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JXB Advance Access published online on October 10, 2007
Journal of Experimental Botany, doi:10.1093/jxb/erm220
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© 2007 The Author(s).
This is an Open Access article distributed under the terms of the Creative Commons Attribution Non-Commercial License (http://creativecommons.org/licenses/by-nc/2.0/uk/) which permits unrestricted non-commercial use, distribution, and reproduction in any medium, provided the original work is properly cited. This paper is available online free of all access charges (see http://jxb.oxfordjournals.org/open_access.html for further details)

RESEARCH PAPER

Low air humidity increases leaf-specific hydraulic conductance of Arabidopsis thaliana (L.) Heynh (Brassicaceae)

Michal Levin1 *, Jorge Hugo Lemcoff2 *, Shabtai Cohen2,{dagger} and Yoram Kapulnik1

1Institute of Field and Garden Crops, The Volcani Center, Bet Dagan 50250, Israel
2Institute of Soil, Water and Environmental Sciences, The Volcani Center, Bet Dagan 50250, Israel

{dagger} To whom correspondence should be addressed. E-mail: vwshep{at}volcani.agri.gov.il

Received 21 June 2007; Revised 15 August 2007 Accepted 22 August 2007


   Abstract
Top
 Abstract
Introduction
 Materials and methods
 Results
 Discussion
 Appendix
 Calculation 1
 Calculation 2
 References
 
The typical isohydric plant response to low relative humidity involves stomatal closure, followed by long-term responses like adjustment of shoot-to-root ratios. Little information is available on the early responses of the root system to exposure of shoots to low humidity, nor is it clear to what extent responses of Arabidopsis thaliana conform to the isohydric model. In this study, A. thaliana plants grown hydroponically at high humidity were exposed to two constant relative humidities, 17% and 77%, while the root system remained in aerated nutrient solution. Leaf conductance (gs), transpiration, water potential ({Psi}l), osmotic potential, and whole plant hydraulic conductance (K) were determined for the following time intervals: 0–10, 10–20, and 20–40 min, and 0–5, 5–10, and 24–29 h. At low relative humidity, no change in gs was detected. {Psi}l decreased by 0.28 MPa during the first 5 h and then remained stable. During the first hour, leaf-specific K averaged 1.6x10–5 kg MPa–1 m–2 s–1 at high humidity. At low humidity it increased >3-fold to 5.8x10–5 kg MPa–1 m–2 s–1. Similar significant differences in K were observed during all time periods. Low concentration mercury amendments in the hydroponic solution (5 µM and 10 µM HgCl2) had no discernible influence, but pre-exposure to 50 µM HgCl2 reduced K differences between humidity treatments. As HgCl2 is known to be a potent inhibitor of aquaporin function, this suggests that aquaporins may have played a role in the fast hydraulic response of plants transferred to low humidity. The rapid hydraulic response and the influence of mercury raise the possibility that an alternative response to atmospheric dryness is increased K modulated by aquaporins.

Key words: Aquaporin, isohydric, leaf conductance, osmotic, water potential, water relations


   Introduction
Top
 Abstract
 Introduction
Materials and methods
 Results
 Discussion
 Appendix
 Calculation 1
 Calculation 2
 References
 
Arabidopsis thaliana is a plant whose genome has been deciphered, and therefore has become a model plant for study of the relationship between plant function and genetics. In nature, A. thaliana grows in a wide range of habitats in temperate regions. Therefore, it may be assumed that it is able to withstand, at least for short periods, arid aerial conditions with low soil water availability. This ability is likely to be associated with characteristic leaf-specific hydraulic conductance (Kl) and responses of leaf stomatal conductance (gs) and water potential ({Psi}l) to environment. Characterizing these responses for wild-type plants is a prerequisite to the study of genetic influences on plant hydraulics. Thus, the current study is a predecessor to work on the genetics associated with the response of A. thaliana plants to low relative humidity.

Typically, water loss is modulated by stomata in order to prevent desiccation of the plant in dry conditions. gs responds to several climatic factors, the main ones being soil water availability, radiation, leaf temperature, and air humidity. gs response in dry conditions has been shown to protect the integrity of the plant's water transport system, either by allowing plant water potential to drop only to fixed values, termed isohydric, or to allow {Psi}l to vary in dry periods but still prevent desiccation in a hydrodynamic response. The latter response has also been shown to be associated with some modulation of plant hydraulic conductance K (Franks et al., 2007).

