Mercury hinders recovery of shoot hydraulic ... - Wiley Online Library

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Mercury hinders recovery of shoot hydraulic conductivity during grapevine rehydration: evidence from a whole-plant approach Blackwell Publishing Ltd

Claudio Lovisolo1 and Andrea Schubert1,2 1

Dipartimento Colture Arboree, Università di Torino, Via Leonardo da Vinci 44, I-10095 Grugliasco, Italy; 2IVV – CNR, Grugliasco (TO), Via Leonardo da

Vinci 44, I-10095 Grugliasco, Italy

Summary Author for correspondence: Claudio Lovisolo Tel: +39 011670 8926 Fax: +39 011236 8926 Email: [email protected] Received: 9 May 2006 Accepted: 15 June 2006

• This experiment aimed to test whether recovery of shoot hydraulic conductivity after drought depends on cellular metabolism in addition to xylem hydraulics. • We rehydrated droughted grapevines (Vitis vinifera) after treating intact plants through the root with 0.5 mM mercuric chloride (a metabolic inhibitor) at the end of the stress period, before rehydration. The contribution of mercury-inhibited water transport in both shoot and root, and the extent of shoot vessel embolization, were assessed. • Drought stress decreased plant water potential and induced embolization of the shoot vessels. The rehydration in Hg-untreated plants re-established both shoot water potential and specific shoot hydraulic conductivity (Kss) at levels comparable with watered controls, and induced recovery of most of the embolisms formed in the shoot during the drought. In contrast, in plants treated with HgCl2, recovery of Kss and root hydraulic conductance were impaired. In rehydrated, Hg-treated plants, the effects of Hg on Kss were reversed when either the shoot or the root was treated with 60 mM β-mercaptoethanol as a mercuric scavenger. • This work suggests that plant cellular metabolism, sensitive to mercuric chloride, affects the recovery of shoot hydraulic conductivity during grapevine rehydration by interfering with embolism removal, and that it involves either the root or the shoot level.

Abbreviations Kss, shoot specific conductivity; calcKss, in vivo calculated Kss; flushedKss, Kss measured after flushing out the embolisms; HgKss, Kss measured after mercuric treatment; MEKss, Kss measured on Hg-treated plants after 60 mM β-mercaptoethanol treatment; Lhr, root hydraulic conductance; HgLhr, Lhr measured after mercuric treatment; MELhr, Lhr measured on Hg-treated plants, after 60 mM β-mercaptoethanol treatment. Key words: aquaporin, drought, embolism, hydraulic conductance, HgCl2, Vitis vinifera, recovery, water channel. New Phytologist (2006) 172: 469–478 © The Authors (2006). Journal compilation © New Phytologist (2006) doi: 10.1111/j.1469-8137.2006.01852.x

Introduction In xylem tissues of plants, water flows under negative pressure along the pathway from root tips to stomata (Pockman et al.,

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1995). The so-called tension forces, originated by water evaporation in the substomatal chambers during transpiration, permit water uptake from the soil and water transport along the plant’s conduction system (Steudle, 2001). This model

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suggests that water columns, in a metastable state when under tension (Tyree, 2003), can be seeded by air bubbles (cavitation) when tensions increase on water stress. This results in the blockage of vascular tissues by embolism formation, causing loss of hydraulic conductance of the plant (Sperry et al., 2002). Hydraulic conductance recovers on plant rehydration, when repair of embolisms can take place via root pressurization of xylem vessels (Cochard et al., 1994), a phenomenon known for grapevine and other woody vines (Clearwater & Clark, 2003); or when vessels are under tension (Holbrook & Zwieniecki, 1999; Tyree et al., 1999). Water permeates the root through apoplastic and transcellular pathways according to the structure and functionality of hydraulic conducting systems. The effects on hydraulic conductance of transcellular water transport via aquaporins have been studied in depth mostly in the root radial pathway ( Javot & Maurel, 2002), while the plant’s ability to respond rapidly to rewetting via a likely aquaporin gating in whole-root (Martre et al., 2002; Vandeleur et al., 2005) or even shoot (De Boer & Volkov, 2003) water pathways has been proposed theoretically and is a subject of current research. Since Maggio & Joly (1995) demonstrated mercuric inhibition of whole-plant water transport for the first time in a tomato whole-root system, HgCl2 has been used extensively at various concentrations (10−6 to 10−3 M) on several plants, as reviewed by Javot & Maurel (2002), to reduce water transport in plant roots and other organs. After this experiment, the use of HgCl2 became a routine tool to investigate membrane water-channel activity; in contrast, today it is assumed that not only water channels, but other cellular metabolic processes putatively affecting water transport, can be negatively influenced by HgCl2 treatments to plants. It is known that HgCl2 rapidly depolarizes the plasmalemma of wheat root cortical cells, potentially altering the osmotic balance across membranes (Zhang & Tyerman, 1999) and related water relations at the interface symplast/ apoplast. Although several published results indirectly suggest that aquaporin-mediated transmembrane water transport or other likely Hg-sensitive cellular metabolisms play an important role in whole-plant recovery after water stress, to date no direct evidence of this has been obtained. The goal of this work was to understand whether recovery of shoot hydraulic conductivity after drought depends on cellular metabolism, as well as on xylem hydraulics, and with this aim we applied a rehydration treatment to water-stressed, Hg-treated grapevine plants. Our work shows that recovery of shoot hydraulic conductivity in droughted–rewetted plants is hindered when Hg-sensitive water transport is lacking, and this phenomenon can be reversed by a mercuric scavenger. In addition, we suggest that the mercuric inhibition cannot be explained only through a direct effect of the Hg on membranes, which would be limiting for the overall water flow, but conductivity reduction is mediated by likely perturbations of water relations at the interface symplast/apoplast. We have been using grape as a model plant for

