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.     

Variation in Mesophyll Cell Number and Size in Wheat Leaves

The numbers of mesophyll cells in wheat leaves were determinedin a variety of wheat species differing in ploidy level andin leaves from different positions on the wheat plant. Leafsize and mesophyll cell number are linearly related in bothcases but differences were observed in mesophyll cell numberper unit leaf area with changing leaf size. Where changes incell size are caused either by nuclear ploidy or leaf position,differences in mesophyll cell number per unit leaf are negativelycorrelated with mesophyll cell plan area. The decrease in cellsize with increasing leaf position also results in a greaternumber of chloroplasts per unit leaf area. These results arediscussed in relation to anatomical variation of the wheat leaf. Mesophyll cell, cell numbers, leaf size, Triticum     

Cellular Levels of Ribulose 1,5 Bisphosphate Carboxylase and Chloroplast Compartment Size in Wheat Mesophyll Cells

Pyke, K. A. and Leech, R. M. 1987. Cellular levels of ribulose1,5 bisphosphate carboxylase and chloroplast compartment sizein wheat mesophyll cells.—J. exp. Bot. 38: 1949–1956. The amount of the photosynthetic enzyme ribulose 1,5 bisphosphatecarboxylase (RUBISCO),as determined in mesophyll cells in primarywheat leaves was related to the size of the chloroplast compartmentwithin the cell for wheat species of three ploidy levels. Asimilar comparison was made for several genotypes of the hexaploidbreadwheat Triticum aestivum. Estimation of total chloroplastvolume per mesophyll cell was made assuming chloroplasts tobe oblate spheroid in shape. A significant correlation was found between the amount of RUBISCOper cell and the total chloroplast volume per cell for diploid,tetraploid and hexaploid wheat species. A significant correlationbetween cellular RUBISCO level and total chloroplast volumeper cell was also observed for a range of genotypes of the hexaploidT. aestivum but these genotypes of T. aestivutn accumulate agreater amount of RUBISCO per unit chloroplast volume than doany other wheat species. For these genotypes of T. aestivumthe stromal concentration of RUBISCO was estimated at 0·5mol m^–3 with a ribulose Msphosphate binding site concentrationof 4·0 mol m^–3. These results are discussed with respect to a gene dosage hypothesisto explain the accumulation of RUBISCO in leaf mesophyll cells. Key words: Ribulose, bisphosphate carboxylase, wheat chloroplasts, mesophyll cells     

Genome-Dependent Factors in the Development of Leaf Phototrophic Tissues in Diploid and Alloploid Wheat Species

Growth and mesostructure of the photosynthetic apparatus were studied in leaves of ten Triticum L. species. Plants with the A^u genome were shown to develop larger leaf assimilation areas due to expanding areas of individual leaves and an increase in the absolute growth rate. Leaf and mesophyll thickness and mesophyll cell size decreased in the G-genome species. Leaf compactness, which depended on cell size and number per unit leaf area and leaf folding, determined the specific patterns of internal leaf organization in wheat species with diverse genotypes. These patterns did not affect cell plastid-to-cytoplasm ratio as shown by the stable indices of cell surface area/cell volume, cell surface area per chloroplast, and cell volume per chloroplast. The structural indices of leaf phototrophic tissues, mesophyll density, and mesophyll CO₂ conductance in alloploids, as compared to diploid species, depended on both ploidy and genome constitution.     

Interpretations on chloroplast reproduction derived from correlations between cells and chloroplasts

Summary The size ranges of chloroplasts in living mesophyll cells of Spinacia oleracea, Allium cepa, Beta vulgaris (Swiss chard and red beet) and Nicotiana glutinosa are extremely wide, e.g., ranging from about 6 µ² to 103 µ² in face area for spinach. Moreover, the size distributions are positively skewed. We interpret the size range and skewed size distributions primarily to reflect an enormous growth of the bulk of the chloroplasts from small, equal-sized chloroplasts produced by fission of a small sub-population of constricted mature chloroplasts. While actual fission has never been observed, a slow division rate of the constricted chloroplasts in N. glutinosa can account for the increase in chloroplast numbers per cell during leaf development and for the presence of small, non-constricted chloroplasts after the small chloroplasts which developed during the initial meristem activity have enlarged. Chloroplast numbers and total amount of chloroplast material per cell face were positively correlated with mesophyll-cell face size. However, the fraction of the cell face occupied with chloroplasts was essentially constant and independent of cell size and cell age while being markedly different for different species of plants. There appear to be some family characteristics in that closely related species have similar size-distributions and ranges of chloroplast sizes. The observations are discussed with respect to the ontogeny of chloroplasts in higher plants.     

