Behavior of chloroplasts and chloroplast nuclei during spermatogenesis in the fern,Pteris vittata L.

Summary The fate of the chloroplasts and chloroplast nuclei (cp-nuclei) was followed during spermatogenesis in the fernPteris vittata L. by epifluorescence microscopy after staining with 4-6-diamidino-2-phenylindole (DAPI) and by quantitation of chloroplast DNA (cp-DNA) by fluorimetry using a video intensified microscope photon counting system (VIMPICS). The spores were grown on solid medium that contained antheridiogen (An_ptd), and formed an antheridium initial on the protonema cell. The antheridium initial divided and produced 16 spermatocytes and 3 surrounding cells. The chloroplasts in the spermatocytes decreased in volume as cell division was repeated, until finally the volume of each chloroplast was 1/15 of that of the primary chloroplasts. The DNA content of the chloroplasts was also reduced to 1/5 of the original value and when the sperm matured, the fluorescence of cp-DNA disappeared. In the 16-cell spermatocyte, the recognition of the fluorescence of chlorophyll in the chloroplasts with a green excitation filter became difficult. But, the plastids could be observed until the final stage of the sperm. From these observations, it appears that there are two steps in the metamorphosis of chloroplasts during spermatogenesis in the fern. The first step involves the decrease in the volume of chloroplasts, accompanied by reduction of the DNA content, and the second step involves the change of the physical state of chloroplasts to amyloplasts and the disappearance of the cp-DNA from the amyloplasts.     

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.     

Isolation and phenotypic characterization ofChlamydomonas reinhardtii mutants defective in chloroplast DNA segregation

Summary Each wild-typeChlamydomonas reinhardtii cell has one large chloroplast containing several nuclei (nucleoids). We used DNA insertional mutagenesis to isolate Chlamydomonas mutants which contain a single, large chloroplast (cp) nucleus and which we namedmoc (monokaryotic chloroplast). DAPI-fluorescence microscopy and microphotometry observations revealed thatmoc mutant cells only contain one cp-nucleus throughout the cell division cycle, and that unequal segregation of cpDNA occurred during cell division in themoc mutant. One cell with a large amount of cpDNA and another with a small amount of cpDNA were produced after the first cell division. Unequal segregation also occurred in the second cell division, producing one cell with a large amount (about 70 copies) of cpDNA and three other cells with a small amount (only 2–8 copies) of cpDNA. However, most individualmoc cells contained several dozen cpDNA copies 12 h after the completion of cell division, suggesting that cpDNA synthesis was activated immediately after chloroplast division. In contrast to the cpDNA, the mitochondrial (mt) DNA of themoc mutants was observed as tiny granules scattered throughout the entire cell. These segregated to each daughter cell equally during cell division. Electron-microscopic observation of the ultrastructure ofmoc mutants showed that a low-electron-density area, which was identified as the cp-nucleus by immunoelectron microscopy with anti-DNA antibody, existed near the pyrenoid. However, there were no other structural differences between the chloroplasts of wild-type cells andmoc mutants. The thylakoid membranes and pyrenoid were identical. Therefore, we propose that the novelmoc mutants are only defective in the dispersion and segregation of cpDNA. This strain should be useful to elucidate the mechanism for the segregation of cpDNA.Abbreviations DAPI 4,6-diamidino-2-phenylindole - VIMPCS video-intensified microscope photon-counting system     

Regulation of Brassica rapa chloroplast proliferation in vivo and in cultured leaf disks

Summary. To understand the regulatory mechanisms of chloroplast proliferation, chloroplast replication was studied in cultured leaf disks cut from plants of 25 species. In leaf disks from Brassica rapa var. perviridis, the number of chloroplasts per cell increased remarkably in culture. We examined chloroplast replication in this plant in vivo and in culture media with and without benzyladenine, a cytokinin. In whole plants, leaf cells undergo two phases from leaf emergence to full expansion: an early proliferative stage, in which mitosis occurs, and a differentiational stage after mitosis has diminished. During the proliferative stage, chloroplast replication keeps pace with cell division. In the differentiational phase, cell division ceases but chloroplast replication continues for two or three more cycles, with the number of chloroplasts per cell reaching about 60. In the leaf disks, the number of chloroplasts per cell increased from about 18 to 300 without benzyladenine, and to over 600 with benzyladenine, indicating that this cytokinin enhances chloroplast replication in cultured tissue. We also studied changes in ploidy and cell volume between in vivo cells and cells grown in culture with and without benzyladenine. Ploidy and cell volume increased in a manner very similar to that of the number of chloroplasts, suggesting a relationship between these phenomena.Correspondence and reprints: Department of Biological Sciences, Graduate School of Science, University of Tokyo, Hongo, Tokyo 113-0033, Japan.     

