Journal of Experimental Botany

JXB Advance Access originally published online on June 18, 2004
Journal of Experimental Botany 2004 55(403):1733-1741; doi:10.1093/jxb/erh189

This Article Abstract FREE Full Text (PDF) All Versions of this Article: 55/403/1733    most recent erh189v1 E-letters: Submit a response Alert me when this article is cited Alert me when E-letters are posted Alert me if a correction is posted Services Email this article to a friend Similar articles in this journal Similar articles in ISI Web of Science Similar articles in PubMed Alert me to new issues of the journal Add to My Personal Archive Download to citation manager Search for citing articles in: ISI Web of Science (17) Request Permissions Disclaimer Google Scholar Articles by Lee, S. H. Articles by Chung, G. C. Search for Related Content PubMed PubMed Citation Articles by Lee, S. H. Articles by Chung, G. C. Agricola Articles by Lee, S. H. Articles by Chung, G. C. Social Bookmarking          What's this?

Journal of Experimental Botany, Vol. 55, No. 403, © Society for Experimental Biology 2004; all rights reserved

RESEARCH PAPER

Rapid accumulation of hydrogen peroxide in cucumber roots due to exposure to low temperature appears to mediate decreases in water transport

Seong Hee Lee¹, Adya P. Singh¹ and Gap Chae Chung^1,*

¹Agricultural Plant Stress Research Center (APSRC), Division of Applied Plant Science, College of Agriculture, Chonnam National University, Gwangju 500-757, South Korea

^* To whom correspondence should be addressed. Fax: +82 62 530 0190. E-mail: gcchung{at}chonnam.ac.kr

Received 17 December 2003; Accepted 29 April 2004

	Abstract

Top Abstract Introduction Materials and methods Results Discussion References

 
Water transport across root systems of young cucumber (Cucumis sativus L.) seedlings was measured following exposure to low temperature (LT, 8–13 °C) for varying periods of time. In addition, the amount of water transported through the stems was evaluated using a heat-balance sap-flow gauge. Following LT treatment, hydrogen peroxide was localized cytochemically in root tissue by the oxidation of cerium (III) chloride. The effects of hydrogen peroxide on the hydraulic conductivity of single cells (Lp) in root tissues, and on the H⁺-ATPase activity of isolated root plasma membrane, have been worked out. Cytochemical evidence suggested that exposure of roots to LT stress caused a release of hydrogen peroxide in the millimolar range in the vicinity of plasma membranes. In response to a low root temperature (8 °C), the hydraulic conductivity of the root (Lp_r) decreased by a factor of 4, and the half-times of water exchange increased by a factor of 5–6. Decreasing root temperatures from 25–13 °C increased the half-times of water exchange in a cell by a factor of 6–9. The measurement of axial water transport with a heat-balance sap-flow gauge showed that only a small amount of water was transported when 8 °C was imposed on cucumber roots. Lp and the H⁺-ATPase activity of the isolated root plasma membrane were very sensitive to externally applied hydrogen peroxide at a concentration of 1–16 mM. These observations suggest that the accumulation of hydrogen peroxide appears to mediate decreases in water transport in cucumber roots under low temperature.

Key words: Cucumis sativus, cytochemical localization, H⁺-ATPase, hydraulic conductivity, hydrogen peroxide, low temperature, plasma membrane, root pressure, turgor pressure, water channel

	Introduction

Top Abstract Introduction Materials and methods Results Discussion References

 
The most common visible consequence of the exposure of root system to a low temperature (LT) is the dehydration of leaves, implying that the tolerance of plants to LT may well be related to their ability to absorb water. Measurements of root hydraulic conductivity (Lp_r) in response to low root temperatures support this hypothesis (Fennel and Markhart, 1998). It has been proposed that major abiotic stresses result in water deficits (Holmberg and Bülow, 1998). However, only a few attempts have been made to relate the LT-tolerance of plants to their ability to take up water directly (Aroca et al., 2001; Vernieri et al., 2001).

Recent studies by Lee et al. (2004) have shown that root pressure (P_r) in an excised cucumber root system, as measured with the root pressure probe at 25 °C of root temperature, was 0.15–0.20 MPa. This value rapidly dropped to zero MPa, when the root temperature was gradually lowered to 8 °C for 15–20 min. The result implied that the cucumber root system was very sensitive to LT. Interestingly, figleaf gourd (Cucurbita ficifolia Bouche), a species tolerant to LT, was able to maintain positive P_r at 8 °C (Lee, 2002). In the absence of transpiration, P_r is given by (Steudle, 1994):

Here is the rate of overall active nutrient uptake by a root in mol m^–2 s^–1, which may be facilitated by a membrane H⁺-ATPase extruding protons into the root medium. _sr is the overall reflection coefficient of the root for nutrients, and P_sr represents the passive permeability of the root to nutrient ions. It is apparent that P_r increases with increasing active ion pumping and reflection coefficient. A reduction in H⁺-ATPase activity in cucumber roots due to exposure to LT has been demonstrated, and Steudle's model helps to explain the observed close relationship between the LT-induced rapid drop in P_r in cucumber and the decline in H⁺-ATPase activity (Steudle, 1994; Ahn et al., 2000; Lee et al., 2004). However, physiological and/or biochemical mechanisms related to the LT-induced reduction of H⁺-ATPase activity in the plasma membrane are not known. It has been suggested that the formation of intermolecular disulphide bonds from thiol groups caused by LT may result in the aggregation of membrane proteins (Levitt, 1980). Conformational alteration of the H⁺-ATPase due to the LT-induced phase change in the plasma membrane is also possible.

