Principles of amyloplast replication in the ovule integuments of Arabidopsis thaliana.

Plastids in vascular plants have various differentiated forms, among which amyloplasts are crucial for starch storage and plant productivity. Despite the vast knowledge of the binary-fission mode of chloroplast division, our understanding of the replication of non-photosynthetic plastids, including amyloplasts, remains limited. Recent studies have suggested the involvement of stromules (stroma-filled tubules) in plastid replication when the division apparatus is faulty. However, details of the underlying mechanism(s) and their relevance to normal processes have yet to be elucidated. Here, we developed a live analysis system for studying amyloplast replication using Arabidopsis (Arabidopsis thaliana) ovule integuments. We showed the full sequence of amyloplast development and demonstrated that wild-type amyloplasts adopt three modes of replication, binary fission, multiple fission, and stromule-mediated fission, via multi-way placement of the FtsZ ring. The minE mutant, with severely inhibited chloroplast division, showed marked heterogeneity in amyloplast size, caused by size-dependent but wild-type modes of plastid fission. The dynamic properties of stromules distinguish the wild-type and minE phenotypes. In minE cells, extended stromules from giant amyloplasts acquired stability, allowing FtsZ ring assembly and constriction, as well as the growth of starch grains therein. Despite hyper-stromule formation, amyloplasts did not proliferate in the ftsZ null mutant. These data clarify the differences between amyloplast and chloroplast replication and demonstrate that the structural plasticity of amyloplasts underlies the multiplicity of their replication processes. Furthermore, this study shows that stromules can generate daughter plastids via assembly of the FtsZ ring.

Spatiotemporal Characteristics Determining the Multifaceted Nature of Reactive Oxygen, Nitrogen, and Sulfur Species in Relation to Proton Homeostasis.

Significance: Reactive oxygen species (ROS), reactive nitrogen species (RNS), and reactive sulfur species (RSS) act as signaling molecules, regulating gene expression, enzyme activity, and physiological responses. However, excessive amounts of these molecular species can lead to deleterious effects, causing cellular damage and death. This dual nature of ROS, RNS, and RSS presents an intriguing conundrum that calls for a new paradigm. Recent Advances: Recent advancements in the study of photosynthesis have offered significant insights at the molecular level and with high temporal resolution into how the photosystem II oxygen-evolving complex manages to prevent harmful ROS production during the water-splitting process. These findings suggest that a dynamic spatiotemporal arrangement of redox reactions, coupled with strict regulation of proton transfer, is crucial for minimizing unnecessary ROS formation. Critical Issues: To better understand the multifaceted nature of these reactive molecular species in biology, it is worth considering a more holistic view that combines ecological and evolutionary perspectives on ROS, RNS, and RSS. By integrating spatiotemporal perspectives into global, cellular, and biochemical events, we discuss local pH or proton availability as a critical determinant associated with the generation and action of ROS, RNS, and RSS in biological systems. Future Directions: The concept of localized proton availability will not only help explain the multifaceted nature of these ubiquitous simple molecules in diverse systems but also provide a basis for new therapeutic strategies to manage and manipulate these reactive species in neural disorders, pathogenic diseases, and antiaging efforts.

Total Synthesis, Absolute Configuration, and Phytotoxic Activity of Foeniculoxin.

Foeniculoxin is a major phytotoxin produced by Italian strains of Phomopsis foeniculi. The first total synthesis is described utilizing the ene reaction and Sonogashira cross-coupling reaction as key steps. The absolute configuration of the C6' was determined using chiral separation and an advanced Mosher's method. The phytotoxicity of the synthesized compound was demonstrated via syringe-based infiltration into Chenopodium album and Arabidopsis thaliana leaves. Synthetic foeniculoxin induced various defects in A. thaliana leaf cells before lesion formation, including protein leakage into the cytoplasm from both chloroplasts and mitochondria and mitochondrial rounding and swelling. Furthermore, foeniculoxin and the antibiotic hygromycin B caused similar agglomeration of mitochondria around chloroplasts, highlighting this event as a common component in the early stages of plant cell death.

