Sensitivity of root system to low temperature appears to be associated with the root hydraulic properties through aquaporin activity

Scientia Horticulturae 105 (2005) 1 11 www.elsevier.com/locate/scihorti Sensitivity of root system to low temperature appears to be associated with the root hydraulic properties through aquaporin activity S.H. Lee, G.C. Chung * Agricultural Plant Stress Research Center, Division of Applied Plant Science, College of Agriculture and Life Sciences, Chonnam National University, Gwangju 500-757, South Korea Received 30 September 2003; received in revised form 26 July 2004; accepted 5 January 2005 Abstract The sensitivity of cucumber and figleaf gourd root system to low temperature was evaluated in terms of water transport capacity. Plants were grown hydroponically and low solution temperature was imposed for different periods. Low temperature severely reduced xylem sap transport in cucumber but not in figleaf gourd to the same extent. Root pressure generated in figleaf gourd during the night under low temperature was able to transport xylem sap but there was little transport in cucumber when measured with heat-balance sap-flow gauge. The different capability in generation of root pressure in two species upon exposure to low temperature was confirmed by measuring the root pressure with excised root system that was related to the activity of plasma membrane H + -ATPase. Measurement of half-times of water exchange and hydraulic conductivity of cortical cells with cell pressure probe showed the insensitivity of figleaf gourd cells to low temperature. External application of HgCl 2, which is known to inhibit the bulk water transport through aquaporins, also showed an insensitivity of individual figleaf gourd cortical cells as well as entire root system while marked reduction in hydraulic conductivity was observed in cucumber plants. Therefore, it is concluded that the activity (open/closed state) of aquaporin may be associated with the rate of water uptake and the sensitivity of root systems to low temperature. # 2005 Elsevier B.V. All rights reserved. Keywords: Aquaporin; Hydraulic conductivity; Low temperature; Root pressure; Turgor pressure; Water uptake * Corresponding author. Tel.: +82 62 530 2063; fax: +82 62 530 0190. E-mail address: gcchung@chonnam.ac.kr (G.C. Chung). 0304-4238/$ see front matter # 2005 Elsevier B.V. All rights reserved. doi:10.1016/j.scienta.2005.01.013

2 S.H. Lee, G.C. Chung / Scientia Horticulturae 105 (2005) 1 11 1. Introduction The grafting between Cucumis (as a scion) and Cucurbita (as a rootstock) species has become a common horticultural practice for successful cucumber (Cucumis sativus L.) production during winter (Lee, 1994), the reason being that Cucurbita species grow well at relatively low root temperature. The most common and rapid visible consequence of low root temperature is wilting of the leaves. It is, therefore, intriguing to note that low temperature as well as other major abiotic stresses result in water-deficit stress as pointed out by Holmberg and Bülow (1998). This clearly implies that the tolerance to low root temperature may be related to ability to control water balance in the plant. Wilting that occurs during low temperature has been attributed to changes in membrane permeability, which allows water and soluble materials to leak out into the intercellular space, where the water is lost through evaporation. In this regard, it has long been known that low temperature-induced wilting is caused by stomatal opening during exposure to low temperature (Lyons, 1973). However, root systems often provide additional resistance to water flow, i.e. there is evidence that the water balance in the plant can be regulated by a capacity of roots to take up water (Steudle, 2000). We have previously shown that the H + -ATPase activity of root plasma membrane is closely involved in low root temperature-tolerant characteristics of plants lowering the osmotic potential for subsequent water uptake (Ahn et al., 1999a, 2000). The hypothesis has been that the low root temperature-tolerant plants are likely to possess the ability of water uptake at low root temperature through active proton pumping. As pointed out by Ahn et al. (1999b), the optimum root temperature for figleaf gourd roots is around 15 8C, a temperature that causes severe growth inhibition of the shoot as well as photosynthesis of leaves. The stomatal resistance was not affected until the root temperature was lowered to 8 8C. We propose that the physiological characteristics of low root temperature-tolerant species may be such that these plants have some ability to absorb water at low root temperature. To confirm this hypothesis, the root and cell pressure probes developed by Steudle (2000) and his co-workers (for example, Steudle and Peterson, 1998) have been adapted to measure the parameters governing root hydraulics (root hydraulic conductivity) as well as those governing root solute transport (solute permeability and reflection coefficient). The hydraulic conductivity (Lp r ) of the root is an important factor in determining the water supply to the root and shoot. According to Steudle (2000), the Lp r is a rather complex parameter that depends on root anatomy, mechanism of water flow, interaction between water and solute flow and activity of water channels. As for the influence of low temperature on root anatomy, we have already reported that the cortical cells of cucumber roots undergo dramatic ultrastructural changes only after 15 min of exposure to 8 8C, which corresponds to rapid drop in the root pressure (P r )(Lee et al., 2002). To measure the P r with the root pressure probe, the excised root is connected to the system with the aid of a silicon seal so that the P r is built up inside the system. The water flow across the root can be induced by either changing the pressure in the pressure chamber with the aid of a metal rod or exchanging the medium in the perfusion chamber with solutions of different osmotic pressures. We suggest that root hydraulics may be an important parameter, determining the uptake of water, i.e. higher Lp r values, under low root temperature conditions. In this regard, we have already shown that the P r in cucumber