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the study of hydraulics under water stress (Lovisolo et al., 2002) because this plant can withstand high levels of water stress and embolization of xylem vessels (Schultz & Matthews, 1988). Being lianas, grapes have stems with high water conductivity and little interference from lateral branches (Lebon et al., 2004), which makes them simplified models for the study of water transport.

Materials and Methods Plant material and growth conditions We used commercial 5-yr-old plants of Vitis vinifera L. cv. Pinot noir PN165, grafted on Vitis riparia × Vitis berlandieri 420A. Plants were grown in 12-l containers filled with a substrate composed of sandy-loam soil/expanded clay/peat mixture (4 : 2 : 1 vol) with a final pH of 7.3. Containers were placed in a glasshouse with no supplementary light or heating. Pots were fertilized once a month with 15 g of a complex (20-10-10) fertilizer. In January, plants were pruned to a single-bud spur. Average budbreak took place on 9 April. The single shoot of each plant was trained vertically on a stick. Lateral shoots and clusters were removed immediately after formation. Plants were watered to container capacity (Ψsoil approx. −0.01 MPa; Lovisolo & Schubert, 1998) each third day until the beginning of the water-stress treatments. Water stress and rehydration treatments Twenty-seven plants (water-stress/rehydration, WS/R treatment) were first subjected to a drought-stress treatment designed to induce embolisms without modifications of xylem development (Schultz & Matthews, 1988; Lovisolo & Schubert, 1998). With this aim, water was withheld from these plants for an 8-d period, starting from 30 June (81 d after budbreak, DAB), while the other plants (irrigated control treatment) were maintained at container capacity as described previously. At the end of the 8-d period, the WS/R plants were subjected to a rehydration period. Roots of intact plants were gently freed from the pot soil underwater, taking care not to break fine roots, and were submerged in aerated water for 24 h. The same procedure was applied to irrigated control plants in order to provide an adequate control at the end of the 24-h rehydration period. Water potential in soil, root-stalk, shoot and leaf Ψsoil was calculated according to soil moisture/water potential curves assessed previously for the pot substrate (Lovisolo & Schubert, 1998). Ψroot-stalk was measured using a Scholander-type pressure chamber (Soil Moisture Equipment Corp., Santa Barbara, CA, USA) on the most basal leaf, wrapped in the evening before measurement with a double-layer bag (inside plastic, outside aluminium), assumed to represent the water

