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

Abstract

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.The attainment of photosynthetic competence in leaves is entirely dependent on the authentic development of chloroplasts. Chloroplasts arise from undifferentiated plastids in small mitotic cells and replicate during normal mesophyll cell development (1, 5, 7). As a result, the number of chloroplasts increases dramatically during mesophyll cell expansion producing a large population of chloroplasts in each mature mesophyll cell. Chloroplast population size is species specific, can be variety specific, and is always closely related to mesophyll cell size (2,4,8 analysis screening after iodine staining of isolated cells (9). The plants were grown as described previously (9) and first leaves were harvested during the course of leaf expansion; for wild type between 9 and 22 d after sowing and between 14 and 33 d after sowing for mutants. The oldest leaves sampled in each line were fully expanded and the mean mesophyll cell plan area for the whole leaf was also maximal. Preparation of Mesophyll Cell Suspensions for Chloroplast CountingThe numbers of chloroplasts in individual isolated fixed mesophyll cells in a representative population from the whole leaf were counted using Nomarski differential interference contrast optics (9). Mesophyll cell plan areas were measured using an image analysis system (Seescan Imaging Ltd, Cambridge, UK) linked to a Nikon Optiphot microscope (9). In mesophyll cells in which chloroplast numbers were counted, plan areas of 5 to 15 individual chloroplasts were also measured using the same image analysis procedures as used for cell plan area measurements. For each cell, the total chloroplast plan area is calculated as number of chloroplasts x mean chloroplast plan area. RESULTSDuring wild-type mesophyll cell development, the number of chloroplasts per cell is closely correlated with the size of the cell (r2 = 0.86, Fig. la). In three different mutants of Arabidopsis (Fig. 1, b-d), this relationship between cell plan area and chloroplast number is significantly different from that shown in wild-type cells. In mutant arcl (Fig. lb), there is an increased rate of chloroplast accumulation during cell expansion compared with wild type, producing an increase in chloroplast number per cell. In contrast, mutant arc2 (Fig. lc) has a reduced rate of chloroplast accumulation during cell expansion, producing fewer chloroplasts per cell. In the most extreme mutant, arc3, chloroplas...

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