GIANT CHLOROPLAST 1 is essential for correct plastid division in Arabidopsis

Plastids are vital plant organelles involved in many essential biological processes. Plastids are not created de novo but divide by binary fission mediated by nuclear-encoded proteins of both prokaryotic and eukaryotic origin. Although several plastid division proteins have been identified in plants, limited information exists regarding possible division control mechanisms. Here, we describe the identification of GIANT CHLOROPLAST 1 (GC1), a new nuclear-encoded protein essential for correct plastid division in Arabidopsis. GC1 is plastid-localized and is anchored to the stromal surface of the chloroplast inner envelope by a C-terminal amphipathic helix. In Arabidopsis, GC1 deficiency results in mesophyll cells harbouring one to two giant chloroplasts, whilst GC1 overexpression has no effect on division. GC1 can form homodimers but does not show any interaction with the Arabidopsis plastid division proteins AtFtsZ1-1, AtFtsZ2-1, AtMinD1, or AtMinE1. Analysis reveals that GC1-deficient giant chloroplasts contain densely packed wild-type-like thylakoid membranes and that GC1-deficient leaves exhibit lower rates of CO(2) assimilation compared to wild-type. Although GC1 shows similarity to a putative cyanobacterial SulA cell division inhibitor, our findings suggest that GC1 does not act as a plastid division inhibitor but, rather, as a positive factor at an early stage of the division process.     

The plastid division protein AtMinD1 is a Ca2+-ATPase stimulated by AtMinE1

Bacteria and plastids divide symmetrically through binary fission by accurately placing the division site at midpoint, a process initiated by FtsZ polymerization, which forms a Z-ring. In Escherichia coli precise Z-ring placement at midcell depends on controlled oscillatory behavior of MinD and MinE: In the presence of ATP MinD interacts with the FtsZ inhibitor MinC and migrates to the membrane where the MinD-MinC complex recruits MinE, followed by MinD-mediated ATP hydrolysis and membrane release. Although correct Z-ring placement during Arabidopsis plastid division depends on the precise localization of the bacterial homologs AtMinD1 and AtMinE1, the underlying mechanism of this process remains unknown. Here we have shown that AtMinD1 is a Ca2+-dependent ATPase and through mutation analysis demonstrated the physiological importance of this activity where loss of ATP hydrolysis results in protein mislocalization within plastids. The observed mislocalization is not due to disrupted AtMinD1 dimerization, however; the active site AtMinD1(K72A) mutant is unable to interact with the topological specificity factor AtMinE1. We have shown that AtMinE1, but not E. coli MinE, stimulates AtMinD1-mediated ATP hydrolysis, but in contrast to prokaryotes stimulation occurs in the absence of membrane lipids. Although AtMinD1 appears highly evolutionarily conserved, we found that important biochemical and cell biological properties have diverged. We propose that correct intraplastidic AtMinD1 localization is dependent on AtMinE1-stimulated, Ca2+-dependent AtMinD1 ATP hydrolysis, ultimately ensuring precise Z-ring placement and symmetric plastid division.     

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.     

In vivo phosphorylation of FtsZ2 in Arabidopsis thaliana

The tubulin-like FtsZ protein initiates assembly of the bacterial and plastid division machineries. In bacteria, phosphorylation of FtsZ impairs GTPase activity, polymerization and interactions with other division proteins. Using a proteomics approach, we have shown that AtFtsZ2 is phosphorylated in vivo in Arabidopsis and that PGK1 (phosphoglycerate kinase 1) interacts with AtFtsZ2 in planta, suggesting a possible role in FtsZ phosphorylation.     

The topological specificity factor AtMinE1 is essential for correct plastid division site placement in Arabidopsis

In plant cells, plastids divide by binary fission involving a complex pathway of events. Although there are clear similarities between bacterial and plastid division, limited information exists regarding the mechanism of plastid division in higher plants. Here we demonstrate that AtMinE1, an Arabidopsis homologue of the bacterial MinE topological specificity factor, is an essential integral component of the plastid division machinery. In prokaryotes MinE imparts topological specificity during cell division by blocking division apparatus assembly at sites other than midcell. We demonstrate that overexpression of AtMinE1 in E. coli results in loss of topological specificity and minicell formation suggesting evolutionary conservation of MinE mode of action. We further show that AtMinE1 can indeed act as a topological specificity factor during plastid division revealing that AtMinE1 overexpression in Arabidopsis seedlings results in division site misplacement giving rise to multiple constrictions along the length of plastids. In agreement with cell division studies in bacteria, AtMinE1 and AtMinD1 show distinct intraplastidic localisation patterns suggestive of dynamic localisation behaviour. Taken together our findings demonstrate that AtMinE1 is an evolutionary conserved topological specificity factor, most probably acting in concert with AtMinD1, required for correct plastid division in Arabidopsis.     