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potential of the corresponding root-stalk xylem (Liu et al., 1978); Ψshoot was measured on bagged leaves at internodes 5, 10 and 15 from the shoot base, representing Ψshoot of the corresponding xylem; Ψleaf was measured on transpiring leaves at nodes 8 and 13 from the shoot base. Hydraulic measurements in shoot and root Hydraulic conductivity of the shoot was first calculated in vivo with the transpirative flux method (Martre et al., 2001; Lovisolo et al., 2002) under steady-state conditions, based on the ratio between Eplant (determined gravimetrically) and the water potential gradient along the shoot (∆Ψ/∆xshoot), where ∆Ψ = Ψroot-stalk − Ψshoot, and ∆xshoot the distance between the most basal and the tenth leaf along the shoot. To normalize conductivity to shoot size, in vivo specific conductivity of the shoot (calcKss) was obtained by dividing the calculated hydraulic conductivity by the shoot cross-sectional area at the tenth internode along the shoot. Hydraulic conductivity of the shoot was then measured on detached shoots through a controlled tension–pressure apparatus (Lovisolo et al., 2002), using the method of Schubert et al. (1999), modified as follows. About 80-cm (80.4 ± 0.29cm)-long shoot portions with leaves were detached from the vine in the central region of the shoot by bending and submerging the central shoot region in a plastic basin. This length was chosen as it has been reported that 90% vessels of Vitis labrusca vines are not longer than 80 cm (Sperry et al., 1987). Moreover, we have previously shown (Lovisolo & Schubert, 1998) that using shoot portions shorter than the maximum vessel length leads to an overestimation of conductivity, but does not affect relative differences among treatments. The basal end of the portion (approx. 15 cm long) was transferred into a pressure chamber filled with tap water, avoiding air contact to the cut basal surface during transfer. The apical end was clamped with a rubber sleeve. First, a measurement of ‘undisturbed’ conductivity was taken by applying apical suction (−80 kPa) for 5 min through the sleeve to the cut shoot portion apical end (Kolb et al., 1996). Before measurement, suction was applied for 10 min to allow stabilization. This tension gradient (−1 kPa cm−1) is close to physiological values, measured under either irrigation or mild stress conditions (Lovisolo & Schubert, 1998), and is expected to give measurements of shoot hydraulic conductivity close to the in vivo measured values. Before applying −1 kPa cm−1, two lower tension gradients (−0.5 and −0.3 kPa cm−1) were also tested in a preliminary experiment, and the flow response to different tension gradients was linear (R 2 = 0.986), although the lower the tension gradient, the higher the variability observed (Fig. 1). In order to estimate the extent of shoot xylem embolization, after the measurement under −80 kPa tension, a basal pressure (+200 kPa) was applied for 5 min to the water in the chamber containing the same basal shoot portion. This

Fig. 1 Flow measurements (Φ) in relation to different tension gradients (∆Ψ) applied to grapevine (Vitis vinifera) shoots to measure shoot hydraulic conductivity. Means ± SE (n = 3).

pressure has been shown to flush out xylem embolisms in grape shoots (Lovisolo et al., 2002). After a further 10 min at −80 kPa to allow stabilization, flow measurement was measured under −1 kPa cm−1 tension gradient. Water exuding from the cut exposed surface was collected and weighed. Specific shoot hydraulic conductivity (Kss) was calculated, following Ohm’s law, dividing flow (volume per time) by tension gradient and by the shoot cross-sectional area at the middle of the shoot portion (Lovisolo & Schubert, 1998). Kss measured after flushing out the embolisms was named flushedKss. Root hydraulic conductance (Lhr) was measured on intact root systems, freed from the pot soil as described above. Roots of intact plants were then transferred to a water basin containing aerated water, and the shoot was cut at its base underwater. The root stalk was then tightly clamped with a rubber sleeve. A −80 kPa suction was applied for 10 min to allow stabilization, then flow was measured for further 5 min at the same tension. Lhr was calculated by dividing flow by applied tension. Mercuric inhibition of water flow In a preliminary experiment, the effects of different concentrations of HgCl2 on shoot specific conductivity and root hydraulic conductance of irrigated grapevines and their reversibility following β-mercaptoethanol (β-ME) treatment, as mercuric scavenger, were studied. For shoot conductivity, the concentrations of HgCl2 tested were: none (control); 0.1 mM, according to the experiences of Wan & Zwiazek (1999); Siemens & Zwiazek (2003) on aspen; 0.25 mM; 0.5 mM, according to the experiment of Maggio & Joly (1995) on tomato; and 1.0 mM. Roots of intact plants were freed from the pot soil as described above, then submerged in the HgCl2 solution for 2 h, while plants were allowed to transpire freely. After this 2-h period the solution bathing the roots was replaced with aerated water, where roots were left for 20 h. At the end of this incubation period, shoots were

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Fig. 2 Rehydration procedures applied to irrigated control plants (a,b) and to water stress/rehydration-treated (WS/R) grapevine (Vitis vinifera) plants (a–d) during 24 h at the end of an 8-d water-stress period. Treatments with water (black bars); 0.5 mM HgCl2 (light grey bars); 60 mM β-ME (dark grey bars). Closed bars, treatments to roots; open bars, treatments to shoots.

