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Cuticular conductance to water (g cw ) is difficult to quantify for stomatous surfaces due to the complexity of separating cuticular and stomatal transpiration, and additional complications arise for determining adaxial and abaxial g cw . This has led to the neglect of g cw as a separate parameter in most common gas exchange measurements. Here, we describe a simple technique to simultaneously estimate adaxial and abaxial values of g cw , tested in two amphistomatous plant species.What we term the 'Red-Light method' is used to estimate g cw from gas exchange measurements and a known CO 2 concentration inside the leaf during photosynthetic induction under red light. We provide an easy-to-use web application to assist with the calculation of g cw .While adaxial and abaxial g cw varies significantly between leaves of the same species we found that the ratio of adaxial/abaxial g cw (γ n ) is stable within a plant species. This has implications for use of generic values of g cw when analysing gas exchange data.The Red-Light method can be used to estimate total cuticular conductance (g cw-T ) accurately with the most common setup of gas exchange instruments, i.e. a chamber mixing the adaxial and abaxial gases, allowing for a wide application of this technique.

We present a robust estimation of the CO 2 concentration at the surface of photosynthetic mesophyll cells (c w ), applicable under reasonable assumptions of assimilation distribution within the leaf. We used Capsicum annuum, Helianthus annuus and Gossypium hirsutumas model plants for our experiments.We introduce calculations to estimate c w using independent adaxial and abaxial gas exchange measurements, and accounting for the mesophyll airspace resistances.The c w was lower than adaxial and abaxial estimated intercellular CO 2 concentrations (c i ). Differences between c w and the c i of each surface were usually larger than 10 μmol mol −1 . Differences between adaxial and abaxial c i ranged from a few μmol mol −1 to almost 50 μmol mol −1 , where the largest differences were found at high air saturation deficits (ASD). Differences between adaxial and abaxial c i and the c i estimated by mixing both fluxes ranged from −30 to +20 μmol mol −1 , where the largest differences were found under high ASD or high ambient CO 2 concentrations.Accounting for c w improves the information that can be extracted from gas exchange experiments, allowing a more detailed description of the CO 2 and water vapor gradients within the leaf.

Measurement of leaf carbon gain and water loss (gas exchange) in planta is a standard procedure in plant science research for attempting to understand physiological traits related to water use and photosynthesis. Leaves carry out gas exchange through the upper (adaxial) and lower (abaxial) surfaces at different magnitudes, depending on the stomatal density, stomatal aperture, cuticular permeability, etc., of each surface, which we account for in gas exchange parameters such as stomatal conductance. Most commercial devices measure leaf gas exchange by combining the adaxial and abaxial fluxes and calculating bulk gas exchange parameters, missing details of the plant's physiological response on each side. Additionally, the widely used equations to estimate gas exchange parameters neglect the contribution of small fluxes such as cuticular conductance, adding extra uncertainties to measurements performed in water-stress or low-light conditions. Accounting for the gas exchange fluxes from each side of the leaf allows us to better describe plants' physiological traits under different environmental conditions and account for genetic variability. Here, apparatus and materials are presented for adapting two LI-6800 Portable Photosynthesis Systems to work as one gas exchange system to measure adaxial and abaxial gas exchange simultaneously. The modification includes a template script with the equations to account for small fluxes. Instructions are provided for incorporating the add-on script into the device's computational sequence, display, variables, and spreadsheet results. We explain the method to obtain an equation to estimate boundary layer conductance to water for the new setup and how to embed this equation in the devices' calculations using the provided add-on script. The apparatus, methods, and protocols presented here provide a simple adaptation combining two LI-6800s to obtain an improved system to measure leaf gas exchange on adaxial and abaxial surfaces. Graphical overview Figure 1. Diagram of the connection of two LI-6800s. Figure adapted from Márquez et al. (2021).