An Arabidopsis homolog of the bacterial cell division inhibitor SulA is involved in plastid division

Plastids have evolved from an endosymbiosis between a cyanobacterial symbiont and a eukaryotic host cell. Their division is mediated both by proteins of the host cell and conserved bacterial division proteins. Here, we identified a new component of the plastid division machinery, Arabidopsis thaliana SulA. Disruption of its cyanobacterial homolog (SSulA) in Synechocystis and overexpression of an AtSulA-green fluorescent protein fusion in Arabidopsis demonstrate that these genes are involved in cell and plastid division, respectively. Overexpression of AtSulA inhibits plastid division in planta but rescues plastid division defects caused by overexpression of AtFtsZ1-1 and AtFtsZ2-1, demonstrating that its role in plastid division may involve an interaction with AtFtsZ1-1 and AtFtsZ2-1.     

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.     

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.     

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.     

Chloroplast targeting of chloroplast division FtsZ2 proteins in Arabidopsis

Plant nuclear genomes encode chloroplast division proteins homologous to the eubacterial cell division protein FtsZ. In higher plants, FtsZ genes constitute a small gene family that consists of two subgroups, FtsZ1 and FtsZ2. It was previously hypothesized that members of one family (FtsZ1) targeted chloroplasts, while members of the other family (FtsZ2) localized in the cytoplasm. We determined the full-length cDNA sequences of two FtsZ2 genes from Arabidopsis thaliana (AtFtsZ2-1 and AtFtsZ2-2) and found that the genes encode polypeptides of 478 and 473 amino acids, respectively, and both contain N-terminal extensions beyond what have previously been predicted. The N-terminal regions of both AtFtsZ2-1 and AtFtsZ2-2 were expressed as green fluorescent protein (GFP) fusions under the cauliflower mosaic virus 35S promoter in bombarded tobacco cells. Confocal laser scanning microscopy revealed both fusions exclusively localized to chloroplasts, demonstrating that the N-terminal regions function as chloroplast-targeting signals in vivo. Thus, FtsZ2 proteins function within chloroplasts.     

Chloroplast division in higher plants requires members of two functionally divergent gene families with homology to bacterial ftsZ.

The division of plastids is critical for viability in photosynthetic eukaryotes, but the mechanisms associated with this process are still poorly understood. We previously identified a nuclear gene from Arabidopsis encoding a chloroplast-localized homolog of the bacterial cell division protein FtsZ, an essential cytoskeletal component of the prokaryotic cell division apparatus. Here, we report the identification of a second nuclear-encoded FtsZ-type protein from Arabidopsis that does not contain a chloroplast targeting sequence or other obvious sorting signals and is not imported into isolated chloroplasts, which strongly suggests that it is localized in the cytosol. We further demonstrate using antisense technology that inhibiting expression of either Arabidopsis FtsZ gene (AtFtsZ1-1 or AtFtsZ2-1) in transgenic plants reduces the number of chloroplasts in mature leaf cells from 100 to one, indicating that both genes are essential for division of higher plant chloroplasts but that each plays a distinct role in the process. Analysis of currently available plant FtsZ sequences further suggests that two functionally divergent FtsZ gene families encoding differentially localized products participate in chloroplast division. Our results provide evidence that both chloroplastic and cytosolic forms of FtsZ are involved in chloroplast division in higher plants and imply that important differences exist between chloroplasts and prokaryotes with regard to the roles played by FtsZ proteins in the division process.     

The assembly of the FtsZ ring at the mid-chloroplast division site depends on a balance between the activities of AtMinE1 and ARC11/AtMinD1