detached from the roots, and Kss measured as described. The solution in the pressure chamber was then susbstituted with 60 mM β-ME, and a further 15 min −80 kPa tension applied to absorb the mercuric scavenger into the shoot portion. The basal end of the shoot was then left submerged in the β-ME solution for 2 h while the shoot portion transpired freely, and the Kss measurement at −80 kPa was then repeated and named MEK . ss Root hydraulic conductance was assessed after treatment with either water (HgCl2 0 mM) or HgCl2 0.5 mM. Roots were detached from shoots at the end of the 20-h incubation in water, and Lhr was measured. Detached roots were then incubated for 2 h in 60 mM β-ME after application of −80 kPa tension through the root stalk for 15 min, and the Lhr measurement was then repeated and named MELhr. Irrigated control plants Eight days after start of the WS/ R experiment, roots of irrigated control plants were gently freed from the pot soil underwater and submerged in a 0.5 mM HgCl2 solution for 2 h, while plants were allowed to transpire freely. At the same time (approx. 10 : 00 h), Hg-untreated roots were submerged in water. Then plants were kept with their roots incubated in aerated water for 22 h (Fig. 2a,b). Shoot conductivity and root conductance measurements on detached shoots and roots were taken at the end of the 8-d period from start of the experiment (contemporary to end stress in WS/R plants) and at the end of the (2 + 22)-h incubation in aerated water (contemporary to end rehydration in WS/R plants). At the same times, measurements of water relations and in vivo calculations of shoot conductivity were carried out on Hg-untreated separate plants. WS/R plants At the end of the 8-d water-stress treatment, roots of WS/R intact plants were freed from the pot soil and submerged in either the a 0.5 mM HgCl2 solution or water,

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as described for irrigated control plants. Plants were then subjected to rehydration for 22 h by keeping the roots submerged in aerated water (Fig. 2a,b). In an aliquot of Hg-treated plants, reversibility after the 0.5 mM HgCl2 treatment was checked using 60 mM β-ME (Maggio & Joly, 1995). β-ME treatment was applied either to intact plants through the root at the beginning of the rehydration period, or to both the excised shoot through the basal cut surface and the excised root at the end of the rehydration period. In the former case, the HgCl2 solution submerging the root of intact plants was replaced with the β-ME solution for a further 2 h, then rehydration continued in aerated water for 20 h (Fig. 2c). In the latter case, at the end of the rehydration treatment in aerated water, shoots were detached from roots and both roots and shoot basal ends were incubated in β-ME solution for 2 h, according to the procedure described in the preliminary experiment (Fig. 2d). MEKss was assessed after both types of β-ME treatment, while MELhr was assessed only after treatment D. Hydraulic measurements and calculations were taken at the end of the 8-d water-stress period and after rehydration, as in irrigated controls. Mercuric content in roots and shoots of Hg-treated plants To check the concentrations of Hg, root and shoot tissue samples of approx. 10 g FW were taken from both irrigated control and WS/R plants at the end of the 2-h period of root Hg feeding. Additional shoot samples were collected at the same time from WS/ R, Hg-untreated plants to check for environmental Hg contamination. Root and shoot samples were washed accurately, then reduced to small pieces using a scalpel, and digested in 6 ml 65% HNO3 and 30% H2O2 at 180°C in a closed vessel at 15 MPa internal pressure for 30 min. The digested extract was taken to 50 ml with distilled water, then Hg concentration in the extract was assessed using a Perkin-Elmer 4100 ZL atomic absorption spectrometer according to the method described by Minoia & Caroli (1989). Experimental design and statistical analysis Three replicate plants per treatment were used for the preliminary experiments designed to test the effects of either different tension gradients (three treatments) or increasing HgCl2 concentrations and β-ME reversibility (five treatments for Kss; two for Lhr) on shoot conductivity. For in vivo measurements and calculations, six replicate plants per water treatment (irrigated control and WS/R) were used. Six of these (three per treatment) were used for measurements at the end of the 8-d drought period, and six more at the end of the following 24-h rehydration period. For measurements on detached roots and shoots, three replicate plants per water


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Fig. 3 Shoot specific conductivity (Kss, a) and root hydraulic conductance (Lhr, b) measured on irrigated control grapevine (Vitis vinifera) plants. Light grey bars (HgKss and HgLhr), measurements taken on plants treated with HgCl2 at increasing concentrations before 60 mM β-mercaptoethanol treatment (–ME). Dark grey bars (MEKss and MELhr), measurements performed on Hg-treated plants after 60 mM β-mercaptoethanol treatment (+ME). Means ± SE. Bars with the same letter are not significantly different at P < 0.05 (n = 3).

treatment (irrigated control and WS/R, both at end of stress period and after rehydration) and per Hg/β-ME treatment (eight treatments) were used. Three separate replicate plants per treatment were used for Hg determination. Experiments were laid down following a randomized design. Results were submitted to one-way ANOVA followed by a Duncan’s test of differences between the means.

