Functional Analysis Of A Peroxisomal-targeted Aldolase OsAld-Y In Rice

Chlorophyll(Chl), one of the most rich organic molecules on earth, is the primary photoreceptor pigment that absorb light energy and drive electron transfer in the reaction centers of the photosynthetic system to synthesize carbohydrates from CO2 and water. Thus, Chl is essential for plant development and agricultural production. Yellow-leaf mutants are considered to be ideal material to explore the molecular mechanism of Chl biosynthesis and chloroplast development. To investigate the molecular nature of rice chlorophyll metabolism, a yellow green and droopy leaf mutant, designated as ygdl-1, was isolated from the T-DNA insertion mutant library. In this research, the synthesis of chlorophyll, the chloroplast ultrastructure as well as carbon metabolism of mutants were investigated compared with the wild-type Zhonghua 11. The main findings are as follows:1. The contents of Chl a, Chl b and carotene in the ygdl-1 mutant were apparently reduced compared to that of the wild-type, indicating the yellow leaves of the ygdl-1 mutant resulted from reduced Chls levels. Examination of the mutant chloroplasts with transmission electron microscopy revealed that the grana stacks in the stroma were highly variable in size and appeared less dense.2. Searching the flanking sequence database RMD revealed that the mutant contained a T-DNA insertion within the promoter of a gene encoding frutose-1,6-bisphosphate aldolase(Os Ald-Y), which dramatically reduced the Os Ald-Y m RNA level. Real-time PCR showed that higher expression levels of Os Ald-Y was detected in potosynthetic organs, including leaf, culm and sheath throughout plant development stages. The preferential expression of Os Ald-Y in autotrophic source tissues strongly suggested that Os Ald-Y functions mainly in photosynthetic source tissue.3. GC-MS analysis of the primary carbon metabolites showed that the levels of sucrose and fructose reduced in the ygdl-1 mutant as compared to that of wild-type,in parallel with the down-regulation of two genes encoding the critical regulatory enzymes in gluconeogenesis. These are strong indications of the role of Os Ald-Y in glycolytic/gluconeogenesis pathways and the accumulation of water soluble carbohydrates in rice.4. The function of a protein is predominantly associated with its localization. In this study, transient expression GFP::Os Ald-Y in tobacco epidermal cells showed that Os Ald-Y was located in peroxisome lacking any recognizable peroxisome targeting signals(PTSs), which raising the possibility that the glycolytic pathwa y is associated with peroxisome.Taken together, these results suggest that the Os Ald-Y is involved in Chl accumulation, chloroplast development and plant growth by influencing the sugar metabolism of rice.