Kl, i.e. the ratio of water flux to the water potential gradient driving that flux, expressed relative to leaf area, is thought to be closely related to plant water stress tolerance (Rieger, 1995; Sperry et al., 1998, 2002; Sobrado, 2003; McClenahan et al., 2004; Sperry, 2004; Tyree et al., 2005). Isohydric and hydrodynamic plant behaviour are tuned to Kl (Hubbard et al., 2001; Schultz, 2003). However, plants grown in non-water-limited environments are likely to grow larger leaf area relative to the plant's water uptake capacity, leading to lower Kl (Hacke et al., 2000; Li et al., 2005). When these plants are exposed to water-limited situations gs usually declines and adjustments in hydraulic architecture may occur in order to maintain hydraulic compatibility between plant and environment (Addington et al., 2006). The adjustments, which involve root growth and/or reductions in leaf area, are relatively slow. It is possible that rapid adjustments occur through active changes in properties of the water transport system, mainly in the symplastic and transcellular pathways, for example, aquaporin frequency and activity in the root membranes (Luu and Maurel, 2005; Aroca et al., 2006).

In many cases an increase in atmospheric evaporative demand precedes a critical decrease in soil water content (Tardieu et al., 1992). A number of studies have focused on the initial effects of reduced relative humidity (increased evaporative demand) on physiological responses of plant water relations parameters like gs changes and guard and epidermal cell osmotic adjustment (Schulze and Hall, 1982; Tardieu and Davies, 1993; Monteith, 1995; Saliendra et al., 1995; Assmann et al., 2000; Xu and Hsiao, 2004; Buckley, 2005). But depending on the genotype, physiological responses may or may not reflect plant water deficit (Bunce, 1996; Tardieu and Simonneau, 1998; Tardieu, 2003).

In this study gs, {Psi}l, leaf osmotic potential and whole plant transpiration and K of A. thaliana plants grown hydroponically at high relative humidity were determined when shoots were exposed to low air humidity. It was hypothesized that plants that had been grown at high humidity would respond to lower humidity by reducing gs and {Psi}l, and possibly with a slow hydraulic response. Once it was established that rapid changes in K occurred, the time-course of the response was studied and the question as to whether the hydraulic adjustment was sensitive to mercurials associated with aquaporin activity was investigated.


   Materials and methods
Top
 Abstract
 Introduction
 Materials and methods
Results
 Discussion
 Appendix
 Calculation 1
 Calculation 2
 References
 
Plant material
Arabidopsis thaliana (L.) Heynh wild-type (ecotype Columbia) plants were grown in a rock wool-based hydroponic system similar to that used by Gibeaut et al. (1997). Plants were seeded in 1000 µl pipette tips cut to a length of 2 cm and loosely filled with rock wool. These were placed in a container filled with nutrient solution consisting of 1.5 mM Ca(NO3)2, 1.25 mM KNO3, 0.75 mM MgSO4, 0.5 mM KH2PO4, 0.0187 g l–1 Sequestrene® 138 Fe, 50 µM KCl, 50 µM H3BO3, 10 µM MnSO4, 2 µM ZnSO4, 1.5 µM CuSO4, and 0.075 µM (H4N)2MO4 at pH 6.0, without Na2SiO3. Seeds were pretreated at 4 °C for 2 d. For germination, the seeded ensembles were transferred to a climate-controlled growth chamber at 23 °C, at a light intensity of 120 µE m–2 s–1 provided by a mixture of fluorescent lamps (Lumilux® Cool White, Warm White, and Daylight; Osram, Germany) in a 12/12 h light–dark regime. After 2 weeks, seedlings were thinned so that each tip contained one sprout. Plants were then transferred to 15 ml Falcon® round-bottom tubes, prepared with holes at the bottom and inserted into Polygal® Standard Sheets prepared with appropriate holes, floating in a 14 l plastic box containing nutrient solution. Plants were grown in the above conditions under continuous aeration for 4 additional weeks. Nutrient solution was replaced every 2 weeks.

