Hydraulic conductance and K+ transport into the xylem depend on radial volume flow, rather than on xylem pressure, in roots of intact, transpiring maize seedlings

Authors

  • Lars H. Wegner,

    1. Lehrstuhl für Biotechnologie, Biozentrum, Universität Würzburg, D-97074 Würzburg, Germany;
    2. Present address: Plant Bioelectrics Group, Karlsruhe Institute of Technology, Campus North, Building 630, Hermann-v-Helmholtz Platz 1, 76344 Eggenstein-Leopoldshafen, Germany
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  • Ulrich Zimmermann

    1. Lehrstuhl für Biotechnologie, Biozentrum, Universität Würzburg, D-97074 Würzburg, Germany;
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Author for correspondence:
Lars H. Wegner
Tel:+49 7247 824302
Fax:+49 7247 822823
Email: lars.wegner@ihm.fzk.de

Summary

  • • The investigation of transport processes in roots has so far been hampered by a lack of adequate methods to study water and solute transport simultaneously in intact, transpiring plants. The role of xylem tension in regulating volume flow and nutrient transport could not be addressed properly.
  • • In order to overcome limitations of conventional, massive-invasive methods, a gravimetric technique was used to measure water uptake by maize roots while simultaneously recording xylem pressure and xylem K+ activity in individual xylem vessels by means of a K+-selective xylem probe. This minimal-invasive approach allowed the calculation of the radial K+flux into the root xylem and the radial root hydraulic conductance on transpiring seedlings.
  • • By changing the light regime or the osmotic pressure of the external solution, radial water and K+ flux could be varied in order to study the interaction between water and solute transport. A major finding was that both radial K+ transport and hydraulic conductance strongly depended on radial volume flow, whereas xylem pressure had little (if any) effect on these parameters.
  • • Results are discussed with respect to relevant membrane transport processes and their regulation by volume flow.

Introduction

Water and solute transport across roots have received much interest in previous decades. Numerous studies have been published on symplastic and apoplastic pathways of ion and water transport, including work that focused on transport processes across membranes of cortex and stelar cells, and on interaction between fluxes (Lopushinski, 1964; Anderson, 1976; Fiscus, 1977; Pitman, 1982; Clarkson, 1993). However, conflicting data have been obtained with different methods on issues that are of paramount importance for our understanding of root transport functions, such as the dependence of root hydraulic conductance on the driving forces (hydrostatic and/or osmotic pressure gradients; Fiscus, 1977; Passioura, 1988), the existence of symplastic ion gradients (Dunlop & Bowling, 1971; Läuchli et al., 1971), or the efficiency of the Casparian band as a barrier to apoplastic water and solute flow into the stele. These questions remained unresolved up to the present day, since no adequate methods were available to study transport processes on intact, transpiring plants. Studies on root function had to rely entirely on techniques that were massive-invasive, casting doubt on the physiological relevance of the data obtained by that means. Our current knowledge is frequently based on work performed on excised roots, despite the fact that root excision will strongly affect root physiology and functioning (Bloom & Caldwell, 1988; Emery & Salon, 2002; but see also Anderson & Allen, 1970). In particular, methods for studying radial volume flow as well as the composition of the xylem sap frequently required pressurization of the root system, leading to an infiltration of air-filled spaces (De Boer & Prins, 1984). This most likely altered transport properties of the tissue (Salim & Pitman, 1984). Obviously, a technique to study these transport processes in the intact, transpiring plants without affecting hydrostatic and osmotic pressure gradients is lacking. In this study, we focussed on radial water and K+ transport in the root, since K+ is a macronutrient that plays an essential role as an osmoticum and is required to maintain the activity of many enzymes. Moreover, radial K+ transport in roots has been studied in detail, and both low-affinity ion channels and high-affinity transporters have been identified and functionally characterized previously (Wegner & Raschke, 1994; Roberts & Tester, 1995; Gaymard et al., 1998; De Boer, 1999; Tester & Leigh, 2001; Very & Sentenac, 2003). Here, we present a means to overcome the limitations of previous methods to study radial water and K+ transport, by making use of multifunctional xylem probes (Wegner & Zimmermann, 2002; Wegner et al., 2007) that allow on-line recording of xylem pressure, electrical potential and ion activities (in this particular case, of K+) in the lumen of a single xylem vessel by impalement with double-barreled microcapillaries, one of them connected to a xylem pressure-potential probe, and the other acting as a K+-selective electrode. This technique is termed minimal-invasive, that is, the plant is still injured by the insertion of microcapillaries, but the injury is local and the interference with plant integrity is definitely much less severe than the damage caused by decapitation of the shoot. A local injury at a leaf midrib to get access to the xylem was also applied by Passioura & Munns (1984). However, with their approach, overpressure still has to be applied to the root system in order to collect xylem sap at that site. Here, we show that radial volume and K+ fluxes in the root and their dependence on xylem pressure, trans-root potential (i.e. the electrical potential difference between the root xylem and an external electrode) and xylem K+ activity can be studied on-line using multifunctional K+ probes, when water uptake is simultaneously measured using a simple gravimetric method.

A re-investigation of root water and ion transport at the level of the intact plant will certainly profit from the tremendous progress that has been made in recent years in unraveling membrane transport processes on a molecular scale (De Boer, 1999; Tester & Leigh, 2001; Very & Sentenac, 2003). By applying the patch-clamp technique to protoplasts derived separately from cortex and stelar cells, respectively, ion channels for ion uptake and release were identified (Wegner & Raschke, 1994; Roberts & Tester, 1995; Wegner & De Boer, 1997, 1999; Maathuis et al., 1998; Köhler & Raschke, 2000; Köhler et al., 2002) and proton pump activity was demonstrated in both cell types (Zhu et al., 2007). The genetic basis of many of these transport proteins has now been resolved, including a K+ outward rectifying ion channel in stelar cells (SKOR) that has been shown to be involved in K+ loading of xylem vessels (Gaymard et al., 1998). Moreover, our understanding of radial water transport in the roots has been revolutionized by the discovery of aquaporines in the plasmalemma and the tonoplast of root cells and their regulation by various environmental factors, such as drought and waterlogging (for reviews, see Javot & Maurel, 2002; Vandeleur et al., 2005); aquaporines facilitate water flow across membranes and thus mediate passage of water through the symplastic pathway.

