Plastid Ontogeny during Petal Development in Arabidopsis

Imaging of chlorophyll autofluorescence by confocal microscopy in intact whole petals of Arabidopsis thaliana has been used to analyze chloroplast development and redifferentiation during petal development. Young petals dissected from unopened buds contained green chloroplasts throughout their structure, but as the upper part of the petal lamina developed and expanded, plastids lost their chlorophyll and redifferentiated into leukoplasts, resulting in a white petal blade. Normal green chloroplasts remained in the stalk of the mature petal. In epidermal cells the chloroplasts were normal and green, in stark contrast with leaf epidermal cell plastids. In addition, the majority of these chloroplasts had dumbbell shapes, typical of dividing chloroplasts, and we suggest that the rapid expansion of petal epidermal cells may be a trigger for the initiation of chloroplast division. In petals of the Arabidopsis plastid division mutant arc6, the conversion of chloroplasts into leukoplasts was unaffected in spite of the greatly enlarged size and reduced number of arc6 chloroplasts in cells in the petal base, resulting in few enlarged leukoplasts in cells from the white lamina of arc6 petals.     

Direct measurement of femtogram amounts of DNA in cells and chloroplasts by quantitative microspectrofluorometry

Absolute DNA amounts of individual chloroplasts were determined by measuring the fluorescence intensity of chloroplasts stained with 4',6-diamidino-2-phenylindole (DAPI) relative to that of the bacterium Pediococcus damnosus (cerevisiae) smeared on the same slide. An absolute DNA content of 7.7 X 10(15) g for a standard P. damnosus cell type was calculated by comparing the relative fluorescence values and frequency of each stage of cellular development in a culture to the average DNA content of all cell types determined by chemical methods. Chlorophyll was extracted from the chloroplasts during fixation so that chlorophyll autofluorescence was not present when DAPI fluorescence was measured. Absolute amounts of DNA could then be determined for single chloroplasts, either within cells that were individually selected from a mixed cell population or in small preparations of isolated chloroplasts. The DNA amounts of chloroplasts from mesophyll cells determined in this way were similar to the values previously determined by bulk averaging methods. Chloroplast DNA amounts from different cell types of the leaf could be measured by microspectrofluorometry, and it was found that chloroplasts from spinach epidermal cells contained about half as much DNA as chloroplasts from adjacent mesophyll cells.     

Characterization of chloroplast division using the Arabidopsis mutant arc5.

arc5 is a chloroplast division mutant of Arabidopsis thaliana. To identify the role of ARC5 in the chloroplast replication process we have followed the changes in arc5 chloroplasts during their perturbed division. ARC5 does not affect proplastid division but functions at a later stage in chloroplast development. Chloroplasts in developing mesophyll cells of arc5 leaves do not increase in number and all of the chloroplasts in mature leaf cells show a central constriction. Young arc5 chloroplasts are capable of initiating the division process but fail to complete daughter-plastid separation. Wild-type plastids increase in number to a mean of 121 after completing the division process, but in the mutant arc5 the approximately 13 plastids per cell are still centrally constricted but much enlarged. As the arc5 chloroplasts expand and elongate without dividing, the internal thylakoid membrane structure becomes flexed into an undulating ribbon. We conclude that the ARC5 gene is necessary for the completion of the last stage of chloroplast division when the narrow isthmus breaks, causing the separation of the daughter plastids.     

