RESUMO
Chloroplast division involves the coordination of protein complexes from the stroma to the cytosol. The Min system of chloroplasts includes multiple stromal proteins that regulate the positioning of the division site. The outer envelope protein PLASTID DIVISION1 (PDV1) was previously reported to recruit the cytosolic chloroplast division protein ACCUMULATION AND REPLICATION OF CHLOROPLAST5 (ARC5). However, we show here that PDV1 is also important for the stability of the inner envelope chloroplast division protein PARALOG OF ARC6 (PARC6), a component of the Min system. We solved the structure of both the C-terminal domain of PARC6 and its complex with the C terminus of PDV1. The formation of an intramolecular disulfide bond within PARC6 under oxidized conditions prevents its interaction with PDV1. Interestingly, this disulfide bond can be reduced by light in planta, thus promoting PDV1-PARC6 interaction and chloroplast division. Interaction with PDV1 can induce the dimerization of PARC6, which is important for chloroplast division. Magnesium ions, whose concentration in chloroplasts increases upon light exposure, also promote the PARC6 dimerization. This study highlights the multilayer regulation of the PDV1-PARC6 interaction as well as chloroplast division.
Assuntos
Proteínas de Arabidopsis , Arabidopsis , Proteínas de Arabidopsis/metabolismo , Arabidopsis/metabolismo , Plastídeos/metabolismo , Cloroplastos/metabolismo , Dissulfetos/metabolismo , Dinaminas/metabolismoRESUMO
Filamentous temperature-sensitive Z (FtsZ) is a tubulin-like GTPase that is highly conserved in bacteria and plants. It polymerizes into a ring at the division site of bacteria and chloroplasts and serves as the scaffold protein of the division complex. While a single FtsZ is present in bacteria and cyanobacteria, there are two subfamilies, FtsZ1 and FtsZ2 in the green lineage, and FtsZA and FtsZB in red algae. In Arabidopsis thaliana, the C-terminal motifs of AtFtsZ1 (Z1C) and AtFtsZ2-1 (Z2C) display distinct functions in the regulation of chloroplast division. Z1C exhibits weak membrane-binding activity, whereas Z2C engages in the interaction with the membrane protein AtARC6. Here, we provide evidence revealing the distinct traits of the C-terminal motifs of FtsZ1 and FtsZ2 throughout the plant evolutionary process. In a range of plant species, the C-terminal motifs of FtsZ1 exhibit diverse membrane-binding properties critical for regulating chloroplast division. In chlorophytes, the C-terminal motifs of FtsZ1 and FtsZ2 exhibit both membrane-binding and protein interaction functions, which are similar to those of cyanobacterial FtsZ and red algal FtsZA. During the transition from algae to land plants, the functions of the C-terminal motifs of FtsZ1 and FtsZ2 exhibit differentiation. FtsZ1 lost the function of interacting with ARC6 in land plants, and the membrane-binding activity of FtsZ2 was lost in ferns. Our findings reveal the functional differentiation of the C-terminal motifs of FtsZs during plant evolution, which is critical for chloroplast division.
