Oligomerization of plant FtsZ1 and FtsZ2 plastid division proteins

FtsZ was identified in bacteria as the first protein to localize mid-cell prior to division and homologs have been found in many plant species. Bacterial studies demonstrated that FtsZ forms a ring structure that is dynamically exchanged with a soluble pool of FtsZ. Our previous work established that Arabidopsis FtsZ1 and FtsZ2-1 are capable of in vitro self-assembly into two distinct filament types, termed type-I and type-II and noted the presence of filament precursor molecules which prompted this investigation. Using a combination of electron microscopy, gel chromatography and native PAGE revealed that (i) prior to FtsZ assembly initiation the pool consists solely of dimers and (ii) during assembly of the Arabidopsis FtsZ type-II filaments the most common intermediate between the dimer and filament state is a tetramer. Three-dimensional reconstructions of the observed dimer and tetramer suggest these oligomeric forms may represent consecutive steps in type-II filament assembly and a mechanism is proposed, which is expanded to include FtsZ assembly into type-I filaments. Finally, the results permit a discussion of the oligomeric nature of the soluble pool in plants.     

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.     

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.     

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.     

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.     

Crystal structure of a conserved domain in the intermembrane space region of the plastid division protein ARC6

The chloroplast division machinery is composed of numerous proteins that assemble as a large complex to divide double‐membraned chloroplasts through binary fission. A key mediator of division‐complex formation is ARC6, a chloroplast inner envelope protein and evolutionary descendant of the cyanobacterial cell division protein Ftn2. ARC6 connects stromal and cytosolic contractile rings across the two membranes through interaction with an outer envelope protein within the intermembrane space (IMS). The ARC6 IMS region bears a structurally uncharacterized domain of unknown function, DUF4101, that is highly conserved among ARC6 and Ftn2 proteins. Here we report the crystal structure of this domain from Arabidopsis thaliana ARC6. The domain forms an α/β barrel open towards the outer envelope membrane but closed towards the inner envelope membrane. These findings provide new clues into how ARC6 and its homologs contribute to chloroplast and cyanobacterial cell division.     

Isolation of two plastid division ftsZ genes from Chlamydomonas reinhardtii and its evolutionary implication for the role of FtsZ in plastid division

In order to elucidate the origin of the plastid division gene ftsZ in green plant lineage, and to understand the significance of this divergence for the function of FtsZ proteins in plants, two full-length cDNAs (accession numbers AF449446 and AB084236) were isolated from Chlamydomonas reinhardtii, a base species of green plant lineage. A phylogenetic analysis based on amino acid sequences of eukaryotic FtsZs reveals that an ancient duplication of the ftsZ gene occurred after the endosymbiotic event. The ancient duplication implies that two ftsZ families might play an indispensable role at the early endosymbiotic stage.     

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.     

PDV1 and PDV2 mediate recruitment of the dynamin-related protein ARC5 to the plastid division site

During plastid division, the dynamin-related protein ACCUMULATION AND REPLICATION OF CHLOROPLASTS5 (ARC5) is recruited from the cytosol to the surface of the outer chloroplast envelope membrane. In Arabidopsis thaliana arc5 mutants, chloroplasts arrest during division site constriction. Analysis of mutants similar to arc5 along with map-based cloning identified PLASTID DIVISION1 (PDV1), an integral outer envelope membrane protein, and its homolog PDV2 as components of the plastid division machinery. Similar to ARC5, PDV1 localized to a discontinuous ring at the division site in wild-type plants. The midplastid PDV1 ring formed in arc5 mutants and the ARC5 ring formed in pdv1 and pdv2 mutants, but not in pdv1 pdv2. Stromal FtsZ ring assembly occurred in pdv1, pdv2, and pdv1 pdv2, as it does in arc5. Topological analysis showed that the large N-terminal region of PDV1 upstream of the transmembrane helix bearing a putative coiled-coil domain is exposed to the cytosol. Mutation of the conserved PDV1 C-terminal Gly residue did not block PDV1 insertion into the outer envelope membrane but did abolish its localization to the division site. Our results indicate that plastid division involves the stepwise localization of FtsZ, PDV1, and ARC5 at the division site and that PDV1 and PDV2 together mediate the recruitment of ARC5 to the midplastid constriction at a late stage of 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.     

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.     

