Reviewer: Dickman LT5
Measurement unit: Mol H2O m-2 s-1; Measurement scale: leaf; Equipment costs: €€–€€€; Running costs: €; Installation effort: low; Maintenance effort: low; Knowledge need: medium; Measurement mode: manual or data logger
Stomatal conductance is a calculation of the influence of stomatal opening on rate of diffusion of CO2 entering or water vapour exiting through the stomata of a leaf. Stomata are an important regulatory point for water movement through the soil–plant–air-continuum (van den Honert, 1948) through varying the diffusion resistance. The conductance of water vapour (i.e. inverse of the diffusion resistance) expresses the regulatory control exerted by stomata through the degree of stomatal opening (Pearcy et al., 1989). Environmental signals such as incident light intensity, CO2 concentration, water availability, vapour pressure deficit (VPD), and leaf temperature affect stomatal aperture (Farquhar et al., 1980). Under favourable conditions of low evaporative demand and high light, maximum stomatal conductance determines the upper limit of CO2 assimilation in a leaf. The ability of a plant to regulate stomatal opening in response to environmental conditions enables it to modulate the rate of transpiration while maintaining carbon uptake (Cowan 1977, Farquhar et al. 1980). Stomatal closure is considered to be the earliest response to drought (Flexas & Medrano, 2002) and other environmental stressors, and is a limitation to photosynthesis. However, dependence of leaf temperature on stomatal conductance occurs through leaf transpiration. Plants must balance lowering stomatal conductance to conserve water with preventing extreme leaf temperatures which affect metabolic rates and physiological processes (De Boeck et al., 2016). As such, stomatal conductance is an important parameter especially in climate-change studies and in process-based models that can be used to predict species responses to climate change (Sperry & Love, 2015; Tai et al., 2018). Stomatal conductance influences both photosynthesis and transpiration, exerting major control on carbon and water cycling, and energy exchange from leaf to landscape level as well as between the Earth’s surface and atmosphere. Because stomata control the rate of water loss in vegetated areas, they affect atmospheric moisture levels and surface temperature. Stomatal conductance is a clearly defined variable that is easily measured for model parameterisation and has substantial biological relevance (Buckley & Mott, 2013). Maximum stomatal conductance is also a function of stomatal density and size (Drake et al., 2013), which vary due to genetic factors or environmental conditions during growth (Bertolino et al., 2019). Stomatal density measurements may, thus, complement conductance measurements to compare species and when treatments can affect leaf development.
5.7.1 What and how to measure?
Diffusion porometers and infrared gas analysers (IRGAs) are the most widely used instruments for quantitatively measuring stomatal conductance and can be used either in the field or laboratory (Bell & Squire, 1981; Kirkham, 2005). These measure the diffusion of water vapour from inside the leaf through the stomata.
Recent IRGAs measure the difference in water vapour concentration between a reference IRGA and sample IRGA as air flows through a chamber that is clamped to the leaf surface. Leaf temperature is measured with a thermocouple held in the bottom of the chamber, minimising error in temperature measurements. To calculate stomatal conductance, transpiration and total conductance are used. Transpiration is a function of the air flow rate, reference and sample water vapour, and leaf area. The total conductance to water vapour includes both stomatal conductance and the boundary layer. The boundary layer is negligible due to air flow through the chamber, thus caution should be used when reporting transpiration, especially in species with large leaves (Meinzer et al., 1995). Total conductance is a function of transpiration and water vapour concentration within the leaf. Temperature is used to estimate the water vapour concentration within the leaf. Stomatal conductance is calculated from total conductance by removing the contribution of the boundary layer. The boundary layer conductance depends on whether the leaf has stomata on one or both sides, thus it is important to accurately input the stomatal ratio (i.e. fraction of stomata on one side of the leaf to the other) before taking measurements (see below Installation, field operation, maintenance and interpretation).
