Transcriptome and WGCNA Analyses Reveal Regulatory Networks and Hub Genes Under Different Durations of Heat Stress in Safflower (Carthamus tinctorius L.)

Safflower (Carthamus tinctorius L.) is an economically important crop, and heat stress has become a major environmental constraint that limits its growth and development under global climate change. However, the molecular mechanisms underlying its response to heat stress remain poorly understood. Here, transcriptome sequencing was performed on safflower leaves exposed to heat stress (42 °C) for 0, 1, 2, 4, 8, and 12 h, with three biological replicates per time point. Compared with the control (0 h), a total of 12,964 differentially expressed genes (DEGs) were identified across the five time points (1, 2, 4, 8, and 12 h) using criteria of |log2 (fold change)| ≥ 1 and false discovery rate (FDR) < 0.05, of which 1097 were common to all comparisons. KEGG enrichment analysis of these DEGs across all five comparison groups consistently showed significant enrichment in plant hormone signal transduction and the MAPK signaling pathway. Furthermore, a total of 750 transcription factors (TFs) were identified as differentially expressed across the five comparison groups, of which 99 were common to all comparisons, with the bHLH, MYB, WRKY, and HSF families being the most abundant. Weighted Gene Co-expression Network Analysis (WGCNA) identified five modules that were significantly associated with different heat stress time points. Furthermore, 13 hub genes were identified as potential targets for future functional studies on heat tolerance in safflower. The reliability of the RNA-seq data was confirmed by qRT-PCR validation of selected hub genes. Notably, a non-specific serine/threonine protein kinase (CtAH03G0292100) from the MEred module, which is also involved in plant hormone signal transduction, emerged as a promising candidate gene for heat tolerance. Collectively, these findings provide candidate genes for future functional studies aimed at further elucidating the mechanisms of heat tolerance in safflower.