JXB Advance Access originally published online on February 25, 2005
Journal of Experimental Botany 2005 56(413):985-995; doi:10.1093/jxb/eri092
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RESEARCH PAPER |
Gating of aquaporins by low temperature in roots of chilling-sensitive cucumber and chilling-tolerant figleaf gourd
1Lehrstuhl Pflanzenökologie, Universität Bayreuth, D-95440 Bayreuth, Germany
2Agricultural Plant Stress Research Center, Division of Applied Plant Science, College of Agriculture and Life Sciences, Chonnam National University, Gwangju 500-757, South Korea
* To whom correspondence should be addressed. Fax: +49 921 552564. E-mail: ernst.steudle{at}uni-bayreuth.de
Received 9 August 2004; Accepted 24 November 2004
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Abstract |
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Effects of low temperature (8 °C) on the hydraulic conductivity of young roots of a chilling-sensitive (cucumber, Cucumis sativus L.) and a chilling-resistant (figleaf gourd, Cucurbita ficifolia Bouché) crop have been measured at the levels of whole root systems (root hydraulic conductivity, Lpr) and of individual cortical cells (cell hydraulic conductivity, Lp). Exposure of roots to low temperature (LRT) for up to 6 d caused a stronger suberization of the endodermis in cucumber compared with figleaf gourd, but no development of exodermal Casparian bands in either species. Changes in anatomy after 6 d of LRT treatment corresponded with a reduction in hydrostatic root Lpr of cucumber roots by a factor of 24, and by a factor of 2 in figleaf gourd. In figleaf gourd, there was a reduction only in hydrostatic Lpr but not in osmotic Lpr suggesting that the activity of water channels was not much affected by LRT treatment in this species. Changes in cell Lp in response to chilling and recovery were similar to the root levels, although they were more intense at the root level. Activation energies (Ea) and Q10 of water flow as measured at the cell level were high in cucumber (Ea=109±13 kJ mol–1; Q10=4.8±0.7; n=6–10 cells), but small in figleaf gourd (Ea=11±2 kJ mol–1; Q10=1.2±0.1; n=6–10 cells). Roots of figleaf gourd recovered better from LRT treatment than those of cucumber. In figleaf gourd, recovery (at both the root and cell level) often resulted in Lp and Lpr values which were even bigger than the original, i.e. there was an overshoot in hydraulic conductivity. These effects were larger for osmotic (representing the cell-to-cell passage of water) than for hydrostatic Lpr. After a short-term (1 d) exposure to 8 °C followed by 1 d at 20 °C, hydrostatic Lpr of cucumber nearly recovered and that of figleaf gourd still remained higher due to the overshoot. By contrast, osmotic Lpr and cell Lp in both species remained high by a factor of 3 compared with the control, possibly due to an increased activity of water channels. After preconditioning of roots at LRT, increased hydraulic conductivity was completely inhibited by HgCl2 at both the root and cell levels. Different from figleaf gourd, recovery from chilling was not complete in cucumber after longer exposure to LRT. It is concluded that at LRT, both changes in the activity of aquaporins (AQPs) and alterations of root anatomy determine the water uptake in both species. The high temperature dependence of cell Lp in cucumber suggests conformational changes of AQPs during LRT treatment which result in channel closure and in a strong gating of AQP activity by low temperature. This mechanism is thought to be different from that in figleaf gourd where AQPs reacted in the conventional way, i.e. low temperature affected the mobility of water molecules in AQPs rather than their open/closed state, and Q10 was low.
Key words: Activation energy, aquaporins, cortical cells, cucumber, figleaf gourd, hydraulic conductivity, low root temperature
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Introduction |
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Abiotic environmental stresses such as low temperature, high salinity, drought, heat, and flooding usually result in water deficits and affect plant growth and productivity (Holmberg and Bülow, 1998
). Stresses induce an increased resistance to water uptake by roots (Fennell and Markhart, 1998; Steudle and Peterson, 1998; Lee et al., 2004a). As a consequence, leaves may wilt or even be injured as previously observed for cucumber and figleaf gourd in response to low root temperature (LRT; Ahn et al., 1999).
