IntroductionTemperature is a critical factor affecting plant growth and development. When ambient temperatures exceed the optimal threshold for plant adaptation, they can retard growth and, under severe conditions, cause plant mortality. Thermotolerance is a complex trait controlled by multiple loci and influenced by environment factors1. However, identifying specific genes associated with thermotolerance in plants remains a significant challenge. Currently, much of the research has focused on rice. For example, TT1 (Thermo-tolerance 1), encoding an α2 subunit of the 26S proteasome, was cloned from heat-tolerant African rice (Oryza glaberrima). Genetic variations in both the promoter and coding region of TT1 contribute to its enhanced thermotolerance compared to other rice species2. Similarly, allelic variation in SLG1 (Slender Guy 1), encoding the cytosolic tRNA 2-thiolation protein 2, impacts thermotolerance in indica rice. The discovery of the tRNA thiolation pathway through SLG1 provides a promising target for the next generation of rice breeding strategies to enhance heat resilience3. These findings underscore the importance of leveraging both heat-tolerant and heat-sensitive germplasms as valuable research materials, as they are highly effective in identifying key thermotolerance genes.Grapevine, known for its economic significance in providing table grapes and serving winemaking, is the largest fruit crop worldwide. However, the escalating impacts of global warming and the expanding grape industry have made heat stress a significant challenge to the sustainable development of the international grape industry. At present, most widely cultivated grape cultivars, primarily derived from Vitis vinifera, exhibit limited thermotolerance. Under high temperatures, these cultivars often show weak bud differentiation, thin leaves, poor fruit flavor, and inadequate coloring. It is anticipated that many international grape cultivars originating from cooler climates may struggle to adapt to extreme heat-stress conditions4. To date, investigations into thermotolerance in grapevines has been focused on physiological and biochemical aspects, using transcriptomic and proteomic approaches. Most studies have centered on V. vinifera5,6,7,8,9,10,11. And significant progress in identifying and characterizing genes associated with thermotolerance in grapevines has been limited. Although ten thermotolerance-related genes (VvHSFA2, VaHSFC1, VvMBF1c, VvDREB2c, VvBAP1, VvbZIP60s, VvHSFB1, VpHSF1, VdHSFA2 and VqHSFB1) have been cloned, their functional validation has largely conducted in model species such as Arabidopsis thaliana or tobacco12,13,14,15,16,17,18. To date, only HSFA2 and HSFB1 have been confirmed to play direct roles in grape16,17. The difficulty in elucidating the molecular mechanisms underlying thermotolerance in grapevines arises from challenges in accurately evaluating phenotypes and the complexity of associated genetic pathways. Previously, we developed a rapid and accurate method to evaluate grape thermotolerance, enabling us to demonstrate that wild species such as V. davidii and V. quinquangularis, native to warmer regions of China, generally exhibit stronger thermotolerance, despite possessing less desirable economic traits19. To advance our understanding of grapevine thermotolerance, it is crucial to explore and evaluate the indigenous diversity of heat-tolerant germplasms.In this study, we identify a key thermotolerance gene, TTC4 (thermotolerance on chromosome 4), which encodes a WRKY transcription factor. TTC4 positively regulates thermotolerance by directly activating expression of HSP18.1 (18.1 kDa class I heat shock protein) and APX3 (L-ascorbate peroxidase 3). A critical discovery from this study is the sequence variation at SNP-T/C (7631) in the TTC4 intron 2. This T to C substitution influences the intron 2 capacity to enhance gene expression and the binding affinity of the negative regulator SPL13 (SQUAMOSA-promoter binding protein-like 13). Consequently, this variation impacts TTC4 expression levels, contributing to differences in thermotolerance among grapevine varieties. TTC4 holds significant potential for grapevine breeding, offering valuable insights and tools to enhance crop resilience under heat stress.ResultsThe identification and characterization of TTC4To investigate the genetic diversity related to thermotolerance in grapevines, we assessed the thermotolerance of 121 grapevine accessions (Supplementary Data 1). Thermotolerance was evaluated by measuring chlorophyll α fluorescence values Fv/Fm (the potential maximum quantum yield of primary photochemistry) of leaves. As expected, the Fv/Fm values of V. vinifera accessions were significantly lower (P