The immense recent progress in unraveling root transport functions on a molecular scale has renewed interest in this topic. However, little progress has been made with respect to the integration of transport processes at the level of the intact plant. This paper will make a contribution to fill this gap. It is shown here that both net K+ release into the xylem and the hydraulic conductance of the root depend on radial volume flow, whereas hydrostatic (xylem) pressure has little, if any, effect on these transport parameters.

Materials and Methods

Plant material

Experiments were performed on 12- to 24-d-old maize seedlings (Zea mays L. cv. Helix) that were grown on aerated hydroponics as described previously (Wegner & Zimmermann, 1998).

The multifunctional xylem pressure probe

For simultaneous recording of xylem pressure, trans-root potential and root xylem K+ activity, a multifunctional xylem pressure probe was inserted into the seminal root of intact maize seedlings at the root base, close to the caryopse. The probe has been described in detail in previous publications (Wegner & Zimmermann, 2002; Wegner et al., 2007). Briefly, it consists of a xylem pressure probe with an integrated potential-sensing electrode and a double-barreled capillary attached to it; the long barrel connected to the perspex body of the probe served for the measurement of xylem pressure and trans-root potential. From the shorter barrel, a K+-selective electrode was prepared for in situ recording of xylem sap K+ activity. To this end, a ‘cocktail’ containing the ionophore valinomycin was dissolved in tetrahydrofuran, back-filled into this barrel and left to dry, so that a pressure-tight plug was formed in the very tip of the barrel. The interior of the perspex body of the probe was filled with 50 mm KCl, whereas the pressure/potential-sensing barrel contained 50 mm NMG-Mes (pH 5.8). Both solutions were filtered (Millex GV filter units, Millipore, Bedford, MA, USA; pore size 0.22 µm) and degassed. The K+-sensing barrel was also back-filled with 50 mm KCl. From the electrical potential recorded by this electrode, the K+ activity in the solution surrounding the tip could be calculated (Wegner & Zimmermann, 2002), provided the electrode had previously been calibrated against solutions with known KCl activities (see later). After mounting the probe on a micromanipulator (Leica, Munich, Germany), an Ag/AgCl electrode was inserted at the open, blunt end of this barrel and connected to a high-impedance differential electrometer (FD223, WPI, Sarasota, FL, USA). The electrode integrated into the probe for registration of the TRP was connected to the second channel of the same amplifier.

Measurement of root water uptake

We have designed a new gravimetric method for measuring water uptake by plant roots that is suitable for combination with the probe technique. A schematic diagram of the setup is shown in Fig. 1. A plastic cuvette containing standard medium (1 mm KCl, 2 mm MgCl2, 2 mm CaCl2, 10 mm Mes/BTP, pH 5.5; π = 22 mosmol kg−1) was placed on digital scales (Heraeus, Hanau, Germany). The root of a maize seedling was fixed to a Teflon rod that could be lowered into the cuvette in a controlled way by two rotary arms made from plastic. A longitudinal notch received the root. At a distance of 5 mm along the notch, small holes (diameter 0.8 mm) had been drilled into the Teflon rod. Because of the hydrophobicity of the material, these holes remained air-filled when the rod was submersed and served as an oxygen reservoir for the root tissue attached to the rod. When the Teflon rod with a root attached to it had been lowered into the medium, the surface of the bath was covered with an approx. 3-mm-thick layer of paraffine oil (Merck, Darmstadt, Germany) to prevent water loss by evaporation. Therefore, a decrease in weight could unequivocally be ascribed to water uptake by the root and subsequent loss to the atmosphere by transpiration, as verified by control experiments in the absence of a seedling (data not shown). The loss of weight of the cuvette with respect to the initial value was sampled every 5 s. From the slope, the water uptake rate could be calculated.

Figure 1.

Setup for measuring root water uptake gravimetrically. The root of an intact seedling supported by a Teflon rod was lowered into a plastic cuvette that was placed on digital scales; the cuvette contained standard nutrient solution. The Teflon rod was fixed by two rotary arms that were attached to an external vertical stand (see front view). A longitudinal notch was engraved into the Teflon rod, which received the root; along the notch, small holes were drilled into the rod at a distance of 5 mm (side view). These holes remained air-filled while the rod was immersed in the medium so that the root attached to the rod was constantly supplied with oxygen. After filling the cuvette with bath medium and lowering the root into the medium, the surface was covered with a 3-mm-thick layer of paraffine oil in order to prevent evaporational water loss from the cuvette. Thus, a decrease in weight registered by the scales could be ascribed unequivocally to water uptake by the root and subsequent transpirational loss to the atmosphere. Optionally, a multifunctional pressure probe (mounted on a micromanipulator) could be inserted into the root during the water uptake measurement (see front view). For further details, see the text.

Simultaneous recording of water uptake, xylem pressure and xylem K+activity

For simultaneous recording of rates of water uptake, xylem pressure and xylem K+ activity, a seedling was fixed to the setup for measuring root water uptake as described in the previous section, and subsequently a multifunctional probe was inserted into the root (see Fig. 1). Before root impalement, the K+-selective barrel had to be calibrated against a range of KCl solutions of different activities (typically ranging from 0.5 to 50 mm). Impalement was performed at least 1 h after installing the plant; the multifunctional probe was mounted on the micromanipulator and inserted into a single xylem vessel of the seminal root by slowly advancing the tip through the tissue until a sudden drop in pressure was observed. The procedure, including possible pitfalls, has been described in detail elsewhere (Balling & Zimmermann, 1990; Wegner & Zimmermann, 1998; Zimmermann et al., 2004; Wegner et al., 2007). At the end of the experiment, the probe was removed and the K+-selective electrode was re-calibrated. When this calibration curve deviated significantly from the one recorded before the impalement, the experiment was discarded. Finally, root and shoot length as well as fresh weight were determined as described previously (Wegner & Zimmermann, 1998, 2002).

