Variation in vein density and mesophyll cell architecture in a rice deletion mutant population.

There is a need to develop rice plants with improved photosynthetic capacity and efficiency in order to enhance potential grain yield. Alterations in internal leaf morphology may be needed to underpin some of these improvements. One target is the production of a 'Kranz-like' anatomy, commonly considered to be required to achieve the desired levels of photosynthesis seen in C(4) crops. Kranz anatomy typically has two or three mesophyll cells interspersing adjacent veins. As a first step to determining the potential for such anatomical modifications in rice leaves, a population of rice deletion mutants was analysed for alterations in vein patterning and mesophyll cells in the interveinal regions. Significant variation is demonstrated in vein arrangement and the sequential distribution of major and minor veins across the leaf width, although there is a significant correlation between the total number of veins present and the width of the leaf. Thus the potential is demonstrated for modifying rice leaf structure. Six distinct rice mutant lines, termed altered leaf morphology (alm) mutants, were analysed for the architecture of their interveinal mesophyll cell arrangement. It is shown that in these mutant lines, the distance between adjacent minor veins and adjacent minor and major veins is essentially determined by the size of the interveinal mesophyll cells rather than changes in mesophyll cell number across this region, and hence interveinal distance changes as a result of cell expansion rather than cell division. This observation will be important when developing screens for traits relevant for the introduction of Kranz anatomy into rice.

Increases in cell elongation, plastid compartment size and phytoene synthase activity underlie the phenotype of the high pigment-1 mutant of tomato.

A characteristic trait of the high pigment-1 ( hp-1) mutant phenotype of tomato ( Lycopersicon esculentum Mill.) is increased pigmentation resulting in darker green leaves and a deeper red fruit. In order to determine the basis for changes in pigmentation in this mutant, cellular and plastid development was analysed during leaf and fruit development, as well as the expression of carotenogenic genes and phytoene synthase enzyme activity. The hp-1 mutation dramatically increases the periclinal elongation of leaf palisade mesophyll cells, which results in increased leaf thickness. In addition, in both palisade and spongy mesophyll cells, the total plan area of chloroplasts per cell is increased compared to the wild type. These two perturbations in leaf development are the primary cause of the darker green hp-1 leaf. In the hp-1 tomato fruit, the total chromoplast area per cell in the pericarp cells of the ripe fruit is also increased. In addition, although expression of phytoene synthase and desaturase is not changed in hp-1 compared to the wild type, the activity of phytoene synthase in ripe fruit is 1.9-fold higher, indicating translational or post-translational control of carotenoid gene expression. The increased plastid compartment size in leaf and fruit cells of hp-1 is novel and provides evidence that the normally tightly controlled relationship between cell expansion and the replication and expansion of plastids can be perturbed and thus could be targeted by genetic manipulation.

A homologue of the bacterial cell division site-determining factor MinD mediates placement of the chloroplast division apparatus.

BACKGROUND: Chloroplast division in plant cells occurs by binary fission, yielding two daughter plastids of equal size. Previously, we reported that two Arabidopsis homologues of FtsZ, a bacterial protein that forms a cytokinetic ring during cell division, are essential for plastid division in plants, and may be involved in the formation of plastid-dividing rings on both the stromal and cytosolic surfaces of the chloroplast envelope membranes. In bacteria, positioning of the FtsZ ring at the center of the cell is mediated in part by the protein MinD. Here, we identified AtMinD1, an Arabidopsis homologue of MinD, and investigated whether positioning of the plastid-division apparatus at the plastid midpoint might involve a mechanism similar to that in bacteria. RESULTS: Sequence analysis and in vitro chloroplast import experiments indicated that AtMinD1 contains a transit peptide that targets it to the chloroplast. Transgenic Arabidopsis plants with reduced AtMinD1 expression exhibited variability in chloroplast size and number and asymmetrically constricted chloroplasts, strongly suggesting that the plastid-division machinery is misplaced. Overexpression of AtMinD1 inhibited chloroplast division. These phenotypes resemble those of bacterial mutants with altered minD expression. CONCLUSIONS: Placement of the plastid-division machinery at the organelle midpoint requires a plastid-targeted form of MinD. The results are consistent with a model whereby assembly of the division apparatus is initiated inside the chloroplast by the plastidic form of FtsZ, and suggest that positioning of the cytosolic components of the apparatus is specified by the position of the plastidic components.

The phosphoenolpyruvate/phosphate translocator is required for phenolic metabolism, palisade cell development, and plastid-dependent nuclear gene expression.