Production of reactive oxygen species (ROS), such as superoxide, hydrogen peroxide, and hydroxyl radicals, is an early response of plants to LT. In cells, the levels of these toxic species are tightly controlled by enzymes such as superoxide dismutase, peroxidases and catalase, as well as several metabolites (Noctor and Foyer, 1998). The sensitivity of the cortical cells of cucumber roots after only 15 min of exposure to LT was seen ultrastructurally (Lee et al., 2002) and this rapid response upon exposure to LT implies involvement of ROS. It is, then, reasonable to assume that ROS generated in excess of normal physiological levels due to LT stress may affect the function(s) of membrane intrinsic proteins, including H⁺-ATPases and aquaporins.

In this study, the water transport across root systems of cucumber was examined using the root pressure probe. P_r and Lp_r were measured following an exposure to LT. In addition to overall measurements of Lp_r, turgor pressure (P) and hydraulic conductivity of individual cells (Lp) were measured to work out the role of aquaporins on water conductivity under conditions of LT stress. The consequence of LT on water transport through the intact plant was assessed using the heat-balance sap-flow gauge (Steinberg et al., 1990). Hydrogen peroxide generated in root tissue was localized using CeCl₃. This was based on the possibility that H₂O₂ produced in excess of normal physiological levels under LT stress may affect the activity of H⁺-ATPases and aquaporins present in the plasma membrane. Evidence is provided that LT-induced rapid reduction in water transport in cucumber plants may be associated with the generation of high concentrations of H₂O₂ in root tissues.

	Materials and methods

Top Abstract Introduction Materials and methods Results Discussion References

 
Plant material
Seeds of cucumber (Cucumis sativus L. cv. Chung-Jang) were germinated for 4 d in an incubator (25 °C) on moist paper towels in plastic trays containing 0.5 mM CaSO₄. After germination, they were transferred to containers with complete nutrient solution (Cooper, 1975) in growth cabinet (Conviron, Canada). The temperature was 20 °C at night and 25 °C during the day. The photoperiod was 12 h, the irradiance about 300 µmol m^–2 s^–1 and the relative humidity 75%. The nutrient solution was regularly replaced to avoid excessive depletion of any particular ion, and oxygen was supplied by vigorous aeration of the nutrient solution. When used for pressure probe measurements, a 14-d-old single cucumber seedling weighed about 4 g FW (fresh weight).

Root pressure probe measurements
Roots of seedlings grown hydroponically for 14 d were excised close to their base and tightly fixed to a root pressure probe using silicon seals (Steudle, 2000). The inner diameters of the seals were adapted to the diameters of the basal parts of root systems and adjusted by screws. The probe was filled with silicon oil and water so that a meniscus formed in the measuring capillary. This meniscus was used as a point of reference. Once stable root pressure developed, normally within 3 h, the temperature of the solution bathing root systems was gradually decreased to 8 °C over a period of about 30 min. Hydrostatic relaxations were performed by changing the root 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 () according to Steudle (2000). After the measurement, the root system was disconnected to measure the surface area. Roots were stained with 0.03% (w/v) Toluidine Blue O. Photographs of the root were recorded with a digital camera (Nikon E990) attached to the microscope and processed with iMT (VT)-size image analysis program (iMTechnology, Korea). Surface areas were calculated from projected areas of roots assuming a cylindrical cross-section. The system was calibrated using metal wires of known length and diameter.

Cell pressure probe measurements
The cell pressure probe was used to measure P and Lp of 14-d-old root cortical cells (Zimmermann et al., 2000). A glass micro-capillary with an outer tip diameter of 7–10 µm was filled with silicone oil (type AS4; Wacker, München, Germany) and attached to the oil-filled pressure chamber of the probe that contained an electronic-pressure transducer. A micromanipulator (Leitz, Germany) allowed careful insertion of the tip into an individual cell by moving the probe. A stationary turgor pressure (P_o) was recorded after puncturing the cell and keeping the position of the meniscus constant for some time. Half-times of water flow equilibrium () were measured after inducing changes in P_o with the aid of a probe and waiting for water flow equilibrium. The cell-elastic modulus ( in MPa) was evaluated by changing the cell volume (V) and recording the resulting changes in cell-turgor pressure (P). Mean-cell volumes (V) were estimated from cross- and longitudinal-sections assuming a cylindrical shape of the cells. Using a micrometer screw, a metal rod could be moved backwards and forwards 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. The Lp was calculated from the half-times of the relaxation (Azaizeh et al., 1992).

Measurement of axial transport of water through main stems
When the diameter of the main stems reached approximately 5 mm, the amount of water transport affected by LT was measured with a heat-balance sap-flow gauge (Dynagage Flow 32, Dynamax, Houston, Texas). Five mm stem gauges were attached to the stem just above the cotyledons. Gauge signals (Steinberg et al., 1990), measured with a data logger (21X, Campbell Scientific, Logan, Utah), were collected every 1 min and averaged over 30 min.

