Cell-type-specific metabolism in plants and metabolic plasticity of C3 and C4 leaves subjected to 13C labelling under dark and light conditions

Abstract

Plants with C3 and C4 metabolisms differ in biochemical, anatomical and physiological aspects. Several factors related to the photosynthesis of these two groups of plants have been well elucidated. However, the distribution of metabolic fluxes after CO2 assimilation is still poorly understood, especially regarding flux distribution through the tricarboxylic acid (TCA) cycle and associated metabolic pathways. Considering the primary role of nitrogen assimilation in plant growth and productivity, our hypothesis is that the greater photosynthetic efficiency of C4 plants can also be explained by a differential regulation of metabolic fluxes through the TCA and glutamine synthetase/glutamate synthase (GS/GOGAT) cycles. In this work, we conducted isotopic labelling experiments by subjecting leaves of C4 plants (maize and sorghum) and a representative species of the C3 group (cowpea) to 13C-HCO3 labelling in the absence and presence of light. The results reported that illuminated leaves of all species showed a significant increase in 13C-labeled metabolites, probably reflecting the activation and increase in the carboxylative activities of the enzymes ribulose-1,5-bisphosphate carboxylase/oxygenase (RuBisCO) and phosphoenolpyruvate carboxylase (PEPc), respectively. However, TCA cycle metabolites showed lower 13C enrichment in cowpea leaves in darkness and light. 13C positional labelling data on malate demonstrate that C4 plants have greater CO2 assimilation mediated by PEPc in light and darkness. According to our hypothesis, 13C enrichment in glutamate was higher in illuminated C4 plants than in cowpea, indicating that C4 plants have a higher carbon flux derived from PEPc activity towards the GS/GOGAT cycle in the light. Furthermore, metabolites related to the TCA cycle and associated pathways presented higher 13C enrichment in C4 plants, especially citrate, which showed enrichment in illuminated and dark exposed C4 leaves. Our results also highlight differences among C4 species. For example, in the presence of light, sorghum displayed higher 13C enrichment in sugars, while maize presented higher enrichment in malate and aspartate, indicating a differential carbon flux mode among C4 species. Our results suggest that C4 plants have greater metabolic flux through the TCA cycle under light and dark conditions and that the distribution of photosynthetic carbon throughout primary metabolism is species- and time-dependent, especially under light conditions.