Chloroplast DNA of Acetabularia mediterranea: Cell cycle related changes in distribution

Chloroplast DNA (cpDNA) distribution in the giant unicellular, uninucleate alga Acetabularia mediterranea was analyzed with the DNA-specific fluorochrome 4'6-diamidino-2-phenylindole (DAPI) at various stages of the cell cycle. The number of chloroplasts exhibiting DNA/DAPI fluorescence changes during the cell's developmental cycle: (1) all chloroplasts in germlings contain DNA; (2) the number of plastids with DNA declines during polar growth of the vegetative cell; (3) it increases again prior to the transition from the vegetative to the generative phase; (4) several nucleoids of low fluorescence intensity are present in the chloroplasts of the gametes. The temporal distribution of the number of chloroplasts with DNA appears to be linked to the different mode of chloroplast division and growth during the various stages of development. The chloroplast cycle in relation to the cell cycle is discussed.Abbreviations cpDNA chloroplast DNA - DAPI 4,6-diamidino-2-phenylindole     

The evolution of the regulatory mechanism of chloroplast division

Chloroplasts arose from a cyanobacterial endosymbiont and multiply by division, reminiscent of their free-living ancestor. However, chloroplasts can not divide by themselves, and the division is performed and controlled by proteins that are encoded by the host nucleus. The continuity of chloroplasts was originally established by synchronization of endosymbiotic cell division with host cell division, as seen in existent algae. In contrast, land plant cells contain multiple chloroplasts, the division of which is not synchronized, even in the same cell. Land plants have evolved cell and chloroplast differentiation systems in which the size and number of chloroplasts (or other types of plastids) change along with their respective cellular function by changes in the division rate. We recently reported that PLASTID DIVISION (PDV) proteins, land-plant specific components of the chloroplast division apparatus, determined the rate of chloroplast division. The level of PDV protein is regulated by the cell differentiation program based on cytokinin, and the increase or decrease of the PDV level gives rise to an increase or decrease in the chloroplast division rate. Thus, the integration of PDV proteins into the chloroplast division machinery enabled land plant cells to change chloroplast size and number in accord with the fate of cell differentiation.Key words: chloroplast division, cell cycle, cell differentiation, cytokinin, endosymbiosis, evolution     

Uncoupling of chloroplast reproductive events from cell cycle division processes by 5-fluorodeoxyuridine in the algaScenedesmus quadricauda

Summary FdUrd (5-fluorodeoxyuridine), a specific inhibitor of thymidylate synthase, was used to study the relationship between reproductive processes in chloroplast and nucleocytoplasmic compartments of the chlorococcal algaScenedesmus quadricauda. The courses of DNA replication and nuclear division in both the compartments were followed in populations synchronised by the alternation of light and dark periods. DAPI-staining of DNA-containing structures was used for their visualisation and quantification. In contrast with cellular reproductive events, those in chloroplasts were not substantially affected by the presence of FdUrd (25 g/ml). It was shown that FdUrd specifically blocked nucDNA replication but not ptDNA replication. Thus, cells which had attained commitment to ptDNA replication, fission of pt-nuclei and chloroplast kinesis triggered and terminated these processes while the corresponding cellular processes were blocked. The courses of reproductive processes in chloroplasts were also substantially unaffected in cells grown in the presence of FdUrd for the whole cell cycle. This provided evidence that attainment of commitment to and termination of the entire sequence of reproductive events, including chloroplast fission, were controlled by different mechanisms than the reproductive processes in the nucleocytoplasmic compartment.Abbreviations DAPI 4,6-diamidino-2-phenylindole - ptDNA DNA of chloroplast nuclei - nucDNA DNA in cell nuclei - FdUrd 5-fluorodeoxyuridine     

Changes in chloroplast number during pea leaf development

Protoplasts were prepared from pea (Pisum sativum L.) leaves throughout development and their contents spread in a monolayer to determine the number of chloroplasts per cell. This approach permitted the rapid analysis of more than 100 cells at each stage of development. The average number of chloroplasts per cell increased from 24±10 to 64±20 during greening and expansion of the first true foliage leaves; all cells containing chloroplasts apparently increase their chloroplast number. A parallel increase in the amount of DNA per nucleus was not observed. As the leaves senesced the chloroplast number gradually decreased to 44±12. We have correlated these changes with our previous results on the percentage of chloroplast DNA per cell. Chloroplast multiplication resulted in a 2.7-fold dilution (from 272 to 102) of the number of copies of the chloroplast DNA molecule per plastid.     

