Genome-wide identification of Hsf gene family in citrus and functional analysis of CsHsfA1a in heat stress response.

key messageGenome-wide identification of Hsf gene family and functional analysis revealed that CsHsfA1a as a positive regulator of heat stress response in citrus. Heat shock transcription factors (Hsfs) are essential for regulating plant stress responses and contributing to growth and development. Although Hsfs have been extensively characterized in several model plant species, there is limited information regarding their presence and function in citrus species. Here, we identified 89 Hsf genes across five citrus genomes and thoroughly analyzed their phylogenetic relationships, gene structures, conserved motifs, and promoter cis-acting elements. qRT-PCR analysis demonstrated that the Hsf gene family in sweet orange exhibited tissue-specific expression patterns and responded to various abiotic stresses and hormonal treatments, with CsHsfA1a consistently showing upregulated expression under heat stress conditions. Subcellular localization and transcriptional activation assays revealed that CsHsfA1a was localized in the nucleus and exhibited transcriptional activation activity. Overexpression of CsHsfA1a remarkably enhanced heat tolerance in tobacco, whereas silencing CsHsfA1a in trifoliate orange increased the sensitivity to heat stress. Furthermore, DAP-qPCR, Y1H, and Dual-Luc assays confirmed that CsHsfA1a functioned as a transcriptional activator of CsHsfA2 by binding to its promoter region. In summary, our findings elucidate the potential roles of CsHsfA1a in response to heat stress, thereby providing valuable targets for enhancing citrus heat tolerance through genetic manipulation.