The increasing frequency of low-temperature events in spring, driven by climate change, poses a serious threat to wheat production in Northern China. Understanding how low-temperature stress affects wheat yield and its components under varying moisture conditions, and exploring the role of irrigation before exposure to low temperatures, is crucial for food security and mitigating agricultural losses. In this study, four wheat cultivars—semi-spring (YZ4110, LK198) and semi-winter (ZM366, FDC21)—were tested across two years under different conditions of soil moisture (irrigation before low-temperature exposure (IBLT) and non-irrigation (NI)) and low temperatures (−2 °C, −4 °C, −6 °C, −8 °C, and −10 °C). The IBLT treatment effectively reduced leaf wilt, stem breakage, and spikelet desiccation. Low-temperature stress adversely impacted the yield per plant—including both original and regenerated yields—and yield components across all wheat varieties. Furthermore, a negative correlation was found between regenerated and original yields. Semi-spring varieties showed greater yield reduction than semi-winter varieties, with a more pronounced impact under NI compared to IBLT. This suggests that the compensatory regenerative yield is more significant in semi-spring varieties and under NI conditions. As low-temperature stress intensified, the primary determinant of yield loss shifted from grain number per spike (GNPS) to spike number per plant (SNPP) beyond a specific temperature threshold. Under NI, this threshold was −6 °C, while it was −8 °C under IBLT. Low-temperature stress led to variability in fruiting rate across different spike positions, with semi-spring varieties and NI conditions showing the most substantial reductions. Sensitivity to low temperatures varied across spikelet positions: Apical spikelets were the most sensitive, followed by basal, while central spikelets showed the largest reduction in grain number as stress levels increased, significantly contributing to reduced overall grain yield. Irrigation, variety, and low temperature had variable impacts on physiological indices in wheat. Structural equation modeling (SEM) analysis revealed that irrigation significantly enhanced wheat’s response to cold tolerance indicators—such as superoxide dismutase (SOD), proline (Pro), and peroxidase (POD)—while reducing malondialdehyde (MDA) levels. Irrigation also improved photosynthesis (Pn), chlorophyll fluorescence (Fv/Fm), and leaf water content (LWC), thereby mitigating the adverse effects of low-temperature stress and supporting grain development in the central spike positions. In summary, IBLT effectively mitigates yield losses due to low-temperature freeze injuries, with distinct yield component contributions under varying stress conditions. Furthermore, this study clarifies the spatial distribution of grain responses across different spike positions under low temperatures, providing insights into the physiological mechanisms by which irrigation mitigates grain loss. These findings provide a theoretical and scientific basis for effective agricultural practices to counter spring freeze damage and predict wheat yield under low-temperature stress.

WRKY (WRKY domain-containing) transcription factors represent one of the largest families of plant-specific transcription regulators and play pivotal roles in plant growth, development, nutrient acquisition, and responses to biotic and abiotic stresses. In this study, the coding sequence of TaWRKY65-3B was cloned from the hexaploid wheat cultivar Hanxuan 10. Domain analysis revealed that TaWRKY65-3B contains a conserved WRKY domain and a typical C₂H₂-type zinc finger motif. The gene spans 1478 bp and comprises two exons and one intron. Promoter analysis identified multiple cis-acting regulatory elements, including those responsive to abscisic acid, methyl jasmonate, drought, and low temperature. Quantitative real-time PCR (qRT-PCR) demonstrated that TaWRKY65-3B was significantly upregulated under polyethylene glycol (PEG)-simulated-drought stress in wheat. Overexpression of TaWRKY65-3B in rice resulted in marked growth inhibition, characterized by reduced plant height, shortened grains, and decreased grain width, perimeter, and surface area. These morphological changes led to a significant reduction in thousand-grain weight and overall grain yield. Mechanistically, TaWRKY65-3B activated the expression of OsCPS2, OsCPS4, and OsKS4, which encode key enzymes in the diterpenoid phytoalexin biosynthetic pathway. Subsequent oxidative modifications catalyzed by the cytochrome P450 monooxygenase OsCYP76M7 facilitated the accumulation of the defensive diterpenoid neoabietic acid. Integrated transcriptomic and metabolomic analyses revealed that constitutive TaWRKY65-3B overexpression reprograms simulated-drought-responsive metabolism in rice, with a diterpenoid metabolite annotated as neoabietic acid as a key correlative feature—though definitive structural confirmation requires authentic standards. These findings identify a WRKY-mediated regulatory module connecting diterpenoid phytoalexin metabolism to simulated-drought stress responses, although the precise molecular cascades governing the growth-defence trade-off between stress adaptation and yield warrant further investigation.