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.     

Triple immunofluorescent labeling of FtsZ, dynamin, and EF-Tu reveals a loose association between the inner and outer membrane mitochondrial division machinery in the red alga Cyanidioschyzon merolae.

In the mitochondria of primitive eukaryotes, FtsZ and dynamin are part of the machinery involved in division of the inner and outer membranes, respectively. These genes also commonly function in the same manner during chloroplast division. In this study, a relationship between the localization of the inner and outer division machinery was directly shown for the first time. Triple immunofluorescent labeling was performed in the red alga Cyanidioschyzon merolae by a device using narrow bandpass filter sets and bright photostable dyes. FtsZ (CmFtsZ1) and dynamin (CmDnm1) localizations were examined simultaneously throughout the mitochondrial division cycle with an alternative mitochondrial marker protein, the mitochondrial translation elongation factor EF-Tu, whose localization was also shown to be identical to the mitochondrial matrix. FtsZ and dynamin did not necessarily co-localize when both were recruited to the mitochondrial constriction site, indicating that inner and outer dividing machineries are not in tight association during the late stage of division.     

An evolutionary puzzle: chloroplast and mitochondrial division rings

Consistent with their bacterial origin, chloroplasts and primitive mitochondria retain a FtsZ ring for division. However, chloroplasts and mitochondria have lost most of the proteins required for bacterial division other than FtsZ and certain homologues of the Min proteins, but they do contain plastid and mitochondrion dividing rings, which were recently shown to be distinct from the FtsZ ring. Moreover, recent studies have revealed that rings of the eukaryote-specific dynamin-related family of GTPases regulate the division of chloroplasts and mitochondria, and these proteins emerged early in eukaryotic evolution. These findings suggest that the division of chloroplasts and primitive mitochondria involve very similar systems, consisting of an amalgamation of rings from bacteria and eukaryotes.     

Origin and evolution of the chloroplast division machinery

Chloroplasts were originally established in eukaryotes by the endosymbiosis of a cyanobacterium; they then spread through diversification of the eukaryotic hosts and subsequent engulfment of eukaryotic algae by previously nonphotosynthetic eukaryotes. The continuity of chloroplasts is maintained by division of preexisting chloroplasts. Like their ancestors, chloroplasts use a bacterial division system based on the FtsZ ring and some associated factors, all of which are now encoded in the host nuclear genome. The majority of bacterial division factors are absent from chloroplasts and several new factors have been added by the eukaryotic host. For example, the ftsZ gene has been duplicated and modified, plastid-dividing (PD) rings were most likely added by the eukaryotic host, and a member of the dynamin family of proteins evolved to regulate chloroplast division. The identification of several additional proteins involved in the division process, along with data from diverse lineages of organisms, our current knowledge of mitochondrial division, and the mining of genomic sequence data have enabled us to begin to understand the universality and evolution of the division system. The principal features of the chloroplast division system thus far identified are conserved across several lineages, including those with secondary chloroplasts, and may reflect primeval features of mitochondrial division. Shin-ya Miyagishima is the recipient of the Botanical Society Award for Young Scientists, 2004.     

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.     

Diverse eukaryotes have retained mitochondrial homologues of the bacterial division protein FtsZ

Mitochondrial fission requires the division of both the inner and outer mitochondrial membranes. Dynamin-related proteins operate in division of the outer membrane of probably all mitochondria, and also that of chloroplasts--organelles that have a bacterial origin like mitochondria. How the inner mitochondrial membrane divides is less well established. Homologues of the major bacterial division protein, FtsZ, are known to reside inside mitochondria of the chromophyte alga Mallomonas, a red alga, and the slime mould Dictyostelium discoideum, where these proteins are likely to act in division of the organelle. Mitochondrial FtsZ is, however, absent from the genomes of higher eukaryotes (animals, fungi, and plants), even though FtsZs are known to be essential for the division of probably all chloroplasts. To begin to understand why higher eukaryotes have lost mitochondrial FtsZ, we have sampled various diverse protists to determine which groups have retained the gene. Database searches and degenerate PCR uncovered genes for likely mitochondrial FtsZs from the glaucocystophyte Cyanophora paradoxa, the oomycete Phytophthora infestans, two haptophyte algae, and two diatoms--one being Thalassiosira pseudonana, the draft genome of which is now available. From Thalassiosira we also identified two chloroplast FtsZs, one of which appears to be undergoing a C-terminal shortening that may be common to many organellar FtsZs. Our data indicate that many protists still employ the FtsZ-based ancestral mitochondrial division mechanism, and that mitochondrial FtsZ has been lost numerous times in the evolution of eukaryotes.     