Arabidopsis EGY1 Is Critical for Chloroplast Development in Leaf Epidermal Guard Cells.

In Arabidopsis thaliana, the Ethylene-dependent Gravitropism-deficient and Yellow-green 1 (EGY1) gene encodes a thylakoid membrane-localized protease involved in chloroplast development in leaf mesophyll cells. Recently, EGY1 was also found to be crucial for the maintenance of grana in mesophyll chloroplasts. To further explore the function of EGY1 in leaf tissues, we examined the phenotype of chloroplasts in the leaf epidermal guard cells and pavement cells of two 40Ar17+ irradiation-derived mutants, Ar50-33-pg1 and egy1-4. Fluorescence microscopy revealed that fully expanded leaves of both egy1 mutants showed severe chlorophyll deficiency in both epidermal cell types. Guard cells in the egy1 mutant exhibited permanent defects in chloroplast formation during leaf expansion. Labeling of plastids with CaMV35S or Protodermal Factor1 (PDF1) promoter-driven stroma-targeted fluorescent proteins revealed that egy1 guard cells contained the normal number of plastids, but with moderately reduced size, compared with wild-type guard cells. Transmission electron microscopy further revealed that the development of thylakoids was impaired in the plastids of egy1 mutant guard mother cells, guard cells, and pavement cells. Collectively, these observations demonstrate that EGY1 is involved in chloroplast formation in the leaf epidermis and is particularly critical for chloroplast differentiation in guard cells.

TGD5 is required for normal morphogenesis of non-mesophyll plastids, but not mesophyll chloroplasts, in Arabidopsis.

Stromules are dynamic membrane-bound tubular structures that emanate from plastids. Stromule formation is triggered in response to various stresses and during plant development, suggesting that stromules may have physiological and developmental roles in these processes. Despite the possible biological importance of stromules and their prevalence in green plants, their exact roles and formation mechanisms remain unclear. To explore these issues, we obtained Arabidopsis thaliana mutants with excess stromule formation in the leaf epidermis by microscopy-based screening. Here, we characterized one of these mutants, stromule biogenesis altered 1 (suba1). suba1 forms plastids with severely altered morphology in a variety of non-mesophyll tissues, such as leaf epidermis, hypocotyl epidermis, floral tissues, and pollen grains, but apparently normal leaf mesophyll chloroplasts. The suba1 mutation causes impaired chloroplast pigmentation and altered chloroplast ultrastructure in stomatal guard cells, as well as the aberrant accumulation of lipid droplets and their autophagic engulfment by the vacuole. The causal defective gene in suba1 is TRIGALACTOSYLDIACYLGLYCEROL5 (TGD5), which encodes a protein putatively involved in the endoplasmic reticulum (ER)-to-plastid lipid trafficking required for the ER pathway of thylakoid lipid assembly. These findings suggest that a non-mesophyll-specific mechanism maintains plastid morphology. The distinct mechanisms maintaining plastid morphology in mesophyll versus non-mesophyll plastids might be attributable, at least in part, to the differential contributions of the plastidial and ER pathways of lipid metabolism between mesophyll and non-mesophyll plastids.

Arabidopsis thaliana Leaf Epidermal Guard Cells: A Model for Studying Chloroplast Proliferation and Partitioning in Plants.

The existence of numerous chloroplasts in photosynthetic cells is a general feature of plants. Chloroplast biogenesis and inheritance involve two distinct mechanisms: proliferation of chloroplasts by binary fission and partitioning of chloroplasts into daughter cells during cell division. The mechanism of chloroplast number coordination in a given cell type is a fundamental question. Stomatal guard cells (GCs) in the plant shoot epidermis generally contain several to tens of chloroplasts per cell. Thus far, chloroplast number at the stomatal (GC pair) level has generally been used as a convenient marker for identifying hybrid species or estimating the ploidy level of a given plant tissue. Here, we report that Arabidopsis thaliana leaf GCs represent a useful system for investigating the unexploited aspects of chloroplast number control in plant cells. In contrast to a general notion based on analyses of leaf mesophyll chloroplasts, a small difference was detected in the GC chloroplast number among three Arabidopsis ecotypes (Columbia, Landsberg erecta, and Wassilewskija). Fluorescence microscopy often detected dividing GC chloroplasts with the FtsZ1 ring not only at the early stage of leaf expansion but also at the late stage. Compensatory chloroplast expansion, a phenomenon well documented in leaf mesophyll cells of chloroplast division mutants and transgenic plants, could take place between paired GCs in wild-type leaves. Furthermore, modest chloroplast number per GC as well as symmetric division of guard mother cells for GC formation suggests that Arabidopsis GCs would facilitate the analysis of chloroplast partitioning, based on chloroplast counting at the individual cell level.

