837

The complete chloroplast genome sequence of the medicinal plant Andrographis paniculata.

PubMed

Ding, Ping; Shao, Yanhua; Li, Qian; Gao, Junli; Zhang, Runjing; Lai, Xiaoping; Wang, Deqin; Zhang, Huiye

2016-07-01

The complete chloroplast genome of Andrographis paniculata, an important medicinal plant with great economic value, has been studied in this article. The genome size is 150,249 bp in length, with 38.3% GC content. A pair of inverted repeats (IRs, 25,300 bp) are separated by a large single copy region (LSC, 82,459 bp) and a small single-copy region (SSC, 17,190 bp). The chloroplast genome contains 114 unique genes, 80 protein-coding genes, 30 tRNA genes and 4 rRNA genes. In these genes, 15 genes contained 1 intron and 3 genes comprised of 2 introns.

An Essential Component in Chloroplast Development and Maintenance at Moderate High Temperature in Higher Plants: Chloroplast-targeted FtsH11 Proteases

USDA-ARS?s Scientific Manuscript database

Among the 12 predicted FtsH proteases in Arabidopsis, AtFtsH11 is the only metalloprotease targeting to both chloroplast and mitochondria and the only one essential for Arabidopsis plant to survive at moderate heat stress at all developmental stages. Under optimal conditions, atftsh11 mutants were...

Identification and Molecular Characterization of the Chloroplast Targeting Domain of Turnip yellow mosaic virus Replication Proteins

PubMed Central

Moriceau, Lucille; Jomat, Lucile; Bressanelli, Stéphane; Alcaide-Loridan, Catherine; Jupin, Isabelle

2017-01-01

Turnip yellow mosaic virus (TYMV) is a positive-strand RNA virus infecting plants. The TYMV 140K replication protein is a key organizer of viral replication complex (VRC) assembly, being responsible for recruitment of the viral polymerase and for targeting the VRCs to the chloroplast envelope where viral replication takes place. However, the structural requirements determining the subcellular localization and membrane association of this essential viral protein have not yet been defined. In this study, we investigated determinants for the in vivo chloroplast targeting of the TYMV 140K replication protein. Subcellular localization studies of deletion mutants identified a 41-residue internal sequence as the chloroplast targeting domain (CTD) of TYMV 140K; this sequence is sufficient to target GFP to the chloroplast envelope. The CTD appears to be located in the C-terminal extension of the methyltransferase domain—a region shared by 140K and its mature cleavage product 98K, which behaves as an integral membrane protein during infection. We predicted the CTD to fold into two amphipathic α-helices—a folding that was confirmed in vitro by circular dichroism spectroscopy analyses of a synthetic peptide. The importance for subcellular localization of the integrity of these amphipathic helices, and the function of 140K/98K, was demonstrated by performing amino acid substitutions that affected chloroplast targeting, membrane association and viral replication. These results establish a short internal α-helical peptide as an unusual signal for targeting proteins to the chloroplast envelope membrane, and provide new insights into membrane targeting of viral replication proteins—a universal feature of positive-strand RNA viruses. PMID:29312393

Studies on the System Regulating Proton Movement across the Chloroplast Envelope 1

PubMed Central

Peters, Jeanne S.; Berkowitz, Gerald A.

1991-01-01

Studies were undertaken to further characterize the spinach (Spinacea oleracea) chloroplast envelope system, which facilitates H+ movement into and out of the stroma, and, hence, modulates photosynthetic activity by regulating stromal pH. It was demonstrated that high envelope-bound Mg2+ causes stromal acidification and photosynthetic inhibition. High envelope-bound Mg2+ was also found to necessitate the activity of a digitoxinand oligomycin-sensitive ATPase for the maintenance of high stromal pH and photosynthesis in the illuminated chloroplast. In chloroplasts that had high envelope Mg2+ and inhibited envelope ATPase activity, 2-(diethylamino)-N-(2,6-dimethylphenyl)acetamide was found to raise stromal pH and stimulate photosynthesis. 2-(Diethylamino)-N-(2,6-dimethylphenyl)acetamide is an amine anesthetic that is known to act as a monovalent cation channel blocker in mammalian systems. We postulate that the system regulating cation and H+ fluxes across the plastid envelope includes a monovalent cation channel in the envelope, some degree of (envelope-bound Mg2+ modulated) H+ flux linked to monovalent cation antiport, and ATPase-dependent H+ efflux. PMID:16668116

A nuclear-encoded chloroplast protein harboring a single CRM domain plays an important role in the Arabidopsis growth and stress response.

PubMed

Lee, Kwanuk; Lee, Hwa Jung; Kim, Dong Hyun; Jeon, Young; Pai, Hyun-Sook; Kang, Hunseung

2014-04-16

Although several chloroplast RNA splicing and ribosome maturation (CRM) domain-containing proteins have been characterized for intron splicing and rRNA processing during chloroplast gene expression, the functional role of a majority of CRM domain proteins in plant growth and development as well as chloroplast RNA metabolism remains largely unknown. Here, we characterized the developmental and stress response roles of a nuclear-encoded chloroplast protein harboring a single CRM domain (At4g39040), designated CFM4, in Arabidopsis thaliana. Analysis of CFM4-GFP fusion proteins revealed that CFM4 is localized to chloroplasts. The loss-of-function T-DNA insertion mutants for CFM4 (cfm4) displayed retarded growth and delayed senescence, suggesting that CFM4 plays a role in growth and development of plants under normal growth conditions. In addition, cfm4 mutants showed retarded seed germination and seedling growth under stress conditions. No alteration in the splicing patterns of intron-containing chloroplast genes was observed in the mutant plants, but the processing of 16S and 4.5S rRNAs was abnormal in the mutant plants. Importantly, CFM4 was determined to possess RNA chaperone activity. These results suggest that the chloroplast-targeted CFM4, one of two Arabidopsis genes encoding a single CRM domain-containing protein, harbors RNA chaperone activity and plays a role in the Arabidopsis growth and stress response by affecting rRNA processing in chloroplasts.

A nuclear-encoded chloroplast protein harboring a single CRM domain plays an important role in the Arabidopsis growth and stress response

PubMed Central

2014-01-01

Background Although several chloroplast RNA splicing and ribosome maturation (CRM) domain-containing proteins have been characterized for intron splicing and rRNA processing during chloroplast gene expression, the functional role of a majority of CRM domain proteins in plant growth and development as well as chloroplast RNA metabolism remains largely unknown. Here, we characterized the developmental and stress response roles of a nuclear-encoded chloroplast protein harboring a single CRM domain (At4g39040), designated CFM4, in Arabidopsis thaliana. Results Analysis of CFM4-GFP fusion proteins revealed that CFM4 is localized to chloroplasts. The loss-of-function T-DNA insertion mutants for CFM4 (cfm4) displayed retarded growth and delayed senescence, suggesting that CFM4 plays a role in growth and development of plants under normal growth conditions. In addition, cfm4 mutants showed retarded seed germination and seedling growth under stress conditions. No alteration in the splicing patterns of intron-containing chloroplast genes was observed in the mutant plants, but the processing of 16S and 4.5S rRNAs was abnormal in the mutant plants. Importantly, CFM4 was determined to possess RNA chaperone activity. Conclusions These results suggest that the chloroplast-targeted CFM4, one of two Arabidopsis genes encoding a single CRM domain-containing protein, harbors RNA chaperone activity and plays a role in the Arabidopsis growth and stress response by affecting rRNA processing in chloroplasts. PMID:24739417

Multimodal lightsheet, structured illumination and Airyscan superresolution microscopy of chloroplast size and its impact on light propagation

USDA-ARS?s Scientific Manuscript database

Altering chloroplast size changes the way light propagates through a leaf by altering light reflectance and transmission as well as absorption by chlorophyll. Thus changing chloroplast size can used to manipulate leaf optical properties to optimize photosynthetic efficiency with the ultimate goal of...

Integrated role of ROS and Ca+2 in blue light-induced chloroplast avoidance movement in leaves of Hydrilla verticillata (L.f.) Royle.

PubMed

Majumdar, Arkajo; Kar, Rup Kumar

2016-11-01

Directional chloroplast photorelocation is a major physio-biochemical mechanism that allows these organelles to realign themselves intracellularly in response to the intensity of the incident light as an adaptive response. Signaling processes involved in blue light (BL)-dependent chloroplast movements were investigated in Hydrilla verticillata (L.f.) Royle leaves. Treatments with antagonists of actin filaments [2,3,5-triiodobenzoic acid (TIBA)] and microtubules (oryzalin) revealed that actin filaments, but not microtubules, play a pivotal role in chloroplast movement. Involvement of reactive oxygen species (ROS) in controlling chloroplast avoidance movement has been demonstrated, as exogenous H 2 O 2 not only accelerated chloroplast avoidance but also could induce chloroplast avoidance even in weak blue light (WBL). Further support came from experiments with different ROS scavengers, i.e., dimethylthiourea (DMTU), KI, and CuCl 2 , which inhibited chloroplast avoidance, and from ROS localization using specific stains. Such avoidance was also partially inhibited by ZnCl 2 , an inhibitor of NADPH oxidase (NOX) as well as 3-(3,4-dichlorophenyl)-1,1-dimethylurea (DCMU), a photosynthetic electron transport chain (ETC) inhibitor at PS II. However, methyl viologen (MV), a PS I ETC inhibitor, rather accelerated avoidance response. Exogenous calcium (Ca +2 ) induced avoidance even in WBL while inhibited chloroplast accumulation partially. On the other hand, chloroplast movements (both accumulation and avoidance) were blocked by Ca +2 antagonists, La 3+ (inhibitor of plasma membrane Ca +2 channel) and ethylene glycol-bis(2-aminoethylether)-N,N,N',N'-tetraacetic acid (EGTA, Ca +2 chelator) while LiCl that affects Ca +2 release from endosomal compartments did not show any effect. A model on integrated role of ROS and Ca +2 (influx from apolastic space) in actin-mediated chloroplast avoidance has been proposed.

Differentiation between Prototheca and morphologically similar green algae in tissue.

PubMed

Chandler, F W; Kaplan, W; Callaway, C S

1978-07-01

Evidence that algae are pathogens was provided by the results of electron microscopic studies of tissues from five cattle and sheep suspected of having green algal infections. Chloroplasts were demonstrated in the algae in each case. Prototheca organisms, considered by some to be achloric mutants of green algae, are causative agents of disease in man and animals and may appear morphologically similar to green algae in tissue. However, electron microscopy showed that chloroplasts were absent in these organisms. Light microscopy revealed not only similarities in size, shape, and mode of reproduction, but also a striking difference between the Prototheca organisms and green algae. Unlike Prototheca, the green algae contained abundant cytoplasmic starch granules that were strongly positive by several staining procedures; these granules, which were PAS-negative following diastase digestion, provide a means of differentiating green algae from Prototheca cells in tissue.

K+ Stimulation of ATPase Activity Associated with the Chloroplast Inner Envelope 1

PubMed Central

Wu, Weihua; Berkowitz, Gerald A.

1992-01-01

Studies were conducted to characterize ATPase activity associated with purified chloroplast inner envelope preparations from spinach (Spinacea oleracea L.) plants. Comparison of free Mg2+ and Mg·ATP complex effects on ATPase activity revealed that any Mg2+ stimulation of activity was likely a function of the use of the Mg·ATP complex as a substrate by the enzyme; free Mg2+ may be inhibitory. In contrast, a marked (one- to twofold) stimulation of ATPase activity was noted in the presence of K+. This stimulation had a pH optimum of approximately pH 8.0, the same pH optimum found for enzyme activity in the absence of K+. K+ stimulation of enzyme activity did not follow simple Michaelis-Menton kinetics. Rather, K+ effects were consistent with a negative cooperativity-type binding of the cation to the enzyme, with the Km increasing at increasing substrate. Of the total ATPase activity associated with the chloroplast inner envelope, the K+-stimulated component was most sensitive to the inhibitors oligomycin and vanadate. It was concluded that K+ effects on this chloroplast envelope ATPase were similar to this cation's effects on other transport ATPases (such as the plasmalemma H+-ATPase). Such ATPases are thought to be indirectly involved in active K+ uptake, which can be facilitated by ATPase-dependent generation of an electrical driving force. Thus, K+ effects on the chloroplast enzyme in vitro were found to be consistent with the hypothesized role of this envelope ATPase in facilitating active cation transport in vivo. ImagesFigure 3 PMID:16668922

Thioredoxin Selectivity for Thiol-based Redox Regulation of Target Proteins in Chloroplasts*

PubMed Central

Yoshida, Keisuke; Hara, Satoshi; Hisabori, Toru

2015-01-01

Redox regulation based on the thioredoxin (Trx) system is believed to ensure light-responsive control of various functions in chloroplasts. Five Trx subtypes have been reported to reside in chloroplasts, but their functional diversity in the redox regulation of Trx target proteins remains poorly clarified. To directly address this issue, we studied the Trx-dependent redox shifts of several chloroplast thiol-modulated enzymes in vitro and in vivo. In vitro assays using a series of Arabidopsis recombinant proteins provided new insights into Trx selectivity for the redox regulation as well as the underpinning for previous suggestions. Most notably, by combining the discrimination of thiol status with mass spectrometry and activity measurement, we identified an uncharacterized aspect of the reductive activation of NADP-malate dehydrogenase; two redox-active Cys pairs harbored in this enzyme were reduced via distinct utilization of Trxs even within a single polypeptide. In our in vitro assays, Trx-f was effective in reducing all thiol-modulated enzymes analyzed here. We then investigated the in vivo physiological relevance of these in vitro findings, using Arabidopsis wild-type and Trx-f-deficient plants. Photoreduction of fructose-1,6-bisphosphatase was partially impaired in Trx-f-deficient plants, but the global impact of Trx-f deficiency on the redox behaviors of thiol-modulated enzymes was not as striking as expected from the in vitro data. Our results provide support for the in vivo functionality of the Trx system and also highlight the complexity and plasticity of the chloroplast redox network. PMID:25878252

Chloroplast Movement May Impact Plant Phenotyping and Photochemistry Results

NASA Astrophysics Data System (ADS)

Malas, J.; Pleban, J. R.; Wang, D. R.; Riley, C.; Mackay, D. S.

2017-12-01

Investigating phenotypic responses of crop species across environmental conditions is vital to improving agricultural productivity. Crop production is closely linked with photosynthetic activity, which can be evaluated using parameters such as relative chlorophyll, SPAD, and variable chlorophyll fluorescence. Recently, a handheld device known as the MultispeQ emerged on the market as an open-source instrument that aims to provide high-output, high-quality field data at a low cost to the plant research community. MultispeQ takes measurements of both environmental conditions (light intensity, temperature, humidity, etc.) and photosynthetic parameters (relative chlorophyll, SPAD, photosystem II quantum efficiency (FII), and non-photochemical quenching (NPQ)). Data are automatically backed up and shared on the PhotosynQ network, which serves as a collaborative platform for researchers and professionals. Here, we used the instrument to quantify photosynthetic time-courses of two Brassica rapa genotypes in response to two contrasting nutrient management strategies (Control; High Nitrogen). Previous research found that chloroplast movement is one strategy plants use to optimize photosynthesis across varying light conditions. We were able to detect chloroplast movement throughout the day using the MultispeQ device. Our results support the idea that chloroplast movement serves both as an intrinsic feature of the circadian clock and as a light avoidance strategy. Under low light conditions (PAR 0-300) more light at the near-infrared and red regions was absorbed than under higher light conditions (PAR 500-800). In one genotype by treatment combination, absorbance at 730nm was around 60% at low light, versus only 30% at high light conditions. In light of our results that relative chlorophyll may change throughout a day, we suggest that it is important to take note of these effects when collecting photosynthesis efficiency data in order to avoid bias in measurements. We also

Characterization of polymorphic SSRs among Prunus chloroplast genomes

USDA-ARS?s Scientific Manuscript database

An in silico mining process yielded 80, 75, and 78 microsatellites in the chloroplast genome of Prunus persica, P. kansuensis, and P. mume. A and T repeats were predominant in the three genomes, accounting for 67.8% on average and most of them were successful in primer design. For the 80 P. persica ...

Photosynthesis-dependent formation of convoluted plasma membrane domains in Chara internodal cells is independent of chloroplast position.

PubMed

Foissner, Ilse; Sommer, Aniela; Hoeftberger, Margit

2015-07-01

The characean green alga Chara australis forms complex plasma membrane convolutions called charasomes when exposed to light. Charasomes are involved in local acidification of the surrounding medium which facilitates carbon uptake required for photosynthesis. They have hitherto been only described in the internodal cells and in close contact with the stationary chloroplasts. Here, we show that charasomes are not only present in the internodal cells of the main axis, side branches, and branchlets but that the plasma membranes of chloroplast-containing nodal cells, protonemata, and rhizoids are also able to invaginate into complex domains. Removal of chloroplasts by local irradiation with intense light revealed that charasomes can develop at chloroplast-free "windows" and that the resulting pH banding pattern is independent of chloroplast or window position. Charasomes were not detected along cell walls containing functional plasmodesmata. However, charasomes formed next to a smooth wound wall which was deposited onto the plasmodesmata-containing wall when the neighboring cell was damaged. In contrast, charasomes were rarely found at uneven, bulged wound walls which protrude into the streaming endoplasm and which were induced by ligation or puncturing. The results of this study show that charasome formation, although dependent on photosynthesis, does not require intimate contact with chloroplasts. Our data suggest further that the presence of plasmodesmata inhibits charasome formation and/or that exposure to the outer medium is a prerequisite for charasome formation. Finally, we hypothesize that the absence of charasomes at bulged wound walls is due to the disturbance of uniform laminar mass streaming.

Knocking Down of Isoprene Emission Modifies the Lipid Matrix of Thylakoid Membranes and Influences the Chloroplast Ultrastructure in Poplar1

PubMed Central

Velikova, Violeta; Müller, Constanze; Ghirardo, Andrea; Rock, Theresa Maria; Aichler, Michaela; Walch, Axel; Schmitt-Kopplin, Philippe

2015-01-01

Isoprene is a small lipophilic molecule with important functions in plant protection against abiotic stresses. Here, we studied the lipid composition of thylakoid membranes and chloroplast ultrastructure in isoprene-emitting (IE) and nonisoprene-emitting (NE) poplar (Populus × canescens). We demonstrated that the total amount of monogalactosyldiacylglycerols, digalactosyldiacylglycerols, phospholipids, and fatty acids is reduced in chloroplasts when isoprene biosynthesis is blocked. A significantly lower amount of unsaturated fatty acids, particularly linolenic acid in NE chloroplasts, was associated with the reduced fluidity of thylakoid membranes, which in turn negatively affects photosystem II photochemical efficiency. The low photosystem II photochemical efficiency in NE plants was negatively correlated with nonphotochemical quenching and the energy-dependent component of nonphotochemical quenching. Transmission electron microscopy revealed alterations in the chloroplast ultrastructure in NE compared with IE plants. NE chloroplasts were more rounded and contained fewer grana stacks and longer stroma thylakoids, more plastoglobules, and larger associative zones between chloroplasts and mitochondria. These results strongly support the idea that in IE species, the function of this molecule is closely associated with the structural organization and functioning of plastidic membranes. PMID:25975835

Complete Chloroplast Genome Sequences of Important Oilseed Crop Sesamum indicum L

PubMed Central

Yi, Dong-Keun; Kim, Ki-Joong

2012-01-01

Sesamum indicum is an important crop plant species for yielding oil. The complete chloroplast (cp) genome of S. indicum (GenBank acc no. JN637766) is 153,324 bp in length, and has a pair of inverted repeat (IR) regions consisting of 25,141 bp each. The lengths of the large single copy (LSC) and the small single copy (SSC) regions are 85,170 bp and 17,872 bp, respectively. Comparative cp DNA sequence analyses of S. indicum with other cp genomes reveal that the genome structure, gene order, gene and intron contents, AT contents, codon usage, and transcription units are similar to the typical angiosperm cp genomes. Nucleotide diversity of the IR region between Sesamum and three other cp genomes is much lower than that of the LSC and SSC regions in both the coding region and noncoding region. As a summary, the regional constraints strongly affect the sequence evolution of the cp genomes, while the functional constraints weakly affect the sequence evolution of cp genomes. Five short inversions associated with short palindromic sequences that form step-loop structures were observed in the chloroplast genome of S. indicum. Twenty-eight different simple sequence repeat loci have been detected in the chloroplast genome of S. indicum. Almost all of the SSR loci were composed of A or T, so this may also contribute to the A-T richness of the cp genome of S. indicum. Seven large repeated loci in the chloroplast genome of S. indicum were also identified and these loci are useful to developing S. indicum-specific cp genome vectors. The complete cp DNA sequences of S. indicum reported in this paper are prerequisite to modifying this important oilseed crop by cp genetic engineering techniques. PMID:22606240

Reference-Free Comparative Genomics of 174 Chloroplasts

PubMed Central

Kua, Chai-Shian; Ruan, Jue; Harting, John; Ye, Cheng-Xi; Helmus, Matthew R.; Yu, Jun; Cannon, Charles H.

2012-01-01

Direct analysis of unassembled genomic data could greatly increase the power of short read DNA sequencing technologies and allow comparative genomics of organisms without a completed reference available. Here, we compare 174 chloroplasts by analyzing the taxanomic distribution of short kmers across genomes [1]. We then assemble de novo contigs centered on informative variation. The localized de novo contigs can be separated into two major classes: tip = unique to a single genome and group = shared by a subset of genomes. Prior to assembly, we found that ∼18% of the chloroplast was duplicated in the inverted repeat (IR) region across a four-fold difference in genome sizes, from a highly reduced parasitic orchid [2] to a massive algal chloroplast [3], including gnetophytes [4] and cycads [5]. The conservation of this ratio between single copy and duplicated sequence was basal among green plants, independent of photosynthesis and mechanism of genome size change, and different in gymnosperms and lower plants. Major lineages in the angiosperm clade differed in the pattern of shared kmers and de novo contigs. For example, parasitic plants demonstrated an expected accelerated overall rate of evolution, while the hemi-parasitic genomes contained a great deal more novel sequence than holo-parasitic plants, suggesting different mechanisms at different stages of genomic contraction. Additionally, the legumes are diverging more quickly and in different ways than other major families. Small duplicated fragments of the rrn23 genes were deeply conserved among seed plants, including among several species without the IR regions, indicating a crucial functional role of this duplication. Localized de novo assembly of informative kmers greatly reduces the complexity of large comparative analyses by confining the analysis to a small partition of data and genomes relevant to the specific question, allowing direct analysis of next-gen sequence data from previously unstudied

Milestones in chloroplast genetic engineering: an environmentally friendly era in biotechnology.

PubMed

Daniell, Henry; Khan, Muhammad S; Allison, Lori

2002-02-01

Chloroplast genomes defied the laws of Mendelian inheritance at the dawn of plant genetics, and continue to defy the mainstream approach to biotechnology, leading the field in an environmentally friendly direction. Recent success in engineering the chloroplast genome for resistance to herbicides, insects, disease and drought, and for production of biopharmaceuticals, has opened the door to a new era in biotechnology. The successful engineering of tomato chromoplasts for high-level transgene expression in fruits, coupled to hyper-expression of vaccine antigens, and the use of plant-derived antibiotic-free selectable markers, augur well for oral delivery of edible vaccines and biopharmaceuticals that are currently beyond the reach of those who need them most.

The complete chloroplast genome of North American ginseng, Panax quinquefolius.

PubMed

Han, Zeng-Jie; Li, Wei; Liu, Yuan; Gao, Li-Zhi

2016-09-01

We report complete nucleotide sequence of the Panax quinquefolius chloroplast genome using next-generation sequencing technology. The genome size is 156 359 bp, including two inverted repeats (IRs) of 52 153 bp, separated by the large single-copy (LSC 86 184 bp) and small single-copy (SSC 18 081 bp) regions. This cp genome encodes 114 unigenes (80 protein-coding genes, four rRNA genes, and 30 tRNA genes), in which 18 are duplicated in the IR regions. Overall GC content of the genome is 38.08%. A phylogenomic analysis of the 10 complete chloroplast genomes from Araliaceae using Daucus carota from Apiaceae as outgroup showed that P. quinquefolius is closely related to the other two members of the genus Panax, P. ginseng and P. notoginseng.

Regulation of Chloroplastic Carbonic Anhydrase 1

PubMed Central

Porter, Michael A.; Grodzinski, Bernard

1983-01-01

It was previously reported that magnesium ion inhibited carbonic anhydrase (Bamberger and Avron 1975 Plant Physiol 56: 481-485). Studies with partially purified carbonic anhydrase from spinach (Spinacia oleracea L.) chloroplasts show that the effect was the result of the chloride counterion and not the magnesium ion. Enzyme activity was reduced 50% upon addition of 3 to 10 millimolar MgCl2 or KCl while all additions of MgSO4 between 0.3 and 10 millimolar were mildly stimulatory. PMID:16663052

Chloroplast DNA codon use: evidence for selection at the psb A locus based on tRNA availability.

PubMed

Morton, B R

1993-09-01

Codon use in the three sequenced chloroplast genomes (Marchantia, Oryza, and Nicotiana) is examined. The chloroplast has a bias in that codons NNA and NNT are favored over synonymous NNC and NNG codons. This appears to be a consequence of an overall high A + T content of the genome. This pattern of codon use is not followed by the psb A gene of all three genomes and other psb A sequences examined. In this gene, the codon use favors NNC over NNT for twofold degenerate amino acids. In each case the only tRNA coded by the genome is complementary to the NNC codon. This codon use is similar to the codon use by chloroplast genes examined from Chlamydomonas reinhardtii. Since psb A is the major translation product of the chloroplast, this suggests that selection is acting on the codon use of this gene to adapt codons to tRNA availability, as previously suggested for unicellular organisms.

Complete chloroplast DNA sequence from a Korean endemic genus, Megaleranthis saniculifolia, and its evolutionary implications.

PubMed

Kim, Young-Kyu; Park, Chong-wook; Kim, Ki-Joong

2009-03-31

The chloroplast DNA sequences of Megaleranthis saniculifolia, an endemic and monotypic endangered plant species, were completed in this study (GenBank FJ597983). The genome is 159,924 bp in length. It harbors a pair of IR regions consisting of 26,608 bp each. The lengths of the LSC and SSC regions are 88,326 bp and 18,382 bp, respectively. The structural organizations, gene and intron contents, gene orders, AT contents, codon usages, and transcription units of the Megaleranthis chloroplast genome are similar to those of typical land plant cp DNAs. However, the detailed features of Megaleranthis chloroplast genomes are substantially different from that of Ranunculus, which belongs to the same family, the Ranunculaceae. First, the Megaleranthis cp DNA was 4,797 bp longer than that of Ranunculus due to an expanded IR region into the SSC region and duplicated sequence elements in several spacer regions of the Megaleranthis cp genome. Second, the chloroplast genomes of Megaleranthis and Ranunculus evidence 5.6% sequence divergence in the coding regions, 8.9% sequence divergence in the intron regions, and 18.7% sequence divergence in the intergenic spacer regions, respectively. In both the coding and noncoding regions, average nucleotide substitution rates differed markedly, depending on the genome position. Our data strongly implicate the positional effects of the evolutionary modes of chloroplast genes. The genes evidencing higher levels of base substitutions also have higher incidences of indel mutations and low Ka/Ks ratios. A total of 54 simple sequence repeat loci were identified from the Megaleranthis cp genome. The existence of rich cp SSR loci in the Megaleranthis cp genome provides a rare opportunity to study the population genetic structures of this endangered species. Our phylogenetic trees based on the two independent markers, the nuclear ITS and chloroplast matK sequences, strongly support the inclusion of the Megaleranthis to the Trollius. Therefore, our

A Comparison of the First Two Sequenced Chloroplast Genomes in Asteraceae: Lettuce and Sunflower

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

Timme, Ruth E.; Kuehl, Jennifer V.; Boore, Jeffrey L.

