A high throughput gas exchange screen for determining rates of photorespiration or regulation of C₄ activity

Large-scale research programmes seeking to characterize the C₄ pathway have a requirement for a simple, high throughput screen that quantifies photorespiratory activity in C₃ and C₄ model systems. At present, approaches rely on model-fitting to assimilatory responses (A/Ci curves, PSII quantum yield) or real-time carbon isotope discrimination, which are complicated and time-consuming. Here we present a method, and the associated theory, to determine the effectiveness of the C₄ carboxylation, carbon concentration mechanism (CCM) by assessing the responsiveness of V _O/V_C, the ratio of RuBisCO oxygenase to carboxylase activity, upon transfer to low O₂. This determination compares concurrent gas exchange and pulse-modulated chlorophyll fluorescence under ambient and low O₂, using widely available equipment. Run time for the procedure can take as little as 6 minutes if plants are pre-adapted. The responsiveness of V_O/V_C is derived for typical C ₃ (tobacco, rice, wheat) and C₄ (maize, Miscanthus, cleome) plants, and compared with full C₃ and C₄ model systems. We also undertake sensitivity analyses to determine the impact of RLIGHT (respiration in the light) and the effectiveness of the light saturating pulse used by fluorescence systems. The results show that the method can readily resolve variations in photorespiratory activity between C₃ and C₄ plants and could be used to rapidly screen large numbers of mutants or transformants in high throughput studies.