Root hydraulic resistances are known to be variable (Weatherley, 1982; Kramer and Boyer, 1995; Steudle and Peterson, 1998). At a given stomatal resistance, the water potential of the shoot will be regulated by the water supply from the root as controlled by its resistance. Hence, root hydraulics play a key role in the plant's water balance which, in turn, affects CO2 assimilation and productivity (Steudle, 2000). At low soil temperature, LRT-sensitive plants develop large hydraulic resistances which tend to disturb the water balance of shoots (Lee et al., 2004b). As root hydraulics is a complex parameter, its regulation is complex as well. It depends on root anatomy, the detailed flow of water across the root cylinder, interactions between water and solute flow and the activity of water channels in root cell membranes (Steudle and Peterson, 1998; Steudle, 2000, 2001).
Regulation of water flow across roots is well described by a composite transport model that identifies hydraulic resistances of components such as individual cells, as well as those of various root tissues (Steudle and Peterson, 1998). In the model, three parallel pathways for water movement in the root cylinder are considered, i.e. the apoplastic, symplastic, and transcellular pathways; the latter two comprise the cell-to-cell path. In the presence of apoplastic barriers such as exo- and endodermal Casparian bands, the amount of water that flows through the apoplast will be limited. On the other hand, the activity of water channels (aquaporins; AQPs) and the formation of suberin lamellae in the inner surface of the cell walls of root cells affect the cell-to-cell component of radial water flow.
The open/closed state of water channels, i.e. their gating, is a crucial factor in determining the capacity of roots to take up water. Water channel activity can be gated by factors such as heavy metals, high concentrations of solute or salinity, temperature, nutrient deprivation, anoxia and oxidative stresses, or by mechanical stimuli (Steudle and Tyerman, 1983; Carvajal et al., 1996; Johansson et al., 1996; Hertel and Steudle, 1997; Henzler et al., 1999; Wan and Zwiazek, 1999; Zhang and Tyerman, 1999; Henzler and Steudle, 2000; Gerbeau et al., 2002; Lee et al., 2004a). Open/closed states of AQPs are known to be under metabolic control as well (phosophorylation of AQPs; Johansson et al., 1996). Early effects of LRT are identified by the wilting of leaves. Hence, it is tempting to propose that the ability of root systems to take up water under conditions of LRT may be a crucial factor to overcome low temperature stress. Recently, Azad et al. (2004) identified temperature as an environmental stimulus that induces phosphorylation or dephosphorylation of AQPs accompanied by changes in water transport. However, more information is required to understand the gating of water channels by LRT in detail. Recently, it has been shown that cucumber roots accumulate hydrogen peroxide, which may be involved in the gating of AQPs (Lee et al., 2004a). On the other hand, the osmotic dehydration of AQPs, in terms of a cohesion/tension mechanism in Chara corallina internodes or the effects of pulses of turgor pressure (energy-injection model) on the open/closed states of corn (Zea mays L.) root cells, indicate that a mechanical gating could be involved as well (Wan et al., 2004; Ye et al., 2004a, b).
In the present study, the effects of LRT were examined on a chilling-sensitive (cucumber) and on a chilling-tolerant (figleaf gourd) species. Both are important crops in Korea. Because of its resistance to low temperature, figleaf gourd is used as a rootstock for cucumber in polythene greenhouses during the cold season (Ahn et al., 1999). In both species, the mechanisms underlying the response to LRT are poorly understood. Responses to LRT in the sensitive and tolerant species were compared in great detail at the level of individual root cells and of whole root systems of young seedlings. The pressure probe and pressure chamber techniques were used to work out detailed information about the mechanisms of water uptake such as the role of water channels.