Chloroplast division machinery as revealed by immunofluorescence and electron microscopy

The division of chloroplasts (plastids) is critical for the viability of photosynthetic eukaryotes. Previously we reported on the chloroplast division apparatus, which consists of inner and outer double or triple rings (PD rings). Chloroplasts are assumed to arise from bacterial endosymbionts, while bacterial division is instigated by a bacterial cytokinesis Z-ring protein (FtsZ). Here we present immunofluorescence and electron-microscopic evidence of chloroplast division via complex machinery involving the FtsZ and PD rings in the higher plant Pelargonium zonale Ait. Prior to invagination, the FtsZ protein was attached to a ring at the stromal division site. Following formation of the FtsZ ring, the inner stromal and outer cytosolic PD rings appeared, signifying the initiation of invagination. The FtsZ ring and the PD rings were found at the leading edge of chloroplast constriction throughout division. During chloroplast division, neither the FtsZ nor the inner rings changed width, but the volume of the outer ring gradually increased. We suggest that the FtsZ ring determines the division region, after which the inner and outer PD rings are formed as a lining for the FtsZ ring. With the outer ring providing the motivating force, the FtsZ and inner PD rings ultimately decompose to their base components.     

The primitive red algae Cyanidium caldarium and Cyanidioschyzon merolae as model system for investigating the dividing apparatus of mitochondria and plastids

The Cyanidiophyceae species Cyanidium caldarium and Cyanidioschyzon merolae have played important roles in showing the division mechanisms of mitochondria and plastids. The apparatus regulating mitochondrial and plastid divisions was formerly unknown. We first identified the division apparatus of plastids, called the plastid-dividing ring (PD ring), in C. caldarium and the division apparatus of mitochondria, called the mitochondrion-dividing ring (MD ring), in C. merolae. Eukaryotic cell division is therefore controlled by at least three dividing apparati (rings)—a contractile ring, an MD ring, and a PD ring—while bacterial division is controlled by a single bacterial contractile FtsZ ring. BioEssays 20 :344-354, 1998.© 1998 John Wiley & Sons, Inc.     

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.     

