Global profiling of alternative splicing landscapes in response to cold stress during the cotyledon stage in cotton

Upland cotton (Gossypium hirsutum L.) is the most important natural fibre crop worldwide. Low-temperature chilling injury during the cotyledon stage severely affects the growth of cotton seedlings. Alternative splicing (AS) plays a significant role in plant responses to stress conditions. However, little is known about the role of AS in response to cold stress in cotyledonary cotton. In this study, we employed nanopore full-length transcriptome sequencing technology to analyse cotton seedlings at the cotyledon stage under low-temperature stress at different time points, to identify AS events, associated key genes, and major pathways involved. When cotton cotyledon-stage seedlings were subjected to cold treatment for 24 h, the seedlings exhibited significant changes in internal physiological indicators while showing no notable phenotypic alterations. Using nanopore full-length transcriptome sequencing, we obtained 64,517 nonredundant unique transcript sequences and identified a total of 21,227 AS events. The genes associated with differentially alternative splicing (DAS) events included primarily the ROS-related genes ALD, CAT, ACX, AGT, and SOD. A total of 60 genes associated with both the CBF-dependent and CBF-independent pathways were identified among the differentially expressed genes (DEGs). Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analysis of both DEGs and DAS genes revealed that plant hormone signal transduction was a shared pathway, primarily involving the auxin (IAA) related genes IAA16 and IAA4. These findings suggest that during the cotyledon stage of cotton, by integrating C-repeat Binding Factor (CBF) dependent and CBF independent transcriptional regulatory networks, such as those involving IAA as a hub, cotton coordinately regulates gene expression and post-transcriptional splicing of downstream physiological processes such as antioxidation, thereby precisely balancing growth inhibition and cold resistance activation. Our study improves the understanding of the role of AS genes in regulating the cold stress response in cotton .