Exposure of the plants to different air relative humidity (RH) values
Plants (stage 5.10: first flower buds visible; Boyes et al., 2001) were transferred 48 h before the experiment was started into new 15 ml black Falcon tubes containing nutrient solution. Tubes were placed in a 14 l plastic container loosely covered with a transparent acrylic cover in order to ensure that plants were at a minimum of 95% RH during the light period. During and after acclimation, the nutrient solution was aerated for 15 min h-1 by pumping air through needles inside the Falcon tubes. These were refilled with water every 5 h during the light cycle (<10% of the total volume needed replacement per event).

One hour after light onset on day 1, Falcon tubes with plants were transferred to two separate 14 l plastic containers hermetically sealed with transparent acrylic covers. RH values of 17±6% (VPD=2.5 kPa) and 77±5% (VPD=0.65 kPa) were created in container head spaces, at temperatures of 24.4±1.0 °C and 23.1±1.1 °C, respectively. 17% RH (RH17) was generated by a saturated KOH solution and 77% RH (RH77) by a saturated NaCl solution (Meites, 1963). RH and temperature were monitored in each container with compact data loggers equipped with LED displays (Microlog, Fourier Systems, Centerport, NY, USA). When the acrylic seal was removed to allow plant manipulation it took <2–3 min for the confined atmosphere to return to the original RH value after the cover was replaced. Containers were aerated continuously with air that had passed, at a flow rate of 0.5 l min–1, through either dry silica gel (RH17) or a saturated NaCl solution (RH77). This ensured atmospheric conditions in the containers were constant.

Mercury treatments
Three additional sets of plants were prepared and acclimated as described above. Thirty minutes after lights were turned on plants were transferred to nutrient solution containing 5, 10, or 50 µM HgCl2. In the 5 µM and 10 µM HgCl2 treatment, measurements were performed as described above for plants not treated with HgCl2 and roots remained in the nutrient solution with HgCl2 during the course of the experiment. In the 50 µM HgCl2 treatment, in order to avoid poisoning, plants were transferred after 30 min of exposure to Hg to new Falcon tubes containing regular nutrient solution. The measurement procedure continued after an additional acclimation time of 30 min.

CO2 concentration measurements
Air samples were collected from both treatments several times and CO2 concentrations were measured with an LI-6200 gas analyser (Li-Cor, Lincoln, NE, USA). Values were within ±10 ppm of ambient (data not shown).

Leaf stomatal conductance (gl) measurements
At 0, 5, 10, 24, and 29 h after transferring the plants to the constant humidity containers, gs was measured in all plants. Measurements were made within 2 min of plant removal from the sealed container; a full mature leaf was measured with a steady-state diffusion porometer (LI-1600 with a narrow aperture cap masked to 0.5 cm2 with PVC electrical adhesive tape; Li-Cor).

Leaf water potential ({Psi}l) measurements
{Psi}l was measured 0, 10, 20, and 40 min and 5, 10, 24, and 29 h after plants were transferred to the constant RH containers. Measurements at 0 h and 24 h were taken 1 h after light onset.

For {Psi}l measurements a plant was taken from the closed container, one fully expanded leaf was sealed rapidly into a polyester film pouch to prevent evaporative water loss, leaving only a 1–2 mm protruding petiole which was cut off at its base. The leaf was then threaded into a 1000 µl pipette tip, cut at both ends to a length of 15 mm, with its protruding petiole towards the narrower edge of the tip. Then the tip was filled completely and sealed tightly in a low-viscosity dental paste (President Light®; Coltene, Altstaetten, Switzerland) as described by Javot et al. (2003) for roots, allowing the cut end of the petiole to emerge 1–2 mm from the tip edge. It took <30 s from the time that the controlled humidity container was opened until leaves were sealed in polyester film pouches. If the increased humidity during that time reduced transpiration and allowed refilling of the leaf, then relative water content (RWC) would have increased <0.4% and {Psi}l would have increased only 0.03 MPa (see calculation in the Appendix), a change that would not have significantly altered the results.