Effects of H₂O₂ on the H⁺-ATPase activity in root plasma membrane and on the cell hydraulic conductivity
Plasma membranes were isolated following the method of Palmgren et al. (1990) and finally resuspended in a medium containing 5 mM K-phosphate (pH 7.8), 5 mM KCl, 1 mM DTT, and 0.1 mM EDTA. H⁺-ATPase activity, with or without various inhibitors, was measured as a marker enzyme for the purity of separation. All preparations were carried out strictly at 2–5 °C and samples were stored at –80 °C.

Plasma membranes suspended in a buffer were incubated at different concentrations of H₂O₂ at 37 °C for 30 min. H⁺-ATPase (EC 3.6.35) activity was measured in an assay system containing MOPS-BTP (pH 6.5), 2.5 mM MgSO₄, 50 mM KCl, 0.02% (w/v) Triton X-100, 2.5 mM TRIS-ATP, and an appropriate amount of enzyme from preincubated buffer. The reaction was carried out for 30 min at 37 °C. 500 µl of 5% cold TCA, and 2 ml of 0.1 M Na-acetate were added to the mixture which was then centrifuged at 2000 g for 10 min. Next, 0.3 ml of 1% ascorbate containing CuSO₄ and 0.3 ml of 1% ammonium molybdate in 0.025 M H₂SO₄ were added. After incubation at 30 °C for 10 min, liberated Pi was measured with a spectrophotometer (Model UV-1201; Shimadzu, Japan) at 720 nm. The protein content was determined using the method of Bradford (1976) and bovine serum albumin as a standard.

In order to measure the effect of H₂O₂ on cell Lp, nutrient solution containing different concentrations of H₂O₂ (0–16 mM) at 25 °C was flushed over the cell for 20 min once a stationary P was obtained. After this treatment, Lp and half-times for water flow were determined.

Cytochemical localization of H₂O₂
Four-day-old seedlings were transferred to an LT incubator set at 8 °C. The method used for cytochemical localization of H₂O₂ was slightly modified from that described by Bestwick et al. (1997). Root segments from seedlings exposed to LT for 15 min, 1 h, 8 h, and 24 h were obtained from the 5 cm region of the tip, corresponding with the mature root-hair-containing part of the root. Right after cutting, sections were placed in freshly prepared 5 mM CeCl₃ in 50 mM MOPS (3-(N-morpholino) propanesulphonic acid) and incubated for 1 h at room temperature at pH 7.2. Samples were briefly washed in 50 mM sodium cacodylate buffer, and fixed in a fixative solution consisting of 2.5% glutaraldehyde and 2% paraformaldehyde (prepared in 50 mM sodium cacodylate buffer) for 4 h at room temperature. They were then washed in the buffer and post-fixed in 1% osmium tetroxide (prepared in 50 mM sodium cacodylate buffer) for 1.5 h at room temperature. After washing with buffer, samples were dehydrated in acetone and embedded in Spurr's low viscosity resin (Spurr, 1969). Ultra-thin sections were cut with a diamond knife on a RMC MTxultramicrotome. Sections were examined with a JEOL 1010 transmission electron microscopy (TEM), unstained or after staining with uranyl acetate for 4 min. The intensity of electron-dense cerium perhydroxide deposition that was formed as a result of the reaction between CeCl₃ and H₂O₂ was determined quantitatively using an image analyser (Image-Pro Plus, Media Cybermetics, Silver Spring, MD). The images of TEM photographs were inverted, and grey levels from zero to 254 in the plasma membrane and cytoplasm in the inverted images were counted using line profile methods. The grey level in the cytoplasm of control roots was assigned to 100 and staining intensity (grey level) of H₂O₂ in the low-temperature-treated roots was counted. At least 10 TEM photographs were examined at each time from three replicate roots per treatment.

	Results

Top Abstract Introduction Materials and methods Results Discussion References

 
Usually, steady P_r was obtained 1–3 h after the excised root systems were attached to the root pressure probe. The P_r of cucumber root systems ranged between 0.15 and 0.20 MPa and gradually decreased when the root temperature was lowered, reaching zero MPa at about 8 °C (Fig. 1). Lp_r was decreased from 7.7x10^–8 m s^–1 MPa^–1 at 25 °C to 1.9x10^–8 m s^–1 MPa^–1 at 8 °C, i.e. by a factor of 4. Half-times of water exchange were 2.5–2.8 s and 5–10 s at 25 °C and 8 °C, respectively.

View larger version (14K): [in this window] [in a new window]   Fig. 1. Effect of low root temperature on the root pressure of cucumber roots. Cucumber plants were hydroponically grown for 14 d and entire root systems (n=10) were cut off and connected to the root pressure probe with the aid of a silicone seal. Once stable, root pressure developed at 25 °C, the temperature of the solution bathing the root system was gradually decreased to 8 °C for about 30 min.

 
Cell turgor pressure ranged between 0.25 and 0.30 MPa at 25 °C. This value was maintained for at least 1 h after cell puncturing (results not shown). Hence various manipulations, including the LT treatment, could be employed. However, when root temperature was lowered to below 8 °C, the half-times of water exchange could not be measured due to the unstable meniscus in the probe. Therefore, 13 °C rather than 8 °C was employed to test the effect of LT on water relations of single cells. Decreasing the temperature from 25 °C to 13 °C increased half-times of water exchange by a factor of 6 to 9, indicating a significant decrease in water permeability (Fig. 2). Turgor pressure, however, was not affected by LT.

