Variation in leaf dark respiration among C₃ and C₄ grasses is associated with use of different substrates

Measurements of respiratory properties have often been made at a single time point either during daytime using dark-adapted leaves or during nighttime. The influence of the day-night cycle on respiratory metabolism has received less attention but is crucial to understand photosynthesis and photorespiration. Here, we examined how CO2- and O-2-based rates of leaf dark respiration (R-dark) differed between midday (after 30-min dark adaptation) and midnight in 8 C-3 and C-4 grasses. We used these data to calculate the respiratory quotient (RQ; ratio of CO2 release to O-2 uptake), and assessed relationships between R-dark and leaf metabolome. R-dark was higher at midday than midnight, especially in C-4 species. The day-night difference in R-dark was more evident when expressed on a CO2 than O-2 basis, with the RQ being higher at midday than midnight in all species, except in rice (Oryza sativa). Metabolomic analyses showed little correlation of R-dark or RQ with leaf carbohydrates (sucrose, glucose, fructose, or starch) but strong multivariate relationships with other metabolites. The results suggest that rates of R-dark and differences in RQ were determined by several concurrent CO2-producing and O-2-consuming metabolic pathways, not only the tricarboxylic acid cycle (organic acids utilization) but also the pentose phosphate pathway, galactose metabolism, and secondary metabolism. As such, R-dark was time-, type- (C-3/C-4) and species-dependent, due to the use of different substrates.