Degradation of chloroplast DNA in second leaves of rice (Oryza sativa) before leaf yellowing

Summary The second leaf ofOryza sativa develops, grows and ages within the 10 days that follow imbibition under our controlled continuous-light conditions. Proplastids in the leaf cells develop, mature to become chloroplasts and then age and disintegrate. In an examination of this life process, we studied first the behavior and the number of copies of plastid DNA and levels of chlorophyll by epifluorescence microscopy after staining with 4,6-diamidino-2-phenylindole (DAPI), and by fluorimetry with a video-intensified microscope photon-counting system (VIMPCS). The results indicated that the number of copies of the plastid DNA per plastid increased and reached to plateau value of approximately 100 at the time when the elongation of the mesophyll cells and the enlargement of chloroplasts ceased 96 h after imbibition. However, 24 h later, the number of copies of plastid DNA per chloroplast began to decrease and fell rapidly to approximately 30 copies within 168 h after imbibition. Our examination of the number of chloroplasts per mesophyll cell indicated that no division of chloroplasts occurred more than 72 h after imbibition. The results suggest that the decrease in number of copies of plastid DNA per chloroplast was not due to an increase in the number of chloroplasts, but that this decrease was caused by degradation by unidentified enzymes. Since visible senescence of leaves, which was characterized by development of a yellowish color, began 168 h after imbibition, the degradation of plastid DNA seemed to occur 48 h before the visible leaf senescence. When we tested the nucleolytic activities in the second leaves after imbibition by digestion of plasmids in vitro and DNA-SDS polyacrylamide gel electrophoresis, five Ca²⁺–, four Zn²⁺–, and four Mn²⁺–dependent nucleases were detected in the leaf blades, and one of the Ca²⁺–, two of the Zn²⁺–, and two of the Mn²⁺–dependent nucleases were also identified in a purified preparation of intact chloroplasts. When the activity of the Zn²⁺–dependent nucleases (51 kDa and 13 kDa) increased markedly, degradation of the plastid DNA occurred. These results suggest that the destruction of chloroplast DNA, which occurs approximately 48 h before leaf yellowing, could be due to the activation of some metallo-nucleases and, furthermore, this enzymatic degradation propels the leaf towards senescence.     

Effect of light on the chloroplast division cycle and DNA synthesis in cultured leaf discs of spinach

The effects of light on both the division cycle of chloroplasts and the synthesis of chloroplast DNA were investigated in cultured discs taken from the distal end of 2-centimeter spinach (Spinacia oleracea) leaves. Comparisons were made of discs cultured for a maximum of 4 days in a shaking liquid medium under continuous white light, darkness, and of discs cultured for 1 day in light following 3 days in darkness. In continuous white light the shortest generation time of chloroplasts observed in this study was 19.4 hours and the duration of spherical, ovoid, and dumbbell-shaped stages in the division cycle were 13.4, 2.8, and 3.1 hours, respectively. In darkness the generation times of chloroplasts extended to 51.5 hours. Under these conditions the duration of spherical, ovoid, and dumbbell-shaped stages were 22.8, 8.4, and 20.2 hours, respectively, suggesting that in darkness the separation of dumbbell-shaped chloroplasts may be the rate limiting step. When discs cultured in the dark were transferred to light, most dumbbell-shaped chloroplasts separated into daughter chloroplasts in less than an hour. Measurements of chloroplast DNA established that the cellular level of chloroplast DNA increased 10-fold over the 4 days of culture in continuous white light. Comparisons of the plastids of dark and light grown discs showed that the synthesis of chloroplast DNA was enhanced by light. Observations of DAPI stained dividing chloroplasts indicate that DNA partitioning can take place during the final stage of chloroplast division and that it does not precede plastid division.     

Chloroplast proliferation based on their distribution and arrangement inAdiantum protonemata growing under red light

Chloroplast proliferation was investigated inAdiantum protonemata growing under continuous red light. Cell division is absent when cells are grown under red light. The chloroplast number increases as the cell length increases, therefore the chloroplasts divide in the absence of cell division. Chloroplasts in the basal part of the filamentous protonemal cell migrate gradually toward the cell apex, but there is no large net migration from the tip to the base or vice versa, indicating that chloroplast division takes place in the apical part of the protonemata. Chloroplast number in the apical 100 μm was maintained at about 200 during cell growth at least over eight days. The chloroplasts were either dumbbell- or ellipsoid-shaped. Dumbbell-shaped chloroplasts are abundant everywhere in a protonema, ranging from 30 to 50% of the total chloroplasts. The dumbbell-shaped chloroplasts attached to or very close to the plasma membrane seem to be the ones that are dividing but the dumbbell-shaped ones in the other regions do not divide. These data support the hypothesis that a signal from the plasma membrane induces the dumbbell-shaped chloroplasts to divide.     

