Xylem Hydraulics - Theory. I. Hydraulic Architecture Definition: i) The structure of water transport system in plants. (Tyree and Ewers 1991) V t

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1 Plant Ecophysiological Measurement Techniques - BOT 6935 January 27, Theory. I. Hydraulic Architecture Definition: i) The structure of water transport system in plants. (Tyree and Ewers 1991) ii) The set of hydraulic characteristics of the conducting tissue of a plant which qualify and quantify the sap flux from roots to leaves. (Cruiziat et al. 1994) This structure can limit plant water relations, gas exchange through the leaves and probably the maximum height that particular tree species can achieve (Sperry 2000; Tyree and Ewers 1991). II. Hydraulic conductivity Hagen-Poiseuille equation - given equal water potential gradients, the flux of water through a xylem conduit is proportional to the 4 th power of the radius of that conduit. This means that a few larger conduits provide much more efficient water transport than many smaller diameter conduits. V t m m = = * Mpa (1) πr dp 8η dl Where: V=volume (m 3 ), t=time (s), r=conduit radius (m), n=viscosity (8.9x10-10 MPa s -1 ), p=pressure (MPa), l=length (m) hydraulic conductivity (k h ) - because of the Hagen-Poiseuille relationship, plants with larger xylem conduits tend to have much higher k h ; you can think of the inverse of k h as the resistance to water flux due to the architectural properties of the xylem. s Mpa * s * m k h = (water flux) water potential difference / segment length (2) k h units = (m 3 s -1 m -2 ) / (MPa / m) = m 2 s -1 MPa -1 leaf-specific hydraulic conductance (k l ) = k h leaf area sapwood-specific hydraulic conductance (k s ) = k h sapwood area Equation (2) suggests methods. See Zimmermann (1978) and Sperry et al. (1988). Whole-tree hydraulic conductance (derivation from Ohm s Law): Page 1 Flux E l = K * Driving Force = K l * Ψ

2 Figure 1. A (Left): Hydraulic conductivity, maximum water velocity and vessel diameter for a range of plant types (from Lambers et al.1998). Note that units have been mis-specified for conductivity, and should be cm 2 s -1 Mpa -1 to obtain the magnitudes shown. B (Right): Variation in water potential as a function of transpiration rate (TFD) for black walnut, a diffuse porous species (circles), white oak, a ring porous species (squares), and eastern redcedar, a conifer (triangles). (Ginter Whitehouse et al. 1983, as shown in Kozlowski and Pallardy 1997) III. Cavitation or embolism Definition: introduction of air into a xylem element - air bubble expands and renders the conduit unable to conduct water. Cavitation due to xylem tension. 1. Cavitation detection by acoustic emissions (Milburn and Johnson 1966, Tyree et al. 1984) When the water columns inside droughted plants come under sufficient tension they should cavitate, and this should be accompanied by a shock-wave detectable as sound (Jackson and Grace, 1996). Figure 2. A (Left): Daily cycles of Cavitation detected by ultrasound acoustic emission sensors (from Jackson and Grace 1996). B (Right): Example of ultrasound acoustic emission sensors installed in mature Scots pine tree. However, acoustic emissions have only a limited use in determining the proportion of embolism in a conducting stem, and other methods are needed to find the percentage reduction in hydraulic conductivity. Page 2

3 2. Xylem vulnerability to cavitation curves Figure 2. Xylem vulnerability curves for P. palustris (longleaf pine) and P. elliottii (slash pine) stem segments (Gonzalez-Benecke et al. 2010) Figure 3 A (Left): Xylem vulnerability curves for trees from a range of environments from xeric to mesic (Sperry et al. 1998). B (Right): Xylem vulnerability curves for loblolly pine roots and branches under different experimental treatments (Ewers et al. 2000). Important parameter from VC-Curves: Ψ 50 (parameter b in Figure 2): The xylem tension at which 50% loss in conductivity occurs (also called as vulnerability to cavitation ). Mechanism for cavitation (Sperry et al. 1996) - "air seeding" - air bubble is pulled through the pit membrane by the difference in pressure between an air-filled conduit and a conduit under tension Page 3