S.H. Lee, G.C. Chung / Scientia Horticulturae 105 (2005) 1 11 3 rapidly dropped as soon as the root temperature was lowered below 25 8C, indicating that the radial transport of water was greatly reduced (Lee et al., 2004b). While data from root hydraulic analysis provided some evidence, the ability of figleaf gourd root system to transport water at low temperature is not yet understood. Recently, Lee et al. (2004b) proposed that there are two different temperature-sensitive effects, one on water permeability of root cell membranes and the other one on P r. Based on the larger reduction in the osmotic component in response to the exposure of the root system to low temperature, it was concluded that water permeability of cucumber root cell membranes was related to changes in the activity (open/closed state) of water channels (aquaporins) that were effectively inhibited at low temperature. While long-term adaptation of plants to drought induces anatomical alterations such as formation of Casparian band in the exodermis, short-term adaptation can be mostly accounted for by changes in cell membrane permeability (Javot and Maurel, 2002). Aquaporins, channel forming integral membrane protein, appear to play central role in root water uptake. In the present study, sensitivity/tolerance of cucumber and figleaf gourd to low temperature was examined in terms of root hydraulics. Xylem sap-flow with heat-balance sap-flow gauge and P r with root pressure probe, respectively, were measured upon exposure to low temperature. Cell turgor pressure was determined in response to the application of HgCl 2, which is known to close aquaporins. It is concluded that the insensitivity of figleaf gourd to low temperature and HgCl 2 indicates that low temperature-induced hydraulic conductivity depends on the activity (open/closed state) of aquaporins. 2. Materials and methods 2.1. Plant materials and culture Cucumber (C. sativus L.) and figleaf gourd (Cucurbita ficifolia Bouché) seeds were germinated in an incubator (25 8C) on moist paper towels in plastic trays containing 0.5 mm CaSO 4. After germination, they were transferred to containers with complete nutrient solution (Cooper, 1975) in a growth cabinet (Conviron, Canada). Temperature was 20 8C at night and 25 8C during the day. The photoperiod was 12 h, the irradiance about 380 mmol m 2 s 1 and the relative humidity 60%. Nutrient solution was regularly replaced to avoid excessive depletion of any particular ion, and oxygen was supplied by vigorous aeration of nutrient solution. Solution temperature was regulated by coolers and heaters in the containers. 2.2. Sap-flow measurements A heat-balance sap-flow gauge (Dynagage Flow 32, Dynamax, Houston, TX) was used to measure sap-flow through the main stems of approximately 25-day-old figleaf gourd and 40-day-old cucumber, respectively. Five to 10 mm stem-gauges covered with weather shield were attached to the stem just above the cotyledons. Gauge signals, recorded with a data logger (21X; Campbell Scientific, Logan, UT), were collected every 1 min and averaged over 15 min. When stable readings from the gauge were obtained, treatment of