2006-01-20

Asteraceae is the second largest family of plants, with over 20,000 species. For the past few decades, numerous phylogenetic studies have contributed to our understanding of the evolutionary relationships within this family, including comparisons of the fast evolving chloroplast gene, ndhF, rbcL, as well as non-coding DNA from the trnL intron plus the trnLtrnF intergenic spacer, matK, and, with lesser resolution, psbA-trnH. This culminated in a study by Panero and Funk in 2002 that used over 13,000 bp per taxon for the largest taxonomic revision of Asteraceae in over a hundred years. Still, some uncertainties remain, and it would bemore » very useful to have more information on the relative rates of sequence evolution among various genes and on genome structure as a potential set of phylogenetic characters to help guide future phylogenetic structures. By way of contributing to this, we report the first two complete chloroplast genome sequences from members of the Asteraceae, those of Helianthus annuus and Lactuca sativa. These plants belong to two distantly related subfamilies, Asteroideae and Cichorioideae, respectively. In addition to these, there is only one other published chloroplast genome sequence for any plant within the larger group called Eusterids II, that of Panax ginseng (Araliaceae, 156,318 bps, AY582139). Early chloroplast genome mapping studies demonstrated that H. annuus and L. sativa share a 22 kb inversion relative to members of the subfamily Barnadesioideae. By comparison to outgroups, this inversion was shown to be derived, indicating that the Asteroideae and Cichorioideae are more closely related than either is to the Barnadesioideae. Later sequencing study found that taxa that share this 22 kb inversion also contain within this region a second, smaller, 3.3 kb inversion. These sequences also enable an analysis of patterns of shared repeats in the genomes at fine level and of RNA editing by comparison to available EST sequences. In addition

Expression of the B subunit of E. coli heat-labile enterotoxin in the chloroplasts of plants and its characterization.

PubMed

Kang, Tae-Jin; Loc, Nguyen-Hoang; Jang, Mi-Ok; Jang, Yong-Suk; Kim, Young-Sook; Seo, Jo-Eun; Yang, Moon-Sik

2003-12-01

Transgenic chloroplasts have become attractive systems for heterologous gene expressions because of unique advantages. Here, we report a feasibility study for producing the nontoxic B subunit of Escherichia coli heat-labile enterotoxin (LTB) via chloroplast transformation of tobacco. Stable site-specific integration of the LTB gene into chloroplast genome was confirmed by PCR and genomic Southern blot analysis in transformed plants. Immunoblot analysis indicated that plant-derived LTB protein was oligomeric, and dissociated after boiling. Pentameric LTB molecules were the dominant molecular species in LTB isolated from transgenic tobacco leaf tissues. The amount of LTB protein detected in transplastomic tobacco leaf was approximately 2.5% of the total soluble plant protein, approximately 250-fold higher than in plants generated via nuclear transformation. The GM1-ELISA binding assay indicated that chloroplast-synthesized LTB protein bound to GM1-ganglioside receptors. LTB protein with biochemical properties identical to native LTB protein in the chloroplast of edible plants opens the way for inexpensive, safe, and effective plant-based edible vaccines for humans and animals.

The first complete chloroplast genome sequence of a lycophyte,Huperzia lucidula (Lycopodiaceae)

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

Wolf, Paul G.; Karol, Kenneth G.; Mandoli, Dina F.

2005-02-01

We used a unique combination of techniques to sequence the first complete chloroplast genome of a lycophyte, Huperzia lucidula. This plant belongs to a significant clade hypothesized to represent the sister group to all other vascular plants. We used fluorescence-activated cell sorting (FACS) to isolate the organelles, rolling circle amplification (RCA) to amplify the genome, and shotgun sequencing to 8x depth coverage to obtain the complete chloroplast genome sequence. The genome is 154,373bp, containing inverted repeats of 15,314 bp each, a large single-copy region of 104,088 bp, and a small single-copy region of 19,671 bp. Gene order is more similarmore » to those of mosses, liverworts, and hornworts than to gene order for other vascular plants. For example, the Huperziachloroplast genome possesses the bryophyte gene order for a previously characterized 30 kb inversion, thus supporting the hypothesis that lycophytes are sister to all other extant vascular plants. The lycophytechloroplast genome data also enable a better reconstruction of the basaltracheophyte genome, which is useful for inferring relationships among bryophyte lineages. Several unique characters are observed in Huperzia, such as movement of the gene ndhF from the small single copy region into the inverted repeat. We present several analyses of evolutionary relationships among land plants by using nucleotide data, amino acid sequences, and by comparing gene arrangements from chloroplast genomes. The results, while still tentative pending the large number of chloroplast genomes from other key lineages that are soon to be sequenced, are intriguing in themselves, and contribute to a growing comparative database of genomic and morphological data across the green plants.« less

Restriction endonuclease analysis of chloroplast DNA in interspecies somatic Hybrids of Petunia.

PubMed

Kumar, A; Cocking, E C; Bovenberg, W A; Kool, A J

1982-12-01

Restriction endonuclease cleavage pattern analysis of chloroplast DNA (cpDNA) of three different interspecific somatic hybrid plants revealed that the cytoplasms of the hybrids contained only cpDNA of P. parodii. The somatic hybrid plants analysed were those between P. parodii (wild type) + P. hybrida (wild type); P. parodii (wild type)+P. inflata (cytoplasmic albino mutant); P. parodii (wild type) + P. parviflora (nuclear albino mutant). The presence of only P. parodii chloroplasts in the somatic hybrid of P. parodii + P. inflata is possibly due to the stringent selection used for somatic hybrid production. However, in the case of the two other somatic hybrids P. parodii + P. hybrida and P. parodii + P. parviflora it was not possible to determine whether the presence of only P. parodii chloroplasts in these somatic hybrid plants was due to the nature of the selection schemes used or simply occurred by chance. The relevance of such somatic hybrid material for the study of genomic-cytoplasmic interaction is discussed, as well as the use of restriction endonuclease fragment patterns for the analysis of taxonomic and evolutionary inter-relationships in the genus Petunia.

Identification of Two Conserved Residues Involved in Copper Release from Chloroplast PIB-1-ATPases*

PubMed Central

Sautron, Emeline; Giustini, Cécile; Dang, ThuyVan; Moyet, Lucas; Salvi, Daniel; Crouzy, Serge; Rolland, Norbert; Catty, Patrice; Seigneurin-Berny, Daphné

2016-01-01

Copper is an essential transition metal for living organisms. In the plant model Arabidopsis thaliana, half of the copper content is localized in the chloroplast, and as a cofactor of plastocyanin, copper is essential for photosynthesis. Within the chloroplast, copper delivery to plastocyanin involves two transporters of the PIB-1-ATPases subfamily: HMA6 at the chloroplast envelope and HMA8 in the thylakoid membranes. Both proteins are high affinity copper transporters but share distinct enzymatic properties. In the present work, the comparison of 140 sequences of PIB-1-ATPases revealed a conserved region unusually rich in histidine and cysteine residues in the TMA-L1 region of eukaryotic chloroplast copper ATPases. To evaluate the role of these residues, we mutated them in HMA6 and HMA8. Mutants of interest were selected from phenotypic tests in yeast and produced in Lactococcus lactis for further biochemical characterizations using phosphorylation assays from ATP and Pi. Combining functional and structural data, we highlight the importance of the cysteine and the first histidine of the CX3HX2H motif in the process of copper release from HMA6 and HMA8 and propose a copper pathway through the membrane domain of these transporters. Finally, our work suggests a more general role of the histidine residue in the transport of copper by PIB-1-ATPases. PMID:27493208

An assessment of Wx microsatellite allele, alkali degradation and differentiation of chloroplast DNA in traditional black rice (Oryza sativa L.) from Thailand and Lao PDR.

PubMed

Prathepha, Preecha

2007-01-15

Thailand and Lao PDR are the country's rich rice diversity. To contribute a significant knowledge for development new rice varieties, a collection of 142 black rice (Oryza sativa) accessions were determined for variation of physico-chemical properties, Wx microsatellite allele, Wx allele and chloroplast DNA type. The results showed that amylose content of black rice accessions were ranged from 1.9 to 6.8%. All of the alkali disintegration types (high, intermediate and low) was observed in these rice with average of 1.75 on the 1-3 digestibility scale. The unique Wx microsatellite allele (CT)17 was found in these samples and all black rice strains carried Wx(b) allele. In addition, all black rice accessions were found the duplication of the 23 bp sequence motif in the exon 2 of the wx gene. This evidence is a common phenomenon in glutinous rice. Based on two growing condition for black rice, rainfed lowland and rainfed upland, chloroplast DNA type was distinct from each other. All rice strains from rainfed lowland was deletion plastotype, but all other rainfed upland strains were non-deletion types.

Transfer of the cytochrome P450-dependent dhurrin pathway from Sorghum bicolor into Nicotiana tabacum chloroplasts for light-driven synthesis

PubMed Central

Gnanasekaran, Thiyagarajan; Karcher, Daniel; Nielsen, Agnieszka Zygadlo; Martens, Helle Juel; Ruf, Stephanie; Kroop, Xenia; Olsen, Carl Erik; Motawie, Mohammed Saddik; Pribil, Mathias; Møller, Birger Lindberg; Bock, Ralph; Jensen, Poul Erik

2016-01-01

Plant chloroplasts are light-driven cell factories that have great potential to act as a chassis for metabolic engineering applications. Using plant chloroplasts, we demonstrate how photosynthetic reducing power can drive a metabolic pathway to synthesise a bio-active natural product. For this purpose, we stably engineered the dhurrin pathway from Sorghum bicolor into the chloroplasts of Nicotiana tabacum (tobacco). Dhurrin is a cyanogenic glucoside and its synthesis from the amino acid tyrosine is catalysed by two membrane-bound cytochrome P450 enzymes (CYP79A1 and CYP71E1) and a soluble glucosyltransferase (UGT85B1), and is dependent on electron transfer from a P450 oxidoreductase. The entire pathway was introduced into the chloroplast by integrating CYP79A1, CYP71E1, and UGT85B1 into a neutral site of the N. tabacum chloroplast genome. The two P450s and the UGT85B1 were functional when expressed in the chloroplasts and converted endogenous tyrosine into dhurrin using electrons derived directly from the photosynthetic electron transport chain, without the need for the presence of an NADPH-dependent P450 oxidoreductase. The dhurrin produced in the engineered plants amounted to 0.1–0.2% of leaf dry weight compared to 6% in sorghum. The results obtained pave the way for plant P450s involved in the synthesis of economically important compounds to be engineered into the thylakoid membrane of chloroplasts, and demonstrate that their full catalytic cycle can be driven directly by photosynthesis-derived electrons. PMID:26969746

A long antisense RNA in plant chloroplasts.

PubMed

Georg, J; Honsel, A; Voss, B; Rennenberg, H; Hess, W R

2010-05-01

Based on computational prediction of RNA secondary structures, a long antisense RNA (asRNA) was found in chloroplasts of Arabidopsis, Nicotiana tabacum and poplar, which occurs in two to three major transcripts. Mapping of primary 5' ends, northern hybridizations and quantitative real-time reverse transcription polymerase chain reaction (qPCR) experiments demonstrated that these transcripts originate from a promoter that is typical for the plastid-encoded RNA polymerase and are over their full length in antisense orientation to the gene ndhB and therefore were designated asRNA_ndhB. The asRNA_ndhB transcripts predominantly accumulate in young leaves and at physiological growth temperatures. Two nucleotide positions in the mRNA that are subject to C-to-U RNA editing and which were previously found to be sensitive to elevated temperatures are covered by asRNA_ndhB. Nevertheless, the correlation between the accumulation of asRNA_ndhB and RNA editing appeared weak in a temperature shift experiment. With asRNA_ndhB, we describe the first asRNA of plant chloroplasts that covers RNA editing sites, as well as a group II intron splice acceptor site, and that is under developmental control, raising the possibility that long asRNAs could be involved in RNA maturation or the control of RNA stability.

"Prokaryotic Pathway" Is Not Prokaryotic: Noncyanobacterial Origin of the Chloroplast Lipid Biosynthetic Pathway Revealed by Comprehensive Phylogenomic Analysis.

PubMed

Sato, Naoki; Awai, Koichiro

2017-11-01

Lipid biosynthesis within the chloroplast, or more generally plastids, was conventionally called "prokaryotic pathway," which produces glycerolipids bearing C18 acids at the sn-1 position and C16 acids at the sn-2 position, as in cyanobacteria such as Anabaena and Synechocystis. This positional specificity is determined during the synthesis of phosphatidate, which is a precursor to diacylglycerol, the acceptor of galactose for the synthesis of galactolipids. The first acylation at sn-1 is catalyzed by glycerol-3-phosphate acyltransferase (GPAT or GPT), whereas the second acylation at sn-2 is performed by lysophosphatidate acyltransferase (LPAAT, AGPAT, or PlsC). Here we present comprehensive phylogenomic analysis of the origins of various acyltransferases involved in the synthesis of phosphatidate, as well as phosphatidate phosphatases in the chloroplasts. The results showed that the enzymes involved in the two steps of acylation in cyanobacteria and chloroplasts are entirely phylogenetically unrelated despite a previous report stating that the chloroplast LPAAT (ATS2) and cyanobacterial PlsC were sister groups. Phosphatidate phosphatases were separated into eukaryotic and prokaryotic clades, and the chloroplast enzymes were not of cyanobacterial origin, in contrast with another previous report. These results indicate that the lipid biosynthetic pathway in the chloroplasts or plastids did not originate from the cyanobacterial endosymbiont and is not "prokaryotic" in the context of endosymbiotic theory of plastid origin. This is another line of evidence for the discontinuity of plastids and cyanobacteria, which has been suggested in the glycolipid biosynthesis. © The Author 2017. Published by Oxford University Press on behalf of the Society for Molecular Biology and Evolution.

Diversity of chloroplast genome among local clones of cocoa (Theobroma cacao, L.) from Central Sulawesi

NASA Astrophysics Data System (ADS)

Suwastika, I. Nengah; Pakawaru, Nurul Aisyah; Rifka, Rahmansyah, Muslimin, Ishizaki, Yoko; Cruz, André Freire; Basri, Zainuddin; Shiina, Takashi

2017-02-01

Chloroplast genomes typically range in size from 120 to 170 kilo base pairs (kb), which relatively conserved among plant species. Recent evaluation on several species, certain unique regions showed high variability which can be utilized in the phylogenetic analysis. Many fragments of coding regions, introns, and intergenic spacers, such as atpB-rbcL, ndhF, rbcL, rpl16, trnH-psbA, trnL-F, trnS-G, etc., have been used for phylogenetic reconstructions at various taxonomic levels. Based on that status, we would like to analysis the diversity of chloroplast genome within species of local cacao (Theobroma cacao L.) from Central Sulawesi. Our recent data showed, there were more than 20 clones from local farming in Central Sulawesi, and it can be detected based on phenotypic and nuclear-genome-based characterization (RAPD- Random Amplified Polymorphic DNA and SSR- Simple Sequences Repeat) markers. In developing DNA marker for this local cacao, here we also included analysis based on the variation of chloroplast genome. At least several regions such as rpl32-TurnL, it can be considered as chloroplast markers on our local clone of cocoa. Furthermore, we could develop phylogenetic analysis in between clones of cocoa.

[Chloroplast ultrastructure and photosynthetic characteristics of five kinds of dandelion (Taraxacum) leaves in northeast China].

PubMed

Ning, Wei; Wu, Jie; Zhao, Ting; Zhao, Xin; Li, Tianlai

2012-05-01

The paper adopted the JEM-100CX II transmission electron microscope to observe chloroplast ultrastructure of five kinds of dandelion (Taraxacum) leaves in northeast, and the LI-6400 portable photosynthesis system was used to compare the chlorophyll fluorescence and the photosynthesis characteristics of five kinds of dandelions in Northeast China. Chloroplast ultrastructure showed: in the five kinds of dandelion, larger chloroplast, grana with more layers, regular thylakoid, without starch grains and so on, these chloroplasts characteristics decided to bigger photosynthetic rate. The five kinds of dandelion P(n) exhibited a "double peak" diurnal curve: stomatal limitation is the main adjustment factors for the midday depression phenomenon. The P(n),G(s),C(i) content of T. mongolicum are the highest, and T. asiaticum are the lowest among them. The relation between P(n) and G(s),C(i) is direct ratio, P(n) and T(r) is in an inverse proportion among the five kinds of dandelion. In addition, P(n) is positively correlated with Chla, Chlb, and the relationship with Chlb is bigger. The paper demonstrates the Mongolian dandelion photosynthetic efficiency is the highest, it is an higher photosynthetic efficiency dandelion,it provide theoretical basis for assessment and use of the resource of dandelion.

Genetic diversity and differentiation in Prunus species (Rosaceae) using chloroplast and mitochondrial DNA CAPS markers.

PubMed

Ben Mustapha, S; Ben Tamarzizt, H; Baraket, G; Abdallah, D; Salhi Hannachi, A

2015-04-27

Chloroplast (cpDNA) and mitochondrial DNA (mtDNA) were analyzed to establish genetic relationships among Tunisian plum cultivars using the polymerase chain reaction restriction fragment length polymorphism (PCR-RFLP) technique. Two mtDNA regions (nad 1 b/c and nad 4 1/2) and a cpDNA region (trnL-trnF) were amplified and digested using restriction enzymes. Seventy and six polymorphic sites were revealed in cpDNA and mtDNA, respectively. As a consequence, cpDNA appears to be more polymorphic than mtDNA. The unweighted pair group method with arithmetic mean (UPGMA) dendrogram showed that accessions were distributed independently of their geographical origin, and introduced and local cultivars appear to be closely related. Both UPGMA and principal component analysis grouped Tunisian plum accessions into similar clusters. The analysis of the pooled sequences allowed the detection of 17 chlorotypes and 12 mitotypes. The unique haplotypes detected for cultivars are valuable for management and preservation of the plum local resources. From this study, PCR-RFLP analysis appears to be a useful approach to detect and identify cytoplasmic variation in plum trees. Our results also provide useful information for the management of genetic resources and to establish a program to improve the genetic resources available for plums.

Specific targeting of proteins to outer envelope membranes of endosymbiotic organelles, chloroplasts, and mitochondria

PubMed Central

Lee, Junho; Kim, Dae Heon; Hwang, Inhwan

2014-01-01

Chloroplasts and mitochondria are endosymbiotic organelles thought to be derived from endosymbiotic bacteria. In present-day eukaryotic cells, these two organelles play pivotal roles in photosynthesis and ATP production. In addition to these major activities, numerous reactions, and cellular processes that are crucial for normal cellular functions occur in chloroplasts and mitochondria. To function properly, these organelles constantly communicate with the surrounding cellular compartments. This communication includes the import of proteins, the exchange of metabolites and ions, and interactions with other organelles, all of which heavily depend on membrane proteins localized to the outer envelope membranes. Therefore, correct and efficient targeting of these membrane proteins, which are encoded by the nuclear genome and translated in the cytosol, is critically important for organellar function. In this review, we summarize the current knowledge of the mechanisms of protein targeting to the outer membranes of mitochondria and chloroplasts in two different directions, as well as targeting signals and cytosolic factors. PMID:24808904

Coherent X-Ray Diffraction Imaging of Chloroplasts from Cyanidioschyzon merolae by Using X-Ray Free Electron Laser.

PubMed

Takayama, Yuki; Inui, Yayoi; Sekiguchi, Yuki; Kobayashi, Amane; Oroguchi, Tomotaka; Yamamoto, Masaki; Matsunaga, Sachihiro; Nakasako, Masayoshi

2015-07-01

Coherent X-ray diffraction imaging (CXDI) is a lens-less technique for visualizing the structures of non-crystalline particles with the dimensions of submicrometer to micrometer at a resolution of several tens of nanometers. We conducted cryogenic CXDI experiments at 66 K to visualize the internal structures of frozen-hydrated chloroplasts of Cyanidioschyzon merolae using X-ray free electron laser (XFEL) as a coherent X-ray source. Chloroplast dispersed specimen disks at a number density of 7/(10×10 µm(2)) were flash-cooled with liquid ethane without staining, sectioning or chemical labeling. Chloroplasts are destroyed at atomic level immediately after the diffraction by XFEL pulses. Thus, diffraction patterns with a good signal-to-noise ratio from single chloroplasts were selected from many diffraction patterns collected through scanning specimen disks to provide fresh specimens into the irradiation area. The electron density maps of single chloroplasts projected along the direction of the incident X-ray beam were reconstructed by using the iterative phase-retrieval method and multivariate analyses. The electron density map at a resolution of 70 nm appeared as a C-shape. In addition, the fluorescence image of proteins stained with Flamingo™ dye also appeared as a C-shape as did the autofluorescence from Chl. The similar images suggest that the thylakoid membranes with an abundance of proteins distribute along the outer membranes of chloroplasts. To confirm the present results statistically, a number of projection structures must be accumulated through high-throughput data collection in the near future. Based on the results, we discuss the feasibility of XFEL-CXDI experiments in the structural analyses of cellular organelles. © The Author 2015. Published by Oxford University Press on behalf of Japanese Society of Plant Physiologists. All rights reserved. For permissions, please email: journals.permissions@oup.com.

Large Diversity of Nonstandard Genes and Dynamic Evolution of Chloroplast Genomes in Siphonous Green Algae (Bryopsidales, Chlorophyta)

PubMed Central

Leliaert, Frederik; Marcelino, Vanessa R

2018-01-01

Abstract Chloroplast genomes have undergone tremendous alterations through the evolutionary history of the green algae (Chloroplastida). This study focuses on the evolution of chloroplast genomes in the siphonous green algae (order Bryopsidales). We present five new chloroplast genomes, which along with existing sequences, yield a data set representing all but one families of the order. Using comparative phylogenetic methods, we investigated the evolutionary dynamics of genomic features in the order. Our results show extensive variation in chloroplast genome architecture and intron content. Variation in genome size is accounted for by the amount of intergenic space and freestanding open reading frames that do not show significant homology to standard plastid genes. We show the diversity of these nonstandard genes based on their conserved protein domains, which are often associated with mobile functions (reverse transcriptase/intron maturase, integrases, phage- or plasmid-DNA primases, transposases, integrases, ligases). Investigation of the introns showed proliferation of group II introns in the early evolution of the order and their subsequent loss in the core Halimedineae, possibly through RT-mediated intron loss. PMID:29635329

Vaccination via Chloroplast Genetics: Affordable Protein Drugs for the Prevention and Treatment of Inherited or Infectious Human Diseases

PubMed Central

Daniell, Henry; Chan, Hui-Ting; Pasoreck, Elise K.

2017-01-01

Plastid-made biopharmaceuticals treat major metabolic or genetic disorders, including Alzheimer’s, diabetes, hypertension, hemophilia, and retinopathy. Booster vaccines made in chloroplasts prevent global infectious diseases, such as tuberculosis, malaria, cholera, and polio, and biological threats, such as anthrax and plague. Recent advances in this field include commercial-scale production of human therapeutic proteins in FDA-approved cGMP facilities, development of tags to deliver protein drugs to targeted human cells or tissues, methods to deliver precise doses, and long-term stability of protein drugs at ambient temperature, maintaining their efficacy. Codon optimization utilizing valuable information from sequenced chloroplast genomes enhanced expression of eukaryotic human or viral genes in chloroplasts and offered unique insights into translation in chloroplasts. Support from major biopharmaceutical companies, development of hydroponic production systems, and evaluation by regulatory agencies, including the CDC, FDA, and USDA, augur well for advancing this novel concept to the clinic and revolutionizing affordable healthcare. PMID:27893966

Arabidopsis nph1 and npl1: blue light receptors that mediate both phototropism and chloroplast relocation.

PubMed

Sakai, T; Kagawa, T; Kasahara, M; Swartz, T E; Christie, J M; Briggs, W R; Wada, M; Okada, K

2001-06-05

UV-A/blue light acts to regulate a number of physiological processes in higher plants. These include light-driven chloroplast movement and phototropism. The NPH1 gene of Arabidopsis encodes an autophosphorylating protein kinase that functions as a photoreceptor for phototropism in response to low-intensity blue light. However, nph1 mutants have been reported to exhibit normal phototropic curvature under high-intensity blue light, indicating the presence of an additional phototropic receptor. A likely candidate is the nph1 homologue, npl1, which has recently been shown to mediate the avoidance response of chloroplasts to high-intensity blue light in Arabidopsis. Here we demonstrate that npl1, like nph1, noncovalently binds the chromophore flavin mononucleotide (FMN) within two specialized PAS domains, termed LOV domains. Furthermore, when expressed in insect cells, npl1, like nph1, undergoes light-dependent autophosphorylation, indicating that npl1 also functions as a light receptor kinase. Consistent with this conclusion, we show that a nph1 npl1 double mutant exhibits an impaired phototropic response under both low- and high-intensity blue light. Hence, npl1 functions as a second phototropic receptor under high fluence rate conditions and is, in part, functionally redundant to nph1. We also demonstrate that both chloroplast accumulation in response to low-intensity light and chloroplast avoidance movement in response to high-intensity light are lacking in the nph1 npl1 double mutant. Our findings therefore indicate that nph1 and npl1 show partially overlapping functions in two different responses, phototropism and chloroplast relocation, in a fluence rate-dependent manner.

The complete chloroplast genome sequence of Epipremnum aureum and its comparative analysis among eight Araceae species

PubMed Central

Han, Limin; Chen, Chen; Wang, Zhezhi

2018-01-01

Epipremnum aureum is an important foliage plant in the Araceae family. In this study, we have sequenced the complete chloroplast genome of E. aureum by using Illumina Hiseq sequencing platforms. This genome is a double-stranded circular DNA sequence of 164,831 bp that contains 35.8% GC. The two inverted repeats (IRa and IRb; 26,606 bp) are spaced by a small single-copy region (22,868 bp) and a large single-copy region (88,751 bp). The chloroplast genome has 131 (113 unique) functional genes, including 86 (79 unique) protein-coding genes, 37 (30 unique) tRNA genes, and eight (four unique) rRNA genes. Tandem repeats comprise the majority of the 43 long repetitive sequences. In addition, 111 simple sequence repeats are present, with mononucleotides being the most common type and di- and tetranucleotides being infrequent events. Positive selection pressure on rps12 in the E. aureum chloroplast has been demonstrated via synonymous and nonsynonymous substitution rates and selection pressure sites analyses. Ycf15 and infA are pseudogenes in this species. We constructed a Maximum Likelihood phylogenetic tree based on the complete chloroplast genomes of 38 species from 13 families. Those results strongly indicated that E. aureum is positioned as the sister of Colocasia esculenta within the Araceae family. This work may provide information for further study of the molecular phylogenetic relationships within Araceae, as well as molecular markers and breeding novel varieties by chloroplast genetic-transformation of E. aureum in particular. PMID:29529038

Effects of plant density on the photosynthetic and chloroplast characteristics of maize under high-yielding conditions

NASA Astrophysics Data System (ADS)

Ren, Baizhao; Liu, Wei; Zhang, Jiwang; Dong, Shuting; Liu, Peng; Zhao, Bin

2017-04-01

Plant density has been recognized as a major factor determining the grain yield. The photosynthetic performance changes as the density increases. The main objective of this research was to evaluate responses of photosynthetic performance and chloroplast ultrastructure to planting densities in two summer maize ( Zea mays L.) hybrids Denghai661 (DH661) and Nongda108 (ND108). DH661 was planted at densities of 30,000, 45,000, 60,000, 75,000, 90,000, 105,000, 120,000, or 135,000 plants ha-1. ND108 was planted at densities of 30,000, 45,000, 60,000, 75,000, or 90,000 plants ha-1. Research variables included leaf area, grain yield, chlorophyll content, leaf gas exchange parameters, number of chloroplasts, and chloroplast ultrastructure. As plant density increased, chlorophyll a and b content significantly decreased; carotenoids initially decreased and then increased; the net photosynthetic rate during each growth period significantly decreased; the membrane structure of mesophyll cells was gradually damaged; the number of chloroplasts significantly decreased; the external form of chloroplasts shifted from long and oval to elliptical or circular; the number of grana significantly decreased, while the number of grana lamellae increased; grana gradually became hypogenetic and eventually dissolved; plot yield increased; and yield per plant significantly decreased. The yield per plant of DH661 at 135,000 plants ha-1 and that of ND108 at 90,000 plants ha-1 decreased by 65.8 and 42.5%, respectively, compared with those at 30,000 plants ha-1.