Results Mercury inhibition of water flow in grapevine shoots In order to choose an HgCl2 concentration that affected water flow in irrigated grapevine shoots reversibly, we tested four increasing concentrations. Treating the shoots of Hg-untreated plants with β-ME 60 mM did not influence shoot specific conductivity (MEKss = 1.04 Kss). Feeding plants with 0.1 and 0.25 mM HgCl2 did not influence Kss, nor could an effect of β-ME be observed. In contrast, 0.5 mM HgCl2 caused a significant decrease of Kss (approx. 20%), which was mostly reversible after application of β-ME 60 mM (MEKss = 0.92 Kss). This concentration of HgCl2 was used for the following

Fig. 4 Water potential (Ψ) measured in soil, root-stalk, stem and leaf at the end of an 8-d water-stress period (a); and at the end of 24-h recovery after stress (b). Closed bars, irrigated control grapevine (Vitis vinifera) plants (IRR); open bars, water stress/rehydration-treated plants (WS/R). Means ± SE. For each series, bars with the same letter are not significantly different at P < 0.05 (n = 3).

experiments. The 1.0-mM HgCl2 treatment decreased Kss to a greater extent, but the effect was not reversible after application of the Hg scavenger (Fig. 3a). In roots, Lhr of plants not treated with Hg was not affected by β-ME. Treatment of the root with 0.5 mM HgCl2 caused a significant decrease in Lhr (20%), which was reversible after application of β-ME (Fig. 3b). Effects of water stress and rehydration on water potential, transpiration and in vivo calculated shoot specific conductivity We imposed on the plants a water stress/rehydration (WS/R) treatment by withholding water for 8 summer days, and immediately thereafter submerging the whole root system in aerated water for 24 h. Irrigated control plants were watered to container capacity. As expected (Lovisolo et al., 2002), at the end of the water-stress period, water potential decreased in soil, root-stalk, shoot and leaves, compared with irrigated controls (Fig. 4a), while at the end of the rehydration period it had increased again to values comparable with irrigated controls (Fig. 4b). Plant transpiration (Eplant) was nearly constant throughout the experiment in control plants, while it decreased during the water-stress period and completely recovered at the end of the rehydration period (Fig. 5a).



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Fig. 5 (a) Whole-plant transpiration (Eplant) and (b) in vivo calculated shoot specific conductivity (calcKss), measured on grapevines (Vitis vinifera) during an 8-d water-stress period (30 June−8 July), followed by a 24-h rehydration period (9 July). , irrigated control plants (IRR); , water stress/rehydration-treated plants (WS/R). Means ± SE. Symbols with the same letter are not significantly different at P < 0.05 (n = 6).

In vivo calculated shoot specific conductivity (calcKss) was nearly constant in irrigated control plants during the experiment, as in the case of Eplant. In contrast, water stress markedly affected calcKss, which decreased to 24.3% of values measured in control plants. Rehydration allowed water-stressed plants to restore conductivity close to the level of control plants (Fig. 5b). Effects of Hg treatment on the recovery of shoot specific conductivity upon rehydration To assess the relevance of Hg-sensitive channel-mediated water transport for hydraulic conductivity recovery in shoots, we subjected water-stressed plants to a rehydration treatment, after treatment with or without HgCl2. In plants not treated with Hg, measurement of specific conductivity in excised plant shoots confirmed the trend observed with in vivo measurements. In irrigated control plants, Kss remained stable during the course of the experiment. In WS/R plants, after the 8-d water-stress period, Kss decreased to 31.9% of irrigated controls, while rehydration allowed a recovery to 80.3% of the conductivity of irrigated control shoots. These changes in conductivity were likely to depend on shoot xylem embolization: in irrigated control plants, pressure flushing did not increase shoot specific conductivity (flushedKss), while in water-stressed and rehydrated New Phytologist (2006) 172: 469– 478

