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Sample records for regulates phototropin-mediated chloroplast

  1. An auxilin-like J-domain protein, JAC1, regulates phototropin-mediated chloroplast movement in Arabidopsis.

    PubMed

    Suetsugu, Noriyuki; Kagawa, Takatoshi; Wada, Masamitsu

    2005-09-01

    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.

  2. Phototropins mediate blue and red light-induced chloroplast movements in Physcomitrella patens.

    PubMed

    Kasahara, Masahiro; Kagawa, Takatoshi; Sato, Yoshikatsu; Kiyosue, Tomohiro; Wada, Masamitsu

    2004-07-01

    Phototropin is the blue-light receptor that mediates phototropism, chloroplast movement, and stomatal opening in Arabidopsis. Blue and red light induce chloroplast movement in the moss Physcomitrella patens. To study the photoreceptors for chloroplast movement in P. patens, four phototropin genes (PHOTA1, PHOTA2, PHOTB1, and PHOTB2) were isolated by screening cDNA libraries. These genes were classified into two groups (PHOTA and PHOTB) on the basis of their deduced amino acid sequences. Then phototropin disruptants were generated by homologous recombination and used for analysis of chloroplast movement. Data revealed that blue light-induced chloroplast movement was mediated by phototropins in P. patens. Both photA and photB groups were able to mediate chloroplast avoidance, as has been reported for Arabidopsis phot2, although the photA group contributed more to the response. Red light-induced chloroplast movement was also significantly reduced in photA2photB1photB2 triple disruptants. Because the primary photoreceptor for red light-induced chloroplast movement in P. patens is phytochrome, phototropins may be downstream components of phytochromes in the signaling pathway. To our knowledge, this work is the first to show a function for the phototropin blue-light receptor in a response to wavelengths that it does not absorb.

  3. Auxin and chloroplast movements.

    PubMed

    Eckstein, Aleksandra; Krzeszowiec, Weronika; Waligórski, Piotr; Gabryś, Halina

    2016-03-01

    Auxin is involved in a wide spectrum of physiological processes in plants, including responses controlled by the blue light photoreceptors phototropins: phototropic bending and stomatal movement. However, the role of auxin in phototropin-mediated chloroplast movements has never been studied. To address this question we searched for potential interactions between auxin and the chloroplast movement signaling pathway using different experimental approaches and two model plants, Arabidopsis thaliana and Nicotiana tabacum. We observed that the disturbance of auxin homeostasis by shoot decapitation caused a decrease in chloroplast movement parameters, which could be rescued by exogenous auxin application. In several cases, the impairment of polar auxin transport, by chemical inhibitors or in auxin carrier mutants, had a similar negative effect on chloroplast movements. This inhibition was not correlated with changes in auxin levels. Chloroplast relocations were also affected by the antiauxin p-chlorophenoxyisobutyric acid and mutations in genes encoding some of the elements of the SCF(TIR1)-Aux/IAA auxin receptor complex. The observed changes in chloroplast movement parameters are not prominent, which points to a modulatory role of auxin in this process. Taken together, the obtained results suggest that auxin acts indirectly to regulate chloroplast movements, presumably by regulating gene expression via the SCF(TIR1)-Aux/IAA-ARF pathway. Auxin does not seem to be involved in controlling the expression of phototropins.

  4. Chloroplast signaling: retrograde regulation revelations.

    PubMed

    Beale, Samuel I

    2011-05-24

    Developing chloroplasts are able to communicate their status to the nucleus and regulate expression of genes whose products are needed for photosynthesis. Heme is revealed to be a signaling molecule for this retrograde communication.

  5. Evolution of the Cp-Actin-based Motility System of Chloroplasts in Green Plants.

    PubMed

    Suetsugu, Noriyuki; Wada, Masamitsu

    2016-01-01

    During the course of green plant evolution, numerous light responses have arisen that optimize their growth under fluctuating light conditions. The blue light receptor phototropin mediates several photomovement responses at the tissue, cellular and organelle levels. Chloroplast photorelocation movement is one such photomovement response, and is found not only in most green plants, but also in some red algae and photosynthetic stramenopiles. In general, chloroplasts move toward weak light to maximally capture photosynthetically active radiation (the chloroplast accumulation response), and they move away from strong light to avoid photodamage (the avoidance response). In land plants, chloroplast movement is dependent on specialized actin filaments, chloroplast-actin filaments (cp-actin filaments). Through molecular genetic analysis using Arabidopsis thaliana, many molecular factors that regulate chloroplast photorelocation were identified. In this Perspective, we discuss the evolutionary history of the molecular mechanism for chloroplast photorelocation movement in green plants in view of cp-actin filaments.

  6. Chloroplast retrograde signal regulates flowering

    PubMed Central

    Feng, Peiqiang; Guo, Hailong; Chi, Wei; Chai, Xin; Sun, Xuwu; Xu, Xiumei; Ma, Jinfang; Rochaix, Jean-David; Leister, Dario; Wang, Haiyang; Lu, Congming; Zhang, Lixin

    2016-01-01

    Light is a major environmental factor regulating flowering time, thus ensuring reproductive success of higher plants. In contrast to our detailed understanding of light quality and photoperiod mechanisms involved, the molecular basis underlying high light-promoted flowering remains elusive. Here we show that, in Arabidopsis, a chloroplast-derived signal is critical for high light-regulated flowering mediated by the FLOWERING LOCUS C (FLC). We also demonstrate that PTM, a PHD transcription factor involved in chloroplast retrograde signaling, perceives such a signal and mediates transcriptional repression of FLC through recruitment of FVE, a component of the histone deacetylase complex. Thus, our data suggest that chloroplasts function as essential sensors of high light to regulate flowering and adaptive responses by triggering nuclear transcriptional changes at the chromatin level. PMID:27601637

  7. Two kinesin-like proteins mediate actin-based chloroplast movement in Arabidopsis thaliana.

    PubMed

    Suetsugu, Noriyuki; Yamada, Noboru; Kagawa, Takatoshi; Yonekura, Hisashi; Uyeda, Taro Q P; Kadota, Akeo; Wada, Masamitsu

    2010-05-11

    Organelle movement is essential for efficient cellular function in eukaryotes. Chloroplast photorelocation movement is important for plant survival as well as for efficient photosynthesis. Chloroplast movement generally is actin dependent and mediated by blue light receptor phototropins. In Arabidopsis thaliana, phototropins mediate chloroplast movement by regulating short actin filaments on chloroplasts (cp-actin filaments), and the chloroplast outer envelope protein CHUP1 is necessary for cp-actin filament accumulation. However, other factors involved in cp-actin filament regulation during chloroplast movement remain to be determined. Here, we report that two kinesin-like proteins, KAC1 and KAC2, are essential for chloroplasts to move and anchor to the plasma membrane. A kac1 mutant showed severely impaired chloroplast accumulation and slow avoidance movement. A kac1kac2 double mutant completely lacked chloroplast photorelocation movement and showed detachment of chloroplasts from the plasma membrane. KAC motor domains are similar to those of the kinesin-14 subfamily (such as Ncd and Kar3) but do not have detectable microtubule-binding activity. The C-terminal domain of KAC1 could interact with F-actin in vitro. Instead of regulating microtubules, KAC proteins mediate chloroplast movement via cp-actin filaments. We conclude that plants have evolved a unique mechanism to regulate actin-based organelle movement using kinesin-like proteins.

  8. REPRESSOR OF ULTRAVIOLET-B PHOTOMORPHOGENESIS function allows efficient phototropin mediated ultraviolet-B phototropism in etiolated seedlings.

    PubMed

    Vanhaelewyn, Lucas; Schumacher, Paolo; Poelman, Dirk; Fankhauser, Christian; Van Der Straeten, Dominique; Vandenbussche, Filip

    2016-11-01

    Ultraviolet B (UV-B) light is a part of the solar radiation which has significant effects on plant morphology, even at low doses. In Arabidopsis, many of these morphological changes have been attributed to a specific UV-B receptor, UV resistance locus 8 (UVR8). Recent findings showed that next to phototropin regulated phototropism, UVR8 mediated signaling is able of inducing directional bending towards UV-B light in etiolated seedlings of Arabidopsis, in a phototropin independent manner. In this study, kinetic analysis of phototropic bending was used to evaluate the relative contribution of each of these pathways in UV-B mediated phototropism. Diminishing UV-B light intensity favors the importance of phototropins. Molecular and genetic analyses suggest that UV-B is capable of inducing phototropin signaling relying on phototropin kinase activity and regulation of NPH3. Moreover, enhanced UVR8 responses in the UV-B hypersensitive rup1rup2 mutants interferes with the fast phototropin mediated phototropism. Together the data suggest that phototropins are the most important receptors for UV-B induced phototropism in etiolated seedlings, and a RUP mediated negative feedback pathway prevents UVR8 signaling to interfere with the phototropin dependent response.

  9. Copper Delivery to Chloroplast Proteins and its Regulation.

    PubMed

    Aguirre, Guadalupe; Pilon, Marinus

    2015-01-01

    Copper is required for photosynthesis in chloroplasts of plants because it is a cofactor of plastocyanin, an essential electron carrier in the thylakoid lumen. Other chloroplast copper proteins are copper/zinc superoxide dismutase and polyphenol oxidase, but these proteins seem to be dispensable under conditions of low copper supply when transcripts for these proteins undergo microRNA-mediated down regulation. Two ATP-driven copper transporters function in tandem to deliver copper to chloroplast compartments. This review seeks to summarize the mechanisms of copper delivery to chloroplast proteins and its regulation. We also delineate some of the unanswered questions that still remain in this field.

  10. Copper Delivery to Chloroplast Proteins and its Regulation

    PubMed Central

    Aguirre, Guadalupe; Pilon, Marinus

    2016-01-01

    Copper is required for photosynthesis in chloroplasts of plants because it is a cofactor of plastocyanin, an essential electron carrier in the thylakoid lumen. Other chloroplast copper proteins are copper/zinc superoxide dismutase and polyphenol oxidase, but these proteins seem to be dispensable under conditions of low copper supply when transcripts for these proteins undergo microRNA-mediated down regulation. Two ATP-driven copper transporters function in tandem to deliver copper to chloroplast compartments. This review seeks to summarize the mechanisms of copper delivery to chloroplast proteins and its regulation. We also delineate some of the unanswered questions that still remain in this field. PMID:26793223

  11. Structure and activity of JAC1 J-domain implicate the involvement of the cochaperone activity with HSC70 in chloroplast photorelocation movement.

    PubMed

    Suetsugu, Noriyuki; Takano, Akira; Kohda, Daisuke; Wada, Masamitsu

    2010-12-01

    Chloroplast photorelocation movement towards weak light and away from strong light is essential for plants to adapt to the fluctuation of ambient light conditions. In the previous study, we showed that blue light receptor phototropins mediated blue light-induced chloroplast movement in Arabidopsis by regulating short actin filaments localized at the chloroplast periphery (cp-actin filaments) rather than actin cables in the cytoplasm. However, the signaling pathway for the chloroplast photorelocation movement is still unclear. We also identified JAC1 (J-domain protein required for chloroplast accumulation response 1) as an essential component for the accumulation response and dark positioning in Arabidopsis. We recently determined the crystal structure of the J-domain of JAC1. The JAC1 J-domain has a positively charged surface, which forms a putative interface with the Hsc70 chaperone by analogy to that of bovine auxilin. Furthermore, the mutation of the highly conserved HPD motif in the JAC1 J-domain impaired the in vivo activity of JAC1. These data suggest that JAC1 cochaperone activity with HSC70 is essential for chloroplast photorelocation movement.

  12. GLK gene pairs regulate chloroplast development in diverse plant species.

    PubMed

    Fitter, David W; Martin, David J; Copley, Martin J; Scotland, Robert W; Langdale, Jane A

    2002-09-01

    Chloroplast biogenesis is a complex process that requires close co-ordination between two genomes. Many of the proteins that accumulate in the chloroplast are encoded by the nuclear genome, and the developmental transition from proplastid to chloroplast is regulated by nuclear genes. Here we show that a pair of Golden 2-like (GLK) genes regulates chloroplast development in Arabidopsis. The GLK proteins are members of the GARP superfamily of transcription factors, and phylogenetic analysis demonstrates that the maize, rice and Arabidopsis GLK gene pairs comprise a distinct group within the GARP superfamily. Further phylogenetic analysis suggests that the gene pairs arose through separate duplication events in the monocot and dicot lineages. As in rice, AtGLK1 and AtGLK2 are expressed in partially overlapping domains in photosynthetic tissue. Insertion mutants demonstrate that this expression pattern reflects a degree of functional redundancy as single mutants display normal phenotypes in most photosynthetic tissues. However, double mutants are pale green in all photosynthetic tissues and chloroplasts exhibit a reduction in granal thylakoids. Products of several genes involved in light harvesting also accumulate at reduced levels in double mutant chloroplasts. GLK genes therefore regulate chloroplast development in diverse plant species.

  13. A chloroplast retrograde signal regulates nuclear alternative splicing

    PubMed Central

    Petrillo, Ezequiel; Herz, Micaela A. Godoy; Fuchs, Armin; Reifer, Dominik; Fuller, John; Yanovsky, Marcelo J.; Simpson, Craig; Brown, John W. S.; Barta, Andrea; Kalyna, Maria; Kornblihtt, Alberto R.

    2015-01-01

    Light is a source of energy and also a regulator of plant physiological adaptations. We show here that light/dark conditions affect alternative splicing of a subset of Arabidopsis genes preferentially encoding proteins involved in RNA processing. The effect requires functional chloroplasts and is also observed in roots when the communication with the photosynthetic tissues is not interrupted, suggesting that a signaling molecule travels through the plant. Using photosynthetic electron transfer inhibitors with different mechanisms of action we deduce that the reduced pool of plastoquinones initiates a chloroplast retrograde signaling that regulates nuclear alternative splicing and is necessary for proper plant responses to varying light conditions. PMID:24763593

  14. Chloroplast Retrograde Regulation of Heat Stress Responses in Plants

    PubMed Central

    Sun, Ai-Zhen; Guo, Fang-Qing

    2016-01-01

    It is well known that intracellular signaling from chloroplast to nucleus plays a vital role in stress responses to survive environmental perturbations. The chloroplasts were proposed as sensors to heat stress since components of the photosynthetic apparatus housed in the chloroplast are the major targets of thermal damage in plants. Thus, communicating subcellular perturbations to the nucleus is critical during exposure to extreme environmental conditions such as heat stress. By coordinating expression of stress specific nuclear genes essential for adaptive responses to hostile environment, plants optimize different cell functions and activate acclimation responses through retrograde signaling pathways. The efficient communication between plastids and the nucleus is highly required for such diverse metabolic and biosynthetic functions during adaptation processes to environmental stresses. In recent years, several putative retrograde signals released from plastids that regulate nuclear genes have been identified and signaling pathways have been proposed. In this review, we provide an update on retrograde signals derived from tetrapyrroles, carotenoids, reactive oxygen species (ROS) and organellar gene expression (OGE) in the context of heat stress responses and address their roles in retrograde regulation of heat-responsive gene expression, systemic acquired acclimation, and cellular coordination in plants. PMID:27066042

  15. Chloroplast anchoring: its implications for the regulation of intracellular chloroplast distribution.

    PubMed

    Takagi, Shingo; Takamatsu, Hideyasu; Sakurai-Ozato, Nami

    2009-01-01

    The intracellular distribution of organelles plays a pivotal role in the maintenance and adaptation of a wide spectrum of cellular activities in plants. Chloroplasts are a special type of organelle able to photosynthesize, capturing light energy to fix atmospheric CO2. Consequently, the intracellular positioning of chloroplasts is crucial for plant growth and development. Knowledge of the photoreceptors and cellular apparatus responsible for chloroplast movement has gradually accumulated over time, yet recent advances have allowed improved understanding. In this article, several aspects of research progress into the mechanisms for maintaining the specific intracellular distribution patterns of chloroplasts, namely, chloroplast anchoring, are summarized, together with a brief consideration of the future prospects of this subject. Our discussion covers developmental, physiological, ecophysiological, and recent cell biological research areas.

  16. A Putative Chloroplast Thylakoid Metalloprotease VIRESCENT3 Regulates Chloroplast Development in Arabidopsis thaliana*

    PubMed Central

    Qi, Yafei; Liu, Xiayan; Liang, Shuang; Wang, Rui; Li, Yuanfeng; Zhao, Jun; Shao, Jingxia; An, Lijun; Yu, Fei

    2016-01-01

    The chloroplast is the site of photosynthesis and many other essential plant metabolic processes, and chloroplast development is an integral part of plant growth and development. Mutants defective in chloroplast development can display various color phenotypes including the intriguing virescence phenotype, which shows yellow/white coloration at the leaf base and greening toward the leaf tip. Through large scale genetic screens, we identified a series of new virescent mutants including virescent3-1 (vir3-1), vir4-1, and vir5-1 in Arabidopsis thaliana. We showed that VIR3 encodes a putative chloroplast metalloprotease by map-based cloning. Through site-directed mutagenesis, we showed that the conserved histidine 235 residue in the zinc binding motif HEAGH of VIR3 is indispensable for VIR3 accumulation in the chloroplast. The chloroplast localization of VIR3 was confirmed by the transient expression of VIR3-GFP in leaf protoplasts. Furthermore, taking advantage of transgenic lines expressing VIR3-FLAG, we demonstrated that VIR3 is an intrinsic thylakoid membrane protein that mainly resides in the stromal lamellae. Moreover, topology analysis using transgenic lines expressing a dual epitope-tagged VIR3 indicated that both the N and C termini of VIR3 are located in the stroma, and the catalytic domain of VIR3 is probably facing the stroma. Blue native gel analysis indicated that VIR3 is likely present as a monomer or part of a small complex in the thylakoid membrane. This work not only implicates VIR3 as a new factor involved in early chloroplast development but also provides more insight into the roles of chloroplast proteases in chloroplast biogenesis. PMID:26702056

  17. Stochastic dynamics of actin filaments in guard cells regulating chloroplast localization during stomatal movement.

    PubMed

    Wang, Xiu-Ling; Gao, Xin-Qi; Wang, Xue-Chen

    2011-08-01

    Actin filaments and chloroplasts in guard cells play roles in stomatal function. However, detailed actin dynamics vary, and the roles that they play in chloroplast localization during stomatal movement remain to be determined. We examined the dynamics of actin filaments and chloroplast localization in transgenic tobacco expressing green fluorescent protein (GFP)-mouse talin in guard cells by time-lapse imaging. Actin filaments showed sliding, bundling and branching dynamics in moving guard cells. During stomatal movement, long filaments can be severed into small fragments, which can form longer filaments by end-joining activities. With chloroplast movement, actin filaments near chloroplasts showed severing and elongation activity in guard cells during stomatal movement. Cytochalasin B treatment abolished elongation, bundling and branching activities of actin filaments in guard cells, and these changes of actin filaments, and as a result, more chloroplasts were localized at the centre of guard cells. However, chloroplast turning to avoid high light, and sliding of actin fragments near the chloroplast, was unaffected following cytochalasin B treatment in guard cells. We suggest that the sliding dynamics of actin may play roles in chloroplast turning in guard cells. Our results indicate that the stochastic dynamics of actin filaments in guard cells regulate chloroplast localization during stomatal movement.

  18. WHITE PANICLE1, a Val-tRNA Synthetase Regulating Chloroplast Ribosome Biogenesis in Rice, Is Essential for Early Chloroplast Development1[OPEN

    PubMed Central

    Wang, Chunming; Zheng, Ming; Lyu, Jia; Xu, Yang; Li, Xiaohui; Niu, Mei; Long, Wuhua; Wang, Di; Wang, Yihua; Wan, Jianmin

    2016-01-01

    Chloroplasts and mitochondria contain their own genomes and transcriptional and translational systems. Establishing these genetic systems is essential for plant growth and development. Here we characterized a mutant form of a Val-tRNA synthetase (OsValRS2) from Oryza sativa that is targeted to both chloroplasts and mitochondria. A single base change in OsValRS2 caused virescent to albino phenotypes in seedlings and white panicles at heading. We therefore named this mutant white panicle 1 (wp1). Chlorophyll autofluorescence observations and transmission electron microscopy analyses indicated that wp1 mutants are defective in early chloroplast development. RNA-seq analysis revealed that expression of nuclear-encoded photosynthetic genes is significantly repressed, while expression of many chloroplast-encoded genes also changed significantly in wp1 mutants. Western-blot analyses of chloroplast-encoded proteins showed that chloroplast protein levels were reduced in wp1 mutants, although mRNA levels of some genes were higher in wp1 than in wild type. We found that wp1 was impaired in chloroplast ribosome biogenesis. Taken together, our results show that OsValRS2 plays an essential role in chloroplast development and regulating chloroplast ribosome biogenesis. PMID:26839129

  19. Regulation of Chloroplast Photosynthetic Activity by Exogenous Magnesium 1

    PubMed Central

    Huber, Steven C.

    1978-01-01

    Magnesium was most inhibitory to photosynthetic reactions by intact chloroplasts when the magnesium was added in the dark before illumination. Two millimolar MgCl2, added in the dark, inhibited CO2-dependent O2 evolution by Hordeum vulgare L. and Spinacia oleracea L. (C3 plants) chloroplasts 70 to 100% and inhibited (pyruvate + oxaloacetate)-dependent O2 evolution by Digitaria sanguinalis L. (C4 plant) mesophyll chloroplasts from 80 to 100%. When Mg2+ was added in the light, O2 evolution was reduced only slightly. O2 evolution in the presence of phosphoglycerate was less sensitive to Mg2+ inhibition than was CO2-dependent O2 evolution. Magnesium prevented the light activation of several photosynthetic enzymes. Two millimolar Mg2+ blocked the light activation of NADP-malate dehydrogenase in D. sanguinalis mesophyll chloroplasts, and the light activation of phosphoribulokinase, NADP-linked glyceraldehyde-3-phosphate dehydrogenase, and fructose 1,6-diphosphatase in barley chloroplasts. The results suggest that Mg2+ inhibits chloroplast photosynthesis by preventing the light activation of certain enzymes. PMID:16660509

  20. 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

  1. GROWTH REGULATING FACTOR5 stimulates Arabidopsis chloroplast division, photosynthesis, and leaf longevity.

    PubMed

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

    2015-03-01

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

  2. Regulation of chloroplast biogenesis: the immutans mutant of Arabidopsis

    SciTech Connect

    Rodermel, Steven

    2015-11-16

    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 to 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.

  3. 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.

  4. THRUMIN1 is a light-regulated actin-bundling protein involved in chloroplast motility.

    PubMed

    Whippo, Craig W; Khurana, Parul; Davis, Phillip A; DeBlasio, Stacy L; DeSloover, Daniel; Staiger, Christopher J; Hangarter, Roger P

    2011-01-11

    Chloroplast movement in response to changing light conditions optimizes photosynthetic light absorption. This repositioning is stimulated by blue light perceived via the phototropin photoreceptors and is transduced to the actin cytoskeleton. Some actin-based motility systems use filament reorganizations rather than myosin-based translocations. Recent research favors the hypothesis that chloroplast movement is driven by actin reorganization at the plasma membrane, but no proteins affecting chloroplast movements have been shown to associate with both the plasma membrane and actin filaments in vivo. Here we identified THRUMIN1 as a critical link between phototropin photoreceptor activity at the plasma membrane and actin-dependent chloroplast movements. THRUMIN1 bundles filamentous actin in vitro, and it localizes to the plasma membrane and displays light- and phototropin-dependent localization to microfilaments in vivo. These results suggest that phototropin-induced actin bundling via THRUMIN1 is important for chloroplast movement. A mammalian homolog of THRUMIN1, GRXCR1, has been implicated in auditory responses and hair cell stereocilla development as a regulator of actin architecture. Studies of THRUMIN1 will help elucidate the function of this family of eukaryotic proteins.

  5. 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

  6. Post-translational Modifications in Regulation of Chloroplast Function: Recent Advances

    PubMed Central

    Grabsztunowicz, Magda; Koskela, Minna M.; Mulo, Paula

    2017-01-01

    Post-translational modifications (PTMs) of proteins enable fast modulation of protein function in response to metabolic and environmental changes. Phosphorylation is known to play a major role in regulating distribution of light energy between the Photosystems (PS) I and II (state transitions) and in PSII repair cycle. In addition, thioredoxin-mediated redox regulation of Calvin cycle enzymes has been shown to determine the efficiency of carbon assimilation. Besides these well characterized modifications, recent methodological progress has enabled identification of numerous other types of PTMs in various plant compartments, including chloroplasts. To date, at least N-terminal and Lys acetylation, Lys methylation, Tyr nitration and S-nitrosylation, glutathionylation, sumoylation and glycosylation of chloroplast proteins have been described. These modifications impact DNA replication, control transcriptional efficiency, regulate translational machinery and affect metabolic activities within the chloroplast. Moreover, light reactions of photosynthesis as well as carbon assimilation are regulated at multiple levels by a number of PTMs. It is likely that future studies will reveal new metabolic pathways to be regulated by PTMs as well as detailed molecular mechanisms of PTM-mediated regulation. PMID:28280500

  7. Circadian regulation of chloroplastic f and m thioredoxins through control of the CCA1 transcription factor

    PubMed Central

    de Dios Barajas-López, Juan; Serrato, Antonio Jesus; Cazalis, Roland; Meyer, Yves; Chueca, Ana; Reichheld, Jean Philippe; Sahrawy, Mariam

    2011-01-01

    Chloroplastic thioredoxins f and m (TRX f and TRX m) mediate light regulation of carbon metabolism through the activation of Calvin cycle enzymes. The role of TRX f and m in the activation of Calvin cycle enzymes is best known among the TRX family. However, the discoveries of new potential targets extend the functions of chloroplastic TRXs to other processes in non-photosynthetic tissues. As occurs with numerous chloroplast proteins, their expression comes under light regulation. Here, the focus is on the light regulation of TRX f and TRX m in pea and Arabidopsis during the day/night cycle that is maintained during the subjective night. In pea (Pisum sativum), TRX f and TRX m1 expression is shown to be governed by a circadian oscillation exerted at both the transcriptional and protein levels. Binding shift assays indicate that this control probably involves the interaction of the CCA1 transcription factor and an evening element (EE) located in the PsTRX f and PsTRX m1 promoters. In Arabidopsis, among the multigene family of TRX f and TRX m, AtTRX f2 and AtTRX m2 mRNA showed similar circadian oscillatory regulation, suggesting that such regulation is conserved in plants. However, this oscillation was disrupted in plants overexpressing CCA1 (cca1-ox) or repressing CCA1 and LHY (cca1-lhy). The physiological role of the oscillatory regulation of chloroplastic TRX f and TRX m in plants during the day/night cycle is discussed. PMID:21196476

  8. Light-regulated differential expression of pea chloroplast and cytosolic fructose-1,6-bisphosphatases.

    PubMed

    Lee, S-W; Hahn, T-R

    2003-02-01

    The light-regulated differential expression of pea chloroplast and cytosolic fructose-1,6-bisphosphatases (FBPase) was investigated using enzyme activity assay, immunoblot, and Northern blot analyses. The enzyme activities of both chloroplast and cytosolic FBPases gradually increased under continuous white light illumination, although the increase in chloroplast FBPase was more drastic. Northern and immunoblot analyses also indicated that light stimulated the expression of both enzymes. Enzyme activity and the transcript levels of both enzymes gradually decreased under the dark treatment, although protein levels were unchanged for up to 24 h following the initiation of culture in the dark, indicating that reversible modifications of the enzymes may occur during the transition from light to dark (or the reverse). Light pulse experiments using blue (420 nm) and red/far-red (660/730 nm) light were carried out to analyze the photoreceptors related to the light-mediated expression of both enzymes. Expression of the chloroplast enzyme was very sensitive to red or far-red light pulses-it was induced by red light, but suppressed by far-red light pulses, as determined by enzyme activity, immunoblot, and Northern blot analyses, suggesting that red light signaling is involved in the control of chloroplast FBPase expression. However, cytosolic FBPase was virtually insensitive to blue or red/far-red light pulses in terms of enzyme activity, as determined by protein and transcript levels, indicating that cytosolic enzyme expression is not directly regulated by light signals. Instead, the expression of the cytosolic enzyme may be closely related to photosynthetic energy conversion accompanied by continuous white light irradiation.

  9. Functional analyses of the Physcomitrella patens phytochromes in regulating chloroplast avoidance movement.

    PubMed

    Uenaka, Hidetoshi; Kadota, Akeo

    2007-09-01

    Red light-induced chloroplast movement in Physcomitrella patens (Pp) is mediated by dichroic phytochrome in the cytoplasm. To analyze the molecular function of the photoreceptor in the cytoplasm, we developed a protoplast system in which chloroplast photomovement was exclusively dependent on the expression of phytochrome cDNA constructs introduced by polyethylene glycol (PEG) transformation. YFP was fused to the phytochrome constructs and their expression was detected by fluorescence. The chloroplast avoidance response was induced in the protoplasts expressing a YFP fusion of PHY1-PHY3, but not of PHY4 or YFP alone. Phy::yfp fluorescence was detected in the cytoplasm. No change in the location of phy1::yfp or phy2::yfp was revealed before and after photomovement. When phy1::yfp and phy2::yfp were targeted to the nucleus by fusing a nuclear localization signal to the constructs, red light avoidance was not induced. To determine the domains of PHY2 essential for avoidance response, various partially-deleted PHY2::YFP constructs were tested. The N-terminal extension domain (NTE) was found to be necessary but the C-terminal histidine kinase-related domain (HKRD) was dispensable. An avoidance response was not induced under expression of phytochrome N-terminal half domain [deleting both the PAS (Per, Arnt, Sim)-related domain (PRD) and HKRD]. GUS fusion of this N-terminal half domain, reported to be fully functional in Arabidopsis for several phyA- and phyB-regulated responses was not effective in chloroplast avoidance movement. Domain requirement and GUS fusion effect were also confirmed in PHY1. These results indicate that Pp phy1-Pp phy3 in the cytoplasm mediate chloroplast avoidance movement, and that NTE and PRD, but not HKRD, are required for their function.

  10. Phototropin encoded by a single-copy gene mediates chloroplast photorelocation movements in the liverwort Marchantia polymorpha.

    PubMed

    Komatsu, Aino; Terai, Mika; Ishizaki, Kimitsune; Suetsugu, Noriyuki; Tsuboi, Hidenori; Nishihama, Ryuichi; Yamato, Katsuyuki T; Wada, Masamitsu; Kohchi, Takayuki

    2014-09-01

    Blue-light-induced chloroplast photorelocation movement is observed in most land plants. Chloroplasts move toward weak-light-irradiated areas to efficiently absorb light (the accumulation response) and escape from strong-light-irradiated areas to avoid photodamage (the avoidance response). The plant-specific kinase phototropin (phot) is the blue-light receptor for chloroplast movements. Although the molecular mechanisms for chloroplast photorelocation movement have been analyzed, the overall aspects of signal transduction common to land plants are still unknown. Here, we show that the liverwort Marchantia polymorpha exhibits the accumulation and avoidance responses exclusively induced by blue light as well as specific chloroplast positioning in the dark. Moreover, in silico and Southern-blot analyses revealed that the M. polymorpha genome encodes a single PHOT gene, MpPHOT, and its knockout line displayed none of the chloroplast photorelocation movements, indicating that the sole MpPHOT gene mediates all types of movement. Mpphot was localized on the plasma membrane and exhibited blue-light-dependent autophosphorylation both in vitro and in vivo. Heterologous expression of MpPHOT rescued the defects in chloroplast movement of phot mutants in the fern Adiantum capillus-veneris and the seed plant Arabidopsis (Arabidopsis thaliana). These results indicate that Mpphot possesses evolutionarily conserved regulatory activities for chloroplast photorelocation movement. M. polymorpha offers a simple and versatile platform for analyzing the fundamental processes of phototropin-mediated chloroplast photorelocation movement common to land plants.

  11. Purine biosynthetic enzyme ATase2 is involved in the regulation of early chloroplast development and chloroplast gene expression in Arabidopsis.

    PubMed

    Yang, Zhipan; Shang, Zengzhen; Wang, Lei; Lu, Qingtao; Wen, Xiaogang; Chi, Wei; Zhang, Lixin; Lu, Congming

    2015-12-01

    To investigate the molecular mechanism of chloroplast biogenesis and development, we characterized an Arabidopsis mutant (dg169, delayed greening 169) which showed growth retardation and delayed greening phenotype in leaves. Newly emerged chlorotic leaves recovered gradually with leaf development in the mutant, and the mature leaves showed similar phenotype to those of wild-typewild-type plants. Compared with wild-type, the chloroplasts were oval-shaped and smaller and the thylakoid membranes were less abundant in yellow section of young leaves of dg169. In addition, the functions of photosystem II (PSII) and photosystem I (PSI) were also impaired. Furthermore, the amount of core subunits of PSII and PSI, as well as PSII and PSI complexes reduced in yellow section of young leaves of dg169. Map-based positional cloning identified that phenotype of dg169 was attributed to a point mutation of ATase2 which converts the conserved Ile-155 residue to Asn. ATase2 catalyzes the first step of de novo purine biosynthesis. This mutation resulted in impaired purine synthesis and a significant decrease in ATP, ADP, GTP and GDP contents. The analysis of ATase2-GFP protein fusion showed that ATase2 was localized to nucleoid of chloroplasts. Our results further demonstrated that the levels of PEP-dependent transcripts in yellow section of young leaves of dg169 were decreased while NEP-dependent and both PEP- and NEP-dependent transcripts and chloroplast DNA replications were increased. The results in this study suggest that ATase2 plays an essential role in early chloroplast development through maintaining PEP function.

  12. 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

  13. Arabidopsis thaliana AMY3 Is a Unique Redox-regulated Chloroplastic α-Amylase*

    PubMed Central

    Seung, David; Thalmann, Matthias; Sparla, Francesca; Abou Hachem, Maher; Lee, Sang Kyu; Issakidis-Bourguet, Emmanuelle; Svensson, Birte; Zeeman, Samuel C.; Santelia, Diana

    2013-01-01

    α-Amylases are glucan hydrolases that cleave α-1,4-glucosidic bonds in starch. In vascular plants, α-amylases can be classified into three subfamilies. Arabidopsis has one member of each subfamily. Among them, only AtAMY3 is localized in the chloroplast. We expressed and purified AtAMY3 from Escherichia coli and carried out a biochemical characterization of the protein to find factors that regulate its activity. Recombinant AtAMY3 was active toward both insoluble starch granules and soluble substrates, with a strong preference for β-limit dextrin over amylopectin. Activity was shown to be dependent on a conserved aspartic acid residue (Asp666), identified as the catalytic nucleophile in other plant α-amylases such as the barley AMY1. AtAMY3 released small linear and branched glucans from Arabidopsis starch granules, and the proportion of branched glucans increased after the predigestion of starch with a β-amylase. Optimal rates of starch digestion in vitro was achieved when both AtAMY3 and β-amylase activities were present, suggesting that the two enzymes work synergistically at the granule surface. We also found that AtAMY3 has unique properties among other characterized plant α-amylases, with a pH optimum of 7.5–8, appropriate for activity in the chloroplast stroma. AtAMY3 is also redox-regulated, and the inactive oxidized form of AtAMY3 could be reactivated by reduced thioredoxins. Site-directed mutagenesis combined with mass spectrometry analysis showed that a disulfide bridge between Cys499 and Cys587 is central to this regulation. This work provides new insights into how α-amylase activity may be regulated in the chloroplast. PMID:24089528

  14. Arabidopsis thaliana AMY3 is a unique redox-regulated chloroplastic α-amylase.

    PubMed

    Seung, David; Thalmann, Matthias; Sparla, Francesca; Abou Hachem, Maher; Lee, Sang Kyu; Issakidis-Bourguet, Emmanuelle; Svensson, Birte; Zeeman, Samuel C; Santelia, Diana

    2013-11-22

    α-Amylases are glucan hydrolases that cleave α-1,4-glucosidic bonds in starch. In vascular plants, α-amylases can be classified into three subfamilies. Arabidopsis has one member of each subfamily. Among them, only AtAMY3 is localized in the chloroplast. We expressed and purified AtAMY3 from Escherichia coli and carried out a biochemical characterization of the protein to find factors that regulate its activity. Recombinant AtAMY3 was active toward both insoluble starch granules and soluble substrates, with a strong preference for β-limit dextrin over amylopectin. Activity was shown to be dependent on a conserved aspartic acid residue (Asp(666)), identified as the catalytic nucleophile in other plant α-amylases such as the barley AMY1. AtAMY3 released small linear and branched glucans from Arabidopsis starch granules, and the proportion of branched glucans increased after the predigestion of starch with a β-amylase. Optimal rates of starch digestion in vitro was achieved when both AtAMY3 and β-amylase activities were present, suggesting that the two enzymes work synergistically at the granule surface. We also found that AtAMY3 has unique properties among other characterized plant α-amylases, with a pH optimum of 7.5-8, appropriate for activity in the chloroplast stroma. AtAMY3 is also redox-regulated, and the inactive oxidized form of AtAMY3 could be reactivated by reduced thioredoxins. Site-directed mutagenesis combined with mass spectrometry analysis showed that a disulfide bridge between Cys(499) and Cys(587) is central to this regulation. This work provides new insights into how α-amylase activity may be regulated in the chloroplast.

  15. A mechanism for regulation of chloroplast LHC II kinase by plastoquinol and thioredoxin.

    PubMed

    Puthiyaveetil, Sujith

    2011-06-23

    State transitions are acclimatory responses to changes in light quality in photosynthesis. They involve the redistribution of absorbed excitation energy between photosystems I and II. In plants and green algae, this redistribution is produced by reversible phosphorylation of the chloroplast light harvesting complex II (LHC II). The LHC II kinase is activated by reduced plastoquinone (PQ) in photosystem II-specific low light. In high light, when PQ is also reduced, LHC II kinase becomes inactivated by thioredoxin. Based on newly identified amino acid sequence features of LHC II kinase and other considerations, a mechanism is suggested for its redox regulation.

  16. Transcriptional regulation of thylakoid galactolipid biosynthesis coordinated with chlorophyll biosynthesis during the development of chloroplasts in Arabidopsis.

    PubMed

    Kobayashi, Koichi; Fujii, Sho; Sasaki, Daichi; Baba, Shinsuke; Ohta, Hiroyuki; Masuda, Tatsuru; Wada, Hajime

    2014-01-01

    Biogenesis of thylakoid membranes in chloroplasts requires the coordinated synthesis of chlorophyll and photosynthetic proteins with the galactolipids monogalactosyldiacylglycerol (MGDG) and digalactosyldiacylglycerol (DGDG), which constitute the bulk of the thylakoid lipid matrix. MGD1 and DGD1 are the key enzymes of MGDG and DGDG synthesis, respectively. We investigated the expression profiles of MGD1 and DGD1 in Arabidopsis to identify the transcriptional regulation that coordinates galactolipid synthesis with the synthesis of chlorophyll and photosynthetic proteins during chloroplast biogenesis. The expression of both MGD1 and DGD1 was repressed in response to defects in chlorophyll synthesis. Moreover, these genes were downregulated by norflurazon-induced chloroplast malfunction via the GENOMES-UNCOUPLED1-mediated plastid signaling pathway. Similar to other photosynthesis-associated nuclear genes, the expression of MGD1 and DGD1 was induced by light, in which both cytokinin signaling and LONG HYPOCOTYL5-mediated light signaling played crucial roles. The expression of these galactolipid-synthesis genes, and particularly that of DGD1 under continuous light, was strongly affected by the activities of the GOLDEN2-LIKE transcription factors, which are potent regulators of chlorophyll synthesis and chloroplast biogenesis. These results suggest tight transcriptional coordination of galactolipid synthesis with the formation of the photosynthetic chlorophyll-protein complexes during leaf development. Meanwhile, unlike the photosynthetic genes, the galactolipid synthesis genes were not upregulated during chloroplast biogenesis in the roots, even though the galactolipids accumulated with chlorophylls, indicating the importance of post-transcriptional regulation of galactolipid synthesis during root greening. Our data suggest that plants utilize complex regulatory mechanisms to modify galactolipid synthesis with chloroplast development during plant growth.

  17. 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

  18. Structural Basis for Redox Regulation of Cytoplasmic and Chloroplastic Triosephosphate Isomerases from Arabidopsis thaliana

    PubMed Central

    López-Castillo, Laura M.; Jiménez-Sandoval, Pedro; Baruch-Torres, Noe; Trasviña-Arenas, Carlos H.; Díaz-Quezada, Corina; Lara-González, Samuel; Winkler, Robert; Brieba, Luis G.

    2016-01-01

    In plants triosephosphate isomerase (TPI) interconverts glyceraldehyde 3-phosphate (G3P) and dihydroxyacetone phosphate (DHAP) during glycolysis, gluconeogenesis, and the Calvin-Benson cycle. The nuclear genome of land plants encodes two tpi genes, one gene product is located in the cytoplasm and the other is imported into the chloroplast. Herein we report the crystal structures of the TPIs from the vascular plant Arabidopsis thaliana (AtTPIs) and address their enzymatic modulation by redox agents. Cytoplasmic TPI (cTPI) and chloroplast TPI (pdTPI) share more than 60% amino acid identity and assemble as (β-α)8 dimers with high structural homology. cTPI and pdTPI harbor two and one accessible thiol groups per monomer respectively. cTPI and pdTPI present a cysteine at an equivalent structural position (C13 and C15 respectively) and cTPI also contains a specific solvent accessible cysteine at residue 218 (cTPI-C218). Site directed mutagenesis of residues pdTPI-C15, cTPI-C13, and cTPI-C218 to serine substantially decreases enzymatic activity, indicating that the structural integrity of these cysteines is necessary for catalysis. AtTPIs exhibit differential responses to oxidative agents, cTPI is susceptible to oxidative agents such as diamide and H2O2, whereas pdTPI is resistant to inhibition. Incubation of AtTPIs with the sulfhydryl conjugating reagents methylmethane thiosulfonate (MMTS) and glutathione inhibits enzymatic activity. However, the concentration necessary to inhibit pdTPI is at least two orders of magnitude higher than the concentration needed to inhibit cTPI. Western-blot analysis indicates that residues cTPI-C13, cTPI-C218, and pdTPI-C15 conjugate with glutathione. In summary, our data indicate that AtTPIs could be redox regulated by the derivatization of specific AtTPI cysteines (cTPI-C13 and pdTPI-C15 and cTPI-C218). Since AtTPIs have evolved by gene duplication, the higher resistance of pdTPI to redox agents may be an adaptive consequence to the

  19. Light-Induced Movements of Chloroplasts and Nuclei Are Regulated in Both Cp-Actin-Filament-Dependent and -Independent Manners in Arabidopsis thaliana

    PubMed Central

    2016-01-01

    Light-induced chloroplast movement and attachment to the plasma membrane are dependent on actin filaments. In Arabidopsis thaliana, the short actin filaments on the chloroplast envelope, cp-actin filaments, are essential for chloroplast movement and positioning. Furthermore, cp-actin-filament-mediated chloroplast movement is necessary for the strong-light-induced nuclear avoidance response. The proteins CHLOROPLAST UNUSUAL POSITIONING 1 (CHUP1), KINESIN-LIKE PROTEIN FOR ACTIN-BASED CHLOROPLAST MOVEMENT 1 (KAC1) and KAC2 are required for the generation and/or maintenance of cp-actin filaments in Arabidopsis. In land plants, CHUP1 and KAC family proteins play pivotal roles in the proper movement of chloroplasts and their attachment to the plasma membrane. Here, we report similar but distinct phenotypes in chloroplast and nuclear photorelocation movements between chup1 and kac1kac2 mutants. Measurement of chloroplast photorelocation movement indicated that kac1kac2, but not chup1, exhibited a clear strong-light-induced increase in leaf transmittance changes. The chloroplast movement in kac1kac2 depended on phototropin 2, CHUP1 and two other regulators for cp-actin filaments, PLASTID MOVEMENT IMPAIRED 1 and THRUMIN 1. Furthermore, kac1kac2 retained a weak but significant nuclear avoidance response although chup1 displayed a severe defect in the nuclear avoidance response. The kac1kac2chup1 triple mutant was completely defective in both chloroplast and nuclear avoidance responses. These results indicate that CHUP1 and the KACs function somewhat independently, but interdependently mediate both chloroplast and nuclear photorelocation movements. PMID:27310016

  20. Light-Induced Movements of Chloroplasts and Nuclei Are Regulated in Both Cp-Actin-Filament-Dependent and -Independent Manners in Arabidopsis thaliana.

    PubMed

    Suetsugu, Noriyuki; Higa, Takeshi; Gotoh, Eiji; Wada, Masamitsu

    2016-01-01

    Light-induced chloroplast movement and attachment to the plasma membrane are dependent on actin filaments. In Arabidopsis thaliana, the short actin filaments on the chloroplast envelope, cp-actin filaments, are essential for chloroplast movement and positioning. Furthermore, cp-actin-filament-mediated chloroplast movement is necessary for the strong-light-induced nuclear avoidance response. The proteins CHLOROPLAST UNUSUAL POSITIONING 1 (CHUP1), KINESIN-LIKE PROTEIN FOR ACTIN-BASED CHLOROPLAST MOVEMENT 1 (KAC1) and KAC2 are required for the generation and/or maintenance of cp-actin filaments in Arabidopsis. In land plants, CHUP1 and KAC family proteins play pivotal roles in the proper movement of chloroplasts and their attachment to the plasma membrane. Here, we report similar but distinct phenotypes in chloroplast and nuclear photorelocation movements between chup1 and kac1kac2 mutants. Measurement of chloroplast photorelocation movement indicated that kac1kac2, but not chup1, exhibited a clear strong-light-induced increase in leaf transmittance changes. The chloroplast movement in kac1kac2 depended on phototropin 2, CHUP1 and two other regulators for cp-actin filaments, PLASTID MOVEMENT IMPAIRED 1 and THRUMIN 1. Furthermore, kac1kac2 retained a weak but significant nuclear avoidance response although chup1 displayed a severe defect in the nuclear avoidance response. The kac1kac2chup1 triple mutant was completely defective in both chloroplast and nuclear avoidance responses. These results indicate that CHUP1 and the KACs function somewhat independently, but interdependently mediate both chloroplast and nuclear photorelocation movements.

  1. Regulation of chloroplast number and DNA synthesis in higher plants. Final report

    SciTech Connect

    Mullet, J.E.

    1995-11-10

    The long term objective of this research is to understand the process of chloroplast development and its coordination with leaf development in higher plants. This is important because the photosynthetic capacity of plants is directly related to leaf and chloroplast development. This research focuses on obtaining a detailed description of leaf development and the early steps in chloroplast development including activation of plastid DNA synthesis, changes in plastid DNA copy number, activation of chloroplast transcription and increases in plastid number per cell. The grant will also begin analysis of specific biochemical mechanisms by isolation of the plastid DNA polymerase, and identification of genetic mutants which are altered in their accumulation of plastid DNA and plastid number per cell.

  2. Regulation of chloroplast number and DNA synthesis in higher plants. Final report

    SciTech Connect

    Mullet, J.E.

    1995-11-10

    The long term objective of this research is to understand the process of chloroplast development and its coordination with leaf development in higher plants. This is important because the photosynthetic capacity of plants is directly related to leaf and chloroplast development. This research focuses on obtaining a detailing description of leaf development and the early steps in chloroplast development including activation of plastid DNA synthesis, changes in plastid DNA copy number, activation of chloroplast transcription and increases in plastid number per cell. The grant will also begin analysis of specific biochemical mechanisms by isolation of the plastid DNA polymerase, and identification of genetic mutants which are altered in their accumulation of plastid DNA and plastid number per cell.

  3. A Chloroplast ABC1-like Kinase Regulates Vitamin E Metabolism in Arabidopsis1[C][W][OA

    PubMed Central

    Martinis, Jacopo; Glauser, Gaétan; Valimareanu, Sergiu; Kessler, Felix

    2013-01-01

    In bacteria and mitochondria, ABC1 (for Activity of bc1 complex)-like kinases regulate ubiquinone synthesis, mutations causing severe respiration defects, including neurological disorders in humans. Little is known about plant ABC1-like kinases; in Arabidopsis (Arabidopsis thaliana), five are predicted in mitochondria but, surprisingly, six are located at lipid droplets in chloroplasts. These are a known site of prenylquinone (including tocopherol [vitamin E], phylloquinone [vitamin K] and plastoquinone) metabolism and contain a large proportion of the tocopherol cyclase (VTE1) required for vitamin E synthesis and recycling. Therefore, ABC1-like kinases may be involved in the regulation of chloroplast prenylquinone metabolism. Using a nontargeted lipidomics approach, we demonstrate that plants lacking the plastoglobule ABC1-like kinase ABC1K3 are defective both for the production of plastochromanol-8 (a plastoquinone-derived lipid antioxidant) and the redox recycling of α-tocopherol, whereas tocopherol production is not affected. All of these pathways require VTE1 activity. However, in the abc1k3 mutant, VTE1 levels are strongly reduced posttranscriptionally. We provide evidence that the ABC1-like kinase ABC1K3 phosphorylates VTE1, possibly stabilizing it at plastoglobules. However, ABC1K3 may also have other targets and be involved in a wider chloroplast regulatory network. PMID:23632854

  4. Post transcriptional regulation of chloroplast gene expression by nuclear encoded gene products

    SciTech Connect

    Kuchka, M.R.

    1992-01-01

    Many individual chloroplast genes require the products of a collection of nuclear genes for their successful expression. These nuclear gene products apparently work with great specificity, each committed to the expression of a single chloroplast gene. We have chosen as a model nuclear mutants of Chlamydomonas affected in different stages in the expression of the chloroplast encoded Photosystem II polypeptide, D2. We have made the progress in understanding how nuclear gene products affect the translation of the D2 encoding MRNA. Two nuclear genes are required for this process which have been mapped genetically. In contrast to other examples of nuclear control of translation in the chloroplast, these nuclear gene products appear to be required either for specific stages in translation elongation or for the post-translational stabilization of the nascent D2 protein. Pseudoreversion analysis has led us to a locus which may be directly involved in D2 expression. We have made considerable progress in pursuing the molecular basis of psbd MRNA stabilization. psbD 5' UTR specific transcripts have been synthesized in vitro and used in gel mobility shift assays. UV-crosslinking studies are underway to identify the transacting factors which bind to these sequences. The continued examination of these mutants will help us to understand how nuclear gene products work in this specific case of chloroplast gene expression, and will elucidate how two distinct genomes can interact generally.

  5. A protein phosphatase 2A catalytic subunit modulates blue light-induced chloroplast avoidance movements through regulating actin cytoskeleton in Arabidopsis.

    PubMed

    Wen, Feng; Wang, Jinqian; Xing, Da

    2012-08-01

    Chloroplast avoidance movements mediated by phototropin 2 (phot2) are one of most important physiological events in the response to high-fluence blue light (BL), which reduces damage to the photosynthetic machinery under excess light. Protein phosphatase 2A-2 (PP2A-2) is an isoform of the catalytic subunit of PP2A, which regulates a number of developmental processes. To investigate whether PP2A-2 was involved in high-fluence BL-induced chloroplast avoidance movements, we first analyzed chloroplast migration in the leaves of the pp2a-2 mutant in response to BL. The data showed that PP2A-2 might act as a positive regulator in phot2-mediated chloroplast avoidance movements, but not in phot1-mediated chloroplast accumulation movements. Then, the effect of okadaic acid (OA) and cantharidin (selective PP2A inhibitors) on high-fluence BL response was further investigated in Arabidopsis thaliana mesophyll cells. Within a certain concentration range, exogenously applied OA or cantharidin inhibited the high-fluence BL-induced chloroplast movements in a concentration-dependent manner. Actin depolymerizing factor (ADF)/cofilin phosphorylation assays demonstrated that PP2A-2 can activate/dephosphorylate ADF/cofilin, an actin-binding protein, in Arabidopsis mesophyll cells. Consistent with this observation, the experiments showed that OA could inhibit ADF1 binding to the actin and suppress the reorganization of the actin cytoskeleton after high-fluence BL irradiation. The adf1 and adf3 mutants also exhibited reduced high-fluence BL-induced chloroplast avoidance movements. In conclusion, we identified that PP2A-2 regulated the activation of ADF/cofilin, which, in turn, regulated actin cytoskeleton remodeling and was involved in phot2-mediated chloroplast avoidance movements.

  6. Why chloroplasts and mitochondria retain their own genomes and genetic systems: Colocation for redox regulation of gene expression

    PubMed Central

    Allen, John F.

    2015-01-01

    Chloroplasts and mitochondria are subcellular bioenergetic organelles with their own genomes and genetic systems. DNA replication and transmission to daughter organelles produces cytoplasmic inheritance of characters associated with primary events in photosynthesis and respiration. The prokaryotic ancestors of chloroplasts and mitochondria were endosymbionts whose genes became copied to the genomes of their cellular hosts. These copies gave rise to nuclear chromosomal genes that encode cytosolic proteins and precursor proteins that are synthesized in the cytosol for import into the organelle into which the endosymbiont evolved. What accounts for the retention of genes for the complete synthesis within chloroplasts and mitochondria of a tiny minority of their protein subunits? One hypothesis is that expression of genes for protein subunits of energy-transducing enzymes must respond to physical environmental change by means of a direct and unconditional regulatory control—control exerted by change in the redox state of the corresponding gene product. This hypothesis proposes that, to preserve function, an entire redox regulatory system has to be retained within its original membrane-bound compartment. Colocation of gene and gene product for redox regulation of gene expression (CoRR) is a hypothesis in agreement with the results of a variety of experiments designed to test it and which seem to have no other satisfactory explanation. Here, I review evidence relating to CoRR and discuss its development, conclusions, and implications. This overview also identifies predictions concerning the results of experiments that may yet prove the hypothesis to be incorrect. PMID:26286985

  7. Why chloroplasts and mitochondria retain their own genomes and genetic systems: Colocation for redox regulation of gene expression.

    PubMed

    Allen, John F

    2015-08-18

    Chloroplasts and mitochondria are subcellular bioenergetic organelles with their own genomes and genetic systems. DNA replication and transmission to daughter organelles produces cytoplasmic inheritance of characters associated with primary events in photosynthesis and respiration. The prokaryotic ancestors of chloroplasts and mitochondria were endosymbionts whose genes became copied to the genomes of their cellular hosts. These copies gave rise to nuclear chromosomal genes that encode cytosolic proteins and precursor proteins that are synthesized in the cytosol for import into the organelle into which the endosymbiont evolved. What accounts for the retention of genes for the complete synthesis within chloroplasts and mitochondria of a tiny minority of their protein subunits? One hypothesis is that expression of genes for protein subunits of energy-transducing enzymes must respond to physical environmental change by means of a direct and unconditional regulatory control--control exerted by change in the redox state of the corresponding gene product. This hypothesis proposes that, to preserve function, an entire redox regulatory system has to be retained within its original membrane-bound compartment. Colocation of gene and gene product for redox regulation of gene expression (CoRR) is a hypothesis in agreement with the results of a variety of experiments designed to test it and which seem to have no other satisfactory explanation. Here, I review evidence relating to CoRR and discuss its development, conclusions, and implications. This overview also identifies predictions concerning the results of experiments that may yet prove the hypothesis to be incorrect.

  8. The chloroplast permease PIC1 regulates plant growth and development by directing homeostasis and transport of iron.

    PubMed

    Duy, Daniela; Stübe, Roland; Wanner, Gerhard; Philippar, Katrin

    2011-04-01

    The membrane-spanning protein PIC1 (for permease in chloroplasts 1) in Arabidopsis (Arabidopsis thaliana) was previously described to mediate iron transport across the inner envelope membrane of chloroplasts. The albino phenotype of pic1 knockout mutants was reminiscent of iron-deficiency symptoms and characterized by severely impaired plastid development and plant growth. In addition, plants lacking PIC1 showed a striking increase in chloroplast ferritin clusters, which function in protection from oxidative stress by sequestering highly reactive free iron in their spherical protein shell. In contrast, PIC1-overexpressing lines (PIC1ox) in this study rather resembled ferritin loss-of-function plants. PIC1ox plants suffered from oxidative stress and leaf chlorosis, most likely originating from iron overload in chloroplasts. Later during growth, plants were characterized by reduced biomass as well as severely defective flower and seed development. As a result of PIC1 protein increase in the inner envelope membrane of plastids, flower tissue showed elevated levels of iron, while the content of other transition metals (copper, zinc, manganese) remained unchanged. Seeds, however, specifically revealed iron deficiency, suggesting that PIC1 overexpression sequestered iron in flower plastids, thereby becoming unavailable for seed iron loading. In addition, expression of genes associated with metal transport and homeostasis as well as photosynthesis was deregulated in PIC1ox plants. Thus, PIC1 function in plastid iron transport is closely linked to ferritin and plastid iron homeostasis. In consequence, PIC1 is crucial for balancing plant iron metabolism in general, thereby regulating plant growth and in particular fruit development.

  9. 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.

  10. Large Scale Comparative Proteomics of a Chloroplast Clp Protease Mutant Reveals Folding Stress, Altered Protein Homeostasis, and Feedback Regulation of Metabolism*

    PubMed Central

    Zybailov, Boris; Friso, Giulia; Kim, Jitae; Rudella, Andrea; Rodríguez, Verenice Ramírez; Asakura, Yukari; Sun, Qi; van Wijk, Klaas J.

    2009-01-01

    The clpr2-1 mutant is delayed in development due to reduction of the chloroplast ClpPR protease complex. To understand the role of Clp proteases in plastid biogenesis and homeostasis, leaf proteomes of young seedlings of clpr2-1 and wild type were compared using large scale mass spectrometry-based quantification using an LTQ-Orbitrap and spectral counting with significance determined by G-tests. Virtually only chloroplast-localized proteins were significantly affected, indicating that the molecular phenotype was confined to the chloroplast. A comparative chloroplast stromal proteome analysis of fully developed plants was used to complement the data set. Chloroplast unfoldase ClpB3 was strongly up-regulated in both young and mature leaves, suggesting widespread and persistent protein folding stress. The importance of ClpB3 in the clp2-1 mutant was demonstrated by the observation that a CLPR2 and CLPB3 double mutant was seedling-lethal. The observed up-regulation of chloroplast chaperones and protein sorting components further illustrated destabilization of protein homeostasis. Delayed rRNA processing and up-regulation of a chloroplast DEAD box RNA helicase and polynucleotide phosphorylase, but no significant change in accumulation of ribosomal subunits, suggested a bottleneck in ribosome assembly or RNA metabolism. Strong up-regulation of a chloroplast translational regulator TypA/BipA GTPase suggested a specific response in plastid gene expression to the distorted homeostasis. The stromal proteases PreP1,2 were up-regulated, likely constituting compensation for reduced Clp protease activity and possibly shared substrates between the ClpP and PreP protease systems. The thylakoid photosynthetic apparatus was decreased in the seedlings, whereas several structural thylakoid-associated plastoglobular proteins were strongly up-regulated. Two thylakoid-associated reductases involved in isoprenoid and chlorophyll synthesis were up-regulated reflecting feedback from rate

  11. Chloroplast Biogenesis-Associated Nuclear Genes: Control by Plastid Signals Evolved Prior to Their Regulation as Part of Photomorphogenesis

    PubMed Central

    Hills, Alison C.; Khan, Safina; López-Juez, Enrique

    2015-01-01

    The assembly of photosynthetically competent chloroplasts occurs in angiosperm seedlings when first exposed to light, and is due to the control by light of photosynthesis-associated nuclear genes (PhANGs), also dependent upon plastid-to-nucleus “biogenic” communication signals. The relationship between light- and plastid signal-regulation of PhANGs is close but poorly understood. In contrast, many conifers green in the dark and the promoter of a pine PhANG, Lhcb, is active in the dark in tobacco. Here, we show that the activity of this promoter in tobacco is sensitive to plastid photobleaching, or to the inhibition of plastid translation in the light or the dark, and the same interventions reduce expression of the native gene in pine seedlings, demonstrating classic plastid biogenic signaling in gymnosperms. Furthermore, Arabidopsis mutations causing defective plastid biogenesis suppress the effect in darkness of mutations in COP1 and DET1, repressors of photomorphogenesis, for the expression of several PhANGs but not a photosynthesis-unrelated, light-regulated gene. GLK transcriptional regulators mediate the response of LHCB but not of other tested PhANGs. We propose the ability to suppress PhANG response to positive plastid biogenic signals in the dark may have contributed to the evolution of light-controlled chloroplast biogenesis. PMID:26697036

  12. Cold-Regulated Cereal Chloroplast Late Embryogenesis Abundant-Like Proteins. Molecular Characterization and Functional Analyses

    PubMed Central

    NDong, Christian; Danyluk, Jean; Wilson, Kenneth E.; Pocock, Tessa; Huner, Norman P.A.; Sarhan, Fathey

    2002-01-01

    Cold acclimation and freezing tolerance are the result of complex interaction between low temperature, light, and photosystem II (PSII) excitation pressure. Previous results have shown that expression of the Wcs19 gene is correlated with PSII excitation pressure measured in vivo as the relative reduction state of PSII. Using cDNA library screening and data mining, we have identified three different groups of proteins, late embryogenesis abundant (LEA) 3-L1, LEA3-L2, and LEA3-L3, sharing identities with WCS19. These groups represent a new class of proteins in cereals related to group 3 LEA proteins. They share important characteristics such as a sorting signal that is predicted to target them to either the chloroplast or mitochondria and a C-terminal sequence that may be involved in oligomerization. The results of subcellular fractionation, immunolocalization by electron microscopy and the analyses of target sequences within the Wcs19 gene are consistent with the localization of WCS19 within the chloroplast stroma of wheat (Triticum aestivum) and rye (Secale cereale). Western analysis showed that the accumulation of chloroplastic LEA3-L2 proteins is correlated with the capacity of different wheat and rye cultivars to develop freezing tolerance. Arabidopsis was transformed with the Wcs19 gene and the transgenic plants showed a significant increase in their freezing tolerance. This increase was only evident in cold-acclimated plants. The putative function of this protein in the enhancement of freezing tolerance is discussed. PMID:12114590

  13. Post transcriptional regulation of chloroplast gene expression by nuclear encoded gene products

    SciTech Connect

    Kuchka, M.R.

    1992-01-01

    The following is a review of research accomplished in the first two years of funding for the above mentioned project. The work performed is a molecular characterization of nuclear mutants of Chlamydomonas reinhardtii which are deficient in different stages in the post-transcriptional expression of a single chloroplast encoded polypeptide, the D2 protein of Photosystem II. Our long-term goals are to understand the molecular mechanisms by which nuclear gene products affect the expression of chloroplast genes. Specifically, we which to understand how specific nuclear gene products affect the turnover rate of the D2 encoding mRNA (psbD), how other nuclear encoded factors work to promote the translation of psbD mRNA and/or stabilize the D2 protein, and what the role of the D2 protein itself is in Photosystem II assembly and in the control of expression of other chloroplast genes. This progress report will be organized into four major sections concerning (I) The characterization of nuclear mutants affected in D2 translation/turnover, (II) The study of trans-acting factors which associate with the 5{prime} end of the psbD mRNA, (III) In vitro mutagenesis of the psbD gene, and (IV) Additional studies.

  14. GOLDEN 2-LIKE transcription factors for chloroplast development affect ozone tolerance through the regulation of stomatal movement

    PubMed Central

    Nagatoshi, Yukari; Mitsuda, Nobutaka; Hayashi, Maki; Inoue, Shin-ichiro; Okuma, Eiji; Kubo, Akihiro; Murata, Yoshiyuki; Seo, Mitsunori; Saji, Hikaru; Kinoshita, Toshinori; Ohme-Takagi, Masaru

    2016-01-01

    Stomatal movements regulate gas exchange, thus directly affecting the efficiency of photosynthesis and the sensitivity of plants to air pollutants such as ozone. The GARP family transcription factors GOLDEN 2-LIKE1 (GLK1) and GLK2 have known functions in chloroplast development. Here, we show that Arabidopsis thaliana (A. thaliana) plants expressing the chimeric repressors for GLK1 and -2 (GLK1/2-SRDX) exhibited a closed-stomata phenotype and strong tolerance to ozone. By contrast, plants that overexpress GLK1/2 exhibited an open-stomata phenotype and higher sensitivity to ozone. The plants expressing GLK1-SRDX had reduced expression of the genes for inwardly rectifying K+ (K+in) channels and reduced K+in channel activity. Abscisic acid treatment did not affect the stomatal phenotype of 35S:GLK1/2-SRDX plants or the transcriptional activity for K+in channel gene, indicating that GLK1/2 act independently of abscisic acid signaling. Our results indicate that GLK1/2 positively regulate the expression of genes for K+in channels and promote stomatal opening. Because the chimeric GLK1-SRDX repressor driven by a guard cell-specific promoter induced a closed-stomata phenotype without affecting chloroplast development in mesophyll cells, modulating GLK1/2 activity may provide an effective tool to control stomatal movements and thus to confer resistance to air pollutants. PMID:27035938

  15. GOLDEN 2-LIKE transcription factors for chloroplast development affect ozone tolerance through the regulation of stomatal movement.

    PubMed

    Nagatoshi, Yukari; Mitsuda, Nobutaka; Hayashi, Maki; Inoue, Shin-Ichiro; Okuma, Eiji; Kubo, Akihiro; Murata, Yoshiyuki; Seo, Mitsunori; Saji, Hikaru; Kinoshita, Toshinori; Ohme-Takagi, Masaru

    2016-04-12

    Stomatal movements regulate gas exchange, thus directly affecting the efficiency of photosynthesis and the sensitivity of plants to air pollutants such as ozone. The GARP family transcription factors GOLDEN 2-LIKE1 (GLK1) and GLK2 have known functions in chloroplast development. Here, we show that Arabidopsis thaliana (A. thaliana) plants expressing the chimeric repressors for GLK1 and -2 (GLK1/2-SRDX) exhibited a closed-stomata phenotype and strong tolerance to ozone. By contrast, plants that overexpress GLK1/2 exhibited an open-stomata phenotype and higher sensitivity to ozone. The plants expressing GLK1-SRDX had reduced expression of the genes for inwardly rectifying K(+) (K(+) in) channels and reduced K(+) in channel activity. Abscisic acid treatment did not affect the stomatal phenotype of 35S:GLK1/2-SRDX plants or the transcriptional activity for K(+) in channel gene, indicating that GLK1/2 act independently of abscisic acid signaling. Our results indicate that GLK1/2 positively regulate the expression of genes for K(+) in channels and promote stomatal opening. Because the chimeric GLK1-SRDX repressor driven by a guard cell-specific promoter induced a closed-stomata phenotype without affecting chloroplast development in mesophyll cells, modulating GLK1/2 activity may provide an effective tool to control stomatal movements and thus to confer resistance to air pollutants.

  16. Chloroplast movement.

    PubMed

    Wada, Masamitsu

    2013-09-01

    Chloroplast movement is important for plant survival under high light and for efficient photosynthesis under low light. This review introduces recent knowledge on chloroplast movement and shows how to analyze the responses and the moving mechanisms, potentially inspiring research in this field. Avoidance from the strong light is mediated by blue light receptor phototropin 2 (phot2) plausibly localized on the chloroplast envelop and accumulation at the week light-irradiated area is mediated by phot1 and phot2 localized on the plasma membrane. Chloroplasts move by chloroplast actin (cp-actin) filaments that must be polymerized by Chloroplast Unusual Positioning1 (CHUP1) at the front side of moving chloroplast. To understand the signal transduction pathways and the mechanism of chloroplast movement, that is, from light capture to motive force-generating mechanism, various methods should be employed based on the various aspects. Observation of chloroplast distribution pattern under different light condition by fixed cell sectioning is somewhat an old-fashioned technique but the most basic and important way. However, most importantly, precise chloroplast behavior during and just after the induction of chloroplast movement by partial cell irradiation using an irradiator with either low light or strong light microbeam should be recorded by time lapse photographs under infrared light and analyzed. Recently various factors involved in chloroplast movement, such as cp-actin filaments and CHUP1, could be traced in Arabidopsis transgenic lines with fluorescent protein tags under a confocal laser scanning microscope (CLSM) and/or a total internal reflection fluorescence microscope (TIRFM). These methods are listed and their advantages and disadvantages are evaluated.

  17. NTRC and chloroplast-generated reactive oxygen species regulate Pseudomonas syringae pv. tomato disease development in tomato and Arabidopsis.

    PubMed

    Ishiga, Yasuhiro; Ishiga, Takako; Wangdi, Tamding; Mysore, Kirankumar S; Uppalapati, Srinivasa Rao

    2012-03-01

    Coronatine (COR)-producing pathovars of Pseudomonas syringae, including pvs. tomato, maculicola, and glycinea, cause important diseases on tomato, crucifers, and soybean, respectively, and produce symptoms with necrotic lesions surrounded by chlorosis. The chlorosis is mainly attributed to COR. However, the significance of COR-induced chlorosis in localized lesion development and the molecular basis of disease-associated cell death is largely unknown. To identify host (chloroplast) genes that play a role in COR-mediated chlorosis, we used a forward genetics approach using Nicotiana benthamiana and virus-induced gene silencing and identified a gene which encodes 2-Cys peroxiredoxin (Prxs) that, when silenced, produced a spreading hypersensitive or necrosis-like phenotype instead of chlorosis after COR application in a COI1-dependent manner. Loss-of-function analysis of Prx and NADPH-dependent thioredoxin reductase C (NTRC), the central players of a chloroplast redox detoxification system, resulted in spreading accelerated P. syringae pv. tomato DC3000 disease-associated cell death with enhanced reactive oxygen species (ROS) accumulation in a COR-dependent manner in tomato and Arabidopsis. Consistent with these results, virulent strain DC3000 suppressed the expression of Prx and NTRC in Arabidopsis and tomato during pathogenesis. However, interestingly, authentic COR suppressed the expression of Prx and NTRC in tomato but not in Arabidopsis, suggesting that COR in conjunction with other effectors may modulate ROS and cell death in different host species. Taken together, these results indicated that NTRC or Prx function as a negative regulator of pathogen-induced cell death in the healthy tissues that surround the lesions, and COR-induced chloroplast-localized ROS play a role in enhancing the disease-associated cell death.