Arabidopsis PARC6 Is Critical for Plastid Morphogenesis in Pavement, Trichome, and Guard Cells in Leaf Epidermis.

Recently, a recessive Arabidopsis thaliana mutant with abundant stromules in leaf epidermal pavement cells was visually screened and isolated. The gene responsible for this mutant phenotype was identified as PARC6, a chloroplast division site regulator gene. The mutant allele parc6-5 carried two point mutations (G62R and W700stop) at the N- and C-terminal ends of the coding sequence, respectively. Here, we further characterized parc6-5 and other parc6 mutant alleles, and showed that PARC6 plays a critical role in plastid morphogenesis in all cell types of the leaf epidermis: pavement cells, trichome cells, and guard cells. Transient expression of PARC6 transit peptide (TP) fused to the green fluorescent protein (GFP) in plant cells showed that the G62R mutation has no or little effect on the TP activity of the PARC6 N-terminal region. Then, plastid morphology was microscopically analyzed in the leaf epidermis of wild-type (WT) and parc6 mutants (parc6-1, parc6-3, parc6-4 and parc6-5) with the aid of stroma-targeted fluorescent proteins. In parc6 pavement cells, plastids often assumed aberrant grape-like morphology, similar to those in severe plastid division mutants, atminE1, and arc6. In parc6 trichome cells, plastids exhibited extreme grape-like aggregations, without the production of giant plastids (>6 µm diameter), as a general phenotype. In parc6 guard cells, plastids exhibited a variety of abnormal phenotypes, including reduced number, enlarged size, and activated stromules, similar to those in atminE1 and arc6 guard cells. Nevertheless, unlike atminE1 and arc6, parc6 exhibited a low number of mini-chloroplasts (< 2 µm diameter) and rarely produced chloroplast-deficient guard cells. Importantly, unlike parc6, the chloroplast division site mutant arc11 exhibited WT-like plastid phenotypes in trichome and guard cells. Finally, observation of parc6 complementation lines expressing a functional PARC6-GFP protein indicated that PARC6-GFP formed a ring-like structure in both constricting and non-constricting chloroplasts, and that PARC6 dynamically changes its configuration during the process of chloroplast division.

The Arabidopsis arc5 and arc6 mutations differentially affect plastid morphology in pavement and guard cells in the leaf epidermis.

Chloroplasts, or photosynthetic plastids, multiply by binary fission, forming a homogeneous population in plant cells. In Arabidopsis thaliana, the division apparatus (or division ring) of mesophyll chloroplasts includes an inner envelope transmembrane protein ARC6, a cytoplasmic dynamin-related protein ARC5 (DRP5B), and members of the FtsZ1 and FtsZ2 families of proteins, which co-assemble in the stromal mid-plastid division ring (FtsZ ring). FtsZ ring placement is controlled by several proteins, including a stromal factor MinE (AtMinE1). During leaf mesophyll development, ARC6 and AtMinE1 are necessary for FtsZ ring formation and thus plastid division initiation, while ARC5 is essential for a later stage of plastid division. Here, we examined plastid morphology in leaf epidermal pavement cells (PCs) and stomatal guard cells (GCs) in the arc5 and arc6 mutants using stroma-targeted fluorescent proteins. The arc5 PC plastids were generally a bit larger than those of the wild type, but most had normal shapes and were division-competent, unlike mutant mesophyll chloroplasts. The arc6 PC plastids were heterogeneous in size and shape, including the formation of giant and mini-plastids, plastids with highly developed stromules, and grape-like plastid clusters, which varied on a cell-by-cell basis. Moreover, unique plastid phenotypes for stomatal GCs were observed in both mutants. The arc5 GCs rarely lacked chlorophyll-bearing plastids (chloroplasts), while they accumulated minute chlorophyll-less plastids, whereas most GCs developed wild type-like chloroplasts. The arc6 GCs produced large chloroplasts and/or chlorophyll-less plastids, as previously observed, but unexpectedly, their chloroplasts/plastids exhibited marked morphological variations. We quantitatively analyzed plastid morphology and partitioning in paired GCs from wild-type, arc5, arc6, and atminE1 plants. Collectively, our results support the notion that ARC5 is dispensable in the process of equal division of epidermal plastids, and indicate that dysfunctions in ARC5 and ARC6 differentially affect plastid replication among mesophyll cells, PCs, and GCs within a single leaf.

