Pangenome-wide identification and analysis of the heat shock transcription factor gene family in tea plant (Camellia sinensis)

Heat shock transcription factors ( Hsf ) are key regulators of plant responses to environmental stresses, but their intraspecific diversity and evolutionary dynamics in the tea plant remain insufficiently understood. In this study, we performed a pangenome-wide identification and systematic analysis of the Hsf gene family across 22 Camellia sinensis genomes. A total of 536 Hsf genes were identified, with gene numbers ranging from 15 in HD to 33 in SCZ, indicating substantial accession-level copy number variation. Phylogenetic analysis classified these genes into three major subfamilies, including Hsf-A, Hsf-B, and Hsf-C, among which Hsf-A was the largest group. Orthogroup-based pangenome analysis assigned the 536 Hsf genes to 21 orthogroups, including 7 core orthogroups and 14 dispensable orthogroups, suggesting that dispensable genes represent an important component of the tea Hsf repertoire. Gene duplication analysis showed that dispersed duplication and WGD/segmental duplication were the major forces driving Hsf family expansion, whereas proximal and tandem duplications contributed relatively little. Copy number variation was widespread among Hsf orthogroups, especially in dispensable orthogroups. Selection pressure analysis revealed that most homologous Hsf gene pairs were under purifying selection, although selection intensity differed among subfamilies. Transcriptome analysis of SCZ showed tissue-preferential expression patterns and PEG-responsive expression divergence. Notably, Hsf-B genes exhibited the highest average expression under PEG stress and occupied a central position in the PCC-based co-expression network. Several genes, including CSS0020980.1 and CSS0019914.1, were identified as candidate PEG-responsive Hsf genes. These results provide a comprehensive pangenome-level framework for understanding Hsf gene evolution and stress-responsive regulation in tea plant.