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.