  18. A novel light-regulated promoter is conserved in cereal and dicot chloroplasts.

    PubMed Central

    Christopher, D A; Kim, M; Mullet, J E

    1992-01-01

    The chloroplast psbD-psbC genes encode D2 and cp43, a reaction center protein and chlorophyll-binding antenna protein of photosystem II, respectively. We have previously shown that differential accumulation of light-induced psbD-psbC mRNAs in barley chloroplasts is due to transcription from a blue light-responsive promoter (LRP). It is hypothesized that the light-induced mRNAs help to maintain levels of the D2 polypeptide, which is photodamaged and degraded in illuminated plants. To determine if light-induced accumulation of psbD-psbC mRNAs was a conserved phenomenon in chloroplasts, the expression of psbD-psbC operons from five cereals (barley, wheat, rice, maize, and sorghum) and three dicot (tobacco, spinach, and pea) species was examined. Cereal and dicot psbD-psbC operons differ due to several DNA rearrangements that moved psbK-psbI proximal to psbD-psbC, allowing cotranscription of these genes and production of several unique transcripts in cereals. Despite differences in the structure and expression of the cereal and dicot psbD-psbC operons, the accumulation of light-induced psbD-psbC mRNAs was conserved in all species studied. An unusual feature of the light-induced mRNAs was the occurrence of 5' end microheterogeneity. The multiple 5' termini were mapped to several consecutive nucleotides (8 to 25 bp) within a highly conserved (61%) DNA region that represents the transcription initiation site for the mRNAs in barley and tobacco. The novel LRP differs in sequence from typical plastid promoters that have prokaryotic "-10" and "-35" elements and is centered 570 bp (cereals), 900 bp (tobacco, spinach), or 1100 bp (pea) upstream from the psbD translational start codon. We propose that physiological and gene regulatory demands of the chloroplast act as constraints that preserved the linkage of the LRP with psbD despite DNA inversions involving the psbD upstream region. PMID:1392595

  19. Chloroplast movement.

    PubMed

    Wada, Masamitsu; Kagawa, Takatoshi; Sato, Yoshikatsu

    2003-01-01

    The study of chloroplast movement made a quantum leap at the beginning of the twenty-first century. Research based on reverse-genetic approaches using targeted mutants has brought new concepts to this field. One of the most exciting findings has been the discovery of photoreceptors for both accumulation and avoidance responses in Arabidopsis and in the fern Adiantum. Evidence for the adaptive advantage of chloroplast avoidance movements in plant survival has also been found. Additional discoveries include mechano-stress-induced chloroplast movement in ferns and mosses, and microtubule-mediated chloroplast movement in the moss Physcomitrella. The possible ecological significance of chloroplast movement is discussed in the final part of this review.

  20. Nuclear gene-regulated expression of chloroplast genes for coupling factor one in maize

    SciTech Connect

    Kobayashi, H.; Bogorad, L.; Miles, C.D.

    1987-11-01

    In order to gain a better understanding of the interaction between the chloroplast and nuclear genomes in controlling the expression of plastid genes and the biosynthesis of chloroplast proteins, maize (Zea mays) nuclear gene mutant hcf*-38, in which ..cap alpha.. and ..beta.. subunits of coupling factor one (CF/sub 1/) are almost completely missing was studied. The mutant possesses all the other subunits of CF/sub 1/ but several peptides of photosystem II are present in reduced amounts. A competitive hybridization experiment showed the presence of the same plastid mRNA species in mutant and wild-type plants except for slightly lower levels of some transcripts in the mutant. Northern hybridization and dot blot hybridization experiments showed the features of transcripts for ..cap alpha.. and ..beta.. subunits of CF/sub 1/ in the mutant to be similar to those in the wild-type maize although their levels are somewhat lower in the mutant. In vivo and in organello protein labeling experiments with L-(/sup 35/S)Met have shown that ..cap alpha.. and ..beta.. subunits of CF/sub 1/ are synthesized, assembled into CF/sub 1/, and probably associated with thylakoid membranes in mutant plants. It is concluded that they are subsequently degraded.

  1. Laminarin modulates the chloroplast antioxidant system to enhance abiotic stress tolerance partially through the regulation of the defensin-like gene expression.

    PubMed

    Wu, Yi-Ru; Lin, Yi-Chen; Chuang, Huey-wen

    2016-06-01

    Algae wall polysaccharide, laminarin (Lam), has an established role on induction of plant disease resistance. In this study, application of Lam increased Arabidopsis fresh weight and enhanced tolerance to salt and heat stress by stabilizing chloroplast under adverse environment. Transcriptome analysis indicated that, in addition to induced a large number of genes associated with the host defense, genes involved in the regulation of abiotic stress tolerance mostly the heat stress response constituted the largest group of the up-regulated genes. Lam induced expression of IRT1, ZIP8, and copper transporters involved in transport of Fe, Zn, Cu ions associated with the activity of chloroplast antioxidant system. Lam also up-regulated genes involved in the synthesis of terpenoid, a plastidial-derived secondary metabolite with antioxidant activity. Overexpression of a Lam-induced defensin like 202 (DEFL202) resulted in increased chloroplast stability under salt stress and increased plant growth activity after heat stress. Expression of antioxidant enzymes including SOD and ascorbate peroxidase (APX), photosystem PsbA-D1 and ABA-dependent responsive to desiccation 22 (RD22) was induced to higher levels in the transgenic seedlings. In sum, our results suggest that Lam is an potent inducer for induction of chloroplastic antioxidant activity. Lam affect plant abiotic stress tolerance partially through regulation of the DEFL-mediated pathway.

  2. Chloroplast localization of methylerythritol 4-phosphate pathway enzymes and regulation of mitochondrial genes in ispD and ispE albino mutants in Arabidopsis.

    PubMed

    Hsieh, Ming-Hsiun; Chang, Chiung-Yun; Hsu, Shih-Jui; Chen, Ju-Jiun

    2008-04-01

    Plant isoprenoids are derived from two independent pathways, the cytosolic mevalonate pathway and the plastid methylerythritol 4-phosphate (MEP) pathway. We used green fluorescent fusion protein assays to demonstrate that the Arabidopsis MEP pathway enzymes are localized to the chloroplast. We have also characterized three Arabidopsis albino mutants, ispD-1, ispD-2 and ispE-1, which have T-DNA insertions in the IspD and IspE genes of the MEP pathway. Levels of photosynthetic pigments are almost undetectable in these albino mutants. Instead of thylakoids, the ispD and ispE mutant chloroplasts are filled with large vesicles. Impairments in chloroplast development and functions may signal changes in the expression of nuclear, chloroplast and mitochondrial genes. We used northern blot analysis to examine the expression of photosynthetic and respiratory genes in the ispD and ispE albino mutants. Steady-state mRNA levels of nucleus- and chloroplast-encoded photosynthetic genes are significantly decreased in the albino mutants. In contrast, transcript levels of nuclear and mitochondrial genes encoding subunits of the mitochondrial electron transport chain are increased or not affected in these mutants. Genomic Southern blot analysis revealed that the DNA amounts of mitochondrial genes are not enhanced in the ispD and ispE albino mutants. These results support the notion that the functional state of chloroplasts may affect the expression of nuclear and mitochondrial genes. The up-regulation of mitochondrial genes in the albino mutants is not caused by changes of mitochondrial DNA copy number in Arabidopsis.

  3. Automatic Chloroplast Movement Analysis.

    PubMed

    Johansson, Henrik; Zeidler, Mathias

    2016-01-01

    In response to low or high intensities of light, the chloroplasts in the mesophyll cells of the leaf are able to increase or decrease their exposure to light by accumulating at the upper and lower sides or along the side walls of the cell respectively. This movement, regulated by the phototropin blue light photoreceptors phot1 and phot2, results in a decreased or increased transmission of light through the leaf. This way the plant is able to optimize harvesting of the incoming light or avoid damage caused by excess light. Here we describe a method that indirectly measures the movement of chloroplasts by taking advantage of the resulting change in leaf transmittance. By using a microplate reader, quantitative measurements of chloroplast accumulation or avoidance can be monitored over time, for multiple samples with relatively little hands-on time.

  4. The kinesin-like proteins, KAC1/2, regulate actin dynamics underlying chloroplast light-avoidance in Physcomitrella patens.

    PubMed

    Shen, Zhiyuan; Liu, Yen-Chen; Bibeau, Jeffrey P; Lemoi, Kyle P; Tüzel, Erkan; Vidali, Luis

    2015-01-01

    In plants, light determines chloroplast position; these organelles show avoidance and accumulation responses in high and low fluence-rate light, respectively. Chloroplast motility in response to light is driven by cytoskeletal elements. The actin cytoskeleton mediates chloroplast photorelocation responses in Arabidopsis thaliana. In contrast, in the moss Physcomitrella patens, both, actin filaments and microtubules can transport chloroplasts. Because of the surprising evidence that two kinesin-like proteins (called KACs) are important for actin-dependent chloroplast photorelocation in vascular plants, we wanted to determine the cytoskeletal system responsible for the function of these proteins in moss. We performed gene-specific silencing using RNA interference in P. patens. We confirmed existing reports using gene knockouts, that PpKAC1 and PpKAC2 are required for chloroplast dispersion under uniform white light conditions, and that the two proteins are functionally equivalent. To address the specific cytoskeletal elements responsible for motility, this loss-of-function approach was combined with cytoskeleton-targeted drug studies. We found that, in P. patens, these KACs mediate the chloroplast light-avoidance response in an actin filament-dependent, rather than a microtubule-dependent manner. Using correlation-decay analysis of cytoskeletal dynamics, we found that PpKAC stabilizes cortical actin filaments, but has no effect on microtubule dynamics.

  5. Chloroplast Activity and 3′phosphadenosine 5′phosphate Signaling Regulate Programmed Cell Death in Arabidopsis1

    PubMed Central

    Mazubert, Christelle; Prunier, Florence; Chan, Kai Xun; Pogson, Barry James; Krieger-Liszkay, Anja; Delarue, Marianne; Benhamed, Moussa; Bergounioux, Catherine; Raynaud, Cécile

    2016-01-01

    Programmed cell death (PCD) is a crucial process both for plant development and responses to biotic and abiotic stress. There is accumulating evidence that chloroplasts may play a central role during plant PCD as for mitochondria in animal cells, but it is still unclear whether they participate in PCD onset, execution, or both. To tackle this question, we have analyzed the contribution of chloroplast function to the cell death phenotype of the myoinositol phosphate synthase1 (mips1) mutant that forms spontaneous lesions in a light-dependent manner. We show that photosynthetically active chloroplasts are required for PCD to occur in mips1, but this process is independent of the redox state of the chloroplast. Systematic genetic analyses with retrograde signaling mutants reveal that 3′-phosphoadenosine 5′-phosphate, a chloroplast retrograde signal that modulates nuclear gene expression in response to stress, can inhibit cell death and compromises plant innate immunity via inhibition of the RNA-processing 5′-3′ exoribonucleases. Our results provide evidence for the role of chloroplast-derived signal and RNA metabolism in the control of cell death and biotic stress response. PMID:26747283

  6. Redox regulation of ascorbate and glutathione by a chloroplastic dehydroascorbate reductase is required for high-light stress tolerance in Arabidopsis.

    PubMed

    Noshi, Masahiro; Hatanaka, Risa; Tanabe, Noriaki; Terai, Yusuke; Maruta, Takanori; Shigeoka, Shigeru

    2016-05-01

    Chloroplasts are a significant site for reactive oxygen species production under illumination and, thus, possess a well-organized antioxidant system involving ascorbate. Ascorbate recycling occurs in different manners in this system, including a dehydroascorbate reductase (DHAR) reaction. We herein investigated the physiological significance of DHAR3 in photo-oxidative stress tolerance in Arabidopsis. GFP-fused DHAR3 protein was targeted to chloroplasts in Arabidopsis leaves. A DHAR3 knockout mutant exhibited sensitivity to high light (HL). Under HL, the ascorbate redox states were similar in mutant and wild-type plants, while total ascorbate content was significantly lower in the mutant, suggesting that DHAR3 contributes, at least to some extent, to ascorbate recycling. Activation of monodehydroascorbate reductase occurred in dhar3 mutant, which might compensate for the lack of DHAR3. Interestingly, glutathione oxidation was consistently inhibited in dhar3 mutant. These findings indicate that DHAR3 regulates both ascorbate and glutathione redox states to acclimate to HL.

  7. Chloroplast signaling and LESION SIMULATING DISEASE1 regulate crosstalk between light acclimation and immunity in Arabidopsis.

    PubMed

    Mühlenbock, Per; Szechynska-Hebda, Magdalena; Plaszczyca, Marian; Baudo, Marcela; Mateo, Alfonso; Mullineaux, Philip M; Parker, Jane E; Karpinska, Barbara; Karpinski, Stanislaw

    2008-09-01

    Plants are simultaneously exposed to abiotic and biotic hazards. Here, we show that local and systemic acclimation in Arabidopsis thaliana leaves in response to excess excitation energy (EEE) is associated with cell death and is regulated by specific redox changes of the plastoquinone (PQ) pool. These redox changes cause a rapid decrease of stomatal conductance, global induction of ASCORBATE PEROXIDASE2 and PATHOGEN RESISTANCE1, and increased production of reactive oxygen species (ROS) and ethylene that signals through ETHYLENE INSENSITIVE2 (EIN2). We provide evidence that multiple hormonal/ROS signaling pathways regulate the plant's response to EEE and that EEE stimulates systemic acquired resistance and basal defenses to virulent biotrophic bacteria. In the Arabidopsis LESION SIMULATING DISEASE1 (lsd1) null mutant that is deregulated for EEE acclimation responses, propagation of EEE-induced programmed cell death depends on the plant defense regulators ENHANCED DISEASE SUSCEPTIBILITY1 (EDS1) and PHYTOALEXIN DEFICIENT4 (PAD4). We find that EDS1 and PAD4 operate upstream of ethylene and ROS production in the EEE response. The data suggest that the balanced activities of LSD1, EDS1, PAD4, and EIN2 regulate signaling of programmed cell death, light acclimation, and holistic defense responses that are initiated, at least in part, by redox changes of the PQ pool.

  8. Roles of Arabidopsis PARC6 in Coordination of the Chloroplast Division Complex and Negative Regulation of FtsZ Assembly1[OPEN

    PubMed Central

    Chen, Cheng; Froehlich, John E.; TerBush, Allan D.

    2016-01-01

    Chloroplast division is driven by the simultaneous constriction of the inner FtsZ ring (Z ring) and the outer DRP5B ring. The assembly and constriction of these rings in Arabidopsis (Arabidopsis thaliana) are coordinated partly through the inner envelope membrane protein ACCUMULATION AND REPLICATION OF CHLOROPLASTS6 (ARC6). Previously, we showed that PARC6 (PARALOG OF ARC6), also in the inner envelope membrane, negatively regulates FtsZ assembly and acts downstream of ARC6 to position the outer envelope membrane protein PLASTID DIVISION1 (PDV1), which functions together with its paralog PDV2 to recruit DYNAMIN-RELATED PROTEIN 5B (DRP5B) from a cytosolic pool to the outer envelope membrane. However, whether PARC6, like ARC6, also functions in coordination of the chloroplast division contractile complexes was unknown. Here, we report a detailed topological analysis of Arabidopsis PARC6, which shows that PARC6 has a single transmembrane domain and a topology resembling that of ARC6. The newly identified stromal region of PARC6 interacts not only with ARC3, a direct inhibitor of Z-ring assembly, but also with the Z-ring protein FtsZ2. Overexpression of PARC6 inhibits FtsZ assembly in Arabidopsis but not in a heterologous yeast system (Schizosaccharomyces pombe), suggesting that the negative regulation of FtsZ assembly by PARC6 is a consequence of its interaction with ARC3. A conserved carboxyl-terminal peptide in FtsZ2 mediates FtsZ2 interaction with both PARC6 and ARC6. Consistent with its role in the positioning of PDV1, the intermembrane space regions of PARC6 and PDV1 interact. These findings provide new insights into the functions of PARC6 and suggest that PARC6 coordinates the inner Z ring and outer DRP5B ring through interaction with FtsZ2 and PDV1 during chloroplast division. PMID:26527658

  9. Synthetic chloroplasts

    SciTech Connect

    Calvin, M.

    1980-06-01

    The principal function of the chloroplast is to capture solar quanta and to store them in some stable form. We are in the process of trying to construct a totally synthetic system that would simulate some of the reactions of the two photosystems which occur in natural chloroplasts. Toward this end, we have demonstrated a number of the reactions required in separated systems. We have shown that it is possible to transfer electrons across an insulating membrane barrier with a surfactant photosensitizer. Others have shown, and we have confirmed, that it is possible to collect the two electrons necessary for the generation of molecular hydrogen on a heterogeneous catalyst suspended in water and similarly to collect the four holes on another heterogeneous catalyst suspended in water for the generation of molecular oxygen. A synthesis of some of these molecular catalysts for both these purposes is underway, with some partial success. When these partial reactions are assembled in a system, the resulting synthetic chloroplasts will not resemble the natural entity in detailed construction as they will contain no protein.

  10. Processing of the 5'-UTR and existence of protein factors that regulate translation of tobacco chloroplast psbN mRNA.

    PubMed

    Kuroda, Hiroshi; Sugiura, Masahiro

    2014-12-01

    The chloroplast psbB operon includes five genes encoding photosystem II and cytochrome b 6 /f complex components. The psbN gene is located on the opposite strand. PsbN is localized in the thylakoid and is present even in the dark, although its level increases upon illumination and then decreases. However, the translation mechanism of the psbN mRNA remains unclear. Using an in vitro translation system from tobacco chloroplasts and a green fluorescent protein as a reporter protein, we show that translation occurs from a tobacco primary psbN 5'-UTR of 47 nucleotides (nt). Unlike many other chloroplast 5'-UTRs, the psbN 5'-UTR has two processing sites, at -39 and -24 upstream from the initiation site. Processing at -39 enhanced the translation rate fivefold. In contrast, processing at -24 did not affect the translation rate. These observations suggest that the two distinct processing events regulate, at least in part, the level of PsbN during development. The psbN 5'-UTR has no Shine-Dalgarno (SD)-like sequence. In vitro translation assays with excess amounts of the psbN 5'-UTR or with deleted psbN 5'-UTR sequences demonstrated that protein factors are required for translation and that their binding site is an 18 nt sequence in the 5'-UTR. Mobility shift assays using 10 other chloroplast 5'-UTRs suggested that common or similar proteins are involved in translation of a set of mRNAs lacking SD-like sequences.

  11. Transient expression of βC1 protein differentially regulates host genes related to stress response, chloroplast and mitochondrial functions

    PubMed Central

    2010-01-01

    Background Geminiviruses are emerging plant pathogens that infect a wide variety of crops including cotton, cassava, vegetables, ornamental plants and cereals. The geminivirus disease complex consists of monopartite begomoviruses that require betasatellites for the expression of disease symptoms. These complexes are widespread throughout the Old World and cause economically important diseases on several crops. A single protein encoded by betasatellites, termed βC1, is a suppressor of gene silencing, inducer of disease symptoms and is possibly involved in virus movement. Studies of the interaction of βC1 with hosts can provide useful insight into virus-host interactions and aid in the development of novel control strategies. We have used the differential display technique to isolate host genes which are differentially regulated upon transient expression of the βC1 protein of chili leaf curl betasatellite (ChLCB) in Nicotiana tabacum. Results Through differential display analysis, eight genes were isolated from Nicotiana tabacum, at two and four days after infitration with βC1 of ChLCB, expressed under the control of the Cauliflower mosaic virus 35S promoter. Cloning and sequence analysis of differentially amplified products suggested that these genes were involved in ATP synthesis, and acted as electron carriers for respiration and photosynthesis processes. These differentially expressed genes (DEGs) play an important role in plant growth and development, cell protection, defence processes, replication mechanisms and detoxification responses. Kegg orthology based annotation system analysis of these DEGs demonstrated that one of the genes, coding for polynucleotide nucleotidyl transferase, is involved in purine and pyrimidine metabolic pathways and is an RNA binding protein which is involved in RNA degradation. Conclusion βC1 differentially regulated genes are mostly involved in chloroplast and mitochondrial functions. βC1 also increases the expression of those

  12. Coordinated regulation of photosynthetic and respiratory components is necessary to maintain chloroplast energy balance in varied growth conditions.

    PubMed

    Dahal, Keshav; Martyn, Greg D; Alber, Nicole A; Vanlerberghe, Greg C

    2016-12-23

    Mitochondria have a non-energy-conserving alternative oxidase (AOX) proposed to support photosynthesis, perhaps by promoting energy balance under varying growth conditions. To investigate this, wild-type (WT) Nicotiana tabacum were compared with AOX knockdown and overexpression lines. In addition, the amount of AOX protein in WT plants was compared with that of chloroplast light-harvesting complex II (LHCB2), whose amount is known to respond to chloroplast energy status. With increased growth irradiance, WT leaves maintained higher rates of respiration in the light (R L), but no differences in R L or photosynthesis were seen between the WT and transgenic lines, suggesting that, under non-stress conditions, AOX was not critical for leaf metabolism, regardless of growth irradiance. However, under drought, the AOX amount became an important determinant of R L, which in turn was an important determinant of chloroplast energy balance (measured as photosystem II excitation pressure, EP), and photosynthetic performance. In the WT, the AOX amount increased and the LHCB2 amount decreased with increased growth irradiance or drought severity. These changes in protein amounts correlated strongly, in opposing ways, with growth EP. This suggests that a signal deriving from the photosynthetic electron transport chain status coordinately controls the amounts of AOX and LHCB2, which then both contribute to maintaining chloroplast energy balance, particularly under stress conditions.

  13. Proteomic Insight into the Response of Arabidopsis Chloroplasts to Darkness

    PubMed Central

    Wang, Jing; Yu, Qingbo; Xiong, Haibo; Wang, Jun; Chen, Sixue; Yang, Zhongnan; Dai, Shaojun

    2016-01-01

    Chloroplast function in photosynthesis is essential for plant growth and development. It is well-known that chloroplasts respond to various light conditions. However, it remains poorly understood about how chloroplasts respond to darkness. In this study, we found 81 darkness-responsive proteins in Arabidopsis chloroplasts under 8 h darkness treatment. Most of the proteins are nucleus-encoded, indicating that chloroplast darkness response is closely regulated by the nucleus. Among them, 17 ribosome proteins were obviously reduced after darkness treatment. The protein expressional patterns and physiological changes revealed the mechanisms in chloroplasts in response to darkness, e.g., (1) inhibition of photosystem II resulted in preferential cyclic electron flow around PSI; (2) promotion of starch degradation; (3) inhibition of chloroplastic translation; and (4) regulation by redox and jasmonate signaling. The results have improved our understanding of molecular regulatory mechanisms in chloroplasts under darkness. PMID:27137770

  14. ADP-Dependent Phosphorylation Regulates Association of a DNA-Binding Complex with the Barley Chloroplast psbD Blue-Light-Responsive Promoter1

    PubMed Central

    Kim, Minkyun; Christopher, David A.; Mullet, John E.

    1999-01-01

    The chloroplast gene psbD encodes D2, a chlorophyll-binding protein located in the photosystem II reaction center. Transcription of psbD in higher plants involves at least three promoters, one of which is regulated by blue light. The psbD blue-light-regulated promoter (BLRP) consists of a −10 promoter element and an activating complex, AGF, that binds immediately upstream of −35. A second sequence-specific DNA-binding complex, PGTF, binds upstream of AGF between −71 and −100 in the barley (Hordeum vulgare) psbD BLRP. In this study we report that ADP-dependent phosphorylation selectively inhibits the binding of PGTF to the barley psbD BLRP. ATP at high concentrations (1–5 mm) inhibits PGTF binding, but in the presence of phosphocreatine and phosphocreatine kinase, this capacity is lost, presumably due to scavenging of ADP. ADP inhibits PGTF binding at relatively low concentrations (0.1 mm), whereas other nucleotides are unable to mediate this response. ADP-mediated inhibition of PGTF binding is reduced in the presence of the protein kinase inhibitor K252a. This and other results suggest that ADP-dependent phosphorylation of PGTF (or some associated protein) inhibits binding of PGTF to the psbD BLRP and reduces transcription. ADP-dependent phosphorylation is expected to increase in darkness in parallel with the rise in ADP levels in chloroplasts. ADP-dependent phosphorylation in chloroplasts may, therefore, in coordination, inactivate enzymes involved in carbon assimilation, protein synthesis, and transcription during diurnal light/dark cycles. PMID:9952463

  15. The Chloroplastic Protein THF1 Interacts with the Coiled-Coil Domain of the Disease Resistance Protein N′ and Regulates Light-Dependent Cell Death1[OPEN

    PubMed Central

    Sekine, Ken-Taro; Wallon, Thérèse; Sugiwaka, Yuji; Kobayashi, Kappei

    2016-01-01

    One branch of plant immunity is mediated through nucleotide-binding/Leu-rich repeat (NB-LRR) family proteins that recognize specific effectors encoded by pathogens. Members of the I2-like family constitute a well-conserved subgroup of NB-LRRs from Solanaceae possessing a coiled-coil (CC) domain at their N termini. We show here that the CC domains of several I2-like proteins are able to induce a hypersensitive response (HR), a form of programmed cell death associated with disease resistance. Using yeast two-hybrid screens, we identified the chloroplastic protein Thylakoid Formation1 (THF1) as an interacting partner for several I2-like CC domains. Co-immunoprecipitations and bimolecular fluorescence complementation assays confirmed that THF1 and I2-like CC domains interact in planta and that these interactions take place in the cytosol. Several HR-inducing I2-like CC domains have a negative effect on the accumulation of THF1, suggesting that the latter is destabilized by active CC domains. To confirm this model, we investigated N′, which recognizes the coat protein of most Tobamoviruses, as a prototypical member of the I2-like family. Transient expression and gene silencing data indicated that THF1 functions as a negative regulator of cell death and that activation of full-length N′ results in the destabilization of THF1. Consistent with the known function of THF1 in maintaining chloroplast homeostasis, we show that the HR induced by N′ is light-dependent. Together, our results define, to our knowledge, novel molecular mechanisms linking light and chloroplasts to the induction of cell death by a subgroup of NB-LRR proteins. PMID:26951433

  16. Insights into chloroplast biogenesis and development.

    PubMed

    Pogson, Barry J; Ganguly, Diep; Albrecht-Borth, Verónica

    2015-09-01

    In recent years many advances have been made to obtain insight into chloroplast biogenesis and development. In plants several plastids types exist such as the proplastid (which is the progenitor of all plastids), leucoplasts (group of colourless plastids important for storage including elaioplasts (lipids), amyloplasts (starch) or proteinoplasts (proteins)), chromoplasts (yellow to orange-coloured due to carotenoids, in flowers or in old leaves as gerontoplasts), and the green chloroplasts. Chloroplasts are indispensable for plant development; not only by performing photosynthesis and thus rendering the plant photoautotrophic, but also for biochemical processes (which in some instances can also take place in other plastids types), such as the synthesis of pigments, lipids, and plant hormones and sensing environmental stimuli. Although we understand many aspects of these processes there are gaps in our understanding of the establishment of functional chloroplasts and their regulation. Why is that so? Even though chloroplast function is comparable in all plants and most of the algae, ferns and moss, detailed analyses have revealed many differences, specifically with respect to its biogenesis. As an update to our prior review on the genetic analysis of chloroplast biogenesis and development [1] herein we will focus on recent advances in Angiosperms (monocotyledonous and dicotyledonous plants) that provide novel insights and highlight the challenges and prospects for unravelling the regulation of chloroplast biogenesis specifically during the establishment of the young plants. This article is part of a Special Issue entitled: Chloroplast Biogenesis.

  17. Calredoxin represents a novel type of calcium-dependent sensor-responder connected to redox regulation in the chloroplast

    PubMed Central

    Hochmal, Ana Karina; Zinzius, Karen; Charoenwattanasatien, Ratana; Gäbelein, Philipp; Mutoh, Risa; Tanaka, Hideaki; Schulze, Stefan; Liu, Gai; Scholz, Martin; Nordhues, André; Offenborn, Jan Niklas; Petroutsos, Dimitris; Finazzi, Giovanni; Fufezan, Christian; Huang, Kaiyao; Kurisu, Genji; Hippler, Michael

    2016-01-01

    Calcium (Ca2+) and redox signalling play important roles in acclimation processes from archaea to eukaryotic organisms. Herein we characterized a unique protein from Chlamydomonas reinhardtii that has the competence to integrate Ca2+- and redox-related signalling. This protein, designated as calredoxin (CRX), combines four Ca2+-binding EF-hands and a thioredoxin (TRX) domain. A crystal structure of CRX, at 1.6 Å resolution, revealed an unusual calmodulin-fold of the Ca2+-binding EF-hands, which is functionally linked via an inter-domain communication path with the enzymatically active TRX domain. CRX is chloroplast-localized and interacted with a chloroplast 2-Cys peroxiredoxin (PRX1). Ca2+-binding to CRX is critical for its TRX activity and for efficient binding and reduction of PRX1. Thereby, CRX represents a new class of Ca2+-dependent ‘sensor-responder' proteins. Genetically engineered Chlamydomonas strains with strongly diminished amounts of CRX revealed altered photosynthetic electron transfer and were affected in oxidative stress response underpinning a function of CRX in stress acclimation. PMID:27297041

  18. Calredoxin represents a novel type of calcium-dependent sensor-responder connected to redox regulation in the chloroplast.

    PubMed

    Hochmal, Ana Karina; Zinzius, Karen; Charoenwattanasatien, Ratana; Gäbelein, Philipp; Mutoh, Risa; Tanaka, Hideaki; Schulze, Stefan; Liu, Gai; Scholz, Martin; Nordhues, André; Offenborn, Jan Niklas; Petroutsos, Dimitris; Finazzi, Giovanni; Fufezan, Christian; Huang, Kaiyao; Kurisu, Genji; Hippler, Michael

    2016-06-14

    Calcium (Ca(2+)) and redox signalling play important roles in acclimation processes from archaea to eukaryotic organisms. Herein we characterized a unique protein from Chlamydomonas reinhardtii that has the competence to integrate Ca(2+)- and redox-related signalling. This protein, designated as calredoxin (CRX), combines four Ca(2+)-binding EF-hands and a thioredoxin (TRX) domain. A crystal structure of CRX, at 1.6 Å resolution, revealed an unusual calmodulin-fold of the Ca(2+)-binding EF-hands, which is functionally linked via an inter-domain communication path with the enzymatically active TRX domain. CRX is chloroplast-localized and interacted with a chloroplast 2-Cys peroxiredoxin (PRX1). Ca(2+)-binding to CRX is critical for its TRX activity and for efficient binding and reduction of PRX1. Thereby, CRX represents a new class of Ca(2+)-dependent 'sensor-responder' proteins. Genetically engineered Chlamydomonas strains with strongly diminished amounts of CRX revealed altered photosynthetic electron transfer and were affected in oxidative stress response underpinning a function of CRX in stress acclimation.

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

    PubMed Central

    Larkin, Robert M.; Stefano, Giovanni; Ruckle, Michael E.; Stavoe, Andrea K.; Sinkler, Christopher A.; Brandizzi, Federica; Malmstrom, Carolyn M.; Osteryoung, Katherine W.

    2016-01-01

    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 to FRIENDLY, which was previously shown to promote the normal distribution of mitochondria in 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 and 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. 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. PMID:26862170

  20. Chloroplast-mediated regulation of CO2-concentrating mechanism by Ca2+-binding protein CAS in the green alga Chlamydomonas reinhardtii

    PubMed Central

    Wang, Lianyong; Yamano, Takashi; Takane, Shunsuke; Niikawa, Yuki; Toyokawa, Chihana; Ozawa, Shin-ichiro; Tokutsu, Ryutaro; Takahashi, Yuichiro; Minagawa, Jun; Kanesaki, Yu; Yoshikawa, Hirofumi; Fukuzawa, Hideya

    2016-01-01

    Aquatic photosynthetic organisms, including the green alga Chlamydomonas reinhardtii, induce a CO2-concentrating mechanism (CCM) to maintain photosynthetic activity in CO2-limiting conditions by sensing environmental CO2 and light availability. Previously, a novel high-CO2–requiring mutant, H82, defective in the induction of the CCM, was isolated. A homolog of calcium (Ca2+)-binding protein CAS, originally found in Arabidopsis thaliana, was disrupted in H82 cells. Although Arabidopsis CAS is reported to be associated with stomatal closure or immune responses via a chloroplast-mediated retrograde signal, the relationship between a Ca2+ signal and the CCM associated with the function of CAS in an aquatic environment is still unclear. In this study, the introduction of an intact CAS gene into H82 cells restored photosynthetic affinity for inorganic carbon, and RNA-seq analyses revealed that CAS could function in maintaining the expression levels of nuclear-encoded CO2-limiting–inducible genes, including the HCO3– transporters high-light activated 3 (HLA3) and low-CO2–inducible gene A (LCIA). CAS changed its localization from dispersed across the thylakoid membrane in high-CO2 conditions or in the dark to being associated with tubule-like structures in the pyrenoid in CO2-limiting conditions, along with a significant increase of the fluorescent signals of the Ca2+ indicator in the pyrenoid. Chlamydomonas CAS had Ca2+-binding activity, and the perturbation of intracellular Ca2+ homeostasis by a Ca2+-chelator or calmodulin antagonist impaired the accumulation of HLA3 and LCIA. These results suggest that Chlamydomonas CAS is a Ca2+-mediated regulator of CCM-related genes via a retrograde signal from the pyrenoid in the chloroplast to the nucleus. PMID:27791081

  1. Analysis of Protein Import into Chloroplasts Isolated from Stressed Plants.

    PubMed

    Ling, Qihua; Jarvis, Paul

    2016-11-01

    Chloroplasts are organelles with many vital roles in plants, which include not only photosynthesis but numerous other metabolic and signaling functions. Furthermore, chloroplasts are critical for plant responses to various abiotic stresses, such as salinity and osmotic stresses. A chloroplast may contain up to ~3,000 different proteins, some of which are encoded by its own genome. However, the majority of chloroplast proteins are encoded in the nucleus and synthesized in the cytosol, and these proteins need to be imported into the chloroplast through translocons at the chloroplast envelope membranes. Recent studies have shown that the chloroplast protein import can be actively regulated by stress. To biochemically investigate such regulation of protein import under stress conditions, we developed the method described here as a quick and straightforward procedure that can easily be achieved in any laboratory. In this method, plants are grown under normal conditions and then exposed to stress conditions in liquid culture. Plant material is collected, and chloroplasts are then released by homogenization. The crude homogenate is separated by density gradient centrifugation, enabling isolation of the intact chloroplasts. Chloroplast yield is assessed by counting, and chloroplast intactness is checked under a microscope. For the protein import assays, purified chloroplasts are incubated with (35)S radiolabeled in vitro translated precursor proteins, and time-course experiments are conducted to enable comparisons of import rates between genotypes under stress conditions. We present data generated using this method which show that the rate of protein import into chloroplasts from a regulatory mutant is specifically altered under osmotic stress conditions.

  2. A role for mechanosensitive channels in chloroplast and bacterial fission.

    PubMed

    Wilson, Margaret; Haswell, Elizabeth

    2012-02-01

    The division site in both chloroplasts and bacteria is established by the medial placement of the FtsZ ring, a process that is in part regulated by the evolutionarily conserved components of the Min system. We recently showed that mechanosensitive ion channels influence FtsZ ring assembly in both Arabidopsis thaliana chloroplasts and in Escherichia coli; in chloroplasts they do so through the same genetic pathway as the Min system. Here we describe the effect of heterologous expression of the Arabidopsis MS channel homolog MSL2 on FtsZ ring placement in E. coli. We also discuss possible molecular mechanisms by which MS channels might influence chloroplast or bacterial division.

  3. New insights into dynamic actin-based chloroplast photorelocation movement.

    PubMed

    Kong, Sam-Geun; Wada, Masamitsu

    2011-09-01

    Chloroplast movement is essential for plants to survive under various environmental light conditions. Phototropins-plant-specific blue-light-activated receptor kinases-mediate the response by perceiving light intensity and direction. Recently, novel chloroplast actin (cp-actin) filaments have been identified as playing a pivotal role in the directional chloroplast photorelocation movement. Encouraging progress has recently been made in this field of research through molecular genetics and cell biological analyses. This review describes factors that have been identified as being involved in chloroplast movement and their roles in the regulation of cp-actin filaments, thus providing a basis for reflection on their biochemical activities and functions.

  4. Manipulating the chloroplast genome of Chlamydomonas: Present realities and future prospects

    SciTech Connect

    Boynton, J.; Gillham, N.; Hauser, C.; Heifetz, P.; Lers, A.; Newman, S.; Osmond, B.

    1992-01-01

    Biotechnology is being applied in vitro modification and stable reintroduction of chloroplast genes in Chlamydomonas reinhardtii and Nicotiana tabacum by homologous recombination. We are attempting the function analyses of plastid encoded proteins involved in photosynthesis, characterization of sequences which regulate expression of plastid genes at the transcriptional and translational levels, targeted disruption of chloroplast genes and molecular analysis of processes involved in chloroplast recombination.

  5. Manipulating the chloroplast genome of Chlamydomonas: Present realities and future prospects

    SciTech Connect

    Boynton, J.; Gillham, N.; Hauser, C.; Heifetz, P.; Lers, A.; Newman, S.; Osmond, B.

    1992-12-31

    Biotechnology is being applied in vitro modification and stable reintroduction of chloroplast genes in Chlamydomonas reinhardtii and Nicotiana tabacum by homologous recombination. We are attempting the function analyses of plastid encoded proteins involved in photosynthesis, characterization of sequences which regulate expression of plastid genes at the transcriptional and translational levels, targeted disruption of chloroplast genes and molecular analysis of processes involved in chloroplast recombination.

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

    Technology Transfer Automated Retrieval System (TEKTRAN)

    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 ...

  7. The Chloroplast-Localized Phospholipases D α4 and α5 Regulate Herbivore-Induced Direct and Indirect Defenses in Rice1[C][W

    PubMed Central

    Qi, Jinfeng; Zhou, Guoxin; Yang, Lijuan; Erb, Matthias; Lu, Yanhua; Sun, Xiaoling; Cheng, Jiaan; Lou, Yonggen

    2011-01-01

    The oxylipin pathway is of central importance for plant defensive responses. Yet, the first step of the pathway, the liberation of linolenic acid following induction, is poorly understood. Phospholipases D (PLDs) have been hypothesized to mediate this process, but data from Arabidopsis (Arabidopsis thaliana) regarding the role of PLDs in plant resistance have remained controversial. Here, we cloned two chloroplast-localized PLD genes from rice (Oryza sativa), OsPLDα4 and OsPLDα5, both of which were up-regulated in response to feeding by the rice striped stem borer (SSB) Chilo suppressalis, mechanical wounding, and treatment with jasmonic acid (JA). Antisense expression of OsPLDα4 and -α5 (as-pld), which resulted in a 50% reduction of the expression of the two genes, reduced elicited levels of linolenic acid, JA, green leaf volatiles, and ethylene and attenuated the SSB-induced expression of a mitogen-activated protein kinase (OsMPK3), a lipoxygenase (OsHI-LOX), a hydroperoxide lyase (OsHPL3), as well as a 1-aminocyclopropane-1-carboxylic acid synthase (OsACS2). The impaired oxylipin and ethylene signaling in as-pld plants decreased the levels of herbivore-induced trypsin protease inhibitors and volatiles, improved the performance of SSB and the rice brown planthopper Nilaparvata lugens, and reduced the attractiveness of plants to a larval parasitoid of SSB, Apanteles chilonis. The production of trypsin protease inhibitors in as-pld plants could be partially restored by JA, while the resistance to rice brown planthopper and SSB was restored by green leaf volatile application. Our results show that phospholipases function as important components of herbivore-induced direct and indirect defenses in rice. PMID:21984727

  8. Arabidopsis Tic62 and Ferredoxin-NADP(H) Oxidoreductase Form Light-Regulated Complexes That Are Integrated into the Chloroplast Redox Poise[C][W

    PubMed Central

    Benz, J.P.; Stengel, A.; Lintala, M.; Lee, Y.-H.; Weber, A.; Philippar, K.; Gügel, I.L.; Kaieda, S.; Ikegami, T.; Mulo, P.; Soll, J.; Bölter, B.

    2009-01-01

    Translocation of nuclear-encoded preproteins across the inner envelope of chloroplasts is catalyzed by the Tic translocon, consisting of Tic110, Tic40, Tic62, Tic55, Tic32, Tic20, and Tic22. Tic62 was proposed to act as a redox sensor of the complex because of its redox-dependent shuttling between envelope and stroma and its specific interaction with the photosynthetic protein ferredoxin-NADP(H) oxidoreductase (FNR). However, the nature of this close relationship so far remained enigmatic. A putative additional localization of Tic62 at the thylakoids mandated further studies examining how this feature might be involved in the respective redox sensing pathway and the interaction with its partner protein. Therefore, both the association with FNR and the physiological role of the third, thylakoid-bound pool of Tic62 were investigated in detail. Coexpression analysis indicates that Tic62 has similar expression patterns as genes involved in photosynthetic functions and protein turnover. At the thylakoids, Tic62 and FNR form high molecular weight complexes that are not involved in photosynthetic electron transfer but are dynamically regulated by light signals and the stromal pH. Structural analyses reveal that Tic62 binds to FNR in a novel binding mode for flavoproteins, with a major contribution from hydrophobic interactions. Moreover, in absence of Tic62, membrane binding and stability of FNR are drastically reduced. We conclude that Tic62 represents a major FNR interaction partner not only at the envelope and in the stroma, but also at the thylakoids of Arabidopsis thaliana and perhaps all flowering plants. Association with Tic62 stabilizes FNR and is involved in its dynamic and light-dependent membrane tethering. PMID:20040542

  9. Melatonin Increases the Chilling Tolerance of Chloroplast in Cucumber Seedlings by Regulating Photosynthetic Electron Flux and the Ascorbate-Glutathione Cycle

    PubMed Central

    Zhao, Hailiang; Ye, Lin; Wang, Yuping; Zhou, Xiaoting; Yang, Junwei; Wang, Jiawei; Cao, Kai; Zou, Zhirong

    2016-01-01

    The aim of the study was to monitor the effects of exogenous melatonin on cucumber (Cucumis sativus L.) chloroplasts and explore the mechanisms through which it mitigates chilling stress. Under chilling stress, chloroplast structure was seriously damaged as a result of over-accumulation of reactive oxygen species (ROS), as evidenced by the high levels of superoxide anion (O2−) and hydrogen peroxide (H2O2). However, pretreatment with 200 μM melatonin effectively mitigated this by suppressing the levels of ROS in chloroplasts. On the one hand, melatonin enhanced the scavenging ability of ROS by stimulating the ascorbate–glutathione (AsA–GSH) cycle in chloroplasts. The application of melatonin led to high levels of AsA and GSH, and increased the activity of total superoxide dismutase (SOD, EC 1.15.1.1), ascorbate peroxidase (APX, EC 1.11.1.11), monodehydroascorbate reductase (MDHAR, EC 1.6.5.4) dehydroascorbate reductase (DHAR, EC 1.5.5.1), glutathione reductase (GR, EC1.6.4.2) in the AsA–GSH cycle. On the other hand, melatonin lessened the production of ROS in chloroplasts by balancing the distribution of photosynthetic electron flux. Melatonin helped maintain a high level of electron flux in the PCR cycle [Je(PCR)] and in the PCO cycle [Je(PCO)], and suppressed the O2-dependent alternative electron flux Ja(O2-dependent) which is one important ROS source. Results indicate that melatonin increased the chilling tolerance of chloroplast in cucumber seedlings by accelerating the AsA–GSH cycle to enhance ROS scavenging ability and by balancing the distribution of photosynthetic electron flux so as to suppress ROS production. PMID:27999581

  10. The chloroplast RNA helicase ISE2 is required for multiple chloroplast RNA processing steps in Arabidopsis thaliana.

    PubMed

    Bobik, Krzysztof; McCray, Tyra N; Ernest, Ben; Fernandez, Jessica C; Howell, Katharine A; Lane, Thomas; Staton, Margaret; Burch-Smith, Tessa M

    2017-03-27

    INCREASED SIZE EXCLUSION LIMIT2 (ISE2) is a chloroplast-localized RNA helicase that is indispensable for proper plant development. Chloroplasts in leaves with reduced ISE2 expression have previously been shown to exhibit reduced thylakoid contents and increased stromal volume, indicative of defective development. It has recently been reported that ISE2 is required for the splicing of group II introns from chloroplast transcripts. The current study extends these findings, and presents evidence for ISE2's role in multiple aspects of chloroplast RNA processing beyond group II intron splicing. Loss of ISE2 from Arabidopsis thaliana leaves resulted in defects in C-to-U RNA editing, altered accumulation of chloroplast transcripts and chloroplast-encoded proteins, and defective processing of chloroplast ribosomal RNAs. Potential ISE2 substrates were identified by RNA immunoprecipitation followed by next-generation sequencing (RIP-seq), and the diversity of RNA species identified supports ISE2's involvement in multiple aspects of chloroplast RNA metabolism. Comprehensive phylogenetic analyses revealed that ISE2 is a non-canonical Ski2-like RNA helicase that represents a separate sub-clade unique to green photosynthetic organisms, consistent with its function as an essential protein. Thus ISE2's evolutionary conservation may be explained by its numerous roles in regulating chloroplast gene expression. This article is protected by copyright. All rights reserved.

  11. [Response of reactive oxygen metabolism in melon chloroplasts to short-term salinity-alkalinity stress regulated by exogenous γ-aminobutyric acid].

    PubMed

    Xiang, Li-xia; Hu, Li-pan; Hu, Xiao-hui; Pan, Xiong-bo; Ren, Wen-qi

    2015-12-01

    The regulatory effect of exogenous γ-aminobutyric acid (GABA) on metabolism of reactive oxygen species (ROS) in melon chloroplasts under short-term salinity-alkalinity stress were investigated in melon variety 'Jinhui No. 1', which was cultured with deep flow hydroponics. The result showed that under salinity-alkalinity stress, the photosynthetic pigment content, MDA content, superoxide anion (O₂·) production rate and hydrogen peroxide (H₂O₂) content in chloroplast increased significantly, the contents of antioxidants ascorbic acid (AsA) and glutathione (GSH) increased, and the activities of H⁺-ATPase and H⁺-PPiase were inhibited obviously. With exogenous GABA application, the accumulations of O₂·, MDA and H₂O₂ induced by salinity-alkalinity stress were inhibited. Exogenous GABA alleviated the increase of photosynthetic pigment content, improved the activity of SOD, enzymes of AsA-GSH cycle, total AsA and total GSH while decreased the AsA/DHA ratio and GSH/GSSH ratio. Foliar GABA could enhance the H⁺-ATPase and H⁺-PPiase activities. Our results suggested that the exogenous GABA could accelerate the ROS metabolism in chloroplast, promote the recycle of AsA-GSH, and maintain the permeability of cell membrane to improve the ability of melon chloroplast against salinity-alkalinity stress.

  12. Heme content and breakdown in developing chloroplasts

    SciTech Connect

    Thomas, J.; Weinstein, J.D. )

    1990-05-01

    Heme regulates tetrapyrrole biosynthesis in plants by inhibition of {delta}-aminolevulinic acid (ALA) synthesis, product inhibition of heme synthesis, and possibly other mechanisms. Plastid heme levels may be modulated by heme synthesis, breakdown and/or efflux. Heme breakdown may be catalyzed by a chloroplast localized heme oxygenase. Chloroplasts isolated from greening cucumber cotyledons were incubated in the presence or absence of various components thought to modulate heme breakdown. Following the incubations, the chloroplasts were broken (freeze-thaw) and then supplemented with horseradish peroxidase apoenzyme. The reconstituted peroxidase activity was used to determine the amount of free heme remaining (Thomas Weinstein (1989) Plant Physiol. 89S: 74). Chloroplasts, freshly isolated from seedlings greened for 16 hours, contained approximately 37 pmol heme/mg protein. When chloroplasts were incubated with 5 mM NADPH for 30 min, the endogenous heme dropped to unmeasurable levels. Exogenous heme was also broken down when NADPH was included in the incubation. Heme levels could be increased by the inclusion of 50 {mu}M ALA and/or p-hydroxymercuribenzoate. The increase due to exogenous ALA was blocked by levulinic acid, an inhibitor of ALA utilization. NADPH-dependent heme breakdown acid was inhibited by p-hydroxymercuribenzoate.

  13. Chloroplast signaling within, between and beyond cells

    PubMed Central

    Bobik, Krzysztof; Burch-Smith, Tessa M.

    2015-01-01

    The most conspicuous function of plastids is the oxygenic photosynthesis of chloroplasts, yet plastids are super-factories that produce a plethora of compounds that are indispensable for proper plant physiology and development. Given their origins as free-living prokaryotes, it is not surprising that plastids possess their own genomes whose expression is essential to plastid function. This semi-autonomous character of plastids requires the existence of sophisticated regulatory mechanisms that provide reliable communication between them and other cellular compartments. Such intracellular signaling is necessary for coordinating whole-cell responses to constantly varying environmental cues and cellular metabolic needs. This is achieved by plastids acting as receivers and transmitters of specific signals that coordinate expression of the nuclear and plastid genomes according to particular needs. In this review we will consider the so-called retrograde signaling occurring between plastids and nuclei, and between plastids and other organelles. Another important role of the plastid we will discuss is the involvement of plastid signaling in biotic and abiotic stress that, in addition to influencing retrograde signaling, has direct effects on several cellular compartments including the cell wall. We will also review recent evidence pointing to an intriguing function of chloroplasts in regulating intercellular symplasmic transport. Finally, we consider an intriguing yet less widely known aspect of plant biology, chloroplast signaling from the perspective of the entire plant. Thus, accumulating evidence highlights that chloroplasts, with their complex signaling pathways, provide a mechanism for exquisite regulation of plant development, metabolism and responses to the environment. As chloroplast processes are targeted for engineering for improved productivity the effect of such modifications on chloroplast signaling will have to be carefully considered in order to avoid

  14. The evolution of chloroplast RNA editing.

    PubMed

    Tillich, Michael; Lehwark, Pascal; Morton, Brian R; Maier, Uwe G

    2006-10-01

    RNA editing alters the nucleotide sequence of an RNA molecule so that it deviates from the sequence of its DNA template. Different RNA-editing systems are found in the major eukaryotic lineages, and these systems are thought to have evolved independently. In this study, we provide a detailed analysis of data on C-to-U editing sites in land plant chloroplasts and propose a model for the evolution of RNA editing in land plants. First, our data suggest that the limited RNA-editing system of seed plants and the much more extensive systems found in hornworts and ferns are of monophyletic origin. Further, although some eukaryotic editing systems appear to have evolved to regulate gene expression, or at least are now involved in gene regulation, there is no evidence that RNA editing plays a role in gene regulation in land plant chloroplasts. Instead, our results suggest that land plant chloroplast C-to-U RNA editing originated as a mechanism to generate variation at the RNA level, which could complement variation at the DNA level. Under this model, many of the original sites, particularly in seed plants, have been subsequently lost due to mutation at the DNA level, and the function of extant sites is merely to conserve certain codons. This is the first comprehensive model for the evolution of the chloroplast RNA-editing system of land plants and may also be applicable to the evolution of RNA editing in plant mitochondria.

  15. Possible association of actin filaments with chloroplasts of spinach mesophyll cells in vivo and in vitro.

    PubMed

    Kumatani, T; Sakurai-Ozato, N; Miyawaki, N; Yokota, E; Shimmen, T; Terashima, I; Takagi, S

    2006-11-01

    In palisade mesophyll cells of spinach (Spinacia oleracea L.) kept under low-intensity white light, chloroplasts were apparently immobile and seemed to be surrounded by fine bundles of actin filaments. High-intensity blue light induced actin-dependent chloroplast movement concomitant with the appearance of a couple of long, straight bundles of actin filaments in each cell, whereas high-intensity red light was essentially ineffective in inducing these responses. The actin organization observed under low-intensity white light has been postulated to function in anchoring chloroplasts at proper intracellular positions through direct interaction with the chloroplasts. Intact chloroplasts, which retained their outer envelopes, were isolated after homogenization of leaves and Percoll centrifugation. No endogenous actin was detected by immunoblotting in the final intact-chloroplast fraction prepared from the leaves kept under low-intensity white light or in darkness. In cosedimentation assays with exogenously added skeletal muscle filamentous actin, however, actin was detected in the intact-chloroplast fraction precipitated after low-speed centrifugation. The association of actin with chloroplasts was apparently dependent on incubation time and chloroplast density. After partial disruption of the outer envelope of isolated chloroplasts by treatment with trypsin, actin was no longer coprecipitated. The results suggest that chloroplasts in spinach leaves can directly interact with actin, and that this interaction may be involved in the regulation of intracellular positioning of chloroplasts.

  16. Nitrogen control of chloroplast differentiation

    SciTech Connect

    Schmidt, G.W.

    1994-11-01

    This project was directed toward understanding how the availability of nitrogen affects the accumulation of chloroplast pigments and proteins that function in energy transduction and carbon metabolism. The availability of this nutrient most pervasively limits plant growth and agricultural productivity but the molecular and physiological consequences of nitrogen-deficiency are poorly understood. The model system for our studies of nitrogen-dependent regulation of chloroplast differentiation is the unicellular green alga Chlamydomonas reinhardtii which is grown phototrophically in a continuous culture system. When 150 {mu}M nitrogen is provided at a dilution rate of 0.25 volumes of the growth medium per day, the cultures are sustained at a density of less than 10{sup 5} cells/ml and chlorophyll deficiency, the classical symptom of nitrogen-deficiency, becomes quite pronounced. We found that there is a concomitant loss of light-harvesting complexes and reduced levels of Photosystem II reaction center complexes while ATP synthetase and Photosystem I reaction centers are maintained at high levels. Moreover, reduced rates of chloroplast protein synthesis are due to differential effects on mRNA translation. In contrast, the deficiency of light-harvesting genes is due to marked reductions of the nuclear-encoded cab mRNAs. Although there is no significant reduction of the amounts of RuBPCase, we also detected substantial changes in the mRNA abundance of the alga`s two small subunit genes. All of the effects of nitrogen-limitation are readily reversible: greening of cells is completed within 24 hours after provision of 10 mM ammonium. During this time, the plastid translational constraints are disengaged and progressive changes in the abundance of nuclear transcripts occur, including a transient 30-fold elevation of {und cab} mRNAs.

  17. 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

  18. 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

  19. Chloroplast movement: dissection of events downstream of photo- and mechano-perception.

    PubMed

    Sato, Yoshikatsu; Kadota, Akeo; Wada, Masamitsu

    2003-02-01

    The study of chloroplast photorelocation movement is progressing rapidly now that mutants for chloroplast movement have become available in Arabidopsis thaliana. However, mechanistic approaches in cell biology still stand to elucidate the mechanisms and regulations of such movement. The fern Adiantum capillus-veneris and the moss Physcomitrella patens are particularly suitable materials for analyzing the kinetics of intracellular chloroplast movement. In these plants, chloroplast movement is induced by red light as well as blue light, mediated by phytochrome and blue light receptor, respectively. In this paper, we review the unique force-generating system for chloroplast motility in P. patens. In addition to light-induced chloroplast movement, we also summarize mechanically induced chloroplast movement in these plants and the motility systems involved. Finally, the different dependency of mechano- and photo-relocation movement on external Ca(2+) is discussed.

  20. Chloroplast: The Trojan Horse in Plant-Virus Interaction.

    PubMed

    Bhattacharyya, Dhriti; Chakraborty, Supriya

    2017-01-05

    Chloroplast is one of the most dynamic organelle of a plant cell. It carries out photosynthesis, synthesizes major phytohormones, takes active part in defence response, and is crucial for inter-organelle signaling. Viruses, on the other hand, are extremely strategic in manipulating the internal environment of the host cell. Chloroplast, a prime target for viruses, undergoes enormous structural and functional damage during viral infection. In fact, large proportions of affected gene products in a virus infected plant are closely associated to chloroplast and photosynthesis process. Although chloroplast is deficient in gene-silencing machinery, it elicits effector-triggered immune response against viral pathogens. Virus infection induces the organelle to produce extensive network of stromules which are involved in both viral propagation and anti-viral defence. From last few decades' study, involvement of chloroplast in regulating plant-virus interaction has become increasingly evident. Current review presents an exhaustive account of these facts, with their implication in pathogenicity. We have attempted to highlight the intricacies of chloroplast-virus interaction and explained the existing gaps in current knowledge, which will promote the virologists to utilize the chloroplast genome-based antiviral resistance in economically important crops. This article is protected by copyright. All rights reserved.

  1. Import of Soluble Proteins into Chloroplasts and Potential Regulatory Mechanisms

    PubMed Central

    Sjuts, Inga; Soll, Jürgen; Bölter, Bettina

    2017-01-01

    Chloroplasts originated from an endosymbiotic event in which a free-living cyanobacterium was engulfed by an ancestral eukaryotic host. During evolution the majority of the chloroplast genetic information was transferred to the host cell nucleus. As a consequence, proteins formerly encoded by the chloroplast genome are now translated in the cytosol and must be subsequently imported into the chloroplast. This process involves three steps: (i) cytosolic sorting procedures, (ii) binding to the designated receptor-equipped target organelle and (iii) the consecutive translocation process. During import, proteins have to overcome the two barriers of the chloroplast envelope, namely the outer envelope membrane (OEM) and the inner envelope membrane (IEM). In the majority of cases, this is facilitated by two distinct multiprotein complexes, located in the OEM and IEM, respectively, designated TOC and TIC. Plants are constantly exposed to fluctuating environmental conditions such as temperature and light and must therefore regulate protein composition within the chloroplast to ensure optimal functioning of elementary processes such as photosynthesis. In this review we will discuss the recent models of each individual import stage with regard to short-term strategies that plants might use to potentially acclimate to changes in their environmental conditions and preserve the chloroplast protein homeostasis. PMID:28228773

  2. [Chloroplast Deg proteases].

    PubMed

    Grabsztunowicz, Magda; Luciński, Robert; Baranek, Małgorzata; Sikora, Bogna; Jackowski, Grzegorz

    2011-01-01

    For some chloroplast proteases ATP binding and hydrolysis is not necessary for their catalytic activity, most probably because even strongly unfolded substrates may penetrate their catalytic chamber. Deg1, 2, 5 and 8 are the best known of Arabidopsis thaliana ATP- independent chloroplast proteases, encoded by orthologues of genes coding for DegP, DegQ and DegS proteases of Escherichia coli. Current awareness in the area of structure and functions of chloroplast Degs is much more limited vs the one about their bacterial counterparts. Deg5 and Deg8 form a catalytic heterododecamer which is loosely attached to luminal side of thylakoid membrane. The complex catalyses--supported by Deg1 and one of FtsH proteases--the degradation of PsbA damaged due to plant exposition to elevated irradiance and thus these protease are of key importance for the plants' sensitivity to photoinhibition. Deg2 role in the disposal of damaged PsbA has not been elucidated. Recombinant Deg1 may degrade PsbO and plastocyanin in vitro but it is not clear whether this reaction is performed in vivo as well.

  3. Immunofluorescent quantitation of chloroplast proteins.

    PubMed

    Leech, R M; Marrison, J L

    1996-12-01

    Using scanning light microscopy software to detect and measure immunofluorescence in leaf sections Rubisco concentration in situ in chloroplasts has been accurately determined throughout development. The fluorescence measurements were calibrated by comparison with values for Rubisco accumulation obtained from rocket immuno-electrophoresis profiles of soluble protein from isolated cells and from chloroplasts using a purified sample of Rubisco as the standard. It has been shown that in situ immunofluorescence can be used for cytoquantitation of proteins within individual chloroplasts to a sensitivity of 1fg and also for the comparison of the protein levels in adjacent chloroplasts and cells. Several important applications of this new technique are discussed.

  4. Photosynthetic light reactions: integral to chloroplast retrograde signalling.

    PubMed

    Gollan, Peter J; Tikkanen, Mikko; Aro, Eva-Mari

    2015-10-01

    Chloroplast retrograde signalling is ultimately dependent on the function of the photosynthetic light reactions and not only guides the acclimation of the photosynthetic apparatus to changing environmental and metabolic cues, but has a much wider influence on the growth and development of plants. New information generated during the past few years about regulation of photosynthetic light reactions and identification of the underlying regulatory proteins has paved the way towards better understanding of the signalling molecules produced in chloroplasts upon changes in the environment. Likewise, the availability of various mutants lacking regulatory functions has made it possible to address the role of excitation energy distribution and electron flow in the thylakoid membrane in inducing the retrograde signals from chloroplasts to the nucleus. Such signalling molecules also induce and interact with hormonal signalling cascades to provide comprehensive information from chloroplasts to the nucleus.

  5. Full transcription of the chloroplast genome in photosynthetic eukaryotes

    PubMed Central

    Shi, Chao; Wang, Shuo; Xia, En-Hua; Jiang, Jian-Jun; Zeng, Fan-Chun; Gao, Li-Zhi

    2016-01-01

    Prokaryotes possess a simple genome transcription system that is different from that of eukaryotes. In chloroplasts (plastids), it is believed that the prokaryotic gene transcription features govern genome transcription. However, the polycistronic operon transcription model cannot account for all the chloroplast genome (plastome) transcription products at whole-genome level, especially regarding various RNA isoforms. By systematically analyzing transcriptomes of plastids of algae and higher plants, and cyanobacteria, we find that the entire plastome is transcribed in photosynthetic green plants, and that this pattern originated from prokaryotic cyanobacteria — ancestor of the chloroplast genomes that diverged about 1 billion years ago. We propose a multiple arrangement transcription model that multiple transcription initiations and terminations combine haphazardly to accomplish the genome transcription followed by subsequent RNA processing events, which explains the full chloroplast genome transcription phenomenon and numerous functional and/or aberrant pre-RNAs. Our findings indicate a complex prokaryotic genome regulation when processing primary transcripts. PMID:27456469

  6. 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.

  7. Establishment of the chloroplast genetic system in rice during early leaf development and at low temperatures

    PubMed Central

    Kusumi, Kensuke; Iba, Koh

    2014-01-01

    Chloroplasts are the central nodes of the metabolic network in leaf cells of higher plants, and the conversion of proplastids into chloroplasts is tightly coupled to leaf development. During early leaf development, the structure and function of the chloroplasts differ greatly from those in a mature leaf, suggesting the existence of a stage-specific mechanism regulating chloroplast development during this period. Here, we discuss the identification of the genes affected in low temperature-conditional mutants of rice (Oryza sativa). These genes encode factors involved in chloroplast rRNA regulation (NUS1), and nucleotide metabolism in mitochondria, chloroplasts, and cytosol (V2, V3, ST1). These genes are all preferentially expressed in the early leaf developmental stage P4, and depleting them causes altered chloroplast transcription and translation, and ultimately leaf chlorosis. Therefore, it is suggested that regulation of cellular nucleotide pools and nucleotide metabolism is indispensable for chloroplast development under low temperatures at this stage. This review summarizes the current understanding of these factors and discusses their roles in chloroplast biogenesis. PMID:25157260

  8. Rapid severing and motility of chloroplast-actin filaments are required for the chloroplast avoidance response in Arabidopsis.

    PubMed

    Kong, Sam-Geun; Arai, Yoshiyuki; Suetsugu, Noriyuki; Yanagida, Toshio; Wada, Masamitsu

    2013-02-01

    Phototropins (phot1 and phot2 in Arabidopsis thaliana) relay blue light intensity information to the chloroplasts, which move toward weak light (the accumulation response) and away from strong light (the avoidance response). Chloroplast-actin (cp-actin) filaments are vital for mediating these chloroplast photorelocation movements. In this report, we examine in detail the cp-actin filament dynamics by which the chloroplast avoidance response is regulated. Although stochastic dynamics of cortical actin fragments are observed on the chloroplasts, the basic mechanisms underlying the disappearance (including severing and turnover) of the cp-actin filaments are regulated differently from those of cortical actin filaments. phot2 plays a pivotal role in the strong blue light-induced severing and random motility of cp-actin filaments, processes that are therefore essential for asymmetric cp-actin formation for the avoidance response. In addition, phot2 functions in the bundling of cp-actin filaments that is induced by dark incubation. By contrast, the function of phot1 is dispensable for these responses. Our findings suggest that phot2 is the primary photoreceptor involved in the rapid reorganization of cp-actin filaments that allows chloroplasts to change direction rapidly and control the velocity of the avoidance movement according to the light's intensity and position.

  9. Chloroplast ribosomes and protein synthesis.

    PubMed Central

    Harris, E H; Boynton, J E; Gillham, N W

    1994-01-01

    Consistent with their postulated origin from endosymbiotic cyanobacteria, chloroplasts of plants and algae have ribosomes whose component RNAs and proteins are strikingly similar to those of eubacteria. Comparison of the secondary structures of 16S rRNAs of chloroplasts and bacteria has been particularly useful in identifying highly conserved regions likely to have essential functions. Comparative analysis of ribosomal protein sequences may likewise prove valuable in determining their roles in protein synthesis. This review is concerned primarily with the RNAs and proteins that constitute the chloroplast ribosome, the genes that encode these components, and their expression. It begins with an overview of chloroplast genome structure in land plants and algae and then presents a brief comparison of chloroplast and prokaryotic protein-synthesizing systems and a more detailed analysis of chloroplast rRNAs and ribosomal proteins. A description of the synthesis and assembly of chloroplast ribosomes follows. The review concludes with discussion of whether chloroplast protein synthesis is essential for cell survival. PMID:7854253

  10. Pb-induced avoidance-like chloroplast movements in fronds of Lemna trisulca L.

    PubMed

    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

  11. 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

  12. Actin-based mechanisms for light-dependent intracellular positioning of nuclei and chloroplasts in Arabidopsis.

    PubMed

    Iwabuchi, Kosei; Takagi, Shingo

    2010-08-01

    The plant organelles, chloroplast and nucleus, change their position in response to light. In Arabidopsis thaliana leaf cells, chloroplasts and nuclei are distributed along the inner periclinal wall in darkness. In strong blue light, they become positioned along the anticlinal wall, while in weak blue light, only chloroplasts are accumulated along the inner and outer periclinal walls. Blue-light dependent positioning of both organelles is mediated by the blue-light receptor phototropin and controlled by the actin cytoskeleton. Interestingly, however, it seems that chloroplast movement requires short, fine actin filaments organized at the chloroplast edge, whereas nuclear movement does cytoplasmic, thick actin bundles intimately associated with the nucleus. Although there are many similarities between photo-relocation movements of chloroplasts and nuclei, plant cells appear to have evolved distinct mechanisms to regulate actin organization required for driving the movements of these organelles.

  13. Chloroplast membrane transport: interplay of prokaryotic and eukaryotic traits.

    PubMed

    Vothknecht, Ute C; Soll, Jürgen

    2005-07-18

    Chloroplasts are specific plant organelles of prokaryotic origin. They are separated from the surrounding cell by a double membrane, which represents an effective barrier for the transport of metabolites and proteins. Specific transporters in the inner envelope membrane have been described, which facilitate the exchange of metabolites. In contrast, the outer envelope has been viewed for a long time as a molecular sieve that offers a mere size constriction to the passage of molecules. This view has been challenged lately, and a number of specific and regulated pore proteins of the outer envelope (OEPs) have been identified. These pores seem to have originated by adaptation of outer membrane proteins of the cyanobacterial ancestor of the chloroplast. In a similar fashion, the transport of proteins across the two envelope membranes is achieved by two hetero-oligomeric protein complexes called Toc (translocon in the outer envelope of chloroplasts) and Tic (translocon in the inner envelope of chloroplasts). The phylogenetic provenance of the translocon components is less clear, but at least the channel protein of the Toc translocon is of cyanobacterial origin. Characteristic of cyanobacteria and chloroplasts is furthermore a specialized internal membrane system, the thylakoids, on which the components of the photosynthetic machinery are located. Despite the importance of this membrane, very little is known about its phylogenetic origin or the manner of its synthesis. Vipp1 appears to be a ubiquitous component of thylakoid formation, while in chloroplasts of land plants, additionally a vesicle transport system of eukaryotic origin might be involved in this process.

  14. Application of HB17, an Arabidopsis class II homeodomain-leucine zipper transcription factor, to regulate chloroplast number and photosynthetic capacity

    PubMed Central

    Kohl, Elizabeth A.; Tiwari, Shiv; Lundgren, Marjorie R.; Channa, Namitha; Creelman, Robert A.