4 S.H. Lee, G.C. Chung / Scientia Horticulturae 105 (2005) 1 11 low root temperature was imposed on three plants. Entire experiments were repeated twice and average values were plotted. 2.3. Measurement of stomatal resistance Cucumber and figleaf gourd plants were grown hydroponically for 25 40 days and different solution temperatures were given for 1 h at 8 8C. Stomatal resistance was measured with a steady-state porometer (LI-1600, LI-COR, USA) clamped onto the second fully expanded leaf until a steady state was recorded. 2.4. Root pressure measurements Roots were excised close to their base and tightly fixed to a root pressure probe (Steudle, 2000). Inner diameters of seals were adapted to diameters of the basal part of root system and adjusted by a screw. The probe was filled with silicon oil and water so that a meniscus formed in the measuring capillary that was used as a point of reference. Once stable P r developed within 2 3 h, P r was measured with a pressure transducer. Hydrostatic relaxation was performed by changing the xylem pressure (moving the metal rod in the probe). Transient changes in pressure were followed which allowed root Lp r to be calculated from half-times of pressure relaxation (T1=2 w ), according to (Steudle, 1993): lnð2þ T w 1=2 ¼ A r DP r DV s Lp r DP r /DV s (in MPa m 3 ) is the elastic coefficient of the measuring system, V s the water volume of the system and A r is the effective surface area of the root. The ratio DP r /DV s was measured by inducing step changes in the volume by moving the metal rod and recording the resulting changes in P r (DP r ). 2.5. Cell pressure measurements The cell pressure probe was used to measure turgor pressure (P) and hydraulic conductivity (L P ) of root cortical cells from 14-day-old seedlings according to the method described by Zimmermann et al. (2000). An oil-filled (type AS4; Wacker, München, Gemany) capillary, with outer tip diameter 7 10 mm, was attached to pressure chamber which contained an electronic transducer. The probe was fixed on a micromanipulator (Leitz, Germany), which allowed insertion of a capillary tip into cortical cells. Cells located at distances of 50 70 mm from the tip and at 3 5 cortical cell layers were punctured. By moving the meniscus formed between oil and sap after the cell was punctured, a stationary turgor pressure (P o ) was recorded. The T1=2 w induced by rapid change in P o was determined from the P o relaxation curves. The cell elastic modulus (e in MPa) was evaluated by changing the cell volume (DV) and recording the resulting changes in cell turgor pressure (DP): e ¼ V DP DV

Cell volumes were estimated from cross-sections and longitudinal sections assuming the shape of cells to be cylindrical. Using a micrometer screw, a metal rod could be moved backward and forward to change the position of the meniscus. Hydrostatic relaxations were performed by moving the meniscus to a new position and keeping it there until a steady pressure was attained. From T1=2 w of the relaxation, the Lp was calculated, as previously described (Azaizeh et al., 1992): Lp ¼ V S.H. Lee, G.C. Chung / Scientia Horticulturae 105 (2005) 1 11 5 lnð2þ AT w 1=2 ðe þ pi o Þ where A denotes the cell surface area. The osmotic pressure of the cell p i owas calculated from P o and from the osmotic pressure of the medium (p o o ). Fig. 1. Effect of root temperature on the xylem sap transport in cucumber (A) and figleaf gourd (B) plants grown either at 25 8C (&) or low temperature of 8 8C (&). Out of six plants grown at 25 8C, three plants were exposed to low temperature from the night of day 1 as indicated by arrow. Signals were collected every 1 min and averaged over 15 min to give hourly mean values. Entire experiments were repeated twice and average values were plotted.

6 S.H. Lee, G.C. Chung / Scientia Horticulturae 105 (2005) 1 11 3. Results Forty- and 25-day-old cucumber and figleaf gourd plants, respectively, transported approximately 25 35 g of xylem sap per hour during the photoperiod (Fig. 1). The amount of xylem sap transported from root to shoot increased from 35 to 70 g h 1 in cucumber and from 25 to 45 g h 1 in figleaf gourd, respectively, during 5 days growth under favorable root temperature. During the night when there was little transpiration, figleaf gourd plants transported about 8 9 gh 1 of sap while cucumber transported about 4 6 gh 1 only during later days of measurement. The amount of xylem sap transported in cucumber plants progressively decreased as roots were exposed to low temperature whereas there was no such reduction in figleaf gourd plants. Cucumber plants exposed to low temperature transported virtually no sap during the dark period. In contrast, figleaf gourd was able to transport (4 5 gh 1 ) during the night even though the root system was exposed to low temperature. Cucumber and figleaf gourd plants of 25 40 days old were treated with 8 8C for 1 h and stomatal resistance was measured. As shown in Fig. 2, low temperature greatly increased stomatal resistances in both species (Fig. 2). The clear differences in root hydraulics between two species can be seen in Table 1 in which P r and T1=2 w were measured at low temperatures. At 25 8C, P r ranged between 0.1 and 0.2 MPa. These values are similar to other plants studied (Steudle and Peterson, 1998). However, there was a strong and immediate effect of temperature on P r of cucumber plants, which was reduced from 0.15 MPa at 25 8C to nearly 0 MPa at 8 8C. T1=2 w gradually increased from 2.8 s at 25 to 11.5 s at 8 8C. Raising temperature back to 25 8C did not result in a complete recovery of P r as well as T1=2 w. Reduction in P r was also noticed by low temperature in figleaf gourd, but positive P r was maintained at 8 8C. Only a small increase in T1=2 w upon exposure to low temperature occurred, i.e. it increased by a factor of 4.6 in Fig. 2. Effect of root temperature on the stomatal resistance in cucumber and figleaf gourd leaves. Low temperature of 8 8C was given for 1 h and resistance was measured with porometer. Means S.D. are shown (n = 10 plants).

