Elucidating the rate limiting components of starch biosynthesis.

摘要

Starch is used as main carbohydrate and energy reserve where it accumulates and stored in plastids. In cereal grains, there are three potential rate limiting steps of starch biosynthesis: production, transport and utilization of ADPglucose (ADPglc). In this dissertation, I present results of my studies on the structure-function relationships of the starch regulatory enzyme ADPglucose pyrophosphorylase (AGPase) at the protein and physiological levels. AGPase possesses a heterotetrameric structure composed of pairs of large (LSs) and small subunits (SSs). Current evidence indicates that the SS possesses a dominant role in catalysis although both AGPase subunits are required for optimal catalytic and allosteric regulatory properties of the heterotetrameric enzyme. Using isothermal titration calorimetry the heterotetrameric enzyme was found to contain two distinct ATP binding sites while the LS and SS homotetramers exhibit only one of the two binding sites with drastically reduced affinity. Hence, the substrate binding properties of the heterotetrameric enzyme is a product of synergy between the two subunit types. In a second study, the potential rate limiting role of ADPglc transport into amyloplast was investigated by analyzing the shrunken 3-OsBt1 mutant (defective in ADPglc transporter) and transgenic wild type and ADPglc-excess CS8 rice lines expressing the maize ZmBt1 gene. Although OsBt1 is essential for ADPglc into amyloplast and starch synthesis, transport of ADPglc was found not to limit carbon flow into starch. These studies indicate the presence of additional regulatory processes within the amyloplasts that restrict maximum carbon flow into starch. RNAseq analysis of CS8 plants showed significant changes in the gene network of primary carbon pathway with conspicuously increased (∼ 5 fold) expression of a starch binding domain containing protein (SBDCP). SBDCP was found to directly interact and inhibit starch synthase IIIa (SSIIIa) in a non-competitive manner. Down regulation of SBDCP activity in CS8 via artificial micro RNA surprisingly restored normal SSIIIa expression, which led to a further increase (∼5%) in seed weight and modification of starch structure. Overall, these results provide novel insights on processes that control starch synthesis and, in turn, regulate sink strength of developing rice seeds.