Reviewer: Dickmann LT3, Estiarte M4,5
Measurement unit: Kleaf in mmol m-2 s-1 MPa-1; Measurement scale: leaf; Equipment costs: €€€; Running costs: €; Installation effort: low; Maintenance effort: low; Knowledge need: medium; Measurement mode: data logger
The leaf hydraulic conductance (Kleaf) quantifies the efficiency of water movement through the leaf, from the petiole to evaporation sites. Thus, Kleaf is influenced by liquid flow through the leaf vein xylem and across the leaf bundle sheath and through the mesophyll, including vapour movement through the airspaces (Sack & Tyree, 2005; Sack & Holbrook, 2006; Buckley et al., 2015, 2017). Kleaf is calculated as the flow rate of water through the leaf (typically normalised by leaf area) for a given water potential driving force. To capture CO2 for photosynthesis, stomata open, exposing the vapour-saturated intercellular airspaces to the dry atmosphere. The higher the Kleaf, the more efficiently leaves can replace water lost via diffusion to the dry atmosphere, allowing stomata to remain open. The maximum leaf hydraulic conductance (i.e. that of a fully hydrated plant; Kmax) varies more than 65-fold across species (Sack & Holbrook, 2006) and constrains both light-saturated photosynthesis and stomatal conductance across diverse and closely related species (Brodribb et al., 2007; Scoffoni et al., 2016). The Kleaf can also be extremely sensitive to internal and environmental factors, such as light (Sack et al., 2002; Cochard et al., 2007; Scoffoni et al., 2008; Guyot et al., 2012) and soil and atmospheric drought (Scoffoni & Sack, 2017). Kleaf is thus an important trait playing a role in determining species plasticity, evolution and distribution across habitats (Blackman et al., 2012, 2014; Scoffoni et al., 2015).
5.10.1 What and how to measure?
While there are several methods currently in use to measure Kleaf, the evaporative flux method (EFM; Sack et al., 2002; Sack & Scoffoni, 2012) has the advantage of mimicking the natural transpirational pathways of water movement in the leaf. The rate of transpiratory water flow is measured via either a 5-decimal analytical balance or a flow meter (for construction details, see Sack et al., 2011). While the flow meter is relatively inexpensive, easily transportable, and completely field deployable, it does require more preparation and knowledge than the balance method. In the EFM, an excised leaf is connected by tubing to a water source on a balance (or to a flowmeter). The leaf is placed under a light source and over a box fan to ensure stomata are open. The loss of water from the balance is recorded at regular intervals (5 to 30 s) to calculate the flow rate through the leaf. When a steady-state is reached (and after a period of > 20 min to ensure leaves are acclimated to light), the leaf is taken off the system, petiole dabbed dry, and placed in a sealable plastic bag previously exhaled into (to create a high humidity and CO2 atmosphere to suppress transpiration) and equilibrated in the dark for > 20 min. The water potential driving force of the flow is then measured using a pressure chamber. Kleaf is calculated by dividing the flow rate by the water potential driving force, and normalising by leaf area and measurement temperature (for step by step protocols and videos, see Sack & Scoffoni, 2012), and therefore highly relevant in a climate-change context.
Branches can be collected in the field the day prior to measurements and placed in large dark plastic bags filled with wet paper towels. Plant material can be transported to the lab in a cooler. In the lab, at least two nodes should be cut under water to ensure the removal of any embolized conduits (as a result of cutting shoots off individuals in air in the field), and the shoots should be left to rehydrate in pure water overnight, with two dark plastic bags filled with wet paper towels covering them, ensuring high atmospheric humidity around the rehydrating samples. Alternatively, if working with greenhouse plants, whole pots can be transported to the lab the evening prior to measurements, watered to saturation and covered in two dark plastic bags filled with wet paper towers.
