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.