By combining measurements using the K+-selective xylem pressure probe with gravimetric recordings of root water uptake, fundamental parameters of plant nutrient and water transport became accessible for the first time in intact, transpiring plants. With respect to plant K+ nutrition, these are the net K+ flux into the root xylem (JK+,cx; also referred to as the net rate of xylem loading of K+) and the export of K+ from the root to the shoot (JK+,exp). Moreover, the data allow the calculation of the radial hydraulic conductance of the root, LP,r, which describes the coupling between radial water (volume) flow, Jv,r, and osmotic and hydraulic pressure gradients between the xylem vessels and the microenvironment of the root.

Theoretical considerations

Calculation of K+ fluxes into the root xylem (JK+,cx)  Calculation of radial K+ fluxes into the root xylem vessels in intact seedlings was based on a simple model, assuming the lumens of the xylem vessels form a homogenous compartment (Fig. 2). Changes in the K+ content of this compartment with time can be expressed as the difference between radial K+ flux from adjacent tissue and K+ export to the shoot by mass flow:

Figure 2.

Diagram showing the model on which calculation of flow parameters is based. The xylem conduit in the root is considered as a homogenous compartment; gradients in xylem pressure and K+ activity in this compartment are not taken into account. aK+,xyl, xylem K+ activity; JK+,cx, net K+ flux into the root xylem; JK+,exp, K+ export into the shoot; Jv,r, radial volume flow, which is set equal to root water uptake; Jv,exp, volume flow from root to shoot; Px, xylem pressure. For further details, see the text.

image(Eqn 1)

dnK+,xyl/dt can be calculated from the changes in xylem K+ activity with time (assuming the K+ activity is numerically equal to the K+ concentration of the xylem sap, which is very low in a transpiring plant) times the total xylem volume, Vxyl:

image(Eqn 2)

K+ export to the shoot by mass flow in xylem vessels is given by:

image(Eqn 3)

with Jv,r being the radial volume flow, which is, to a first approximation, equal to root water uptake. Insertion of Eqns 2 and 3 into Eqn 1 and rearrangement gives:

image(Eqn 4)

Note that the parameters required to calculate JK+,cx are experimentally accessible by the approach used here. The first derivative of the K+ activity (daK+,xyl/dt) was obtained numerically using the computer program ‘Origin’ (OriginLabCorp, Northampton, MA, USA). The total xylem volume that was involved in long-distance transport was estimated from anatomical data of maize roots. To this end, the number of early and late metaxylem elements and their cross-sectional areas were determined on freehand thin cross-sections (n = 5 roots). For calculation of early metaxylem volume, it was assumed that xylem vessels were mature and conducting, starting at 3 cm above the root tip (Steudle & Peterson, 1998). Maturation of late metaxylem that can be quite variable and depends on growth conditions was estimated by cutting the root into 3 cm sections and imposing an overpressure of 0.1 MPa on one end using a home-built pressure bomb, while the other end was covered with a droplet of water and inspected with a stereomicroscope. Conducting late metaxylem vessels were identified by the release of air bubbles. This approach will yield only a rough estimate of Vxyl (and, hence, of daK+/dt × Vxyl), but even omitting this component of Eqn 4 will lead to an error of < 5% under the conditions applied here (see later).

For comparison, K+ fluxes were normalized to the root fresh weight.

Calculation of the radial hydraulic conductance of the root (LP,r)  A radial hydraulic conductance of the root has been defined based on the concept of root tissue (including cortex, endodermis and xylem parenchyma) forming a single, uniform barrier to radial water flow into the xylem vessels (Fiscus, 1977; Zimmermann et al., 2001). It has to be noted that this is a purely phenomenological parameter that reflects various structural and physiological properties of the root, such as, for example, the presence or absence of an exodermis or the expression of aquaporins in various tissues.

This approach justifies the adaptation of the general water flow equation (Zimmermann & Steudle, 1978) to the maize seminal root (Fiscus, 1977) according to the following:

image(Eqn 5)

ΔPr, σr and Δπr are, respectively, the pressure gradient between the xylem vessel and the ambient medium, the apparent radial reflection coefficient of the root and the difference in osmotic pressure between the xylem sap and the ambient medium. (Following Miller (1985a,b), ΔPr is defined as atmospheric minus xylem pressure; accordingly Δπr is defined as bath minus xylem osmotic pressure. Hence, Jvr values with a positive sign are equivalent to water uptake.) LP,r is accessible by the experimental setup for simultaneous xylem probing and gravimetric recording of root water uptake (Fig. 1) based on a few generalizing assumptions. First of all, it is assumed that xylem pressures (which are recorded in a single vessel only) are the same in all functional vessels that contribute to volume flow to the shoot. Previous work provides evidence in favor of this assumption (Schneider et al., 1997a,b). Moreover, longitudinal pressure gradients in the root xylem vessels are supposed to be so shallow that the xylem pressure can be considered constant along the root to a first approximation. On the basis of these assumptions, the radial hydrostatic pressure drop, ΔPr, corresponds to the xylem tension value measured by the probe. Unlike the hydrostatic pressure the osmotic pressure of the xylem sap (and, hence, the osmotic driving force for water flow across the root, Δπr) is not directly accessible by the experimental approach presented here. However, based on earlier work on maize roots (Miller, 1985a), the concentration of osmotically active compounds is estimated to be about four times the xylem K+ activity. Furthermore, the assumptions made for the hydrostatic xylem pressure, as outlined earlier, also apply to the osmotic pressure of the xylem sap.

The radial reflection coefficient of maize roots, σr, is also a variable parameter, which is affected by environmental conditions. For maize seedlings, Schneider et al. (1997b) observed an increase of σr with light irradiation below c. 700 µmol m−2 s−1, starting from a value of 0.15 at a low light regime to a maximum value close to 1. This increase was apparently correlated with light-induced radial volume flow in the root. If not stated otherwise, we interpolated the σr-values required here for the calculation of LP,r from Schneider et al. (1997b).