Isolation and analysis of a stromule-overproducing Arabidopsis mutant suggest the role of PARC6 in plastid morphology maintenance in the leaf epidermis.

Stromules, or stroma-filled tubules, are thin extensions of the plastid envelope membrane that are most frequently observed in undifferentiated or non-mesophyll cells. The formation of stromules is developmentally regulated and responsive to biotic and abiotic stress; however, the physiological roles and molecular mechanisms of the stromule formation remain enigmatic. Accordingly, we attempted to obtain Arabidopsis thaliana mutants with aberrant stromule biogenesis in the leaf epidermis. Here, we characterize one of the obtained mutants. Plastids in the leaf epidermis of this mutant were giant and pleomorphic, typically having one or more constrictions that indicated arrested plastid division, and usually possessed one or more extremely long stromules, which indicated the deregulation of stromule formation. Genetic mapping, whole-genome resequencing-aided exome analysis, and gene complementation identified PARC6/CDP1/ARC6H, which encodes a vascular plant-specific, chloroplast division site-positioning factor, as the causal gene for the stromule phenotype. Yeast two-hybrid assay and double mutant analysis also identified a possible interaction between PARC6 and MinD1, another known chloroplast division site-positioning factor, during the morphogenesis of leaf epidermal plastids. To the best of our knowledge, PARC6 is the only known A. thaliana chloroplast division factor whose mutations more extensively affect the morphology of plastids in non-mesophyll tissue than in mesophyll tissue. Therefore, the present study demonstrates that PARC6 plays a pivotal role in the morphology maintenance and stromule regulation of non-mesophyll plastids.

The Arabidopsis minD mutation causes aberrant FtsZ1 ring placement and moderate heterogeneity of chloroplasts in the leaf epidermis.

Symmetric division of leaf mesophyll chloroplasts requires MinD and MinE, which work together to suppress division other than at the mid-chloroplast. arc11 is a MinD loss-of-function mutant of Arabidopsis thaliana. In arc11 plants, asymmetric chloroplast division, as well as its delay or arrest, results in extreme size polymorphism of chloroplasts in mature mesophyll cells. The current study examined chloroplast phenotypes in the epidermis of arc11 leaves. Fluorescence microscopy analysis revealed that epidermal chloroplasts in mature leaves exhibited moderate heterogeneity in size. This probably resulted from completion of many of the previous non-equatorial or multiple division events in expanding leaves. Additionally, analyses of plastids found that epidermal chloroplasts in arc11 mutants showed several phenotypes that have not previously been described.

Heavy-ion beam mutagenesis identified an essential gene for chloroplast development under cold stress conditions during both early growth and tillering stages in rice.

We isolated a cold sensitive virescent1 (csv1) mutant from a rice (Oryza sativa L.) population mutagenized by carbon ion irradiation. The mutant exhibited chlorotic leaves during the early growth stages, and produced normal green leaves as it grew. The growth of csv1 plants displayed sensitivity to low temperatures. In addition, the mutant plants that were transferred to low temperatures at the fifth leaf stage produced chlorotic leaves subsequently. Genetic and molecular analyses revealed translocation of a 13-kb genomic fragment that disrupted the causative gene (CSV1; LOC_Os05g34040). CSV1 encodes a plastid-targeted oxidoreductase-like protein conserved among land plants, green algae, and cyanobacteria. Furthermore, CSV1 transcripts were more abundant in immature than in mature leaves, and they did not markedly increase or decrease with temperature. Taken together, our results indicate that CSV1 supports chloroplast development under cold stress conditions, in both the early growth and tillering stages in rice.

