The first committed step in the endosperm starch biosynthetic pathway is catalyzed by the cytosolic glucose-1-phosphate adenylyl transferase (AGPase) comprising large and small subunits encoded by the OsAPL2 and OsAPS2b genes, respectively. OsAPL2 is expressed solely in the endosperm so we hypothesized that mutating this gene would block starch biosynthesis in the endosperm without affecting the leaves. We used CRISPR/Cas9 to create two heterozygous mutants, one with a severely truncated and nonfunctional AGPase and the other with a C-terminal structural modification causing a partial loss of activity. Unexpectedly, we observed starch depletion in the leaves of both mutants and a corresponding increase in the level of soluble sugars. This reflected the unanticipated expression of both OsAPL2 and OsAPS2b in the leaves, generating a complete ectopic AGPase in the leaf cytosol, and a corresponding decrease in the expression of the plastidial small subunit OsAPS2a that was only partially complemented by an increase in the expression of OsAPS1. The new cytosolic AGPase was not sufficient to compensate for the loss of plastidial AGPase, most likely because there is no wider starch biosynthesis pathway in the leaf cytosol and because pathway intermediates are not shuttled between the two compartments.
Keywords: AGPase large subunit gene; CRISPR/Cas9; Ectopic expression; Monoallelic mutations; Rice.