A Genetic Analysis of Chloroplast Division and Expansion in Arabidopsis thaliana

A nuclear recessive mutant of Arabidopsis thaliana, arc5, has been isolated in which there is no significant increase in chloroplast number during leaf mesophyll cell expansion and in which there are only 13 chloroplasts per mesophyll cell compared with 121 in wild-type cells. Mature arc5 chloroplasts in fully expanded mesophyll cells are 6-fold larger than in wild-type cells. A large proportion of arc5 chloroplasts also show some degree of central constriction, suggesting that the mutation has prevented the completion of the chloroplast division process. To examine the interaction of arc loci, a double mutant was constructed between arc1, a mutant possessing many small chloroplasts, and arc5. A second double mutant was also constructed between arc3, a previously discovered mutant also possessing few large chloroplasts per cell, and arc1. Analysis of these double mutants shows that chloroplast number per mesophyll cell is greater when arc5 and arc3 mutations are expressed in the arc1 background than when expressed alone. The cell-specific nature of arc mutants was also analyzed. The phenotypic traits characteristic of arc3 and arc5 are a reduction in chloroplast number and an increase in chloroplast size in mesophyll cells: these changes are also observed in reduced form in the epidermal and guard cell chloroplasts of arc3 and arc5 plants. Analysis of parenchyma sheath cell chloroplasts suggests that in leaves of arc1 plants the normal developmental distinction between mesophyll and parenchyma sheath chloroplasts is perturbed. The relevance of these findings to the analysis of the control of chloroplast division in mesophyll cells is discussed.     

Membrane-bound Plastid Inclusions and Chloroplast Thylakoid Formation in Sunflower (Helianthus annuus L.)

The ultrastructure, distribution and frequency of membrane-boundplastid inclusions present in the epidermal cells of leavesof intact sunflower plants (Helianthus annuus L.) and in theepidermal and mesophyll cells of sunflower leaf discs culturedin darkness have been studied. These inclusions appear to bedilated thylakoids containing a granular material which, undernormal conditions, is probably involved in chloroplast membraneformation. It is suggested that this material accumulates, andinclusions form, in the chloroplasts of sunflower leaves intwo specific situations. Firstly, in the completely differentiatedcells of the epidermis where the chloroplasts, although at arelatively immature stage, have nevertheless reached a terminalstage of development. Secondly, in the mesophyll cells of youngleaves when chloroplast development has been arrested at animmature stage by a 5-day dark period. In the latter situationthe material can be remobilized if plastid development is restimulated.The plastids of sunflower leaf discs cultured in darkness containboth membrane-bound inclusions and prolamellar bodies, indicatingthat they are separate and distinct structures possibly containingdifferent membrane components. Helianthus annuus L., sunflower, chloroplast, ultrastructure, plastid inclusions, thylakoid formation     

Chloroplast nucleoids are highly dynamic in ploidy,number, and structure during angiosperm leaf development

Chloroplast nucleoids are large, compact nucleoprotein structures containing multiple copies of the plastid genome. Studies on structural and quantitative changes of plastid DNA (ptDNA) during leaf development are scarce and have produced controversial data. We have systematically investigated nucleoid dynamics and ptDNA quantities in the mesophyll of Arabidopsis, tobacco, sugar beet, and maize from the early post‐meristematic stage until necrosis. DNA of individual nucleoids was quantified by DAPI‐based supersensitive epifluorescence microscopy. Nucleoids occurred in scattered, stacked, or ring‐shaped arrangements and in recurring patterns during leaf development that was remarkably similar between the species studied. Nucleoids per organelle varied from a few in meristematic plastids to >30 in mature chloroplasts (corresponding to about 20–750 nucleoids per cell). Nucleoid ploidies ranged from haploid to >20‐fold even within individual organelles, with average values between 2.6‐fold and 6.7‐fold and little changes during leaf development. DNA quantities per organelle increased gradually from about a dozen plastome copies in tiny plastids of apex cells to 70–130 copies in chloroplasts of about 7 μm diameter in mature mesophyll tissue, and from about 80 plastome copies in meristematic cells to 2600–3300 copies in mature diploid mesophyll cells without conspicuous decline during leaf development. Pulsed‐field electrophoresis, restriction of high‐molecular‐weight DNA from chloroplasts and gerontoplasts, and CsCl equilibrium centrifugation of single‐stranded and double‐stranded ptDNA revealed no noticeable fragmentation of the organelle DNA during leaf development, implying that plastid genomes in mesophyll tissues are remarkably stable until senescence.     

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.     