In accordance with the K+ fluxes and following Miller (1985b), Lp,r values were normalized to the root fresh weight. According to the usual definition, the hydraulic conductance is related to a unit area (Zimmermann & Steudle, 1978); however, it did not seem meaningful to normalize the water flow into the xylem with respect to an area such as the root surface that is difficult to assess from an experimental viewpoint. Therefore, LP,r values are given in terms of µl min−1 MPa−1 gFW−1.

Results

Evaluation of the gravimetric method for root water uptake

In a first set of experiments, the gravimetric technique for measuring root water uptake (cf. Fig. 1) was tested. In the absence of a maize seedling, the weight registered by the scales remained constant with time (data not shown). When a seedling had been mounted on the setup, a continuous decrease in weight (expressed with respect to the starting value that was set to zero) was monitored at a light irradiation of 300 µmol m−2 s−1 (Fig. 3). Subsequently, the shoot was cut at the base with a razor blade and removed, and the cut surface of the stump that remained attached to the root was covered with a droplet of silicon oil; immediately afterwards, the weight remained at a constant value. The same observation was made on three other seedlings. This result confirmed that evaporational water loss from the surface of the cuvette was totally eliminated by the paraffine layer. Therefore, the permanent decrease of weight observed when the plant was still intact could unequivocally be ascribed to transpiration-induced water uptake by the root. The experiment also demonstrated that the weight of the shoot that was received by the rotary arm of the plant support did not affect the value registered by the scales; the same can be inferred for the root, which was tightly fixed to the Teflon rod of the plant holder.

Figure 3.

Effect of excision of the shoot on root water uptake by a 24-d-old maize (Zea mays) seedling as measured gravimetrically using the setup depicted in Fig. 1. The plot shows the decrease in the bath volume (recorded by the decrease in weight; thick line) and the radial volume flow into the root (thin line; normalized to the root fresh weight; 0.439 g) calculated from the slope of the volume decrease. The arrow indicates cutting of the shoot; the cut surface was immediately covered with a droplet of silicone oil. Note that a continuous volume decrease was registered as long as the seedling was intact; this was the result of water uptake by the root in order to compensate for transpirational water loss to the atmosphere (light irradiation: 300 µmol m−2 s−1). As soon as the shoot was excised, transpiration stopped and the volume of the cuvette remained at a constant value; hence, radial volume flow into the root dropped to zero.

From the change of weight with time, the radial water uptake rate of the root normalized to the root fresh weight could be calculated. A typical example out of four independent experiments is shown in Fig. 3. The water uptake rate fluctuated somewhat, c. 6.5 µl min−1 gFW−1 for the intact seedling in the experiment shown here (mean value 9.6 ± 3.6 µl min−1 gFW−1 for n = 4 plants). In all four plants tested, it dropped immediately to zero upon removal of the shoot.

Root K+ and water transport parameters with a varying light regime but constant osmotic conditions

A second type of experiment that comprised measurements of water uptake following impalement by the K+-selective xylem probe at the root base is presented in Fig. 4 (a typical example out of five independent experiments). The seedling was exposed to three different intensities of light irradiation (10, 70 and 300 µmol m−2 s−1). Upon insertion of the probe tip into a vessel at the low-light regime, the pressure registered by the probe dropped immediately from about atmospheric (+0.1 MPa) to +0.045 MPa and relaxed slowly to a steady value of +0.03 MPa. When light irradiation was successively increased to 70 and 300 µmol m−2 s−1, respectively, the pressure decreased below vacuum to –0.04 and –0.11 MPa. Establishment of a new steady-state value took c. 20 min at the medium light intensity and 10 min at the high light intensity. When the light regime was subsequently reduced again, the pressure increased to –0.05 MPa (70 µmol m−2 s−1) and +0.025 MPa (10 µmol m−2 s−1), indicating that the effect of irradiation on pressure was reversible. These results are in agreement with previous reports (Wegner & Zimmermann, 2002; Wegner et al., 2007). No undershoot of pressure, as reported in those previous publications, was observed here; it did, however, occur in other experiments.

Figure 4.

Response of xylem pressure (Px), radial volume flow (Jv,r), xylem K+ activity (aK+,xyl), K+ flux into the xylem (JK+,cx) and K+ export to the shoot (JK+,exp) to a stepwise increase and subsequent decrease in light irradiation. Representative traces measured on a 12-d-old maize (Zea mays) seedling are shown. To this end, the root xylem was impaled with a multifunctional K+ probe, and radial volume flow was recorded simultaneously using the setup introduced in Fig. 1. Bars on top of the figures indicate the time schedule of light regime changes (in µmol m−2 s−1). (a) The time course of xylem pressure. Impalement of a xylem vessel (indicated by asterisk) in laboratory light (10 µmol m-2 s−1) was associated with a pressure drop from atmospheric to 0.44 MPa. In (b), the radial volume flow (top trace), xylem K+ activity (middle trace), K+ flux into the root xylem (bottom, thin line, calculated according to Eqn 4) and K+ export to the shoot (bottom, thick line, calculated according to Eqn 3) are plotted with time for the same experiment. Note that for technical reasons, recording of Jv,r started with a delay of c. 15 min with respect to impalement. For this (triangle apex down) as well as four other seedlings, dependence of JK+,cx on Jv,r and Px is shown in (c) and (d), respectively. The sequences of light regimes for these experiments were (irradiance in µmol m−2 s−1): 10–70–300–70–10 (circles, triangles apex down, triangles apex up, half-shaded circles); 10–70–300 (squares). The index ‘1’ indicates the starting value for the respective experiment. Despite some hysteresis, plants could be divided into two groups with respect to the dependence of JK+,cx on Jv,r, as indicated by shaded areas in (c). In three plants, K+ flux was close to zero in the absence of volume flow, whereas in two further plants, extrapolation yielded a considerable rate of xylem loading at zero volume flow (c. 15 nmol min−1 gFW−1).

