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

The splicing of pre-mRNA can be constitutive or alternative. The resulting transcripts may play significant roles in plant development and responses to environmental changes. Wild soybean, as a wild relative of cultivated soybean, has excellent traits such as alkali tolerance and is an ideal material for studying stress mechanisms and mining resistant genes. In this study, we identified a splicing factor GsSCL30a of the SR family that depends on the GsSnRK1 protein kinase. Y2H and BiFC assays verified the interaction between GsSnRK1 and GsSCL30a. In vitro phosphorylation experiments confirmed that GsSnRK1 could phosphorylate GsSCL30a, and six phosphorylation sites of GsSCL30a by GsSnRK1 were determined. qRT-PCR results showed that the expression level of GsSCL30a was highest in wild soybean leaves, and its transcription level was upregulated under alkali stress. Splicing factor reporter analysis found that GsSCL30a could self-splice the third intron, which contains multiple conserved regions of the GAAG motif. In addition, the phosphorylation of GsSCL30a by GsSnRK1 promoted its self-splicing. Y2H experiments proved that there is a physical interaction between GsSCL30a and the U1 snRNP-specific protein U1-70K, indicating that GsSCL30a plays an important role at the 5' splicing site. We overexpressed GsSCL30a-GsSnRK1 in soybean hairy roots. It was found that OE# GsSCL30a-GsSnRK1 could synergistically enhance the tolerance of soybean to alkali stress. Our study revealed the response mechanism of GsSCL30a in soybean under alkali stress conditions, that is, GsSCL30a relies on GsSnRK1 for phosphorylation modification during the alkali stress process, which affects the activity of the splicing factor and then regulates the plant's response to alkali stress, providing a new idea for improving the stress resistance ability of crops.