Discussion
The grain size and weight of rice are complex traits, which involves in
grain development and filling process (mainly the sucrose metabolism and
starch biosynthesis), and is controlled by both the genetic and the
environmental factors. In last decades, several research groups have
provided genetic and molecular evidence that grain size and filling
processes are controlled by many genes (see review by Zuo & Li, 2014;
Sun et al., 2018). All these researches provide rice breeders
opportunities to improve rice grain yield by manipulating these genes.
However, it still has a long way to go before understanding the complex
regulatory networks involving grain development and filling processes.
The heterotrimeic G protein (hereafter G protein) is well known to play
important roles in plant growth and development (Botella, 2012; Sun et
al., 2018). Recently, several rice genes/QTLs encoding G protein
subunits have been shown to control grain size and shape of rice,
including: qPE9-1/DEP1 , GS3 , RGB1 and RGA1(Oki et al., 2005; Huang et al., 2009; Zhou et al. , 2009; Mao et
al., 2010; Liu et al., 2018; Miao et al., 2018; Sun et al., 2018).qPE9-1/DEP1 encoding a Gγ subunit, positively regulates grain
size (Huang et al., 2009; Zhou et al., 2009); GS3, another Gγ subunit,
negatively regulate grain size and shape (Mao et al., 2010; Sun et al.,
2018); RGB1 and RGA1 , encoding Gβ and Gα subunit,
respectively, positively regulates grain size and shape (Fujisawa et
al. , 1999; Utsunomiya et al., 2011; Sun et al., 2018). However,
the functions and molecular mechanisms of G protein to regulate grain
development and starch biosynthesis are largely unknown.
Rice grain filling is a very complicated process that involves
photoassimilate translocation from photosynthetic sources (i.e., leaves
and stem-sheaths), sucrose degradation, transmembrane transport and
starch synthesis in the grains (Liang et al., 2001; Lü et al., 2008;
Tang et al., 2009). Approximately twenty enzymes/proteins have been
reported to be involved in these biochemical processes (Zhu et
al. , 2003; Tetlow et al., 2004; Ohdan et al., 2005). The results
of our lab and several other groups showed that sucrose synthase (SUS),
invertase (INV), ADP-glucose pyrophosphorylase (AGP), soluble starch
synthase (SSS) and grannuel-bound starch synthase (GBSS), branching
enzyme (BE) and debranching enzyme (DBE) play key roles in the
regulation of sucrose metabolism and starch biosynthesis (Liang et al.,
2001; Lü et al. , 2008; Tang et al., 2009; also see reviewed by
Tetlow et al., 2004; Keeling & Myers, 2010; Zeeman et al., 2010). The
grain-filling process is also a highly regulated process in which both
genetic and environmental factors are involved. It is well known that
plant hormones play important roles in grain development and filling
process (Liang et al. , 2001; Yang et al., 2006; Tang et al.,
2009; see also reviewed by Basunia & Nonhebel, 2019). Understandably,
rice grain size, not like the wheat grains, is physically limited by the
spikelet hull, of which the final size is almost determined upon
flowering. In this sense, the endosperm cell proliferation and
elongation as well as the accumulation of starch and other storage
compounds after flowering are of crucial importance for final grain
yield and quality. Our results showed that grain size, including the
length, width and thickness of grains reduced, and as a result, the
final grain weight and starch content reduced significantly inRGB1 knock-down lines (Figure 1). This reduction was mainly due
to the delay of caryopsis development and the lower starch accumulation
at the early stage of grain filling. As we known, the rice grain filling
process is in fact the process of sucrose metabolism and starch
biosynthesis occurred in endosperm cells, in which many enzymes/proteins
are involved. So, it is expected that the expression levels of genes
encoding enzymes catalyzing sucrose metabolism and starch biosynthesis
during grain filling are closely related to the starch accumulation and
grain weight. In RGB1 knock-down lines, the expression of genes
encoding sucrose metabolism and starch biosynthesis was either initiated
later or much lower at the early filling stage, which can well explain
the results observed here (Figure 1 and 2) and imply that suppression ofRGB1 expression could down-regulate the expression of genes
encoding enzymes catalyzing sucrose metabolism and starch biosynthesis.
However, we still unknown the molecular mechanisms of RGB1regulation on the expression of these genes.