4 Figure 4. Illustration of air seeding between a cavitated xylem vessel and a vessel under tension (top) or air seeding between a pressurized, air filled vessel and a water-filled conduit under no tension (bottom) (Sperry et al. 1996) Page 4

5 Measurement of xylem hydraulic conductivity and vulnerability to cavitation Methods for generating vulnerability curves 1. Generate a range of stem water potentials, measuring hydraulic conductance at each. a. Generate range of stem water potentials by bench drying (Sperry and Pockman 1993) b. Generate range of stem water potentials by spinning. (Holbrook et al. 1995, Pockman et al. 1995) Figure 5. Centrifuge apparatus used to induce tension in branches for generating xylem vulnerability curves. (From J.S. Sperry lab website). 2. Use positive pressure to generate pressure difference across pit membranes to reproduce effect of lowering stem water potential (Figure 4; Salleo et al. 1992, Sperry et al. 1996) Page 5

6 Measurement of xylem hydraulic conductivity and vulnerability to cavitation Xylem hydraulic conductivity (k, kg water s -1 MPa -1 m) is calculated according to Darcy s law as the flow rate of water (Kg s -1 ) for a given pressure gradient through a segment of known length (MPa m -1 ) (Tyree and Zimmermann, 2002). Procedure: Note: Always cut the segments and attach tubing UNDER WATER. This will minimize cavitation. Before start, need to prepare 20 mm KCl solution (degased and filtered at 0.2 µm). About 6 liters is enough for many samples. Fill KCl solution reservoir (Mariotte bottle is recommended). Also need to prepare several connectors using Luer Fittings and tubing ready to be installed. 1) Place the cut end of a stem segment (branch, trunk or, root) under water, and recut the segment at a point where it is approximately 2-10 mm in diameter. Using razor blade, make a second cut to produce a segment that is about 15 cm long (for some angiosperms need longer segments). Keep track of the upstream or "proximal" end of the branch, as we will want our water flow to go the same direction as it did in the intact branch (from "distal" to "proximal"). 2) Keep sections cut at each end, about 0.5 to 1.0 mm width, to determine area using image analyzer (example: image J: Need to scan both sections. 3) Place the tube from the reservoir under water. Affix the tubing with connectors to the upstream or proximal end of the segment. Use a clamp to tighten the tubing onto the segment to assure a leak-free seal. 4) Place another tubing with connectors onto the downstream or distal end of the segment. 5) Bring the segment out of the tub. 6) Ensure that tubing connections are filled with KCl solution (no air in the line). Refill with KCl solution using syringe with rounded needle. 7) Connect segment with connectors to hydraulic conductivity apparatus ( Sperry tubing apparatus ): Proximal end to tubing from KCl solution source (Mariotte Bottle), and Distal end to tubing that goes to beaker on electronic balance. 8) Ensure that all tubing connections are filled with KCl solution (no air in the line). If needed, refill with KCl solution using syringe with rounded needle. 9) Open KCl solution source from Mariotte bottle using stopcock in Lauer fitting. 10) Record height of water level in beaker on electronic balance. 11) Start program that connects electronic balance with computer. Need to input: segment length; segment distal and proximal areas; height of water level in beaker on electronic balance; height of water column (from bench top, to air-entry point in Mariotte bottle). If no segment length and areas are known yet, you can input a reference number and change later when segment-specific values are known. Note that height from bench top to air-entry point in Mariotte bottle is constant for all measurements, even if water level changes inside Mariotte bottle. Page 6

