Phenotypic Variation in Water-Use Efficiency, Heat Tolerance, and Carbon Isotope Discrimination Across Canadian Spring Wheat Cultivars Under Climate Stress

Understanding phenotypic variation in traits associated with drought and heat tolerance is essential for developing climate-resilient spring wheat cultivars under increasingly variable environmental conditions. To evaluate phenotypic and physiological variation in water-use efficiency (WUE), carbon isotope discrimination (δ13C), and heat tolerance, 198 Canadian spring wheat cultivars representing diverse breeding backgrounds were assessed under controlled drought and high-temperature conditions. Traits measured included whole-plant water-use efficiency (WUEWP), carbon isotope composition (δ13C), biomass accumulation, water use per plant, and chlorophyll fluorescence across six developmental stages. Whole-plant WUE ranged from 3.07 to 7.81 g L−1, while δ13C values ranged from −24.06‰ to −29.33‰. Biomass accumulation and water use were strongly positively correlated (r = 0.94, p < 0.001), indicating that greater biomass production was associated with increased water consumption. In contrast, the relationship between WUEWP and δ13C was weak (r = −0.09), suggesting that δ13C alone may not be a reliable proxy for WUEWP under combined drought and heat stress conditions. Phenotypic diversity across the cultivar panel was relatively low to moderate (Shannon diversity index, H′ = 1.88–2.62), indicating limited adaptive capacity within the evaluated germplasm. Principal component analysis explained 76.6% of the total variation and effectively differentiated cultivar responses to stress. Chlorophyll fluorescence, particularly the maximum quantum efficiency of PSII photochemistry (FV/FM), was highly sensitive to stress-induced reductions in photosynthetic performance. Measurements obtained during reproductive drought and heat stress stages showed stronger associations with biomass, water use, WUEWP, and δ13C than measurements collected during non-stress periods, indicating that FV/FM can be a reliable physiological indicator for screening drought and heat tolerance. Overall, the results revealed detectable phenotypic variation but relatively modest diversity and generally weak to moderate trait associations, highlighting the potential value of incorporating diverse germplasm and integrated phenotyping approaches to improve climate resilience in Canadian spring wheat.