Figure 4(b) shows the corresponding traces of the volume flow (Jv,r) and the xylem K+ activity, as well as the time courses of the K+ flux into the xylem (JK+,cx; calculated according to Eqn 4) and the K+ export to the shoot (JK+,exp; Eqn 3). At the lowest light irradiation, radial volume flow (top trace) was extremely low (< 0.1 µl min−1 gFW−1). When irradiation was increased to 70 µmol m−2 s−1, the volume flow increased gradually to c. 2.5 µl min−1 gFW−1; initially, the volume flow tended to oscillate slightly. Upon a further increase of the light regime to 300 µmol m−2 s−1, Jv,r was subsequently up-regulated to a value of c. 8 µl min−1 gFW−1 and decreased accordingly when the light irradiation was stepped down to the medium intensity and, with some delay and slight oscillations, upon return to the low value. (Even with constant environmental conditions, some fluctuations were inherent to all the parameters measured here. It has to be kept in mind that the experiments were performed on living organisms that were operating away from thermodynamic equilibrium. Hence, no ‘steady state’ in the strict sense of the word was established here.)

The middle trace of Fig. 4(b) shows the time course of the xylem K+ activity for this experiment. K+ activity attained values between 2.0 and 2.3 mm, with a tendency to increase when the experiment was started at the low light intensity. Raising light intensity reduced K+activity to 1.9 mm (70 µmol m−2 s−1) and further to 1.3 mm (300 µmol m−2 s−1), but aK+,xyl hardly responded when the light regime was down-stepped to the initial value. It was only 1.5 mm at the end of the experiment when the initial photon flux densitiy was restored. A similar response was observed in two further experiments, whereas in three other seedlings subjected to the same light regime, the effect of irradiation on aK+,xyl was reversible. In the bottom panel of Fig. 4(b), K+ flux into the xylem (JK+,cx) and K+ export to the shoot (JK+,exp) are depicted; note that the traces are nearly identical. K+ flux fluctuated considerably with time; nevertheless, some trends could be clearly identified. It increased from a low value at 10 µmol m−2 s−1 background photon flux density (c. 2 nmol min−1 gFW−1), to 7 nmol min−1 gFW−1 at 70 µmol m−2 s−1, and further to 9 nmol min−1 gFW−1 at 300 µmol m−2 s−1. Subsequent lowering of the light intensity to 70 µmol m−2 s−1 led to a pronounced drop of JK+,cx to 2.5 nmol min−1 gFW−1. When the light was reduced further, JK+,cx returned, after some fluctuations, to the initial value. This time course suggests that net K+ flux into the xylem was positively correlated with photon flux density, roughly in parallel with the volume flow. In Fig. 4(c), steady-state JK+,cx values are plotted against Jv,r for five independent experiments (including the one shown in Fig. 4a,b). In all experiments JK+,cx increased with Jv,r, (shaded areas) irrespective of whether the ‘background K+ flow’ extrapolated at zero volume flow was high (c. 15 nmol min−1 gFW−1; two plants) or very low (three plants). Positive correlation of K+and volume flow into the xylem became obvious despite some hysteresis with ascending and descending irradiation; correlation coefficients ranged between r = 0.845 and 0.995 for the individual experiments. When JK+,cx was plotted against Px (Fig. 4d), data scattered considerably, but a negative correlation between these parameters could be established. With one exception, this correlation was weaker than that between JK+,cx and Jv,r; correlation coefficients ranged between r = −0.772 and −0.996 for the individual experiments. It should also be noted that in one further experiment of this type that is not shown here, no pronounced dependence of JK+,cx on Px and Jv,r was observed. However, other experiments that comprised only two light regimes (not shown) rendered results that were in agreement with those shown in Fig. 4(c,d).

Hence, while showing that K+ loading into the root xylem was strongly affected by changes in environmental conditions, this experiment was not conclusive with respect to the parameters that control net radial K+ flux in the root.

As pointed out above, the experimental approach introduced here was also suitable to assess the radial hydraulic conductance of the root, LP,r, in intact maize seedlings under truly physiological conditions. Figure 5(a) shows steady-state values for the hydrostatic driving force (PatmosPx) and the osmotic driving force (–σrext–πx)) for radial water transport in the root measured on a maize seedling at a varying light regime (same experiment as documented in Fig. 4a,b). It is obvious that both forces were opposite in sign, but that the hydrostatic driving force that mediates net water uptake was by far the dominating one. In Fig. 5(b), steady-state values of LP,r calculated according to Eqn 5 are plotted as a function of photon flux density for the same experiment. Interestingly, LP,r increased from 8 µl min−1 MPa−1 gFW−1 at background light irradiation (10 µmol m−2 s−1) to 56 µl min−1 gFW−1 at maximum light irradiation (300 µmol m−2 s−1) and decreased when the photon flux density was lowered again. This result suggested that LP,r increased with the radial volume flow (cf. Fig. 4b); positive correlation was indeed confirmed by plotting these parameters against each other for five independent experiments (Fig. 5c). Correlation coefficients for the individual experiments ranged between r = 0.851 and 0.996. When LP,r was re-plotted against xylem pressure (Fig. 5d), the quality of the correlation decreased (with one exception) and scattering was more pronounced. Correlation coefficients ranged from −0.781 to −0.957.

Figure 5.

Root hydraulic conductance of intact maize seedlings as a function of driving forces and radial volume flow in a varying light regime. In (a), steady-state gradients of hydrostatic pressure (PatmosPx; shaded columns) and osmotic pressure (–σrext–πxyl); black columns) that drive radial volume flow between bath and root xylem are plotted for a sequence of photon flux densities imposed on the same seedling as shown in Fig. 4(a,b). From radial volume flow (cf. Fig. 4b, top trace) and the driving forces, the root hydraulic conductance was calculated according to Eqn 5; in (b), steady-state LP,r values are plotted against photon flux density for the same experiment. In (c) and (d), LP,r is shown as a function of radial volume flow and xylem pressure, respectively, for this (triangles apex down) as well as four other seedlings. The sequences of light regimes for these experiments were (irradiance in µmol m−2 s−1): 10–70–300–70–10 (circles, triangles apex down, half-shaded circles); 10–70–300 (squares); 10–70–300–70–300 (diamonds). The index ‘1’ indicates the starting value for the respective experiment.

