RGB1 enhances the expression of genes associated with sucrose metabolism and starch biosynthesis
To investigate how RGB1 regulates the grain development and starch biosynthesis, the expression profiles of genes in caryopsis ofWYJ8 and RGB1Ri-5 line at 5 and 10 DAF were analyzed, using the RNA-seq assay. Approximately 3216 and 3363 genes that were up- or down-regulated, respectively, in RGB1Ri-5 as compared withWYJ8 , were identified at 5 DAF. The numbers of up-regulated genes at 10 DAF decreased to 2832 and those of down-regulated genes increased to 3398, respectively (Figure S1a). The Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment analysis showed that these differently expressed genes (DEGs) were significantly enriched for “sucrose metabolism and starch biosynthesis” and “plant hormone transduction” (Figure S1b), suggesting that RGB1 may regulate sucrose metabolism and grain starch synthesis through manipulating hormone homeostasis and/or hormone-mediated signaling.
To validate above hypothesis, we analyzed the expression patterns of DEGs for sucrose metabolism and starch synthesis (29 genes) and grain development regulation (17 genes). The results indicated that the expression of about 70% of the sucrose metabolism and starch biosynthesis-related genes and all the grain development-related genes were up-regulated in WYJ8 , as compared with that inRGB1Ri-5 line (Figure 2a). These results were consistent with our observation that RGB1 positively regulate grain filling and starch accumulation. We further analyzed the dynamic expression patterns of these genes in grains of WYJ8 and RGB1Ri lines during grain filling period using both real-time quantitative PCR and western blotting assay. As shown in Figure 2b, the dynamic expression patterns of all twelve sucrose metabolism and starch biosynthesis-related genes could be roughly divided into three groups. OsAGPS2a ,OsAGPS2b , OsISA2 , OsSSSI , OsSSSIIa andOsPUL belonged to the first group, of which their transcript levels rapidly increased after flowering, and reached their maximal levels at 20 DAF, and then decreased both in WYJ8 and inRGB1Ri lines. However, the expression levels of these six genes in grains of WYJ8 were higher than in RGB1Ri lines, especially during early stage of grain filling. The second group included OsISA1 , OsBE1 and OsBEIIb, and the transcript levels of these three genes increased in grains of all genotypes, but the time reached maximal levels was different between genotypes. The maximal transcript levels of these genes in WYJ8grains were detected at 10 DAF, which was much earlier than those ofRGB1Ri lines. OsAGPL2 , OsSSSIIIa and OsGBSSIgenes belonged to the third group, of which the transcript level in grains of WYJ8 line was higher at the early stage of grain filling, whereas the transcript levels in grains of RGB1Ri lines increased rapidly after 10 DAF and kept very higher levels thereafter. The western blotting results were well consistent with the RT-PCR results, of which the protein level of OsSSSIIa, OsBEI, OsBEIIb, OsSSSI and OsGBSSI were much lower in grains of RGB1Ri-5 line at the early stage of filling (Figure 2c).Taken together, these results indicate that RGB1 play key roles in controlling grain filling through its effects on the expression of genes encoding sucrose metabolism and starch biosynthesis-related enzymes, especially at the early stage of grain filling.