Chloroplast division comprises a sequence of events that facilitatesymmetric binary fission and that involve prokaryotic-like stromaldivision factors such as tubulin-like GTPase FtsZ and the divisionsite regulator MinD. In Arabidopsis, a nuclear-encoded prokaryoticMinE homolog, AtMinE1, has been characterized in terms of itseffects on a dividing or terminal chloroplast state in a limitedseries of leaf tissues. However, the relationship between AtMinE1expression and chloroplast phenotype remains to be fully elucidated.Here, we demonstrate that a T-DNA insertion mutation in AtMinE1results in a severe inhibition of chloroplast division, producingmotile dots and short filaments of FtsZ. In AtMinE1 sense (overexpressor)plants, dividing chloroplasts possess either single or multipleFtsZ rings located at random intervals and showing constrictiondepth, mainly along the chloroplast polarity axis. The AtMinE1sense plants displayed equivalent chloroplast phenotypes toarc11, a loss-of-function mutant of AtMinD1 which forms replicatingmini-chloroplasts. Furthermore, a certain population of FtsZrings formed within developing chloroplasts failed to initiateor progress the membrane constriction of chloroplasts and consequentiallyto complete chloroplast fission in both AtMinE1 sense and arc11/atminD1plants. Our present data thus demonstrate that the chloroplastdivision site placement involves a balance between the opposingactivities of AtMinE1 and AtMinD1, which acts to prevent FtsZring formation anywhere outside of the mid-chloroplast. In addition,the imbalance caused by an AtMinE1 dominance causes multiple,non-synchronous division events at the single chloroplast level,as well as division arrest, which becomes apparent as the chloroplastsmature, in spite of the presence of FtsZ rings.     

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.     

Colocalization of Plastid Division Proteins in the Chloroplast Stromal Compartment Establishes a New Functional Relationship between FtsZ1 and FtsZ2 in Higher Plants

Chloroplast division is driven by a macromolecular complex containing components that are positioned on the cytosolic surface of the outer envelope, the stromal surface of the inner envelope, and in the intermembrane space. The only constituents of the division apparatus identified thus far are the tubulin-like proteins FtsZ1 and FtsZ2, which colocalize to rings at the plastid division site. However, the precise positioning of these rings relative to the envelope membranes and to each other has not been previously defined. Using newly isolated cDNAs with open reading frames longer than those reported previously, we demonstrate here that both FtsZ2 proteins in Arabidopsis, like FtsZ1 proteins, contain cleavable transit peptides that target them across the outer envelope membrane. To determine their topological arrangement, protease protection experiments designed to distinguish between stromal and intermembrane space localization were performed on both in vitro imported and endogenous forms of FtsZ1 and FtsZ2. Both proteins were shown to reside in the stromal compartment of the chloroplast, indicating that the FtsZ1- and FtsZ2-containing rings have similar topologies and may physically interact. Consistent with this hypothesis, double immunofluorescence labeling of various plastid division mutants revealed precise colocalization of FtsZ1 and FtsZ2, even when their levels and assembly patterns were perturbed. Overexpression of FtsZ2 in transgenic Arabidopsis inhibited plastid division in a dose-dependent manner, suggesting that the stoichiometry between FtsZ1 and FtsZ2 is an important aspect of their function. These studies raise new questions concerning the functional and evolutionary significance of two distinct but colocalized forms of FtsZ in plants and establish a revised framework within which to understand the molecular architecture of the plastid division apparatus in higher plants.     

Analysis of the chloroplast proteome in arc mutants and identification of novel protein components associated with FtsZ2

Chloroplasts are descendants of cyanobacteria and divide by binary fission. The number of chloroplasts is regulated in a cell type-specific manner to ensure that specialized cell types can perform their functions optimally. Several protein components of the chloroplast division apparatus have been identified in the past several years, but how this process is regulated in response to developmental status, environmental signals and stress is still unknown. To begin to address this we undertook a proteomic analysis of three accumulation and replication of chloroplasts mutants that show a spectrum of plastid division perturbations. We show that defects in the chloroplast division process results in changes in the abundance of proteins when compared to wild type, but that the profile of the native stromal and membrane complexes remains unchanged. Furthermore, by combining BN-PAGE with protein interaction assays we show that AtFtsZ2-1 and AtFtsZ2-2 assemble together with rpl12A and EF-Tu into a novel chloroplast membrane complex.     

Effects of mutations in Arabidopsis FtsZ1 on plastid division, FtsZ ring formation and positioning, and FtsZ filament morphology in vivo