S.H. Lee, G.C. Chung / Scientia Horticulturae 105 (2005) 1 11 7 Table 1 Effect of low temperature on the P r and T1=2 w in cucumber and figleaf gourd roots Temperature (8C) Cucumber Figleaf gourd P r (MPa) T1=2 w (s) P r (MPa) T1=2 w (s) Decreasing temperature 25 0.15 0.015 2.8 0.30 0.16 0.015 3.2 0.02 19 0.10 0.010 4.8 0.41 0.12 0.012 3.2 0.02 14 0.03 0.001 6.3 0.58 0.09 0.010 4.0 0.03 10 0.02 0.001 8.9 0.72 0.07 0.010 4.1 0.03 8 0.01 0.001 11.5 0.97 0.06 0.010 4.1 0.03 Increasing temperature 10 0.01 0.001 10.5 0.95 0.07 0.010 3.9 0.03 14 0.03 0.001 8.2 0.64 0.11 0.010 3.5 0.02 19 0.08 0.010 7.4 0.45 0.14 0.014 2.4 0.02 25 0.15 0.015 5.3 0.48 0.17 0.015 2.2 0.02 Plants were grown hydroponically and entire root systems were cut and connected to the root pressure probe with the aid of a silicon seal, so that the root pressure developed in the root cylinder could be measured. Then, attached root systems were exposed to each temperature for 1 h followed by gradual decrease or increase for a period of 30 min to attain next level of temperature. Values are means S.D. (n = 8 roots). cucumber but only by a factor of 1.3 in figleaf gourd. In addition, raising temperature back to 25 8C from 8 8C resulted in a complete recovery of P r and T1=2 w. Once the root system was connected to the root pressure probe, P r between 0.1 and 0.2 MPa was established in both species (Fig. 3). As soon as stable P r was established, nutrient solution containing 50 mm HgCl 2 was flushed over the root system for 30 40 min and resulting T1=2 w and Lp r were calculated. Clear differences in response to applied HgCl 2 exist between two species in that T1=2 w in cucumber increased by a factor of 2.5 3 and hence Lp r decreased by same factor. However, there were no alterations in T1=2 w in figleaf gourd plants. Such insensitivity of figleaf gourd and sensitivity of cucumber in response to HgCl 2 were also confirmed by cell pressure probe as shown in Fig. 4. Different concentrations of HgCl 2 were flushed over the cell to measure the Lp. As shown in Fig. 4, cucumber cell showed a rapid reduction in Lp, as low as 25 mm HgCl 2, whereas figleaf gourd maintained high conductivity even at 100 mm HgCl 2. 4. Discussion In the absence of transpiration during the night, or when excised root is used to measure P r with a root pressure probe as in the present study, steady P r is given by (Steudle, 1993): P r ¼ s sr RTJ sr P sr Here J sr is the rate of active nutrient uptake by a root in mol m 2 s 1 which should be largely related to the action of H + -ATPase that provides the energy (electrochemical gradient) by an extrusion of protons from the root. s sr is the root reflection coefficient and P sr represents the permeability of the root to nutrient ions in m s 1. Therefore, P r increases with increasing active nutrient ion pumping and reflection coefficient. It decreases with increasing permeability of the root to nutrient ions, i.e. with increasing leakiness of roots to

8 S.H. Lee, G.C. Chung / Scientia Horticulturae 105 (2005) 1 11 Fig. 3. Effect of mercury chloride on the half-times of water exchange in cucumber (A) and figleaf gourd roots (B). Once a stationary root pressure was obtained, nutrient solution containing 50 mm HgCl 2 was flushed over entire root and resulting changes of half-times of water exchange and of hydraulic conductivity were measured. ions. Therefore, the activity of H + -ATPase is an important determinant of maintaining P r by which sap transport occurs during the night when there is no transpiration. We have already reported that H + -ATPase activity of cucumber is sensitive to low temperature while that of figleaf gourd is tolerant (Ahn et al., 1999a, 2000). It was concluded that low temperature effect on root hydraulics is attributable to factors such as proton pumping by H + -ATPase (Lee et al., 2004b). The present study confirms such conclusion by measuring the sap transport with intact plant during the night (Fig. 1) and direct measurement of P r with root pressure probe (Table 1). Furthermore, species differences in root hydraulics upon exposure to low temperature become evident. Stomatal resistances in both species were greatly increased when the root system was exposed to low temperature (Fig. 2). In plants, the highest resistance in soil plant air-continuum occurs in stomata where output of water is controlled and hence it appears that the control of output was effectively operating in both species in the present study. However, the function of input of water appears different between two species because figleaf gourd was able to maintain sap-flow