The Arabidopsis minE mutation causes new plastid and FtsZ1 localization phenotypes in the leaf epidermis.

Plastids in the leaf epidermal cells of plants are regarded as immature chloroplasts that, like mesophyll chloroplasts, undergo binary fission. While mesophyll chloroplasts have generally been used to study plastid division, recent studies have suggested the presence of tissue- or plastid type-dependent regulation of plastid division. Here, we report the detailed morphology of plastids and their stromules, and the intraplastidic localization of the chloroplast division-related protein AtFtsZ1-1, in the leaf epidermis of an Arabidopsis mutant that harbors a mutation in the chloroplast division site determinant gene AtMinE1. In atminE1, the size and shape of epidermal plastids varied widely, which contrasts with the plastid phenotype observed in atminE1 mesophyll cells. In particular, atminE1 epidermal plastids occasionally displayed grape-like morphology, a novel phenotype induced by a plastid division mutation. Observation of an atminE1 transgenic line harboring an AtMinE1 promoter::AtMinE1-yellow fluorescent protein fusion gene confirmed the expression and plastidic localization of AtMinE1 in the leaf epidermis. Further examination revealed that constriction of plastids and stromules mediated by the FtsZ1 ring contributed to the plastid pleomorphism in the atminE1 epidermis. These results illustrate that a single plastid division mutation can have dramatic consequences for epidermal plastid morphology, thereby implying that plastid division and morphogenesis are differentially regulated in epidermal and mesophyll plastids.

Organ-level analysis of idioblast patterning in Egeria densa Planch. leaves.

Leaf tissues of plants usually contain several types of idioblasts, defined as specialized cells whose shape and contents differ from the surrounding homogeneous cells. The spatial patterning of idioblasts, particularly of trichomes and guard cells, across the leaf epidermis has received considerable attention as it offers a useful biological model for studying the intercellular regulation of cell fate and patterning. Excretory idioblasts in the leaves of the aquatic monocotyledonous plant Egeria densa produced light blue autofluorescence when irradiated with ultraviolet light. The use of epifluorescence microscopy to detect this autofluorescence provided a simple and convenient method for detecting excretory idioblasts and allowed tracking of those cells across the leaf surfaces, enabling quantitative measurement of the clustering and spacing patterns of idioblasts at the whole leaf level. Occurrence of idioblasts was coordinated along the proximal-distal, medial-lateral, and adaxial-abaxial axes, producing a recognizable consensus spatial pattern of idioblast formation among fully expanded leaves. Idioblast clusters, which comprised up to nine cells aligned along the proximal-distal axis, showed no positional bias or regularity in idioblast-forming areas when compared with singlet idioblasts. Up to 75% of idioblasts existed as clusters on every leaf side examined. The idioblast-forming areas varied between leaves, implying phenotypic plasticity. Furthermore, in young expanding leaves, autofluorescence was occasionally detected in a single giant vesicle or else in one or more small vesicles, which eventually grew to occupy a large portion of the idioblast volume as a central vacuole. Differentiation of vacuoles by accumulating the fluorescence substance might be an integral part of idioblast differentiation. Red autofluorescence from chloroplasts was not detected in idioblasts of young expanding leaves, suggesting idioblast differentiation involves an arrest in chloroplast development at a very early stage, rather than transdifferentiation of chloroplast-containing epidermal cells.

Visualization of plastid movement in the pollen tube of Arabidopsis thaliana.

