Carbon isotope composition of soluble sugars in leaves of C3 and C4 grasses

This study examined the carbon isotope composition (δ13C) of soluble sugars (glucose, fructose, sucrose) and cellulose in leaves of C3 and C4 grasses, including NAD-ME and NADP-ME subtypes, grown under controlled conditions. Using a novel compound-specific isotope analysis, we investigated post-photosynthetic fractionation and its relationship to stomatal conductance and intrinsic water-use efficiency (WUEi). C3 species were more 13C-depleted than C4 species due to differences in photosynthetic pathways. In C3 grasses, sucrose, glucose, and cellulose were enriched in 13C relative to bulk tissue, while fructose was more depleted, likely reflecting isotope discrimination by invertase. In C4 grasses, all sugars were more enriched in 13C than bulk tissue, especially sucrose. This pattern reflects the spatial separation of sugar synthesis and transport between mesophyll and bundle sheath cells, causing isotopic shifts during transport and metabolic cycling. Fructose and glucose δ13C showed opposite discrimination patterns between NADP ME and NAD ME species, indicating subtype specific differences in carbohydrate processing, including sucrose turnover and invertase related fractionation. These contrasting signals highlight key metabolic divergence between C4 subtypes with sucrose δ13C unchanged across subtypes. Across C4 grasses, δ13C did not correlate with WUEi, confirming that the CO2-concentrating mechanism decouples δ13C from Anet. However, bulk leaf and cellulose δ13C correlated negatively with stomatal conductance (gs), indicating that δ13C reflects long-term gs rather than instantaneous gas exchange. Thus, δ13C can indicate integrated stomatal behavior but not WUEi. This study provides the first compound-specific δ13C evidence of metabolic divergence between photosynthetic types and C4 subtypes. Fructose versus glucose emerged as key metabolites for tracing differences in carbohydrate metabolism, offering new potential for improving crop water-use efficiency through isotopic and genetic screening.