Photosynthetic Characteristics and Chloroplast Ultrastructure of Summer Maize Response to Different Nitrogen Supplies

PubMed Central

Liu, Zheng; Gao, Jia; Gao, Fei; Liu, Peng; Zhao, Bin; Zhang, Jiwang

2018-01-01

Maize (Zea mays L.) is the important crop over the world. Nitrogen (N) as necessary element affects photosynthetic characteristics and grain yield of summer maize. In this study, N0 (0 kg N ha-1), N1 (129 kg N ha-1), N2 (185 kg N ha-1), and N3 (300 kg N ha-1) was conducted using hybrid ‘ZhengDan958’ at Dawenkou research field (36°11′N, 117°06′E, 178 m altitude) in the North China Plain to explore the effects of N rate on photosynthetic characteristics and chloroplast ultrastructure. Gas exchange parameters, chlorophyll fluorescence parameters, leaf area index (LAI), chlorophyll SPAD value, chloroplast ultrastructure, dry matter weight and grain yield were measured. At physiological maturity stage, dry matter weight and grain yield of N2 increased by 33–52% (P ≤ 0.05) and 6–32% (P ≤ 0.05), respectively, compared with other treatments. During the growing from silking (R1) to milk (R3) stage, LAI of N0 and N1 were 35–38% (P ≤ 0.05) and 9–23% (P ≤ 0.05) less than that of N2, respectively. Chlorophyll SPAD value of N0 and N1 were 13–22% (P ≤ 0.05) and 5–11% (P ≤ 0.05) lower than that of N2. There was no significant difference in LAI and chlorophyll SPAD value between N2 and N3 during the period from R1 to R3 (P > 0.05). The net photosynthetic rate (Pn), maximal quantum efficiency of PSII (Fv/Fm) and quantum efficiency of PSII (ΦPSII) were higher with the increase of N rate up to N2 (P ≤ 0.05), and those of N3 were significantly less than N2 (P ≤ 0.05). In compared with N2, the chloroplast configuration of N0 and N1 became elliptical, almost circular or irregular. The membrane of chloroplast and thylakoid resolved with growing stage, and the number of chloroplast per cell and lamellae per grana decreased under N0 and N1 treatment (P ≤ 0.05). Under N0 and N1 treatments, summer maize had more negative photosynthetic characteristics. The more number of osmium granule and vesicle and the larger gap between lamellae were shown in N3

Photosynthetic Characteristics and Chloroplast Ultrastructure of Summer Maize Response to Different Nitrogen Supplies.

PubMed

Liu, Zheng; Gao, Jia; Gao, Fei; Liu, Peng; Zhao, Bin; Zhang, Jiwang

2018-01-01

Maize ( Zea mays L.) is the important crop over the world. Nitrogen (N) as necessary element affects photosynthetic characteristics and grain yield of summer maize. In this study, N0 (0 kg N ha -1 ), N1 (129 kg N ha -1 ), N2 (185 kg N ha -1 ), and N3 (300 kg N ha -1 ) was conducted using hybrid 'ZhengDan958' at Dawenkou research field (36°11'N, 117°06'E, 178 m altitude) in the North China Plain to explore the effects of N rate on photosynthetic characteristics and chloroplast ultrastructure. Gas exchange parameters, chlorophyll fluorescence parameters, leaf area index (LAI), chlorophyll SPAD value, chloroplast ultrastructure, dry matter weight and grain yield were measured. At physiological maturity stage, dry matter weight and grain yield of N2 increased by 33-52% ( P ≤ 0.05) and 6-32% ( P ≤ 0.05), respectively, compared with other treatments. During the growing from silking (R1) to milk (R3) stage, LAI of N0 and N1 were 35-38% ( P ≤ 0.05) and 9-23% ( P ≤ 0.05) less than that of N2, respectively. Chlorophyll SPAD value of N0 and N1 were 13-22% ( P ≤ 0.05) and 5-11% ( P ≤ 0.05) lower than that of N2. There was no significant difference in LAI and chlorophyll SPAD value between N2 and N3 during the period from R1 to R3 ( P > 0.05). The net photosynthetic rate ( P n ), maximal quantum efficiency of PSII ( F v / F m ) and quantum efficiency of PSII (Φ PSII ) were higher with the increase of N rate up to N2 ( P ≤ 0.05), and those of N3 were significantly less than N2 ( P ≤ 0.05). In compared with N2, the chloroplast configuration of N0 and N1 became elliptical, almost circular or irregular. The membrane of chloroplast and thylakoid resolved with growing stage, and the number of chloroplast per cell and lamellae per grana decreased under N0 and N1 treatment ( P ≤ 0.05). Under N0 and N1 treatments, summer maize had more negative photosynthetic characteristics. The more number of osmium granule and vesicle and the larger gap between lamellae were

The role of heterologous chloroplast sequence elements in transgene integration and expression.

PubMed

Ruhlman, Tracey; Verma, Dheeraj; Samson, Nalapalli; Daniell, Henry

2010-04-01

Heterologous regulatory elements and flanking sequences have been used in chloroplast transformation of several crop species, but their roles and mechanisms have not yet been investigated. Nucleotide sequence identity in the photosystem II protein D1 (psbA) upstream region is 59% across all taxa; similar variation was consistent across all genes and taxa examined. Secondary structure and predicted Gibbs free energy values of the psbA 5' untranslated region (UTR) among different families reflected this variation. Therefore, chloroplast transformation vectors were made for tobacco (Nicotiana tabacum) and lettuce (Lactuca sativa), with endogenous (Nt-Nt, Ls-Ls) or heterologous (Nt-Ls, Ls-Nt) psbA promoter, 5' UTR and 3' UTR, regulating expression of the anthrax protective antigen (PA) or human proinsulin (Pins) fused with the cholera toxin B-subunit (CTB). Unique lettuce flanking sequences were completely eliminated during homologous recombination in the transplastomic tobacco genomes but not unique tobacco sequences. Nt-Ls or Ls-Nt transplastomic lines showed reduction of 80% PA and 97% CTB-Pins expression when compared with endogenous psbA regulatory elements, which accumulated up to 29.6% total soluble protein PA and 72.0% total leaf protein CTB-Pins, 2-fold higher than Rubisco. Transgene transcripts were reduced by 84% in Ls-Nt-CTB-Pins and by 72% in Nt-Ls-PA lines. Transcripts containing endogenous 5' UTR were stabilized in nonpolysomal fractions. Stromal RNA-binding proteins were preferentially associated with endogenous psbA 5' UTR. A rapid and reproducible regeneration system was developed for lettuce commercial cultivars by optimizing plant growth regulators. These findings underscore the need for sequencing complete crop chloroplast genomes, utilization of endogenous regulatory elements and flanking sequences, as well as optimization of plant growth regulators for efficient chloroplast transformation.

Chloroplastic and cytoplasmic overexpression of sheep serotonin N-acetyltransferase in transgenic rice plants is associated with low melatonin production despite high enzyme activity.

PubMed

Byeon, Yeong; Lee, Hyoung Yool; Back, Kyoungwhan

2015-05-01

Serotonin N-acetyltransferase (SNAT), the penultimate enzyme in melatonin biosynthesis, catalyzes the conversion of serotonin into N-acetylserotonin. Plant SNAT is localized in chloroplasts. To test SNAT localization effects on melatonin synthesis, we generated transgenic rice plants overexpressing a sheep (Ovis aries) SNAT (OaSNAT) in their chloroplasts and compared melatonin biosynthesis with that of transgenic rice plants overexpressing OaSNAT in their cytoplasm. To localize the OaSNAT in chloroplasts, we used a chloroplast targeting sequence (CTS) from tobacco protoporphyrinogen IX oxidase (PPO), which expresses in chloroplasts. The purified recombinant CTS:OaSNAT fusion protein was enzymatically functional and localized in chloroplasts as confirmed by confocal microscopic analysis. The chloroplast-targeted CTS:OaSNAT lines and cytoplasm-expressed OaSNAT lines had similarly high SNAT enzyme activities. However, after cadmium and butafenacil treatments, melatonin production in rice leaves was severalfold lower in the CTS:OaSNAT lines than in the OaSNAT lines. Notably, enhanced SNAT enzyme activity was not directly proportional to the production of N-acetylserotonin, melatonin, or 2-hydroxymelatonin, suggesting that plant SNAT has a role in the homeostatic regulation of melatonin rather than in accelerating melatonin synthesis. © 2015 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.

Transfer of the cytochrome P450-dependent dhurrin pathway from Sorghum bicolor into Nicotiana tabacum chloroplasts for light-driven synthesis.

PubMed

Gnanasekaran, Thiyagarajan; Karcher, Daniel; Nielsen, Agnieszka Zygadlo; Martens, Helle Juel; Ruf, Stephanie; Kroop, Xenia; Olsen, Carl Erik; Motawie, Mohammed Saddik; Pribil, Mathias; Møller, Birger Lindberg; Bock, Ralph; Jensen, Poul Erik

2016-04-01

Plant chloroplasts are light-driven cell factories that have great potential to act as a chassis for metabolic engineering applications. Using plant chloroplasts, we demonstrate how photosynthetic reducing power can drive a metabolic pathway to synthesise a bio-active natural product. For this purpose, we stably engineered the dhurrin pathway from Sorghum bicolor into the chloroplasts of Nicotiana tabacum (tobacco). Dhurrin is a cyanogenic glucoside and its synthesis from the amino acid tyrosine is catalysed by two membrane-bound cytochrome P450 enzymes (CYP79A1 and CYP71E1) and a soluble glucosyltransferase (UGT85B1), and is dependent on electron transfer from a P450 oxidoreductase. The entire pathway was introduced into the chloroplast by integrating CYP79A1, CYP71E1, and UGT85B1 into a neutral site of the N. tabacum chloroplast genome. The two P450s and the UGT85B1 were functional when expressed in the chloroplasts and converted endogenous tyrosine into dhurrin using electrons derived directly from the photosynthetic electron transport chain, without the need for the presence of an NADPH-dependent P450 oxidoreductase. The dhurrin produced in the engineered plants amounted to 0.1-0.2% of leaf dry weight compared to 6% in sorghum. The results obtained pave the way for plant P450s involved in the synthesis of economically important compounds to be engineered into the thylakoid membrane of chloroplasts, and demonstrate that their full catalytic cycle can be driven directly by photosynthesis-derived electrons. © The Author 2016. Published by Oxford University Press on behalf of the Society for Experimental Biology.

Analysis of Complete Nucleotide Sequences of 12 Gossypium Chloroplast Genomes: Origin and Evolution of Allotetraploids

PubMed Central

Xu, Qin; Xiong, Guanjun; Li, Pengbo; He, Fei; Huang, Yi; Wang, Kunbo; Li, Zhaohu; Hua, Jinping

2012-01-01

Background Cotton (Gossypium spp.) is a model system for the analysis of polyploidization. Although ascertaining the donor species of allotetraploid cotton has been intensively studied, sequence comparison of Gossypium chloroplast genomes is still of interest to understand the mechanisms underlining the evolution of Gossypium allotetraploids, while it is generally accepted that the parents were A- and D-genome containing species. Here we performed a comparative analysis of 13 Gossypium chloroplast genomes, twelve of which are presented here for the first time. Methodology/Principal Findings The size of 12 chloroplast genomes under study varied from 159,959 bp to 160,433 bp. The chromosomes were highly similar having >98% sequence identity. They encoded the same set of 112 unique genes which occurred in a uniform order with only slightly different boundary junctions. Divergence due to indels as well as substitutions was examined separately for genome, coding and noncoding sequences. The genome divergence was estimated as 0.374% to 0.583% between allotetraploid species and A-genome, and 0.159% to 0.454% within allotetraploids. Forty protein-coding genes were completely identical at the protein level, and 20 intergenic sequences were completely conserved. The 9 allotetraploids shared 5 insertions and 9 deletions in whole genome, and 7-bp substitutions in protein-coding genes. The phylogenetic tree confirmed a close relationship between allotetraploids and the ancestor of A-genome, and the allotetraploids were divided into four separate groups. Progenitor allotetraploid cotton originated 0.43–0.68 million years ago (MYA). Conclusion Despite high degree of conservation between the Gossypium chloroplast genomes, sequence variations among species could still be detected. Gossypium chloroplast genomes preferred for 5-bp indels and 1–3-bp indels are mainly attributed to the SSR polymorphisms. This study supports that the common ancestor of diploid A-genome species in

The complete chloroplast genome sequence of Gossypium hirsutum: organization and phylogenetic relationships to other angiosperms

PubMed Central

Lee, Seung-Bum; Kaittanis, Charalambos; Jansen, Robert K; Hostetler, Jessica B; Tallon, Luke J; Town, Christopher D; Daniell, Henry

2006-01-01

Background Cotton (Gossypium hirsutum) is the most important fiber crop grown in 90 countries. In 2004–2005, US farmers planted 79% of the 5.7-million hectares of nuclear transgenic cotton. Unfortunately, genetically modified cotton has the potential to hybridize with other cultivated and wild relatives, resulting in geographical restrictions to cultivation. However, chloroplast genetic engineering offers the possibility of containment because of maternal inheritance of transgenes. The complete chloroplast genome of cotton provides essential information required for genetic engineering. In addition, the sequence data were used to assess phylogenetic relationships among the major clades of rosids using cotton and 25 other completely sequenced angiosperm chloroplast genomes. Results The complete cotton chloroplast genome is 160,301 bp in length, with 112 unique genes and 19 duplicated genes within the IR, containing a total of 131 genes. There are four ribosomal RNAs, 30 distinct tRNA genes and 17 intron-containing genes. The gene order in cotton is identical to that of tobacco but lacks rpl22 and infA. There are 30 direct and 24 inverted repeats 30 bp or longer with a sequence identity ≥ 90%. Most of the direct repeats are within intergenic spacer regions, introns and a 72 bp-long direct repeat is within the psaA and psaB genes. Comparison of protein coding sequences with expressed sequence tags (ESTs) revealed nucleotide substitutions resulting in amino acid changes in ndhC, rpl23, rpl20, rps3 and clpP. Phylogenetic analysis of a data set including 61 protein-coding genes using both maximum likelihood and maximum parsimony were performed for 28 taxa, including cotton and five other angiosperm chloroplast genomes that were not included in any previous phylogenies. Conclusion Cotton chloroplast genome lacks rpl22 and infA and contains a number of dispersed direct and inverted repeats. RNA editing resulted in amino acid changes with significant impact on their

Chloroplast microsatellite markers for Artocarpus (Moraceae) developed from transcriptome sequences

USDA-ARS?s Scientific Manuscript database

Premise of the study: Chloroplast microsatellite loci were characterized from transcriptomes of Artocarpus (A.) altilis (breadfruit) and A. camansi (breadnut). They were tested in A. odoratissimus (terap) and A. altilis and evaluated in silico for two congeners. Methods and Results: 15 simple seque...

Learning the Languages of the Chloroplast: Retrograde Signaling and Beyond.

PubMed

Chan, Kai Xun; Phua, Su Yin; Crisp, Peter; McQuinn, Ryan; Pogson, Barry J

2016-04-29

The chloroplast can act as an environmental sensor, communicating with the cell during biogenesis and operation to change the expression of thousands of proteins. This process, termed retrograde signaling, regulates expression in response to developmental cues and stresses that affect photosynthesis and yield. Recent advances have identified many signals and pathways-including carotenoid derivatives, isoprenes, phosphoadenosines, tetrapyrroles, and heme, together with reactive oxygen species and proteins-that build a communication network to regulate gene expression, RNA turnover, and splicing. However, retrograde signaling pathways have been viewed largely as a means of bilateral communication between organelles and nuclei, ignoring their potential to interact with hormone signaling and the cell as a whole to regulate plant form and function. Here, we discuss new findings on the processes by which organelle communication is initiated, transmitted, and perceived, not only to regulate chloroplastic processes but also to intersect with cellular signaling and alter physiological responses.

Modified Clp Protease Complex in the ClpP3 Null Mutant and Consequences for Chloroplast Development and Function in Arabidopsis1[C][W][OA

PubMed Central

Kim, Jitae; Olinares, Paul Dominic; Oh, Soo-hyun; Ghisaura, Stefania; Poliakov, Anton; Ponnala, Lalit; van Wijk, Klaas J.

2013-01-01

The plastid ClpPRT protease consists of two heptameric rings of ClpP1/ClpR1/ClpR2/ClpR3/ClpR4 (the R-ring) and ClpP3/ClpP4/ClpP5/ClpP6 (the P-ring) and peripherally associated ClpT1/ClpT2 subunits. Here, we address the contributions of ClpP3 and ClpP4 to ClpPRT core organization and function in Arabidopsis (Arabidopsis thaliana). ClpP4 is strictly required for embryogenesis, similar to ClpP5. In contrast, loss of ClpP3 (clpp3-1) leads to arrest at the hypocotyl stage; this developmental arrest can be removed by supplementation with sucrose or glucose. Heterotrophically grown clpp3-1 can be transferred to soil and generate viable seed, which is surprising, since we previously showed that CLPR2 and CLPR4 null alleles are always sterile and die on soil. Based on native gels and mass spectrometry-based quantification, we show that despite the loss of ClpP3, modified ClpPR core(s) could be formed, albeit at strongly reduced levels. A large portion of ClpPR subunits accumulated in heptameric rings, with overaccumulation of ClpP1/ClpP5/ClpP6 and ClpR3. Remarkably, the association of ClpT1 to the modified Clp core was unchanged. Large-scale quantitative proteomics assays of clpp3-1 showed a 50% loss of photosynthetic capacity and the up-regulation of plastoglobules and all chloroplast stromal chaperone systems. Specific chloroplast proteases were significantly up-regulated, whereas the major thylakoid protease (FtsH1/FtsH2/FtsH5/FtsH8) was clearly unchanged, indicating a controlled protease network response. clpp3-1 showed a systematic decrease of chloroplast-encoded proteins that are part of the photosynthetic apparatus but not of chloroplast-encoded proteins with other functions. Candidate substrates and an explanation for the differential phenotypes between the CLPP3, CLPP4, and CLPP5 null mutants are discussed. PMID:23548781

Complete Chloroplast Genome Sequence of Coptis chinensis Franch. and Its Evolutionary History

PubMed Central

He, Yang; Deng, Cao; Fan, Gang; Qin, Shishang

2017-01-01

The Coptis chinensis Franch. is an important medicinal plant from the Ranunculales. We used next generation sequencing technology to determine the complete chloroplast genome of C. chinensis. This genome is 155,484 bp long with 38.17% GC content. Two 26,758 bp long inverted repeats separated the genome into a typical quadripartite structure. The C. chinensis chloroplast genome consists of 128 gene loci, including eight rRNA gene loci, 28 tRNA gene loci, and 92 protein-coding gene loci. Most of the SSRs in C. chinensis are poly-A/T. The numbers of mononucleotide SSRs in C. chinensis and other Ranunculaceae species are fewer than those in Berberidaceae species, while the number of dinucleotide SSRs is greater than that in the Berberidaceae. C. chinensis diverged from other Ranunculaceae species an estimated 81 million years ago (Mya). The divergence between Ranunculaceae and Berberidaceae was ~111 Mya, while the Ranunculales and Magnoliaceae shared a common ancestor during the Jurassic, ~153 Mya. Position 104 of the C. chinensis ndhG protein was identified as a positively selected site, indicating possible selection for the photosystem-chlororespiration system in C. chinensis. In summary, the complete sequencing and annotation of the C. chinensis chloroplast genome will facilitate future studies on this important medicinal species. PMID:28698879

Genetic structure of Eurasian and North American Leymus (Triticeae) wildryes assessed by chloroplast DNA sequences and AFLP profiles

Treesearch

C. Mae Culumber; Steve R. Larson; Kevin B. Jensen; Thomas A. Jones

2011-01-01

Leymus is a genomically defined allopolyploid of genus Triticeae with two distinct subgenomes. Chloroplast DNA sequences of Eurasian and North American species are distinct and polyphyletic. However, phylogenies derived from chloroplast and nuclear DNA sequences are confounded by polyploidy and lack of polymorphism among many taxa. The AFLP technique can resolve...

Identification of Highly Divergent Diatom-Derived Chloroplasts in Dinoflagellates, Including a Description of Durinskia kwazulunatalensis sp. nov. (Peridiniales, Dinophyceae).

PubMed

Yamada, Norico; Sym, Stuart D; Horiguchi, Takeo

2017-06-01

Dinoflagellates are known to possess chloroplasts of multiple origins derived from a red alga, a green alga, haptophytes, or diatoms. The monophyletic "dinotoms" harbor a chloroplast of diatom origin, but their chloroplasts are polyphyletic belonging to one of four genera: Chaetoceros, Cyclotella, Discostella, or Nitzschia. It has been speculated that serial replacement of diatom-derived chloroplasts by other diatoms has caused this diversity of chloroplasts. Although previous work suggested that the endosymbionts of Nitzschia origin might not be monophyletic, this has not been seriously investigated. To infer the number of replacements of diatom-derived chloroplasts in dinotoms, we analyzed the phylogenetic affinities of 14 species of dinotoms based on the endosymbiotic rbcL gene and SSU rDNA, and the host SSU rDNA. Resultant phylogenetic trees revealed that six species of Nitzschia were taken up by eight marine dinoflagellate species. Our phylogenies also indicate that four separate diatom species belonging to three genera were incorporated into the five freshwater dinotoms. Particular attention was paid to two crucially closely related species, Durinskia capensis and a novel species, D. kwazulunatalensis, because they possess distantly related Nitzschia species. This study clarified that any of a total of at least 11 diatom species in five genera are employed as an endosymbiont by 14 dinotoms, which infers a more frequent replacement of endosymbionts in the world of dinotoms than previously envisaged. © The Author 2017. Published by Oxford University Press on behalf of the Society for Molecular Biology and Evolution. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.

“Prokaryotic Pathway” Is Not Prokaryotic: Noncyanobacterial Origin of the Chloroplast Lipid Biosynthetic Pathway Revealed by Comprehensive Phylogenomic Analysis

PubMed Central

Awai, Koichiro

2017-01-01

Abstract Lipid biosynthesis within the chloroplast, or more generally plastids, was conventionally called “prokaryotic pathway,” which produces glycerolipids bearing C18 acids at the sn-1 position and C16 acids at the sn-2 position, as in cyanobacteria such as Anabaena and Synechocystis. This positional specificity is determined during the synthesis of phosphatidate, which is a precursor to diacylglycerol, the acceptor of galactose for the synthesis of galactolipids. The first acylation at sn-1 is catalyzed by glycerol-3-phosphate acyltransferase (GPAT or GPT), whereas the second acylation at sn-2 is performed by lysophosphatidate acyltransferase (LPAAT, AGPAT, or PlsC). Here we present comprehensive phylogenomic analysis of the origins of various acyltransferases involved in the synthesis of phosphatidate, as well as phosphatidate phosphatases in the chloroplasts. The results showed that the enzymes involved in the two steps of acylation in cyanobacteria and chloroplasts are entirely phylogenetically unrelated despite a previous report stating that the chloroplast LPAAT (ATS2) and cyanobacterial PlsC were sister groups. Phosphatidate phosphatases were separated into eukaryotic and prokaryotic clades, and the chloroplast enzymes were not of cyanobacterial origin, in contrast with another previous report. These results indicate that the lipid biosynthetic pathway in the chloroplasts or plastids did not originate from the cyanobacterial endosymbiont and is not “prokaryotic” in the context of endosymbiotic theory of plastid origin. This is another line of evidence for the discontinuity of plastids and cyanobacteria, which has been suggested in the glycolipid biosynthesis. PMID:29145606

Loss‐of‐function mutation of rice SLAC7 decreases chloroplast stability and induces a photoprotection mechanism in rice

PubMed Central

Fan, Xiaolei; Wu, Jiemin; Chen, Taiyu; Tie, Weiwei; Chen, Hao; Zhou, Fei

2015-01-01

Abstract Plants absorb sunlight to power the photochemical reactions of photosynthesis, which can potentially damage the photosynthetic machinery. However, the mechanism that protects chloroplasts from the damage remains unclear. In this work, we demonstrated that rice (Oryza sativa L.) SLAC7 is a generally expressed membrane protein. Loss‐of‐function of SLAC7 caused continuous damage to the chloroplasts of mutant leaves under normal light conditions. Ion leakage indicators related to leaf damage such as H2O2 and abscisic acid levels were significantly higher in slac7‐1 than in the wild type. Consistently, the photosynthesis efficiency and Fv/Fm ratio of slac7‐1 were significantly decreased (similar to photoinhibition). In response to chloroplast damage, slac7‐1 altered its leaf morphology (curled or fused leaf) by the synergy between plant hormones and transcriptional factors to decrease the absorption of light, suggesting that a photoprotection mechanism for chloroplast damage was activated in slac7‐1. When grown in dark conditions, slac7‐1 displayed a normal phenotype. SLAC7 under the control of the AtSLAC1 promoter could partially complement the phenotypes of Arabidopsis slac1 mutants, indicating a partial conservation of SLAC protein functions. These results suggest that SLAC7 is essential for maintaining the chloroplast stability in rice. PMID:25739330

A set of primers for analyzing chloroplast DNA diversity in Citrus and related genera.

PubMed

Cheng, Yunjiang; de Vicente, M Carmen; Meng, Haijun; Guo, Wenwu; Tao, Nengguo; Deng, Xiuxin

2005-06-01

Chloroplast simple sequence repeat (cpSSR) markers in Citrus were developed and used to analyze chloroplast diversity of Citrus and closely related genera. Fourteen cpSSR primer pairs from the chloroplast genomes of tobacco (Nicotiana tabacum L.) and Arabidopsis were found useful for analyzing the Citrus chloroplast genome (cpDNA) and recoded with the prefix SPCC (SSR Primers for Citrus Chloroplast). Eleven of the 14 primer pairs revealed some degree of polymorphism among 34 genotypes of Citrus, Fortunella, Poncirus and some of their hybrids, with polymorphism information content (PIC) values ranging from 0.057 to 0.732, and 18 haplotypes were identified. The cpSSR data were analyzed with NTSYS-pc software, and the genetic relationships suggested by the unweighted pair group method based on arithmetic means (UPGMA) dendrogram were congruent with previous taxonomic investigations: the results showed that all samples fell into seven major clusters, i.e., Citrus medica L., Poncirus, Fortunella, C. ichangensis Blanco, C. reticulata Swingle, C. aurantifolia (Christm.) Swingle and C. grandis (L.) Osbeck. The results of previous studies combined with our cpSSR analyses revealed that: (1) Calamondin (C. madurensis Swingle) is the result of hybridization between kumquat (Fortunella) and mandarin (C. reticulata), where kumquat acted as the female parent; (2) Ichang papeda (C. ichangensis) has a unique taxonomic status; and (3) although Bendiguangju mandarin (C. reticulata) and Satsuma mandarin (C. reticulata) are similar in fruit shape and leaf morphology, they have different maternal parents. Bendiguangju mandarin has the same cytoplasm as sweet orange (C. sinensis), whereas Satsuma mandarin has the cytoplasm of C. reticulata. Seventeen PCR products from SPCC1 and 21 from SPCC11 were cloned and sequenced. The results revealed that mononucleotide repeats as well as insertions and deletions of small segments of DNA were associated with SPCC1 polymorphism, whereas polymorphism

Disordered Cold Regulated15 Proteins Protect Chloroplast Membranes during Freezing through Binding and Folding, But Do Not Stabilize Chloroplast Enzymes in Vivo1[W][OPEN

PubMed Central

Thalhammer, Anja; Bryant, Gary; Sulpice, Ronan; Hincha, Dirk K.

2014-01-01

Freezing can severely damage plants, limiting geographical distribution of natural populations and leading to major agronomical losses. Plants native to cold climates acquire increased freezing tolerance during exposure to low nonfreezing temperatures in a process termed cold acclimation. This involves many adaptative responses, including global changes in metabolite content and gene expression, and the accumulation of cold-regulated (COR) proteins, whose functions are largely unknown. Here we report that the chloroplast proteins COR15A and COR15B are necessary for full cold acclimation in Arabidopsis (Arabidopsis thaliana). They protect cell membranes, as indicated by electrolyte leakage and chlorophyll fluorescence measurements. Recombinant COR15 proteins stabilize lactate dehydrogenase during freezing in vitro. However, a transgenic approach shows that they have no influence on the stability of selected plastidic enzymes in vivo, although cold acclimation results in increased enzyme stability. This indicates that enzymes are stabilized by other mechanisms. Recombinant COR15 proteins are disordered in water, but fold into amphipathic α-helices at high osmolyte concentrations in the presence of membranes, a condition mimicking molecular crowding induced by dehydration during freezing. X-ray scattering experiments indicate protein-membrane interactions specifically under such crowding conditions. The COR15-membrane interactions lead to liposome stabilization during freezing. Collectively, our data demonstrate the requirement for COR15 accumulation for full cold acclimation of Arabidopsis. The function of these intrinsically disordered proteins is the stabilization of chloroplast membranes during freezing through a folding and binding mechanism, but not the stabilization of chloroplastic enzymes. This indicates a high functional specificity of these disordered plant proteins. PMID:25096979

Chloroplast DNA Structural Variation, Phylogeny, and Age of Divergence among Diploid Cotton Species.

