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.     

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.     

Chloroplast division in cultured cells of the hornwortAnthoceros punctatus

Using cultured cells of the hornwortAnthoceros punctatus, the change in the relative chloroplast DNA content in each stage of chloroplast division was investigated to clarify the relationship between the division cycle of a chloroplast and a cell nucleus. Samples of cultured cells were stained with 4′,6-diamidino-2-phenylindole (DAPI) and then observed with an epifluorescence microscope and a chromosome image analyzing system (CHIAS). A chloropiast in cultured cells duplicated DNA with an increase in size. When a chloroplast began to divide, it was constricted in the middle, taking a dumbbell shape, and then divided into two daughter chloroplasts. In cultured cells of this species, the pattern of quantitative change of chloroplast DNA, that is, the DNA replication pattern of chloroplasts, corresponded to that of cell nuclear DNA in mitosis.     

Random chloroplast segregation and frequent mtDNA rearrangements in fertile somatic hybrids between Nicotiana tabacum L. and N. glutinosa L.

Patterns of organelle inheritance were examined among fertile somatic hybrids between allotetraploid Nicotiana tabacum L. (2n=4x=48) and a diploid wild relative N. glutinosa L. (2n=2x=24). Seventy somatic hybrids resistant to methotrexate and kanamycin were recovered following fusion of leaf mesophyll protoplasts of transgenic methotrexate-resistant N. tabacum and kanamycin-resistant N. glutinosa. Evidence for hybridization of nuclear genomes was obtained by analysis of glutamate oxaloacetate transaminase and peroxidase isoenzymes and by restriction fragment length polymorphism (RFLP) analysis using a heterologous nuclear ribosomal DNA probe. Analysis of chloroplast genomes in a population of 41 hybrids revealed a random segregation of chloroplasts since 25 possessed N. glutinosa chloroplasts and 16 possessed N. tabacum chloroplasts. This contrasts with the markedly non-random segregation of plastids in N. tabacum (+)N. rustica and N. tabacum (+) N. debneyi somatic hybrids which we described previously and which were recovered using the same conditions for fusion and selection. The organization of the mitochondrial DNA (mtDNA) in 40 individuals was examined by RFLP analysis with a heterologous cytochrome B gene. Thirty-eight somatic hybrids possessed mitochondrial genomes which were rearranged with respect to the parental genomes, two carried mtDNA similar to N. tabacum, while none had mtDNA identical to N. glutinosa. The somatic hybrids were self-fertile and fertile in backcrosses with the tobacco parent.Contribution No. 1487 Plant Research Centre     

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.     

Comparative study of chloroplast morphology and ontogeny in <Emphasis Type="Italic">Asterochloris</Emphasis> (Trebouxiophyceae,Chlorophyta)

Confocal laser scanning microscopy was utilized to compare the chloroplast morphology and ontogeny among five strains of the green alga Asterochloris. Parsimony analysis inferred from the rDNA ITS sequences confirmed their placement in three distinct lineages: Asterochloris phycobiontica, Trebouxia pyriformis and Asterochloris sp. Examination by confocal microscopy revealed the existence of interspecific differences in the chloroplast ontogeny of Asterochloris; this was based upon either specific chloroplast structures observed in a single species, or on the differential timing of particular ontogenetic sequences. The occurrence of flat parietal chloroplasts prior to cell division, considered as a basic morphological discriminative character of Asterochloris, was clearly associated with the process of aplanosporogenesis. By contrast, chloroplast transformation prior to the formation of autospores proceeded simply by the multiple fission of the chloroplast matrix in the cell lumen. Presented at the International Symposium Biology and Taxonomy of Green Algae V, Smolenice, June 26–29, 2007, Slovakia.     

Structural and functional properties of the coleoptile chloroplast: Photosynthesis and photosensory transduction

