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.

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.

SUMMARYChloroplast 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 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.Evolved from prokaryotic endosymbionts (Martin and Herrmann, 1998;Gray, 1999;McFadden, 1999), plastids divide by binary fission, whereby a constriction forms at the division plane, progressively tightening to separate the new organelles. Although plastids differ structurally from their cyanobacterial ancestors (Douglas, 1998), the division processes in both share mechanistic similarities. In bacteria, the tubulin-like GTPase FtsZ assembles into a dynamic circular structure termed the Z-ring at the cell center, forming a cytoskeletal framework to which all other cell division proteins are recruited (for review, see Rothfield et al., 1999) and probably providing the force that powers the contractile machinery (Lu et al., 2000;Erickson, 2001). It is now established that homologs of FtsZ also are essential for chloroplast division in land plants (Osteryoung and Vierling, 1995;Osteryoung et al., 1998; Strepp et al., 1998). In vascular plants, plastid division entails the participation of two distinct, highly conserved nuclear-encoded FtsZ protein families, FtsZ1 and FtsZ2 (Osteryoung et al., 1998;Osteryoung and McAndrew, 2001). Immu...

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 first identified proteins of the chloroplast division machinery were homologs of the essential bacterial cell division protein FtsZ (Osteryoung and Vierling, 1995; Osteryoung et al., 1998;Strepp et al., 1998). In contrast with most bacterial genomes that contain only a single gene encoding FtsZ, the nuclear genome of Arabidopsis contains at least three FtsZ genes encoding members of two distinct protein families, FtsZ1 and FtsZ2. AtFtsZ1-1 and AtFtsZ2-1, members of the FtsZ1 and FtsZ2 families, respectively, have been shown to be essential for chloroplast division. When the level of either gene is diminished, chloroplast division is inhibited, yielding cells with as few as one very large chloroplast. One important difference between AtFtsZ1-1 and AtFtsZ2-1 is their predicted localization. AtFtsZ1-1 is targeted to the chloroplast, as is a closely related FtsZ protein from pea (Gaikwad et al., 2000), and is thought to be a component of a division ring that forms on the stromal side of the inner envelope membrane. AtFtsZ2-1, in contrast, lacks a chloroplast transit peptide, is not targeted to the chloroplast in vitro, and is hypothesized to be a constituent of a division ring that assembles on the cytoplasmic surface of the outer envelope membrane. The two rings together are postulated to coordinate chloroplast division (Osteryoung et ...

Chloroplasts are descendants of cyanobacteria and divide by binary fission. Several components of the division apparatus have been identified in the past several years and we are beginning to appreciate the plastid division process at a mechanistic level. In this review, we attempt to summarize the most recent developments in the field and assemble these observations into a working model of plastid division in plants. Plastids: Characteristics, Origins and DistributionWithout chloroplasts, plant and animal life would not exist. Plastids have been studied most extensively with regard to their role in photosynthesis, and are essential to the plant itself, being required for a select of amino acid and lipid metabolism, assimilation of nitrogen and sulfur into organic compounds, signaling in response to environmental cues and biosynthesis of plant hormones (1)(2)(3)(4)(5). Despite all we have learned over the past century regarding this organelle, we still know relatively little about its replication-which, superficially, seems a simple process of binary fission. Because plastid division and biogenesis are an integral part of plant growth and development, understanding of the division process is paramount to our understanding of plant and organelle biology.Plastids arose from an endosymbiotic relationship between a primitive biciliate protozoan and an ancient cyanobacterium that began 1.2-1.5 billion years ago (Figure 1) (6,7). Initially, each partner derived some mutual benefit: the cyanobacterium occupied and exploited an untapped protective niche and the host extracted reduced carbon and other nutrients from its new tenant. Time, selective pressure and mutation bring us to the present day where the chloroplast is a prisoner in its own home, having lost more than 90% of the gene content present in its free-living ancestor. Most of the plastid division genes identified to date are cyanobacterial in origin and encode division proteins localized inside the organelle. These are directed to the plastid post-translationally by N-terminal transit peptides that are cleaved upon import. A few components of the division complex were invented by the host after endosymbiosis and function on the surface of the organelle in contact with the cytosol; the mechanisms targeting these proteins to the chloroplast surface are unknown. In land plants, all known plastid division genes reside in the nucleus-none are found in the plastid genome-although in at least one unicellular green alga two plastid division genes remain associated with the plastid genome (8).The terms chloroplast and plastid are often used interchangeably, but in vascular plants chloroplasts are actually a subset of plastids, each of which is specialized for a given set of functions within a specific cell type. All plastids are surrounded by two envelope membranes and are derived by division from a population of proplastids within the meristematic (stem) cells of the plant. Beyond land plants, red algae, green algae and diatoms also contain plastids. Even apicomplex...