    2013-01-01

    Transcription factors are proposed as suitable targets for the control of traits such as yield or food quality in plants. This study reports the results of a functional genomics research effort that identified ATHB17, a transcription factor from the homeodomain-leucine zipper class II family, as a novel target for the enhancement of photosynthetic capacity. It was shown that ATHB17 is expressed natively in the root quiescent centre (QC) from Arabidopsis embryos and seedlings. Analysis of the functional composition of genes differentially expressed in the QC from a knockout mutant (athb17-1) compared with its wild-type sibling revealed the over-representation of genes involved in auxin stimulus, embryo development, axis polarity specification, and plastid-related processes. While no other phenotypes were observed in athb17-1 plants, overexpression of ATHB17 produced a number of phenotypes in Arabidopsis including enhanced chlorophyll content. Image analysis of isolated mesophyll cells of 35S::ATHB17 lines revealed an increase in the number of chloroplasts per unit cell size, which is probably due to an increase in the number of proplastids per meristematic cell. Leaf physiological measurements provided evidence of improved photosynthetic capacity in 35S::ATHB17 lines on a per unit leaf area basis. Estimates of the capacity for ribulose-1,5-bisphosphate-saturated and -limited photosynthesis were significantly higher in 35S::ATHB17 lines. PMID:24006420

  15. Application of HB17, an Arabidopsis class II homeodomain-leucine zipper transcription factor, to regulate chloroplast number and photosynthetic capacity.

    PubMed

    Hymus, Graham J; Cai, Suqin; Kohl, Elizabeth A; Holtan, Hans E; Marion, Colleen M; Tiwari, Shiv; Maszle, Don R; Lundgren, Marjorie R; Hong, Melissa C; Channa, Namitha; Loida, Paul; Thompson, Rebecca; Taylor, J Philip; Rice, Elena; Repetti, Peter P; Ratcliffe, Oliver J; Reuber, T Lynne; Creelman, Robert A

    2013-11-01

    Transcription factors are proposed as suitable targets for the control of traits such as yield or food quality in plants. This study reports the results of a functional genomics research effort that identified ATHB17, a transcription factor from the homeodomain-leucine zipper class II family, as a novel target for the enhancement of photosynthetic capacity. It was shown that ATHB17 is expressed natively in the root quiescent centre (QC) from Arabidopsis embryos and seedlings. Analysis of the functional composition of genes differentially expressed in the QC from a knockout mutant (athb17-1) compared with its wild-type sibling revealed the over-representation of genes involved in auxin stimulus, embryo development, axis polarity specification, and plastid-related processes. While no other phenotypes were observed in athb17-1 plants, overexpression of ATHB17 produced a number of phenotypes in Arabidopsis including enhanced chlorophyll content. Image analysis of isolated mesophyll cells of 35S::ATHB17 lines revealed an increase in the number of chloroplasts per unit cell size, which is probably due to an increase in the number of proplastids per meristematic cell. Leaf physiological measurements provided evidence of improved photosynthetic capacity in 35S::ATHB17 lines on a per unit leaf area basis. Estimates of the capacity for ribulose-1,5-bisphosphate-saturated and -limited photosynthesis were significantly higher in 35S::ATHB17 lines.

  16. Tagetitoxin inhibits chloroplast RNA synthesis

    SciTech Connect

    Mathews, D.E.; Durbin, R.D.

    1987-04-01

    Tagetitoxin is a non-host specific phytotoxin which inhibits chloroplast development. Chloroplast encoded gene products as well as their transcripts are conspicuously depleted in toxin-treated tissue. Intact chloroplasts from 8-9 day old peas were incubated for 60 min. in the presence of tagetitoxin. This treatment reduced RNA synthesis but did not affect protein synthesis as measured by the incorporation of radiolabeled uridine or methionine, respectively. Tagetitoxin also inhibited chloroplast RNA synthesis in vitro. Total UTP incorporation was reduced 50% by 0.5..mu..M tagetitoxin in transcriptionally active chloroplast extracts containing 5mg/ml protein. In vitro transcription with purified E. coli RNA polymerase was also inhibited by tagetitoxin, yet wheat germ RNA polymerase II and several bacteriophage RNA polymerase enzymes were unaffected. Recent evidence suggests that RNA polymerase from chloroplasts and prokaryotes may share extensive homology. In light of this evidence and the authors own data, they propose that tagetitoxin directly inhibits chloroplast RNA polymerase.

  17. Genetic Analysis of Chloroplast Translation

    SciTech Connect

    Barkan, Alice

    2005-08-15

    The assembly of the photosynthetic apparatus requires the concerted action of hundreds of genes distributed between the two physically separate genomes in the nucleus and chloroplast. Nuclear genes coordinate this process by controlling the expression of chloroplast genes in response to developmental and environmental cues. However, few regulatory factors have been identified. We used mutant phenotypes to identify nuclear genes in maize that modulate chloroplast translation, a key control point in chloroplast gene expression. This project focused on the nuclear gene crp1, required for the translation of two chloroplast mRNAs. CRP1 is related to fungal proteins involved in the translation of mitochondrial mRNAs, and is the founding member of a large gene family in plants, with {approx}450 members. Members of the CRP1 family are defined by a repeated 35 amino acid motif called a ''PPR'' motif. The PPR motif is closely related to the TPR motif, which mediates protein-protein interactions. We and others have speculated that PPR tracts adopt a structure similar to that of TPR tracts, but with a substrate binding surface adapted to bind RNA instead of protein. To understand how CRP1 influences the translation of specific chloroplast mRNAs, we sought proteins that interact with CRP1, and identified the RNAs associated with CRP1 in vivo. We showed that CRP1 is associated in vivo with the mRNAs whose translation it activates. To explore the functions of PPR proteins more generally, we sought mutations in other PPR-encoding genes: mutations in the maize PPR2 and PPR4 were shown to disrupt chloroplast ribosome biogenesis and chloroplast trans-splicing, respectively. These and other results suggest that the nuclear-encoded PPR family plays a major role in modulating the expression of the chloroplast genome in higher plants.

  18. Dynamics of Chloroplast Translation during Chloroplast Differentiation in Maize

    PubMed Central

    Chotewutmontri, Prakitchai; Barkan, Alice

    2016-01-01

    Chloroplast genomes in land plants contain approximately 100 genes, the majority of which reside in polycistronic transcription units derived from cyanobacterial operons. The expression of chloroplast genes is integrated into developmental programs underlying the differentiation of photosynthetic cells from non-photosynthetic progenitors. In C4 plants, the partitioning of photosynthesis between two cell types, bundle sheath and mesophyll, adds an additional layer of complexity. We used ribosome profiling and RNA-seq to generate a comprehensive description of chloroplast gene expression at four stages of chloroplast differentiation, as displayed along the maize seedling leaf blade. The rate of protein output of most genes increases early in development and declines once the photosynthetic apparatus is mature. The developmental dynamics of protein output fall into several patterns. Programmed changes in mRNA abundance make a strong contribution to the developmental shifts in protein output, but output is further adjusted by changes in translational efficiency. RNAs with prioritized translation early in development are largely involved in chloroplast gene expression, whereas those with prioritized translation in photosynthetic tissues are generally involved in photosynthesis. Differential gene expression in bundle sheath and mesophyll chloroplasts results primarily from differences in mRNA abundance, but differences in translational efficiency amplify mRNA-level effects in some instances. In most cases, rates of protein output approximate steady-state protein stoichiometries, implying a limited role for proteolysis in eliminating unassembled or damaged proteins under non-stress conditions. Tuned protein output results from gene-specific trade-offs between translational efficiency and mRNA abundance, both of which span a large dynamic range. Analysis of ribosome footprints at sites of RNA editing showed that the chloroplast translation machinery does not generally

  19. 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.

  20. 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

  1. Heme content in developing chloroplasts

    SciTech Connect

    Thomas, J.; Weinstein, J.D. )

    1991-05-01

    Heme regulates tetrapyrrole biosynthesis by inhibition of {delta}-aminolevulinic acid synthesis, product inhibition of heme synthesis, and possibly other mechanisms. Determination of the physiological relevance of this inhibition requires a sensitive measurement which can distinguish regulatory free heme from heme which is an integral part of functional hemoproteins. A preliminary estimate was provided by reconstituting peroxidase activity from apo-peroxidase and the heme contained in broken plastids. However, subsequent experiments have suggested that this initial estimate was too large due to reconstitution of apo-peroxidase with heme from functional hemoproteins (i.e. heme stealing). The authors have now refined the measurement techniques to greatly reduce the extent of this heme stealing. Incubation of broken plastids with apo-peroxidase at 10C resolves the kinetics of reconstitution into two components. A fast component levels off after 100 min, and a slow component increases linearly for up to 6 hours. They believe that the heme which reconstitutes during the fast phase represents free heme, and the linear slow component represents heme stealing. In support of this theory, incubation at 15C increases the rate of both components. However, extrapolation to zero time of the slow components of the 10C and 15C time courses results in equivalent amounts of heme. Based on this kinetic differentiation between free heme and hemoprotein heme, chloroplasts isolated from cucumber cotyledons after 30 h of greening contain substantially greater amounts of free heme than etioplasts.

  2. The Physcomitrella patens Chloroplast Proteome Changes in Response to Protoplastation

    PubMed Central

    Fesenko, Igor; Seredina, Anna; Arapidi, Georgij; Ptushenko, Vasily; Urban, Anatoly; Butenko, Ivan; Kovalchuk, Sergey; Babalyan, Konstantin; Knyazev, Andrey; Khazigaleeva, Regina; Pushkova, Elena; Anikanov, Nikolai; Ivanov, Vadim; Govorun, Vadim M.

    2016-01-01

    Plant protoplasts are widely used for genetic manipulation and functional studies in transient expression systems. However, little is known about the molecular pathways involved in a cell response to the combined stress factors resulted from protoplast generation. Plants often face more than one type of stress at a time, and how plants respond to combined stress factors is therefore of great interest. Here, we used protoplasts of the moss Physcomitrella patens as a model to study the effects of short-term stress on the chloroplast proteome. Using label-free comparative quantitative proteomic analysis (SWATH-MS), we quantified 479 chloroplast proteins, 219 of which showed a more than 1.4-fold change in abundance in protoplasts. We additionally quantified 1451 chloroplast proteins using emPAI. We observed degradation of a significant portion of the chloroplast proteome following the first hour of stress imposed by the protoplast isolation process. Electron-transport chain (ETC) components underwent the heaviest degradation, resulting in the decline of photosynthetic activity. We also compared the proteome changes to those in the transcriptional level of nuclear-encoded chloroplast genes. Globally, the levels of the quantified proteins and their corresponding mRNAs showed limited correlation. Genes involved in the biosynthesis of chlorophyll and components of the outer chloroplast membrane showed decreases in both transcript and protein abundance. However, proteins like dehydroascorbate reductase 1 and 2-cys peroxiredoxin B responsible for ROS detoxification increased in abundance. Further, genes such as thylakoid ascorbate peroxidase were induced at the transcriptional level but down-regulated at the proteomic level. Together, our results demonstrate that the initial chloroplast reaction to stress is due changes at the proteomic level. PMID:27867392

  3. Red light, Phot1 and JAC1 modulate Phot2-dependent reorganization of chloroplast actin filaments and chloroplast avoidance movement.

    PubMed

    Ichikawa, Satoshi; Yamada, Noboru; Suetsugu, Noriyuki; Wada, Masamitsu; Kadota, Akeo

    2011-08-01

    The phototropin (phot)-dependent intracellular relocation of chloroplasts is a ubiquitous phenomenon in plants. We have previously revealed the involvement of a short cp-actin (chloroplast actin) filament-based mechanism in this movement. Here, the reorganization of cp-actin filaments during the avoidance movement of chloroplasts was analyzed in higher time resolution under blue GFP (green fluorescent protein) excitation light in an actin filament-visualized line of Arabidopsis thaliana. Under standard background red light of 89 μmol m(-2) s(-1), cp-actin filaments transiently disappeared at approximately 30 s and reappeared in a biased configuration on chloroplasts approximately 70 s after blue excitation light irradiation. The timing of biased cp-actin reappearance was delayed under the background of strong red light or in the absence of red light. Consistently, chloroplast movement was delayed under these conditions. In phot1 mutants, acceleration of both the disappearance and reappearance of cp-actin filaments occurred, indicating an inhibitory action of phot1 on reorganization of cp-actin filaments. Avoidance movements began sooner in phot1 than in wild-type plants. No reorganization of cp-actin filaments was seen in phot2 or phot1phot2 mutants lacking phot2, which is responsible for avoidance movements. Surprisingly, jac1 (j-domain protein required for chloroplast accumulation response 1) mutants, lacking the accumulation response, showed no avoidance movements under the whole-cell irradiation condition for GFP observation. Cp-actin filaments in jac1 did not show a biased distribution, with a small or almost no transient decrease in the number. These results indicate a close association between the biased distribution of cp-actin filaments and chloroplast movement. Further, JAC1 is suggested to function in the biased cp-actin filament distribution by regulating their appearance and disappearance.

  4. 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.

  5. IM30 triggers membrane fusion in cyanobacteria and chloroplasts.

    PubMed

    Hennig, Raoul; Heidrich, Jennifer; Saur, Michael; Schmüser, Lars; Roeters, Steven J; Hellmann, Nadja; Woutersen, Sander; Bonn, Mischa; Weidner, Tobias; Markl, Jürgen; Schneider, Dirk

    2015-05-08

    The thylakoid membrane of chloroplasts and cyanobacteria is a unique internal membrane system harbouring the complexes of the photosynthetic electron transfer chain. Despite their apparent importance, little is known about the biogenesis and maintenance of thylakoid membranes. Although membrane fusion events are essential for the formation of thylakoid membranes, proteins involved in membrane fusion have yet to be identified in photosynthetic cells or organelles. Here we show that IM30, a conserved chloroplast and cyanobacterial protein of approximately 30 kDa binds as an oligomeric ring in a well-defined geometry specifically to membranes containing anionic lipids. Triggered by Mg(2+), membrane binding causes destabilization and eventually results in membrane fusion. We propose that IM30 establishes contacts between internal membrane sites and promotes fusion to enable regulated exchange of proteins and/or lipids in cyanobacteria and chloroplasts.

  6. Chloroplast in Plant-Virus Interaction

    PubMed Central

    Zhao, Jinping; Zhang, Xian; Hong, Yiguo; Liu, Yule

    2016-01-01

    In plants, the chloroplast is the organelle that conducts photosynthesis. It has been known that chloroplast is involved in virus infection of plants for approximate 70 years. Recently, the subject of chloroplast-virus interplay is getting more and more attention. In this article we discuss the different aspects of chloroplast-virus interaction into three sections: the effect of virus infection on the structure and function of chloroplast, the role of chloroplast in virus infection cycle, and the function of chloroplast in host defense against viruses. In particular, we focus on the characterization of chloroplast protein-viral protein interactions that underlie the interplay between chloroplast and virus. It can be summarized that chloroplast is a common target of plant viruses for viral pathogenesis or propagation; and conversely, chloroplast and its components also can play active roles in plant defense against viruses. Chloroplast photosynthesis-related genes/proteins (CPRGs/CPRPs) are suggested to play a central role during the complex chloroplast-virus interaction. PMID:27757106

  7. 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

  8. AKR2A-mediated import of chloroplast outer membrane proteins is essential for chloroplast biogenesis.

    PubMed

    Bae, Wonsil; Lee, Yong Jik; Kim, Dae Heon; Lee, Junho; Kim, Soojin; Sohn, Eun Ju; Hwang, Inhwan

    2008-02-01

    In plant cells, chloroplasts have essential roles in many biochemical reactions and physiological responses. Chloroplasts require numerous protein components, but only a fraction of these proteins are encoded by the chloroplast genome. Instead, most are encoded by the nuclear genome and imported into chloroplasts from the cytoplasm post-translationally. Membrane proteins located in the chloroplast outer envelope membrane (OEM) have a critical function in the import of proteins into the chloroplast. However, the biogenesis of chloroplast OEM proteins remains poorly understood. Here, we report that an Arabidopsis ankyrin repeat protein, AKR2A, plays an essential role in the biogenesis of the chloroplast OEM proteins. AKR2A binds to chloroplast OEM protein targeting signals, as well as to chloroplasts. It also displays chaperone activity towards chloroplast OEM proteins, and facilitates the targeting of OEP7 to chloroplasts in vitro. AKR2A RNAi in plants with an akr2b knockout background showed greatly reduced levels of chloroplast proteins, including OEM proteins, and chloroplast biogenesis was also defective. Thus, AKR2A functions as a cytosolic mediator for sorting and targeting of nascent chloroplast OEM proteins to the chloroplast.

  9. Structural relationship in chloroplast membranes. Final report, August 1, 1977-August 31, 1980

    SciTech Connect

    1980-09-01

    Methodology has been developed for the isolation and characterization of pigment-proteins from chloroplast membranes. Characterization of these pigment-proteins has increased our understanding of mechanisms regulating the efficiency of photosynthetic light harvesting during photosynthesis. Incorporation of isolated pigment-protein complexes into model membranes has allowed simulation of grana stacks; these structural features of chloroplasts play a key role in maintaining appropriate interactions among light-harvesting assembles to regulate photosynthetic solar energy conversion.

  10. Chloroplast evolution: secondary symbiogenesis and multiple losses.

    PubMed

    Cavalier-Smith, T

    2002-01-22

    Chloroplasts originated from cyanobacteria only once, but have been laterally transferred to other lineages by symbiogenetic cell mergers. Such secondary symbiogenesis is rarer and chloroplast losses commoner than often assumed.

  11. Mutation of the rice ASL2 gene encoding plastid ribosomal protein L21 causes chloroplast developmental defects and seedling death.

    PubMed

    Lin, D; Jiang, Q; Zheng, K; Chen, S; Zhou, H; Gong, X; Xu, J; Teng, S; Dong, Y

    2015-05-01

    The plastid ribosome proteins (PRPs) play important roles in plastid protein biosynthesis, chloroplast differentiation and early chloroplast development. However, the specialised functions of individual protein components of the chloroplast ribosome in rice (Oryza sativa) remain unresolved. In this paper, we identified a novel rice PRP mutant named asl2 (Albino seedling lethality 2) exhibiting an albino, seedling death phenotype. In asl2 mutants, the alteration of leaf colour was associated with chlorophyll (Chl) content and abnormal chloroplast development. Through map-based cloning and complementation, the mutated ASL2 gene was isolated and found to encode the chloroplast 50S ribosome protein L21 (RPL21c), a component of the chloroplast ribosome large subunit, which was localised in chloroplasts. ASL2 was expressed at a higher level in the plumule and leaves, implying its tissue-specific expression. Additionally, the expression of ASL2 was regulated by light. The transcript levels of the majority of genes for Chl biosynthesis, photosynthesis and chloroplast development were strongly affected in asl2 mutants. Collectively, the absence of functional ASL2 caused chloroplast developmental defects and seedling death. This report establishes the important role of RPL21c in chloroplast development in rice.

  12. Chloroplast Proteases: Updates on Proteolysis within and across Suborganellar Compartments1[OPEN

    PubMed Central

    Nishimura, Kenji

    2016-01-01

    Chloroplasts originated from the endosymbiosis of ancestral cyanobacteria and maintain transcription and translation machineries for around 100 proteins. Most endosymbiont genes, however, have been transferred to the host nucleus, and the majority of the chloroplast proteome is composed of nucleus-encoded proteins that are biosynthesized in the cytosol and then imported into chloroplasts. How chloroplasts and the nucleus communicate to control the plastid proteome remains an important question. Protein-degrading machineries play key roles in chloroplast proteome biogenesis, remodeling, and maintenance. Research in the past few decades has revealed more than 20 chloroplast proteases, which are localized to specific suborganellar locations. In particular, two energy-dependent processive proteases of bacterial origin, Clp and FtsH, are central to protein homeostasis. Processing endopeptidases such as stromal processing peptidase and thylakoidal processing peptidase are involved in the maturation of precursor proteins imported into chloroplasts by cleaving off the amino-terminal transit peptides. Presequence peptidases and organellar oligopeptidase subsequently degrade the cleaved targeting peptides. Recent findings have indicated that not only intraplastidic but also extraplastidic processive protein-degrading systems participate in the regulation and quality control of protein translocation across the envelopes. In this review, we summarize current knowledge of the major chloroplast proteases in terms of type, suborganellar localization, and diversification. We present details of these degradation processes as case studies according to suborganellar compartment (envelope, stroma, and thylakoids). Key questions and future directions in this field are discussed. PMID:27288365

  13. Chloroplast photorelocation movement mediated by phototropin family proteins in green plants.

    PubMed

    Suetsugu, Noriyuki; Wada, Masamitsu

    2007-09-01

    Chloroplasts gather in areas irradiated with weak light to maximize photosynthesis (the accumulation response). They move away from areas irradiated with strong light to minimize damage of the photosynthetic apparatus (the avoidance response). The processes underlying these chloroplast movements can be divided into three parts: photoperception, signal transduction, and chloroplast movement. Photoreceptors for chloroplast movement have been identified recently in various plant species. A blue light receptor phototropin (phot) mediates chloroplast photorelocation movement in the seed plant Arabidopsis thaliana, the fern Adiantum capillus-veneris, the moss Physcomitrella patens and possibly the green alga Mougeotia scalaris. A chimeric photoreceptor between phytochrome and phototropin, neochrome (neo), was found in some advanced ferns and in the green alga M. scalaris. While the mechanism of chloroplast movement is not well understood, it is known that actin filaments play an important role in this process. To understand the molecular mechanisms associated with chloroplast movement, several mutants were isolated in A. thaliana (jac1 and chup1) and the corresponding genes were cloned. In this review, recent progress in photoreceptor research into chloroplast movement in various plant species and the possible factors functioning in signal transduction or the regulation of actin filaments identified in A. thaliana is discussed.

  14. Short actin-based mechanism for light-directed chloroplast movement in Arabidopsis.

    PubMed

    Kadota, Akeo; Yamada, Noboru; Suetsugu, Noriyuki; Hirose, Mana; Saito, Chieko; Shoda, Keiko; Ichikawa, Satoshi; Kagawa, Takatoshi; Nakano, Akihiko; Wada, Masamitsu

    2009-08-04

    Organelle movement is essential for proper function of living cells. In plants, these movements generally depend on actin filaments, but the underlying mechanism is unknown. Here, in Arabidopsis, we identify associations of short actin filaments along the chloroplast periphery on the plasma membrane side associated with chloroplast photorelocation and anchoring to the plasma membrane. We have termed these chloroplast-actin filaments (cp-actin filaments). Cp-actin filaments emerge from the chloroplast edge and exhibit rapid turnover. The presence of cp-actin filaments depends on an actin-binding protein, chloroplast unusual positioning1 (CHUP1), localized on the chloroplast envelope. chup1 mutant lacked cp-actin filaments but showed normal cytoplasmic actin filaments. When irradiated with blue light to induce chloroplast movement, cp-actin filaments relocalize to the leading edge of chloroplasts before and during photorelocation and are regulated by 2 phototropins, phot1 and phot2. Our findings suggest that plants evolved a unique actin-based mechanism for organelle movement.

  15. 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.

  16. Short actin-based mechanism for light-directed chloroplast movement in Arabidopsis

    PubMed Central

    Kadota, Akeo; Yamada, Noboru; Suetsugu, Noriyuki; Hirose, Mana; Saito, Chieko; Shoda, Keiko; Ichikawa, Satoshi; Kagawa, Takatoshi; Nakano, Akihiko; Wada, Masamitsu

    2009-01-01

    Organelle movement is essential for proper function of living cells. In plants, these movements generally depend on actin filaments, but the underlying mechanism is unknown. Here, in Arabidopsis, we identify associations of short actin filaments along the chloroplast periphery on the plasma membrane side associated with chloroplast photorelocation and anchoring to the plasma membrane. We have termed these chloroplast-actin filaments (cp-actin filaments). Cp-actin filaments emerge from the chloroplast edge and exhibit rapid turnover. The presence of cp-actin filaments depends on an actin-binding protein, chloroplast unusual positioning1 (CHUP1), localized on the chloroplast envelope. chup1 mutant lacked cp-actin filaments but showed normal cytoplasmic actin filaments. When irradiated with blue light to induce chloroplast movement, cp-actin filaments relocalize to the leading edge of chloroplasts before and during photorelocation and are regulated by 2 phototropins, phot1 and phot2. Our findings suggest that plants evolved a unique actin-based mechanism for organelle movement. PMID:19620714

  17. Isolation of chloroplastic phosphoglycerate kinase

    SciTech Connect

    Macioszek, J.; Anderson, L.E. ); Anderson, J.B. )

    1990-09-01

    We report here a method for the isolation of high specific activity phosphoglycerate kinase (EC 2.7.2.3) from chloroplasts. The enzyme has been purified over 200-fold from pea (Pisum sativum L.) stromal extracts to apparent homogeneity with 23% recovery. Negative cooperativity is observed with the two enzyme phosphoglycerate kinase/glyceraldehyde-3-P dehydrogenase (EC 1.2.1.13) couple restored from the purified enzymes when NADPH is the reducing pyridine nucleotide, consistent with earlier results obtained with crude chloroplastic extracts. Michaelis Menten kinetics are observed when 3-phosphoglycerate is held constant and phosphoglycerate kinase is varied, which suggests that phosphoglycerate kinase-bound 1,3-bisphosphoglycerate may be the preferred substrate for glyceraldehyde-3-P dehydrogenase in the chloroplast.

  18. Heterologous expression of a chloroplast outer envelope protein from Suaeda salsa confers oxidative stress tolerance and induces chloroplast aggregation in transgenic Arabidopsis plants.

    PubMed

    Wang, Fang; Yang, Chun-Lin; Wang, Li-Li; Zhong, Nai-Qin; Wu, Xiao-Min; Han, Li-Bo; Xia, Gui-Xian

    2012-03-01

    Suaeda salsa is a euhalophytic plant that is tolerant to coastal seawater salinity. In this study, we cloned a cDNA encoding an 8.4 kDa chloroplast outer envelope protein (designated as SsOEP8) from S. salsa and characterized its cellular function. Steady-state transcript levels of SsOEP8 in S. salsa were up-regulated in response to oxidative stress. Consistently, ectopic expression of SsOEP8 conferred enhanced oxidative stress tolerance in transgenic Bright Yellow 2 (BY-2) cells and Arabidopsis, in which H(2) O(2) content was reduced significantly in leaf cells. Further studies revealed that chloroplasts aggregated to the sides of mesophyll cells in transgenic Arabidopsis leaves, and this event was accompanied by inhibited expression of genes encoding proteins for chloroplast movements such as AtCHUP1, a protein involved in actin-based chloroplast positioning and movement. Moreover, organization of actin cytoskeleton was found to be altered in transgenic BY-2 cells. Together, these results suggest that SsOEP8 may play a critical role in oxidative stress tolerance by changing actin cytoskeleton-dependent chloroplast distribution, which may consequently lead to the suppressed production of reactive oxygen species (ROS) in chloroplasts. One significantly novel aspect of this study is the finding that the small chloroplast envelope protein is involved in oxidative stress tolerance.

  19. CHLOROPLAST DEVELOPMENT IN OCHROMONAS DANICA

    PubMed Central

    Gibbs, Sarah P.

    1962-01-01

    When dark-grown cells of Ochromonas danica are placed in the light, the amount of chlorophyll a per cell increases 82-fold; the content of carotenoid pigment, 24-fold. Concomitantly with this increase in chlorophyll and carotenoid pigment, the small proplastid of dark-grown cells develops into a large lamellate chloroplast. During the first 12 hours in the light, vesicles appear within the loose clusters of dense chloroplast granules, enlarge, align themselves into rows (plates in three dimensions), and fuse into discs. Double discs may form from the more or less simultaneous fusion of two adjacent plates of vesicles or by the addition of vesicles to an already formed single disc. Three-disc bands arise by the addition of a disc to an already formed two-disc band through the approach and fusion of more vesicles. After 24 hours in the light, most of the chloroplast bands contain three discs, but the chloroplasts are still small. After 48 hours in the light, almost all the cells contain full-sized chloroplasts with a full complement of three-disc bands. However, at this time the amount of chlorophyll a and carotenoid pigment is only one-half of maximum. During the next 3 days in the light, as the number of chlorophyll and carotenoid molecules per chloroplast approximately doubles, there is a compression of the discs in each band (from 180 to 130 A) and a precise alignment of their membranes. Changes also occur in the nucleus when dark-grown cells are placed in the light. There is an increase in the number of small nucleolar bodies, many of which lie directly against the nuclear envelope, and in a few cells a dense mass of granules is seen between the two membranes of the nuclear envelope. PMID:13947686

  20. Nitrogen control of chloroplast differentiation

    SciTech Connect

    Schmidt, G.W.

    1992-07-01

    This project is directed toward understanding how the availability of nitrogen affects the accumulation of chloroplast pigments and proteins functioning in energy transduction and carbon metabolism. Molecular analyses performed with Chlamydomonas reinhardtii grown in a continuous culture system such that ammonium concentration is maintained at a low steady-state concentration so as to limit cell division. As compared to chloroplasts from cells of non-limiting nitrogen provisions, chloroplasts of N-limited cells are profoundly chlorophyll-deficient but still assimilate carbon for deposition of as starch and as storage lipids. Chlorophyll deficiency arises by limiting accumulation of appropriate nuclear-encoded mRNAs of and by depressed rates of translation of chloroplast mRNAs for apoproteins of reaction centers. Chloroplast translational effects can be partially ascribed to diminished rates of chlorophyll biosynthesis in N-limited cells, but pigment levels are not determinants for expression of the nuclear light-harvesting protein genes. Consequently, other signals that are responsive to nitrogen availability mediate transcriptional or post-transcriptional processes for accumulation of the mRNAs for LHC apoproteins and other mRNAs whose abundance is dependent upon high nitrogen levels. Conversely, limited nitrogen availability promotes accumulation of other proteins involved in carbon metabolism and oxidative electron transport in chloroplasts. Hence, thylakoids of N-limited cells exhibit enhanced chlororespiratory activities wherein oxygen serves as the electron acceptor in a pathway that involves plastoquinone and other electron carrier proteins that remain to be thoroughly characterized. Ongoing and future studies are also outlined.

  1. Protein import into chloroplasts requires a chloroplast ATPase

    SciTech Connect

    Pain, D.; Blobel, G.

    1987-05-01

    The authors have transcribed mRNA from a cDNA clone coding for pea ribulose-1,5-bisphosphate carboxylase, translated the mRNA in a wheat germ cell-free system, and studied the energy requirement for posttranslational import of the (/sup 35/S)methionine-labeled protein into the stroma of pea chloroplasts. They found that import depends on ATP hydrolysis within the stroma. Import is not inhibited when H/sup +/, K/sup +/, Na/sup +/, or divalent cation gradients across the chloroplast membranes are dissipated by ionophores, as long as exogenously added ATP is also present during the import reaction. The data suggest that protein import into the chloroplast stroma requires a chloroplast ATPase that does not function to generate a membrane potential for driving the import reaction but that exerts its effect in another, yet-to-be-determined, mode. They have carried out a preliminary characterization of this ATPase regarding its nucleotide specificity and the effects of various ATPase inhibitors.

  2. Both phototropin 1 and 2 localize on the chloroplast outer membrane with distinct localization activity.

    PubMed

    Kong, Sam-Geun; Suetsugu, Noriyuki; Kikuchi, Shingo; Nakai, Masato; Nagatani, Akira; Wada, Masamitsu

    2013-01-01

    Chloroplasts change their position to adapt cellular activities to fluctuating environmental light conditions. Phototropins (phot1 and phot2 in Arabidopsis) are plant-specific blue light photoreceptors that perceive changes in light intensity and direction, and mediate actin-based chloroplast photorelocation movements. Both phot1 and phot2 regulate the chloroplast accumulation response, while phot2 is mostly responsible for the regulation of the avoidance response. Although it has been widely accepted that distinct intracellular localizations of phototropins are implicated in the specificity, the mechanism underlying the phot2-specific avoidance response has remained elusive. In this study, we examined the relationship of the phot2 localization pattern to the chloroplast photorelocation movement. First, the fusion of a nuclear localization signal with phot2, which effectively reduced the amount of phot2 in the cytoplasm, retained the activity for both the accumulation and avoidance responses, indicating that membrane-localized phot2 but not cytoplasmic phot2 is functional to mediate the responses. Importantly, some fractions of phot2, and of phot1 to a lesser extent, were localized on the chloroplast outer membrane. Moreover, the deletion of the C-terminal region of phot2, which was previously shown to be defective in blue light-induced Golgi localization and avoidance response, affected the localization pattern on the chloroplast outer membrane. Taken together, these results suggest that dynamic phot2 trafficking from the plasma membrane to the Golgi apparatus and the chloroplast outer membrane might be involved in the avoidance response.

  3. Chloroplasts extend stromules independently and in response to internal redox signals.

    PubMed

    Brunkard, Jacob O; Runkel, Anne M; Zambryski, Patricia C

    2015-08-11

    A fundamental mystery of plant cell biology is the occurrence of "stromules," stroma-filled tubular extensions from plastids (such as chloroplasts) that are universally observed in plants but whose functions are, in effect, completely unknown. One prevalent hypothesis is that stromules exchange signals or metabolites between plastids and other subcellular compartments, and that stromules are induced during stress. Until now, no signaling mechanisms originating within the plastid have been identified that regulate stromule activity, a critical missing link in this hypothesis. Using confocal and superresolution 3D microscopy, we have shown that stromules form in response to light-sensitive redox signals within the chloroplast. Stromule frequency increased during the day or after treatment with chemicals that produce reactive oxygen species specifically in the chloroplast. Silencing expression of the chloroplast NADPH-dependent thioredoxin reductase, a central hub in chloroplast redox signaling pathways, increased chloroplast stromule frequency, whereas silencing expression of nuclear genes related to plastid genome expression and tetrapyrrole biosynthesis had no impact on stromules. Leucoplasts, which are not photosynthetic, also made more stromules in the daytime. Leucoplasts did not respond to the same redox signaling pathway but instead increased stromule formation when exposed to sucrose, a major product of photosynthesis, although sucrose has no impact on chloroplast stromule frequency. Thus, different types of plastids make stromules in response to distinct signals. Finally, isolated chloroplasts could make stromules independently after extraction from the cytoplasm, suggesting that chloroplast-associated factors are sufficient to generate stromules. These discoveries demonstrate that chloroplasts are remarkably autonomous organelles that alter their stromule frequency in reaction to internal signal transduction pathways.

  4. Functional characterization of the GATA transcription factors GNC and CGA1 reveals their key role in chloroplast development, growth, and division in Arabidopsis.

    PubMed

    Chiang, Yi-Hsuan; Zubo, Yan O; Tapken, Wiebke; Kim, Hyo Jung; Lavanway, Ann M; Howard, Louisa; Pilon, Marinus; Kieber, Joseph J; Schaller, G Eric

    2012-09-01

    Chloroplasts develop from proplastids in a process that requires the interplay of nuclear and chloroplast genomes, but key steps in this developmental process have yet to be elucidated. Here, we show that the nucleus-localized transcription factors GATA NITRATE-INDUCIBLE CARBON-METABOLISM-INVOLVED (GNC) and CYTOKININ-RESPONSIVE GATA1 (CGA1) regulate chloroplast development, growth, and division in Arabidopsis (Arabidopsis thaliana). GNC and CGA1 are highly expressed in green tissues, and the phytohormone cytokinin regulates their expression. A gnc cga1 mutant exhibits a reduction in overall chlorophyll levels as well as in chloroplast size in the hypocotyl. Ectopic overexpression of either GNC or CGA1 promotes chloroplast biogenesis in hypocotyl cortex and root pericycle cells, based on increases in the number and size of the chloroplasts, and also results in expanded zones of chloroplast production into the epidermis of hypocotyls and cotyledons and into the cortex of roots. Ectopic overexpression also promotes the development of etioplasts from proplastids in dark-grown seedlings, subsequently enhancing the deetiolation process. Inducible expression of GNC demonstrates that GNC-mediated chloroplast biogenesis can be regulated postembryonically, notably so for chloroplast production in cotyledon epidermal cells. Analysis of the gnc cga1 loss-of-function and overexpression lines supports a role for these transcription factors in regulating the effects of cytokinin on chloroplast division. These data support a model in which GNC and CGA1 serve as two of the master transcriptional regulators of chloroplast biogenesis, acting downstream of cytokinin and mediating the development of chloroplasts from proplastids and enhancing chloroplast growth and division in specific tissues.

  5. (Calcium gating of proton fluxes in chloroplasts)

    SciTech Connect

    Dilley, R.A.

    1991-01-01

    Work supported by this grant has been aimed at better understanding the still-mysterious phenomenon of sequestered proton gradients which strong evidence suggests can energize ATP formation in chloroplast organelle membranes. Results from several laboratories support the notion that chloroplasts can couple the energy of H{sup +} gradients. Research in may lab has, since 1983, been deeply involved in a two-pronged approach, (A) identifying and quantitating sequestered proton buffering groups and (B) trying to elucidate just how the sequestered H{sup +} gradient is maintained and what regulates the switching between the localized and delocalized coupling modes. One aspect we have worked on under DOE auspices is the question whether the localized H{sup +} coupling, routinely detected in my lab by a protocol which measures the number of single-turnover flashes (usually fired at 5 Hz) required to reach the energization threshold for ATP formation, can continue in steady illumination. It is possible to consider that a localized coupling response could be observed in the initial energization transient followed obligatorily by the sustained H{sup +} gradient equilibrating with the lumen bulk phase. If that occurred, it would have very important ramifications as to how one evaluates the possible physiological meaning of localized'' coupling. To test this, we developed a steady illumination protocol which is briefly discussed.

  6. Ribonucleic acid synthesis in chloroplasts

    PubMed Central

    Hartley, M. R.; Ellis, R. J.

    1973-01-01

    Chloroplasts isolated from young spinach leaves incorporate [3H]uridine into RNA. This incorporation shows an absolute requirement for light and does not occur in lysed chloroplasts. Fractionation by polyacrylamide-gel electrophoresis of the RNA synthesized in vitro reveals a major discrete product of molecular weight 2.7×106 and two minor products of molecular weight 1.2×106 and 0.47×106. These discrete products are super-imposed on a background of polydisperse RNA. The incorporation of 32Pi into chloroplast rRNA species (mol.wt. 1.05×106 and 0.56×106) in excised spinach leaves proceeds after a distinct lag period compared with the incorporation into cytoplasmic rRNA species (mol.wt. 1.34×106 and 0.7×106). Incorporation of 32Pi into chloroplast RNA species of molecular weight 2.7×106, 1.2×106, 0.65×106 and 0.47×106 proceeds without such a time-lag. The kinetics of labelling of the individual RNA components is consistent with the rapidly labelled RNA species of molecular weight 1.2×106 and 0.65×106 being precursors to the more slowly labelled rRNA species of molecular weight 1.05×106 and 0.56×106 respectively. PMID:4723226

  7. 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.

  8. Chloroplast outer envelope protein CHUP1 is essential for chloroplast anchorage to the plasma membrane and chloroplast movement.

    PubMed

    Oikawa, Kazusato; Yamasato, Akihiro; Kong, Sam-Geun; Kasahara, Masahiro; Nakai, Masato; Takahashi, Fumio; Ogura, Yasunobu; Kagawa, Takatoshi; Wada, Masamitsu

    2008-10-01

    Chloroplasts change their intracellular distribution in response to light intensity. Previously, we isolated the chloroplast unusual positioning1 (chup1) mutant of Arabidopsis (Arabidopsis thaliana). This mutant is defective in normal chloroplast relocation movement and shows aggregation of chloroplasts at the bottom of palisade mesophyll cells. The isolated gene encodes a protein with an actin-binding motif. Here, we used biochemical analyses to determine the subcellular localization of full-length CHUP1 on the chloroplast outer envelope. A CHUP1-green fluorescent protein (GFP) fusion, which was detected at the outermost part of mesophyll cell chloroplasts, complemented the chup1 phenotype, but GFP-CHUP1, which was localized mainly in the cytosol, did not. Overexpression of the N-terminal hydrophobic region (NtHR) of CHUP1 fused with GFP (NtHR-GFP) induced a chup1-like phenotype, indicating a dominant-negative effect on chloroplast relocation movement. A similar pattern was found in chloroplast OUTER ENVELOPE PROTEIN7 (OEP7)-GFP transformants, and a protein containing OEP7 in place of NtHR complemented the mutant phenotype. Physiological analyses of transgenic Arabidopsis plants expressing truncated CHUP1 in a chup1 mutant background and cytoskeletal inhibitor experiments showed that the coiled-coil region of CHUP1 anchors chloroplasts firmly on the plasma membrane, consistent with the localization of coiled-coil GFP on the plasma membrane. Thus, CHUP1 localization on chloroplasts, with the N terminus inserted into the chloroplast outer envelope and the C terminus facing the cytosol, is essential for CHUP1 function, and the coiled-coil region of CHUP1 prevents chloroplast aggregation and participates in chloroplast relocation movement.

  9. Photosynthesis of root chloroplasts developed in Arabidopsis lines overexpressing GOLDEN2-LIKE transcription factors.

    PubMed

    Kobayashi, Koichi; Sasaki, Daichi; Noguchi, Ko; Fujinuma, Daiki; Komatsu, Hirohisa; Kobayashi, Masami; Sato, Mayuko; Toyooka, Kiminori; Sugimoto, Keiko; Niyogi, Krishna K; Wada, Hajime; Masuda, Tatsuru

    2013-08-01

    In plants, genes involved in photosynthesis are encoded separately in nuclei and plastids, and tight cooperation between these two genomes is therefore required for the development of functional chloroplasts. Golden2-like (GLK) transcription factors are involved in chloroplast development, directly targeting photosynthesis-associated nuclear genes for up-regulation. Although overexpression of GLKs leads to chloroplast development in non-photosynthetic organs, the mechanisms of coordination between the nuclear gene expression influenced by GLKs and the photosynthetic processes inside chloroplasts are largely unknown. To elucidate the impact of GLK-induced expression of photosynthesis-associated nuclear genes on the construction of photosynthetic systems, chloroplast morphology and photosynthetic characteristics in greenish roots of Arabidopsis thaliana lines overexpressing GLKs were compared with those in wild-type roots and leaves. Overexpression of GLKs caused up-regulation of not only their direct targets but also non-target nuclear and plastid genes, leading to global induction of chloroplast biogenesis in the root. Large antennae relative to reaction centers were observed in wild-type roots and were further enhanced by GLK overexpression due to the increased expression of target genes associated with peripheral light-harvesting antennae. Photochemical efficiency was lower in the root chloroplasts than in leaf chloroplasts, suggesting that the imbalance in the photosynthetic machinery decreases the efficiency of light utilization in root chloroplasts. Despite the low photochemical efficiency, root photosynthesis contributed to carbon assimilation in Arabidopsis. Moreover, GLK overexpression increased CO₂ fixation and promoted phototrophic performance of the root, showing the potential of root photosynthesis to improve effective carbon utilization in plants.

  10. Functional characterization of the chloroplast ferric chelate oxidoreductase enzyme.

    PubMed

    Solti, Adám; Müller, Brigitta; Czech, Viktória; Sárvári, Éva; Fodor, Ferenc

    2014-05-01

    Iron (Fe) has an essential role in the biosynthesis of chlorophylls and redox cofactors, and thus chloroplast iron uptake is a process of special importance. The chloroplast ferric chelate oxidoreductase (cFRO) has a crucial role in this process but it is poorly characterized. To study the localization and mechanism of action of cFRO, sugar beet (Beta vulgaris cv Orbis) chloroplast envelope fractions were isolated by gradient ultracentrifugation, and their purity was tested by western blotting against different marker proteins. The ferric chelate reductase (FCR) activity of envelope fractions was studied in the presence of NAD(P)H (reductants) and FAD coenzymes. Reduction of Fe(III)-ethylenediaminetetraacetic acid was monitored spectrophotometrically by the Fe(II)-bathophenanthroline disulfonate complex formation. FCR activity, that is production of free Fe(II) for Fe uptake, showed biphasic saturation kinetics, and was clearly associated only to chloroplast inner envelope (cIE) vesicles. The reaction rate was > 2.5 times higher with NADPH than with NADH, which indicates the natural coenzyme preference of cFRO activity and its dependence on photosynthesis. FCR activity of cIE vesicles isolated from Fe-deficient plants also showed clear biphasic kinetics, where the KM of the low affinity component was elevated, and thus this component was down-regulated.

  11. Darkness affects differentially the expression of plastid-encoded genes and delays the senescence-induced down-regulation of chloroplast transcription in cotyledons of Cucurbita pepo L. (Zucchini).

    PubMed

    Mishev, Kiril; Dimitrova, Anna; Ananiev, Evguéni D

    2011-01-01

    In contrast to differentiated leaves, the regulatory mechanisms of chloroplast gene expression in darkened cotyledons have not been elucidated. Although some results have been reported indicating accelerated senescence in Arabidopsis upon reillumination, the capacity of cotyledons to recover after dark stress remains unclear. We analysed the effect of two-days dark stress, applied locally or at the whole-plant level, on plastid gene expression in zucchini cotyledons. Our results showed that in the dark the overall chloroplast transcription rate was much more inhibited than the nuclear run-on transcription. While the activities of the plastid-encoded RNA polymerase (PEP) and nuclear RNA polymerase II were strongly reduced, the activities of the nuclear-encoded plastid RNA polymerase (NEP) and nuclear RNA polymerase I were less affected. During recovery upon reillumination, chloroplast transcription in the cotyledons was strongly stimulated (3-fold) compared with the naturally senescing controls, suggesting delayed senescence. Northern blot and dot blot analyses of the expression of key chloroplast-encoded photosynthetic genes showed that in contrast to psbA, which remained almost unaffected, both the transcription rate and mRNA content of psaB and rbcL were substantially decreased.

  12. Abscisic acid and blue light signaling pathways in chloroplast movements in Arabidopsis mesophyll.

    PubMed

    Eckstein, Aleksandra; Krzeszowiec, Weronika; Banaś, Agnieszka Katarzyna; Janowiak, Franciszek; Gabryś, Halina

    2016-01-01

    Abscisic acid (ABA) and phototropins act antagonistically to control stomatal movements. Here, we investigated the role of ABA in phototropin-directed chloroplast movements in mesophyll cells of Arabidopsis thaliana. We analyzed the expression of phototropins at mRNA and protein level under the influence of ABA. PHOT1 mRNA level was decreased by ABA in the dark while it was insensitive to ABA in light. PHOT2 mRNA level was independent of the hormone treatment. The levels of phototropin proteins were down-regulated by ABA, both in darkness and light. No impact of exogenous ABA on amplitudes and kinetics of chloroplast movements was detected. Chloroplast responses in wild type Arabidopsis and three mutants, abi4, abi2 (abscisic acid insensitive4, 2) and aba1 (abscisic acid1), were measured to account for endogenous ABA signaling. The chloroplast responses were slightly reduced in abi2 and aba1 mutants in strong light. To further investigate the effect, abi2 and aba1 mutants were supplemented with exogenous ABA. In the aba1 mutant, the reaction was rescued but in abi2 it was unaffected. Our results show that ABA is not directly involved in phototropin-controlled chloroplast responses in mature leaves of Arabidopsis. However, the disturbance of ABA biosynthesis and signaling in mutants affects some elements of the chloroplast movement mechanism. In line with its role as a stress hormone, ABA appears to enhance plant sensitivity to light and promote the chloroplast avoidance response.

  13. Albino Leaf 2 is involved in the splicing of chloroplast group I and II introns in rice

    PubMed Central

    Liu, Changhong; Zhu, Haitao; Xing, Yi; Tan, Jianjie; Chen, Xionghui; Zhang, Jianjun; Peng, Haifeng; Xie, Qingjun; Zhang, Zemin

    2016-01-01

    Chloroplasts play an essential role in plant growth and development through manipulating photosynthesis and the production of hormones and metabolites. Although many genes or regulators involved in chloroplast biogenesis and development have been isolated and characterized, identification of novel components is still lacking. We isolated a rice (Oryza sativa) mutant, termed albino leaf 2 (al2), using genetic screening. Phenotypic analysis revealed that the al2 mutation caused obvious albino leaves at the early developmental stage, eventually leading to al2 seedling death. Electron microscopy investigations indicated that the chloroplast structure was disrupted in the al2 mutants at an early developmental stage and subsequently resulted in the breakdown of the entire chloroplast. Molecular cloning illustrated that AL2 encodes a chloroplast group IIA intron splicing facilitator (CRS1) in rice, which was confirmed by a genetic complementation experiment. Moreover, our results demonstrated that AL2 was constitutively expressed in various tissues, including green and non-green tissues. Interestingly, we found that the expression levels of a subset of chloroplast genes that contain group IIA and IIB introns were significantly reduced in the al2 mutant compared to that in the wild type, suggesting that AL2 is a functional CRS1 in rice. Differing from the orthologous CRS1 in maize and Arabidopsis that only regulates splicing of the chloroplast group II intron, our results demonstrated that the AL2 gene is also likely to be involved in the splicing of the chloroplast group I intron. They also showed that disruption of AL2 results in the altered expression of chloroplast-associated genes, including chlorophyll biosynthetic genes, plastid-encoded polymerases and nuclear-encoded chloroplast genes. Taken together, these findings shed new light on the function of nuclear-encoded chloroplast group I and II intron splicing factors in rice. PMID:27543605

  14. Preparation and proteomic analysis of chloroplast ribosomes.

    PubMed

    Yamaguchi, Kenichi

    2011-01-01

    Proteomics of chloroplast ribosomes in spinach and Chlamydomonas revealed unique protein composition and structures of plastid ribosomes. These studies have suggested the presence of some ribosomal proteins unique to plastid ribosomes which may be involved in plastid-unique translation regulation. Considering the strong background of genetic analysis and molecular biology in Arabidopsis, the in-depth proteomic characterization of Arabidopsis plastid ribosomes would facilitate further understanding of plastid translation in higher plants. Here, I describe simple and rapid methods for the preparation of plastid ribosomes from Chlamydomonas and Arabidopsis using sucrose gradients. I also describe purity criteria and methods for yield estimation of the purified plastid ribosomes and subunits, methods for the preparation of plastid ribosomal proteins, as well as the identification of some Arabidopsis plastid ribosomal proteins by matrix-assisted laser desorption/ionization mass spectrometry.

  15. Chloroplast avoidance movement reduces photodamage in plants.

    PubMed

    Kasahara, Masahiro; Kagawa, Takatoshi; Oikawa, Kazusato; Suetsugu, Noriyuki; Miyao, Mitsue; Wada, Masamitsu

    When plants are exposed to light levels higher than those required for photosynthesis, reactive oxygen species are generated in the chloroplasts and cause photodamage. This can occur even under natural growth conditions. To mitigate photodamage, plants have developed several protective mechanisms. One is chloroplast avoidance movement, in which chloroplasts move from the cell surface to the side walls of cells under high light conditions, although experimental support is still awaited. Here, using different classes of mutant defective in chloroplast avoidance movement, we show that these mutants are more susceptible to damage in high light than wild-type plants. Damage of the photosynthetic apparatus and subsequent bleaching of leaf colour and necrosis occur faster under high light conditions in the mutants than in wild-type plants. We conclude that chloroplast avoidance movement actually decreases the amount of light absorption by chloroplasts, and might therefore be important to the survival of plants under natural growth conditions.

  16. Chloroplast actin filaments organize meshwork on the photorelocated chloroplasts in the moss Physcomitrella patens.

    PubMed

    Yamashita, Hiroko; Sato, Yoshikatsu; Kanegae, Takeshi; Kagawa, Takatoshi; Wada, Masamitsu; Kadota, Akeo

    2011-02-01

    Cytoskeleton dynamics during phototropin-dependent chloroplast photorelocation movement was analyzed in protonemal cells of actin- and microtubule-visualized lines of Physcomitrella patens expressing GFP- or tdTomato-talin and GFP-tubulin. Using newly developed epi- and trans-microbeam irradiation systems that permit fluorescence observation of the cell under blue microbeam irradiation inducing chloroplast relocation, it was revealed that meshwork of actin filaments formed at the chloroplast-accumulating area both in the avoidance and accumulation movements. The structure disappeared soon when blue microbeam was turned off, and it was not induced under red microbeam irradiation that did not evoke chloroplast relocation movement. In contrast, no apparent change in microtubule organization was detected during the movements. The actin meshwork was composed of short actin filaments distinct from the cytoplasmic long actin cables and was present between the chloroplasts and plasma membrane. The short actin filaments emerged from around the chloroplast periphery towards the center of chloroplast. Showing highly dynamic behavior, the chloroplast actin filaments (cp-actin filaments) were rapidly organized into meshwork on the chloroplast surface facing plasma membrane. The actin filament configuration on a chloroplast led to the formation of actin meshwork area in the cell as the chloroplasts arrived at and occupied the area. After establishment of the meshwork, cp-actin filaments were still highly dynamic, showing appearance, disappearance, severing and bundling of filaments. These results indicate that the cp-actin filaments have significant roles in the chloroplast movement and positioning in the cell.

  17. Chloroplast research in the genomic age.

    PubMed

    Leister, Dario

    2003-01-01

    Chloroplast research takes significant advantage of genomics and genome sequencing, and a new picture is emerging of how the chloroplast functions and communicates with other cellular compartments. In terms of evolution, it is now known that only a fraction of the many proteins of cyanobacterial origin were rerouted to higher plant plastids. Reverse genetics and novel mutant screens are providing a growing catalogue of chloroplast protein-function relationships, and the characterization of plastid-to-nucleus signalling mutants reveals cell-organelle interactions. Recent advances in transcriptomics and proteomics of the chloroplast make this organelle one of the best understood of all plant cell compartments.

  18. Decoding the role of phosphoinositides in phototropin signaling involved in chloroplast movements.

    PubMed

    Aggarwal, Chhavi; Labuz, Justyna; Gabryś, Halina

    2013-08-01

    In angiosperms, light-dependent chloroplast movements are exclusively mediated by UVA/blue light receptors - phototropins. The two photoreceptors of Arabidopsis thaliana, phot1 and phot2, have overlapping roles in the control of these movements. Experiments performed in different plant species point to the participation of phosphoinositides in blue light-controlled chloroplast relocations. Here, we report a summary of recent findings presenting the involvement of phosphatidylinositol 4,5-bisphosphate as well as phosphatidylinositol 3- and 4-phosphates in weak blue light-mediated (accumulation) and strong blue light-mediated (avoidance) responses of chloroplasts. The blue light-activated alterations in phosphoinositide concentration are partly responsible for cytosolic Ca (2+) changes. Ca (2+) influx from apoplast does not seem to be involved in the mechanism of movement responses. In summary, interplay between phosphoinositides and intracellular Ca (2+) regulates chloroplast redistribution in response to blue light in higher plants.

  19. The chloroplast outer membrane protein CHUP1 interacts with actin and profilin.

    PubMed

    Schmidt von Braun, Serena; Schleiff, Enrico

    2008-04-01

    Chloroplasts accumulate in response to low light, whereas high light induces an actin-dependent avoidance movement. This is a long known process, but its molecular base is barely understood. Only recently first components of the blue light perceiving signal cascade initiating this process were described. Among these, a protein was identified by the analysis of a deletion mutant in the corresponding gene resulting in a chloroplast unusual positioning phenotype. The protein was termed CHUP1 and initial results suggested chloroplast localization. We demonstrate that the protein is indeed exclusively and directly targeted to the chloroplast surface. The analysis of the deletion mutant of CHUP1 using microarray analysis shows an influence on the expression of genes found to be up-regulated, but not on genes found to be down-regulated upon high light exposure in wild-type. Analyzing a putative role of CHUP1 as a linker between chloroplasts and the cytoskeleton, we demonstrate an interaction with actin, which is independent on the filamentation status of actin. Moreover, binding of CHUP1 to profilin -- an actin modifying protein -- could be shown and an enhancing effect of CHUP1 on the interaction of profilin to actin is demonstrated. Therefore, a role of CHUP1 in bridging chloroplasts to actin filaments and a regulatory function in actin polymerization can be discussed.

  20. 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

  1. External Ca(2+) is essential for chloroplast movement induced by mechanical stimulation but not by light stimulation.

    PubMed

    Sato, Y; Wada, M; Kadota, A

    2001-10-01

    In the fern Adiantum capillus-veneris, chloroplast movement is induced by mechanical stimulation as well as by light stimulation. Directional movement of both types depends on an actin-based motile system. To investigate the physiological relationship between mechanical and light signaling in the regulation of chloroplast movement, we examined the mechano-response of chloroplasts whose motility had been already restricted after photo-relocation. Chloroplast mechano-avoidance movement was induced under all of the photo-relocation conditions tested, indicating that mechano-specific signals generated by mechanical stimulation dominate over the light signals and reactivate the motility of chloroplasts. When the effects of external Ca(2+) on the induction of mechano- and light responses were examined, strikingly different requirements of external Ca(2+) were found for each. In medium without Ca(2+), the mechano-response was suppressed but no effects were observed on photo-response. Mechano-relocation movement of chloroplasts was inhibited by 100 microM lanthanum (La(3+)), a plasma membrane calcium channel blocker, and by 10 microM gadolinium (Gd(3+)), a stretch-activated channel blocker. However, the same concentrations of these drugs did not affect the photo-relocation movement at all. These results suggest that the influx of external Ca(2+) is crucial for the early signaling step of chloroplast mechano-relocation but not for that of photo-relocation. This is the first report showing the separation of signaling pathways in mechano- and photo-relocation of chloroplasts.

  2. A large population of small chloroplasts in tobacco leaf cells allows more effective chloroplast movement than a few enlarged chloroplasts.

    PubMed

    Jeong, Won Joong; Park, Youn-Il; Suh, KyeHong; Raven, John A; Yoo, Ook Joon; Liu, Jang Ryol

    2002-05-01

    We generated transgenic tobacco (Nicotiana tabacum cv Xanthi) plants that contained only one to three enlarged chloroplasts per leaf mesophyll cell by introducing NtFtsZ1-2, a cDNA for plastid division. These plants were used to investigate the advantages of having a large population of small chloroplasts rather than a few enlarged chloroplasts in a leaf mesophyll cell. Despite the similarities in photosynthetic components and ultrastructure of photosynthetic machinery between wild-type and transgenic plants, the overall growth of transgenic plants under low- and high-light conditions was retarded. In wild-type plants, the chloroplasts moved toward the face position under low light and toward the profile position under high-light conditions. However, chloroplast rearrangement in transgenic plants in response to light conditions was not evident. In addition, transgenic plant leaves showed greatly diminished changes in leaf transmittance values under both light conditions, indicating that chloroplast rearrangement was severely retarded. Therefore, under low-light conditions the incomplete face position of the enlarged chloroplasts results in decreased absorbance of light energy. This, in turn, reduces plant growth. Under high-light conditions, the amount of absorbed light exceeds the photosynthetic utilization capacity due to the incomplete profile position of the enlarged chloroplasts, resulting in photodamage to the photosynthetic machinery, and decreased growth. The presence of a large number of small and/or rapidly moving chloroplasts in the cells of higher land plants permits more effective chloroplast phototaxis and, hence, allows more efficient utilization of low-incident photon flux densities. The photosynthetic apparatus is, consequently, protected from damage under high-incident photon flux densities.

  3. Molecular basis of chloroplast photorelocation movement.

    PubMed

    Kong, Sam-Geun; Wada, Masamitsu

    2016-03-01

    Chloroplast photorelocation movement is an essential physiological response for sessile plant survival and the optimization of photosynthetic ability. Simple but effective experiments on the physiological, cell biological and molecular genetic aspects have been widely used to investigate the signaling components of chloroplast photorelocation movement in Arabidopsis for the past few decades. Although recent knowledge on chloroplast photorelocation movement has led us to a deeper understanding of its physiological and molecular basis, the biochemical roles of the downstream factors remain largely unknown. In this review, we briefly summarize recent advances regarding chloroplast photorelocation movement and propose that a new high-resolution approach is necessary to investigate the molecular mechanism underlying actin-based chloroplast photorelocation movement.

  4. Ribonuclease J is required for chloroplast and embryo development in Arabidopsis.

    PubMed

    Chen, Hongyu; Zou, Wenxuan; Zhao, Jie

    2015-04-01

    Chloroplasts perform many essential metabolic functions and their proper development is critically important in embryogenesis. However, little is known about how chloroplasts function in embryogenesis and more relevant components need to be characterized. In this study, we show that Arabidopsis Ribonuclease J (RNase J) is required for chloroplast and embryo development. Mutation of AtRNJ led to albino ovules containing aborted embryos; the morphological development of rnj embryos was disturbed after the globular stage. Observation of ultrastructures indicated that these aborted embryos may result from impaired chloroplast development. Furthermore, by analyzing the molecular markers of cell fate decisions (STM, FIL, ML1, SCR, and WOX5) in rnj embryos, we found that this impairment of chloroplast development may lead to aberrant embryo patterning along the apical-basal axis, indicating that AtRNJ is important in initiating and maintaining the organization of shoot apical meristems (SAMs), cotyledons, and hypocotyls. Moreover, the transport and response of auxin in rnj embryos was found to be disrupted, suggesting that AtRNJ may be involved in auxin-mediated pathways during embryogenesis. Therefore, we speculate that RNJ plays a vital role in embryo morphogenesis and apical-basal pattern formation by regulating chloroplast development.

  5. Functional Differentiation of Bundle Sheath and Mesophyll Maize Chloroplasts Determined by Comparative ProteomicsW⃞

    PubMed Central

    Majeran, Wojciech; Cai, Yang; Sun, Qi; van Wijk, Klaas J.

    2005-01-01

    Chloroplasts of maize (Zea mays) leaves differentiate into specific bundle sheath (BS) and mesophyll (M) types to accommodate C4 photosynthesis. Consequences for other plastid functions are not well understood but are addressed here through a quantitative comparative proteome analysis of purified M and BS chloroplast stroma. Three independent techniques were used, including cleavable stable isotope coded affinity tags. Enzymes involved in lipid biosynthesis, nitrogen import, and tetrapyrrole and isoprenoid biosynthesis are preferentially located in the M chloroplasts. By contrast, enzymes involved in starch synthesis and sulfur import preferentially accumulate in BS chloroplasts. The different soluble antioxidative systems, in particular peroxiredoxins, accumulate at higher levels in M chloroplasts. We also observed differential accumulation of proteins involved in expression of plastid-encoded proteins (e.g., EF-Tu, EF-G, and mRNA binding proteins) and thylakoid formation (VIPP1), whereas others were equally distributed. Enzymes related to the C4 shuttle, the carboxylation and regeneration phase of the Calvin cycle, and several regulators (e.g., CP12) distributed as expected. However, enzymes involved in triose phosphate reduction and triose phosphate isomerase are primarily located in the M chloroplasts, indicating that the M-localized triose phosphate shuttle should be viewed as part of the BS-localized Calvin cycle, rather than a parallel pathway. PMID:16243905

  6. A geminivirus betasatellite damages the structural and functional integrity of chloroplasts leading to symptom formation and inhibition of photosynthesis

    PubMed Central

    Bhattacharyya, Dhriti; Gnanasekaran, Prabu; Kumar, Reddy Kishore; Kushwaha, Nirbhay Kumar; Sharma, Veerendra Kumar; Yusuf, Mohd Aslam; Chakraborty, Supriya

    2015-01-01

    Geminivirus infection often causes severe vein clearing symptoms in hosts. Recently a betasatellite has emerged as a key regulator of symptom induction. To understand the host–betasatellite interactions in the process of symptom development, a systematic study was carried out involving symptoms induced by a betasatellite associated with radish leaf curl disease (RaLCB) in Nicotiana benthamiana. It has been found that βC1 protein localized to chloroplasts of host cells, and RaLCB lacking βC1, which failed to produce symptoms, had no effect on chloroplast ultrastructure. Vein flecking induced by transiently expressed βC1 was associated with chloroplast ultrastructure. In addition, the betasatellite down-regulates expression of genes involved in chlorophyll biosynthesis as well as genes involved in chloroplast development and plastid translocation. Interestingly, the expression of key host genes involved in chlorophyll degradation remains unaffected. Betasatellite infection drastically reduced the numbers of active reaction centres and the plastoquinol pool size in leaves exhibiting vein clearing symptoms. Betasatellite-mediated impediments at different stages of chloroplast functionality affect the photosynthetic efficiency of N. benthamiana. To the best of the authors’ knowledge, this is the first evidence of a chloroplast-targeting protein encoded by a DNA virus which induces vein clearing and structurally and functionally damages chloroplasts in plants. PMID:26113193

  7. Evolutionary rewiring: a modified prokaryotic gene-regulatory pathway in chloroplasts

    PubMed Central

    Puthiyaveetil, Sujith; Ibrahim, Iskander M.; Allen, John F.

    2013-01-01

    Photosynthetic electron transport regulates chloroplast gene transcription through the action of a bacterial-type sensor kinase known as chloroplast sensor kinase (CSK). CSK represses photosystem I (PS I) gene transcription in PS I light and thus initiates photosystem stoichiometry adjustment. In cyanobacteria and in non-green algae, CSK homologues co-exist with their response regulator partners in canonical bacterial two-component systems. In green algae and plants, however, no response regulator partner of CSK is found. Yeast two-hybrid analysis has revealed interaction of CSK with sigma factor 1 (SIG1) of chloroplast RNA polymerase. Here we present further evidence for the interaction between CSK and SIG1. We also show that CSK interacts with quinone. Arabidopsis SIG1 becomes phosphorylated in PS I light, which then specifically represses transcription of PS I genes. In view of the identical signalling properties of CSK and SIG1 and of their interactions, we suggest that CSK is a SIG1 kinase. We propose that the selective repression of PS I genes arises from the operation of a gene-regulatory phosphoswitch in SIG1. The CSK-SIG1 system represents a novel, rewired chloroplast-signalling pathway created by evolutionary tinkering. This regulatory system supports a proposal for the selection pressure behind the evolutionary stasis of chloroplast genes. PMID:23754813

  8. Evolutionary rewiring: a modified prokaryotic gene-regulatory pathway in chloroplasts.

    PubMed

    Puthiyaveetil, Sujith; Ibrahim, Iskander M; Allen, John F

    2013-07-19

    Photosynthetic electron transport regulates chloroplast gene transcription through the action of a bacterial-type sensor kinase known as chloroplast sensor kinase (CSK). CSK represses photosystem I (PS I) gene transcription in PS I light and thus initiates photosystem stoichiometry adjustment. In cyanobacteria and in non-green algae, CSK homologues co-exist with their response regulator partners in canonical bacterial two-component systems. In green algae and plants, however, no response regulator partner of CSK is found. Yeast two-hybrid analysis has revealed interaction of CSK with sigma factor 1 (SIG1) of chloroplast RNA polymerase. Here we present further evidence for the interaction between CSK and SIG1. We also show that CSK interacts with quinone. Arabidopsis SIG1 becomes phosphorylated in PS I light, which then specifically represses transcription of PS I genes. In view of the identical signalling properties of CSK and SIG1 and of their interactions, we suggest that CSK is a SIG1 kinase. We propose that the selective repression of PS I genes arises from the operation of a gene-regulatory phosphoswitch in SIG1. The CSK-SIG1 system represents a novel, rewired chloroplast-signalling pathway created by evolutionary tinkering. This regulatory system supports a proposal for the selection pressure behind the evolutionary stasis of chloroplast genes.

  9. Analysis of chloroplast movement and relocation in Arabidopsis.

    PubMed

    Wada, Masamitsu; Kong, Sam-Geun

    2011-01-01

    Chloroplast photorelocation movement is essential for the sessile plant survival and plays a role for efficient photosynthesis and avoiding photodamage of chloroplasts. There are several ways to observe or detect chloroplast movement directly or indirectly. Here, techniques for the induction of chloroplast movement and how to detect the responses, as well as various points of attention and advice for the experiments, are described.

  10. Rapid isolation of intact chloroplasts from spinach leaves.

    PubMed

    Joly, David; Carpentier, Robert

    2011-01-01

    In this chapter, a rapid method to isolate intact chloroplasts from spinach leaves is described. Intact chloroplasts are isolated using two short centrifugation steps and avoiding the use of percoll gradient. Intactness of chloroplast is evaluated by the inability of potassium ferricyanide to enter inside the chloroplasts and to act as an electron acceptor for photosystem II.