Recent studies have shown that guard cell and coleoptile chloroplasts appear to be involved in blue light photoreception during blue light-dependent stomatal opening and phototropic bending. The guard cell chloroplast has been studied in detail but the coleoptile chloroplast is poorly understood. The present study was aimed at the characterization of the corn coleoptile chloroplast, and its comparison with mesophyll and guard cell chloroplasts. Coleoptile chloroplasts operated the xanthophyll cycle, and their zeaxanthin content tracked incident rates of solar radiation throughout the day. Zeaxanthin formation was very sensitive to low incident fluence rates, and saturated at around 800–1000 mol m^–2 s^–1. Zeaxanthin formation in corn mesophyll chloroplasts was insensitive to low fluence rates and saturated at around 1800 mol m^–2 s^–1. Quenching rates of chlorophyll a fluorescence transients from coleoptile chloroplasts induced by saturating fluence rates of actinic red light increased as a function of zeaxanthin content. This implies that zeaxanthin plays a photoprotective role in the coleoptile chloroplast. Addition of low fluence rates of blue light to saturating red light also increased quenching rates in a zeaxanthin-dependent fashion. This blue light response of the coleoptile chloroplast is analogous to that of the guard cell chloroplast, and implicates these organelles in the sensory transduction of blue light. On a chlorophyll basis, coleoptile chloroplasts had high rates of photosynthetic oxygen evolution and low rates of photosynthetic carbon fixation, as compared with mesophyll chloroplasts. In contrast with the uniform chloroplast distribution in the leaf, coleoptile chloroplasts were predominately found in the outer cell layers of the coleoptile cortex, and had large starch grains and a moderate amount of stacked grana and stroma lamellae. Several key properties of the coleoptile chloroplast were different from those of mesophyll chloroplasts and resembled those of guard cell chloroplasts. We propose that the common properties of guard cell and coleoptile chloroplasts define a functional pattern characteristic of chloroplasts specialized in photosensory transduction.Abbreviations Ant or A antheraxanthin - dv/dt fluorescence quenching rate - Fm maximum yield of fluorescence with all PS II reaction centers closed - Fo yield of instantaneous fluorescence with all PS II reaction centers open - Vio or V violaxanthin - Zea or Z zeaxanthin     

A rapid method for isolation of purified,physiologically active chloroplasts,used to study the intracellular distribution of amino acids in pea leaves

A procedure is described for the rapid (<5 min) isolation of purified, physiologically active chloroplasts from Pisum sativum L. Mitochondrial and microbody contamination is substantially reduced and broken chloroplasts are excluded by washing through a layer containing a treated silica sol. On average the preparations contain 93% intact chloroplasts and show high rates of ¹⁴CO₂ fixation and CO₂-dependent O₂ evolution (over 100 mol/mg chlorophyll(chl)/h); they are also able to carry out light-driven incorporation of leucine into protein (4 nmol/mg chl/h). The amino-acid contents of chloroplasts prepared from leaves and from leaf protoplasts have been determined. Asparagine is the most abundant amino acid in the pea chloroplast (>240 nmol/mg chl), even thought it is proportionately lower in the chloroplast relative to the rest of the cell. The chloroplasts contain about 20% of many of the amino acids of the cell, but for individual amino acids the percentage in the chloroplast ranges from 8 to 40% of the cell total. Glutamic acid, glutamine and aspartic acid are enriched in the chloroplasts, while asparagine, homoserine and -(isoxazolin-5-one-2-yl)-alanine are relatively lower. Leakage of amino acids from the chloroplast during preparation or repeated washing was ca. 20%. Some differences exist between the amino-acid composition of chloroplasts isolated from intact leaves and from protoplasts. In particular, -aminobutyric acid accumulates to high levels, while homoserine and glutamic acid decrease, during protoplast formation and breakage.     

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

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

The control of chloroplast number in wheat mesophyll cells

Chloroplast number per cell and mesophyll cell plan area were determined in populations of separated cells from the primary leaves of different wheat species representing three levels of ploidy. Mean chloroplast number per cell increases with ploidy level as mean cell size increases. But in addition the analysis of individual cells clearly shows that cells of a similar size but from species of different ploidies have similar numbers of chloroplasts. We conclude that the number of chloroplasts within a cell is closely correlated (P<0.001) with the size of the cell and this relationship is consistent for species of different ploidies over a wide range of cell sizes. These results are discussed in relation to the hypothesis that chloroplast number in leaf mesophyll cells is determined by the size of the cell.     

Sodium,potassium, chloride and proline concentrations of chloroplasts isolated from a halophyte,Mesembryanthemum crystallinum L.

Concentrations of four major solutes (Na⁺, K⁺, Cl^-, proline) were determined in isolated, intact chloroplasts from the halophyte Mesembryanthemum crystallinum L. following long-term exposure of plants to three levels of NaCl salinity in the rooting medium. Chloroplasts were obtained by gentle rupture of leaf protoplasts. There was either no or only small leakage of inorganic ions from the chloroplasts to the medium during three rapidly performed washing steps involving precipitation and re-suspension of chloroplast pellets. Increasing NaCl salinity of the rooting medium resulted in a rise of Na⁺ und Cl^- in the total leaf sap, up to approximately 500 and 400 mM, respectively, for plants grown at 400 mM NaCl. However, chloroplast levels of Na⁺ und Cl^- did not exceed 160–230 and 40–60 mM, respectively, based upon a chloroplast osmotic volume of 20–30 l per mg chlorophyll. At 20 mM NaCl in the rooting medium, the Na⁺/K⁺ ratio of the chloroplasts was about 1; at 400 mM NaCl the ratio was about 5. Growth at 400 mM NaCl led to markedly increased concentrations of proline in the leaf sap (8 mM) compared with the leaf sap of plants grown in culture solution without added NaCl (proline 0.25 mM). Although proline was fivefold more concentrated in the chloroplasts than in the total leaf sap of plants treated with 400 mM NaCl, the overall contribution of proline to the osmotic adjustment of chloroplasts was small. The capacity to limit chloroplast Cl^- concentrations under conditions of high external salinity was in contrast to an apparent affinity of chloroplasts for Cl^- under conditions of low Cl^- availability.Abbreviation Chl chlorophyll     