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Materials and methods |
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Plant material
Seeds of cucumber and figleaf gourd were germinated for 2–3 d at 23 °C in the dark on filter paper soaked with tap water. After germination, seedlings were transferred to containers (5.0 l with 8 seedlings per container), with aerated 1/5 strength Cooper solution (Cooper, 1975
) in a growth chamber (day/night cycle: 12/12 h; temperature: 23/21 °C; light intensity: 300 µmol m–2 s–1 PAR). To avoid excessive depletion of any particular nutrient ion, the solution was replaced frequently. Cucumber and figleaf gourd seedlings used in the experiments were 14–20-d and 7–14-d-old, respectively, including the time for germination. Different ages were used because root growth rates of the two species were different. Using different ages, root systems were comparable in size (length of root systems: cucumber, 450–550 mm; figleaf gourd, 400–500 mm). At the base, root thickness was 1.5–2.0 mm for both species. A cooler (Haake, Berlin, Germany) was used to provide a temperature of 8 °C for a period of up to 6 d in the container in a controlled environment chamber when necessary. After LRT treatment, root growth of cucumber almost ceased, but that of figleaf gourd was slightly increased (2.5%). Following LRT treatment, the hydraulic root systems and root cells were measured at 8 °C and 20 °C. The consequences of acclimation to low temperature (preconditioning of the root system) are frequently seen when the low temperature is raised back to normal. In order to simulate such instances, different periods of optimal temperature at 20 °C were given after termination of the LRT treatments, and hydraulic conductivity of the root systems measured again after such periods.
Root anatomy and surface area of root systems
Freehand cross-sections were taken at 25, 100, and 200 mm from the root tip for both species. At 25 mm, there were no suberin lamellae in the endodermis (data not shown). At 200 mm, the endodermis was fully developed in both species and root diameters were similar (about 1.3 mm). In cucumber, the number of passage cells was smaller than in figleaf gourd. In this paper, only sections at 200 mm were compared for a better contrast.
Sections were stained for 1 h either with 0.1% berberine hemisulphate (w/v) or 0.01% fluorol yellow 088 (w/v) and subsequently for 30 min with 0.5% aniline blue (w/v) at room temperature (Brundrett et al., 1988, 1991). Sections were viewed with an epifluorescence microscope (Zeiss Standard 18, Oberkochen, Germany) using an ultraviolet filter set (excitation filter BP 365, dichroitic mirror FT 395, barrier filter LP 397, Zeiss, Oberkochen, Germany). The surface areas of the root systems were determined using a video camera and image analysing software (Skye Instruments, Llandrindod Wells, UK). For enhanced contrast, roots were stained with 0.03% toluidine blue O (w/v) prior to measurement. Surface areas were calculated from projected areas of roots assuming a cylindrical shape. The system was calibrated using metal wires of known length and diameter and was continuously checked. Surface areas of root systems from cucumber and figleaf gourd were (3.3±0.7)x10–2 m2 and (2.9±0.9)x10–2 m2, respectively.
Root exudation driven by gradients of osmotic pressure
Shoots were cut with a razor blade 30–60 mm distal to the base. With the aid of a microsyringe, xylem sap exuding from the cut surface was collected at given time intervals and weighed in Eppendorf tubes (Miyamoto et al., 2001). In the absence of hydrostatic pressure gradients, differences in osmotic pressure [
=RT(Ci–C°)] between the medium (osmotic concentration: C°) and xylem sap (osmotic concentration: Ci) drove the water uptake by the root, i.e.:
 | (1) |
sr denotes the reflection coefficient of solutes of the nutrient solution measured with the root pressure probe. To calculate root Lpr, a value of sr=0.4 and sr=0.5 were used for cucumber and figleaf gourd, respectively. Osmotic concentrations were measured using a freezing-point depression osmometer (Osmomat 030; Gonotec, Berlin, Germany). The osmotic pressure of the nutrient solution was 0.046 MPa, which is equivalent to 18 mOsmol of osmotic concentration.
Root exudation in the presence of hydrostatic pressure gradients
During these measurements, roots were enclosed in a steel chamber to apply pneumatic pressure to the root medium (Miyamoto et al., 2001). The base was fixed using silicon seals (Xantopren plus from Bayer, Leverkusen, Germany). For a tight sealing, flexible rubber material (Terostat, Heidelberg, Germany) was used, which was placed in the two halves of the seal. The pressure in the chamber was raised in steps of 0.05 MPa up to 0.3 MPa above atmospheric. With the aid of a microsyringe, exuded sap was collected at given time intervals and weighed. For a given applied gas pressure (Pgas in MPa), the volume exuded from the root system (V in m3) was plotted against time. The slopes of these relations were calculated and referred to unit root surface area, which yielded the volume flow (JVr in m3 m–2 s–1). Root Lpr was calculated from the slopes of JVr against the driving force (Pgas+sr) according to Miyamoto et al. (2001):
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In the range of values of Pgas >0.15–0.20 MPa, the effect of the osmotic term was not very pronounced, and the JVr/Pgas curves were linear to a good approximation. Measured values of were used to calculate the overall driving force in equation (1). Alternatively, Lpr was determined from plots of JVr against Pgas. Slopes of the plots were nonlinear in the range of low values of Pgas, i.e. root Lpr depended on the magnitude of water flow as found for other species (Fiscus, 1975; Rüdinger et al., 1994). Lpr was determined from the linear part of the pressure/flow curves (high pressure gradients, Pgas), where the osmotic component of the driving force was small due to dilution effects.
