Wheat is a cornerstone of global food security, with its production being pivotal in both China and the international community. With global climate change, the threat of high temperature has become increasingly prominent, posing a significant challenge to wheat cultivation. The strategic identification and selection of heat-tolerant germplasm, coupled with the exploration of genes associated with heat resistance, are crucial steps. These efforts are essential for broadening the genetic diversity of heat tolerance in wheat within China, providing prerequisites for breeding heat-tolerant wheat varieties and ultimately contributing to the safeguarding of our nation's food security in the face of a warming climate.

In this study, a natural population of 331 wheat accessions was utilized, and artificial climate chambers were employed to simulate high temperatures conditions. The heat tolerance of wheat seedlings was assessed by monitoring their survival rate under various durations of treatment, using heat resistance grade as the evaluative metric. Meanwhile, a genome-wide association study (GWAS) was conducted using the 55K SNP chip to identify genetic loci associated with heat tolerance. Expression data from multiple tissues, including roots, leaves under heat stress were analyzed, leading to the selection of genes related to heat tolerance. Subsequently, qPCR validation of candidate genes was performed using the extremely heat-tolerant accession Xinong 889 and the heat-sensitive accession Chinese Spring (CS) as materials.

Under high-temperature stress, significant variations in survival rates were observed among different wheat accessions. The extremely heat-tolerant, moderately heat-tolerant, moderately heat-sensitive, and extremely heat-sensitive germplasm accounted for 110, 104, 110, and 7, respectively, representing 33.23%, 31.42%, 33.23%, and 2.12% of the total. Heat-tolerant germplasms, including Xinong 889, Zhengmai 7698, Zhongmai 895, Zhoumai 18, and Fengchan 3, were identified. Through GWAS, a total of 293 SNP loci significantly associated with the 12-hour survival rates (SR) and heat resistance grades (HRG) were detected, with the phenotypic variation explained ranging from 4.40% to 12.46%. Among these, 200 loci were related to the 12-hour survival rates, and 257 were related to the heat resistance grades, with 164 loci identified as the same heat-related loci. Based on significantly associated SNP markers, 313 heat-related genes were predicted. According to gene annotation information and expression data under heat stress, 23 heat tolerance candidates were selected, and after qPCR validation of differentially expressed candidate's genes, 20 key heat tolerance candidate genes were identified.

At the seedling stage, 331 wheat germplasms were identified for heat tolerance. A rapid method was developed for determining the survival rate of wheat seedlings subjected to treatments of varying durations at 45 ℃ to assess their heat tolerance In total, 38 heat-tolerant germplasms and 293 loci significantly associated with seedling heat tolerance were screened. Also, TraesCS1A02G355900, TraesCS1A02G389500, TraesCS5A02G550700, TraesCS5D02G557100, TraesCS6D02G402500 and TraesCS7A02G232500 represented as candidate genes were filtered out.

The GRAS family constitutes a unique class of plant-specific transcription factors that play a pivotal role in plant development and stress response. To elucidate the function of GRAS family genes in wheat heat tolerance, which can provide genetic resources and theoretical foundation for wheat heat-resistant breeding.

A potential heat stress-responsive transcription factor gene, TaGRAS34-5A, was identified through transcriptome analysis of TAM107 and Chinese spring wheat seedlings under high-temperature conditions. Subsequently, a bioinformatics analysis was performed on TaGRAS34-5A, and a phylogenetic tree was constructed to elucidate its molecular characteristics. The expression pattern of TaGRAS34-5A under various stresses, including high temperature, abscisic acid (ABA), ethylene (ETH), and salicylic acid (SA) treatments, were examined using real-time quantitative reverse transcription polymerase chain reaction (qRT-PCR) method. The subcellular localization of the TaGRAS34-5A protein was determined using wheat protoplast transient expression technique. Furthermore, the heat tolerance function of TaGRAS34-5A was validated using the heterologous expression system of Saccharomyces cerevisiae and the BSMV:VIGS (Barley stripe mosaic virus: Virus-Induced Gene Silencing) silencing technique. potential interacting proteins of TaGRAS34-5A were screened using yeast two-hybrid technology, and the heat tolerance function was verified, providing preliminary insights into its heat tolerance mechanism.

TaGRAS34-5A, equipped with a characteristic GRAS domain and belongs to the GRAS transcription factor family, is localized to both the cell nucleus and cytoplasm. Bioinformatics analysis indicates that the TaGRAS34-5A promoter contains a large number of hormone response elements and light response elements, and it is most closely related to TaSCL14, OsGRAS23, and AtSCL14 in terms of phylogenetic relationships, suggesting its potential function in responding to oxidative stress. Its expression is upregulated under high-temperature, ethylene (ETH), abscisic acid (ABA), and salicylic acid (SA) treatments, peaking at 4, 6, 0.5, and 12 hours post-treatment, respectively, with the most significant induction observed under heat stress and SA. Functional assays in yeast demonstrated that heterologous expression of TaGRAS34-5A enhances the heat tolerance of the yeast. The results of BSMV:VIGS transient silencing experiment showed that after the 42 ℃ high-temperature treatment, TaGRAS34-5A silenced plants exhibited decreased chlorophyll content, reduced POD enzyme activity, increased cellular peroxidation, and decreased heat tolerance compared to the control. Preliminary studies on the heat tolerance mechanism suggest that TaGRAS34-5A exhibits strong transcriptional self-activation activity.it may modulate wheat heat tolerance by interacting with proteins such as the bZIP family transcription factor HBP-1b and the E3 ubiquitin ligase hel2, thereby regulating cellular redox homeostasis and detoxification processes, positively influencing the heat tolerance of wheat.

TaGRAS34-5A is induced by heat, ABA, ETH, and SA, and its encoded protein is located in the nucleus and cytoplasm. It exhibits transcriptional activation activity. Heterologous overexpression of TaGRAS34-5A enhances the heat tolerance of Saccharomyces cerevisiae. Silencing TaGRAS34-5A in wheat plants increases cellular peroxidation, decreases chlorophyll content, and reduces heat tolerance. TaGRAS34-5A may regulate the heat tolerance of wheat by modulating cellular redox state and detoxification processes.