Journal of Experimental Botany Journal of Experimental Botany, Vol. 48, No. 307, pp. 333-336, February 1997 A technique to measure root tip hydraulic conductivity and root water potential simultaneously Maciej A. Zwieniecki1'3 and Larry Boersma2 1 Forest Science Department, Peavy Hall 154, Oregon State University, Corvallis, OR 97331, USA 2 Crop and Soil Science Department, Oregon State University, Corvallis, OR 97331, USA Downloaded from https://academic.oup.com/jxb/article-abstract/48/2/333/652852 by guest on 12 May 2020 Received 29 November 1995; Accepted 4 September 1996 hydraulic conductivity of roots can be measured separately using root segments separated from the plant. Measurements of axial hydraulic conductivity are rela- tively simple (Chiu and Evers, 1993), whereas measure- ments of the radial component require more sophisticated equipment, for example, a modified cell-pressure probe (Melchior and Steudle, 1993). Root water potential can be measured with a modified Scholander pressure bomb (Nobel et al, 1991), or with a hygrometer for small segments of root apices (Rhizopoulou and Davies, 1991). Each of these measurements has certain limitations and a fully satisfactory theory to explain the results is not available (Nobel et al, 1991). Recently Simonneau and Habib (1994) reported a method using root suckers. This method is only suitable for woody plants. A simple and accurate method to measure hydraulic properties of root remains a research objective (Nobel et al, 1991; Tardieu et al, 1992). Here, a method is presented for the simultaneous measurement of root hydraulic conductivity and xylem root water potential which is based on the traditional potometer method. Abstract A procedure for the simultaneous measurement of hydraulic conductivity and xylem water potential of roots is presented. Roots remain intact and attached to the transpiring plant during measurement. The rate of water uptake by roots is measured at different water potential gradients along the root radial axis, obtained by placing them in solutions with different osmotic potentials. Hydraulic conductivity and xylem water potential are calculated by regression analysis of the relationship between water uptake rate and osmotic potential of the bathing solution, assuming that xylem water potential and reflection coefficient remain con- stant during measurement. Results for tomato plants experiencing drought are presented and discussed. Key words: Root, hydraulic conductivity, water potential. Introduction Hydraulic conductivity of roots has traditionally been measured by using the potometer technique. This method has a long history, and has been used by many scientists including Rosene (1937), Brewig (1939) and Brouwer (1951). The method consists of placing the root or root segment in a small chamber and then measuring the rate of water loss from this chamber as the water is absorbed by the root segment. Brouwer (1951) studied the effect of water demand on root water conductivity by placing roots in osmotic solutions with a range of potentials. The main interest of this work was in a method which can be used simultaneously to measure the hydraulic conductivity and xylem water potential of roots while still attached to the transpiring plant. Axial and radial Theory An evaluation of the potometer technique suggested a new method for the analysis of the data obtained which yields the root xylem water potential as well as the hydraulic conductivity. The analysis uses the general flow equation: J\ — LP(fR— !rs) (1) where iv(ms ') is flux, LP is hydraulic conductivity (ms"1 MPa"1), and fs and WR are total water poten- tials (MPa) at the root surface and in the root xylem, 3 To whom correspondence should be addressed at: OEB, Harvard University Biological Laboratories, Cambridge, Massachusetts, MA 02138, USA. Fax: +1 617 496 5854. Oxford University Press 1997