Organelle dynamics in the plant male gametophyte has received attention for its importance in pollen tube growth and cytoplasmic inheritance. We recently revealed the dynamic behaviors of plastids in living Arabidopsis pollen grains and tubes, using an inherent promoter-driven FtsZ1-green fluorescent protein (GFP) fusion. Here, we further monitored the movement of pollen tube plastids with an actin1 promoter-driven, stroma-targeted yellow fluorescent protein (YFP). In elongating pollen tubes, most plastids localized to the tube shank, where they displayed either retarded and unsteady motion, or fast, directional, and long-distance movement along the tube polarity. Efficient plastid tracking further revealed a population of tip-forwarding plastids that undergo a fluctuating motion(s) before traveling backwards. The behavior of YFP-labeled plastids in pollen basically resembled that of FtsZ1-GFP-labeled plastids, thus validating the use of FtsZ1-GFP for simultaneous visualization of the stroma and the plastid-dividing FtsZ ring.

Dynamic morphologies of pollen plastids visualised by vegetative-specific FtsZ1-GFP in Arabidopsis thaliana.

The behaviour and multiplication of pollen plastids have remained elusive despite their crucial involvement in cytoplasmic inheritance. Here, we present live images of plastids in pollen grains and growing tubes from transgenic Arabidopsis thaliana lines expressing stroma-localised FtsZ1-green-fluorescent protein fusion in a vegetative cell-specific manner. Vegetative cells in mature pollen contained a morphologically heterogeneous population of round to ellipsoidal plastids, whilst those in late-developing (maturing) pollen included plastids that could have one or two constriction sites. Furthermore, plastids in pollen tubes exhibited remarkable tubulation, stromule (stroma-filled tubule) extension, and back-and-forth movement along the direction of tube growth. Plastid division, which involves the FtsZ1 ring, was rarely observed in mature pollen grains.

Chemical induction of rapid and reversible plastid filamentation in Arabidopsis thaliana roots.

Plastids assume various morphologies depending on their developmental status, but the basis for developmentally regulated plastid morphogenesis is poorly understood. Chemical induction of alterations in plastid morphology would be a useful tool for studying this; however, no such chemicals have been identified. Here, we show that antimycin A, an effective respiratory inhibitor, can change plastid morphology rapidly and reversibly in Arabidopsis thaliana. In the root cortex, hypocotyls, cotyledon epidermis and true leaf epidermis, significant differences in mitochondrial morphology were not observed between antimycin-treated and untreated tissues. In contrast, antimycin caused extreme filamentation of plastids in the mature cortices of main roots. This phenomenon was specifically observed in the mature root cortex. Other mitochondrial respiratory inhibitors (rotenone and carbonyl cyanide m-chlorophenylhydrazone), hydrogen peroxide, S-nitroso-N-acetylpenicillamine [a nitric oxide (NO) donor] and 3-(3,4-dichlorophenyl)-1,1-dimethylurea did not mimic the phenomenon under the present study conditions. Antimycin-induced plastid filamentation was initiated within 5 min after the onset of chemical treatment and appeared to complete within 1 h. Plastid morphology was restored within 7 h after the washout of antimycin, suggesting that the filamentation was reversible. Co-applications of antimycin and cytoskeletal inhibitors (demecolcine or latrunculin B) or protein synthesis inhibitors (cycloheximide or chloramphenicol) still caused plastid filamentation. Antimycin A was also effective for plastid filamentation in the chloroplast division mutants atftsZ1-1 and atminE1. Salicylhydroxamic acid, an alternative oxidase inhibitor, was solely found to suppress the filamentation, implying the possibility that this phenomenon was partly mediated by an antimycin-activated alternative oxidase in the mitochondria.

Involvement of AtMinE1 in plastid morphogenesis in various tissues of Arabidopsis thaliana.

While it has been established that binary fission of leaf chloroplasts requires the prokaryote-derived, division site determinant protein MinE, it remains to be clarified whether chloroplast division in non-leaf tissues and the division of non-colored plastids also involve the MinE protein. In an attempt to address this issue, plastids of cotyledons, floral organs, and roots were examined in the Arabidopsis thaliana mutant of the MinE (AtMinE1) gene, which was modified to express the plastid-targeted cyan fluorescent protein constitutively, and were quantitatively compared with those in the wild type. In the cotyledons, floral organs, and root columella, the plastid size in the atminE1 mutant was significantly larger than in the wild type, while the plastid number per cell in atminE1 appeared to be inversely smaller than that in the wild type. In addition, formation of the stroma-containing plastid protrusions (stromules) in the cotyledon epidermis, petal tip, and root cells was more active in atminE1 than in the wild type.

