Map-based Cloning And Function Analysis Of Yellow Leaf Mutant Yl1 In Rice

Rice is one of the world’s main food crops. By screening leaf colour mutants and cloning the mutation genes, we can investigate chlorophyll metabolism and photosynthesis mechanism to improve rice photosynthetic efficiency, which is of great significance to increase the yield of rice.Leaf, a crucial organ for photosynthesis, provides the basis of crop yield formation. Leaf colour mutation is one of the most common mutation types and is mainly divided into albefaction, etiolation, virescent, stripe and spot. The causes of leaf color mutation include chlorophyll metabolism disruption, chloroplast development defects, and the cytoplasm and nuclear signal transduction blocked etc. The chlorophyll synthesis disruption is one of the important factors. As the crucial pigment of plant photosynthesis, chlorophylls, including chlorophyll a and b, etc, can capture light energy and transfer energy to the reaction center. The chlorophyll synthesis enzyme mutation is the key reason of chlorophyll synthesis blocked. In addition to as a trait marker in breedings, leaf color mutants in crops are one of the prerequisites for investigation in chloroplast development and cultivation of new germplasms with higher photosynthetic efficiency.We isolated a yellow leaf mutant(yl1) from a pool of japonica rice Kitaake mutagenized with ethyl methanesulfonate(EMS). The yl1 mutant shows lower plant height, less grain number and less 1000-seed weight compared with wild type. The chlorophyll b content of yl1 mutant leaf is significantly lower than that of wild type, and chloroplast thylakoid is thinning, starch grains significantly decrease in yl1 mutant leaf. Genetic analysis showed that yellow leaf phenotype in yl1 was caused by a single recessive gene. Map-based cloning demonstrated that the coding region of Os CAO1(LOC_Os10g41780), encoding chlorophyllide a oxygenase, has a G to A point mutation site(Glycine 396 to Arginine in amino acid sequence) in yl1. Meanwhile, we ordered the yl2 mutant whith T-DNA insertion in the first intron of the Os CAO1. The yl2 mutant and yl1 mutant has the same yellow leaf phenotype and significant reduction of chlorophyll b content. Sequence analysis and GFP experiment indicate the mutation site G396 R of CAO1 in yl1 is in catalytic domain, and make no change in CAO1 location in chloroplast. Therefore, G396 might be the key point for Os CAO1 enzyme activity as G396 is highly conserved in different species.Furthermore,we isolated the Os CAO1 interacting protein Os CBSX3 through yeast two hybrid method. Os CBSX3 protein only contain CBS domain and the domain helps in maintain the intracellular redox balance. A total of six CBSXs have been identified in the Arabidopsis genome. CBSX1 and OCSX2 locate in the chloroplast and help to regulate cellular redox levels. In Oryza, a total of 22 CBSX proteins are encoded by 12 genes through the alternate splicing mechanism, and Os CBSX3 has the highest homology with At CBSX1 and At CBSX2. Os CBSX3 might protect the rice leaf from oxidative damage. Presently, we only preliminary tested Os CAO1 interactions with Os CBSX3 in yeast and the point mutation of Os CAO1 can lead to their interaction dropped significantly.