Root Acclimation to Chilling Root Temperature in Figleaf Gourd Plants: The Role of Root Plasma Membrane H+-ATPase

Sung Ju Ahn, Yang Ju Im, Gap Chae Chung, and Baik Ho Cho

Department of Horticulture and Dept. of Agricultural Biology, Institute of Biotechnology, College of Agriculture, Chonnam National University, Kwangju 500-757, Korea

Additional index words. ATPase, Cucurbita ficifolia, figleaf gourd, H+-pumping, leaf temperature, root acclimation

Abstract. Figleaf gourd (Cucurbita ficifolia Bouché) plants were grown under different root temperatures and the acclimation process of roots was investigated. Conditioning of roots by gradual decrease (2 oC·h­1) of root temperature to 6 oC considerably lowered the electrolyte leakage of the leaves compared to nonconditioned roots and no wilting of the leaves was observed. Root temperature of 6 oC was critical in terms of the activation of H+-ATPase (enzyme classification = in the plasma membrane of root and such activation was abolished by the addition of cycloheximide in the root medium. Increased activity disappeared when roots were transferred back to 20 oC for 1 day. H+-transport rate of the vesicles were also activated by low root temperature. Leaf temperature measured with infrared thermometer confirmed the involvement of H+-ATPase in root acclimation process.

This work was supported by the Korea Science and Engineering Foundation (95-0402-08-01-3) and in part by a special grant from Chonnam National University to G.C.C. (1995). Critical review by C. Larsson in the Department of Plant Biochemistry, Lund University, Sweden, is greatly appreciated.

1Corresponding author G.C. Chung;



The use of Cucurbita species as rootstocks for Cucumis species has been a generalized practice for successful cucumber production (Lee, 1994). Cucurbita species grow well under relatively low root temperature, a characteristic that is particularly useful when grafted cucumber plants are cultivated during winter (Tachibana, 1987). Physiological basis of higher absorption rate of water and nutrients by Cucurbita species at low root temperature has not, however, been fully understood although increased cytochrome respiration (Tachibana, 1989) and the activation of H+-ATPase in the plasma membrane of root (Choi et al., 1995) have been suggested.

The acclimation process of roots in response to low root temperature has not attracted much attention despite the vital role of the root in absorbing water and nutrients. Zhao et al. (1995) demonstrated that conditioning of jack pine seedlings to low temperature increased survival rate accompanied by the changes in two root-plasma mem

brane-associated enzymes, H+-ATPase and NADH-dependent ferricyanide reductase. For herbaceous plants of subtropical origin, the immediate response is wilting of the leaves when plants are transferred from optimum to a low root temperature. Adaptation of roots, if any, to low root temperature requires, therefore, mechanism by which normal water absorption should proceed. Water uptake is maintained through a water potential gradient (y) between plant cells and soil solution, and y is established by the H+ electrochemical potential gradient created for solute transport across the roots (Sze, 1985). If root acclimation process does exist, it should involve the H+-ATPase activity for solute transport and resultant establishment of y.

In most studies where the response of plasma membrane H+-ATPase has been examined, the whole plant was exposed to a designated temperature (Prasad et al., 1994; Zhou et al., 1994). Under these circumstances, the alteration of H+-ATPase activity becomes unclear due to the interdependence of leaf and root. For example, Binzel (1995), demonstrated that H+-ATPase in tomato leaves increased to higher levels in response to NaCl treatment of the roots. This is a clear indication of the close interdependence of root and


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shoot. Therefore, the present study was undertaken to find out how the plasma membrane H+-ATPase activities of roots of figleaf gourd plants responded to low root temperatures while aerial temperatures were maintained optimum.

Materials and methods

Plant materials. Figleaf gourd (Cucurbita ficifolia) seeds were sown in plastic trays in vermiculite in a temperature-controlled greenhouse (18 oC at night and 28 oC in the daytime) with bottom heat (30 oC). After germination, seedlings were transferred to 20 L containers that contained an aerated nutrient solution (Cooper, 1975). Seedlings were grown for a different number of days until the start of low-temperature treatment. Oxygen was supplied by continuous aeration of nutrient solution. The solution was replaced at weekly intervals to avoid the excessive depletion of any particular ion. Different solution temperatures were maintained using coolers and heaters in the containers.