Assuntos
Proteínas de Arabidopsis , Proteínas de Arabidopsis/metabolismo , Proteínas de Arabidopsis/genética , Motivos de Aminoácidos , Arabidopsis/genética , Arabidopsis/metabolismo , Evolução Molecular , Cloroplastos/metabolismo , Evolução BiológicaRESUMO
The division of cyanobacteria and their chloroplast descendants is orchestrated by filamenting temperature-sensitive Z (FtsZ), a cytoskeletal GTPase that polymerizes into protofilaments that form a "Z ring" at the division site. The Z ring has both a scaffolding function for division-complex assembly and a GTPase-dependent contractile function that drives cell or organelle constriction. A single FtsZ performs these functions in bacteria, whereas in chloroplasts, they are performed by two copolymerizing FtsZs, called AtFtsZ2 and AtFtsZ1 in Arabidopsis thaliana, which promote protofilament stability and dynamics, respectively. To probe the differences between cyanobacterial and chloroplast FtsZs, we used light scattering to characterize the in vitro protofilament dynamics of FtsZ from the cyanobacterium Synechococcus elongatus PCC 7942 (SeFtsZ) and investigate how coassembly of AtFtsZ2 or AtFtsZ1 with SeFtsZ influences overall dynamics. SeFtsZ protofilaments assembled rapidly and began disassembling before GTP depletion, whereas AtFtsZ2 protofilaments were far more stable, persisting beyond GTP depletion. Coassembled SeFtsZ-AtFtsZ2 protofilaments began disassembling before GTP depletion, similar to SeFtsZ. In contrast, AtFtsZ1 did not alter disassembly onset when coassembled with SeFtsZ, but fluorescence recovery after photobleaching analysis showed it increased the turnover of SeFtsZ subunits from SeFtsZ-AtFtsZ1 protofilaments, mirroring its effect upon coassembly with AtFtsZ2. Comparisons of our findings with previous work revealed consistent differences between cyanobacterial and chloroplast FtsZ dynamics and suggest that the scaffolding and dynamics-promoting functions were partially separated during evolution of two chloroplast FtsZs from their cyanobacterial predecessor. They also suggest that chloroplasts may have evolved a mechanism distinct from that in cyanobacteria for promoting FtsZ protofilament dynamics.
Assuntos
Proteínas do Citoesqueleto , Synechococcus , Arabidopsis/genética , Proteínas de Bactérias/genética , Cloroplastos , GTP Fosfo-Hidrolases/genética , Guanosina Trifosfato , Synechococcus/genética , Temperatura , Proteínas do Citoesqueleto/metabolismoRESUMO
The peptidoglycan (PG) layer, a core component of the bacterial cell wall, has been retained in the Physcomitrium patens chloroplasts. The PG layer entirely encompasses the P. patens chloroplast, including the division site, but how PG biosynthesis cooperates with the constriction of two envelope membranes at the chloroplast division site remains elusive. Here, focusing on the PG synthase penicillin-binding protein (PBP), we performed cytological and molecular analyses to dissect the mechanism of chloroplast division in P. patens. We showed that PBP, acting in the final step of PG biosynthesis, is likely a chloroplast inner envelope protein that can aggregate at mid-chloroplasts during chloroplast division. Physcomitrium patens had five orthologs of PLASTID DIVISION2 (PDV2), an outer envelope component of the chloroplast division complex. Our data indicated that PpPDV2 proteins interact with PpPBP and are responsible for recruiting PpPBP to the chloroplast division site, in addition to PpDRP5B. Furthermore, we found that PBP deletion and carbenicillin application restrain constriction of the chloroplast division complex, rather than its assembly. This work provides direct molecular evidence for a link between chloroplast division of P. patens and PG biosynthesis and indicates that PG biosynthesis is required for the constriction of the chloroplast division apparatus in P. patens.
Assuntos
Peptidoglicano , Plastídeos , Peptidoglicano/metabolismo , Plastídeos/metabolismo , Cloroplastos/metabolismo , Parede Celular/metabolismoRESUMO
Chloroplasts are double membrane bound organelles that are found in plants and algae. Their division requires a number of proteins to assemble into rings along the center of the organelle and to constrict in synchrony. Chloroplasts possess a third membrane system, the thylakoids, which house the majority of proteins responsible for the light-dependent reactions. The mechanism that allows chloroplasts to sort out and separate the intricate thylakoid membrane structures during organelle division remain unknown. By characterizing the sizes of thylakoids found in a number of different chloroplast division mutants in Arabidopsis, we show that thylakoids do not divide independently of the chloroplast division cycle. More specifically, we show that thylakoid division requires the formation of both the inner and the outer contractile rings of the chloroplast.