PubMed

Chen, Zhiwen; Feng, Kun; Grover, Corrinne E; Li, Pengbo; Liu, Fang; Wang, Yumei; Xu, Qin; Shang, Mingzhao; Zhou, Zhongli; Cai, Xiaoyan; Wang, Xingxing; Wendel, Jonathan F; Wang, Kunbo; Hua, Jinping

2016-01-01

The cotton genus (Gossypium spp.) contains 8 monophyletic diploid genome groups (A, B, C, D, E, F, G, K) and a single allotetraploid clade (AD). To gain insight into the phylogeny of Gossypium and molecular evolution of the chloroplast genome in this group, we performed a comparative analysis of 19 Gossypium chloroplast genomes, six reported here for the first time. Nucleotide distance in non-coding regions was about three times that of coding regions. As expected, distances were smaller within than among genome groups. Phylogenetic topologies based on nucleotide and indel data support for the resolution of the 8 genome groups into 6 clades. Phylogenetic analysis of indel distribution among the 19 genomes demonstrates contrasting evolutionary dynamics in different clades, with a parallel genome downsizing in two genome groups and a biased accumulation of insertions in the clade containing the cultivated cottons leading to large (for Gossypium) chloroplast genomes. Divergence time estimates derived from the cpDNA sequence suggest that the major diploid clades had diverged approximately 10 to 11 million years ago. The complete nucleotide sequences of 6 cpDNA genomes are provided, offering a resource for cytonuclear studies in Gossypium.

Connectivity between electron transport complexes and modulation of photosystem II activity in chloroplasts.

PubMed

Tikhonov, Alexander N; Vershubskii, Alexey V

2017-09-01

In chloroplasts, photosynthetic electron transport complexes interact with each other via the mobile electron carriers (plastoquinone and plastocyanin) which are in surplus amounts with respect to photosystem I and photosystem II (PSI and PSII), and the cytochrome b 6 f complex. In this work, we analyze experimental data on the light-induced redox transients of photoreaction center P 700 in chloroplasts within the framework of our mathematical model. This analysis suggests that during the action of a strong actinic light, even significant attenuation of PSII [for instance, in the result of inhibition of a part of PSII complexes by DCMU or due to non-photochemical quenching (NPQ)] will not cause drastic shortage of electron flow through PSI. This can be explained by "electronic" and/or "excitonic" connectivity between different PSII units. At strong AL, the overall flux of electrons between PSII and PSI will maintain at a high level even with the attenuation of PSII activity, provided the rate-limiting step of electron transfer is beyond the stage of PQH 2 formation. Results of our study are briefly discussed in the context of NPQ-dependent mechanism of chloroplast protection against light stress.

Chloroplast DNA Structural Variation, Phylogeny, and Age of Divergence among Diploid Cotton Species

PubMed Central

Li, Pengbo; Liu, Fang; Wang, Yumei; Xu, Qin; Shang, Mingzhao; Zhou, Zhongli; Cai, Xiaoyan; Wang, Xingxing; Wendel, Jonathan F.; Wang, Kunbo

2016-01-01

The cotton genus (Gossypium spp.) contains 8 monophyletic diploid genome groups (A, B, C, D, E, F, G, K) and a single allotetraploid clade (AD). To gain insight into the phylogeny of Gossypium and molecular evolution of the chloroplast genome in this group, we performed a comparative analysis of 19 Gossypium chloroplast genomes, six reported here for the first time. Nucleotide distance in non-coding regions was about three times that of coding regions. As expected, distances were smaller within than among genome groups. Phylogenetic topologies based on nucleotide and indel data support for the resolution of the 8 genome groups into 6 clades. Phylogenetic analysis of indel distribution among the 19 genomes demonstrates contrasting evolutionary dynamics in different clades, with a parallel genome downsizing in two genome groups and a biased accumulation of insertions in the clade containing the cultivated cottons leading to large (for Gossypium) chloroplast genomes. Divergence time estimates derived from the cpDNA sequence suggest that the major diploid clades had diverged approximately 10 to 11 million years ago. The complete nucleotide sequences of 6 cpDNA genomes are provided, offering a resource for cytonuclear studies in Gossypium. PMID:27309527

Identification of Simple Sequence Repeats in Chloroplast Genomes of Magnoliids Through Bioinformatics Approach.

PubMed

Srivastava, Deepika; Shanker, Asheesh

2016-12-01

Basal angiosperms or Magnoliids is an important clade of commercially important plants which mainly include spices and edible fruits. In this study, 17 chloroplast genome sequences belonging to clade Magnoliids were screened for the identification of chloroplast simple sequence repeats (cpSSRs). Simple sequence repeats or microsatellites are short stretches of DNA up to 1-6 base pair in length. These repeats are ubiquitous and play important role in the development of molecular markers and to study the mapping of traits of economic, medical or ecological interest. A total of 479 SSRs were detected, showing average density of 1 SSR/6.91 kb. Depending on the repeat units, the length of SSRs ranged from 12 to 24 bp for mono-, 12 to 18 bp for di-, 12 to 26 bp for tri-, 12 to 24 bp for tetra-, 15 bp for penta- and 18 bp for hexanucleotide repeats. Mononucleotide repeats were the most frequent (207, 43.21 %) followed by tetranucleotide repeats (130, 27.13 %). Penta- and hexanucleotide repeats were least frequent or absent in these chloroplast genomes.

Arabidopsis nph1 and npl1: Blue light receptors that mediate both phototropism and chloroplast relocation

PubMed Central

Sakai, Tatsuya; Kagawa, Takatoshi; Kasahara, Masahiro; Swartz, Trevor E.; Christie, John M.; Briggs, Winslow R.; Wada, Masamitsu; Okada, Kiyotaka

2001-01-01

UV-A/blue light acts to regulate a number of physiological processes in higher plants. These include light-driven chloroplast movement and phototropism. The NPH1 gene of Arabidopsis encodes an autophosphorylating protein kinase that functions as a photoreceptor for phototropism in response to low-intensity blue light. However, nph1 mutants have been reported to exhibit normal phototropic curvature under high-intensity blue light, indicating the presence of an additional phototropic receptor. A likely candidate is the nph1 homologue, npl1, which has recently been shown to mediate the avoidance response of chloroplasts to high-intensity blue light in Arabidopsis. Here we demonstrate that npl1, like nph1, noncovalently binds the chromophore flavin mononucleotide (FMN) within two specialized PAS domains, termed LOV domains. Furthermore, when expressed in insect cells, npl1, like nph1, undergoes light-dependent autophosphorylation, indicating that npl1 also functions as a light receptor kinase. Consistent with this conclusion, we show that a nph1npl1 double mutant exhibits an impaired phototropic response under both low- and high-intensity blue light. Hence, npl1 functions as a second phototropic receptor under high fluence rate conditions and is, in part, functionally redundant to nph1. We also demonstrate that both chloroplast accumulation in response to low-intensity light and chloroplast avoidance movement in response to high-intensity light are lacking in the nph1npl1 double mutant. Our findings therefore indicate that nph1 and npl1 show partially overlapping functions in two different responses, phototropism and chloroplast relocation, in a fluence rate-dependent manner. PMID:11371609

The complete chloroplast genome sequence of American bird pepper (Capsicum annuum var. glabriusculum).

PubMed

Zeng, Fan-chun; Gao, Cheng-wen; Gao, Li-zhi

2016-01-01

The complete chloroplast genome sequence of American bird pepper (Capsicum annuum var. glabriusculum) is reported and characterized in this study. The genome size is 156,612 bp, containing a pair of inverted repeats (IRs) of 25,776 bp separated by a large single-copy region of 87,213 bp and a small single-copy region of 17,851 bp. The chloroplast genome harbors 130 known genes, including 89 protein-coding genes, 8 ribosomal RNA genes, and 37 tRNA genes. A total of 18 of these genes are duplicated in the inverted repeat regions, 16 genes contain 1 intron, and 2 genes and one ycf have 2 introns.

Stomatal responses to CO/sub 2/ in Paphiopedilum and Phragmipedium: role of the guard cell chloroplast

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

Assmann, S.M.; Zeiger, E.

A role of the guard cell chloroplasts in the CO/sub 2/ response of stomata was investigated through a comparison of the leaf gas exchange characteristics of two closely related orchids: Paphiopedilum harrisianum, which lacks guard cell chloroplasts and Phragmipedium longifolium, which has chlorophyllous guard cells. Leaves of both species had an apparent quantum yield for assimilation of about 0.05, with photosynthesis saturating at 0.300 to 0.400 millimoles per square meter per second. The response of assimilation to changes in CO/sub 2/ was similar in the two species, but the response of conductance was consistently weaker in Paphiopedilum than in Phragmipedium.more » The data suggest involvement of guard cell chloroplasts in the stomatal response to CO/sub 2/ and in the coupling of assimilation and conductance in the intact leaf.« less

Transcriptional regulation and DNA methylation in plastids during transitional conversion of chloroplasts to chromoplasts.

PubMed Central

Kobayashi, H; Ngernprasirtsiri, J; Akazawa, T

1990-01-01

During transitional conversion of chloroplasts to chromoplasts in ripening tomato (Lycopersicon esculentum) fruits, transcripts for several plastid genes for photosynthesis decreased to undetectable levels. Run-on transcription of plastids indicated that transcriptional regulation operated as a predominant factor. We found that most of the genes in chloroplasts were actively transcribed in vitro by Escherichia coli and soluble plastid RNA polymerases, but some genes in chromoplasts seemed to be silent when assayed by the in vitro systems. The regulatory step, therefore, was ascribed to DNA templates. The analysis of modified base composition revealed the presence of methylated bases in chromoplast DNA, in which 5-methylcytosine was most abundant. The presence of 5-methylcytosine detected by isoschizomeric endonucleases and Southern hybridization was correlated with the undetectable transcription activity of each gene in the run-on assay and in vitro transcription experiments. It is thus concluded that the suppression of transcription mediated by DNA methylation is one of the mechanisms governing gene expression in plastids converting from chloroplasts to chromoplasts. Images Fig. 1 Fig. 2 Fig. 3. Fig. 4. Fig. 5. PMID:2303026

Phylogeography of Camellia taliensis (Theaceae) inferred from chloroplast and nuclear DNA: insights into evolutionary history and conservation.

PubMed

Liu, Yang; Yang, Shi-xiong; Ji, Peng-zhang; Gao, Li-zhi

2012-06-21

As one of the most important but seriously endangered wild relatives of the cultivated tea, Camellia taliensis harbors valuable gene resources for tea tree improvement in the future. The knowledge of genetic variation and population structure may provide insights into evolutionary history and germplasm conservation of the species. Here, we sampled 21 natural populations from the species' range in China and performed the phylogeography of C. taliensis by using the nuclear PAL gene fragment and chloroplast rpl32-trnL intergenic spacer. Levels of haplotype diversity and nucleotide diversity detected at rpl32-trnL (h = 0.841; π = 0.00314) were almost as high as at PAL (h = 0.836; π = 0.00417). Significant chloroplast DNA population subdivision was detected (GST = 0.988; NST = 0.989), suggesting fairly high genetic differentiation and low levels of recurrent gene flow through seeds among populations. Nested clade phylogeographic analysis of chlorotypes suggests that population genetic structure in C. taliensis has been affected by habitat fragmentation in the past. However, the detection of a moderate nrDNA population subdivision (GST = 0.222; NST = 0.301) provided the evidence of efficient pollen-mediated gene flow among populations and significant phylogeographical structure (NST > GST; P < 0.01). The analysis of PAL haplotypes indicates that phylogeographical pattern of nrDNA haplotypes might be caused by restricted gene flow with isolation by distance, which was also supported by Mantel's test of nrDNA haplotypes (r = 0.234, P < 0.001). We found that chlorotype C1 was fixed in seven populations of Lancang River Region, implying that the Lancang River might have provided a corridor for the long-distance dispersal of the species. We found that C. taliensis showed fairly high genetic differentiation resulting from restricted gene flow and habitat fragmentation. This phylogeographical study gives us deep insights into

Evidence for an operative glutamine translocator in chloroplasts from maritime pine (Pinus pinaster Ait.) cotyledons.

PubMed

Claros, M G; Aguilar, M L; Cánovas, F M

2010-09-01

In higher plants, ammonium is assimilated into amino acids through the glutamine synthetase (GS)/glutamate synthase (GOGAT) cycle. This metabolic cycle is distributed in different cellular compartments in conifer seedlings: glutamine synthesis occurs in the cytosol and glutamate synthesis within the chloroplast. A method for preparing intact chloroplasts of pine cotyledons is presented with the aim of identifying a glutamine-glutamate translocator. Glutamine-glutamate exchange has been studied using the double silicone layer system, suggesting the existence of a translocator that imports glutamine into the chloroplast and exports glutamate to the cytoplasm. The translocator identified is specific for glutamine and glutamate, and the kinetic constants for both substrates indicate that it is unsaturated at intracellular concentrations. Thus, the experimental evidence obtained supports the model of the GS/GOGAT cycle in developing pine seedlings that accounts for the stoichiometric balance of metabolites. As a result, the efficient assimilation of free ammonia produced by photorespiration, nitrate reduction, storage protein mobilisation, phenylpropanoid pathway or S-adenosylmethionine synthesis is guaranteed.

THE WATER-WATER CYCLE IN CHLOROPLASTS: Scavenging of Active Oxygens and Dissipation of Excess Photons.

PubMed

Asada, Kozi

1999-06-01

Photoreduction of dioxygen in photosystem I (PSI) of chloroplasts generates superoxide radicals as the primary product. In intact chloroplasts, the superoxide and the hydrogen peroxide produced via the disproportionation of superoxide are so rapidly scavenged at the site of their generation that the active oxygens do not inactivate the PSI complex, the stromal enzymes, or the scavenging system itself. The overall reaction for scavenging of active oxygens is the photoreduction of dioxygen to water via superoxide and hydrogen peroxide in PSI by the electrons derived from water in PSII, and the water-water cycle is proposed for these sequences. An overview is given of the molecular mechanism of the water-water cycle and microcompartmentalization of the enzymes participating in it. Whenever the water-water cycle operates properly for scavenging of active oxygens in chloroplasts, it also effectively dissipates excess excitation energy under environmental stress. The dual functions of the water-water cycle for protection from photoinihibition are discussed.

The C-type Arabidopsis thioredoxin reductase ANTR-C acts as an electron donor to 2-Cys peroxiredoxins in chloroplasts

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

Moon, Jeong Chan; Jang, Ho Hee; Chae, Ho Byoung

2006-09-22

2-Cys peroxiredoxins (Prxs) play important roles in the antioxidative defense systems of plant chloroplasts. In order to determine the interaction partner for these proteins in Arabidopsis, we used a yeast two-hybrid screening procedure with a C175S-mutant of Arabidopsis 2-Cys Prx-A as bait. A cDNA encoding an NADPH-dependent thioredoxin reductase (NTR) isotype C was identified and designated ANTR-C. We demonstrated that this protein effected efficient transfer of electrons from NADPH to the 2-Cys Prxs of chloroplasts. Interaction between 2-Cys Prx-A and ANTR-C was confirmed by a pull-down experiment. ANTR-C contained N-terminal TR and C-terminal Trx domains. It exhibited both TR andmore » Trx activities and co-localized with 2-Cys Prx-A in chloroplasts. These results suggest that ANTR-C functions as an electron donor for plastidial 2-Cys Prxs and represents the NADPH-dependent TR/Trx system in chloroplasts.« less

Expression of dengue-3 premembrane and envelope polyprotein in lettuce chloroplasts

PubMed Central

Kanagaraj, Anderson Paul; Verma, Dheeraj

2012-01-01

Dengue is an acute febrile viral disease with >100 million infections occurring each year and more than half of the world population is at risk. Global resurgence of dengue in many urban centers of the tropics is a major concern. Therefore, development of a successful vaccine is urgently needed that is economical and provide long-lasting protection from dengue virus infections. In this manuscript, we report expression of dengue-3 serotype polyprotein (prM/E) consisting of part of capsid, complete premembrane (prM) and truncated envelope (E) protein in an edible crop lettuce. The dengue sequence was controlled by endogenous Lactuca sativa psbA regulatory elements. PCR and Southern blot analysis confirmed transgene integration into the lettuce chloroplast genome via homologous recombination at the trnI/trnA intergenic spacer region. Western blot analysis showed expression of polyprotein prM/E in different forms as monomers (~65 kDa) or possibly heterodimers (~130 kDa) or multimers. Multimers were solubilized into monomers using guanidine hydrochloride. Transplastomic lettuce plants expressing dengue prM/E vaccine antigens grew normally and transgenes were inherited in the T1 progeny without any segregation. Transmission electron microscopy showed the presence of virus-like particles of ~20 nm diameter in chloroplast extracts of transplastomic lettuce expressing prM/E proteins, but not in untransformed plants. The prM/E antigens expressed in lettuce chloroplasts should offer a potential source for investigating an oral Dengue vaccine. PMID:21431782

Physical, chemical and biochemical studies with isolated chloroplasts and purified enzymes

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

El-Badry, A. M.

1971-01-01

Fructose-1,6-diphosphatase has been isolated, purified, and crystallized from previously isolated spinach chloroplasts. The effects of various anions, cations, and sulfhydryl reagent were tested, and activation by Mg ++, glycine, HCO 3 -, and sulfhydryl reagent is described.

Phosphatidylglycerol Synthesis in Spinach Chloroplasts: Characterization of the Newly Synthesized Molecule

PubMed Central

Sparace, Salvatore A.; Mudd, J. Brian

1982-01-01

Intact chloroplasts from spinach (Spinacia oleracea L., hybrid 424) readily incorporate [14C]glycerol-3-phosphate and [14C]acetate into diacylglycerol, monoacylglycerol, diacylglycrol, free fatty acids (only when acetate is the precursor), phosphatidic acid, phosphatidylcholine, and most notably phosphatidylglycerol. The fraction of phosphatidylglycerol synthesized is greatly increased by the presence of manganese chloride in the reaction mixture. Glycerol-3-phosphate-labeled phosphatidylglycerol is equally labeled in the two glycerol moieties of the molecule. Acetate-labeled phosphatidylglycerol is equally labeled in both acyl groups. Position one contains primarily oleate, linoleate and small amounts of palmitate. Position two contains primarily palmitate. No radioactive trans-Δ3-hexadecenoate was detected. The labeling patterns indicate that the radioactive phosphatidylglycerol is the product of de novo chloroplast lipid biosynthesis and furthermore, phosphatidylglycerol may be a substrate for fatty acid desaturation. Images Fig. 1 PMID:16662664

Overproduction of Petunia Chloroplastic Copper/Zinc Superoxide Dismutase Does Not Confer Ozone Tolerance in Transgenic Tobacco 1

PubMed Central

Pitcher, Lynne H.; Brennan, Eileen; Hurley, Arthur; Dunsmuir, Pamela; Tepperman, James M.; Zilinskas, Barbara A.

1991-01-01

Transgenic tobacco (Nicotiana tabacum cultivar W38) plants that overproduce petunia chloroplastic Cu/Zn superoxide dismutase were exposed to ozone dosages that injure control tobacco plants. Based on foliar injury ratings, there was no consistent protection provided to the transgenic plants. These data indicate that an increase in the chloroplastic Cu/Zn superoxide dismutase alone is not sufficient to reduce ozone toxicity. ImagesFigure 1 PMID:16668407

New chloroplast microsatellite markers suitable for assessing genetic diversity of Lolium perenne and other related grass species

PubMed Central

Diekmann, Kerstin; Hodkinson, Trevor R.; Barth, Susanne

2012-01-01

Background and Aims Lolium perenne (perennial ryegrass) is the most important forage grass species of temperate regions. We have previously released the chloroplast genome sequence of L. perenne ‘Cashel’. Here nine chloroplast microsatellite markers are published, which were designed based on knowledge about genetically variable regions within the L. perenne chloroplast genome. These markers were successfully used for characterizing the genetic diversity in Lolium and different grass species. Methods Chloroplast genomes of 14 Poaceae taxa were screened for mononucleotide microsatellite repeat regions and primers designed for their amplification from nine loci. The potential of these markers to assess genetic diversity was evaluated on a set of 16 Irish and 15 European L. perenne ecotypes, nine L. perenne cultivars, other Lolium taxa and other grass species. Key Results All analysed Poaceae chloroplast genomes contained more than 200 mononucleotide repeats (chloroplast simple sequence repeats, cpSSRs) of at least 7 bp in length, concentrated mainly in the large single copy region of the genome. Nucleotide composition varied considerably among subfamilies (with Pooideae biased towards poly A repeats). The nine new markers distinguish L. perenne from all non-Lolium taxa. TeaCpSSR28 was able to distinguish between all Lolium species and Lolium multiflorum due to an elongation of an A8 mononucleotide repeat in L. multiflorum. TeaCpSSR31 detected a considerable degree of microsatellite length variation and single nucleotide polymorphism. TeaCpSSR27 revealed variation within some L. perenne accessions due to a 44-bp indel and was hence readily detected by simple agarose gel electrophoresis. Smaller insertion/deletion events or single nucleotide polymorphisms detected by these new markers could be visualized by polyacrylamide gel electrophoresis or DNA sequencing, respectively. Conclusions The new markers are a valuable tool for plant breeding companies, seed testing

New chloroplast microsatellite markers suitable for assessing genetic diversity of Lolium perenne and other related grass species.

PubMed

Diekmann, Kerstin; Hodkinson, Trevor R; Barth, Susanne

2012-11-01

Lolium perenne (perennial ryegrass) is the most important forage grass species of temperate regions. We have previously released the chloroplast genome sequence of L. perenne 'Cashel'. Here nine chloroplast microsatellite markers are published, which were designed based on knowledge about genetically variable regions within the L. perenne chloroplast genome. These markers were successfully used for characterizing the genetic diversity in Lolium and different grass species. Chloroplast genomes of 14 Poaceae taxa were screened for mononucleotide microsatellite repeat regions and primers designed for their amplification from nine loci. The potential of these markers to assess genetic diversity was evaluated on a set of 16 Irish and 15 European L. perenne ecotypes, nine L. perenne cultivars, other Lolium taxa and other grass species. All analysed Poaceae chloroplast genomes contained more than 200 mononucleotide repeats (chloroplast simple sequence repeats, cpSSRs) of at least 7 bp in length, concentrated mainly in the large single copy region of the genome. Nucleotide composition varied considerably among subfamilies (with Pooideae biased towards poly A repeats). The nine new markers distinguish L. perenne from all non-Lolium taxa. TeaCpSSR28 was able to distinguish between all Lolium species and Lolium multiflorum due to an elongation of an A(8) mononucleotide repeat in L. multiflorum. TeaCpSSR31 detected a considerable degree of microsatellite length variation and single nucleotide polymorphism. TeaCpSSR27 revealed variation within some L. perenne accessions due to a 44-bp indel and was hence readily detected by simple agarose gel electrophoresis. Smaller insertion/deletion events or single nucleotide polymorphisms detected by these new markers could be visualized by polyacrylamide gel electrophoresis or DNA sequencing, respectively. The new markers are a valuable tool for plant breeding companies, seed testing agencies and the wider scientific community due to

A chloroplast membrane protein LTO1/AtVKOR involving in redox regulation and ROS homeostasis.

PubMed

Lu, Ying; Wang, Hua-Rong; Li, Han; Cui, Hao-Ran; Feng, Yue-Guang; Wang, Xiao-Yun

2013-09-01

The role of LTO1/ At VKOR-DsbA in ROS homeostasis and in redox regulation of cysteine-containing proteins in chloroplast was studied in lto1 - 2 mutant, and a potential target of LTO1 was captured. A chloroplast membrane protein LTO1/AtVKOR-DsbA encoded by the gene At4g35760 was recently found to be an oxidoreductase and involved in assembly of PSII. Here, the growth of a mutant lto1-2 line of Arabidopsis was found to be severely stunted and transgenic complementation ultimately demonstrated the phenotype changes were due to this gene. A proteomic experiment identified 23 proteins presenting a differential abundance in lto1-2 compared with wild-type plants, including components in PSII and proteins scavenging active oxygen. Three scavengers of active oxygen, L-ascorbate peroxidase 1, peroxisomal catalase 2, dehydroascorbate reductase 1, are reduced in lto1-2 plants, corresponding to high levels of accumulation of reactive oxygen species (ROS). The photosynthetic activities of PSII and the quantity of core protein D1 decreased significantly in lto1-2. Further investigation showed the synthesis of D1 was not affected in mutants both at transcription and translation levels. The soluble DsbA-like domain of LTO1 was found to have reduction, oxidation and isomerization activities, and could promote the formation of disulfide bonds in a lumenal protein, FKBP13. A potential target of LTO1 was captured which was involving in chlorophyll degradation and photooxidative stress response. Experimental results imply that LTO1 plays important roles in redox regulation, ROS homeostasis and maintenance of PSII.

The complete chloroplast genome sequence of strawberry (Fragaria  × ananassa Duch.) and comparison with related species of Rosaceae

PubMed Central

Cheng, Hui; Li, Jinfeng; Zhang, Hong; Cai, Binhua; Gao, Zhihong

2017-01-01

Compared with other members of the family Rosaceae, the chloroplast genomes of Fragaria species exhibit low variation, and this situation has limited phylogenetic analyses; thus, complete chloroplast genome sequencing of Fragaria species is needed. In this study, we sequenced the complete chloroplast genome of F. × ananassa ‘Benihoppe’ using the Illumina HiSeq 2500-PE150 platform and then performed a combination of de novo assembly and reference-guided mapping of contigs to generate complete chloroplast genome sequences. The chloroplast genome exhibits a typical quadripartite structure with a pair of inverted repeats (IRs, 25,936 bp) separated by large (LSC, 85,531 bp) and small (SSC, 18,146 bp) single-copy (SC) regions. The length of the F. × ananassa ‘Benihoppe’ chloroplast genome is 155,549 bp, representing the smallest Fragaria chloroplast genome observed to date. The genome encodes 112 unique genes, comprising 78 protein-coding genes, 30 tRNA genes and four rRNA genes. Comparative analysis of the overall nucleotide sequence identity among ten complete chloroplast genomes confirmed that for both coding and non-coding regions in Rosaceae, SC regions exhibit higher sequence variation than IRs. The Ka/Ks ratio of most genes was less than 1, suggesting that most genes are under purifying selection. Moreover, the mVISTA results also showed a high degree of conservation in genome structure, gene order and gene content in Fragaria, particularly among three octoploid strawberries which were F. × ananassa ‘Benihoppe’, F. chiloensis (GP33) and F. virginiana (O477). However, when the sequences of the coding and non-coding regions of F. × ananassa ‘Benihoppe’ were compared in detail with those of F. chiloensis (GP33) and F. virginiana (O477), a number of SNPs and InDels were revealed by MEGA 7. Six non-coding regions (trnK-matK, trnS-trnG, atpF-atpH, trnC-petN, trnT-psbD and trnP-psaJ) with a percentage of variable sites greater than 1% and no less

Chloroplast-to-nucleus communication: current knowledge, experimental strategies and relationship to drought stress signaling.

PubMed

Chan, Kai Xun; Crisp, Peter Alexander; Estavillo, Gonzalo Martin; Pogson, Barry James

2010-12-01

In order for plant cells to function efficiently under different environmental conditions, chloroplastic processes have to be tightly regulated by the nucleus. It is widely believed that there is inter-organelle communication from the chloroplast to the nucleus, called retrograde signaling. Although some pathways of communication have been identified, the actual signals that move between the two cellular compartments are largely unknown. This review provides an overview of retrograde signaling including its importance to the cell, candidate signals, recent advances, and current experimental systems. In addition, we highlight the potential of using drought stress as a model for studying retrograde signaling.

Capturing chloroplast variation for molecular ecology studies: a simple next generation sequencing approach applied to a rainforest tree

PubMed Central

2013-01-01

Background With high quantity and quality data production and low cost, next generation sequencing has the potential to provide new opportunities for plant phylogeographic studies on single and multiple species. Here we present an approach for in silicio chloroplast DNA assembly and single nucleotide polymorphism detection from short-read shotgun sequencing. The approach is simple and effective and can be implemented using standard bioinformatic tools. Results The chloroplast genome of Toona ciliata (Meliaceae), 159,514 base pairs long, was assembled from shotgun sequencing on the Illumina platform using de novo assembly of contigs. To evaluate its practicality, value and quality, we compared the short read assembly with an assembly completed using 454 data obtained after chloroplast DNA isolation. Sanger sequence verifications indicated that the Illumina dataset outperformed the longer read 454 data. Pooling of several individuals during preparation of the shotgun library enabled detection of informative chloroplast SNP markers. Following validation, we used the identified SNPs for a preliminary phylogeographic study of T. ciliata in Australia and to confirm low diversity across the distribution. Conclusions Our approach provides a simple method for construction of whole chloroplast genomes from shotgun sequencing of whole genomic DNA using short-read data and no available closely related reference genome (e.g. from the same species or genus). The high coverage of Illumina sequence data also renders this method appropriate for multiplexing and SNP discovery and therefore a useful approach for landscape level studies of evolutionary ecology. PMID:23497206

Chloroplast targeting of FanC, the major antigenic subunit of Escherichia coli K99 fimbriae, in transgenic soybean.