Fig. 6 Shoot specific conductivity (Kss) measured on irrigated control grapevine (Vitis vinifera) plants (IRR) and on water stress/ rehydration-treated plants (WS/R) at the end of an 8-d stress period and at the end of a subsequent 24-h rehydration period. Filled bars, measurements performed by depressurizing the excised shoot with a vacuum application of −80 kPa at the apical cut. Open bars, measurements performed after imposing a pressure flushing of +200 kPa to the shoot (with basal cut end submerged in water) to eject out air embolisms (flushedKss). Black bars, measurements taken on Hg-untreated plants; light grey bars, measurements taken on plants treated with 0.5 HgCl2 at the end of drought stress (HgKss); dark grey bars, measurements taken on Hg-treated plants after a 60 mM β-mercaptoethanol treatment (MEKss) to the root (rehydration procedure C in Fig. 2), and shoot (rehydration procedure D in Fig. 2). Upper-case letters (A–D) refer to rehydration procedures in Fig. 2. Means ± SE. Bars with the same letter are not significantly different at P < 0.05 (n = 3).

plants it allowed a recovery of flushedKss to values similar to those in irrigated controls. This meant that conductivity values observed after pressure flushing were similar in all treatments (Fig. 6). In Hg-treated, irrigated control plants after rehydration, Kss was approx. 80% of Hg-untreated plants, confirming data obtained in the preliminary experiment; recovery after β-ME treatment was therefore not tested again in these plants. In WS/R, Hg-treated plants after rehydration, Kss was approx. 38.4% of that in Hg-untreated, irrigated controls. This means a very slight increase on the value measured at the end of water stress, suggesting an inhibition of Kss recovery during rehydration. This lack of recovery was mostly caused by an inhibition in recovery of embolisms, as shown by the fact that flushedK of Hg-treated WS/R plants at the end of recovery was ss similar to that in irrigated control plants (Fig. 6). When WS/R, Hg-treated plants were treated with β-ME, applied either to roots of intact plants at the beginning of the recovery period (rehydration procedure C, Fig. 2) or to shoot portions at the end of the recovery period (rehydration procedure D, Fig. 2), specific conductivity after rehydration (MEKss) increased by more than twice the value observed in the absence of β-ME, reaching approx. 75% of Hg-untreated, irrigated controls. The two different β-ME treatments gave results that showed no significant differences (Fig. 6).


Research Table 1 Mercury content in root and shoot of irrigated control (IRR) and water stress/rehydration (WS/R) plants treated via roots with 0.5 mM HgCl2 at the end of the water-stress period; and in shoots of Hg-untreated plants

Fig. 7 Root hydraulic conductance measured on irrigated control grapevine (Vitis vinifera) plants (IRR), and on water stress/ rehydration-treated plants (WS/R) both at the end of an 8-d-long stress period and at the end of a subsequent 24-h rehydration period. Measurements taken on Hg-untreated plants (Lhr) (black bars); plants treated with Hg at the end of drought stress (HgLhr) (light grey bars); plants after a 60 mM β-mercaptoethanol treatment (MELhr) (dark grey bars). Means ± SE. Bars with the same letter are not significantly different at P < 0.05 (n = 3).

The application of β-ME to Hg-treated, WS/R plants after rehydration reduced the differences between flushedKss and Kss at levels comparable with those observed in irrigated control plants (Fig. 6). Effects of Hg treatment on root hydraulic conductance In irrigated control plants, Lhr did not change during the course of the experiment. In WS/R plants, after the 8-d water-stress period, Lhr decreased to 42.2% of irrigated controls, while rehydration allowed a recovery to 78.4% of the conductance of irrigated control roots. In Hg-treated, irrigated control plants after rehydration, Lhr was approx. 80% of that in Hg-untreated plants, confirming data obtained in the preliminary experiment; as in the case of shoots, recovery after β-ME treatment was therefore not tested again in these plants. In contrast, in WS/R Hg-treated plants no recovery of Lhr was observed, and root hydraulic conductance remained even lower than at the end of water stress. When, after the Hg treatment, roots were incubated for 2 h in a β-ME solution, Lhr recovered to values similar to those of Hguntreated plants (Fig. 7). Mercury absorption from the root and movement to the shoot In plants not treated with HgCl2, shoot Hg content was lower than instrument sensitivity, showing an absence of environmental contamination. Mercuric content in the root did not differ in irrigated controls and in WS/R plants. In the shoot of Hg-treated plants, Hg was present at concentrations