  11. Plastidic Isoprenoid Synthesis during Chloroplast Development 1

    PubMed Central

    Heintze, Adolf; Görlach, Jörn; Leuschner, Carola; Hoppe, Petra; Hagelstein, Petra; Schulze-Siebert, Detlef; Schultz, Gernot

    1990-01-01

    The chloroplast isoprenoid synthesis of very young leaves is supplied by the plastidic CO2 → pyruvate → acetyl-coenzyme A (C3 → C2) metabolism (D Schulze-Siebert, G Schultz [1987] Plant Physiol 84: 1233-1237) and occurs via the plastidic mevalonate pathway. The plastidic C3 → C2 metabolism and/or plastidic mevalonate pathway of barley (Hordeum vulgare L.) seedlings changes from maximal activity at the leaf base (containing developing chloroplasts with incomplete thylakoid stacking but a considerable rate of photosynthetic CO2-fixation) almost to ineffectivity at the leaf tip (containing mature chloroplasts with maximal photosynthetic activity). The ability to import isopentenyl diphosphate from the extraplastidic space gradually increases to substitute for the loss of endogenous intermediate supply for chloroplast isoprenoid synthesis (change from autonomic to division-of-labor stage). Fatty acid synthesis from NaH14CO3 decreases in the same manner as shown for leaf sections and chloroplasts isolated from these. Evidence has been obtained for a drastic decrease of pyruvate decarboxylase-dehydrogenase activity during chloroplast development compared with other anabolic chloroplast pathways (synthesis of aromatic amino acid and branched chain amino acids). The noncompetition of pyruvate and acetate in isotopic dilution studies indicates that both a pyruvate-derived and an acetate-derived compound are simultaneously needed to form introductory intermediates of the mevalonate pathway, presumably acetoacetyl-coenzyme A. PMID:16667567

  12. In vitro comparative kinetic analysis of the chloroplast Toc GTPases.

    PubMed

    Reddick, L Evan; Vaughn, Michael D; Wright, Sarah J; Campbell, Ian M; Bruce, Barry D

    2007-04-13

    A unique aspect of protein transport into plastids is the coordinate involvement of two GTPases in the translocon of the outer chloroplast membrane (Toc). There are two subfamilies in Arabidopsis, the small GTPases (Toc33 and Toc34) and the large acidic GTPases (Toc90, Toc120, Toc132, and Toc159). In chloroplasts, Toc34 and Toc159 are implicated in precursor binding, yet mechanistic details are poorly understood. How the GTPase cycle is modulated by precursor binding is complex and in need of careful dissection. To this end, we have developed novel in vitro assays to quantitate nucleotide binding and hydrolysis of the Toc GTPases. Here we present the first systematic kinetic characterization of four Toc GTPases (cytosolic domains of atToc33, atToc34, psToc34, and the GTPase domain of atToc159) to permit their direct comparison. We report the KM, Vmax, and Ea values for GTP hydrolysis and the Kd value for nucleotide binding for each protein. We demonstrate that GTP hydrolysis by psToc34 is stimulated by chloroplast transit peptides; however, this activity is not stimulated by homodimerization and is abolished by the R133A mutation. Furthermore, we show peptide stimulation of hydrolytic rates are not because of accelerated nucleotide exchange, indicating that transit peptides function as GTPase-activating proteins and not guanine nucleotide exchange factors in modulating the activity of psToc34. Finally, by using the psToc34 structure, we have developed molecular models for atToc33, atToc34, and atToc159G. By combining these models with the measured enzymatic properties of the Toc GTPases, we provide new insights of how the chloroplast protein import cycle may be regulated.

  13. Nitrogen control of chloroplast development and differentiation

    SciTech Connect

    Schmidt, G.W.

    1991-12-01

    The growth and development of plants and photosynthetic microorganisms is commonly limited by the availability of nitrogen. Our work concerns understanding the mechanisms by which plants and algae that are subjected to nitrogen deprivation alter the composition of photosynthetic membranes and enzymes involved in photosynthetic carbon metabolism. Toward these ends, we study biosynthetic and gene expression processes in the unicellular green alga Chlamydomonas reinhardtii which is grown in an ammonium-limited continuous culture system. We have found that the expression of nuclear genes, including those encoding for light-harvesting proteins, are severely repressed in nitrogen-limited cells whereas, in general, chloroplast protein synthesis is attenuated primarily at the level of mRNA translation. Conversely, nitrogen deprivation appears to lead to enhanced synthesis of enzymes that are involved in starch and storage lipid deposition. In addition, as a possible means by which photosynthetic electron transport activities and ATP synthesis is sustained during chronic periods of nitrogen deprivation, thylakoid membranes become enriched with components for chlororespiration. Characterization of the chlororespiratory electron transport constituents, including cytochrome complexes and NAD(P)H dehydrogenase is a major current effort. Also, we are striving to isolate the genes encoding chlororespiration proteins toward determining how they and others that are strongly responsive to nutrient availability are regulated.

  14. Nitrogen control of chloroplast differentiation. Final report

    SciTech Connect

    Schmidt, G.W.

    1998-05-01

    This project was directed toward understanding at the physiological, biochemical and molecular levels of how photosynthetic organisms adapt to long-term nitrogen-deficiency conditions is quite incomplete even though limitation of this nutrient is the most commonly restricts plant growth and development. For our work on this problem, the unicellular green alga, Chlamydomonas reinhardtii, was grown in continuous cultures in which steady-state levels of nitrogen can be precisely controlled. N-limited cells exhibit the classical symptoms of deficiency of this nutrient, chlorosis and slow growth rates, and respond to nitrogen provision by rapid greening and chloroplast differentiation. We have addressed three aspects of this problem: (1) the regulation of pigment synthesis; (2) control of expression of nuclear genes encoding photosynthetic proteins; (3) changes in metabolic and electron transport pathways that enable sustained CO{sub 2} fixation even though they cannot be readily converted into amino and nucleic acids. For the last, principle components are: (a) enhanced mitochondrial respiratory activity intimately associated with photosynthates, and (b) the occurrence in thylakoids of a supplemental electron transport pathway that facilitates reduction of the plastoquinone pool. Together, these distinguishing features of N-limited cells are likely to enable cell survival, especially under conditions of high irradiance stress.

  15. Export of carbon from chloroplasts at night

    SciTech Connect

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

    1998-12-01

    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 at 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.

  16. Identification of Putative Substrates of SEC2, a Chloroplast Inner Envelope Translocase1[OPEN

    PubMed Central

    2017-01-01

    Most chloroplast proteins are synthesized in the cytosol and imported into chloroplasts. Many imported proteins are further targeted to the thylakoid membrane and lumen by the SEC1, TAT, or SRP/ALB3 translocases. Others are targeted to the inner chloroplast envelope membrane by undescribed translocases. Recently, a second SEC system (SEC2) consisting of SCY2, SECE2, and SECA2 was found in the chloroplast envelope. Null mutants of SCY2 in Arabidopsis (Arabidopsis thaliana) exhibit a severe embryo-lethal phenotype. To investigate the function of the SEC2 system in plants, we used inducible RNA interference to knock down SCY2 in Arabidopsis. Seedlings cultured with inducer were chlorotic with aberrant chloroplasts and undeveloped thylakoids, indicating an essential role for SCY2 in chloroplast biogenesis beyond embryo development. In SCY2 down-regulated seedlings, several thylakoid membrane proteins, including SCY1, ALB3, and TATC, and inner envelope membrane proteins, including TIC40, TIC110, and FTSH12, were reduced substantially, suggesting that they may be SEC2 substrates. Additional insight was achieved by the in vitro reconstitution of protein integration into chloroplast membranes. The results show that SCY1 and ALB3 target directly to the thylakoid membrane and are likely independent of SEC2. FTSH12 was integrated into the envelope membrane in a coupled import-integration reaction that was impaired by the SECA inhibitor sodium azide. The stromal intermediate of TIC40 integrated into the envelope in a reaction that was largely inhibited when antibodies against epitope-tagged SCY2 or SECE2 were applied. These data demonstrate that the SEC2 translocase likely integrates a subset of inner envelope membrane proteins, such as FTSH12 and TIC40. PMID:28213560

  17. Activation of polyphenol oxidase of chloroplasts.

    PubMed

    Tolbert, N E

    1973-02-01

    Polyphenol oxidase of leaves is located mainly in chloroplasts isolated by differential or sucrose density gradient centrifugation. This activity is part of the lamellar structure that is not lost on repeated washing of the plastids. The oxidase activity was stable during prolonged storage of the particles at 4 C or -18 C. The Km (dihydroxyphenylalanine) for spinach leaf polyphenol oxidase was 7 mm by a spectrophotometric assay and 2 mm by the manometric assay. Polyphenol oxidase activity in the leaf peroxisomal fraction, after isopycnic centrifugation on a linear sucrose gradient, did not coincide with the peroxisomal enzymes but was attributed to proplastids at nearly the same specific density.Plants were grouped by the latency properties for polyphenol oxidase in their isolated chloroplasts. In a group including spinach, Swiss chard, and beet leaves the plastids immediately after preparation from fresh leaves required a small amount of light for maximal rates of oxidation of dihydroxyphenylalanine. Polyphenol oxidase activity in the dark or light increased many fold during aging of these chloroplasts for 1 to 5 days. Soluble polyphenol oxidase of the cytoplasm was not so stimulated. Chloroplasts prepared from stored leaves were also much more active than from fresh leaves. Maximum rates of dihydroxyphenylalanine oxidation were 2 to 6 mmoles x mg(-1) chlorophyll x hr(-1). Equal stimulation of latent polyphenol oxidase in fresh or aged chloroplasts in this group was obtained by either light, an aged trypsin digest, 3-(4-chlorophenyl)-1, 1-dimethylurea, or antimycin A. A variety of other treatments did not activate or had little effect on the oxidase, including various peptides, salts, detergents, and other proteolytic enzymes.Activation of latent polyphenol oxidase in spinach chloroplasts by trypsin amounted to as much as 30-fold. The trypsin activation occurred even after the trypsin had been treated with 10% trichloroacetic acid, 1.0 n HCl or boiled for 30

  18. 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.

  19. 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.

  20. Roles of chloroplast RNA polymerase sigma factors in chloroplast development and stress response in higher plants.

    PubMed

    Kanamaru, Kengo; Tanaka, Kan

    2004-11-01

    Chloroplast transcription in higher plants is performed by two types of RNA polymerases, plastid-encoded RNA polymerase (PEP) and nuclear-encoded RNA polymerase (NEP). PEP is a eubacteria-type multisubunit enzyme whose catalytic core subunits are encoded by the chloroplast genome, whereas NEP is the nuclear encoded T7 phage-type single subunit enzyme. PEP is critical for the biogenesis and maintenance of chloroplasts, and is finely tuned by the nuclear encoded sigma subunits. Of the six Arabidopsis sigma subunits, SIG2 is involved in the transcription of several chloroplast tRNA genes, including trnE encoding tRNA-Glu. SIG2 possibly couples translation and pigment synthesis in chloroplasts. On the other hand, SIG5 is induced by various stresses and contributes to repair of damaged photosystem II (PSII) through transcription of the psbD and psbC genes. Thus target genes and the physiological role of each sigma subunit are becoming clearer.

  1. Novel protein-protein interaction family proteins involved in chloroplast movement response.

    PubMed

    Kodama, Yutaka; Suetsugu, Noriyuki; Wada, Masamitsu

    2011-04-01

    To optimize photosynthetic activity, chloroplasts change their intracellular location in response to ambient light conditions; chloroplasts move toward low intensity light to maximize light capture, and away from high intensity light to avoid photodamage. Although several proteins have been reported to be involved in the chloroplast photorelocation movement response, any physical interaction among them was not found so far. We recently found a physical interaction between two plant-specific coiled-coil proteins, WEB1 (Weak Chloroplast Movement under Blue Light 1) and PMI2 (Plastid Movement Impaired 2), that were identified to regulate chloroplast movement velocity. Since the both coiled-coil regions of WEB1 and PMI2 were classified into an uncharacterized protein family having DUF827 (DUF: Domain of Unknown Function) domain, it was the first report that DUF827 proteins could mediate protein-protein interaction. In this mini-review article, we discuss regarding molecular function of WEB1 and PMI2, and also define a novel protein family composed of WEB1, PMI2 and WEB1/PMI2-like proteins for protein-protein interaction in land plants.

  2. ChloroSeq, an Optimized Chloroplast RNA-Seq Bioinformatic Pipeline, Reveals Remodeling of the Organellar Transcriptome Under Heat Stress

    PubMed Central

    Castandet, Benoît; Hotto, Amber M.; Strickler, Susan R.; Stern, David B.

    2016-01-01

    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 Arabidopsis 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. ChloroSeq thus represents a unique method for streamlining RNA-Seq data interpretation of the chloroplast transcriptome and its regulators. PMID:27402360

  3. Comparative proteomics of chloroplasts envelopes from bundle sheath and mesophyll chloroplasts reveals novel membrane proteins with a possible role in c4-related metabolite fluxes and development.

    PubMed

    Manandhar-Shrestha, K; Tamot, B; Pratt, E P S; Saitie, S; Bräutigam, A; Weber, A P M; Hoffmann-Benning, Susanne

    2013-01-01

    As the world population grows, our need for food increases drastically. Limited amounts of arable land lead to a competition between food and fuel crops, while changes in the global climate may impact future crop yields. Thus, a second "green revolution" will need a better understanding of the processes essential for plant growth and development. One approach toward the solution of this problem is to better understand regulatory and transport processes in C4 plants. C4 plants display an up to 10-fold higher apparent CO2 assimilation and higher yields while maintaining high water use efficiency. This requires differential regulation of mesophyll (M) and bundle sheath (BS) chloroplast development as well as higher metabolic fluxes of photosynthetic intermediates between cells and particularly across chloroplast envelopes. While previous analyses of overall chloroplast membranes have yielded significant insight, our comparative proteomics approach using enriched BS and M chloroplast envelopes of Zea mays allowed us to identify 37 proteins of unknown function that have not been seen in these earlier studies. We identified 280 proteins, 84% of which are known/predicted to be present in chloroplasts. Seventy-four percent have a known or predicted membrane association. Twenty-one membrane proteins were 2-15 times more abundant in BS cells, while 36 of the proteins were more abundant in M chloroplast envelopes. These proteins could represent additional candidates of proteins essential for development or metabolite transport processes in C4 plants. RT-PCR confirmed differential expression of 13 candidate genes. Chloroplast association for seven proteins was confirmed using YFP/GFP labeling. Gene expression of four putative transporters was examined throughout the leaf and during the greening of leaves. Genes for a PIC-like protein and an ER-AP-like protein show an early transient increase in gene expression during the transition to light. In addition, PIC gene expression is

  4. PLASTID MOVEMENT IMPAIRED1 and PLASTID MOVEMENT IMPAIRED1-RELATED1 Mediate Photorelocation Movements of Both Chloroplasts and Nuclei.

    PubMed

    Suetsugu, Noriyuki; Higa, Takeshi; Kong, Sam-Geun; Wada, Masamitsu

    2015-10-01

    Organelle movement and positioning play important roles in fundamental cellular activities and adaptive responses to environmental stress in plants. To optimize photosynthetic light utilization, chloroplasts move toward weak blue light (the accumulation response) and escape from strong blue light (the avoidance response). Nuclei also move in response to strong blue light by utilizing the light-induced movement of attached plastids in leaf cells. Blue light receptor phototropins and several factors for chloroplast photorelocation movement have been identified through molecular genetic analysis of Arabidopsis (Arabidopsis thaliana). PLASTID MOVEMENT IMPAIRED1 (PMI1) is a plant-specific C2-domain protein that is required for efficient chloroplast photorelocation movement. There are two PLASTID MOVEMENT IMPAIRED1-RELATED (PMIR) genes, PMIR1 and PMIR2, in the Arabidopsis genome. However, the mechanism in which PMI1 regulates chloroplast and nuclear photorelocation movements and the involvement of PMIR1 and PMIR2 in these organelle movements remained unknown. Here, we analyzed chloroplast and nuclear photorelocation movements in mutant lines of PMI1, PMIR1, and PMIR2. In mesophyll cells, the pmi1 single mutant showed severe defects in both chloroplast and nuclear photorelocation movements resulting from the impaired regulation of chloroplast-actin filaments. In pavement cells, pmi1 mutant plants were partially defective in both plastid and nuclear photorelocation movements, but pmi1pmir1 and pmi1pmir1pmir2 mutant lines lacked the blue light-induced movement responses of plastids and nuclei completely. These results indicated that PMI1 is essential for chloroplast and nuclear photorelocation movements in mesophyll cells and that both PMI1 and PMIR1 are indispensable for photorelocation movements of plastids and thus, nuclei in pavement cells.

  5. Glycolate transporter of the pea chloroplast envelope

    SciTech Connect

    Howitz, K.T.

    1985-01-01

    The discovery of a glycolate transporter in the pea (Pisum sativum) chloroplast envelope is described. Several novel silicone oil centrifugation methods were developed to resolve the initial rate kinetics of (/sup 14/C)glycolate transport by isolated, intact pea chloroplasts. Chloroplast glycolate transport was found to be carrier mediated. Transport rates saturated with increasing glycolate concentration. N-Ethylmaleimide (NEM) pretreatment of chloroplasts inhibited transport, an inhibition prevented by glycolate. Glycolate distributed across the envelope in a way which equalized stromal and medium glycolic acid concentrations, limiting possible transport mechanisms to facilitated glycolic acid diffusion, proton symport or hydroxyl antiport. The effects of stomal and medium pH's on the K/sub m/ and V/sub max/ fit the predictions of mobile carrier kinetic models of hydroxyl antiport or proton symport (H/sup +/ binds first). The carrier mediated transport was fast enough to be consistent with in vivo rates of photorespiration. The 2-hydroxymonocarboxylates, glycerate, lactate and glyoxylate are competitive inhibitors of chloroplast glycolate uptake. Glyoxylate, D-lactate and D-glycerate cause glycolate counterflow, indicating that they are also substrates of the glycolate carrier. This finding was confirmed for D-glycerate by studies on glycolate effects on (1-/sup 14/C)D-glycerate transport.

  6. Isolation and Suborganellar Fractionation of Arabidopsis Chloroplasts.

    PubMed

    Flores-Pérez, Úrsula; Jarvis, Paul

    2017-01-01

    Chloroplasts are structurally complex organelles containing ~2000-3000 proteins. They are delimited by a double membrane system or envelope, have an inner aqueous compartment called the stroma, and possess a second internal membrane system called the thylakoids. Thus, determining the suborganellar location of a chloroplast protein is vital to understanding or verifying its function. One way in which protein localization can be addressed is through fractionation. Here we present two rapid and simple methods that may be applied sequentially on the same day: (a) The isolation of intact chloroplasts from Arabidopsis thaliana plants that may be used directly (e.g., for functional studies such as protein import analysis), or for further processing as follows; (b) separation of isolated chloroplasts into three suborganellar fractions (envelope membranes, a soluble fraction containing stromal proteins, and the thylakoids). These methods are routinely used in our laboratory, and they provide a good yield of isolated chloroplasts and suborganellar fractions that can be used for various downstream applications.

  7. Vectorial photocurrents and photoconductivity in metalized chloroplasts

    SciTech Connect

    Greenbaum, E. )

    1990-08-09

    A novel photobiophysical phenomenon was observed in isolated spinach chloroplasts that were metalized by precipitating colloidal platinum onto the surface of the thylakoid membranes. A two-point irradiation and detection system was constructed in which a continuous-beam helium-neon laser ({lambda} = 632.8 nm) was used to irradiate the platinized chloroplasts at varying perpendicular distances (Figure 1) from a single linear platinum electrode in pressure contact with the platinized chloroplasts. No external voltage bias was applied to the system. The key objective of the experiments reported in this report was to measure the relative photoconductivity of the chloroplast-metal composite matrix. Unlike conventional photosynthetic electrochemical cells, in which irradiated chloroplasts are in close proximity to an electrode or linked to the electrode by an electrode-active mediator, the flow of photocurrent was through the biocomposite material. A sustained steady-state vectorial flow of current in the plane of the entrapped composite from the point of laser irradiation to the wire electrode was measured.

  8. Betaine synthesis in chenopods: localization in chloroplasts

    SciTech Connect

    Hanson, A.D.; May A.M.; Grumet, R.; Bode, J.; Jamieson, G.C.; Rhodes, D.

    1985-06-01

    Plants from several families (Chenopodiaceae, Gramineae, Compositae) accumulate betaine (glycine betaine) in response to salt or water stress via the pathway: choline betainal (betaine aldehyde) betaine. Betaine accumulation is probably a metabolic adaptation to stress. Intact protoplasts from leaves of spinach (Spinacia oleracea) oxidized ( UC)choline to betainal and betaine, as did protoplast lysates. Upon differential centrifugation, the ( UC)choline-oxidizing activity of lysates sedimented with chloroplasts. Chloroplasts purified from protoplast lysates by a Percoll cushion procedure retained strong ( UC)choline-oxidizing activity, although the proportion of the intermediate, ( UC)betainal, in the reaction products was usually higher than for protoplasts. Isolated chloroplasts also readily oxidized ( UC)betainal to betaine. Light increased the oxidation of both ( UC)choline and ( UC)betainal by isolated chloroplasts. Similar results were obtained with another chenopod (Beta vulgaris) but not with pea (Pisum sativum), a species that accumulates no betaine. The chloroplast site for betaine synthesis in chenopods contrasts with the mitochondrial site in mammals.

  9. 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.

  10. Non-invasive, whole-plant imaging of chloroplast movement and chlorophyll fluorescence reveals photosynthetic phenotypes independent of chloroplast photorelocation defects in chloroplast division mutants.

    PubMed

    Dutta, Siddhartha; Cruz, Jeffrey A; Jiao, Yuhua; Chen, Jin; Kramer, David M; Osteryoung, Katherine W

    2015-10-01

    Leaf chloroplast movement is thought to optimize light capture and to minimize photodamage. To better understand the impact of chloroplast movement on photosynthesis, we developed a technique based on the imaging of reflectance from leaf surfaces that enables continuous, high-sensitivity, non-invasive measurements of chloroplast movement in multiple intact plants under white actinic light. We validated the method by measuring photorelocation responses in Arabidopsis chloroplast division mutants with drastically enlarged chloroplasts, and in phototropin mutants with impaired photorelocation but normal chloroplast morphology, under different light regimes. Additionally, we expanded our platform to permit simultaneous image-based measurements of chlorophyll fluorescence and chloroplast movement. We show that chloroplast division mutants with enlarged, less-mobile chloroplasts exhibit greater photosystem II photodamage than is observed in the wild type, particularly under fluctuating high levels of light. Comparison between division mutants and the severe photorelocation mutant phot1-5 phot2-1 showed that these effects are not entirely attributable to diminished photorelocation responses, as previously hypothesized, implying that altered chloroplast morphology affects other photosynthetic processes. Our dual-imaging platform also allowed us to develop a straightforward approach to correct non-photochemical quenching (NPQ) calculations for interference from chloroplast movement. This correction method should be generally useful when fluorescence and reflectance are measured in the same experiments. The corrected data indicate that the energy-dependent (qE) and photoinhibitory (qI) components of NPQ contribute differentially to the NPQ phenotypes of the chloroplast division and photorelocation mutants. This imaging technology thus provides a platform for analyzing the contributions of chloroplast movement, chloroplast morphology and other phenotypic attributes to the

  11. Choice of tracks, microtubules and/or actin filaments for chloroplast photo-movement is differentially controlled by phytochrome and a blue light receptor.

    PubMed

    Sato, Y; Wada, M; Kadota, A

    2001-01-01

    Light induced chloroplast movement has been studied as a model system for photoreception and actin microfilament (MF)-based intracellular motilities in plants. Chloroplast photo-accumulation and -avoidance movement is mediated by phytochrome as well as blue light (BL) receptor in the moss Physcomitrella patens. Here we report the discovery of an involvement of a microtubule (MT)-based system in addition to an MF-based system in photorelocation of chloroplasts in this moss. In the dark, MTs provided tracks for rapid movement of chloroplasts in a longitudinal direction and MFs contributed the tracks for slow movement in any direction. We found that phytochrome responses utilized only the MT-based system, while BL responses had an alternative way of moving, either along MTs or MFs. MT-based systems were mediated by both photoreceptors, but chloroplasts showed movements with different velocity and pattern between them. No apparent difference in the behavior of chloroplast movement between the accumulation and avoidance movement was detected in phytochrome responses or BL responses, except for the direction of the movement. The results presented here demonstrate that chloroplasts use both MTs and MFs for motility and that phytochrome and a BL receptor control directional photo-movement of chloroplasts through the differential regulation of these motile systems.

  12. 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

  13. 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.

  14. Protein methylation reactions in intact pea chloroplasts

    SciTech Connect

    Niemi, K.J. )

    1989-04-01

    Post-translational protein methylation was investigated in Pisum sativum chloroplasts. Intact pea chloroplasts were incubated with ({sup 3}H-methyl)-S-adenosylmethionine under various conditions. The chloroplasts were then separated into stromal and thylakoid fractions and analyzed for radioactivity transferred to protein. Light enhanced the magnitude of labeling in both fractions. One thylakoid polypeptide with an apparent molecular mass of 43 kDa was labeled only in the light. Several other thylakoid and stromal proteins were labeled in both light and dark-labeling conditions. Both base-labile methylation, carboxy-methylesters and base-stable groups, N-methylations were found. Further characterization of the methyl-transfer reactions will be presented.

  15. 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

  16. The chloroplast genome exists in multimeric forms

    SciTech Connect

    Deng, Xingwang; Wing, R.A.; Gruissem, W. )

    1989-06-01

    Chloroplast DNA conformation was analyzed by pulse-field gel electrophoresis. The authors found that spinach leaf chloroplast DNA molecules exist in at least four distinct forms with the apparent molecular weights of monomer, dimer, trimer, and tetramer. Two-dimensional gel analysis of DNA after UV nicking and in the presence of ethidium bromide indicates that they are not isomers that differ in superhelical density. DNA gyrase decatenation analysis demonstrates that the majority of the DNA molecules are oligomers rather than catenanes. The relative amounts of monomer, dimer, trimer, and tetramer forms, quantitated by molecular hybridization, are 1, 1/3, 1/9, and 1/27, respectively, and do not change during leaf maturation. The possible mechanisms of chloroplast DNA oligomer formation are discussed.

  17. Chloroplasts can move in any direction to avoid strong light.

    PubMed

    Tsuboi, Hidenori; Wada, Masamitsu

    2011-01-01

    Chloroplasts migrate in response to different light intensities. Under weak light, chloroplasts gather at an illuminated area to maximize light absorption and photosynthesis rates (the accumulation response). In contrast, chloroplasts escape from strong light to avoid photodamage (the avoidance response). Photoreceptors involved in these phenomena have been identified in Arabidopsis thaliana and Adiantum capillus-veneris. Chloroplast behavior has been studied in detail during the accumulation response, but not for the avoidance response. Hence, we analyzed the chloroplast avoidance response in detail using dark-adapted Adiantum capillus-veneris gametophyte cells and partial cell irradiation with a microbeam of blue light. Chloroplasts escaped from an irradiated spot. Both duration of this response and the distance of the migrated chloroplasts were proportional to the total fluence irradiated. The speed of movement during the avoidance response was dependent on the fluence rate, but the speed of the accumulation response towards the microbeam from cell periphery was constant irrespective of fluence rate. When a chloroplast was only partially irradiated with a strong microbeam, it moved away towards the non-irradiated region within a few minutes. During this avoidance response two additional microbeam irradiations were applied to different locus of the same chloroplast. Under these conditions the chloroplast changed the moving direction after a lag time of a few minutes without rolling. Taken together, these findings indicate that chloroplasts can move in any direction and never have an intrinsic polarity. Similar phenomenon was observed in chloroplasts of Arabidopsis thaliana palisade cells.

  18. Integration of Phot1, Phot2, and PhyB signalling in light-induced chloroplast movements.

    PubMed

    Luesse, Darron R; DeBlasio, Stacy L; Hangarter, Roger P

    2010-10-01

    In Arabidopsis thaliana, chloroplasts move towards the periclinal cell walls upon exposure to low blue light intensities and to anticlinal walls under high light. The regulation of these chloroplast movements involves members of both the phototropin and phytochrome families of photoreceptors. Examination of fluence-rate response dependencies in phot1 and phot2 mutants revealed that although both photoreceptors are capable of inducing chloroplast accumulation under low-light conditions, the signals from these photoreceptors appear to be antagonistic. Chloroplast movements in wild-type plants were intermediate between those of the single phot mutants, consistent with each operating through separate signalling cascades. Mutants in phot2 showed transient chloroplast avoidance responses upon exposure to intense blue light, and slow but sustained chloroplast avoidance under intense white light, indicating that in the absence of phot2, phot1 is capable of generating both a low and a high-light response signal. Mutations in phytochrome B (phyB) caused an enhanced avoidance response at intermediate and high light intensities. Examination of phyB, phot1phyB, and phot2phyB mutants indicated that this enhancement is caused by PhyB inhibition of the high-light avoidance response in wild-type plants. In addition, our results suggest that the inhibition by PhyB is not exclusive to either of the phot1 or phot2 signalling pathways.

  19. Chloroplast RNA-Binding Protein RBD1 Promotes Chilling Tolerance through 23S rRNA Processing in Arabidopsis

    PubMed Central

    Yang, Leiyun; Yang, Fen; Wang, Yi; Zhu, Jian-Kang; Hua, Jian

    2016-01-01

    Plants have varying abilities to tolerate chilling (low but not freezing temperatures), and it is largely unknown how plants such as Arabidopsis thaliana achieve chilling tolerance. Here, we describe a genome-wide screen for genes important for chilling tolerance by their putative knockout mutants in Arabidopsis thaliana. Out of 11,000 T-DNA insertion mutant lines representing half of the genome, 54 lines associated with disruption of 49 genes had a drastic chilling sensitive phenotype. Sixteen of these genes encode proteins with chloroplast localization, suggesting a critical role of chloroplast function in chilling tolerance. Study of one of these proteins RBD1 with an RNA binding domain further reveals the importance of chloroplast translation in chilling tolerance. RBD1 is expressed in the green tissues and is localized in the chloroplast nucleoid. It binds directly to 23S rRNA and the binding is stronger under chilling than at normal growth temperatures. The rbd1 mutants are defective in generating mature 23S rRNAs and deficient in chloroplast protein synthesis especially under chilling conditions. Together, our study identifies RBD1 as a regulator of 23S rRNA processing and reveals the importance of chloroplast function especially protein translation in chilling tolerance. PMID:27138552

  20. Two interacting coiled-coil proteins, WEB1 and PMI2, maintain the chloroplast photorelocation movement velocity in Arabidopsis.

    PubMed

    Kodama, Yutaka; Suetsugu, Noriyuki; Kong, Sam-Geun; Wada, Masamitsu

    2010-11-09

    Chloroplasts move toward weak light (accumulation response) and away from strong light (avoidance response). The fast and accurate movement of chloroplasts in response to ambient light conditions is essential for efficient photosynthesis and photodamage prevention in chloroplasts. Here, we report that two Arabidopsis mutants, weak chloroplast movement under blue light 1 (web1) and web2, are defective in both the avoidance and the accumulation responses. Map-based cloning revealed that both genes encode coiled-coil proteins and that WEB2 is identical to the plastid movement impaired 2 (PMI2) gene. The velocities of chloroplast movement in web1 and pmi2 were approximately threefold lower than that in the wild type. Defects in the avoidance response of web1 and pmi2 were suppressed by mutation of the J-domain protein required for chloroplast accumulation response 1 (JAC1) gene, which is essential for the accumulation response; these results indicate that WEB1 and PMI2 play a role in suppressing JAC1 under strong light conditions. A yeast two-hybrid analysis and a nuclear recruitment assay identified a physical interaction between WEB1 and PMI2, and transient expression analysis of CFP-WEB1 and YFP-PMI2 revealed that they colocalized in the cytosol. Bimolecular fluorescence complementation analysis confirmed the interaction of these proteins in the cytosol. Blue light-induced changes in short chloroplast actin filaments (cp-actin filaments) were impaired in both web1 and pmi2. Our findings suggest that a cytosolic WEB1-PMI2 complex maintains the velocity of chloroplast photorelocation movement via cp-actin filament regulation.

  1. Photosynthesis-dependent but neochrome1-independent light positioning of chloroplasts and nuclei in the fern Adiantum capillus-veneris.

    PubMed

    Sugiyama, Yuka; Kadota, Akeo

    2011-03-01

    Chloroplasts change their positions in the cell depending on the light conditions. In the dark, chloroplasts in fern prothallia locate along the anticlinal wall (dark position). However, chloroplasts become relocated to the periclinal wall (light position) when the light shines perpendicularly to the prothallia. Red light is effective in inducing this relocation in Adiantum capillus-veneris, and neochrome1 (neo1) has been identified as the red light receptor regulating this movement. Nevertheless, we found here that chloroplasts in neo1 mutants still become relocated from the dark position to the light position under red light. We tested four neo1 mutant alleles (neo1-1, neo1-2, neo1-3, and neo1-4), and all of them showed the red-light-induced chloroplast relocation. Furthermore, chloroplast light positioning under red light occurred also in Pteris vittata, another fern species naturally lacking the neo1-dependent phenomenon. The light positioning of chloroplasts occurred independently of the direction of red light, a response different to that of the neo1-dependent movement. Photosynthesis inhibitors 3-(3,4 dichlorophenyl)-1,1-dimethylurea or 2,5-dibromo-3-isopropyl-6-methyl-p-benzoquinone blocked this movement. Addition of sucrose (Suc) or glucose to the culture medium induced migration of the chloroplasts to the periclinal wall in darkness. Furthermore, Suc could override the effects of 3-(3,4 dichlorophenyl)-1,1-dimethylurea. Interestingly, the same light positioning was evident for nuclei under red light in the neo1 mutant. The nuclear light positioning was also induced in darkness with the addition of Suc or glucose. These results indicate that photosynthesis-dependent nondirectional movement contributes to the light positioning of these organelles in addition to the neo1-dependent directional movement toward light.

  2. Effects of exogenous spermine on chlorophyll fluorescence, antioxidant system and ultrastructure of chloroplasts in Cucumis sativus L. under salt stress.

    PubMed

    Shu, Sheng; Yuan, Ling-Yun; Guo, Shi-Rong; Sun, Jin; Yuan, Ying-Hui

    2013-02-01

    The effects of exogenous spermine (Spm) on plant growth, chlorophyll fluorescence, ultrastructure and anti-oxidative metabolism of chloroplasts were investigated in Cucumis sativus L. under NaCl stress. Salt stress significantly reduced plant growth, chlorophylls content and F(v)/F(m). These changes could be alleviated by foliar spraying with Spm. Salt stress caused an increase in malondialdehyde (MDA) content and superoxide anion [Formula: see text] generation rate in chloroplasts. Application of Spm significantly increased activities of superoxidase dismutase (SOD, EC 1.15.1.1), peroxidase (POD, EC 1.11.1.7), and ascorbate peroxidase (APX, EC 1.11.1.11) which decreased the levels of [Formula: see text] and MDA in the salt-stressed chloroplasts. Salt stress decreased the activities of dehydroascorbate reductase (DHAR, EC 1.8.5.1) and glutathione reductase (GR, EC 1.6.4.2) in the chloroplasts and reduced the contents of dehydroascorbate (DAsA) and glutathione (GSH), but increased monodehydroascorbate reductase (MDAR, EC 1.6.5.4) activity. On the other hand, Spm significantly increased the activities of antioxidant enzymes and levels of antioxidants in the salt-stressed chloroplasts. Further analysis of the ultrastructure of chloroplasts indicated that salinity induced destruction of the chloroplast envelope and increased the number of plastoglobuli with aberrations in thylakoid membranes. However, Spm application to salt-stressed plant leaves counteracted the adverse effects of salinity on the structure of the photosynthetic apparatus. These results suggest that Spm alleviates salt-induced oxidative stress through regulating antioxidant systems in chloroplasts of cucumber seedlings, which is associated with an improvement of the photochemical efficiency of PSII.

  3. The Arabidopsis Chloroplast Stromal N-Terminome: Complexities of Amino-Terminal Protein Maturation and Stability.

    PubMed

    Rowland, Elden; Kim, Jitae; Bhuiyan, Nazmul H; van Wijk, Klaas J

    2015-11-01

    Protein amino (N) termini are prone to modifications and are major determinants of protein stability in bacteria, eukaryotes, and perhaps also in chloroplasts. Most chloroplast proteins undergo N-terminal maturation, but this is poorly understood due to insufficient experimental information. Consequently, N termini of mature chloroplast proteins cannot be accurately predicted. This motivated an extensive characterization of chloroplast protein N termini in Arabidopsis (Arabidopsis thaliana) using terminal amine isotopic labeling of substrates and mass spectrometry, generating nearly 14,000 tandem mass spectrometry spectra matching to protein N termini. Many nucleus-encoded plastid proteins accumulated with two or three different N termini; we evaluated the significance of these different proteoforms. Alanine, valine, threonine (often in N-α-acetylated form), and serine were by far the most observed N-terminal residues, even after normalization for their frequency in the plastid proteome, while other residues were absent or highly underrepresented. Plastid-encoded proteins showed a comparable distribution of N-terminal residues, but with a higher frequency of methionine. Infrequent residues (e.g. isoleucine, arginine, cysteine, proline, aspartate, and glutamate) were observed for several abundant proteins (e.g. heat shock proteins 70 and 90, Rubisco large subunit, and ferredoxin-glutamate synthase), likely reflecting functional regulation through their N termini. In contrast, the thylakoid lumenal proteome showed a wide diversity of N-terminal residues, including those typically associated with instability (aspartate, glutamate, leucine, and phenylalanine). We propose that, after cleavage of the chloroplast transit peptide by stromal processing peptidase, additional processing by unidentified peptidases occurs to avoid unstable or otherwise unfavorable N-terminal residues. The possibility of a chloroplast N-end rule is discussed.

  4. Sensing and signaling of oxidative stress in chloroplasts by inactivation of the SAL1 phosphoadenosine phosphatase

    PubMed Central

    Mabbitt, Peter D.; Phua, Su Yin; Mueller, Jonathan W.; Nisar, Nazia; Gigolashvili, Tamara; Stroeher, Elke; Grassl, Julia; Arlt, Wiebke; Estavillo, Gonzalo M.; Jackson, Colin J.; Pogson, Barry J.

    2016-01-01

    Intracellular signaling during oxidative stress is complex, with organelle-to-nucleus retrograde communication pathways ill-defined or incomplete. Here we identify the 3′-phosphoadenosine 5′-phosphate (PAP) phosphatase SAL1 as a previously unidentified and conserved oxidative stress sensor in plant chloroplasts. Arabidopsis thaliana SAL1 (AtSAL1) senses changes in photosynthetic redox poise, hydrogen peroxide, and superoxide concentrations in chloroplasts via redox regulatory mechanisms. AtSAL1 phosphatase activity is suppressed by dimerization, intramolecular disulfide formation, and glutathionylation, allowing accumulation of its substrate, PAP, a chloroplast stress retrograde signal that regulates expression of plastid redox associated nuclear genes (PRANGs). This redox regulation of SAL1 for activation of chloroplast signaling is conserved in the plant kingdom, and the plant protein has evolved enhanced redox sensitivity compared with its yeast ortholog. Our results indicate that in addition to sulfur metabolism, SAL1 orthologs have evolved secondary functions in oxidative stress sensing in the plant kingdom. PMID:27432987

  5. Role of membrane glycerolipids in photosynthesis, thylakoid biogenesis and chloroplast development.

    PubMed

    Kobayashi, Koichi

    2016-07-01

    The lipid bilayer of the thylakoid membrane in plant chloroplasts and cyanobacterial cells is predominantly composed of four unique lipid classes; monogalactosyldiacylglycerol (MGDG), digalactosyldiacylglycerol (DGDG), sulfoquinovosyldiacylglycerol (SQDG) and phosphatidylglycerol (PG). MGDG and DGDG are uncharged galactolipids that constitute the bulk of thylakoid membrane lipids and provide a lipid bilayer matrix for photosynthetic complexes as the main constituents. The glycolipid SQDG and phospholipid PG are anionic lipids with a negative charge on their head groups. SQDG and PG substitute for each other to maintain the amount of total anionic lipids in the thylakoid membrane, with PG having indispensable functions in photosynthesis. In addition to biochemical studies, extensive analyses of mutants deficient in thylakoid lipids have revealed important roles of these lipids in photosynthesis and thylakoid membrane biogenesis. Moreover, recent studies of Arabidopsis thaliana suggest that thylakoid lipid biosynthesis triggers the expression of photosynthesis-associated genes in both the nucleus and plastids and activates the formation of photosynthetic machineries and chloroplast development. Meanwhile, galactolipid biosynthesis is regulated in response to chloroplast functionality and lipid metabolism at transcriptional and post-translational levels. This review summarizes the roles of thylakoid lipids with their biosynthetic pathways in plants and discusses the coordinated regulation of thylakoid lipid biosynthesis with the development of photosynthetic machinery during chloroplast biogenesis.

  6. Velocity of chloroplast avoidance movement is fluence rate dependent.

    PubMed

    Kagawa, Takatoshi; Wada, Masamitsu

    2004-06-01

    In Arabidopsis leaves, chloroplast movement is fluence rate dependent. At optimal, lower light fluences, chloroplasts accumulate at the cell surface to maximize photosynthetic potential. Under high fluence rates, chloroplasts avoid incident light to escape photodamage. In this paper, we examine the phenomenon of chloroplast avoidance movement in greater detail and demonstrate a proportional relationship between fluence rate and the velocity of chloroplast avoidance. In addition we show that the amount of light-activated phototropin2, the photoreceptor for the avoidance response, likely plays a role in this phenomenon, as heterozygous mutant plants show a reduced avoidance velocity compared to that of homozygous wild type plants.

  7. Chloroplast DNA codes for transfer RNA.

    PubMed Central

    McCrea, J M; Hershberger, C L

    1976-01-01

    Transfer RNA's were isolated from Euglena gracilis. Chloroplast cistrons for tRNA were quantitated by hybridizing tRNA to ct DNA. Species of tRNA hybridizing to ct DNA were partially purified by hybridization-chromatography. The tRNA's hybridizing to ct DNA and nuclear DNA appear to be different. Total cellular tRNA was hybridized to ct DNA to an equivalent of approximately 25 cistrons. The total cellular tRNA was also separated into 2 fractions by chromatography on dihydroxyboryl substituted amino ethyl cellulose. Fraction I hybridized to both nuclear and ct DNA. Hybridizations to ct DNA indicated approximately 18 cistrons. Fraction II-tRNA hybridized only to ct DNA, saturating at a level of approximately 7 cistrons. The tRNA from isolated chloroplasts hybridized to both chloroplast and nuclear DNA. The level of hybridization to ct DNA indicated approximately 18 cistrons. Fraction II-type tRNA could not be detected in the isolated chloroplasts. PMID:823529

  8. Ascorbate-independent carotenoid de-epoxidation in intact spinach chloroplasts.

    PubMed

    Sokolove, P M; Marsho, T V

    1976-05-14

    Slow (greater 1 s) light-induced absorbance changes in the 475-5300 nm spectral region were examined in Type A chloroplasts from spinach. The most prominent absorption change occurred at 505 nm. The difference spectrum for this light-induced increase, its absence in osmotically shocked chloroplasts and restoration by ascorbate, and its sensitivity to dithiothreitol indicate that the absorption change is due to carotenoid de-epoxidatiion. The reaction in intact chloroplasts is characterized by its independence of exogenous ascorbate and a rate constant 3- to 8-fold higher than that reported previously for chloroplasts supplemented with ascorbate. The relevance of carotenoid de-epoxidation to other photosynthetic processes was examined by comparing their sensitivities to dithiothreitol. Levels of dithiothreitol that eliminate the 505 nm shift are without effect on saturated rates of CO2 fixation and do not appreciably inhibit fluorescence quenching. We conclude that carotenoid de-epoxidation is not directly involved in the reactions of photosynthesis or in the regulation of excitation allocation between the photosystems.

  9. Arabidopsis MDA1, a nuclear-encoded protein, functions in chloroplast development and abiotic stress responses.

    PubMed

    Robles, Pedro; Micol, José Luis; Quesada, Víctor

    2012-01-01

    Most chloroplast and mitochondrial proteins are encoded by nuclear genes, whose functions remain largely unknown because mutant alleles are lacking. A reverse genetics screen for mutations affecting the mitochondrial transcription termination factor (mTERF) family in Arabidopsis thaliana allowed us to identify 75 lines carrying T-DNA insertions. Two of them were homozygous for insertions in the At4g14605 gene, which we dubbed MDA1 (MTERF DEFECTIVE IN Arabidopsis1). The mda1 mutants exhibited altered chloroplast morphology and plant growth, and reduced pigmentation of cotyledons, leaves, stems and sepals. The mda1 mutations enhanced salt and osmotic stress tolerance and altered sugar responses during seedling establishment, possibly as a result of reduced ABA sensitivity. Loss of MDA1 function caused up-regulation of the RpoTp/SCA3 nuclear gene encoding a plastid RNA polymerase and modified the steady-state levels of chloroplast gene transcripts. Double mutant analyses indicated that MDA1 and the previously described mTERF genes SOLDAT10 and RUG2 act in different pathways. Our findings reveal a new role for mTERF proteins in the response to abiotic stress, probably through perturbed ABA retrograde signalling resulting from a disruption in chloroplast homeostasis.

  10. 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.

  11. Brassinosteroid-induced CO{sub 2} assimilation is associated with increased stability of redox-sensitive photosynthetic enzymes in the chloroplasts in cucumber plants

    SciTech Connect

    Jiang, Yu Ping; Cheng, Fei; Zhou, Yan Hong; Xia, Xiao Jian; Mao, Wei Hua; Shi, Kai; Chen, Zhi Xiang; Yu, Jing Quan

    2012-09-28

    Highlights: Black-Right-Pointing-Pointer Activity of certain Calvin cycle enzymes and CO{sub 2} assimilation are induced by BRs. Black-Right-Pointing-Pointer BRs upregulate the activity of the ascorbate-glutathione cycle in the chloroplasts. Black-Right-Pointing-Pointer BRs increase the chloroplast thiol reduction state. Black-Right-Pointing-Pointer A BR-induced reducing environment increases the stability of photosynthetic enzymes. -- Abstract: Brassinosteroids (BRs) play important roles in plant growth, development, photosynthesis and stress tolerance; however, the mechanism underlying BR-enhanced photosynthesis is currently unclear. Here, we provide evidence that an increase in the BR level increased the quantum yield of PSII, activities of Rubisco activase (RCA) and fructose-1,6-bisphosphatase (FBPase), and CO{sub 2} assimilation. BRs upregulated the transcript levels of genes and activity of enzymes involved in the ascorbate-glutathione cycle in the chloroplasts, leading to an increased ratio of reduced (GSH) to oxidized (GSSG) glutathione in the chloroplasts. An increased GSH/GSSG ratio protected RCA from proteolytic digestion and increased the stability of redox-sensitive enzymes in the chloroplasts. These results strongly suggest that BRs are capable of regulating the glutathione redox state in the chloroplasts through the activation of the ascorbate-glutathione cycle. The resulting increase in the chloroplast thiol reduction state promotes CO{sub 2} assimilation, at least in part, by enhancing the stability and activity of redox-sensitive photosynthetic enzymes through post-translational modifications.

  12. Direct Chloroplast Sequencing: Comparison of Sequencing Platforms and Analysis Tools for Whole Chloroplast Barcoding

    PubMed Central

    Brozynska, Marta; Furtado, Agnelo; Henry, Robert James

    2014-01-01

    Direct sequencing of total plant DNA using next generation sequencing technologies generates a whole chloroplast genome sequence that has the potential to provide a barcode for use in plant and food identification. Advances in DNA sequencing platforms may make this an attractive approach for routine plant identification. The HiSeq (Illumina) and Ion Torrent (Life Technology) sequencing platforms were used to sequence total DNA from rice to identify polymorphisms in the whole chloroplast genome sequence of a wild rice plant relative to cultivated rice (cv. Nipponbare). Consensus chloroplast sequences were produced by mapping sequence reads to the reference rice chloroplast genome or by de novo assembly and mapping of the resulting contigs to the reference sequence. A total of 122 polymorphisms (SNPs and indels) between the wild and cultivated rice chloroplasts were predicted by these different sequencing and analysis methods. Of these, a total of 102 polymorphisms including 90 SNPs were predicted by both platforms. Indels were more variable with different sequencing methods, with almost all discrepancies found in homopolymers. The Ion Torrent platform gave no apparent false SNP but was less reliable for indels. The methods should be suitable for routine barcoding using appropriate combinations of sequencing platform and data analysis. PMID:25329378

  13. Protein trafficking to the complex chloroplasts of Euglena.

    PubMed

    Vacula, Rostislav; Sláviková, Silvia; Schwartzbach, Steven D

    2007-01-01

    Proteins are delivered to Euglena chloroplasts using the secretory pathway. We describe analytical methods to study the intracellular trafficking of Euglena chloroplast proteins and a method to isolate preparative amounts of intact import competent chloroplasts for biochemical studies. Cells are pulse labeled with 35S-sulfate and chased with unlabeled sulfate allowing the trafficking and posttranslational processing of the labeled protein to be followed. Sucrose gradients are used to separate a 35S-labeled cell lysate into cytoplasmic, endoplasmic reticuum (ER), Golgi apparatus, chloroplast and mitochondrial fractions. Immunoprecipitation of each gradient fraction allows identification of the intracellular compartment containing a specific 35S-labeled protein at different times after synthesis delineating the trafficking pathway. Because sucrose gradients cannot be used to isolate preparative amounts of highly purified chloroplasts for biochemical characterization, a preparative high-yield procedure using Percoll gradients to isolate highly purified import competent chloroplasts is also presented.

  14. Chloroplast targeting factor AKR2 evolved from an ankyrin repeat domain coincidentally binds two chloroplast lipids

    PubMed Central

    Kim, Dae Heon; Park, Mi-Jeong; Gwon, Gwang Hyeon; Silkov, Antonina; Xu, Zheng-Yi; Yang, Eun Chan; Song, Seohyeon; Song, Kyungyoung; Kim, Younghyun; Yoon, Hwan Su; Honig, Barry; Cho, Wonhwa; Cho, Yunje; Hwang, Inhwan

    2014-01-01

    SUMMARY In organellogenesis of the chloroplast from endosymbiotic cyanobacterium, the establishment of protein targeting mechanisms to the chloroplast should have been pivotal. However, it is still mysterious how these mechanisms were established and how they work in plant cells. Here, we show that AKR2A, the cytosolic targeting factor for chloroplast outer membrane (COM) proteins, evolved from the ankyrin repeat domain (ARD) of the host cell by stepwise extensions of its N-terminal domain, and two lipids monogalactosyldiacylglycerol (MGDG) and phosphatidylglycerol (PG) of the endosymbiont were selected to function as the AKR2A receptor. Structural analysis, molecular modeling and mutational analysis of the ARD identified two adjacent sites for coincidental and synergistic binding of MGDG and PG. Based on these findings, we propose that the targeting mechanism of COM proteins was established using components from both the endosymbiont and host cell through a modification of the protein-protein interacting ARD into a lipid binding domain. PMID:25203210

  15. Mitochondria, Chloroplasts in Animal and Plant Cells: Significance of Conformational Matching.

    PubMed

    Stefano, George B; Snyder, Christopher; Kream, Richard M

    2015-07-17

    Many commonalities between chloroplasts and mitochondria exist, thereby suggesting a common origin via a bacterial ancestor capable of enhanced ATP-dependent energy production functionally linked to cellular respiration and photosynthesis. Accordingly, the molecular evolution/retention of the catalytic Qo quinol oxidation site of cytochrome b complexes as the tetrapeptide PEWY sequence functionally underlies the common retention of a chemiosmotic proton gradient mechanism for ATP synthesis in cellular respiration and photosynthesis. Furthermore, the dual regulatory targeting of mitochondrial and chloroplast gene expression by mitochondrial transcription termination factor (MTERF) proteins to promote optimal energy production and oxygen consumption further advances these evolutionary contentions. As a functional consequence of enhanced oxygen utilization and production, significant levels of reactive oxygen species (ROS) may be generated within mitochondria and chloroplasts, which may effectively compromise cellular energy production following prolonged stress/inflammationary conditions. Interestingly, both types of organelles have been identified in selected animal cells, most notably specialized digestive cells lining the gut of several species of Sacoglossan sea slugs. Termed kleptoplasty or kleptoplastic endosymbiosis, functional chloroplasts from algal food sources are internalized and stored within digestive cells to provide the host with dual energy sources derived from mitochondrial and photosynthetic processes. Recently, the observation of internalized algae within embryonic tissues of the spotted salamander strongly suggest that developmental processes within a vertebrate organism may require photosynthetic endosymbiosis as an internal regulator. The dual presence of mitochondria and functional chloroplasts within specialized animal cells indicates a high degree of biochemical identity, stereoselectivity, and conformational matching that are the likely

  16. Below-ambient levels of UV induce chloroplast structural change and alter starch metabolism.

    PubMed

    Fagerberg, W R

    2007-01-01

    Electromagnetic radiation (EMR) in the 400-700 nm bandwidth of photosynthetically active radiation (PAR) has been established as an important source of energy for photosynthesis and environmental signals regulating many aspects of green-plant life. Above-ambient levels of UV-B radiation (290-320 nm) under high-PAR conditions have been shown to elicit responses in chloroplasts of Brassica napus similar to those of chloroplasts at low-PAR exposure (W. Fagerberg and J. Bornman, Physiol. Plant. 101: 833-844, 1997). The question arises as to whether UV at normal levels can also evoke similar responses. Here we provide evidence that even below-ambient levels of UV-B (1/28 ambient; Durham, N.H., U.S.A., 1200 hours, March) were capable of inducing an increase in thylakoid surface area relative to the chloroplast volume typical of a low-PAR response (shade response) in sunflowers. This response occurred even though leaves were concurrently exposed to PAR levels that normally induce a "sun" or high-PAR response in the absence of UV-B. Subambient levels of UV-B were also associated with a decrease in chloroplast and starch volume. Exposure to levels of UV-A 1/10 of ambient appeared to enhance the high-PAR response of the chloroplast, characterized by an increase in the amounts of stored starch, an increase in chloroplast volume density ratio values, and a decrease in thylakoid surface area density ratios relative to the high-light controls. These effects were opposite to those seen in UV-B-exposed tissue. In a general sense, subambient levels of UV-B evoked a response similar to that elicited by low-PAR irradiance, while subambient UV-A elicited responses similar to those typical of high-PAR irradiance. The fact that below-ambient levels of UV altered a normal chloroplast structural response to PAR provides evidence that UV may be an important environmental signal for plants.

  17. Oxidation-reduction signalling components in regulatory pathways of state transitions and photosystem stoichiometry adjustment in chloroplasts.

    PubMed

    Puthiyaveetil, Sujith; Ibrahim, Iskander M; Allen, John F

    2012-02-01

    State transitions and photosystem stoichiometry adjustment are two oxidation-reduction (redox)-regulated acclimatory responses in photosynthesis. State transitions are short-term adaptations that, in chloroplasts, involve reversible post-translational modification by phosphorylation of light-harvesting complex II (LHC II). Photosystem stoichiometry adjustments are long-term responses involving transcriptional regulation of reaction centre genes. Both responses are initiated by changes in light quality and are regulated by the redox state of plastoquinone (PQ). The LHC II kinase involved in the state 2 transition is a serine/threonine kinase known as STT7 in Chlamydomonas, and as STN7 in Arabidopsis. The phospho-LHC II phosphatase that produces the state 1 transition is a PP2C-type protein phosphatase currently termed both TAP38 and PPH1. In plants and algae, photosystem stoichiometry adjustment is governed by a modified two-component sensor kinase of cyanobacterial origin - chloroplast sensor kinase (CSK). CSK is a sensor of the PQ redox state. Chloroplast sigma factor 1 (SIG1) and plastid transcription kinase (PTK) are the functional partners of CSK in chloroplast gene regulation. We suggest a signalling pathway for photosystem stoichiometry adjustment. The signalling pathways of state transitions and photosystem stoichiometry adjustments are proposed to be distinct, with the two pathways sensing PQ redox state independently of each other.

  18. The complete chloroplast genome of Phalaenopsis "Tiny Star".

    PubMed

    Kim, Goon-Bo; Kwon, Youngeun; Yu, Hee-Ju; Lim, Ki-Byung; Seo, Jae-Hwan; Mun, Jeong-Hwan

    2016-01-01

    We determined the complete chloroplast DNA sequence of Phalaenopsis "Tiny Star" based on Illumina sequencing. The total length of the chloroplast genome is 148,918 bp long with GC content of 36.7%. It contains 70 protein-coding genes, 30 tRNA genes, and 4 rRNA genes. Comparative analysis with the reported orchid chloroplast sequences identified unique InDel variations in the "Tiny Star" chloroplast genome that have potential as genetic markers to investigate the maternal lineage of Phalaenopsis and Doritaenopsis cultivars.

  19. Nanophotonics of Chloroplasts for Bio-Inspired Solar Energy Materials

    NASA Astrophysics Data System (ADS)

    Gourley, Paul L.; Gourley, Cheryl R.

    2011-03-01

    In the search for new energy sources, lessons can be learned from chloroplast photonics. The nano-architecture of chloroplasts is remarkably well-adapted to mediate sunlight interactions for efficient energy conversion. We carried out experiments with chloroplasts isolated from spinach and leaf lettuce to elucidate the relationship between nano-architecture, biomolecular composition and photonic properties. We obtained high-resolution microscopic images of single chloroplasts to identify geometries of chloroplasts and interior grana. We performed micro-spectroscopy to identify strengths of absorption and fluorescence transitions and related them to broadband reflectance and transmittance spectra of whole leaf structures. Finally, the nonlinear optical properties were investigated with nanolaser spectroscopy by placing chloroplasts into micro-resonators and optically pumping. These spectra reveal chloroplast photonic modes and allow measurement of single chloroplast light scattering cross section, polarizability, and refractive index. The nanolaser spectra recorded at increasing pump powers enabled us to observe non-linear optics, photon dynamics, and stimulated emission from single chloroplasts. All of these experiments provide insight into plant photonics and inspiration of paradigms for synthetic biomaterials to harness sunlight in new ways.

  20. Complex chloroplast RNA metabolism: just debugging the genetic programme?

    PubMed Central

    Maier, Uwe G; Bozarth, Andrew; Funk, Helena T; Zauner, Stefan; Rensing, Stefan A; Schmitz-Linneweber, Christian; Börner, Thomas; Tillich, Michael

    2008-01-01

    Background The gene expression system of chloroplasts is far more complex than that of their cyanobacterial progenitor. This gain in complexity affects in particular RNA metabolism, specifically the transcription and maturation of RNA. Mature chloroplast RNA is generated by a plethora of nuclear-encoded proteins acquired or recruited during plant evolution, comprising additional RNA polymerases and sigma factors, and sequence-specific RNA maturation factors promoting RNA splicing, editing, end formation and translatability. Despite years of intensive research, we still lack a comprehensive explanation for this complexity. Results We inspected the available literature and genome databases for information on components of RNA metabolism in land plant chloroplasts. In particular, new inventions of chloroplast-specific mechanisms and the expansion of some gene/protein families detected in land plants lead us to suggest that the primary function of the additional nuclear-encoded components found in chloroplasts is the transgenomic suppression of point mutations, fixation of which occurred due to an enhanced genetic drift exhibited by chloroplast genomes. We further speculate that a fast evolution of transgenomic suppressors occurred after the water-to-land transition of plants. Conclusion Our inspections indicate that several chloroplast-specific mechanisms evolved in land plants to remedy point mutations that occurred after the water-to-land transition. Thus, the complexity of chloroplast gene expression evolved to guarantee the functionality of chloroplast genetic information and may not, with some exceptions, be involved in regulatory functions. PMID:18755031

  1. A mutation in the FZL gene of Arabidopsis causing alteration in chloroplast morphology results in a lesion mimic phenotype

    PubMed Central

    Landoni, Michela

    2013-01-01

    Lesion mimic mutants (LMMs) are a class of mutants in which hypersensitive cell death and defence responses are constitutively activated in the absence of pathogen attack. Various signalling molecules, such as salicylic acid (SA), reactive oxygen species (ROS), nitric oxide (NO), Ca2+, ethylene, and jasmonate, are involved in the regulation of multiple pathways controlling hypersensitive response (HR) activation, and LMMs are considered useful tools to understand the role played by the key elements of the HR cell death signalling cascade. Here the characterization of an Arabidopsis LMM lacking the function of the FZL gene is reported. This gene encodes a membrane-remodelling GTPase playing an essential role in the determination of thylakoid and chloroplast morphology. The mutant displayed alteration in chloroplast number, size, and shape, and the typical characteristics of an LMM, namely development of chlorotic lesions on rosette leaves and constitutive expression of genetic and biochemical markers associated with defence responses. The chloroplasts are a major source of ROS, and the characterization of this mutant suggests that their accumulation, triggered by damage to the chloroplast membranes, is a signal sufficient to start the HR signalling cascade, thus confirming the central role of the chloroplast in HR activation. PMID:23963675

  2. PLASTID MOVEMENT IMPAIRED1 and PLASTID MOVEMENT IMPAIRED1-RELATED1 Mediate Photorelocation Movements of Both Chloroplasts and Nuclei1[OPEN

    PubMed Central

    Suetsugu, Noriyuki; Higa, Takeshi; Kong, Sam-Geun; Wada, Masamitsu

    2015-01-01

    Organelle movement and positioning play important roles in fundamental cellular activities and adaptive responses to environmental stress in plants. To optimize photosynthetic light utilization, chloroplasts move toward weak blue light (the accumulation response) and escape from strong blue light (the avoidance response). Nuclei also move in response to strong blue light by utilizing the light-induced movement of attached plastids in leaf cells. Blue light receptor phototropins and several factors for chloroplast photorelocation movement have been identified through molecular genetic analysis of Arabidopsis (Arabidopsis thaliana). PLASTID MOVEMENT IMPAIRED1 (PMI1) is a plant-specific C2-domain protein that is required for efficient chloroplast photorelocation movement. There are two PLASTID MOVEMENT IMPAIRED1-RELATED (PMIR) genes, PMIR1 and PMIR2, in the Arabidopsis genome. However, the mechanism in which PMI1 regulates chloroplast and nuclear photorelocation movements and the involvement of PMIR1 and PMIR2 in these organelle movements remained unknown. Here, we analyzed chloroplast and nuclear photorelocation movements in mutant lines of PMI1, PMIR1, and PMIR2. In mesophyll cells, the pmi1 single mutant showed severe defects in both chloroplast and nuclear photorelocation movements resulting from the impaired regulation of chloroplast-actin filaments. In pavement cells, pmi1 mutant plants were partially defective in both plastid and nuclear photorelocation movements, but pmi1pmir1 and pmi1pmir1pmir2 mutant lines lacked the blue light-induced movement responses of plastids and nuclei completely. These results indicated that PMI1 is essential for chloroplast and nuclear photorelocation movements in mesophyll cells and that both PMI1 and PMIR1 are indispensable for photorelocation movements of plastids and thus, nuclei in pavement cells. PMID:26324877

  3. PLASTID MOVEMENT IMPAIRED1 mediates ABA sensitivity during germination and implicates ABA in light-mediated Chloroplast movements.

    PubMed

    Rojas-Pierce, Marcela; Whippo, Craig W; Davis, Phillip A; Hangarter, Roger P; Springer, Patricia S

    2014-10-01

    The plant hormone abscisic acid (ABA) controls many aspects of plant growth and development, including seed development, germination and responses to water-deficit stress. A complex ABA signaling network integrates environmental signals including water availability and light intensity and quality to fine-tune the response to a changing environment. To further define the regulatory pathways that control water-deficit and ABA responses, we carried out a gene-trap tagging screen for water-deficit-regulated genes in Arabidopsis thaliana. This screen identified PLASTID MOVEMENT IMPAIRED1 (PMI1), a gene involved in blue-light-induced chloroplast movement, as functioning in ABA-response pathways. We provide evidence that PMI1 is involved in the regulation of seed germination by ABA, acting upstream of the intersection between ABA and low-glucose signaling pathways. Furthermore, PMI1 participates in the regulation of ABA accumulation during periods of water deficit at the seedling stage. The combined phenotypes of pmi1 mutants in chloroplast movement and ABA responses indicate that ABA signaling may modulate chloroplast motility. This result was further supported by the detection of altered chloroplast movements in the ABA mutants aba1-6, aba2-1 and abi1-1.

  4. Proton Gradients and Proton-Dependent Transport Processes in the Chloroplast

    PubMed Central

    Höhner, Ricarda; Aboukila, Ali; Kunz, Hans-Henning; Venema, Kees

    2016-01-01

    Proton gradients are fundamental to chloroplast function. Across thylakoid membranes, the light induced -proton gradient is essential for ATP synthesis. As a result of proton pumping into the thylakoid lumen, an alkaline stromal pH develops, which is required for full activation of pH-dependent Calvin Benson cycle enzymes. This implies that a pH gradient between the cytosol (pH 7) and the stroma (pH 8) is established upon illumination. To maintain this pH gradient chloroplasts actively extrude protons. More than 30 years ago it was already established that these proton fluxes are electrically counterbalanced by Mg2+, K+, or Cl- fluxes, but only recently the first transport systems that regulate the pH gradient were identified. Notably several (Na+,K+)/H+ antiporter systems where identified, that play a role in pH gradient regulation, ion homeostasis, osmoregulation, or coupling of secondary active transport. The established pH gradients are important to drive uptake of essential ions and solutes, but not many transporters involved have been identified to date. In this mini review we summarize the current status in the field and the open questions that need to be addressed in order to understand how pH gradients are maintained, how this is interconnected with other transport processes and what this means for chloroplast function. PMID:26973667

  5. Posttranslational Modifications of FERREDOXIN-NADP+ OXIDOREDUCTASE in Arabidopsis Chloroplasts1[W][OPEN

    PubMed Central

    Lehtimäki, Nina; Koskela, Minna M.; Dahlström, Käthe M.; Pakula, Eveliina; Lintala, Minna; Scholz, Martin; Hippler, Michael; Hanke, Guy T.; Rokka, Anne; Battchikova, Natalia; Salminen, Tiina A.; Mulo, Paula

    2014-01-01

    Rapid responses of chloroplast metabolism and adjustments to photosynthetic machinery are of utmost importance for plants’ survival in a fluctuating environment. These changes may be achieved through posttranslational modifications of proteins, which are known to affect the activity, interactions, and localization of proteins. Recent studies have accumulated evidence about the crucial role of a multitude of modifications, including acetylation, methylation, and glycosylation, in the regulation of chloroplast proteins. Both of the Arabidopsis (Arabidopsis thaliana) leaf-type FERREDOXIN-NADP+ OXIDOREDUCTASE (FNR) isoforms, the key enzymes linking the light reactions of photosynthesis to carbon assimilation, exist as two distinct forms with different isoelectric points. We show that both AtFNR isoforms contain multiple alternative amino termini and undergo light-responsive addition of an acetyl group to the α-amino group of the amino-terminal amino acid of proteins, which causes the change in isoelectric point. Both isoforms were also found to contain acetylation of a conserved lysine residue near the active site, while no evidence for in vivo phosphorylation or glycosylation was detected. The dynamic, multilayer regulation of AtFNR exemplifies the complex regulatory network systems controlling chloroplast proteins by a range of posttranslational modifications, which continues to emerge as a novel area within photosynthesis research. PMID:25301888

  6. Ion Channels in Native Chloroplast Membranes: Challenges and Potential for Direct Patch-Clamp Studies

    PubMed Central

    Pottosin, Igor; Dobrovinskaya, Oxana

    2015-01-01

    Photosynthesis without any doubt depends on the activity of the chloroplast ion channels. The thylakoid ion channels participate in the fine partitioning of the light-generated proton-motive force (p.m.f.). By regulating, therefore, luminal pH, they affect the linear electron flow and non-photochemical quenching. Stromal ion homeostasis and signaling, on the other hand, depend on the activity of both thylakoid and envelope ion channels. Experimentally, intact chloroplasts and swollen thylakoids were proven to be suitable for direct measurements of the ion channels activity via conventional patch-clamp technique; yet, such studies became infrequent, although their potential is far from being exhausted. In this paper we wish to summarize existing challenges for direct patch-clamping of native chloroplast membranes as well as present available results on the activity of thylakoid Cl− (ClC?) and divalent cation-permeable channels, along with their tentative roles in the p.m.f. partitioning, volume regulation, and stromal Ca2+ and Mg2+ dynamics. Patch-clamping of the intact envelope revealed both large-conductance porin-like channels, likely located in the outer envelope membrane and smaller conductance channels, more compatible with the inner envelope location. Possible equivalent model for the sandwich-like arrangement of the two envelope membranes within the patch electrode will be discussed, along with peculiar properties of the fast-activated cation channel in the context of the stromal pH control. PMID:26733887

  7. 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.

  8. The Chlamydomonas reinhardtii Nar1 Gene Encodes a Chloroplast Membrane Protein Involved in Nitrite Transport

    PubMed Central

    Rexach, Jesus; Fernández, Emilio; Galván, Aurora

    2000-01-01

    A key step for nitrate assimilation in photosynthetic eukaryotes occurs within chloroplasts, where nitrite is reduced to ammonium, which is incorporated into carbon skeletons. The Nar1 gene from Chlamydomonas reinhardtii is clustered with five other genes for nitrate assimilation, all of them regulated by nitrate. Sequence analysis of genomic DNA and cDNA of Nar1 and comparative studies of strains having or lacking Nar1 have been performed. The deduced amino acid sequence indicates that Nar1 encodes a chloroplast membrane protein with substantial identity to putative formate and nitrite transporters in bacteria. Use of antibodies against NAR1 has corroborated its location in the plastidic membrane. Characterization of strains having or lacking this gene suggests that NAR1 is involved in nitrite transport in plastids, which is critical for cell survival under limiting nitrate conditions, and controls the amount of nitrate incorporated by the cells under limiting CO2 conditions. PMID:10948261

  9. The chloroplast ATP-dependent Clp protease in vascular plants - new dimensions and future challenges.

    PubMed

    Clarke, Adrian K

    2012-05-01

    The ATP-dependent Clp protease is by far the most intricate protease in chloroplasts of vascular plants. Structurally, it is particularly complex with a proteolytic core complex containing 11 distinct subunits along with three potential chaperone partners. The Clp protease is also essential for chloroplast development and overall plant viability. Over the past decade, many of the important characteristics of this crucial protease have been revealed in the model plant species Arabidopsis thaliana. Despite this, challenges still remain in fully resolving certain key features, in particular, how the assembly of this multisubunit protease is regulated, the full range of native protein substrates and how they are targeted for degradation and how this complicated enzyme might have developed from simpler bacterial forms. This article focuses upon the recent advances in revealing the details underlying these important features. It also take the opportunity to speculate upon many of these findings in the hope of stimulating further investigation.