Lamelloplasts and minichloroplasts in Begoniaceae: iridescence and photosynthetic functioning

Iridoplasts (modified plastids in adaxial epidermal cells) reported from Begonia were originally hypothesized to cause iridescence, which was broadly accepted for decades. However, several species of Begonia with iridoplasts are not iridescent causing confusion. Here chloroplast ultrastructure was observed in 40 taxa of Begoniaceae to explore the phenomenon of iridescence. However, 22 Begonias and Hillebrandia were found to have iridoplasts, but only nine display visually iridescent blue to blue-green leaves. Unexpectedly, a new type of plastid, a ‘minichloroplast,’ was found in the abaxial epidermal cells of all taxa, but was present in adaxial epidermal cells only if iridoplasts were absent. Comparative ultrastructural study of iridoplasts and a shading experiment of selected taxa show that a taxon with iridoplasts does not inevitably have visual iridescence, but iridescence is greatly affected by the spacing between thylakoid lamellae (stoma spacing). Thus, we propose instead the name ‘lamelloplast’ for plastids filled entirely with regular lamellae to avoid prejudging their function. To evaluate photosynthetic performance, chlorophyll fluorescence (F _v /F _m ) was measured separately from the chloroplasts in the adaxial epidermis and lower leaf tissues by using leaf dermal peels. Lamelloplasts and minichloroplasts have much lower photosynthetic efficiency than mesophyll chloroplasts. Nevertheless, photosynthetic proteins (psbA protein of PSII, RuBisCo and ATPase) were detected in both plastids as well as mesophyll chloroplasts in an immunogold labeling. Spectrometry revealed additional blue to blue-green peaks in visually iridescent leaves. Micro-spectrometry detected a blue peak from single blue spots in adaxial epidermal cells confirming that the color is derived from lamelloplasts. Presence of lamelloplasts or minichloroplasts is species specific and exclusive. High prevalence of lamelloplasts in Begoniaceae, including the basal clade Hillebrandia, highlights a unique evolutionary development. These new findings clarify the association between iridescence and lamelloplasts, and with implications for new directions in the study of plastid morphogenesis.     

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.     

Marking cell layers with spectinomycin provides a new tool for monitoring cell fate during leaf development

Spectinomycin, an inhibitor of plastid protein synthesis, can be used to mark specific cell layers in the shoot meristem of Brassica napus. Pale yellow-green (YG) plants resulting from spectinomycin-treatment can be propagated indefinitely in vitro. Microscopic examination showed that YG-plants result from inactivation of plastids in the L2 and L3 layers and are composed of a pale green epidermis covering a white mesophyll layer. Epidermal cells of YG and normal green plants are similar and contain 10-20 small pale green plastids. YG plants are equivalent to periclinal chimeras with the important distinction that there is no genotypic difference between the white and green cell layers. Periclinal divisions of epidermal cells take place at all stages of leaf development to produce invaginations of green mesophyll located in sectors of widely varying sizes. A periclinal division rate of 1 in 3000-4000 anticlinal divisions for the adaxial epidermis, was 2-3-fold higher than that estimated for the abaxial epidermis. Analysis of white and green mesophyll showed that chloroplasts are essential for palisade cell differentiation and this requirement is cell-autonomous. Stable marking of cell lineages with spectinomycin is simple, rapid and reveals the requirement for functional plastids in cellular differentiation.     

Changes in the Number and Composition of Chloroplasts during Senescence of Mesophyll Cells of Attached and Detached Primary Leaves of Wheat (Triticum aestivum L.)

Changes in the number and composition of chloroplasts of mesophyll cells were followed during senescence of the primary leaf of wheat (Triticum aestivum L.). Senescence was due to the natural pattern of leaf ontogeny or was either induced by leaf detachment and incubation in darkness, or incubation of attached leaves in the dark. In each case discrete sections (1 centimeter) of the leaf, representing mesophyll cells of the basal, middle, and tip regions, were examined. For all treatments, senescence was characterized by a loss of chlorophyll and the protein ribulose 1,5-bisphosphate carboxylase (RuBPCase). Chloroplast number per mesophyll cell remained essentially constant during senescence. It was not until more than 80% of the plastid chlorophyll and RuBPCase was degraded that some reduction (22%) in chloroplast number per mesophyll cell was recorded and this was invariably in the mesophyll cells of the leaf tip. We conclude that these data are consistent with the idea that degradation occurs within the chloroplast and that all chloroplasts in a mesophyll cell senesce with a high degree of synchrony rather than each chloroplast senescing sequentially.     