Assuntos
Proteínas de Arabidopsis , Arabidopsis , Tilacoides/metabolismo , Arabidopsis/genética , Arabidopsis/metabolismo , Cloroplastos/metabolismo , Organelas/metabolismo , Proteínas de Arabidopsis/genética , Proteínas de Arabidopsis/metabolismo , Plantas/metabolismoRESUMO
It is widely believed that cleavage-furrow formation during cytokinesis is driven by the contraction of a ring containing F-actin and type-II myosin. However, even in cells that have such rings, they are not always essential for furrow formation. Moreover, many taxonomically diverse eukaryotic cells divide by furrowing but have no type-II myosin, making it unlikely that an actomyosin ring drives furrowing. To explore this issue further, we have used one such organism, the green alga Chlamydomonas reinhardtii We found that although F-actin is associated with the furrow region, none of the three myosins (of types VIII and XI) is localized there. Moreover, when F-actin was eliminated through a combination of a mutation and a drug, furrows still formed and the cells divided, although somewhat less efficiently than normal. Unexpectedly, division of the large Chlamydomonas chloroplast was delayed in the cells lacking F-actin; as this organelle lies directly in the path of the cleavage furrow, this delay may explain, at least in part, the delay in cytokinesis itself. Earlier studies had shown an association of microtubules with the cleavage furrow, and we used a fluorescently tagged EB1 protein to show that microtubules are still associated with the furrows in the absence of F-actin, consistent with the possibility that the microtubules are important for furrow formation. We suggest that the actomyosin ring evolved as one way to improve the efficiency of a core process for furrow formation that was already present in ancestral eukaryotes.
Assuntos
Actinas/metabolismo , Chlamydomonas/citologia , Chlamydomonas/metabolismo , Citoesqueleto de Actina/química , Citoesqueleto de Actina/metabolismo , Actinas/química , Divisão Celular , Chlamydomonas/química , Citocinese , Microtúbulos/metabolismo , Miosinas/química , Miosinas/metabolismo , Ligação ProteicaRESUMO
KEY MESSAGE: Homologous genes for the peptidoglycan precursor flippase MurJ, and peptidoglycan hydrolases: lytic transglycosylase MltB, and DD-carboxypeptidase VanY are required for chloroplast division in the moss Physcomitrella patens. The moss Physcomitrella patens is used as a model plant to study plastid peptidoglycan biosynthesis. In bacteria, MurJ flippase transports peptidoglycan precursors from the cytoplasm to the periplasm. In this study, we identified a MurJ homolog (PpMurJ) in the P. patens genome. Bacteria employ peptidoglycan degradation and recycling pathways for cell division. We also searched the P. patens genome for genes homologous to bacterial peptidoglycan hydrolases and identified genes homologous for the lytic transglycosylase mltB, N-acetylglucosaminidase nagZ, and LD-carboxypeptidase ldcA in addition to a putative DD-carboxypeptidase vanY reported previously. Moreover, we found a ß-lactamase-like gene (Pplactamase). GFP fusion proteins with either PpMltB or PpVanY were detected in the chloroplasts, whereas fusion proteins with PpNagZ, PpLdcA, or Pplactamase localized in the cytoplasm. Experiments seeking PpMurJ-GFP fusion proteins failed. PpMurJ gene disruption in P. patens resulted in the appearance of macrochloroplasts in protonemal cells. Compared with the numbers of chloroplasts in wild-type plants (38.9 ± 4.9), PpMltB knockout and PpVanY knockout had lower numbers of chloroplasts (14.3 ± 6.7 and 28.1 ± 5.9, respectively). No differences in chloroplast numbers were observed after PpNagZ, PpLdcA, or Pplactamase single-knockout. Chloroplast numbers in PpMltB/PpVanY double-knockout cells were similar to those in PpMltB single-knockout cells. Zymogram analysis of the recombinant PpMltB protein revealed its peptidoglycan hydrolase activity. Our results imply that PpMurJ, PpMltB and PpVanY play a critical role in chloroplast division in the moss P. patens.
