Analyses of starch biosynthetic protein complexes and starch properties from developing mutant rice seeds with...

Starch is the major component of cereal grains and is composed of essentially linear amylose and highly branched amylopectin. The properties and composition of starch determine the use and value of grains and their products. Starch synthase (SS) I, SSIIa, and SSIIIa play central roles in amylopectin biosynthesis. These three SS isozymes also affect seed development, as complete loss of both SSI and SSIIIa under reduced SSIIa activity in rice lead to sterility, whereas presence of minimal SSI or SSIIIa activity is sufficient for generating fertile seeds. SSs, branching enzymes, and/or debranching enzymes form protein complexes in cereal. However, the relationship between starch properties and the formation of protein complexes remain largely unknown. To better understand this phenomenon, properties of starch and protein complex formation were analyzed using developing mutant rice seeds (ss1 ^L /ss2a ^L /ss3a) in which all three major SS activities were reduced.

The SS activity of ss1 ^L /ss2a ^L /ss3a was 25%-30% that of the wild-type. However, the grain weight of ss1 ^L /ss2a ^L /ss3a was 89% of the wild-type, 55% of which was starch, showing considerable starch synthesis. The reduction of soluble SS activity in ss1 ^L /ss2a ^L /ss3a resulted in increased levels of ADP-glucose pyrophosphorylase and granule-bound starch synthase I, which are responsible for substrate synthesis and amylose synthesis, respectively. Together, these features led to an increase in apparent amylose content (34%) in ss1 ^L /ss2a ^L /ss3a compared with wild-type (20%). Gel filtration chromatography of the soluble proteins in ss1 ^L /ss2a ^L /ss3a showed that the majority of the starch biosynthetic enzymes maintained the similar elution patterns as wild-type, except that the amounts of high-molecular-weight SSI (> 300 kDa) were reduced and SSIIa of approximately 200-300 kDa were present instead of those > 440 kDa, which predominate in wild-type. Immuno-precipitation analyses suggested that the interaction between the starch biosynthetic enzymes maybe reduced or weaker than in wild-type.

Although major SS isozymes were simultaneously reduced in ss1 ^L /ss2a ^L /ss3a rice, active protein complexes were formed with a slightly altered pattern, suggesting that the assembly of protein complexes may be complemented among the SS isozymes. In addition, ss1 ^L /ss2a ^L /ss3a maintained the ability to synthesize starch and accumulated less amylopectin and more amylose in starch.