View larger version (12K): [in this window] [in a new window]   Fig. 2. The effect of low root temperature on the cell turgor pressure. The excised root (n=12) was placed in a chamber and, once a stationary P was obtained after puncturing the cell, the nutrient solution, with different temperatures, was circulated. The half-times of the water exchange between cell and medium were recorded.

 
Forty-day-old seedlings transported a maximum of 20–30 g h^–1 water (Fig. 3). As soon as the solution temperature was lowered to 8 °C (see arrow 1 in Fig. 3), there was an immediate decrease in water transport. After 1 h at this temperature, there was virtually no water uptake. On day 2, although the solution temperature was raised back to 25 °C (see arrow 2 in Fig. 3), water transport increased only marginally. This may imply that the water transport capacity of the root system was severely affected by LT.

View larger version (16K): [in this window] [in a new window]   Fig. 3. The effect of low root temperature on the axial water transport in cucumber plants. Plants were either grown at 25 °C (filled squares), or given 8 °C (open squares) as indicated by arrow 1. On day 2, the solution temperature was raised back to 25 °C, as indicated by arrow 2. Signals were collected every 1 min from three replicate plants and averaged over 15 min to give hourly mean values.

 
The cortical cells of cucumber roots maintained a stable P for several hours after the puncturing. During this time interval, different concentrations of H₂O₂ with nutrient solution were applied. Usually, it was possible to treat the same cell with all concentrations of H₂O₂ (0, 0.5, 1, 2, 4, 8, and 16 mM). Even in the presence of the highest concentrations of H₂O₂ (8 mM or 16 mM), P was not affected (results not shown). However, there were significant differences in half-times in the presence of concentrations of more than 2 mM H₂O₂ (Table 1). The addition of 2 mM H₂O₂ increased the half-times by a factor of about 3.

View this table: [in this window] [in a new window]   Table 1. Effect of H2O2 concentrations on the half-times for water exchange and hydraulic conductivity of cucumber cortical cells

 
Plasma membrane preparations of high purity were obtained by partitioning in aqueous-polymer two-phase systems as reported previously (Ahn et al., 2000). Inhibition by vanadate, a slightly acidic pH optimum, and insensitivity to azide, molybdate, and nitrate, confirmed the purity of the preparations (results not shown). The activity of H⁺-ATPase was measured after the incubation of isolated plasma membrane with various concentrations of H₂O₂ for 30 min at 37 °C. As shown in Table 2, the activity was considerably decreased to values of less than 1 mM. However, the activity after incubation with 2 mM H₂O₂ showed somewhat higher values than at 1 mM. At 8 mM H₂O₂, the activity was only 36% compared with the control.

View this table: [in this window] [in a new window]   Table 2. Effect of H2O2 on the H+-ATPase activity of cucumber root plasma membranes

 
Cytochemical localization of H₂O₂ using the cerium chloride technique provided evidence of an enhanced cerium (IV) perhydroxide staining of cell walls and plasma membranes initially and, later, of the cytoplasm in the cortical cells as well within only 15 min of exposure of roots to LT. Although samples were also prepared for longer periods of LT exposure of up to 24 h, illustrations are presented only from the earliest period of exposure (15 min) causing a noticeable increase in the accumulation of H₂O₂.

The low magnification TEM micrographs shown in Fig. 4 provide a comparison of cortical cells from the control (unexposed material) and LT-exposed roots. In the control, the staining of cell wall and plasma membrane due to cerium (IV) perhydroxide deposits was rather weak and fairly uniform (Fig. 4A). The staining of these cell structures was noticeably more pronounced in the root that had been exposed to LT (Fig. 4B). Figures 5B and C reveal these features in greater detail, where dense granular particles can be seen along the plasma membrane and also within adjacent cell wall regions in the LT-exposed cell, but not in the control cell (Fig. 5A). Figure 5D shows the presence of dense particles also in the cortical cytoplasm, which is filled with membraneous vesicles, and showed signs of degeneration. Quantitative assessment of electron-dense deposits of cerium perhydroxide also showed greater accumulations of deposits along the plasma membrane and in cell walls in the LT-exposed cells compared with the control cells (Table 3).

View larger version (125K): [in this window] [in a new window]   Fig. 4. A transverse section through cortical cells from a control (unexposed, A) root, and low-temperature exposed (15 min) (B) roots. The staining of the cell wall and plasma membrane due to cerium perhydroxide deposits in the control is weak. However, in exposed roots, the staining is intense. TEM. Bar=1 µm.

 

View larger version (86K): [in this window] [in a new window]   Fig. 5. A high magnification view of parts of cortical cells from a control root (unexposed, A) and low temperature exposed (15 min) (B, C, D) root. Dense granular particles of cerium perhydroxide are present all along the plasma membrane and adjoining cell-wall regions (B, C) in low-temperature-exposed roots. Dense granular particles of cerium perhydroxide (D) are present throughout the cytoplasm (asterisks). TEM. Bar=500 nm.

