A high throughput gas exchange screen for determining rates of photorespiration or regulation of C(4) activity

Large-scale research programmes seeking to characterize the C(4) pathway have a requirement for a simple, high throughput screen that quantifies photorespiratory activity in C(3) and C(4) model systems. At present, approaches rely on model-fitting to assimilatory responses (A/C (i) 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 C(4) 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 O(2). This determination compares concurrent gas exchange and pulse-modulated chlorophyll fluorescence under ambient and low O(2), 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 C(4) (maize, Miscanthus, cleome) plants, and compared with full C(3) and C(4) model systems. We also undertake sensitivity analyses to determine the impact of R (LIGHT) (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 C(3) and C(4) plants and could be used to rapidly screen large numbers of mutants or transformants in high throughput studies.