Measuring Kleaf using the EFM with a balance
A custom-built top is needed for the balance that will stably secure a metal or glass tube inside a container of water on the balance. Water will then flow from the container, up through the tube, and then into plastic tubing to the leaf. Prior to measurements, beakers filled with wet paper towels should be placed inside the balance, and the balance sealed, such that high humidity within the balance prevents evaporation from the container of water: verify no drift in the mass value on the balance. Next, under pure water, cut a leaf off a fully hydrated branch and rapidly wrap parafilm around its petiole. Recut the tip of the petiole under pure water using a fresh razor blade and connect it (while under water) to tubing that runs to the water source on the balance (after making sure no air bubbles are trapped anywhere in the system). You can gently lift the connection out of water to check for leaks by raising the sample above the pressure head. If no air bubble appears in the tubing, then the seal is good. Place the leaf adaxial surface upwards in wooden frames strung with fishing line to stabilise the leaf, above a large box fan, and under at least 1000 μmol m-2 s-1 photosynthetically active radiation. Maintain leaf temperature around 25 °C throughout the experiment. Allow leaves to transpire on the apparatus for at least 30 min (to allow leaves sufficient time to acclimate to high irradiance) and until flow rate has stabilised – no upward or downward trend – and with a coefficient of variation < 5% for at least five measurements made at 30 sec flow intervals. Discard measurements if the flow suddenly changes, either due to apparent leakage from the seal or blockage in the system by particles or air bubbles. When flow stabilises, record leaf temperature with a thermocouple and quickly remove the leaf from the tubing. Dab dry the petiole and place the leaf into a sealable bag which has been previously exhaled into to halt transpiration. Let the leaf sit for at least 30 min to equilibrate. Measure the final leaf water potential on the equilibrated leaf using a pressure chamber, and the leaf area with a scanner and image analysis, or a leaf area meter.
Measuring Kleaf using a flowmeter
A pressure-drop flowmeter can be built following detailed steps available online (Sack et al., 2011). Every morning prior to measurements, calibrate the transducers of the flowmeter by plotting the voltage against known positive pressures, obtained by placing the water source connected to the flowmeter at different heights. The slope and intercept are used to convert the voltage into pressure units. To check accuracy, move the water source to an intermediate height and ensure transducers values are comparable and are the same as the pressure generated from the height. If one or both transducers are not responding to applied pressure, first check for a loose connection, for example ensure wires are making contact with the data acquisition board transmitting transducer input to the computer. Tighten if necessary. If there is still no response, check for air bubbles in the system and flush, and check for air bubbles in the transducers themselves. Connect the leaf following the same recommendations as for the balance method. At the end of the measurement, in addition to measuring leaf temperature, measure the water temperature before and after the resistor of the flowmeter. You will need this to adjust the resistance value for differences in water viscosity. The flow rate is calculated by the difference in pressure between the two transducers divided by the resistor (which you can measure using either a balance or a pipette, measuring the flow rate through the resistor at different heights). The Kleaf (mmol m-2 s-1 MPa-1) is calculated by dividing the flow rate (normalised by leaf temperature and size) by the final leaf water potential driving force (measured at the end of the experiment using a pressure chamber).
Where to start
Brodribb et al. (2007), Sack & Holbrook (2006), Sack & Scoffoni (2012), Sack et al. (2002, 2011), Scoffoni et al. (2016)
5.10.2 Special cases, emerging issues, and challenges
The EFM can also be used to measure stomatal conductance, by recording the air temperature and relative humidity around the leaf during steady-state flow at the end of the measurement. These parameters can be used to measure the vapour pressure difference driving diffusion of water vapour out of the leaf through stomata. Stomatal conductance (gs; mmol m-2 s-1) is then calculated by dividing the flow rate through the leaf by the vapour pressure difference expressed as a mole fraction. The ratio of hydraulic supply over demand (Kleaf / gs) can then also be calculated (Sack & Scoffoni, 2012); a higher supply over demand would enable species to maintain stomata open during rapid changes in vapor pressure differences (VPD) during the day (Carins Murphy et al., 2012; Scoffoni et al., 2015, 2016). The system can also be modified to measure Kleaf under varying environmental factors, such as light (Scoffoni et al., 2008). Some species can rapidly enhance their Kleaf values by substantial amounts under high light (Sack et al., 2002; Cochard et al., 2007; Scoffoni et al., 2008; Voicu et al., 2008; Guyot et al., 2012) and under shifts that are associated with changes in the expression and activation of aquaporins (Cochard et al., 2007; Shatil-Cohen et al., 2011; Shatil-Cohen & Moshelion, 2012; Laur & Hacke, 2014a, 2014b; Sade et al., 2014).
The use of a digital pressure chamber and stereoscope is highly recommended for more accurate water potential measurement, especially at high water potentials.