Assuntos
Bryopsida/genética , Cloroplastos/genética , N-Acetil-Muramil-L-Alanina Amidase/genética , Proteínas de Transferência de Fosfolipídeos/genética , Proteínas de Plantas/genética , Uridina Difosfato Ácido N-Acetilmurâmico/análogos & derivados , Bryopsida/metabolismo , Cloroplastos/metabolismo , Eletroforese em Gel de Poliacrilamida , Regulação da Expressão Gênica de Plantas , Técnicas de Inativação de Genes , Proteínas de Fluorescência Verde/genética , Proteínas de Fluorescência Verde/metabolismo , N-Acetil-Muramil-L-Alanina Amidase/metabolismo , Peptidoglicano/metabolismo , Proteínas de Transferência de Fosfolipídeos/metabolismo , Proteínas de Plantas/metabolismo , Plantas Geneticamente Modificadas , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/metabolismo , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Uridina Difosfato Ácido N-Acetilmurâmico/metabolismoRESUMO
BACKGROUND: Maize bsd2 (bundle sheath defective2) is a classical C4 mutant with defective C4 photosynthesis, accompanied with reduced accumulation of Rubisco (ribulose bisphosphate carboxylase oxygenase) and aberrant mature chloroplast morphology in the bundle sheath (BS) cells. However, as a hypothetical chloroplast chaperone, the effects of BSD2 on C4 chloroplast development have not been fully examined yet, which precludes a full appreciation of BSD2 function in C4 photosynthesis. The aims of our study are to find out the role ofBSD2 in regulating chloroplasts development in maize leaves, and to add new insights into our understanding of C4 biology. RESULTS: We found that at the chloroplast maturation stage, the thylakoid membranes of chloroplasts in the BS and mesophyll (M) cells became significantly looser, and the granaof chloroplasts in the M cells became thinner stacking in the bsd2 mutant when compared with the wildtype plant. Moreover, at the early chloroplast development stage, the number of dividing chloroplasts and the chloroplast division rate are both reduced in the bsd2 mutant, compared with wild type. Quantitative reverse transcriptase-PCR analysis revealed that the expression of both thylakoid formation-related genesand chloroplast division-related genes is significantly reduced in the bsd2 mutants. Further, we showed that BSD2 interacts physically with the large submit of Rubisco (LS) in Bimolecular Fluorescence Complementation assay. CONCLUSIONS: Our combined results suggest that BSD2 plays an essential role in regulating the division and differentiation of the dimorphic BS and M chloroplasts, and that it acts at a post-transcriptional level to regulate LS stability or assembly of Rubisco.
Assuntos
Cloroplastos/ultraestrutura , Folhas de Planta/citologia , Proteínas de Plantas/genética , Zea mays , Cloroplastos/metabolismo , Mutação , Folhas de Planta/metabolismo , Proteínas de Plantas/metabolismo , Ribulose-Bifosfato Carboxilase/metabolismo , Zea mays/citologia , Zea mays/genética , Zea mays/metabolismo , Zea mays/ultraestruturaRESUMO
Chloroplast division is a critical process for the maintenance of appropriate chloroplast number in plant cells. It is known that in some plant species and cell types, environmental stresses can affect chloroplast division, differentiation and morphology, however the significance and regulation of these processes are largely unknown. Here we investigated the regulation of salt stress-induced chloroplast division in protonemal cells of the moss, Physcomitrella patens, and found that, salt stress as one of the major abiotic stresses, induced chloroplast division and resulted in increased chloroplast numbers. We further identified three APETALA2/ETHYLENE RESPONSIVE FACTOR (AP2/ERF) transcription factors (TFs) that were responsible for this regulation. These AP2/ERF genes were up-regulated under salt stress, and amino acid sequences and phylogenetic analyses indicated that all TFs possess only one conserved AP2 domain and likely belong to the same subgroup of ERF-B3 in the AP2/ERF superfamily. Overexpression of these TFs significantly increased the chloroplast number even in the absence of NaCl stress. On the contrary, inducible overexpression of the dominant repressor form of these TFs suppressed salt stress-induced chloroplast division. Thus, our results suggest that salt stress induced-chloroplast division is regulated through members of the AP2/ERF TF superfamily.