7 12) Record for 2 to 5 minutes as needed. 13) Stop flow of KCl solution using stopcock in Lauer fitting from Mariotte bottle. 14) Continue recording, this is the background measurement (about 1-2 minutes). This measurement is discounted from value recorded in step ) Re-open KCl solution source from Mariotte bottle using stopcock in Lauer fitting and check if hydraulic conductivity comes back to pre-background measurement. 16) Stop program that connects electronic balance with computer. 17) Save excel file with proper name. Additional Notes In many studies, after the initial hydraulic conductance measurement, the stem segment is flushed with higher pressure fluid or using vacuum soaking, to sweep away existing embolisms. A subsequent conductance measurement then reveals a "maximum" conductance. The water head or hydrostatic pressure head or gravitational pressure difference can be calculated as ρ*g*h where ρ is the density of water (998.2 kg m -3 ), g is acceleration due to gravity (9.81 m s -2 ) and h is height above the reference plane. For a 1 m height above the reference plane, this works out to kg m -3 * 9.81 m s -2 * 1 m = 9792 kg m -1 s -2 = 9792 Pa = MPa. In excel file you just need to input h. Depending on each species (xylem cell diameter), an adequate hydrostatic pressure head should be defined. For pines, a water head between 0.5 and 1.0 m is OK. For poplar, we recommend a water head of about 0.5 m. Check literature for the species you are working with. Native hydraulic conductivity (k nat ) : Is the k measured at field conditions (reflect the amount of water stress of the plant when the segment was cut). Maximum hydraulic conductivity (k max ) : Is the k measured after removing native embolism (using KCl solution flushing at high pressure or using vacuum soaking). If sapwood area of the segment is known, k is expressed per unit sapwwod area and is called: sapwood-specific hydraulic conductivity (k s, kg water s -1 m -2 sapwood MPa -1 m). If leaf area distal to the segment is known, k is expressed per unit leaf area and is called: leaf-specific hydraulic conductivity (k l, kg water s -1 m -2 leaf area MPa -1 m) Page 7

8 Xylem vulnerability to cavitation. 1) Measure k nat using procedure previously described. 2) Remove native embolism using vacuum soaking for 24 h (Domec and Gartner, 2001; Domec et al. 2005): Fill a bottle that allows vacuum with KCl solution. Insert stem segments properly labeled. Connect vacuum pump. Check vacuum with vacuum meter. Continue soaking under vacuum for 24 h. Then remove sampled and place in tub with KCl solution. 3) Measure k max using procedure previously described on sampled that were vacuum-soaked. 4) The vulnerability to cavitation curve (VC-curve; Sperry and Tyree 1988) is determined by measuring k under different levels of Ψ xylem. The more negative Ψ xylem, the larger the water stress and therefore larger cavitation and loss of hydraulic conductivity. 5) After measuring k max, place the segment, both ends protruding, in a double-ended pressure chamber (for this class we will use pressure chamber constructed with portable pressure chamber caps with compression gland cover of a PMS Instrument Co. Scholander chamber, and a custom designed aluminum body; there are commercial pressure chamber that can be used as well). 6) Using a needle, pinch two shallow (0.5 mm deep) notches into opposite sides of the xylem about 5 cm apart in the center of the segment in order to ensure entry of air into the xylem inside the pressure chamber. 7) After the chamber is tightly closed, increase air pressure inside the chamber to a prescribed value (start at 0.1 to 0.5 MPa steps) and held for 10 minutes. Then lowered back air pressure (depressurized) and wait for 1-3 minutes. 8) Remove segment from cavitation chamber and seal the notches using parafilm. 9) Re-connect tubings and re-introduce flow by opening the valve and re-measure hydraulic conductivity at the level of pressure previously applied (see page 6). 10) Exposure of the segment to progressively higher air pressures continuing until hydraulic conductance measurements are at least 95% below the initial value (k max ) using 0.5 MPa steps. Before and after each k measurement, background flow rates should be determined. Note: The range of pressure applied to reach more than 95% cavitation is different for each species. We recommend carrying out pilot measurements in some spare samples in order to determine the level of starting pressure and forthcoming increasing pressure steps needed. Save the excel file after each measurement. Create a new spreadsheet with the proper name indicating the sample ID and the corresponding pressure applied. Page 8