Measurements of electrolyte leakage. Leaves from figleaf gourd plants grown under different root temperatures were excised and washed twice with tap water and deionized water. After drying with filter paper, leaves were cut into small pieces (2 cm2) and placed in flasks with 60 mL deionized water. Flasks were shaken on a rotary shaker at 26 oC for 2 h (Reyes and Jennings, 1994). Electrical conductivity of the solution was measured with a conductivity meter (model 4070, Ele, England). Values were expressed on a fresh weight basis. Three independent experiments were conducted with five replications.

Preparation of plasma membranes. Plasma membranes were isolated following the method of Palmgren et al. (1990). The roots were ground in the presence of insoluble polyvinylpyrrolidone with a homogenizing buffer which consisted of 330 mm sucrose, 50 mm Mops-BTP (pH 7.5), 5 mm EDTA, 5 mm DTT, 0.5 mm PMSF, 0.2% (w/v) BSA (Sigma, Protease free), and 0.2% casein. The homogenate was filtered through four layers of cheesecloth and centrifuged at 10,000 gn for 15 min. The supernatant was again centrifuged at 30,000 gn for 2 h and the resulting precipitate was suspended with a glass homogenizer in a suspen

sion buffer that consisted of 330 mm sucrose, 5 mm potassium phosphate (pH 7.8), 5 mm KCl, 1 mm DTT, and 0.1 mm EDTA. The homogenate was loaded on a two phase system, which contained 6.5% (W/W) Dextran T500, 6.5% (W/W) polyethylene glycol 3350, 330 mm sucrose, 5 mm potassium phosphate (pH 7.8), 5 mm KCl, 1 mm DTT, and 0.1 mm EDTA. After the three-step batch procedure, the resulting upper phase was diluted with suspension buffer and centrifuged at 100,000 gn for 1 h. The plasma membranes were pelleted and suspended with a medium containing 5 mm potassium phosphate (pH 7.8), 5 mm KCl, 1 mm DTT, and 0.1 mm EDTA. H+-ATPase activities were measured immediately after preparation, and the samples were otherwise stored at ­80 oC. All the preparations were carried out strictly under 2 to 5 oC.

Measurement of h+-atpase activity. H+-ATPase (EC 3. 6. 1. 35) activity was measured in an assay system containing Mops-BTP (pH 6.5), 2.5 mm MgSO4, 50 mm KCl, 0.02% Triton X-100, 2.5 mm Tris-ATP, and an appropriate amount of enzyme. The reaction was carried out for 30 min at 37 oC. Five-hundred microliters of 5% cold TCA and 2 mL of 0.1 m sodium acetate was added to mixture and centrifuged at 3,000 rpm for 10 min, and 0.3 mL of 1% ascorbate containing CuSO4 and 0.3 mL of 1% ammonium molybdate in 0.025 m H2SO4 was added again. After incubation at 30 oC for 10 min, the liberated Pi was measured with spectrophotometer (model UV-1201, Shimadzu, Japan) at 720 nm. Protein was determined by the method of Lowry et al. (1951).

Measurement of h+ pumping. The highly purified right-side-out plasma membrane vesicles were frozen and thawed to produce a mixture of inside-out and right-side-out vesicles. Typically, portions of 1 mL were frozen in liquid N2 and thawed in water at 20 oC a total of four times. Then proton uptake into the vesicles was monitored as the absorbance decrease at 495 nm of the pH probe acridine orange (Palmgren et al., 1990). The assay medium was essentially consisted of 20 mm acridine orange, 2 mm ATP-BTP (pH 7.0), 140 mm KCl, 1 mm EDTA, 1 mm DTT, 1 mg·mL­1 BSA (essentially fatty acid free), 50 µm gramicidine, and 50 µg·mL­1 plasma membrane protein in a total vol

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Measurement of leaf temperature. Before the root system was harvested for the preparation of plasma membrane, leaf temperatures were measured with an infrared thermometer (model 510, Everest Interscience) with a 15o field of view. Temperature of second leaf from apex was obtained by hand-holding the thermometer directly over the leaf at 20 cm above the leaf surface.

