A guide to photosynthetic gas exchange measurements: Fundamental principles, best practice and potential pitfalls.

Gas exchange measurements enable mechanistic insights into the processes that underpin carbon and water fluxes in plant leaves which in turn inform understanding of related processes at a range of scales from individual cells to entire ecosytems. Given the importance of photosynthesis for the global climate discussion it is important to (a) foster a basic understanding of the fundamental principles underpinning the experimental methods used by the broad community, and (b) ensure best practice and correct data interpretation within the research community. In this review, we outline the biochemical and biophysical parameters of photosynthesis that can be investigated with gas exchange measurements and we provide step-by-step guidance on how to reliably measure them. We advise on best practices for using gas exchange equipment and highlight potential pitfalls in experimental design and data interpretation. The Supporting Information contains exemplary data sets, experimental protocols and data-modelling routines. This review is a community effort to equip both the experimental researcher and the data modeller with a solid understanding of the theoretical basis of gas-exchange measurements, the rationale behind different experimental protocols and the approaches to data interpretation.

A comparison of stomatal conductance responses to blue and red light between C3 and C4 photosynthetic species in three phylogenetically-controlled experiments.

Introduction: C4 photosynthesis is an adaptation that has independently evolved at least 66 times in angiosperms. C4 plants, unlike their C3 ancestral, have a carbon concentrating mechanism which suppresses photorespiration, often resulting in faster photosynthetic rates, higher yields, and enhanced water use efficiency. Moreover, the presence of C4 photosynthesis greatly alters the relation between CO2 assimilation and stomatal conductance. Previous papers have suggested that the adjustment involves a decrease in stomatal density. Here, we tested if C4 species also have differing stomatal responses to environmental cues, to accommodate the modified CO2 assimilation patterns compared to C3 species. Methods: To test this hypothesis, stomatal responses to blue and red-light were analysed in three phylogenetically linked pairs of C3 and C4 species from the Cleomaceae (Gynandropsis and Tarenaya), Flaveria, and Alloteropsis, that use either C3 or C4 photosynthesis. Results: The results showed strongly decreased stomatal sensitivity to blue light in C4 dicots, compared to their C3 counterparts, which exhibited significant blue light responses. In contrast, in C3 and C4 subspecies of the monocot A. semialata, the blue light response was observed regardless of photosynthetic type. Further, the quantitative red-light response varied across species, but the presence or absence of a significant stomatal red-light response was not directly associated with differences in photosynthetic pathway. Interestingly, stomatal density and morphology patterns observed across the three comparisons were also not consistent with patterns commonly asserted for C3 and C4 species. Discussion: The strongly diminished blue-light sensitivity of stomatal responses in C4 species across two of the comparisons suggests a common C4 feature that may have functional implications. Altogether, the strong prevalence of species-specific effects clearly emphasizes the importance of phylogenetic controls in comparisons between C3 and C4 photosynthetic pathways.

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

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.

Corrigendum: Activation of CO2 assimilation during photosynthetic induction is slower in C4 than in C3 photosynthesis in three phylogenetically controlled experiments.

[This corrects the article DOI: 10.3389/fpls.2022.1091115.].

Activation of CO2 assimilation during photosynthetic induction is slower in C4 than in C3 photosynthesis in three phylogenetically controlled experiments.

Introduction: Despite their importance for the global carbon cycle and crop production, species with C4 photosynthesis are still somewhat understudied relative to C3 species. Although the benefits of the C4 carbon concentrating mechanism are readily observable under optimal steady state conditions, it is less clear how the presence of C4 affects activation of CO2 assimilation during photosynthetic induction. Methods: In this study we aimed to characterise differences between C4 and C3 photosynthetic induction responses by analysing steady state photosynthesis and photosynthetic induction in three phylogenetically linked pairs of C3 and C4 species from Alloteropsis, Flaveria, and Cleome genera. Experiments were conducted both at 21% and 2% O2 to evaluate the role of photorespiration during photosynthetic induction. Results: Our results confirm C4 species have slower activation of CO2 assimilation during photosynthetic induction than C3 species, but the apparent mechanism behind these differences varied between genera. Incomplete suppression of photorespiration was found to impact photosynthetic induction significantly in C4 Flaveria bidentis, whereas in the Cleome and Alloteropsis C4 species, delayed activation of the C3 cycle appeared to limit induction and a potentially supporting role for photorespiration was also identified. Discussion: The sheer variation in photosynthetic induction responses observed in our limited sample of species highlights the importance of controlling for evolutionary distance when comparing C3 and C4 photosynthetic pathways.