The division of pleomorphic plastids with multiple FtsZ rings in tobacco BY-2 cells

Plastids, an essential group of plant cellular organelles, proliferate by division to maintain continuity through cell lineages in plants. In recent years, it was revealed that the bacterial cell division protein FtsZ is encoded in the nuclear genome of plant cells, and plays a major role in the plastid division process forming a ring along the center of plastids. Although the best-characterized type of plastid division so far is the division with a single FtsZ ring at the plastid midpoint, it was recently reported that in some plant organs and tissues, plastids are pleomorphic and form multiple FtsZ rings. However, the pleomorphic plastid division mechanism, such as the formation of multiple FtsZ rings, the constriction of plastids and the behavior of plastid (pt) nucleoids, remains totally unclear. To elucidate these points, we used the cultured cell line, tobacco (Nicotiana tabacum L.) Bright Yellow-2, in which plastids are pleomorphic and show dynamic morphological changes during culture. As a result, it was revealed that as the plastid elongates from an ellipsoid shape to a string shape after medium renewal, FtsZ rings are multiplied almost orderly and perpendicularly to the long axis of plastids. Active DNA synthesis of pt nucleoids is induced by medium transfer, and the division and the distribution of pt nucleoids occur along with plastid elongation. Although it was thought that the plastid divides with simultaneous multiple constrictions at all the FtsZ ring sites, giving rise to many small plastids, we found that the plastids generally divide constricting at only one FtsZ ring site. Moreover, using electron microscopy, we revealed that plastid-dividing (PD) rings are observed only at the constriction site, and not at swollen regions. These results indicate that in the pleomorphic plastid division with multiple FtsZ rings, the formation of PD rings occurs at a limited FtsZ ring site for one division. Multiplied FtsZ rings seem to localize in advance at the expected sites of division, and the formation of a PD ring at each FtsZ ring site occurs in a certain order, not simultaneously. Based on these results, a novel model for the pleomorphic plastid division with multiple FtsZ rings is proposed.     

ARC3 is a stromal Z-ring accessory protein essential for plastid division

In plants, chloroplast division is an integral part of development, and these vital organelles arise by binary fission from pre-existing cytosolic plastids. Chloroplasts arose by endosymbiosis and although they have retained elements of the bacterial cell division machinery to execute plastid division, they have evolved to require two functionally distinct forms of the FtsZ protein and have lost elements of the Min machinery required for Z-ring placement. Here, we analyse the plastid division component accumulation and replication of chloroplasts 3 (ARC3) and show that ARC3 forms part of the stromal plastid division machinery. ARC3 interacts specifically with AtFtsZ1, acting as a Z-ring accessory protein and defining a unique function for this family of FtsZ proteins. ARC3 is involved in division site placement, suggesting that it might functionally replace MinC, representing an important advance in our understanding of the mechanism of chloroplast division and the evolution of the chloroplast division machinery.     

Manipulation of starch granule size distribution in potato tubers by modulation of plastid division

Starch granule size is an important parameter for starch applications in industry. Starch granules are formed in amyloplasts, which are, like chloroplasts, derived from proplastids. Division processes and associated machinery are likely to be similar for all plastids. Essential roles for FtsZ proteins in plastid division in land plants have been revealed. FtsZ forms the so-called Z ring which, together with inner and outer plastid division rings, brings about constriction of the plastid. It has been shown that modulation of the expression level of FtsZ may result in altered chloroplast size and number. To test whether FtsZ is also involved in amyloplast division and whether this, in turn, may affect the starch granule size in crop plants, FtsZ protein levels were either reduced or increased in potato. As shown previously in other plant species, decreased StFtsZ1 protein levels in leaves resulted in a decrease in the number of chloroplasts in guard cells. More interestingly, plants with increased StFtsZ1 protein levels in tubers resulted in less, but larger, starch granules. This suggests that the stoichiometry between StFtsZ1 and other components of the plastid division machinery is important for its function. Starch from these tubers also had altered pasting properties and phosphate content. The importance of our results for the starch industry is discussed.     

