Abstract
Global warming poses a substantial threat to crop productivity, yet the genetic basis of thermotolerance in wheat remains poorly understood. Here we cloned a heat stress tolerance (HST) gene, TaHST2, and revealed that it underwent functional silencing during wheat domestication. As a negative regulator of basal HST, TaHST2 was progressively suppressed through intronic sequence polymorphisms and epigenetic modifications, which might be an evolutionary consequence of hexaploidization. Haplotype analysis suggests strong artificial selection against TaHST2 expression, favouring improved thermotolerance in cultivated wheat. Further studies demonstrated that TaHST2 encodes a ubiquitin hydrolase that stabilizes HST repression proteins TaHSC701 and TaHSC702, thereby modulating heat response pathways. Our findings uncover a potential key genetic event in wheat evolution and offer new strategies for utilizing synthetic hexaploidy and octoploid wheat to breed heat-resilient varieties.
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Data availability
The transcriptomic raw sequencing data have been deposited in the NCBI Sequence Read Archive database under accession numbers PRJNA1236532 and PRJNA1344897. The MS proteomics data have been deposited to the ProteomeXchange Consortium via the PRIDE partner repository with the dataset identifier PXD062212. Source data are provided with this paper. All other data needed to evaluate the conclusions in the paper are available in the Article and Supplementary Information.
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Acknowledgements
We thank Z. Wang (Xinjiang Academy of Agricultural Sciences) for providing the breeding lines derived from crosses between SHW and common wheat. We thank H. Li (Henan University) for providing A. tauschii accessions. We thank N. Song (China Agricultural University) for helping with wheat transformations. We thank Y. Guo, T. Zhang (China Agricultural University) and Q. Song (Nanjing Agricultural University) for reading and participating in the discussions relating to this study. Our confocal microscopy work was performed at the CAB Public Instrument Platform of Chinese Agricultural University, and we thank C. Sun and C. Yin (China Agricultural University) for their professional support. This work was supported by grants from the National Natural Science Foundation of China to Z.N. (no. 32530075), STI 2030-Major Projects to B.L. (no. 2023ZD0406903), the National Key Research and Development Program of China to J.X. (no. 2023YFF1000601), the National Natural Science Foundation of China to H.Z. (no. 32372210) and G. Liu (no. 32401832), and the China Postdoctoral Science Foundation to R.Z. (nos 2023M743818 and GZC20233050).
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B.L. conceived the project. B.L., Z.N. and J.X. designed the research procedure. R.Z. and G.L. performed the experiments. S.Z., X.M., S.W., X.L., W.H., H.L. and T.S. took part in the phenotype screening and field experiments. J.Y., Yuqi Zhang and W.G. contributed to the bioinformatics analysis. R.L., J.M., M.Y., C.X., Yufeng Zhang and H.Z. helped analyse the experimental data. R.Z. and G.L. wrote the manuscript. B.L., Z.N., J.X., J.L., Z.H. and Q.S. revised the manuscript.
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Extended data
Extended Data Fig. 1 Heat stress tolerance identification of parental plants and mapping strategy for TaHST2.
a, Grain morphology of XX329 and TAA10 under normal and HS conditions. TAA10 and XX329 plants were cultivated under normal conditions (16 h 23 °C/8 h 20 °C in green house), heat stress was conducted after anthesis (16 h 37 °C/8 h 25 °C in growth chamber) lasted for 18 d. Scale bars, 5 mm. b, Schematic diagram of mapping strategy for TaHST2.
Extended Data Fig. 2 The exon-intron structures of TaHST2 homologs in the reference genomes of different species.
The distribution and evolutionary trajectory of TaHST2 homlos.
Extended Data Fig. 3 Genotypic and phenotypic analyses of TaHST2 transgenic lines.
a, TaHST2 expression level in parental plants (TAA10 and XX329) and other spring wheat varieties. Wheat GAPDH was used as reference transcript, data are mean ± SD. b, Generation of tahst2 mutants using the CRISPR/Cas9 strategy. The positions and sequences of target sites for gene editing are shown above. The symbols ‘+’ and ‘-’ separately indicate the nucleotide insertion and deletion, and the base numbers of insertion/deletion are shown behind. The green blocks represent coding regions (CDS) and blue blocks represent untranslated regions (UTRs). c, Growth status of Fielder and TaHST2 knockout lines under normal (16 h 23 °C/8 h 20 °C) and HS (16 h 37 °C/8 h 25 °C, 30 d in growth chamber) condition. d, Comparison of SPAD value and plant height (PH) in c. e, Comparison of grain length (GL) between Fielder and TaHST2 transgenic lines under normal (16 h 23 °C/8 h 20 °C in green house) and HS condition. The plants were grown in green house (16 h 23 °C/8 h 20 °C) before treatment and subjected to heat stress after flowering (thermal stress tent in the field) lasted for 27 d. f, Comparison of TaHST2 expression level between WT and TaHST2 overexpression lines (**P < 0.01; two-tailed Student’s t-test; data are mean ± SD). Wheat GAPDH was used as reference transcript. g, TaHST2 protein abundance in WT and TaHST2 overexpression lines. In d,e, letters indicate significant differences (P < 0.05, ANOVA, Duncan’s multiple range test; data are mean ± SD). In a,d,e,f, n represents numbers of biologically independent samples. Scale bars, 20 cm (c).