Assuntos
Bryopsida , Cloroplastos , Fatores de Transcrição , Bryopsida/genética , Bryopsida/metabolismo , Cloroplastos/metabolismo , Etilenos , Regulação da Expressão Gênica de Plantas , Filogenia , Proteínas de Plantas/genética , Proteínas de Plantas/metabolismo , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismoRESUMO
Mitochondria, which evolved from a free-living bacterial ancestor, contain their own genomes and genetic systems and are produced from preexisting mitochondria by binary division. The mitochondrion-dividing (MD) ring is the main skeletal structure of the mitochondrial division machinery. However, the assembly mechanism and molecular identity of the MD ring are unknown. Multi-omics analysis of isolated mitochondrial division machinery from the unicellular alga Cyanidioschyzon merolae revealed an uncharacterized glycosyltransferase, MITOCHONDRION-DIVIDING RING1 (MDR1), which is specifically expressed during mitochondrial division and forms a single ring at the mitochondrial division site. Nanoscale imaging using immunoelectron microscopy and componential analysis demonstrated that MDR1 is involved in MD ring formation and that the MD ring filaments are composed of glycosylated MDR1 and polymeric glucose nanofilaments. Down-regulation of MDR1 strongly interrupted mitochondrial division and obstructed MD ring assembly. Taken together, our results suggest that MDR1 mediates the synthesis of polyglucan nanofilaments that assemble to form the MD ring. Given that a homolog of MDR1 performs similar functions in chloroplast division, the establishment of MDR1 family proteins appears to have been a singular, crucial event for the emergence of endosymbiotic organelles.
Assuntos
Glicosiltransferases/metabolismo , Biogênese de Organelas , Proteínas de Plantas/metabolismo , Rodófitas/metabolismo , Glucanos/metabolismo , Glicosiltransferases/genética , Mitocôndrias/metabolismo , Mitocôndrias/fisiologia , Mitocôndrias/ultraestrutura , Proteínas de Plantas/genética , Rodófitas/ultraestruturaRESUMO
Chloroplasts host photosynthesis and fulfill other metabolic functions that are essential to plant life. They have to divide by binary fission to maintain their numbers throughout cycles of cell division. Chloroplast division is achieved by a complex ring-shaped division machinery located on both the inner (stromal) and the outer (cytosolic) side of the chloroplast envelope. The inner division ring (termed the Z ring) is formed by the assembly of tubulin-like FtsZ1 and FtsZ2 proteins. ARC6 is a key chloroplast division protein that interacts with the Z ring. ARC6 spans the inner envelope membrane, is known to stabilize or maintain the Z ring, and anchors the Z ring to the inner membrane through interaction with FtsZ2. The underlying mechanism of Z ring stabilization is not well-understood. Here, biochemical and structural characterization of ARC6 was conducted using light scattering, sedimentation, and light and transmission EM. The recombinant protein was purified as a dimer. The results indicated that a truncated form of ARC6 (tARC6), representing the stromal portion of ARC6, affects FtsZ2 assembly without forming higher-order structures and exerts its effect via FtsZ2 dynamics. tARC6 prevented GDP-induced FtsZ2 disassembly and caused a significant net increase in FtsZ2 assembly when GDP was present. Single particle analysis and 3D reconstruction were performed to elucidate the structural basis of ARC6 activity. Together, the data reveal that a dimeric form of tARC6 binds to FtsZ2 filaments and does not increase FtsZ polymerization rates but rather inhibits GDP-associated FtsZ2 disassembly.
Assuntos
Proteínas de Arabidopsis/antagonistas & inibidores , Proteínas de Arabidopsis/química , Proteínas de Arabidopsis/metabolismo , Arabidopsis/metabolismo , Cloroplastos/metabolismo , Guanosina Difosfato/metabolismo , Proteínas de Arabidopsis/genética , Cristalografia por Raios X , Conformação Proteica , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismoRESUMO
Chloroplasts evolved from cyanobacterial endosymbiotic ancestors and their division is a complex process initiated by the assembly of cytoskeletal FtsZ (Filamentous temperature sensitive Z) proteins into a ring structure at the division site (Z-ring). The cyanobacterial Z-ring positioning system (MinCDE proteins) is also conserved in chloroplasts, except that MinC was lost and replaced by the eukaryotic ARC3 (accumulation and replication of chloroplasts). Both MinC and ARC3 act as negative regulators of FtsZ assembly, but ARC3 bears little sequence similarity with MinC. Here, light scattering assays, co-sedimentation, GTPase assay and transmission electron microscopy in conjunction with single-particle analysis have been used to elucidate the structure of ARC3 and its effect on its main target in chloroplast division, FtsZ2. Analysis of FtsZ2 in vitro assembly reactions in the presence and absence of GMPCPP showed that ARC3 promotes FtsZ2 debundling and disassembly of existing filaments in a concentration-dependent manner and requires GTP hydrolysis. Three-dimensional reconstruction of ARC3 revealed an almost circular molecule in which the FtsZ-binding N-terminus and the C-terminal PARC6 (paralog of ARC6)-binding MORN (Membrane Occupation and Recognition Nexus) domain are in close proximity and suggest a model for PARC6-enabled binding of ARC3 to FtsZ2. The latter is corroborated by in vivo data.