334 Zwieniecki et al. then inserted into the L-shaped connector. After the rubber stopper is firmly secured in the wall of the container and a seal around the root is obtained, the volume of the container with the capillary tube forms a self-contained chamber which can be filled with solutions with predetermined water potentials. Solutions are prepared by adding solutes such as mannitol or PEG to distilled water in the required concentrations. These volumes are filled to overflow through the capillary tubes (5 [A capacity) by attaching a small funnel to them, After allowing about 5 min for the system to relax, funnels are removed and a meniscus is established at the top of the capillary tubes. Water uptake by the root from each chamber is recorded by noting the position of the meniscus several times. Usually a set of measurements is completed in about 30 min. The connectors are then drained and a solution with different Vs is added. This procedure can be repeated several times. When the experiment is completed, roots are removed and the length and average diameter is measured; the surface area of the roots can be calculated using these measurements. Water potential of the root xylem is obtained from Equation (1) by solving it for the flow equal to zero. In order to assure constant "PR during the measurement an equilibration time of 1 h is allowed after set-up. respectively (Nobel, 1991). The driving force for the flow is the difference in solution osmotic potential and water potential in the root xylem, which is the sum of negative hydrostatic potential caused by transpiration and osmotic potential. Equation (1) contains the two unknowns "FR and LP and can thus be solved by multiple measurements. /v is measured at different values of *PS. Slopes and intercepts of the linear relationship between Jv and Ws are obtained where the slope is equal to LP and the intercept is fR. The reflection coefficient is assumed to be 0.85 for the PEG 2000 solution. This value was chosen on the basis of the literature, reporting a value of 0.82 for PEG 1000 for maize roots (Steudle, 1989). Downloaded from https://academic.oup.com/jxb/article-abstract/48/2/333/652852 by guest on 12 May 2020 Materials and methods The experimental device is a container with ports which can receive a stopper into which an L-shaped connector, with a capillary riser attached to it, is inserted (Fig. 1). The plant is placed in the container and roots of similar size, order, and distance from the stem are selected and threaded through the rubber stopper sealed with high pressure silicon paste, which is Application Measurements esculentum Mill.). Plants were grown for 2 months in a greenhouse in 1 1 pots filled with a mixture of vermiculite and soil. Plants were then moved outside and irrigated daily to prevent water stress. Twelve plants were randomly assigned to two treatments, namely well-watered (watered daily to full soil water-holding capacity) and water-stressed (watered every other day to half soil-water capacity). Stressed plants were watered 2 h before measurement to regain turgor. Measurements were made on four plants at the beginning of the experimental period. After 7 d exposure to the treatments, measurements were made on two well-watered and two stressed plants. Measurements were made on four roots per plant for a total of 16 roots. The measurements were repeated on the remaining plants after 14 d of treatment. Soil was washed from the roots and plants were placed in the container prepared to accept four connector assemblies (Fig. 1). Four healthy roots of the same order, similar size, and similar distance from the stem were chosen and threaded through the rubber stopper with the root tip protruding for 5 cm. The rubber stopper was pushed into the connector and the assembly was secured in the wall of the container. Chambers formed by L-shaped connectors were filled with either distilled water, a PEG 2000 solution (Sigma) with Ws= -0.097 MPa, or with Vs= -0.193 MPa (at 20°C). The sequence in which water or solution was added to each root was chosen randomly to account for any possible influence of sequence on water status of the plant. Osmotic potential of solutions was verified with an osmometer. were made with tomato (Lycoperisicon removable funnel meniscus capillary tube L-shape connector volume=2.5 ml PEG solution or distilled water root valve to allow solute replacement in connector rubber stopper, root sealed with silicon paste rubber stopper Results distilled water Plots of Jy versus ys showed the linear relationship of Equation (1) (Fig. 2). Flows between different roots varied so that parameters were calculated separately for each root. Recorded root uptake was constant during each set of measurements. No observable trends were one litre pot Fig. 1. Cross-section of the experimental arrangement. The volume of the L-shaped connectors is 2.5 ml, that of the capillary tube 5 /J, and the volume of the container is chosen based on the size of the root system.
