Rice LIKE EARLY STARVATION1 cooperates with FLOURY ENDOSPERM6 to modulate starch biosynthesis and endosperm development.

In cereal grains, starch is synthesized by the concerted actions of multiple enzymes on the surface of starch granules within the amyloplast. However, little is known about how starch-synthesizing enzymes access starch granules, especially for amylopectin biosynthesis. Here, we show that the rice (Oryza sativa) floury endosperm9 (flo9) mutant is defective in amylopectin biosynthesis, leading to grains exhibiting a floury endosperm with a hollow core. Molecular cloning revealed that FLO9 encodes a plant-specific protein homologous to Arabidopsis (Arabidopsis thaliana) LIKE EARLY STARVATION1 (LESV). Unlike Arabidopsis LESV, which is involved in starch metabolism in leaves, OsLESV is required for starch granule initiation in the endosperm. OsLESV can directly bind to starch by its C-terminal tryptophan (Trp)-rich region. Cellular and biochemical evidence suggests that OsLESV interacts with the starch-binding protein FLO6, and loss-of-function mutations of either gene impair ISOAMYLASE1 (ISA1) targeting to starch granules. Genetically, OsLESV acts synergistically with FLO6 to regulate starch biosynthesis and endosperm development. Together, our results identify OsLESV-FLO6 as a non-enzymatic molecular module responsible for ISA1 localization on starch granules, and present a target gene for use in biotechnology to control starch content and composition in rice endosperm.

SUBSTANDARD STARCH GRAIN7 regulates starch grain size and endosperm development in rice.

Starch is synthesized as insoluble, semicrystalline particles within plant chloroplast and amyloplast, which are referred to as starch grains (SGs). The size and morphology of SGs in the cereal endosperm are diverse and species-specific, representing a key determinant of the suitability of starch for industrial applications. However, the molecular mechanisms modulating SG size in cereal endosperm remain elusive. Here, we functionally characterized the rice (Oryza sativa) mutant substandard starch grain7 (ssg7), which exhibits enlarged SGs and defective endosperm development. SSG7 encodes a plant-specific DUF1001 domain-containing protein homologous to Arabidopsis (Arabidopsis thaliana) CRUMPLED LEAF (AtCRL). SSG7 localizes to the amyloplast membrane in developing endosperm. Several lines of evidence suggest that SSG7 functions together with SSG4 and SSG6, known as two regulators essential for SG development, to control SG size, by interacting with translocon-associated components, which unveils a molecular link between SG development and protein import. Genetically, SSG7 acts synergistically with SSG4 and appears to be functional redundancy with SSG6 in modulating SG size and endosperm development. Collectively, our findings uncover a multimeric functional protein complex involved in SG development in rice. SSG7 represents a promising target gene for the biotechnological modification of SG size, particularly for breeding programs aimed at improving starch quality.

Du13 encodes a C2 H2 zinc-finger protein that regulates Wxb pre-mRNA splicing and microRNA biogenesis in rice endosperm.

Amylose content is a crucial physicochemical property responsible for the eating and cooking quality of rice (Oryza sativa L.) grain and is mainly controlled by the Waxy (Wx) gene. Previous studies have identified several Dull genes that modulate the expression of the Wxb allele in japonica rice by affecting the splicing efficiency of the Wxb pre-mRNA. Here, we uncover dual roles for a novel Dull gene in pre-mRNA splicing and microRNA processing. We isolated the dull mutant, du13, with a dull endosperm and low amylose content. Map-based cloning showed that Du13 encodes a C2 H2 zinc-finger protein. Du13 coordinates with the nuclear cap-binding complex to regulate the splicing of Wxb transcripts in rice endosperm. Moreover, Du13 also regulates alternative splicing of other protein-coding transcripts and affects the biogenesis of a subset of microRNAs. Our results reveal an evolutionarily conserved link between pre-mRNA splicing and microRNA biogenesis in rice endosperm. Our findings also provide new insights into the functions of Dull genes in rice and expand our knowledge of microRNA biogenesis in monocots.