Extended Data Fig. 4 Association analysis between variation sites of TaHST2 and its expression levels.
a, TaHST2 expression level in a panel of wheat germplasms. Wheat GAPDH was used as reference transcript. Values are mean ± SD, n represents numbers of biologically independent samples. b, Genotype of wheat germplasms in upstream polymeric structure and downstream SNP of TaHST2 and their expression level of TaHST2. The green blocks represent coding regions (CDS) and blue blocks represent untranslated regions (UTRs). c, PCR products of Xcau4D525-1 and Xcau4D525-2 in several hexaploid wheat accessions. d, Statistical comparison of TaHST2 expression levels between germplasms carrying the TaHST2S and TaHST2T haplotypes (**P < 0.01; two-tailed Student’s t-test; data are mean ± SD). n represents numbers of biologically independent samples.
Extended Data Fig. 5 Schematic diagrams of the reporter constructs used in the dual-luciferase transcriptional activity assays.
SI: containing the 4th and 5th exons along with the 4th intron of TaHST2; SII: containing the 5th and 6th exons along with the 5th intron of TaHST2; SIII: containing both SI and SII.
Extended Data Fig. 6 Temperature records from Beijing and thousand grain weight (TGW) of wheat sown at the different times.
a, Temperature data obtained in Beijing, China, between May and July 2024. Data from Global Historical Climatology Network hourly (GHCNh). The black solid lines correspond to the grain-filling periods of the wheat sown at autumn (September 26, 2023) and late-spring (March 29, 2024). b, TGW of TaHST2S and TaHST2T haplotype wheat lines under normal (autumn-sown) and HS (late spring-sown) condition. n represents the numbers of biologically independent samples. Data are mean ± SD.
Extended Data Fig. 7 Subcellular localization of TaHST2 and physical interactions between TaHST2 and TaHSC701 or TaHSC702.
a, Subcellular localization of TaHST2 in wheat leaf protoplasts under normal (28 °C) and HS (37 °C/7 h) condition. Before observation, the wheat protoplasts were transfected with constructs and incubated at 28°C for 16 h, followed by normal condition (28°C) or heat stress (37 °C) for 7 h. HDEL and AtRBP47 fused to mCherry were used as the marker for endoplasmic reticulum (ER) and stress granule (SG) localization, respectively. b, Bimolecular fluorescence complementation (BiFC) analysis of the interaction between TaHST2 and TaHSC701 or TaHSC702 in wheat leaf protoplasts under HS (37 °C/7 h) condition. Before observation, the wheat protoplasts were transfected with constructs and incubated at 28°C for 16 h, followed by heat stress (37 °C) for 7 h. AtRBP47 fused to mCherry was used as the marker for SG localization. c, Immunoblot analysis of the recombinant proteins obtained from the coimmunoprecipitation (Co-IP) assay. Red arrows indicate the TaHST2-GFP band. The tobacco leaves were infiltrated with Agrobacterium and incubated at 25°C for 55 h, leaf samples were then collected. In a-c, three biological replicates were performed, yielding similar results. Scale bars (a,b), 10 μm.
Extended Data Fig. 8 The ubiquitination levels of TaHSC701/702 after co-incubating with cell extracts derived from wild type and the TaHST2 overexpressing lines.
The purified recombinant His-TaHSC701 or His-TaHSC702 were incubated with cell extracts, then the His-TaHSC701/His-TaHSC701-(Ub)n or His-TaHSC702/His-TaHSC702-(Ub)n were purified with Ni-NTA Agarose and detected by immunoblotting with anti-His and anti-ubiquitin antibodies. Three biological replicates were performed, yielding similar results.
Extended Data Fig. 9 Genotypic analyses of TaHSC701 overexpression lines and expression levels of heat-stress responsive genes downstream of TaHSC701/702.
a, Comparison of TaHSC701 expression level between WT and TaHSC701 overexpression lines. n represents the numbers of biologically independent samples; data are mean ± SD, P-values were calculated by two-tailed Student’s t-test, **P < 0.01. Wheat GAPDH was used as reference transcript. b, TaHSC701 protein abundance in WT and TaHSC701 overexpression lines. c, Expression levels of heat-stress responsive genes downstream of TaHSC701/702 in XX329 and TaHSC702 mutants. In a, c, n represents the numbers of biologically independent samples; data are mean ± SD, P-values were calculated by two-tailed Student’s t-test, **P < 0.01; *P < 0.05; ns, not significant.
Extended Data Fig. 10 The auto-activation and DNA pull down assays for K4T-SIII and A8S-SIII.
a, Auto-activation assays for K4T-SIII and A8S-SIII. b, Venn diagram showing the numbers of proteins pulled down by K4T-SIII and A8S-SIII.
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Zhang, R., Liu, G., Zhai, S. et al. TaHST2 silencing shapes basal heat tolerance in allohexaploid wheat. Nat. Plants (2026). https://doi.org/10.1038/s41477-026-02257-0
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DOI: https://doi.org/10.1038/s41477-026-02257-0
