The <scp>E3</scp> Ubiquitin Ligases <scp>SINA3</scp> and <scp>SINA5</scp> Control <scp>DEP2</scp> Ubiquitination and Proteasomal Degradation to Regulate Grain Size and Weight in Rice

Grain size is a critical agronomic trait, yet the molecular mechanisms governing its determination in crops remain incompletely understood. While recent studies revealed that OsRING80 facilitates DENSE AND ERECT PANICLE 2 (DEP2) degradation to mediate immunity without impacting growth and development, our work identifies a distinct regulatory pathway controlling grain development. We demonstrate that two RING finger E3 ubiquitin ligases, SEVEN IN ABSENTIA 3 (SINA3) and SINA5, post-translationally regulate the stability of DEP2 (also known as SRS1/EP2/OsRELA/SUG1) to modulate grain size. SINA3 and SINA5 physically interact with DEP2, specifically mediated through the C1 structural region of DEP2 containing a coiled-coil domain. These E3 ligases promote K48-linked polyubiquitination of DEP2, targeting it for proteasomal degradation. Liquid chromatography-mass spectrometry (LC-MS) analysis identified six critical lysine residues (K399, K722, K746, K958, K962 and K1344) within DEP2 that are essential for its ubiquitylation and subsequent destabilization. Our genetic evidence further supports this regulatory module: knockout of SINA3 and/or SINA5 leads to DEP2 accumulation, concomitantly increasing grain size and 1000-grain weight significantly, without altering other agronomic traits; conversely, overexpression of SINA3 or SINA5 reduces DEP2 protein levels and diminishes grain size and weight. Therefore, our study uncovers a novel post-translational regulatory module where SINA3 and SINA5 control DEP2 stability to fine-tune grain development. These findings present a promising strategy for optimizing grain yield by manipulating this post-translational regulatory node.