Abstract:【Objective】The purpose of this study was to identify the members of the Hsf gene family in citrus species, including sweet orange, mandarin, and pomelo at the whole-genome level, and to investigate the expression characteristics of CsHsfs in sunburned fruit peel of satsuma mandarin and heat-stressed peel of kumquat, as well as to explore the key Hsf genes possibly involved in citrus fruit sunburn occurrence/ kumquat to heat stress.【Methods】The Hsf family in citrus was identified, and its physicochemical properties, evolutionary development, structural features, cis-acting elements, collinearity, transcriptome, and expression patterns in different tissues of sunburned fruit peel and heat-treated kumquat peel were analyzed.【Results】In this study, 14, 20, and 16 Hsf genes were identified from the genomes of sweet orange, clementine mandarin, and wanbai pomelo respectively. Physicochemical property analysis revealed that the protein lengths and molecular weights were similar among the three citrus varieties, with protein lengths ranging from 271 to 517 amino acids for sweet orange, 208 to 505 amino acids for clementine mandarin, and 261 to 516 amino acids for wanbai pomelo. The molecular weight ranges were 30.1 to 56.3 kDa for sweet orange, 24.1 to 56.2 kDa for clementine mandarin, and 30.1 to 56.1 kDa for wanbai pomelo. Subcellular localization prediction indicated that all 50 Hsf genes from the three citrus species were localized in the cell nucleus, consistent with their transcription factor characteristics. Additionally, evolutionary analysis of Hsf genes of sweet orange, clementine mandarin, wanbai pomelo, as well as Arabidopsis, which categorized into three clusters (HSFA, HSFB, and HSFC). The number of members in each cluster followed the pattern HSFA > HSFB > HSFC, with the fewest members in cluster C, where each species had only one member. Gene structure analysis revealed that members within the same cluster exhibited similar and conserved structural features. Additionally, certain motifs were specific to particular clusters; for instance, motifs 6, 8, and 17 were exclusively present in cluster-A members, while motif 15 was unique to cluster-B members. Motif 5 was found only in cluster-A and cluster-B members, whereas motif 4 occurred solely in clusters-A and clusters-C. Furthermore, the majority of members contained only 2 exons, with a few exceptions such as CsHsf9, CcHsf1, CcHsf4, CcHsf16, and CgHsf14, which harbored 3 exons. The analysis of cis-acting elements revealed that among the 9 hormone-responsive elements, the methyl jasmonate-responsive elements (TGACG-motif, CGTCA-motif), and abscisic acid-responsive elements (ABRE) were the most abundant in the Hsfs promoters. Specifically, CsHsf2, CsHsf4, CcHsf3, CcHsf13, and CgHsf10 contained 8-12 methyl jasmonate-responsive elements, while CsHsf2, CsHsf6, CsHsf8, CsHsf9, CcHsf4, and CgHsf7 contained 8-9 ABA-responsive elements. Among the 5 stress-responsive elements, the drought-inducible response element (MBS) was the most prominent in the Hsfs promoters. Notably, CsHsf8, CsHsf11, CsHsf13, CcHsf11, CcHsf12, CcHsf14, CcHsf15, CcHsf18, and CgHsf9 contained 3-5 MBS elements. The collinearity analysis revealed that segmental duplication events played a predominant role in the expansion of the citrus Hsf family. Three segmental duplication events were identified in each of the sweet orange, clementine mandarin, and wanbai pomelo species. Specifically, in sweet orange, these events involved CsHsf5/7, CsHsf8/13, and CsHsf10/14; in clementine mandarin, they involved CcHsf9/10, CcHsf13/18, and CcHsf15/17; and in wanbai pomelo, they involved CgHsf6/7, CgHsf8/16, and CgHsf10/12. Additionally, interspecies collinearity relationships were observed, with 11, 15, 16, 17, 20, and 20 collinear gene pairs identified between sweet orange and Arabidopsis, Clementine mandarin and Arabidopsis, wanbai pomelo and Arabidopsis, sweet orange and clementine mandarin, sweet orange and wanbai pomelo, and clementine mandarin and wanbai pomelo, respectively. Transcriptome analysis revealed that the majority of CsHsf members exhibited specific expression in the outer peel or mesocarp of ‘Fengjie’ navel oranges. Additionally, nearly all CsHsf members showed differential expression compared to normal untreated leaves after exposure to light and heat stress in trifoliate orange leaflets. Specifically, CsHsf4, CsHsf5, CsHsf9, CsHsf10, CsHsf11, and CsHsf13 were significantly upregulated at different stages in the mesocarp of rough-skinned large fruits compared to normal fruits, with CsHsf5 showing upregulation throughout the entire fruit development period of Peel Roughing-Disordered large fruits compared to normal fruits. RT-qPCR analysis revealed significant differential expression of nearly all CsHsf genes in the sunburned peel of satsuma mandarin compared to normal peel, and CsHsf1, CsHsf5, CsHsf6, and CsHsf8 exhibited significant differential expression between heat-stressed and control kumquat peel. Finally, through comparison of expression patterns in multiple tissues and under stress conditions in leaves or citrus peels, CsHsf5 was found to be enriched in the sunburned peel of satsuma mandarin, heat-stressed kumquat peel, and Peel Roughing-Disordered large fruits. Therefore, CsHsf5 was selected as a key candidate gene possibly involved in citrus fruit sunburn occurrence.【Conclusion】this study identified and analyzed the members of the Hsf gene family in citrus species, clarifying their expression patterns in sunburned peel and heat-stressed peel tissues. These findings would provide a theoretical foundation for systematically unraveling the functions of Hsf genes in citrus and the molecular mechanisms underlying citrus fruit sunburn.
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