Genome-wide identification of the HSF gene family in Chinese chestnut and functional characterization of CmHSF4 under temperature stress

Xiurong Xu¹ZiQi Wu²Shiming Cheng^1*

• ¹Zhejiang Academy of Forestry, Hangzhou, China
• ²Zhengzhou University, Zhengzhou, China

Heat shock transcription factors (HSFs) are key regulatory factors involved in plant responses to abiotic stress. To explore the HSF gene family characteristics in the Chinese chestnut (Castanea mollissima) (Cm) and its role in abiotic stress responses, we conducted a systematic analysis at the whole-genome level. Using bioinformatic methods, 18 CmHSF genes in the Chinese chestnut genome were identified, and these were unevenly distributed across 12 chromosomes. Phylogenetic analysis classified them into three subfamilies, A, B, and C, with the largest number of members (12) in A. Protein-conserved motif and gene structure analyses indicated that the internal structures of each subfamily were conserved, whereas significant differences existed among subfamilies, suggesting functional differentiation. Synteny analysis revealed that segmental duplication was the main driving force for the expansion of this family and that the genes were under strong purifying selection. Cis-acting element analysis showed that the promoter regions of CmHSF genes were rich in elements related to abiotic stress (such as hypoxia and low temperature) and hormonal responses, suggesting their involvement in complex stress regulatory networks. Expression pattern analysis further revealed the diversity of CmHSF gene functions. The transcriptome data indicated that different members exhibited complex and specific expression patterns under shade, low temperature, high temperature, and drought stress. Among them, the CmHSF4 gene was particularly prominent under high-temperature stress, and its expression level increased sharply by 324 times at 4 h, indicating that it is a core candidate gene for understanding Chinese chestnut responses to heat stress. Subcellular localization experiments confirmed that CmHSF4 is localized in the nucleus, which is consistent with its characteristics as a transcription factor. Preliminary functional verification in transgenic tobacco showed that high-temperature stress induced significant oxidative damage (increased MDA and H₂O₂ content), laying the foundation for further in-depth research on the stress resistance function of CmHSF4.