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

ABSTRACT 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.