Disentangling leaf day respiration from photorespiration in C3 leaves: A single-curve An/Ca approach validated under photorespiration-suppressed conditions

Leaf day respiratory rate (Rd), a pivotal component of plant carbon metabolism that directly modulates net photosynthetic carbon gain and plant fitness, has long been challenging to accurately quantify due to overlapping CO2 fluxes with photosynthesis and photorespiration (Rp). This study aimed to disentangle Rd from Rp in C3 crops using a high-CO2/low-O2 (>1200 μmol mol-1 CO2 + 2% O2) An/Ca (net photosynthetic rate-ambient CO2 concentration) model. We integrated two datasets: An/Ca curve data of four C3 species (Trifolium pratense, Silphium perfoliatum, Lolium perenne, Triticum aestivum) under ambient O2 (21% O2) from Ye et al. (2025) and newly collected data under low O2 (2% O2) for the same species. A dedicated model (Eqn 1) was developed to extract key parameters [e.g., the maximum net photosynthetic rate (Anmax), CO2 compensation point (Γ) in the presence of Rd, photorespiratory rate at Ca = 0 μmol mol-1 (RC0)] from An/Ca curves, with ANOVA confirming no significant differences between calculated and observed parameter values (p > 0.05). Under 2% O2, Rp was strongly suppressed, making the gross photorespiratory rate (RC = Rp + Rd) a reliable proxy for approximate Rd. Results showed that Rd estimates from the high-CO2/low-O2 approach (0.74-1.12 μmol m-2 s-1) were consistent with literature ranges (0.74-2.89 μmol m-2 s-1) for four C3 crops. Interspecific differences in Rd were observed: L. perenne had the highest Rd, while T. aestivum had the lowest, which correlated with species-specific photosynthetic traits (e.g., carboxylation efficiency). Rd remained stable across O2 levels (21% vs. 2%), indicating independence from short-term photorespiratory flux changes. The RC ≈ Rd assumption is supported by the substantial decrease in Γ (50-67%) and RCO (40-55%) under 2% O2, and by the consistency of Rd estimates with literature ranges, although independent ground-truth measurements remain to be conducted. This study suggests the high-CO2/low-O2 An/Ca model as a new tool for Rd quantification in C3 crops. The findings enhance understanding of non-photosynthetic carbon flux dynamics and provide critical parameters for refining crop carbon cycling models to predict growth and yield under current and future atmospheric conditions.