Developmentally regulated association of plastid division protein FtsZ1 with thylakoid membranes in Arabidopsis thaliana

FtsZ is a key protein involved in bacterial and organellar division. Bacteria have only one ftsZ gene, while chlorophytes (higher plants and green alga) have two distinct FtsZ gene families, named FtsZ1 and FtsZ2. This raises the question of why chloroplasts in these organisms need distinct FtsZ proteins to divide. In order to unravel new functions associated with FtsZ proteins, we have identified and characterized an Arabidopsis thaliana FtsZ1 loss-of-function mutant. ftsZ1-knockout mutants are impeded in chloroplast division, and division is restored when FtsZ1 is expressed at a low level. FtsZ1-overexpressing plants show a drastic inhibition of chloroplast division. Chloroplast morphology is altered in ftsZ1, with chloroplasts having abnormalities in the thylakoid membrane network. Overexpression of FtsZ1 also induced defects in thylakoid organization with an increased network of twisting thylakoids and larger grana. We show that FtsZ1, in addition to being present in the stroma, is tightly associated with the thylakoid fraction. This association is developmentally regulated since FtsZ1 is found in the thylakoid fraction of young developing plant leaves but not in mature and old plant leaves. Our results suggest that plastid division protein FtsZ1 may have a function during leaf development in thylakoid organization, thus highlighting new functions for green plastid FtsZ.     

The ultrastructural features and division of secondary plastids

Plastids in heterokonts, cryptophytes, haptophytes, dinoflagellates, chlorarachniophytes, euglenoids, and apicomplexan parasites derive from secondary symbiogenesis. These plastids are surrounded by one or two additional membranes covering the plastid-envelope double membranes. Consequently, nuclear-encoded plastid division proteins have to be targeted into the division site through the additional surrounding membranes. Electron microscopic observations suggest that the additional surrounding membranes are severed by mechanisms distinct from those for the division of the plastid envelope. In heterokonts, cryptophytes and haptophytes, the outermost surrounding membrane (epiplastid rough endoplasmic reticulum, EPrER) is studded with cytoplasmic ribosomes and connected to the rER and the outer nuclear envelope. In monoplastidic species belonging to these three groups, the EPrER and the outer nuclear envelope are directly connected to form a sac enclosing the plastid and the nucleus. This nuclear-plastid connection, referred to as the nucleus-plastid consortium (NPC), may be significant to ensure the transmission of the plastids during cell division. The plastid dividing-ring (PD-ring) is a conserved component of the division machinery for both primary and secondary plastids. Also, homologues of the bacterial cell division protein, FtsZ, may be involved in the division of secondary plastids as well as primary plastids, though in secondary plastids they have not yet been localized to the division site. It remains to be examined whether or not dynamin-like proteins and other protein components known to function in the division of primary plastids are used also in secondary plastids. The nearly completed sequencing of the nuclear genome of the diatom Thalassiosira pseudonana will give impetus to molecular and cell biological studies on the division of secondary plastids.     

Rings and networks: the amazing complexity of FtsZ in chloroplasts

Bacteria have proteins that can form filaments and rings, and these are thought to be the evolutionary progenitors of actin and tubulin. Plant homologues of the most intensively studied bacterial FtsZ protein are nuclear-encoded by a small gene family, are plastid-bound and participate in the plastid division process. The hypothesis is put forward that FtsZ and other proteins form a filamentous network in plastids, a plastoskeleton, which keeps these organelles in shape and helps them to divide.     

Two Types of FtsZ Proteins in Mitochondria and Red-Lineage Chloroplasts: The Duplication of FtsZ Is Implicated in Endosymbiosis