In summary, the experiment clearly showed nonlinear coupling between radial volume flow and driving forces (which is expressed by a variable hydraulic conductance), but, once again, from the results no conclusion could be drawn concerning the mechanism underlying this nonlinearity.

Root K+ and water transport parameters with a constant light regime but varying osmotic conditions

In order to decide whether K+ flux into the root xylem and the radial hydraulic conductance depended on hydrostatic pressure (gradients) or rather on radial volume flow, another experiment was performed that involved changes to the osmotic pressure of the ambient medium at a constant irradiation (70 µmol m−2 s−1). Part of such an experiment is shown in Fig. 6. Here, the effect of decreasing the osmolality of the external medium is demonstrated. Initially, the root was immersed in standard medium that had been adjusted to a final osmolality of 182 mosmol kg−1 by adding mannitol, a sugar that is ‘physiologically inert’ and is not taken up by the root or metabolized at a timescale of minutes. Subsequently, the bath was substituted by mannitol-free standard medium using bath perfusion, starting at t = 184 min. After perfusion, the osmolality of the bath had decreased to 31 mosmol. This decrease in the osmotic pressure of the bath was associated with an increase in xylem pressure from –0.088 MPa to +0.013 MPa, that is, by 0.101 MPa (Fig. 6a). Dividing the hydrostatic pressure change by the difference in osmotic pressure (151 mosmol kg−1, corresponding to a pressure drop of 0.378 MPa), the radial reflection coefficient of the root, σr, could be calculated (Schneider et al., 1997b) as an operational parameter; for this experiment the value was 0.27. Figure 6b shows the time courses of Jv,r, aK+,xyl and JK+,cx for the same experiment. Note that during bath perfusion, flow could not be monitored for technical reasons. Jv,r (upper trace) was more variable at the high osmolality but hardly responded to the osmolality change. Mean values were 34 ± 7 nmol min−1 gFW−1 before and 43 ± 4 nmol min−1 gFW−1 after bath perfusion (P > 0.05 with Student's t-test). The trace below shows the K+ activity, which increased slightly from 2.4 to 2.9 mm upon reduction of bath osmolality. JK+,cx was also hardly affected by the strong increase in xylem pressure. This result is clearly at variance with a short-term regulation of K+ loading into the xylem by xylem pressure and related parameters, such as turgor pressure of adjacent cells, or hydrostatic pressure gradients (see the Discussion section), at least in the pressure range tested here. Taking into account the data shown in Fig. 4, it appears that JK+,cx responds to changes in Jv,r rather than Px. Qualitatively similar results were obtained in two further independent experiments.

Figure 6.

Changing the osmotic bath pressure at a constant light irradiation strongly affects Px, but has only minor effects on Jv,r, aK+,xyl and JK+,cx. Here, a 17-d-old maize (Zea mays) seedling was exposed to a reduction of the osmotic bath pressure from 182 to 31 mosmol kg−1 (see bars attached to the time axes in a and b; arrows mark the time required for bath perfusion) at a photon flux density of 70 µmol m−2 s−1. As a consequence, the xylem pressure increased from −0.088 to +0.013 MPa (Fig. 6a), but the radial volume flow (Jv,r) was hardly affected after bath perfusion (b, top trace; note that during perfusion, volume flow could not be recorded for technical reasons). Xylem K+ activity (aK+,xyl) increased only slightly from 2.4 to 2.9 mm (b, middle trace). Hence, K+ flux into the xylem (JK+,cx), which was calculated from Jv,r and aK+,xyl according to Eqn 4, also remained nearly constant before and after bath perfusion (b, bottom trace). For more details and interpretation, see the text.

The same experiment also offered the possibility to test whether LP,r was under control of xylem pressure (or of the radial hydrostatic pressure gradient). At the high external osmolality, the hydrostatic pressure gradient and the effective osmotic pressure gradient were 0.188 (cf. Fig. 6a) and –0.130 MPa, respectively. After washing the mannitol away, the hydrostatic pressure gradient reduced to 0.087 MPa and the effective osmotic gradient decreased to −0.014 MPa. By contrast, the effective driving force for radial water flow into the xylem (ΔPr–σrΔπr) remained constant upon removal of mannitol; that is, the decrease in effective osmotic pressure was outbalanced by the concomitant increase in xylem pressure. (Numerically, a slight increase in the net driving force is obtained, since the calculation of the radial reflection coefficient from osmotically induced xylem pressure changes implies that the osmotic pressure of the xylem sap remains constant; slight dilution or up-concentration of the xylem sap by water-shifting is not taken into account.) Since Jv,r also hardly responded to the change in osmotic bath pressure (Fig. 6b), the steady-state hydraulic conductance likewise remained largely unaffected. Despite the strong increase in xylem pressure (Fig. 6a), LP,r decreased only slightly from 227 to 193 µl min−1 MPa−1 gFW−1. Two further independent experiments supported these findings. From these data, it can be concluded that the pronounced changes in LP,r with irradiation depicted in Fig. 5(b) were not the result of a regulatory function of (cellular or xylem) hydrostatic pressure, but rather were associated with the increase in Jv,r (Fig. 5c).

Discussion

In this paper, radial transport of K+into the root xylem as well as radial volume (water) flow in the root were investigated using a new experimental approach that allows the simultaneous study of these transport processes and their driving forces in intact, transpiring maize seedlings. This was achieved by combining a gravimetric method for measuring root water uptake with recordings of xylem pressure, trans-root potential (not shown here) and xylem K+ activity in a single root xylem vessel by using the multifunctional xylem K+ probe.

