RGB1 stimulates accumulation of IAA by regulating the expression of auxin biosynthesis-related genes
It is well known that grain development and sucrose metabolism and starch biosynthesis are highly regulated by both the genetic and the environmental cues. Plant hormones are involved in the regulation of many aspects of grain development and sucrose metabolism and starch biosynthesis (Tang et al., 2009; Yang & Zhang, 2010; Hu, et al., 2013; Zhang et al., 2014; Zuo & Li, 2014). Our previous study and other research groups’ results clearly showed that auxin plays important roles in these processes (Zhao, 2008; Ghorbani et al., 2011; Abu-Zaitoon et al. 2012; Zhao et al. 2013; Tamaki et al., 2015; McAdam et al., 2017). However, the molecular basis of auxin accumulation in rice grains is largely unclear. Based on the results of RNA-seq assay and the KEGG pathway enrichment analysis, we found that there were 77 DGEs involved in auxin biosynthesis and metabolism, transport, and signaling during grain filling period (Figure S2). Among them, six auxin biosynthesis genes were up-regulated in WYJ8 grains at 10 DAF, as compared with those in RGB1Ri-5 line (Figure 3a), implying thatRGB1 positively regulates sucrose metabolism and starch biosynthesis probably through controlling IAA biosynthesis in grains.
To validate this hypothesis, we measured the endogenous IAA content of grains during grain filling period. The results showed that theWYJ8 grain IAA content increased almost linearly after flowering and reached its maximal value at 20 DAF, whereas those of RGB1Rilines increased much slowly, especially at the early stage of grain development, and kept much lower levels throughout all experimental period (Figure 3b). Application of NAA after flowering accelerated starch accumulation and enhanced the final grain weight significantly ofRGB1Ri-5/6 lines (Figure 3c-3d). NAA application also stimulated the expression of OsSSSI, OsSSSIIa , OsGBSSI,OsBEIIb and OsAGPS2a, but had no effects on the expression of other seven genes in Figure 2, as compared with the no-NAA treatedRGB1Ri-5 line (Figure 3d). All these indicate positive correlations between IAA and sucrose metabolism and starch biosynthesis and between RGB1 expression and grain IAA level. So, it is reasonable to assume that RGB1 stimulates the grain filling and sucrose metabolism and starch biosynthesis largely through influencing auxin biosynthesis.
In higher plants, the de novo IAA biosynthesis is viaTAR/YUCCA pathway. Our RNA-seq analysis results revealed that several auxin biosynthesis genes, including YUCs and TARs , were down-regulated in grains of RGB1Ri lines either at 5 DAF or 10 DAF (Figure 3a). There is evidence that IAA plays several distinct roles at different stages of endosperm development (Figueiredo & Köhler, 2018; Basunia & Nonhebel, 2019). These differing roles require strong localized control of IAA biosynthesis by specific enzymes. According to the public available expression data, three genes, OsYUC9, OsTAR1and OsYUC11 , are specifically and highly expressed in rice endosperm cells (Figure 4a) .Our qRT-PCR results showed that the transcript levels of all three genes increased at the early filling stage of wildtype grains, whereas those in RGB1Ri lines kept lower at the same stage of grain filling and increased till 15 DAF (Figure 4b and Figure S3a), suggesting their involvement in IAA biosynthesis in grains. However, among three genes, only the expression of OsYUC11 was significantly correlated to the grain IAA levles (Figure 4c and Figure S3b). If we considered the temporal expression patterns of OsYUC11 combined with the results of the initiation of rapid accumulation of IAA and starch in grains of both wildtype andRGB1Ri lines (Figure 1i and 3b), it is reasonable to assume that various auxin biosynthesizing genes may control auxin production in different organs/tissues and the endosperm-specific OsYUC11 gene is the most important one that is responsible for auxin biosynthesis of grains during filling stage, and that RGB1 stimulates auxin accumulation through enhancing the expression of OsYUC11 gene in grains.