Double mutants of granule-bound starch synthases, GBSSI and GBSSII, revealed their roles and the existence of amylose-like branched α-glucans in rice

Two isozymes of granule-bound starch synthase (GBSS), GBSSI and GBSSII, are present in rice. While GBSSI is solely responsible for amylose synthesis in endosperm and gbss1 mutants accumulate glutinous starch in grains, the role of GBSSII remains unclear. In this study, rice gbss2 mutants were isolated for the first time in cereals and gbss1 gbss2 double mutants were generated to clarify the respective roles of GBSS isozymes. Iodine staining of starch confirmed that amylose in pollen is synthesized by GBSSI and those in leaf and root tip are by GBSSII. Gel filtration chromatography of debranched starch from endosperm and leaf sheath revealed that GBSSI and GBSSII appear to be the primary amylose synthases in endosperm and in leaf sheath, respectively. However, the absence of the primary GBSS in the respective organs did not result in the complete elimination of amylose. A small peak detected in the fractions corresponding to amylose was shown to be α-glucans by glucoamylase digestion, and the presence of branches was revealed by detailed structure analyses. The amylose-like, branched α-glucans found in the double mutants, which probably also existed in single mutants and wild-type starches, were suggested to be synthesized by other enzymes than GBSSs.