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.

Fiber length (FL) and strength (FS) are the core indicators for evaluating cotton fiber quality. The corresponding stages of fiber elongation and secondary wall thickening are of great significance in determining FL and FS formation, respectively. QTL mapping and high-throughput sequencing technology have been applied to dissect the molecular mechanism of fiber development. In this study, 15 cotton chromosome segment substitution lines (CSSLs) with significant differences in FL and FS, together with their recurrent parental Gossypium hirsutum line CCRI45 and donor parent G. barbadense line Hai1, were chosen to conduct RNA-seq on developing fiber samples at 10 days post anthesis (DPA) and 20 DPA. Differentially expressed genes (DEGs) were obtained via pairwise comparisons among all 24 samples (each one with three biological repeats). A total of 969 DEGs related to FL-high, 1285 DEGs to FS-high, and 997 DEGs to FQ-high were identified. The functional enrichment analyses of them indicated that the GO terms of cell wall structure and ROS, carbohydrate, and phenylpropanoid metabolism were significantly enriched, while the GO terms of glucose and polysaccharide biosynthesis, and brassinosteroid and glycosylphosphatidylinositol metabolism could make great contributions to FL and FS formation, respectively. Weighted gene co-expressed network analyses (WGCNA) were separately conducted for analyzing FL and FS traits, and their corresponding hub DEGs were screened in significantly correlated expression modules, such as EXPA8, XTH, and HMA in the fiber elongation and WRKY, TDT, and RAC-like 2 during secondary wall thickening. An integrated analysis of these hub DEGs with previous QTL identification results successfully identified a total of 33 candidate introgressive DEGs with non-synonymous mutations between the Gh and Gb species. A common DEG encoding receptor-like protein kinase 1 was reported to likely participate in fiber secondary cell thickening regulation by brassionsteroid signaling. Such valuable information was conducive to enlightening the developing mechanism of cotton fiber and also provided an abundant gene pool for further molecular breeding.

Verticillium wilt (VW) caused by Verticillium dahliae (Vd) is a devastating fungal cotton disease characterized by high pathogenicity, widespread distribution, and frequent variation. It leads to significant losses in both the yield and quality of cotton. Identifying key non-synonymous single nucleotide polymorphism (SNP) markers and crucial genes associated with VW resistance in Gossypium hirsutum and Gossypium barbadense, and subsequently breeding new disease-resistant varieties, are essential for VW management. Here, we sequenced the transcriptome and metabolome of roots of TM-1 (G. hirsutum) and Hai7124 (G. barbadense) after 0, 1, and 2 days of V991 inoculation. Transcriptome analysis identified a total of 72,752 genes, with 5814 differentially expressed genes (DEGs) determined through multiple group comparisons. KEGG enrichment analysis revealed that the key pathways enriched by DEGs obtained from both longitudinal and transverse comparisons contained the glutathione metabolism pathway. Metabolome analysis identified 995 metabolites, and 22 differentially accumulated metabolites (DAMs), which were correlated to pathways including glutathione metabolism, degradation of valine, leucine, and isoleucine, and biosynthesis of terpenoids, alkaloids, pyridine, and piperidine. The conjoint analysis of transcriptomic and metabolomic sequencing revealed DAMs and DEGs associated with the glutathione metabolism pathway, and the key candidate gene GH_D11G2329 (glutathione S-transferase, GSTF8) potentially associated with cotton response to VW infection was selected. These findings establish a basis for investigating the mechanisms underlying the cotton plant’s resistance to VW.

Leucine-rich repeat extensins (LRXs) are essential regulators of plant development, cell wall integrity, and stress responses. However, genome-wide LRX studies in cotton are limited. Analysis of four Gossypium species identified 29, 28, 16, and 16 LRX genes in G. hirsutum, G. barbadense, G. arboreum, and G. raimondii, respectively. Phylogenetic analysis resolved these 89 genes into four subfamilies (I–IV). Structural annotation revealed that cotton LRX family members exhibit conserved domain architectures. This finding was corroborated by motif analysis, which revealed notable conservation in the motif compositions of most cotton LRX proteins, suggesting functional conservation across evolutionary lineages. Distinct spatiotemporal expression patterns were uncovered between G. hirsutum and G. barbadense. Prolonged exposure to extreme temperatures induced widespread down-regulation of most GhLRX genes, whereas genes in subgroup IV were significantly up-regulated under salt and drought stress conditions, respectively. Notably, GhLRX7 showed a more proactive responding profile to Verticillium wilt (VW) infection, which was therefore selected for functional validation employing virus-induced gene silencing in the cotton cultivars MBI9626 and CCRI36. Phenotypic analysis of silenced plants revealed exacerbated disease symptoms compared to wild-type controls, providing direct evidence implicating GhLRX7 as a key contributor to defense against VW.