9 Data analysis: A VC-curve is later constructed for each segment showing the cumulative percentage decrease in hydraulic conductivity versus the negative of air-injection pressure applied. Percentage loss of conductivity (PLC) at a given pressure is calculated using the equation given by Sperry and Tyree (1988 and 1990): PLC Ψ = k (Ψ) k max Where PLC Ψ is the percentage loss of conductivity at pressure Ψ, k (Ψ) is the hydraulic conductivity measured after apply pressure Ψ and k max is the maximum hydraulic conductivity measured previously after vacuum soaking. The plot of these data is the VC-curve (Sperry and Tyree 1988). We recommend using a sigmoidal equation (Pammenter and Vander Willinger, 1998) to calculate biological parameters from the VC-curve: PLC = e a (Ψ b) where a is an indicator of the slope and b represents the pressure applied at which 50% loss of conductivity occurred. Several parameters can be calculated in order to compare different curves (Domec and Gartner 2001): Ψ 50 = b where Ψ 50 is the xylem tension (MPa) at which 50% of loss of conductivity occurs. Ψ air = a 2 + b where Ψ air is the air entry point, an estimate of xylem tension (MPa) when cavitation starts and pit membrane is overcome. 2 Ψ max = - + b a where Ψ max is the full embolism point, an estimate of the maximum tension (MPa) in the xylem before failing and becoming non-conductive. s = 25 a where s is the slope of the linear portion of the VC-curve (% loss of k MPa -1 ), an estimate of rate of change in loss of k per unit change in xylem tension. Page 9

10 Procedure to prepare 20 mm KCl solution: 1. Get a 2000 ml graduated cylinder 2. Add ~1900 ml deionized (DI) H Add g of KCl (KCl molar mass is g/mol: 0.02 mol/litre x g/mol = g KCl per litre = g KCl per 2 litres H 2 0) 4. Stir with magnetic stirrer on plate for 10 minutes 5. Fill graduated cylinder to 2000 ml with DI H Degas with Helium for 10 minutes 7. Filter with 0.2 µm filter Page 10

11 Useful References Cochard, H., E. Badel1, S. Herbette, S. Delzon, B. Choat and S. Jansen Methods for measuring plant vulnerability to cavitation: a critical review. Journal of Experimental Botany doi: /jxb/ert193 Cochard, H., and S. Delzon Misunderstanding sap ascent in trees. Journal of Plant Hydraulics. e0001. Cochard, H The basics of plant hydraulics. Journal of Plant Hydraulics. e0004. Cruiziat, P., H. Cochard and T. Ameglio Hydraulic architecture of trees: main concepts and results. Annals of Forest Science 59: , Domec, J.C. and B.L. Gartner Cavitation and water storage capacity in bole xylem segments of mature and young Douglas-fir trees. Trees 15: Domec J.C., King J.S., Noormets A., Treasure E.A., Gavazzi M.J., Sun G., McNulty S.G Hydraulic redistribution of soil water by roots affects whole stand evapotranpiration and net ecosystem carbon exchange. New Phytologist 187: Domec J.C., Ogée J, Noormets A., Jouangy J. Gavazzi M., Treasure E., Sun G., McNulty S. and J.S.King Interactive effects of nocturnal transpiration and climate change on the root hydraulic redistribution and carbon and water budgets of Southern US pine plantations. Tree Physiology 32(6): Ewers, F.W. and M.H. Zimmermann The hydraulic architecture of balsam fir (Abies balsamea). Physiologia Plantarum 60: Ewers, B.E., R. Oren and J.S. Sperry Influence of nutrient versus water supply on hydraulic architecture and water balance in Pinus taeda. Plant Cell Environ 23: Gonzalez-Benecke, C.A. and T.A. Martin Water availability and genetic effects on water relations of loblolly pine (Pinus taeda) stands, Tree Physiology 30: Gonzalez-Benecke, C.A., T.A. Martin and G.F. Peter Hydraulic architecture and tracheid allometry in mature Pinus palustris and Pinus elliottii trees. Tree Physiology 30: Holbrook, N.M., M.J. Burns and C.B. Field Negative xylem pressures in plants: a test of the balancing pressure technique. Science 270: Holbrook, N. M. and M. A. Zwieniecki Embolism repair and xylem tension: Do we need a miracle? Plant Physiology 120:7-10. Jackson, G.E. and J. Grace Field measurements of xylem cavitation: are acoustic emissions useful? Journal of Experimental Botany. 47: McDowell, Nate G., Rosie A. Fisher, Chonggang Xu, J. C. Domec, Teemu Hölttä, D. Scott Mackay, John S. Sperry et al "Evaluating theories of drought induced vegetation mortality using a multimodel experiment framework." New Phytologist 200: Milburn, J.A. and R.P.C. Johnson The conduction of sap. II. Detection of vibrations produced by sap cavitation in Ricinus xylem. Planta 66: Pallardy, S.G., J. Cermak, F.W. Ewers, M.R. Kaufmann, W.C. Parker and J.S. Sperry Water transport dynamics in trees and stands. In: W.K. Smith and T.M. Hinckley (Eds.), Resource Physiology of Conifers. Academic Press, San Diego. pp Pammenter N.W. and C. Vander Willigen.1998 A mathematical and statistical analysis of the curves illustrating vulnerability of xylem to cavitation. Tree Physiology 18: Pittermann, J. and J.S. Sperry Tracheid diameter is the key trait determining the extent of freezinginduced embolism in conifers. Tree Physiology 23: Page 11