A number of additional methods exist for the determination of Kleaf. A version of the evaporative flux method can be applied with leaves still attached, by determining transpiration rate using a gas exchange or sapflow system, and dividing by the driving force estimated from the difference in water potentials between transpiring and non-transpiring (bagged) leaves (Sack & Tyree, 2005). The vacuum pump method involves measuring the flow rate of water through a leaf under varying levels of subatmospheric pressures (partial vacuum), and Kleaf is calculated from the slope of flow against pressure. The high-pressure flow method calculates Kleaf from the flow rate of water delivered through a leaf at a known pressure. Finally, the rehydration kinetics method estimates Kleaf as the inverse of the resistance in series with a charging capacitor as a leaf recovers from rehydration (Brodribb & Holbrook, 2003; Blackman & Brodribb, 2011). However, with that method, it is not easy to light acclimate the samples prior to measurements, which would underestimate Kleaf for many species (Scoffoni et al., 2008). Obtaining Kleaf values above -0.5 MPa is difficult (Scoffoni et al., 2008), and most importantly, the pathways of water movement in rehydrating leaves could be very distinct from those of a transpiring leaf (Zwieniecki et al., 2007). While all these various methods have yielded comparable maximum Kleaf values to the EFM in several species it remains unclear whether the pathways for water movement are equivalent across methods (e.g. Tsuda & Tyree, 2000; Nardini et al., 2001, 2010; Sack et al., 2002; Scoffoni et al., 2008; Blackman & Brodribb, 2011; Hernandez-Santana et al., 2016).
Theory, significance, and large datasets
Carins Murphy et al. (2012), Laur & Hacke (2014b), Nardini et al. (2010), Sack & Scoffoni (2012), Sack & Tyree (2005), Sack et al. (2002)
More on methods and existing protocols
Guyot et al. (2012), Sack & Scoffoni (2012), Scoffoni et al. (2008)
Blackman, C. J., & Brodribb, T. J. (2011). Two measures of leaf capacitance: insights into the water transport pathway and hydraulic conductance in leaves. Functional Plant Biology, 38(2), 118-126.
Blackman, C. J., Brodribb, T. J., & Jordan, G. J. (2012). Leaf hydraulic vulnerability influences species’ bioclimatic limits in a diverse group of woody angiosperms. Oecologia, 168(1), 1-10.
Blackman, C. J., Gleason, S. M., Chang, Y., Cook, A. M., Laws, C., & Westoby, M. (2014). Leaf hydraulic vulnerability to drought is linked to site water availability across a broad range of species and climates. Annals of Botany, 114(3), 435-440.
Brodribb, T. J., & Holbrook, N. M. (2003). Stomatal closure during leaf dehydration, correlation with other leaf physiological traits. Plant Physiology, 132(4), 2166-2173.
Brodribb, T. J., Field, T. S., & Jordan, G. J. (2007). Leaf maximum photosynthetic rate and venation are linked by hydraulics. Plant Physiology, 144(4), 1890-1898.
Buckley, T. N., John, G. P., Scoffoni, C., & Sack, L. (2015). How does leaf anatomy influence water transport outside the xylem? Plant Physiology, 168(4), 1616-1635.
Buckley, T. N., John, G. P., Scoffoni, C., & Sack, L. (2017). The sites of evaporation within leaves. Plant Physiology, 173, 1763-1782.
Carins Murphy, M. R., Jordan, G. J., & Brodribb, T. J. (2012). Differential leaf expansion can enable hydraulic acclimation to sun and shade. Plant, Cell & Environment, 35(8), 1407-1418.
Cochard, H., Venisse, J. S., Barigah, T. S., Brunel, N., Herbette, S., Guilliot, A., … Sakr, S. (2007). Putative role of aquaporins in variable hydraulic conductance of leaves in response to light. Plant Physiology, 143(1), 122-133.
Guyot, G., Scoffoni, C., & Sack, L. (2012). Combined impacts of irradiance and dehydration on leaf hydraulic conductance: insights into vulnerability and stomatal control. Plant, Cell & Environment, 35, 857-871.
Hernandez-Santana, V., Rodriguez-Dominguez, C. M., Fernández, J. E., & Diaz-Espejo, A. (2016). Role of leaf hydraulic conductance in the regulation of stomatal conductance in almond and olive in response to water stress. Tree Physiology, 36(6): 725-735.
Laur, J., & Hacke, U. G. (2014a). Exploring Picea glauca aquaporins in the context of needle water uptake and xylem refilling. New Phytologist, 203(2), 388-400.
Laur, J., & Hacke, U. G. (2014b). The role of water channel proteins in facilitating recovery of leaf hydraulic conductance from water stress in Populus trichocarpa. PLoS One, 9(11), e111751.