Chloroplast Growth and Replication in Germinating Spinach Cotyledons following Massive gamma-Irradiation of the Seed

Spinach seeds (Spinacia oleracea L.) given massive doses of γ-irradiation (500 krad) germinate and form a seedling with two green cotyledons and a radicle, but develop no further. Irradiated cotyledons show no increase in cell number or total DNA over a 7-day period in the light, while in control cotyledons there is a small increase in cell number and large increases in total DNA and chloroplast number. The chloroplasts of irradiated cotyledons are delayed in their division, become greatly enlarged and contain large amounts of starch. The whole population of chloroplasts subsequently undergoes a wave of division. The daughter chloroplasts show normal thylakoid development, but have some abnormal structural features caused by the radiation stress. Information on the effect of X-irradiation, ultraviolet irradiation, and 5-fluorodeoxyuridine on chloroplast replication and on chloroplast and nuclear DNA synthesis was obtained from cultured spinach leaf discs. It appears that chloroplast replication is more resistant to ionizing radiation than cell division and can proceed in the absence of nuclear DNA synthesis and greatly reduced chloroplast DNA synthesis.     

Cell size and chloroplast size in relation to chloroplast replication in light-grown wheat leaves

As part of an investigation into the control of chloroplast replication the number and size of chloroplasts in mesophyll cells was examined in relation to the size of the cells. In first leaves of Triticum aestivum L. and T. monococcum L. the number of chloroplasts in fully expanded mesophyll cells is positively correlated with the plan area of the cells. The linear relationship between chloroplast number per cell and cell plan area is also consistent over a fivefold range of cell size in isogenic diploid and tetraploid T. monococcum. In T. aestivum the chloroplast number per unit cell plan area varies among cells in relation to the size of the chloroplasts. Those cells containing chloroplasts with a relatively small face area have a correspondingly higher density of chloroplasts, and consequently, the total chloroplast area per unit cell plan area is very similar in all the cells. The results indicate that the proportion of the cell surface area covered by chloroplasts is precisely regulated, and that this is achieved during cell development by growth and replication of the chloroplasts.     

Treatment with antibiotics that interfere with peptidoglycan biosynthesis inhibits chloroplast division in the desmid Closterium

Charophytes is a green algal group closely related to land plants. We investigated the effects of antibiotics that interfere with peptidoglycan biosynthesis on chloroplast division in the desmid Closterium peracerosum-strigosum-littorale complex. To detect cells just after division, we used colchicine, which inhibits Closterium cell elongation after division. Although normal Closterium cells had two chloroplasts before and after cell division, cells treated with ampicillin, D-cycloserine, or fosfomycin had only one chloroplast after cell division, suggesting that the cells divided without chloroplast division. The antibiotics bacitracin and vancomycin showed no obvious effect. Electron microscopic observation showed that irregular-shaped chloroplasts existed in ampicillin-treated Closterium cells. Because antibiotic treatments resulted in the appearance of long cells with irregular chloroplasts and cell death, we counted cell types in the culture. The results suggested that cells with one chloroplast appeared first and then a huge chloroplast was generated that inhibited cell division, causing elongation followed by cell death.     

Division of chloroplast nucleoids and replication of chloroplast DNA during the cell cycle ofDunaliella salina grown under blue and red light

Summary Synchronous cultures of the algaDunaliella salina were grown in blue or red light. The relationships between replication of chloroplast DNA, cell size, cell age and the number of chloroplast nucleoids were studied. The replication of chloroplast DNA and the division of chloroplast nucleoids occurred in two separate periods of the chloroplast cycle. DNA replication was concomitant with that in the nucleocytoplasmic compartment but nucleoid division occurred several hours earlier than nuclear division. Red-light-grown cells were bigger and grew more rapidly than those grown in blue light. In newly formed daughter cells, the chloroplast nucleoids were small and spherical and they were localized around the pyrenoid. During the cell cycle they spread to other parts of the chloroplast. The number of DNA molecules per nucleoid doubled during DNA replication in the first third of the cell cycle but decreased several hours later when the nucleoids divided. Their number was fairly constant independent of the different light quality. Cells grown in red light replicated their chl-DNA and divided their nucleoids before those grown in blue light and their daughter cells possessed about 25 nucleoids as opposed to 15.Abbreviations DAPI 4,6-diamidino-2-phenylindole - chl-DNA chloroplast DNA - PAR photosynthetically active radiation     