A technique to measure root tip hydraulic conductivity and root water potential simultaneously 335 plants was —0.53 MPa and for plants exposed to stress -0.51 MPa. 1 5e-7 well-watered plant well-watered plant stressed plant stressed plant • regressions i r 1 3e-7 Discussion Results of the experiments are consistent with those of previous studies, which have shown that root hydraulic conductivity of numerous species is decreased by pro- longed exposure to water stress (Nobel and North, 1993). Cruz (1991) found that when plants were grown under water stress, the hydraulic conductivity of roots decreased by 70%. The reductions were attributed to embolism of xylem, cell dehydration, and air gaps between the roots and the soil (Turner, 1986). In this case, the plants were immersed in water during the measurements, so it is less likely that these explanations are satisfactory. Some specific morphological adaptations are suggested, such as thickening of cell walls or changes in the number of protein water channels in the cell membranes (Henzler and Steudle, 1995). Such responses can be important to protect the root from losing water to dry soil (Baker and van Bavel, 1986). Measured root water potential varied by as much as 0.1 MPa between the root segments. The average value for each plant was about —0.3 MPa and was similar to those reported by Rhizopoulou and Davies (1991) and Tardieu et al. (1992). The root hydraulic conductivity, obtained by the pro- posed method are higher than results obtained with osmotically driven flow on cut sections of roots, e.g. Steudle (1989), Melchior and Steudle (1993) and Tyree et al. (1994). This difference could be the result of the difference in experimental technique. In these experiments, the water in the root was under negative pressure caused by transpiration. The results obtained with the proposed procedure remain to be compared with the results obtained by techniques which employ flow driven by positive pressure (Steudle, 1989; Brouwer, 1951). It is also noted that a dependence of LP on the rate of flow is often found, e.g. Mees and Weatherley (1957). Fiscus (1975) provided an explanation for this observation based on the theoretical analysis of flow across membranes which showed that the dependence decreases with increas- ing flow rates. The lack of the relationship between /v and LP in the experiments here may be due to the high flow rates. An important concern regarding the method is whether the xylem water potential remains constant during the experiment. It is noted that the rate of water uptake (7V) remained constant during the 30 min of measurements, indicating that WR did not change (Fig. 2). This assump- tion may be justified because it is reported that the majority of the resistance resides in the radial not in the longitudinal resistance (Peterson and Steudle, 1993). Thus, xylem water potential was regulated by the entire root system which was kept in constant equilibrium - 7 5e-8 3 0 0e+0 -0 3 -0 2 -0 1 0 0 Downloaded from https://academic.oup.com/jxb/article-abstract/48/2/333/652852 by guest on 12 May 2020 Solution water potential (4>s) |MPa| Fig. 2. Rate of water uptake by roots as a function of the water potential in the L-shaped connectors. Results are shown for two roots of well-watered plants and two roots of plants subject to water stress during 14 d of experiment duration. noticed. Measurements made at the beginning of the experiment, after 7 d, and again after 14 d showed that LP of plants grown under well-watered condition remained constant and ranged from 3.70 x 10~7 to 3.97xl0-7[m3 plants decreased from 3.82 x 10~7 to 1.41 x 10~7 [m m~2 MPa"1], a decrease of about 60% (Fig. 3). Multiple regression analysis confirmed that LP changed over time due to the stress treatment (P< 0.0001, /?2 = 0.98). Water potential of root xylem was obtained from the intercept of Jv versus *FS (Fig. 2) which represents root water potential according to Equation (1). Values ranged from -0.18 to -0.31 MPa and did not differ between treat- ments or plants. Leaf water potentials were measured for both treat- ments. Values were not different between the two treat- ments, probably because the plants were allowed to regain turgor, and later, the entire root system remained in the water bath with fs = 0 MPa during the sets of measure- ments. The average leaf water potential for well-watered s-'m-2 m3 s 'm 2 MPa 1]. LP of the water-stressed 3s"' 4.5e-7 4.0e-7 3.5e-7 3.0e-7 Z.Se-7 I _ _ — — " 1. <1 1 E 'A. 2.0e-7 1.5c-7 l.Oe-7 5.0e-8 O.Oe+0 1 a. - -A- well-watered plants —• - stressed plants 0 7 Days 14 Fig. 3. Hydraulic conductivity of roots as a function of number of days since water stress was imposed. Results are shown for well-watered plants and plants stressed for 14 d. Vertical bars are standard deviation from the mean.