The procedure was repeated for all plants. Six minutes later, the conditioned leaf in the pipette tip was sealed into a pressure chamber (Arimad-2, Kibbutz Kfar Charuv/MRD, Israel) with the cut end protruding from the tip edge. Nitrogen gas was used to apply pressure, which was increased gradually until a neat water front was observed with the help of a binocular microscope (WILD® M8, Wild-Heerbrugg, Switzerland) and cool lights (Intralux® 6000, Volpi, Switzerland) directed towards the petiole. Then the pressure was released and the procedure was repeated. The second reading was taken as the final measurement, and differed at most by <2% from the first reading (data not shown). After {Psi}l was measured, microscopic inspection of the cut end of the leaf petiole and longitudinal sections of the petiole did not reveal crushed vessels.

Leaf relative water content (RWC)
Leaf water status was also determined from measurements of RWC, after Weatherley (1950). Leaf samples were weighed (fresh mass) immediately after harvesting, then floated in a Petri dish containing distilled water, and placed for 1 h inside a vacuum chamber at 30 kPa. Once removed, they were gently blotted with tissue paper and weighed (turgid mass). Samples were then dried in an oven at 65 °C for 48 h and their dry mass was determined. RWC was calculated as [(fresh mass–dry mass)/(turgid mass–dry mass)]x100.

Plant water uptake measurements
In order to determine water uptake by the plants during specific time periods the Falcon tubes containing plants were weighed on an electronic analytical balance accurate to 100 µg (Sartorius®, BP2215; Sartorius AG, Goettingen, Germany) at the beginning and at the end of each time period. At those times the controlled humidity containers were opened, leaves were cut for {Psi}l measurement, plants were weighed and replaced in the container in <1 min, and then the container was closed and re-sealed. Identical tubes that did not contain any plants were placed in each container as blanks. The mass difference of the Falcon tubes containing plants minus the average evaporation from blanks containing no plants (three per container) was assumed to be the water transpired by the plants.

Leaf osmotic potential measurements
At 0 h and 29 h after transferring the plants to the controlled humidity containers, leaf osmotic potential at full turgor was measured. In each treatment three full mature leaves were collected from each plant, placed in a Petri dish with distilled water and transferred to a vacuum chamber at 30 kPa for 15 min. Afterwards, leaves were removed, gently blotted with tissue paper, put into a 1.5 ml Eppendorf tube, and frozen in liquid nitrogen. The tube was punctured at the base, placed into another Eppendorf tube, centrifuged at 1466 g for 8 min (Centrifuge SIGMA® 1–15K, Sigma Laborzentrifugen, Osterode am Harz, Germany) and the collected liquid was measured with an osmometer (Micro Osmometer µOsmette® 5004, Precision Systems, Natick, MA, USA).

Leaf area measurements
Leaf areas of all leaves used for {Psi}l measurements and of leaves remaining at the end of the experiment were determined. The area was measured by scanning the leaves with a digital scanner (HP Scanjet® 3970; Hewlett Packard, Palo Alto, CA, USA) and the surface area was computed from image analysis with ImageJ® software (ImageJ Shareware, NIH, Bethesda, MD, USA).

Leaf specific hydraulic conductance (Kl)
Kl was estimated for each interval from the water transpired by the plants, their actual leaf area, and the driving force developed (average of initial and final {Psi}l), this last corrected by {Psi}l at predawn in a 100% RH atmosphere (–0.18±0.02 MPa), as in David et al. (2004). A second correction was applied when at the end of an interval {Psi}l decreased, indicating a significant reduction in leaf RWC. This implied that in the specific interval some water lost by transpiration did not pass through the roots and the hypocotyls' xylem. In other words, the decrease in relative water content leading to the final {Psi}l was assumed not to have come from flow through the plant during the interval, but from loss of water from the leaf tissue. This quantity of water (computed from a leaf water retention curve made with A. thaliana plants of equivalent age; data not shown) was subtracted from the gravimetric water loss. Corrections were smaller than 3.8% for the 5 h intervals, but as large as 50% for the 10–20 min intervals (see the Appendix).

Due to leaf excision for {Psi}l measurement, leaf area was reduced during the experiments by as much as 24±3% and 27±3% in the RH77 and RH17 treatments, respectively.

