Plastid Division Research Articles

Septum Formation in Amyloplasts Produces Compound Granules in the Rice Endosperm and is Regulated by Plastid Division Proteins

Storage tissues such as seed endosperm and tubers store starch in the form of granules in the amyloplast. In the rice (Oryza sativa) endosperm, each amyloplast produces compound granules consisting of several dozen polyhedral, sharp-edged and easily separable granules; whereas in other cereals, including wheat (Triticum aestivum), barley (Hordeum vulgare) and maize (Zea mays), each amyloplast synthesizes one granule. Despite extensive studies on mutants of starch synthesis in cereals, the molecular mechanisms involved in compound granule synthesis in rice have remained elusive. In this study, we expressed green fluorescent protein (GFP) fused to rice Brittle1 (BT1), an inner envelope membrane protein, to characterize dividing amyloplasts in the rice endosperm. Confocal microscopic analyses revealed that a septum-like structure, or cross-wall, containing BT1-GFP divides granules in the amyloplast. Plastid division proteins including FtsZ, Min and PDV2 play significant roles not only in amyloplast division, but also in septum synthesis, suggesting that amyloplast division and septum synthesis are related processes that share common factors. We propose that successive septum syntheses which create sections inside the amyloplast and de novo granule synthesis in each section are primarily responsible for the synthesis of compound granules.

The complexity and evolution of the plastid-division machinery

Plastids are vital organelles, fulfilling important metabolic functions that greatly influence plant growth and productivity. In order to both regulate and harness the metabolic output of plastids, it is vital that the process of plastid division is carefully controlled. This is essential, not only to ensure persistence in dividing plant cells and that optimal numbers of plastids are obtained in specialized cell types, but also to allow the cell to act in response to developmental signals and environmental changes. How this control is exerted by the host nucleus has remained elusive. Plastids evolved by endosymbiosis and during the establishment of a permanent endosymbiosis they retained elements of the bacterial cell-division machinery. Through evolution the photosynthetic eukaryotes have increased dramatically in complexity, from single-cell green algae to multicellular non-vascular and vascular plants. Reflected with this is an increasing complexity of the division machinery and recent findings also suggest increasing complexity in the molecular mechanisms used by the host cell to control the process of plastid division. In the present paper, we explore the current understanding of the process of plastid division at the molecular and cellular level, with particular respect to the evolution of the division machinery and levels of control exerted on the process.

The Conservative Function of Chloroplast Division Associated CrMinD Protein

The interacting of MinD and MinC and MinE proteins of Escherichia coli are required for proper placement of the division septum at midcell, The MinD protein plays a key role in the course of bacterial cell and plant chloroplast division.The over production of MinD protein can block the normal cell division and lead to a filamentation cell. In plant cells, There are clear similarities between bacterial and plastid division, but limited information exists regarding the mechanism of plastid division in higher plants. Here we constructed a expression plasmid encoding the full-length CrMinD, an Chlamydomonas reinhardtii homologue of the bacterial MinD. In agreement with cell division studies in bacteria over expression of CrMinD in E.coli results in filamentation cell formation. The sesuggestion that CrMinD is an evolutionary conserved cell division protein. Meanwhile, the result lays a certain foundation for studying on the division mechanism of plant chloroplast.

Dynamic morphologies of pollen plastids visualised by vegetative-specific FtsZ1–GFP in Arabidopsis thaliana

The behaviour and multiplication of pollen plastids have remained elusive despite their crucial involvement in cytoplasmic inheritance. Here, we present live images of plastids in pollen grains and growing tubes from transgenic Arabidopsis thaliana lines expressing stroma-localised FtsZ1-green-fluorescent protein fusion in a vegetative cell-specific manner. Vegetative cells in mature pollen contained a morphologically heterogeneous population of round to ellipsoidal plastids, whilst those in late-developing (maturing) pollen included plastids that could have one or two constriction sites. Furthermore, plastids in pollen tubes exhibited remarkable tubulation, stromule (stroma-filled tubule) extension, and back-and-forth movement along the direction of tube growth. Plastid division, which involves the FtsZ1 ring, was rarely observed in mature pollen grains.