336 Zwieniecki et al. condition throughout the measurement period. However, changes of xylem water potential due to experimental conditions cannot be completely excluded and so measure- ments should not be taken over an extended period of time. The proposed method seems to be promising for relatively non-invasive measurements of root hydraulic properties. The low cost of the method and its simplicity and ease of use should permit its wide use in laboratories and in the field as a complementary method to other techniques. The method has an advantage that water flow occurs under a gradient of natural negative potentials which are difficult to obtain by other methods (Steudle, 1994). Mees GC, Weatherley PE. 1957. The mechanism of water absorption by roots. 1. Preliminary studies on the effects of hydrostatic pressure gradients. Proceedings of the Royal Society of London, Series B 147, 367-80. Melchior VV, Steudle E. 1993. Water transport in onion (Allium cepa L.) roots. Plant Physiology 101, 1305-15. Nobel PS. 1991. Phvsicochemical and environmental plant physiology. San Diego: Academic Press. Inc. Nobel PS,'Lopez FB, Aim DM. 1991. Water uptake and respiration of root system of two cacti: observation and predictions based on individual roots. Journal of Experimental Botany 42, 1215-23. Nobel PS, North GB. 1993. Rectifier-like behaviour of root-soil system: new insights from desert succulents. In: Smith JAC, Griffiths H, eds. Water deficit: plant response from cell to community. Oxford: BIOS Scientific Publishers Limited, 163-76. Peterson CA, Steudle E. 1993. Lateral hydraulic conductivity of early metaxylem vessels in Zea mays L. roots. Planta 189, 288-97. Rhizopoulou S, Davies WJ. 1991. Influence of soil drying on root development, water relations and leaf growth of Ceratonia siliqua L. Oecologia 88, 41-7. Rosene HF. 1937. Distribution of the velocities of absorption of water in the onion root. Plant Physiology 12, 1-9. Simonneau T, Habib R. 1994. Water uptake regulation in peach trees with split-root systems. Plant, Cell and Environment 17, 379-88. Steudle E. 1989. Water flow in plants and its coupling to other processes: an overview. Methods in Enzvmologv 179, 183-225. Steudle E. 1994. Water transport across the roots. Plant and Soil 167, 79-90. Tardieu F, Bruckler L, Lafolie F. 1992. Root clumping may affect the root water potential and the resistance to soil-root water transport. Plant and Soil 140, 291-301. Turner NC. 1986. Adaptation to water deficits: a changing perspective. Australian Journal of Plant Physiology 13, 175-90. Tyree MT, Yang S, Cruiziat P, Sinclair B. 1994. Novel methods of measuring hydraulic conductivity of tree root systems and interpretation using AMAIZED. Plant Physiology 104, 189-99. Downloaded from https://academic.oup.com/jxb/article-abstract/48/2/333/652852 by guest on 12 May 2020 References Baker JM, van Bavel CHM. 1986. Resistance of plant roots to water loss. Agronomy Journal 78, 641-4. Brouwer R. 1951. Water absorption by the roots of Vicia faba at various transpiration strengths. Proceedings Koningklijke Nederlandse Akademie van Wetenschappen, Serie C56, 106-15. Brewig A. 1939. Auslosung leichter Wasserdurchlassigkeit an Wurzeln von Vicia faba. Planta 29, 341-60. Chiu S, Evers FW. 1993. The effect of segment length on conductance measurements in Lonicera fragrantissima. Journal of Experimental Botany 44, 175-81. Cruz RT. 1991. Structural and hydraulic properties of Sorghum bicolor (L.) roots following exposure to water deficit. Thesis, Texas A&M University USA. Fiscus EL. 1975. The interaction between osmotic and pressure- induced water flow in plant roots. Plant Physiology 55, 917-22. Henzler T, Steudle E. 1995. Reversible closing of water channels in Chara internodes provides evidence for a composite transport model of the plasma membrane. Journal of Experimental Botany 46, 199-209.