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.     

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.     

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

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

The 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.     

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.     

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.     

Plastid division is mediated by combinatorial assembly of plastid division proteins

Plastids arise by division from pre-existing organelles, and with the recent characterization of several new components of plastid division our understanding of the division process in higher plants has improved dramatically. However, it is still not known how these different protein components act together during division. Here we analyse protein-protein interactions between all known stromal plastid division proteins. Using a combination of quantitative yeast two-hybrid assays, in planta co-localization studies, fluorescence resonance energy transfer and bimolecular fluorescence complementation assays we show that these proteins do not act in isolation but rather in protein complexes to govern appropriate plastid division. We have previously shown that AtMinD1 forms functional homodimers and we show here that in addition to homodimerization AtMinD1 also interacts with AtMinE1. Furthermore, AtMinE1 has the ability to homodimerize. We also demonstrate that proteins from both FtsZ families (AtFtsZ1-1 and AtFtsZ2-1) not only interact with themselves but also with each other, and we show that these interactions are not dependent on correct Z-ring formation. Further to this we demonstrate that ARC6 specifically interacts with the core domain of AtFtsZ2-1, but not with AtFtsZ1-1, providing in planta evidence for a functional difference between the two FtsZ protein families in plants. Our studies have enabled us to construct a meaningful intraplastidic protein-protein interaction map of all known stromal plastid division proteins in Arabidopsis.     

MULTIPLE FtsZ RING FORMATION AND REDUPLICATED CHLOROPLAST DNA IN NANNOCHLORIS BACILLARIS (CHLOROPHYTA,TREBOUXIOPHYCEAE) UNDER PHOSPHATE‐ENRICHED CULTURE1

We examined the effects of phosphate enrichment on chloroplasts of the unicellular green alga Nannochloris bacillaris Naumann. The doubling time of cells was similar in phosphate‐limited (no β‐glycerophosphate) and phosphate‐enriched (2 mM β‐glycerophosphate) media. The lengths of cells and chloroplasts were similar, regardless of phosphate concentration. The relationship between the ring formation of the prokaryote‐derived chloroplast division protein FtsZ and phosphate concentration was examined using indirect fluorescent antibody staining. The number of FtsZ rings increased as the phosphate concentration of the medium increased. Multiple FtsZ rings were formed in cells in phosphate‐enriched medium; up to six FtsZ rings per chloroplast were observed. The number of FtsZ rings increased as the chloroplast grew. The FtsZ ring located near the center of the chloroplast had the strongest fluorescence. The FtsZ ring at the relative center of all FtsZ rings was used for division. Plastid division rings did not multiply in phosphate‐enriched culture. The chloroplast DNA content was 2.3 times greater in phosphate‐enriched than in phosphate‐limited culture and decreased in cells cultured in phosphate‐enriched medium containing 5‐fluorodeoxyuridine (FdUr). In the presence of FdUr, only one FtsZ ring formed, even under phosphate enrichment. This finding suggests that excessive chloroplast DNA replication induces multiple FtsZ ring formation in phosphate‐enriched culture. We propose a multiple FtsZ ring formation model under phosphate enrichment.     

ROLE OF MULTIPLE FTSZ RINGS IN CHLOROPLAST DIVISION UNDER OLIGOTROPHIC AND EUTROPHIC CONDITIONS IN THE UNICELLULAR GREEN ALGA NANNOCHLORIS BACILLARIS (CHLOROPHYTA,TREBOUXIOPHYCEAE)1

Chloroplasts of the unicellular green alga Nannochloris bacillaris Naumann cultured under nutrient‐enriched conditions have multiple rings of FtsZ, a prokaryote‐derived chloroplast division protein. We previously reported that synthesis of excess chloroplast DNA and formation of multiple FtsZ rings occur simultaneously. To clarify the role of multiple FtsZ rings in chloroplast division, we investigated chloroplast DNA synthesis and ring formation in cells cultured under various culture conditions. Cells transferred from a nutrient‐enriched medium to an inorganic medium in the light showed a drop in cell division rate, a reduction in chloroplast DNA content, and changes in the shape of chloroplast nucleoids as cells divided. We then examined DNA synthesis by immunodetecting BrdU incorporated into DNA strands using the anti‐BrdU antibody. BrdU‐labeled nuclei were clearly observed in cells 48 h after transfer into the inorganic medium, while only weak punctate signals were visible in the chloroplasts. In parallel, the number of FtsZ rings decreased from 6 to only 1. When the cells were transferred from an inorganic medium to a nutrient‐enriched medium, the number of cells increased only slightly in the first 12 h after transfer; after this time, however, they started to divide more quickly and increased exponentially. Chloroplast nucleoids changed from punctate to rod‐like structures, and active chloroplast DNA synthesis and FtsZ ring formation were observed. On the basis of our results, we conclude that multiple FtsZ ring assembly and chloroplast DNA duplication under nutrient‐rich conditions facilitate chloroplast division after transfer to oligotrophic conditions without further duplication of chloroplast DNA and formation of new FtsZ rings.     