Division of cell nuclei, mitochondria, plastids, and microbodies mediated by mitotic spindle poles in the primitive red alga Cyanidioschyzon merolae

To understand the cell cycle, we must understand not only mitotic division but also organelle division cycles. Plant and animal cells contain many organelles which divide randomly; therefore, it has been difficult to elucidate these organelle division cycles. We used the primitive red alga Cyanidioschyzon merolae, as it contains a single mitochondrion and plastid per cell, and organelle division can be highly synchronized by a light/dark cycle. We demonstrated that mitochondria and plastids multiplied by independent division cycles (organelle G1, S, G2 and M phases) and organelle division occurred before cell-nuclear division. Additionally, organelle division was found to be dependent on microtubules as well as cell-nuclear division. We have observed five stages of microtubule dynamics: (1) the microtubule disappears during the G1 phase; (2) alpha-tubulin is dispersed within the cytoplasm without forming microtubules during the S phase; (3) alpha-tubulin is assembled into spindle poles during the G2 phase; (4) polar microtubules are organized along the mitochondrion during prophase; and (5) mitotic spindles in cell nuclei are organized during the M phase. Microfluorometry demonstrated that the intensity peak of localization of alpha-tubulin changed in the order to spindle poles, mitochondria, spindle poles, and central spindle area, but total fluorescent intensity did not change remarkably throughout mitotic phases suggesting that division and separation of the cell nucleus and mitochondrion is mediated by spindle pole bodies. Inhibition of microtubule organization induced cell-nuclear division, mitochondria separation, and division of a single membrane-bound microbody, suggesting that similar to cell-nuclear division, mitochondrion separation and microbody division are dependent on microtubules.

The evolution of the regulatory mechanism of chloroplast division

Chloroplasts arose from a cyanobacterial endosymbiont and multiply by division, reminiscent of their free-living ancestor. However, chloroplasts can not divide by themselves, and the division is performed and controlled by proteins that are encoded by the host nucleus. The continuity of chloroplasts was originally established by synchronization of endosymbiotic cell division with host cell division, as seen in existent algae. In contrast, land plant cells contain multiple chloroplasts, the division of which is not synchronized, even in the same cell. Land plants have evolved cell and chloroplast differentiation systems in which the size and number of chloroplasts (or other types of plastids) change along with their respective cellular function by changes in the division rate. We recently reported that PLASTID DIVISION (PDV) proteins, land-plant specific components of the chloroplast division apparatus, determined the rate of chloroplast division. The level of PDV protein is regulated by the cell differentiation program based on cytokinin, and the increase or decrease of the PDV level gives rise to an increase or decrease in the chloroplast division rate. Thus, the integration of PDV proteins into the chloroplast division machinery enabled land plant cells to change chloroplast size and number in accord with the fate of cell differentiation.

Plastid division

Plastids undergo a process of binary fission in order to replicate. Plastid replication is required at two distinct stages of plant growth: during cell division to ensure correct plastid segregation, and during cell expansion and development to generate large populations of functional plastids, as in leaf mesophyll cells. This review considers some of the recent advances in the understanding of how plastids undergo binary fission, a process which uses several different proteins, both internal and external to the plastid, which have been derived from the original endosymbiont's genome as well as new proteins that have been recruited from the host genome. Several of the proteins currently used in this process in higher plants have homologues in modern-day bacteria. An alternative mode of replication by a budding-type mechanism also appears to be used in some circumstances. The review also highlights how most of our knowledge of plastid division is centred on the chloroplast developing in leaf mesophyll cells and a role for plastid division during the development of other plastid types is poorly understood. Whilst models for a protein-based mechanism have been devised, exactly how the division process is controlled at the plastid level and at the plastid population level is poorly understood.