12 Pockman, W.T., J.S. Sperry and J.W. O'Leary Sustained and significant negative water pressure in xylem. Nature 378: Salleo, S., T.M. Hinckley, S.B. Kikuta, M.A. Lo Gullo, P. Weilgony, T.-M. Yoon and H. Richter A method for inducing xylem emboli in situ: experiments with a field-grown tree. Plant, Cell and Environment 15: Sperry, J.S Limitations on stem water transport and their consequences. In B.L. Gartner (Ed.). Plant Stems. Physiology and Functional Morphology. Academic Press, San Diego, pp Sperry, J.S Hydraulic constraints on plant gas exchange. Agricultural and Forest Meteorology 104: Sperry, J.S. and W.T. Pockman Limitation of transpiration by hydraulic conductance and xylem cavitation in Betula occidentalis. Plant, Cell and Environment 16: Sperry, J.S. and N.Z. Saliendra Intra- and inter-plant variation in xylem cavitation in Betula occidentalis. Plant, Cell and Environment 17: Sperry, J.S. and J.E.M. Sullivan Xylem embolism in response to freeze-thaw cycles and water stress in ring-porous, diffuse-porous, and conifer species. Plant Physiology 100: Sperry, J.S. and M.T. Tyree Mechanism of water-stress induced xylem embolism. Plant Physiology 88: Sperry, J.S. and M.T. Tyree Water-stress-induced xylem embolism in three species of conifers. Plant, Cell and Environment 13: Sperry, J.S., J.R. Donnelly and M.T. Tyree A method for measuring hydraulic conductivity and embolism in xylem. Plant Cell Environ 11: Sperry, J.S., N.Z. Saliendra, W.T. Pockman, H. Cochard, P. Cuizat, S.D. Davis, F.W. Ewers and M.T. Tyree New evidence for large negative xylem pressures and their measurement by the pressure chamber technique. Plant, Cell and Environment 19: Tyree, M.T. and M.A. Dixon Water stress induced cavitation and embolism in some woody plants. Physiologia Plantarum 66: Tyree, M.T. and J.S. Sperry Do woody plants operate near the point of catastrophic xylem dysfunction caused by dynamic water stress? Answers from a model. Plant Physiology 88: Tyree, M. T., S. Salleo, A. Nardini, M. A. Lo Gullo and R. Mosca Refilling of Embolized Vessels in Young Stems of Laurel. Do We Need a New Paradigm? Plant Physiology 120: Tyree, M.T., M.A. Dixon, E.L. Tyree and R. Johnson Ultrasonic acoustics emissions from the sapwood of cedar and hemlock: an examination of three hypotheses concerning cavitation. Plant Physiology 75: Tyree M.T. and F.W. Ewers The hydraulic architecture of trees and other woody plants. New Phytologist 119: Ward, E.J., R. Oren, D.M. Bell, J.S. Clark, H.R. McCarthy, H. Seok-Kim and J.-C. Domec The effects of elevated CO2 and nitrogen fertilization on stomatal conductance estimated from scaled sap flux measurements at Duke FACE. Tree Physiology, 33 (2): Way D.A., Domec JC and R.B. Jackson Elevated growth temperatures alter hydraulic characteristics in trembling aspen (Populus tremuloides) seedlings: implications for tree drought tolerance. Plant, Cell and Environment : 36: Zimmermann, M.H Hydraulic architecture of some diffuse-porous trees. Canadian Journal of Botany 56: Page 12