Assuntos
Proteínas de Arabidopsis/metabolismo , Arabidopsis/genética , Cloroplastos/metabolismo , Regulação da Expressão Gênica de Plantas , Guanosina Trifosfato/metabolismo , Arabidopsis/efeitos dos fármacos , Arabidopsis/metabolismo , Proteínas de Arabidopsis/química , Proteínas de Arabidopsis/genética , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Divisão Celular , Cloroplastos/efeitos dos fármacos , Cloroplastos/genética , Cloroplastos/ultraestrutura , Citoesqueleto/metabolismo , Citoesqueleto/ultraestrutura , Células Eucarióticas/metabolismo , Células Eucarióticas/ultraestrutura , Guanosina Trifosfato/análogos & derivados , Guanosina Trifosfato/farmacologia , Cinética , Proteínas Nucleares/química , Proteínas Nucleares/genética , Proteínas Nucleares/metabolismo , Pichia/genética , Pichia/metabolismo , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismoRESUMO
Chloroplasts evolved from a cyanobacterial endosymbiont. It is believed that the synchronization of endosymbiotic and host cell division, as is commonly seen in existing algae, was a critical step in establishing the permanent organelle. Algal cells typically contain one or only a small number of chloroplasts that divide once per host cell cycle. This division is based partly on the S-phase-specific expression of nucleus-encoded proteins that constitute the chloroplast-division machinery. In this study, using the red alga Cyanidioschyzon merolae, we show that cell-cycle progression is arrested at the prophase when chloroplast division is blocked before the formation of the chloroplast-division machinery by the overexpression of Filamenting temperature-sensitive (Fts) Z2-1 (Fts72-1), but the cell cycle progresses when chloroplast division is blocked during division-site constriction by the overexpression of either FtsZ2-1 or a dominant-negative form of dynamin-related protein 5B (DRP5B). In the cells arrested in the prophase, the increase in the cyclin B level and the migration of cyclin-dependent kinase B (CDKB) were blocked. These results suggest that chloroplast division restricts host cell-cycle progression so that the cell cycle progresses to the metaphase only when chloroplast division has commenced. Thus, chloroplast division and host cell-cycle progression are synchronized by an interactive restriction that takes place between the nucleus and the chloroplast. In addition, we observed a similar pattern of cell-cycle arrest upon the blockage of chloroplast division in the glaucophyte alga Cyanophora paradoxa, raising the possibility that the chloroplast division checkpoint contributed to the establishment of the permanent organelle.
Assuntos
Cloroplastos/fisiologia , Proteínas de Plantas/metabolismo , Rodófitas/fisiologia , Ciclo Celular , Núcleo Celular/genética , Núcleo Celular/metabolismo , Proteínas de Plantas/genética , Regulação para CimaRESUMO
Chloroplasts (plastids) and mitochondria evolved from endosymbiotic bacteria. These organelles perform vital functions in photosynthetic eukaryotes, such as harvesting and converting energy for use in biological processes. Consistent with their evolutionary origins, plastids and mitochondria proliferate by the binary fission of pre-existing organelles. Here, I review the structures and functions of the supramolecular machineries driving plastid and mitochondrial division, which were discovered and first studied in the primitive red alga Cyanidioschyzon merolae. In the past decade, intact division machineries have been isolated from plastids and mitochondria and examined to investigate their underlying structure and molecular mechanisms. A series of studies has elucidated how these division machineries assemble and transform during the fission of these organelles, and which of the component proteins generate the motive force for their contraction. Plastid- and mitochondrial-division machineries have important similarities in their structures and mechanisms despite sharing no component proteins, implying that these division machineries evolved in parallel. The establishment of these division machineries might have enabled the host eukaryotic ancestor to permanently retain these endosymbiotic organelles by regulating their binary fission and the equal distribution of resources to daughter cells. These findings provide key insights into the establishment of endosymbiotic organelles and have opened new avenues of research into their evolution and mechanisms of proliferation.