Chloroplast biogenesis during the early stage of leaf development in rice

In rice, the developmental process in leaf formation can be divided into 7 stages (stages P0 to P6). We investigated chloroplast biogenesis and physiological changes in the developing leaves at the stage P4, during which leaf blade elongation and establishment of basic leaf blade structure occur. Chlorophyll content was negligible in the leaves early in the P4 stage and increased rapidly as they enters the late P4 phase. Chlorophyll fluorescence ratio (Fv/Fm) also increased markedly and the final value was comparable with that of mature leaves. Gene expression analysis showed that during the P4 stage, chloroplasts in the leaf cell undergo all three steps of differentiation: (i) plastid division and DNA replication (ii) establishment of plastid genetic system (iii) activation of photosynthetic apparatus. These observations suggest that the P4 is key in the development of a leaf, during which leaf rapidly differentiated both morphologically and physiologically, and that the P4 leaf is suitable for investigation of physiological relationships between chloroplast and leaf development.

Involvement ofAtMinE1in Plastid Morphogenesis in Various Tissues ofArabidopsis thaliana

While it has been established that binary fission of leaf chloroplasts requires the prokaryote-derived, division site determinant protein MinE, it remains to be clarified whether chloroplast division in non-leaf tissues and the division of non-colored plastids also involve the MinE protein. In an attempt to address this issue, plastids of cotyledons, floral organs, and roots were examined in the Arabidopsis thaliana mutant of the MinE (AtMinE1) gene, which was modified to express the plastid-targeted cyan fluorescent protein constitutively, and were quantitatively compared with those in the wild type. In the cotyledons, floral organs, and root columella, the plastid size in the atminE1 mutant was significantly larger than in the wild type, while the plastid number per cell in atminE1 appeared to be inversely smaller than that in the wild type. In addition, formation of the stroma-containing plastid protrusions (stromules) in the cotyledon epidermis, petal tip, and root cells was more active in atminE1 than in the wild type.

Plant FtsZ1 and FtsZ2 expressed in a eukaryotic host: GTPase activity and self-assembly

Plants and algae contain the FtsZ1 and FtsZ2 protein families that perform specific, non-redundant functions in plastid division. In vitro studies of chloroplast division have been hampered by the lack of a suitable expression system. Here we report the expression and purification of FtsZ1-1 and FtsZ2-1 from Arabidopsis thaliana using a eukaryotic host . Specific GTPase activities were determined and found to be different for FtsZ1-1 vs. FtsZ2-1. The purified proteins readily assembled into previously unreported assembly products named type-I and -II filaments. In contrast to bacterial FtsZ, the Arabidopsis proteins do not form bundled sheets in the presence of Ca 2+.

Targeted Gene Knockouts Reveal Overlapping Functions of the Five Physcomitrella patens FtsZ Isoforms in Chloroplast Division, Chloroplast Shaping, Cell Patterning, Plant Development, and Gravity Sensing

Chloroplasts and bacterial cells divide by binary fission. The key protein in this constriction division is FtsZ, a self-assembling GTPase similar to eukaryotic tubulin. In prokaryotes, FtsZ is almost always encoded by a single gene, whereas plants harbor several nuclear-encoded FtsZ homologs. In seed plants, these proteins group in two families and all are exclusively imported into plastids. In contrast, the basal land plant Physcomitrella patens, a moss, encodes a third FtsZ family with one member. This protein is dually targeted to the plastids and to the cytosol. Here, we report on the targeted gene disruption of all ftsZ genes in P. patens. Subsequent analysis of single and double knockout mutants revealed a complex interaction of the different FtsZ isoforms not only in plastid division, but also in chloroplast shaping, cell patterning, plant development, and gravity sensing. These results support the concept of a plastoskeleton and its functional integration into the cytoskeleton, at least in the moss P. patens.

