Diurnal and Phenological Modulation of Canopy Temperature in Wheat Breeding Under Mediterranean Conditions

Canopy temperature (CT) is widely used to assess crop water and heat status, but it is often recorded at a single hour, implicitly treating CT as a stable trait. Here, we show that canopy cooling is a dynamic phenotype whose expression depends on time of day, phenological stage, and environment. First, we monitored 184 spring wheat (Triticum aestivum L.) genotypes in two Mediterranean environments (fully irrigated vs. rainfed, contrasting atmospheric demand) using UAV-based thermal imaging. CT was measured six times per day (10:30-17:30 h) at four reproductive stages (anthesis, milk-grain, milk-dough, and dough), enabling quantification of diurnal plasticity, seasonal shifts, and environmental effects on canopy cooling. Second, repeated-measures mixed models confirmed that Location, Stage, and Time of day, and all interactions, were highly significant (p < 0.001). Variance-component analyses showed a strong genetic signal within each Stage × Environment combination, with 87.6-97.7% of total variance attributable to genotypic effects pooled across hours. Third, the optimal phenotyping window was context dependent: under rainfed conditions, genotypic discrimination consistently peaked around mid-afternoon (~15:00 h), whereas under irrigation, the optimal window shifted with stage (13:30-15:00 h). Genotype rankings were also markedly less stable across hours under rainfed conditions, indicating substantial within-day re-ranking as atmospheric demand increased. Finally, thermal exposure analyses showed that exceeding a physiologically relevant threshold (CT > 32 °C) depended strongly on time of day and stage; maximum CT captured short heat events missed by daily means. Clustering and alluvial analyses revealed frequent reclassification across stages, with only a small subset remaining consistently cooler, particularly under stress. Random regression of CT on vapor pressure deficit (VPD) indicated that CT-VPD sensitivity was largely environment-dependent and showed weak cross-environment correspondence (Spearman ρ = -0.166). Overall, single-time-point CT phenotyping provides an incomplete view of thermal status, underscoring the need for multi-temporal protocols and context-specific measurement windows for breeding and physiological interpretation under drought and heat.