The Dynamic Assimilation Technique measures photosynthetic CO2 response curves with similar fidelity to steady-state approaches in half the time

Abstract

The net CO2 assimilation (A) response to intercellular CO2 concentration (Ci) is a fundamental measurement in photosynthesis and plant physiology research. The conventional A/Ci protocols rely on steady-state measurements and take 15-40 min per measurement, limiting data resolution or biological replication. Additionally, there are several CO2 protocols employed across the literature, without clear consensus as to the optimal protocol or systematic biases in their estimations. We compared the non-steady-state Dynamic Assimilation Technique (DAT) protocol and the three most used CO2 protocols in steady-state measurements, and tested whether different CO2 protocols lead to systematic differences in estimations of the biochemical limitations to photosynthesis. The DAT protocol reduced the measurement time by almost half without compromising estimation accuracy or precision. The monotonic protocol was the fastest steady-state method. Estimations of biochemical limitations to photosynthesis were very consistent across all CO2 protocols, with slight differences in Rubisco carboxylation limitation. The A/Ci curves were not affected by the direction of the change of CO2 concentration but rather the time spent under triose phosphate utilization (TPU)-limited conditions. Our results suggest that the maximum rate of Rubisco carboxylation (Vcmax), linear electron flow for NADPH supply (J), and TPU measured using different protocols within the literature are comparable, or at least not systematically different based on the measurement protocol used.

Keywords: A/Ci response; RuBP regeneration limitation; Rubisco carboxylation limitation; gas exchange methods; photosynthesis; triose phosphate limitation.

Fig. 1.

Net CO₂ assimilation (A_net) response to intercellular CO₂ concentration (C_i) measured on apple (A), Arabidopsis (B), potato (C), soybean (D), and tobacco (E) using the non-steady-state Dynamic Assimilation Technique (DAT) and four steady-state methods (monotonic, split down–up, split up–down, and random). Steady-state protocols are based on the progression of the CO₂ concentration within the sequence. Vertical and horizontal error bars correspond to ±1 standard error of the mean. Note that horizontal bars may be covered by data points. *Indicates significant differences between CO₂ protocols (P-value <0.05).

Fig. 2.

Comparison of the biochemical limitations to photosynthesis estimated from A/C_i data measured on apple, Arabidopsis, potato, soybean, and tobacco using the non-steady-state Dynamic Assimilation technique (DAT) and three steady-state methods (monotonic, split down–up, and split up–down). Solid lines and equations correspond to the Deming linear regression between the steady-state and DAT estimations of the maximum rate of Rubisco carboxylation (V_cmax; A), linear electron flow for NADPH supply (J; B), and triose phosphate utilization (TPU; C). The dashed line represents the 1:1 line. None of the slope or intercept estimations was significantly different from one or zero, respectively (P>0.1). Biochemical limitations to photosynthesis were estimated using the Farquhar–von Caemmerer–Berry model of C₃ photosynthesis; see Supplementary Fig. S4 for comparison of respiration in the light, mesophyll conductance, and carbon flow out of photorespiration as glycine or serine.