Ultrastructural Study of Proplastid Ontogeny in Tobacco Mesophyll Cells in Vitro

Since the discovery of plastid DNA the continuity of plastids has well been established. It is known that in plant cultures a form of plastid can differentiate into others. However, only a little has been made in studing chloroplast dedifferentiation in vitro. In the work present here, we reported on ultrastructural changes of chloroplasts dedifferentiation and the proplastid origin in the mesophyll cells of cultured tobacco leaf explant. Fully expanded leaves of haploid tobacco (cv. Ge Xin No. 1) were cut into pieces of 5–6 mm width. These were inoculated on MS medium supplemented with 1 mg/L 2,4-D and 1 mg/l kinetin. The cultures were maintained at (30±2) ℃ and illuminatied by a bank of fluorescent lamps. For electronmicroseopic investigation, after 0, 1, 2, 3, 6 days of culture small leaf fragments were cut off along the cut edges of the explants. The samples were fixed and processed in the manner as described earlier. The sections were examined with a Hitachi HU-11A or a JEM-100CX electronmicroscope. Electronmicroscopic observation shows that the uncultured mesophyll cells are highly vacuolete, with a thin peripheral layer of cytoplasm in which a nucleus and some chloroplasts and other organelles are found in it. But these cells do not contain proplastids (Fig. l). In the explants cultured for 1 day there are no obviously changes in mesophyll cells, except a few cytoplasmic strands extend from periphery to central vacuole. At 2 days of culture quite obvious changes can be detected. A increase in the amount of cytoplasm becomes apparent and transvacuolar cytoplasmic strands grow up. Following cytoplasmic growth, the nucleus and chloroplasts move away from the peripheral cytoplasm and enter the central vacuolate zone (Fig. 2). At this stage some of mesophyll cells have completed the first cell division. After 3 days of culture numerous mesophyll cells have undergone several divisions and formed multicellular masses. In those subdivided cells a more important change of the chloroplasts is the occurrence of protrusions which we call proplastid buds. This phenomenon has also been named as chloroplast budding. According to observations on a large amount of sections chloroplast budding is a common phenomenon in the dedifferentiating mesophyll cells of tobacco leaf explants. Fig ure 3 exhibits a typical profile of a chloroplast with a proplastid bud. The proplastid buds observed are generally long-oval in shape and 1.0–2.5 μm long and about 0.5–0.7 μm thick. These dimensions agree with those of proplastids in meristematie cells. Inside of proplastids ribosomes and electron opaque areas containing DNA fibrils can be seen (Fig. 3). Near the proplastid buds proplastids can often be found (Fig.5). According to above observations we can conclude that the proplastids in dedifferentiating mesophyll cells originate from the proplastid buds by chloroplast budding. The newly formed proplastids usually surround the nucleus and sometimes undergo equal division to increase their number (Figs.5, 6). There are no inner membranes in the newly formed proplastids except vesicles connected with inner membrane of the envelope (Fig.7). While the proplastids are continuously produced, the chloroplasts themselves are filled with starch and gradually turned to large amyloplasts (Fig.5). On the other hand, a few of chloroplasts can divide into equal parts following the chloroplast budding (Fig.4). Israel and Steward (1967) suggested that when cultured carrot cells developed into plantlets the chloroplasts turned into leucoplastids, chromoplastids or proplastids. However, they did not describe how chloroplast became a proplastid. Several investigators reported that the chloroplasts in the dedifferentiating cells gradually lost their grana and intergranal lamellae and then became eueoplasts or proplastids. But according to our observation in tobacco explants, the initiation of proplastids is due to unequal division of chloroplasts, i.e. “budding fission” as described by Malzan and Miihlethaler in Splachnum ampullaceum. Since the proplastid is an organelle characteristic of meristematie cells, the ontogeny of proplastids and its control mechanism should be very important in studing cell dedifferentiation.     

Nuclear DNA content and the control of chloroplast replication in wheat leaves

During development of the first leaf of breadwheat (Triticum aestivum L.) the number of chloroplasts per mesophyll cell increases between three- and four-fold. To establish if chloroplast replication is accompanied by endoreduplication, the nuclear DNA content of the cells was determined by chemical assay of isolated nuclei from mesophyll protoplasts and by microdensitometry of nuclei in mesophyll tissue. The DNA content of the nuclei was constant (27 to 32 pg) at each phase of chloroplast replication. Approximately 93% of the cells had a nuclear DNA content close to the 2C value of 32 pg. It is concluded that chloroplast replication is not dependent on nuclear endoreduplication in seedling leaves of wheat.     

