Eriobotrya japonica (loquat) is an evergreen fruit tree of the apple tribe in Rosaceae with high edible and medicinal value. The yield and quality of loquat fruits are significantly influenced by environmental stress, particularly heat stress during fruit ripening. In this study, thirty EjHSFs were identified in the loquat genome. Twenty-nine EjHSFs were unevenly distributed across sixteen chromosomes, except Chr-6. A synteny analysis revealed that twenty-six EjHSF genes had undergone duplication events. Twenty-nine EjHSF genes were found to be in sync with HSF in apples while also diverging with other Rosaceae species. A phylogenetic analysis revealed that EjHSFs could be divided into three categories, including eighteen EjHSF-A, ten EjHSF-B, and two EjHSF-C. Twenty-nine members of the EjHSF family showed high homology to those of Malus domestica and Gillenia trifoliate. A promoter analysis retrieved thirty-three CAREs that were functionally relevant and connected to the expression of HSFs. Among these, the promoters of twenty-three EjHSF genes possessed at least one STRE element that could be activated by heat shock. Eleven of these EjHSFs were highly expressed in loquat tissues, namely EjHSF-B4a, EjHSF-A4a, EjHSF-A4d, and EjHSF-C1a in roots; EjHSF-B4b in roots and inflorescence; EjHSF-C1b in stems and roots; EjHSF-A2a in three tissues; EjHSF-A8b in four tissues; and EjHSF-A4c, EjHSF-B1a, and EjHSF-B2b in six tissues. Moreover, fifteen EjHSFs were differentially expressed (eleven upregulated and four downregulated) in fruits under heat stress treatment in the color-changing period. Among them, EjHSF-A2a and EjHSF-A2b upregulated transcriptional abundance over 300 times after heat treatment; EjHSF-B2b consistently displayed an increasing trend over time; and EjHSF-B1a was significantly downregulated. Hence, these results suggest that EjHSFs take part in loquat tissue development and may be involved in the fruit’s heat stress response. These findings enhance the understanding of EjHSFs’ role within loquats and the heat stress response of its fruit and provide target genes for heat stress improvement.
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