Structure, function and evolution of the mitochondrial division apparatus

Mitochondria are derived from free-living alpha-proteobacteria that were engulfed by eukaryotic host cells through the process of endosymbiosis, and therefore have their own DNA which is organized using basic proteins to form organelle nuclei (nucleoids). Mitochondria divide and are split amongst the daughter cells during cell proliferation. Their division can be separated into two main events: division of the mitochondrial nuclei and division of the matrix (the so-called mitochondrial division, or mitochondriokinesis). In this review, we first focus on the cytogenetical relationships between mitochondrial nuclear division and mitochondriokinesis. Mitochondriokinesis occurs after mitochondrial nuclear division, similar to bacterial cytokinesis. We then describe the fine structure and dynamics of the mitochondrial division ring (MD ring) as a basic morphological background for mitochondriokinesis. Electron microscopy studies first identified a small electron-dense MD ring in the cytoplasm at the constriction sites of dividing mitochondria in the slime mold Physarum polycephalum, and then two large MD rings (with outer cytoplasmic and inner matrix sides) in the red alga Cyanidioschyzon merolae. Now MD rings have been found in all eukaryotes. In the third section, we describe the relationships between the MD ring and the FtsZ ring descended from ancestral bacteria. Other than the GTPase, FtsZ, mitochondria have lost most of the proteins required for bacterial cytokinesis as a consequence of endosymbiosis. The FtsZ protein forms an electron transparent ring (FtsZ or Z ring) in the matrix inside the inner MD ring. For the fourth section, we describe the dynamic association between the outer MD ring with a ring composed of the eukaryote-specific GTPase dynamin. Recent studies have revealed that eukaryote-specific GTPase dynamins form an electron transparent ring between the outer membrane and the MD ring. Thus, mitochondriokinesis is thought to be controlled by a mitochondrial division (MD) apparatus including a dynamic trio, namely the FtsZ, MD and dynamin rings, which consist of a chimera of rings from bacteria and eukaryotes in primitive organisms. Since the genes for the MD ring and dynamin rings are not found in the prokaryotic genome, the host genomes may make these rings to actively control mitochondrial division. In the fifth part, we focus on the dynamic changes in the formation and disassembly of the FtsZ, MD and dynamin rings. FtsZ rings are digested during a later period of mitochondrial division and then finally the MD and dynamin ring apparatuses pinched off the daughter mitochondria, supporting the idea that the host genomes are responsible for the ultimate control of mitochondrial division. We discuss the evolution, from the original vesicle division (VD) apparatuses to VD apparatuses including classical dynamin rings and MD apparatuses. It is likely that the MD apparatuses involving the dynamic trio evolved into the plastid division (PD) apparatus in Bikonta, while in Opisthokonta, the MD apparatus was simplified during evolution and may have branched into the mitochondrial fusion apparatus. Finally, we describe the possibility of intact isolation of large MD/PD apparatuses, the identification of all their proteins and their related genes using C. merolae genome information and TOF-MS analyses. These results will assist in elucidating the universal mechanism and evolution of MD, PD and VD apparatuses.     

Mitochondrial division by an electron-dense ring inCyanidioschyzon merolae

Summary The first identification of a mitochondria-dividing ring (MD-ring), which is located in the cytoplasm near the outer envelope membrane at the constricted isthmus of dividing mitochondria in the red algaCyanidioschyzon merolae, is reported. The MD-ring is about 50 nm wide and 10 nm thick at early stage of mitochondrial constriction and is a somewhat electron-dense circular bundle. The MD-ring is believed to be essential for the division of mitochondrion (mitochondriokinesis) since the ring appears at the equatorial region of the mitochondria just before the initiation of mitochondrial division and can be observed throughout mitochondrial division. The MD-ring has features comparable to that of the plastid-dividing (PD) ring.Abbreviations MD mitochondria-dividing - PD plastid-dividing     

Organelle fission in eukaryotes

The cellular machineries that power chloroplast and mitochondrial division in eukaryotes carry out the topologically challenging job of constricting and severing these double-membraned organelles. Consistent with their endosymbiotic origins, mitochondria in protists and chloroplasts in photosynthetic eukaryotes have evolved organelle-targeted forms of FtsZ, the prokaryotic ancestor of tubulin, as key components of their fission complexes. In fungi, animals and plants, mitochondria no longer utilize FtsZ for division, but several mitochondrial division proteins that localize to the outer membrane and intermembrane space, including two related to the filament-forming dynamins, have been identified in yeast and animals. Although the reactions that mediate organelle division are not yet understood, recent progress in uncovering the constituents of the organelle division machineries promises rapid advancement in our understanding of the biochemical mechanisms underlying the distinct but related processes of chloroplast and mitochondrial division in eukaryotes.     

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.     

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.     

Two mechanosensitive channel homologs influence division ring placement in Arabidopsis chloroplasts

Chloroplasts must divide repeatedly to maintain their population during plant growth and development. A number of proteins required for chloroplast division have been identified, and the functional relationships between them are beginning to be elucidated. In both chloroplasts and bacteria, the future site of division is specified by placement of the Filamentous temperature sensitive Z (FtsZ) ring, and the Min system serves to restrict FtsZ ring formation to mid-chloroplast or mid-cell. How the Min system is regulated in response to environmental and developmental factors is largely unstudied. Here, we investigated the role in chloroplast division played by two Arabidopsis thaliana homologs of the bacterial mechanosensitive (MS) channel MscS: MscS-Like 2 (MSL2) and MSL3. Immunofluorescence microscopy and live imaging approaches demonstrated that msl2 msl3 double mutants have enlarged chloroplasts containing multiple FtsZ rings. Genetic analyses indicate that MSL2, MSL3, and components of the Min system function in the same pathway to regulate chloroplast size and FtsZ ring formation. In addition, an Escherichia coli strain lacking MS channels also showed aberrant FtsZ ring assembly. These results establish MS channels as components of the chloroplast division machinery and suggest that their role is evolutionarily conserved.     