Steady-state porometers are typically less expensive and even more lightweight and portable than most IRGAs. They measure the water vapour flux and gradient near a leaf. A chamber is clamped to the leaf surface and the vapour pressure at two different fixed locations in the diffusion path is measured. Leaf temperature is measured with infrared thermometers or a thermocouple held in the bottom of the leaf chamber. Stomatal conductance is calculated from the vapour pressure measurements, the known conductance of the diffusion path, and temperature. Steady-state diffusion porometers are calibrated directly against relative humidity. However, any error in the humidity sensor will result in stomatal conductance calculation errors. At relative humidity of 50%, these errors are low, but rise dramatically above 80% relative humidity (Pearcy et al., 1989).
Installation, field operation, maintenance, interpretation
Instructions are provided for taking measurements with an IRGA. Set-up and calibration may differ based on the instrument used and should follow the manual. These instructions assume the IRGA has been properly calibrated. Once the system is ready, the basic procedure for taking measurements is simple. Set the desired environmental conditions within the chamber. Depending on the question, these may follow ambient conditions or may have pre-determined values. Flow rate is typically fixed to 500 umol s-1 but may be adjusted if desired. Light level may be modified if using an LED source attached to the chamber (ambient is a good value to start with as it will not be an abrupt change for the leaf). If an LED source is not used, orientate the chamber so that shading of the leaf by the chamber walls does not occur. In direct sun, the leaf fan can be used to control the temperature. Humidity and CO2 interact with the sample cell CO2. Due to the relationship between stomatal conductance and CO2 assimilation, having stable values of H20 and CO2 are important in obtaining reliable measurements. The desiccant setting controls the humidity within the chamber from ambient down to zero. Drierite (drying agent) is used as the desiccant and should be adjusted according to required conditions. Humidity within the chamber is a balance between water vapour coming from the leaf and air flow rate. Desiccant set to zero will result in a large vapour pressure deficit at the leaf surface and may affect stomatal conductance. Controlling CO2 with a CO2 mixer provides more accurate and easily obtained measurements. CO2 is typically adjusted to ambient levels and soda lime is used to scrub ambient CO2. The soda lime-scrub setting controls reference CO2 from ambient down to zero and can be adjusted accordingly if not using a CO2 mixer. Insert the leaf into the chamber and close it. Check the latch to ensure a good seal. Check to ensure that the proper leaf area has been set based on the area exposed inside the chamber. On some IRGAs you may be able to adjust stomatal ratio (i.e. estimate of the ratio of stomata on one side of the leaf to the other). Use 1 if stomatal density on the top and bottom are equal, use 0 if stomata are only present on one side, and use 0.5 if you are not sure. Because the calculation of stomatal conductance includes stomatal ratio, incorrect values will yield measurement errors. Observe the CO2 and H2O concentrations for the reference and the sample and ensure that they have stabilised. CO2 concentrations of the sample should stabilise within 30 seconds of clamping onto the leaf and should be lower than the reference CO2 under conditions where photosynthesis exceeds respiration. Record the value for stomatal conductance and depending on your experiment, proceed to the next leaf.
Where to start
Bell & Squire (1981), Drake et al. (2013), Flexas & Medrano (2002), Kirkham (2005), Pearcy et al. (1989)
5.7.2 Special cases, emerging issues, and challenges
Stomatal conductance is highly dynamic on a diurnal basis as well as throughout the growing season requiring frequent measurements in time and space to thoroughly assess conductance relative to temporal shifts in environmental parameters. Minimum steady-state stomatal conductance (gmin) occurs in the morning prior to light exposure (Drake et al., 2013). Maximum stomatal opening generally occurs in the early portion of the day, so measurements comparing stomatal conductance of different species or under different treatment conditions are typically taken mid to late morning (~8:00 to 10:00 a.m. depending on species and site). Timing may differ based on site location and environmental conditions such as temperature, vapour pressure, soil water availability, and so on.