 

View this table: [in this window] [in a new window]   Table 3. Quantitative assessment of staining with CeCl3 to reveal H2O2 accumulation in cucumber root cortical cells

 

	Discussion

Top Abstract Introduction Materials and methods Results Discussion References

 
The present study shows that LT causes a significant reduction in Lp and in the activity of plasma membrane-associated H⁺-ATPase in the root cells of cucumber seedlings. There appeared to be a relationship between this phenomenon and the elevated production of H₂O₂ as indicated by cerium perhydroxide deposits. The results also show a correspondence between a decrease in Lp_r in cucumber due to LT and H₂O₂ production. The cytochemical method employed here has been used to study the role of H₂O₂ in plant–microbe interactions (Bestwick et al., 1997), and during extension growth of plants (Rodriguez et al., 2002). Penetration of cerium chloride across a mass of tissues such as epidermal and cortical cells, as a result of the reaction between CeCl₃ and H₂O₂, is likely to take some time and, therefore, this method could not be used to study dynamic processes related to hydrogen-peroxide generation and gradient across the plasma membrane. However, it has been proved to be a useful tool in assessing major concentration differences in hydrogen peroxide at one point in time (Bestwick et al., 1997).

In cucumber roots, H₂O₂ accumulated rapidly in the cell wall and along the plasma membrane within the short period of only 15 min following exposure to LT. This correlated with adverse effects of LT on Lp_r and H⁺-ATPase. The presence of highest concentrations of H₂O₂, initially in cell walls and along the plasma membrane, suggested that both the cell wall and the plasma membrane may be the major source of H₂O₂, as suggested by Ranieri et al. (2003). Oxidative burst generating ROS is known to occur in response to both biotic (Bolwell et al., 2002) and abiotic (Vacca et al., 2004) stresses. ROS can lead to successful defence responses against biotic factors, adaptation to abiotic factors in tolerant plant species, and can cause injury to susceptible plants (Prasad et al., 1994). Although a direct relationship has yet to be demonstrated, it is likely that elevated level of H₂O₂ causes alterations in permeability properties of the plasma membrane, and its integral proteins and protein complexes may also be affected. The H₂O₂-mediated decrease in hydraulic conductivity in wheat (Triticum aestivum L.) roots in relation to salt stress has recently been reported (Ktitorova et al., 2002), and the results, provided in the present study, showing a close correlation between H₂O₂ level and Lp and H⁺-ATPase activity in isolated plasma membrane, support this view.

Considerable reductions of Lp_r occurred at concentrations of H₂O₂ as low as 2 mM. This concentration appears to be within the range that may occur in the apoplast. Prasad et al. (1994) showed that, when corn (Zea mays L.) plants were grown under favourable environmental conditions, the H₂O₂ concentration was kept low, ranging between 0.1 and 2 mM. However, during chilling stress it may rapidly increase up to 10 mM. Frahry and Schopfer (1998) also showed that in the soybean (Glycine max L.) root H₂O₂ production could be stimulated a 10-fold by exogenous NADH or NADPH. By contrast, Chara corallina internodes have been shown to tolerate up to 350 mM of H₂O₂ for quite some time (Henzler and Steudle, 2000). The examples show that, depending on the species, the resistance to tolerate toxic stresses of high H₂O₂ may be quite different. It appears that cucumber is quite sensitive, and this high sensitivity correlates with a high sensitivity to LT.

H₂O₂ accumulation in cells is likely to be a complex event, and the mechanism underlying H₂O₂-mediated reduction in the Lp in cucumber remains to be clarified. The rapid reduction of cell Lp suggests that, in addition to the H⁺-ATPases, the water-channel proteins, aquaporins, may be affected. Reduction in the activity of the H⁺-ATPases may lower cytoplasmic pH, and in turn cause a decrease in Lp. There are indications that acidic cytoplasmic pH inhibits aquaporin activity (Javot and Maurel, 2002). The sensitivity of water-channel activity to environmental conditions may affect the overall water uptake by roots and the regulation of the water balance of plants (Javot and Maurel, 2002). The activity of aquaporins in response to LT has been studied with the cell pressure probe in algae and in the root cortical cell of several species (Hertel and Steudle, 1997; Zhang and Tyerman, 1999). Although it has been shown that changes in Lp_r result from expression of aquaporins (Javot and Maurel, 2002), the very rapid response of cucumber roots to LT makes it unlikely that the response in cucumber was mediated by gene expression. Water transport is inhibited by mercurial agents such as HgCl₂, which reacts with sulphydryl groups in proteins. Therefore, the application of HgCl₂ to roots at micromolar concentrations usually closes the channels reducing hydraulic conductivity (Wan and Zwiazek, 1999). It may be that H₂O₂ at elevated levels interferes with the activity of both H⁺-ATPase and aquaporins. However, a direct relationship of the activity of aquaporins (open/closed state) to H₂O₂ remains to be experimentally demonstrated.

The gradual decrease in P_r soon after lowering of root temperature from 25 °C (Fig. 1) suggests that aquaporins may start to close below this temperature. However, since the growth of cucumber plants is not adversely affected by 20 °C of root temperature (Ahn et al., 1999), it is unlikely that aquaporins close at this temperature. It is not yet known what the critical threshold temperature is for the closure of aquaporins. In addition, there may be a reduction in the hydraulic conductivity due to the increase in the viscosity of water with lowering of temperature, which may affect water uptake, but this is usually small (Q₁₀ of 1.25; Cochard et al., 2000). Wan et al. (2001) have shown that LT significantly increased the resistance to water flow through the roots of aspen (Populus tremuloides Michx.) seedlings, and this increased resistance to water flow could not be fully explained by the corresponding increase in the viscosity of water. The results shown in Fig. 3 provide support for this view. Raising the temperature back to 25 °C after 22 h of LT treatment failed to restore the original rate of water transport through the main stem. Lee et al. (2004) have shown that the half-times and Lp_r of cucumber did not recover fully even after short-term exposure of root system to LT. Confirmation that LT affects the architecture and function of aquaporins will require further studies.