Microspectrofluorometric Measurement of Chloroplast DNA in Dividing and Expanding Leaf Cells of Spinacia oleracea

Absolute DNA amounts of individual chloroplasts from mesophyll and epidermal cells of developing spinach leaves were measured by microspectrofluorometry using the DNA-specific stain, 4,6-diamidino-2-phenyl indole, and the bacterium, Pediococcus damnosus, as an internal standard. Values obtained by this method showed that DNA amounts of individual chloroplasts from mesophyll cells fell within a normal distribution curve, although mean DNA amounts changed during leaf development and also differed from the levels in epidermal chloroplasts. There was no evidence in the data of plastids containing either the high or low levels of DNA which would be indicative of discontinuous polyploidy of plastids, or of division occurring in only a small subpopulation of chloroplasts. By contrast, the distribution of nuclear DNA amounts in the same leaf tissues in which cell division was known to be occurring showed a clear bimodal distribution. We consider that the distribution of chloroplast DNA in the plastid population shows that there is no S-phase of chloroplast DNA synthesis, all chloroplasts in the population in young leaf cells synthesize DNA, and all chloroplasts divide.     

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.     

Analysis of chloroplast and mitochondrial DNA in asymmetric somatic hybrids betweenSolanum tuberosum and irradiatedS. brevidens

Organellar DNA of asymmetric somatic hybrids betweenSolanum tuberosum and irradiatedS. brevidens were analysed by DNA hybridization methods using the spinach chloroplast probepSBD, wheat mitochondrial genenad5 and petunia mitochondrial geneorf25. Eight of the 12 asymmetric hybrid plants hadS. tuberosum chloroplast DNA and the remaining fourS. brevidens chloroplast DNA. A novel mitochondrial hybridization pattern was present in eight out of the 17 hybrids tested. In six hybrids, novel combinations of chloroplasts and mitochondria were found, indicating that both organelle types sorted out independently.     

Constancy of organellar genome copy numbers during leaf development and senescence in higher plants

In higher plants, plastid and mitochondrial genomes occur at high copy numbers per cell. Several recent publications have suggested that, in higher plants like Arabidopsis and maize, chloroplast DNA is virtually absent in mature and old leaves. This conclusion was mainly based on DAPI staining of isolated chloroplasts. If correct, the finding that chloroplasts in mature leaves lack DNA would change dramatically our understanding of gene expression, mRNA stability and protein stability in chloroplasts. In view of the wide implications that the disposal of chloroplast DNA during leaf development would have, we have reinvestigated the age dependency of genome copy numbers in chloroplasts and, in addition, tested for possible changes in mitochondrial genome copy number during plant development. Analyzing chloroplast and mitochondrial DNA amounts in Arabidopsis and tobacco plants, we find that organellar genome copy numbers remain remarkably constant during leaf development and are present in essentially unchanged numbers even in the senescing leaves. We conclude that, during leaf development, organellar gene expression in higher plants is not significantly regulated at the level of genome copy number and we discuss possible explanations for the failure to detect DNA in isolated chloroplasts stained with DAPI.     

Giant chloroplast development in ethylene response1-1 is caused by a second mutation in ACCUMULATION AND REPLICATION OF CHLOROPLAST3 in Arabidopsis

The higher plants of today array a large number of small chloroplasts in their photosynthetic cells. This array of small chloroplasts results from organelle division via prokaryotic binary fission in a eukaryotic plant cell environment. Functional abnormalities of the tightly coordinated biochemical event of chloroplast division lead to abnormal chloroplast development in plants. Here, we described an abnormal chloroplast phenotype in an ethylene insensitive ethylene response1-1 (etr1-1) of Arabidopsis thaliana. Extensive transgenic and genetic analyses revealed that this organelle abnormality was not linked to etr1-1 or ethylene signaling, but linked to a second mutation in ACCUMULATION AND REPLICATION3 (ARC3), which was further verified by genetic complementation analysis. Despite the normal expression of other plastid division-related genes, the loss of ARC3 caused the enlargement of chloroplasts as well as the diminution of a photosynthetic protein Rubisco in etr1-1. Our study has suggested that the increased size of the abnormal chloroplasts may not be able to fully compensate for the loss of a greater array of small chloroplasts in higher plants.