Results and discussion

Effects of low-temperature conditioning of roots on the electrolyte leakage of leaves. Responses of leaves when low root temperature is imposed may be found by measuring the electrolyte leakage (Jennings and Saltveit, 1994). When figleaf gourd plants were transferred directly from 20 to 6 oC root temperature (nonconditioned), leaves started to wilt within one h. Electrolyte leakage from the leaves was rapidly increased up to 3 h of transferring the plants (Figure 1). However, if the root temperature was gradually decreased (2 oC·h­1) to 6 oC (conditioned), no wilting of the leaves occurred and the extent of electrolyte leakage was not altered. Wilting of the leaves was not only dependent on the root temperature but also on the aerial temperature. Wilting was partially recovered when aerial tempera

Figure 1. Effects of low-temperature conditioning of roots on the electrolyte leakage of leaves. Root temperature was gradually lowered (2 oC·h­1) from 20 to 6 oC (l), or plants grown under 20 oC of root temperature were directly transferred to 6 oC of root temperature (m) while control plant were grown under 20 oC (q). Values are the means of five replications with three independent experiments.

ume of 2 mL. After 5 min preincubation at 20 oC, the reaction was initiated by the addition of ATP. The rate of H+ accumulation was estimated from the initial slope of absorbance quenching (495) of acridine orange.

Protein extraction. About 400 mg (fresh weight) of the plant root tissues was powdered under liquid nitrogen, and then dissolved in 150 µL of cold O'Farrell (Laemmli, 1970) lysis buffer (50 mm Tris-Cl, pH 6.8, 20% NP-40, 10 mm b-mercaptoethanol, 8 mm leupeptin). The homogenate was clarified several times by centrifugation (13,000 rpm) in a microfuge for 10 min at 4 oC. Protein content was determined by the method of Bradford (1976) using bovine serum albumin as a standard.

Discontinuous sds-page. Equal amounts of proteins (50 µg) were loaded on 12% polyacrylamide slab gels (5% stacking gel) and subjected to electrophoresis under constant current of 150 V per gel. Gels were fixed and stained with 0.1% (w/v) Coomassie blue prepared in 40% (V/V) methanol and 10% (w/v) acetic acid. They were destained in 40% methanol and 10% acetic acid.

Table 1. The effect of different root temperatures on the activities of H+-ATPase in the plasma membrane of figleaf gourd root systems. Plants were grown at each temperature (±0.5 oC) for 1 day and plasma membranes were isolated by two-phase partitioning. Measurements were repeated 3 to 4 times without significant differences and standard errors were <10% of the given values.

Temp ATPase activity

(oC) (µmol Pi/mg protein per h)

22 31.2 ± 3.2

12 29.7 ± 1.7

10 28.3 ± 0.7

8 25.6 ± 0.9

6 56.4 ± 3.2

2 20.6 ± 2.5

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shown). The optimal pH of H+-ATPase activity was investigated between pH 4 to 9 at intervals of 0.5. The pH curves were almost symmetrical and the highest activity was obtained at pH 6.0 (data not shown), which has been observed by others for cucumber plants (Memon et al., 1987) and for rice (Ros et al., 1991). There was a large increase in H+-ATPase activity with the addition of up to 0.02% of Triton X-100 (data not shown), an indication of sealed and right-side-out vesicles in the membrane preparations (Sandstrom et al., 1987). Taken together, the preparations of the root plasma membrane in the figleaf gourd are of high purity.

