Carbon isotope discrimination as a diagnostic tool for C₄ photosynthesis in C₃-C₄ intermediate species

The presence and activity of the C₄ cycle in C₃-C₄ intermediate species have proven difficult to analyze, especially when such activity is low. This study proposes a strategy to detect C₄ activity and estimate its contribution to overall photosynthesis in intermediate plants, by using tunable diode laser absorption spectroscopy (TDLAS) coupled to gas exchange systems to simultaneously measure the CO₂ responses of CO₂ assimilation (A) and carbon isotope discrimination (Δ) under low O₂ partial pressure. Mathematical models of C₃-C₄ photosynthesis and Δ are then fitted concurrently to both responses using the same set of constants. This strategy was applied to the intermediate species Flaveria floridana and F. brownii, and to F. pringlei and F. bidentis as C₃ and C₄ controls, respectively. Our results support the presence of a functional C₄ cycle in F. floridana, that can fix 12-21% of carbon. In F. brownii, 75-100% of carbon is fixed via the C₄ cycle, and the contribution of mesophyll Rubisco to overall carbon assimilation increases with CO₂ partial pressure in both intermediate plants. Combined gas exchange and Δ measurement and modeling is a powerful diagnostic tool for C₄ photosynthesis.