Cell pressure probe measurement and application of HgCl2
A cell-pressure probe was used to measure half-times of pressure relaxations 
elastic moduli (
), turgor pressure (P), and the hydraulic conductivity of individual cortical cells (cell Lp) in the primary roots of cucumber and figleaf gourd (Azaizeh et al., 1992). The probe was filled with silicon oil (type AS4; Wacker, München, Germany) up to the tip (diameter: 7–10 µm). Each excised root was vertically fixed on a metal sledge by magnets. The nutrient solution, as used for hydroponic culture of the seedlings, flowed along each root. Using a micromanipulator, the microcapillary of the cell-pressure probe was inserted into cells of the second to fourth layers of the cortex at distances from the root apex of between 50–70 mm. The position of cortical cells was estimated from the depth of the insertion of the microcapillary tip inside the root. When a cell was punctured, cell sap formed a meniscus with the oil. The meniscus was gently pushed to a position close to the surface of the root to restore the original cell volume. Cell turgor became steady within about 5 min and then hydraulic parameters of the cell were determined according to procedures which have been explained in earlier publications (Steudle, 1993; Wan et al., 2004). In order to measure cell Lp, pressure relaxations were produced and recorded on a computer. To produce relaxations, pressure pulses (P) of 0.05–0.10 MPa were applied by rapidly moving the meniscus using a micrometer screw to a new position and keeping it there until a steady pressure was attained.
From the half-times of pressure relaxations cell Lp was calculated according to equation (3) (Azaizeh et al., 1992):
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), was evaluated from cell turgor and from the osmotic pressure of the medium. The cell elastic moduli (=1–7 MPa) were measured from cell volumes and from changes in cell volumes (V), which caused changes in cell turgor (P) (Azaizeh et al., 1992):
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When cells had attained steady half-times, 50 µM of the water channel blocker HgCl2 was added to the circulating medium. The data of cell Lp were compared using a paired t-test to determine any effects on the 
(Lp) values before and after treatments using the same individual cells. Results were considered as significantly different at P 
0.05.
Temperature dependence of cell Lp
The dependencies of cell 
on temperature were measured in the range between 281 and 295 K (8–22 °C). The Q10 values given in this paper refer to the range between 283 and 293 K. In order to evaluate activation energies of water, the natural log of cell 
was plotted against the inverse of absolute temperature (Arrhenius plots), since:
 | (5) |
represents the per mole difference in enthalpy of a molecule which is necessary to overcome transport barriers during its passage across the membrane. This process is equivalent to the formation of transition complexes during a chemical reaction. It should depend on the structure of channels and on interactions between water molecules traversing them. In order to evaluate Ea from Q10 values, a modified equation (6) was used:
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Results |
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Root anatomy
When grown at optimal root temperature (20 °C), there were no visible differences in root anatomy of cucumber and figleaf gourd (Fig. 1A, E). However, exposure of root systems to LRT (8 °C) for up to 6 d resulted in clear anatomical differences between the species. In cucumber roots, LRT initiated a strong development of suberin layers in the endodermis, but did not overly affect the epidermis (Fig. 1C, G). In figleaf gourd roots, there was only a slight endodermal suberization (Fig. 1F). At a distance of 200 mm from the root apex, few endodermal passage cells (n=2–5) were observed in cucumber (Fig. 1B), but in figleaf gourd, clusters of passage cells (n=20–24) were found close to the protoxylem (Fig. 1F). In both species, LRT caused a reduction in the number of passage cells. LRT did not induce the development of hypodermal Casparian bands (exodermis). Casparian bands were found in the endodermis of both species (Fig. 1D, H).
