Quantitative metabolic and lipidomic landscapes reveal the molecular phenotype of C4 evolution in genus Flaveria

C4 photosynthesis possess complex traits associated with high efficiencies in light, nitrogen and water utilization. To investigate the coupling of carbon and nitrogen stoichiometry within the systematic metabolism along the evolutionary path from C3 to C4 photosynthesis, we applied the nuclear magnetic resonance (NMR)-based metabolomics and advanced lipidomic approaches to profile 38 metabolites and 246 lipids in leaves of representative C3 ancestral, C3–C4 intermediate, and C4 species in the genus Flaveria. Our results revealed that progressive changes in metabolic profiles associated with amino acids/amines, organic acids, choline/its derivatives, and phospholipids along the C3 to C4 transition, implicating these pathways in cellular responses functionally oriented toward protein synthesis and degradation, tricarboxylic acid (TCA) cycle activity, and oxidative stress. In contrast, the metabolism related to carbohydrate and lipid biosynthesis exhibited a declining trend from C3 to C4 species. Among C3–C4 intermediates, the type II F. ramosissima displayed a unique metabolic profile, combining characteristics of both C3 species (elevated carbohydrate levels) and C4 species (elevated amino acid and organic acid levels). Taking together, these comprehensive metabolic phenotypes delineate distinct biochemical signatures associated with photosynthetic types in Flaveria, providing novel insights into the metabolic reprogramming underpinning C4 photosynthetic evolution. C4 photosynthetic evolution in Flaveriais characterized by a progressive metabolic shift from carbohydrate and lipid biosynthesis towards enhanced nitrogen-rich compound accumulation, delineating a biochemical phenotype across C3, intermediate, and C4 species.