Directing a colleague: LIKE EARLY STARVATION with FLOURY ENDOSPERM 6 targets isoamylase 1 to rice starch granules

Starch is one of the most abundant biopolymers on Earth, constituting 80-90%, of the rice grain endosperm weight, thereby greatly affecting crop yield and quality.In plants, starch is stored in the form of starch granules.Amylopectin and amylose polymers assemble in crystalline and amorphous regions of the starch granules.There are three classes of enzymes required for amylopectin biosynthesis: starch synthases (SSs), which elongate linear glucan chains, starch branching enzymes (BEs) responsible for adding α-1,6 branch points, and the starch debranching enzymes named isoamylases (ISAs), which remove improper branch points.In addition to these enzymes, non-enzymatic proteins with starch-binding domains are emerging to be important for starch synthesis.An example is, LIKE EARLY STARVATION (LESV), a widely conserved non-enzymatic protein with a C-terminal region rich in aromatic and acidic amino acid residues, originally discovered in Arabidopsis (Feike et al. 2016).Several roles have been proposed for LESV, including a role in the assembly of amylopectin helices into starch granules (Liu et al. 2023), and in modifying the surface structure of starch granules to influence enzymatic activities (Singh et al. 2022).In this issue of The Plant Cell, Haigang Yan, Wenwei Zhang, Yihua Wang, and colleagues (Yan et al. 2024) identified a rice mutant, floury endosperm 9 (flo9), that is defective in starch accumulation from an N-methyl-N-nitrosourea-mutagenized mutant library.They showed that the flo9 mutation was in the rice ortholog of LESV, providing an opportunity to investigate the role of LESV in the context of storage starch synthesis in grain, which differs significantly from leaf starch metabolism in Arabidopsis.Using immunoblot analysis; authors showed that OsLESV exhibits a gradient increase from the exterior to the interior of the grain endosperm.The mutation in OsLESV resulted in grain endosperms with a translucent periphery, a floury-white intermediate region, and a hollow core region (Figure ) Mutant grain showed a decrease in total starch and amylopectin content, but unaltered amylose and protein content relative to the wild type.The chain-length distribution of total glucans was altered in the mutant grain, exhibiting a pronounced increase in the central regions of grain endosperm.Following these observations, the authors studied starch granule initiation during rice grain development after flowering in peripheral, intermediate, and central regions of grains.They noted atypical amyloplasts in mutant grains without compound granules in the central regions, akin to what was observed in flo6 mutants (Peng et al. 2014), with negligible effects on the peripheral region and loosely bound, tiny granules in intermediate regions.Localization studies of LESV in rice protoplasts with different combinations of domain truncations indicated that LESV is associated with starch granules.This was further confirmed through immunoblot analysis of subcellular fractionations and using in vitro glucan binding assays.Since the LESV mutant grains partially phenocopied isa1 mutants, the authors investigated protein interactions and confirmed that LESV interacts with ISA1 through its N-terminal region (Figure).The