  10. Arabidopsis thaliana leaves with altered chloroplast numbers and chloroplast movement exhibit impaired adjustments to both low and high light.

    PubMed

    Königer, Martina; Delamaide, Joy A; Marlow, Elizabeth D; Harris, Gary C

    2008-01-01

    The effects of chloroplast number and size on the capacity for blue light-dependent chloroplast movement, the ability to increase light absorption under low light, and the susceptibility to photoinhibition were investigated in Arabidopsis thaliana. Leaves of wild-type and chloroplast number mutants with mean chloroplast numbers ranging from 120 to two per mesophyll cell were analysed. Chloroplast movement was monitored as changes in light transmission through the leaves. Light transmission was used as an indicator of the ability of leaves to optimize light absorption. The ability of leaves to deal with 3 h of high light stress at 10 degrees C and their capacity to recover in low light was determined by measuring photochemical efficiencies of PSII using chlorophyll a fluorescence. Chloroplast movement was comparable in leaves ranging in chloroplast numbers from 120 to 30 per mesophyll cell: the final light transmission levels after exposure to 0.1 (accumulation response) and 100 micromol photons m(-2) s(-1) (avoidance response) were indistinguishable, the chloroplasts responded quickly to small increases in light intensity and the kinetics of movement were similar. However, when chloroplast numbers per mesophyll cell decreased to 18 or below, the accumulation response was significantly reduced. The avoidance response was only impaired in mutants with nine or fewer chloroplasts, both in terms of final transmission levels and the speed of movement. Only mutants lacking both blue light receptors (phot1/phot2) or those with drastically reduced chloroplast numbers and severely impacted avoidance responses showed a reduced ability to recover from high light stress.

  11. Why have chloroplasts developed a unique motility system?

    PubMed

    Suetsugu, Noriyuki; Dolja, Valerian V; Wada, Masamitsu

    2010-10-01

    Organelle movement in plants is dependent on actin filaments with most of the organelles being transported along the actin cables by class XI myosins. Although chloroplast movement is also actin filament-dependent, a potential role of myosin motors in this process is poorly understood. Interestingly, chloroplasts can move in any direction, and change the direction within short time periods, suggesting that chloroplasts use the newly formed actin filaments rather than preexisting actin cables. Furthermore, the data on myosin gene knockouts and knockdowns in Arabidopsis and tobacco do not support myosins' XI role in chloroplast movement. Our recent studies revealed that chloroplast movement and positioning are mediated by the short actin filaments localized at chloroplast periphery (cp-actin filaments) rather than cytoplasmic actin cables. The accumulation of cp-actin filaments depends on kinesin-like proteins, KAC1 and KAC2, as well as on a chloroplast outer membrane protein CHUP1. We propose that plants evolved a myosin XI-independent mechanism of the actin-based chloroplast movement that is distinct from the mechanism used by other organelles.

  12. Membrane heredity and early chloroplast evolution.

    PubMed

    Cavalier-Smith, T

    2000-04-01

    Membrane heredity was central to the unique symbiogenetic origin from cyanobacteria of chloroplasts in the ancestor of Plantae (green plants, red algae, glaucophytes) and to subsequent lateral transfers of plastids to form even more complex photosynthetic chimeras. Each symbiogenesis integrated disparate genomes and several radically different genetic membranes into a more complex cell. The common ancestor of Plantae evolved transit machinery for plastid protein import. In later secondary symbiogeneses, signal sequences were added to target proteins across host perialgal membranes: independently into green algal plastids (euglenoids, chlorarachneans) and red algal plastids (alveolates, chromists). Conservatism and innovation during early plastid diversification are discussed.

  13. Noncoding chloroplast DNA variation in Mexican pines.

    PubMed

    Perez de la Rosa, J; Harris, S A; Farjon, A

    1995-11-01

    Universal primers were used for PCR amplification of three noncoding regions of chloroplast DNA in order to study restriction site variation in 12 Mexican pine species. Two length mutations were identified that are of diagnostic value for two subgenera or sections of the genus. Phylogenetic analysis of the restriction site and length variation showed patterns of variation largely consistent with previous arrangements of these pines, except for the position of Pinus nelsonii, indicating that Pinus section Parraya Mayr, as circumscribed by Little and Critchfield (1969) and later authors, is not a monophyletic group.

  14. 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.

  15. Non-contact intracellular binding of chloroplasts in vivo

    NASA Astrophysics Data System (ADS)

    Li, Yuchao; Xin, Hongbao; Liu, Xiaoshuai; Li, Baojun

    2015-06-01

    Non-contact intracellular binding and controllable manipulation of chloroplasts in vivo was demonstrated using an optical fiber probe. Launching a 980-nm laser beam into a fiber, which was placed about 3 μm above the surface of a living plant (Hydrilla verticillata) leaf, enabled stable binding of different numbers of chloroplasts, as well as their arrangement into one-dimensional chains and two-dimensional arrays inside the leaf without damaging the chloroplasts. Additionally, the formed chloroplast chains were controllably transported inside the living cells. The optical force exerted on the chloroplasts was calculated to explain the experimental results. This method provides a flexible method for studying intracellular organelle interaction with highly organized organelle-organelle contact in vivo in a non-contact manner.

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

    Technology Transfer Automated Retrieval System (TEKTRAN)

    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...

  17. Importance of the green color, absorption gradient, and spectral absorption of chloroplasts for the radiative energy balance of leaves.

    PubMed

    Kume, Atsushi

    2017-03-14

    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 CO2 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

  18. Albino Leaf1 That Encodes the Sole Octotricopeptide Repeat Protein Is Responsible for Chloroplast Development1[OPEN

    PubMed Central

    Tan, Jianjie; Xing, Yi; Liu, Changhong; Chen, Qiaoling; Zhu, Haitao; Wang, Jiang; Zhang, Jingliu; Zhang, Guiquan

    2016-01-01

    Chloroplast, the photosynthetic organelle in plants, plays a crucial role in plant development and growth through manipulating the capacity of photosynthesis. However, the regulatory mechanism of chloroplast development still remains elusive. Here, we characterized a mutant with defective chloroplasts in rice (Oryza sativa), termed albino leaf1 (al1), which exhibits a distinct albino phenotype in leaves, eventually leading to al1 seedling lethality. Electronic microscopy observation demonstrated that the number of thylakoids was reduced and the structure of thylakoids was disrupted in the al1 mutant during rice development, which eventually led to the breakdown of chloroplast. Molecular cloning revealed that AL1 encodes the sole octotricopeptide repeat protein (RAP) in rice. Genetic complementation of Arabidopsis (Arabidopsis thaliana) rap mutants indicated that the AL1 protein is a functional RAP. Further analysis illustrated that three transcript variants were present in the AL1 gene, and the altered splices occurred at the 3′ untranslated region of the AL1 transcript. In addition, our results also indicate that disruption of the AL1 gene results in an altered expression of chloroplast-associated genes. Consistently, proteomic analysis demonstrated that the abundance of photosynthesis-associated proteins is altered significantly, as is that of a group of metabolism-associated proteins. More specifically, we found that the loss of AL1 resulted in altered abundances of ribosomal proteins, suggesting that RAP likely also regulates the homeostasis of ribosomal proteins in rice in addition to the ribosomal RNA. Taken together, we propose that AL1, particularly the AL1a and AL1c isoforms, plays an essential role in chloroplast development in rice. PMID:27208287

  19. The Arabidopsis Chloroplast Stromal N-Terminome: Complexities of Amino-Terminal Protein Maturation and Stability1[OPEN

    PubMed Central

    Rowland, Elden; Kim, Jitae; Bhuiyan, Nazmul H.; van Wijk, Klaas J.

    2015-01-01

    Protein amino (N) termini are prone to modifications and are major determinants of protein stability in bacteria, eukaryotes, and perhaps also in chloroplasts. Most chloroplast proteins undergo N-terminal maturation, but this is poorly understood due to insufficient experimental information. Consequently, N termini of mature chloroplast proteins cannot be accurately predicted. This motivated an extensive characterization of chloroplast protein N termini in Arabidopsis (Arabidopsis thaliana) using terminal amine isotopic labeling of substrates and mass spectrometry, generating nearly 14,000 tandem mass spectrometry spectra matching to protein N termini. Many nucleus-encoded plastid proteins accumulated with two or three different N termini; we evaluated the significance of these different proteoforms. Alanine, valine, threonine (often in N-α-acetylated form), and serine were by far the most observed N-terminal residues, even after normalization for their frequency in the plastid proteome, while other residues were absent or highly underrepresented. Plastid-encoded proteins showed a comparable distribution of N-terminal residues, but with a higher frequency of methionine. Infrequent residues (e.g. isoleucine, arginine, cysteine, proline, aspartate, and glutamate) were observed for several abundant proteins (e.g. heat shock proteins 70 and 90, Rubisco large subunit, and ferredoxin-glutamate synthase), likely reflecting functional regulation through their N termini. In contrast, the thylakoid lumenal proteome showed a wide diversity of N-terminal residues, including those typically associated with instability (aspartate, glutamate, leucine, and phenylalanine). We propose that, after cleavage of the chloroplast transit peptide by stromal processing peptidase, additional processing by unidentified peptidases occurs to avoid unstable or otherwise unfavorable N-terminal residues. The possibility of a chloroplast N-end rule is discussed. PMID:26371235

  20. Differential Replication of Two Chloroplast Genome Forms in Heteroplasmic Chlamydomonas reinhardtii Gametes Contributes to Alternative Inheritance Patterns

    PubMed Central

    Nishimura, Yoshiki; Stern, David B.

    2010-01-01

    Two mechanisms for chloroplast DNA replication have been revealed through the study of an unusual heteroplasmic strain of the green alga Chlamydomonas reinhardtii. Heteroplasmy is a state in which more than one genome type occurs in a mitochondrion or chloroplast. The Chlamydomonas strain spa19 bears two distinct chloroplast genomes, termed PS+ and PS−. PS+ genomes predominate and are stably maintained in vegetative cells, despite their lack of known replication origins. In sexual crosses with spa19 as the mating type plus parent, however, PS+ genomes are transmitted in only ∼25% of tetrads, whereas the PS− genomes are faithfully inherited in all progeny. In this research, we have explored the mechanism underlying this biased uniparental inheritance. We show that the relative reduction and dilution of PS+ vs. PS− genomes takes place during gametogenesis. Bromodeoxyuridine labeling, followed by immunoprecipitation and PCR, was used to compare replication activities of PS+ and PS− genomes. We found that the replication of PS+ genomes is specifically suppressed during gametogenesis and germination of zygospores, a phenomenon that also was observed when spa19 cells were treated with rifampicin, an inhibitor of the chloroplast RNA polymerase. Furthermore, when bromodeoxyuridine incorporation was compared at 11 sites within the chloroplast genome between vegetative cells, gametes, and rifampicin-treated cells by quantitative PCR, we found that incorporation was often reduced at the same sites in gametes that were also sensitive to rifampicin treatment. We conclude that a transcription-mediated form of DNA replication priming, which may be downregulated during gametogenesis, is indispensable for robust maintenance of PS+ genomes. These results highlight the potential for chloroplast genome copy number regulation through alternative replication strategies. PMID:20519744

  1. Differential replication of two chloroplast genome forms in heteroplasmic Chlamydomonas reinhardtii gametes contributes to alternative inheritance patterns.

    PubMed

    Nishimura, Yoshiki; Stern, David B

    2010-08-01

    Two mechanisms for chloroplast DNA replication have been revealed through the study of an unusual heteroplasmic strain of the green alga Chlamydomonas reinhardtii. Heteroplasmy is a state in which more than one genome type occurs in a mitochondrion or chloroplast. The Chlamydomonas strain spa19 bears two distinct chloroplast genomes, termed PS+ and PS-. PS+ genomes predominate and are stably maintained in vegetative cells, despite their lack of known replication origins. In sexual crosses with spa19 as the mating type plus parent, however, PS+ genomes are transmitted in only approximately 25% of tetrads, whereas the PS- genomes are faithfully inherited in all progeny. In this research, we have explored the mechanism underlying this biased uniparental inheritance. We show that the relative reduction and dilution of PS+ vs. PS- genomes takes place during gametogenesis. Bromodeoxyuridine labeling, followed by immunoprecipitation and PCR, was used to compare replication activities of PS+ and PS- genomes. We found that the replication of PS+ genomes is specifically suppressed during gametogenesis and germination of zygospores, a phenomenon that also was observed when spa19 cells were treated with rifampicin, an inhibitor of the chloroplast RNA polymerase. Furthermore, when bromodeoxyuridine incorporation was compared at 11 sites within the chloroplast genome between vegetative cells, gametes, and rifampicin-treated cells by quantitative PCR, we found that incorporation was often reduced at the same sites in gametes that were also sensitive to rifampicin treatment. We conclude that a transcription-mediated form of DNA replication priming, which may be downregulated during gametogenesis, is indispensable for robust maintenance of PS+ genomes. These results highlight the potential for chloroplast genome copy number regulation through alternative replication strategies.

  2. Ferredoxin-linked chloroplast enzymes. Progress report

    SciTech Connect

    1993-12-31

    This report summarizes research on ferredoxin:NADP{sup +} oxidoreductase and ferredoxin:thioredoxin reductase. One of the primary goals of the original proposal was to map the ferredoxin-binding sites on three soluble enzymes that are located in spinach chloroplasts and utilize ferredoxin as an electron donor:Ferredoxin:NADP{sup +} oxidoreductase (FNR); ferredoxin:thioredoxin reductase (FTR) and glutamate synthase. As the availability of amino acid sequences for the enzymes are important in such studies, it was proposed that the amino acid sequence of glutamate synthase be determined. The amino acid sequences of FNR, FTR and ferredoxin are already known. An aim related to elucidating the binding sites on these enzymes for ferredoxin was to determine whether there is a common site on ferredoxin involved in binding to all of these ferredoxin-dependent chloroplast enzymes and, if so, to map it. One additional aim was to characterize thioredoxin binding by FTR and determine whether the same site on FTR is involved in binding both ferredoxin and thioredoxin. Considerable progress has been made on most of these original projects, although work conducted on FTR is still in its preliminary stages.

  3. 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

  4. Complete sequence of Euglena gracilis chloroplast DNA.

    PubMed Central

    Hallick, R B; Hong, L; Drager, R G; Favreau, M R; Monfort, A; Orsat, B; Spielmann, A; Stutz, E

    1993-01-01

    We report the complete DNA sequence of the Euglena gracilis, Pringsheim strain Z chloroplast genome. This circular DNA is 143,170 bp, counting only one copy of a 54 bp tandem repeat sequence that is present in variable copy number within a single culture. The overall organization of the genome involves a tandem array of three complete and one partial ribosomal RNA operons, and a large single copy region. There are genes for the 16S, 5S, and 23S rRNAs of the 70S chloroplast ribosomes, 27 different tRNA species, 21 ribosomal proteins plus the gene for elongation factor EF-Tu, three RNA polymerase subunits, and 27 known photosynthesis-related polypeptides. Several putative genes of unknown function have also been identified, including five within large introns, and five with amino acid sequence similarity to genes in other organisms. This genome contains at least 149 introns. There are 72 individual group II introns, 46 individual group III introns, 10 group II introns and 18 group III introns that are components of twintrons (introns-within-introns), and three additional introns suspected to be twintrons composed of multiple group II and/or group III introns, but not yet characterized. At least 54,804 bp, or 38.3% of the total DNA content is represented by introns. PMID:8346031

  5. Heteroplasmy of chloroplast DNA in Medicago.

    PubMed

    Johnson, L B; Palmer, J D

    1989-01-01

    Two chloroplast DNA (cpDNA) regions exhibiting a high frequency of intra- or inter-species variation were identified in 12 accessions of the genus Medicago. Restriction maps of both regions were prepared for alfalfa, and the probable nature of the events causing the DNA differences was identified. Specific DNA fragments were then cloned for use in identification of variants in each region. Two each of M. sativa ssp. varia and ssp. caerulea and one of six M. sativa ssp. sativa single plants examined possessed cpDNA heterogeneity as identified by screening extracts for fragments generated by the presence and absence of a specific Xba I restriction site. Three plants of M. sativa ssp. sativa, two of each of sspp. varia and caerulea, and three M. scutellata were also examined for single-plant cpDNA heterogeneity at a hypervariable region where differences resulted from small insertion-deletion events. A single M. scutellata plant with mixed cpDNAs was identified. Sorting out was seen when one spp. sativa plant with mixed plastid types identifiable by the Xba I restriction site difference was vegetatively propagated. This indicated that the initial stock plant was heteroplastidic. Controlled crosses will be required in order to test whether heteroplasmy results from chloroplast transmission in the pollen and to examine the dynamic of sorting out. However, heteroplasmy is apparently not a rare situation in Medicago.

  6. 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

  7. The workflow for quantitative proteome analysis of chloroplast development and differentiation, chloroplast mutants, and protein interactions by spectral counting.

    PubMed

    Friso, Giulia; Olinares, Paul Dominic B; van Wijk, Klaas J

    2011-01-01

    This chapter outlines a quantitative proteomics workflow using a label-free spectral counting technique. The workflow has been tested on different aspects of chloroplast biology in maize and Arabidopsis, including chloroplast mutant analysis, cell-type specific chloroplast differentiation, and the proplastid-to-chloroplast transition. The workflow involves one-dimensional SDS-PAGE of the proteomes of leaves or chloroplast subfractions, tryptic digestions, online LC-MS/MS using a mass spectrometer with high mass accuracy and duty cycle, followed by semiautomatic data processing. The bioinformatics analysis can effectively select best gene models and deals with quantification of closely related proteins; the workflow avoids overidentification of proteins and results in more accurate protein quantification. The final output includes pairwise comparative quantitative analysis, as well as hierarchical clustering for discovery of temporal and spatial patterns of protein accumulation. A brief discussion about potential pitfalls, as well as the advantages and disadvantages of spectral counting, is provided.

  8. The chloroplast genome of Pellia endiviifolia: gene content, RNA-editing pattern, and the origin of chloroplast editing.

    PubMed

    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.

  9. Antisense Transcript and RNA Processing Alterations Suppress Instability of Polyadenylated mRNA in Chlamydomonas Chloroplasts

    PubMed Central

    Nishimura, Yoshiki; Kikis, Elise A.; Zimmer, Sara L.; Komine, Yutaka; Stern, David B.

    2004-01-01

    In chloroplasts, the control of mRNA stability is of critical importance for proper regulation of gene expression. The Chlamydomonas reinhardtii strain Δ26pAtE is engineered such that the atpB mRNA terminates with an mRNA destabilizing polyadenylate tract, resulting in this strain being unable to conduct photosynthesis. A collection of photosynthetic revertants was obtained from Δ26pAtE, and gel blot hybridizations revealed RNA processing alterations in the majority of these suppressor of polyadenylation (spa) strains, resulting in a failure to expose the atpB mRNA 3′ poly(A) tail. Two exceptions were spa19 and spa23, which maintained unusual heteroplasmic chloroplast genomes. One genome type, termed PS+, conferred photosynthetic competence by contributing to the stability of atpB mRNA; the other, termed PS−, was required for viability but could not produce stable atpB transcripts. Based on strand-specific RT-PCR, S1 nuclease protection, and RNA gel blots, evidence was obtained that the PS+ genome stabilizes atpB mRNA by generating an atpB antisense transcript, which attenuates the degradation of the polyadenylated form. The accumulation of double-stranded RNA was confirmed by insensitivity of atpB mRNA from PS+ genome-containing cells to S1 nuclease digestion. To obtain additional evidence for antisense RNA function in chloroplasts, we used strain Δ26, in which atpB mRNA is unstable because of the lack of a 3′ stem-loop structure. In this context, when a 121-nucleotide segment of atpB antisense RNA was expressed from an ectopic site, an elevated accumulation of atpB mRNA resulted. Finally, when spa19 was placed in a genetic background in which expression of the chloroplast exoribonuclease polynucleotide phosphorylase was diminished, the PS+ genome and the antisense transcript were no longer required for photosynthesis. Taken together, our results suggest that antisense RNA in chloroplasts can protect otherwise unstable transcripts from 3′→5

  10. Transposon-induced nuclear mutations that alter chloroplast gene expression

    SciTech Connect

    Barkan, A.

    1992-01-01

    The goal of this project is to use mutant phenotypes as a guide to nuclear genes that determine the timing and localization of chloroplast development The immediate goals are to identify nuclear mutants with defects in chloroplast gene expression from maize lines harboring active Mu transposons; characterize their phenotypes to determine the precise defect in gene expression; clone several of the most interesting mutations by exploiting the transposon tag; and use the clones to further define the roles of these genes in modulating chloroplast gene expression. Three mutants were described earlier that had global defects in chloroplast gene expression. We have found that two of these mutations are allelic. Both alleles have global defects in chloroplast translation initiation, as revealed by the failure to assemble chloroplast mRNAs into polysomes. We have isolated and characterized three new mutants from Mu lines that have novel defects in chloroplast RNA metabolism. We are now ready to begin the task of cloning several of these genes, by using the Mu transposon tag.

  11. Does Chloroplast Size Influence Photosynthetic Nitrogen Use Efficiency?

    PubMed Central

    Li, Yong; Ren, Binbin; Ding, Lei; Shen, Qirong; Peng, Shaobing; Guo, Shiwei

    2013-01-01

    High nitrogen (N) supply frequently results in a decreased photosynthetic N-use efficiency (PNUE), which indicates a less efficient use of accumulated Ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco). Chloroplasts are the location of Rubisco and the endpoint of CO2 diffusion, and they play a vital important role in photosynthesis. However, the effects of chloroplast development on photosynthesis are poorly explored. In the present study, rice seedlings (Oryza sativa L., cv. ‘Shanyou 63’, and ‘Yangdao 6’) were grown hydroponically with three different N levels, morphological characteristics, photosynthetic variables and chloroplast size were measured. In Shanyou 63, a negative relationship between chloroplast size and PNUE was observed across three different N levels. Here, plants with larger chloroplasts had a decreased ratio of mesophyll conductance (gm) to Rubisco content (gm/Rubisco) and a lower Rubisco specific activity. In Yangdao 6, there was no change in chloroplast size and no decline in PNUE or gm/Rubisco ratio under high N supply. It is suggested that large chloroplasts under high N supply is correlated with the decreased Rubisco specific activity and PNUE. PMID:23620801

  12. Chloroplasts move towards the nearest anticlinal walls under dark condition.

    PubMed

    Tsuboi, Hidenori; Wada, Masamitsu

    2012-03-01

    Chloroplasts change their intracellular positions in response to their light environment. Under darkness, chloroplasts assume special positions that are different from those under light conditions. Here, we analyzed chloroplast dark positioning using Adiantum capillus-veneris gametophyte cells. When chloroplasts were transferred into darkness, during the first 1-5 h, they moved towards the anticlinal cell walls bordering the adjacent cells rather rapidly. Then, they slowed down and accumulated at the anticlinal walls gradually over the following 24-36 h. The chloroplast movements could be roughly classified into two different categories: initial rapid straight movement and later, slow staggering movement. When the chloroplast accumulation response was induced in dark-adapted cells by partial cell irradiation with a microbeam targeted to the center of the cells, chloroplasts moved towards the beam spot from the anticlinal walls. However, when the microbeam was switched off, they moved to the nearest anticlinal walls and not to their original positions if they were not the closest, indicating that they know the direction of the nearest anticlinal wall and do not have particular areas that they migrate to during dark positioning.

  13. Differential Subplastidial Localization and Turnover of Enzymes Involved in Isoprenoid Biosynthesis in Chloroplasts

    PubMed Central

    Perello, Catalina; Llamas, Ernesto; Burlat, Vincent; Ortiz-Alcaide, Miriam; Phillips, Michael A.; Pulido, Pablo; Rodriguez-Concepcion, Manuel

    2016-01-01

    Plastidial isoprenoids are a diverse group of metabolites with roles in photosynthesis, growth regulation, and interaction with the environment. The methylerythritol 4-phosphate (MEP) pathway produces the metabolic precursors of all types of plastidial isoprenoids. Proteomics studies in Arabidopsis thaliana have shown that all the enzymes of the MEP pathway are localized in the plastid stroma. However, immunoblot analysis of chloroplast subfractions showed that the first two enzymes of the pathway, deoxyxylulose 5-phosphate synthase (DXS) and reductoisomerase (DXR), can also be found in non-stromal fractions. Both transient and stable expression of GFP-tagged DXS and DXR proteins confirmed the presence of the fusion proteins in distinct subplastidial compartments. In particular, DXR-GFP was found to accumulate in relatively large vesicles that could eventually be released from chloroplasts, presumably to be degraded by an autophagy-independent process. Together, we propose that protein-specific mechanisms control the localization and turnover of the first two enzymes of the MEP pathway in Arabidopsis chloroplasts. PMID:26919668

  14. 2010 GORDON RESEARCH CONFERENCE ON MITOCHONDRIA & CHLOROPLASTS, LUCCA, ITALY, JULY 11-16, 2010

    SciTech Connect

    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 organelle 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.

  15. Complete Arabis alpina chloroplast genome sequence and insight into its polymorphism☆

    PubMed Central

    Melodelima, Christelle; Lobréaux, Stéphane

    2013-01-01

    The alpine plant Arabis alpina (alpine rock-cress) is a thoroughly studied species in the fields of perennial plant flowering regulation, phylogeography, and adaptation to harsh alpine climatic conditions. We report the complete A. alpina chloroplast genome sequence obtained through de novo assembly of Illumina paired-end reads produced by total DNA sequencing. The A. alpina cp circular genome is 152,866 bp in length and built of two inverted repeats of 26,933 bp separated by unique regions: a large single copy of 82,338 bp and a small single copy of 17,938 bp. The genome contains 131 genes, some of them being duplicated in the inverted repeats. Seventy-nine unique protein-coding genes were annotated, together with 29 tRNA genes and 4 ribosomal RNA genes. Sequencing and mapping of 23 additional A. alpina DNA samples enabled to gain insight into the intraspecies polymorphism of the sequenced cp genome. Genetic variability among genomes was detected as 44 indels, most of them being located in noncoding regions, and 130 single-nucleotide polymorphisms, 37 of them corresponding to mutations in coding regions. A. alpina chloroplast genome sequence will be helpful in population studies or investigations of chloroplast functions of this alpine plant species. PMID:25606376

  16. Physiological and proteomic analysis in chloroplasts of Solanum lycopersicum L. under silicon efficiency and salinity stress.

    PubMed

    Muneer, Sowbiya; Park, Yoo Gyeong; Manivannan, Abinaya; Soundararajan, Prabhakaran; Jeong, Byoung Ryong

    2014-11-26

    Tomato plants often grow in saline environments in Mediterranean countries where salt accumulation in the soil is a major abiotic stress that limits its productivity. However, silicon (Si) supplementation has been reported to improve tolerance against several forms of abiotic stress. The primary aim of our study was to investigate, using comparative physiological and proteomic approaches, salinity stress in chloroplasts of tomato under silicon supplementation. Tomato seedlings (Solanum lycopersicum L.) were grown in nutrient media in the presence or absence of NaCl and supplemented with silicon for 5 days. Salinity stress caused oxidative damage, followed by a decrease in silicon concentrations in the leaves of the tomato plants. However, supplementation with silicon had an overall protective effect against this stress. The major physiological parameters measured in our studies including total chlorophyll and carotenoid content were largely decreased under salinity stress, but were recovered in the presence of silicon. Insufficient levels of net-photosynthesis, transpiration and stomatal conductance were also largely improved by silicon supplementation. Proteomics analysis of chloroplasts analyzed by 2D-BN-PAGE (second-dimensional blue native polyacrylamide-gel electrophoresis) revealed a high sensitivity of multiprotein complex proteins (MCPs) such as photosystems I (PSI) and II (PSII) to the presence of saline. A significant reduction in cytochrome b6/f and the ATP-synthase complex was also alleviated by silicon during salinity stress, while the complex forms of light harvesting complex trimers and monomers (LHCs) were rapidly up-regulated. Our results suggest that silicon plays an important role in moderating damage to chloroplasts and their metabolism in saline environments. We therefore hypothesize that tomato plants have a greater capacity for tolerating saline stress through the improvement of photosynthetic metabolism and chloroplast proteome expression

  17. The Role of Pea Chloroplast [alpha]-Glucosidase in Transitory Starch Degradation.

    PubMed

    Sun, Z.; Duke, S. H.; Henson, C. A.

    1995-05-01

    Pea chloroplastic [alpha]-glucosidase (EC 3.2.1.20) involved in transitory starch degradation was purified to apparent homogeneity by ion exchange, reactive dye, hydroxylapatite, hydrophobic interaction, and gel filtration column chromatography. The native molecular mass and the subunit molecular mass were about 49.1 and 24.4 kD, respectively, suggesting that the enzyme is a homodimer. The enzyme had a Km of 7.18 mM for maltose. The enzyme's maximal activity at pH 7.0 and stability at pH 6.5 are compatible with the diurnal oscillations of the chloroplastic stromal pH and transitory starch accumulation. This pH modulation of the [alpha]-glucosidase's activity and stability is the only mechanism known to regulate starch degradative enzymes in leaves. Although the enzyme was specific for the [alpha]-D-glucose in the nonreducing end as the glycon, the aglycon moieties could be composed of a variety of groups. However, the hydrolysis rate was greatly influenced by the aglycon residues. Also, the enzyme could hydrolyze glucans in which carbon 1 of the glycon was linked to different carbon positions of the penultimate glucose residue. The ability of the [alpha]-glucosidase to hydrolyze [alpha]-1,2- and [alpha]-1,3-glucosidic bonds may be vital if these bonds exist in starch granules because they would be barriers to other starch degradative enzymes. This purified pea chloroplastic [alpha]-glucosidase was demonstrated to initiate attacks on native transitory chloroplastic starch granules.

  18. Polyphenol oxidase in leaves: is there any significance to the chloroplastic localization?

    PubMed

    Boeckx, Tinne; Winters, Ana L; Webb, K Judith; Kingston-Smith, Alison H

    2015-06-01

    Polyphenol oxidase (PPO) catalyses the oxidation of monophenols and/or o-diphenols to o-quinones with the concomitant reduction of oxygen to water which results in protein complexing and the formation of brown melanin pigments. The most frequently suggested role for PPO in plants has been in defence against herbivores and pathogens, based on the physical separation of the chloroplast-localized enzyme from the vacuole-localized substrates. The o-quinone-protein complexes, formed as a consequence of cell damage, may reduce the nutritional value of the tissue and thereby reduce predation but can also participate in the formation of structural barriers against invading pathogens. However, since a sufficient level of compartmentation-based regulation could be accomplished if PPO was targeted to the cytosol, the benefit derived by some plant species in having PPO present in the chloroplast lumen remains an intriguing question. So is there more to the chloroplastic location of PPO? An interaction between PPO activity and photosynthesis has been proposed on more than one occasion but, to date, evidence either for or against direct involvement has been equivocal, and the lack of identified chloroplastic substrates remains an issue. Similarly, PPO has been suggested to have both pro- and anti-oxidant functions. Nevertheless, several independent lines of evidence suggest that PPO responds to environmental conditions and could be involved in the response of plants to abiotic stress. This review highlights our current understanding of the in vivo functions of PPO and considers the potential opportunities it presents for exploitation to increase stress tolerance in food crops.

  19. Changes in the alternative electron sinks and antioxidant defence in chloroplasts of the extreme halophyte Eutrema parvulum (Thellungiella parvula) under salinity

    PubMed Central

    Uzilday, Baris; Ozgur, Rengin; Sekmen, A. Hediye; Yildiztugay, Evren; Turkan, Ismail

    2015-01-01

    Background and Aims Eutrema parvulum (synonym, Thellungiella parvula) is an extreme halophyte that thrives in high salt concentrations (100–150 mm) and is closely related to Arabidopsis thaliana. The main aim of this study was to determine how E. parvulum uses reactive oxygen species (ROS) production, antioxidant systems and redox regulation of the electron transport system in chloroplasts to tolerate salinity. Methods Plants of E. parvulum were grown for 30 d and then treated with either 50, 200 or 300 mm NaCl. Physiological parameters including growth and water relationships were measured. Activities of antioxidant enzymes were determined in whole leaves and chloroplasts. In addition, expressions of chloroplastic redox components such as ferrodoxin thioredoxin reductases (FTR), NADPH thioredoxin reductases (NTRC), thioredoxins (TRXs) and peroxiredoxins (PRXs), as well as genes encoding enzymes of the water–water cycle and proline biosynthesis were measured. Key Results Salt treatment affected water relationships negatively and the accumulation of proline was increased by salinity. E. parvulum was able to tolerate 300 mm NaCl over long periods, as evidenced by H2O2 content and lipid peroxidation. While Ca2+ and K+ concentrations were decreased by salinity, Na+ and Cl– concentrations increased. Efficient induction of activities and expressions of water–water cycle enzymes might prevent accumulation of excess ROS in chloroplasts and therefore protect the photosynthetic machinery in E. parvulum. The redox homeostasis in chloroplasts might be achieved by efficient induction of expressions of redox regulatory enzymes such as FTR, NTRC, TRXs and PRXs under salinity. Conclusions E. parvulum was able to adapt to osmotic stress by an efficient osmotic adjustment mechanism involving proline and was able to regulate its ion homeostasis. In addition, efficient induction of water–water cycle enzymes and other redox regulatory components such as TRXs and PRXs in

  20. Production of therapeutic proteins in the chloroplast of Chlamydomonas reinhardtii

    PubMed Central

    2014-01-01

    Chloroplast transformation in the photosynthetic alga Chlamydomonas reinhardtii has been used to explore the potential to use it as an inexpensive and easily scalable system for the production of therapeutic recombinant proteins. Diverse proteins, such as bacterial and viral antigens, antibodies and, immunotoxins have been successfully expressed in the chloroplast using endogenous and chimeric promoter sequences. In some cases, proteins have accumulated to high level, demonstrating that this technology could compete with current production platforms. This review focuses on the works that have engineered the chloroplast of C. reinhardtii with the aim of producing recombinant proteins intended for therapeutical use in humans or animals. PMID:25136510

  1. Production of therapeutic proteins in the chloroplast of Chlamydomonas reinhardtii.

    PubMed

    Almaraz-Delgado, Alma Lorena; Flores-Uribe, José; Pérez-España, Víctor Hugo; Salgado-Manjarrez, Edgar; Badillo-Corona, Jesús Agustín

    2014-01-01

    Chloroplast transformation in the photosynthetic alga Chlamydomonas reinhardtii has been used to explore the potential to use it as an inexpensive and easily scalable system for the production of therapeutic recombinant proteins. Diverse proteins, such as bacterial and viral antigens, antibodies and, immunotoxins have been successfully expressed in the chloroplast using endogenous and chimeric promoter sequences. In some cases, proteins have accumulated to high level, demonstrating that this technology could compete with current production platforms. This review focuses on the works that have engineered the chloroplast of C. reinhardtii with the aim of producing recombinant proteins intended for therapeutical use in humans or animals.

  2. Origins of prokaryotes, eukaryotes, mitochondria, and chloroplasts

    NASA Technical Reports Server (NTRS)

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

    1978-01-01

    A computer branching model is used to analyze cellular evolution. Attention is given to certain key amino acids and nucleotide residues (ferredoxin, 5s ribosomal RNA, and c-type cytochromes) because of their commonality over a wide variety of cell types. Each amino acid or nucleotide residue is a sequence in an inherited biological trait; and the branching method is employed to align sequences so that changes reflect substitution of one residue for another. Based on the computer analysis, the symbiotic theory of cellular evolution is considered the most probable. This theory holds that organelles, e.g., mitochondria and chloroplasts invaded larger bodies, e.g., bacteria, and combined functions to form eucaryotic cells.

  3. Chloroplast Phylogenomic Inference of Green Algae Relationships.

    PubMed

    Sun, Linhua; Fang, Ling; Zhang, Zhenhua; Chang, Xin; Penny, David; Zhong, Bojian

    2016-02-05

    The green algal phylum Chlorophyta has six diverse classes, but the phylogenetic relationship of the classes within Chlorophyta remains uncertain. In order to better understand the ancient Chlorophyta evolution, we have applied a site pattern sorting method to study compositional heterogeneity and the model fit in the green algal chloroplast genomic data. We show that the fastest-evolving sites are significantly correlated with among-site compositional heterogeneity, and these sites have a much poorer fit to the evolutionary model. Our phylogenomic analyses suggest that the class Chlorophyceae is a monophyletic group, and the classes Ulvophyceae, Trebouxiophyceae and Prasinophyceae are non-monophyletic groups. Our proposed phylogenetic tree of Chlorophyta will offer new insights to investigate ancient green algae evolution, and our analytical framework will provide a useful approach for evaluating and mitigating the potential errors of phylogenomic inferences.

  4. An organelle-free assay for pea chloroplast Mg-chelatase: Resolution of the activity into soluble and membrane bound fractions

    SciTech Connect

    Walker, C.J.; Weinstein, J.D. )

    1991-05-01

    Mg-chelatase, which catalyzes the insertion of magnesium into protoporphyrin, lies at the branchpoint of heme and chlorophyll biosynthesis in chloroplasts. Since magnesium chelation is the first step unique to chlorophyll synthesis, one would expect this step to be highly regulated. However, to date little is known about the enzymology or regulation of Mg-chelatase due mostly to an inability to assay it's activity outside of the intact plastid. Here the authors report the first truly in vitro i.e. organelle-free, assay for Mg-chelatase. Mg-chelatase activity in intact pea chloroplasts which is 3 to 4 fold higher than in cucumber chloroplasts, survived chloroplast lysis and could be fractionated, by centrifugation, into supernatant and pellet components. Both of these fractions were required to reconstitute Mg-chelatase activity and both were inactivated by boiling; indicating that the enzyme is composed of soluble and membrane bound protein(s). The specific activity of the reconstituted system was typically 1 nmol Mg-Deuteroporphyrin/h/mg protein and activity was linear for at least 60 min under our assay conditions. ATP and magnesium were required for Mg-chelatase activity. The soluble component could be fractionated with ammonium sulfate. The product of the reaction was confirmed fluorometrically as the magnesium chelate of the porphyrin substrate. Crude separation of chloroplast membranes into thylakoids and envelopes, suggested that the membrane-bound component of Mg-chelatase is probably located in the envelope.

  5. Chloroplast dysfunction causes multiple defects in cell cycle progression in the Arabidopsis crumpled leaf mutant.

    PubMed

    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

    2014-09-01

    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.

  6. Transport of Ions Across the Inner Envelope Membrane of Chloroplasts

    SciTech Connect

    McCarty, R. E.

    2004-06-02

    The technical report outlines the results of nine years of research on how ions cross the inner envelope membrane of chloroplasts. The ions include protons, nitrite, calcium and ferrous iron. Bicarbonate transport was also studied.

  7. Synthesis of Proteins by Isolated Euglena gracilis Chloroplasts 1

    PubMed Central

    Vasconcelos, Aurea C.

    1976-01-01

    Intact Euglena gracilis chloroplasts, which had been purified on gradients of silica sol, incorporated [35S]methionine or [3H]leucine into soluble and membrane-bound products, using light as the only source of energy. The chloroplasts were osmotically shocked, fractionated on discontinuous gradients of sucrose, and the products of protein synthesis of the different fractions characterized by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The soluble fraction resolved into three zones of radioactivity, the major one corresponding to the large subunit or ribulose diphosphate carboxylase. The thylakoid membrane fraction contained nine labeled polypeptides, the two most prominent in the region of 31 and 42 kilodaltons. The envelope fraction contained a major radioactive peak of about 48 kilodaltons and four other minor peaks. The patterns of protein synthesis by isolated Euglena chloroplasts are broadly similar to those observed with chloroplasts of spinach and pea. PMID:16659752

  8. 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)

  9. Nitrogen control of chloroplast development and differentiation. Annual progress report

    SciTech Connect

    Schmidt, G.W.

    1991-12-01

    The growth and development of plants and photosynthetic microorganisms is commonly limited by the availability of nitrogen. Our work concerns understanding the mechanisms by which plants and algae that are subjected to nitrogen deprivation alter the composition of photosynthetic membranes and enzymes involved in photosynthetic carbon metabolism. Toward these ends, we study biosynthetic and gene expression processes in the unicellular green alga Chlamydomonas reinhardtii which is grown in an ammonium-limited continuous culture system. We have found that the expression of nuclear genes, including those encoding for light-harvesting proteins, are severely repressed in nitrogen-limited cells whereas, in general, chloroplast protein synthesis is attenuated primarily at the level of mRNA translation. Conversely, nitrogen deprivation appears to lead to enhanced synthesis of enzymes that are involved in starch and storage lipid deposition. In addition, as a possible means by which photosynthetic electron transport activities and ATP synthesis is sustained during chronic periods of nitrogen deprivation, thylakoid membranes become enriched with components for chlororespiration. Characterization of the chlororespiratory electron transport constituents, including cytochrome complexes and NAD(P)H dehydrogenase is a major current effort. Also, we are striving to isolate the genes encoding chlororespiration proteins toward determining how they and others that are strongly responsive to nutrient availability are regulated.

  10. A Nucleus-Encoded Chloroplast Protein YL1 Is Involved in Chloroplast Development and Efficient Biogenesis of Chloroplast ATP Synthase in Rice

    PubMed Central

    Chen, Fei; Dong, Guojun; Wu, Limin; Wang, Fang; Yang, Xingzheng; Ma, Xiaohui; Wang, Haili; Wu, Jiahuan; Zhang, Yanli; Wang, Huizhong; Qian, Qian; Yu, Yanchun

    2016-01-01

    Chloroplast ATP synthase (cpATPase) is an importance thylakoid membrane-associated photosynthetic complex involved in the light-dependent reactions of photosynthesis. In this study, we isolated and characterized a rice (Oryza sativa) mutant yellow leaf 1 (yl1), which exhibits chlorotic leaves throughout developmental stages. The YL1 mutation showed reduced chlorophyll contents, abnormal chloroplast morphology, and decreased photochemical efficiency. Moreover, YL1 deficiency disrupts the expression of genes associated with chloroplast development and photosynthesis. Molecular and genetic analyses revealed that YL1 is a nucleus-encoded protein with a predicted transmembrane domain in its carboxyl-terminus that is conserved in the higher plant kingdom. YL1 localizes to chloroplasts and is preferentially expressed in green tissues containing chloroplasts. Immunoblot analyses showed that inactivation of YL1 leads to drastically reduced accumulation of AtpA (α) and AtpB (β), two core subunits of CF1αβ subcomplex of cpATPase, meanwhile, a severe decrease (ca. 41.7%) in cpATPase activity was observed in the yl1-1 mutant compared with the wild type. Furthermore, yeast two-hybrid and bimolecular fluorescence complementation assays revealed a specific interaction between YL1 and AtpB subunit of cpATPase. Taken together, our results suggest that YL1 is a plant lineage-specific auxiliary factor involved in the biogenesis of the cpATPase complex, possibly via interacting with the β-subunit. PMID:27585744

  11. Complete Chloroplast Genome of Tanaecium tetragonolobum: The First Bignoniaceae Plastome

    PubMed Central

    Nazareno, Alison Gonçalves; Carlsen, Monica; Lohmann, Lúcia Garcez

    2015-01-01

    Bignoniaceae is a Pantropical plant family that is especially abundant in the Neotropics. Members of the Bignoniaceae are diverse in many ecosystems and represent key components of the Tropical flora. Despite the ecological importance of the Bignoniaceae and all the efforts to reconstruct the phylogeny of this group, whole chloroplast genome information has not yet been reported for any members of the family. Here, we report the complete chloroplast genome sequence of Tanaecium tetragonolobum (Jacq.) L.G. Lohmann, which was reconstructed using de novo and referenced-based assembly of single-end reads generated by shotgun sequencing of total genomic DNA in an Illumina platform. The gene order and organization of the chloroplast genome of T. tetragonolobum exhibits the general structure of flowering plants, and is similar to other Lamiales chloroplast genomes. The chloroplast genome of T. tetragonolobum is a circular molecule of 153,776 base pairs (bp) with a quadripartite structure containing two single copy regions, a large single copy region (LSC, 84,612 bp) and a small single copy region (SSC, 17,586 bp) separated by inverted repeat regions (IRs, 25,789 bp). In addition, the chloroplast genome of T. tetragonolobum has 38.3% GC content and includes 121 genes, of which 86 are protein-coding, 31 are transfer RNA, and four are ribosomal RNA. The chloroplast genome of T. tetragonolobum presents a total of 47 tandem repeats and 347 simple sequence repeats (SSRs) with mononucleotides being the most common and di-, tri-, tetra-, and hexanucleotides occurring with less frequency. The results obtained here were compared to other chloroplast genomes of Lamiales available to date, providing new insight into the evolution of chloroplast genomes within Lamiales. Overall, the evolutionary rates of genes in Lamiales are lineage-, locus-, and region-specific, indicating that the evolutionary pattern of nucleotide substitution in chloroplast genomes of flowering plants is complex

  12. 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.

  13. [Chloroplast genetic engineering: a new approach in plant biotechnology].

    PubMed

    Su, Tao; Zhan, Ya-Guang; Han, Mei; Hao, Ai-Ping

    2005-07-01

    Chloroplast genetic engineering, offers several advantages over nuclear transformation, including high level of gene expression, increased biosafety, remedying some limitations associated with nuclear genetic transformation, such as gene silencing and the stability of transformed genes. It is now regarded as an attractive new transgenic technique and further development of biotechnology in agriculture. In this article we reviewed the characteristics, applications of chloroplast genetic engineering and its promising prospects were discussed.

  14. Surveying the Oligomeric State of Arabidopsis thaliana Chloroplasts.

    PubMed

    Lundquist, Peter K; Mantegazza, Otho; Stefanski, Anja; Stühler, Kai; Weber, Andreas P M

    2017-01-09

    Blue native-PAGE (BN-PAGE) resolves protein complexes in their native state. When combined with immunoblotting, it can be used to identify the presence of high molecular weight complexes at high resolution for any protein, given a suitable antibody. To identify proteins in high molecular weight complexes on a large scale and to bypass the requirement for specific antibodies, we applied a tandem mass spectrometry (MS/MS) approach to BN-PAGE-resolved chloroplasts. Fractionation of the gel into six bands allowed identification and label-free quantification of 1000 chloroplast proteins with native molecular weight separation. Significantly, this approach achieves a depth of identification comparable with traditional shotgun proteomic analyses of chloroplasts, indicating much of the known chloroplast proteome is amenable to MS/MS identification under our fractionation scheme. By limiting the number of fractionation bands to six, we facilitate scaled-up comparative analyses, as we demonstrate with the reticulata chloroplast mutant displaying a reticulated leaf phenotype. Our comparative proteomics approach identified a candidate interacting protein of RETICULATA as well as effects on lipid remodeling proteins, amino acid metabolic enzymes, and plastid division machinery. We additionally highlight selected proteins from each sub-compartment of the chloroplast that provide novel insight on known or hypothesized protein complexes to further illustrate the utility of this approach. Our results demonstrate the high sensitivity and reproducibility of this technique, which is anticipated to be widely adaptable to other sub-cellular compartments.

  15. Role of mitochondria in sulfolipid biosynthesis by Euglena chloroplasts

    SciTech Connect

    Saidha, T.; Schiff, J.A.

    1987-04-01

    Sulfate activation occurs in Euglena mitochondria the authors now find that the sulfate activating enzymes are absent from Euglena chloroplasts. Cells of mutant W/sub 10/BSmL lacking plastids also lack detectable sulfolipid (SL) when grown on /sup 35/SO/sub 4//sup 2 -/ indicating that SL is absent from the mitochondria and is exclusively in the plastids. Plastids alone will convert /sup 35/S-cysteine to /sup 35/SL in the presence of ATP and Mg/sup 2 +/; light is stimulatory. Under similar conditions, chloroplasts and mitochondria incubated together convert /sup 35/SO/sub 4//sup 2 -/ to plastid-localized /sup 35/SL but either organelle incubated alone fails to effect this conversion. Unlabeled cysteine blocks SL labeling from sulfate in the mixed incubation; since cysteine is formed from sulfate by Euglena mitochrondria, cysteine (and other compounds) may move from the mitochondrion to the chloroplast to provide the sulfo group for SL formation. Although mitochondria form labeled protein from /sup 35/SO/sub 4//sup 2 -/ via cysteine, chloroplasts alone do not form labeled protein from /sup 35/SO/sub 4//sup 2 -/, ATP and Mg/sup 2 +/ in light or darkness; incubation of chloroplasts plus mitochondria under these conditions labels chloroplast protein.

  16. Chloroplasts continuously monitor photoreceptor signals during accumulation movement.

    PubMed

    Tsuboi, Hidenori; Wada, Masamitsu

    2013-07-01

    Under low light conditions, chloroplasts gather at a cell surface to maximize light absorption for efficient photosynthesis, which is called the accumulation response. Phototropin1 (phot1) and phototropin2 (phot2) were identified as blue light photoreceptors in the accumulation response that occurs in Arabidopsis thaliana and Adiantum capillus-veneris with neochrome1 (neo1) as a red light photoreceptor in A. capillus-veneris. However, the signal molecule that is emitted from the photoreceptors and transmitted to the chloroplasts is not known. To investigate this topic, the accumulation response was induced by partial cell irradiation with a microbeam of red, blue and far-red light in A. capillus-veneris gametophyte cells. Chloroplasts moved towards the irradiated region and were able to sense the signal as long as its signal flowed. The signal from neo1 had a longer life than the signal that came from phototropins. When two microbeams with the same wavelength and the same fluence rate were placed 20 μm apart from each other and were applied to a dark-adapted cell, chloroplasts at an equidistant position always moved towards the center (midpoint) of the two microbeams, but not towards either one. This result indicates that chloroplasts are detecting the concentration of the signal but not the direction of signal flow. Chloroplasts repeatedly move and stop at roughly 10 s intervals during the accumulation response, suggesting that they monitor the intermittent signal waves from photoreceptors.

  17. Interaction of actin and the chloroplast protein import apparatus.

    PubMed

    Jouhet, Juliette; Gray, John C

    2009-07-10

    Actin filaments are major components of the cytoskeleton and play numerous essential roles, including chloroplast positioning and plastid stromule movement, in plant cells. Actin is present in pea chloroplast envelope membrane preparations and is localized at the surface of the chloroplasts, as shown by agglutination of intact isolated chloroplasts by antibodies to actin. To identify chloroplast envelope proteins involved in actin binding, we have carried out actin co-immunoprecipitation and co-sedimentation experiments on detergent-solubilized pea chloroplast envelope membranes. Proteins co-immunoprecipitated with actin were identified by mass spectrometry and by Western blotting and included the Toc159, Toc75, Toc34, and Tic110 components of the TOC-TIC protein import apparatus. A direct interaction of actin with Escherichia coli-expressed Toc159, but not Toc33, was shown by co-sedimentation experiments, suggesting that Toc159 is the component of the TOC complex that interacts with actin on the cytosolic side of the outer envelope membrane. The physiological significance of this interaction is unknown, but it may play a role in the import of nuclear-encoded photosynthesis proteins.

  18. Speed of signal transfer in the chloroplast accumulation response.

    PubMed

    Tsuboi, Hidenori; Wada, Masamitsu

    2010-05-01

    Chloroplast photorelocation movement is important for plants to perform efficient photosynthesis. Phototropins were identified as blue-light receptors for chloroplast movement in Arabidopsis thaliana and in the fern Adiantum capillus-veneris, whereas neochrome functions as a dual red/blue light receptor in the latter. However, the signal transduction pathways involved in chloroplast movement remain to be clarified. To investigate the kinetic properties of signalling from these photoreceptors to the chloroplasts, we deduced the speed of signal transfer using Adiantum capillus-veneris gametophytes. When a region of dark-adapted gametophyte cells was subjected to microbeam irradiation, chloroplasts moved towards the irradiated area even in subsequent darkness. We therefore recorded the movement and calculated the speeds of signal transfer by time-lapse imaging. Movement speeds under red or blue light were similar, e.g., about 1.0 microm min(-1) in prothallial cells. However, speeds varied according to cell polarity in protonemal cells. The speed of signal transfer from the protonemal apex to the base was approximately 0.7 microm min(-1), but roughly 2.3 microm min(-1) in the opposite direction. The speed of signal transfer in Arabidopsis thaliana mesophyll cells was approximately 0.8 microm min(-1) by comparison. Surprisingly, chloroplasts located farthest away from the microbeam were found to move faster than those in close proximity to the site of irradiation both in Adiantum capillus-veneris and A. thaliana.

  19. Novel mechanisms for maturation of chloroplast transfer RNA precursors

    PubMed Central

    Wang, Ming Jing; Davis, N. Wayne; Gegenheimer, Peter

    1988-01-01

    Despite the prokaryotic origins of chloroplasts, a plant chloroplast tRNA precursor is processed in a homologous in vitro system by a pathway distinct from that observed in Escherichia coli, but identical to that utilized for maturation of nuclear pre-tRNAs. The mature tRNA 5' terminus is generated by the site-specific endonucleolytic cleavage of an RNase P (or P-type) activity. The 3' end is likewise produced by a single precise endonucleolytic cut at the 3' terminus of the encoded tRNA domain. This is the first complete structural characterization of an organellar tRNA processing system using a homologous substrate. In contrast to eubacterial RNase P, chloroplast RNase P does not appear to contain an RNA subunit. The chloroplast activity bands with bulk protein at 1.28 g/ml in CsCI density gradients, whereas E.coli RNase P bands as ribonucleoprotein at 1.73 g/ml. Chloroplast RNase P activity survives treatment with micrococcal nuclease (MN) at levels 10- to 100-fold higher than those required to totally inactivate the E.coli enzyme. The chloroplast system is sensitive to a suppression of tRNA processing, caused by binding of inactive MN to pre-tRNA substrate, which is readily overcome by addition of carrier RNA to the assay. Images PMID:16453848

  20. Evidence that sigma factors are components of chloroplast RNA polymerase.

    PubMed Central

    Troxler, R F; Zhang, F; Hu, J; Bogorad, L

    1994-01-01

    Plastid genes are transcribed by DNA-dependent RNA polymerase(s), which have been incompletely characterized and have been examined in a limited number of species. Plastid genomes contain rpoA, rpoB, rpoC1, and rpoC2 coding for alpha, beta, beta', and beta" RNA polymerase subunits that are homologous to the alpha, beta, and beta' subunits that constitute the core moiety of RNA polymerase in bacteria. However, genes with homology to sigma subunits in bacteria have not been found in plastid genomes. An antibody directed against the principal sigma subunit of RNA polymerase from the cyanobacterium Anabaena sp. PCC 7120 was used to probe western blots of purified chloroplast RNA polymerase from maize, rice, Chlamydomonas reinhardtii, and Cyanidium caldarium. Chloroplast RNA polymerase from maize and rice contained an immunoreactive 64-kD protein. Chloroplast RNA polymerase from C. reinhardtii contained immunoreactive 100- and 82-kD proteins, and chloroplast RNA polymerase from C. caldarium contained an immunoreactive 32-kD protein. The elution profile of enzyme activity of both algal chloroplast RNA polymerases coeluted from DEAE with the respective immunoreactive proteins, indicating that they are components of the enzyme. These results provide immunological evidence for sigma-like factors in chloroplast RNA polymerase in higher plants and algae. PMID:8159791

  1. Thiol-Based Peroxidases and Ascorbate Peroxidases: Why Plants Rely on Multiple Peroxidase Systems in the Photosynthesizing Chloroplast?

    PubMed

    Dietz, Karl-Josef

    2016-01-01

    Photosynthesis is a highly robust process allowing for rapid adjustment to changing environmental conditions. The efficient acclimation depends on balanced redox metabolism and control of reactive oxygen species release which triggers signaling cascades and potentially detrimental oxidation reactions. Thiol peroxidases of the peroxiredoxin and glutathione peroxidase type, and ascorbate peroxidases are the main peroxide detoxifying enzymes of the chloroplast. They use different electron donors and are linked to distinct redox networks. In addition, the peroxiredoxins serve functions in redox regulation and retrograde signaling. The complexity of plastid peroxidases is discussed in context of suborganellar localization, substrate preference, metabolic coupling, protein abundance, activity regulation, interactions, signaling functions, and the conditional requirement for high antioxidant capacity. Thus the review provides an opinion on the advantage of linking detoxification of peroxides to different enzymatic systems and implementing mechanisms for their inactivation to enforce signal propagation within and from the chloroplast.

  2. Thiol-Based Peroxidases and Ascorbate Peroxidases: Why Plants Rely on Multiple Peroxidase Systems in the Photosynthesizing Chloroplast?

    PubMed Central

    Dietz, Karl-Josef

    2016-01-01

    Photosynthesis is a highly robust process allowing for rapid adjustment to changing environmental conditions. The efficient acclimation depends on balanced redox metabolism and control of reactive oxygen species release which triggers signaling cascades and potentially detrimental oxidation reactions. Thiol peroxidases of the peroxiredoxin and glutathione peroxidase type, and ascorbate peroxidases are the main peroxide detoxifying enzymes of the chloroplast. They use different electron donors and are linked to distinct redox networks. In addition, the peroxiredoxins serve functions in redox regulation and retrograde signaling. The complexity of plastid peroxidases is discussed in context of suborganellar localization, substrate preference, metabolic coupling, protein abundance, activity regulation, interactions, signaling functions, and the conditional requirement for high antioxidant capacity. Thus the review provides an opinion on the advantage of linking detoxification of peroxides to different enzymatic systems and implementing mechanisms for their inactivation to enforce signal propagation within and from the chloroplast. PMID:26810073

  3. Chloroplast microsatellite markers for Pseudotaxus chienii developed from the whole chloroplast genome of Taxus chinensis var. mairei (Taxaceae)1

    PubMed Central

    Deng, Qi; Zhang, Hanrui; He, Yipeng; Wang, Ting; Su, Yingjuan

    2017-01-01

    Premise of the study: Pseudotaxus chienii (Taxaceae) is an old rare species endemic to China that has adapted well to ecological heterogeneity with high genetic diversity in its nuclear genome. However, the genetic variation in its chloroplast genome is unknown. Methods and Results: Eighteen chloroplast microsatellite markers (cpSSRs) were developed from the whole chloroplast genome of Taxus chinensis var. mairei and successfully amplified in four P. chienii populations and one T. chinensis var. mairei population. Of these loci, 10 were polymorphic in P. chienii, whereas six were polymorphic in T. chinensis var. mairei. The unbiased haploid diversity per locus ranged from 0.000 to 0.641 and 0.000 to 0.545 for P. chienii and T. chinensis var. mairei, respectively. Conclusions: The 18 cpSSRs will be used to further investigate the chloroplast genetic structure and adaptive evolution in P. chienii populations. PMID:28337394

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

    PubMed

    Suetsugu, Noriyuki; Higa, Takeshi; Wada, Masamitsu

    2016-11-17

    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.

  5. Evidence for chloroplastic succinate dehydrogenase participating in the chloroplastic respiratory and photosynthetic electron transport chains of Chlamydomonas reinhardtii

    SciTech Connect

    Willeford, K.O.; Gombos, Z.; Gibbs, M. )

    1989-07-01

    A method for isolating intact chloroplasts from Chlamydomonas reinhardtii F-60 was developed from the Klein, Chen, Gibbs, Platt-Aloia procedure. Protoplasts, generated by treatment with autolysine, were lysed with a solution of digitonin and fractionated on Percoll step gradients. The chloroplasts were assessed to be 90% intact (ferricyanide assay) and free from cytoplasmic contamination (NADP isocitrate dehydrogenase activity) and to range from 2 to 5% in mitochondrial contamination (cytochrome c oxidase activity). About 25% of the cellular succinate dehydrogenase activity (21.6 micromoles per milligram chlorophyll per hour, as determined enzymically) was placed within the chloroplast. Chloroplastic succinate dehydrogenase had a K{sub m} for succinate of 0.55 millimolar and was associated with the thylakoidal material derived from the intact chloroplasts. This same thylakoidal material, with an enzymic assay of 21.6 micromoles per milligram chlorophyll per hour was able to initiate a light-dependent uptake of oxygen at a rate of 16.4 micromoles per milligram chlorophyll per hour when supplied with succinate and methyl viologen. Malonate was an apparent competitive inhibitor of this reaction. The succinate dehydrogenase activity present in the chloroplast was sufficient to account for the photoanaerobic rate of acetate dissimilation in H{sub 2} adapted Chlamydomonas.

  6. Hydrogen peroxide generated by NADPH oxidase is involved in high blue-light-induced chloroplast avoidance movements in Arabidopsis

    NASA Astrophysics Data System (ADS)

    Wen, Feng; Xing, Da; Zhang, Lingrui

    2009-08-01

    One of the most important functions of blue light is to induce chloroplast movements by reducing the damage to photosynthetic machinery under excess light. Hydrogen peroxide (H2O2), generated by various environmental stimuli, can act as a signaling molecule that regulates a number of developmental processes and environmental responses. To investigate whether H2O2 is involved in high blue light-induced chloroplast avoidance movements, we use luminescence spectrometer to observe H2O2 generation with the assistance of the fluorescence probe dichlorofluorescin diacetate (H2DCF-DA). After treatment with high blue light, a large quantity of H2O2 indicated by the fluorescence intensity of DCF is produced in a dose-dependent manner in leaf strip of Arabidopsis. Enzymatic assay shows that the activity of NADPH oxidase, which is a major site for H2O2 generation, also rapidly increases in treated strips. Exogenously applied H2O2 can promote the high blue light-induced chloroplast movements. Moreover, high blue light-induced H2O2 generation can be abolished completely by addition of exogenous catalase (CAT), and partly by diphenylene iodonium (DPI) and dichlorophenyl dimethylurea (DCMU), which are an NADPH oxidase inhibitor and a blocker of electron transport chain. And subsequent chloroplast movements can be abolished by CAT and DPI, but not by DCMU. These results presented here suggested that high blue light can induce oxidative burst, and NADPH oxidase as a major producer for H2O2 is involved in blue light-induced chloroplast avoidance movements.

  7. A chloroplast lipoxygenase is required for wound-induced jasmonic acid accumulation in Arabidopsis.

    PubMed

    Bell, E; Creelman, R A; Mullet, J E

    1995-09-12

    Plant lipoxygenases are thought to be involved in the biosynthesis of lipid-derived signaling molecules. The potential involvement of a specific Arabidopsis thaliana lipoxygenase isozyme, LOX2, in the biosynthesis of the plant growth regulators jasmonic acid (JA) and abscisic acid was investigated. Our characterization of LOX2 indicates that the protein is targeted to chloroplasts. The physiological role of this chloroplast lipoxygenase was analyzed in transgenic plants where cosuppression reduced LOX2 accumulation. The reduction in LOX2 levels caused no obvious changes in plant growth or in the accumulation of abscisic acid. However, the wound-induced accumulation of JA observed in control plants was absent in leaves of transgenic plants that lacked LOX2. Thus, LOX2 is required for the wound-induced synthesis of the plant growth regulator JA in leaves. We also examined the expression of a wound- and JA-inducible Arabidopsis gene, vsp, in transgenic and control plants. Leaves of transgenic plants lacking LOX2 accumulated less vsp mRNA than did control leaves in response to wounding. This result suggests that wound-induced JA (or some other LOX2-requiring component of the wound response pathway) is involved in the wound-induced regulation of this gene.

  8. Dimorphic chloroplasts in the epidermis of Podostemoideae, a subfamily of the unique aquatic angiosperm family Podostemaceae.

    PubMed

    Fujinami, Rieko; Yoshihama, Isao; Imaichi, Ryoko

    2011-09-01

    Plants of the Podostemoideae, a subfamily of the unique aquatic angiosperm family Podostemaceae, which are found in rapids and waterfalls of the tropics and subtropics, have two different sizes of chloroplasts in their epidermis. These small and large chloroplasts are located separately in each epidermal cell along its upper and inner tangential walls, respectively. This is the first case of the chloroplast dimorphism in a single epidermal cell of angiosperms. While the large chloroplasts have well developed starch grains, the small chloroplasts have a normal granal ultrastructure but very few starch grains. This suggests that the small chloroplasts mainly function in CO(2) uptake for photosynthesis from torrential water.

  9. Nucleus-encoded light-harvesting chlorophyll a/b proteins are imported normally into chlorophyll b-free chloroplasts of Arabidopsis.

    PubMed

    Nick, Sabine; Meurer, Jörg; Soll, Jürgen; Ankele, Elisabeth

    2013-05-01

    Chloroplast-located proteins which are encoded by the nuclear genome have to be imported from the cytosol into the organelle in a posttranslational manner. Among these nuclear-encoded chloroplast proteins are the light-harvesting chlorophyll a/b-binding proteins (LHCPs). After translation in the cytosol, precursor proteins of LHCPs are imported via the TOC/TIC translocase, processed to their mature size to insert into thylakoid membranes where they recruit chlorophylls a and b to form pigment-protein complexes. The translocation of proteins is a highly regulated process which employs several regulators. To analyze whether CAO (chlorophyll a oxigenase) which converts chlorophyll a to chlorophyll b at the inner chloroplast membrane, is one of these regulators, we performed import reactions utilizing a homozygous loss-of-function mutant (cao-1). We imported in vitro translated and (35)S-labeled precursor proteins of light-harvesting proteins of photosystem II LHCB1, LHCB4, and LHCB5 into chloroplasts isolated from cao-1 and show that import of precursor proteins and their processing to mature forms are not impaired in the mutant. Therefore, regulation of the import machinery cannot be responsible for the decreased steady-state levels of light-harvesting complex (LHC) proteins. Regulation does not take place at the transcriptional level either, because Lhcb mRNAs are not down-regulated. Additionally, reduced steady-state levels of LHCPs also do not occur due to posttranslational turnover of non-functional LHCPs in chloroplasts. Taken together, our data show that plants in the absence of CAO and therefore devoid of chlorophyll b are not influenced in their import behavior of LHC proteins.

  10. The stay-green phenotype of TaNAM-RNAi wheat plants is associated with maintenance of chloroplast structure and high enzymatic antioxidant activity.

    PubMed

    Checovich, Mariana L; Galatro, Andrea; Moriconi, Jorge I; Simontacchi, Marcela; Dubcovsky, Jorge; Santa-María, Guillermo E

    2016-07-01

    TaNAM transcription factors play an important role in controlling senescence, which in turn, influences the delivery of nitrogen, iron and other elements to the grain of wheat (Triticum aestivum) plants, thus contributing to grain nutritional value. While lack or diminished expression of TaNAMs determines a stay-green phenotype, the precise effect of these factors on chloroplast structure has not been studied. In this work we focused on the events undergone by chloroplasts in two wheat lines having either control or diminished TaNAM expression due to RNA interference (RNAi). It was found that in RNAi plants maintenance of chlorophyll levels and maximal photochemical efficiency of photosystem II were associated with lack of chloroplast dismantling. Flow cytometer studies and electron microscope analysis showed that RNAi plants conserved organelle ultrastructure and complexity. It was also found that senescence in control plants was accompanied by a low leaf enzymatic antioxidant activity. Lack of chloroplast dismantling in RNAi plants was associated with maintenance of protein and iron concentration in the flag leaf, the opposite being observed in control plants. These data provide a structural basis for the observation that down regulation of TaNAMs confers a functional stay-green phenotype and indicate that the low export of iron and nitrogen from the flag leaf of these plants is concomitant, within the developmental window studied, with lack of chloroplast degradation and high enzymatic antioxidant activity.

  11. Chloroplast genome structure in Ilex (Aquifoliaceae)

    PubMed Central

    Yao, Xin; Tan, Yun-Hong; Liu, Ying-Ying; Song, Yu; Yang, Jun-Bo; Corlett, Richard T.