ARC3, a chloroplast division factor, is a chimera of prokaryotic FtsZ and part of eukaryotic phosphatidylinositol-4-phosphate 5-kinase

The arc3 (accumulation and replication of chloroplast) mutant of Arabidopsis thaliana has a small number of abnormally large chloroplasts in the cell, suggesting that chloroplast division is arrested in the mutant and ARC3 has an important role in the initiation of chloroplast division. To elucidate the role of ARC3, first we identified the ARC3 gene, and determined the location of ARC3 protein during chloroplast division because the localization and spatial orientation of such division factors are vital for correct chloroplast division. Sequencing analysis showed that ARC3 was a fusion of the prokaryotic FtsZ and part of the eukaryotic phosphatidylinositol-4-phosphate 5-kinase (PIP5K) genes. The PIP5K-homologous region of ARC3 had no catalytic domain but a membrane-occupation-and-recognition-nexus (MORN) repeat motif. Immunofluorescence microscopy, Western blotting analysis and in vitro chloroplast import and protease protection assays revealed that ARC3 protein was soluble, and located on the outer surface of the chloroplast in a ring-like structure at the early stage of chloroplast division. Prokaryotes have one FtsZ as a gene for division but have no ARC3 counterparts, the chimera of FtsZ and PIP5K, suggesting that the ARC3 gene might have been generated from FtsZ as another division factor during the evolution of chloroplast by endosymbiosis.     

The structure of FtsZ filaments in vivo suggests a force-generating role in cell division

In prokaryotes, FtsZ (the filamentous temperature sensitive protein Z) is a nearly ubiquitous GTPase that localizes in a ring at the leading edge of constricting plasma membranes during cell division. Here we report electron cryotomographic reconstructions of dividing Caulobacter crescentus cells wherein individual arc-like filaments were resolved just underneath the inner membrane at constriction sites. The filaments' position, orientation, time of appearance, and resistance to A22 all suggested that they were FtsZ. Predictable changes in the number, length, and distribution of filaments in cells where the expression levels and stability of FtsZ were altered supported that conclusion. In contrast to the thick, closed-ring-like structure suggested by fluorescence light microscopy, throughout the constriction process the Z-ring was seen here to consist of just a few short (approximately 100 nm) filaments spaced erratically near the division site. Additional densities connecting filaments to the cell wall, occasional straight segments, and abrupt kinks were also seen. An 'iterative pinching' model is proposed wherein FtsZ itself generates the force that constricts the membrane in a GTP-hydrolysis-driven cycle of polymerization, membrane attachment, conformational change, depolymerization, and nucleotide exchange.     

Conserved relationship between FtsZ and peptidoglycan in the cyanelles of Cyanophora paradoxa similar to that in bacterial cell division

Cyanelles of the biflagellate protist Cyanophora paradoxa have retained the peptidoglycan layer, which is critical for division, as indicated by the inhibitory effects of β-lactam antibiotics. An FtsZ ring is formed at the division site during cyanelle division. We used immunofluorescence microscopy to observe the process of FtsZ ring formation, which is expected to lead cyanelle division, and demonstrated that an FtsZ arc and a split FtsZ ring emerge during the early and late stages of cyanelle division, respectively. We used an anti-FtsZ antibody to observe cyanelle FtsZ rings. We observed bright, ring-shaped fluorescence of FtsZ in cyanelles. Cyanelles were kidney-shaped shortly after division. Fluorescence indicated that FtsZ did not surround the division plane at an early stage of division, but rather formed an FtsZ arc localized at the constriction site. The constriction spread around the cyanelle, which gradually became dumbbell shaped. After the envelope’s invagination, the ring split parallel to the cyanelle division plane without disappearing. Treatment of C. paradoxa cells with ampicillin, a β-lactam antibiotic, resulted in spherical cyanelles with an FtsZ arc or ring on the division plane. Transmission electron microscopy of the ampicillin-treated cyanelle envelope membrane revealed that the surface was not smooth. Thus, the inhibition of peptidoglycan synthesis by ampicillin causes the inhibition of septum formation and a marked delay in constriction development. The formation of the FtsZ arc and FtsZ ring is the earliest sign of cyanelle division, followed by constriction and septum formation.     