A role for mechanosensitive channels in chloroplast and bacterial fission

The division site in both chloroplasts and bacteria is established by the medial placement of the FtsZ ring, a process that is in part regulated by the evolutionarily conserved components of the Min system. We recently showed that mechanosensitive ion channels influence FtsZ ring assembly in both Arabidopsis thaliana chloroplasts and in Escherichia coli; in chloroplasts they do so through the same genetic pathway as the Min system. Here we describe the effect of heterologous expression of the Arabidopsis MS channel homolog MSL2 on FtsZ ring placement in E. coli. We also discuss possible molecular mechanisms by which MS channels might influence chloroplast or bacterial division.     

Plastid division: evolution, mechanism and complexity

BACKGROUND: The continuity of chloroplasts is maintained by division of pre-existing chloroplasts. Chloroplasts originated as bacterial endosymbionts; however, the majority of bacterial division factors are absent from chloroplasts and the eukaryotic host has added several new components. For example, the ftsZ gene has been duplicated and modified, and the Min system has retained MinE and MinD but lost MinC, acquiring at least one new component ARC3. Further, the mechanism has evolved to include two members of the dynamin protein family, ARC5 and FZL, and plastid-dividing (PD) rings were most probably added by the eukaryotic host. SCOPE: Deciphering how the division of plastids is coordinated and controlled by nuclear-encoded factors is key to our understanding of this important biological process. Through a number of molecular-genetic and biochemical approaches, it is evident that FtsZ initiates plastid division where the coordinated action of MinD and MinE ensures correct FtsZ (Z)-ring placement. Although the classical FtsZ antagonist MinC does not exist in plants, ARC3 may fulfil this role. Together with other prokaryotic-derived proteins such as ARC6 and GC1 and key eukaryotic-derived proteins such as ARC5 and FZL, these proteins make up a sophisticated division machinery. The regulation of plastid division in a cellular context is largely unknown; however, recent microarray data shed light on this. Here the current understanding of the mechanism of chloroplast division in higher plants is reviewed with an emphasis on how recent findings are beginning to shape our understanding of the function and evolution of the components. CONCLUSIONS: Extrapolation from the mechanism of bacterial cell division provides valuable clues as to how the chloroplast division process is achieved in plant cells. However, it is becoming increasingly clear that the highly regulated mechanism of plastid division within the host cell has led to the evolution of features unique to the plastid division process.     

Visualization of an FtsZ ring in chloroplasts of Lilium longiflorum leaves.

FtsZ is a bacterial division protein which forms a ring at the leading edge of the cell division site. To date, a hypothesis that the plant FtsZ forms the same structure in chloroplast division is proposed, but has not been demonstrated yet. In this study, recombinant LlFtsZ (Lilium longiflorum FtsZ) protein was produced from a previously isolated ftsZ cDNA clone [Mori and Tanaka (2000) Protoplasma 214: 57] and used to raise polyclonal anti-LlFtsZ antibodies in rabbits. In immunoblot analysis with the total protein extracted from L. longiflorum leaves, purified antibodies specifically recognized LlFtsZ whose molecular mass was approximately 43 kDa. This size corresponded to that of the recombinant LlFtsZ protein lacking N-terminal sequence, which suggests that the full-length LlFtsZ translation product has a putative N-terminal signal peptide. Moreover, fluorescent and electron microscopy revealed that the anti-LlFtsZ antibodies recognized ring structures at stromal side of the constriction point of dividing chloroplasts. Here, we show direct evidence that FtsZ ring is involved in chloroplast division.     

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.     

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.