Although an alternative pathway has been suggested, the prevailing view is that starch synthesis in cereal endosperm is controlled by the activity of the cytosolic isoform of ADPglucose pyrophosphorylase (AGPase). In rice, the cytosolic AGPase isoform is encoded by the OsAGPS2b and OsAGPL2 genes, which code for the small (S2b) and large (L2) subunits of the heterotetrameric enzyme, respectively. In this study, we isolated several allelic missense and nonsense OsAGPL2 mutants by N-methyl-N-nitrosourea (MNU) treatment of fertilized egg cells and by TILLING (Targeting Induced Local Lesions in Genomes). Interestingly, seeds from three of the missense mutants (two containing T139I and A171V) were severely shriveled and had seed weight and starch content comparable with the shriveled seeds from OsAGPL2 null mutants. Results from kinetic analysis of the purified recombinant enzymes revealed that the catalytic and allosteric regulatory properties of these mutant enzymes were significantly impaired. The missense heterotetramer enzymes and the S2b homotetramer had lower specific (catalytic) activities and affinities for the activator 3-phosphoglycerate (3-PGA). The missense heterotetramer enzymes showed more sensitivity to inhibition by the inhibitor inorganic phosphate (Pi) than the wild-type AGPase, while the S2b homotetramer was profoundly tolerant to Pi inhibition. Thus, our results provide definitive evidence that starch biosynthesis during rice endosperm development is controlled predominantly by the catalytic activity of the cytoplasmic AGPase and its allosteric regulation by the effectors. Moreover, our results show that the L2 subunit is essential for both catalysis and allosteric regulatory properties of the heterotetramer enzyme.

Amylopectin has a highly regulated branched structure called cluster structure, which causes the glucan to be hydrophobic and to form a semicrystalline architecture of starch granules. It is known that lesions of the isoamylase1 (ISA1) gene result in accumulation of a water‐soluble glucan (WSG) called phytoglycogen instead of amylopectin in various plant species. This type of cereal mutant is referred to as sugary‐1 and accumulates a large amount of phytoglycogen in the endosperm. In this study, another WSG‐synthesizing mutant has been isolated from japonica rice. This mutant accumulated a significant amount of WSG in the center of endosperm. No significant changes were found in activities of ISA and pullulanase and in the compositions of ISA1 homomer and ISA1‐ISA2 heteromer. In addition, activities of starch branching enzyme and starch synthase isoforms were not altered. The accumulated WSG had a specific fine structure, that differed from that of phytoglycogen. Thus, we designated this new mutant as the sugary‐2 mutant. Our study results strongly suggest that the Sugary‐2 gene product plays an important role in amylopectin synthesis of rice endosperm and identifies a new factor that controls the normal amylopectin structure, especially at the early developmental stage of the endosperm.

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