PubMed

Garg, Renu; Tolbert, Melanie; Oakes, Judy L; Clemente, Thomas E; Bost, Kenneth L; Piller, Kenneth J

2007-07-01

Enterotoxigenic Escherichia coli (ETEC) strains are a major cause of enteric diseases affecting livestock and humans. Edible transgenic plants producing E. coli fimbrial subunit proteins have the potential to vaccinate against these diseases, but have not reached their full potential as a renewable source of oral vaccines due in part to insufficient levels of recombinant protein accumulation. Previously, we reported that cytosol targeting of the E. coli K99 fimbrial subunit antigen resulted in FanC accumulation to approximately 0.4% of total soluble protein in soybean leaves (Piller et al. in Planta 222:6-18, 2005). In this study, we report on the subcellular targeting of FanC to chloroplasts. Twenty-two transgenic T1 progeny derived from seven individual T0 transformation events were characterized, and 17 accumulated transgenic FanC. All of the characterized events displayed relatively low T-DNA complexity, and all exhibited proper targeting of FanC to the chloroplast. Accumulation of chloroplast-targeted FanC was approximately 0.08% of total soluble leaf protein, or approximately 5-fold less than cytosol-targeted FanC. Protein analysis of leaves at various stages of maturity suggested stability of chloroplast-targeted FanC throughout leaf maturation. Furthermore, mice immunized intraperitoneally with protein extract derived from transgenic leaves expressing chloroplast-targeted FanC developed significant antibody titers against FanC. This is the first report of subcellular targeting of a vaccine subunit antigen in soybean.

Mutations Altering Chloroplast Ribosome Phenotype in Chlamydomonas, I. Non-Mendelian Mutations*

PubMed Central

Gillham, Nicholas W.; Boynton, John E.; Burkholder, Barbara

1970-01-01

Uniparentally inherited mutations to antibiotic resistance and dependence in Chlamydomonas reinhardi exhibit an altered chloroplast ribosome phenotype. Genetic studies demonstrate an absolute correlation between the drug resistance or dependence and the ribosome phenotype in two such mutants. Images PMID:5289000

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

Chloroplast Omp85 proteins change orientation during evolution

PubMed Central

Sommer, Maik S.; Daum, Bertram; Gross, Lucia E.; Weis, Benjamin L. M.; Mirus, Oliver; Abram, Lars; Maier, Uwe-G.; Kühlbrandt, Werner; Schleiff, Enrico

2011-01-01

The majority of outer membrane proteins (OMPs) from Gram-negative bacteria and many of mitochondria and chloroplasts are β-barrels. Insertion and assembly of these proteins are catalyzed by the Omp85 protein family in a seemingly conserved process. All members of this family exhibit a characteristic N-terminal polypeptide-transport–associated (POTRA) and a C-terminal 16-stranded β-barrel domain. In plants, two phylogenetically distinct and essential Omp85's exist in the chloroplast outer membrane, namely Toc75-III and Toc75-V. Whereas Toc75-V, similar to the mitochondrial Sam50, is thought to possess the original bacterial function, its homolog, Toc75-III, evolved to the pore-forming unit of the TOC translocon for preprotein import. In all current models of OMP biogenesis and preprotein translocation, a topology of Omp85 with the POTRA domain in the periplasm or intermembrane space is assumed. Using self-assembly GFP-based in vivo experiments and in situ topology studies by electron cryotomography, we show that the POTRA domains of both Toc75-III and Toc75-V are exposed to the cytoplasm. This unexpected finding explains many experimental observations and requires a reevaluation of current models of OMP biogenesis and TOC complex function. PMID:21825140

Distinct Pseudomonas type-III effectors use a cleavable transit peptide to target chloroplasts.

PubMed

Li, Guangyong; Froehlich, John E; Elowsky, Christian; Msanne, Joseph; Ostosh, Andrew C; Zhang, Chi; Awada, Tala; Alfano, James R

2014-01-01

The pathogen Pseudomonas syringae requires a type-III protein secretion system and the effector proteins it injects into plant cells for pathogenesis. The primary role for P. syringae type-III effectors is the suppression of plant immunity. The P. syringae pv. tomato DC3000 HopK1 type-III effector was known to suppress the hypersensitive response (HR), a programmed cell death response associated with effector-triggered immunity. Here we show that DC3000 hopK1 mutants are reduced in their ability to grow in Arabidopsis, and produce reduced disease symptoms. Arabidopsis transgenically expressing HopK1 are reduced in PAMP-triggered immune responses compared with wild-type plants. An N-terminal region of HopK1 shares similarity with the corresponding region in the well-studied type-III effector AvrRps4; however, their C-terminal regions are dissimilar, indicating that they have different effector activities. HopK1 is processed in planta at the same processing site found in AvrRps4. The processed forms of HopK1 and AvrRps4 are chloroplast localized, indicating that the shared N-terminal regions of these type-III effectors represent a chloroplast transit peptide. The HopK1 contribution to virulence and the ability of HopK1 and AvrRps4 to suppress immunity required their respective transit peptides, but the AvrRps4-induced HR did not. Our results suggest that a primary virulence target of these type-III effectors resides in chloroplasts, and that the recognition of AvrRps4 by the plant immune system occurs elsewhere. Moreover, our results reveal that distinct type-III effectors use a cleavable transit peptide to localize to chloroplasts, and that targets within this organelle are important for immunity. © 2013 The Authors The Plant Journal © 2013 John Wiley & Sons Ltd.

Expression of the recombinant bacterial outer surface protein A in tobacco chloroplasts leads to thylakoid localization and loss of photosynthesis.

PubMed

Hennig, Anna; Bonfig, Katharina; Roitsch, Thomas; Warzecha, Heribert

2007-11-01

Bacterial lipoproteins play crucial roles in host-pathogen interactions and pathogenesis and are important targets for the immune system. A prominent example is the outer surface protein A (OspA) of Borrelia burgdorferi, which has been efficiently used as a vaccine for the prevention of Lyme disease. In a previous study, OspA could be produced in tobacco chloroplasts in a lipidated and immunogenic form. To further explore the potential of chloroplasts for the production of bacterial lipoproteins, the role of the N-terminal leader sequence was investigated. The amount of recombinant OspA could be increased up to ten-fold by the variation of the insertion site in the chloroplast genome. Analysis of OspA mutants revealed that replacement of the invariant cysteine residue as well as deletion of the leader sequence abolishes palmitolyation of OspA. Also, decoration of OspA with an N-terminal eukaryotic lipidation motif does not lead to palmitoylation in chloroplasts. Strikingly, the bacterial signal peptide of OspA efficiently targets the protein to thylakoids, and causes a mutant phenotype. Plants accumulating OspA at 10% total soluble protein could not grow without exogenously supplied sugars and rapidly died after transfer to soil under greenhouse conditions. The plants were found to be strongly affected in photosystem II, as revealed by the analyses of temporal and spatial dynamics of photosynthetic activity by chlorophyll fluorescence imaging. Thus, overexpression of OspA in chloroplasts is limited by its concentration-dependent interference with essential functions of chloroplastic membranes required for primary metabolism.

Complete chloroplast genome sequence of MD-2 pineapple and its comparative analysis among nine other plants from the subclass Commelinidae.

PubMed

Redwan, R M; Saidin, A; Kumar, S V

2015-08-12

Pineapple (Ananas comosus var. comosus) is known as the king of fruits for its crown and is the third most important tropical fruit after banana and citrus. The plant, which is indigenous to South America, is the most important species in the Bromeliaceae family and is largely traded for fresh fruit consumption. Here, we report the complete chloroplast sequence of the MD-2 pineapple that was sequenced using the PacBio sequencing technology. In this study, the high error rate of PacBio long sequence reads of A. comosus's total genomic DNA were improved by leveraging on the high accuracy but short Illumina reads for error-correction via the latest error correction module from Novocraft. Error corrected long PacBio reads were assembled by using a single tool to produce a contig representing the pineapple chloroplast genome. The genome of 159,636 bp in length is featured with the conserved quadripartite structure of chloroplast containing a large single copy region (LSC) with a size of 87,482 bp, a small single copy region (SSC) with a size of 18,622 bp and two inverted repeat regions (IRA and IRB) each with the size of 26,766 bp. Overall, the genome contained 117 unique coding regions and 30 were repeated in the IR region with its genes contents, structure and arrangement similar to its sister taxon, Typha latifolia. A total of 35 repeats structure were detected in both the coding and non-coding regions with a majority being tandem repeats. In addition, 205 SSRs were detected in the genome with six protein-coding genes contained more than two SSRs. Comparative chloroplast genomes from the subclass Commelinidae revealed a conservative protein coding gene albeit located in a highly divergence region. Analysis of selection pressure on protein-coding genes using Ka/Ks ratio showed significant positive selection exerted on the rps7 gene of the pineapple chloroplast with P less than 0.05. Phylogenetic analysis confirmed the recent taxonomical relation among the member of

Combined effects of lanthanum ion and acid rain on growth, photosynthesis and chloroplast ultrastructure in soybean seedlings.

PubMed

Wen, Kejia; Liang, Chanjuan; Wang, Lihong; Hu, Gang; Zhou, Qing

2011-07-01

Rare earth elements (REEs) have been accumulated in the agricultural environment. Acid rain is a serious environmental issue. In the present work, the effects of lanthanum ion (La(3+)) and acid rain on the growth, photosynthesis and chloroplast ultrastructure in soybean seedlings were investigated using the gas exchange measurements system, chlorophyll fluorometer, transmission electron microscopy and some biochemical techniques. It was found that although the growth and photosynthesis of soybean seedlings treated with the low concentration of La(3+) was improved, the growth and photosynthesis of soybean seedlings were obviously inhibited in the combined treatment with the low concentration of La(3+) and acid rain. At the same time, the chloroplast ultrastructure in the cell of soybean seedlings was destroyed. Under the combined treatment with the high concentration of La(3+) and acid rain, the chloroplast ultrastructure in the cell of soybean seedlings was seriously destroyed, and the growth and of photosynthesis were greatly decreased compared with those of the control, the single treatment with the high concentration of La(3+) and the single treatment with acid rain, respectively. The degree of decrease and destruction on chloroplast ultrastructure depended on the increases in the concentration of La(3+) and acid rain (H(+)). In conclusion, the combined pollution of La(3+) and acid rain obviously destroyed the chloroplast ultrastructure of cell and aggravated the harmful effect of the single La(3+) and acid rain on soybean seedlings. As a new combined pollutant, the harmful effect of REEs ions and acid rain on plant should be paid attention to. Copyright © 2011 Elsevier Ltd. All rights reserved.

Phytoremediation of Mercury and Organomercurials in Chloroplast Transgenic Plants: Enhanced Root Uptake, Translocation to Shoots, and Volatilization

PubMed Central

Hussein, Hussein S.; Ruiz, Oscar N.; Terry, Norman; Daniell, Henry

2008-01-01

Transgenic tobacco plants engineered with bacterial merA and merB genes via the chloroplast genome were investigated to study the uptake, translocation of different forms of mercury (Hg) from roots to shoots, and their volatilization. Untransformed plants, regardless of the form of Hg supplied, reached a saturation point at 200 µM of phenylmercuric acetate (PMA) or HgCl2, accumulating Hg concentrations up to 500 µg g−1 with significant reduction in growth. In contrast, chloroplast transgenic lines continued to grow well with Hg concentrations in root tissues up to 2000 µg g−1. Chloroplast transgenic lines accumulated both the organic and inorganic Hg forms to levels surpassing the concentrations found in the soil. The organic-Hg form was absorbed and translocated more efficiently than the inorganic-Hg form in transgenic lines, whereas no such difference was observed in untransformed plants. Chloroplast-transgenic lines showed about 100-fold increase in the efficiency of Hg accumulation in shoots compared to untransformed plants. This is the first report of such high levels of Hg accumulation in green leaves or tissues. Transgenic plants attained a maximum rate of elemental-Hg volatilization in two days when supplied with PMA and in three days when supplied with inorganic-Hg, attaining complete volatilization within a week. The combined expression of merAB via the chloroplast genome enhanced conversion of Hg2+ into Hg,0 conferred tolerance by rapid volatilization and increased uptake of different forms of mercury, surpassing the concentrations found in the soil. These investigations provide novel insights for improvement of plant tolerance and detoxification of mercury. PMID:18200876

Characterization and Comparative Analysis of the Complete Chloroplast Genome of the Critically Endangered Species Streptocarpus teitensis (Gesneriaceae).

PubMed

Kyalo, Cornelius M; Gichira, Andrew W; Li, Zhi-Zhong; Saina, Josphat K; Malombe, Itambo; Hu, Guang-Wan; Wang, Qing-Feng

2018-01-01

Streptocarpus teitensis (Gesneriaceae) is an endemic species listed as critically endangered in the International Union for Conservation of Nature (IUCN) red list of threatened species. However, the sequence and genome information of this species remains to be limited. In this article, we present the complete chloroplast genome structure of Streptocarpus teitensis and its evolution inferred through comparative studies with other related species. S. teitensis displayed a chloroplast genome size of 153,207 bp, sheltering a pair of inverted repeats (IR) of 25,402 bp each split by small and large single-copy (SSC and LSC) regions of 18,300 and 84,103 bp, respectively. The chloroplast genome was observed to contain 116 unique genes, of which 80 are protein-coding, 32 are transfer RNAs, and four are ribosomal RNAs. In addition, a total of 196 SSR markers were detected in the chloroplast genome of Streptocarpus teitensis with mononucleotides (57.1%) being the majority, followed by trinucleotides (33.2%) and dinucleotides and tetranucleotides (both 4.1%), and pentanucleotides being the least (1.5%). Genome alignment indicated that this genome was comparable to other sequenced members of order Lamiales. The phylogenetic analysis suggested that Streptocarpus teitensis is closely related to Lysionotus pauciflorus and Dorcoceras hygrometricum .

The complete chloroplast DNA sequences of the charophycean green algae Staurastrum and Zygnema reveal that the chloroplast genome underwent extensive changes during the evolution of the Zygnematales

PubMed Central

Turmel, Monique; Otis, Christian; Lemieux, Claude

2005-01-01

Background The Streptophyta comprise all land plants and six monophyletic groups of charophycean green algae. Phylogenetic analyses of four genes from three cellular compartments support the following branching order for these algal lineages: Mesostigmatales, Chlorokybales, Klebsormidiales, Zygnematales, Coleochaetales and Charales, with the last lineage being sister to land plants. Comparative analyses of the Mesostigma viride (Mesostigmatales) and land plant chloroplast genome sequences revealed that this genome experienced many gene losses, intron insertions and gene rearrangements during the evolution of charophyceans. On the other hand, the chloroplast genome of Chaetosphaeridium globosum (Coleochaetales) is highly similar to its land plant counterparts in terms of gene content, intron composition and gene order, indicating that most of the features characteristic of land plant chloroplast DNA (cpDNA) were acquired from charophycean green algae. To gain further insight into when the highly conservative pattern displayed by land plant cpDNAs originated in the Streptophyta, we have determined the cpDNA sequences of the distantly related zygnematalean algae Staurastrum punctulatum and Zygnema circumcarinatum. Results The 157,089 bp Staurastrum and 165,372 bp Zygnema cpDNAs encode 121 and 125 genes, respectively. Although both cpDNAs lack an rRNA-encoding inverted repeat (IR), they are substantially larger than Chaetosphaeridium and land plant cpDNAs. This increased size is explained by the expansion of intergenic spacers and introns. The Staurastrum and Zygnema genomes differ extensively from one another and from their streptophyte counterparts at the level of gene order, with the Staurastrum genome more closely resembling its land plant counterparts than does Zygnema cpDNA. Many intergenic regions in Zygnema cpDNA harbor tandem repeats. The introns in both Staurastrum (8 introns) and Zygnema (13 introns) cpDNAs represent subsets of those found in land plant cp

Importance of the green color, absorption gradient, and spectral absorption of chloroplasts for the radiative energy balance of leaves.

PubMed

Kume, Atsushi

2017-05-01

Terrestrial green plants absorb photosynthetically active radiation (PAR; 400-700 nm) but do not absorb photons evenly across the PAR waveband. The spectral absorbance of photosystems and chloroplasts is lowest for green light, which occurs within the highest irradiance waveband of direct solar radiation. We demonstrate a close relationship between this phenomenon and the safe and efficient utilization of direct solar radiation in simple biophysiological models. The effects of spectral absorptance on the photon and irradiance absorption processes are evaluated using the spectra of direct and diffuse solar radiation. The radiation absorption of a leaf arises as a consequence of the absorption of chloroplasts. The photon absorption of chloroplasts is strongly dependent on the distribution of pigment concentrations and their absorbance spectra. While chloroplast movements in response to light are important mechanisms controlling PAR absorption, they are not effective for green light because chloroplasts have the lowest spectral absorptance in the waveband. With the development of palisade tissue, the incident photons per total palisade cell surface area and the absorbed photons per chloroplast decrease. The spectral absorbance of carotenoids is effective in eliminating shortwave PAR (<520 nm), which contains much of the surplus energy that is not used for photosynthesis and is dissipated as heat. The PAR absorptance of a whole leaf shows no substantial difference based on the spectra of direct or diffuse solar radiation. However, most of the near infrared radiation is unabsorbed and heat stress is greatly reduced. The incident solar radiation is too strong to be utilized for photosynthesis under the current CO 2 concentration in the terrestrial environment. Therefore, the photon absorption of a whole leaf is efficiently regulated by photosynthetic pigments with low spectral absorptance in the highest irradiance waveband and through a combination of pigment density

Chloroplast biogenesis at cold-hardening temperatures. Kinetics of trans-Δ3-hexadecenoic acid accumulation and the assembly of LHCII.

PubMed

Krol, M; Huner, N P; Williams, J P; Maissan, E

1988-02-01

Etiolated seedlings developed at cold-hardening temperatures (5°C) exhibited etioplasts with considerable vesiculation of internal membranes compared to etioplasts developed at 20°C regardless of the osmotic concentration employed during sample preparation. This vesiculation disappeared during exposure to continuous light at 5°C. This transformation of 5°C and 20°C etioplasts to chloroplasts under continuous light at 5° and 20°C respectively proceeded normally with the initial development of non-appressed lamellae and the subsequent appearance of granal stacks. However, chloroplasts developed at 5°C exhibited fewer lamellae per granum than chloroplasts developed at 20°C.Although the polypeptide complements of etioplasts and chloroplasts developed at 5° or 20°C were not significantly different, monomeric light harvesting complex (LHCII3) was assembled into oligomeric light harvesting complex (LHCII1) during chloroplast biogenesis at 20°C (oligomer:monomer =1.8) whereas monomeric LHCII predominated at 5°C (oligomer:monomer =0.3). Low temperature fluorescence emission spectra of isolated thylakoids indicated that both the F685/F735 and F695/F735 were significantly higher after greening at 5°C than at 20°C. In addition, chloroplast biogenesis at 5°C was associated with a low ratio of trans-Δ3-hexadecenoic acid (0.5) in phosphatidylglycerol whereas at 20°C biogenesis was associated with a high ratio (1.6). Comparative kinetics indicated that the maximization of the trans-Δ3-hexadecenoic acid level precedes the assembly of monomeric LHCII into oligomeric LHCII during biogenesis at 20°C. It is suggested that low developmental temperatures modulate the assembly of LHCII by reducing the trans-Δ3-hexadecenoic acid content of phosphatidylglycerol such that monomeric or some intermediate form of LHCII predominates.

The complete chloroplast genome sequences of Lychnis wilfordii and Silene capitata and comparative analyses with other Caryophyllaceae genomes.

PubMed

Kang, Jong-Soo; Lee, Byoung Yoon; Kwak, Myounghai

2017-01-01

The complete chloroplast genomes of Lychnis wilfordii and Silene capitata were determined and compared with ten previously reported Caryophyllaceae chloroplast genomes. The chloroplast genome sequences of L. wilfordii and S. capitata contain 152,320 bp and 150,224 bp, respectively. The gene contents and orders among 12 Caryophyllaceae species are consistent, but several microstructural changes have occurred. Expansion of the inverted repeat (IR) regions at the large single copy (LSC)/IRb and small single copy (SSC)/IR boundaries led to partial or entire gene duplications. Additionally, rearrangements of the LSC region were caused by gene inversions and/or transpositions. The 18 kb inversions, which occurred three times in different lineages of tribe Sileneae, were thought to be facilitated by the intermolecular duplicated sequences. Sequence analyses of the L. wilfordii and S. capitata genomes revealed 39 and 43 repeats, respectively, including forward, palindromic, and reverse repeats. In addition, a total of 67 and 56 simple sequence repeats were discovered in the L. wilfordii and S. capitata chloroplast genomes, respectively. Finally, we constructed phylogenetic trees of the 12 Caryophyllaceae species and two Amaranthaceae species based on 73 protein-coding genes using both maximum parsimony and likelihood methods.

Starch synthase 4 is essential for coordination of starch granule formation with chloroplast division during Arabidopsis leaf expansion

PubMed Central

Crumpton-Taylor, Matilda; Pike, Marilyn; Lu, Kuan-Jen; Hylton, Christopher M; Feil, Regina; Eicke, Simona; Lunn, John E; Zeeman, Samuel C; Smith, Alison M

2013-01-01

Arabidopsis thaliana mutants lacking the SS4 isoform of starch synthase have strongly reduced numbers of starch granules per chloroplast, suggesting that SS4 is necessary for the normal generation of starch granules. To establish whether it plays a direct role in this process, we investigated the circumstances in which granules are formed in ss4 mutants. Starch granule numbers and distribution and the accumulation of starch synthase substrates and products were investigated during ss4 leaf development, and in ss4 mutants carrying mutations or transgenes that affect starch turnover or chloroplast volume. We found that immature ss4 leaves have no starch granules, but accumulate high concentrations of the starch synthase substrate ADPglucose. Granule numbers are partially restored by elevating the capacity for glucan synthesis (via expression of bacterial glycogen synthase) or by increasing the volumes of individual chloroplasts (via introduction of arc mutations). However, these granules are abnormal in distribution, size and shape. SS4 is an essential component of a mechanism that coordinates granule formation with chloroplast division during leaf expansion and determines the abundance and the flattened, discoid shape of leaf starch granules. PMID:23952675

Chloroplastic responses of ponderosa pine (Pinus ponderosa) seedlings to ozone exposure.

PubMed

Anderson, Paul D; Palmer, Brent; Houpis, James L J; Smith, Mary K; Pushnik, James C

2003-06-01

Integrity of chloroplast membranes is essential to photosynthesis. Loss of thylakoid membrane integrity has been proposed as a consequence of ozone (O(3)) exposure and therefore may be a mechanistic basis for decreased photosynthetic rates commonly associated with ozone exposure. To investigate this hypothesis, Pinus ponderosa seedlings were exposed to ambient air or ozone concentrations maintained at 0.15 or 0.30 microliter l(-1) for 10 h day(-1) for 51 days during their second growing season. Over the course of the study, foliage samples were periodically collected for thylakoid membrane, chlorophyll and protein analyses. Additionally, gas-exchange measurements were made in conjunction with foliage sampling to verify that observed chloroplastic responses were associated with ozone-induced changes in photosynthesis. Needles exposed to elevated ozone exhibited decreases in chlorophyll a and b content. The decreases were dependent on the duration and intensity of ozone exposure. When based on equal amounts of chlorophyll, ozone-exposed sample tissue exhibited an increase in total protein. When based on equal amounts of protein, ozone-exposed samples exhibited an increase in 37 kDa proteins, possibly consisting of breakdown products, and a possible decrease in 68 kDa proteins, Rubisco small subunit. There was also a change in the ratio of Photosystem I protein complexes CPI and CPII that may have contributed to decreased photosynthesis. Net photosynthetic rates were decreased in the high ozone treatment suggesting that observed structural and biochemical changes in the chloroplast were associated with alterations of the photosynthetic process.

Identification of two structurally related proteins involved in proteolytic processing of precursors targeted to the chloroplast.

PubMed Central

Oblong, J E; Lamppa, G K

1992-01-01

Two proteins of 145 and 143 kDa were identified in pea which co-purify with a chloroplast processing activity that cleaves the precursor for the major light-harvesting chlorophyll binding protein (preLHCP). Antiserum generated against the 145/143 kDa doublet recognizes only these two polypeptides in a chloroplast soluble extract. In immunodepletion experiments the antiserum removed the doublet, and there was a concomitant loss of cleavage of preLHCP as well as of precursors for the small subunit of Rubisco and the acyl carrier protein. The 145 and 143 kDa proteins co-eluted in parallel with the peak of processing activity during all fractionation procedures, but they were not detectable as a homo- or heterodimeric complex. The 145 and 143 kDa proteins were used separately to affinity purify immunoglobulins; each preparation recognized both polypeptides, indicating that they are antigenically related. Wheat chloroplasts contain a soluble species similar in size to the 145/143 kDa doublet. Images PMID:1385116

Expression of eukaryotic polypeptides in chloroplasts

DOEpatents

Mayfield, Stephen P.

2013-06-04

The present invention relates to a gene expression system in eukaryotic and prokaryotic cells, preferably plant cells and intact plants. In particular, the invention relates to an expression system having a RB47 binding site upstream of a translation initiation site for regulation of translation mediated by binding of RB47 protein, a member of the poly(A) binding protein family. Regulation is further effected by RB60, a protein disulfide isomerase. The expression system is capable of functioning in the nuclear/cytoplasm of cells and in the chloroplast of plants. Translation regulation of a desired molecule is enhanced approximately 100 fold over that obtained without RB47 binding site activation.

Temperature-induced lipocalin (TIL) is translocated under salt stress and protects chloroplasts from ion toxicity.

PubMed

Abo-Ogiala, Atef; Carsjens, Caroline; Diekmann, Heike; Fayyaz, Payam; Herrfurth, Cornelia; Feussner, Ivo; Polle, Andrea

2014-02-15

Temperature-induced lipocalins (TIL) have been invoked in the defense from heat, cold and oxidative stress. Here we document a function of TIL for basal protection from salinity stress. Heterologous expression of TIL from the salt resistant poplar Populus euphratica did not rescue growth but prevented chlorophyll b destruction in salt-exposed Arabidopsis thaliana. The protein was localized to the plasma membrane but was re-translocated to the symplast under salt stress. The A. thaliana knock out and knock down lines Attil1-1 and Attil1-2 showed stronger stress symptoms and stronger chlorophyll b degradation than the wildtype (WT) under excess salinity. They accumulated more chloride and sodium in chloroplasts than the WT. Chloroplast chloride accumulation was found even in the absence of salt stress. Since lipocalins are known to bind regulatory fatty acids of channel proteins as well as iron, we suggest that the salt-induced trafficking of TIL may be required for protection of chloroplasts by affecting ion homeostasis. Copyright © 2013 Elsevier GmbH. All rights reserved.

A Reliable and Non-destructive Method for Monitoring the Stromal pH in Isolated Chloroplasts Using a Fluorescent pH Probe.

PubMed

Su, Pai-Hsiang; Lai, Yen-Hsun

2017-01-01

The proton gradient established by the pH difference across a biological membrane is essential for many physiological processes, including ATP synthesis and ion and metabolite transport. Currently, ionophores are used to study proton gradients, and determine their importance to biological functions of interest. Because of the lack of an easy method for monitoring the proton gradient across the inner envelope membrane of chloroplasts (ΔpH env ), whether the concentration of ionophores used can effectively abolish the ΔpH env is not proven for most experiments. To overcome this hindrance, we tried to setup an easy method for real-time monitoring of the stromal pH in buffered, isolated chloroplasts by using fluorescent pH probes; using this method the ΔpH env can be calculated by subtracting the buffer pH from the measured stromal pH. When three fluorescent dyes, BCECF-AM [2',7'-bis-(2-carboxyethyl)-5-(and-6)-carboxyfluorescein acetoxymethyl ester], CFDA-SE [5(6)-Carboxyfluorescein diacetate succinimidyl ester] and SNARF-1 carboxylic acid acetate succinimidyl ester were incubated with isolated chloroplasts, BCECF-AM and CFDA-SE, but not the ester-formed SNARF-1 were taken up by chloroplasts and digested with esterase to release high levels of fluorescence. According to its relatively higher pKa value (6.98, near the physiological pH of the stroma), BCECF was chosen for further development. Due to shielding of the excitation and emission lights by chloroplast pigments, the ratiometric fluorescence of BCECF was highly dependent on the concentration of chloroplasts. By using a fixed concentration of chloroplasts, a highly correlated standard curve of pH to the BCECF ratiometric fluorescence with an r -square value of 0.98 was obtained, indicating the reliability of this method. Consistent with previous reports, the light-dependent formation of ΔpH env can be detected ranging from 0.15 to 0.33 pH units upon illumination. The concentration of the ionophore nigericin

Ruminal protozoal contribution to the duodenal flow of fatty acids following feeding of steers on forages differing in chloroplast content.