On the basis of these measurements and a few general assumptions (see the Materials and Methods and Theoretical considerations sections), radial net flux of K+ into the xylem and the radial hydraulic conductance of the root could be calculated per root fresh weight. Data for both parameters varied strongly among individual plants irrespective of age and developmental status; in Table 1, they are compared with values published previously on detopped maize seedlings. The hydraulic conductance determined on intact seedlings roughly matched values reported by Melkonian et al. (2004) for this parameter under control conditions (in comparison to seedlings exposed to chilling stress); they were about one order of magnitude lower than those communicated by Miller (1980, 1985a,b). For the radial net flux of K+ into the root xylem, results reported by Davis & Higinbotham (1976) fall in the range of values found in this study, but Engels & Marschner (1992) and Engels et al. (1992) found flow rates that were somewhat higher. These authors manipulated root transport by varying the temperature at the shoot base. In the experiments reported here, the driving forces for radial transport were adjusted by changing the photon flux density and/or the osmotic pressure of the bath medium by adding mannitol. A major finding was that K+ flux into the xylem vessels was strongly affected by Jv,r (i.e. by root water uptake), whereas xylem pressure had only a minor (if any) effect on K+ release into the xylem. Radial hydraulic conductance was also largely pressure-independent, but increased with root water uptake.

Table 1.  Literature survey of the K+ flux rate into the xylem (JK+,cx) and the radial hydraulic conductance (LP,r) in maize (Zea mays)
 JK+,cx (nmol  min−1 gFw−1)LP,r (µl min−1  MPa−1 gFW−1)Method
  • a

    Strongly depending on radial volume flow.

This paper1–80a5–100aXylem K+ probe, gravimetric measurement of flow; intact seedlings
Miller (1985b) 300–360Exudation from pressurized roots of detopped seedlings
Miller (1980) 246Exudation from pressurized roots of detopped seedlings
Melkonian et al. (2004) 60Exudation from pressurized roots of detopped seedlings
Davis & Higinbotham (1976)16 Tracer flux measurements (42K+), detopped seedlings
Engels et al. (1992)161 Root pressure exudation, detopped seedlings
Engels & Marschner (1992)99 Root pressure exudation, detopped seedlings

Dependence of K+transport into the root xylem on volume flow has been observed previously (Lopushinski, 1964), but these investigations relied entirely on work performed on excised roots. The relevance of those data to the physiology of the intact, transpiring plants appeared to be dubious, since removal of the shoot was shown to have a strong effect on root transport properties (see the Introduction). Moreover, conventional techniques are based on adjustment of radial volume flow by imposing overpressure on the root system (Passioura, 1988); this may lead to an infiltration of air-filled spaces, thus completely changing root hydraulic properties and transport pathways. The present study confirms strong interaction of radial K+ flux and water flow for the root of intact maize plants. This could be interpreted in terms of a coupling of water and K+ flow, most likely in the apoplastic cell wall pores, following the classical framework of the thermodynamics of irreversible processes (Dainty, 1963; Zimmermann & Steudle, 1978). Low root reflection coefficients (ranging from 0.15 to 0.7 under the conditions applied here; cf. Schneider et al., 1997a,b) would be in agreement with this explanation (see also Steudle & Peterson, 1998). However, Schneider et al. (1997a,b) also found that the root responded to a stepwise change in external osmotic pressure (by adding either NaCl or mannitol), almost like a perfect osmometer (reflection coefficient close to unity), when irradiation was increased further (up to c. 700 µmol m−2 s−1), providing evidence against a significant apoplastic bypass for water and solute transport in the intact plant. Low reflection coefficients observed at a low-light regime were attributed to concentration-polarization effects in the root tissue (Schneider et al., 1997b). This is supported by reports in the literature, indicating that the Casparian band is a very effective barrier against radial apoplastic flow of ions (Clarkson, 1993), and that ions enter the stele predominantly via the symplastic route. Additional evidence in favor of this interpretation of the data came in the form of measurements of the radial electrical resistance of the root using the xylem pressure-potential probe (Wegner & Zimmermann, 1998); very high values, c. 1 MΩ, were reported for maize roots. Rather than being mediated by an apoplastic bypass, K+ flux into the xylem is predominantly mediated by K+ outward rectifying channels in the plasma membrane of xylem parenchyma cells that border on the root xylem vessels (KORCs; Wegner & Raschke, 1994; Wegner & De Boer, 1997); the gene encoding for this channel has been cloned in Arabidopsis and was assigned the name SKOR (Gaymard et al., 1998). Using a pharmacological approach, Wegner & De Boer (1997) demonstrated for barley roots that c. 95% of the K+ transported to the shoot via the transpiration stream is released by this route. An increase in the rate of K+ release to the xylem with volume flow can readily be explained by the observation that, in roots of transpiring maize plants, a radial symplastic K+ gradient is generated (Läuchli et al., 1971) that is associated with an accumulation of K+ in xylem parenchyma cells. The symplastic gradient of K+ (and of other solutes, as reflected by the cellular osmotic pressure) immediately breaks down upon cutting off the shoot (Rygol et al., 1993) and is thus absent in excised roots (Dunlop & Bowling, 1971). This may be result, at least in part, of a K+ depletion at the sites of water uptake (rhizodermis and cortex) by a sweep-away effect and of a retention of K+ at the inner symplast/apoplast boundary in the stele when water is passing through these cells. Retention of K+ in xylem parenchyma cells when a stream of water passes through these cells will lead to an increased driving force for K+ release across the plasma membrane, and will thus enhance the rate of K+ efflux from the cells. Admittedly, this explanation somewhat oversimplifies the highly regulated process of K+ release into the xylem sap. Recently, Liu et al. (2006) reported that SKOR channel activity is enhanced by an increase in the intracellular K+ concentration in a voltage-independent manner. From this effect described by Liu et al. (2006), an even more pronounced coupling between K+ accumulation in xylem parenchyma cells and the K+ efflux rate from these cells is expected. Gating of K+ outward rectifiers in this cell type was also shown to be strongly affected by the extracellular (xylem) K+ concentration (Roberts & Tester, 1995; Wegner & De Boer, 1997, 1999). This led Wegner & De Boer (1997) to speculate that K+ recirculated via the phloem could increase K+ concentration in the xylem and, in turn, reduce K+ release by xylem parenchyma cells into the xylem sap. However, the present data did not provide any evidence to support this hypothesis; a negative correlation between K+ flux and xylem K+ concentration would have been predicted from the model, but these parameters were found to be only weakly correlated, if at all (data not shown). Apparently, changes in xylem K+ activity were not pronounced enough to affect rates of K+ release into the xylem under the conditions applied here. Further experimental work is required to elucidate precisely the mechanisms of coupling between water and K+ transport in roots.

