Systematic analysis of the rice E2 ubiquitin-conjugating enzyme family identifies seven genes essential for seed germination

Rice seed germination marks the start of cultivation and influences subsequent seedling growth, and is affected by hormones and environmental factors. Ubiquitination plays a critical role in this process by regulating hormonal homeostasis. In the ubiquitination cascade, ubiquitin-conjugating enzymes (UBCs) function as ubiquitin carriers to determine linkage specificity of ubiquitin chains. In rice ( Oryza sativa ), 39 UBC genes are identified, but only one gene OsUBC12 has been functionally studied to promote seed germination under low-temperatures in japonica rice. To elucidate the role of UBCs in seed germination, we generated CRISPR-Cas9 mutants for 23 UBC genes and overexpressed 20 members in rice. Among them, seven UBC genes ( OsUBC4 / 6 / 7 / 12 / 25 / 27 / 48 ) were found to regulate seed germination, with OsUBC27 and OsUBC48 acting through the ABA pathway. Exogenous ABA inhibitors restored the germination rate of osubc27 CR . RT-qPCR analysis revealed that the ABA synthesis genes OsNCED1 - 5 were significantly upregulated in the mutants. Further differential ubiquitination proteomics in knockout mutants and wild-type plants showed that OsUBC27 regulates ABA homeostasis by modulating ubiquitination of the ABA-degrading protein OsABA8ox1, thereby balancing seed dormancy and germination. Sequence analysis identified distinct haplotypes of the seven OsUBCs that showed differential distribution between japonica and indica subspecies. Our study provides valuable molecular targets for developing rice varieties resistant to seed vivipary