Structure of the Photosynthetic Apparatus in Leaves of Freshwater Hydrophytes: 1. General Characteristics of the Leaf Mesophyll and a Comparison with Terrestrial Plants

The structure of photosynthetic elements was investigated in leaves of 42 boreal plant species featuring different degrees of submergence (helophytes, neustophytes, and hydatophytes). The mesophyll structure types were identified for all these species. Chlorenchyma tissues and phototrophic cells were quantitatively described by such characteristics as the sizes of cells and chloroplasts in the mesophyll and epidermis, the abundance of cells and chloroplasts in these tissues, the total surface area of cells and chloroplasts per unit leaf area, the number of plastids per cell, etc. The hydrophytes typically had thick leaves (200–350 m) with a well-developed aerenchyma; their specific density per unit area (100–200 mg/dm²) was lower than in terrestrial plants. Mesophyll cells in aquatic plants occupied a larger volume (5–20 × 10³m³) than epidermal cells (1–15 × 10³m³). The number of mesophyll cells per unit leaf area was nearly 1.5 times higher than that of epidermal cells. Chloroplasts were present in the epidermis of almost all species, including emergent leaves, but the ratio of the chloroplast total number to the number of all plastids varied depending on the degree of leaf submergence. The total number of plastids per unit leaf area (2–6 × 10⁶/cm²) and the surface of chloroplasts per unit leaf area (2–6 cm²/cm²) were lower in hydrophytes than in terrestrial plants from climatically similar habitats. The functional relations between mesophyll parameters were similar for hydrophytes and terrestrial plants (a positive correlation between the leaf weight per unit area, leaf thickness, and the number of mesophyll cells per unit leaf area), although no correlation was found in hydrophytes between the volume of mesophyll cells and the leaf thickness. Phototrophic tissues in aquatic plants contributed a larger fraction to the leaf weight than in terrestrial plants, because the mechanical tissues were less developed in hydrophytes. The CO₂assimilation rates by leaves were lower in hydrophytes than in terrestrial plants, because the total surface area of chloroplasts per unit leaf area is comparatively small in hydrophytes, which reduces the conductivity for carbon dioxide diffusion towards the carboxylation sites.     

Synchronous Growth and Plastid Replication in the Naturally Wall-less Alga Olisthodiscus luteus

Olisthodiscus luteus is a unicellular biflagellate alga which contains many small discoidal chloroplasts. This naturally wall-less organism can be axenically maintained on a defined nonprecipitating artificial seawater medium. Sufficient light, the presence of bicarbonate, minimum mechanical turbulence, and the addition of vitamin B₁₂ to the culture medium are important factors in the maintenance of a good growth response. Cells can be induced to divide synchronously when subject to a 12-hour light/12-hour dark cycle. The chronology of cell division, DNA synthesis, and plastid replication has been studied during this synchronous growth cycle. Cell division begins at hour 4 in the dark and terminates at hour 3 in the light, whereas DNA synthesis initiates 3 hours prior to cell division and terminates at hour 10 in the dark. Synchronous replication of the cell's numerous chloroplasts begins at hour 10 in the light and terminates almost 8 hours before cell division is completed. The average number of chloroplasts found in an exponentially growing synchronous culture is rather stringently maintained at 20 to 21 plastids per cell, although a large variability in plastid complement (4-50) is observed within individual cells of the population. A change in the physiological condition of an Olisthodiscus cell may cause an alteration of this chloroplast complement. For example, during the linear growth period, chloroplast number is reduced to 14 plastids per cell. In addition, when Olisthodiscus cells are grown in medium lacking vitamin B₁₂, plastid replication continues in the absence of cell division thereby increasing the cell's plastid complement significantly.     

Regulation of leucoplast morphology in roots: Interorganellar signaling from mitochondria?