Kl was estimated for two experiments, one with two time scales and a second with different HgCl2 concentrations in the nutrient solution. In the first experiment, measurements were performed for a short-term time scale: the first 10 min (0–10 min), the following 10 min (10–20 min), and the following 20 min (20–40 min) of exposure to RH17 and RH77; and for a long-term time scale: the first 5 h (0–5 h), the following 5 h (5–10 h), and the first 5 h of the following light cycle (24–29 h). In the second experiment with nutrient solution containing HgCl2 (at concentrations of 0, 5, 10, and 50 µM) Kl measurements were made over the 0–5 h period.

Statistical analysis
All experiments were repeated at least twice and values were subjected to statistical analysis using JMP® software from SAS, release 6 (SAS Institute Inc., Cary, NC, USA). Two-way analysis of variance and Tukey–Kramer multiple comparison tests were used to analyse the effect of air relative humidity and time on the dependent variables, {Psi}l, gs, and Kl, with an {alpha} level of 0.05 unless otherwise specified. When necessary, log transformations were done for homoscedasticity.


   Results
Top
 Abstract
 Introduction
 Materials and methods
 Results
Discussion
 Appendix
 Calculation 1
 Calculation 2
 References
 
Leaf stomatal conductance (gs), water potential ({Psi}l), and osmotic potential
gs was measured at the beginning and end of each measurement interval. Results for both RH treatments are shown in Fig. 1. No interaction between RH and time was detected (P=0.72). gs changed significantly over time (P <0.001), but differences between RH treatments were not significant at any time during the experiment (P=0.24).


Figure 1
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  		Fig. 1. Leaf stomatal conductance measured at different time points during exposure to 17% and 77% relative humidity. n=6 plants. Vertical bars represent ±one standard error of the mean. Time points with the same letter are not significantly different (P >0.05).

 
In {Psi}l a significant interaction between air relative humidity and time was detected (P <0.05). At high humidity (RH77) {Psi}l did not change significantly (P=0.81) during the experiment, while at low humidity (RH17) {Psi}l changed significantly with time and was highest at 0 h and lowest at 10 h and 29 h. Only initial (0 h) RH17 values were similar to those for RH77 (P=0.82) (Fig. 2). After 5 h, {Psi}l at RH17 was significantly (P <0.02) lower than at RH77 and did not change afterwards. The average difference between treatments was 0.28±0.02 MPa.


Figure 2
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  		Fig. 2. Water potential measured at different time points during exposure to 17% and 77% relative humidity. n=6 plants. *, Significant difference between RH17 and RH77 treatments at P <0.05; ns, non-significant difference. Vertical bars represent ±one standard error of the mean.

 
Leaf osmotic potential at full turgor, measured at 29 h, was similar between treatments, i.e. 0.75±0.02 and 0.74±0.03 MPa for RH77 and RH17, respectively. Thus, leaf osmotic adjustment was minimal. Maximum leaf turgor pressure was 0.49 MPa. This was calculated from predawn values of {Psi}l, –0.18±0.02 MPa, and leaf osmotic potential, –0.67±0.04 MPa. These values are similar to previous measurements of Arabidopsis (Martre et al., 2002).

Leaf-specific hydraulic conductance (Kl)
Kl values for the experiment without HgCl2 were of the same order of magnitude in all cases, ranging from 1.5x10–5 to 7.5x10–5 kg MPa–1 m–2 s–1 (Fig. 3). After log transformation (Bartlett's test, P=0.96), no interaction between relative humidity and time was detected and both factors were significant (P <0.01). Kl of RH17 was higher than for RH77. Kl for the 10–20 min interval was significantly higher than that obtained for time periods after 5 h, and Kl for the 10–20 and 20–40 min intervals was significantly higher than that at 5–10 h.


Figure 3
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  		Fig. 3. Leaf-specific hydraulic conductance calculated for different time periods of exposure to 17% and 77% relative humidity. n=3 plants for short times (<1 h) and n=6 plants for the longer experiments. Vertical bars represent ±one standard error of the mean. For 17% relative humidity, time points with the same letter are not significantly different (P >0.05).

 
The second experiment was with plants treated with 0, 5, 10, or 50 µM HgCl2. Figure 4 shows Kl for these plants for the first 5 h (0–5 h interval) of exposure to 17% and 77% relative humidity. The interaction between relative humidity and HgCl2 concentration was marginally significant (P=0.09). Kl values at high RH (RH77) were significantly lower than at RH17 for all HgCl2 concentrations, except for the 50 µM HgCl2 treatment, where differences were not significant.