    2016-01-01

    Aquifoliaceae is the largest family in the campanulid order Aquifoliales. It consists of a single genus, Ilex, the hollies, which is the largest woody dioecious genus in the angiosperms. Most species are in East Asia or South America. The taxonomy and evolutionary history remain unclear due to the lack of a robust species-level phylogeny. We produced the first complete chloroplast genomes in this family, including seven Ilex species, by Illumina sequencing of long-range PCR products and subsequent reference-guided de novo assembly. These genomes have a typical bicyclic structure with a conserved genome arrangement and moderate divergence. The total length is 157,741 bp and there is one large single-copy region (LSC) with 87,109 bp, one small single-copy with 18,436 bp, and a pair of inverted repeat regions (IR) with 52,196 bp. A total of 144 genes were identified, including 96 protein-coding genes, 40 tRNA and 8 rRNA. Thirty-four repetitive sequences were identified in Ilex pubescens, with lengths >14 bp and identity >90%, and 11 divergence hotspot regions that could be targeted for phylogenetic markers. This study will contribute to improved resolution of deep branches of the Ilex phylogeny and facilitate identification of Ilex species. PMID:27378489

  12. Photonic multilayer structure of Begonia chloroplasts enhances photosynthetic efficiency.

    PubMed

    Jacobs, Matthew; Lopez-Garcia, Martin; Phrathep, O-Phart; Lawson, Tracy; Oulton, Ruth; Whitney, Heather M

    2016-10-24

    Enhanced light harvesting is an area of interest for optimizing both natural photosynthesis and artificial solar energy capture(1,2). Iridescence has been shown to exist widely and in diverse forms in plants and other photosynthetic organisms and symbioses(3,4), but there has yet to be any direct link demonstrated between iridescence and photosynthesis. Here we show that epidermal chloroplasts, also known as iridoplasts, in shade-dwelling species of Begonia(5), notable for their brilliant blue iridescence, have a photonic crystal structure formed from a periodic arrangement of the light-absorbing thylakoid tissue itself. This structure enhances photosynthesis in two ways: by increasing light capture at the predominantly green wavelengths available in shade conditions, and by directly enhancing quantum yield by 5-10% under low-light conditions. These findings together imply that the iridoplast is a highly modified chloroplast structure adapted to make best use of the extremely low-light conditions in the tropical forest understorey in which it is found(5,6). A phylogenetically diverse range of shade-dwelling plant species has been found to produce similarly structured chloroplasts(7-9), suggesting that the ability to produce chloroplasts whose membranes are organized as a multilayer with photonic properties may be widespread. In fact, given the well-established diversity and plasticity of chloroplasts(10,11), our results imply that photonic effects may be important even in plants that do not show any obvious signs of iridescence to the naked eye but where a highly ordered chloroplast structure may present a clear blue reflectance at the microscale. Chloroplasts are generally thought of as purely photochemical; we suggest that one should also think of them as a photonic structure with a complex interplay between control of light propagation, light capture and photochemistry.

  13. Chloroplast DNA Sequence Homologies among Vascular Plants 1

    PubMed Central

    Lamppa, Gayle K.; Bendich, Arnold J.

    1979-01-01

    The extent of sequence conservation in the chloroplast genome of higher plants has been investigated. Supercoiled chloroplast DNA, prepared from pea seedlings, was labeled in vitro and used as a probe in reassociation experiments with a high concentration of total DNAs extracted from several angiosperms, gymnosperms, and lower vascular plants. In each case the probe reassociation was accelerated, demonstrating that some chloroplast sequences have been highly conserved throughout the evolution of vascular plants. Only among the flowering plants were distinct levels of cross-reaction with the pea chloroplast probe evident; broad bean and barley exhibited the highest and lowest levels, respectively. With the hydroxylapatite assay these levels decreased with a decrease in probe fragment length (from 1,860 to 735 bases), indicating that many conserved sequences in the chloroplast genome are separated by divergent sequences on a rather fine scale. Despite differences observed in levels of homology with the hydroxylapatite assay, S1 nuclease analysis of heteroduplexes showed that outside of the pea family the extent of sequence relatedness between the probe and various heterologous DNAs is approximately the same: 30%. In our interpretation, the fundamental changes in the chloroplast genome during angiosperm evolution involved the rearrangement of this 30% with respect to the more rapidly changing sequences of the genome. These rearrangements may have been more extensive in dicotyledons than in monocotyledons. We have estimated the amount of conserved and divergent DNA interspersed between one another. From the reassociation experiments, determinations were made of the percentage of chloroplast DNA in total DNA extracts from different higher plants; this value remained relatively constant when compared with the large variation in the diploid genome size of the plants. PMID:16660786

  14. Transplastomic integration of a cyanobacterial bicarbonate transporter into tobacco chloroplasts.

    PubMed

    Pengelly, J J L; Förster, B; von Caemmerer, S; Badger, M R; Price, G D; Whitney, S M

    2014-07-01

    Improving global yields of agricultural crops is a complex challenge with evidence indicating benefits in productivity are achieved by enhancing photosynthetic carbon assimilation. Towards improving rates of CO2 capture within leaf chloroplasts, this study shows the versatility of plastome transformation for expressing the Synechococcus PCC7002 BicA bicarbonate transporter within tobacco plastids. Fractionation of chloroplast membranes from transplastomic tob(BicA) lines showed that ~75% of the BicA localized to the thylakoid membranes and ~25% to the chloroplast envelope. BicA levels were highest in young emerging tob(BicA) leaves (0.12 μmol m(-2), ≈7mg m(-2)) accounting for ~0.1% (w/w) of the leaf protein. In these leaves, the molar amount of BicA was 16-fold lower than the abundant thylakoid photosystem II D1 protein (~1.9 μmol m(-2)) which was comparable to the 9:1 molar ratio of D1:BicA measured in air-grown Synechococcus PCC7002 cells. The BicA produced had no discernible effect on chloroplast ultrastructure, photosynthetic CO2-assimilation rates, carbon isotope discrimination, or growth of the tob(BicA) plants, implying that the bicarbonate transporter had little or no activity. These findings demonstrate the utility of plastome transformation for targeting bicarbonate transporter proteins into the chloroplast membranes without impeding growth or plastid ultrastructure. This study establishes the span of experimental measurements required to verify heterologous bicarbonate transporter function and location in chloroplasts and underscores the need for more detailed understanding of BicA structure and function to identify solutions for enabling its activation and operation in leaf chloroplasts.

  15. Chloroplasts as a nitric oxide cellular source. Effect of reactive nitrogen species on chloroplastic lipids and proteins.

    PubMed

    Jasid, Sebastián; Simontacchi, Marcela; Bartoli, Carlos G; Puntarulo, Susana

    2006-11-01

    Nitric oxide (NO) generation by soybean (Glycine max var. ADM 4800) chloroplasts was studied as an endogenous product assessed by the electron paramagnetic resonance spin-trapping technique. Nitrite and l-arginine (Arg) are substrates for enzymatic activities considered to be the possible sources of NO in plants. Soybean chloroplasts showed a NO production of 3.2 +/- 0.2 nmol min(-1) mg(-1) protein in the presence of 1 mm NaNO(2). Inhibition of photosynthetic electron flow by 3-(3,4-dichlorophenyl)-1,1-dimethyl urea resulted in a lower rate (1.21 +/- 0.04 nmol min(-1) mg(-1) protein) of NO generation. Chloroplasts incubated with 1 mm Arg showed NO production of 0.76 +/- 0.04 nmol min(-1) mg(-1) protein that was not affected either by omission of Ca(2+) or by supplementation with Ca(2+) and calmodulin to the incubation medium. This production was inhibited when chloroplasts were incubated in the presence of NO synthase inhibitors N(omega)-nitro-l-Arg methyl ester hydrochloride and N(omega)-nitro-l-Arg. In vitro exposure of chloroplasts to an NO donor (250 mum S-nitrosoglutathione) decreased lipid radical content in membranes by 29%; however, incubation in the presence of 25 mum peroxynitrite (ONOO(-)) led to an increase in lipid-derived radicals (34%). The effect of ONOO(-) on protein oxidation was determined by western blotting, showing an increase in carbonyl content either in stroma or thylakoid proteins as compared to controls. Moreover, ONOO(-) treatment significantly affected both O(2) evolution and chlorophyll fluorescence in thylakoids. Data reported here suggest that NO is an endogenous metabolite in soybean chloroplasts and that reactive nitrogen species could exert either antioxidant or prooxidant effects on chloroplast macromolecules.

  16. The complete chloroplast genome of Capsicum frutescens (Solanaceae)1

    PubMed Central

    Shim, Donghwan; Raveendar, Sebastin; Lee, Jung-Ro; Lee, Gi-An; Ro, Na-Young; Jeon, Young-Ah; Cho, Gyu-Taek; Lee, Ho-Sun; Ma, Kyung-Ho; Chung, Jong-Wook

    2016-01-01

    Premise of the study: We report the complete sequence of the chloroplast genome of Capsicum frutescens (Solanaceae), a species of chili pepper. Methods and Results: Using an Illumina platform, we sequenced the chloroplast genome of C. frutescens. The total length of the genome is 156,817 bp, and the overall GC content is 37.7%. A pair of 25,792-bp inverted repeats is separated by small (17,853 bp) and large (87,380 bp) single-copy regions. The C. frutescens chloroplast genome encodes 132 unique genes, including 87 protein-coding genes, 37 transfer RNA (tRNA) genes, and eight ribosomal RNA (rRNA) genes. Of these, seven genes are duplicated in the inverted repeats and 12 genes contain one or two introns. Comparative analysis with the reference chloroplast genome revealed 125 simple sequence repeat motifs and 34 variants, mostly located in the noncoding regions. Conclusions: The complete chloroplast genome sequence of C. frutescens reported here is a valuable genetic resource for Capsicum species. PMID:27213127

  17. 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

  18. The chloroplast view of the evolution of polyploid wheat.

    PubMed

    Gornicki, Piotr; Zhu, Huilan; Wang, Junwei; Challa, Ghana S; Zhang, Zhengzhi; Gill, Bikram S; Li, Wanlong

    2014-11-01

    Polyploid wheats comprise four species: Triticum turgidum (AABB genomes) and T. aestivum (AABBDD) in the Emmer lineage, and T. timopheevii (AAGG) and T. zhukovskyi (AAGGA(m) A(m) ) in the Timopheevi lineage. Genetic relationships between chloroplast genomes were studied to trace the evolutionary history of the species. Twenty-five chloroplast genomes were sequenced, and 1127 plant accessions were genotyped, representing 13 Triticum and Aegilops species. The A. speltoides (SS genome) diverged before the divergence of T. urartu (AA), A. tauschii (DD) and the Aegilops species of the Sitopsis section. Aegilops speltoides forms a monophyletic clade with the polyploid Emmer and Timopheevi wheats, which originated within the last 0.7 and 0.4 Myr, respectively. The geographic distribution of chloroplast haplotypes of the wild tetraploid wheats and A. speltoides illustrates the possible geographic origin of the Emmer lineage in the southern Levant and the Timopheevi lineage in northern Iraq. Aegilops speltoides is the closest relative of the diploid donor of the chloroplast (cytoplasm), as well as the B and G genomes to Timopheevi and Emmer lineages. Chloroplast haplotypes were often shared by species or subspecies within major lineages and between the lineages, indicating the contribution of introgression to the evolution and domestication of polyploid wheats.

  19. Comparison of intraspecific, interspecific and intergeneric chloroplast diversity in Cycads.

    PubMed

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

    2016-08-25

    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.

  20. Inhibition of chloroplastic respiration by osmotic dehydration. [Spinacia oleracea L

    SciTech Connect

    Willeford, K.O.; Ahluwalia, K.J.K.; Gibbs, M. )

    1989-04-01

    The respiratory capacity of isolated spinach (Spinacia oleracea L.) chloroplasts, measured as the rate of {sup 14}CO{sub 2} evolved from the oxidative pentose phosphate cycle in darkened chloroplasts exogenously supplied with ({sup 14}C)glucose, was progressively diminished by escalating osmotic dehydration with betaine or sorbitol. Comparing the inhibitions of CO{sub 2} evolution generated by osmotic dehydration in chloroplasts given C-1 and C-6 labeled glucose, 54% and 84%, respectively, indicates that osmotic dehydration effects to a greater extent the recycling of the oxidative pentose phosphate intermediates, fructose-6P and glyceraldehyde-3P. Respiratory inhibition in the darkened chloroplast could be alleviated by addition of NH{sub 4}Cl (a stromal alkylating agent), iodoacetamide (an inhibitor of glyceraldehyde-3P dehydrogenase), or glycolate-2P (an inhibitor of phosphofructokinase). It is concluded that the site which primarily mediates respiratory inhibition in the darkened chloroplast occurs at the fructose 1,6-bisphosphatase/phosphofructokinase junction.

  1. Purification of intact chloroplasts from Arabidopsis and spinach leaves by isopycnic centrifugation.

    PubMed

    Seigneurin-Berny, Daphné; Salvi, Daniel; Joyard, Jacques; Rolland, Norbert

    2008-09-01

    Chloroplasts are plant-specific organelles. They are the site of photosynthesis but also of many other essential metabolic pathways, such as syntheses of amino acids, vitamins, lipids, and pigments. This unit describes the isolation and purification of chloroplasts from Arabidopsis and spinach leaves. Differential centrifugation is first used to obtain a suspension enriched in chloroplasts (crude chloroplasts extract). In a second step, Percoll density gradient centrifugation is used to recover pure and intact chloroplasts. The Basic Protocol describes the purification of chloroplasts from Arabidopsis leaves. This small flowering plant is now widely used as a model organism in plant biology as it offers important advantages for basic research in genetics and molecular biology. The Alternate Protocol describes the purification of chloroplasts from spinach leaves. Spinach, easily available all through the year, remains a model of choice for the large-scale preparation of pure chloroplasts with a high degree of intactness.

  2. 5-aminolevulinic acid alleviates the salinity-induced changes in Brassica napus as revealed by the ultrastructural study of chloroplast.

    PubMed

    Naeem, Muhammad S; Warusawitharana, Hasitha; Liu, Hongbo; Liu, Dan; Ahmad, Rashid; Waraich, Ejaz Ahmad; Xu, Ling; Zhou, Weijun

    2012-08-01

    5-Aminolevulinic acid (ALA) is an important plant growth regulator which is derived from 5-carbon aliphatic amino acid. The present study investigates the interaction of increasing NaCl-salinity and ALA on plant growth, leaf pigment composition, leaf and root Na(+)/K(+) ratio and chloroplast ultrastructure in mesophyll cells of oilseed rape (Brassica napus) leaves. The plants were treated hydroponically with three different salinity levels (0, 100, 200 mM) and foliar application of ALA (30 mg l(-1)) simultaneously. Ten days after treatment, higher NaCl-salinity significantly reduced the plant biomass and height. However, ALA application restored the plant biomass and plant height under saline conditions. A concentration-dependent increase in Na(+) uptake was observed in the aerial parts of B. napus plants. On the other hand, ALA reduced Na(+) uptake, leading to a significant decrease in Na(+)/K(+) ratio. Accumulation of Na(+) augmented the oxidative stress, which was evident by electron microscopic images, highlighting several changes in cell shape and size, chloroplast swelling, increased number of plastogloubli, reduced starch granules and dilations of the thylakoids. Foliar application of ALA improved the energy supply and investment in mechanisms (higher chlorophyll and carotenoid contents, enhanced photosynthetic efficiency), reduced the oxidative stress as evident by the regular shaped chloroplasts with more intact thylakoids. On the basis of these results we can suggest that ALA is a promising plant growth regulator which can improve plant survival under salinity.

  3. The physiological roles and metabolism of ascorbate in chloroplasts.

    PubMed

    Tóth, Szilvia Z; Schansker, Gert; Garab, Győző

    2013-06-01

    Ascorbate is a multifunctional metabolite in plants. It is essential for growth control, involving cell division and cell wall synthesis and also involved in redox signaling, in the modulation of gene expression and regulation of enzymatic activities. Ascorbate also fulfills crucial roles in scavenging reactive oxygen species, both enzymatically and nonenzymatically, a well-established phenomenon in the chloroplasts stroma. We give an overview on these important physiological functions and would like to give emphasis to less well-known roles of ascorbate, in the thylakoid lumen, where it also plays multiple roles. It is essential for photoprotection as a cofactor for violaxanthin de-epoxidase, a key enzyme in the formation of nonphotochemical quenching. Lumenal ascorbate has recently also been shown to act as an alternative electron donor of photosystem II once the oxygen-evolving complex is inactivated and to protect the photosynthetic machinery by slowing down donor-side induced photoinactivation; it is yet to be established if ascorbate has a similar role in the case of other stress effects, such as high light and UV-B stress. In bundle sheath cells, deficient in oxygen evolution, ascorbate provides electrons to photosystem II, thereby poising cyclic electron transport around photosystem I. It has also been shown that, by supporting linear electron transport through photosystem II in sulfur-deprived Chlamydomonas reinhardtii cells, in which oxygen evolution is largely inhibited, externally added ascorbate enhances hydrogen production. For fulfilling its multiple roles, Asc has to be transported into the thylakoid lumen and efficiently regenerated; however, very little is known yet about these processes.

  4. Oryza sativa Chloroplast Signal Recognition Particle 43 (OscpSRP43) Is Required for Chloroplast Development and Photosynthesis

    PubMed Central

    Xu, Xia; Wei, Yan-lin; Wang, Hui-mei; Zhang, Xiao-bo; Wu, Jian-li

    2015-01-01

    A rice chlorophyll-deficient mutant w67 was isolated from an ethyl methane sulfonate (EMS)–induced IR64 (Oryza sativa L. ssp. indica) mutant bank. The mutant exhibited a distinct yellow-green leaf phenotype in the whole plant growth duration with significantly reduced levels of chlorophyll and carotenoid, impaired chloroplast development and lowered capacity of photosynthesis compared with the wild-type IR64. Expression of a number of genes associated with chlorophyll metabolism, chloroplast biogenesis and photosynthesis was significantly altered in the mutant. Genetic analysis indicated that the yellow-green phenotype was controlled by a single recessive nuclear gene located on the short arm of chromosome 3. Using map-based strategy, the mutation was isolated and predicted to encode a chloroplast signal recognition particle 43 KD protein (cpSRP43) with 388 amino acid residuals. A single base substitution from A to T at position 160 resulted in a premature stop codon. OscpSRP43 was constitutively expressed in various organs with the highest level in the leaf. Functional complementation could rescue the mutant phenotype and subcellular localization showed that the cpSRP43:GFP fusion protein was targeted to the chloroplast. The data suggested that Oryza sativa cpSRP43 (OscpSRP43) was required for the normal development of chloroplasts and photosynthesis in rice. PMID:26600124

  5. Oryza sativa Chloroplast Signal Recognition Particle 43 (OscpSRP43) Is Required for Chloroplast Development and Photosynthesis.

    PubMed

    Lv, Xiang-guang; Shi, Yong-feng; Xu, Xia; Wei, Yan-lin; Wang, Hui-mei; Zhang, Xiao-bo; Wu, Jian-li

    2015-01-01

    A rice chlorophyll-deficient mutant w67 was isolated from an ethyl methane sulfonate (EMS)-induced IR64 (Oryza sativa L. ssp. indica) mutant bank. The mutant exhibited a distinct yellow-green leaf phenotype in the whole plant growth duration with significantly reduced levels of chlorophyll and carotenoid, impaired chloroplast development and lowered capacity of photosynthesis compared with the wild-type IR64. Expression of a number of genes associated with chlorophyll metabolism, chloroplast biogenesis and photosynthesis was significantly altered in the mutant. Genetic analysis indicated that the yellow-green phenotype was controlled by a single recessive nuclear gene located on the short arm of chromosome 3. Using map-based strategy, the mutation was isolated and predicted to encode a chloroplast signal recognition particle 43 KD protein (cpSRP43) with 388 amino acid residuals. A single base substitution from A to T at position 160 resulted in a premature stop codon. OscpSRP43 was constitutively expressed in various organs with the highest level in the leaf. Functional complementation could rescue the mutant phenotype and subcellular localization showed that the cpSRP43:GFP fusion protein was targeted to the chloroplast. The data suggested that Oryza sativa cpSRP43 (OscpSRP43) was required for the normal development of chloroplasts and photosynthesis in rice.

  6. Post-Transcriptional Control of Chloroplast Gene Expression

    PubMed Central

    del Campo, Eva M.

    2009-01-01

    Chloroplasts contain their own genome, organized as operons, which are generally transcribed as polycistronic transcriptional units. These primary transcripts are processed into smaller RNAs, which are further modified to produce functional RNAs. The RNA processing mechanisms remain largely unknown and represent an important step in the control of chloroplast gene expression. Such mechanisms include RNA cleavage of pre-existing RNAs, RNA stabilization, intron splicing, and RNA editing. Recently, several nuclear-encoded proteins that participate in diverse plastid RNA processing events have been characterised. Many of them seem to belong to the pentatricopeptide repeat (PPR) protein family that is implicated in many crucial functions including organelle biogenesis and plant development. This review will provide an overview of current knowledge of the post-transcriptional processing in chloroplasts. PMID:19838333

  7. Purification and Characterization of Pea Chloroplastic Phosphoriboisomerase 12

    PubMed Central

    Skrukrud, Cynthia L.; Gordon, Ilana M.; Dorwin, Sally; Yuan, Xiao-Hua; Johansson, Göte; Anderson, Louise E.

    1991-01-01

    Pea (Pisum sativum L.) chloroplastic phosphoriboisomerase (EC 5.3.1.6) can be purified to apparent homogeneity in less than 2 days time with a 53% yield. Important steps in the purification include heat treatment and pseudoaffinity chromatography on Red H-3BN Sepharose. The purified isomerase has a subunit molecular mass of 26.4 kD. The N-terminal sequence has been determined through 34 residues. pH optima are 7.8 (ribose-5-phosphate) and 7.7 (ribulose-5-phosphate); Km values are 0.9 millimolar (ribose-5-phosphate) and 0.6 millimolar (ribulose-5-phosphate). The enzyme is inhibited by erythrose-4-phosphate, sedoheptulosebisphosphate, glyceraldehyde-3-phosphate, and 3-phosphoglycerate at concentrations close to those found in photosynthesizing chloroplasts. Countercurrent phase partitioning experiments indicate that the pea chloroplastic phosphoriboisomerase interacts physically with phosphoribulokinase. ImagesFigure 1 PMID:16668459

  8. The complete chloroplast genome sequence of Alocasia macrorrhizos.

    PubMed

    Wang, Bin; Han, Limin

    2016-09-01

    The complete chloroplast sequence of Alocasia macrorrhizos is 154 995 bp in length, containing a pair of inverted repeats of 25 944 bp separated by a large single-copy (LSC) region and a small single-copy (SSC) region of 87 366 bp and 15 741 bp, respectively. The chloroplast genome encodes 132 predicted functional genes, including 87 protein-coding genes, four ribosomal RNA genes, and 37 transfer RNA genes, 18 of which are duplicated in the inverted repeat regions. In these genes, 16 genes contained single intron and two genes comprising double introns. A maximum-likelihood phylogenetic analysis using complete chloroplast genome revealed that A. macrorrhizos does not belong to Araceae family, which infers that the A. macrorrhizos is distant from the species in Araceae family.

  9. 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

  10. Actin-dependence of the chloroplast cold positioning response in the liverwort Marchantia polymorpha L.

    PubMed

    Kimura, Shun; Kodama, Yutaka

    2016-01-01

    The subcellular positioning of chloroplasts can be changed by alterations in the environment such as light and temperature. For example, in leaf mesophyll cells, chloroplasts localize along anticlinal cell walls under high-intensity light, and along periclinal cell walls under low-intensity light. These types of positioning responses are involved in photosynthetic optimization. In light-mediated chloroplast positioning responses, chloroplasts move to the appropriate positions in an actin-dependent manner, although some exceptions also depend on microtubule. Even under low-intensity light, at low temperature (e.g., 5°C), chloroplasts localize along anticlinal cell walls; this phenomenon is termed chloroplast cold positioning. In this study, we analyzed whether chloroplast cold positioning is dependent on actin filaments and/or microtubules in the liverwort Marchantia polymorpha L. When liverwort cells were treated with drugs for the de-polymerization of actin filaments, chloroplast cold positioning was completely inhibited. In contrast, chloroplast cold positioning was not affected by treatment with a drug for the de-polymerization of microtubules. These observations indicate the actin-dependence of chloroplast cold positioning in M. polymorpha. Actin filaments during the chloroplast cold positioning response were visualized by using fluorescent probes based on fluorescent proteins in living liverwort cells, and thus, their behavior during the chloroplast cold positioning response was documented.

  11. Salinity induces membrane structure and lipid changes in maize mesophyll and bundle sheath chloroplasts.

    PubMed

    Omoto, Eiji; Iwasaki, Yugo; Miyake, Hiroshi; Taniguchi, Mitsutaka

    2016-05-01

    The membranes of Zea mays (maize) mesophyll cell (MC) chloroplasts are more vulnerable to salinity stress than are those of bundle sheath cell (BSC) chloroplasts. To clarify the mechanism underlying this difference in salt sensitivity, we monitored changes in the glycerolipid and fatty acid compositions of both types of chloroplast upon exposure to salinity stress. The monogalactosyldiacylglycerol (MGDG) and digalactosyldiacylglycerol (DGDG) contents were higher in MC chloroplasts than in BSC chloroplasts, in both the presence and absence of salt treatment. Under salt conditions, the MGDG level in MC chloroplasts was significantly lower than under normal conditions, while it was unchanged in BSC chloroplasts. In both types of chloroplast, the contents of DGDG, phosphatidylglycerol and phosphatidylinositol remained at the same levels in control and salt-treated plants, whereas sulfoquinovosyldiacylglycerol and phosphatidylcholine were significantly lower and higher, respectively, upon salt treatment. In addition, the fatty acid composition and double bond index of individual lipid classes were changed by salt treatment in both BSC and MC chloroplasts, although these factors had no effect on glycerolipid content. These findings suggest that the difference in salt sensitivity of MC and BSC chloroplast membranes is related to differences in MGDG responses to salinity. Thus, we propose that the low MGDG content and the low sensitivity of MGDG to salinity in BSC chloroplasts render them more tolerant than MC chloroplasts to salinity stress.

  12. Actin-dependence of the chloroplast cold positioning response in the liverwort Marchantia polymorpha L.

    PubMed Central

    Kimura, Shun

    2016-01-01

    The subcellular positioning of chloroplasts can be changed by alterations in the environment such as light and temperature. For example, in leaf mesophyll cells, chloroplasts localize along anticlinal cell walls under high-intensity light, and along periclinal cell walls under low-intensity light. These types of positioning responses are involved in photosynthetic optimization. In light-mediated chloroplast positioning responses, chloroplasts move to the appropriate positions in an actin-dependent manner, although some exceptions also depend on microtubule. Even under low-intensity light, at low temperature (e.g., 5°C), chloroplasts localize along anticlinal cell walls; this phenomenon is termed chloroplast cold positioning. In this study, we analyzed whether chloroplast cold positioning is dependent on actin filaments and/or microtubules in the liverwort Marchantia polymorpha L. When liverwort cells were treated with drugs for the de-polymerization of actin filaments, chloroplast cold positioning was completely inhibited. In contrast, chloroplast cold positioning was not affected by treatment with a drug for the de-polymerization of microtubules. These observations indicate the actin-dependence of chloroplast cold positioning in M. polymorpha. Actin filaments during the chloroplast cold positioning response were visualized by using fluorescent probes based on fluorescent proteins in living liverwort cells, and thus, their behavior during the chloroplast cold positioning response was documented. PMID:27703856

  13. Nitrogen control of chloroplast differentiation. Annual progress report

    SciTech Connect

    Schmidt, G.W.

    1992-07-01

    This project is directed toward understanding how the availability of nitrogen affects the accumulation of chloroplast pigments and proteins functioning in energy transduction and carbon metabolism. Molecular analyses performed with Chlamydomonas reinhardtii grown in a continuous culture system such that ammonium concentration is maintained at a low steady-state concentration so as to limit cell division. As compared to chloroplasts from cells of non-limiting nitrogen provisions, chloroplasts of N-limited cells are profoundly chlorophyll-deficient but still assimilate carbon for deposition of as starch and as storage lipids. Chlorophyll deficiency arises by limiting accumulation of appropriate nuclear-encoded mRNAs of and by depressed rates of translation of chloroplast mRNAs for apoproteins of reaction centers. Chloroplast translational effects can be partially ascribed to diminished rates of chlorophyll biosynthesis in N-limited cells, but pigment levels are not determinants for expression of the nuclear light-harvesting protein genes. Consequently, other signals that are responsive to nitrogen availability mediate transcriptional or post-transcriptional processes for accumulation of the mRNAs for LHC apoproteins and other mRNAs whose abundance is dependent upon high nitrogen levels. Conversely, limited nitrogen availability promotes accumulation of other proteins involved in carbon metabolism and oxidative electron transport in chloroplasts. Hence, thylakoids of N-limited cells exhibit enhanced chlororespiratory activities wherein oxygen serves as the electron acceptor in a pathway that involves plastoquinone and other electron carrier proteins that remain to be thoroughly characterized. Ongoing and future studies are also outlined.

  14. Localization of phosphatidylcholine in outer envelope membrane of spinach chloroplasts

    PubMed Central

    1985-01-01

    We have examined the effects of phospholipase C from Bacillus cereus on the extent of phospholipid hydrolysis in envelope membrane vesicles and in intact chloroplasts. When isolated envelope vesicles were incubated in presence of phospholipase C, phosphatidylcholine and phosphatidylglycerol, but not phosphatidylinositol, were totally converted into diacylglycerol if they were available to the enzyme (i.e., when the vesicles were sonicated in presence of phospholipase C). These experiments demonstrate that phospholipase C can be used to probe the availability of phosphatidylcholine and phosphatidylglycerol in the cytosolic leaflet of the outer envelope membrane from spinach chloroplasts. When isolated, purified, intact chloroplasts were incubated with low amounts of phospholipase C (0.3 U/mg chlorophyll) under very mild conditions (12 degrees C for 1 min), greater than 80% of phosphatidylcholine molecules and almost none of phosphatidylglycerol molecules were hydrolyzed. Since we have also demonstrated, by using several different methods (phase-contrast and electron microscopy, immunochemical and electrophoretic analyses) that isolated spinach chloroplasts, and especially their outer envelope membrane, remained intact after mild treatment with phospholipase C, we can conclude that there is a marked asymmetric distribution of phospholipids across the outer envelope membrane of spinach chloroplasts. Phosphatidylcholine, the major polar lipid of the outer envelope membrane, is almost entirely accessible from the cytosolic side of the membrane and therefore is probably localized in the outer leaflet of the outer envelope bilayer. On the contrary, phosphatidylglycerol, the major polar lipid in the inner envelope membrane and the thylakoids, is probably not accessible to phospholipase C from the cytosol and therefore is probably localized mostly in the inner leaflet of the outer envelope membrane and in the other chloroplast membranes. PMID:3988805

  15. 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.

  16. 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.

  17. Protein transport in chloroplasts - targeting to the intermembrane space.

    PubMed

    Vojta, Lea; Soll, Jürgen; Bölter, Bettina

    2007-10-01

    The import of proteins destined for the intermembrane space of chloroplasts has not been investigated in detail up to now. By investigating energy requirements and time courses, as well as performing competition experiments, we show that the two intermembrane space components Tic22 and MGD1 (E.C. 2.4.1.46) both engage the Toc machinery for crossing the outer envelope, whereas their pathways diverge thereafter. Although MGD1 appears to at least partly cross the inner envelope, Tic22 very likely reaches its mature form in the intermembrane space without involving stromal components. Thus, different pathways for intermembrane space targeting probably exist in chloroplasts.

  18. The complete chloroplast genome sequence of desert poplar (Populus euphratica).

    PubMed

    Zhang, Qun-jie; Gao, Li-zhi

    2016-01-01

    The complete chloroplast sequence of the desert poplar (Populus euphratica), a plant well-adapted to salt stress, was determined in this study. The genome consists of 156,766 bp containing a pair of inverted repeats (IRs) of 16,591 bp separated by a large single-copy region and a small single-copy region of 84,888 bp and 27,646 bp, respectively. The chloroplast genome contains 130 known genes, including 89 protein-coding genes, 8 ribosomal RNA genes, and 37 tRNA genes; 18 of these are located in the inverted repeat region.

  19. Oxidation versus reductive detoxification of SO sub 2 by chloroplasts

    SciTech Connect

    Ghisi, R.; Dittrich, A.P.M.; Heber, U. )

    1990-03-01

    Intact chloroplasts isolated from spinach (Spinacia oleracea L. cv Yates) both oxidized and reduced added sulfite in the light. Oxidation was fast only when endogenous superoxide dismutase was inhibited by cyanide. It was largely suppressed by scavengers of oxygen radicals. After addition of O-acetylserine, chloroplasts reduced sulfite to cysteine and exhibited sulfite-dependent oxygen evolution. Cysteine synthesis from sulfite was faster than from sulfate. The results are discussed in relation to species-specific differences in the phytotoxicity of SO{sub 2}.

  20. Changing the light environment: chloroplast signalling and response mechanisms.

    PubMed

    Spetea, Cornelia; Rintamäki, Eevi; Schoefs, Benoît

    2014-04-19

    Light is an essential environmental factor required for photosynthesis, but it also mediates signals to control plant development and growth and induces stress tolerance. The photosynthetic organelle (chloroplast) is a key component in the signalling and response network in plants. This theme issue of Philosophical Transactions of the Royal Society of London B: Biology provides updates, highlights and summaries of the most recent findings on chloroplast-initiated signalling cascades and responses to environmental changes, including light and biotic stress. Besides plant molecular cell biology and physiology, the theme issue includes aspects from the cross-disciplinary fields of environmental adaptation, ecology and agronomy.

  1. Chloroplast membranes of the green alga Acetabularia mediterranea. II. Topography of the chloroplast membrane.

    PubMed

    Apel, K; Miller, K R; Bogorad, L; Miller, G J

    1976-12-01

    The localization of the chlorophyll-protein complexes inside the thylakoid membrane of Acetabularia mediterranea was determined by fractionating the chloroplast membrane with EDTA and Triton X-100, by using pronase treatment, and by labeling the surface-exposed proteins with 125I. The effects of the various treatments were established by electrophoresis of the solubilized membrane fractions and electron microscopy. After EDTA and pronase treatment, the membrane structure was still intact. Only the two chlorophyll-protein complexes of 67,000 and 152,000 daltons and an additional polypeptides were found in the membrane before the EDTA and pronase treatment. The 125,000 dalton complex seems to be buried inside the lipid layer. The 23,000 dalton subunit of the 67,000 dalton complex is largely exposed to the surface of the EDTA-insoluble membrane and only the chlorophyll-binding subunit of 21,500 daltons is buried inside the lipid layer.

  2. Defective chloroplast development inhibits maintenance of normal levels of abscisic acid in a mutant of the Arabidopsis RH3 DEAD-box protein during early post-germination growth.

    PubMed

    Lee, Kwang-Hee; Park, Jiyoung; Williams, Donna S; Xiong, Yuqing; Hwang, Inhwan; Kang, Byung-Ho

    2013-03-01

    The plastid has its own translation system, and its ribosomes are assembled through a complex process in which rRNA precursors are processed and ribosomal proteins are inserted into the rRNA backbone. DEAD-box proteins have been shown to play roles in multiple steps in ribosome biogenesis. To investigate the cellular and physiological roles of an Arabidopsis DEAD-box protein, RH3, we examined its expression and localization and the phenotypes of rh3-4, a T-DNA insertion mutant allele of RH3. The promoter activity of RH3 is strongest in the greening tissues of 3-day and 1-week-old seedlings but reduced afterwards. Cotyledons were pale and seedling growth was retarded in the mutant. The most obvious abnormality in the mutant chloroplasts was their lack of normal ribosomes. Electron tomography analysis indicated that ribosome density in the 3-day-old mutant chloroplasts is only 20% that of wild-type chloroplasts, and the ribosomes in the mutant are smaller. These chloroplast defects in rh3-4 were alleviated in 2-week-old cotyledons and true leaves. Interestingly, rh3-4 seedlings have lower amounts of abscisic acid prior to recovery of their chloroplasts, and were more sensitive to abiotic stresses. Transcriptomic analysis indicated that nuclear genes for chloroplast proteins are down-regulated, and proteins mediating chloroplast-localized steps of abscisic acid biosynthesis are expressed to a lower extent in 1-week-old rh3-4 seedlings. Taken together, these results suggest that conversion of eoplasts into chloroplasts in young seedlings is critical for the seedlings to start carbon fixation as well as for maintenance of abscisic acid levels for responding to environmental challenges.

  3. Myosin inhibitors block accumulation movement of chloroplasts in Arabidopsis thaliana leaf cells.

    PubMed

    Paves, H; Truve, E

    2007-01-01

    Chloroplasts alter their distribution within plant cells depending on the external light conditions. Myosin inhibitors 2,3-butanedione monoxime (BDM), N-ethylmaleimide (NEM), and 1-(5-iodonaphthalene-1-sulfonyl)-1H-hexahydro-1,4-diazepine hydrochloride (ML-7) were used to study the possible role of myosins in chloroplast photorelocation in Arabidopsis thaliana mesophyll cells. None of these agents had an effect on the chloroplast high-fluence-rate avoidance movement but all of the three myosin inhibitors blocked the accumulation movement of chloroplasts after a high-fluence-rate irradiation of the leaves. The results suggest that myosins have a role in A. thaliana chloroplast photorelocation.

  4. Diversity of a ribonucleoprotein family in tobacco chloroplasts: two new chloroplast ribonucleoproteins and a phylogenetic tree of ten chloroplast RNA-binding domains.

    PubMed Central

    Ye, L H; Li, Y Q; Fukami-Kobayashi, K; Go, M; Konishi, T; Watanabe, A; Sugiura, M

    1991-01-01

    Two new ribonucleoproteins (RNPs) have been identified from a tobacco chloroplast lysate. These two proteins (cp29A and cp29B) are nuclear-encoded and have a less affinity to single-stranded DNA as compared with three other chloroplast RNPs (cp28, cp31 and cp33) previously isolated. DNA sequencing revealed that both contain two consensus sequence-type homologous RNA-binding domains (CS-RBDs) and a very acidic amino-terminal domain but shorter than that of cp28, cp31 and cp33. Comparison of cp29A and cp29B showed a 19 amino acid insertion in the region separating the two CS-RBDs in cp29B. This insertion results in three tandem repeats of a glycine-rich sequence of 10 amino acids, which is a novel feature in RNPs. The two proteins are encoded by different single nuclear genes and no alternatively spliced transcripts could be identified. We constructed a phylogenetic tree for the ten chloroplast CS-RBDs. These results suggest that there is a sizable RNP family in chloroplasts and the diversity was mainly generated through a series of gene duplications rather than through alternative pre-mRNA splicing. The gene for cp29B contains three introns. The first and second introns interrupt the first CS-RBD and the third intron does the second CS-RBD. The position of the first intron site is the same as that in the human hnRNP A1 protein gene. Images PMID:1721701

  5. A small multifunctional pentatricopeptide repeat protein in the chloroplast of Chlamydomonas reinhardtii.

    PubMed

    Jalal, Abdullah; Schwarz, Christian; Schmitz-Linneweber, Christian; Vallon, Olivier; Nickelsen, Jörg; Bohne, Alexandra-Viola

    2015-03-01

    Organellar biogenesis is mainly regulated by nucleus-encoded factors, which act on various steps of gene expression including RNA editing, processing, splicing, stabilization, and translation initiation. Among these regulatory factors, pentatricopeptide repeat (PPR) proteins form the largest family of RNA binding proteins, with hundreds of members in flowering plants. In striking contrast, the genome of the unicellular green alga Chlamydomonas reinhardtii encodes only 14 such proteins. In this study, we analyzed PPR7, the smallest and most highly expressed PPR protein in C. reinhardtii. Green fluorescent protein-based localization and gel-filtration analysis revealed that PPR7 forms a part of a high-molecular-weight ribonucleoprotein complex in the chloroplast stroma. RIP-chip analysis of PPR7-bound RNAs demonstrated that the protein associates with a diverse set of chloroplast transcripts in vivo, i.e. rrnS, psbH, rpoC2, rbcL, atpA, cemA-atpH, tscA, and atpI-psaJ. Furthermore, the investigation of PPR7 RNAi strains revealed that depletion of PPR7 results in a light-sensitive phenotype, accompanied by altered levels of its target RNAs that are compatible with the defects in their maturation or stabilization. PPR7 is thus an unusual type of small multifunctional PPR protein, which interacts, probably in conjunction with other RNA binding proteins, with numerous target RNAs to promote a variety of post-transcriptional events.

  6. [Calcium gating of proton fluxes in chloroplasts]. Progress report, April 1989--April 1991

    SciTech Connect

    Dilley, R.A.

    1991-12-31

    Work supported by this grant has been aimed at better understanding the still-mysterious phenomenon of sequestered proton gradients which strong evidence suggests can energize ATP formation in chloroplast organelle membranes. Results from several laboratories support the notion that chloroplasts can couple the energy of H{sup +} gradients. Research in may lab has, since 1983, been deeply involved in a two-pronged approach, (A) identifying and quantitating sequestered proton buffering groups and (B) trying to elucidate just how the sequestered H{sup +} gradient is maintained and what regulates the switching between the localized and delocalized coupling modes. One aspect we have worked on under DOE auspices is the question whether the localized H{sup +} coupling, routinely detected in my lab by a protocol which measures the number of single-turnover flashes (usually fired at 5 Hz) required to reach the energization threshold for ATP formation, can continue in steady illumination. It is possible to consider that a localized coupling response could be observed in the initial energization transient followed obligatorily by the sustained H{sup +} gradient equilibrating with the lumen bulk phase. If that occurred, it would have very important ramifications as to how one evaluates the possible physiological meaning of ``localized`` coupling. To test this, we developed a steady illumination protocol which is briefly discussed.

  7. 2012 MITOCHONDRIA AND CHLOROPLASTS GORDON RESEARCH CONFERENCE & GORDON RESEARCH SEMINAR, JULY 29 - AUGUST 3, 2012

    SciTech Connect

    Barkan, Alice

    2012-08-03

    The 2012 Gordon Research Conference on Mitochondria and Chloroplasts will assemble an international group of scientists investigating fundamental properties of these organelles, and their integration into broader physiological processes. The conference will emphasize the many commonalities between mitochondria and chloroplasts: their evolution from bacterial endosymbionts, their genomes and gene expression systems, their energy transducing membranes whose proteins derive from both nuclear and organellar genes, the challenge of maintaining organelle integrity in the presence of the reactive oxygen species that are generated during energy transduction, their incorporation into organismal signaling pathways, and more. The conference will bring together investigators working in animal, plant, fungal and protozoan systems who specialize in cell biology, genetics, biochemistry, physiology, proteomics, genomics, and structural biology. As such, this conference will provide a unique forum that engenders cross-disciplinary discussions concerning the biogenesis, dynamics, and regulation of these key cellular structures. By fostering interactions among mammalian, fungal and plant organellar biologists, this conference also provides a conduit for the transmission of mechanistic insights obtained in model organisms to applications in medicine and agriculture. The 2012 conference will highlight areas that are moving rapidly and emerging themes. These include new insights into the ultrastructure and organization of the energy transducing membranes, the coupling of organellar gene expression with the assembly of photosynthetic and respiratory complexes, the regulatory networks that couple organelle biogenesis with developmental and physiological signals, the signaling events through which organellar physiology influences nuclear gene expression, and the roles of organelles in disease and development.

  8. Distribution of Pyruvate Dehydrogenase Complex Activities between Chloroplasts and Mitochondria from Leaves of Different Species.

    PubMed Central

    Lernmark, U.; Gardestrom, P.

    1994-01-01

    Protoplasts from barley (Hordeum vulgare), pea (Pisum sativum), wheat (Triticum aestivum), and spinach (Spinacia oleracea) leaves were fractionated into chloroplast- and mitochondrion-enriched fractions. Pyruvate dehydrogenase complex capacities in mitochondria (mtPDC) and chloroplasts (cpPDC) were measured in appropriate fractions under conditions optimal for each isozyme. The total cellular capacity of PDC was similar in barley and pea but about 50% lower in wheat and spinach. In pea a distribution of 87% mtPDC and 13% cpPDC was found on a cellular basis. In barley, wheat, and spinach the subcellular distribution was the opposite, with about 15% mtPDC and 85% cpPDC. cpPDC activity was constant at about 0.1 nmol cell-1 h-1 in cells from different regions along the developing barley leaf and showed no correlation with developmental patterns of photosynthetic parameters, such as increasing Chl and NADP-glyceraldehyde-3-phosphate dehydrogenase activity. Similarly, the capacity of the mitochondrial isoform did not change during barley leaf development and had a developmental pattern similar to that of citrate synthase and fumarase. Differences in subcellular distribution of PDCs in barley and pea are proposed to be due to differences in regulation, not to changes in isozyme proportions during leaf development or to species-specific differences in phosphorylation state of mtPDC after organelle separation. PMID:12232437

  9. Complete Chloroplast Genome Sequence of Dendrobium nobile from Northeastern India

    PubMed Central

    Parameswaran, Sriram; Sundar, Durai

    2016-01-01

    The orchid species Dendrobium nobile belonging to the family Orchidaceae and genus Dendrobium (a vast genus that encompasses nearly 1,200 species) has an herbal medicinal history of about 2000 years in east and south Asian countries. Here, we report the complete chloroplast genome sequence of D. nobile from northeastern India for the first time. PMID:27795255

  10. Fatty acid phytyl ester synthesis in chloroplasts of Arabidopsis.

    PubMed

    Lippold, Felix; vom Dorp, Katharina; Abraham, Marion; Hölzl, Georg; Wewer, Vera; Yilmaz, Jenny Lindberg; Lager, Ida; Montandon, Cyrille; Besagni, Céline; Kessler, Felix; Stymne, Sten; Dörmann, Peter

    2012-05-01

    During stress or senescence, thylakoid membranes in chloroplasts are disintegrated, and chlorophyll and galactolipid are broken down, resulting in the accumulation of toxic intermediates, i.e., tetrapyrroles, free phytol, and free fatty acids. Chlorophyll degradation has been studied in detail, but the catabolic pathways for phytol and fatty acids remain unclear. A large proportion of phytol and fatty acids is converted into fatty acid phytyl esters and triacylglycerol during stress or senescence in chloroplasts. We isolated two genes (PHYTYL ESTER SYNTHASE1 [PES1] and PES2) of the esterase/lipase/thioesterase family of acyltransferases from Arabidopsis thaliana that are involved in fatty acid phytyl ester synthesis in chloroplasts. The two proteins are highly expressed during senescence and nitrogen deprivation. Heterologous expression in yeast revealed that PES1 and PES2 have phytyl ester synthesis and diacylglycerol acyltransferase activities. The enzymes show broad substrate specificities and can employ acyl-CoAs, acyl carrier proteins, and galactolipids as acyl donors. Double mutant plants (pes1 pes2) grow normally but show reduced phytyl ester and triacylglycerol accumulation. These results demonstrate that PES1 and PES2 are involved in the deposition of free phytol and free fatty acids in the form of phytyl esters in chloroplasts, a process involved in maintaining the integrity of the photosynthetic membrane during abiotic stress and senescence.

  11. Targeting of nucleus-encoded proteins to chloroplasts in plants.

    PubMed

    Jarvis, Paul

    2008-07-01

    Most chloroplast proteins are encoded in the nucleus and synthesized on free, cytosolic ribosomes in precursor form. Each precursor has an amino-terminal extension called a transit peptide, which directs the protein through a post-translational targeting pathway and is removed upon arrival inside the organelle. This 'protein import' process is mediated by the coordinate action of two multiprotein complexes, one in each of the envelope membranes: the TOC and TIC (Translocon at the Outer/ Inner envelope membrane of Chloroplasts) machines. Many components of these complexes have been identified biochemically in pea; these include transit peptide receptors, channel proteins, and molecular chaperones. Intriguingly, the Arabidopsis genome encodes multiple, homologous genes for receptor components of the TOC complex. Careful analysis indicated that the different receptor isoforms operate in different import pathways with distinct precursor recognition specificities. These 'substrate-specific' import pathways might play a role in the differentiation of different plastid types, and/or act to prevent deleterious competition effects between abundant and nonabundant precursors. Until recently, all proteins destined for internal chloroplast compartments were thought to possess a cleavable transit peptide, and to engage the TOC/TIC machinery. New studies using proteomics and other approaches have revealed that this is far from true. Remarkably, a significant number of chloroplast proteins are transported via a pathway that involves the endoplasmic reticulum and Golgi apparatus. Other recent reports have elucidated an intriguing array of protein targeting routes leading to the envelope membranes themselves.

  12. Chloroplast microsatellite markers for Artocarpus (Moraceae) developed from transcriptome sequences

    Technology Transfer Automated Retrieval System (TEKTRAN)

    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...

  13. The molecular integrity of chloroplast ribosomal ribonucleic acid.

    PubMed

    Leaver, C J; Ingle, J

    1971-06-01

    Instability of chloroplast rRNA has been observed with essentially all chloroplast RNA preparations. This paper describes experiments that show that, under normal conditions of preparation and fractionation, only the heavy chloroplast component (mol.wt. 1.1x10(6)) is unstable, the light chloroplast rRNA (mol.wt. 0.56x10(6)) and the cytoplasmic rRNA species (mol.wt. 1.3x10(6) and 0.70x10(6)) being stable. The stability of the 1.1x10(6)-mol. wt. molecule varies with different plant species, as also does the size and the number of fragments produced. Cleavages in three particular regions of the molecule are very frequent within the range of tissues studied. The 1.1x10(6)-mol.wt. rRNA is, however, stabilized by the presence of Mg(2+) during the preparation and fractionation of the RNA.

  14. Protein disorder in plants: a view from the chloroplast

    PubMed Central

    2012-01-01

    Background The intrinsically unstructured state of some proteins, observed in all living organisms, is essential for basic cellular functions. In this field the available information from plants is limited but it has been reached a point where these proteins can be comprehensively classified on the basis of disorder, function and evolution. Results Our analysis of plant genomes confirms that nuclear-encoded proteins follow the same trend than other multi-cellular eukaryotes; however, chloroplast- and mitochondria- encoded proteins conserve the patterns of Archaea and Bacteria, in agreement with their phylogenetic origin. Based on current knowledge about gene transference from the chloroplast to the nucleus, we report a strong correlation between the rate of disorder of transferred and nuclear-encoded proteins, even for polypeptides that play functional roles back in the chloroplast. We further investigate this trend by reviewing the set of chloroplast ribosomal proteins, one of the most representative transferred gene clusters, finding that the ribosomal large subunit, assembled from a majority of nuclear-encoded proteins, is clearly more unstructured than the small one, which integrates mostly plastid-encoded proteins. Conclusions Our observations suggest that the evolutionary dynamics of the plant nucleus adds disordered segments to genes alike, regardless of their origin, with the notable exception of proteins currently encoded in both genomes, probably due to functional constraints. PMID:22970728

  15. Structure of "Arabidopsis" chloroplastic monothiol glutaredoxin AtGRXcp

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Monothiol glutaredoxins (Grxs) play important roles in maintaining redox homeostasis in living cells and are conserved across species. "Arabidopsis thaliana" monothiol glutaredoxin AtGRXcp, is critical for protection from oxidative stress in chloroplasts. The crystal structure of AtGRXcp has been de...

  16. GRANULES ASSOCIATED WITH THE CHLOROPLAST LAMELLAE OF PORPHYRIDIUM CRUENTUM

    PubMed Central

    Gantt, E.; Conti, S. F.

    1966-01-01

    Small granules with a diameter of approximately 350 A are attached to the chloroplast lamellae of the red alga Porphyridium cruentum. To some extent, their size depends on the culture conditions and the age of the cell. It was possible to preserve the granules only with aldehyde prefixation. It can be seen that fixed or negatively stained granules are comprised of smaller subunits. The granules are arranged regularly on the lamellae in repeating rows with a center-to-center granule distance of 400 to 500 A. Attempts at characterization of these chloroplast granules revealed that they are resistant to hydrolysis by ribonuclease and appear to be structurally unaffected by methanol-acetone extraction. Because of their close association with the chloroplast lamellae, they are considered as possible sites of phycobilin concentration. This possibility is supported by two observations: when the phycobilins are removed, the granules disappear; and, when the chlorophyll and stainable membrane portions are selectively removed, the phycobilins and granules are still present. It was found that all other marine red algae examined had granules which were associated with the chloroplast lamellae. PMID:5962937

  17. Choline oxidation by intact chloroplasts isolated directly from spinach leaves

    SciTech Connect

    Weigel, P.; Hanson, A.D.

    1986-04-01

    Illuminated chloroplasts derived from spinach leaf protoplasts synthesize betaine from choline via the intermediate betaine aldehyde (BAL) (PNAS 82:3678). Photosynthetically active chloroplasts isolated directly from spinach leaves oxidized (/sup 14/C)choline in the light at rates 10 times higher (25-80 nmol/mg chl b) than protoplast-derived chloroplasts. Up to 20% of the (/sup 14/C)choline supplied during a 30 min incubation was oxidized in the light; the main product was (/sup 14/C)BAL. Rates of (/sup 14/C)choline oxidation in darkness were only 5-30% of rates in light. Light-dependent (/sup 14/C)choline oxidation was abolished by DCMU and 5 mM DTT. Pre-illumination of the chloroplasts did not promote (/sup 14/C)choline oxidation in darkness. The uncouplers nigericin and CCCP at concentrations which eliminated CO/sub 2/-dependent O/sub 2/ evolution did not affect (/sup 14/C)choline oxidation in the light. They hypothesize that (/sup 14/C)choline oxidation is not dependent upon light activation of an enzymatic system or upon the electrochemical proton gradient but requires an oxidant generated in the light.

  18. Choline oxidation by intact spinach chloroplasts. [Spinacia oleracea L

    SciTech Connect

    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 greatly 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.

  19. Senescence-Associated Vacuoles, a Specific Lytic Compartment for Degradation of Chloroplast Proteins?

    PubMed

    Carrión, Cristian A; Martínez, Dana E; Costa, M Lorenza; Guiamet, Juan José

    2014-11-11

    Degradation of chloroplasts and chloroplast components is a distinctive feature of leaf senescence. In spite of its importance in the nutrient economy of plants, knowledge about the mechanism(s) involved in the breakdown of chloroplast proteins is incomplete. A novel class of vacuoles, "senescence-associated vacuoles" (SAVs), characterized by intense proteolytic activity appear during senescence in chloroplast-containing cells of leaves. Since SAVs contain some chloroplast proteins, they are candidate organelles to participate in chloroplast breakdown. In this review we discuss the characteristics of SAVs, and their possible involvement in the degradation of Rubisco, the most abundant chloroplast protein. Finally, SAVs are compared with other extra-plastidial protein degradation pathways operating in senescing leaves.

  20. Senescence-Associated Vacuoles, a Specific Lytic Compartment for Degradation of Chloroplast Proteins?

    PubMed Central

    Carrión, Cristian A.; Martínez, Dana E.; Costa, M. Lorenza; Guiamet, Juan José

    2014-01-01

    Degradation of chloroplasts and chloroplast components is a distinctive feature of leaf senescence. In spite of its importance in the nutrient economy of plants, knowledge about the mechanism(s) involved in the breakdown of chloroplast proteins is incomplete. A novel class of vacuoles, “senescence-associated vacuoles” (SAVs), characterized by intense proteolytic activity appear during senescence in chloroplast-containing cells of leaves. Since SAVs contain some chloroplast proteins, they are candidate organelles to participate in chloroplast breakdown. In this review we discuss the characteristics of SAVs, and their possible involvement in the degradation of Rubisco, the most abundant chloroplast protein. Finally, SAVs are compared with other extra-plastidial protein degradation pathways operating in senescing leaves. PMID:27135516

  1. Targeting and biogenesis of transporters and channels in chloroplast envelope membranes: Unsolved questions.

    PubMed

    Oh, Young Jun; Hwang, Inhwan

    2015-07-01

    Chloroplasts produce carbohydrates, hormones, vitamins, amino acids, pigments, nucleotides, ATP, and secondary metabolites. Channels and transporters are required for the movement of molecules across the two chloroplast envelope membranes. These transporters and channel proteins are grouped into two different types, including β-barrel proteins and transmembrane-domain (TMD) containing proteins. Most β-barrel proteins are localized at the outer chloroplast membrane, and TMD-containing proteins are localized at the inner chloroplast membrane. Many of these transporters and channels are encoded by nuclear genes; therefore, they have to be imported into chloroplasts after translation on cytosolic ribosomes. These proteins should have specific targeting signals for their final destination in the chloroplast membrane and for assembly into specific complexes. In this review, we summarize recent progress in the identification, functional characterization, and biogenesis of transporters and channels at the chloroplast envelope membranes, and discuss outstanding questions regarding transporter and channel protein biogenesis.

  2. Distribution pattern changes of actin filaments during chloroplast movement in Adiantum capillus-veneris.

    PubMed

    Tsuboi, Hidenori; Wada, Masamitsu

    2012-05-01

    Chloroplasts change their positions in a cell in response to light intensities. The photoreceptors involved in chloroplast photo-relocation movements and the behavior of chloroplasts during their migration were identified in our previous studies, but the mechanism of movement has yet to be clarified. In this study, the behavior of actin filaments under various light conditions was observed in Adiantum capillus-veneris gametophytes. In chloroplasts staying in one place under a weak light condition and not moving, circular structures composed of actin filaments were observed around the chloroplast periphery. In contrast, short actin filaments were observed at the leading edge of moving chloroplasts induced by partial cell irradiation. In the dark, the circular structures found under the weak light condition disappeared and then reappeared around the moving chloroplasts. Mutant analyses revealed that the disappearance of the circular actin structure was mediated by the blue light photoreceptor, phototropin2.

  3. Differential positioning of chloroplasts in C4 mesophyll and bundle sheath cells.

    PubMed

    Maai, Eri; Miyake, Hiroshi; Taniguchi, Mitsutaka

    2011-08-01

    Chloroplast photorelocation movement is extensively studied in C3 but not C4 plants. C4 plants have 2 types of photosynthetic cells: mesophyll and bundle sheath cells. Mesophyll chloroplasts are randomly distributed along cell walls, whereas bundle sheath chloroplasts are located close to the vascular tissues or mesophyll cells depending on the plant species. The cell-specific C 4 chloroplast arrangement is established during cell maturation, and is maintained throughout the life of the cell. However, only mesophyll chloroplasts can change their positions in response to environmental stresses. The migration pattern is unique to C4 plants and differs from that of C3 chloroplasts. In this mini-review, we highlight the cell-specific disposition of chloroplasts in C4 plants and discuss the possible physiological significances.

  4. Reinvestigation of the triplet-minus-singlet spectrum of chloroplasts

    NASA Astrophysics Data System (ADS)

    Jávorfi, T.; Garab, G.; Razi Naqvi, K.

    2000-01-01

    A comparison of the triplet-minus-singlet (TmS) absorption spectrum of spinach chloroplasts, recorded some thirty years ago, with the more recently published TmS spectrum of isolated Chl a/ b LHCII (light-harvesting complexes associated with photosystem II of higher plants) shows that the two spectra are very similar, which is to be expected, since only the carotenoid pigments contribute to each spectrum. Be that as it may, the comparison also reveals a dissimilarity: photoexcitation of the sample does, or does not, affect the absorbance in the Qy region (650-700 nm), depending on whether the sample is a suspension of chloroplasts or of isolated LHCII. The Qy-signal in the TmS spectrum of LHCII decays, it should be noted, at the same rate as the rest of the difference spectrum, and its most prominent feature is a negative peak. As the carotenoids do not absorb in the Qy region, the presence of a signal in this region calls for an explanation: van der Vos, Carbonera and Hoff, the first to find as well as fathom the phenomenon, attributed the Qy-signal to a change, in the absorption spectrum of a chlorophyll a (Chl a) molecule, brought about by the presence of triplet excitation on a neighbouring carotenoid (Car). The difference in the behaviours of chloroplasts and LHCII, if reproducible, would imply that the Car triplets which give rise to the TmS spectrum of chloroplasts do not influence the absorption spectra of their Chl a neighbours. With a view to reaching a firm conclusion about this vexed issue, spinach chloroplasts and thylakoids have been examined with the aid of the same kinetic spectrometer as that used for investigating LHCII; the TmS spectra of both chloroplasts and thylakoids contain prominent bleaching signals centred at 680 nm, and the triplet decay time in each case is comparable to that of the Chl a/ b LHCII triplets. Results pertaining to other closely related systems are recalled, and it is concluded that, so far as the overall appearance of the

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

    PubMed Central

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

    2015-01-01

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

  6. Molecular characterization of a positively photoregulated nuclear gene for a chloroplast RNA polymerase sigma factor in Cyanidium caldarium.

    PubMed Central

    Liu, B; Troxler, R F

    1996-01-01

    We have cloned the gene for a putative chloroplast RNA polymerase sigma factor from the unicellular rhodophyte Cyanidium caldarium. This gene contains an open reading frame encoding a protein of 609 amino acids with domains highly homologous to all four conserved regions found in bacterial and cyanobacterial sigma 70-type subunits. When Southern blots of genomic DNA were hybridized to the "rpoD box" oligonucleotide probe, up to six hybridizing hands were observed. Transcripts of the sigma factor gene were undetectable in RNA from dark-grown cells but were abundant in the poly(A)+ fraction of RNA from illuminated cells. The sigma factor gene was expressed in Escherichia coli, and antibodies against the expressed sigma factor fusion protein cross-reacted with a 55-kDa protein in partially purified chloroplast RNA polymerase. Antibodies directed against a cyanobacterial RNA polymerase sigma factor also cross-reacted with a 55-kDa protein in the same enzyme preparation. Immunoprecipitation experiments showed that this enzyme preparation contains proteins with the same molecular weights as the alpha, beta, beta', and beta" subunits of chloroplast RNA polymerase in higher plants. This study identifies a gene for a plastid RNA polymerase sigma factor and indicates that there may be a family of nuclear-encoded sigma factors that recognize promoters in subsets of plastid genes and regulate differential gene expression at the transcriptional level. Images Fig. 1 Fig. 4 Fig. 6 PMID:8622935

  7. Linking chloroplast antioxidant defense to carbohydrate availability: the transcript abundance of stromal ascorbate peroxidase is sugar-controlled via ascorbate biosynthesis.

    PubMed

    Heiber, Isabelle; Cai, Wenguo; Baier, Margarete

    2014-01-01

    All genes encoding chloroplast antioxidant enzymes are nuclear-encoded and posttranscriptionally targeted to chloroplasts. The transcript levels of most of them decreased upon sucrose feeding like the transcript levels of many genes encoding components of the photosynthetic electron transport chain. However, the transcript abundance of stromal ascorbate peroxidase (s-APX; At4g08390) increased. Due to mild sugar application conditions, the plants kept the phosphorylation status of the ADP+ATP pool and the redox states of the NADPH+NADP+ and the ascorbate pools under control, which excludes them as signals in s-APX regulation. Correlation with ascorbate pool size regulation and comparison of transcript abundance regulation in the starch-biosynthetic mutant adg1, the ascorbate biosynthesis mutant vtc1, and the abscisic acid (ABA) biosynthetic mutant aba2 showed a link between sugar induction of s-APX and ascorbate biosynthesis.

  8. The contribution of NADPH thioredoxin reductase C (NTRC) and sulfiredoxin to 2-Cys peroxiredoxin overoxidation in Arabidopsis thaliana chloroplasts.

    PubMed

    Puerto-Galán, Leonor; Pérez-Ruiz, Juan M; Guinea, Manuel; Cejudo, Francisco Javier

    2015-05-01

    Hydrogen peroxide is a harmful by-product of photosynthesis, which also has important signalling activity. Therefore, the level of hydrogen peroxide needs to be tightly controlled. Chloroplasts harbour different antioxidant systems including enzymes such as the 2-Cys peroxiredoxins (2-Cys Prxs). Under oxidizing conditions, 2-Cys Prxs are susceptible to inactivation by overoxidation of their peroxidatic cysteine, which is enzymatically reverted by sulfiredoxin (Srx). In chloroplasts, the redox status of 2-Cys Prxs is highly dependent on NADPH-thioredoxin reductase C (NTRC) and Srx; however, the relationship of these activities in determining the level of 2-Cys Prx overoxidation is unknown. Here we have addressed this question by a combination of genetic and biochemical approaches. An Arabidopsis thaliana double knockout mutant lacking NTRC and Srx shows a phenotype similar to the ntrc mutant, while the srx mutant resembles wild-type plants. The deficiency of NTRC causes reduced overoxidation of 2-Cys Prxs, whereas the deficiency of Srx has the opposite effect. Moreover, in vitro analyses show that the disulfide bond linking the resolving and peroxidatic cysteines protects the latter from overoxidation, thus explaining the dominant role of NTRC on the level of 2-Cys Prx overoxidation in vivo. The overoxidation of chloroplast 2-Cys Prxs shows no circadian oscillation, in agreement with the fact that neither the NTRC nor the SRX genes show circadian regulation of expression. Additionally, the low level of 2-Cys Prx overoxidation in the ntrc mutant is light dependent, suggesting that the redox status of 2-Cys Prxs in chloroplasts depends on light rather than the circadian clock.

  9. Chloroplast biogenesis during rehydration of the resurrection plant Xerophyta humilis: parallels to the etioplast-chloroplast transition.

    PubMed

    Ingle, Robert A; Collett, Helen; Cooper, Keren; Takahashi, Yuichiro; Farrant, Jill M; Illing, Nicola

    2008-12-01

    De-etiolation of dark-grown seedlings is a commonly used experimental system to study the mechanisms of chloroplast biogenesis, including the stacking of thylakoid membranes into grana, the response of the nuclear-chloroplast transcriptome to light, and the ordered synthesis and assembly of photosystem II (PSII). Here, we present the xeroplast to chloroplast transition during rehydration of the resurrection plant Xerophyta humilis as a novel system for studying chloroplast biogenesis, and investigate the role of light in this process. Xeroplasts are characterized by the presence of numerous large and small membrane-bound vesicles and the complete absence of thylakoid membranes. While the initial assembly of stromal thylakoid membranes occurs independently of light, the formation of grana is light dependent. Recovery of photosynthetic activity is rapid in plants rehydrated in the light and correlates with the light-dependent synthesis of the D1 protein, but does not require de novo chlorophyll biosynthesis. Light-dependent synthesis of the chlorophyll-binding protein Lhcb2 and digalactosyldiacylglycerol synthase 1 correlated with the formation of grana and with the increased PSII activity. Our results suggest that the molecular mechanisms underlying photomorphogenic development may also function in desiccation tolerance in poikilochlorophyllous resurrection plants.

  10. Programmed cell death activated by Rose Bengal in Arabidopsis thaliana cell suspension cultures requires functional chloroplasts

    PubMed Central

    Gutiérrez, Jorge; González-Pérez, Sergio; García-García, Francisco; Daly, Cara T.; Lorenzo, Óscar; Revuelta, José L.; McCabe, Paul F.; Arellano, Juan B.

    2014-01-01

    Light-grown Arabidopsis thaliana cell suspension culture (ACSC) were subjected to mild photooxidative damage with Rose Bengal (RB) with the aim of gaining a better understanding of singlet oxygen-mediated defence responses in plants. Additionally, ACSC were treated with H2O2 at concentrations that induced comparable levels of protein oxidation damage. Under low to medium light conditions, both RB and H2O2 treatments activated transcriptional defence responses and inhibited photosynthetic activity, but they differed in that programmed cell death (PCD) was only observed in cells treated with RB. When dark-grown ACSC were subjected to RB in the light, PCD was suppressed, indicating that the singlet oxygen-mediated signalling pathway in ACSC requires functional chloroplasts. Analysis of up-regulated transcripts in light-grown ACSC, treated with RB in the light, showed that both singlet oxygen-responsive transcripts and transcripts with a key role in hormone-activated PCD (i.e. ethylene and jasmonic acid) were present. A co-regulation analysis proved that ACSC treated with RB exhibited higher correlation with the conditional fluorescence (flu) mutant than with other singlet oxygen-producing mutants or wild-type plants subjected to high light. However, there was no evidence for the up-regulation of EDS1, suggesting that activation of PCD was not associated with the EXECUTER- and EDS1-dependent signalling pathway described in the flu mutant. Indigo Carmine and Methylene Violet, two photosensitizers unable to enter chloroplasts, did not activate transcriptional defence responses in ACSC; however, whether this was due to their location or to their inherently low singlet oxygen quantum efficiencies was not determined. PMID:24723397

  11. Differential positioning of C4 mesophyll and bundle sheath chloroplasts: aggregative movement of C4 mesophyll chloroplasts in response to environmental stresses.

    PubMed

    Yamada, Masahiro; Kawasaki, Michio; Sugiyama, Tatsuo; Miyake, Hiroshi; Taniguchi, Mitsutaka

    2009-10-01

    In C(4) plants, mesophyll (M) chloroplasts are randomly distributed along the cell walls, while bundle sheath (BS) chloroplasts are typically located in either a centripetal or centrifugal position. We investigated whether these intracellular positions are affected by environmental stresses. When mature leaves of finger millet (Eleusine coracana) were exposed to extremely high intensity light, most M chloroplasts aggregatively re-distributed to the BS side, whereas the intracellular arrangement of BS chloroplasts was unaffected. Compared with the homologous light-avoidance movement of M chloroplasts in C(3) plants, it requires extremely high light (3,000-4,000 micromol m(-2) s(-1)) and responds more slowly (distinctive movement observed in 1 h). The high light-induced movement of M chloroplasts was also observed in maize (Zea mays), another C(4) species, but with a distinct pattern of redistribution along the sides of anticlinal walls, analogous to C(3) plants. The aggregative movement of M chloroplasts occurred at normal light intensities (250-500 micromol m(-2) s(-1)) in response to environmental stresses, such as drought, salinity and hyperosmosis. Moreover, the re-arrangement of M chloroplasts was observed in field-grown C(4) plants when exposed to mid-day sunlight, but also under midsummer drought conditions. The migration of M chloroplasts was controlled by actin filaments and also induced in a light-dependent fashion upon incubation with ABA, which may be the physiological signal transducer. Together these results suggest that M and BS cells of C(4) plants have different mechanisms controlling intracellular chloroplast positioning, and that the aggregative movement of C(4) M chloroplasts is thought to be a protective response under environmental stress conditions.

