For decades, gas exchange techniques using infrared gas analysers (IRGAs) have been widely used to measure fluxes of CO2 and H2O into and out of leaves and, less frequently, nonfoliar tissues. These measurements allow for assessments of the physiological performance of leaves and to benchmark the biochemical capacity for photosynthesis . They have enabled measurements of in situ photosynthetic CO2 assimilation (A) and stomatal conductance (gs) in diverse ecosystems from the tropics (e.g., Carter et al., 2021; Slot & Winter, 2017) to the arctic (Rogers et al., 2019), and from grasslands to forests to agricultural fields (e.g., Coast et al., 2021; Dillaway & Kruger, 2010; Koester et al., 2016; Wohlfahrt et al., 1999).

Gas exchange measurements can test how fluxes of CO2 and H2O between the leaf and the atmosphere change as environmental conditions in the leaf chamber are manipulated (e.g., Ball et al., 1987; Leakey et al., 2006; Li et al., 2021; Miner et al., 2017; Wolz et al., 2017) and how long-term growth under different environmental conditions impacts rates, biochemical capacity and diffusional limitations of photosynthesis (see Ainsworth & Long, 2005; Medlyn et al., 2002; Wittig et al., 2007; Yan et al., 2016 for meta-analyses). Gas exchange measurements have also improved our understanding of the physiology of species operating different types of photosynthesis (such as C3, C4, CAM) (Hogewoning et al., 2021; Lundgren et al., 2016; Sage & Kubien, 2007; Schuster & Monson, 1990).

Several introductory guides to gas exchange have been published, mostly focusing on the details of a particular measurement technique (Busch, 2018; Evans & Santiago, 2014; Haworth et al., 2018; Long & Bernacchi, 2003; Parsons et al., 1997). In the following, we give an overview of the biochemical and biophysical photosynthetic processes that can be investigated with gas exchange measurements and outline the parameters that can be estimated. We then discuss suitable approaches for quantifying these parameters with photosynthetic gas exchange techniques in C3 plants, which operate the dominant photosynthetic pathway. However, in many cases the approaches can be applied to C4 or CAM species with appropriate modifications, some of which are discussed in this article.

While recording gas exchange data with current commercially available equipment is relatively easy, the obtained data are not necessarily meaningful. Both the correct experimental design and the correct data interpretation are crucial for obtaining high quality information. We therefore include here “how-to" tips that have not been part of prior guides. Of course, the specific design of commercial gas exchange equipment varies between manufacturers and the reader should refer to the manufacturer manuals for their operation. 

2 KEY PARAMETERS OBTAINED FROM GAS EXCHANGE MEASUREMENTS

Photosynthetic CO2 fixation is the result of a complex set of biochemical and biophysical processes, many of which can be probed with gas exchange techniques. Gas exchange measurements inform us about diverse aspects of carbon and water relations, ranging from processes associated with the light-dependent and light-independent reactions to CO2/H2O diffusion and the sink capacity of a leaf. The only information that gas exchange measurements give us directly are concentrations of CO2 and H2O and the associated net fluxes. However, a wide range of parameters can be estimated from these values indirectly, based on certain assumptions. These parameters can be crudely divided into three categories: The first category informs on the ‘maximum’ capacities of the leaf, and its parameters describe the intrinsic properties of a leaf's investment into different photosynthetic aspects. These parameters may have a fixed temperature response or may change throughout leaf development but can otherwise be treated as constants. The second category informs on the current physiological state of a leaf, that is, the ‘instantaneous’ or ‘effective’ rates that are realised during the instance of the measurement. These are variables that can be expected to change in the short-term (seconds to hours) with changing environmental conditions, such as light intensity, atmospheric [CO2] or water status. The third category includes parameters that describe some aspect of photosynthetic performance, but may not directly correspond to a single identifiable physiological trait. Examples include the CO2 concentration at which A transitions from one biochemical limitation to another, or the relative contribution of different physiological processes limiting A.