Figure 4
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  		Fig. 4. Leaf-specific hydraulic conductance of A. thaliana WT plants exposed to 0, 5, 10, and 50 µM HgCl2, calculated for the first 5 h of exposure to 17% and 77% relative humidity. n=3 plants. *, Significant difference between RH17 and RH77 treatments at P <0.05; ns, non-significant difference. Vertical bars represent ±one standard error of the mean.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
Appendix
 Calculation 1
 Calculation 2
 References
 
Physiological responses of A. thaliana plants to reduced air relative humidity were studied when roots were in optimal conditions. Experiments were conducted for up to 29 h and included short (minute) and long-term (hour) time scales. The involvement of mercury-sensitive aquaporins in the hydraulic response was investigated by adding mercurials to the nutrient solution. In these conditions, exposure of A. thaliana shoots to low humidity (RH=17%) did not affect gs. The initial low gs values of both treatments might be a consequence of the relatively high humidity (95%) during the acclimation period before the experiment (Turner et al., 1984; Aphalo and Jarvis, 1991; Bunce, 1997). Low humidity caused a significant decrease in {Psi}l. Simultaneous measurement of water loss, {Psi}l, and leaf area enabled the whole plant Kl to be calculated.

There have been few other reports of {Psi}l and K in A. thaliana. This may be due to the technical difficulty in sealing small leaves and plants in high pressure apparatus required for these measurements. The innovation used to rapidly seal the tiny A. thaliana leaves in pipette tips solved this problem, and reliable and repeatable measurements of {Psi}l were obtained. This technique should prove useful in subsequent studies of A. thaliana and other plants with small leaves and/or small and weak petioles.

Changes in Kl were significant during the first 10 min interval, when RH17 values were more than twice those for RH77. Significant 2–4-fold differences between the two humidity values were sustained for the duration of the experiment. The large difference in vapour pressure deficit between the two treatments, i.e. 2.5 kPa for RH17 versus 0.7 kPa at RH77 at 23–24 °C, would be enough to influence gs significantly in isohydric species (Tardieu and Simonneau, 1998; Dewar, 2002). The lack of response of gs (Fig. 1) indicates that A. thaliana is either anisohydric (Bunce, 2006) or hydrodynamic. Such behaviour (i.e. no reduction in gs in dry conditions) could endanger the plant's hydraulic system during dry periods if no other hydraulic response occurs. Therefore the lack of a significant gs response is compatible with the large response in hydraulic conductance observed (Franks et al., 2007).

Kl values reported here are much lower (around one-sixth to one-half) than those reported by Martre et al. (2002) for the same species. However, their plants were grown at higher light levels (photosynthetic photon flux density 200 µmol m–2 s–1) and in soil (vermiculite and Sphagnum peat moss). Both of those factors should have led to lower shoot-to-root ratios and therefore to the higher Kl values. The present values of Kl were one order of magnitude lower than those reported for cotton by Li et al. (2005), who calculated Kl from transpiration measured with sap flow sensors and water potential measured with a dew point psychrometer, and for sunflower by Nardini et al. (2005), who measured with a high pressure flow meter.

Short-term changes in hydraulic conductance under constant environmental conditions, as a consequence of changes in xylem sap composition, expression of aquaporins, or other endogenous processes, have been observed in various species. Henzler et al. (1999) and Clarkson et al. (2000) reported circadian rhythms in K of detached root systems of Lotus japonicus. Similarly, in Helianthus annuus both root and shoot resistances were found to change over the course of the day (Tsuda and Tyree, 2000) and K was 30–40% lower at night (Nardini et al., 2005). Circadian rhythms in K (with an amplitude of two to three times) have also been observed in excised root systems of tobacco and apple seedlings (M Tyree, unpublished data). These observations indicate that root and/or shoot system K is probably much less constant than previously thought, and the magnitude of the reported rhythms is similar to the magnitude of the response of whole-plant K to atmospheric humidity found in the current study.