Selection of leaves
Measurements are most commonly made on fully exposed sun leaves of whole plants. Because stomatal development depends on leaf age, care should be taken to standardise measurements across species and treatments, typically on mature leaves. With diffusion and steady-state porometers, ambient light conditions should also be standardised, preferably using light-saturated canopy leaves. Measurements with IRGAs allow for adjustment of PAR, and are commonly set with either ambient PAR values or saturating PAR (≥ 1500 µmol m-2 s-1 for most species) depending on the question of interest. With porometers that do not enclose the whole leaf, measurements are most commonly taken from the abaxial leaf surface where stomatal density is highest (Meidner & Mansfield, 1968), but stomata may occur on both sides of the leaf, depending on species (Smith et al., 1997) and this should be taken into consideration when taking measurements. It is not recommended to detach leaves for stomatal conductance measurements on angiosperms as this can cause stomata to close; however, conifers can be detached and measured within 20 mins. In the lab, it is ideal to take stomatal conductance measurements under saturating light conditions.
The boundary layer is a thin layer of still air around all surfaces. The thickness of the boundary layer above and below the leaf surface affects diffusion of gas and water vapour through the stomata and is determined by surface roughness, leaf characteristic dimension (i.e. leaf thickness in the direction of the wind), and wind speed. A leaf with high pubescence is rougher than one with a smooth waxy cuticle and will have a thicker boundary layer. Increased wind speed results in a thinner boundary layer. Most instruments for calculating stomatal conductance pass air through the chamber, thus removing any resistance from the boundary layer. Measurements should not be taken on windy days when the boundary air layer is thinner as evaporative demand is higher, potentially reducing stomatal conductance. Likewise, since the calculation for stomatal conductance uses a water vapour gradient, measurements should not be taken on wet leaves. Instead, wet leaves should be gently blotted with tissue paper to remove any surface moisture prior to measurement.
Stomatal response curves are becoming more common in the literature, particularly the response of stomata to increasing VPD (Woodruff et al., 2010; Ocheltree et al., 2014). This requires an instrument that allows for precise control over VPD in the sample chamber (e.g. LI-COR LI-6800, PP Systems CIRAS-3).
Stomata play a critical role in controlling both water loss and CO2 uptake, thus modelling stomatal conductance under various environmental conditions is of interest in many disciplines. Damour et al. (2010), Buckley & Mott (2013), and Buckley (2017) review in depth many of the available stomatal conductance models. Models of stomatal conductance occur at multiple spatial levels, from subcellular to Earth system processes. Empirical models were the first developed and were used to account for stomatal responses to light intensity, VPD, air temperature, CO2 concentration, and leaf water potential (e.g. Jarvis, 1976; more models reviewed in Damour et al., 2010). Numerous models have been developed based on the relationship between stomatal conductance (gs) and net photosynthetic rate (Anet). The empirical Ball-Berry stomatal conductance model (Ball et al., 1987) is one of the most commonly used models and is often used in land surface climate models to simulate regulation of evapotranspiration (Bonan et al., 2014). Empirical models are often simpler and used in larger canopy or global level processes (Buckley & Mott, 2013). With a better understanding of plant physiology, mechanistic or process-based models have been proposed. Mechanistic models tend to be mathematically complex and are often used in investigating cellular and subcellular processes in environmental sensing (Buckley & Mott, 2013). Mechanistic models include stomatal response to abscisic acid (ABA; e.g. Davies & Zhang, 1991; Tardieu & Simonneau, 1998), transpiration that includes hydraulic architecture (e.g. Tyree & Sperry, 1988), and predictions of photosynthesis under fluctuating conditions (reviewed in Buckley & Mott, 2013). Currently, most stomatal conductance models are adapted for well-watered conditions and do not sufficiently account for multiple environmental influences, especially drought conditions (Damour et al., 2010; Bonan et al., 2014).