PARC6, a novel chloroplast division factor, influences FtsZ assembly and is required for recruitment of PDV1 during chloroplast division in Arabidopsis

Chloroplast division in plant cells is accomplished through the coordinated action of the tubulin-like FtsZ ring inside the organelle and the dynamin-like ARC5 ring outside the organelle. This coordination is facilitated by ARC6, an inner envelope protein required for both assembly of FtsZ and recruitment of ARC5. Recently, we showed that ARC6 specifies the mid-plastid positioning of the outer envelope proteins PDV1 and PDV2, which have parallel functions in dynamin recruitment. PDV2 positioning involves direct ARC6–PDV2 interaction, but PDV1 and ARC6 do not interact indicating that an additional factor functions downstream of ARC6 to position PDV1. Here, we show that PARC6 (paralog of ARC6), an ARC6-like protein unique to vascular plants, fulfills this role. Like ARC6, PARC6 is an inner envelope protein with its N-terminus exposed to the stroma and Arabidopsis parc6 mutants exhibit defects of chloroplast and FtsZ filament morphology. However, whereas ARC6 promotes FtsZ assembly, PARC6 appears to inhibit FtsZ assembly, suggesting that ARC6 and PARC6 function as antagonistic regulators of FtsZ dynamics. The FtsZ inhibitory activity of PARC6 may involve its interaction with the FtsZ-positioning factor ARC3. A PARC6–GFP fusion protein localizes both to the mid-plastid and to a single spot at one pole, reminiscent of the localization of ARC3, PDV1 and ARC5. Although PARC6 localizes PDV1, it is not required for PDV2 localization or ARC5 recruitment. Our findings indicate that PARC6, like ARC6, plays a role in coordinating the internal and external components of the chloroplast division complex, but that PARC6 has evolved distinct functions in the division process.     

Chloroplast Division Protein ARC3 Regulates Chloroplast FtsZ-Ring Assembly and Positioning in Arabidopsis through Interaction with FtsZ2

Chloroplast division is initiated by assembly of a mid-chloroplast FtsZ (Z) ring comprising two cytoskeletal proteins, FtsZ1 and FtsZ2. The division-site regulators ACCUMULATION AND REPLICATION OF CHLOROPLASTS3 (ARC3), MinD1, and MinE1 restrict division to the mid-plastid, but their roles are poorly understood. Using genetic analyses in Arabidopsis thaliana, we show that ARC3 mediates division-site placement by inhibiting Z-ring assembly, and MinD1 and MinE1 function through ARC3. ftsZ1 null mutants exhibited some mid-plastid FtsZ2 rings and constrictions, whereas neither constrictions nor FtsZ1 rings were observed in mutants lacking FtsZ2, suggesting FtsZ2 is the primary determinant of Z-ring assembly in vivo. arc3 ftsZ1 double mutants exhibited multiple parallel but no mid-plastid FtsZ2 rings, resembling the Z-ring phenotype in arc3 single mutants and showing that ARC3 affects positioning of FtsZ2 rings as well as Z rings. ARC3 overexpression in the wild type and ftsZ1 inhibited Z-ring and FtsZ2-ring assembly, respectively. Consistent with its effects in vivo, ARC3 interacted with FtsZ2 in two-hybrid assays and inhibited FtsZ2 assembly in a heterologous system. Our studies are consistent with a model wherein ARC3 directly inhibits Z-ring assembly in vivo primarily through interaction with FtsZ2 in heteropolymers and suggest that ARC3 activity is spatially regulated by MinD1 and MinE1 to permit Z-ring assembly at the mid-plastid.     

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 Ca²⁺.     

Bcr and Raf form a complex in vivo via 14-3-3 proteins.

In a yeast two-hybrid screen we identified a member of the 14-3-3 family of proteins that can bind to Bcr. 14-3-3 beta binds to the serine/threonine rich region B in the kinase domain encoded by the first exon. In this paper we show by co-immunoprecipitation that Bcr binds to Raf in vivo and we argue that this interaction is mediated by 14-3-3 dimers, based on the following findings. First, 14-3-3 isoforms bind to both Raf and Bcr. Second, Bcr does not bind to Raf directly in the two-hybrid system, but co-expression of 14-3-3 beta allows complex formation. Third, Bcr, 14-3-3 proteins and Raf co-elute in gel filtration and in sequential ion exchange chromatography and the three proteins can be co-immunoprecipitated from the the separate fractions, indicating that they are present in a ternary complex. Moreover, approximately 10 times more Raf is bound to Bcr, and vice versa, in the membrane fraction (where Raf is activated) than in the cytosolic fraction. We suggest a new function for 14-3-3 proteins as a novel type of new function for 14-3-3 proteins as a novel type of adaptor which acts by dimerization and binding to different proteins.     

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.