The new experimental approach applied in this study also allowed us to investigate the dependence of radial volume flow on the driving forces, that is, hydrostatic and (effective) osmotic pressure gradients between the xylem and the ambient medium, in roots of intact seedlings. It was found that the relationship was nonlinear, that is, an increase in the hydrostatic pressure gradient had a more pronounced effect on volume flow than expected for a linear relationship. This is expressed by a variable radial hydraulic conductance, LP,r, of the root. A nonlinear relationship between radial water flow and hydrostatic pressure gradient has been observed before in various species, but was ascribed to osmotic effects associated with a progressive dilution of the xylem sap (Fiscus, 1977; Passioura, 1988; but see also Emery & Salon, 2002). This explanation can be excluded here, since changes in the osmotic pressure of the xylem sap resulting from a dilution effect could be estimated by monitoring the xylem K+ activity. Furthermore, by changing the bath osmotic pressure, it could be shown that LP,r in intact maize seedlings depended on the radial volume flow, but not on xylem pressure (nor on turgor pressure of xylem parenchyma cells that is tightly coupled to xylem pressure; Zimmermann et al., 2004). This is at variance with data obtained on excised maize roots (Miller, 1985a,b; Zimmermann & Steudle, 1998), which exhibited constant hydraulic conductance at varying hydrostatic pressure gradients (i.e. a linear relationship between pressure and flow). These opposing results highlight the necessity of performing work on intact plants to obtain information on root water transport that is of relevance to the physiology of the whole organism. So far, different explanations have been put forward to explain variability of LP,r (Weatherley, 1982). Steudle & Peterson (1998), referring to data obtained by root pressurization, suggested that air entrapped in intercellular spaces is removed when a steeper hydrostatic gradient is imposed, thus increasing the cross-sectional area for apoplastic water flow. However, this explanation does not apply to the experiments reported in this paper; it does not seem likely that an increase in xylem tension is associated with a re-filling of apoplastic air spaces. In view of the finding that mercuric chloride, an inhibitor of aquaporins, strongly inhibits root water uptake, it seems more likely that transcellular water permeability is a variable parameter, most likely the result of the regulation of aquaporins. Emery & Salon (2002) found a sigmoidal dependence of volume flow on the applied pressure in detached root systems of tomato and soybean with saturation of flow at elevated pressures. This was explained by properties of water flow through aquaporins. Emery & Salon (2002) and Beaudette et al. (2007) argued that Eqn 5, which deals with water flow across roots in terms of a process of ordinary diffusion, does not describe this process adequately but should be replaced by a formalism based on Michaelis–Menten kinetics, reflecting transport properties of aquaporins. In maize roots, different aquaporins have been identified and characterized on a molecular scale (Chaumont et al., 2001), and some information on the regulation of different aquaporins in maize roots is available from their expression pattern, which has been studied in detail (Hachez et al., 2006). Moreover, mechanisms of plant aquaporin gating have recently been unraveled using X-ray diffraction analysis of crystallized proteins (Törnrodt-Horsefield et al., 2006). The data presented here argue against a dependence of aquaporin gating on hydrostatic pressure (i.e. the turgor pressure of root cortex or xylem parenchyma cells), since root hydraulic conductance remained more or less constant when hydrostatic pressure in the xylem was lowered by adding mannitol to the bath. Rather, gating of aquaporins may be affected by the flow rate in such a way that a greater fraction of water channels remains in the open state when volume flow increases (see also Emery & Salon, 2002). Wan et al. (2004), using the turgor pressure probe to study the hydraulic conductance of maize root cortex cells, arrived at the opposite conclusion. They applied pressure pulses of different magnitude and found that the time constant of the subsequent pressure relaxation increased with the size of the pressure jump, independent of the polarity of the pulse; this was interpreted in terms of a closure of aquaporins induced by volume flow. However, the experimental approach used by Wan et al. (2004) is quite nonphysiological; plant cells will never experience a sudden drop or increase in pressure of 0.2 MPa or more. Another concern is that the hydraulic conductance is likely to be underestimated when calculated from pressure relaxations as a consequence of concentration-polarization effects, which will increase with the magnitude of the pressure pulse (and the volume flow induced by it). Recently, Knipfer et al. (2007) revisited possible errors in the determination of LP,r of excised corn roots when water flow is forced through the root tissue with the root pressure probe, either by applying pressure pulses or by performing pressure clamp experiments. There is currently some debate as to the interpretation of multiphasic relaxations of pressure or volume with regard to the contribution of concentration-polarization effects and pressure propagation in the xylem (Bramley et al., 2007; Knipfer et al., 2007). These potential sources of error can be totally avoided when LP,r is calculated from truly steady-state volume flow and pressure data, as has been done in this study.

We have proposed molecular mechanisms here for the observed dependence of JK+,cx and LP,r on radial volume flow that need further testing, for example, by using transgenic plants that lack certain aquaporins and/or K+ channels. Further work is also required to refine the experimental approach presented here, for example, to separate apoplastic and symplastic pathways of water and ion transport, and to adjust the technique for use in potted plants in order to establish a model system that allows the simulation of field conditions. Generally speaking, the technique outlined here offers the opportunity to generate a database for a mechanistic rather than a purely empirical modeling of transport processes in intact, transpiring plants that is highly relevant for application in agronomy and ecology.

Acknowledgements

This work was supported by grants of the Deutsche Forschungsgemeinschaft to LHW and UZ. We wish to thank Dr Sergey Shabala at the University of Tasmania, Hobart, for critical reading of the manuscript.

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