Table 2_Expression and interaction of AGPase subunits reveal functional enzyme complexes in barley.docx

In the starch biosynthetic pathway of Poaceae plants, ADP-glucose pyrophosphorylase (AGPase) serves as the rate-limiting enzyme that catalyzes the conversion of glucose-1-phosphate (G1P) and ATP to ADP-glucose, the immediate precursor for starch synthesis. Despite its fundamental role, the molecular characteristics and regulation of AGPase in barley (Hordeum vulgare L.) remain poorly understood. This study systematically investigated the expression dynamics during barley grain development and subunit interactions of AGPase in vitro. Our findings revealed distinct spatiotemporal expression patterns among AGPase, with preferential accumulation during late grain-filling stages. Co-immunoprecipitation coupled with mass spectrometry (Co-IP/MS) demonstrated specific physical interactions between small (AGPS) and large (AGPL) subunits, confirming the heterotetrameric architecture of functional AGPase complexes in barley. Enzymatic characterization showed that particular subunit combinations (AGPS1-AGPL1 and AGPS2b-AGPL2) exhibited significantly higher catalytic activity compared to other permutations. These results demonstrate that AGPase expression is developmentally regulated, specific inter-subunit interactions determine enzymatic efficiency, and optimal activity requires precise stoichiometric assembly. The demonstrated spatiotemporal coordination of AGPase subunits provides mechanistic insight into the control of starch biosynthesis during the late stage of grain filling. These results also provide a potential key target to improve barley starch synthesis and metabolism.

在禾本科植物的淀粉生物合成途径中，腺苷二磷酸葡萄糖焦磷酸化酶（ADP-glucose pyrophosphorylase，AGPase）作为限速酶，可催化葡萄糖-1-磷酸（glucose-1-phosphate，G1P）与ATP反应生成腺苷二磷酸葡萄糖，后者为淀粉合成的直接前体。尽管AGPase在淀粉合成中发挥核心功能，但大麦（Hordeum vulgare L.）中AGPase的分子特性与调控机制仍不甚明晰。本研究系统探究了大麦籽粒发育进程中AGPase的表达动态，以及其体外亚基互作情况。研究结果显示，AGPase各亚基呈现独特的时空表达模式，且在籽粒灌浆后期的积累水平更高。免疫共沉淀结合质谱（Co-immunoprecipitation coupled with mass spectrometry，Co-IP/MS）实验证实，AGPase的小亚基（AGPS）与大亚基（AGPL）之间存在特异性物理互作，明确了大麦功能性AGPase复合物为异四聚体结构。酶学特性分析表明，特定亚基组合（AGPS1-AGPL1与AGPS2b-AGPL2）的催化活性显著高于其他组合形式。上述结果证实，AGPase的表达受发育进程调控，亚基间的特异性互作决定酶促效率，且其最优活性依赖于精准的化学计量组装。AGPase亚基的时空协同特性，为解析籽粒灌浆后期淀粉生物合成的调控机制提供了新的机制视角。本研究结果同时为改良大麦淀粉合成与代谢途径提供了潜在的关键靶标。