Assuntos
Organelas/ultraestrutura , Rodófitas/ultraestrutura , Simbiose , Divisão Celular , Cloroplastos/fisiologia , Cloroplastos/ultraestrutura , Mitocôndrias/fisiologia , Mitocôndrias/ultraestrutura , Organelas/fisiologia , Plastídeos/fisiologia , Plastídeos/ultraestrutura , Rodófitas/fisiologiaRESUMO
The endosymbiosis of a free-living cyanobacterium into an ancestral eukaryote led to the evolution of the chloroplast (plastid) more than one billion years ago. Given their independent origins, plastid proliferation is restricted to the binary fission of pre-existing plastids within a cell. In the last 25 years, the structure of the supramolecular machinery regulating plastid division has been discovered, and some of its component proteins identified. More recently, isolated plastid-division machineries have been examined to elucidate their structural and mechanistic details. Furthermore, complex studies have revealed how the plastid-division machinery morphologically transforms during plastid division, and which of its component proteins play a critical role in generating the contractile force. Identifying the three-dimensional structures and putative functional domains of the component proteins has given us hints about the mechanisms driving the machinery. Surprisingly, the mechanisms driving plastid division resemble those of mitochondrial division, indicating that these division machineries likely developed from the same evolutionary origin, providing a key insight into how endosymbiotic organelles were established. These findings have opened new avenues of research into organelle proliferation mechanisms and the evolution of organelles.
Assuntos
Cloroplastos/metabolismo , Biogênese de Organelas , Proteínas de Arabidopsis/química , Proteínas de Arabidopsis/genética , Proteínas de Arabidopsis/metabolismo , Cloroplastos/fisiologia , Dinaminas/química , Dinaminas/genética , Dinaminas/metabolismoRESUMO
Plant chloroplasts originate from the symbiotic relationship between ancient free-living cyanobacteria and ancestral eukaryotic cells. Since the discovery of the bacterial derivative FtsZ gene-which encodes a tubulin homolog responsible for the formation of the chloroplast inner division ring (Z ring)-in the Arabidopsis genome in 1995, many components of the chloroplast division machinery were successively identified. The knowledge of these components continues to expand; however, the mode of action of the chloroplast dividing system remains unknown (compared to bacterial cell division), owing to the complexities faced in in planta analyses. To date, yeast and bacterial heterologous expression systems have been developed for the reconstitution of Z ring-like structures formed by chloroplast FtsZ. In this review, we especially focus on recent progress of our bacterial system using the model bacterium Escherichia coli to dissect and understand the chloroplast division machinery-an evolutionary hybrid structure composed of both bacterial (inner) and host-derived (outer) components.
Assuntos
Proteínas de Cloroplastos/genética , Cloroplastos/metabolismo , Escherichia coli/genética , Transgenes , Proteínas de Cloroplastos/metabolismo , Cloroplastos/fisiologia , Escherichia coli/metabolismoRESUMO
Arogenate dehydratases (ADTs) catalyze the final step in phenylalanine biosynthesis in plants. The Arabidopsis thaliana genome encodes a family of six ADTs capable of decarboxylating/dehydrating arogenate into phenylalanine. Using cyan fluorescent protein (CFP)-tagged proteins, the subcellular localization patterns of all six A. thaliana ADTs were investigated in intact Nicotiana benthamiana and A. thaliana leaf cells. We show that A. thaliana ADTs localize to stroma and stromules (stroma-filled tubules) of chloroplasts. This localization pattern is consistent with the enzymatic function of ADTs as many enzymes required for amino acid biosynthesis are primarily localized to chloroplasts, and stromules are thought to increase metabolite transport from chloroplasts to other cellular compartments. Furthermore, we provide evidence that ADTs have additional, non-enzymatic roles. ADT2 localizes in a ring around the equatorial plane of chloroplasts or to a chloroplast pole, which suggests that ADT2 is a component of the chloroplast division machinery. In addition to chloroplasts, ADT5 was also found in nuclei, again suggesting a non-enzymatic role for ADT5. We also show evidence that ADT5 is transported to the nucleus via stromules. We propose that ADT2 and ADT5 are moonlighting proteins that play an enzymatic role in phenylalanine biosynthesis and a second role in chloroplast division or transcriptional regulation, respectively.
