Map-Based Cloning And Functional Study Of WP1 Gene In Rice

Chloroplasts are organelles for photosynthesis, and it is also a place for the synthesis and metabolism of some important substances. So the chloroplast development is very important to the plant growth and development. Studies on chloroplast development are mainly focused on the Arabidopsis thaliana, while the studies on the monocots are relatively rare. It is different between monocot and dicot chloroplast development. As a monocot model system it was important to study rice chloroplast development. And rice is one of the world’s most important grain crop, study of rice chloroplast development will cultivate high luminous efficiency of the super rice lay theoretical foundation. In this research, a rice white panicle mutant wpl (white panicle 1) was studied. The study was carried out from the aspects of cytology, genetics and functional genomics. The main results are as follows:1. wpl shown white stripe and the albino phenotype at seedling stage, and shown white panicle phenotype at heading. The chlorophyll content of these tissues was decreased significantly in the wpl mutant seedlings and the panicle. Then we observed the ultrastructure of chloroplasts in these tissues, and found that the chloroplast structure in the wpl mutant was severely damaged. And then we observed the process of chloroplast at the early stage of wpl mutant by chlorophyll fluorescence and transmission electron microscopy. We found that the chloroplasts are severely damaged during early chloroplast development in wpl mutant.2. We constructed the mapping populations to map the WP1 locus. We found a possible mutation of the gene LOC_Os07g06940 by sequencing the 5 ORFs (open reading frames)in 43 kb region. Then we further proved that the mutation of this gene led to a series of color phenotypes in wpl mutants.3. We analysed the WP1 protein sequences, found WP1 encoding a valine tRNA synthase named OsValRS2. Then further analysis indicated that there is another functional valine tRNA synthase (OsVa1RS1) in rice. In Arabidopsis, there were homologous genes of the two proteins. We analyzed the expression of WP1 and found that it was expressed in all tissues, and the expression level was higher in green tissues. Another valine tRNA synthase was expressed in all tissues in rice, and the expression was very high in all tissues. Then we observed subcellular localization of valine tRNA synthase in Rice. We found that WP1 was dual targeted to chloroplasts and mitochondria, and OsValRS1 was targeted to cytoplasm.WP1 and OsValRS1 are responsible for the protein synthesis of three organelles, and these two proteins are not redundant in their functions.4. In order to study how WP1 affect the chloroplast development. We detected the gene expression difference between wild type and wp1 mutant using RNA-seq. In wp1s, many genes involved in photosynthesis were significantly repressed, including genes encoding proteins of the light-harvesting complex, photosystems Ⅰ and Ⅱ (PSⅠ and PSⅡ), electron carriers, ATP synthase, ferredoxin-NADP+ reductase and proteins involved in carbon fixation, which are necessary for photosynthesis. In wpls the expression levels of gene transcripted by NEP were strongly increased, while genes transcripted by NEP were strongly decreased. These results suggested that wp1 was defective in PEP activity which will lead defective in the abnormal early chloroplast development5. We also analyzed contents of the plastid encoding proteins in the wpl mutant, and found that the contents of the detected proteins were significantly decreased in the wp1 mutant.Then we detected the mRNA levels of these proteins and found that the expression level of these proteins is repressed. So we thought that the protein synthesis was imparied in wp1 mutant. Then we analyze the ribosome of the wpl mutant. We could not detect rRNA and ribosomal proteins in the wp1 seedlings and panicle. These results indicate that the damage of ribosome biosynthesis in the wpl mutant leads to the inhibition of protein synthesis, and we thought that WP1 may be an indirect effect on the ribosomal biosynthesis.