Differential Positioning of C4 Mesophyll and Bundle Sheath Chloroplasts: Recovery of Chloroplast Positioning Requires the Actomyosin System

In C₄ plants, bundle sheath (BS) chloroplasts are arranged inthe centripetal position or in the centrifugal position, althoughmesophyll (M) chloroplasts are evenly distributed along cellmembranes. To examine the molecular mechanism for the intracellulardisposition of these chloroplasts, we observed the distributionof actin filaments in BS and M cells of the C₄ plants fingermillet (Eleusine coracana) and maize (Zea mays) using immunofluorescence.Fine actin filaments encircled chloroplasts in both cell types,and an actin network was observed adjacent to plasma membranes.The intracellular disposition of both chloroplasts in fingermillet was disrupted by centrifugal force but recovered within2 h in the dark. Actin filaments remained associated with chloroplastsduring recovery. We also examined the effects of inhibitorson the rearrangement of chloroplasts. Inhibitors of actin polymerization,myosin-based activities and cytosolic protein synthesis blockedmigration of chloroplasts. In contrast, a microtubule-depolymerizingdrug had no effect. These results show that C₄ plants possessa mechanism for keeping chloroplasts in the home position whichis dependent on the actomyosin system and cytosolic proteinsynthesis but not tubulin or light.     

Overexpression of the Arabidopsis thaliana MinD1 gene alters chloroplast size and number in transgenic tobacco plants

The Arabidopsis thaliana (L.) Heynh. minD gene (AtMinD1) was isolated and constitutively expressed in tobacco (Nicotiana tabacum L.) plants using the CaMV 35S promoter. Confocal and electron-microscopic analysis of the AtMinD1 transgenic tobacco lines revealed that the chloroplasts were abnormally large and fewer in number compared with wild-type tobacco plants. The abnormal chloroplasts were less prevalent in guard cells than in mesophyll cells. Chloroplast and nuclear gene expression was not significantly different in AtMinD1-overexpressing plants relative to wild-type tobacco plants. Chloroplast DNA copy number was not affected, based on the relative level of the rbcL gene in transgenic plants. Transgenic tobacco plants constitutively overexpressing AtMinD1 were completely normal phenotypically with respect to growth and development, and also displayed normal photosynthetic electron transport rates. These results show that the Arabidopsis MinD1 gene also functions in a heterologous system and confirm the role of the MinD protein in regulation of chloroplast division.     

烟草NtFtsZ2-1基因参与叶绿体分裂和扩大的调节

叶绿体虽然是植物细胞内一种极其重要的细胞器,但其分裂的分子机制尚不很清楚。已经证明FtsZ蛋白作为真核细胞分裂装置的一个关键成分,参与叶绿体的分裂过程。烟草的FtsZ基因属于2个不同的家族,在对NtFtsZ1家族成员研究的基础上,用正义和反义表达技术研究了NtFtsZ2家族成员NtFtsZ2-1基因在转基因烟草中的功能。显微分析结果表明NtFtsZ2-1基因的表达水平异常增强或减弱都会严重干扰叶绿体的正常分裂过程,导致叶绿体在形态和数目上的异常（体积明显增大,数目显著减少）,而单个叶肉细胞中叶绿体的总表面积在正反义转基因烟草和野生型烟草之间保持了相对稳定,没有发生明显的变化。同时还证明NtFtsZ2-1基因表达的变化对叶绿素含量和叶绿体的光合作用能力没有直接的影响。据此我们认为NtFtsZ2-1基因参与叶绿体的分裂和体积的扩大,其表达水平的波动会改变植物中叶绿体的数目和大小,而且在叶绿体的数目与体积之间可能存在一种补偿机制,保证叶绿体能最大限度地吸收光能,从而使光合作用得以正常进行。     

FtsHi1/ARC1 is an essential gene in Arabidopsis that links chloroplast biogenesis and division

