Rachel M. Leech

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.

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.

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.

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.

Significant changes in cell and chloroplast development in young wheat leaves (Triticum aestivum cv Hereward) grown in elevated CO2

Cell and chloroplast development were characterized in young Triticum aestivum cv Hereward leaves grown at ambient (350 [mu]L L-1) or at elevated (650 [mu]L L-1) CO2. In elevated CO2, cell and chloroplast expansion was accelerated by 10 and 25%, respectively, in the first leaf of 7-d-old wheat plants without disruption to the leaf developmental pattern. Elevated CO2 did not affect the number of chloroplasts in relation to mesophyll cell size or the linear relationship between chloroplast number or size and mesophyll cell size. No major changes in leaf anatomy or in chloroplast ultrastructure were detected as a result of growth in elevated CO2, but there was a marked reduction in starch accumulation. In leaf sections fluorescently tagged antisera were used to visualize and quantitate the amount of cytochrome f, the [alpha]- and [beta]-subunits of the coupling factor 1 in ATP synthase, D1 protein of the photosystem II reaction center, the 33-kD protein of the extrinsic oxygen-evolving complex, subunit II of photosystem I, and ribulose-1,5-bisphosphate carboxylase/oxygenase. A significant finding was that in 10 to 20% of the mesophyll cells grown in elevated CO2 the 33-kD protein of the extrinsic oxygen-evolving complex of photosystem II and cytochrome f were deficient by 75%, but the other proteins accumulated normally.

Mitochondria Increase Three-Fold and Mitochondrial Proteins and Lipid Change Dramatically in Postmeristematic Cells in Young Wheat Leaves Grown in Elevated CO2.

A dramatic stimulation in mitochondrial biogenesis during the very early stages of leaf development was observed in young wheat plants (Triticum aestivum cv Hereward) grown in elevated CO2 (650 [mu]L L-1). An almost 3-fold increase in the number of mitochondria was observed in the very young leaf cells at the base of the first leaf of a 7-d-old wheat plant. In the same cells large increases in the accumulation of a mitochondrial chaperonin protein and the mitochondrial 2-oxoglutarate dehydrogenase complex and pyruvate dehydrogenase complex were detected by immunolabeling. Furthermore, the basal segment also shows a large increase in the rate of radiolabeling of diphosphatidylglycerol, a lipid confined to the inner mitochondrial membrane. This dramatic response in very young leaf cells to elevated CO2 suggests that the numerous documented positive effects of elevated CO2 on wheat leaf development are initiated as early as 12 h postmitosis.

The pathway of [14C]bicarbonate incorporation into lipids in isolated photosynthesising spinach chloroplasts

Intact photosynthesising spinach chloroplasts incorporate [14C]bicarbonate, [2-14C]pyruvate and [1-14C] acetate into acyl lipids [ 1 ]. With each substrate, free fatty acids, monoacylglycerols and diacylglycerols become radioactively labelled and rates of incorporation increase after the chloroplasts are purified on a sorbitol density gradient. These results suggest that acetyl-CoA can be synthesised from bicarbonate within the chloroplast. The details of this chloroplast-located biosynthesis of acetyl-CoA are unknown, but one possible pathway would be via photosynthetically-reduced 3-phosphoglycerate and phosphoenolpyruvate [1,2]. We present here evidence from a comparison of labelling patterns of lipids and fatty acids from the different postulated precursors and also evidence from isotope competition experiments which are consistent with the operation of a pathway from CO2 via 3-PGA, PEP and pyruvate to acetyl-CoA in isolated spinach chloroplasts.

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.

Lipid biosynthesis by chloroplasts isolated from developing Zea mays

Abstract Fatty acid biosynthesis by isolated plastids has been examined in relation to chloroplast development and differentiation in leaves of maize plants grown in light for 7 days. Biosynthesis of fatty acids from acetate by proplastids prepared from the basal regions of the leaf was low and mainly palmitate was synthesized. The greatly increased utilization of acetate for fatty acid biosynthesis as the plastids increased in size was due to an increased synthesis of oleate. The maximum synthesis of total fatty acids and monoenoic fatty acids was obtained in chloroplasts prepared from leaf tissue 6–8 cm from the base of the plant where granal formation was most active. Fully-developed chloroplasts prepared from distal regions of the leaf were less active in fatty acid biosynthesis. Maize chloroplasts failed to synthesize fatty acids when isolated by methods commonly used to prepare active spinach chloroplasts. The method of isolation which included a density gradient gave a high proportion of Class I chloroplasts from maize leaves and incorporated up to about 10% of the acetate used. Biosynthesis of unsaturated fatty acids, especially with chloroplasts prepared from the most mature tissue, was increased by the addition of both mitochondrial and microsomal fractions. Increases in polyunsaturated fatty acids were also obtained but the proportions in the newly-synthesized fatty acids were well below the endogenous levels. Monoenoic synthesis was greatly stimulated by increasing the pH in the range 7·0–8·0 and also the highest proportions of unsaturated fatty acids were obtained at short incubation times.

Evidence for a type II topoisomerase in wheat chloroplasts

Western blotting of total cell extracts from the young developing wheat leaf with an antibody to yeast topoisomerase II locates two proteins of molecular mass 96–101 kDa which also appear on blots of chloroplast proteins. The maximal levels of both proteins are present in chloroplasts in which chloroplast DNA replication is occurring prior to chloroplast division.