PubMed

Huws, S A; Lee, M R F; Kingston-Smith, A H; Kim, E J; Scott, M B; Tweed, J K S; Scollan, N D

2012-12-28

Ruminant products are criticised for their SFA content relative to PUFA, although n-6:n-3 PUFA is desirable for human health ( < 4). Rumen protozoa are rich in unsaturated fatty acids due to engulfment of PUFA-rich chloroplasts. Increasing the chloroplast content of rumen protozoa offers a potentially novel approach to enhance PUFA flow to the duodenum and subsequent incorporation into meat and milk. We evaluated protozoal contribution to duodenal n-3 PUFA flow due to intracellular chloroplast content. A total of six Holstein × Friesian steers were fed, in a two-period changeover design, either straw:concentrate (S:C, 60:40; DM basis; S:C, low chloroplast) or fresh perennial ryegrass (PRG; high chloroplast). Following 12 d adaptation to diet, ruminal protozoal and whole duodenal samples were obtained. N and fatty acid content of whole duodenum and rumen protozoal samples were assessed and protozoal 18S rDNA quantitative PCR performed, enabling calculation of protozoal N flow. The ratio of individual fatty acids:N in rumen protozoal samples was calculated to obtain protozoal fatty acid flows. Based on total fatty acid flow, contribution (%) of protozoa to individual fatty acid flows was calculated. Protozoal fatty acid data and microscopical observations revealed that protozoa were enriched with 18 : 3n-3 following PRG feeding, compared with the S:C diet, due to increased intracellular chloroplast content. However, duodenal protozoal 18S rDNA concentration post PRG feeding was low, indicating rumen retention of the protozoa. Nutrition influences the 18 : 3n-3 content of protozoa; the challenge is to increase protozoal flow to the small intestine, while maintaining sustainable rumen densities.

Megadalton Complexes in the Chloroplast Stroma of Arabidopsis thaliana Characterized by Size Exclusion Chromatography, Mass Spectrometry, and Hierarchical Clustering*

PubMed Central

Olinares, Paul Dominic B.; Ponnala, Lalit; van Wijk, Klaas J.

2010-01-01

To characterize MDa-sized macromolecular chloroplast stroma protein assemblies and to extend coverage of the chloroplast stroma proteome, we fractionated soluble chloroplast stroma in the non-denatured state by size exclusion chromatography with a size separation range up to ∼5 MDa. To maximize protein complex stability and resolution of megadalton complexes, ionic strength and composition were optimized. Subsequent high accuracy tandem mass spectrometry analysis (LTQ-Orbitrap) identified 1081 proteins across the complete native mass range. Protein complexes and assembly states above 0.8 MDa were resolved using hierarchical clustering, and protein heat maps were generated from normalized protein spectral counts for each of the size exclusion chromatography fractions; this complemented previous analysis of stromal complexes up to 0.8 MDa (Peltier, J. B., Cai, Y., Sun, Q., Zabrouskov, V., Giacomelli, L., Rudella, A., Ytterberg, A. J., Rutschow, H., and van Wijk, K. J. (2006) The oligomeric stromal proteome of Arabidopsis thaliana chloroplasts. Mol. Cell. Proteomics 5, 114–133). This combined experimental and bioinformatics analyses resolved chloroplast ribosomes in different assembly and functional states (e.g. 30, 50, and 70 S), which enabled the identification of plastid homologues of prokaryotic ribosome assembly factors as well as proteins involved in co-translational modifications, targeting, and folding. The roles of these ribosome-associating proteins will be discussed. Known RNA splice factors (e.g. CAF1/WTF1/RNC1) as well as uncharacterized proteins with RNA-binding domains (pentatricopeptide repeat, RNA recognition motif, and chloroplast ribosome maturation), RNases, and DEAD box helicases were found in various sized complexes. Chloroplast DNA (>3 MDa) was found in association with the complete heteromeric plastid-encoded DNA polymerase complex, and a dozen other DNA-binding proteins, e.g. DNA gyrase, topoisomerase, and various DNA repair enzymes. The

Chloroplast genes as genetic markers for inferring patterns of change, maternal ancestry and phylogenetic relationships among Eleusine species

PubMed Central

Agrawal, Renuka; Agrawal, Nitin; Tandon, Rajesh; Raina, Soom Nath

2013-01-01

Assessment of phylogenetic relationships is an important component of any successful crop improvement programme, as wild relatives of the crop species often carry agronomically beneficial traits. Since its domestication in East Africa, Eleusine coracana (2n = 4x = 36), a species belonging to the genus Eleusine (x = 8, 9, 10), has held a prominent place in the semi-arid regions of India, Nepal and Africa. The patterns of variation between the cultivated and wild species reported so far and the interpretations based upon them have been considered primarily in terms of nuclear events. We analysed, for the first time, the phylogenetic relationship between finger millet (E. coracana) and its wild relatives by species-specific chloroplast deoxyribonucleic acid (cpDNA) polymerase chain reaction–restriction fragment length polymorphism (PCR–RFLP) and chloroplast simple sequence repeat (cpSSR) markers/sequences. Restriction fragment length polymorphism of the seven amplified chloroplast genes/intergenic spacers (trnK, psbD, psaA, trnH–trnK, trnL–trnF, 16S and trnS–psbC), nucleotide sequencing of the chloroplast trnK gene and chloroplast microsatellite polymorphism were analysed in all nine known species of Eleusine. The RFLP of all seven amplified chloroplast genes/intergenic spacers and trnK gene sequences in the diploid (2n = 16, 18, 20) and allotetraploid (2n = 36, 38) species resulted in well-resolved phylogenetic trees with high bootstrap values. Eleusine coracana, E. africana, E. tristachya, E. indica and E. kigeziensis did not show even a single change in restriction site. Eleusine intermedia and E. floccifolia were also shown to have identical cpDNA fragment patterns. The cpDNA diversity in Eleusine multiflora was found to be more extensive than that of the other eight species. The trnK gene sequence data complemented the results obtained by PCR–RFLP. The maternal lineage of all three allotetraploid species (AABB, AADD) was the same, with E. indica being

Chloroplast genes as genetic markers for inferring patterns of change, maternal ancestry and phylogenetic relationships among Eleusine species.

PubMed

Agrawal, Renuka; Agrawal, Nitin; Tandon, Rajesh; Raina, Soom Nath

2014-01-01

Assessment of phylogenetic relationships is an important component of any successful crop improvement programme, as wild relatives of the crop species often carry agronomically beneficial traits. Since its domestication in East Africa, Eleusine coracana (2n = 4x = 36), a species belonging to the genus Eleusine (x = 8, 9, 10), has held a prominent place in the semi-arid regions of India, Nepal and Africa. The patterns of variation between the cultivated and wild species reported so far and the interpretations based upon them have been considered primarily in terms of nuclear events. We analysed, for the first time, the phylogenetic relationship between finger millet (E. coracana) and its wild relatives by species-specific chloroplast deoxyribonucleic acid (cpDNA) polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) and chloroplast simple sequence repeat (cpSSR) markers/sequences. Restriction fragment length polymorphism of the seven amplified chloroplast genes/intergenic spacers (trnK, psbD, psaA, trnH-trnK, trnL-trnF, 16S and trnS-psbC), nucleotide sequencing of the chloroplast trnK gene and chloroplast microsatellite polymorphism were analysed in all nine known species of Eleusine. The RFLP of all seven amplified chloroplast genes/intergenic spacers and trnK gene sequences in the diploid (2n = 16, 18, 20) and allotetraploid (2n = 36, 38) species resulted in well-resolved phylogenetic trees with high bootstrap values. Eleusine coracana, E. africana, E. tristachya, E. indica and E. kigeziensis did not show even a single change in restriction site. Eleusine intermedia and E. floccifolia were also shown to have identical cpDNA fragment patterns. The cpDNA diversity in Eleusine multiflora was found to be more extensive than that of the other eight species. The trnK gene sequence data complemented the results obtained by PCR-RFLP. The maternal lineage of all three allotetraploid species (AABB, AADD) was the same, with E. indica being the

Characterization of mango (Mangifera indica L.) transcriptome and chloroplast genome.

PubMed

Azim, M Kamran; Khan, Ishtaiq A; Zhang, Yong

2014-05-01

We characterized mango leaf transcriptome and chloroplast genome using next generation DNA sequencing. The RNA-seq output of mango transcriptome generated >12 million reads (total nucleotides sequenced >1 Gb). De novo transcriptome assembly generated 30,509 unigenes with lengths in the range of 300 to ≥3,000 nt and 67× depth of coverage. Blast searching against nonredundant nucleotide databases and several Viridiplantae genomic datasets annotated 24,593 mango unigenes (80% of total) and identified Citrus sinensis as closest neighbor of mango with 9,141 (37%) matched sequences. The annotation with gene ontology and Clusters of Orthologous Group terms categorized unigene sequences into 57 and 25 classes, respectively. More than 13,500 unigenes were assigned to 293 KEGG pathways. Besides major plant biology related pathways, KEGG based gene annotation pointed out active presence of an array of biochemical pathways involved in (a) biosynthesis of bioactive flavonoids, flavones and flavonols, (b) biosynthesis of terpenoids and lignins and (c) plant hormone signal transduction. The mango transcriptome sequences revealed 235 proteases belonging to five catalytic classes of proteolytic enzymes. The draft genome of mango chloroplast (cp) was obtained by a combination of Sanger and next generation sequencing. The draft mango cp genome size is 151,173 bp with a pair of inverted repeats of 27,093 bp separated by small and large single copy regions, respectively. Out of 139 genes in mango cp genome, 91 found to be protein coding. Sequence analysis revealed cp genome of C. sinensis as closest neighbor of mango. We found 51 short repeats in mango cp genome supposed to be associated with extensive rearrangements. This is the first report of transcriptome and chloroplast genome analysis of any Anacardiaceae family member.

Most of ADP·glucose linked to starch biosynthesis occurs outside the chloroplast in source leaves

PubMed Central

Baroja-Fernández, Edurne; Muñoz, Francisco José; Zandueta-Criado, Aitor; Morán-Zorzano, María Teresa; Viale, Alejandro Miguel; Alonso-Casajús, Nora; Pozueta-Romero, Javier

2004-01-01

Sucrose and starch are end products of two segregated gluconeogenic pathways, and their production takes place in the cytosol and chloroplast of green leaves, respectively. According to this view, the plastidial ADP·glucose (ADPG) pyrophosphorylase (AGP) is the sole enzyme catalyzing the synthesis of the starch precursor molecule ADPG. However, a growing body of evidences indicates that starch formation involves the import of cytosolic ADPG to the chloroplast. This evidence is consistent with the idea that synthesis of the ADPG linked to starch biosynthesis takes place in the cytosol by means of sucrose synthase, whereas AGP channels the glucose units derived from the starch breakdown. To test this hypothesis, we first investigated the subcellular localization of ADPG. Toward this end, we constructed transgenic potato plants that expressed the ADPG-cleaving adenosine diphosphate sugar pyrophosphatase (ASPP) from Escherichia coli either in the chloroplast or in the cytosol. Source leaves from plants expressing ASPP in the chloroplast exhibited reduced starch and normal ADPG content as compared with control plants. Most importantly however, leaves from plants expressing ASPP in the cytosol showed a large reduction of the levels of both ADPG and starch, whereas hexose phosphates increased as compared with control plants. No pleiotropic changes in photosynthetic parameters and maximum catalytic activities of enzymes closely linked to starch and sucrose metabolism could be detected in the leaves expressing ASPP in the cytosol. The overall results show that, essentially similar to cereal endosperms, most of the ADPG linked to starch biosynthesis in source leaves occurs in the cytosol. PMID:15326306

The Arabidopsis ppi1 Mutant Is Specifically Defective in the Expression, Chloroplast Import, and Accumulation of Photosynthetic ProteinsW⃞

PubMed Central

Kubis, Sybille; Baldwin, Amy; Patel, Ramesh; Razzaq, Azam; Dupree, Paul; Lilley, Kathryn; Kurth, Joachim; Leister, Dario; Jarvis, Paul

2003-01-01

The import of nucleus-encoded proteins into chloroplasts is mediated by translocon complexes in the envelope membranes. A component of the translocon in the outer envelope membrane, Toc34, is encoded in Arabidopsis by two homologous genes, atTOC33 and atTOC34. Whereas atTOC34 displays relatively uniform expression throughout development, atTOC33 is strongly upregulated in rapidly growing, photosynthetic tissues. To understand the reason for the existence of these two related genes, we characterized the atTOC33 knockout mutant ppi1. Immunoblotting and proteomics revealed that components of the photosynthetic apparatus are deficient in ppi1 chloroplasts and that nonphotosynthetic chloroplast proteins are unchanged or enriched slightly. Furthermore, DNA array analysis of 3292 transcripts revealed that photosynthetic genes are moderately, but specifically, downregulated in ppi1. Proteome differences in ppi1 could be correlated with protein import rates: ppi1 chloroplasts imported the ribulose-1,5-bisphosphate carboxylase/oxygenase small subunit and 33-kD oxygen-evolving complex precursors at significantly reduced rates, but the import of a 50S ribosomal subunit precursor was largely unaffected. The ppi1 import defect occurred at the level of preprotein binding, which is consistent with a role for atToc33 during preprotein recognition. The data suggest that atToc33 is involved preferentially in the import of photosynthetic proteins and, by extension, that atToc34 is involved in the import of nonphotosynthetic chloroplast proteins. PMID:12897258

Gain and Loss of Photosynthetic Membranes during Plastid Differentiation in the Shoot Apex of Arabidopsis[W

PubMed Central

Charuvi, Dana; Kiss, Vladimir; Nevo, Reinat; Shimoni, Eyal; Adam, Zach; Reich, Ziv

2012-01-01

Chloroplasts of higher plants develop from proplastids, which are undifferentiated plastids that lack photosynthetic (thylakoid) membranes. In flowering plants, the proplastid-chloroplast transition takes place at the shoot apex, which consists of the shoot apical meristem (SAM) and the flanking leaf primordia. It has been believed that the SAM contains only proplastids and that these become chloroplasts only in the primordial leaves. Here, we show that plastids of the SAM are neither homogeneous nor necessarily null. Rather, their developmental state varies with the specific region and/or layer of the SAM in which they are found. Plastids throughout the L1 and L3 layers of the SAM possess fairly developed thylakoid networks. However, many of these plastids eventually lose their thylakoids during leaf maturation. By contrast, plastids at the central, stem cell–harboring region of the L2 layer of the SAM lack thylakoid membranes; these appear only at the periphery, near the leaf primordia. Thus, plastids in the SAM undergo distinct differentiation processes that, depending on their lineage and position, lead to either development or loss of thylakoid membranes. These processes continue along the course of leaf maturation. PMID:22438022

Electrons and proton transfer in chloroplasts in silico: 1. The effect of topological factors on energy coupling in chloroplasts with a nonuniform distribution of protein complexes

NASA Astrophysics Data System (ADS)

Vershubskii, A. V.; Tikhonov, A. N.

2017-05-01

This paper presents a theoretical study of the effects of topological factors (density of thylakoid packing in grana) on the efficiency of energy coupling in chloroplasts. The study is based on a mathematical model of electron and proton transport processes coupled to ATP synthesis in chloroplasts. The model was developed by the authors earlier, and the nonuniform distribution of electron transport and ATP synthase complexes in the membranes of granal and intergranal thylakoids was taken into account in the model. The results of numerical experiments enabled the analysis of the distribution of lateral profiles of the transmembrane pH difference and the concentrations of mobile plastoquinone and plastocyanin electron transporters in granal and intergranal thylakoids and the dependence of this distribution on the metabolic state of class B chloroplasts (photosynthetic control state or the conditions of intensive ATP synthesis). Moreover, the influence of topological factors (the density of thylakoid packing in grana and the degree of thylakoid swelling) that affect the rate of diffusion of protons and mobile electron carriers in the intrathylakoid space and in the interthylakoidal gap was investigated. The results of numerical experiments that involved the variation of geometric parameters of the system revealed the influence of thylakoid thickness and the distance between the granal thylakoids on the lateral pH profiles inside the thylakoids (pHi) and in the interthylakoidal gap (pHo). Acidification of the intrathylakoid space characterized by the pHi value increased concomitantly to the increase of the width of the interthylakoidal gap l o and decreased concomitantly to the increase of the width of the intrathylakoidal space l i.

The complete chloroplast genome sequence of Dodonaea viscosa: comparative and phylogenetic analyses.

PubMed

Saina, Josphat K; Gichira, Andrew W; Li, Zhi-Zhong; Hu, Guang-Wan; Wang, Qing-Feng; Liao, Kuo

2018-02-01

The plant chloroplast (cp) genome is a highly conserved structure which is beneficial for evolution and systematic research. Currently, numerous complete cp genome sequences have been reported due to high throughput sequencing technology. However, there is no complete chloroplast genome of genus Dodonaea that has been reported before. To better understand the molecular basis of Dodonaea viscosa chloroplast, we used Illumina sequencing technology to sequence its complete genome. The whole length of the cp genome is 159,375 base pairs (bp), with a pair of inverted repeats (IRs) of 27,099 bp separated by a large single copy (LSC) 87,204 bp, and small single copy (SSC) 17,972 bp. The annotation analysis revealed a total of 115 unique genes of which 81 were protein coding, 30 tRNA, and four ribosomal RNA genes. Comparative genome analysis with other closely related Sapindaceae members showed conserved gene order in the inverted and single copy regions. Phylogenetic analysis clustered D. viscosa with other species of Sapindaceae with strong bootstrap support. Finally, a total of 249 SSRs were detected. Moreover, a comparison of the synonymous (Ks) and nonsynonymous (Ka) substitution rates in D. viscosa showed very low values. The availability of cp genome reported here provides a valuable genetic resource for comprehensive further studies in genetic variation, taxonomy and phylogenetic evolution of Sapindaceae family. In addition, SSR markers detected will be used in further phylogeographic and population structure studies of the species in this genus.

Complete Chloroplast Genome of the Wollemi Pine (Wollemia nobilis): Structure and Evolution.

PubMed

Yap, Jia-Yee S; Rohner, Thore; Greenfield, Abigail; Van Der Merwe, Marlien; McPherson, Hannah; Glenn, Wendy; Kornfeld, Geoff; Marendy, Elessa; Pan, Annie Y H; Wilton, Alan; Wilkins, Marc R; Rossetto, Maurizio; Delaney, Sven K

2015-01-01

The Wollemi pine (Wollemia nobilis) is a rare Southern conifer with striking morphological similarity to fossil pines. A small population of W. nobilis was discovered in 1994 in a remote canyon system in the Wollemi National Park (near Sydney, Australia). This population contains fewer than 100 individuals and is critically endangered. Previous genetic studies of the Wollemi pine have investigated its evolutionary relationship with other pines in the family Araucariaceae, and have suggested that the Wollemi pine genome contains little or no variation. However, these studies were performed prior to the widespread use of genome sequencing, and their conclusions were based on a limited fraction of the Wollemi pine genome. In this study, we address this problem by determining the entire sequence of the W. nobilis chloroplast genome. A detailed analysis of the structure of the genome is presented, and the evolution of the genome is inferred by comparison with the chloroplast sequences of other members of the Araucariaceae and the related family Podocarpaceae. Pairwise alignments of whole genome sequences, and the presence of unique pseudogenes, gene duplications and insertions in W. nobilis and Araucariaceae, indicate that the W. nobilis chloroplast genome is most similar to that of its sister taxon Agathis. However, the W. nobilis genome contains an unusually high number of repetitive sequences, and these could be used in future studies to investigate and conserve any remnant genetic diversity in the Wollemi pine.

Complete Chloroplast Genome of the Wollemi Pine (Wollemia nobilis): Structure and Evolution

PubMed Central

Yap, Jia-Yee S.; Rohner, Thore; Greenfield, Abigail; Van Der Merwe, Marlien; McPherson, Hannah; Glenn, Wendy; Kornfeld, Geoff; Marendy, Elessa; Pan, Annie Y. H.; Wilkins, Marc R.; Rossetto, Maurizio; Delaney, Sven K.

2015-01-01

The Wollemi pine (Wollemia nobilis) is a rare Southern conifer with striking morphological similarity to fossil pines. A small population of W. nobilis was discovered in 1994 in a remote canyon system in the Wollemi National Park (near Sydney, Australia). This population contains fewer than 100 individuals and is critically endangered. Previous genetic studies of the Wollemi pine have investigated its evolutionary relationship with other pines in the family Araucariaceae, and have suggested that the Wollemi pine genome contains little or no variation. However, these studies were performed prior to the widespread use of genome sequencing, and their conclusions were based on a limited fraction of the Wollemi pine genome. In this study, we address this problem by determining the entire sequence of the W. nobilis chloroplast genome. A detailed analysis of the structure of the genome is presented, and the evolution of the genome is inferred by comparison with the chloroplast sequences of other members of the Araucariaceae and the related family Podocarpaceae. Pairwise alignments of whole genome sequences, and the presence of unique pseudogenes, gene duplications and insertions in W. nobilis and Araucariaceae, indicate that the W. nobilis chloroplast genome is most similar to that of its sister taxon Agathis. However, the W. nobilis genome contains an unusually high number of repetitive sequences, and these could be used in future studies to investigate and conserve any remnant genetic diversity in the Wollemi pine. PMID:26061691

Chloroplast microsatellite markers for Artocarpus (Moraceae) developed from transcriptome sequences1

PubMed Central

Gardner, Elliot M.; Laricchia, Kristen M.; Murphy, Matthew; Ragone, Diane; Scheffler, Brian E.; Simpson, Sheron; Williams, Evelyn W.; Zerega, Nyree J. C.

2015-01-01

Premise of the study: Chloroplast microsatellite loci were characterized from transcriptomes of Artocarpus altilis (breadfruit) and A. camansi (breadnut). They were tested in A. odoratissimus (terap) and A. altilis and evaluated in silico for two congeners. Methods and Results: Fifteen simple sequence repeats (SSRs) were identified in chloroplast sequences from four Artocarpus transcriptome assemblies. The markers were evaluated using capillary electrophoresis in A. odoratissimus (105 accessions) and A. altilis (73). They were also evaluated in silico in A. altilis (10), A. camansi (6), and A. altilis × A. mariannensis (7) transcriptomes. All loci were polymorphic in at least one species, with all 15 polymorphic in A. camansi. Per species, average alleles per locus ranged between 2.2 and 2.5. Three loci had evidence of fragment-length homoplasy. Conclusions: These markers will complement existing nuclear markers by enabling confident identification of maternal and clone lines, which are often important in vegetatively propagated crops such as breadfruit. PMID:26421253

Role of Temperature Stress on Chloroplast Biogenesis and Protein Import in Pea1[OA

PubMed Central

Dutta, Siddhartha; Mohanty, Sasmita; Tripathy, Baishnab C.

2009-01-01

Modulation of photosynthesis and chloroplast biogenesis, by low and high temperatures, was studied in 12-d-old pea (Pisum sativum) plants grown at 25°C and subsequently exposed to 7°C or 40°C up to 48 h. The decline in variable chlorophyll a fluorescence/maximum chlorophyll a fluorescence and estimated electron transport rate in temperature-stressed plants was substantially restored when they were transferred to room temperature. The ATP-driven import of precursor of small subunit of Rubisco (pRSS) into plastids was down-regulated by 67% and 49% in heat-stressed and chill-stressed plants, respectively. Reduction in binding of the pRSS to the chloroplast envelope membranes in heat-stressed plants could be due to the down-regulation of Toc159 gene/protein expression. In addition to impaired binding, reduced protein import into chloroplast in heat-stressed plants was likely due to decreased gene/protein expression of certain components of the TOC complex (Toc75), the TIC complex (Tic20, Tic32, Tic55, and Tic62), stromal Hsp93, and stromal processing peptidase. In chill-stressed plants, the gene/protein expression of most of the components of protein import apparatus other than Tic110 and Tic40 were not affected, suggesting the central role of Tic110 and Tic40 in inhibition of protein import at low temperature. Heating of intact chloroplasts at 35°C for 10 min inhibited protein import, implying a low thermal stability of the protein import apparatus. Results demonstrate that in addition to decreased gene and protein expression, down-regulation of photosynthesis in temperature-stressed plants is caused by reduced posttranslational import of plastidic proteins required for the replacement of impaired proteins coded by nuclear genome. PMID:19403728

Chloroplast avoidance movement as a sensitive indicator of relative water content during leaf desiccation in the dark.

PubMed

Nauš, Jan; Šmecko, Slavomír; Špundová, Martina

2016-08-01

In the context of global climate change, drought is one of the major stress factors with negative effect on photosynthesis and plant productivity. Currently, chlorophyll fluorescence parameters are widely used as indicators of plant stress, mainly owing to the rapid, non-destructive and simple measurements this technique allows. However, these parameters have been shown to have limited sensitivity for the monitoring of water deficit as leaf desiccation has relatively small effect on photosystem II photochemistry. In this study, we found that blue light-induced increase in leaf transmittance reflecting chloroplast avoidance movement was much more sensitive to a decrease in relative water content (RWC) than chlorophyll fluorescence parameters in dark-desiccating leaves of tobacco (Nicotiana tabacum L.) and barley (Hordeum vulgare L.). Whereas the inhibition of chloroplast avoidance movement was detectable in leaves even with a small RWC decrease, the chlorophyll fluorescence parameters (F V/F M, V J, Ф PSII, NPQ) changed markedly only when RWC dropped below 70 %. For this reason, we propose light-induced chloroplast avoidance movement as a sensitive indicator of the decrease in leaf RWC. As our measurement of chloroplast movement using collimated transmittance is simple and non-destructive, it may be more suitable in some cases for the detection of plant stresses including water deficit than the conventionally used chlorophyll fluorescence methods.

2010 GORDON RESEARCH CONFERENCE ON MITOCHONDRIA & CHLOROPLASTS, LUCCA, ITALY, JULY 11-16, 2010

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

Alice Barkan

2010-07-16

The 2010 GRC on Mitochondria & Chloroplasts will assemble an international group of molecular, structural and cellular biologists, biochemists and geneticists investigating a broad spectrum of fundamental problems related to the biology of these organelles in animal, plant and fungal cells. This field has witnessed an extraordinary expansion in recent years, fueled by the discovery of the role of mitochondria in human disease and ageing, and of the synergy of chloroplasts and mitochondria in energetic output, the identification of novel factors involved in organelle division, movement, signaling and acclimation to changing environmental conditions, and by the powerful tools of organellemore » proteomics. The 2010 GRC will highlight advances in the elucidation of molecular mechanisms of organelle biogenesis including regulation of genome structure, evolution and expression, organellar protein import, assembly and turnover of respiratory and photosynthetic complexes, bidirectional signaling between organelles and nucleus, organelle morphology and dynamics, and the integration of cellular metabolism. We will also explore progress in mechanisms of disease and ageing/ senescence in animals and plants. The organellar field has forged new fronts toward a global and comprehensive understanding of mitochondrial and chloroplast biology at the molecular level. Many of the molecules under study in model organisms are responsible for human diseases, providing significant impetus for a meeting that encourages interactions between mammalian, fungal and plant organellar biologists.« less

Comparative Chloroplast Genomes of Photosynthetic Orchids: Insights into Evolution of the Orchidaceae and Development of Molecular Markers for Phylogenetic Applications

PubMed Central

Niu, Zhi-Tao; Liu, Wei; Xue, Qing-Yun; Ding, Xiao-Yu

2014-01-01

The orchid family Orchidaceae is one of the largest angiosperm families, including many species of important economic value. While chloroplast genomes are very informative for systematics and species identification, there is very limited information available on chloroplast genomes in the Orchidaceae. Here, we report the complete chloroplast genomes of the medicinal plant Dendrobium officinale and the ornamental orchid Cypripedium macranthos, demonstrating their gene content and order and potential RNA editing sites. The chloroplast genomes of the above two species and five known photosynthetic orchids showed similarities in structure as well as gene order and content, but differences in the organization of the inverted repeat/small single-copy junction and ndh genes. The organization of the inverted repeat/small single-copy junctions in the chloroplast genomes of these orchids was classified into four types; we propose that inverted repeats flanking the small single-copy region underwent expansion or contraction among Orchidaceae. The AT-rich regions of the ycf1 gene in orchids could be linked to the recombination of inverted repeat/small single-copy junctions. Relative species in orchids displayed similar patterns of variation in ndh gene contents. Furthermore, fifteen highly divergent protein-coding genes were identified, which are useful for phylogenetic analyses in orchids. To test the efficiency of these genes serving as markers in phylogenetic analyses, coding regions of four genes (accD, ccsA, matK, and ycf1) were used as a case study to construct phylogenetic trees in the subfamily Epidendroideae. High support was obtained for placement of previously unlocated subtribes Collabiinae and Dendrobiinae in the subfamily Epidendroideae. Our findings expand understanding of the diversity of orchid chloroplast genomes and provide a reference for study of the molecular systematics of this family. PMID:24911363

Comparative chloroplast genomes of photosynthetic orchids: insights into evolution of the Orchidaceae and development of molecular markers for phylogenetic applications.

