Phosphorylation of Wild Soybean (<i>Glycine soja</i>) Splicing Factor GsSCL30a by GsSnRK1 Regulates Soybean Tolerance to Alkali Stress

Pre-mRNA splicing is a fundamental process in plant gene expression, and the resulting transcripts may play significant roles in plant development and responses to environmental changes. Wild soybean (Glycine soja), as a close relative of cultivated soybean (Glycine max), has excellent traits such as alkali tolerance and is an ideal material for mining stress-resistant genes. Previous phosphoproteomic analysis identified SR protein GsSCL30a as a potential phosphorylation substrate of GsSnRK1 kinase. Y2H and BiFC assays confirmed their interaction. In vitro phosphorylation and mass spectrometry showed GsSnRK1 phosphorylates six residues in GsSCL30a. RT-qPCR revealed GsSCL30a is ubiquitously expressed, with alkali stress regulating its transcript levels. Alternative splicing analysis showed GsSCL30a acts as a splicing factor, self-splicing its third intron with conserved GAAG motifs and GsSnRK1 phosphorylation enhances this activity. GsSCL30a interacts with U1-70K, suggesting involvement in U1-RNP complex at 5' splice sites. Soybean hairy root overexpression showed GsSCL30a-GsSnRK1 synergistically enhances alkali tolerance. This study revealed the molecular mechanism of GsSnRK1-GsSCL30a module in response to alkali stress and provides a new idea to breed the high-resistant crops.