Arabidopsis FtsZ2-1 and FtsZ2-2 Are Functionally Redundant, But FtsZ-Based Plastid Division Is Not Essential for Chloroplast Partitioning or Plant Growth and Development

FtsZ1 and FtsZ2 are phylogenetically distinct families of FtsZ in plants that co-localize to mid-plastid rings and facilitate division of chloroplasts. In plants, altered levels of either FtsZ1 or FtsZ2 cause dose-dependent defects in chloroplast division; thus, studies on the functional relationship between FtsZ genes require careful manipulation of FtsZ levels in vivo. To define the functional relationship between the two FtsZ2 genes in Arabidopsis thaliana, FtsZ2-1 and FtsZ2-2, we expressed FtsZ2-1 in an ftsZ2-2 null mutant, and vice versa, and determined whether the chloroplast division defects were rescued in plants expressing different total levels of FtsZ2. Full rescue was observed when either the FtsZ2-1 or FtsZ2-2 level approximated total FtsZ2 levels in wild-type (WT). Additionally, FtsZ2-2 interacts with ARC6, as shown previously for FtsZ2-1. These data indicate that FtsZ2-1 and FtsZ2-2 are functionally redundant for chloroplast division in Arabidopsis. To rigorously validate the requirement of each FtsZ family for chloroplast division, we replaced FtsZ1 with FtsZ2 in vivo, and vice versa, while maintaining the FtsZ level in the transgenic plants equal to that of the total level in WT. Chloroplast division defects were not rescued, demonstrating conclusively that FtsZ1 and FtsZ2 are non-redundant for maintenance of WT chloroplast numbers. Finally, we generated ftsZ triple null mutants and show that plants completely devoid of FtsZ protein are viable and fertile. As plastids are presumably essential organelles, these findings suggest that an FtsZ-independent mode of plastid partitioning may occur in higher plants.

The infective cycle of Cabbage leaf curl virus (CaLCuV) is affected by CRUMPLED LEAF (CRL) gene in Arabidopsis thaliana

BackgroundGeminiviruses are single-stranded DNA viruses that cause serious crop losses worldwide. Successful infection by these pathogens depends extensively on virus-host intermolecular interactions that allow them to express their gene products, to replicate their genomes and to move to adjacent cells and throughout the plant.ResultsTo identify host genes that show an altered regulation in response to Cabbage leaf curl virus (CaLCuV) infection, a screening of transposant Arabidopsis thaliana lines was carried out. Several genes were identified to be virus responsive and one, Crumpled leaf (CRL) gene, was selected for further characterization. CRL was previously reported by Asano et al., (2004) to affect the morphogenesis of all plant organs and the division of plastids. We report here that CRL expression, during CaLCuV infection, shows a short but strong induction at an early stage (3-5 days post inoculation, dpi). To study the role of CRL in CaLCuV infection, CRL over-expressing and silenced transgenic plants were generated. We compared the replication, movement and infectivity of CaLCuV in transgenic and wild type plants.ConclusionOur results showed that CRL over-expressing plants showed an increased susceptibility to CaLCuV infection (as compared to wt plants) whereas CRL-silenced plants, on the contrary, presented a reduced susceptibility to viral infection. The possible role of CRL in the CaLCuV infection cycle is discussed.

The Peptidoglycan Biosynthesis Genes MurA and MraY are Related to Chloroplast Division in the Moss Physcomitrella patens