Maherali et al. (1997) reported reductions, in K in response to low atmospheric humidity in maple saplings but their measurements were made after longer periods of acclimation. Changes in root conductance have been reported for A. thaliana and other higher plants under different stresses (Javot and Maurel, 2002), and, as far as is known, those changes were not related to changes in shoot-to-root ratios.

When HgCl2 was introduced into the root medium differences in Kl between RH77 and RH17 were significant at the lower concentrations (Fig. 4). However, at 50 µM HgCl2 they were not significant. As HgCl2 is known to be a potent inhibitor or blocker of many plant aquaporins (Wan and Zwiazek, 1999; Javot and Maurel, 2002) this observation indicates that aquaporins could have been involved in the fast hydraulic response of the plants transferred to low humidity conditions.

The concentrations of HgCl2 used here to block aquaporins (5–50 µM) are at the low end of the range used in other studies. Lovisolo and Schubert (2006) used up to 500 µM in Vitis vinifera and Wan and Zwiazek (1999) used up to 100 µM in aspen. The concentrations used in the present study were selected because it was thought that Arabidopsis is a delicate plant and might therefore respond to lower concentrations, and because experiments were set to run for longer periods, i.e. >2 h or 3 h, and mercurial effects should increase with time. Low concentrations were also expected to prevent artefacts related to toxicity and, thus, non-selectivity of the HgCl2 (Barrowclough et al., 2000; Martre et al., 2001). Wan and Zwiazek (1999) reported that 25 µM HgCl2 ‘acted relatively slowly on the steady-state flow rate and was less effective than the higher concentration treatments’. This may be the reason that the only significant response found in the present study was at 50 µM and indicates that higher concentrations should be used in the future. Additional questions as to the efficacy of pre-treatment, continuous treatment, and the influence of the rate of HgCl2 uptake on K were beyond the scope of this study.

The role of aquaporins in plant water relations was more evident in other studies. Fluctuations in hydraulic conductivity were observed during nutrient deprivation by Carvajal et al. (1996) and after exposure to salinity by Carvajal et al. (1999), and they suggested that water channel regulation could account for these fluctuations. In rice (Oryza sativa) seedlings, the application of 500 µM HgCl2 had an effect on whole-plant conductance only when plants were water-stressed by the presence of PEG (polyethylene glycol), but not in control conditions (Lu and Neumann, 1999). This may mean that apoplastic transport predominated in normal conditions and that water channels might be up-regulated under water stress. Various factors have been shown to regulate plant hydraulic conductance by controlling, among other things, aquaporin gene transcription and protein abundance, stimulus-induced aquaporin subcellular relocalization, and channel gating, i.e. adjusting the water transport properties of the membranes (Luu and Maurel, 2005; Aroca et al., 2006).

Different abiotic stresses influence aquaporin expression. Kawasaki et al. (2001) recognized two aquaporin encoding transcripts in a salt-insensitive rice cultivar which were found to be down-regulated during the first 15 min and 60 min of exposure to high salinity. The transcript level then recovered and, after 7 d, expression levels became higher than in standard conditions. The up- or down-regulation of aquaporin homologues has been described in the roots of many plant species including Helianthus annuus under water stress (Sarda et al., 1999), Mesembryanthemum crystallinum under salt stress (Yamada et al., 1995), and rice (Liu et al., 1994) and Craterostigma plantagineum after desiccation (Mariaux et al., 1998). Short-term effects (within 1–3 h) of ABA on root aquaporin expression have also been described for rice (Liu et al., 1994), C. plantagineum (Mariaux et al., 1998), and A. thaliana (Weig et al., 1997). Thus, transcriptional regulation of aquaporin genes can operate in <1 h, but may be combined with other regulatory mechanisms of aquaporin activity like phosphorylation, Ca2+, and pH.

The results of the current study imply that rapid changes in A. thaliana hydraulic properties occur in response to changes in atmospheric evaporative demand and one mechanism that can explain this phenomenon is aquaporin modulation. It is not clear how the changes in relative humidity at the shoot of the plant could provoke such large and rapid changes in aquaporin activity. One possibility is that roots have water potential- or hydraulic conductance-sensing mechanisms which track changes in hydraulic parameters of the plant and trigger processes in the roots to prevent future stress. Another option could be that chemical signals generated in the shoot in response to low air relative humidity could be the messengers that transfer information on changing conditions to the roots. No reports were found that addressed the root response to changes in hydraulic factors and the possible signals that might be involved, and therefore it would be interesting to expand the study to the investigation of the signalling mechanisms involved in the response of Arabidopsis to low relative humidity.


