Genome-Wide Identification of Ovarian Tumor Proteases Gene Family and Knockout of TaOTU6 Increases Grain Width and Weight in Wheat

Deubiquitinating enzymes (DUBs) play essential roles in diverse plant biological processes, yet the ovarian tumor proteases (OTUs), a major DUB subfamily, have not been systematically characterized in wheat, and their functions in grain development remain unclear. Here, we identified 49 <i>OTU</i> genes (<i>TaOTUs</i>) in the hexaploid wheat genome and classified them into four subfamilies based on phylogenetic relationships, with nomenclature assigned according to homology. <i>TaOTU6-7B</i> was highly expressed during early and mid-grain development and was responsive to gibberellin and jasmonic acid. Its expression differed significantly between large-grain wheat Pindong34 (PD34) and small-grain wheat MY11847 at 7 and 11 days after flowering. To elucidate its function, we used CRISPR/Cas9 to generate loss-of-function mutants by knocking out the three homoeologs (<i>TaOTU6-7A</i>, <i>-7B</i>, and <i>-7D</i>). These mutants exhibited significantly increased grain width and weight relative to wild type. Moreover, <i>TaOTU6-7B</i> directly interacted with <i>TaUBC13</i>, whose expression was markedly elevated in the <i>TaOTU6</i> knockout background, suggesting that <i>TaUBC13</i> may positively regulate wheat grain size. Collectively, this study establishes the <i>TaOTU</i> gene family in wheat, reveals <i>TaOTU6</i> as a negative regulator of grain width and weight, and provides valuable genetic resources and a theoretical foundation for high-yield wheat breeding.