In the moss Physcomitrella patens, 10 Mur genes involved in peptidoglycan biosynthesis were found, and the MurE and Pbp genes are related to plastid division. Although the MraY and MurG genes were missing in our previous expressed sequence tag screening, they were discovered in the P. patens genome in this study, indicating that P. patens has a full set of genes capable of synthesizing peptidoglycan. In addition, a second MurA gene (PpMurA2) was found. Whereas Northern analyses indicated that PpMurA1, PpMurG and PpMraY were expressed, transcripts of PpMurA2 were detected only when RT-PCR was employed. Whereas GFP fusion proteins with either PpMurA1 or PpMraY were detected in chloroplasts, the PpMurA2 fusion proteins were located in the cytoplasm. Protonema cells in the wild-type plants had an average of 46 chloroplasts. PpMurA1 gene-disrupted lines had <10 chloroplasts, whereas approximately 30 chloroplasts existed in the PpMurA2 knockout lines. The PpMurA1/A2 double-knockout lines had only a few macrochloroplasts, suggesting a redundant function for these two genes. Disruption of the PpMraY gene in P. patens resulted in the appearance of macrochloroplasts. Anabaena MraY fused to the N-terminal region of PpMraY and A. thaliana MraY could complement the macrochloroplast phenotype in the PpMraY knockout line. Electron microscopic observations showed no obvious differences in the shape or stacking of thylakoid membranes between all knockout transformants and wild-type plants, suggesting that these Mur genes are related only to plastid division in moss.

Arabidopsis Plastid Division Proteins FtsZ1 and FtsZ2: Macromolecular Assembly and Subunit Exchange Dynamics

Extended abstract of a paper presented at Microscopy and Microanalysis 2009 in Richmond, Virginia, USA, July 26 – July 30, 2009

The PLASTID DIVISION1 and 2 components of the chloroplast division machinery determine the rate of chloroplast division in land plant cell differentiation.

In most algae, the chloroplast division rate is held constant to maintain the proper number of chloroplasts per cell. By contrast, land plants evolved cell and chloroplast differentiation systems in which the size and number of chloroplasts change along with their respective cellular function by regulation of the division rate. Here, we show that PLASTID DIVISION (PDV) proteins, land plant-specific components of the division apparatus, determine the rate of chloroplast division. Overexpression of PDV proteins in the angiosperm Arabidopsis thaliana and the moss Physcomitrella patens increased the number but decreased the size of chloroplasts; reduction of PDV levels resulted in the opposite effect. The level of PDV proteins, but not other division components, decreased during leaf development, during which the chloroplast division rate also decreased. Exogenous cytokinins or overexpression of the cytokinin-responsive transcription factor CYTOKININ RESPONSE FACTOR2 increased the chloroplast division rate, where PDV proteins, but not other components of the division apparatus, were upregulated. These results suggest that the integration of PDV proteins into the division machinery enabled land plant cells to change chloroplast size and number in accord with the fate of cell differentiation.

Plastid chaperonin proteins Cpn60α and Cpn60β are required for plastid division in Arabidopsis thaliana

BackgroundPlastids arose from a free-living cyanobacterial endosymbiont and multiply by binary division as do cyanobacteria. Plastid division involves nucleus-encoded homologs of cyanobacterial division proteins such as FtsZ, MinD, MinE, and ARC6. However, homologs of many other cyanobacterial division genes are missing in plant genomes and proteins of host eukaryotic origin, such as a dynamin-related protein, PDV1 and PDV2 are involved in the division process. Recent identification of plastid division proteins has started to elucidate the similarities and differences between plastid division and cyanobacterial cell division. To further identify new proteins that are required for plastid division, we characterized previously and newly isolated plastid division mutants of Arabidopsis thaliana.ResultsLeaf cells of two mutants, br04 and arc2, contain fewer, larger chloroplasts than those of wild type. We found that ARC2 and BR04 are identical to nuclear genes encoding the plastid chaperonin 60α (ptCpn60α) and chaperonin 60β (ptCpn60β) proteins, respectively. In both mutants, plastid division FtsZ ring formation was partially perturbed though the level of FtsZ2-1 protein in plastids of ptcpn60β mutants was similar to that in wild type. Phylogenetic analyses showed that both ptCpn60 proteins are derived from ancestral cyanobacterial proteins. The A. thaliana genome encodes two members of ptCpn60α family and four members of ptCpn60β family respectively. We found that a null mutation in ptCpn60α abolished greening of plastids and resulted in an albino phenotype while a weaker mutation impairs plastid division and reduced chlorophyll levels. The functions of at least two ptCpn60β proteins are redundant and the appearance of chloroplast division defects is dependent on the number of mutant alleles.ConclusionOur results suggest that both ptCpn60α and ptCpn60β are required for the formation of a normal plastid division apparatus, as the prokaryotic counterparts are required for assembly of the cell division apparatus. Since moderate reduction of ptCpn60 levels impaired normal FtsZ ring formation but not import of FtsZ into plastids, it is suggested that the proper levels of ptCpn60 are required for folding of stromal plastid division proteins and/or regulation of FtsZ polymer dynamics.