Assuntos
Proteínas de Arabidopsis/genética , Arabidopsis/genética , Hidroliases/genética , Arabidopsis/enzimologia , Proteínas de Arabidopsis/metabolismo , Cloroplastos/enzimologia , Proteínas de Fluorescência Verde , Hidroliases/metabolismo , Folhas de Planta/enzimologia , Plantas Geneticamente Modificadas/enzimologia , Plantas Geneticamente Modificadas/genética , Nicotiana/enzimologia , Nicotiana/genéticaRESUMO
Chloroplasts divide to maintain consistent size, shape, and number in leaf mesophyll cells. Altered expression of chloroplast division proteins in Arabidopsis results in abnormal chloroplast morphology. To better understand the influence of chloroplast morphology on chloroplast movement and photosynthesis, we compared the chloroplast photorelocation and photosynthetic responses of a series of Arabidopsis chloroplast division mutants with a wide variety of chloroplast phenotypes. Chloroplast movement was monitored by red light reflectance imaging of whole plants under increasing intensities of white light. The accumulation and avoidance responses were differentially affected in different mutants and depended on both chloroplast number and morphological heterogeneity. Chlorophyll fluorescence measurements during 5 d light experiments demonstrated that mutants with large-chloroplast phenotypes generally exhibited greater PSII photodamage than those with intermediate phenotypes. No abnormalities in photorelocation efficiency or photosynthetic capacity were observed in plants with small-chloroplast phenotypes. Simultaneous measurement of chloroplast movement and chlorophyll fluorescence indicated that the energy-dependent (qE) and long-lived components of non-photochemical quenching that reflect photoinhibition are affected differentially in different division mutants exposed to high or fluctuating light intensities. We conclude that chloroplast division mutants with abnormal chloroplast morphologies differ markedly from the wild type in their light adaptation capabilities, which may decrease their relative fitness in nature.
Assuntos
Arabidopsis/metabolismo , Clorofila/metabolismo , Cloroplastos/metabolismo , Fluorescência , Luz , Arabidopsis/genética , Genótipo , FotossínteseRESUMO
KEY MESSAGE: PDV2 has a dosage effect on chloroplast division in Arabidopsis thaliana , but this effect may vary in different plants. Chloroplasts have to be divided as plants grow to maintain an optimized number in the cell. Chloroplasts are divided by protein complexes across the double membranes from the stroma side to the cytosolic side. PDV2 is a chloroplast division protein on the chloroplast outer membrane. It recruits the dynamin-related GTPase ARC5 to the division site. The C-terminus of PDV2 and the C-terminus of ARC6 interact in the intermembrane space, which is important for the localization of PDV2. Previously, it was shown that overexpression of PDV2 can increase the division of chloroplasts in Arabidopsis and moss, so the authors concluded that PDV2 determines the rate of chloroplast division in land plants. PDV2 was also shown to inhibit the GTPase activity of ARC5 by in vitro experiment. These results look to be contradictory. Here, we identified a null allele of PDV2 in Arabidopsis and studied plants with different levels of PDV2. Our results suggested that the chloroplast division phenotype in Arabidopsis is sensitive to the level of PDV2, while this is not the case for ARC6. The level of PDV2 protein is reduced sharply in fast-growing leaves, while the level of ARC6 is not. The levels of PDV2 and ARC6 in several other plant species at different developmental stages were also investigated. The results indicated that their expression pattern varies in different species. Thus, PDV2 is an important positive factor of chloroplast division with an apparent dosage effect in Arabidopsis, but this effect for different chloroplast division proteins in different plants may vary.