The Arabidopsis chlorophyll a/b binding protein (CAB) gene underexpressed 1 (cue1) mutant underexpresses light-regulated nuclear genes encoding chloroplast-localized proteins. cue1 also exhibits mesophyll-specific chloroplast and cellular defects, resulting in reticulate leaves. Both the gene underexpression and the leaf cell morphology phenotypes are dependent on light intensity. In this study, we determine that CUE1 encodes the plastid inner envelope phosphoenolpyruvate/phosphate translocator (PPT) and define amino acid residues that are critical for translocator function. The biosynthesis of aromatics is compromised in cue1, and the reticulate phenotype can be rescued by feeding aromatic amino acids. Determining that CUE1 encodes PPT indicates the in vivo role of the translocator in metabolic partitioning and reveals a mesophyll cell-specific requirement for the translocator in Arabidopsis leaves. The nuclear gene expression defects in cue1 suggest that a light intensity-dependent interorganellar signal is modulated through metabolites dependent on a plastid supply of phosphoenolpyruvate.

Bundle sheath cells and cell-specific plastid development in Arabidopsis leaves.

Bundle sheath cells form a sheath around the entire vascular tissue in Arabidopsis leaves and constitute a distinct leaf cell type, as defined by their elongate morphology, their position adjacent to the vein and by differences in their chloroplast development compared to mesophyll cells. They constitute about 15% of chloroplast-containing cells in the leaf. In order to identify genes which play a role in the differential development of bundle sheath and mesophyll cell chloroplasts, a screen of reticulate leaf mutants of Arabidopsis was used to identify a new class of mutants termed dov (differential development of vascular-associated cells). The dov1 mutant clearly demonstrates a cell-specific difference in chloroplast development. Mutant leaves are highly reticulate with a green vascular pattern. The underlying bundle sheath cells always contain normal chloroplasts, whereas chloroplasts in mesophyll cells are abnormal, reduced in number per cell and seriously perturbed in morphology at the ultrastructural level. This demonstrates that differential chloroplast development occurs between the bundle sheath and mesophyll cells in the Arabidopsis leaf.

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.

Plastid division: evidence for a prokaryotically derived mechanism.

Plastid division is a critical process in plant cell biology but it is poorly understood. Recent studies combining mutant analysis, gene cloning, and exploitation of genomic resources have revealed that the molecular machinery associated with plastid division is derived evolutionarily from the bacterial cell division apparatus. Comparison of the two processes provides a basis for identifying new components of the plastid division mechanism, but also serves to highlight the differences, not least of which is the nuclear control of the plastid division process.

arc6, an extreme chloroplast division mutant of Arabidopsis also alters proplastid proliferation and morphology in shoot and root apices.

The arc6 (accumulation and replication of chloroplasts) mutant of Arabidopsis has only two greatly enlarged chloroplasts per mature leaf mesophyll cell compared with ninety chloroplasts per cell in the wild type. The mutation is a single nuclear gene and the plant phenotype is normal. Shoot and root apical meristems of arc6 plants have been examined to determine how early during plastid development the mutant arc6 phenotype can be recognised. In the cells of the arc6 apical meristem there are only two proplastids, which are larger than wild type with a highly variable morphology. In the cells of the leaf primordia where differentiation of proplastids to chloroplasts occurs arc6 plastids are larger and at a more advanced developmental stage than wild-type plastids. In arc6 root cells statoliths and other plastids also show grossly abnormal morphology and the statoliths are greatly increased in size. During arc6 stomatal guard cell development the perturbation in proplastid population dynamics affects plastid segregation and 30% of stomata lack plastids in one or both guard cells. Our evidence would suggest that ARC6 is expressed throughout the vegetative cells of the Arabidopsis seedling with major effects on both the proplastid phenotype and the proplastid population. ARC6 is the first gene to be identified in Arabidopsis which has a global effect on plastid development in cells arising from both the shoot and root meristems, and is of major importance in the nuclear control of plastid differentiation in higher plants.

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.

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.

Chloroplast Division and Expansion Is Radically Altered by Nuclear Mutations in Arabidopsis thaliana.

We have isolated three mutants of Arabidopsis thaliana in which there is a sevenfold change in chloroplast number in fully expanded leaf mesophyll cells and increases and decreases in chloroplast number are compensated for by changes in chloroplast size. The changes are stably inherited in sexual crosses for three generations and mutant phenotypes are effected by changes at single recessive nuclear loci, termed arc loci. This is the first report of large, stably inherited changes in chloroplast number in higher plants, and represents a major advance toward the genetic dissection of the control of chloroplast division.

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.

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.