In plants, chloroplast division FtsZ proteins have diverged into two families, FtsZ1 and FtsZ2. FtsZ1 is more divergent from its bacterial counterparts and lacks a C-terminal motif conserved in most other FtsZs. To begin investigating FtsZ1 structure-function relationships, we first identified a T-DNA insertion mutation in the single FtsZ1 gene in Arabidopsis thaliana, AtFtsZ1-1. Homozygotes null for FtsZ1, though impaired in chloroplast division, could be isolated and set seed normally, indicating that FtsZ1 is not essential for viability. We then mapped five additional atftsZ1-1 alleles onto an FtsZ1 structural model and characterized chloroplast morphologies, FtsZ protein levels and FtsZ filament morphologies in young and mature leaves of the corresponding mutants. atftsZ1-1(G267R), atftsZ1-1(R298Q) and atftsZ1-1(Delta404-433) exhibit reduced FtsZ1 accumulation but wild-type FtsZ2 levels. The semi-dominant atftsZ1-1(G267R) mutation caused the most severe phenotype, altering a conserved residue in the predicted T7 loop. atftsZ1-1(G267R) protein accumulates normally in young leaves but is not detected in rings or filaments. atftsZ1-1(R298Q) has midplastid FtsZ1-containing rings in young leaves, indicating that R298 is not critical for ring formation or positioning despite its conservation. atftsZ1-1(D159N) and atftsZ1-1(G366A) both have overly long, sometimes spiral-like FtsZ filaments, suggesting that FtsZ dynamics are altered in these mutants. However, atftsZ1-1(D159N) exhibits loss of proper midplastid FtsZ positioning while atftsZ1-1(G366A) does not. Finally, truncation of the FtsZ1 C-terminus in atftsZ1-1(Delta404-433) impairs chloroplast division somewhat but does not prevent midplastid Z ring formation. These alleles will facilitate understanding of how the in vitro biochemical properties of FtsZ1 are related to its in vivo function.     

FtsZ ring formation at the chloroplast division site in plants

Among the events that accompanied the evolution of chloroplasts from their endosymbiotic ancestors was the host cell recruitment of the prokaryotic cell division protein FtsZ to function in chloroplast division. FtsZ, a structural homologue of tubulin, mediates cell division in bacteria by assembling into a ring at the midcell division site. In higher plants, two nuclear-encoded forms of FtsZ, FtsZ1 and FtsZ2, play essential and functionally distinct roles in chloroplast division, but whether this involves ring formation at the division site has not been determined previously. Using immunofluorescence microscopy and expression of green fluorescent protein fusion proteins in Arabidopsis thaliana, we demonstrate here that FtsZ1 and FtsZ2 localize to coaligned rings at the chloroplast midpoint. Antibodies specific for recognition of FtsZ1 or FtsZ2 proteins in Arabidopsis also recognize related polypeptides and detect midplastid rings in pea and tobacco, suggesting that midplastid ring formation by FtsZ1 and FtsZ2 is universal among flowering plants. Perturbation in the level of either protein in transgenic plants is accompanied by plastid division defects and assembly of FtsZ1 and FtsZ2 into filaments and filament networks not observed in wild-type, suggesting that previously described FtsZ-containing cytoskeletal-like networks in chloroplasts may be artifacts of FtsZ overexpression.     

In vivo quantitative relationship between plastid division proteins FtsZ1 and FtsZ2 and identification of ARC6 and ARC3 in a native FtsZ complex

FtsZ1 and FtsZ2 are phylogenetically distinct homologues of the tubulin-like bacterial cell division protein FtsZ that play major roles in the initiation and progression of plastid division in plant cells. Both proteins are components of a mid-plastid ring, the Z-ring, which functions as a contractile ring on the stromal surface of the chloroplast IEM (inner envelope membrane). FtsZ1 and FtsZ2 have been shown to interact, but their in vivo biochemical properties are largely unknown. To gain insight into the in vivo biochemical relationship between FtsZ1 and FtsZ2, in the present study we investigated their molecular levels in wild-type Arabidopsis thaliana plants and endogenous interactions in Arabidopsis and pea. Quantitative immunoblotting and morphometric analysis showed that the average total FtsZ concentration in chloroplasts of 3-week-old Arabidopsis plants is comparable with that in Escherichia coli. FtsZ levels declined as plants matured, but the molar ratio between FtsZ1 and FtsZ2 remained constant at approx. 1:2, suggesting that this stoichiometry is regulated and functionally important. Density-gradient centrifugation, native gel electrophoresis, gel filtration and co-immunoprecipitation experiments showed that a portion of the FtsZ1 and FtsZ2 in Arabidopsis and pea chloroplasts is stably associated in a complex of approximately 200-245 kDa. This complex also contains the FtsZ2-interacting protein ARC6 (accumulation and replicatioin of chloroplasts 6), an IEM protein, and analysis of density-gradient fractions suggests the presence of the FtsZ1-interacting protein ARC3. Based on the mid-plastid localization of ARC6 and ARC3 and their postulated roles in promoting and inhibiting chloroplast FtsZ polymer formation respectively, we hypothesize that the FtsZ1-FtsZ2-ARC3-ARC6 complex represents an unpolymerized IEM-associated pool of FtsZ that contributes to the dynamic regulation of Z-ring assembly and remodelling at the plastid division site in vivo.     

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 .     

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.