Physiological and Transcriptome Analyses Provide Insights into the Response of Grain Filling to High Temperature in Male-Sterile Wheat (Triticum aestivum L.) Lines

High-temperature (HT) stress frequently affects the early and middle stages of grain filling in hybrid seed production regions. Photo-thermo-sensitive male-sterile (PTMS) wheat lines, which play a critical role as female parents in hybrid seed production, face challenges under HT conditions. However, the mechanisms governing grain filling in PTMS lines under HT stress remain poorly understood. This study used the BS253 line to investigate the effects of HT on grain filling, primarily focusing on the transition from sucrose unloading to starch synthesis. The findings indicated that HT significantly reduced the grain starch content and weight by 7.65% and 36.35% at maturity, respectively. Further analysis revealed that the expression levels of TaSUT1 and TaSWEETs in grains initially increased after HT stress, paralleling the rise in sucrose content during the same period. The activities of ADP-glucose pyrophosphorylase, UDP-glucose pyrophosphorylase, granule-bound starch synthase, and soluble starch synthase were markedly decreased, indicating that impaired starch synthesis was a key factor limiting grain filling immediately after HT exposure. A total of 41 key regulatory genes involved in sucrose-to-starch metabolism were identified, with HT significantly reducing the expression of genes associated with pathways from sucrose unloading to starch synthesis during the middle and late stages post-HT. Based on the observed ultrastructural changes in the abdominal phloem and sucrose transporter expression levels under HT, we concluded that limited sucrose supply, degradation, and inhibition of starch synthesis collectively constrained grain filling during these stages. Additionally, 11 heat shock proteins and two catalase genes were identified and significantly upregulated during the initial phase post-HT, suggesting their potential role in enhancing sucrose supply at this critical time. More importantly, seven key genes involved in the sucrose-to-starch pathway were identified by weighted gene co-expression network analysis (WGCNA), which provides target genes for their functional research for starch synthase. These findings provide a comprehensive understanding of how HT limits grain filling, identify several genes involved in the sucrose-to-starch pathway, and offer a novel perspective for future research on HT-restricted grain filling across the entire process from sucrose unloading to starch synthesis in developing grains.