Structural Adaptation of the Leaf Mesophyll to Shading

Structural characteristics of the mesophyll were studied in five boreal grass species experiencing a wide range of light and water supply conditions. Quantitative indices of the palisade and spongy mesophyll tissues (cell and chloroplast sizes, the number of chloroplasts per cell, the total cell and chloroplast surface area per unit leaf surface area) were determined in leaves of each of the species. The cell surface area and the cell volume in spongy mesophyll were determined with a novel method based on stereological analysis of cell projections. An important role of spongy parenchyma in the photosynthetic apparatus was demonstrated. In leaves of the species studied, the spongy parenchyma constituted about 50% of the total volume and 40% of the total surface area of mesophyll cells. The proportion of the palisade to spongy mesophyll tissues varied with plant species and growth conditions. In a xerophyte Genista tinctoria, the total cell volume, cell abundance, and the total surface area of cells and chloroplasts were 30–40% larger in the palisade than in the spongy mesophyll. In contrast, in a shade-loving species Veronica chamaedris, the spongy mesophyll was 1.5–2 times more developed than the palisade mesophyll. In mesophyte species grown under high light conditions, the cell abundance and the total cell surface area were 10–20% greater in the palisade mesophyll than in the spongy parenchyma. In shaded habitats, these indices were similar in the palisade and spongy mesophyll or were 10–20% lower in the palisade mesophyll. In mesophytes, CO₂ conductance of the spongy mesophyll accounted for about 50% of the total mesophyll conductance, as calculated from the structural characteristics, with the mesophyll CO₂ conductance increasing with leaf shading.     

Plastid division and morphology in the genus Peperomia

We have investigated several factors determining plastid size and number in Peperomia, a genus in the Piperaceae family whose species naturally display great interspecific variation in chloroplast size and number per cell. Using microscopic techniques, we show that chloroplast size and number are differently regulated in the palisade parenchyma and the spongy parenchyma, suggesting that chloroplast division in these cell types is controlled in different ways. Microscopic studies of iodine-stained root cells revealed a correlation between amyloplast size in root cells and chloroplast size in palisade parenchyma cells. However, despite substantial variation in chloroplast number in leaf mesophyll cells, amyloplast number in root cells was very similar in all species. The results suggest that organelle size and number are regulated in a tissue-specific manner rather than in dependency on the plastid type. We also demonstrate that plastid size determines the size but not the number of starch grains in root amyloplasts.     

Shading-induced changes in the leaf mesophyll of plants of different functional types

Changes in the structural characteristics of mesophyll induced by shading were investigated in ten species of wild plants of diverse functional types. In all plant types, shading reduced leaf thickness and density by 30–50% and total surface of mesophyll, by 30–70%. The extent and mechanisms of mesophyll structural rearrangement depended on the plant functional type. In the ruderal plants, integral parameters of mesophyll, such as the surface of cells and chloroplasts and mesophyll resistance, changed threefold predominantly because of changes in the dimensions of the cells and chloroplasts. In these plants, shading reduced the volume of chloroplasts by 30%, and the chloroplast numbers per cell declined. The competitor plants showed a twofold increase in mesophyll resistance due to a decrease in the number of photosynthesizing cells per leaf area unit. Moreover, these plants maintained constant dimensions of mesophyll cells, ratios mesophyll surface/mesophyll volume and chloroplast surface/cell surface. In stress-tolerant plants, diffusion resistance of mesophyll remained the same irrespective of the growing conditions, and mesophyll rearrangement was associated with inversely proportional changes in the dimensions of the cells and cell volume per chloroplast. Noteworthy of these plants were relatively constant chloroplasts number per cell, per leaf area unit and total surface area of chloroplasts. The nature of relationship between the mesophyll diffusion resistance and structural parameters of leaf mesophyll differed in plants of diverse functional types.     

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.     

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.     