Recently, several research groups have reported that an accumulation of
auxin immediately before the starch biosynthesis in rice grains
(Abu-Zaiton et al., 2012). Furthermore, application of exogenous auxin
also had a positive effect on starch accumulation. These results
suggested that auxin may involve in the regulation of starch
biosynthesis in rice grains. RNA-seq assay and auxin quantitation also
showed a great difference in the expression of auxin biosynthesis
related genes and IAA contents in grains between RGB1 knocking
down lines and wildtype during filling stage. The expression of
endosperm-specific genes for auxin biosynthesis was down-regulated and
endogenous IAA content significantly reduced in the grains ofRGB1 knocking down lines (Figure 3). The assumption that RGB1
involvement in the regulation of starch biosynthesis is through changing
auxin homeostasis of grains was further validated according to the
results of exogenous application of IAA on starch accumulation and the
expression of sucrose metabolism and starch biosynthesis related genes
during grain filling stage (Figure 3).
Auxin biosynthesis in higher plants is catalyzed by a large number ofTARs (encoding tryptophan aminotransferase) and YUCs(encoding indole-3-pyruvate mono-oxygenases) with differing patterns of
spatiotemporal expression, which allows for multiple roles. Different
genes may be responsible for the auxin production in different time
and/or in different tissues, and therefore, play various roles in
regulating plant growth and development. In rice, tissue-specific
expression of these genes showed that OsTAR1 , OsYUC9 andOsYUC11 genes expressed highly in the endosperm cells (Figure
4a), suggesting these three genes might play important roles in
controlling auxin biosynthesis in grains. However, only the expression
of OsYUC11 was well correlated with the grain IAA content (Figure
4c). Based on these results, we hypothesize that OsYUC11 were
mainly and especially responsible for the auxin biosynthesis in
endosperm cells of rice and other auxin biosynthesis related genes may
participate in separate signaling processes.
It is clear that the delay of caryopsis development and the lower starch
accumulation and grain weight in RGB1 knocking down lines are due
to the lower auxin content in grains caused by the lower expression ofOsYUC11 during grain filling stage. Furthermore, OsYUC11also plays a positive regulatory role in the starch biosynthesis pathway
by up-regulating the expression of several sucrose metabolism and starch
biosynthesis related genes. Collectively, our study suggests thatOsYUC11 is of crucial importance in regulating grain development
and starch biosynthesis by controlling auxin content during grain
filling stage. However, we still need direct evidence to verify that
OsYUC11 is the key enzyme in controlling the level of IAA in rice grain.
We tried to knock out OsYUC11 in WYJ8, but failed to obtain the
regeneration plantlet from callus, likely because of disrupting the
balance of auxin and cytokinin supplemented in medium. Next, we will
modify the ratio of auxin and cytokinin in regeneration medium to create
the null mutant of OsYUC11 .
In eukaryotes, transcription of genes is regulated by various
transcription factors. Our results showed that OsYUC11 promoter
may interact with several families of transcript factors, including
MADS, MYB, CCAAT, etc. (Table S1), suggesting that the regulation ofOsYUC11 expression is very complicated and involves signaling
networks. However, if we considered the results of RNA-seq analysis that
showed the differences of the expression of various transcription
factors between RGB1Ri line and WYJ8 plant during grain
filling stage, and the results of tissue-specific analysis of gene
expression of these transcript factors, it is reasonable to assume thatOsNF-YB1 might involve in the regulation of the expression ofOsYUC11 . Our present results provide biochemical evidence to
support the conclusion that OsNF-YB1 was crucial importance in
regulating OsYUC11 expression, and finally the auxin content in
grains. OsNF-YB1 has been reported to activate the expression of
sucrose transporters and waxy gene, and finally regulate the
endosperms development. Knockout of OsNF-YB1 led to defective
grains with chalky endosperms and significantly decreased grain weight
(Bai et al. , 2016; Bello et al. , 2018). Our results
provide a new insight that OsNF-YB1 regulates grain development
not only through directly activating the sucrose metabolism and starch
biosynthesis genes, but also through controlling the auxin accumulation.
In summary, according to our present results, a working model is
proposed to illustrate the roles of RGB1 in regulating grain development
and grain filling process. RGB1 may positively regulate\souts
expression of transcription factor OsNF-YB1 , which activates theOsYUC11 transcription by interacting with its promoter, and leads
to an increase in auxin level. The increased auxin then stimulates the
expression of sucrose metabolism and starch biosynthesis related genes
in endosperm cells, as a consequence, the biosynthesis of starch and
grain filling. However, the mechanism underlying how RGB1 regulates the
expression of OsNF-YB1 remains to be further studied (Figure 6).