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Steady-state root hydraulics (hydrostatic and osmotic root Lpr)
At a given applied pressure gradient (Pgas), exuded volumes of xylem sap (V) increased linearly with time (Fig. 2A). In Fig. 2B, water flow densities (JVr) are plotted against both the gas pressure applied to the root systems (Pgas) and the overall driving force Pgas+
sr. As Pgas increased, slopes (JVr) became steeper. This meant that Lpr was low at low Pgas or flow rates and increased with increasing driving force to reach a maximum value usually at pressure differences of 0.05–0.15 MPa (Fig. 2B). The resulting non-linear response of water flow to the overall driving force could have been either due to the decrease of one driving force () with increases in the other (Pgas; Fiscus, 1977), or due to an increase of the apoplastic component of water flow as compared to the cell-to-cell component (composite transport model). A linear relationship was used to calculate the hydrostatic Lpr from slopes between 0.15–0.30 MPa of applied air pressure (equation 2). At these ranges of high water flow, slopes were similar because the xylem sap was strongly diluted and the osmotic term did not contribute much to the overall driving force. Increased flow of both root systems through the application of pressure (0.20 MPa) brought about a decrease of internal concentration (Ci) below external concentration (C°).
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Data of hydraulic conductivity of root systems of cucumber and figleaf gourd grown at LRT of 8 °C for up to 6 d are summarized in Table 1. They were measured in the presence of both hydrostatic and osmotic driving forces. Osmotic Lpr at LRT could not be measured for cucumber because osmotic exudation ceased at LRT, due to a lack of active pumping of ions to the xylem (see Discussion). LRT for 1 d reduced hydrostatic Lpr in cucumber by a factor of 10, while that of figleaf gourd was reduced by a factor of only 2. At an exposure to LRT of 3–6 d, hydrostatic Lpr of cucumber decreased further, probably in response to anatomical alterations of the roots. However, further reduction in root Lpr of figleaf gourd was not observed even though plants were kept at 8 °C for up to 6 d. Root systems of both species were exposed to LRT for different periods of time before raising the temperature back to 20 °C for 5 h. This was followed by the measurement of root Lpr (Table 2). In cucumber, after 1 d exposure to LRT, the hydrostatic Lpr almost completely recovered to the control level. However, such a recovery was not observed if the duration of LRT was extended to 3 d or 6 d. Large increases in both hydrostatic (by a factor of 3) and osmotic (by a factor of 13) Lpr were observed in figleaf gourd roots, when LRT lasted for 1 d and temperature was raised back to 20 °C for 5 h of recovery. Interestingly, longer durations of LRT for 3 d or 6 d brought the hydrostatic Lpr back only to that of the control, but osmotic Lpr remained considerably higher than that of the control. When root systems of cucumber were kept at 20 °C for 1 d following 1 d at LRT, the osmotic Lpr increased by a factor of 3 of the control (Table 3). Huge increases by a factor of 10 after a recovery at 20 °C for 5 h reduced to a factor of 3, when roots of figleaf gourd were allowed to recover for a longer time (1 d at 20 °C).
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Inhibition of water channels by HgCl2: effects at the root and cell level
At the root level, addition of HgCl2 to root systems of both species grown at 20 °C caused a slight decline in root Lpr which was not significantly different from the control (unpaired t-test, P >0.05; Table 4). However, the previously observed increase in root Lpr in figleaf gourd after preconditioning roots at LRT (see Table 2) was substantially inhibited by 50 µM HgCl2 (unpaired t-test, P <0.0002; Table 4). Measurements of cell Lp after preconditioning roots for different durations showed a pattern similar to that observed at the whole root level. Cucumber cortical cells were more sensitive to LRT than those of figleaf gourd (Table 5). However, when root systems were kept at 20 °C for 1 d following LRT, cell Lp of cucumber became higher than the controls and the effects of HgCl2 treatment significant (paired t-test, P
0.05). Increases resulted from LRT with figleaf gourd cells, but these depended on the duration of temperature treatment. Sensitivity to HgCl2 after preconditioning was apparent. No responses of cucumber and figleaf gourd cell Lp were found when HgCl2was added after LRT treatment (Table 5).