Stomatal density refers to the number of stomata per area of leaf and is related to stomatal conductance and thus the CO2 and water fluxes through plants. Stomatal density can provide additional information as to differences in conductance among plants. Stomatal density can be made through impressions of adaxial and/or abaxial epidermis of a leaf using cyanoacrylate adhesive (Wilson, Pusey and Otto, 1981). A microscope slide is immediately pressed against the glue and held firmly for 90 sec. The slide is then gently peeled away from the skin. With this technique, a sheet of the outermost two to three layers of cells adheres to the slide. Clear nailpolish can also be used by painting onto the leaf surface and peeling away after it has dried. Features of the epidermis, including individual cells, stomata and trichomes are visible at most magnifications with these methods. Impressions can be examined with a light microscope. Stomata are counted in a known field of view to calculate stomatal density. Epidermal cells can also be calcualted and used with stomata to calculate a stomatal index as [s/(e+s)] x 100, where s is number of stomata and e is number of epidermal cells (Salisbury, 1928).
Theory, significance, and large datasets
Buckley & Mott (2013), Cowan (1977), Farquhar et al. (1980), Pearcy et al. (1989), van den Honert (1948)
More on methods and existing protocols
Buckley (2017), Buckley & Mott (2013), Kirkham (2005), Pearcy et al. (1989), Tardieu & Simonneau (1998)
Ball J. T., Woodrow, I. E., & Berry, J. A. (1987). A model predicting stomatal conductance and its contribution to the control of photosynthesis under different environmental conditions. In J. Biggens (Ed.), Progress in Photosynthesis Research (pp. 221-224). Netherlands: Martinus Nijhoff Publishers.
Bell, C. J., & Squire, G. R. (1981). Comparative measurements with two water vapour diffusion porometers (dynamic and steady-state). Journal of Experimental Botany, 32(131), 1143-1156.
Bertolini, L.T., Caine, R. S., & Gray, J.E. (2019). Impact of stomatal density and morphology on water-use efficiency in a changing world. Frontiers in Plant Science, 10, 225.
Bonan, G. B., Wiliams, M., Fisher, R. A., & Oleson, K. W. (2014). Modeling stomatal conductance in the earth system: linking leaf water-use efficiency and water transport along the soil–plant–atmosphere continuum. Geoscientific Model Development, 7, 2193-2222.
Buckley, T. N. (2017). Modeling stomatal conductance. Plant Physiology, 174, 572-582.
Buckley, T. N., & Mott, K. A. (2013). Modelling stomatal conductance in response to environmental factors. Plant, Cell, & Environment, 36(9), 1691-1699.
Cowan, I. R. (1977). Stomatal behavior and environment. Advances in Botanical Research, 4, 117-228.
Damour, G., Simonneau, T., Cochard, H., & Urban, L. (2010). An overview of models of stomatal conductance at the leaf level. Plant, Cell, & Environment, 33, 1419-1438.
Davies, W. J., & Zhang, J. H. (1991). Root signals and the regulation of growth and development of plants in drying soil. Annual Review of Plant Physiology and Plant Molecular Biology, 42, 55-76.
De Boeck, H. J., van de Velde, H., de Groote, T., & Nijs, I. (2016). Ideas and perspectives: heat stress: more than hot air. Biogeosciences, 13(20), 5821-5825.
Drake, P. L., Froend, R. H., & Franks, P. J. (2013). Smaller, faster stomata: scaling of stomatal size, rate of response, and stomatal conductance. Journal of Experimental Botany, 64(2), 495-505.
Farquhar, G.D., von Caemmerer, S., & Berry, J.A. (1980). A biochemical model of photosynthetic CO2 assimilation in leaves of C3 species. Planta, 149(1), 78-90.
Flexas, J., & Medrano, H. (2002). Drought-inhibition of photosynthesis in C3 plant: stomatal and non-stomatal limitations revisited. Annals of Botany, 89(2), 183-189.