S.H. Lee, G.C. Chung / Scientia Horticulturae 105 (2005) 1 11 9 Fig. 4. Effect of mercury chloride on the hydraulic conductivity of cucumber (A) and figleaf gourd (B) cortical cells. Once a stationary turgor pressure was obtained after puncturing the cells, nutrient solutions with different concentrations of HgCl 2 were flushed over the cells and resulting changes of half-times of water exchange and of hydraulic conductivity were measured. Since the values of hydraulic conductivity were different for each cells, control values at 0 mm HgCl 2 were measured first and different concentrations of HgCl 2 were flushed. Means S.D. are shown (n = 5 cells). Hydraulic conductivity of cucumber cells was significantly reduced in all concentrations of HgCl 2 applied whereas figleaf gourd cells were not. while cucumber showed drastic reduction. In addition, figleaf gourd was able to transport sap during the night when transpirational force was absent. The ability of figleaf gourd to maintain positive P r, when measured with root pressure probe, is a representative characteristic in terms of low temperature-tolerance while cucumber root lost entire P r at low temperature. Water transport protein, aquaporins, are membrane proteins that belong to the major intrinsic protein found in nearly all living organisms and mercury is known to reversibly inhibit the bulk water transport by binding to sulfhydryl group of water channels (Wan and Zwiazek, 1999). Hence, 50 90% of reduction in water transport occurs by the application

10 S.H. Lee, G.C. Chung / Scientia Horticulturae 105 (2005) 1 11 of mercury depending on the species (Javot and Maurel, 2002). Most of the evidence suggests that the cell-to-cell pathway through aquaporins plays a major role in overall water uptake (Tyerman et al., 2002). Insensitivity of figleaf gourd root as well as a cell upon the addition of HgCl 2 indicates that the figleaf gourd either lacks sulfhydryl group in aquaporins, or have different aquaporin families. Plants are known to have a particularly large number of membrane intrinsic protein homologues (Javot and Maurel, 2002). However, biological significance of mercury insensitivity in plants is not clear yet (Baiges et al., 2002). According to Lee and Ahn (2004), there were no significant differences in root anatomy between cucumber and figleaf gourd and hence permeation of mercurial solution through the root system should not differ between two species. Alternatively, the activity (open/closed state) of aquaporin was not sensitive to the concentration of HgCl 2 used in the present study. Moreover, exposing the root system to 8 8C for 2 days did not affect aquaporin activity if cell turgor was measured at 25 8C just after the termination of the low temperature treatment. These findings suggest that figleaf gourd aquaporins may not close and able to maintain the activity of water transport at low temperature. The strategy adapted by plants when confronted with water stress, low temperatureinduced water stress in the present study, may not be same throughout plant species. There are reports that aquaporin gene expression is induced by water stress, which indicates the necessity of facilitated water transport to meet the requirement of the shoot (Yamada et al., 1997). However, plants evolved in a place where water-shortage prevails need to conserve water by reducing aquaporin activity (Oshima et al., 2001). Therefore, lower expression of aquaporin genes may be beneficial to preserve water. In the present study where short-term response to low temperature was studied, the maintenance of P r in figleaf gourd root during the night when no transpiration occurred and insensitivity to HgCl 2 up to high concentration in root system as well as a cell clearly indicates that the activity of aquaporin determines the water uptake and temperature sensitivity of the root system. Gating mechanism of the aquaporins is not known but enhanced production of hydrogen peroxide in the vicinity of the plasma membrane has been suggested as a short-term response to close aquaporins (Lee et al., 2004a). Fennel and Markhart (1998) also suggested that root acclimation to low temperature may be related to changes in the activity of water channel protein. Contrasting results obtained from low temperature-sensitive cucumber plants support such conclusion. Studies on the detailed mechanism involved in aquaporin opening/closing are in progress. Acknowledgement This work was supported by Korea Science and Engineering Foundation through Agricultural Plant Stress Research Center (APSRC, R11-2001-003104-0) of Chonnam National University, Gwangju, Korea. References Ahn, S.J., Im, Y.J., Chung, G.C., Cho, B.H., 1999a. Inducible expression of plasmamembrane H + -ATPase in the roots of figleaf gourd plants under chilling root temperature. Physiol. Plant 106, 35 40.

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