Heat stress is a major threat to maize (Zea mays L.) production worldwide. Heat shock transcription factors (HSFs) play vital roles in plant responses to heat stress. However, the molecular and genetic mechanisms underlying HSF-meditated thermotolerance in maize remain largely unexplored. In this study, we demonstrate that the alternative splicing of ZmHsf23 modulates heat stress tolerance in maize. Hsf23 produced two functional transcripts, Hsf23L and Hsf23S, which differ by the presence of a cryptic mini-exon in Hsf23L that is spliced out in Hsf23S. Both transcripts were strongly induced by heat stress. Mutants lacking Hsf23L alone (hsf23l) or both Hsf23L and Hsf23S (hsf23l23s) exhibited increased susceptibility to heat stress, whereas overexpression of Hsf23S enhanced heat stress tolerance in maize. Subsequently, we found that Hsf23S positively regulates heat stress tolerance by directly activating the transcription of three sHSP genes (Hsp16.9, Hsp17.2, and Hsp18a) and TIL1 gene. In addition, Hsf23L physically interacted with Hsf23S and enhanced the transcriptional activation of Hsf23S on the sHSPs and TIL1 promoters. Notably, genetic analysis suggested that co-overexpression of Hsf23L and Hsf23S further improves heat tolerance of the transgenic plants. Taken together, these results reveal two splicing variants of ZmHsf23 cooperatively regulate maize heat tolerance, thus highlighting potential value of ZmHsf23 in breeding heat-tolerant maize varieties.

GTs (Glycosyltransferases) are important in plant growth and abiotic stresses. However, its role in maize heat response is far from clear. Here, we describe the constitutively expressed UDP-glycosyltransferase ZmUGT92A1, which has a highly conserved PSPG box and is localized in chloroplasts, is induced under heat stress. Functional disruption of ZmUGT92A1 leads to heat sensitivity and reactive oxygen species accumulation in maize. Metabolomics analysis revealed that ZmUGT92A1 affected multiple metabolic pathways and altered the metabolic homeostasis of flavonoids under heat stress. In vitro assay showed ZmUGT92A1 exhibits glycosyltransferase activity on flavonoids and hormones. Additionally, we identified a rapidly heat-induced transcription factor, ZmHSF08, which can directly bind and repress the promoter region of ZmUGT92A1. The ZmHSF08 overexpression line exhibits heat sensitivity and reactive oxygen species accumulation. These findings reveal that the ZmHSF08-ZmUGT92A1 module plays a role in heat tolerance in maize and provide candidate strategies for the development of heat-tolerant varieties.