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

Abstract

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/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 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 C3 (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 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 C3 and C4 plants and could be used to rapidly screen large numbers of mutants or transformants in high throughput studies.

Keywords: C3; C4; Cleome gynandra; Miscanthus.; RuBisCO; carbon concentration mechanism (CCM); carboxylation; maize; oxygenation; photosynthesis; rice; wheat.

Fig. 1.

Summary of experimental approach. One representative dataset from C₃ tobacco is presented. Once stable assimilatory conditions are reached, a first set of data are recorded (left hatched area). The background gas is then switched from ambient to 2% O₂. After a suitable acclimation time to allow flushing of the cuvette and reacclimation (c. 6min), a second set of data are recorded (right hatched area). The response of assimilation (triangles) and Photosystem II yield Y(II) (squares) during the experiment are shown.

Fig. 2.

V_O/V _C measured under different CO₂ concentrations in the substomatal cavity (C_i), obtained by imposing reference CO₂ concentrations of 400, 300, 200, 150, 100, and 50 μmol mol^–1 for C₃ tobacco (triangles) and C₄ maize (squares). Data are compared with simulated V _O/V _C using the validated von Caemmerer C₃ and C₄ models (lines, see also Table 3 and 4). With decreasing C _i, V _O/V _C gets progressively higher in tobacco but it is only marginally affected in maize, CO₂ concentration can therefore be used to control the resolution of the method. All data shown, n=4.

Fig. 3.

Sensitivity to errors in the determination of R _LIGHT. True values were simulated by calculating equation 8 for R _LIGHT=1 μmol m^–2 s^–1, V _O/V _C = 0.2, and Y(II)=0.65 at variable assimilation (A) values. Test values of V _O/V _C were then calculated by solving equation 8 at different values for R _LIGHT: 2 μmol m^–2 s^–1 (+100%), 1.5 μmol m^–2 s^–1 (+50%), 1.2 μmol m^–2 s^–1 (+20%), 0.8 μmol m^–2 s^–1 (–20%), 0.5 μmol m^–2 s^–1 (–50%), 0 μmol m^–2 s^–1 (–100%, GA=A). The difference in V _O/V _C between the test minus the true value was expressed as relative to the true value.

Fig. 4.

Sensitivity to errors in the determination of Fm′. True values were simulated by calculating equation 8 for R _LIGHT=1 μmol m^–2 s^–1, V _O/V _C=0.2 and A=5 μmol m^–2 s^–1 at different Y(II) values. Test values of V _O/V _C were then calculated by solving equation 8 introducing increasing Fm′ underestimation: –1, –2, –3, and –5%. The difference in V _O/V _C between the test minus the true value was expressed as relative to the true value.