As observed here, H₂O₂ caused a reduction in the activity of H⁺-ATPases in isolated plasma membranes. However, the relationship of this to water channel activity is as yet unclear, although the extrusion of protons from cells should be linked to the uptake of nutrient ions. Zhang and Tyerman (1999) have shown that the Lp was not affected by K⁺-channel blocker tetraethylammonium at concentrations that normally block K⁺ channels. Hence, the parallel inhibition by H₂O₂ simply means that H₂O₂ treatment is less selective on different transporters. In an earlier study, it was shown that the rapid drop in P_r in response to LT was largely caused by a reduction in the activity of the plasma membrane H⁺-ATPase activity (Lee et al., 2004). In the present work, preincubation of plasma membrane vesicles with 1 mM H₂O₂ for 30 min, reduced the H⁺-ATPase, suggesting that the reduced H⁺-ATPase due to LT may be mediated by H₂O₂ production in root tissues. It is interesting to note that, in cucumber, a similar concentration of H₂O₂ affected both Lp (2 mM) and H⁺-ATPase activity (1 mM). The ways in which short-term exposure of root system to LT inhibit H⁺-ATPase are not yet known. H₂O₂ may interfere with ATP hydrolysis (Feng and Forgac, 1994) and/or disulphide exchange of oxidized glutathione with the reactive cysteine in V-ATPase (Wang and Floor, 1998). With jack pine (Pinus banksiana Lamb.) seedlings, it has been shown that the inhibition of the plasma membrane H⁺-ATPase activity by direct freeze and thaw was caused by the thiol oxidation of plasma membrane proteins (Zhao and Blumwald, 1998). Reduced glutathione prevented lipid peroxidation through a glutathione-mediated free-radical scavenging system (Kumar and Knowles, 1996). Finally, as plasmodesmata can also facilitate water movement between adjoining cells, LT-induced closure of plasmodesmata may be another reason for the observed reduction in Lp. A low (non-freezing) temperature has been shown to cause a rapid reversible closure of plasmodesmata (Holdaway-Clark et al., 2000). Rapid accumulation of H₂O₂ initially at the plasma membrane may also be relevant in this regard.

In conclusion, this study has provided evidence for a reduction in water transport in cucumber roots due to exposure to LT. LT treatment also caused an inhibition of H⁺-ATPase activity and a rapid elevation in H₂O₂ concentration, particularly in the region of the cell wall–plasma membrane interface, and there appeared to be a relationship between H₂O₂ concentration and hydraulic conductivity, and H⁺-ATPase activity. Higher concentration of H₂O₂ may react with a Fenton catalyst, such as iron ions, in the bathing medium, generating reactive hydroxyl radicals which may affect aquaporin activity. Further detailed biochemical approaches will be needed to understand the possible mechanisms underlying H₂O₂-mediated reduction in the hydraulic conductivity.

	Acknowledgements

 
This work was supported by grants from the Korea Science and Engineering Foundation (KOSEF) to the Agricultural Plant Stress Research Center (APSRC, R11-2001-09201004-0) of Chonnam National University, Korea. Adya P Singh is grateful for Brain Pool Scientist Awards from KOSEF.

	Footnotes

 
Abbreviations: LT, low temperature; Lp_r, hydraulic conductivity of root; Lp, hydraulic conductivity of a cell; P_r, root pressure; P, turgor pressure; ROS, reactive oxygen species; TEM, transmission electron microscopy

	References

Top Abstract Introduction Materials and methods Results Discussion References

 
Ahn SJ, Im YJ, Chung GC, Cho BH, Suh SR. 1999. Physiological responses of grafted cucumber leaves and rootstock roots affected by low root temperature. Scientia HorticuLTurae 81, 397–408.[CrossRef]

Ahn SJ, Im YJ, Chung GC, Seong KY, Cho BH. 2000. Sensitivity of plasma membrane H⁺-ATPase of cucumber root system in response to low root temperature. Plant Cell Reports 19, 831–835.[CrossRef]

Aroca R, Tognoni F, Irigoyen JJ, Sanchez-Diaz M, Pardossi A. 2001. Different root low temperature response to two maize genotypes differing in chilling sensitivity. Plant Physiology and Biochemistry 39, 1067–1073.[CrossRef]

Azaizeh H, Gunse B, Steudle E. 1992. Effect of NaCl and CaCl₂ on water transport across root cells of maize (Zea mays L.) seedlings. Plant Physiology 99, 886–894.[Abstract/Free Full Text]

Bestwick CS, Brown IR, Bennet MHR, Mansfield JW. 1997. Localization of hydrogen peroxide accumulation during the hypersensitive reaction of lettuce cells to Pseudomonas syringae pv phaseolicola. The Plant Cell 9, 209–221.[Abstract]

Bolwell GP, Bindschedler LV, Blee KA, Butt VS, Davies DR, Gardner SL, Gerrish C, Minibayeva F. 2002. The apoplastic oxidative burst in response to biotic stress in plants: a three-component system. Journal of Experimental Botany 53, 1367–1376.[Abstract/Free Full Text]

Bradford MM. 1976. A rapid and sensitive method for the quantification of microgram quantities of protein utilizing the principle of protein–dye binding. Analytical Biochemistry 72, 248–254.[CrossRef][ISI][Medline]

Cochard H, Martin R, Gross P, Bogeat-Triboulot MB. 2000. Temperature effects on hydraulic conductance and water relations of Quercus robur L. Journal of Experimental Botany 51, 1255–1259.[Abstract/Free Full Text]

Cooper AJ. 1975. Crop production in recirculating nutrient solution. Scientia HorticuLTurae 3, 251–258.