The ancestors of plastids and mitochondria were once free-living bacteria that became organelles as a result of endosymbiosis. According to this theory, a key bacterial division protein, FtsZ, plays a role in plastid division in algae and plants as well as in mitochondrial division in lower eukaryotes. Recent studies have shown that organelle division is a process that combines features derived from the bacterial division system with features contributed by host eukaryotic cells. Two nonredundant versions of FtsZ, FtsZ1 and FtsZ2, have been identified in green-lineage plastids, whereas most bacteria have a single ftsZ gene. To examine whether there is also more than one type of FtsZ in red-lineage chloroplasts (red algal chloroplasts and chloroplasts that originated from the secondary endosymbiosis of red algae) and in mitochondria, we obtained FtsZ sequences from the complete sequence of the primitive red alga Cyanidioschyzon merolae and the draft sequence of the stramenopile (heterokont) Thalassiosira pseudonana. Phylogenetic analyses that included known FtsZ proteins identified two types of chloroplast FtsZ in red algae (FtsZA and FtsZB) and stramenopiles (FtsZA and FtsZC). These analyses also showed that FtsZB emerged after the red and green lineages diverged, while FtsZC arose by the duplication of an ftsZA gene that in turn descended from a red alga engulfed by the ancestor of stramenopiles. A comparison of the predicted proteins showed that like bacterial FtsZ and green-lineage FtsZ2, FtsZA has a short conserved C-termmal sequence (the C-terminal core domain), whereas FtsZB and FtsZC, like the green-lineage FtsZ1, lack this sequence. In addition, the Cyanidioschyzon and Dictyostelium genomes encode two types of mitochondrial FtsZ proteins, one of which lacks the C-terminal variable domain. These results suggest that the acquisition of an additional FtsZ protein with a modified C terminus was common to the primary and secondary endosymbioses that produced plastids and that this also occurred during the establishment of mitochondria, presumably to regulate the multiplication of these organelles.     

Plastid division is driven by a complex mechanism that involves differential transition of the bacterial and eukaryotic division rings

During plastid division, two structures have been detected at the division site in separate analyses. The plastid-dividing ring can be detected by transmission electron microscopy as two (or three) electron-dense rings: an outer ring on the cytosolic face of the outer envelope, occasionally a middle ring in the intermembrane space, and an inner ring on the stromal face of the inner envelope. The FtsZ ring, which plays a central role in bacterial division, also is involved in plastid division and is believed to have descended to plastids from cyanobacterial endosymbiosis. The relationship between the two structures is not known, although there is discussion regarding whether they are identical. Biochemical and immunocytochemical investigations, using synchronized chloroplasts of the red alga Cyanidioschyzon merolae, showed that the plastid FtsZ ring is distinct and separable from the plastid-dividing ring. The FtsZ ring localizes in stroma and faces the inner plastid-dividing ring at the far side from the inner envelope. The FtsZ ring and the inner and outer plastid-dividing rings form in that order before plastid division. The FtsZ ring disappears at the late stage of constriction before dissociation of the plastid-dividing ring, when the constriction is still in progress. Our results suggest that the FtsZ ring;-based system, which originated from a plastid ancestor, cyanobacteria, and the plastid-dividing ring;-based system, which probably originated from host eukaryotic cells, form a complex and are involved in plastid division by distinct modes.     

Filamentous temperature-sensitive Z (FtsZ) isoforms specifically interact in the chloroplasts and in the cytosol of Physcomitrella patens

Plant filamentous temperature-sensitive Z (FtsZ) proteins have been reported to be involved in biological processes related to plastids. However, the precise functions of distinct isoforms are still elusive. Here, the intracellular localization of the FtsZ1-1 isoform in a moss, Physcomitrella patens, was examined. Furthermore, the in vivo interaction behaviour of four distinct FtsZ isoforms was investigated. Localization studies of green fluorescent protein (GFP)-tagged FtsZ1-1 and fluorescence resonance energy transfer (FRET) analyses employing all dual combinations of four FtsZ isoforms were performed in transient protoplast transformation assays. FtsZ1-1 is localized to network structures inside the chloroplasts and exerts influence on plastid division. Interactions between FtsZ isoforms occur in distinct ordered structures in the chloroplasts as well as in the cytosol. The results expand the view of the involvement of Physcomitrella FtsZ proteins in chloroplast and cell division. It is concluded that duplication and diversification of ftsZ genes during plant evolution were the main prerequisites for the successful remodelling and integration of the prokaryotic FtsZ-dependent division mechanism into the cellular machineries of distinct complex processes in plants.     