   Appendix
Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 Appendix
Calculation 1
 Calculation 2
 References
 
The appendix gives two calculations of possible errors in {Psi}l and hydraulic conductance. The second calculation was applied routinely in the study.


   Calculation 1
Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 Appendix
 Calculation 1
Calculation 2
 References
 
This calculates the change in relative water content ({Delta}RWC) in 30 s, when transpiration is stopped, to estimate the error in water potential that might occur due to the increase in humidity when the vessel with 17% RH is opened in order to sample the leaves for water potential ({Psi}l). Leaves were sampled within 30 s.

The amount of water, Jw, that would flow during the 30 s period can be expressed as

Formula (A1)
Assuming that leaf dry matter content (LDMC), which expresses the ratio of dry matter to fresh mass is measured on turgid leaves, 1–LDMC is the ratio of turgid leaf water mass to total leaf fresh mass. Specific leaf area (SLA) is the ratio of leaf area to leaf dry mass. Using these parameters, {Delta}RWC can be computed from:

Formula (A2)
Taking Kl=3 e-05 kg m–2 s –1 MPa–1, {Delta}{Psi}=1 MPa, and t=30 s, equation A1 gives Jw=9 e-4 kg m–2 of water. SLA (specific leaf area) was taken as 345 cm2 g–1 (Gibeaut et al., 1997), and LDMC was taken as 0.11 g dry mass (DM) g–1 fresh mass (FM) (Garnier et al., 2001), giving {Delta}RWC=0.384%. Pressure–volume curves for A. thaliana leaves (data not shown) give RWC=({Psi}l+7.572)/0.073; r2=0.74, n=12. Thus, a change of 0.4% in RWC implies a change of 0.03 MPa in {Psi}l, which is negligible in this study.


   Calculation 2
Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 Appendix
 Calculation 1
 Calculation 2
References
 
When water potential changed in a time interval, the change in water content of the leaf represents water that did not pass through the xylem, and therefore should be subtracted from the transpiration during that interval before calculating hydraulic conductance. The following are examples of calculations of the change (reduction) in leaf- and root-specific hydraulic conductance ({Delta}Kl and {Delta}Kroot) when transpiration reduces {Psi}l significantly.

Formula (A3)
where {Delta}Wtreatment is the mass change of the Falcon tube containing a plant during the period; {Delta}Wblank is the mass change of the Falcon tube not containing a plant during the period; {Delta}Wtissue is the change in the plant's mass, representing water that did not pass through the xylem during that period, estimated from the conversion of {Delta}{Psi}l to {Delta}RWC, using the PV curve measured for A. thaliana leaves (see above) normalized to the leaf area; {Psi}l avg is the average between initial and final {Psi}l of the period (MPa); {Psi}l predawn is the value of a plant at steady-state in a 100% RH atmosphere, equivalent to a predawn measurement (MPa); t is time (s); LA is leaf area (m2).

For example, during a 5 h interval {Psi}l changed from –0.73 to –1.01 MPa, equivalent to a 3.8% reduction in leaf RWC. Leaf water content was 0.240 kg water m–2, thus 0.0092 kg water m–2 was lost from the leaf. Multiplying by the remaining leaf area gives the amount of water lost from the plant that did not pass through the xylem. Thus, the correction for K is 1.9%, which is negligible. A second example is for a 10 min interval. A {Psi}l change from –0.60 to –0.95 MPa is equivalent to a 4.8% reduction in leaf RWC, meaning 0.0115 kg water m–2. In this case the correction for K is not negligible and quite large, i.e. 50.5%.


   Acknowledgements
 
We thank Smadar Wininger for technical assistance and Dr Eran Raveh of the Gilat Research Station of the ARO for measuring CO2 concentrations. This project was supported by grant number 259-0146-03 from the Chief Scientist of the Israeli Ministry of Agriculture.


   Footnotes
 
* Both to be considered as first authors. Back


   References
Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 Appendix
 Calculation 1
 Calculation 2
 References
 
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