Genome-wide characterization of HSP40 family and the HSF-HSP40 regulatory cascade underlying high temperature and high humidity stress tolerance in Zingiber officinale Roscoe

As a high-value industrial crop widely used in food and pharmaceutical fields, Zingiber officinale Roscoe is highly susceptible to combined high temperature and high humidity (HTHH) stress, but the molecular basis of HTHH tolerance remains poorly understood. Here, 97 ZoHSP40 genes were identified in ginger and classified into three subfamilies, with segmental duplication as the major driver of family expansion. Transcriptome and RT-qPCR analyses revealed that ZoHSP40–59 was one of the most strongly HTHH-induced members, showing approximately 10.94-, 18.09-, and 14.80-fold upregulation in roots, stems, and leaves, respectively. Functional assays using virus-induced gene silencing and overexpression demonstrated that ZoHSP40–59 positively regulates ginger HTHH tolerance by enhancing antioxidant enzyme activities and reducing oxidative damage. Mechanistically, the 1972 bp ZoHSP40–59 promoter drove stress-inducible GUS activity, with stronger activation under combined HTHH stress than under single HT or HH stress. ZoHSF02 and ZoHSF05 directly bound to distinct HSE-containing promoter fragments and positively activated ZoHSP40–59 transcription. In addition, ZoHSF05 physically interacted with ZoHSF13 and ZoHSF20, suggesting an HSF-mediated regulatory cascade that reinforces ZoHSP40–59 expression and antioxidant defense under HTHH stress. Collectively, this study establishes an ZoHSF-ZoHSP40 regulatory module underlying ginger adaptation to combined HTHH stress and provides candidate genetic resources for breeding stress-tolerant ginger cultivars.