Different root temperatures were given to figleaf gourd root systems for 24 h and H+-ATPase activities were measured (Table 1). It is quite clear that temperatures of 6 oC for figleaf gourd roots were critical in terms of the activation of H+-ATPase activities. The activation of the H+-ATPase of figleaf gourd roots has been reported (Choi et al., 1995), but a rather long period (13 days) was necessary when roots were grown at a 12 oC root temperature. Root temperatures of 6 oC for figleaf gourd were imposed for various hours as indicated in Figure 2, and H+-ATPase activities of root plasma membranes were measured. The assay was repeated a number of times and the activation of H+-ATPase occurred sometimes as early as after 2 to 4 h of low-temperature treatment. Repeated experiments confirmed that consistent and highest activation occurred after 24 h and the low root temperature-induced increase in the H+-ATPase activity was abolished by the addition of 50 µm cycloheximide in the root medium (Figure 2), suggesting that the activation of H+-ATPase in the plasma membrane of figleaf gourd root system requires the synthesis of protein and mRNA. Increased activity disappeared when roots were transferred back to 20 oC for 1 day (Figure 3), and electrophoretic results also confirmed induction and disappearance of 100 kD protein band by

Figure 2. Effects of low root temperature on the plasma membrane H+-ATPase activity of figleaf gourd roots. Root temperatures of 6 oC (l) or 20 oC (q) were given for different hours and plasma membranes were isolated by two phase partitioning.

ture became low in the afternoon, a fact that could be seen by a decrease in electrolyte leakage after 3 h of transferring plants as shown in Figure 1. No further increase in electrolyte leakage was observed thereafter, an indication of a possible acclimation process when aerial temperature was low. Electrolyte leakages of the leaves from nonconditioned roots were consistently higher than conditioned roots. Measurement of the electrolyte leakage from roots after low-temperature treatment may indicate the sensitivity of roots (Reyes and Jennings, 1994). Electrolyte leakage from the conditioned and nonconditioned roots did not, however, differ in the present experiment (data not shown).

Effects of low root temperature on the h+-atpase activities. The method of the plasma membrane isolated from cucumber roots (Memon et al., 1987) was used to purify the plasma membrane in the figleaf gourd roots since they belong to same family. H+-ATPase activities of root plasma membranes were compared in the presence and absence of various inhibitors. Vanadate, which is a specific inhibitor of plasma membrane ATPase activity, inhibited 90% of its activity, while nitrate and azide achieved little inhibition (data not

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for solute uptake, a water potential gradient will be established enabling roots to absorb water. Transpiration, as a consequence of the established water potential gradient, may proceed normally, lowering leaf temperature due to high heat of vaporization of water on the leaf surface. Wilting is an early response of plants, as pointed out earlier, when they are grown at low root temperatures. Once acclimation is accomplished, leaves regain full turgidity. If water absorption proceeds normally after cold acclimation, leaf temperatures may not differ between low- and optimal-temperature-treated plants due to the normal transpiration process.

The temperature difference between leaf and air was read using an infrared thermometer, and the values obtained from control plants were sub

Figure 3. Induction and disappearance of the plasma membrane H+-ATPase activity in figleaf gourd roots. After exposure to 6 oC for 1 day (l), roots were transferred back to 20 oC for 1 day. Control root was grown at 20 oC (q) continuously.

Figure 4. SDS-PAGE profile of total protein fractions from root tissues. (A) C = control at 20 oC ; CA = cold acclimation for 1 day at 6 oC; DA = deacclimation after 1 day at 20 oC. (B) C = control at 20 oC; CA = cold acclimation for 1 day at 6 oC; HS = heat shock for 1 day at 35 oC; SA = salt stress for 1 day in 200 mm NaCl, Proteins were solubilized in O'Farrell buffer, electrophoresed on a 12% polyacrylamide gel and stained with Coomassie blue. Standard proteins are shown on the left with open arrows. Full arrows indicate induced bands of 100-kD protein in roots.

temperature treatment (Figure 4A). Interestingly, induction of 100 kD protein was specific to low root temperature (Figure 4B).