A homologue of the bacterial cell division site-determining factor MinD mediates placement of the chloroplast division apparatus

BACKGROUND: Chloroplast division in plant cells occurs by binary fission, yielding two daughter plastids of equal size. Previously, we reported that two Arabidopsis homologues of FtsZ, a bacterial protein that forms a cytokinetic ring during cell division, are essential for plastid division in plants, and may be involved in the formation of plastid-dividing rings on both the stromal and cytosolic surfaces of the chloroplast envelope membranes. In bacteria, positioning of the FtsZ ring at the center of the cell is mediated in part by the protein MinD. Here, we identified AtMinD1, an Arabidopsis homologue of MinD, and investigated whether positioning of the plastid-division apparatus at the plastid midpoint might involve a mechanism similar to that in bacteria. RESULTS: Sequence analysis and in vitro chloroplast import experiments indicated that AtMinD1 contains a transit peptide that targets it to the chloroplast. Transgenic Arabidopsis plants with reduced AtMinD1 expression exhibited variability in chloroplast size and number and asymmetrically constricted chloroplasts, strongly suggesting that the plastid-division machinery is misplaced. Overexpression of AtMinD1 inhibited chloroplast division. These phenotypes resemble those of bacterial mutants with altered minD expression. CONCLUSIONS: Placement of the plastid-division machinery at the organelle midpoint requires a plastid-targeted form of MinD. The results are consistent with a model whereby assembly of the division apparatus is initiated inside the chloroplast by the plastidic form of FtsZ, and suggest that positioning of the cytosolic components of the apparatus is specified by the position of the plastidic components.     

Visualization of a cytoskeleton-like FtsZ network in chloroplasts

It has been a long-standing dogma in life sciences that only eukaryotic organisms possess a cytoskeleton. Recently, this belief was questioned by the finding that the bacterial cell division protein FtsZ resembles tubulin in sequence and structure and, thus, may be the progenitor of this major eukaryotic cytoskeletal element. Here, we report two nuclear-encoded plant ftsZ genes which are highly conserved in coding sequence and intron structure. Both their encoded proteins are imported into plastids and there, like in bacteria, they act on the division process in a dose-dependent manner. Whereas in bacteria FtsZ only transiently polymerizes to a ring-like structure, in chloroplasts we identified persistent, highly organized filamentous scaffolds that are most likely involved in the maintenance of plastid integrity and in plastid division. As these networks resemble the eukaryotic cytoskeleton in form and function, we suggest the term "plastoskeleton" for this newly described subcellular structure.     

Regulation of leucoplast morphology in roots: Interorganellar signaling from mitochondria?

The appearance of leaf mesophyll chloroplasts in angiosperms is characterized by their uniform and static shape, which is molded by symmetric division of the preexisting organelles, involving three prokaryote-derived proteins: the division executor protein, FtsZ, and the division site positioning proteins, MinD and MinE. On the other hand, noncolored plastids in roots, where the involvement of the known chloroplast division factors in plastid morphogenesis is yet unclear, are morphologically heterogeneous and transform dynamically. This is further emphasized by the active formation of long tubular protrusions called stromules from the main body of those plastids. Molecular regulation and physiological significance of such dynamic morphology of root plastids also remain unknown. In this context, we have recently demonstrated that the mitochondrial respiratory inhibitor antimycin A induces rapid and reversible filamentation of root plastids (leucoplasts) in Arabidopsis thaliana. In contrast, the same treatment with antimycin A did not affect the morphology of amyloplasts in the columella cells at the root tip. The alternative oxidase inhibitor salicylhydroxamic acid suppresses the antimycin-induced plastid filamentation, perhaps implying an alternative oxidase-mediated interorganellar signaling between the mitochondria and the leucoplasts in the root cells. Our data may provide some clues as to how the formation of stromules is initiated.Key words: antimycin A, interorganellar crosstalk, plastid morphology, respiration, stress response, stromule     

A uniquely high number of ftsZ genes in the moss Physcomitrella patens

Plant FtsZ proteins are encoded by two small nuclear gene families (FtsZ1 and FtsZ2) and are involved in chloroplast division. From the moss Physcomitrella patens , four FtsZ proteins, two in each nuclear gene family, have been characterised and described so far. In the recently sequenced P. patens genome, we have now found a fifth fts Z gene. This novel gene has a genomic structure similar to Pp fts Z1-1. According to phylogenetic analysis, the encoded protein is a member of the FtsZ1 family, while PpFtsZ1-2, together with an orthologue from Selaginella moellendorffii , forms a separate clade. Further, this new gene is expressed in different gametophytic tissues and the encoded protein forms filamentous networks in chloroplasts, is found in stromules, and acts in plastid division. Based on all these results, we have renamed the PpFtsZ proteins of family 1 and suggest the existence of a third FtsZ family. No species is known to encode more FtsZ proteins per haploid genome than P. patens .     