Jarvis, P. G. (1976). The interpretation of the variations in leaf water potential and stomatal conductance found in canopies in the field. Philosophical Transactions of the Royal Society of London Series B, 273, 593-610.
Kirkham, M. B. (2005). Stomata and measurement of stomatal resistance In Principles of Soil and Plant Water Relations. (pp. 379-401). Cambridge, MA: Academic Press.
Meidner, H., & Mansfield, T. A. (1968). Physiology of Stomata. New York: McGraw-Hill.
Meinzer, F. C., Goldstein, G., Jackson, P., Holbrook, N. M., Gutierrez, M. V., & Cavelier, J. (1995). Environmental and physiological regulation of transpiration in tropical forest gap species: the influence of boundary layer and hydraulic properties. Oecologia, 101(4), 514-522.
Ocheltree, T. W., Nippert, J. B., & Prasad, P. V. V. (2014). Stomatal responses to changes in vapor pressure deficit reflect tissue‐specific differences in hydraulic conductance. Plant, Cell & Environment, 37(1), 132-139.
Pearcy, R. W., Schulze, E.-D., & Zimmerman, R. (1989). Measurement of transpiration and leaf conductance. In R. W. Pearcy, J. Ehleringer, H. A. Mooney, & P. W. Rundel (Eds.), Plant Physiological Ecology: Field Methods and Instrumentation (pp. 137-160). New York: Chapman and Hall.
Salisbury, E. J. (1928). On the causes and ecological significance of stomatal frequency, with special reference to the woodland flora. Philosophical Transactions of the Royal Society of London, B216(431-439), 1-65.
Smith, W. K., Vogelmann, T. C., DeLucia, E. H., Bell, D. T., & Shepherd, K. A. (1997). Leaf form and photosynthesis. Bioscience, 47(11), 785-793.
Sperry, J. S. & Love, D. M. (2015). What plant hydraulics can tell us about responses to climate‐change droughts. New Phytologist, 207(1), 14-27.
Tai, X., Mackay, D. S., Sperry, J. S., Brooks, P., Anderegg, W. R., Flanagan, L. B., … Hopkinson, C. (2018). Distributed plant hydraulic and hydrological modeling to understand the susceptibility of riparian woodland trees to drought‐induced mortality. Water Resources Research, 54(7), 4901-4915.
Tardieu, F. & Simonneau, T. (1998). Variability among species of stomatal control under fluctuating soil water status and evaporative demand: modelling isohydric and anisohydric behaviours. Journal of Experimental Botany, 49, 419-432.
Tyree, M. T. & Sperry, J. S. (1988). Do woody plants operate near the point of catastrophic xylem dysfunction caused by dynamic water stress? Plant Physiology, 88, 574-580.
van der Honert, T. H. (1948). Water transport in plants as a catenary process. Discussions of the Faraday Society, 3, 146-153.
Wilson, C. K. L., Pusey, P. L. & Otto, B. E. (1981). Plant epidermal sections and imprints using cyanoacrylate adhesives. Canadian Journal of Plant Science, 61(3), 781-783.
Woodruff, D. R., Meinzer, F. C., & McCulloh, K. A. (2010). Height-related trends in stomatal sensitivity to leaf-to-air vapour pressure deficit in a tall conifer. Journal of Experimental Botany, 61(1), 203-210.
Authors: Zinnert J1, Estiarte M2,3, Johnson DM4
Reviewer: Dickman LT5
1 Department of Biology, Virginia Commonwealth University, Richmond, USA
2 CSIC, Global Ecology Unit CREAF-CSIC-UAB, Bellaterra, Spain
3 CREAF, Cerdanyola del Vallès, Spain
4 Warnell School of Forestry and Natural Resources, University of Georgia, Athens, USA
5 Earth and Environmental Sciences Division, Los Alamos National Laboratory, Los Alamos, USA