Differences in specificity and compensatory functions among three major starch synthases determine the structure of amylopectin in rice endosperm.

The lengths of amylopectin-branched chains are precise and influence the physicochemical properties of starch, which determine starch functionality. Three major isozymes of starch synthases (SSs), SSI, SSII(a), and SSIII(a), are primarily responsible for amylopectin chain elongation in the storage tissues of plants. To date, the majority of reported rice mutants were generated using japonica cultivars, which have almost inactive SSIIa. Although three SSs share some overlapping chain length preferences, whether they complement each other remains unknown due to the absence of suitable genetic combinations of materials. In this study, rice ss1/SS2a/SS3a and SS1/SS2a/ss3a were newly generated, and the chain length distribution patterns of all the possible combinations of presence and absence of SSI, SSIIa, and SSIIIa activities were compared. This study demonstrated that SSIIa can complement most SSI functions that use glucan chains with DP 6-7 to generate DP 8-12 chains but cannot fully compensate for the elongation of DP 16-19 chains. This suggests that SSIIa preferentially elongates outer but not inner chains of amylopectin. In addition, the results revealed that neither SSI nor SSIIIa compensate for SSIIa. Neither SSI nor SSIIa compensate for elongation of DP >30 by SSIIIa. SSIIa could not resolve the pleiotropic increase of SSI caused by the absence of SSIIIa; instead, SSIIa further elongated those branches elongated by SSI. These results revealed compensatory differences among three major SS isozymes responsible for lengths of amylopectin branches.