Acetyl coenzyme A carboxylase in species of Triticum of different ploidy

The cellular amounts and cellular activities of acetyl CoA carboxylase (ACC; EC 6.4.1.2.) were determined in the first leaves of diploid, tetraploid and hexaploid species of Triticum (wheat). Per leaf the ACC activities were very similar in T. monococcum (2 ), T. dicoccum (4 ) and T. aestivum (6 ). The ACC activity per chloroplast also showed little variation between species of different ploidy but since chloroplast number increases with ploidy, the ACC activities and ACC amounts per cell also increased with ploidy. These cellular increases in ACC amounts associated with increases in gene dosage were highly co-ordinated in the diploids T. monococcum and T. tauschii and their respective autotetraploids so the specific activity of ACC was highly conserved in these plants. The relevance of these findings to attempts to genetically manipulate lipid biosynthesis in chloroplasts is discussed.Abbreviation ACC acetyl CoA carboxylase We are very grateful to Dr. Kevin Pyke and Miss Jo Marrison for many helpful discussions and to Dr. Collin Law for the generous gift of seeds.     

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.     

Photosynthesis in <Emphasis Type="Italic">Arabidopsis thaliana</Emphasis> Mutants with Reduced Chloroplast Number

We have used a class of Arabidopsis mutants altered in the accumulation and replication of chloroplasts (arc mutants) to investigate the effect of reduced chloroplast number on the photosynthetic competence of leaves. Each of the arc mutants examined (arc3, arc5, and arc6) accumulate only a few (2–15) large chloroplasts per mesophyll cell [K.A. Pyke and R.M. Leech (1992) Plant Physiology 99: 1005–1008]. The increased plastid size maintains a constant plastid to mesophyll cell volume, which has been suggested to compensate for the lower chloroplast number. In fact, we find that reduced chloroplast number has an effect on both the composition and structure of the photosynthetic apparatus, and that each arc mutant has an altered photosynthetic capacity, and we conclude that photosynthetic competence is dependent on proper chloroplast division and development.     

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.     

Leaf structure and specific leaf mass: the alpine desert plants of the Eastern Pamirs, Tadjikistan

This study examines interrelationships between eight leaf attributes (specific leaf mass, area, dry mass, lamina thickness, mesophyll cell number per cm², mesophyll cell volume, chloroplast volume, and number of chloroplasts per mesophyll cell) in field-grown plants of 94 species from the Eastern Pamir Mountains, at elevations between 3800 and 4750 m. Unlike most other mountain areas, the Eastern Pamirs, Karakorum system, Tadjikistan provide localities where low temperatures and radiation combine with moisture stress at high altitudes. For all the attributes measured, significant differences were found between plants with different mesophyll types. Leaves with dorsiventral palisade structure (dorsal palisade, ventral spongy mesophyll cells) had thicker leaves with larger but fewer mesophyll cells, containing more and larger chloroplasts. These differences in mesophyll type are reflected in differences in the total surface of mesophyll cells per unit leaf area ( A _mes/ A ) or volume ( A _mes/ V ). Plants with isopalisade leaf structure (palisade cells under both dorsal and ventral surfaces) are more commonly xerophytes and their increased values of A _mes/ A and A _mes/ V decrease CO₂ mesophyll resistance, which is an important adaptation to drought. Path analysis shows the critical importance of mesophyll cell volume in leading to the covariance between the different leaf attributes and hence to specific leaf mass (SLM), even though mesophyll cell volume is not itself strongly correlated with SLM. This is because mesophyll cell volume increases SLM through its effects on leaf thickness and chloroplast number per cell, but decreases SLM through its negative effect on mesophyll cell density.     

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.     

Over-expression of gsh1 in the cytosol affects the photosynthetic apparatus and improves the performance of transgenic poplars on heavy metal-contaminated soil