Organ

Water treatment

HgCl2 application

Hg content (mg kg−1 FW)

Root Root Shoot Shoot Shoot

IRR WS/R IRR WS/R WS/R

Hg-treated Hg-treated Hg-treated Hg-treated Hg-untreated

1030 ± 153.8 a 1169 ± 166.7 a 30.2 ± 7.1 b 28.7 ± 14.0 b nd

Means ± SE. Numbers ± SE followed by the same letter are not significantly different at P < 0.05 (n = 3). nd, Not determined.

two orders of magnitude lower than in the root, in both control and WS/R plants (Table 1).

Discussion The drought stress applied to plants in our experiment induced a significant decrease in soil, root-stalk, stem and leaf water potentials, in calculated shoot specific hydraulic conductivity, and in plant transpiration. The rehydration treatment following drought stress caused an increase in these parameters to values similar to those observed in irrigated controls. Direct measurement of Kss on cut shoot portions confirmed the effects of drought and rehydration we had observed in vivo. The drought stress strongly increased the extent of embolisms, in agreement with results obtained on grapevine (Schultz & Matthews, 1988; Schultz, 2003). The rehydration treatment conversely acted by restoring water transport in most of the vessels that had embolized during the water-stress treatment, confirming previous results obtained on grapevine by a NMR-based technique (Holbrook et al., 2001). In plants treated with HgCl2 at the end of rehydration after drought stress, recovery of Kss was impaired, and this effect could be reversed by treatment with β-ME. The exact extent of embolism recovery that took place in these plants cannot be measured directly from our data, as their low shoot hydraulic conductivity after rewetting was caused by a mixture of hindered embolism recovery and likely reduction of the Hg-inhibited overall water transport. The process of embolism recovery after water stress has been studied in several plants at the physiological level (Salleo et al., 2004), and it has been suggested that it could entail increased aquaporin-mediated transmembrane water transport (De Boer & Volkov, 2003). Activation during embolism refilling of aquaporin genes, the products of which localize in vessel parenchyma cells, has been demonstrated (Sakr et al., 2003). In Hg-untreated, WS/R plants, Kss observed after rehydration was approx. 20% lower than observed in irrigated control



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plants after rehydration. This suggests that, in these conditions, rehydration needs longer than 24 h to be fully accomplished. However, in Hg-treated, WS/R plants, Kss after rehydration reverted following β-ME application to approx. 60% of that in irrigated plants after rehydration, both when β-ME was applied to the root (treatment C, Fig. 2) and when it was applied to the shoot (treatment D, Fig. 2). So the β-ME treatment lasted only 2 h and was sufficient to reach only a slightly (not significantly) lower recovery of Kss than observed in non-Hg-treated plants in 24 h. This suggests that unblocking of plant processes inhibited by Hg is a rather fast phenomenon, but is not sufficient to cause complete conductivity recovery. Our experiments provide evidence that Hg-sensitive water transport affects the recovery of shoot hydraulic conductivity during rehydration. In principle, recovery can be caused by root pressure pushing embolisms out of the xylem vessels, as has been reported for seasonal embolism recovery after summer drought and/or after winter freezing in woody plants such as Vitis (Sperry et al., 1987; Tibbetts & Ewers, 2000) and vinelike bamboo (Cochard et al., 1994), and trees (Sperry et al., 1988; Améglio et al., 2002; Hacke & Sperry, 2003). As an alternative, it has been shown that conductivity recovery can take place while significant tension exists in the xylem (Holbrook & Zwieniecki, 1999), suggesting mechanisms of embolism repair. According to Salleo et al. (2004), at least three mechanisms can be hypothesized for embolism reversal: the osmotic mechanism; the reverse osmotic mechanism; and phloem-driven xylem refilling. All these mechanisms suggest roles of living cells of the shoot, which can be facilitated by the presence of water channels. In our experiment, recovery of hydraulic conductivity in the shoot was affected by HgCl2 treatment to the root. Two hypotheses could explain this effect: (i) that HgCl2 inhibited cellular water channel activity and/or other metabolic process(es) at the root level, reducing root conductance, as has been shown in several papers (Tyerman et al., 2002); and/or (ii) that HgCl2 acted at the level of shoot living cell metabolism, which may be involved in facilitated water transport towards the xylem elements. The first hypothesis finds support in the impairment of root hydraulic conductance after mercuric treatment (HgLhr). Decrease of HgLhr, even if significant, was only approx. 20% under irrigated conditions; this result could have been influenced by the 20-h period of recovery, which could have been long enough even for new lateral roots to begin emerging. However, in this case we should hypothesize that the contribution of new roots in Hg-treated plants was higher than in non-Hg-treated plants. Moreover, we tested only three replicates, which may not be fully representative of putatively different root growths. Part of the decrease in HgLhr can probably be ascribed to a reduction in radial aquaporin-mediated water transport in the root. However, this suffers from the fact that sulfhydryl reagents such as HgCl2 are, in general, nonspecifically toxic, and not only in root cell membranes (Verkman, 2001).