The appearance of leaf mesophyll chloroplasts in angiosperms is characterized by their uniform and static shape, which is molded by symmetric division of the preexisting organelles, involving three prokaryote-derived proteins: the division executor protein, FtsZ, and the division site positioning proteins, MinD and MinE. On the other hand, noncolored plastids in roots, where the involvement of the known chloroplast division factors in plastid morphogenesis is yet unclear, are morphologically heterogeneous and transform dynamically. This is further emphasized by the active formation of long tubular protrusions called stromules from the main body of those plastids. Molecular regulation and physiological significance of such dynamic morphology of root plastids also remain unknown. In this context, we have recently demonstrated that the mitochondrial respiratory inhibitor antimycin A induces rapid and reversible filamentation of root plastids (leucoplasts) in Arabidopsis thaliana. In contrast, the same treatment with antimycin A did not affect the morphology of amyloplasts in the columella cells at the root tip. The alternative oxidase inhibitor salicylhydroxamic acid suppresses the antimycin-induced plastid filamentation, perhaps implying an alternative oxidase-mediated interorganellar signaling between the mitochondria and the leucoplasts in the root cells. Our data may provide some clues as to how the formation of stromules is initiated.Key words: antimycin A, interorganellar crosstalk, plastid morphology, respiration, stress response, stromule     

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     

Rapid Image Analysis Screening Procedure for Identifying Chloroplast Number Mutants in Mesophyll Cells of Arabidopsis thaliana (L.) Heynh

To analyze the genetic control of the process of chloroplast division, a direct image analysis screening procedure has been developed in which mutants of Arabidopsis thaliana (L.) Heynh. var Landsberg erecta are selected on the basis of abnormal chloroplast number. The selection procedure is based on image analysis thresholding after iodine staining, which facilitates the automatic counting of chloroplasts in isolated mesophyll cells. M2 seedlings are screened for significant deviation from the wild type relationship between mesophyll cell size and chloroplast number. Mutants with both abnormally high and abnormally low chloroplast numbers were identified. Of 3500 individual M2 seedlings screened, 18 mutant lines have been isolated and shown to be stably inherited in three subsequent generations. The most extreme phenotypes show an 80% reduction or a 50% increase in chloroplast number per mesophyll cell.     

arc6, A Fertile Arabidopsis Mutant with Only Two Mesophyll Cell Chloroplasts

A novel mutant of Arabidopsis thaliana, arc6 (accumulation and replication of chloroplasts), has been isolated from a transfer DNA-mutagenized population of Arabidopsis seedlings. arc6 has the most extreme arc mutant phenotype we have yet described, with only one to three chloroplasts per leaf mesophyll cell compared to a mean of 83 in cells of the wild-type var Wassilewskija. The chloroplasts of arc6 are 20-fold larger than wild-type chloroplasts.Chloroplast division is almost certainly precluded in arc6 mesophyll cells, since chloroplast number per cell does not increase during mesophyll cell expansion. arc6 chloroplasts are long and thin in cross-section and only one-half the width of wild-type chloroplasts and the arrangement of thylakoid membranes is largely unaltered. arc6 segregates as a monogenic recessive nuclear mutation in a normal Mendelian manner and the arc6 phenotype is stably inherited for at least four generations. arc6 plants grow normally and are fertile, although the rosette leaves appear curled and twisted. arc6 plants accumulate 70 to 75% of the biomass of wild type. The phenotype of this novel mutant is discussed in relation to the nature of the control of chloroplast division in leaf cells.     

Changes in chloroplast number per cell during leaf development in spinach

Summary The amounts of chlorophyll and nitrogen and the numbers of cells per unit area change as the green leaves of spinach plants grow and increase in size in the light. The changes in the numbers of chloroplasts per cell were measured by a new method. A 5-fold increase in the numbers of chloroplasts per cell took place in both palisade and mesophyll cells over a growing period of 10 days during which time the area of the leaves increased from 1 to 50 cm². Proplastids were not present in the young green leaves but electron-microscope and phase-contrast observations showed the presence of grana-containing chloroplasts, many of which appeared to be undergoing division by constriction. It is suggested that the large increase in chloroplast numbers as leaf cells grow and expand in the light is from the division of differentiated chloroplasts containing grana.