Lessons from relatives: C4 photosynthesis enhances CO2 assimilation during the low-light phase of fluctuations

Abstract

Despite the global importance of species with C4 photosynthesis, there is a lack of consensus regarding C4 performance under fluctuating light. Contrasting hypotheses and experimental evidence suggest that C4 photosynthesis is either less or more efficient in fixing carbon under fluctuating light than the ancestral C3 form. Two main issues have been identified that may underly the lack of consensus: neglect of evolutionary distance between selected C3 and C4 species and use of contrasting fluctuating light treatments. To circumvent these issues, we measured photosynthetic responses to fluctuating light across 3 independent phylogenetically controlled comparisons between C3 and C4 species from Alloteropsis, Flaveria, and Cleome genera under 21% and 2% O2. Leaves were subjected to repetitive stepwise changes in light intensity (800 and 100 µmol m-2 s-1 photon flux density) with 3 contrasting durations: 6, 30, and 300 s. These experiments reconciled the opposing results found across previous studies and showed that (i) stimulation of CO2 assimilation in C4 species during the low-light phase was both stronger and more sustained than in C3 species; (ii) CO2 assimilation patterns during the high-light phase could be attributable to species or C4 subtype differences rather than photosynthetic pathway; and (iii) the duration of each light step in the fluctuation regime can strongly influence experimental outcomes.

Figure 1.

Light response curves of phylogenetically linked C₃ and C₄Alloteropsis, Flaveria, and Cleome species at 21% and 2% O₂. As a function of absorbed photon flux density (PFD_abs), plots show net CO₂ assimilation (A_CO2, A, D), intercellular CO₂ concentration (C_i, B, E), and stomatal conductance to water vapor (g_sw, C, F). Ribbons represent standard error of the mean (n = 5).

Figure 2.

Net CO₂ assimilation (A_CO2) in phylogenetically linked C₃ and C₄Alloteropsis, Flaveria, and Cleome species under 3 different fluctuating light regimes at 21% (A-I) and 2% O₂ (J-R). Each light regime consisted of alternating 800 and 100 µmol m⁻² s⁻¹ PFD periods, where each light step lasted 6, 30, or 300 s before changing. Treatments were started after leaves were acclimated at 150 µmol m⁻² s⁻¹ PFD and lasted 1 h, data was analyzed from minutes 50 to 60 of each treatment. Ribbons represent the standard error of the mean (n = 5). The full time series is shown in Supplemental Fig. S1.

Figure 3.

Net CO₂ assimilation (A_CO2) relative to steady state (%) across the 800 and 100 µmol m⁻² s⁻¹ PFD light steps of differing lengths starting at the 50 min mark, in white and shaded, respectively. Depending on the fluctuating light treatment, subplots are showing one complete fluctuation of 12, 60, or 600 s. Values represent A_CO2 at a given point in the fluctuating light treatment relative to A_CO2 obtained from steady-state light response curves at the light intensity of each period in phylogenetically linked C₃ and C₄Alloteropsis, Flaveria, and Cleome species at 21% (A-I) and 2% O₂ (J-R). The dashed line represents 100%, where assimilation would be exactly that of steady state. Ribbons represent the standard error of the mean (n = 5). The corresponding absolute assimilation values are provided in Supplemental Fig. S2.

Figure 4.

Boxplots of net CO₂ assimilation (A_CO2) relative to steady state (%) under the 800 (A–C) and 100 µmol m⁻² s⁻¹ PFD (D–F) light steps of the fluctuating light regimes. Each regime consisted of alternating 800 and 100 µmol m⁻² s⁻¹ PFD periods, where each light step lasted 6, 30, or 300 s. For each period, A_CO2 across the time series for phylogenetically linked C₃ and C₄Alloteropsis, Flaveria, and Cleome species at 21% or 2% O₂ was calculated as a percentage of steady state values obtained from light response curves at the same light intensity and O₂ concentration. The dashed line represents 100%, where the assimilation rate would equal steady state. Box edges represent the lower and upper quartiles, the solid line indicates the median, and points represent outliers beyond 1.5 times the interquartile range (n = 5 for each combination of species/oxygen). Three-way ANOVA (Table 4) was used to test the effect of photosynthetic pathway, fluctuating length, O₂ concentration, and their interaction on A_CO2 relative to steady state in Alloteropsis, Flaveria, and Cleome. The corresponding absolute assimilation values are shown in Supplemental Fig. S4.

Figure 5.

Boxplots of the quantum yield of carbon assimilation (ΦCO₂) under the 800 (A–C) and 100 (E–H) µmol m⁻² s⁻¹ PFD periods of the fluctuating light regimes or from steady-state measurements (D, H). Each fluctuating regime consisted of alternating 800 and 100 µmol m⁻² s⁻¹ PFD periods, where each light step lasted 6, 30, or 300 s. Box edges represent the lower and upper quartiles, the solid line indicates the median, and points represent outliers beyond 1.5 times the interquartile range (n = 5 for each combination of species/measurement condition). Three-way ANOVA was used to test the effect of photosynthetic pathway, fluctuating length, O₂ concentration, and their interaction on ΦCO₂ in Alloteropsis, Flaveria, and Cleome (results shown in Table 5).