Tissue-Specific RNA-Seq Analysis of Cotton Roots’ Response to Compound Saline-Alkali Stress and the Functional Validation of the Key Gene GhERF2

Saline-alkali stress is one of the major abiotic stresses threatening crop growth. Cotton, as a “pioneer crop” that can grow in saline and alkali lands, is of great significance for understanding the regulatory mechanisms of plant response to stresses. Upland cotton has thus become a model plant for researchers to explore plant responses to saline-alkali stresses. In this study, RNA sequencing was employed to analyze tissue-specific expression of root tissues of TM-1 seedlings 20 min after exposure to compound saline-alkali stress. The RNA-Seq results revealed significant molecular differences in the responses of different root regions to the stress treatment. A total of 3939 differentially expressed genes (DEGs) were identified from pairwise comparisons between the non-root tip and root tip samples, which were primarily enriched in pathways including plant hormone signal transduction, MAPK signaling, and cysteine and methionine metabolism. Combined with the expression pattern investigation by quantitative real-time PCR (qRT-PCR) experiments, a key gene, GhERF2 (GH_A08G1918, ethylene-responsive transcription factor 2-like), was identified to be associated with saline-alkali tolerance. Through virus-induced gene silencing (VIGS), the GhERF2-silenced plants exhibited a more severe wilting phenotype under combined salt-alkali stress, along with a significant reduction in leaf chlorophyll content and fresh weights of plants and roots. Additionally, these plants showed greater cellular damage and a lower ability to scavenge reactive oxygen species (ROS) when exposed to the stress. These findings suggest that the GhERF2 gene may play a positive regulatory role in cotton responses to salt-alkali stress. These findings not only enhance our understanding of the molecular mechanisms underlying cotton response to compound saline-alkali stress, but also provide a foundation for future molecular breeding efforts aimed at improving cotton saline-alkali tolerance.