Effect of low root temperature on the h+ pumping activities. Figleaf gourd plants were grown under 6 oC of root temperature and the H+ transport rate of plasma membranes was measured as described in Materials and Methods. Gramicidine at 50 µm dissipated the pH and quenching of acridine orange did not occur without ATP, suggesting that vesicles prepared from figleaf gourd roots are not leaky to proton and can transport H+ into the vesicles in an ATP-dependent manner (Figure 5). The H+ transport activity of vesicles prepared from the low-temperature-treated roots at day 1 was more than twice that of control roots, which is consistent with H+-ATPase activities as shown in Figure 2. As an adaptive mechanism, the increase in H+ transport rate of tonoplast vesicles has been reported when barley plants were subjected to NaCl stress (DuPont, 1992; Matsumoto and Chung, 1988).

Effects of low root temperature on leaf temperature. Assuming that the H+ concentration gradient created by H+-ATPase was used

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since the H+ concentration gradient created by H+-ATPase is used for solute uptake (Sze, 1985). Reports on the activity of root plasma membrane H+-ATPase at different root temperatures are , however, scarce. The survival of low-temperature-conditioned jack pine seedlings was markedly increased following exposure to freezing temperatures, which coincided with recovery of root plasma membrane H+-ATPase activity (Zhao et al., 1995). Choi et al. (1995) also demonstrated that figleaf gourd root systems exhibited an higher ATPase activity after low-temperature acclimation. The present study showed the activations of H+-ATPase (Figure 2) and the H+ pumping (Figure 5), due to low-temperature treatment and the abolishment of such activation by cycloheximide

Figure 5. Proton transport rate of plasma membrane vesicles (50 µg proteins) prepared from figleaf gourd roots grown under 20 oC (C) or 6 oC (L) for 1 day. When ATP or Mg was not included, no proton transport was observed. Absorbance was reversed by the addition of gramicidin (G).

tracted from treated plants. The subtracted values were then plotted against the ratio of H+-ATPase activity of low-temperature-treated and control plants as shown in Figure 6. When values along the x-axis are greater than one, they indicate an activation of H+-ATPase influenced by low root temperature. When values along the y-axis become more negative, they indicate a high leaf temperature due to restricted transpiration compared to the control plants. A clear positive relationship can be seen in Figure 6, an indication of the involvement of H+-ATPase in cold acclimation.

The acclimation processes of roots may be equally important to leaf acclimation for whole plant survival since the uptake of water and mineral nutrients is carried out by root system. H+-ATPase in root plasma membranes may, therefore, be involved in the acclimation process of roots

Figure 6. Relationships between the ratio of the H+-ATPase activity of root plasma membranes (activity of low-temperature-treated roots and control roots) and leaf temperature differences. Leaf temperature difference was calculated as the differences between leaf temperature and air temperature for control plants minus the differences between leaf temperature and air temperature for low-temperature-treated plant. 6 oC were applied for 1 day while control plants were grown at 20 oC.

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(Figure 2). The results of our study also provide indirect evidence, by measuring leaf temperature, that an H+ concentration gradient created by the H+-ATPase activity may have reestablished the water potential gradient, a fact that was inferred from wilting recovery. One proposition is that H+ concentration gradient is coupled to solute uptake, generating lower water potential in the cell. Water uptake then proceeds normally followed by transpiration, lowering leaf temperature (Figure 6). Leaf wilting was an early response when root systems were treated with low root temperature, indicating that the plants had to resort to immediate defensive measures. In this regard, it is interesting to note that H+-ATPase activities were activated as early as 2 to 4 h after low-temperature treatment (Figure 2), whereupon they recovered from leaf wilting. Figleaf gourd plants consumed >4 L of water per day after 2 months of growth, suggesting the importance of a water uptake for their normal growth. Rapid establishment of a water potential gradient in case of water shortage, resulting from low root temperature in the present experiment, would be essential for their survival. The H+-ATPase activity in the root plasma membrane may be of paramount importance in this respect.

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