REDUCED CHLOROPLAST COVERAGE genes from Arabidopsis thaliana help to establish the size of the chloroplast compartment

Eukaryotic cells require mechanisms to establish the proportion of cellular volume devoted to particular organelles. These mechanisms are poorly understood. From a screen for plastid-to-nucleus signaling mutants in Arabidopsis thaliana, we cloned a mutant allele of a gene that encodes a protein of unknown function that is homologous to two other Arabidopsis genes of unknown function and to FRIENDLY, which was previously shown to promote the normal distribution of mitochondria in Arabidopsis. In contrast to FRIENDLY, these three homologs of FRIENDLY are found only in photosynthetic organisms. Based on these data, we proposed that FRIENDLY expanded into a small gene family to help regulate the energy metabolism of cells that contain both mitochondria and chloroplasts. Indeed, we found that knocking out these genes caused a number of chloroplast phenotypes, including a reduction in the proportion of cellular volume devoted to chloroplasts to 50% of wild type. Thus, we refer to these genes as REDUCED CHLOROPLAST COVERAGE (REC). The size of the chloroplast compartment was reduced most in rec1 mutants. The REC1 protein accumulated in the cytosol and the nucleus. REC1 was excluded from the nucleus when plants were treated with amitrole, which inhibits cell expansion and chloroplast function. We conclude that REC1 is an extraplastidic protein that helps to establish the size of the chloroplast compartment, and that signals derived from cell expansion or chloroplasts may regulate REC1.Chloroplasts drive plant growth, development, and reproduction by converting solar energy into biologically useful forms of energy. Thus, the biogenesis and function of chloroplasts underpin crop yields and, indeed, life on Earth. Chloroplasts develop from proplastids during germination and leaf development (1). After chloroplast biogenesis, chloroplasts divide by binary fission. A number of mutant alleles enhance or reduce the size of individual chloroplasts by attenuating or promoting chloroplast division (2). Regardless of the size of individual chloroplasts, the proportion of cellular volume devoted to all chloroplasts appears indistinguishable from wild type in these mutants (3–5). Thus, the mechanism that establishes the size of the chloroplast compartment appears distinct from the mechanisms of chloroplast division.The cell expansion that drives the expansion of leaves also drives the proliferation of chloroplasts. Indeed, the proliferation of chloroplasts is so tightly correlated with cell expansion that the ratio of the size of the chloroplast compartment to the size of mesophyll cells is constant, regardless of cell size (2, 6, 7). Cell type exerts a major influence over the proportion of cellular volume devoted to the chloroplast. For instance, the size of the chloroplast compartment in mesophyll cells is larger than in epidermal cells. Thus, an extraplastidic mechanism appears to determine the size of the chloroplast compartment (6). However, during the expansion of leaves, chloroplasts are not completely submissive to the cell. Indeed, chloroplast dysfunction inhibits the expansion of leaves (8).Although mechanisms that establish the proportion of cellular volume devoted to particular organelles are of fundamental importance to biology, these mechanisms remain poorly understood (2). In the particular case of chloroplasts, understanding these mechanisms may lead to significant advances for agriculture. For example, introducing C₄ photosynthesis into rice, a plant that performs C₃ photosynthesis, is one strategy for potentially increasing yields from this important crop (9). C₃ and C₄ leaves are distinct at the metabolic, cellular, and anatomical levels (9, 10). One of the conserved features of C₄ leaves is the increase and decrease in the size of the chloroplast compartment in bundle sheath and mesophyll cells, respectively, relative to C₃ leaves (10). The engineering of C₄ photosynthesis in important C₃ crops, such as rice, is thought to depend on the ability to rationally manipulate the size of the chloroplast compartment (11).We performed a screen for plastid-to-nucleus signaling mutants in Arabidopsis thaliana (12). From this screen, we obtained one mutant allele of a gene that encodes a protein of unknown function. This gene is homologous to two Arabidopsis genes that encode proteins of unknown function and to FRIENDLY. FRIENDLY and its orthologs promote the normal distribution of mitochondria in Arabidopsis and in nonphotosynthetic organisms (13). However, these three Arabidopsis homologs of FRIENDLY are found only in photosynthetic organisms. Based on these data, we thought that FRIENDLY may have expanded into a small gene family to help manage the energy metabolism of cells that contain both chloroplasts and mitochondria. We tested this idea by examining the phenotypes of mutants in which one, two, three, or all four of these genes are knocked out. We found that these mutants exhibited a number of chloroplast phenotypes, including a smaller chloroplast compartment relative to wild type. Thus, we named these genes REDUCED CHLOROPLAST COVERAGE (REC). We also found that the protein that contributes most to establishing the size of the chloroplast compartment, REC1, localizes to both the nucleus and the cytosol, and we provide evidence that signals derived from dysfunctional chloroplasts or the inhibition of cell expansion may regulate the nucleocytoplasmic partitioning of REC1.