Re‐programming of gene expression in the CS8 rice line over‐expressing ADPglucose pyrophosphorylase induces a suppressor of starch biosynthesis

Summary The CS 8 transgenic rice ( Oryza sativa L.) lines expressing an up‐regulated glgC gene produced higher levels of ADP glucose ( ADP glc), the substrate for starch synthases. However, the increase in grain weight was much less than the increase in ADP glc levels suggesting one or more downstream rate‐limiting steps. Endosperm starch levels were not further enhanced in double transgenic plants expressing both glgC and the maize brittle‐1 gene, the latter responsible for transport of ADP glc into the amyloplast. These studies demonstrate that critical processes within the amyloplast stroma restrict maximum carbon flow into starch. RNA‐ seq analysis showed extensive re‐programming of gene expression in the CS 8 with 2073 genes up‐regulated and 140 down‐regulated. One conspicuous gene, up‐regulated ~15‐fold, coded for a biochemically uncharacterized starch binding domain‐containing protein ( SBDCP 1) possessing a plastid transit peptide. Confocal microscopy and transmission electron microscopy analysis confirmed that SBDCP 1 was located in the amyloplasts. Reciprocal immunoprecipitation and pull‐down assays indicated an interaction between SBDCP 1 and starch synthase III a ( SSIII a), which was down‐regulated at the protein level in the CS 8 line. Furthermore, binding by SBDCP 1 inhibited SSIII a starch polymerization activity in a non‐competitive manner. Surprisingly, artificial micro RNA gene suppression of SBDCP 1 restored protein expression levels of SSIII a in the CS 8 line resulting in starch with lower amylose content and increased amylopectin chains with a higher degree of polymerization. Collectively, our results support the involvement of additional non‐enzymatic factors such as SBDCP in starch biosynthesis.