Further evaluation of the localization and functionality of hemagglutinin epitope- and fluorescent protein-tagged AtMinD1 in Arabidopsis thaliana.

Symmetric chloroplast division requires a prokaryote-derived division regulator protein MinD, whose subchloroplastic localization remains to be completely established. We investigated the localization and functionality of AtMinD1 (Arabidopsis thaliana MinD) fused with a dual hemagglutinin epitope (dHA) or a yellow fluorescent protein (YFP). AtMinD1-dHA, which successfully complemented the arc11/atminD1 mutant phenotype, was predominantly located at the envelope membrane and the mid-chloroplast constriction site. Meanwhile, AtMinD1-YFP was non-functional and showed suborganellar localization partly similar to that of AtMinD1-dHA. This prompts us to reevaluate earlier transgenic and transient expression studies using fluorescent protein-tagged AtMinD1.

Live imaging of chloroplast FtsZ1 filaments, rings, spirals, and motile dot structures in the AtMinE1 mutant and overexpressor of Arabidopsis thaliana.

Chloroplast division involves the tubulin-related GTPase FtsZ that assembles into a ring structure (Z-ring) at the mid-chloroplast division site, which is where invagination and constriction of the envelope membranes occur. Z-ring assembly is usually confined to the mid-chloroplast site by a well balanced counteraction of the stromal proteins MinD and MinE. The in vivo mechanisms by which FtsZ nucleates at specific sites, polymerises into a protofilament and organizes a closed ring of filament bundles remain largely unknown. To clarify the dynamic aspects of FtsZ, we developed a living cell system for simultaneous visualisation of various FtsZ configurations, utilising the Arabidopsis thaliana overexpressor and mutant of the MinE (AtMinE1) gene, which were modified to weakly express green fluorescent protein (GFP) fused to AtFtsZ1-1. Time-lapse observation in the chloroplasts of both plants revealed disorderly movement of the dots and short filaments of FtsZ. The short filaments often appeared to emanate from the dots and to converge with a long filament, producing a thick cable. In the AtMinE1 overexpressor, we also observed spirals along the longitudinal axis of the organelle that often rolled the closed rings together. In the atminE1 mutant, we visualised the 'isolated' rings with a maximum diameter of approximately 2 mum that did not encircle the organelle periphery, but appeared to be suspended in the stroma. Our observations further demonstrated heterogeneity in chloroplast shapes and concurrently altered configurations of FtsZ in the mutant.

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 facilitate symmetric binary fission and that involve prokaryotic-like stromal division factors such as tubulin-like GTPase FtsZ and the division site regulator MinD. In Arabidopsis, a nuclear-encoded prokaryotic MinE homolog, AtMinE1, has been characterized in terms of its effects on a dividing or terminal chloroplast state in a limited series of leaf tissues. However, the relationship between AtMinE1 expression and chloroplast phenotype remains to be fully elucidated. Here, we demonstrate that a T-DNA insertion mutation in AtMinE1 results in a severe inhibition of chloroplast division, producing motile dots and short filaments of FtsZ. In AtMinE1 sense (overexpressor) plants, dividing chloroplasts possess either single or multiple FtsZ rings located at random intervals and showing constriction depth, mainly along the chloroplast polarity axis. The AtMinE1 sense plants displayed equivalent chloroplast phenotypes to arc11, a loss-of-function mutant of AtMinD1 which forms replicating mini-chloroplasts. Furthermore, a certain population of FtsZ rings formed within developing chloroplasts failed to initiate or progress the membrane constriction of chloroplasts and consequentially to complete chloroplast fission in both AtMinE1 sense and arc11/atminD1 plants. Our present data thus demonstrate that the chloroplast division site placement involves a balance between the opposing activities of AtMinE1 and AtMinD1, which acts to prevent FtsZ ring 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 chloroplasts mature, in spite of the presence of FtsZ rings.