Fine Chloroplast Structure in Cucumber and Pea Leaves Developed under Red Light

Photosynthetic activity, the content of various photosynthetic pigments, and the chloroplast ultrastructure were examined in the leaves of cucumber (Cucumis sativus L.) and pea (Pisum sativum L.) plants of different ages grown under red light (600–700 nm, 100 W/m²). In pea leaves tolerant to red-light irradiation, chloroplast ultrastructure did not essentially change. In the first true leaves of cucumber plants susceptible to red-light irradiation, we observed a considerable increase in the number and size of plastoglobules, the appearance of chloroplasts lacking grana or containing only infrequent grana, and stromal thylakoids. In the upper leaves of 22-day-old cucumber plants, the chloroplast structure was essentially similar to that of the control chloroplasts in white light, and we therefore suppose that these plants have acclimated to red light.     

AUTORADIOGRAPHIC EVIDENCE FOR MANY SEGREGATING DNA MOLECULES IN THE CHLOROPLAST OF OCHROMONAS DANICA

Light-grown cells of Ochromonas danica, which contain a single chloroplast per cell, were labeled with [methyl-³H]thymidine for 3 h (0.36 generations) and the distribution of labeled DNA among the progeny chloroplasts was followed during exponential growth in unlabeled medium for a further 3.3 generations using light microscope autoradiography of serial sections of entire chloroplasts. Thymidine was specifically incorporated into DNA in both nuclei and chloroplasts. Essentially all the chloroplasts incorporated label in the 3-h labeling period, indicating that chloroplast DNA is synthesized throughout the cell cycle. Nuclear DNA has a more limited S period. Both chloroplast DNA and nuclear DNA are conserved during 3.3 generations. After 3.3 generations in unlabeled medium, grains per chloroplast followed a Poisson distribution indicating essentially equal labeling of all progeny chloroplasts. It is concluded that the average chloroplast in cells of Ochromonas growing exponentially in the light contains at least 10 segregating DNA molecules.     

Die Chloroplastendrehung beiMougeotia

Summary Mougeotia cells with chloroplasts oriented in profile have been irradiated with small spots of monochromatic red polarized light in order to induce chloroplast movement.In these experiments, four factors have been varied: 1. the orientation of the vibration plane of the light in relation to the cell axis, 2. the localization of the spot, i. e. irradiation of the chloroplast or the cytoplasm, 3. the spot size, and 4. the duration of the irradiation.As a result of our experiments, we conclude that the photoreceptor molecules responsible for the light-induced chloroplast movement are localized in the cytoplasm.As the photoreceptor of this reaction is the well known phytochromesystem, we may assume that also in other plants the phytochrome is localized in the cytoplasm rather than in the chloroplast.

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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     

THE ULTRAMICRO STRUCTURE OF STARCH-FREE CHLOROPLASTS OF FULLY EXPANDED LEAVES OF NICOTIANA RUSTICA

Weier , T. Elliot . (U. California, Davis.) The ultramicro structure of starch-free chloroplasts of fully expanded leaves of Nicotiana rustica. Amer. Jour. Bot. 48(7): 615–630. Illus. 1961.—The grana of starch-free chloroplasts of fully expanded leaves of Nicotiana rustica are distinct, compartmented, subplastid entities. They vary in size, shape, orientation and in the distinctness with which their compartments are delineated. It has not been possible to equate accurately their micro and ultramicro appearances. At the ultramicro level, the grana are connected with each other at irregular intervals by a system of anastomosing channels. The partitions forming the compartments of the grana may be coarse or very fine but are constant in appearance in any given chloroplast. The loculi enclosed by the partitions may vary in size with a granum, depending upon their location or upon the physiological activity of the chloroplast. The stroma does not penetrate the grana; it may be relatively fluid and the grana-fretwork system may move within it. A double envelope, which may have pores connecting stroma and hyaloplasm, surrounds the chloroplasts. Materials may collect between the surfaces of the envelope. There is considerable variation in the ultramicro details of chloroplast structure of Nicotiana rustica. It is not yet possible to distinguish accurately between those variations which may be of physiological significance and those which may be induced by processing.     