Feng Y, Forgac M. 1994. Inhibition of vacuolar H⁺-ATPase by disulfide bond formation between cysteine 254 and cysteine 532 in subunit A. Journal of Biological Chemistry 269, 13224–13230.[Abstract/Free Full Text]

Fennell A, Markhart AH. 1998. Rapid acclimation of root hydraulic conductivity to low temperature. Journal of Experimental Botany 49, 879–884.[Abstract/Free Full Text]

Frahry G, Schopfer P. 1998. Hydrogen peroxide production by roots and its stimulation by exogenous NADH. Physiologia Plantarum 103, 395–404.[CrossRef]

Henzler T, Steudle E. 2000. Transport and metabolic degradation of hydrogen peroxide in Chara corallina: model calculations and measurements with the pressure probe suggest transport of H₂O₂ across water channels. Journal of Experimental Botany 51, 2053–2066.[Abstract/Free Full Text]

Hertel A, Steudle E. 1997. The function of water channels in Chara: the temperature dependences of water and solute flows provides evidence for composite membrane transport and for a slippage of small organic solutes across water channel. Planta 202, 324–335.[CrossRef]

Holdaway-Clarke TL, Walker NA, Helper PK, Overall RL. 2000. Physiological elevations in cytoplasmic free calcium by cold or iron injection result in transient closure of higher plant plasmodesmata. Planta 210, 329–335.[CrossRef][ISI][Medline]

Holmberg N, Bülow L. 1998. Improving stress tolerance in plants by gene transfer. Trends in Plant Science 3, 61–66.

Javot H, Maurel C. 2002. The role of aquaporins in root water uptake. Annals of Botany 90, 301–313.[Abstract/Free Full Text]

Ktitorova IN, Skobeleva OV, Sharova EI, Ermakov EI. 2002. Hydrogen peroxide appears to mediate a decrease in hydraulic conductivity in wheat roots under salt stress. Russian Journal of Plant Physiology 49, 369–380.[CrossRef]

Kumar GNM, Knowles NR. 1996. Oxidative stress results in increased sinks for metabolic energy during ageing and sprouting of potato seed-tubers. Plant Physiology 112, 1301–1313.[Abstract]

Lee SH. 2002. Hydraulic characteristics in tomato (Lycopersicon esculentum Mill) shoot, and in cucumber (Cucumis sativus L.) and figleaf gourd (Cucurbita ficifolia Bouché) root system under environmental stresses. PhD thesis, Chonnam National University, Korea.

Lee SH, Sing AP, Chung GC, Kim YS, Kong IB. 2002. Chilling root temperature causes rapid ultrastructural changes in cortical cells of cucumber (Cucumis sativus L.) root tips. Journal of Experimental Botany 53, 2225–2237.[Abstract/Free Full Text]

Lee SH, Singh AP, Chung GC, Ahn SJ, Noh EK, Steudle E. 2004. Exposure of roots of cucumber (Cucumis sativus L.) to low temperature severely reduces root pressure, hydraulic conductivity and active transport of nutrients. Physiologia Plantarum 120, 413–420.[CrossRef][Medline]

Levitt J. 1980. Responses of plants to environmental stresses. Vol. 1. Chilling, freezing and high temperature stresses. New York, NY: Academic Press, 248–344.

Noctor G, Foyer CH. 1998. Ascorbate and glutathione: keeping active oxygen under control. Annual Review of Plant Physiology and Plant Molecular Biology 49, 249–279.[CrossRef][ISI]

Palmgren MG, Askerlund P, Fredrikson K, Widell S, Sommarin M, Larsson C. 1990. Sealed inside-out and right-side-out plasma membrane vesicles. Optimal conditions for formation and separation. Plant Physiology 92, 871–880.[Abstract/Free Full Text]

Prasad TK, Anderson MD, Martin BA, Stewart CR. 1994. Evidence for chilling-induced oxidative stress in maize seedlings and a regulatory role for hydrogen peroxide. The Plant Cell 6, 65–74.[Abstract]

Ranieri A, Castagna A, Pacini J, Baldan B, Sodi AM, Soldatini GF. 2003. Early production and scavenging of hydrogen peroxide in the apoplast of sunflower plants exposed to ozone. Journal of Experimental Botany 54, 2529–2540.[Abstract/Free Full Text]

Rodriguez AA, Grunberg KA, Taleisnik EL. 2002. Reactive oxygen species in the elongation zone of maize leaves are necessary for leaf extension. Plant Physiology 129, 1627–1632.[Abstract/Free Full Text]

Spurr AR. 1969. A low viscosity embedding medium for electron microscopy. Journal of ULTrastructure Research 26, 31–43.[CrossRef][ISI][Medline]

