Comparative Ubiquitome Analysis Reveals Diverse Functions of Ubiquitination in Rice Seed Development under High-Temperature Stress

Protein ubiquitination plays vital roles in regulation of diverse cellular processes during plant growth and development. However, how protein ubiquitination regulates seed development in high-temperature environments is less understood. Here, a label-free quantification identified 488 lysine modification sites in 246 ubiquitinated proteins in the endosperm of two rice varieties, 9311 and Guangluai4 (GLA4). Under high-temperature stress, the number of significantly up-regulated sites was far greater than down-regulated sites, and 37 ubiquitinated proteins were commonly regulated with the same trend in the two varieties. The sucrose and starch metabolism were greatly over-represented by functional and pathway enrichment analyses. The key functions of ubiquitinated proteins related to starch metabolism are SUS1, SUS2, SUS3, FK and UGPase for sucrose hydrolysis, and AGPL2, AGPL3, AGPS1, AGPS2, GBSSI, BEI, BEIIb, PUL and Pho1 for starch synthesis. Most lysine modification sites were first identified in rice and tended to be up-regulated under heat stress, providing evidence for decreased protein abundance of starch synthesis related enzyme at the ubiquitination level. Predicted 3D models of GBSSI revealed an important role of ubiquitylation sites K462 involved in the interaction between the GBSSI and ligands (SO₄ and ADP). Our study provides the first comprehensive view of the ubiquitome in rice seeds, which will provide important insight into the mechanism underlying seed development and grain quality improvement under high-temperature stress.