SPAD chlorophyll meter reading can be pronouncedly affected by chloroplast movement

Non-destructive assessment of chlorophyll content has recently been widely done by chlorophyll meters based on measurement of leaf transmittance (e.g. the SPAD-502 chlorophyll meter measures the leaf transmittance at 650 and 940 nm). However, the leaf transmittance depends not only on the content of chlorophylls but also on their distribution in leaves. The chlorophyll distribution within leaves is co-determined by chloroplast arrangement in cells that depends on light conditions. When tobacco leaves were exposed to a strong blue light (about 340 μmol of photons m⁻² s⁻¹), a very pronounced increase in the leaf transmittance was observed as chloroplasts migrated from face position (along cell walls perpendicular to the incident light) to side position (along cell walls parallel to the incoming light) and the SPAD reading decreased markedly. This effect was more pronounced in the leaves of young tobacco plants compared with old ones; the difference between SPAD values in face and side position reached even about 35%. It is shown how the chloroplast movement changes a relationship between the SPAD readings and real chlorophyll content. For an elimination of the chloroplast movement effect, it can be recommended to measure the SPAD values in leaves with a defined chloroplasts arrangement.     

Biochemical Oxidation of Reduced-form Cytochrome c by Mercuric Ions

A simple method has been developed for DNA isolation from purified chloroplasts of Marchantia polymorpha L. (liverwort) cell suspension cultures. Purified chloroplasts exhibited ribulose-bisphosphate carboxylase activity comparable to that of Fraction 1 protein obtained from Nicotiana tabacum. Fraction 1 protein isolated from purified chloroplasts clearly showed large and small subunits when subjected to isoelectric focussing. These results indicate that the purified chloroplasts are intact. DNA isolated from purified chloroplasts showed a covalently closed circular form, and restriction endonuclease digestions of the chloroplast DNA showed clear fragmentation indicating that the DNA was sufficiently free from those of other organelles.     

Arrangement of the ribosomal RNA genes in chloroplast DNA of Leguminosae

Summary The circular chloroplast DNA from three species of plants in the taxonomic family Leguminosae were examined using electron microscopic techniques and restriction endonuclease digestion. Chloroplast DNAs from chickpea (Cicer arietinum), mung bean (Vigna radiata), and soy bean (Glycine max) were found to range in size from 119–151 kilobase pairs by contour length measurements. Sizes of the chloroplast DNAs have been further confirmed using different restriction endonucleases. Two of the chloroplast DNAs examined, soy bean and mung bean, contain a region approximately 15.9–18% of their monomer length that is repeated in reverse polarity. This repeated region separates a small unique region that ranges in size from 18.75–20.4 kilobase pairs and a large unique region that ranges in size from 73.4–85 kbp. This feature was not found in the chloroplast DNA of chickpea. R-loop hybridizations performed using chloroplast ribosomal RNAs demonstrate that the two ribosomal gene sets of the mung been and soy bean are arranged in inverted orientation within this repeated region. In contrast, the chickpea chloroplast DNA posesses a single ribosomal RNA gene set in the circular molecule. In all three chloroplast DNAs examined, the genes encoding the chloroplast 23S and 16S ribosomal RNA genes are separated by a spacer region which ranges in size from 2.2 to 2.48 kbp.     

The apparent absence of a pathway for synthesis of acetyl coenzyme A in pea chloroplasts: Lack of 14CO2 incorporation into lipids by the isolated organelle

Summary We have examined the extent to which isotopic lable derived from photosynthetically fixed ¹⁴CO₂ can be transferred to lipids by aqueously isolated chloroplasts of Pisum sativum. Although photosynthetically active, chloroplast preparations incubated with ¹⁴CO₂ showed little or no accumulation of label in lipids under any condition tested. Under identical conditions the chloroplasts were readily able to incorporate [¹⁴C]acetate into the lipid fraction; a fatty-acid synthesizing system was therefore operative in these chloroplasts.The essential failure of the isolated chloroplasts to incorporate label from fixed ¹⁴CO₂ into fatty acids supports the view that the organelle itself does not possess a self-contained pathway for the synthesis of acetyl coenzyme A, and favours the possibility that a shuttle mechanism involving the participation of extra-chloroplastic enzymes may be responsible for supplying the chloroplast with acetyl coenzyme A in vivo.