  12. 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

  13. Completion of the Chloroplast Genomes of Five Chinese Juglans and Their Contribution to Chloroplast Phylogeny

    PubMed Central

    Hu, Yiheng; Woeste, Keith E.; Zhao, Peng

    2017-01-01

    Juglans L. (walnuts and butternuts) is an economically and ecologically important genus in the family Juglandaceae. All Juglans are important nut and timber trees. Juglans regia (Common walnut), J. sigillata (Iron walnut), J. cathayensis (Chinese walnut), J. hopeiensis (Ma walnut), and J. mandshurica (Manchurian walnut) are native to or naturalized in China. A strongly supported phylogeny of these five species is not available due to a lack of informative molecular markers. We compared complete chloroplast genomes and determined the phylogenetic relationships among the five Chinese Juglans using IIumina sequencing. The plastid genomes ranged from 159,714 to 160,367 bp encoding 128 functional genes, including 88 protein-coding genes and 40 tRNA genes each. A complete map of the variability across the genomes of the five Juglans species was produced that included single nucleotide variants, indels (insertions and deletions), and large structural variants, as well as differences in simple sequence repeats (SSR) and repeat sequences. Molecular phylogeny strongly supported division of the five walnut species into two previously recognized sections (Juglans/Dioscaryon and Cardiocaryon) with a 100% bootstrap (BS) value using the complete cp genomes, protein coding sequences (CDS), and the introns and spacers (IGS) data. The availability of these genomes will provide genetic information for identifying species and hybrids, taxonomy, phylogeny, and evolution in Juglans, and also provide insight into utilization of Juglans plants. PMID:28111577

  14. Completion of the Chloroplast Genomes of Five Chinese Juglans and Their Contribution to Chloroplast Phylogeny.

    PubMed

    Hu, Yiheng; Woeste, Keith E; Zhao, Peng

    2016-01-01

    Juglans L. (walnuts and butternuts) is an economically and ecologically important genus in the family Juglandaceae. All Juglans are important nut and timber trees. Juglans regia (Common walnut), J. sigillata (Iron walnut), J. cathayensis (Chinese walnut), J. hopeiensis (Ma walnut), and J. mandshurica (Manchurian walnut) are native to or naturalized in China. A strongly supported phylogeny of these five species is not available due to a lack of informative molecular markers. We compared complete chloroplast genomes and determined the phylogenetic relationships among the five Chinese Juglans using IIumina sequencing. The plastid genomes ranged from 159,714 to 160,367 bp encoding 128 functional genes, including 88 protein-coding genes and 40 tRNA genes each. A complete map of the variability across the genomes of the five Juglans species was produced that included single nucleotide variants, indels (insertions and deletions), and large structural variants, as well as differences in simple sequence repeats (SSR) and repeat sequences. Molecular phylogeny strongly supported division of the five walnut species into two previously recognized sections (Juglans/Dioscaryon and Cardiocaryon) with a 100% bootstrap (BS) value using the complete cp genomes, protein coding sequences (CDS), and the introns and spacers (IGS) data. The availability of these genomes will provide genetic information for identifying species and hybrids, taxonomy, phylogeny, and evolution in Juglans, and also provide insight into utilization of Juglans plants.

  15. A novel chloroplast localized Rab GTPase protein CPRabA5e is involved in stress, development, thylakoid biogenesis and vesicle transport in Arabidopsis.

    PubMed

    Karim, Sazzad; Alezzawi, Mohamed; Garcia-Petit, Christel; Solymosi, Katalin; Khan, Nadir Zaman; Lindquist, Emelie; Dahl, Peter; Hohmann, Stefan; Aronsson, Henrik

    2014-04-01

    A novel Rab GTPase protein in Arabidopsis thaliana, CPRabA5e (CP = chloroplast localized) is located in chloroplasts and has a role in transport. Transient expression of CPRabA5e:EGFP fusion protein in tobacco (Nicotiana tabacum) leaves, and immunoblotting using Arabidopsis showed localization of CPRabA5e in chloroplasts (stroma and thylakoids). Ypt31/32 in the yeast Saccharomyces cerevisiae are involved in regulating vesicle transport, and CPRabA5e a close homolog of Ypt31/32, restores the growth of the ypt31Δ ypt32(ts) mutant at 37 °C in yeast complementation. Knockout mutants of CPRabA5e displayed delayed seed germination and growth arrest during oxidative stress. Ultrastructural studies revealed that after preincubation at 4 °C mutant chloroplasts contained larger plastoglobules, lower grana, and more vesicles close to the envelopes compared to wild type, and vesicle formation being enhanced under oxidative stress. This indicated altered thylakoid development and organization of the mutants. A yeast-two-hybrid screen with CPRabA5e as bait revealed 13 interacting partner proteins, mainly located in thylakoids and plastoglobules. These proteins are known or predicted to be involved in development, stress responses, and photosynthesis related processes, consistent with the stress phenotypes observed. The results observed suggest a role of CPRabA5e in transport to and from thylakoids, similar to cytosolic Rab proteins involved in vesicle transport.

  16. 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

  17. 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.

  18. 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

  19. Phosphorylation of the transit sequence of chloroplast precursor proteins.

    PubMed

    Waegemann, K; Soll, J

    1996-03-15

    A protein kinase was located in the cytosol of pea mesophyll cells. The protein kinase phosphorylates, in an ATP-dependent manner, chloroplast-destined precursor proteins but not precursor proteins, which are located to plant mitochondria or plant peroxisomes. The phosphorylation occurs on either serine or threonine residues, depending on the precursor protein used. We demonstrate the specific phosphorylation of the precursor forms of the chloroplast stroma proteins ferredoxin (preFd), small subunit of ribulose-bisphosphate-carboxylase (preSSU), the thylakoid localized light-harvesting chlorophyll a/b-binding protein (preLHCP), and the thylakoid lumen-localized proteins of the oxygen-evolving complex of 23 kDa (preOE23) and 33 kDa (preOE33). In the case of thylakoid lumen proteins which possess bipartite transit sequences, the phosphorylation occurs within the stroma-targeting domain. By using single amino acid substitution within the presequences of preSSU, preOE23, and preOE33, we were able to tentatively identify a consensus motif for the precursor protein protein kinase. This motif is (P/G)X(n)(R/K)X(n)(S/T)X(n) (S*/T*), were n = 0-3 amino acids spacer and S*/T* represents the phosphate acceptor. The precursor protein protein kinase is present only in plant extracts, e.g. wheat germ and pea, but not in a reticulocyte lysate. Protein import experiments into chloroplasts revealed that phosphorylated preSSU binds to the organelles, but dephosphorylation seems required to complete the translocation process and to obtain complete import. These results suggest that a precursor protein protein phosphatase is involved in chloroplast import and represents a so far unidentified component of the import machinery. In contrast to sucrose synthase, a cytosolic marker protein, the precursor protein protein kinase seems to adhere partially to the chloroplast surface. A phosphorylation-dephosphorylation cycle of chloroplast-destined precursor proteins might represent one step

  20. Chloroplast Dynamics and Photosynthetic Efficiency: Final Technical Report

    SciTech Connect

    Hanson, Maureen

    2016-11-03

    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 in 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 line

  1. Chloroplast remodeling during state transitions in Chlamydomonas reinhardtii as revealed by noninvasive techniques in vivo

    PubMed Central

    Nagy, Gergely; Ünnep, Renáta; Zsiros, Ottó; Tokutsu, Ryutaro; Takizawa, Kenji; Porcar, Lionel; Moyet, Lucas; Petroutsos, Dimitris; Garab, Győző; Finazzi, Giovanni; Minagawa, Jun

    2014-01-01

    Plants respond to changes in light quality by regulating the absorption capacity of their photosystems. These short-term adaptations use redox-controlled, reversible phosphorylation of the light-harvesting complexes (LHCIIs) to regulate the relative absorption cross-section of the two photosystems (PSs), commonly referred to as state transitions. It is acknowledged that state transitions induce substantial reorganizations of the PSs. However, their consequences on the chloroplast structure are more controversial. Here, we investigate how state transitions affect the chloroplast structure and function using complementary approaches for the living cells of Chlamydomonas reinhardtii. Using small-angle neutron scattering, we found a strong periodicity of the thylakoids in state 1, with characteristic repeat distances of ∼200 Å, which was almost completely lost in state 2. As revealed by circular dichroism, changes in the thylakoid periodicity were paralleled by modifications in the long-range order arrangement of the photosynthetic complexes, which was reduced by ∼20% in state 2 compared with state 1, but was not abolished. Furthermore, absorption spectroscopy reveals that the enhancement of PSI antenna size during state 1 to state 2 transition (∼20%) is not commensurate to the decrease in PSII antenna size (∼70%), leading to the possibility that a large part of the phosphorylated LHCIIs do not bind to PSI, but instead form energetically quenched complexes, which were shown to be either associated with PSII supercomplexes or in a free form. Altogether these noninvasive in vivo approaches allow us to present a more likely scenario for state transitions that explains their molecular mechanism and physiological consequences. PMID:24639515

  2. Polyphyletism of Celastrales deduced from a chloroplast noncoding DNA region.

    PubMed

    Savolainen, V; Spichiger, R; Manen, J F

    1997-04-01

    In a previous study we examined the phylogeny of four families related to the angiosperm order Celastrales based on chloroplast rbcL 5' flanking sequences. We have added here several additional dicots, sampled from 6 of the 7 families of Celastrales sensu Cronquist and 19 putatively related genera. Based on a cladistic analysis of these DNA sequences, the order Celastrales appears polyphyletic: it is here restricted to Celastraceae (including Hippocrateaceae and Brexia) with Parnassia as sister; Aquifoliaceae plus Helwingia are included in Asteridae. Neither Salvadoraceae nor Geissolomataceae, Icacinaceae, Phellinaceae, Aextoxicaceae, Corynocarpaceae, Dichapetalaceae, Stackhousiaceae, or Goupiaceae are related to Celastrales. The usefulness of this noncoding region is discussed and the influence of the A + T content of neighboring bases on the increase of transversions is also observed as previously shown in chloroplast noncoding regions of monocots.

  3. 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.

  4. The complete chloroplast genome sequence of Safflower (Carthamus tinctorius L.).

    PubMed

    Lu, Chaolong; Shen, Qi; Yang, Jun; Wang, Bo; Song, Chi

    2016-09-01

    Safflower (Carthamus tinctorius L.) is a traditional medical plants of Asia. In this study, the complete chloroplast genome of safflower was presented. The total genome size was 153,675 bp in length, containing a pair of inverted repeats (IRs) of 25,407 bp, separated by large single copy (LSC) and small single copy (SSC) of 83,606 bp and 19,156 bp, respectively. Overall GC content of the genome was 37.4%. The chloroplast genome harbored 127 annotated genes, including 89 protein coding genes, 30 tRNA genes and 8 rRNA genes. A total of 7 of these genes were duplicated in the inverted repeat regions. Twelve genes contained one intron.

  5. The whole chloroplast genome of shrub willows (Salix suchowensis).

    PubMed

    Wu, Zhiqiang

    2016-05-01

    The whole chloroplast genome of Salix suchowensis was determined in this study using next generation sequencing data. The total genome size was 155,214 bp in length, containing a pair of inverted repeats (IRs) of 27,459 bp, which were separated by large single copy (LSC) and small single copy (SSC) of 84,077 bp and 16,219 bp, respectively. The overall GC contents of the chloroplast genome were 36.73%. One hundred and ten unique genes were annotated, including 76 protein coding genes, 30 tRNA genes and 4 rRNA genes. Among these, 18 are duplicated in the inverted repeat regions, 14 genes contained 1 intron, and 3 genes (rps12, clpP and ycf3) comprised of 2 introns.

  6. Toxic effects of copper on photosystem II of spinach chloroplasts

    SciTech Connect

    Hsu, Bandar; Lee, Jeeyau )

    1988-05-01

    The room temperature fluorescence induction of chloroplasts was utilized as a probe to locate the site of inhibition on PSII by copper. It was found that, while the initial fluorescence yield was hardly affected, the variable fluorescence yield was lowered without significant change in its kinetics. Addition of DCMU, or abolishing oxygen evolution capability by Tris treatment, did not alter this basic inhibition pattern. Copper was also found to lower the fluorescence yield of chloroplasts treated with linolenic acid which inhibited the secondary electron transport on both oxidizing and reducing sides of PSII. The data indicate that copper adversely affects the primary change separation at the PSII reaction center. We suggest that the inhibition is due to creation of a lesion close to the reaction center, leading to increased dissipation of incoming excitation energy to heat.

  7. Chloroplast gene sequences and the study of plant evolution.

    PubMed Central

    Clegg, M T

    1993-01-01

    A large body of sequence data has accumulated for the chloroplast-encoded gene ribulose-1,5-biphosphate carboxylase/oxygenase (rbcL) as the result of a cooperative effort involving many laboratories. The data span all seed plants, including most major lineages from the angiosperms, and as such they provide an unprecedented opportunity to study plant evolutionary history. The full analysis of this large data set poses many problems and opportunities for plant evolutionary biologists and for biostatisticians. PMID:8421667

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

    PubMed Central

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

    2016-01-01

    ABSTRACT 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. PMID:27700374

  9. Chloroplasts play a central role in plant defence and are targeted by pathogen effectors.

    PubMed

    de Torres Zabala, Marta; Littlejohn, George; Jayaraman, Siddharth; Studholme, David; Bailey, Trevor; Lawson, Tracy; Tillich, Michael; Licht, Dirk; Bölter, Bettina; Delfino, Laura; Truman, William; Mansfield, John; Smirnoff, Nicholas; Grant, Murray

    2015-06-01

    Microbe associated molecular pattern (MAMP) receptors in plants recognize MAMPs and activate basal defences; however a complete understanding of the molecular and physiological mechanisms conferring immunity remains elusive. Pathogens suppress active defence in plants through the combined action of effector proteins. Here we show that the chloroplast is a key component of early immune responses. MAMP perception triggers the rapid, large-scale suppression of nuclear encoded chloroplast-targeted genes (NECGs). Virulent Pseudomonas syringae effectors reprogramme NECG expression in Arabidopsis, target the chloroplast and inhibit photosynthetic CO2 assimilation through disruption of photosystem II. This activity prevents a chloroplastic reactive oxygen burst. These physiological changes precede bacterial multiplication and coincide with pathogen-induced abscisic acid (ABA) accumulation. MAMP pretreatment protects chloroplasts from effector manipulation, whereas application of ABA or the inhibitor of photosynthetic electron transport, DCMU, abolishes the MAMP-induced chloroplastic reactive oxygen burst, and enhances growth of a P. syringae hrpA mutant that fails to secrete effectors.

  10. Chloroplast Structure and Function Is Altered in the NCS2 Maize Mitochondrial Mutant 1

    PubMed Central

    Roussell, Deborah L.; Thompson, Deborah L.; Pallardy, Steve G.; Miles, Donald; Newton, Kathleen J.

    1991-01-01

    The nonchromosomal stripe 2 (NCS2) mutant of maize (Zea mays L.) has a DNA rearrangement in the mitochondrial genome that segregates with the abnormal growth phenotype. Yet, the NCS2 characteristic phenotype includes striped sectors of pale-green tissue on the leaves. This suggests a chloroplast abnormality. To characterize the chloroplasts present in the mutant sectors, we examined the chloroplast structure by electron microscopy, chloroplast function by radiolabeled carbon dioxide fixation and fluorescence induction kinetics, and thylakoid protein composition by polyacrylamide gel electrophoresis. The data from these analyses suggest abnormal or prematurely arrested chloroplast development. Deleterious effects of the NCS2 mutant mitochondria upon the cells of the leaf include structural and functional alterations in the both the bundle sheath and mesophyll chloroplasts. ImagesFigure 1Figure 2Figure 3Figure 5Figure 6 PMID:16668157

  11. Photoprotective function of chloroplast avoidance movement: in vivo chlorophyll fluorescence study.

    PubMed

    Sztatelman, Olga; Waloszek, Andrzej; Banaś, Agnieszka Katarzyna; Gabryś, Halina

    2010-06-15

    Light-induced chloroplast avoidance movement has long been considered to be a photoprotective mechanism. Here, we present an experimental model in which this function can be shown for wild type Arabidopsis thaliana. We used blue light of different fluence rates for chloroplast positioning, and strong red light inactive in chloroplast positioning as a stressing light. The performance of photosystem II was measured by means of chlorophyll fluorescence. After stressing light treatment, a smaller decrease in photosystem II quantum yield was observed for leaves with chloroplasts in profile position as compared with leaves with chloroplasts in face position. Three Arabidopsis mutants, phot2 (no avoidance response), npq1 (impaired zeaxanhtin accumulation) and stn7 (no state transition), were examined for their chloroplast positioning and chlorophyll fluorescence parameters under identical experimental conditions. The results obtained for these mutants revealed additional stressing effects of blue light as compared with red light.

  12. Chloroplast Distribution in Arabidopsis thaliana (L.) Depends on Light Conditions during Growth.

    PubMed Central

    Trojan, A.; Gabrys, H.

    1996-01-01

    Chloroplasts of Arabidopsis thaliana move in response to blue light. Sensitivity to light and the range of fluence rates to which the chloroplasts respond were found to be comparable to those of other higher plants studied. We investigated typical chloroplast distributions in Arabidopsis grown under three different light conditions:standard-light conditions, similar to natural light intensities; weak-light intensities, close to the compensation point of photosynthesis; and strong-light intensities, close to the saturation of the light-response curve of photosynthesis. We observed a striking difference in chloroplast arrangement in darkness between plants grown under weak- and strong-light conditions. There was a slight difference after weak-light pretreatment, and the arrangements of chloroplasts after strong-light pretreatment in both plant groups were very similar. These results support the ecological significance of chloroplast movements. PMID:12226297

  13. A Role for Membrane Lipid Polyunsaturation in Chloroplast Biogenesis at Low Temperature 1

    PubMed Central

    Hugly, Suzanne; Somerville, Chris

    1992-01-01

    Two different mutants of Arabidopsis thaliana deficient in chloroplast membrane lipid polyunsaturation were indistinguishable in appearance from the wild-type when grown at 22°C. By contrast, leaf tissues of the mutants that developed during growth at 5°C were chlorotic, whereas the wild type was not. This is the first direct evidence that chloroplast lipid polyunsaturation contributes to low-temperature fitness. Chloroplasts from mutant lines grown at 5°C were much smaller than those of the wild-type, and the thylakoid membrane content was reduced by up to 70%. However, there was no discernible effect of low temperature on chloroplasts that developed prior to exposure to low temperatures. These and related observations suggest that the high degree of chloroplast membrane lipid polyunsaturation is required for some aspect of chloroplast biogenesis. ImagesFigure 3Figure 4 PMID:16668849

  14. 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 F1 hybrids and recovery in the F2 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.

  15. Functional analysis and expression characteristics of chloroplastic Prx IIE.

    PubMed

    Gama, Filipe; Bréhélin, Claire; Gelhaye, Eric; Meyer, Yves; Jacquot, Jean-Pierre; Rey, Pascal; Rouhier, Nicolas

    2008-07-01

    Peroxiredoxins (Prxs) are ubiquitous thiol-dependent peroxidases capable of eliminating a variety of peroxides through reactive catalytic cysteines, which are regenerated by reducing systems. Based on amino acid sequences and their mode of catalysis, five groups of thiol peroxidases have been distinguished in plants, and type II Prx is one of them with representatives in many sub-cellular compartments. The mature form of poplar chloroplastic Prx IIE was expressed as a recombinant protein in Escherichia coli. The protein is able to reduce H2O2 and tert-butyl hydroperoxide and is regenerated by both glutaredoxin (Grx) and thioredoxin (Trx) systems. Nevertheless, compared with Trxs, Grxs, and more especially chloroplastic Grx S12, are far more efficient reductants towards Prx IIE. The expression of Prx IIE at both the mRNA and protein levels as a function of organ type and abiotic stress conditions was investigated. Western blot analysis revealed that Prx IIE gene is constitutively expressed in Arabidopsis thaliana, mostly in young and mature leaves and in flowers. Under photo-oxidative treatment and water deficit, almost no change was observed in the abundance of Prx IIE in A. thaliana, while the level of Prx Q (one of the two other chloroplastic Prxs with 2-Cys Prx) increased in response to both stresses, indicating that plastidic members of the Prx family exhibit specific patterns of expression under stress.

  16. 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.

  17. An exogenous chloroplast genome for complex sequence manipulation in algae

    PubMed Central

    O'Neill, Bryan M.; Mikkelson, Kari L.; Gutierrez, Noel M.; Cunningham, Jennifer L.; Wolff, Kari L.; Szyjka, Shawn J.; Yohn, Christopher B.; Redding, Kevin E.; Mendez, Michael J.

    2012-01-01

    We demonstrate a system for cloning and modifying the chloroplast genome from the green alga, Chlamydomonas reinhardtii. Through extensive use of sequence stabilization strategies, the ex vivo genome is assembled in yeast from a collection of overlapping fragments. The assembled genome is then moved into bacteria for large-scale preparations and transformed into C. reinhardtii cells. This system also allows for the generation of simultaneous, systematic and complex genetic modifications at multiple loci in vivo. We use this system to substitute genes encoding core subunits of the photosynthetic apparatus with orthologs from a related alga, Scenedesmus obliquus. Once transformed into algae, the substituted genome recombines with the endogenous genome, resulting in a hybrid plastome comprising modifications in disparate loci. The in vivo function of the genomes described herein demonstrates that simultaneous engineering of multiple sites within the chloroplast genome is now possible. This work represents the first steps toward a novel approach for creating genetic diversity in any or all regions of a chloroplast genome. PMID:22116061

  18. 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.

  19. Longevity of guard cell chloroplasts in falling leaves: implication for stomatal function and cellular aging

    SciTech Connect

    Zeiger, E.; Schwartz, A.

    1982-11-12

    Guard cell chloroplasts in senescing leaves from 12 species of perennial trees and three species of annual plants survived considerably longer than their mesophyll counterparts. In Ginkgo biloba, stomata from yellow leaves opened during the day and closed at night; guard cell chloroplasts from these leaves showed fluorescence transients associated with electron transport and photophosphorylation. These findings indicate that guard cell chloroplasts are highly conserved throughout the life-span of the leaf and that leaves retain stomatal control during senescence.

  20. 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.

  1. Production of Recombinant Proteins in the Chloroplast of the Green Alga Chlamydomonas reinhardtii.

    PubMed

    Guzmán-Zapata, Daniel; Macedo-Osorio, Karla Soledad; Almaraz-Delgado, Alma Lorena; Durán-Figueroa, Noé; Badillo-Corona, Jesus Agustín

    2016-01-01

    Chloroplast transformation in the green algae Chlamydomonas reinhardtii can be used for the production of valuable recombinant proteins. Here, we describe chloroplast transformation of C. reinhardtii followed by protein detection. Genes of interest integrate stably by homologous recombination into the chloroplast genome following introduction by particle bombardment. Genes are inherited and expressed in lines recovered after selection in the presence of an antibiotic. Recombinant proteins can be detected by conventional techniques like immunoblotting and purified from liquid cultures.

  2. A point mutation in atpC1 raises the redox potential of the Arabidopsis chloroplast ATP synthase gamma-subunit regulatory disulfide above the range of thioredoxin modulation

    Technology Transfer Automated Retrieval System (TEKTRAN)

    The light-dependent regulation of chloroplast ATP synthase activity depends on an intricate but ill-defined interplay between the proton electrochemical potential across the thylakoid membrane and thioredoxin-mediated redox modulation of a cysteine bridge located on the ATP synthase gamma-subunit. T...

  3. 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.

  4. A plant-specific protein essential for blue-light-induced chloroplast movements.

    PubMed

    DeBlasio, Stacy L; Luesse, Darron L; Hangarter, Roger P

    2005-09-01

    In Arabidopsis (Arabidopsis thaliana), light-dependent chloroplast movements are induced by blue light. When exposed to low fluence rates of light, chloroplasts accumulate in periclinal layers perpendicular to the direction of light, presumably to optimize light absorption by exposing more chloroplast area to the light. Under high light conditions, chloroplasts become positioned parallel to the incoming light in a response that can reduce exposure to light intensities that may damage the photosynthetic machinery. To identify components of the pathway downstream of the photoreceptors that mediate chloroplast movements (i.e. phototropins), we conducted a mutant screen that has led to the isolation of several Arabidopsis mutants displaying altered chloroplast movements. The plastid movement impaired1 (pmi1) mutant exhibits severely attenuated chloroplast movements under all tested fluence rates of light, suggesting that it is a necessary component for both the low- and high-light-dependant chloroplast movement responses. Analysis of pmi1 leaf cross sections revealed that regardless of the light condition, chloroplasts are more evenly distributed in leaf mesophyll cells than in the wild type. The pmi1-1 mutant was found to contain a single nonsense mutation within the open reading frame of At1g42550. This gene encodes a plant-specific protein of unknown function that appears to be conserved among angiosperms. Sequence analysis of the protein suggests that it may be involved in calcium-mediated signal transduction, possibly through protein-protein interactions.

  5. Final Step of Phosphatidic Acid Synthesis in Pea Chloroplasts Occurs in the Inner Envelope Membrane 1

    PubMed Central

    Andrews, Jaen; Ohlrogge, John B.; Keegstra, Kenneth

    1985-01-01

    The second enzyme of phosphatidic acid synthesis from glycerol-3-phosphate, 1-acylglycerophospate acyltransferase, was localized to the inner envelope membrane of pea chloroplasts. The activity of this enzyme was measured by both a coupled enzyme assay and a direct enzyme assay. Using the coupled enzyme assay, phosphatidic acid phosphatase was also localized to the inner envelope membrane, although this enzyme has very low activity in pea chloroplasts. The addition of UDP-galactose to unfractionated pea chloroplast envelope preparations did not result in significant conversion of newly synthesized diacylglycerol to monogalactosyldiacylglycerol. Thus, the envelope synthesized phosphatidic acid may not be involved in galactolipid synthesis in pea chloroplasts. PMID:16664266

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

    SciTech Connect

    Angelova, Angelina; Park, Sang-Hycuk; Kyndt, John; Fitzsimmons, Kevin; Brown, Judith K

    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. 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.

  7. Integrated analysis of transcriptome and metabolome of Arabidopsis albino or pale green mutants with disrupted nuclear-encoded chloroplast proteins.

    PubMed

    Satou, Masakazu; Enoki, Harumi; Oikawa, Akira; Ohta, Daisaku; Saito, Kazunori; Hachiya, Takushi; Sakakibara, Hitoshi; Kusano, Miyako; Fukushima, Atsushi; Saito, Kazuki; Kobayashi, Masatomo; Nagata, Noriko; Myouga, Fumiyoshi; Shinozaki, Kazuo; Motohashi, Reiko

    2014-07-01

    We used four mutants having albino or pale green phenotypes with disrupted nuclear-encoded chloroplast proteins to analyze the regulatory system of metabolites in chloroplast. We performed an integrated analyses of transcriptomes and metabolomes of the four mutants. Transcriptome analysis was carried out using the Agilent Arabidopsis 2 Oligo Microarray, and metabolome analysis with two mass spectrometers; a direct-infusion Fourier transform ion cyclotron resonance mass spectrometer (FT-ICR/MS) and a gas chromatograph-time of flight mass spectrometer. Among approximately 200 known metabolites detected by the FT-ICR/MS, 71 metabolites showed significant changes in the mutants when compared with controls (Ds donor plants). Significant accumulation of several amino acids (glutamine, glutamate and asparagine) was observed in the albino and pale green mutants. Transcriptome analysis revealed altered expressions of genes in several metabolic pathways. For example, genes involved in the tricarboxylic acid cycle, the oxidative pentose phosphate pathway, and the de novo purine nucleotide biosynthetic pathway were up-regulated. These results suggest that nitrogen assimilation is constitutively promoted in the albino and pale green mutants. The accumulation of ammonium ions in the albino and pale green mutants was consistently higher than in Ds donor lines. Furthermore, genes related to pyridoxin accumulation and the de novo purine nucleotide biosynthetic pathway were up-regulated, which may have occurred as a result of the accumulation of glutamine in the albino and pale green mutants. The difference in metabolic profiles seems to be correlated with the disruption of chloroplast internal membrane structures in the mutants. In albino mutants, the alteration of metabolites accumulation and genes expression is stronger than pale green mutants.

  8. Transcript abundance supercedes editing efficiency as a factor in developmental variation of chloroplast gene expression.

    PubMed Central

    Peeters, Nemo M; Hanson, Maureen R

    2002-01-01

    In maize plastids, transcripts are known to be modified at 27 C-to-U RNA editing sites, affecting the expression-of 15 different genes. The relative contribution of editing efficiency versus transcript abundance in regulation of chloroplast gene expression has previously been analyzed for only a few genes. We undertook a comprehensive analysis of the editing efficiency of each of the 27 maize editing sites in 10 different maize tissues, which contain a range of plastid types including chloroplasts, etioplasts, and amyloplasts. Using a reproducible poisoned primer extension assay, we detected variation between RNA editing extent of different sites in the same transcript in the same tissue, and between the same site in different tissues. The most striking editing deficiency is in an editing site in ndhB that is edited at only 8% and 1% in roots and callus plastids respectively, whereas green leaf chloroplasts edit this site at 100%. Editing efficiencies of some sites are not affected by the developmental stages we examined and are always edited close to 80-100%. The relative amounts of transcripts of each of the 10 genes that exhibited variable editing extents were determined by real-time PCR. Seven genes exhibited over 100 times lower transcript abundance in either roots or tissue-cultured cells relative to green leaf tissue. The quantitative analysis indicates that a particular editing site can be efficiently edited over a large range of transcript abundance, resulting in no general correlation of transcript abundance and editing extent. The independent variation of editing efficiency of different sites within the same transcript fits with a model that postulates individual trans-acting factors specific to each editing site. Because tissues where editing efficiency at certain sites is low invariably also exhibited greatly decreased abundance of the transcripts carrying those sites, decrease in the amounts of particular RNAs rather than a lack of editing is

  9. Editing of the chloroplast rpoB transcript is independent of chloroplast translation and shows different patterns in barley and maize.

    PubMed Central

    Zeltz, P; Hess, W R; Neckermann, K; Börner, T; Kössel, H

    1993-01-01

    Sequence analysis of amplified cDNAs derived from the maize chloroplast rpoB transcript which encodes the beta subunit of a chloroplast specific, DNA dependent RNA polymerase reveals four C-to-U editing sites clustered within 150 nucleotides of the 5' terminal region of the rpoB message. These newly identified editing sites confirm the bias of chloroplast editing for certain codon transitions and for second codon positions which both appear suggestive for an involvement of the translational apparatus in the editing process. This supposition prompted us to investigate editing of the rpoB transcript from ribosome deficient, and hence protein synthesis deficient, plastids of the barley mutant albostrians. In this mutant editing is, however, not impaired at any of the editing sites functional in the barley wild type rpoB transcript. This demonstrates that chloroplast editing is neither linked to nor dependent on the chloroplast translational apparatus. As a further consequence any peptide components required for chloroplast editing must be encoded in the nuclear genome. In spite of strong sequence conservation only three of the four editing sites identified in the maize rpoB transcript are functional in barley. This indicates that sequences surrounding an editing site alone are not sufficient as determinants for the editing process in chloroplasts, but that trans-acting templates carrying the editing information for each individual site may also be required. Images PMID:8223439

  10. Euglena gracilis chloroplast ribosomal protein operon: a new chloroplast gene for ribosomal protein L5 and description of a novel organelle intron category designated group III.

    PubMed Central

    Christopher, D A; Hallick, R B

    1989-01-01

    We describe the structure (3840 bp) of a novel Euglena gracilis chloroplast ribosomal protein operon that encodes the five genes rpl16-rpl14-rpl5-rps8-rpl36. The gene organization resembles the spc and the 3'-end of the S10 ribosomal protein operons of E. coli. The rpl5 is a new chloroplast gene not previously reported for any chloroplast genome to date and also not described as a nuclear-encoded, chloroplast protein gene. The operon contains at least 7 introns. We present evidence from primer extension analysis of chloroplast RNA for the correct in vivo splicing of five of the introns. Two of the introns within the rps8 gene flank an 8 bp exon, the smallest exon yet characterized in a chloroplast gene. Three introns resemble the classical group II introns of organelle genomes. The remaining 4 introns appear to be unique to the Euglena chloroplast DNA. They are uniform in size (95-109 nt), share common features with each other and are distinct from both group I and group II introns. We designate this new intron category as 'group III'. Images PMID:2477800

  11. Spontaneous capture of oilseed rape (Brassica napus) chloroplasts by wild B. rapa: implications for the use of chloroplast transformation for biocontainment.

    PubMed

    Haider, Nadia; Allainguillaume, Joel; Wilkinson, Mike J

    2009-04-01

    Environmental concerns over the cultivation of Genetically Modified (GM) crops largely centre on the ecological consequences following gene flow to wild relatives. One attractive solution is to deploy biocontainment measures that prevent hybridization. Chloroplast transformation is the most advanced biocontainment method but is compromised by chloroplast capture (hybridization through the maternal lineage). To date, however, there is a paucity of information on the frequency of chloroplast capture in the wild. Oilseed rape (Brassica napus, AACC) frequently hybridises with wild Brassica rapa (AA, as paternal parent) and yields B. rapa-like introgressed individuals after only two generations. In this study we used chloroplast CAPS markers that differentiate between the two species to survey wild and weedy populations of B. rapa for the capture of B. napus chloroplasts. A total of 464 B. rapa plants belonging to 14 populations growing either in close proximity to B. napus (i.e. sympatric <5 m) or else were allopatric from the crop (>1 km) were assessed for chloroplast capture using PCR (trnL-F) and CAPS (trnT-L-Xba I) markers. The screen revealed that two sympatric B. rapa populations included 53 plants that possessed the chloroplast of B. napus. In order to discount these B. rapa plants as F(1) crop-wild hybrids, we used a C-genome-specific marker and found that 45 out of 53 plants lacked the C-genome and so were at least second generation introgressants. The most plausible explanation is that these individuals represent multiple cases of chloroplast capture following introgressive hybridisation through the female germ line from the crop. The abundance of such plants in sympatric sites thereby questions whether the use of chloroplast transformation would provide a sufficient biocontainment for GM oilseed rape in the United Kingdom.

  12. Photochemical Properties of Mesophyll and Bundle Sheath Chloroplasts of Maize 1

    PubMed Central

    Bazzaz, Maarib Bakri; Govindjee

    1973-01-01

    Several photochemical and spectral properties of maize (Zea mays) bundle sheath and mesophyll chloroplasts are reported that provide a better understanding of the photosynthetic apparatus of C4 plants. The difference absorption spectrum at 298 K and the fluorescence excitation and emission spectra of chlorophyll at 298 K and 77 K provide new information on the different forms of chlorophyll a in bundle sheath and mesophyll chloroplasts: the former contain, relative to short wavelength chlorophyll a forms, more long wavelength chlorophyll a form (e.g. chlorophyll a 693 and chlorophyll a 705) and less chlorophyll b than the latter. The degree of polarization of chlorophyll a fluorescence is 6% in bundle sheath and 4% in mesophyll chloroplasts. This result is consistent with the presence of relatively high amounts of oriented long wavelength forms of chlorophyll a in bundle sheath compared to mesophyll chloroplasts. The relative yield of variable, with respect to constant, chorophyll a fluorescence in mesophyll chloroplasts is more than twice that in bundle sheath chloroplast. Furthermore, the relative yield of total chlorophyll a fluorescence is 40% lower in bundle sheath compared to that in mesophyll chloroplasts. This is in agreement with the presence of the higher ratio of the weakly fluorescent pigment system I to pigment system II in bundle sheath than in mesophyll chloroplast. The efficiency of energy transfer from chlorophyll b and carotenoids to chlorophyll a are calculated to be 100 and 50%, respectively, in both types of chloroplasts. Fluorescence quenching of atebrin, reflecting high energy state of chloroplasts, is 10 times higher in mesophyll chloroplasts than in bundle sheath chloroplasts during noncyclic electron flow but is equal during cyclic flow. The entire electron transport chain is shown to be present in both types of chloroplasts, as inferred from the antagonistic effect of red (650 nm) and far red (710 nm) lights on the absorbance changes at

  13. Whole genome sequencing of enriched chloroplast DNA using the Illumina GAII platform

    PubMed Central

    2010-01-01

    Background Complete chloroplast genome sequences provide a valuable source of molecular markers for studies in molecular ecology and evolution of plants. To obtain complete genome sequences, recent studies have made use of the polymerase chain reaction to amplify overlapping fragments from conserved gene loci. However, this approach is time consuming and can be more difficult to implement where gene organisation differs among plants. An alternative approach is to first isolate chloroplasts and then use the capacity of high-throughput sequencing to obtain complete genome sequences. We report our findings from studies of the latter approach, which used a simple chloroplast isolation procedure, multiply-primed rolling circle amplification of chloroplast DNA, Illumina Genome Analyzer II sequencing, and de novo assembly of paired-end sequence reads. Results A modified rapid chloroplast isolation protocol was used to obtain plant DNA that was enriched for chloroplast DNA, but nevertheless contained nuclear and mitochondrial DNA. Multiply-primed rolling circle amplification of this mixed template produced sufficient quantities of chloroplast DNA, even when the amount of starting material was small, and improved the template quality for Illumina Genome Analyzer II (hereafter Illumina GAII) sequencing. We demonstrate, using independent samples of karaka (Corynocarpus laevigatus), that there is high fidelity in the sequence obtained from this template. Although less than 20% of our sequenced reads could be mapped to chloroplast genome, it was relatively easy to assemble complete chloroplast genome sequences from the mixture of nuclear, mitochondrial and chloroplast reads. Conclusions We report successful whole genome sequencing of chloroplast DNA from karaka, obtained efficiently and with high fidelity. PMID:20920211

  14. Identification of nuclear genes encoding chloroplast-localized proteins required for embryo development in Arabidopsis.

    PubMed

    Bryant, Nicole; Lloyd, Johnny; Sweeney, Colleen; Myouga, Fumiyoshi; Meinke, David

    2011-04-01

    We describe here the diversity of chloroplast proteins required for embryo development in Arabidopsis (Arabidopsis thaliana). Interfering with certain chloroplast functions has long been known to result in embryo lethality. What has not been reported before is a comprehensive screen for embryo-defective (emb) mutants altered in chloroplast proteins. From a collection of transposon and T-DNA insertion lines at the RIKEN chloroplast function database (http://rarge.psc.riken.jp/chloroplast/) that initially appeared to lack homozygotes and segregate for defective seeds, we identified 23 additional examples of EMB genes that likely encode chloroplast-localized proteins. Fourteen gene identities were confirmed with allelism tests involving duplicate mutant alleles. We then queried journal publications and the SeedGenes database (www.seedgenes.org) to establish a comprehensive dataset of 381 nuclear genes encoding chloroplast proteins of Arabidopsis associated with embryo-defective (119 genes), plant pigment (121 genes), gametophyte (three genes), and alternate (138 genes) phenotypes. Loci were ranked based on the level of certainty that the gene responsible for the phenotype had been identified and the protein product localized to chloroplasts. Embryo development is frequently arrested when amino acid, vitamin, or nucleotide biosynthesis is disrupted but proceeds when photosynthesis is compromised and when levels of chlorophyll, carotenoids, or terpenoids are reduced. Chloroplast translation is also required for embryo development, with genes encoding chloroplast ribosomal and pentatricopeptide repeat proteins well represented among EMB datasets. The chloroplast accD locus, which is necessary for fatty acid biosynthesis, is essential in Arabidopsis but not in Brassica napus or maize (Zea mays), where duplicated nuclear genes compensate for its absence or loss of function.

  15. pCyP B: a chloroplast-localized, heat shock-responsive cyclophilin from fava bean.

    PubMed

    Luan, S; Lane, W S; Schreiber, S L

    1994-06-01

    When the immunosuppressants cyclosporin A (CsA) and FK506 bind to their intracellular receptors (immunophillins), they form complexes that bind to calcineurin and block calcineurin-dependent signaling pathways in immune cells. Previously, we reported that higher plants also express immunophilins and have a Ca(2+)-dependent signaling pathway sensitive to immunophilin-ligand complexes. Based on an N-terminal peptide sequence of a chloroplast-localized cyclophilin (pCyP B), we isolated a cDNA clone encoding the preprotein of the cyclophilin. The deduced amino acid sequence of this cDNA starts with a putative transit sequence for chloroplast targeting. The mature pCyP B protein has rotamase activity with low-substrate specificity. Enzyme activity was inhibited by CsA with an inhibition constant of 3.9 nM. Similar to other CyPs from mammalian cells, pCyP B, when complexed with CsA, inhibited the phosphatase activity of bovine calcineurin. The mRNA level of pCyP B was high in leaf tissue but was not detectable in roots. Expression of the transcript in the leaf tissues was regulated by light and induced by heat shock. These findings illustrate the conserved nature of cyclophilin proteins among all of the eukaryotes and suggest that cyclophilins have a unique mode of regulation in higher plants.

  16. A purification strategy for analysis of the DNA/RNA-associated sub-proteome from chloroplasts of mustard cotyledons

    PubMed Central

    Schröter, Yvonne; Steiner, Sebastian; Weisheit, Wolfram; Mittag, Maria; Pfannschmidt, Thomas

    2014-01-01

    Plant cotyledons are a tissue that is particularly active in plastid gene expression in order to develop functional chloroplasts from pro-plastids, the plastid precursor stage in plant embryos. Cotyledons, therefore, represent a material being ideal for the study of composition, function and regulation of protein complexes involved in plastid gene expression. Here, we present a pilot study that uses heparin-Sepharose and phospho-cellulose chromatography in combination with isoelectric focussing and denaturing SDS gel electrophoresis (two-dimensional gel electrophoresis) for investigating the nucleic acids binding sub-proteome of mustard chloroplasts purified from cotyledons. We describe the technical requirements for a highly resolved biochemical purification of several hundreds of protein spots obtained from such samples. Subsequent mass spectrometry of peptides isolated out of cut spots that had been treated with trypsin identified 58 different proteins within 180 distinct spots. Our analyses indicate a high enrichment of proteins involved in transcription and translation and, in addition, the presence of massive post-translational modification of this plastid protein sub-fraction. The study provides an extended catalog of plastid proteins from mustard being involved in gene expression and its regulation and describes a suitable purification strategy for further analysis of low abundant gene expression related proteins. PMID:25400643

  17. TCA1, a single nuclear-encoded translational activator specific for petA mRNA in Chlamydomonas reinhardtii chloroplast.

    PubMed Central

    Wostrikoff, K; Choquet, Y; Wollman, F A; Girard-Bascou, J

    2001-01-01

    We isolated seven allelic nuclear mutants of Chlamydomonas reinhardtii specifically blocked in the translation of cytochrome f, a major chloroplast-encoded subunit of the photosynthetic electron transport chain encoded by the petA gene. We recovered one chloroplast suppressor in which the coding region of petA was now expressed under the control of a duplicated 5' untranslated region from another open reading frame of presently unknown function. Since we also recovered 14 nuclear intragenic suppressors, we ended up with 21 alleles of a single nuclear gene we called TCA1 for translation of cytochrome b(6)f complex petA mRNA. The high number of TCA1 alleles, together with the absence of genetic evidence for other nuclear loci controlling translation of the chloroplast petA gene, strongly suggests that TCA1 is the only trans-acting factor. We studied the assembly-dependent regulation of cytochrome f translation--known as the CES process--in TCA1-mutated contexts. In the presence of a leaky tca1 allele, we observed that the regulation of cytochrome f translation was now exerted within the limits of the restricted translational activation conferred by the altered version of TCA1 as predicted if TCA1 was the ternary effector involved in the CES process. PMID:11560891

  18. Defining the Core Proteome of the Chloroplast Envelope Membranes

    PubMed Central

    Simm, Stefan; Papasotiriou, Dimitrios G.; Ibrahim, Mohamed; Leisegang, Matthias S.; Müller, Bernd; Schorge, Tobias; Karas, Michael; Mirus, Oliver; Sommer, Maik S.; Schleiff, Enrico

    2013-01-01

    High-throughput protein localization studies require multiple strategies. Mass spectrometric analysis of defined cellular fractions is one of the complementary approaches to a diverse array of cell biological methods. In recent years, the protein content of different cellular (sub-)compartments was approached. Despite of all the efforts made, the analysis of membrane fractions remains difficult, in that the dissection of the proteomes of the envelope membranes of chloroplasts or mitochondria is often not reliable because sample purity is not always warranted. Moreover, proteomic studies are often restricted to single (model) species, and therefore limited in respect to differential individual evolution. In this study we analyzed the chloroplast envelope proteomes of different plant species, namely, the individual proteomes of inner and outer envelope (OE) membrane of Pisum sativum and the mixed envelope proteomes of Arabidopsis thaliana and Medicago sativa. The analysis of all three species yielded 341 identified proteins in total, 247 of them being unique. 39 proteins were genuine envelope proteins found in at least two species. Based on this and previous envelope studies we defined the core envelope proteome of chloroplasts. Comparing the general overlap of the available six independent studies (including ours) revealed only a number of 27 envelope proteins. Depending on the stringency of applied selection criteria we found 231 envelope proteins, while less stringent criteria increases this number to 649 putative envelope proteins. Based on the latter we provide a map of the outer and inner envelope core proteome, which includes many yet uncharacterized proteins predicted to be involved in transport, signaling, and response. Furthermore, a foundation for the functional characterization of yet unidentified functions of the inner and OE for further analyses is provided. PMID:23390424

  19. Selenocystamine improves protein accumulation in chloroplasts of eukaryotic green algae.

    PubMed

    Ferreira-Camargo, Livia S; Tran, Miller; Beld, Joris; Burkart, Michael D; Mayfield, Stephen P

    2015-12-01

    Eukaryotic green algae have become an increasingly popular platform for recombinant proteins production. In particular, Chlamydomonas reinhardtii, has garnered increased attention for having the necessary biochemical machinery to produce vaccines, human antibodies and next generation cancer targeting immunotoxins. While it has been shown that chloroplasts contain chaperones, peptidyl prolylisomerases and protein disulfide isomerases that facilitate these complex proteins folding and assembly, little has been done to determine which processes serve as rate-limiting steps for protein accumulation. In other expression systems, as Escherichia coli, Chinese hamster ovary cells, and insect cells, recombinant protein accumulation can be hampered by cell's inability to fold the target polypeptide into the native state, resulting in aggregation and degradation. To determine if chloroplasts' ability to oxidize proteins that require disulfide bonds into a stable conformation is a rate-limiting step of protein accumulation, three recombinant strains, each expressing a different recombinant protein, were analyzed. These recombinant proteins included fluorescent GFP, a reporter containing no disulfide bonds; Gaussia princeps luciferase, a luminescent reporter containing disulfide bonds; and an immunotoxin, an antibody-fusion protein containing disulfide bonds. Each strain was analyzed for its ability to accumulate proteins when supplemented with selenocystamine, a small molecule capable of catalyzing the formation of disulfide bonds. Selenocystamine supplementation led to an increase in luciferase and immunotoxin but not GFP accumulation. These results demonstrated that selenocystamine can increase the accumulation of proteins containing disulfide bonds and suggests that a rate-limiting step in chloroplast protein accumulation is the disulfide bonds formation in recombinant proteins native structure.

  20. 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

  1. A comparative approach to elucidate chloroplast genome replication

    PubMed Central

    Krishnan, Neeraja M; Rao, Basuthkar J

    2009-01-01

    Background Electron microscopy analyses of replicating chloroplast molecules earlier predicted bidirectional Cairns replication as the prevalent mechanism, perhaps followed by rounds of a rolling circle mechanism. This standard model is being challenged by the recent proposition of homologous recombination-mediated replication in chloroplasts. Results We address this issue in our current study by analyzing nucleotide composition in genome regions between known replication origins, with an aim to reveal any adenine to guanine deamination gradients. These gradual linear gradients typically result from the accumulation of deaminations over the time spent single-stranded by one of the strands of the circular molecule during replication and can, therefore, be used to model the course of replication. Our linear regression analyses on the nucleotide compositions of the non-coding regions and the synonymous third codon position of coding regions, between pairs of replication origins, reveal the existence of significant adenine to guanine deamination gradients in portions overlapping the Small Single Copy (SSC) and the Large Single Copy (LSC) regions between inverted repeats. These gradients increase bi-directionally from the center of each region towards the respective ends, suggesting that both the strands were left single-stranded during replication. Conclusion Single-stranded regions of the genome and gradients in time that these regions are left single-stranded, as revealed by our nucleotide composition analyses, appear to converge with the original bi-directional dual displacement loop model and restore evidence for its existence as the primary mechanism. Other proposed faster modes such as homologous recombination and rolling circle initiation could exist in addition to this primary mechanism to facilitate homoplasmy among the intra-cellular chloroplast population PMID:19457260

  2. 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.

  3. Nucleotide specificity of the RNA editing reaction in pea chloroplasts.

    PubMed

    Nakajima, Yuki; Mulligan, R Michael

    2005-12-01

    A sensitive in vitro editing assay for the pea chloroplast petB editing site has been developed and utilized to study the mechanism of C-to-U editing in chloroplast extracts. The in vitro editing assay was characterized by several criteria including: linearity with extract amount; linearity over time; dependence on assay components; and specificity of editing site conversion. The increase in the extent C-to-U conversion of the petB editing site was nearly linear with the amount chloroplast protein extract added, although the reaction appeared to decline in rate after approximately 30 min. The assay was tested for the importance of various assay components, and the omission of protease inhibitor and ATP was shown to dramatically reduce the extent of the editing reaction. Sequence analysis of cDNA clones obtained after an in vitro editing reaction demonstrated that 12 of 17 (71%) clones were edited, and that no other nucleotide changes in these cDNAs were detected. Thus, the fidelity and specificity of the in vitro editing system appears to be excellent, and this system should be suitable to study both mechanism of the editing reaction and editing site selection. The in vitro editing reaction was strongly stimulated by the addition of ATP, and all four NTPs and dNTPs stimulated the editing reaction except for rGTP, which had no effect. Thus, the nucleotide specificity of the editing reaction is broad, and is similar in this respect to the mitochondrial editing system. Most enzyme or processes specifically utilize ATP or GTP for phosphorylation and the ability to substitute other NTPs and dNTPs is unusual. RNA helicases have a similar broad nucleotide specificity and this may reflect the involvement of an RNA helicase in plant organelle editing.

  4. Carbonic anhydrase activity in isolated chloroplasts of chlamydomonas reinhardtii

    SciTech Connect

    Katzman, G.; Togasaki, R.K. ); Marcus, Y. ); Moroney, J.V. )

    1989-04-01

    In a new assay of carbonic anhydrase, NaH{sup 14}CO{sub 3} solution at the bottom of a sealed vessel releases {sup 14}CO{sub 3} which diffuses to the top of the vessel to be assimilated by actively photosynthesizing Chlamydomonas cells. The assay is initiated by illuminating cells and stopped by turning the light off and killing the cells with acid. Enzyme activity was estimated from acid stable radioactivity above the uncatalyzed background level. With bovine carbonic anhydrase, 1.5 Wilbur Anderson Unit (WAU) can be consistantly measured at 5-6 fold above background. Sonicated whole cells of air adapted wild type (+)gave 741.1 {plus minus} 12.4 WAU/mg chl. Intact washed cells of mixotrophically grown wall-less mutant CWD(-) and a high CO2 requiring wall-less double mutant CIA-3/CW15 (-) gave 7.1 {plus minus} 1.9 and 2.8 {plus minus} 7.8 WAU/mg chl respectively. Chloroplasts isolated from CWD and CIA-3/CW15 and subsequently disrupted gave 64.0 {plus minus} 14.7 and 2.8 {plus minus} 3.2 WAU/mg chl respectively. Chloroplast sonicate from another wall-less mutant CW15(-) gave activity comparable to CWD. Thus on a chlorophyll basis, enzyme activity in chloroplasts from mixotrophically grown cells is about 1/10th of the level found in air adapted wild type cells. CIA-3 seems to lack this activity.

  5. Development and use of chlorotetracycline fluorescence as a measurement assay of chloroplast envelope-bound mg.

    PubMed

    Gupta, A S; Berkowitz, G A

    1989-03-01

    Experiments were conducted to develop chlorotetracycline (CTC) fluorescence as an assay of Mg(2+) bound to the envelope of the intact chloroplast. This assay technique has been widely used to measure envelope associated divalent cations in animal cell and subcellular systems, but has not been used with chloroplasts. Chloroplast envelope-associated Mg(2+) was altered by pretreatment with Mg(2+) and divalent cation chelating agents and by additions of Mg(2+) to the CTC assay medium. Results indicated that for a given chloroplast preparation, relative changes in envelope-associated Mg(2+) can be effectively monitored with CTC fluorescence. It was concluded that the limitations of this assay system are: (a) chlorophyll strongly quenches CTC fluorescence signal, so a constant chlorophyll concentration must be maintained, (b) measurements must be made quickly, and (c) use of the technique to compare different chloroplast preparations may not be valid. Studies with (28)Mg(2+) confirmed our interpretation of the fluorescence results, and also suggested that the chloroplast envelope is fairly impermeable to Mg(2+). It was concluded that changes in Mg(2+) associated with the chloroplast due to incubation of plastids in solutions containing up to 5 millimolar Mg(2+) may be exclusively due to increased envelope-associated Mg(2+). The CTC assay was used in experiments to demonstrate that increases in chloroplast envelope-associated Mg(2+) inhibit photosynthetic capacity. This inhibition can be partially overcome by the presence of K(+) in the photosynthetic reaction media.

  6. Accumulation response of chloroplasts induced by mechanical stimulation in bryophyte cells.

    PubMed

    Sato, Yoshikatsu; Wada, Masamitsu; Kadota, Akeo

    2003-03-01

    Chloroplast movement has been studied in many plants but mainly as a model system for light signaling. However, we recently showed that the avoidance response of chloroplasts is also induced by mechanical stimulation in fern protonemal cells. Here we report the discovery of a mechanically induced accumulation response of chloroplasts in bryophytes. When mechanical stimulation was directly applied with a capillary to a part of a cell, chloroplasts moved towards and accumulated at the pressed site within 30 min after the onset of stimulation in all species tested. The accumulation movement of chloroplasts was inhibited by Cremart but not by cytochalasin B in red-light-grown protonemata of Physcomitrella patens (Hedw.) B., S. & G. To determine the contribution of external Ca(2+) to the response, we examined the effects on the accumulation movement of gadolinium (Ga(3+)), an inhibitor of stretch-activated ion channels, and lanthanum (La(3+)), a potent inhibitor of calcium channels. Mechano-relocation of chloroplasts was abolished by these drugs, but no effects were observed on photo-relocation of chloroplasts, irrespective of light colors and intensity. These results suggest that influx of external Ca(2+) through the plasma membrane is essential for the early steps in signaling of mechano-relocation of chloroplasts whose motility system is dependent on microtubules.

  7. The Arabidopsis YELLOW STRIPE LIKE4 and 6 transporters control iron release from the chloroplast.

    PubMed

    Divol, Fanchon; Couch, Daniel; Conéjéro, Geneviève; Roschzttardtz, Hannetz; Mari, Stéphane; Curie, Catherine

    2013-03-01

    In most plant cell types, the chloroplast represents the largest sink for iron, which is both essential for chloroplast metabolism and prone to cause oxidative damage. Here, we show that to buffer the potentially harmful effects of iron, besides ferritins for storage, the chloroplast is equipped with specific iron transporters that respond to iron toxicity by removing iron from the chloroplast. We describe two transporters of the YELLOW STRIPE1-LIKE family from Arabidopsis thaliana, YSL4 and YSL6, which are likely to fulfill this function. Knocking out both YSL4 and YSL6 greatly reduces the plant's ability to cope with excess iron. Biochemical and immunolocalization analyses showed that YSL6 resides in the chloroplast envelope. Elemental analysis and histochemical staining indicate that iron is trapped in the chloroplasts of the ysl4 ysl6 double mutants, which also accumulate ferritins. Also, vacuolar iron remobilization and NRAMP3/4 expression are inhibited. Furthermore, ubiquitous expression of YSL4 or YSL6 dramatically reduces plant tolerance to iron deficiency and decreases chloroplastic iron content. These data demonstrate a fundamental role for YSL4 and YSL6 in managing chloroplastic iron. YSL4 and YSL6 expression patterns support their physiological role in detoxifying iron during plastid dedifferentiation occurring in embryogenesis and senescence.

  8. Conservation and differences of the Min system in the chloroplast and bacterial division site placement

    PubMed Central

    Suzuki, Kenji; Kabeya, Yukihiro; Okazaki, Kumiko; Miyagishima, Shin-ya

    2009-01-01

    Chloroplasts are descended from a cyanobacterial endosymbiont and divide by binary fission. Reminiscent of the process in their bacterial ancestor, chloroplast division involves a part of cyanobacteria-derived division machineries in addition to those acquired during chloroplast evolution.1,2 In both bacterial and chloroplast division, formation of the FtsZ ring at the mid position is required for subsequent constriction and fission at the mid division site.1–4 As in bacteria, positioning of the FtsZ ring at the mid-chloroplast is mediated by the Min system.1,2 Recently, we identified the MCD1 protein, a plant-specific component of the Min system in Arabidopsis thaliana chloroplasts.5 Unlike other division components that have been acquired after endosymbiosis and function outside of the chloroplasts (i.e., in/on the outer envelope membrane),6–9 MCD1 functions inside the chloroplast. Since we already discussed about the function and significance of MCD1 as a division component of plant origin,5 here we focus on and discuss about the diversity and evolution of the Min system. PMID:19907699

  9. Changes in leaf optical properties associated with light-dependent chloroplast movements.

    PubMed

    Davis, Phillip A; Caylor, Steven; Whippo, Craig W; Hangarter, Roger P

    2011-12-01

    We surveyed 24 plant species to examine how leaf anatomy influenced chloroplast movement and how the optical properties of leaves change with chloroplast position. All species examined exhibited light-dependent chloroplast movements but the associated changes in leaf absorptance varied considerably in magnitude. Chloroplast movement-dependent changes in leaf absorptance were greatest in shade species, in which absorptance changes of >10% were observed between high- and low-light treatments. Using the Kubelka-Munk theory, we found that changes in the absorption (k) and chlorophyll a absorption efficiency (k*) associated with chloroplast movement correlated with cell diameter, such that the narrower, more columnar cells found in sun leaves restricted the ability of chloroplasts to move. The broader, more spherical cells of shade leaves allowed greater chloroplast rearrangements and in low-light conditions allowed efficient light capture. Across the species tested, light-dependent chloroplast movements modulated leaf optical properties and light absorption efficiency by manipulating the package (sieve or flattening) effect but not the detour (path lengthening) effect.

  10. Hartmut Lichtenthaler: an authority on chloroplast structure and isoprenoid biochemistry.

    PubMed

    Sharkey, Thomas D; Govindjee

    2016-05-01

    We pay tribute to Hartmut Lichtenthaler for making important contributions to the field of photosynthesis research. He was recently recognized for ground-breaking discoveries in chloroplast structure and isoprenoid biochemistry by the Rebeiz Foundation for Basic Research (RFBR; http://vlpbp.org/ ), receiving a 2014 Lifetime Achievement Award for Photosynthesis. The ceremony, held in Champaign, Illinois, was attended by many prominent researchers in the photosynthesis field. We provide below a brief note on his education, and then describe some of the areas in which Hartmut Lichtenthaler has been a pioneer.

  11. The complete chloroplast genome sequence of Hibiscus syriacus.

    PubMed

    Kwon, Hae-Yun; Kim, Joon-Hyeok; Kim, Sea-Hyun; Park, Ji-Min; Lee, Hyoshin

    2016-09-01

    The complete chloroplast genome sequence of Hibiscus syriacus L. is presented in this study. The genome is composed of 161 019 bp in length, with a typical circular structure containing a pair of inverted repeats of 25 745 bp of length separated by a large single-copy region and a small single-copy region of 89 698 bp and 19 831 bp of length, respectively. The overall GC content is 36.8%. One hundred and fourteen genes were annotated, including 81 protein-coding genes, 4 ribosomal RNA genes and 29 transfer RNA genes.

  12. Chloroplast protein import inhibition by a soluble factor from wheat germ lysate.

    PubMed

    Schleiff, Enrico; Motzkus, Michael; Soll, Jürgen

    2002-09-01

    Protein import into chloroplasts occurs post-translationally in vitro. The precursor proteins are generally synthesised in a reticulocyte lysate- or wheat germ lysate-derived system and imported out of this system into chloroplast. These complex soluble protein mixtures are likely to contain factors, which influence somehow the import competence and import efficiency. Here we describe a heat-stable soluble proteinaceaous factor, which inhibits protein import into chloroplasts in vitro. The inhibitor interacts directly with the precursor protein and renders it import incompetent. This mode of action is supported by two observations: firstly, binding of the precursor to the chloroplast surface is diminished in the presence of the inhibitor. Secondly, when chloroplasts were loaded with precursor proteins under conditions, which allow only binding but not import the inhibitor was unable to abolish the subsequent translocation step.

  13. The Effect of Gametogenesis Regimes on the Chloroplast Genetic System of CHLAMYDOMONAS REINHARDTII

    PubMed Central

    Sears, Barbara B.; Boynton, John E.; Gillham, Nicholas W.

    1980-01-01

    In Chlamydomonas reinhardtii, gamete differentiation is induced by nitrogen deprivation. While cellular nitrogen content and amount of chloroplast DNA in cells of both mating types are reduced during gametogenesis, the spontaneous transmission of paternal (mt-) chloroplast alleles in crosses is specifically affected by the stringency of the nitrogen starvation regime used for pregrowth and gametogenesis of the mt- parent. In all cases, reciprocal crosses yielded biparental zygospores whose clones contain predominantly cells expressing only the chloroplast alleles from the maternal (mt+) parent. No differences attributable to strain divergence were seen in chloroplast gene inheritance pattern, DNA content, or the relative frequency of transmission of paternal chloroplast alleles to progeny of biparental zygospores. PMID:17249065

  14. Ubiquitin facilitates a quality-control pathway that removes damaged chloroplasts

    SciTech Connect

    Woodson, Jesse D.; Joens, Matthew S.; Sinson, Andrew B.; Gilkerson, Jonathan; Salome, Patrice A.; Weigel, Detlef; Fitzpatrick, James A.; Chory, Joanne

    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 targeted removal of ROS-overproducing chloroplasts.

  15. Three-dimensional reconstruction of anomalous chloroplasts in transgenic ipt tobacco.

    PubMed

    Synková, Helena; Schnablová, Renáta; Polanská, Lenka; Husák, Michal; Siffel, Pavel; Vácha, Frantisek; Malbeck, Jirí; Machácková, Ivana; Nebesárová, Jana

    2006-03-01

    Anomalies in the ultrastructure of chloroplasts, from transgenic ipt tobacco, overproducing endogenous cytokinins (CKs) were studied. Detailed analyses of CKs and their metabolites showed that Pssu-ipt tobacco contained enhanced contents of CKs both in leaves and in isolated chloroplasts. The role of CKs in the formation of anomalous structures is suggested. Pssu-ipt chloroplasts frequently formed the distinct peripheral reticulum with a system of caverns that often involved mitochondria and/or peroxisomes. Large crystalloids, which were found in chloroplasts of Pssu-ipt, occupied up to 16% of chloroplast volume. We suggested that the crystalloids were formed by LHC II aggregates. This was supported by analysis of the fluorescence emission spectra at 77 degrees K, chlorophyll a/b ratio, immunogold staining of the structures, and crystallographic unit size analysis.

  16. Update on chloroplast research: new tools, new topics, and new trends.

    PubMed

    Armbruster, Ute; Pesaresi, Paolo; Pribil, Mathias; Hertle, Alexander; Leister, Dario

    2011-01-01

    Chloroplasts, the green differentiation form of plastids, are the sites of photosynthesis and other important plant functions. Genetic and genomic technologies have greatly boosted the rate of discovery and functional characterization of chloroplast proteins during the past decade. Indeed, data obtained using high-throughput methodologies, in particular proteomics and transcriptomics, are now routinely used to assign functions to chloroplast proteins. Our knowledge of many chloroplast processes, notably photosynthesis and photorespiration, has reached such an advanced state that biotechnological approaches to crop improvement now seem feasible. Meanwhile, efforts to identify the entire complement of chloroplast proteins and their interactions are progressing rapidly, making the organelle a prime target for systems biology research in plants.

  17. 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.

  18. The complete chloroplast genome sequence of Dendropanax morbifera (Léveillé).

    PubMed

    Kim, Kyunghee; Lee, Sang-Choon; Yang, Tae-Jin

    2016-07-01

    The complete chloroplast genome sequence of Dendropanax morbifera, an economically and medicinally important endemic tree species in Korea, was obtained by de novo assembly with whole-genome sequence data and manual correction. A circular 156 366-bp chloroplast genome showed typical chloroplast genome structure comprising a large single copy region of 86 475 bp, a small single copy region of 18 125 bp, and a pair of inverted repeats of 25 883 bp. The chloroplast genome harbored 87 protein-coding genes. Phylogenetic analysis with the chloroplast genome revealed that D. morbifera is most closely related to Dendropanax dentiger, an evergreen tree species in China and Southeastern Asia.

  19. 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

  20. Circadian Rhythms of Chloroplast Orientation and Photosynthetic Capacity in Ulva123

    PubMed Central

    Britz, Steven J.; Briggs, Winslow R.

    1976-01-01

    Ulva lactuca L. var. latissima (L.) Decandolle and var. rigida (C. Agardh) Le Jolis and U. mutabilis Foyn have a circadian rhythm of chloroplast orientation which results in large changes in the light-absorption properties of the thallus. During the day, the chloroplasts cover the outer face of the cells and absorbance is high. At night, the chloroplasts are along the side walls and absorbance is low. Enteromorpha linza (L.) J. Agardh, E. intestinalis (L.) Link, E. sp., and Monostroma grevillei (Thuret) Wittrock, members of the Ulvales, were not observed to have this rhythmic movement. Chloroplasts, when in the face position, could not be induced to move to the sides by high intensity light up to 80,000 lux. Unrelated to chloroplast position per se and light-absorption efficiency, there is a rhythm of photosynthetic capacity which peaks just before midday and which continues in constant darkness. Images PMID:16659613

  1. Chloroplast genome sequence of the moss Tortula ruralis: gene content, polymorphism, and structural arrangement relative to other green plant chloroplast genomes

    PubMed Central

    2010-01-01

    Background Tortula ruralis, a widely distributed species in the moss family Pottiaceae, is increasingly used as a model organism for the study of desiccation tolerance and mechanisms of cellular repair. In this paper, we present the chloroplast genome sequence of T. ruralis, only the second published chloroplast genome for a moss, and the first for a vegetatively desiccation-tolerant plant. Results The Tortula chloroplast genome is ~123,500 bp, and differs in a number of ways from that of Physcomitrella patens, the first published moss chloroplast genome. For example, Tortula lacks the ~71 kb inversion found in the large single copy region of the Physcomitrella genome and other members of the Funariales. Also, the Tortula chloroplast genome lacks petN, a gene found in all known land plant plastid genomes. In addition, an unusual case of nucleotide polymorphism was discovered. Conclusions Although the chloroplast genome of Tortula ruralis differs from that of the only other sequenced moss, Physcomitrella patens, we have yet to determine the biological significance of the differences. The polymorphisms we have uncovered in the sequencing of the genome offer a rare possibility (for mosses) of the generation of DNA markers for fine-level phylogenetic studies, or to investigate individual variation within populations. PMID:20187961

  2. Protein synthesis in chloroplasts. Characteristics and products of protein synthesis in vitro in etioplasts and developing chloroplasts from pea leaves.

    PubMed Central

    Siddell, S G; Ellis, R J

    1975-01-01

    The function of plastid ribosomes in pea (Pisum sativum L.) was investigated by characterizing the products of protein synthesis in vitro in plastids isolated at different stages during the transition from etioplast to chloroplast. Etioplasts and plastids isolated after 24, 48 and 96h of greening in continuous white light, use added ATP to incorporate labelled amino acids into protein. Plastids isolated from greening leaves can also use light as the source of energy for protein synthesis. The labelled polypeptides synthesized in isolated plastids were analysed by electrophoresis in sodium dodecyl sulphate-ureapolyacrylamide gels. Six polypeptides are synthesized in etioplasts with ATP as energy source. Only one of these polypeptides is present in a 150 000g supernatant fraction. This polypeptide has been identified as the large subunit of Fraction I protein (3-phospho-D-glycerate carboxylyase EC 4.1.1.39) by comparing the tryptic 'map' of its L-(35S)methionine-labelled peptides with the tryptic 'map' of large subunit peptides from Fraction I labelled with L-(35S)methionine in vivo. The same gel pattern of six polypeptides is seen when plastids isolated from greening leaves are incubated with either added ATP or light as the energy source. However, the rates of synthesis of particular polypeptides are different in plastids isolated at different stages of the etioplast to chloroplast transition. The results support the idea that plastid ribosomes synthesize only a small number of proteins, and that the number and molecular weight of these proteins does not alter during the formation of chloroplasts from etioplasts. Images PLATE 1 PMID:1147911

  3. 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.

