Diel-driven variations of leaf dark respiration and metabolite levels in C3 and C4 grasses

Leaf dark respiration (Rdark) is often measured in artificially dark-adapted samples at a single time point during the day, with temperature-normalised rates often assumed to be constant throughout a 24-hour cycle. However, the extent to which the 24-hour cycle influences leaf Rdark and respiratory metabolic profile remains unclear, particularly in C4 plants. We quantified O2-based leaf Rdark and metabolites at six time points over a diel (i.e. 24-hour) cycle in leaves of four grass species (one C3 and three C4 species). We found that Rdark and leaf metabolites varied among species, and between dark-adapted day-sampled and night-sampled leaves. In general, C4 plants contained a relatively higher content of organic acids and soluble sugars than C3 plants. Across the four species, variations in Rdark were associated with changes in the abundance of metabolites involved in the mitochondrial tricarboxylic acid pathway (malate, fumarate, succinate and citrate), amino acid metabolism (alanine and asparagine) and sugar interconversion (lactose and mannose). Multivariate statistics suggested that Rdark of the examined species is influenced more by the relative contribution of multiple concurrent metabolic pathways across the diel cycle than by C3 and C4 photosynthetic types. We suggest that Rdark and metabolite profiles measured during daytime on dark-adapted leaves are not good surrogates for nighttime respiratory properties. Understanding how the supply of respiratory substrates varies during the diel cycle would lead to a more accurate prediction of Rdark over the course of a day.