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Activation energies and Q10
Figure 3 shows how temperature affected cell Lp in cucumber and figleaf gourd. Due to differences between cells, values of cell Lp were normalized, taking the Lp at 20 °C as the reference (100%). This allowed direct comparison of data from all cells. Arrhenius plots of
versus 1/T yielded activation energies (Ea) of water transport from the slopes of graphs. The Q10 values for the range between T=283 and 293 K were obtained (Fig. 3). The mean value of Q10 of Lp of the cucumber and figleaf gourd were 4.8±0.7 and 1.2±0.2, respectively (Table 6). The Ea and Q10 values of cell Lp for figleaf gourd were similar to those for the viscous flow of water (Q10=1.25; Nobel, 1999). Corresponding values of cucumber were quite high compared with most of the literature values for Lp (Pf) (Table 6; see Discussion). The values of Ea/Q10 for cucumber are on the upper edge of the range of data known so far for plant and animal cells. The data for figleaf gourd, however, are small and similar to literature data obtained for the passage of water across AQPs (see Discussion).
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in cucumber and figleaf gourd (T=281–295 K). A typical Arrhenius plot is shown for a cortex cell of cucumber and figleaf gourd. Activation energies (Ea) for the two cells shown were 102 kJ mol–1 and 13 kJ mol–1 for cucumber and figleaf gourd, respectively.
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Discussion |
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For the first time, the results of this work present a rigorous comparison of root hydraulics for a chilling-sensitive and a chilling-resistant species both at the whole root and root cell levels. Comparisons in anatomy are provided as well. The data indicate substantial differences in the changes of both the activity of AQPs and in root anatomy. The latter were largely due to the formation of apoplastic barriers during chilling stress (Casparian bands, suberin lamellae). At normal temperature, freehand cross-sections of the two species neither differed much and nor did the hydraulic data at the whole root and cell levels. Hydraulic data were in a range similar to others reported so far for roots of herbaceous plants (Steudle and Peterson, 1998
; Zimmermann and Steudle, 1998; Steudle, 2000, 2001; Javot et al., 2003; Wan et al., 2004).
When LRT was imposed, depositions of suberin occurred in epi- and endodermal cell walls of both species. However, at least in the range of up to 200 mm from the apex, the sensitive species (cucumber) developed suberin lamellae and Casparian bands much faster (within 3 d) and more intensely than figleaf gourd. The more intense expression of apoplastic barriers in cucumber should have reduced the apoplastic and cell-to-cell passages of water and contributed to the stronger reduction of overall root Lpr as compared with figleaf gourd. Perhaps, the most interesting anatomical finding was that the number of passage cells in the endodermis of cucumber was greatly reduced. This effect of LRT on root anatomy was less pronounced in figleaf gourd. Due to differences in suberization between species, the cell-to-cell (osmotic) water flow should have been more strongly affected in cucumber than in figleaf gourd. However, in the latter, LRT even increased osmotic water flow. This may be due to the fact that either the formation of suberin lamellae did not hinder the cell-to-cell flow of water very much or that the increased activity of aquaporins overcompensated for this effect, or both. In both species, LRT treatment did not cause the development of exodermal Casparian bands. This finding differs from the results of previous work on other genera (Sorghum, onion, Agave, corn), which showed that environmental stresses initiated development of Casparian bands in the exodermis and subsequently decreased root Lpr (Cruz et al., 1992; Stasovsky and Peterson, 1993; North and Nobel, 1995, 1996; Zimmermann and Steudle, 1998; Zimmermann et al., 2000). Overall, it is concluded that the formation of apoplastic barriers and the existence of passage cells contributed to the observed changes in root hydraulic conductivity along with changes in the AQPs activity.
By a factor of about 3, hydrostatic root Lpr was larger than the osmotic Lpr for both species at the control temperature. Similar substantial differences have also been found for other species. Ratios between the hydrostatic and osmotic Lpr measured so far range between unity and several hundred depending on the species (Rüdinger et al., 1994; Steudle and Peterson, 1998; Zimmermann and Steudle, 1998; Miyamoto et al., 2001). They have been explained by the composite transport model of the root in terms of a preferred apoplastic water flow in the presence of hydrostatic pressure gradients, although hydrostatic water flow involves membrane components as well (see Introduction). During osmotic water flow, the apoplastic path can be largely discounted because there are no membranes along this passage. Hence, osmotic water flow should largely represent the transmembrane component of water flow.