PubMed

Luo, Jing; Hou, Bei-Wei; Niu, Zhi-Tao; Liu, Wei; Xue, Qing-Yun; Ding, Xiao-Yu

2014-01-01

The orchid family Orchidaceae is one of the largest angiosperm families, including many species of important economic value. While chloroplast genomes are very informative for systematics and species identification, there is very limited information available on chloroplast genomes in the Orchidaceae. Here, we report the complete chloroplast genomes of the medicinal plant Dendrobium officinale and the ornamental orchid Cypripedium macranthos, demonstrating their gene content and order and potential RNA editing sites. The chloroplast genomes of the above two species and five known photosynthetic orchids showed similarities in structure as well as gene order and content, but differences in the organization of the inverted repeat/small single-copy junction and ndh genes. The organization of the inverted repeat/small single-copy junctions in the chloroplast genomes of these orchids was classified into four types; we propose that inverted repeats flanking the small single-copy region underwent expansion or contraction among Orchidaceae. The AT-rich regions of the ycf1 gene in orchids could be linked to the recombination of inverted repeat/small single-copy junctions. Relative species in orchids displayed similar patterns of variation in ndh gene contents. Furthermore, fifteen highly divergent protein-coding genes were identified, which are useful for phylogenetic analyses in orchids. To test the efficiency of these genes serving as markers in phylogenetic analyses, coding regions of four genes (accD, ccsA, matK, and ycf1) were used as a case study to construct phylogenetic trees in the subfamily Epidendroideae. High support was obtained for placement of previously unlocated subtribes Collabiinae and Dendrobiinae in the subfamily Epidendroideae. Our findings expand understanding of the diversity of orchid chloroplast genomes and provide a reference for study of the molecular systematics of this family.

Effect of cationic plastoquinone SkQ1 on electron transfer reactions in chloroplasts and mitochondria from pea seedlings.

PubMed

Samuilov, V D; Kiselevsky, D B

2015-04-01

Plastoquinone bound with decyltriphenylphosphonium cation (SkQ1) penetrating through the membrane in nanomolar concentrations inhibited H2O2 generation in cells of epidermis of pea seedling leaves that was detected by the fluorescence of 2',7'-dichlorofluorescein. Photosynthetic electron transfer in chloroplasts isolated from pea leaves is suppressed by SkQ1 at micromolar concentrations: the electron transfer in chloroplasts under the action of photosystem II or I (with silicomolybdate or methyl viologen as electron acceptors, respectively) is more sensitive to SkQ1 than under the action of photosystem II + I (with ferricyanide or p-benzoquinone as electron acceptors). SkQ1 reduced by borohydride is oxidized by ferricyanide, p-benzoquinone, and, to a lesser extent, by silicomolybdate, but not by methyl viologen. SkQ1 is not effective as an electron acceptor supporting O2 evolution from water in illuminated chloroplasts. The data on suppression of photosynthetic O2 evolution or consumption show that SkQ1, similarly to phenazine methosulfate, causes conversion of the chloroplast redox-chain from non-cyclic electron transfer mode to the cyclic mode without O2 evolution. Oxidation of NADH or succinate in mitochondria isolated from pea roots is stimulated by SkQ1.

Interactions of Lipoidal Materials and a Pyridazinone Inhibitor of Chloroplast Development

PubMed Central

Hilton, J. L.; John, J. B. St.; Christiansen, M. N.; Norris, K. H.

1971-01-01

Formation of chloroplast pigments was inhibited, and free fatty acids accumulated in mustard (Brassica juncea [L.] Coss.) cotyledons and in barley (Hordeum vulgare L.) first leaves developed after treatment with 4-chloro-5- (dimethylamino)-2- (α, α, α-trifluoro-m-tolyl) -3 (2H) -pyridazinone. The inhibitor reduced the amount of fatty acids found in polar lipids (galactolipids) of barley chloroplasts and increased the amount in nonpolar lipids while having little effect on total content of bound fatty acids. The inhibition of chlorophyll formation was circumvented by D-α-tocopherol acetate, phytol, farnesol, and squalene, and by unsaturated fatty acids and their methyl esters. The protective action can be explained partially by an interaction external to the plant whereby 4-chloro-5- (dimethylamino) -2- (α, α, α-trifluoro-m-tolyl) -3 (2H) -pyridazinone partitioned out of the aqueous phase and into the lipid phase, thus limiting availability of the inhibitor to plants. However, the amount of inhibitor reaching the cotyledons of tocopherol-protected mustard seedlngs was still in excess of the amount necessary to cause white foliage, but it failed to produce the effect. Tocopherol treatment did not prevent the 4-chloro-5- (dimethylamino) -2- (α, α, α-trifluoro-m-tolyl) -3 (2H) -pyridazinone-induced buildup of fatty acids in mustard cotyledons but did partially circumvent the effect in barley leaves. The amount of linolenic acid relative to linoleic acid was reduced in barley leaves and chloroplasts by 4-chloro-5- (dimethylamino) -2- (α, α, α-trifluoro-m-tolyl) -3 (2H) -pyridazinone action and this effect was circumvented by tocopherol. PMID:16657757

Methionine biosynthesis in higher plants. II. Purification and characterization of cystathionine beta-lyase from spinach chloroplasts.

PubMed

Droux, M; Ravanel, S; Douce, R

1995-01-10

Cystathionine beta-lyase, the second enzyme of the transsulfuration pathway leading to homocysteine synthesis was purified over 16,000-fold from spinach (Spinacia oleracea L.) leaf chloroplasts (soluble fraction). Enzyme activity was followed along the purification scheme by either a colorimetric method for the determination of cysteine or by fluorescence detection of the bimane derivative of L-homocysteine after reverse-phase HPLC. Cystathionine beta-lyase has a molecular mass of 170,000 +/- 5000 Da and consists of four identical subunits of 44,000 Da. The enzyme exhibits an absorption spectrum in the visible range with a maximum at 418 nm due to pyridoxal 5'-phosphate. The chloroplastic enzyme catalyzes alpha,beta-cleavage of the thioether L-cystathionine and the dithioacetal L-djenkolate with apparent Km values of 0.15 and 0.34 mM, respectively, and apparent Vm values corresponding to a specific activity of 13 Units mg-1. However, no activity was detected toward the disulfide L-cysteine. With either L-cystathionine and L-djenkolate as substrate, maximal activity was obtained between pH 8.3 and pH 9.0. Besides the chloroplastic enzyme form, anion exchange chromatography of a total spinach leaf extract allowed the detection of a second pool of cystathionine beta-lyase activity that is associated with the cytosolic compartment and eluted at a lower salt concentration than the chloroplastic isoform. Kinetics of inactivation of cystathionine beta-lyase by the L-alpha-(2-aminoethoxyvinyl) glycine (AVG), an analogue of L-cystathionine, are consistent with the existence of an intermediate reversible enzyme inhibitor complex (apparent inhibition constant Kappi of 110 microM) preceding the irreversible formation of a final inactivated state of the enzyme (kd = 4.8 x 10(-3) s-1). Pyridoxal 5'-phosphate free in solution binds AVG with an apparent dissociation constant Kapp in the order of 350 microM. The comparison between the Kapp (free pyridoxal 5'-phosphate) and Kappi

Chloroplast genome expansion by intron multiplication in the basal psychrophilic euglenoid Eutreptiella pomquetensis

PubMed Central

Bennett, Matthew S.; Triemer, Richard E.; Preisfeld, Angelika

2017-01-01

Background Over the last few years multiple studies have been published showing a great diversity in size of chloroplast genomes (cpGenomes), and in the arrangement of gene clusters, in the Euglenales. However, while these genomes provided important insights into the evolution of cpGenomes across the Euglenales and within their genera, only two genomes were analyzed in regard to genomic variability between and within Euglenales and Eutreptiales. To better understand the dynamics of chloroplast genome evolution in early evolving Eutreptiales, this study focused on the cpGenome of Eutreptiella pomquetensis, and the spread and peculiarities of introns. Methods The Etl. pomquetensis cpGenome was sequenced, annotated and afterwards examined in structure, size, gene order and intron content. These features were compared with other euglenoid cpGenomes as well as those of prasinophyte green algae, including Pyramimonas parkeae. Results and Discussion With about 130,561 bp the chloroplast genome of Etl. pomquetensis, a basal taxon in the phototrophic euglenoids, was considerably larger than the two other Eutreptiales cpGenomes sequenced so far. Although the detected quadripartite structure resembled most green algae and plant chloroplast genomes, the gene content of the single copy regions in Etl. pomquetensis was completely different from those observed in green algae and plants. The gene composition of Etl. pomquetensis was extensively changed and turned out to be almost identical to other Eutreptiales and Euglenales, and not to P. parkeae. Furthermore, the cpGenome of Etl. pomquetensis was unexpectedly permeated by a high number of introns, which led to a substantially larger genome. The 51 identified introns of Etl. pomquetensis showed two major unique features: (i) more than half of the introns displayed a high level of pairwise identities; (ii) no group III introns could be identified in the protein coding genes. These findings support the hypothesis that group III

CpGAVAS, an integrated web server for the annotation, visualization, analysis, and GenBank submission of completely sequenced chloroplast genome sequences

PubMed Central

2012-01-01

Background The complete sequences of chloroplast genomes provide wealthy information regarding the evolutionary history of species. With the advance of next-generation sequencing technology, the number of completely sequenced chloroplast genomes is expected to increase exponentially, powerful computational tools annotating the genome sequences are in urgent need. Results We have developed a web server CPGAVAS. The server accepts a complete chloroplast genome sequence as input. First, it predicts protein-coding and rRNA genes based on the identification and mapping of the most similar, full-length protein, cDNA and rRNA sequences by integrating results from Blastx, Blastn, protein2genome and est2genome programs. Second, tRNA genes and inverted repeats (IR) are identified using tRNAscan, ARAGORN and vmatch respectively. Third, it calculates the summary statistics for the annotated genome. Fourth, it generates a circular map ready for publication. Fifth, it can create a Sequin file for GenBank submission. Last, it allows the extractions of protein and mRNA sequences for given list of genes and species. The annotation results in GFF3 format can be edited using any compatible annotation editing tools. The edited annotations can then be uploaded to CPGAVAS for update and re-analyses repeatedly. Using known chloroplast genome sequences as test set, we show that CPGAVAS performs comparably to another application DOGMA, while having several superior functionalities. Conclusions CPGAVAS allows the semi-automatic and complete annotation of a chloroplast genome sequence, and the visualization, editing and analysis of the annotation results. It will become an indispensible tool for researchers studying chloroplast genomes. The software is freely accessible from http://www.herbalgenomics.org/cpgavas. PMID:23256920

Arrangement of photosystem II and ATP synthase in chloroplast membranes of spinach and pea.

PubMed

Daum, Bertram; Nicastro, Daniela; Austin, Jotham; McIntosh, J Richard; Kühlbrandt, Werner

2010-04-01

We used cryoelectron tomography to reveal the arrangements of photosystem II (PSII) and ATP synthase in vitreous sections of intact chloroplasts and plunge-frozen suspensions of isolated thylakoid membranes. We found that stroma and grana thylakoids are connected at the grana margins by staggered lamellar membrane protrusions. The stacking repeat of grana membranes in frozen-hydrated chloroplasts is 15.7 nm, with a 4.5-nm lumenal space and a 3.2-nm distance between the flat stromal surfaces. The chloroplast ATP synthase is confined to minimally curved regions at the grana end membranes and stroma lamellae, where it covers 20% of the surface area. In total, 85% of the ATP synthases are monomers and the remainder form random assemblies of two or more copies. Supercomplexes of PSII and light-harvesting complex II (LHCII) occasionally form ordered arrays in appressed grana thylakoids, whereas this order is lost in destacked membranes. In the ordered arrays, each membrane on either side of the stromal gap contains a two-dimensional crystal of supercomplexes, with the two lattices arranged such that PSII cores, LHCII trimers, and minor LHCs each face a complex of the same kind in the opposite membrane. Grana formation is likely to result from electrostatic interactions between these complexes across the stromal gap.

Effects of inorganic phosphate on the light dependent thylakoid energization of intact spinach chloroplasts

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

Heineke, D.; Heldt, H.W.; Stitt, M.

1989-09-01

The light dependent energization of the thylakoid membrane was analyzed in isolated intact spinach (Spinacia oleracea L.) chloroplasts incubated with different concentrations of inorganic phosphate (Pi). Two independent methods were used: (a) the accumulation of ({sup 14}C)5,5-dimethyl-2,4-oxazolidinedione and ({sup 14}C)methylamine; (b) the energy dependent chlorophyll fluorescence quenching. The inhibition of CO{sub 2} fixation by superoptimal medium Pi or by adding glyceraldehyde - an inhibitor of the Calvin cycle - leads to an increased energization of the thylakoid membrane; however, the membrane energization decreases when chloroplasts are inhibited by suboptimal Pi. This specific low phosphate effect could be partially reversed bymore » adding oxaloacetate, which regenerates the electron acceptor NADP{sup +} and stimulates linear electron transport. The energization seen in low Pi is, however, always lower than in superoptimal Pi, even in the presence of oxaloacetate. Energization recovers in the presence of low amounts of N,N{prime}-dicyclohexylcarbodiimide, which reacts with proton channels including the coupling factor 1 ATP synthase. N,N{prime}-Dicyclohexylcarbodiimide has no effect on energization of chloroplasts in superoptimal Pi. These results suggest there is a specific low phosphate proton leak in the thylakoids, and its origin is discussed.« less

The Role of Light-Dark Regulation of the Chloroplast ATP Synthase.

PubMed

Kohzuma, Kaori; Froehlich, John E; Davis, Geoffry A; Temple, Joshua A; Minhas, Deepika; Dhingra, Amit; Cruz, Jeffrey A; Kramer, David M

2017-01-01

The chloroplast ATP synthase catalyzes the light-driven synthesis of ATP and is activated in the light and inactivated in the dark by redox-modulation through the thioredoxin system. It has been proposed that this down-regulation is important for preventing wasteful hydrolysis of ATP in the dark. To test this proposal, we compared the effects of extended dark exposure in Arabidopsis lines expressing the wild-type and mutant forms of ATP synthase that are redox regulated or constitutively active. In contrast to the predictions of the model, we observed that plants with wild-type redox regulation lost photosynthetic capacity rapidly in darkness, whereas those expressing redox-insensitive form were far more stable. To explain these results, we propose that in wild-type plants, down-regulation of ATP synthase inhibits ATP hydrolysis, leading to dissipation of thylakoid proton motive force (pmf) and subsequent inhibition of protein transport across the thylakoid through the twin arginine transporter (Tat)-dependent and Sec-dependent import pathways, resulting in the selective loss of specific protein complexes. By contrast, in mutants with a redox-insensitive ATP synthase, pmf is maintained by ATP hydrolysis, thus allowing protein transport to maintain photosynthetic activities for extended periods in the dark. Hence, a basal level of Tat-dependent, as well as, Sec-dependent import activity, in the dark helps replenishes certain components of the photosynthetic complexes and thereby aids in maintaining overall complex activity. However, the influence of a dark pmf on thylakoid protein import, by itself, could not explain all the effects we observed in this study. For example, we also observed in wild type plants a large transient buildup of thylakoid pmf and nonphotochemical exciton quenching upon sudden illumination of dark adapted plants. Therefore, we conclude that down-regulation of the ATP synthase is probably not related to preventing loss of ATP per se . Instead

The Role of Light–Dark Regulation of the Chloroplast ATP Synthase

PubMed Central

Kohzuma, Kaori; Froehlich, John E.; Davis, Geoffry A.; Temple, Joshua A.; Minhas, Deepika; Dhingra, Amit; Cruz, Jeffrey A.; Kramer, David M.

2017-01-01

The chloroplast ATP synthase catalyzes the light-driven synthesis of ATP and is activated in the light and inactivated in the dark by redox-modulation through the thioredoxin system. It has been proposed that this down-regulation is important for preventing wasteful hydrolysis of ATP in the dark. To test this proposal, we compared the effects of extended dark exposure in Arabidopsis lines expressing the wild-type and mutant forms of ATP synthase that are redox regulated or constitutively active. In contrast to the predictions of the model, we observed that plants with wild-type redox regulation lost photosynthetic capacity rapidly in darkness, whereas those expressing redox-insensitive form were far more stable. To explain these results, we propose that in wild-type plants, down-regulation of ATP synthase inhibits ATP hydrolysis, leading to dissipation of thylakoid proton motive force (pmf) and subsequent inhibition of protein transport across the thylakoid through the twin arginine transporter (Tat)-dependent and Sec-dependent import pathways, resulting in the selective loss of specific protein complexes. By contrast, in mutants with a redox-insensitive ATP synthase, pmf is maintained by ATP hydrolysis, thus allowing protein transport to maintain photosynthetic activities for extended periods in the dark. Hence, a basal level of Tat-dependent, as well as, Sec-dependent import activity, in the dark helps replenishes certain components of the photosynthetic complexes and thereby aids in maintaining overall complex activity. However, the influence of a dark pmf on thylakoid protein import, by itself, could not explain all the effects we observed in this study. For example, we also observed in wild type plants a large transient buildup of thylakoid pmf and nonphotochemical exciton quenching upon sudden illumination of dark adapted plants. Therefore, we conclude that down-regulation of the ATP synthase is probably not related to preventing loss of ATP per se. Instead

Multi-functional roles for the polypeptide transport associated domains of Toc75 in chloroplast protein import

PubMed Central

Paila, Yamuna D; Richardson, Lynn GL; Inoue, Hitoshi; Parks, Elizabeth S; McMahon, James; Inoue, Kentaro; Schnell, Danny J

2016-01-01

Toc75 plays a central role in chloroplast biogenesis in plants as the membrane channel of the protein import translocon at the outer envelope of chloroplasts (TOC). Toc75 is a member of the Omp85 family of bacterial and organellar membrane insertases, characterized by N-terminal POTRA (polypeptide-transport associated) domains and C-terminal membrane-integrated β-barrels. We demonstrate that the Toc75 POTRA domains are essential for protein import and contribute to interactions with TOC receptors, thereby coupling preprotein recognition at the chloroplast surface with membrane translocation. The POTRA domains also interact with preproteins and mediate the recruitment of molecular chaperones in the intermembrane space to facilitate membrane transport. Our studies are consistent with the multi-functional roles of POTRA domains observed in other Omp85 family members and demonstrate that the domains of Toc75 have evolved unique properties specific to the acquisition of protein import during endosymbiotic evolution of the TOC system in plastids. DOI: http://dx.doi.org/10.7554/eLife.12631.001 PMID:26999824

Reduced starch granule number per chloroplast in the dpe2/phs1 mutant is dependent on initiation of starch degradation

PubMed Central

Malinova, Irina

2017-01-01

An Arabidopsis double knock-out mutant lacking cytosolic disproportionating enzyme 2 (DPE2) and the plastidial phosphorylase (PHS1) revealed a dwarf-growth phenotype, reduced starch content, an uneven distribution of starch within the plant rosette, and a reduced number of starch granules per chloroplast under standard growth conditions. In contrast, the wild type contained 5–7 starch granules per chloroplast. Mature and old leaves of the double mutant were essentially starch free and showed plastidial disintegration. Several analyses revealed that the number of starch granules per chloroplast was affected by the dark phase. So far, it was unclear if it was the dark phase per se or starch degradation in the dark that was connected to the observed decrease in the number of starch granules per chloroplast. Therefore, in the background of the double mutant dpe2/phs1, a triple mutant was generated lacking the initial starch degrading enzyme glucan, water dikinase (GWD). The triple mutant showed improved plant growth, a starch-excess phenotype, and a homogeneous starch distribution. Furthermore, the number of starch granules per chloroplast was increased and was similar to wild type. However, starch granule morphology was only slightly affected by the lack of GWD as in the triple mutant and, like in dpe2/phs1, more spherical starch granules were observed. The characterized triple mutant was discussed in the context of the generation of starch granules and the formation of starch granule morphology. PMID:29155859

Reduced starch granule number per chloroplast in the dpe2/phs1 mutant is dependent on initiation of starch degradation.

PubMed

Malinova, Irina; Fettke, Joerg

2017-01-01

An Arabidopsis double knock-out mutant lacking cytosolic disproportionating enzyme 2 (DPE2) and the plastidial phosphorylase (PHS1) revealed a dwarf-growth phenotype, reduced starch content, an uneven distribution of starch within the plant rosette, and a reduced number of starch granules per chloroplast under standard growth conditions. In contrast, the wild type contained 5-7 starch granules per chloroplast. Mature and old leaves of the double mutant were essentially starch free and showed plastidial disintegration. Several analyses revealed that the number of starch granules per chloroplast was affected by the dark phase. So far, it was unclear if it was the dark phase per se or starch degradation in the dark that was connected to the observed decrease in the number of starch granules per chloroplast. Therefore, in the background of the double mutant dpe2/phs1, a triple mutant was generated lacking the initial starch degrading enzyme glucan, water dikinase (GWD). The triple mutant showed improved plant growth, a starch-excess phenotype, and a homogeneous starch distribution. Furthermore, the number of starch granules per chloroplast was increased and was similar to wild type. However, starch granule morphology was only slightly affected by the lack of GWD as in the triple mutant and, like in dpe2/phs1, more spherical starch granules were observed. The characterized triple mutant was discussed in the context of the generation of starch granules and the formation of starch granule morphology.

Characterization of the complete chloroplast genome of Platycarya strobilacea (Juglandaceae)

Treesearch

Jing Yan; Kai Han; Shuyun Zeng; Peng Zhao; Keith Woeste; Jianfang Li; Zhan-Lin Liu

2017-01-01

The whole chloroplast genome (cp genome) sequence of Platycarya strobilacea was characterized from Illumina pair-end sequencing data. The complete cp genome was 160,994 bp in length and contained a large single copy region (LSC) of 90,225 bp and a small single copy region (SSC) of 18,371 bp, which were separated by a pair of inverted repeat regions...

Development of Chloroplast and Nuclear DNA Markers for Chinese Oaks (Quercus Subgenus Quercus) and Assessment of Their Utility as DNA Barcodes

PubMed Central

Yang, Jia; Vázquez, Lucía; Chen, Xiaodan; Li, Huimin; Zhang, Hao; Liu, Zhanlin; Zhao, Guifang

2017-01-01

Chloroplast DNA (cpDNA) is frequently used for species demography, evolution, and species discrimination of plants. However, the lack of efficient and universal markers often brings particular challenges for genetic studies across different plant groups. In this study, chloroplast genomes from two closely related species (Quercus rubra and Castanea mollissima) in Fagaceae were compared to explore universal cpDNA markers for the Chinese oak species in Quercus subgenus Quercus, a diverse species group without sufficient molecular differentiation. With the comparison, nine and 14 plastid markers were selected as barcoding and phylogeographic candidates for the Chinese oaks. Five (psbA-trnH, matK-trnK, ycf3-trnS, matK, and ycf1) of the nine plastid candidate barcodes, with the addition of newly designed ITS and a single-copy nuclear gene (SAP), were then tested on 35 Chinese oak species employing four different barcoding approaches (genetic distance-, BLAST-, character-, and tree-based methods). The four methods showed different species identification powers with character-based method performing the best. Of the seven barcodes tested, a barcoding gap was absent in all of them across the Chinese oaks, while ITS and psbA-trnH provided the highest species resolution (30.30%) with the character- and BLAST-based methods, respectively. The six-marker combination (psbA-trnH + matK-trnK + matK + ycf1 + ITS + SAP) showed the best species resolution (84.85%) using the character-based method for barcoding the Chinese oaks. The barcoding results provided additional implications for taxonomy of the Chinese oaks in subg. Quercus, basically identifying three major infrageneric clades of the Chinese oaks (corresponding to Groups Quercus, Cerris, and Ilex) referenced to previous phylogenetic classification of Quercus. While the morphology-based allocations proposed for the Chinese oaks in subg. Quercus were challenged. A low variation rate of the chloroplast genome, and complex

Changes induced by the Pepper mild mottle tobamovirus on the chloroplast proteome of Nicotiana benthamiana.

PubMed

Pineda, M; Sajnani, C; Barón, M

2010-01-01

We have analyzed the chloroplast proteome of Nicotiana benthamiana using two-dimensional gel electrophoresis and mass spectrometry followed by a database search. In order to improve the resolution of the two-dimensional electrophoresis gels, we have made separate maps for the low and the high pH range. At least 200 spots were detected. We identified 72 polypeptides, some being isoforms of different multiprotein families. In addition, changes in this chloroplast proteome induced by the infection with the Spanish strain of the Pepper mild mottle virus were investigated. Viral infection induced the down-regulation of several chloroplastidic proteins involved in both the photosynthetic electron-transport chain and the Benson-Calvin cycle.

[Effects of light intensities after anthesis on the photosynthetic characteristics and chloroplast ultrastructure in mesophyll cell of summer maize (Zea mays L. )].

PubMed

Gao, Jia; Cui, Hai Yan; Shi, Jian Guo; Dong, Shu Ting; Liu, Peng; Zhao, Bin; Zhang, Ji Wang

2018-03-01

We examined the changes of photosynthetic characteristics and chloroplast ultrastructure in mesophyll cell of summer maize in response to different light intensities in the field, with the summer maize hybrid Denghai 605 as experimental material. Two treatments of both shading (S) and increasing light (L) from flowering to physiological maturity stage were designed, with the ambient sunlight treatment as control (CK). Under shading treatment, poorly developed thylakoid structure, blurry lamellar structure, loose granum, large gap between slices and warping granum were the major characteristics in chloroplast. Meanwhile, photosynthetic rate (P n ), transpiration rate, stomatal conductance, chlorophyll content, and actual photo-chemical efficiency (Φ PSII ) decreased, whereas the maximal photochemical efficiency and non-photochemical quenching increased, which resulted in decreases in grain yield under shading treatment. However, a better development was observed in chloroplasts for L treatment, with the number of grana and lamellae increased and lamellae arranged compactly. In addition, P n and Φ PSII increased under L treatment, which increased grain yield. The chloroplast arrangement dispersed in mesophyll cells and chloroplast ultrastructure was destroyed after shading, and then chlorophyll synthesis per unit leaf area and photosynthetic capacity decreased. In contrast, the number of grana and lamellae increased and lamellae arranged compactly after increasing light, which are beneficial for corn yield.

