INTRODUCTION
The rice grain weight is determined by both the grain sink size (numbers and size of endosperm cells) and the physiological and biochemical activities (sucrose metabolism and starch biosynthesis) of the endosperm cells for cereals (Liang et al., 2001). The rice grain is composed of the embryo and the endosperm, which is enclosed by a thin seed coat and covered by the spikelet hull (the husk), which physically restricts the size of caryopsis. The endosperm, which is the major sites storing starches and other nutritious compounds, occupies the bulk of the caryopsis. In this sense, the size and weight of the mature grain are mainly determined by the shape and size of the spikelet hull and the filling degrees of caryopsis (mainly endosperm), both of which varied greatly and controlled by different genetic and environmental factors. Rapid advances in rice functional genomics and the advent of next generation sequencing technologies have led to the recent cloning of a series of quantitative trait loci (QTLs)/genes to control grain size (length, width, thickness) and grain weight (See: Reviewed by Li et al., 2018; Table S1). However, the underlying molecular mechanism regulating the grain development and grain filling processes remains elusive.
Heterotrimeric G protein (hereafter G protein)-mediated signal transduction pathway is considered as one of the most important signaling mechanisms that regulates various important physiological and molecular processes both in animals and plants (Jones & Assmann, 2004; Urano et al., 2013; Urano & Jones, 2014 ). In general, G protein is composed of three subunits, Gα, Gβ and Gγ. Several researches showed that the Gβ and Gγ works as a single functional subunit (dimer) to regulate organ size (Urano et al., 2013). Rice genome encodes one Gα (RGA1), one Gβ (RGB1), and five Gγs (RGG1, RGG2, GS3, DEP1/qPE9-1 and GGC2), and evidence have shown all these G protein subunits play important roles in regulating rice grain size ( Ashikari et al., 1999; Fujisawa et al., 1999; Huang et al., 2009; Zhou et al., 2009; Mao et al., 2010; Trusov et al., 2012; Liu et al., 2018; Sun et al., 2018; Zhang et al., 2019). RGA1 and two Gγ subunits, GGC2 and DEP1/qPE9-1, positively regulate rice grain size, whereas other three Gγ subunits, RGG1, RGG2 and GS3, play opposite roles in grain size regulation ( Oki et al., 2005; Huang et al., 2009; Zhou et al., 2009; Mao et al., 2010; Liu et al., 2018; Sun et al., 2018). Interestingly, bothRGB1 -knocking down lines and RGB1 -overexpressing lines show smaller rice grain size as compared with the wildtype (Utsunomiya et al., 2011; Liu et al., 2018; Sun et al., 2018). A possible explanation is that Gγ and Gβ function as a dimer and their roles in regulating rice grain size depend on competitively coupling of Gβ with different Gγ subunits. Of course, other factors cannot be ruled out, such as MADS-domain transcription factor, BR transcription factor, BES1, etc (Liu et al., 2018; Zhang et al., 2018).
Although G proteins play important roles in regulating grain size, the molecular mechanisms underlying controlling grain filling process by G protein are still largely unclear. In the present study, we provide both genetic and biochemical evidence to show that, RGB1 controls not only the grain size, but the grain filling process as well. Knock-down ofRGB1 significantly delayed grain development and reduced starch accumulation and grain weight, which is closely related to the delayed and lower expression of genes encoding sucrose metabolism and starch biosynthesis related enzymes during grain filling stage. The lower auxin levels in the grains of the RGB1-k nocking down lines are due to the lower expression of OsYUC11 controlled by OsNF-YB1, a transcript factor, and NAA application can partially recovered the grain filling through stimulating the expression of sucrose metabolism and starch biosynthesis related genes.