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

Leaf day respiratory rate ( R _d ), 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 CO ₂ fluxes with photosynthesis and photorespiration ( R _p ). This study aimed to disentangle R _d from R _p in C ₃ crops using a high-CO ₂ /low-O ₂ (>1200 μmol mol ^–1 CO ₂  + 2% O ₂ ) A _n /C _a (net photosynthetic rate-ambient CO ₂ concentration) model. We integrated two datasets: A _n /C _a curve data of four C ₃ species ( Trifolium pratense , Silphium perfoliatum , Lolium perenne , Triticum aestivum ) under ambient O ₂ (21% O ₂ ) from Ye et al. (2025) and newly collected data under low O ₂ (2% O ₂ ) for the same species. A dedicated model (Eqn 1) was developed to extract key parameters [e.g., the maximum net photosynthetic rate ( A _nmax ), CO ₂ compensation point ( Γ ) in the presence of R _d , photorespiratory rate at C _a  = 0 μmol mol ^–1 ( R _C0 )] from A _n /C _a curves, with ANOVA confirming no significant differences between calculated and observed parameter values ( p  > 0.05). Under 2% O ₂ , R _p was strongly suppressed, making the gross photorespiratory rate ( R _C  =  R _p  +  R _d ) a reliable proxy for approximate R _d . Results showed that R _d estimates from the high-CO ₂ /low-O ₂ 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 C ₃ crops. Interspecific differences in R _d were observed: L. perenne had the highest R _d , while T. aestivum had the lowest, which correlated with species-specific photosynthetic traits (e.g., carboxylation efficiency). R _d remained stable across O ₂ levels (21% vs . 2%), indicating independence from short-term photorespiratory flux changes. The R _C  ≈  R _d assumption is supported by the substantial decrease in Γ (50–67%) and R _CO (40–55%) under 2% O ₂ , and by the consistency of R _d estimates with literature ranges, although independent ground-truth measurements remain to be conducted. This study suggests the high-CO ₂ /low-O ₂ A _n /C _a model as a new tool for R _d quantification in C ₃ 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.