Yield improvement has long been an important task for soybean breeding in the world in order to meet the increasing demand for food and animal feed. miR396 genes have been shown to negatively regulate grain size in rice, but whether miR396 family members may function in a similar manner in soybean is unknown. Here, we generated eight soybean mutants harboring different combinations of homozygous mutations in the six soybean miR396 genes through genome editing with clustered regularly interspaced palindromic repeats (CRISPR)/CRISPR‐associated nuclease (Cas)12SF01 in the elite soybean cultivar Zhonghuang 302 (ZH302). Four triple mutants (mir396aci, mir396acd, mir396adf, and mir396cdf), two quadruple mutants (mir396abcd and mir396acfi), and two quintuple mutants (mir396abcdf and mir396bcdfi) were characterized. We found that plants of all the mir396 mutants produced larger seeds compared to ZH302 plants. Field tests showed that mir396adf and mir396cdf plants have significantly increased yield in growth zones with relatively high latitude which are suited for ZH302 and moderately increased yield in lower latitude. In contrast, mir396abcdf and mir396bcdfi plants have increased plant height and decreased yield in growth zones with relatively high latitude due to lodging issues, but they are suited for low latitude growth zones with increased yield without lodging problems. Taken together, our study demonstrated that loss‐of‐function of miR396 genes leads to significantly enlarged seed size and increased yield in soybean, providing valuable germplasms for breeding high‐yield soybean.

Cold stress is a major environmental factor limiting the productivity and geographical distribution of horticultural crops. Here, we investigated the role of the spliceosome U4/U6.U5 tri-snRNP-associated protein 1 (UAP1) in modulating low-temperature responses across three dicotyledonous species: tomato (Solanum lycopersicum), soybean (Glycine max), and Arabidopsis thaliana. Using CRISPR/Cas9-mediated gene editing, we generated UAP1 knockout orthologs in two dicotyledonous species: tomato and soybean. The sluap1 and gmuap1 mutants exhibited significantly enhanced cold resilience, characterized by reduced membrane damage, lower reactive oxygen species accumulation, and higher survival rates after cold stress. To elucidate the underlying molecular mechanism, we further characterized the uap1 mutants in Arabidopsis, which displayed alterations in cold-regulated hypocotyl elongation. Transcriptome-wide analysis revealed that the loss of AtUAP1 results in widespread differential alternative splicing under cold stress. Notably, we identified a critical intron retention event in the circadian clock gene AtPRR3, leading to a truncated, non-functional protein. Genetic evidence confirmed that atprr3 mutants mimic the low-temperature growth phenotype of atuap1, and heterologous expression of SlUAP1 or GmUAP1 effectively rescued the atuap1 mutant, demonstrating high functional conservation at the molecular level. Our findings establish the UAP1-mediated splicing module participates in cold-regulated growth and stress responses in dicotyledonous plants, providing insights into post-transcriptional regulation under low temperature.