Protein Interaction Network Analysis of Soluble Starch Synthase 3 Reveals Its Composition of the Starch Synthase Complex in Barley

Starch synthase collaboratively accomplishes the synthesis of starch by forming a protein complex. In barley (Hordeum vulgare), the interaction mechanisms within the soluble starch synthase 3 (SSIII) protein complex and their specific roles in starch biosynthesis remain poorly understood. To investigate proteins interacting with SSIII isoforms (SSIIIa/SSIIIb), the polyclonal antibodies against HvSSIIIa and HvSSIIIb were generated in rabbits. Co-immunoprecipitation (Co-IP) was employed to enrich HvSSIIIa- and HvSSIIIb-associated protein complexes from developing barley seeds. The potential interacting proteins were identified using mass spectrometry coupled with database searches. The results demonstrated the successful production of highly specific polyclonal antibodies against HvSSIIIa and HvSSIIIb. These antibodies were used to analyze the expression levels of HvSSIIIa and HvSSIIIb across different barley tissue and seed developmental stages. Both isoforms exhibited peak expression during early seed development. Further Co-IP-based screening revealed several candidate interacting proteins, and the interacting proteins had some overlaps. Among them, adenosine diphosphate glucose pyrophosphorylase, as a key rate-limiting enzyme in starch synthesis, interacts with SSIII to jointly regulate the supply of ADP-glucose precursors. Differential proteins show functional differentiation. In SSIIIa, the interacting protein DELLA protein SLR1-like is involved in processes such as hormone regulation, and in SSIIIb, the interacting protein 14-3-3 is related to metabolism. These findings provide new insights into the composition and function of the SSIII protein complex in barley, facilitating future studies on its regulatory role in starch biosynthesis.