For cucumber roots, the decrease in hydrostatic root Lpr due to LRT was much stronger than could be explained by just a reduction in the viscosity of water flowing through the porous apoplast. From 30 °C to 10 °C, the viscosity of water increases by only 25% which cannot explain the observed responses in root Lpr. Hence, there was either a stronger effect on the cell-to-cell component or the development of apoplastic barriers during chilling contributed substantially, or both. It is interesting that figleaf gourd exhibited a much smaller decrease in hydrostatic root Lpr which corresponded to a less pronounced development of barriers. Accordingly, the recovery was better for figleaf gourd compared with cucumber. For figleaf gourd, raising the temperature back to the original even caused a transient overshoot in hydrostatic root Lpr which may be explained by a transient increase in water channel activity. This may also explain the fact that, unlike cucumber, the osmotic root Lpr of figleaf gourd was increasing rather than decreasing during LRT plus some time for recovery.
Unfortunately, responses in osmotic Lpr to LRT could not be measured for cucumber in longer terms. For this species, osmotic water flow ceased when the temperature was lowered because active ion pumping ceased and so the root pressure necessary to obtain the measurements was absent (Steudle, 1994; Lee et al., 2004b). Overall, the data shown in Tables 2 to 4
can be explained in terms of both effects on water channel activity and the development of apoplastic barriers, namely in cucumber. The latter should have been irreversible even after longer periods of recovery.
At the level of individual cells, as measured in layers two to four of the cortex, effects of LRT were most pronounced in cucumber. A reduction of Lp as large as a factor of 16 was observed after treating the roots for only 0.5–1 h at LRT. In figleaf gourd, differences were not significant. As at the root level, there was an overshoot in responses when roots of figleaf recovered at 20 °C. In both cucumber and figleaf gourd, the water channel inhibitor HgCl2 reduced cell Lp. However, effects were less pronounced than those observed with root cells of other species or with isolated cells (Steudle and Henzler, 1995; Maurel, 1997; Schütz and Tyerman, 1997; Tyerman et al., 1999; Zhang and Tyerman, 1999; Javot et al., 2003). This may be due to the fact that HgCl2 would have to diffuse across suberized root tissue to reach the cells in layers two to four. It is plausible though that changes were relatively small when cell Lp was already low following LRT. This indicated that an AQP closure was involved in the reduction of cell Lp during LRT. Closed channels no longer reacted to the mercurial.
At first sight, the high Q10 value of 4.8 for cucumber at the root cell level may not fit into the picture of a substantial contribution of AQP activity. Usually, Q10 values for water transport across AQPs are much smaller (Hertel and Steudle, 1997; Maurel, 1997; Tyerman et al., 1999, 2002; Javot and Maurel, 2002). They are similar or even smaller than that for the viscous flow of water (Q10=1.25). This has been interpreted by the fact that, during its passage through AQPs, water molecules see a surrounding similar to that in bulk water. The high Q10 value for the cell Lp of cucumber roots is explained by a high temperature dependence for conformational changes of AQPs in this species which, in turn, results in channel closure at LRT and low cell Lp. Figleaf gourd, on the other hand, exhibits the usual low Q10 of around 1.2 which is in agreement with other (although not all) literature data on the temperature dependence of water transport across cell membranes (AQPs). The findings indicate more stable AQPs in figleaf gourd than in cucumber. Besides the effect of suberization, it was concluded that this is the major reason for the differences found in the temperature dependence of root hydraulics between the two species.
It may be argued that the strong temperature dependence found in cucumber for the cell Lp (Q10 of 4.8) could be explained by a preferred transport of water across the bilayer rather than across AQPs in this species. If true, changes in cell Lp would then be due to phase transitions in the bilayer as suggested in work from the early literature (Petersen, 1980; Palta et al., 1993; Carvajal et al., 1998). However, this is unlikely since for biological membranes most of the water transport is considered to occur via AQPs, unless they are gated shut or expressed at very low levels. Even if the bilayer passage were rather temperature-dependent, its contribution to the overall cell Lp could not have been very large. Furthermore, Q10 values of bilayer transport are usally smaller than the obtained value of 4.8. They ranged between 1.8 to 2.4 (Cohen, 1975; Petersen, 1980; Hertel and Steudle, 1997; Finkelstein, 1987). Hence, a contribution of the bilayer passage should have been rather small if any.