f Botryococcus braunii is a colonial green alga whose cells associate via a complex extracellular matrix (ECM) and produce prodigious amounts of liquid hydrocarbons that can be readily converted into conventional combustion engine fuels. We used quickfreeze deep-etch electron microscopy and biochemical/histochemical analysis to elucidate many new features of B. braunii cell/ colony organization and composition. Intracellular lipid bodies associate with the chloroplast and endoplasmic reticulum (ER) but show no evidence of being secreted. The ER displays striking fenestrations and forms a continuous subcortical system in direct contact with the cell membrane. The ECM has three distinct components. (i) Each cell is surrounded by a fibrous ␤-1, 4-and/or ␤-1, 3-glucan-containing cell wall. (ii) The intracolonial ECM space is filled with a cross-linked hydrocarbon network permeated with liquid hydrocarbons. (iii) Colonies are enclosed in a retaining wall festooned with a fibrillar sheath dominated by arabinose-galactose polysaccharides, which sequesters ECM liquid hydrocarbons. Each cell apex associates with the retaining wall and contributes to its synthesis. Retaining-wall domains also form "drapes" between cells, with some folding in on themselves and penetrating the hydrocarbon interior of a mother colony, partitioning it into daughter colonies. We propose that retaining-wall components are synthesized in the apical Golgi apparatus, delivered to apical ER fenestrations, and assembled on the surfaces of apical cell walls, where a proteinaceous granular layer apparently participates in fibril morphogenesis. We further propose that hydrocarbons are produced by the nonapical ER, directly delivered to the contiguous cell membrane, and pass across the nonapical cell wall into the hydrocarbon-based ECM.T he trebouxiophyte green fresh-to brackish-water alga Botryococcus braunii has a unique colonial organization: individual cells of the colony are embedded in an extracellular matrix (ECM) composed of polymerized and liquid hydrocarbons (LH) (29,35,(46)(47)(48). Although they may serve additional functions, these hydrocarbons notably allow B. braunii colonies to float, presumably to increase exposure to light for photosynthesis at the surfaces of ponds or lakes (4). The presence of B. braunii oils and ECM fossils in petroleum, coal deposits, and oil shale suggests that their hydrocarbon products once contributed to these reserves (1,2,8,10,16,17,34,36,52,62,63,69,70) and that these hydrocarbons could be used as a source of renewable energy. In fact, B. braunii hydrocarbons can be readily converted into petroleum equivalent, drop-in transportation fuels by conventional petroleum-processing techniques (22, 28).The three races of B. braunii are classified on the basis of the chemical nature of the liquid hydrocarbons they produce. The A race primarily produces alkadienes and alkatrienes derived from fatty acids (38,49,65,67); the B race (the focus of this study) produces two triterpenoids, tetramethylsqualene as a minor comp...

Holins are small phage-encoded proteins that accumulate harmlessly in the cytoplasmic membrane during the infection cycle until suddenly, at an allele-specific time, triggering to form lethal lesions, or "holes." In the phages λ and T4, the holes have been shown to be large enough to allow release of prefolded active endolysin from the cytoplasm, which results in destruction of the cell wall, followed by lysis within seconds. Here, the holes caused by S105, the λ-holin, have been captured in vivo by cryo-EM. Surprisingly, the scale of the holes is at least an order of magnitude greater than any previously described membrane channel, with an average diameter of 340 nm and some exceeding 1 μm. Most cells exhibit only one hole, randomly positioned in the membrane, irrespective of its size. Moreover, on coexpression of holin and endolysin, the degradation of the cell wall leads to spherically shaped cells and a collapsed inner membrane sac. To obtain a 3D view of the hole by cryo-electron tomography, we needed to reduce the average size of the cells significantly. By taking advantage of the coupling of bacterial cell size and growth rate, we achieved an 80% reduction in cell mass by shifting to succinate minimal medium for inductions of the S105 gene. Cryotomographic analysis of the holes revealed that they were irregular in shape and showed no evidence of membrane invagination. The unexpected scale of these holes has implications for models of holin function.bacteriophage | cryoelectron tomography | Escherichia coli | holin | lambda B acteriophage lysis, the most frequent cytolethal event in the biosphere, is a precisely scheduled process controlled by proteins of the holin family (1). Holins are an extremely diverse class of small phage-encoded membrane proteins (2). The best studied holin is S105, a 105-residue polypeptide with three transmembrane domains (TMDs) encoded by the S gene of phage λ (3). Throughout the period of late gene expression and particle assembly, S105 accumulates in the cytoplasmic membrane of Escherichia coli without any effect on its integrity (4). Suddenly, at a programmed time, S105 triggers to form a lesion, or hole, in the membrane; this allows the λ-endolysin, R, to escape from the cytoplasm and attack the cell wall (2). In phages of Gram-negative hosts, there is a third step to complete the lysis pathway involving a protein or protein complex, the spanin, which connects the cytoplasmic and outer membranes (5, 6). In λ, the spanin complex consists of the cytoplasmic membrane protein, Rz, and the outer membrane lipoprotein, Rz1. This complex is essential for lysis in media containing millimolar concentrations of divalent cations, and thus is thought to act by disrupting the outer membrane, possibly by fusion with the inner membrane (6).Although the S105 holin has been extensively studied using genetic and biochemical approaches (3, 4, 7-9), nothing is known about the membrane holes except that they are nonspecific and large enough to allow escape of fully folded tetrameric R-β-galactosi...