Steinberg SL, van Barel CHM, McFarland MJ. 1990. Improved sap flow gauge for woody and herbaceous plants. Agronomy Journal 82, 851–854.[Abstract/Free Full Text]

Steudle E. 1994. Water transport across roots. Plant and Soil 167, 79–90.[CrossRef][ISI]

Steudle E. 2000. Water uptake by plant roots: an integration of views. Plant and Soil 226, 45–56.[CrossRef][ISI]

Vacca RA, de Pinto MC, Valenti D, Passarella S, Marra E, Gara LD. 2004. Production of reactive oxygen species, alteration of cytosolic ascorbate peroxidase, and impairment of mitochondrial metabolism, are early events in heat shock-induced programmed cell death in tobacco bright-yellow 2 cells. Plant Physiology 134, 1100–1112.[Abstract/Free Full Text]

Vernieri P, Lenzi A, Figaro M, Tognoni F, Pardossi A. 2001. How the roots contribute to the ability of Phaseolus vulgaris L. to cope with chilling-induced water stress. Journal of Experimental Botany 52, 2199–2206.[Abstract/Free Full Text]

Wan XC, Zwiazek JJ. 1999. Mercuric chloride effects on root water transport in aspen seedlings. Plant Physiology 121, 939–946.[Abstract/Free Full Text]

Wan XC, Zwiazek JJ, Lieffers VJ, Landhausser SM. 2001. Hydraulic conductance in aspen (Populus tremuloides) seedlings exposed to low root temperature. Tree Physiology 21, 691–696.[ISI][Medline]

Wang Y, Floor E. 1998. Hydrogen peroxide inhibits the vacuolar H⁺-ATPase in brain synaptic vesicles at micromolar concentrations. Journal of Neurochemistry 70, 646–652.[ISI][Medline]

Zhang WH, Tyerman SD. 1999. Inhibition of water channels by HgCl₂ in intact wheat root cells. Plant Physiology 120, 849–857.[Abstract/Free Full Text]

Zhao S, Blumwald E. 1998. Changes in oxidation-reduction state and antioxidant enzymes in the roots of jack pine seedlings during cold acclimation. Physiologia Plantarum 104, 134–142.[CrossRef]

Zimmermann HM, Hartmann K, Schreiber L, Steudle E. 2000. Chemical composition of apoplastic transport barriers in relation to radial hydraulic conductivity of corn roots (Zea mays L.). Planta 210, 302–311.[CrossRef][ISI][Medline]

CiteULike    Connotea    Del.icio.us    What's this?

This article has been cited by other articles:

				M. Murai-Hatano, T. Kuwagata, J. Sakurai, H. Nonami, A. Ahamed, K. Nagasuga, T. Matsunami, K. Fukushi, M. Maeshima, and M. Okada Effect of Low Root Temperature on Hydraulic Conductivity of Rice Plants and the Possible Role of Aquaporins Plant Cell Physiol., September 1, 2008; 49(9): 1294 - 1305. [Abstract] [Full Text] [PDF]

				S. Prak, S. Hem, J. Boudet, G. Viennois, N. Sommerer, M. Rossignol, C. Maurel, and V. Santoni Multiple Phosphorylations in the C-terminal Tail of Plant Plasma Membrane Aquaporins: Role in Subcellular Trafficking of AtPIP2;1 in Response to Salt Stress Mol. Cell. Proteomics, June 1, 2008; 7(6): 1019 - 1030. [Abstract] [Full Text] [PDF]

				Y. Zhou, L. Huang, Y. Zhang, K. Shi, J. Yu, and S. Nogues Chill-Induced Decrease in Capacity of RuBP Carboxylation and Associated H2O2 Accumulation in Cucumber Leaves are Alleviated by Grafting onto Figleaf Gourd Ann. Bot., October 1, 2007; 100(4): 839 - 848. [Abstract] [Full Text] [PDF]

				S. H. Lee, G. C. Chung, and E. Steudle Gating of aquaporins by low temperature in roots of chilling-sensitive cucumber and chilling-tolerant figleaf gourd J. Exp. Bot., March 1, 2005; 56(413): 985 - 995. [Abstract] [Full Text] [PDF]

				R. Aroca, G. Amodeo, S. Fernandez-Illescas, E. M. Herman, F. Chaumont, and M. J. Chrispeels The Role of Aquaporins and Membrane Damage in Chilling and Hydrogen Peroxide Induced Changes in the Hydraulic Conductance of Maize Roots Plant Physiology, January 1, 2005; 137(1): 341 - 353. [Abstract] [Full Text] [PDF]

This Article Abstract FREE Full Text (PDF) All Versions of this Article: 55/403/1733    most recent erh189v1 E-letters: Submit a response Alert me when this article is cited Alert me when E-letters are posted Alert me if a correction is posted Services Email this article to a friend Similar articles in this journal Similar articles in ISI Web of Science Similar articles in PubMed Alert me to new issues of the journal Add to My Personal Archive Download to citation manager Search for citing articles in: ISI Web of Science (17) Request Permissions Disclaimer Google Scholar Articles by Lee, S. H. Articles by Chung, G. C. Search for Related Content PubMed PubMed Citation Articles by Lee, S. H. Articles by Chung, G. C. Agricola Articles by Lee, S. H. Articles by Chung, G. C. Social Bookmarking