High Basal Expression and Dual Stress Responsiveness of Soybean (Glycine max) Resistance Gene SRC4

Genes involved in disease resistance are crucial for plant immune systems, yet their transcriptional regulatory mechanisms remain poorly understood. <i>SRC4</i>, a key member of the soybean mosaic virus resistance cluster (SRC), encodes a Ca²⁺-binding EF-hand domain and possesses antiviral activity, but its expression regulation is unclear. Here, we systematically analyzed 4085 soybean (<i>Glycine max</i>) transcriptome datasets and conducted SMV inoculation experiments to characterize <i>SRC4</i> expression patterns. <i>Cis</i>-acting element analysis identified 12 regulatory elements in the <i>SRC4</i> promoter, including salicylic acid (SA)-responsive elements. Furthermore, a <i>Pro_SRC4</i>::<i>GUS</i> reporter vector was constructed and functional analysis was performed in tobacco (<i>Nicotiana benthamiana</i>) and transgenic <i>Arabidopsis thaliana</i>. <i>SRC4</i> exhibited significantly higher basal expression than typical resistance genes (<i>R</i> genes) and was induced by SMV infection, SA treatment, and Ca²⁺ supplementation, with peak expression at 2-5 h post-treatment (hpi). In transgenic tobacco overexpressing <i>NahG</i>, neither SMV nor Ca²⁺ could induce <i>Pro_SRC4</i>::<i>GUS</i> expression, demonstrating that <i>SRC4</i> transcriptional regulation is mediated through SA signaling pathways. <i>SRC4</i> showed predominant expression in roots and leaves and responded to temperature stress. Transgenic plants overexpressing <i>SRC4</i> exhibited enhanced tolerance to both 12 °C and 37 °C temperature stress. This study elucidates the molecular mechanisms underlying <i>SRC4</i> transcriptional regulation through Ca²⁺ and SA signaling pathways, revealing its dual role in both biotic and abiotic stress responses, especially in temperature stress.