Recent studies of transgenic poplars over‐expressing the genes gsh1 and gsh2 encoding γ‐glutamylcysteine synthetase (γ‐ECS) and glutathione synthetase, respectively, provided detailed information on regulation of GSH synthesis, enzymes activities and mRNA expression. In this experiment, we studied quantitative parameters of leaves, assimilating tissues, cells and chloroplasts, mesophyll resistance for CO₂ diffusion, chlorophyll and carbohydrate content in wild‐type poplar and transgenic plants over‐expressing gsh1 in the cytosol after 3 years of growth in relatively clean (control) or heavy metal‐contaminated soil in the field. Over‐expression of gsh1 in the cytosol led to a twofold increase of intrafoliar GSH concentration and influenced the photosynthetic apparatus at different levels of organisation, i.e., leaves, photosynthetic cells and chloroplasts. At the control site, transgenic poplars had a twofold smaller total leaf area per plant and a 1.6‐fold leaf area per leaf compared to wild‐type controls. Annual aboveground biomass gain was reduced by 50% in the transgenic plants. The reduction of leaf area of the transformants was accompanied by a significant decline in total cell number per leaf, indicating suppression of cell division. Over‐expression of γ‐ECS in the cytosol also caused changes in mesophyll structure, i.e., a 20% decrease in cell and chloroplast number per leaf area, but also an enhanced volume share of chloroplasts and intercellular airspaces in the leaves. Transgenic and wild poplars did not exhibit differences in chlorophyll and carotenoid content of leaves, but transformants had 1.3‐fold fewer soluble carbohydrates. Cultivation on contaminated soil caused a reduction of palisade cell volume and chloroplast number, both per cell and leaf area, in wild‐type plants but not in transformants. Biomass accumulation of wild‐type poplars decreased in contaminated soil by more than 30‐fold, whereas transformants showed a twofold decrease compared to the control site. Thus, poplars over‐expressing γ‐ECS in the cytosol were more tolerant to heavy metal stress under field conditions than wild‐type plants according to the parameters analysed. Correlation analysis revealed strong dependence of cell number per leaf area unit, chloroplast parameters and mesophyll resistance with the GSH level in poplar leaves.     

Arabidopsis thaliana leaves with altered chloroplast numbers and chloroplast movement exhibit impaired adjustments to both low and high light

The effects of chloroplast number and size on the capacity for blue light-dependent chloroplast movement, the ability to increase light absorption under low light, and the susceptibility to photoinhibition were investigated in Arabidopsis thaliana. Leaves of wild-type and chloroplast number mutants with mean chloroplast numbers ranging from 120 to two per mesophyll cell were analysed. Chloroplast movement was monitored as changes in light transmission through the leaves. Light transmission was used as an indicator of the ability of leaves to optimize light absorption. The ability of leaves to deal with 3 h of high light stress at 10 degrees C and their capacity to recover in low light was determined by measuring photochemical efficiencies of PSII using chlorophyll a fluorescence. Chloroplast movement was comparable in leaves ranging in chloroplast numbers from 120 to 30 per mesophyll cell: the final light transmission levels after exposure to 0.1 (accumulation response) and 100 micromol photons m(-2) s(-1) (avoidance response) were indistinguishable, the chloroplasts responded quickly to small increases in light intensity and the kinetics of movement were similar. However, when chloroplast numbers per mesophyll cell decreased to 18 or below, the accumulation response was significantly reduced. The avoidance response was only impaired in mutants with nine or fewer chloroplasts, both in terms of final transmission levels and the speed of movement. Only mutants lacking both blue light receptors (phot1/phot2) or those with drastically reduced chloroplast numbers and severely impacted avoidance responses showed a reduced ability to recover from high light stress.     

Mesophyll cells of C4 plants have fewer chloroplasts than those of closely related C3 plants

The evolution of C₄ photosynthesis from C₃ ancestors eliminates ribulose bisphosphate carboxylation in the mesophyll (M) cell chloroplast while activating phosphoenolpyruvate (PEP) carboxylation in the cytosol. These changes may lead to fewer chloroplasts and different chloroplast positioning within M cells. To evaluate these possibilities, we compared chloroplast number, size and position in M cells of closely related C₃, C₃–C₄ intermediate and C₄ species from 12 lineages of C₄ evolution. All C₃ species had more chloroplasts per M cell area than their C₄ relatives in high‐light growth conditions. C₃ species also had higher chloroplast coverage of the M cell periphery than C₄ species, particularly opposite intercellular air spaces. In M cells from 10 of the 12 C₄ lineages, a greater fraction of the chloroplast envelope was pulled away from the plasmalemma in the C₄ species than their C₃ relatives. C₃–C₄ intermediate species generally exhibited similar patterns as their C₃ relatives. We interpret these results to reflect adaptive shifts that facilitate efficient C₄ function by enhancing diffusive access to the site of primary carbon fixation in the cytosol. Fewer chloroplasts in C₄ M cells would also reduce shading of the bundle sheath chloroplasts, which also generate energy required by C₄ photosynthesis.     

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.