Decreases in cell Lp and root Lpr in response to different stresses have already been measured in the past (Azaizeh et al., 1992; Birner and Steudle, 1993; Zhang and Tyerman, 1999; Henzler et al., 2004; Lee et al., 2004b). Yet the mechanisms by which the different stresses affect the closing of AQPs are not always clear. Heavy metals, such as Hg2+, are thought to bind to SH-groups, which results in a reversible change of protein conformation and channel closure. The action of a high concentration of solute, on the other hand, has been explained in terms of a cohesion/tension model which provides a reversible collapse of the conformation caused by osmotic pore dehydration (see Introduction; Ye et al., 2004a, b). Another mechanical model was provided by Wan et al. (2004) who showed that the injection of kinetic energy into water channels of corn root membranes may be sufficient to cause a channel closure, again indicating a reversible change in the protein conformation. In the case of root cells of cucumber, the conformational change could be caused by changes in thermal energy. If true, the differences between chilling-resistant and chilling-sensitive species would be due to differences in the stability of the open-state conformation of AQPs (AQPs of figleaf gourd were more stable than those of cucumber), which may be related to their anchorage in the bilayer. While water transport through the lipid bilayer accounts for only a minor portion (see above), it is not known whether membrane fluidity affects open/closed states of aquaporins (Carvajal et al., 1998). Freeze-tolerant species of Solanum commerson and S. tuberosum have a higher ratio of unsaturated to saturated fatty acids (Palta et al., 1993). With figleaf gourd plants, bond index in root plasma membrane upon exposure to LRT rapidly increased, due to an increase in linolenic acid (GC Chung, unpublished results). It may be speculated that, unlike figleaf gourd, the AQPs of cucumber could also be easily affected by other means of energy injection, such as by the mechanical perturbations just mentioned (Wan et al., 2004). Experiments are underway to test this.
In conclusion, results from different experimental approaches in the present work indicate a significant cell-to-cell water flow in roots of both cucumber and figleaf gourd, and this flow was differently affected by the events of chilling. Water channels of cucumber root cells were more sensitive to LRT than those of figleaf gourd. The Q10 value for the change in cell Lp for cells of cucumber was unusually high (Q10=4.8); for figleaf gourd, it was typical (Q10=1.2). This indicated that responses in cell Lp of cucumber were due to conformational changes of water-channel protein rather than being caused by the temperature dependence of water transport across open pores. In cucumber, the number of open pores decreased rather than the hydraulic conductivity of individual pores (as in figleaf gourd). In both species, there was a recovery at the cell and root level. In figleaf gourd, there was even an overcompensation of the effects of LRT when cells were allowed to recover at optimal temperature. Recovery at optimal temperature was faster and more intense in figleaf gourd than in cucumber. Water channel activity was inhibited by the AQP blocker HgCl2 in both species. In figleaf gourd, LRT caused a reduction of root hydraulic conductivity in hydrostatic Lpr but an increase in osmotic root Lpr. This suggested that the activity of water channels was even stimulated by LRT in this species rather than decreased. Unlike figleaf gourd, LRT induced a strong development of suberin layers in the endodermis of cucumber root suggesting a substantial reduction of root Lpr along the apoplastic and cell-to-cell pathways, in addition to the effect on AQP activity. It is concluded that the LRT-tolerant figleaf gourd has an ability to respond to chilling by an increased absorption of water through the water channels. Unlike the chilling-sensitive cucumber, the apoplastic passage is kept more open at low temperature in figleaf gourd. The occurrence of a greater number of passage cells in the endodermis of figleaf gourd should also have an ameliorative effect. These results indicate that the differences in root hydraulics in response to chilling between the two species are related both to changes in the activity of AQPs and in root anatomy.
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We thank Professor Carol A Peterson and Chris Meyer, University of Waterloo, Ontario, Canada, for critically reading the manuscript and for valuable suggestions. The excellent technical support of Burkhard Stumpf (Department of Plant Ecology, University of Bayreuth) is acknowledged. This work was supported by the Korea Science and Engineering Foundation (KOSEF) to Agricultural Plant Stress Research Center (APSRC, R11-2001-09201004-0) of Chonnam National University. Financial support by KOSEF to SH Lee as post-doctoral fellowship is greatly appreciated.
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