A pumpkin heat shock factor CmHSF30 positively regulates thermotolerance in transgenic plants

Heat shock factors (HSFs) play a central role in regulating the responses of plants to various stresses. However, the function and regulation of HSFs in pumpkins remains largely unknown. In this study, an HSF, CmHSF30 was identified in Cucurbiamoschata, which belongs to the HSFA subfamily. The expression level of CmHSF30 was significantly upregulated in response to heat stress and exogenous phytohormone treatments, including ABA, GA, IAA, and SA. The CmHSF30 was localized in the nucleus and functions as a transcriptional activator. By overexpressing CmHSF30 in Arabidopsis and pumpkin, the function and regulation of CmHSF30 in response to heat stress were studied. The overexpression of CmHSF30 in Arabidopsis enhanced plant thermotolerance by increased germination rate and survival rate under heat stress, as evidenced by the elevated of contents chlorophyll and GSH, and SOD activity, and decreased contents of H₂O₂ and MDA. Furthermore, the overexpression of CmHSF30 in pumpkins also enhanced the thermotolerance of transgenic pumpkins by reducing cell death. In contrast, CRISPR/Cas9 mediated knockout of CmHSF30 decreased pumpkin thermotolerance. Besides, RT-qPCR analysis revealed that CmHSF30 plays a positive role in regulating the expression of stress-related genes, including AtHSP18.2, AtHSP20, AtHSP70, AtPP2C, and AtMYB82 from Arabidopsis and CmHSP18.2, CmHSP20, CmHSP70, CmPP2C, and CmMYB46 from pumpkin. Yeast two-hybrid showed that CmHSF30 interacts with CmMYB46. The results indicate that CmHSF30 functions as a positive regulator, enhancing plant thermotolerance by regulating target genes and reducing ROS accumulation.