Isolation of mitochondrial and plastid ftsZ genes and analysis of the organelle targeting sequence in the diatom Chaetoceros neogracile (Diatoms,Bacillariophyceae)

Mitochondria and plastids multiply by division in eukaryotic cells. Recently, the eukaryotic homolog of the bacterial cell division protein FtsZ was identified and shown to play an important role in the organelle division process inside the inner membrane. To explore the evolution of FtsZ proteins, and to accumulate data on the protein import system in mitochondria and plastids of the red algal lineage, one mitochondrial and three plastid ftsZ genes were isolated from the diatom Chaetoceros neogracile, whose plastids were acquired by secondary endosymbiotic uptake of a red alga. Protein import into organelles depends on the N‐terminal organelle targeting sequences. N‐terminal bipartite presequences consisting of an endoplasmic reticulum signal peptide and a plastid transit peptide are required for protein import into diatom plastids. To characterize the organelle targeting peptides of C. neogracile, we observed the localization of each green fluorescent protein‐tagged predicted organelle targeting peptide in cultured tobacco cells and diatom cells. Our data suggested that each targeting sequences functioned both in tobacco cultured cells and diatom cells.     

高等植物质体的分裂

质体来源于早期具光合能力的原核生物与原始真核生物的内共生事件。原核起源的蛋白以及真核寄主起源的蛋白共同参与了质体的分裂过程。以原核生物的细胞分裂蛋白为蓝本, 近些年在植物中陆续鉴定出几种主要的原核生物细胞分裂蛋白的同源物, 如FtsZ、MinD和MinE蛋白。然而, 除此之外, 原核细胞大多数分裂相关因子在植物中找不到其同源物, 但却鉴定了许多真核寄主来源的分裂相关蛋白。当前研究的重点是剖析各种质体分裂蛋白协同作用的机制, 业已证明MinD和MinE的协同作用保证了FtsZ(Z)环的正确定位。尽管经典的FtsZ的抑制因子MinC在植物中不存在, 但实验表明ARC3在拟南芥中具有类似MinC的功能。ARC3蛋白与真核起源的蛋白如ARC5、ARTEMIS、FZL和PD环以及其它原核起源的蛋白如ARC6和GC1等共同构成了一个复杂的植物质体分裂调控系统。     

高等植物质体的分裂

质体来源于早期具光合能力的原核生物与原始真核生物的内共生事件。原核起源的蛋白以及真核寄主起源的蛋白共同参与了质体的分裂过程。以原核生物的细胞分裂蛋白为蓝本,近些年在植物中陆续鉴定出几种主要的原核生物细胞分裂蛋白的同源物,如FtsZ、MinD和MinE蛋白。然而,除此之外,原核细胞大多数分裂相关因子在植物中找不到其同源物,但却鉴定了许多真核寄主来源的分裂相关蛋白。当前研究的重点是剖析各种质体分裂蛋白协同作用的机制,业已证明MinD和Mine的协同作用保证了FtsZ（Z）环的正确定位。尽管经典的FtsZ的抑制因子MinC在植物中不存在,但实验表明ARC3在拟南芥中具有类似MinC的功能。ARC3蛋白与真核起源的蛋白如ARC5、ARTEMIS、FZL和PD环以及其它原核起源的蛋白如ARC6和GC1等共同构成了一个复杂的植物质体分裂调控系统。     

Chloroplast division and morphology are differentially affected by overexpression of FtsZ1 and FtsZ2 genes in Arabidopsis