Nardini, A,, Tyree, M. T., & Salleo, S. (2001). Xylem cavitation in the leaf of Prunus laurocerasus and its impact on leaf hydraulics. Plant Physiology, 125(4), 1700-1709.
Nardini, A., Raimondo, F., Lo Gullo, M. A., & Salleo, S. (2010). Leafminers help us understand leaf hydraulic design. Plant, Cell & Environment, 33(7), 1091-1100.
Sack, L., & Holbrook, N. M. (2006). Leaf hydraulics. Annual Review of Plant Biology, 57, 361-381.
Sack, L., & Scoffoni, C. (2012). Measurement of leaf hydraulic conductance and stomatal conductance and their responses to irradiance and dehydration using the evaporative flux methods (EFM). Journal of Visualized Experiments, (70), e4179.
Sack, L., & Tyree, M. T. (2005). Leaf hydraulics and its implications in plant structure and function. In N. M. Holbrook, & M. A. Zweiniecki (Eds.), Vascular Transport in Plants (pp. 93-114). Oxford: Elsevier/Academic Press.
Sack, L., Melcher, P. J., Zwieniecki, M. A., & Holbrook, N. M. (2002). The hydraulic conductance of the angiosperm leaf lamina: a comparison of three measurement methods. Journal of Experimental Botany, 53(378), 2177-2184.
Sack, L., Bartlett, M., Creese, C., Guyot, G., & Scoffoni, C. (2011). Constructing and operating a hydraulics flow meter. http://prometheuswiki.org/tiki-index.php?page=Constructing+and+operating+a+hydraulics+flow+meter: Prometheus Wiki.
Sade, N., Shatil-Cohen, A., Attia, Z., Maurel, C., Boursiac, Y., Kelly, G., … Moshelion, M. (2014). The role of plasma membrane aquaporins in regulating the bundle sheath-mesophyll continuum and leaf hydraulics. Plant Physiology, 166(3), 1609-1620.
Scoffoni, C., & Sack, L. (2017). The causes and consequences of leaf hydraulic decline with dehydration. Journal of Experimental Botany, 68(16), 4479-4496.
Scoffoni, C., Pou, A., Aasamaa, K., & Sack, L. (2008). The rapid light response of leaf hydraulic conductance: new evidence from two experimental methods. Plant, Cell & Environment, 31(12), 1803-1812.
Scoffoni, C., Kunkle, J., Pasquet-Kok, J., Vuong, C., Patel, A. J., Montgomery, R. A., … Sack, L. (2015). Light-induced plasticity in leaf hydraulics, venation, anatomy and gas exchange in ecologically diverse Hawaiian lobeliads. New Phytologist, 207, 43-58.
Scoffoni, C., Chatelet, D. S., Pasquet-Kok, J., Rawls, M., Donoghue, M. J., Edwards, E. J., & Sack, L. (2016). Hydraulic basis for the evolution of photosynthetic productivity. Nature Plants, 2, a16072.
Shatil-Cohen, A., & Moshelion, M. (2012). Smart pipes: the bundle sheath role as xylem-mesophyll barrier. Plant Signaling & Behavior, 7(9), 1088-1091.
Shatil-Cohen, A., Attia, Z., & Moshelion, M. (2011). Bundle-sheath cell regulation of xylem-mesophyll water transport via aquaporins under drought stress: a target of xylem-borne ABA? Plant Journal, 67(1), 72-80.
Tsuda, M., & Tyree, M. T. (2000). Plant hydraulic conductance measured by the high pressure flow meter in crop plants. Journal of Experimental Botany, 51(345), 823-828.
Voicu, M. C., Zwiazek, J. J., & Tyree, M. T. (2008). Light response of hydraulic conductance in bur oak (Quercus macrocarpa) leaves. Tree Physiology, 28(7), 1007-1015.
Zwieniecki, M. A., Brodribb, T. J., & Holbrook, N. M. (2007). Hydraulic design of leaves: insights from rehydration kinetics. Plant, Cell & Environment, 30(8), 910-921.
Authors: Scoffoni C1, Sack L2
Reviewer: Dickmann LT3, Estiarte M4,5
1 Department of Biological Sciences, California State University, Los Angeles, USA
2 Department of Ecology and Evolutionary Biology, University of California Los Angeles, Los Angeles, USA
3 Earth and Environmental Sciences Division, Los Alamos National Laboratory, Los Alamos, USA
4 CSIC, Global Ecology Unit CREAF-CSIC-UAB, Bellaterra, Spain
5 CREAF, Cerdanyola del Vallès, Spain