The Arabidopsis arc1 (accumulation and replication of chloroplasts 1) mutant has pale seedlings and smaller, more numerous chloroplasts than the wild type. Previous work has suggested that arc1 affects the timing of chloroplast division but does not function directly in the division process. We isolated ARC1 by map‐based cloning and discovered it encodes FtsHi1 (At4g23940), one of several FtsHi proteins in Arabidopsis. These poorly studied proteins resemble FtsH metalloproteases important for organelle biogenesis and protein quality control but are presumed to be proteolytically inactive. FtsHi1 bears a predicted chloroplast transit peptide and localizes to the chloroplast envelope membrane. Phenotypic studies showed that arc1 (hereafter ftsHi1‐1), which bears a missense mutation, is a weak allele of FtsHi1 that disrupts thylakoid development and reduces de‐etiolation efficiency in seedlings, suggesting that FtsHi1 is important for chloroplast biogenesis. Consistent with this finding, transgenic plants suppressed for accumulation of an FtsHi1 fusion protein were often variegated. A strong T‐DNA insertion allele, ftsHi1‐2, caused embryo‐lethality, indicating that FtsHi1 is an essential gene product. A wild‐type FtsHi1 transgene rescued both the chloroplast division and pale phenotypes of ftsHi1‐1 and the embryo‐lethal phenotype of ftsHi1‐2. FtsHi1 overexpression produced a subtle increase in chloroplast size and decrease in chloroplast number in wild‐type plants while suppression led to increased numbers of small chloroplasts, providing new evidence that FtsHi1 negatively influences chloroplast division. Taken together, our analyses reveal that FtsHi1 functions in an essential, envelope‐associated process that may couple plastid development with division.     

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.     

A Novel Nuclear-Encoded Protein, NDH-Dependent Cyclic Electron Flow 5, is Essential for the Accumulation of Chloroplast NAD(P)H Dehydrogenase Complexes

The chloroplast NAD(P)H dehydrogenase (NDH) complex, which reducesplastoquinones in thylakoid membranes, is involved in PSI cyclicelectron flow and chlororespiration. In addition to land plants,the NDH complex is conserved in cyanobacteria. In this study,we identified a novel NDH-related gene of Arabidopsis, NDH-dependentcyclic electron flow 5 (NDF5, At1g55370). Post-illuminationincreases in chlorophyll fluorescence were absent in ndf5 mutantplants, which indicated that NDF5 is essential for NDH activity.Sequence analysis did not reveal any known functional motifsin NDF5, but there was some homology in amino acid sequencebetween NDF5 and NDF2, a known NDH subunit. NDF5 and NDF2 homologswere present in higher plants, but not cyanobacteria. A singlehomolog, which had similarity to both NDF5 and NDF2, was identifiedin the moss Physcomitrella patens. Immunoblot analysis showedthat NDF5 localizes to membrane fractions of chloroplasts. Thestability of NdhH, a subunit of the NDH complex, as well asNDF5 and NDF2, was decreased in ndf5, ndf2 and double ndf2/ndf5mutants, resulting in a loss of NDH activity in these mutants.These results indicated that both NDF5 and NDF2 have essentialfunctions in the stabilization of the NDH complex. We proposethat NDF5 and NDF2 were acquired by land plants during evolution,and that in higher plants both NDF5 and NDF2 are critical toregulate NDH activity and each other's protein stability, aswell as the stability of additional NDH subunits.     

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.     

A Chloroplast Protein Homologous to the Eubacterial Topological Specificity Factor MinE Plays a Role in Chloroplast Division

We report the identification of a nucleus-encoded minE gene, designated AtMinE1, of Arabidopsis. The encoded AtMinE1 protein possesses both N- and C-terminal extensions, relative to the eubacterial and algal chloroplast-encoded MinE proteins. The N-terminal extension functioned as a chloroplast-targeting transit peptide, as revealed by a transient expression assay using an N terminus:green fluorescent protein fusion. Histochemical beta-glucuronidase staining of transgenic Arabidopsis lines harboring an AtMinE1 promoter::uidA reporter fusion unveiled specific activation of the promoter in green tissues, especially at the shoot apex, which suggests a requirement for cell division-associated AtMinE1 expression for proplastid division in green tissues. In addition, we generated transgenic plants overexpressing a full-length AtMinE1 cDNA and examined the subcellular structures of those plants. Giant heteromorphic chloroplasts were observed in transgenic plants, with a reduced number per cell, whereas mitochondrial morphology remained similar to that of wild-type plants. Taken together, these observations suggest that MinE is the third conserved component involved in chloroplast division.