In higher plants, two nuclear gene families, FtsZ1 and FtsZ2, encode homologs of the bacterial protein FtsZ, a key component of the prokaryotic cell division machinery. We previously demonstrated that members of both gene families are essential for plastid division, but are functionally distinct. To further explore differences between FtsZ1 and FtsZ2 proteins we investigated the phenotypes of transgenic plants overexpressing AtFtsZ1-1 or AtFtsZ2-1, Arabidopsis members of the FtsZ1 and FtsZ2 families, respectively. Increasing the level of AtFtsZ1-1 protein as little as 3-fold inhibited chloroplast division. Plants with the most severe plastid division defects had 13- to 26-fold increases in AtFtsZ1-1 levels over wild type, and some of these also exhibited a novel chloroplast morphology. Quantitative immunoblotting revealed a correlation between the degree of plastid division inhibition and the extent to which the AtFtsZ1-1 protein level was elevated. In contrast, expression of an AtFtsZ2-1 sense transgene had no obvious effect on plastid division or morphology, though AtFtsZ2-1 protein levels were elevated only slightly over wild-type levels. This may indicate that AtFtsZ2-1 accumulation is more tightly regulated than that of AtFtsZ1-1. Plants expressing the AtFtsZ2-1 transgene did accumulate a form of the protein smaller than those detected in wild-type plants. AtFtsZ2-1 levels were unaffected by increased or decreased accumulation of AtFtsZ1-1 and vice versa, suggesting that the levels of these two plastid division proteins are regulated independently. Taken together, our results provide additional evidence for the functional divergence of the FtsZ1 and FtsZ2 plant gene families.     

ARC6 is a J-domain plastid division protein and an evolutionary descendant of the cyanobacterial cell division protein Ftn2

Replication of chloroplasts is essential for achieving and maintaining optimal plastid numbers in plant cells. The plastid division machinery contains components of both endosymbiotic and host cell origin, but little is known about the regulation and molecular mechanisms that govern the division process. The Arabidopsis mutant arc6 is defective in plastid division, and its leaf mesophyll cells contain only one or two grossly enlarged chloroplasts. We show here that arc6 chloroplasts also exhibit abnormal localization of the key plastid division proteins FtsZ1 and FtsZ2. Whereas in wild-type plants, the FtsZ proteins assemble into a ring at the plastid division site, chloroplasts in the arc6 mutant contain numerous short, disorganized FtsZ filament fragments. We identified the mutation in arc6 and show that the ARC6 gene encodes a chloroplast-targeted DnaJ-like protein localized to the plastid envelope membrane. An ARC6-green fluorescent protein fusion protein was localized to a ring at the center of the chloroplasts and rescued the chloroplast division defect in the arc6 mutant. The ARC6 gene product is related closely to Ftn2, a prokaryotic cell division protein unique to cyanobacteria. Based on the FtsZ filament morphology observed in the arc6 mutant and in plants that overexpress ARC6, we hypothesize that ARC6 functions in the assembly and/or stabilization of the plastid-dividing FtsZ ring. We also analyzed FtsZ localization patterns in transgenic plants in which plastid division was blocked by altered expression of the division site-determining factor AtMinD. Our results indicate that MinD and ARC6 act in opposite directions: ARC6 promotes and MinD inhibits FtsZ filament formation in the chloroplast.     

The molecular biology of plastid division in higher plants

Plastids are essential plant organelles vital for life on earth, responsible not only for photosynthesis but for many fundamental intermediary metabolic reactions. Plastids are not formed de novo but arise by binary fission from pre-existing plastids, and plastid division therefore represents an important process for the maintenance of appropriate plastid populations in plant cells. Plastid division comprises an elaborate pathway of co-ordinated events which include division machinery assembly at the division site, the constriction of envelope membranes, membrane fusion and, ultimately, the separation of the two new organelles. Because of their prokaryotic origin bacterial cell division has been successfully used as a paradigm for plastid division. This has resulted in the identification of the key plastid division components FtsZ, MinD, and MinE, as well as novel proteins with similarities to prokaryotic cell division proteins. Through a combination of approaches involving molecular genetics, cell biology, and biochemistry, it is now becoming clear that these proteins act in concert during plastid division, exhibiting both similarities and differences compared with their bacterial counterparts. Recent efforts in the cloning of the disrupted loci in several of the accumulation and replication of chloroplasts mutants has further revealed that the division of plastids is controlled by a combination of prokaryote-derived and host eukaryote-derived proteins residing not only in the plastid stroma but also in the cytoplasm. Based on the available data to date, a working model is presented showing the protein components involved in plastid division, their subcellular localization, and their protein interaction properties.