  4. The complete chloroplast genome provides insight into the evolution and polymorphism of Panax ginseng

    PubMed Central

    Zhao, Yongbing; Yin, Jinlong; Guo, Haiyan; Zhang, Yuyu; Xiao, Wen; Sun, Chen; Wu, Jiayan; Qu, Xiaobo; Yu, Jun; Wang, Xumin; Xiao, Jingfa

    2015-01-01

    Panax ginseng C.A. Meyer (P. ginseng) is an important medicinal plant and is often used in traditional Chinese medicine. With next generation sequencing (NGS) technology, we determined the complete chloroplast genome sequences for four Chinese P. ginseng strains, which are Damaya (DMY), Ermaya (EMY), Gaolishen (GLS), and Yeshanshen (YSS). The total chloroplast genome sequence length for DMY, EMY, and GLS was 156,354 bp, while that for YSS was 156,355 bp. Comparative genomic analysis of the chloroplast genome sequences indicate that gene content, GC content, and gene order in DMY are quite similar to its relative species, and nucleotide sequence diversity of inverted repeat region (IR) is lower than that of its counterparts, large single copy region (LSC) and small single copy region (SSC). A comparison among these four P. ginseng strains revealed that the chloroplast genome sequences of DMY, EMY, and GLS were identical and YSS had a 1-bp insertion at base 5472. To further study the heterogeneity in chloroplast genome during domestication, high-resolution reads were mapped to the genome sequences to investigate the differences at the minor allele level; 208 minor allele sites with minor allele frequencies (MAF) of ≥0.05 were identified. The polymorphism site numbers per kb of chloroplast genome sequence for DMY, EMY, GLS, and YSS were 0.74, 0.59, 0.97, and 1.23, respectively. All the minor allele sites located in LSC and IR regions, and the four strains showed the same variation types (substitution base or indel) at all identified polymorphism sites. Comparison results of heterogeneity in the chloroplast genome sequences showed that the minor allele sites on the chloroplast genome were undergoing purifying selection to adapt to changing environment during domestication process. A study of P. ginseng chloroplast genome with particular focus on minor allele sites would aid in investigating the dynamics on the chloroplast genomes and different P. ginseng strains

  5. Salt stress affects mRNA editing in soybean chloroplasts.

    PubMed

    Rodrigues, Nureyev F; Fonseca, Guilherme C da; Kulcheski, Franceli R; Margis, Rogério

    2017-03-02

    Soybean, a crop known by its economic and nutritional importance, has been the subject of several studies that assess the impact and the effective plant responses to abiotic stresses. Salt stress is one of the main environmental stresses and negatively impacts crop growth and yield. In this work, the RNA editing process in the chloroplast of soybean plants was evaluated in response to a salt stress. Bioinformatics approach using sRNA and mRNA libraries were employed to detect specific sites showing differences in editing efficiency. RT-qPCR was used to measure editing efficiency at selected sites. We observed that transcripts of NDHA, NDHB, RPS14 and RPS16 genes presented differences in coverage and editing rates between control and salt-treated libraries. RT-qPCR assays demonstrated an increase in editing efficiency of selected genes. The salt stress enhanced the RNA editing process in transcripts, indicating responses to components of the electron transfer chain, photosystem and translation complexes. These increases can be a response to keep the homeostasis of chloroplast protein functions in response to salt stress.

  6. Lateral distribution and diffusion of plastocyanin in chloroplast thylakoids

    PubMed Central

    1989-01-01

    The lateral distribution of plastocyanin in the thylakoid lumen of spinach and pea chloroplasts was studied by combining immunocytochemical localization and kinetic measurements of P700+ reduction at high time resolution. In dark-adapted chloroplasts, the concentration of plastocyanin in the photosystem I containing stroma membranes exceeds that in photosystem II containing grana membranes by a factor of about two. Under these conditions, the reduction of P700+ with a halftime of 12 microseconds after a laser flash of saturating intensity indicates that to greater than 95% of total photosystem I a plastocyanin molecule is bound. An analysis of the labeling densities, the length of the different lumenal regions, and the total amounts of plastocyanin and P700 shows that most of the remaining presumable mobile plastocyanin is found in the granal lumen. This distribution of plastocyanin is consistent with a more negative surface charge density in the stromal than in the granal lumen. During illumination the concentration of plastocyanin in grana increases at the expense of that in stroma lamellae, indicating a light-driven diffusion from stroma to grana regions. Our observations provide evidence that a high concentration of plastocyanin in grana in the light favors the lateral electron transport from cytochrome b6/f complexes in appressed grana across the long distance to photosystem I in nonappressed stroma membranes. PMID:2647767

  7. Changes in chloroplast pigments of olive varieties during fruit ripening.

    PubMed

    Roca, M; Mínguez-Mosquera, M I

    2001-02-01

    Changes in chlorophyll and carotenoid pigments of five olive (Olea europaea L.) varieties destined for milling were investigated at six consecutive ripening stages. There was a manifest dependence between olive variety, moment of picking, and chloroplast pigment composition of the fruits. Although the content of chlorophylls and carotenoids differed with fruit variety, ripening always involved their gradual loss, which becames more pronounced with increased presence of anthocyanin compounds. The relative rates of disappearance of chlorophylls and carotenoids were markedly different between varieties, implying that the catabolism of these pigments takes place at a relative rate inherent to each variety. The varieties less rich in pigments showed the most extreme behavior. The highest relative rate of disappearance was observed in fruits of the Blanqueta variety, and the lowest was observed in those of Arbequina. The chlorophyll a/chlorophyll b ratio remained practically constant during ripening, with a value very similar for Hojiblanca, Picual, Cornicabra, and Blanqueta, but much higher for Arbequina, implying that the structure of the photosynthetic apparatus is different in the latter variety. In the five varieties studied, lutein was the slowest carotenoid to be degraded, so that its percentage in the fruits increased with ripening, whereas beta-carotene was the fastest to disappear. In ripe fruits covered with anthocyanins, chloroplast pigments were retained in both skin and pulp, with the rate of disappearance being much higher in the latter.

  8. Euglena mitochondria and chloroplasts form tyrosine-O-sulfate

    SciTech Connect

    Saidha, T.; Hanfstingl, U.; Schiff, J.A. )

    1989-04-01

    Mitochondria from light-grown wild-type Euglena gracilis var. bacillaris Cori or dark-grown mutant W{sub 10}BSmL incubated with {sup 35}SO{sub 4}{sup 2{minus}} and ATP, or with {sup 14}C-tyrosine, non-radioactive sulfate and ATP accumulate a labeled compound in the medium. Since this compound shows exact coelectrophoresis with tyrosine-O-sulfate (TOS) at pH 2.0, 5.8 or 8.0., yields sulfate and tyrosine on acid hydrolysis, and treatment with aryl sulfatase from Aerobacter aerogenes yields sulfate and tyrosine but no tyrosine methyl ester, it is identified as TOS. No TOS is found outside purified developing chloroplasts incubated with {sup 35}SO{sub 4}{sup 2{minus}} and ATP, but both chloroplasts and mitochondria form to {sup 35}S externally when incubated with adenosine 3{prime} phosphate 5{prime}phospho({sup 35}S) sulfate (PAP{sup 35}S). Since no tyrosine need be added, tyrosine is provided from endogenous sources. Although TOS is found in the free pool of Euglena cells it cannot be detected in proteins of cells or mucus ruling our sulfation of tyrosine of protein or incorporation of TOS into proteins. The system forming TOS is membrane-bound and may be involved in tyrosine transport.

  9. Chloroplast DNA evolution and phylogenetic relationships in Lycopersicon.

    PubMed

    Palmer, J D; Zamir, D

    1982-08-01

    Chloroplast DNA was purified from 12 accessions that represent most of the species diversity in the genus Lycopersicon (family Solanaceae) and from 3 closely related species in the genus Solanum. Fragment patterns produced by digestion of these DNAs with 25 different restriction endonucleases were analyzed by agarose gel electrophoresis. In all 15 DNAs, a total of only 39 restriction site mutations were detected among 484 restriction sites surveyed, representing 2,800 base pairs of sequence information. This low rate of base sequence change is paralleled by an extremely low rate of convergent change in restriction sites; only 1 of the 39 mutations appears to have occurred independently in two different lineages. Parsimony analysis of shared mutations has allowed the construction of a maternal phylogeny for the 15 accessions. This phylogeny is generally consistent with relationships based on morphology and crossability but provides more detailed resolution at several places. All accessions within Lycopersicon form a coherent group, with two of the three species of Solanum as outside reference points. Chloroplast DNA analysis places S. pennellii firmly within Lycopersicon, confirming recent studies that have removed it from Solanum. Red-orange fruit color is shown to be a monophyletic trait in three species of Lycopersicon, including the cultivated tomato, L. esculentum. Analysis of six accessions within L. peruvianum reveals a limited amount of intraspecific polymorphism which, however, encompasses all the variation observed in L. chilense and L. chmielewskii. It is suggested that these latter two accessions be relegated to positions within the L. peruvianum complex.

  10. Light-harvesting superstructures of green plant chloroplasts lacking photosystems.

    PubMed

    Belgio, Erica; Ungerer, Petra; Ruban, Alexander V

    2015-10-01

    The light-harvesting antenna of higher plant photosystem II (LHCII) is the major photosynthetic membrane component encoded by an entire family of homologous nuclear genes. On the contrary, the great majority of proteins of photosystems and electron transport components are encoded by the chloroplast genome. In this work, we succeeded in gradually inhibiting the expression of the chloroplast genes that led to the disappearance of the photosystem complexes, mimicking almost total photoinhibition. The treated plants, despite displaying only some early signs of senescence, sustained their metabolism and growth for several weeks. The only major remaining membrane component was LHCII antenna that formed superstructures - stacks of dozens of thylakoids or supergrana. Freeze-fracture electron microscopy revealed specific organization, directly displaying frequently bifurcated membranes with reduced or totally absent photosystem II (PSII) reaction centre complexes. Our findings show that it is possible to accumulate large amounts of light-harvesting membranes, organized into three-dimensional structures, in the absence of reaction centre complexes. This points to the reciprocal role of LHCII and PSII in self-assembly of the three-dimensional matrix of the photosynthetic membrane, dictating its size and flexible adaptation to the light environment.

  11. SsTypA1, a chloroplast-specific TypA/BipA-type GTPase from the halophytic plant Suaeda salsa, plays a role in oxidative stress tolerance.

    PubMed

    Wang, Fang; Zhong, Nai-Qin; Gao, Peng; Wang, Gui-Ling; Wang, Hai-Yun; Xia, Gui-Xian

    2008-07-01

    Suaeda salsa is a leaf-succulent euhalophytic plant capable of surviving under seawater salinity. Here, we report the isolation and functional analysis of a novel Suaeda gene (designated as SsTypA1) encoding a member of the TypA/BipA GTPase gene family. The steady-state transcript level of SsTypA1 in S. salsa was up-regulated in response to various external stressors. Expression of SsTypA1 was restricted to the epidermal layers of the leaf and stem in S. salsa, and SsTypA1-green fluorescence protein (GFP) fusion proteins were targeted to the chloroplasts of tobacco leaves. Ectopic over-expression of SsTypA1 rendered the transgenic tobacco plants with significantly increased tolerance to oxidative stress, and this was accompanied by a reduction in H(2)O(2) content. Enzymatic and Western blot analyses revealed that the activity and amount of the thylakoid-bound NAD(P)H dehydrogenase (NDH) complex in the chloroplasts of leaf cells were enhanced. Additionally, an in vitro assay demonstrated that SsTypA1 bound to GTP and possessed GTPase activity that was stimulated by the presence of chloroplast 70S ribosomes. Together, these results suggest that SsTypA1 may play a critical role in the development of oxidative stress tolerance, perhaps as a translational regulator of the stress-responsive proteins involved in reactive oxygen species (ROS) suppression in chloroplast.

  12. 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

  13. The complete chloroplast genome sequence of Helwingia himalaica (Helwingiaceae, Aquifoliales) and a chloroplast phylogenomic analysis of the Campanulidae

    PubMed Central

    Liu, Ying-Ying; Song, Yu

    2016-01-01

    Complete chloroplast genome sequences have been very useful for understanding phylogenetic relationships in angiosperms at the family level and above, but there are currently large gaps in coverage. We report the chloroplast genome for Helwingia himalaica, the first in the distinctive family Helwingiaceae and only the second genus to be sequenced in the order Aquifoliales. We then combine this with 36 published sequences in the large (c. 35,000 species) subclass Campanulidae in order to investigate relationships at the order and family levels. The Helwingia genome consists of 158,362 bp containing a pair of inverted repeat (IR) regions of 25,996 bp separated by a large single-copy (LSC) region and a small single-copy (SSC) region which are 87,810 and 18,560 bp, respectively. There are 142 known genes, including 94 protein-coding genes, eight ribosomal RNA genes, and 40 tRNA genes. The topology of the phylogenetic relationships between Apiales, Asterales, and Dipsacales differed between analyses based on complete genome sequences and on 36 shared protein-coding genes, showing that further studies of campanulid phylogeny are needed. PMID:27917320

  14. Light-Dependent Isoprene Emission (Characterization of a Thylakoid-Bound Isoprene Synthase in Salix discolor Chloroplasts).

    PubMed Central

    Wildermuth, M. C.; Fall, R.

    1996-01-01

    Isoprene synthase is an enzyme that is responsible for the production of the volatile C5 hydrocarbon, isoprene, in plant leaves. Isoprene formation in numerous C3 plants is interesting because (a) large quantities of isoprene are emitted, 5 x 1014 g of C annually, (b) a plant may release 1 to 8% of its fixed C as isoprene, and (c) the function of plant isoprene production is unknown. Because of the dependence of foliar isoprene emission on light, the existence of a plastidic isoprene synthase has been postulated. To pursue this idea, a method to isolate chloroplasts from Salix discolor was developed and shows a plastidic isoprene synthase that is tightly bound to the thylakoid membrane and accessible to trypsin inactivation. The thylakoid-bound isoprene synthase has catalytic properties similar to known soluble isoprene synthases; however, the relationship between these enzymes is unknown. The discovery of a thylakoid-bound isoprene synthase with a stromal-facing domain places it in the chloroplast, where it may be subject to numerous direct and indirect light-mediated effects. Implications for the light-dependent regulation of foliar isoprene production and its function are presented. PMID:12226383

  15. Phylogeography of Thlaspi arvense (Brassicaceae) in China Inferred from Chloroplast and Nuclear DNA Sequences and Ecological Niche Modeling

    PubMed Central

    An, Miao; Zeng, Liyan; Zhang, Ticao; Zhong, Yang

    2015-01-01

    Thlaspi arvense is a well-known annual farmland weed with worldwide distribution, which can be found from sea level to above 4000 m high on the Qinghai-Tibetan Plateau (QTP). In this paper, a phylogeographic history of T. arvense including 19 populations from China was inferred by using three chloroplast (cp) DNA segments (trnL-trnF, rpl32-trnL and rps16) and one nuclear (n) DNA segment (Fe-regulated transporter-like protein, ZIP). A total of 11 chloroplast haplotypes and six nuclear alleles were identified, and haplotypes unique to the QTP were recognized (C4, C5, C7 and N4). On the basis of molecular dating, haplotypes C4, C5 and C7 have separated from others around 1.58 Ma for cpDNA, which corresponds to the QTP uplift. In addition, this article suggests that the T. arvense populations in China are a mixture of diverged subpopulations as inferred by hT/vT test (hT ≤ vT, cpDNA) and positive Tajima’s D values (1.87, 0.05 < p < 0.10 for cpDNA and 3.37, p < 0.01 for nDNA). Multimodality mismatch distribution curves and a relatively large shared area of suitable environmental conditions between the Last Glacial Maximum (LGM) as well as the present time recognized by MaxEnt software reject the sudden expansion population model. PMID:26110380

  16. Repression of Essential Chloroplast Genes Reveals New Signaling Pathways and Regulatory Feedback Loops in Chlamydomonas[W

    PubMed Central

    Ramundo, Silvia; Rahire, Michèle; Schaad, Olivier; Rochaix, Jean-David

    2013-01-01

    Although reverse genetics has been used to elucidate the function of numerous chloroplast proteins, the characterization of essential plastid genes and their role in chloroplast biogenesis and cell survival has not yet been achieved. Therefore, we developed a robust repressible chloroplast gene expression system in the unicellular alga Chlamydomonas reinhardtii based mainly on a vitamin-repressible riboswitch, and we used this system to study the role of two essential chloroplast genes: ribosomal protein S12 (rps12), encoding a plastid ribosomal protein, and rpoA, encoding the α-subunit of chloroplast bacterial-like RNA polymerase. Repression of either of these two genes leads to the arrest of cell growth, and it induces a response that involves changes in expression of nuclear genes implicated in chloroplast biogenesis, protein turnover, and stress. This response also leads to the overaccumulation of several plastid transcripts and reveals the existence of multiple negative regulatory feedback loops in the chloroplast gene circuitry. PMID:23292734

  17. Comparative Analysis of the Complete Chloroplast Genomes of Five Quercus Species

    PubMed Central

    Yang, Yanci; Zhou, Tao; Duan, Dong; Yang, Jia; Feng, Li; Zhao, Guifang

    2016-01-01

    Quercus is considered economically and ecologically one of the most important genera in the Northern Hemisphere. Oaks are taxonomically perplexing because of shared interspecific morphological traits and intraspecific morphological variation, which are mainly attributed to hybridization. Universal plastid markers cannot provide a sufficient number of variable sites to explore the phylogeny of this genus, and chloroplast genome-scale data have proven to be useful in resolving intractable phylogenetic relationships. In this study, the complete chloroplast genomes of four Quercus species were sequenced, and one published chloroplast genome of Quercus baronii was retrieved for comparative analyses. The five chloroplast genomes ranged from 161,072 bp (Q. baronii) to 161,237 bp (Q. dolicholepis) in length, and their gene organization and order, and GC content, were similar to those of other Fagaceae species. We analyzed nucleotide substitutions, indels, and repeats in the chloroplast genomes, and found 19 relatively highly variable regions that will potentially provide plastid markers for further taxonomic and phylogenetic studies within Quercus. We observed that four genes (ndhA, ndhK, petA, and ycf1) were subject to positive selection. The phylogenetic relationships of the Quercus species inferred from the chloroplast genomes obtained moderate-to-high support, indicating that chloroplast genome data may be useful in resolving relationships in this genus. PMID:27446185

  18. Nucleotide sequence of a preferred maize chloroplast genome template for in vitro DNA synthesis.

    PubMed Central

    Gold, B; Carrillo, N; Tewari, K K; Bogorad, L

    1987-01-01

    Maize chloroplast DNA sequences representing 94% of the chromosome have been surveyed for their activity as autonomously replicating sequences in yeast and as templates for DNA synthesis in vitro by a partially purified chloroplast DNA polymerase. A maize chloroplast DNA region extending over about 9 kilobase pairs is especially active as a template for the DNA synthesis reaction. Fragments from within this region are much more active than DNA from elsewhere in the chromosome and 50- to 100-fold more active than DNA of the cloning vector pBR322. The smallest of the strongly active subfragments that we have studied, the 1368-base-pair EcoRI fragment x, has been sequenced and found to contain the coding region of chloroplast ribosomal protein L16. EcoRI fragment x shows sequence homology with a portion of the Chlamydomonas reinhardtii chloroplast chromosome that forms a displacement loop [Wang, X.-M., Chang, C.H., Waddell, J. & Wu, M. (1984) Nucleic Acids Res. 12, 3857-3872]. Maize chloroplast DNA fragments that permit autonomous replication of DNA in yeast are not active as templates for DNA synthesis in the in vitro assay. The template active region we have identified may represent one of the origins of replication of maize chloroplast DNA. Images PMID:3025853

  19. Is chloroplast movement in tobacco plants influenced systemically after local illumination or burning stress?

    PubMed

    Naus, Jan; Rolencová, Monika; Hlavácková, Vladimíra

    2008-10-01

    Chloroplast movement has been studied in many plants mainly in relation to the local light, mechanical or stress effects. Here we investigated possible systemic responses of chloroplast movement to local light or burning stress in tobacco plants (Nicotiana tabacum cv. Samsun). Chloroplast movement was measured using two independent methods: one with a SPAD 502 Chlorophyll meter and another by collimated transmittance at a selected wavelength (676 nm). A sensitive periodic movement of chloroplasts was used in high or low (2 000 or 50 micromol/m(2) per s photosynthetically active radiation, respectively) cold white light with periods of 50 or 130 min. Measurements were carried out in the irradiated area, in the non-irradiated area of the same leaf or in the leaf located on the stem below the irradiated or burned one. No significant changes in systemic chloroplast movement in non-irradiated parts of the leaf and in the non-treated leaf were detected. Our data indicate that chloroplast movement in tobacco is dependent dominantly on the intensity and spectral composition of the incident light and on the local stimulation and state of the target tissue. No systemic signal was strong enough to evoke a detectable systemic response in chloroplast movement in distant untreated tissues of tobacco plants.

  20. 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.

  1. 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.

  2. Comparative analysis of microsatellites in chloroplast genomes of lower and higher plants.

    PubMed

    George, Biju; Bhatt, Bhavin S; Awasthi, Mayur; George, Binu; Singh, Achuit K

    2015-11-01

    Microsatellites, or simple sequence repeats (SSRs), contain repetitive DNA sequence where tandem repeats of one to six base pairs are present number of times. Chloroplast genome sequences have been  shown to possess extensive variations in the length, number and distribution of SSRs. However, a comparative analysis of chloroplast microsatellites is not available. Considering their potential importance in generating genomic diversity, we have systematically analysed the abundance and distribution of simple and compound microsatellites in 164 sequenced chloroplast genomes from wide range of plants. The key findings of these studies are (1) a large number of mononucleotide repeats as compared to SSR(2-6)(di-, tri-, tetra-, penta-, hexanucleotide repeats) are present in all chloroplast genomes investigated, (2) lower plants such as algae show wide variation in relative abundance, density and distribution of microsatellite repeats as compared to flowering plants, (3) longer SSRs are excluded from coding regions of most chloroplast genomes, (4) GC content has a weak influence on number, relative abundance and relative density of mononucleotide as well as SSR(2-6). However, GC content strongly showed negative correlation with relative density (R (2) = 0.5, P < 0.05) and relative abundance (R (2) = 0.6, P < 0.05) of cSSRs. In summary, our comparative studies of chloroplast genomes illustrate the variable distribution of microsatellites and revealed that chloroplast genome of smaller plants possesses relatively more genomic diversity compared to higher plants.

  3. Search for an endotherm in chloroplast lamellar membranes associated with chilling-inhibition of photosynthesis.

    PubMed

    Low, P S; Ort, D R; Cramer, W A; Whitmarsh, J; Martin, B

    1984-06-01

    The phase transition of chloroplast lamellar membrane lipids has been proposed to be the underlying cause of chilling-induced inhibition of photosynthesis in sensitive plants. Differential scanning calorimetry has been used to search for any endotherms arising from lipid state changes in chloroplast lamellar membranes of the chilling-sensitive plants cantaloupe , kidney bean, domestic tomato, and soybean. For comparison, calorimetric scans of chloroplast lamellar membranes from the chilling-insensitive plants spinach, pea, and wild tomato were made. A large reversible endotherm, extending from below 10 degrees to nearly 40 degrees C, was observed in chloroplast membranes from tomatoes of both chilling-sensitive (Lycopersicon esculentum Mill. cv. Floramerica ) and chilling-insensitive (L. hirsutum LA 1361) species. A much smaller endotherm, approximately 5 to 10% of the area of that seen in the two tomato species, and extending over a similar temperature range, was detected in chloroplasts from chilling-insensitive spinach and peas, and also was generally observed in chloroplasts from chilling-sensitive cantaloupe , kidney bean, and soybean. The enthalpy of these smaller endotherms indicates that, if the endotherm arose entirely from a lipid transition, then it corresponded to the melting of less than about 10% of the total membrane polar lipid. On the basis of these data it is concluded that there is no correlation between chilling sensitivity of photosynthesis and the presence or absence of a phase transition of bulk membrane lipids of the chloroplast lamellar membrane at temperatures above 5 degrees C.

  4. 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

  5. Misexpression of a Chloroplast Aspartyl Protease Leads to Severe Growth Defects and Alters Carbohydrate Metabolism in Arabidopsis1[C][W

    PubMed Central

    Paparelli, Eleonora; Gonzali, Silvia; Parlanti, Sandro; Novi, Giacomo; Giorgi, Federico M.; Licausi, Francesco; Kosmacz, Monika; Feil, Regina; Lunn, John E.; Brust, Henrike; van Dongen, Joost T.; Steup, Martin; Perata, Pierdomenico

    2012-01-01

    The crucial role of carbohydrate in plant growth and morphogenesis is widely recognized. In this study, we describe the characterization of nana, a dwarf Arabidopsis (Arabidopsis thaliana) mutant impaired in carbohydrate metabolism. We show that the nana dwarf phenotype was accompanied by altered leaf morphology and a delayed flowering time. Our genetic and molecular data indicate that the mutation in nana is due to a transfer DNA insertion in the promoter region of a gene encoding a chloroplast-located aspartyl protease that alters its pattern of expression. Overexpression of the gene (oxNANA) phenocopies the mutation. Both nana and oxNANA display alterations in carbohydrate content, and the extent of these changes varies depending on growth light intensity. In particular, in low light, soluble sugar levels are lower and do not show the daily fluctuations observed in wild-type plants. Moreover, nana and oxNANA are defective in the expression of some genes implicated in sugar metabolism and photosynthetic light harvesting. Interestingly, some chloroplast-encoded genes as well as genes whose products seem to be involved in retrograde signaling appear to be down-regulated. These findings suggest that the NANA aspartic protease has an important regulatory function in chloroplasts that not only influences photosynthetic carbon metabolism but also plastid and nuclear gene expression. PMID:22987884

  6. Chloroplast gene arrangement variation within a closely related group of green algae (Trebouxiophyceae, Chlorophyta).

    PubMed

    Letsch, Molly R; Lewis, Louise A

    2012-09-01

    The 22 published chloroplast genomes of green algae, representing sparse taxonomic sampling of diverse lineages that span over one billion years of evolution, each possess a unique gene arrangement. In contrast, many of the >190 published embryophyte (land plant) chloroplast genomes have relatively conserved architectures. To determine the phylogenetic depth at which chloroplast gene rearrangements occur in green algae, a 1.5-4 kb segment of the chloroplast genome was compared across nine species in three closely related genera of Trebouxiophyceae (Chlorophyta). In total, four distinct gene arrangements were obtained for the three genera Elliptochloris, Hemichloris, and Coccomyxa. In Elliptochloris, three distinct chloroplast gene arrangements were detected, one of which is shared with members of its sister genus Hemichloris. Both species of Coccomyxa examined share the fourth arrangement of this genome region, one characterized by very long spacers. Next, the order of genes found in this segment of the chloroplast genome was compared across green algae and land plants. As taxonomic ranks are not equivalent among different groups of organisms, the maximum molecular divergence among taxa sharing a common gene arrangement in this genome segment was compared. Well-supported clades possessing a single gene order had similar phylogenetic depth in green algae and embryophytes. When the dominant gene order of this chloroplast segment in embryophytes was assumed to be ancestral for land plants, the maximum molecular divergence was found to be over two times greater in embryophytes than in trebouxiophyte green algae. This study greatly expands information about chloroplast genome variation in green algae, is the first to demonstrate such variation among congeneric green algae, and further illustrates the fluidity of green algal chloroplast genome architecture in comparison to that of many embryophytes.

  7. 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

  8. Effect of Salts and Electron Transport on the Conformation of Isolated Chloroplasts. II. Electron Microscopy 1

    PubMed Central

    Izawa, Seikichi; Good, Norman E.

    1966-01-01

    Spinach chloroplasts isolated in media containing salts and the rare chloroplasts which are still within their envelopes alike retain grana similar to those seen in chloroplasts in situ. Chloroplasts isolated in low-salt media lose their grana without losing any chlorophyll. These grana-free chloroplasts are considerably swollen and consist almost entirely of continuous sheets of paired-membrane structures. These double structures, the lamellae, are only loosely held together, primarily at the edges, by tenuous material which does not react with permanganate. Addition of salts (methylamine hydrochloride, NaCl, MgCl2) to the grana-free low-salt chloroplasts provide strong interlamellar attractions. These attractions result in a stacking of the lamellae which is sometimes almost random but sometimes results in regular structures indistinguishable from the original grana. The phosphorylation-uncoupler atebrin causes further swelling of the chloroplasts in the absence of electron transport by increasing the space between the paired membranes of the lamellae. The rapid electron transport (Hill reaction) made possible by atebrin-uncoupling is associated with a great decrease in chloroplast volume. This decrease results from a collapsing together of the widely separated lamellar membrane pairs. The pairs approach each other so closely that they usually appear as a single membrane when viewed with the electron microscope. The much slower electron transport which occurs in the absence of uncouplers is associated with a similar but smaller decrease in the space between the lamellar membrane pairs. Chloroplasts swell during the rapid electron transport made possible by the phosphorylation-uncoupler methylamine. This swelling is accompanied by a degree of membrane distortion which precludes an interpretation of the mechanism. As with atebrin-faciliated electron transport, obviously paired membranes disappear but it is not yet clear whether this is by association or

  9. Arabidopsis S-Sulfocysteine Synthase Activity Is Essential for Chloroplast Function and Long-Day Light-Dependent Redox Control[W

    PubMed Central

    Bermúdez, Maria Angeles; Páez-Ochoa, Maria Angeles; Gotor, Cecilia; Romero, Luis C.

    2010-01-01

    In bacteria, the biosynthesis of Cys is accomplished by two enzymes that are encoded by the cysK and cysM genes. CysM is also able to use thiosulfate as a substrate to produce S-sulfocysteine. In plant cells, the biosynthesis of Cys occurs in the cytosol, mitochondria, and chloroplasts. Chloroplasts contain two O-acetylserine(thiol)lyase homologs, which are encoded by the OAS-B and CS26 genes in Arabidopsis thaliana. An in vitro enzymatic analysis of the recombinant CS26 protein demonstrated that this isoform possesses S-sulfocysteine synthase activity and lacks O-acetylserine(thiol)lyase activity. In vivo functional analysis of this enzyme in knockout mutants demonstrated that mutation of CS26 suppressed the S-sulfocysteine synthase activity that was detected in the wild type; furthermore, the cs26 mutants exhibited a reduction in size and showed paleness, but penetrance of the growth phenotype depended on the light regime. The cs26 mutant plants also had reductions in chlorophyll content and photosynthetic activity (neither of which were observed in oas-b mutants) as well as elevated glutathione levels. However, cs26 leaves were not able to properly detoxify reactive oxygen species, which accumulated to high levels under long-day growth conditions. The transcriptional profile of the cs26 mutant revealed that the mutation had a pleiotropic effect on many cellular and metabolic processes. Our findings reveal that S-sulfocysteine and the activity of S-sulfocysteine synthase play important roles in chloroplast function and are essential for light-dependent redox regulation within the chloroplast. PMID:20179139

  10. Photoreduction of Sulfur Dioxide by Spinach Leaves and Isolated Spinach Chloroplasts

    PubMed Central

    Silvius, John E.; Baer, Charles H.; Dodrill, Sherman; Patrick, Homer

    1976-01-01

    Labeled sulfur dioxide was found to be extensively absorbed by spinach (Spinacea oleracea L.) leaves. Labeled sulfides detected in leaf blades following fumigations with sulfur dioxide in light indicated that photoreduction of sulfur dioxide had occurred. Measurable proportions of this labeled sulfur was localized within the chloroplast fraction. Suspensions of isolated chloroplasts supplied with labeled sulfur dioxide contained labeled sulfides following a 30-minute illumination period in water-cooled reaction vessels. With reference to recent studies of the chloroplast sulfur reduction pathway, probable points of entry for sulfur dioxide and the subsequent release of hydrogen sulfide are discussed. PMID:16659572

  11. Granal stacking of thylakoid membranes in higher plant chloroplasts: the physicochemical forces at work and the functional consequences that ensue.

    PubMed

    Chow, Wah Soon; Kim, Eun-Ha; Horton, Peter; Anderson, Jan M

    2005-12-01

    The formation of grana in chloroplasts of higher plants is examined in terms of the subtle interplay of physicochemical forces of attraction and repulsion. The attractive forces between two adjacent membranes comprise (1) van der Waals attraction that depends on the abundance and type of atoms in each membrane, on the distance between the membranes and on the dielectric constant, (2) depletion attraction that generates local order by granal stacking at the expense of greater disorder (i.e. entropy) in the stroma, and (3) an electrostatic attraction of opposite charges located on adjacent membranes. The repulsive forces comprise (1) electrostatic repulsion due to the net negative charge on the outer surface of thylakoid membranes, (2) hydration repulsion that operates at small separations between thylakoid membranes due to layers of bound water molecules, and (3) steric hindrance due to bulky protrusions of Photosystem I (PSI) and ATP synthase into the stroma. In addition, specific interactions may occur, but they await experimental demonstration. Although grana are not essential for photosynthesis, they are ubiquitous in higher plants. Grana may have been selected during evolution for the functional advantages that they confer on higher plants. The functional consequences of grana stacking include (1) enhancement of light capture through a vastly increased area-to-volume ratio and connectivity of several PSIIs with large functional antenna size, (2) the ability to control the lateral separation of PSI from PSII and, therefore, the balanced distribution of excitation energy between two photosystems working in series, (3) the reversible fine-tuning of energy distribution between the photosystems by State 1-State 2 transitions, (4) the ability to regulate light-harvesting via controlled thermal dissipation of excess excitation energy, detected as non-photochemical quenching, (5) dynamic flexibility in the light reactions mediated by a granal structure in response to

  12. Functional analysis of chloroplast early light inducible proteins (ELIPs)

    SciTech Connect

    Wetzel, Carolyn M

    2005-02-22

    The objectives of this project were to characterize gene expression patterns of early light inducible protein (ELIP) genes in Arabidopsis thaliana and in Lycopersicon esculentum, to identify knock mutants of the 2 ELIP genes in Arabidopsis, and to characterize the effects of the knockouts. Expression in Arabidopsis was studied in response to thylakoid electron transport chain (PETC) capacity, where it was found that there is a signal for expression associated with reduction of the PETC. Expression in response to salt was also studied, with different responses of the two gene copies. Knockout lines for ELIP1 and ELIP2 have been identified and are being characterized. In tomato, it was found that the single-copy ELIP gene is highly expressed in ripening fruit during the chloroplast-to-chromoplast transition. Studies of expression in tomato ripening mutants are ongoing.

  13. Chloroplast-mitochondria cross-talk in diatoms.

    PubMed

    Prihoda, Judit; Tanaka, Atsuko; de Paula, Wilson B M; Allen, John F; Tirichine, Leïla; Bowler, Chris

    2012-02-01

    Diatoms are unicellular, mainly photosynthetic, eukaryotes living within elaborate silicified cell walls and believed to be responsible for around 40% of global primary productivity in the oceans. Their abundance in aquatic ecosystems is such that they have on different occasions been described as the insects, the weeds, or the cancer cells of the ocean. In contrast to higher plants and green algae which derive from a primary endosymbiosis, diatoms are now believed to originate from a serial secondary endosymbiosis involving both green and red algae and a heterotrophic exosymbiont host. As a consequence of their dynamic evolutionary history, they appear to have red algal-derived chloroplasts empowered largely by green algal proteins, working alongside mitochondria derived from the non-photosynthetic exosymbiont. This review will discuss the evidence for such an unusual assemblage of organelles in diatoms, and will present the evidence implying that it has enabled them with unorthodox metabolisms that may have contributed to their profound ecological success.

  14. Requirements for a conservative protein translocation pathway in chloroplasts.

    PubMed

    Vojta, Lea; Soll, Jürgen; Bölter, Bettina

    2007-06-12

    The chloroplast inner envelope translocon subunit Tic110 is imported via a soluble stromal translocation intermediate. In this study an in-organellar import system is established which allows for an accumulation of this intermediate in order to analyze its requirements for reexport. All results demonstrate that the re-export of Tic110 from the soluble intermediate stage into the inner envelope requires ATP hydrolysis, which cannot be replaced by other NTPs. Furthermore, the molecular chaperone Hsp93 seems prominently involved in the reexport pathway of Tic110, because other stromal intermediates like that of the oxygen evolving complex subunit OE33 (iOE33) en route to the thylakoid lumen interacts preferentially with Hsp70.

  15. Cryoprotection by Glucose, Sucrose, and Raffinose to Chloroplast Thylakoids 1

    PubMed Central

    Lineberger, R. Daniel; Steponkus, Peter L.

    1980-01-01

    Differential cryoprotection is afforded to chloroplast thylakoids against freeze-induced uncoupling of cyclic photophosphorylation by equimolar concentrations of glucose, sucrose, and raffinose. This differential protective effect appears to be due to nonideal activity-concentration profiles exhibited by the sugars during freezing. When cryoprotection is analyzed as a function of the mole fraction of NaCl to which the membranes are exposed during freezing, the pattern of protection to cyclic photophosphorylation and its component reactions is not dependent upon the chemical identity of the protective solute. Cryoprotective efficiency of glucose, sucrose, and raffinose can be accounted for by proposing an activity dependent alteration in the freezing environment rather than specific solute-membrane interactions. PMID:16661177

  16. The complete chloroplast genomes of Cannabis sativa and Humulus lupulus.

    PubMed

    Vergara, Daniela; White, Kristin H; Keepers, Kyle G; Kane, Nolan C

    2016-09-01

    Cannabis and Humulus are sister genera comprising the entirety of the Cannabaceae sensu stricto, including C. sativa L. (marijuana, hemp), and H. lupulus L. (hops) as two economically important crops. These two plants have been used by humans for many purposes including as a fiber, food, medicine, or inebriant in the case of C. sativa, and as a flavoring component in beer brewing in the case of H. lupulus. In this study, we report the complete chloroplast genomes for two distinct hemp varieties of C. sativa, Italian "Carmagnola" and Russian "Dagestani", and one Czech variety of H. lupulus "Saazer". Both C. sativa genomes are 153 871 bp in length, while the H. lupulus genome is 153 751 bp. The genomes from the two C. sativa varieties differ in 16 single nucleotide polymorphisms (SNPs), while the H. lupulus genome differs in 1722 SNPs from both C. sativa cultivars.

  17. The chloroplast ATP synthase: structural changes during catalysis.

    PubMed

    Richter, M L; Gao, F

    1996-10-01

    This article summarizes some of the evidence for the existence of light-driven structural changes in the epsilon and gamma subunits of the chloroplast ATP synthase. Formation of a transmembrane proton gradient results in: (1) a changed in the position of the epsilon subunit such that it becomes exposed to polyclonal antibodies and to reagents which selectively modify epsilon Lys109; (2) enhanced solvent accessibility of several sulfhydryl residues on the gamma subunit; and (3) release/exchange of tightly bound ADP from the enzyme. Theses and related experimental observations can, at least partially, be explained in terms of two different bound conformational states of the epsilon subunit. Evidence for structural changes in the enzyme which are driven by light or nucleotide binding is discussed with special reference to the popular rotational model for catalysis.

  18. The complete chloroplast genome of Sinopodophyllum hexandrum (Berberidaceae).

    PubMed

    Li, Huie; Guo, Qiqiang

    2016-07-01

    The complete chloroplast (cp) genome of the Sinopodophyllum hexandrum (Berberidaceae) was determined in this study. The circular genome is 157,940 bp in size, and comprises a pair of inverted repeat (IR) regions of 26,077 bp each, a large single-copy (LSC) region of 86,460 bp and a small single-copy (SSC) region of 19,326 bp. The GC content of the whole cp genome was 38.5%. A total of 133 genes were identified, including 88 protein-coding genes, 37 tRNA genes and eight rRNA genes. The whole cp genome consists of 114 unique genes, and 19 genes are duplicated in the IR regions. The phylogenetic analysis revealed that S. hexandrum is closely related to Nandina domestica within the family Berberidaceae.

  19. The evolution of chloroplast genes and genomes in ferns.

    PubMed

    Wolf, Paul G; Der, Joshua P; Duffy, Aaron M; Davidson, Jacob B; Grusz, Amanda L; Pryer, Kathleen M

    2011-07-01

    Most of the publicly available data on chloroplast (plastid) genes and genomes come from seed plants, with relatively little information from their sister group, the ferns. Here we describe several broad evolutionary patterns and processes in fern plastid genomes (plastomes), and we include some new plastome sequence data. We review what we know about the evolutionary history of plastome structure across the fern phylogeny and we compare plastome organization and patterns of evolution in ferns to those in seed plants. A large clade of ferns is characterized by a plastome that has been reorganized with respect to the ancestral gene order (a similar order that is ancestral in seed plants). We review the sequence of inversions that gave rise to this organization. We also explore global nucleotide substitution patterns in ferns versus those found in seed plants across plastid genes, and we review the high levels of RNA editing observed in fern plastomes.

  20. [Study of Chloroplast DNA Polymorphism in the Sunflower (Helianthus L.)].

    PubMed

    Markina, N V; Usatov, A V; Logacheva, M D; Azarin, K V; Gorbachenko, C F; Kornienko, I V; Gavrilova, V A; Tihobaeva, V E

    2015-08-01

    The polymorphism of microsatellite loci of chloroplast genome in six Helianthus species and 46 lines of cultivated sunflower H. annuus (17 CMS lines and 29 Rf-lines) were studied. The differences between species are confined to four SSR loci. Within cultivated forms of the sunflower H. annuus, the polymorphism is absent. A comparative analysis was performed on sequences of the cpDNA inbred line 3629, line 398941 of the wild sunflower, and the American line HA383 H. annuus. As a result, 52 polymorphic loci represented by 27 SSR and 25 SNP were found; they can be used for genotyping of H. annuus samples, including cultural varieties: twelve polymorphic positions, of which eight are SSR and four are SNP.

  1. Development of novel chloroplast microsatellite markers for Ginkgo biloba.

    PubMed

    Xu, M; Xu, L A; Cao, F L; Zhang, H J; Yu, F X

    2015-07-13

    Ginkgo biloba is considered to be a living fossil that can be used to understand the ancient evolutionary history of gymnosperms, but little attention has been given to the study of its population genetics, molecular phylogeography, and genetic resources assessment. Chloroplast simple sequence repeat (cpSSR) markers are powerful tools for genetic studies of plants. In this study, a total of 30 perfect cpSSRs of Ginkgo were identified and characterized, including di-, tri, tetra-, penta-, and hexanucleotide repeats. Fifteen of 21 designed primer pairs were successfully amplified to yield specific polymerase chain reaction products from 16 Ginkgo cultivars. Polymorphic cpSSRs were further applied to determine the genetic variation of 116 individuals in 5 populations of G. biloba. The results showed that 24 and 76% genetic variation existed within and among populations of this species, respectively. These polymorphic and monomorphic cpSSR markers can be used to trace the origin and evolutionary history of Ginkgo.

  2. Chloroplast RNA editing going extreme: more than 3400 events of C-to-U editing in the chloroplast transcriptome of the lycophyte Selaginella uncinata.

    PubMed

    Oldenkott, Bastian; Yamaguchi, Kazuo; Tsuji-Tsukinoki, Sumika; Knie, Nils; Knoop, Volker

    2014-10-01

    RNA editing in chloroplasts and mitochondria of land plants differs significantly in abundance. For example, some 200-500 sites of cytidine-to-uridine RNA editing exist in flowering plant mitochondria as opposed to only 30-50 such C-to-U editing events in their chloroplasts. In contrast, we predicted significantly more chloroplast RNA editing for the protein-coding genes in the available complete plastome sequences of two species of the spike moss genus Selaginella (Lycopodiophyta). To evaluate these predictions we investigated the Selaginella uncinata chloroplast transcriptome. Our exhaustive cDNA studies identified the extraordinary number of 3415 RNA-editing events, exclusively of the C-to-U type, in the 74 mRNAs encoding intact reading frames in the S. uncinata chloroplast. We find the overwhelming majority (61%) of the 428 silent editing events leaving codon meanings unaltered directly neighboring other editing events, possibly suggesting a sterically more flexible RNA-editing deaminase activity in Selaginella. No evidence of RNA editing was found for tRNAs or rRNAs but we identified a total of 74 editing sites in cDNA sequences of four group II introns (petBi6g2, petDi8g2, ycf3i124g2, and ycf3i354g2) retained in partially matured transcripts, which strongly contribute to improved base-pairing in the intron secondary structures as a likely prerequisite for their splicing.

  3. Chloroplast RNA editing going extreme: more than 3400 events of C-to-U editing in the chloroplast transcriptome of the lycophyte Selaginella uncinata

    PubMed Central

    Oldenkott, Bastian; Yamaguchi, Kazuo; Tsuji-Tsukinoki, Sumika; Knie, Nils

    2014-01-01

    RNA editing in chloroplasts and mitochondria of land plants differs significantly in abundance. For example, some 200–500 sites of cytidine-to-uridine RNA editing exist in flowering plant mitochondria as opposed to only 30–50 such C-to-U editing events in their chloroplasts. In contrast, we predicted significantly more chloroplast RNA editing for the protein-coding genes in the available complete plastome sequences of two species of the spike moss genus Selaginella (Lycopodiophyta). To evaluate these predictions we investigated the Selaginella uncinata chloroplast transcriptome. Our exhaustive cDNA studies identified the extraordinary number of 3415 RNA-editing events, exclusively of the C-to-U type, in the 74 mRNAs encoding intact reading frames in the S. uncinata chloroplast. We find the overwhelming majority (61%) of the 428 silent editing events leaving codon meanings unaltered directly neighboring other editing events, possibly suggesting a sterically more flexible RNA-editing deaminase activity in Selaginella. No evidence of RNA editing was found for tRNAs or rRNAs but we identified a total of 74 editing sites in cDNA sequences of four group II introns (petBi6g2, petDi8g2, ycf3i124g2, and ycf3i354g2) retained in partially matured transcripts, which strongly contribute to improved base-pairing in the intron secondary structures as a likely prerequisite for their splicing. PMID:25142065

  4. Metallothionein expression in chloroplasts enhances mercury accumulation and phytoremediation capability.

    PubMed

    Ruiz, Oscar N; Alvarez, Derry; Torres, Cesar; Roman, Laura; Daniell, Henry

    2011-06-01

    Genetic engineering to enhance mercury phytoremediation has been accomplished by expression of the merAB genes that protects the cell by converting Hg[II] into Hg[0] which volatilizes from the cell. A drawback of this approach is that toxic Hg is released back into the environment. A better phytoremediation strategy would be to accumulate mercury inside plants for subsequent retrieval. We report here the development of a transplastomic approach to express the mouse metallothionein gene (mt1) and accumulate mercury in high concentrations within plant cells. Real-time PCR analysis showed that up to 1284 copies of the mt1 gene were found per cell when compared with 1326 copies of the 16S rrn gene, thereby attaining homoplasmy. Past studies in chloroplast transformation used qualitative Southern blots to evaluate indirectly transgene copy number, whereas we used real-time PCR for the first time to establish homoplasmy and estimate transgene copy number and transcript levels. The mt1 transcript levels were very high with 183,000 copies per ng of RNA or 41% the abundance of the 16S rrn transcripts. The transplastomic lines were resistant up to 20 μm mercury and maintained high chlorophyll content and biomass. Although the transgenic plants accumulated high concentrations of mercury in all tissues, leaves accumulated up to 106 ng, indicating active phytoremediation and translocation of mercury. Such accumulation of mercury in plant tissues facilitates proper disposal or recycling. This study reports, for the first time, the use of metallothioneins in plants for mercury phytoremediation. Chloroplast genetic engineering approach is useful to express metal-scavenging proteins for phytoremediation.

  5. The oligomeric stromal proteome of Arabidopsis thaliana chloroplasts.

    PubMed

    Peltier, Jean-Benoit; Cai, Yang; Sun, Qi; Zabrouskov, Vladimir; Giacomelli, Lisa; Rudella, Andrea; Ytterberg, A Jimmy; Rutschow, Heidi; van Wijk, Klaas J

    2006-01-01

    This study presents an analysis of the stromal proteome in its oligomeric state extracted from highly purified chloroplasts of Arabidopsis thaliana. 241 proteins (88% with predicted cTP), mostly assembled in oligomeric complexes, were identified by mass spectrometry with emphasis on distinguishing between paralogues. This is critical because different paralogues in a gene family often have different subcellular localizations and/or different expression patterns and functions. The native protein masses were determined for all identified proteins. Comparison with the few well characterized stromal complexes from A. thaliana confirmed the accuracy of the native mass determination, and by extension, the usefulness of the native mass data for future in-depth protein interaction studies. Resolved protein interactions are discussed and compared with an extensive collection of native mass data of orthologues in other plants and bacteria. Relative protein expression levels were estimated from spot intensities and also provided estimates of relative concentrations of individual proteins. No such quantification has been reported so far. Surprisingly proteins dedicated to chloroplast protein synthesis, biogenesis, and fate represented nearly 10% of the total stroma protein mass. Oxidative pentose phosphate pathway, glycolysis, and Calvin cycle represented together about 75%, nitrogen assimilation represented 5-7%, and all other pathways such as biosynthesis of e.g. fatty acids, amino acids, nucleotides, tetrapyrroles, and vitamins B(1) and B(2) each represented less than 1% of total protein mass. Several proteins with diverse functions outside primary carbon metabolism, such as the isomerase ROC4, lipoxygenase 2 involved in jasmonic acid biosynthesis, and a carbonic anhydrase (CA1), were surprisingly abundant in the range of 0.75-1.5% of the total stromal mass. Native images with associated information are available via the Plastid Proteome Database.

  6. Chloroplast DNA restriction site variation and phylogeny of the Berberidaceae.

    PubMed

    Kim, Y D; Jansen, R K

    1998-12-01

    Comparative restriction site mapping of the chloroplast genome was performed to examine phylogenetic relationships among 27 species representing 16 genera of the Berberidaceae and two outgroups. Chloroplast genomes of the species included in this study showed no major structural rearrangements (i.e., they are collinear to tobacco cpDNA) except for the extension of the inverted repeat in species of Berberis and Mahonia. Excluding several regions that exhibited severe length variation, a total of 501 phylogenetically informative sites was mapped for ten restriction enzymes. The strict consensus tree of 14 equally parsimonious trees indicated that some berberidaceous genera (Berberis, Mahonia, Diphylleia) are not monophyletic. To explore phylogenetic utility of different parsimony methods phylogenetic trees were generated using Wagner, Dollo, and weighted parsimony for a reduced data set that included 18 species. One of the most significant results was the recognition of the four chromosomal groups, which were strongly supported regardless of the parsimony method used. The most notable difference among the trees produced by the three parsimony methods was the relationships among the four chromosomal groups. The cpDNA trees also strongly supported a close relationship of several generic pairs (e.g., Berberis-Mahonia, Epimedium-Vancouveria, etc.). Maximum likelihood values were computed for the four different tree topologies of the chromosomal groups, two Wagner, one Dollo, and one weighted topology. The results indicate that the weighted tree has the highest likelihood value. The lowest likelihood value was obtained for the Dollo tree, which had the highest bootstrap and decay values. Separate analyses using only the Inverted Repeat (IR) region resulted in a tree that is identical to the weighted tree. Poor resolution and/or support for the relationships among the four chromosomal lineages of the Berberidaceae indicate that they may have radiated from an ancestral

  7. Energetic factors affecting carbon dioxide fixation in isolated chloroplasts

    SciTech Connect

    Slovacek, R.E.; Hind, G.

    1980-03-01

    Light- and HCO/sub 3/-saturated (10 millimolar) rates of O/sub 2/ evolution (120 to 220 micromoles O/sub 2/ per milligram chlorophyll per hour), obtained with intact spinach chloroplasts, are decreased up to 3-fold by changes in assay conditions such as omission of catalase from the medium, the use of high (greater than or equal to 1 millimolar) inorganic phosphate, inclusion of NO/sub 2/- as an electron acceptor, or bright illumination at low partial pressures of O/sub 2/. These inhibitions may be reversed by addition of uncoupling levels of NH/sub 4/Cl or of antimycin concentrations that partially block cyclic electron transfer between cytochrome b/sub 6/ and cytochrome f. Measurements of the pH gradient across the thylakoid membrane with the fluorescent probe, 9-aminoacridine, indicate that changes in ..delta..pH are sufficient to account for both the inhibited and restored rates of electron transport. It follows that the rate of HCO/sub 3/-saturated photosynthesis may be restricted by a proton gradient back pressure under these conditions. The rate of O/sub 2/ evolution is also decreased 3-fold when ambient CO/sub 2/ (0.63 millimolar HCO/sub 3/- at pH 8.1) is used in place of saturating HCO/sub 3/- and chloroplasts are illuminated aerobically with catalase and a low level (0.25 millimolar) of K/sub 2/HPO/sub 4/. Only inhibitory effects are observed with additions of antimycin or NH/sub 4/Cl. Under these conditions, excessive photophosphorylation or a large pH gradient does not limit the rate of photosynthesis.

  8. The complete chloroplast genome of Origanum vulgare L. (Lamiaceae).

    PubMed

    Lukas, Brigitte; Novak, Johannes

    2013-10-10

    Oregano (Origanum vulgare L., Lamiaceae) is a medicinal and aromatic plant maybe best known for flavouring pizza. New applications e.g. as natural antioxidants for food are emerging due to the plants' high antibacterial and antioxidant activity. The complete chloroplast (cp) genome of Origanum vulgare (GenBank/EBML/DDBJ accession number: JX880022) consists of 151,935 bp and includes a pair of inverted repeats (IR) of 25,527 bp separated by one small and one large single copy region (SSC and LSC) of 17,745 and 83,136 bp, respectively. The genome with an overall GC content of 38% hosts 114 genes that covering 63% of the genome of which 8% were introns. The comparison of the Origanum cp genome with the cp genomes of two other core lamiales (Salvia miltiorrhiza Bunge and Sesamum indicum L.) revealed completely conserved protein-coding regions in the IR region but also in the LSC and SSC regions. Phylogenetic analysis of the lamiids based on 56 protein-coding genes give a hint at the basic structure of the Lamiales. However, further genomes will be necessary to clarify this taxonomically complicated order. The variability of the cp within the genus Origanum, studied exemplarily on 16 different chloroplast DNA regions, demonstrated that in 14 regions analyzed, the variability was extremely low (max. 0.7%), while only two regions showed a moderate variability of up to 2.3%. The cp genome of Origanum vulgare contains 27 perfect mononucleotide repeats (number of repeats>9) consisting exclusively of the nucleotides A or T. 34 perfect repeats (repeat lengths>1 and number of repeats>3) were found, of which 32 were di-, and 2 were trinucleotide repeats.

  9. Signal Transduction Pathways that Regulate CAB Gene Expression

    SciTech Connect

    Chory, Joanne

    2004-12-31

    The process of chloroplast differentiation, involves the coordinate regulation of many nuclear and chloroplast genes. The cues for the initiation of this developmental program are both extrinsic (e.g., light) and intrinsic (cell-type and plastid signals). During this project period, we utilized a molecular genetic approach to select for Arabidopsis mutants that did not respond properly to environmental light conditions, as well as mutants that were unable to perceive plastid damage. These latter mutants, called gun mutants, define two retrograde signaling pathways that regulate nuclear gene expression in response to chloroplasts. A major finding was to identify a signal from chloroplasts that regulates nuclear gene transcription. This signal is the build-up of Mg-Protoporphyrin IX, a key intermediate of the chlorophyll biosynthetic pathway. The signaling pathways downstream of this signal are currently being studied. Completion of this project has provided an increased understanding of the input signals and retrograde signaling pathways that control nuclear gene expression in response to the functional state of chloroplasts. These studies should ultimately influence our abilities to manipulate plant growth and development, and will aid in the understanding of the developmental control of photosynthesis.

  10. Signal Transduction Pathways that Regulate CAB Gene Expression

    SciTech Connect

    Chory, Joanne

    2006-01-16

    The process of chloroplast differentiation, involves the coordinate regulation of many nuclear and chloroplast genes. The cues for the initiation of this developmental program are both extrinsic (e.g., light) and intrinsic (cell-type and plastid signals). During this project period, we utilized a molecular genetic approach to select for Arabidopsis mutants that did not respond properly to environmental light conditions, as well as mutants that were unable to perceive plastid damage. These latter mutants, called gun mutants, define two retrograde signaling pathways that regulate nuclear gene expression in response to chloroplasts. A major finding was to identify a signal from chloroplasts that regulates nuclear gene transcription. This signal is the build-up of Mg-Protoporphyrin IX, a key intermediate of the chlorophyll biosynthetic pathway. The signaling pathways downstream of this signal are currently being studied. Completion of this project has provided an increased understanding of the input signals and retrograde signaling pathways that control nuclear gene expression in response to the functional state of chloroplasts. These studies should ultimately influence our abilities to manipulate plant growth and development, and will aid in the understanding of the developmental control of photosynthesis.

  11. The avoidance and aggregative movements of mesophyll chloroplasts in C(4) monocots in response to blue light and abscisic acid.

    PubMed

    Maai, Eri; Shimada, Shouu; Yamada, Masahiro; Sugiyama, Tatsuo; Miyake, Hiroshi; Taniguchi, Mitsutaka

    2011-05-01

    In C(4) plants, mesophyll (M) chloroplasts are randomly distributed along the cell walls, whereas bundle sheath chloroplasts are located in either a centripetal or centrifugal position. It was reported previously that only M chloroplasts aggregatively redistribute to the bundle sheath side in response to extremely strong light or environmental stresses. The aggregative movement of M chloroplasts is also induced in a light-dependent fashion upon incubation with abscisic acid (ABA). The involvement of reactive oxygen species (ROS) and red/blue light in the aggregative movement of M chloroplasts are examined here in two distinct subtypes of C(4) plants, finger millet and maize. Exogenously applied hydrogen peroxide or ROS scavengers could not change the response patterns of M chloroplast movement to light and ABA. Blue light irradiation essentially induced the rearrangement of M chloroplasts along the sides of anticlinal walls, parallel to the direction of the incident light, which is analogous to the avoidance movement of C(3) chloroplasts. In the presence of ABA, most of the M chloroplasts showed the aggregative movement in response to blue light but not red light. Together these results suggest that ROS are not involved in signal transduction for the aggregative movement, and ABA can shift the blue light-induced avoidance movement of C(4)-M chloroplasts to the aggregative movement.

  12. Arabidopsis Chloroplast Mini-Ribonuclease III Participates in rRNA Maturation and Intron Recycling

    PubMed Central

    Hotto, Amber M.; Castandet, Benoît; Gilet, Laetitia; Higdon, Andrea; Condon, Ciarán; Stern, David B.

    2015-01-01

    RNase III proteins recognize double-stranded RNA structures and catalyze endoribonucleolytic cleavages that often regulate gene expression. Here, we characterize the functions of RNC3 and RNC4, two Arabidopsis thaliana chloroplast Mini-RNase III-like enzymes sharing 75% amino acid sequence identity. Whereas rnc3 and rnc4 null mutants have no visible phenotype, rnc3/rnc4 (rnc3/4) double mutants are slightly smaller and chlorotic compared with the wild type. In Bacillus subtilis, the RNase Mini-III is integral to 23S rRNA maturation. In Arabidopsis, we observed imprecise maturation of 23S rRNA in the rnc3/4 double mutant, suggesting that exoribonucleases generated staggered ends in the absence of specific Mini-III-catalyzed cleavages. A similar phenotype was found at the 3′ end of the 16S rRNA, and the primary 4.5S rRNA transcript contained 3′ extensions, suggesting that Mini-III catalyzes several processing events of the polycistronic rRNA precursor. The rnc3/4 mutant showed overaccumulation of a noncoding RNA complementary to the 4.5S-5S rRNA intergenic region, and its presence correlated with that of the extended 4.5S rRNA precursor. Finally, we found rnc3/4-specific intron degradation intermediates that are probable substrates for Mini-III and show that B. subtilis Mini-III is also involved in intron regulation. Overall, this study extends our knowledge of the key role of Mini-III in intron and noncoding RNA regulation and provides important insight into plastid rRNA maturation. PMID:25724636

  13. Arabidopsis chloroplast mini-ribonuclease III participates in rRNA maturation and intron recycling.

    PubMed

    Hotto, Amber M; Castandet, Benoît; Gilet, Laetitia; Higdon, Andrea; Condon, Ciarán; Stern, David B

    2015-03-01

    RNase III proteins recognize double-stranded RNA structures and catalyze endoribonucleolytic cleavages that often regulate gene expression. Here, we characterize the functions of RNC3 and RNC4, two Arabidopsis thaliana chloroplast Mini-RNase III-like enzymes sharing 75% amino acid sequence identity. Whereas rnc3 and rnc4 null mutants have no visible phenotype, rnc3/rnc4 (rnc3/4) double mutants are slightly smaller and chlorotic compared with the wild type. In Bacillus subtilis, the RNase Mini-III is integral to 23S rRNA maturation. In Arabidopsis, we observed imprecise maturation of 23S rRNA in the rnc3/4 double mutant, suggesting that exoribonucleases generated staggered ends in the absence of specific Mini-III-catalyzed cleavages. A similar phenotype was found at the 3' end of the 16S rRNA, and the primary 4.5S rRNA transcript contained 3' extensions, suggesting that Mini-III catalyzes several processing events of the polycistronic rRNA precursor. The rnc3/4 mutant showed overaccumulation of a noncoding RNA complementary to the 4.5S-5S rRNA intergenic region, and its presence correlated with that of the extended 4.5S rRNA precursor. Finally, we found rnc3/4-specific intron degradation intermediates that are probable substrates for Mini-III and show that B. subtilis Mini-III is also involved in intron regulation. Overall, this study extends our knowledge of the key role of Mini-III in intron and noncoding RNA regulation and provides important insight into plastid rRNA maturation.

  14. Echinochloa Chloroplast Genomes: Insights into the Evolution and Taxonomic Identification of Two Weedy Species

    PubMed Central

    Li, Gengmi; Wang, Ying-Ying; Qiu, Jie; Fu, Fei; Zhang, Haiqiang; Chen, Li; Ye, Sisi; Song, Weijie; Jin, Gulei; Zhu, Jinwen; Lu, Yongliang; Guo, Longbiao; Fan, Longjiang

    2014-01-01

    The genus Echinochloa (Poaceae) includes numerous problematic weeds that cause the reduction of crop yield worldwide. To date, DNA sequence information is still limited in the genus Echinochloa. In this study, we completed the entire chloroplast genomes of two Echinochloa species (Echinochloa oryzicola and Echinochloa crus-galli) based on high-throughput sequencing data from their fresh green leaves. The two Echinochloa chloroplast genomes are 139,891 and 139,800 base pairs in length, respectively, and contain 131 protein-coding genes, 79 indels and 466 substitutions helpful for discrimination of the two species. The divergence between the genus Echinochloa and Panicum occurred about 21.6 million years ago, whereas the divergence between E. oryzicola and E. crus-galli chloroplast genes occurred about 3.3 million years ago. The two reported Echinochloa chloroplast genome sequences contribute to better understanding of the diversification of this genus. PMID:25427255

  15. The complete chloroplast genome sequence of an important medicinal plant Cynanchum wilfordii (Maxim.) Hemsl. (Apocynaceae).

    PubMed

    Park, Hyun-Seung; Kim, Kyu-Yeob; Kim, Kyunghee; Lee, Sang-Choon; Lee, Junki; Seong, Rack Seon; Shim, Young Hun; Sung, Sang Hyun; Yang, Tae-Jin

    2016-09-01

    Cynanchum wilfordii (Maxim.) Hemsl. is a traditional medicinal herb belonging to the Asclepiadoideae subfamily, whose dried roots have been used as traditional medicine in Asia. The complete chloroplast genome of C. wilfordii was generated by de novo assembly using the small amount of whole genome sequencing data. The chloroplast genome of C. wilfordii was 161 241 bp long, composed of large single copy region (91 995 bp), small single copy region (19 930 bp) and a pair of inverted repeat regions (24 658 bp). The overall GC contents of the chloroplast genome was 37.8%. A total of 114 genes were annotated, which included 80 protein-coding genes, 30 tRNA genes and 4 rRNA genes. Phylogenetic analysis with the reported chloroplast genomes revealed that C. wilfordii is most closely related to Asclepias nivea (Caribbean milkweed) and Asclepias syriaca (common milkweed) within the Asclepiadoideae subfamily.

  16. Confocal laser scanning microscopy detection of chlorophylls and carotenoids in chloroplasts and chromoplasts of tomato fruit.

    PubMed

    D'Andrea, Lucio; Amenós, Montse; Rodríguez-Concepción, Manuel

    2014-01-01

    Plant cells are unique among eukaryotic cells because of the presence of plastids, including chloroplasts and chromoplasts. Chloroplasts are found in green tissues and harbor the photosynthetic machinery (including chlorophyll molecules), while chromoplasts are present in non-photosynthetic tissues and accumulate large amounts of carotenoids. During tomato fruit development, chloroplasts are converted into chromoplasts that accumulate high levels of lycopene, a linear carotenoid responsible for the characteristic red color of ripe fruit. Here, we describe a simple and fast method to detect both types of fully differentiated plastids (chloroplasts and chromoplasts), as well as intermediate stages, in fresh tomato fruits. The method is based on the differential autofluorescence of chlorophylls and carotenoids (lycopene) detected by Confocal Laser Scanning Microscopy.

  17. Complete Chloroplast Genome Sequence of the Early Diverging Green Alga Palmophyllum crassum

    PubMed Central

    Furukawa, Ryo; Kunugi, Motoshi; Ihara, Kunio; Tanaka, Ayumi

    2017-01-01

    ABSTRACT Palmophyllum crassum is a little-known green alga, with a unique evolutionary position and distinctive photosynthetic features. Here, we present the complete chloroplast genome sequence of Palmophyllum crassum. PMID:28280029

  18. Complete chloroplast genome of the orchid Cattleya crispata (Orchidaceae:Laeliinae), a Neotropical rupiculous species.

    PubMed

    da Rocha Perini, Violeta; Leles, Bruno; Furtado, Carolina; Prosdocimi, Francisco

    2016-11-01

    A partial genome dataset was sequenced for the orchid Cattleya crispata using both Illumina and 454 technologies. The chloroplast genome was assembled using iterative runs of MIRA software that yielded a circular molecule with 148,343 bp in length and deposited in GenBank database (Accession Number KP168671). The plastid genome conserved the quadripartite structure present in most Orchidaceae chloroplasts and was composed by 79 protein-coding genes, 39 tRNAs and 8 rRNAs. Genome structure, gene order and orientation were similar to previously described chloroplasts for Cymbidium orchids, differing in gene order for petN and psbM genes. Data described here contain the first report of a complete chloroplast for the Neotropical subtribe Laeliinae and may contribute to improve the phylogenetic resolution and allow the development of new molecular markers for population genetic studies of orchids.

  19. Programmed chloroplast destruction during leaf senescence involves 13-lipoxygenase (13-LOX)

    PubMed Central

    Springer, Armin; Kang, ChulHee; Rustgi, Sachin; von Wettstein, Diter; Reinbothe, Christiane; Pollmann, Stephan; Reinbothe, Steffen

    2016-01-01

    Leaf senescence is the terminal stage in the development of perennial plants. Massive physiological changes occur that lead to the shut down of photosynthesis and a cessation of growth. Leaf senescence involves the selective destruction of the chloroplast as the site of photosynthesis. Here, we show that 13-lipoxygenase (13-LOX) accomplishes a key role in the destruction of chloroplasts in senescing plants and propose a critical role of its NH2-terminal chloroplast transit peptide. The 13-LOX enzyme identified here accumulated in the plastid envelope and catalyzed the dioxygenation of unsaturated membrane fatty acids, leading to a selective destruction of the chloroplast and the release of stromal constituents. Because 13-LOX pathway products comprise compounds involved in insect deterrence and pathogen defense (volatile aldehydes and oxylipins), a mechanism of unmolested nitrogen and carbon relocation is suggested that occurs from leaves to seeds and roots during fall. PMID:26969728

  20. Chloroplast phosphoglycerate kinase from Euglena gracilis: endosymbiotic gene replacement going against the tide.

    PubMed

    Nowitzki, Ulrich; Gelius-Dietrich, Gabriel; Schwieger, Maike; Henze, Katrin; Martin, William

    2004-10-01

    Two chloroplast phosphoglycerate kinase isoforms from the photosynthetic flagellate Euglena gracilis were purified to homogeneity, partially sequenced, and subsequently cDNAs encoding phosphoglycerate kinase isoenzymes from both the chloroplast and cytosol of E. gracilis were cloned and sequenced. Chloroplast phosphoglycerate kinase, a monomeric enzyme, was encoded as a polyprotein precursor of at least four mature subunits that were separated by conserved tetrapeptides. In a Neighbor-Net analysis of sequence similarity with homologues from numerous prokaryotes and eukaryotes, cytosolic phosphoglycerate kinase of E. gracilis showed the highest similarity to cytosolic and glycosomal homologues from the Kinetoplastida. The chloroplast isoenzyme of E. gracilis did not show a close relationship to sequences from other photosynthetic organisms but was most closely related to cytosolic homologues from animals and fungi.