Plant Cells Without Detectable Plastids are Generated in the crumpled leaf Mutant of Arabidopsis thaliana

Plastids are maintained in cells by proliferating prior to cell division and being partitioned to each daughter cell during cell division. It is unclear, however, whether cells without plastids are generated when plastid division is suppressed. The crumpled leaf (crl) mutant of Arabidopsis thaliana is a plastid division mutant that displays severe abnormalities in plastid division and plant development. We show that the crl mutant contains cells lacking detectable plastids; this situation probably results from an unequal partitioning of plastids to each daughter cell. Our results suggest that crl has a partial defect in plastid expansion, which is suggested to be important in the partitioning of plastids to daughter cells when plastid division is suppressed. The absence of cells without detectable plastids in the accumulation and replication of chloroplasts 6 (arc6) mutant, another plastid division mutant of A. thaliana having no significant defects in plant morphology, suggests that the generation of cells without detectable plastids is one of the causes of the developmental abnormalities seen in crl plants. We also demonstrate that plastids with trace or undetectable amounts of chlorophyll are generated from enlarged plastids by a non-binary fission mode of plastid replication in both crl and arc6.

Visualization of Plastids in Pollen Grains: Involvement of FtsZ1 in Pollen Plastid Division

Visualizing organelles in living cells is a powerful method to analyze their intrinsic mechanisms. Easy observation of chlorophyll facilitates the study of the underlying mechanisms in chloroplasts, but not in other plastid types. Here, we constructed a transgenic plant enabling visualization of plastids in pollen grains. Combination of a plastid-targeted fluorescent protein with a pollen-specific promoter allowed us to observe the precise number, size and morphology of plastids in pollen grains of the wild type and the ftsZ1 mutant, whose responsible gene plays a central role in chloroplast division. The transgenic material presented in this work is useful for studying the division mechanism of pollen plastids.

Periodic Gene Expression Patterns during the Highly Synchronized Cell Nucleus and Organelle Division Cycles in the Unicellular Red Alga Cyanidioschyzon merolae

Previous cell cycle studies have been based on cell-nuclear proliferation only. Eukaryotic cells, however, have double membranes-bound organelles, such as the cell nucleus, mitochondrion, plastids and single-membrane-bound organelles such as ER, the Golgi body, vacuoles (lysosomes) and microbodies. Organelle proliferations, which are very important for cell functions, are poorly understood. To clarify this, we performed a microarray analysis during the cell cycle of Cyanidioschyzon merolae. C. merolae cells contain a minimum set of organelles that divide synchronously. The nuclear, mitochondrial and plastid genomes were completely sequenced. The results showed that, of 158 genes induced during the S or G2-M phase, 93 were known and contained genes related to mitochondrial division, ftsZ1-1, ftsz1-2 and mda1, and plastid division, ftsZ2-1, ftsZ2-2 and cmdnm2. Moreover, three genes, involved in vesicle trafficking between the single-membrane organelles such as vps29 and the Rab family protein, were identified and might be related to partitioning of single-membrane-bound organelles. In other genes, 46 were hypothetical and 19 were hypothetical conserved. The possibility of finding novel organelle division genes from hypothetical and hypothetical conserved genes in the S and G2-M expression groups is discussed.