Carbon Dioxide Fixation in Isolated Kalanchoe Chloroplasts 1

PubMed Central

Levi, Carolyn; Gibbs, Martin

1975-01-01

Chloroplasts isolated from Kalanchoe diagremontiana leaves were capable of photosynthesizing at a rate of 5.4 μmoles of CO2 per milligram of chlorophyll per hour. The dark rate of fixation was about 1% of the light rate. A high photosynthetic rate was associated with low starch content of the leaves. Ribose 5-phosphate, fructose 1,6-diphosphate, and dithiothreitol stimulated fixation, whereas phosphoenolpyruvate and azide were inhibitors. The products of CO2 fixation were primarily those of the photosynthetic carbon reduction cycle. PMID:16659249

The radical induced cell death protein 1 (RCD1) supports transcriptional activation of genes for chloroplast antioxidant enzymes

PubMed Central

Hiltscher, Heiko; Rudnik, Radoslaw; Shaikhali, Jehad; Heiber, Isabelle; Mellenthin, Marina; Meirelles Duarte, Iuri; Schuster, Günter; Kahmann, Uwe; Baier, Margarete

2014-01-01

The rimb1 (redox imbalanced 1) mutation was mapped to the RCD1 locus (radical-induced cell death 1; At1g32230) demonstrating that a major factor involved in redox-regulation genes for chloroplast antioxidant enzymes and protection against photooxidative stress, RIMB1, is identical to the regulator of disease response reactions and cell death, RCD1. Discovering this link let to our investigation of its regulatory mechanism. We show in yeast that RCD1 can physically interact with the transcription factor Rap2.4a which provides redox-sensitivity to nuclear expression of genes for chloroplast antioxidant enzymes. In the rimb1 (rcd1-6) mutant, a single nucleotide exchange results in a truncated RCD1 protein lacking the transcription factor binding site. Protein-protein interaction between full-length RCD1 and Rap2.4a is supported by H2O2, but not sensitive to the antioxidants dithiotreitol and ascorbate. In combination with transcript abundance analysis in Arabidopsis, it is concluded that RCD1 stabilizes the Rap2.4-dependent redox-regulation of the genes encoding chloroplast antioxidant enzymes in a widely redox-independent manner. Over the years, rcd1-mutant alleles have been described to develop symptoms like chlorosis, lesions along the leaf rims and in the mesophyll and (secondary) induction of extra- and intra-plastidic antioxidant defense mechanisms. All these rcd1 mutant characteristics were observed in rcd1-6 to succeed low activation of the chloroplast antioxidant system and glutathione biosynthesis. We conclude that RCD1 protects plant cells from running into reactive oxygen species (ROS)-triggered programs, such as cell death and activation of pathogen-responsive genes (PR genes) and extra-plastidic antioxidant enzymes, by supporting the induction of the chloroplast antioxidant system. PMID:25295044

Authentication Markers for Five Major Panax Species Developed via Comparative Analysis of Complete Chloroplast Genome Sequences.

PubMed

Nguyen, Van Binh; Park, Hyun-Seung; Lee, Sang-Choon; Lee, Junki; Park, Jee Young; Yang, Tae-Jin

2017-08-02

Ginseng represents a set of high-value medicinal plants of different species: Panax ginseng (Asian ginseng), Panax quinquefolius (American ginseng), Panax notoginseng (Chinese ginseng), Panax japonicus (Bamboo ginseng), and Panax vietnamensis (Vietnamese ginseng). Each species is pharmacologically and economically important, with differences in efficacy and price. Accordingly, an authentication system is needed to combat economically motivated adulteration of Panax products. We conducted comparative analysis of the chloroplast genome sequences of these five species, identifying 34-124 InDels and 141-560 SNPs. Fourteen InDel markers were developed to authenticate the Panax species. Among these, eight were species-unique markers that successfully differentiated one species from the others. We generated at least one species-unique marker for each of the five species, and any of the species can be authenticated by selection among these markers. The markers are reliable, easily detectable, and valuable for applications in the ginseng industry as well as in related research.

Establishment of monitoring methods for autophagy in rice reveals autophagic recycling of chloroplasts and root plastids during energy limitation.

PubMed

Izumi, Masanori; Hidema, Jun; Wada, Shinya; Kondo, Eri; Kurusu, Takamitsu; Kuchitsu, Kazuyuki; Makino, Amane; Ishida, Hiroyuki

2015-04-01

Autophagy is an intracellular process leading to vacuolar or lysosomal degradation of cytoplasmic components in eukaryotes. Establishment of proper methods to monitor autophagy was a key step in uncovering its role in organisms, such as yeast (Saccharomyces cerevisiae), mammals, and Arabidopsis (Arabidopsis thaliana), in which chloroplastic proteins were found to be recycled by autophagy. Chloroplast recycling has been predicted to function in nutrient remobilization for growing organs or grain filling in cereal crops. Here, to develop our understanding of autophagy in cereals, we established monitoring methods for chloroplast autophagy in rice (Oryza sativa). We generated transgenic rice-expressing fluorescent protein (FP) OsAuTophaGy8 (OsATG8) fusions as autophagy markers. FP-ATG8 signals were delivered into the vacuolar lumen in living cells of roots and leaves mainly as vesicles corresponding to autophagic bodies. This phenomenon was not observed upon the addition of wortmannin, an inhibitor of autophagy, or in an ATG7 knockout mutant. Markers for the chloroplast stroma, stromal FP, and FP-labeled Rubisco were delivered by a type of autophagic body called the Rubisco-containing body (RCB) in the same manner. RCB production in excised leaves was suppressed by supply of external sucrose or light. The release of free FP caused by autophagy-dependent breakdown of FP-labeled Rubisco was induced during accelerated senescence in individually darkened leaves. In roots, nongreen plastids underwent both RCB-mediated and entire organelle types of autophagy. Therefore, our newly developed methods to monitor autophagy directly showed autophagic degradation of leaf chloroplasts and root plastids in rice plants and its induction during energy limitation. © 2015 American Society of Plant Biologists. All Rights Reserved.

Overexpression of a natural chloroplast-encoded antisense RNA in tobacco destabilizes 5S rRNA and retards plant growth.

PubMed

Hotto, Amber M; Huston, Zoe E; Stern, David B

2010-09-29

The roles of non-coding RNAs in regulating gene expression have been extensively studied in both prokaryotes and eukaryotes, however few reports exist as to their roles in organellar gene regulation. Evidence for accumulation of natural antisense RNAs (asRNAs) in chloroplasts comes from the expressed sequence tag database and cDNA libraries, while functional data have been largely obtained from artificial asRNAs. In this study, we used Nicotiana tabacum to investigate the effect on sense strand transcripts of overexpressing a natural chloroplast asRNA, AS5, which is complementary to the region which encodes the 5S rRNA and tRNAArg. AS5-overexpressing (AS5ox) plants obtained by chloroplast transformation exhibited slower growth and slightly pale green leaves. Analysis of AS5 transcripts revealed four distinct species in wild-type (WT) and AS5ox plants, and additional AS5ox-specific products. Of the corresponding sense strand transcripts, tRNAArg overaccumulated several-fold in transgenic plants whereas 5S rRNA was unaffected. However, run-on transcription showed that the 5S-trnR region was transcribed four-fold more in the AS5ox plants compared to WT, indicating that overexpression of AS5 was associated with decreased stability of 5S rRNA. In addition, polysome analysis of the transformants showed less 5S rRNA and rbcL mRNA associated with ribosomes. Our results suggest that AS5 can modulate 5S rRNA levels, giving it the potential to affect Chloroplast translation and plant growth. More globally, overexpression of asRNAs via chloroplast transformation may be a useful strategy for defining their functions.

Chloroplast- or Mitochondria-Targeted DEAD-Box RNA Helicases Play Essential Roles in Organellar RNA Metabolism and Abiotic Stress Responses

PubMed Central

Nawaz, Ghazala; Kang, Hunseung

2017-01-01

The yields and productivity of crops are greatly diminished by various abiotic stresses, including drought, cold, heat, and high salinity. Chloroplasts and mitochondria are cellular organelles that can sense diverse environmental stimuli and alter gene expression to cope with adverse environmental stresses. Organellar gene expression is mainly regulated at posttranscriptional levels, including RNA processing, intron splicing, RNA editing, RNA turnover, and translational control, during which a variety of nucleus-encoded RNA-binding proteins (RBPs) are targeted to chloroplasts or mitochondria where they play essential roles in organellar RNA metabolism. DEAD-box RNA helicases (RHs) are enzymes that can alter RNA structures and affect RNA metabolism in all living organisms. Although a number of DEAD-box RHs have been found to play important roles in RNA metabolism in the nucleus and cytoplasm, our understanding on the roles of DEAD-box RHs in the regulation of RNA metabolism in chloroplasts and mitochondria is only at the beginning. Considering that organellar RNA metabolism and gene expression are tightly regulated by anterograde signaling from the nucleus, it is imperative to determine the functions of nucleus-encoded organellar RBPs. In this review, we summarize the emerging roles of nucleus-encoded chloroplast- or mitochondria-targeted DEAD-box RHs in organellar RNA metabolism and plant response to diverse abiotic stresses. PMID:28596782

WHITE STRIPE LEAF4 Encodes a Novel P-Type PPR Protein Required for Chloroplast Biogenesis during Early Leaf Development

PubMed Central

Wang, Ying; Ren, Yulong; Zhou, Kunneng; Liu, Linglong; Wang, Jiulin; Xu, Yang; Zhang, Huan; Zhang, Long; Feng, Zhiming; Wang, Liwei; Ma, Weiwei; Wang, Yunlong; Guo, Xiuping; Zhang, Xin; Lei, Cailin; Cheng, Zhijun; Wan, Jianmin

2017-01-01

Pentatricopeptide repeat (PPR) proteins comprise a large family in higher plants and perform diverse functions in organellar RNA metabolism. Despite the rice genome encodes 477 PRR proteins, the regulatory effects of PRR proteins on chloroplast development remains unknown. In this study, we report the functional characterization of the rice white stripe leaf4 (wsl4) mutant. The wsl4 mutant develops white-striped leaves during early leaf development, characterized by decreased chlorophyll content and malformed chloroplasts. Positional cloning of the WSL4 gene, together with complementation and RNA-interference tests, reveal that it encodes a novel P-family PPR protein with 12 PPR motifs, and is localized to chloroplast nucleoids. Quantitative RT-PCR analyses demonstrate that WSL4 is a low temperature response gene abundantly expressed in young leaves. Further expression analyses show that many nuclear- and plastid-encoded genes in the wsl4 mutant are significantly affected at the RNA and protein levels. Notably, the wsl4 mutant causes defects in the splicing of atpF, ndhA, rpl2, and rps12. Our findings identify WSL4 as a novel P-family PPR protein essential for chloroplast RNA group II intron splicing during early leaf development in rice. PMID:28694820

Identification of the "A" genome of finger millet using chloroplast DNA.

PubMed

Hilu, K W

1988-01-01

Finger millet (Eleusine corocana subsp. coracana), an important cereal in East Africa and India, is a tetraploid species with unknown genomic components. A recent cytogenetic study confirmed the direct origin of this millet from the tetraploid E. coracana subsp. africana but questioned Eleusine indica as a genomic donor. Chloroplast (ct) DNA sequence analysis using restriction fragment pattern was used to examine the phylogenetic relationships between E. coracana subsp. coracana (domesticated finger millet), E. coracana subspecies africana (wild finger millet), and E. indica. Eleusine tristachya was included since it is the only other annual diploid species in the genus with a basic chromosome number of x = 9 like finger millet. Eight of the ten restriction endonucleases used had 16 to over 30 restriction sites per genome and were informative. E. coracana subsp. coracana and subsp. africana and E. indica were identical in all the restriction sites surveyed, while the ct genome of E, tristachya differed consistently by at least one mutational event for each restriction enzyme surveyed. This random survey of the ct genomes of these species points out E. indica as one of the genome donors (maternal genome donor) of domesticated finger millet contrary to a previous cytogenetic study. The data also substantiate E. coracana subsp. africana as the progenitor of domesticated finger millet. The disparity between the cytogenetic and the molecular approaches is discussed in light of the problems associated with chromosome pairing and polyploidy.

Identification of the ``a'' Genome of Finger Millet Using Chloroplast DNA

PubMed Central

Hilu, K. W.

1988-01-01

Finger millet (Eleusine corocana subsp. coracana), an important cereal in East Africa and India, is a tetraploid species with unknown genomic components. A recent cytogenetic study confirmed the direct origin of this millet from the tetraploid E. coracana subsp. africana but questioned Eleusine indica as a genomic donor. Chloroplast (ct) DNA sequence analysis using restriction fragment pattern was used to examine the phylogenetic relationships between E. coracana subsp. coracana (domesticated finger millet), E. coracana subspecies africana (wild finger millet), and E. indica. Eleusine tristachya was included since it is the only other annual diploid species in the genus with a basic chromosome number of x = 9 like finger millet. Eight of the ten restriction endonucleases used had 16 to over 30 restriction sites per genome and were informative. E. coracana subsp. coracana and subsp. africana and E. indica were identical in all the restriction sites surveyed, while the ct genome of E. tristachya differed consistently by at least one mutational event for each restriction enzyme surveyed. This random survey of the ct genomes of these species points out E. indica as one of the genome donors (maternal genome donor) of domesticated finger millet contrary to a previous cytogenetic study. The data also substantiate E. coracana subsp. africana as the progenitor of domesticated finger millet. The disparity between the cytogenetic and the molecular approaches is discussed in light of the problems associated with chromosome pairing and polyploidy. PMID:8608927

The complete chloroplast genome sequence of Dendrobium officinale.

PubMed

Yang, Pei; Zhou, Hong; Qian, Jun; Xu, Haibin; Shao, Qingsong; Li, Yonghua; Yao, Hui

2016-01-01

The complete chloroplast sequence of Dendrobium officinale, an endangered and economically important traditional Chinese medicine, was reported and characterized. The genome size is 152,018 bp, with 37.5% GC content. A pair of inverted repeats (IRs) of 26,284 bp are separated by a large single-copy region (LSC, 84,944 bp) and a small single-copy region (SSC, 14,506 bp). The complete cp DNA contains 83 protein-coding genes, 39 tRNA genes and 8 rRNA genes. Fourteen genes contained one or two introns.

The Role of Heterologous Chloroplast Sequence Elements in Transgene Integration and Expression1[W][OA

PubMed Central

Ruhlman, Tracey; Verma, Dheeraj; Samson, Nalapalli; Daniell, Henry

2010-01-01

Heterologous regulatory elements and flanking sequences have been used in chloroplast transformation of several crop species, but their roles and mechanisms have not yet been investigated. Nucleotide sequence identity in the photosystem II protein D1 (psbA) upstream region is 59% across all taxa; similar variation was consistent across all genes and taxa examined. Secondary structure and predicted Gibbs free energy values of the psbA 5′ untranslated region (UTR) among different families reflected this variation. Therefore, chloroplast transformation vectors were made for tobacco (Nicotiana tabacum) and lettuce (Lactuca sativa), with endogenous (Nt-Nt, Ls-Ls) or heterologous (Nt-Ls, Ls-Nt) psbA promoter, 5′ UTR and 3′ UTR, regulating expression of the anthrax protective antigen (PA) or human proinsulin (Pins) fused with the cholera toxin B-subunit (CTB). Unique lettuce flanking sequences were completely eliminated during homologous recombination in the transplastomic tobacco genomes but not unique tobacco sequences. Nt-Ls or Ls-Nt transplastomic lines showed reduction of 80% PA and 97% CTB-Pins expression when compared with endogenous psbA regulatory elements, which accumulated up to 29.6% total soluble protein PA and 72.0% total leaf protein CTB-Pins, 2-fold higher than Rubisco. Transgene transcripts were reduced by 84% in Ls-Nt-CTB-Pins and by 72% in Nt-Ls-PA lines. Transcripts containing endogenous 5′ UTR were stabilized in nonpolysomal fractions. Stromal RNA-binding proteins were preferentially associated with endogenous psbA 5′ UTR. A rapid and reproducible regeneration system was developed for lettuce commercial cultivars by optimizing plant growth regulators. These findings underscore the need for sequencing complete crop chloroplast genomes, utilization of endogenous regulatory elements and flanking sequences, as well as optimization of plant growth regulators for efficient chloroplast transformation. PMID:20130101

ALA10, a Phospholipid Flippase, Controls FAD2/FAD3 Desaturation of Phosphatidylcholine in the ER and Affects Chloroplast Lipid Composition in Arabidopsis thaliana1

PubMed Central

Sautron, Emeline; Boudiere, Laurence; Michaud, Morgane; Dubots, Emmanuelle; Albrieux, Catherine; Marechal, Eric; Jouhet, Juliette

2016-01-01

The biogenesis of photosynthetic membranes relies on galactoglycerolipids, which are synthesized via pathways that are dispatched over several cell compartments. This membrane biogenesis requires both trafficking of lipid intermediates and a tight homeostatic regulation. In this work, we address the role of ALA10 (for aminophospholipid ATPase), a P4-type ATPase, in a process counteracting the monogalactosyldiacylglycerol (MGDG) shortage in Arabidopsis (Arabidopsis thaliana) leaves. ALA10 can interact with protein partners, ALIS1 (for ALA-interacting subunit1) or ALIS5, leading to differential endomembrane localizations of the interacting proteins, close to the plasma membrane with ALIS1 or to chloroplasts with ALIS5. ALA10 interacts also with FATTY ACID DESATURASE2 (FAD2), and modification of ALA10 expression affects phosphatidylcholine (PC) fatty acyl desaturation by disturbing the balance between FAD2 and FAD3 activities. Modulation of ALA10 expression downstream impacts the fatty acyl composition of chloroplast PC. ALA10 expression also enhances leaf growth and improves the MGDG-PC ratio, possibly through MGDG SYNTHASE1 (MGD1) activation by phosphatidic acid. The positive effect of ALA10 on leaf development is significant in conditions such as upon treatment of plants with Galvestine-1, an inhibitor of MGDG synthases, or when plants are grown at chilling temperature. PMID:26620528

Phylogenetic analysis of 47 chloroplast genomes clarifies the contribution of wild species to the domesticated apple maternal line.

PubMed

Nikiforova, Svetlana V; Cavalieri, Duccio; Velasco, Riccardo; Goremykin, Vadim

2013-08-01

Both the origin of domesticated apple and the overall phylogeny of the genus Malus are still not completely resolved. Having this as a target, we built a 134,553-position-long alignment including two previously published chloroplast DNAs (cpDNAs) and 45 de novo sequenced, fully colinear chloroplast genomes from cultivated apple varieties and wild apple species. The data produced are free from compositional heterogeneity and from substitutional saturation, which can adversely affect phylogeny reconstruction. Phylogenetic analyses based on this alignment recovered a branch, having the maximum bootstrap support, subtending a large group of the cultivated apple sorts together with all analyzed European wild apple (Malus sylvestris) accessions. One apple cultivar was embedded in a monophylum comprising wild M. sieversii accessions and other Asian apple species. The data demonstrate that M. sylvestris has contributed chloroplast genome to a substantial fraction of domesticated apple varieties, supporting the conclusion that different wild species should have contributed the organelle and nuclear genomes to the domesticated apple.

Chloroplast Transcription at Different Light Intensities. Glutathione-Mediated Phosphorylation of the Major RNA Polymerase Involved in Redox-Regulated Organellar Gene Expression1

PubMed Central

Baena-González, Elena; Baginsky, Sacha; Mulo, Paula; Summer, Holger; Aro, Eva-Mari; Link, Gerhard

2001-01-01

Previous studies using purified RNA polymerase from mustard (Sinapis alba) chloroplasts showed control of transcription by an associated protein kinase. This kinase was found to respond to reversible thiol/disulfide formation mediated by glutathione (GSH), although at concentrations exceeding those thought to exist in vivo. In the present study, several lines of evidence are presented to substantiate the functioning of this regulation mechanism, also in vivo: (a) Studies on the polymerase-associated transcription kinase revealed that at appropriate ATP levels, GSH concentrations similar to those in vivo are sufficient to modulate the kinase activity; (b) GSH measurements from isolated mustard chloroplasts showed considerable differences in response to light intensity; (c) this was reflected by run-on transcription rates in isolated chloroplasts that were generally higher if organelles were prepared from seedlings incubated under high-light as compared with growth-light conditions; (d) the notion of a general transcriptional switch was strengthened by in vitro experiments showing that the kinase not only affects the transcription of a photosynthetic gene (psbA) but also that of a non-photosynthetic gene (trnQ); and (e) the polymerase-kinase complex revealed specific differences in the phosphorylation state of polypeptides depending on the light intensity to which the seedlings had been exposed prior to chloroplast isolation. Taken together, these data are consistent with GSH and phosphorylation-dependent regulation of chloroplast transcription in vivo. PMID:11706185

The complete chloroplast genome sequence of Citrus sinensis (L.) Osbeck var 'Ridge Pineapple': organization and phylogenetic relationships to other angiosperms

PubMed Central

Bausher, Michael G; Singh, Nameirakpam D; Lee, Seung-Bum; Jansen, Robert K; Daniell, Henry

2006-01-01

Background The production of Citrus, the largest fruit crop of international economic value, has recently been imperiled due to the introduction of the bacterial disease Citrus canker. No significant improvements have been made to combat this disease by plant breeding and nuclear transgenic approaches. Chloroplast genetic engineering has a number of advantages over nuclear transformation; it not only increases transgene expression but also facilitates transgene containment, which is one of the major impediments for development of transgenic trees. We have sequenced the Citrus chloroplast genome to facilitate genetic improvement of this crop and to assess phylogenetic relationships among major lineages of angiosperms. Results The complete chloroplast genome sequence of Citrus sinensis is 160,129 bp in length, and contains 133 genes (89 protein-coding, 4 rRNAs and 30 distinct tRNAs). Genome organization is very similar to the inferred ancestral angiosperm chloroplast genome. However, in Citrus the infA gene is absent. The inverted repeat region has expanded to duplicate rps19 and the first 84 amino acids of rpl22. The rpl22 gene in the IRb region has a nonsense mutation resulting in 9 stop codons. This was confirmed by PCR amplification and sequencing using primers that flank the IR/LSC boundaries. Repeat analysis identified 29 direct and inverted repeats 30 bp or longer with a sequence identity ≥ 90%. Comparison of protein-coding sequences with expressed sequence tags revealed six putative RNA edits, five of which resulted in non-synonymous modifications in petL, psbH, ycf2 and ndhA. Phylogenetic analyses using maximum parsimony (MP) and maximum likelihood (ML) methods of a dataset composed of 61 protein-coding genes for 30 taxa provide strong support for the monophyly of several major clades of angiosperms, including monocots, eudicots, rosids and asterids. The MP and ML trees are incongruent in three areas: the position of Amborella and Nymphaeales, relationship

Intraspecific and heteroplasmic variations, gene losses and inversions in the chloroplast genome of Astragalus membranaceus.

PubMed

Lei, Wanjun; Ni, Dapeng; Wang, Yujun; Shao, Junjie; Wang, Xincun; Yang, Dan; Wang, Jinsheng; Chen, Haimei; Liu, Chang

2016-02-22

Astragalus membranaceus is an important medicinal plant in Asia. Several of its varieties have been used interchangeably as raw materials for commercial production. High resolution genetic markers are in urgent need to distinguish these varieties. Here, we sequenced and analyzed the chloroplast genome of A. membranaceus (Fisch.) Bunge var. mongholicus (Bunge) P.K. Hsiao using the next generation DNA sequencing technology. The genome was assembled using Abyss and then subjected to gene prediction using CPGAVAS and repeat analysis using MISA, Tandem Repeats Finder, and REPuter. Finally, the genome was subjected phylogenetic and comparative genomic analyses. The complete genome is 123,582 bp long, containing only one copy of the inverted repeat. Gene prediction revealed 110 genes encoding 76 proteins, 30 tRNAs, and four rRNAs. Five intra-specific hypermutation loci were identified, three of which are heteroplasmic. Furthermore, three gene losses and two large inversions were identified. Comparative genomic analyses demonstrated the dynamic nature of the Papilionoideae chloroplast genomes, which showed occurrence of numerous hypermutation loci, frequent gene losses, and fragment inversions. Results obtained herein elucidate the complex evolutionary history of chloroplast genomes and have laid the foundation for the identification of genetic markers to distinguish A. membranaceus varieties.

TGD4 involved in endoplasmic reticulum-to-chloroplast lipid trafficking is a phosphatidic acid binding protein

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

Wang Z.; Xu C.; Benning, C.

The synthesis of galactoglycerolipids, which are prevalent in photosynthetic membranes, involves enzymes at the endoplasmic reticulum (ER) and the chloroplast envelope membranes. Genetic analysis of trigalactosyldiacylglycerol (TGD) proteins in Arabidopsis has demonstrated their role in polar lipid transfer from the ER to the chloroplast. The TGD1, 2, and 3 proteins resemble components of a bacterial-type ATP-binding cassette (ABC) transporter, with TGD1 representing the permease, TGD2 the substrate binding protein, and TGD3 the ATPase. However, the function of the TGD4 protein in this process is less clear and its location in plant cells remains to be firmly determined. The predicted C-terminalmore » {beta}-barrel structure of TGD4 is weakly similar to proteins of the outer cell membrane of Gram-negative bacteria. Here, we show that, like TGD2, the TGD4 protein when fused to DsRED specifically binds phosphatidic acid (PtdOH). As previously shown for tgd1 mutants, tgd4 mutants have elevated PtdOH content, probably in extraplastidic membranes. Using highly purified and specific antibodies to probe different cell fractions, we demonstrated that the TGD4 protein was present in the outer envelope membrane of chloroplasts, where it appeared to be deeply buried within the membrane except for the N-terminus, which was found to be exposed to the cytosol. It is proposed that TGD4 is either directly involved in the transfer of polar lipids, possibly PtdOH, from the ER to the outer chloroplast envelope membrane or in the transfer of PtdOH through the outer envelope membrane.« less

Investigating the Production of Foreign Membrane Proteins in Tobacco Chloroplasts: Expression of an Algal Plastid Terminal Oxidase

PubMed Central

Ahmad, Niaz; Michoux, Franck; Nixon, Peter J.

2012-01-01

Chloroplast transformation provides an inexpensive, easily scalable production platform for expression of recombinant proteins in plants. However, this technology has been largely limited to the production of soluble proteins. Here we have tested the ability of tobacco chloroplasts to express a membrane protein, namely plastid terminal oxidase 1 from the green alga Chlamydomonas reinhardtii (Cr-PTOX1), which is predicted to function as a plastoquinol oxidase. A homoplastomic plant containing a codon-optimised version of the nuclear gene encoding PTOX1, driven by the 16S rRNA promoter and 5′UTR of gene 10 from phage T7, was generated using a particle delivery system. Accumulation of Cr-PTOX1 was shown by immunoblotting and expression in an enzymatically active form was confirmed by using chlorophyll fluorescence to measure changes in the redox state of the plastoquinone pool in leaves. Growth of Cr-PTOX1 expressing plants was, however, more sensitive to high light than WT. Overall our results confirm the feasibility of using plastid transformation as a means of expressing foreign membrane proteins in the chloroplast. PMID:22848578

Dual-targeted tRNA-dependent amidotransferase ensures both mitochondrial and chloroplastic Gln-tRNAGln synthesis in plants

PubMed Central

Pujol, Claire; Bailly, Marc; Kern, Daniel; Maréchal-Drouard, Laurence; Becker, Hubert; Duchêne, Anne-Marie

2008-01-01

Aminoacyl-tRNAs are generally formed by direct attachment of an amino acid to tRNAs by aminoacyl-tRNA synthetases, but Gln-tRNA is an exception to this rule. Gln-tRNAGln is formed by this direct pathway in the eukaryotic cytosol and in protists or fungi mitochondria but is formed by an indirect transamidation pathway in most of bacteria, archaea, and chloroplasts. We show here that the formation of Gln-tRNAGln is also achieved by the indirect pathway in plant mitochondria. The mitochondrial-encoded tRNAGln, which is the only tRNAGln present in mitochondria, is first charged with glutamate by a nondiscriminating GluRS, then is converted into Gln-tRNAGln by a tRNA-dependent amidotransferase (AdT). The three subunits GatA, GatB, and GatC are imported into mitochondria and assemble into a functional GatCAB AdT. Moreover, the mitochondrial pathway of Gln-tRNAGln formation is shared with chloroplasts as both the GluRS, and the three AdT subunits are dual-imported into mitochondria and chloroplasts. PMID:18441100

Crystallization of the c14-rotor of the chloroplast ATP synthase reveals that it contains pigments

PubMed Central

Varco-Merth, Benjamin; Fromme, Raimund; Wang, Meitian; Fromme, Petra

2012-01-01

The ATP synthase is one of the most important enzymes on earth as it couples the transmembrane electrochemical potential of protons to the synthesis of ATP from ADP and inorganic phosphate, providing the main ATP source of almost all higher life on earth. During ATP synthesis, stepwise protonation of a conserved carboxylate on each protein subunit of an oligomeric ring of 10–15 c-subunits is commonly thought to drive rotation of the rotor moiety (c10–14γε) relative to stator moiety (α3β3δab2). Here we report the isolation and crystallization of the c14-ring of subunit c from the spinach chloroplast enzyme diffracting as far as 2.8 Å. Though ATP synthase was not previously known to contain any pigments, the crystals of the c-subunit possessed a strong yellow color. The pigment analysis revealed that they contain 1 chlorophyll and 2 carotenoids, thereby showing for the first time that the chloroplast ATP synthase contains cofactors, leading to the question of the possible roles of the functions of the pigments in the chloroplast ATP synthase. PMID:18515064