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In contrast, despite its shortcomings, the use of HgCl2 is still considered a reliable method to block the contribution of aquaporins to water movement in plant organs in vivo, provided concentrations as low as possible are used, and that reversibility after application of a thiol reagent is demonstrated ( Javot & Maurel, 2002). In our experiment, the HgCl2 concentration that effectively and reversibly decreased shoot hydraulic conductivity was 0.5 mM. This concentration is higher than that used in similar root treatments to woody plants (aspen and elm: Wan & Zwiazek, 1999; Muhsin & Zwiazek, 2002; Siemens & Zwiazek, 2003, 2004). In these experiments, however, HgCl2 was applied to roots at 0.1 mM concentration, but water flow through the plant was forced by applying a positive pressure ranging from 0.3 to 1.0 MPa for up to 60 min. In the four experiments cited, HgCl2 caused approx. 50, 70, 15.5 and 29% reversible decrease in water flow, respectively, in the root system of seedlings of irrigated aspen (Wan & Zwiazek, 1999), irrigated elm (Muhsin & Zwiazek, 2002) and mildly stressed aspen (Siemens & Zwiazek, 2003, 2004). Based on the flow data given in those reports (approx. 2.0, 191.0, 13.2 and 5.0 × 10−8 m3 m−2 s−1), this means that approx. 7.2, 690.0, 47.5 and 18.1 µmol HgCl2 m−2 root surface had to be taken to observe this inhibition. In WS/R plants in our experiment, the HgCl2 concentration and exposure time were higher (0.5 mM and 2 h, respectively), but water flow was lower (approx. 1.63 × 10−8 m3 m−2 s−1, obtained by dividing Eplant of Fig. 4a by 0.46 ± 0.02 m2 average root surface), which resulted in approx. 58.7 µmol HgCl2 m−2 root surface being taken in order to observe a reversible inhibition of water flow. Thus, while we used higher HgCl2 concentrations than in those experiments, the actual amount of the inhibitor at root level (and, given the similarity in root anatomy in grapevine and aspen, possibly also at shoot level) was of the same order of magnitude. Moreover, the decrease in shoot conductivity after HgCl2 treatment to the root was approx. 90% reversible after treatment with 60 mM β-mercaptoethanol in both irrigated and WS/R plants. Reversibility following treatment with reducing agents is an important control of the validity of the Hg treatment, and either full or partial reversibility has been reported previously (Javot & Maurel, 2002). In the absence of HgCl2 treatment, Lhr decreased following water stress and increased again after rehydration. In contrast, in Hg-treated plants, at the end of the rehydration period Lhr was even lower than in water-stressed Hg-untreated plants, suggesting that Hg effects on reducing hydraulic conductance were more effective than effects induced by water stress. Our Lhr measurements are less confident than Kss ones, as Lhr was assessed only at −80 kPa. We did not test increasing vacuum tensions, as this effect happens in vivo when IRR situations move progressively to WS situations, so a comparison between the different water conditions could be misleading. However, effects of HgCl2 are evident in each water treatment. We can suppose that, in HgCl2-treated plants, a reduction in root


Research

conductance during rehydration may have maintained lower water potentials in shoots, hindering the recovery of hydraulic conductivity under tension in the shoot vessels; or it may have prevented positive root pressures from developing in the vessels, again hindering recovery. While grapevines show root pressure after winter freezing (Sperry et al., 1987), root pressures in grapevine have not been observed after drought stress (Holbrook et al., 2001). Moreover, root pressure is not a general phenomenon (Salleo et al., 2004). In this experiment we made no direct measurement of root pressure in our plants, although we never observed water exudation from the cut surface of shoots or root stalks. The apparent sensitivity of Lhr to Hg is clearly different between irrigated and water-stressed plants. Therefore the relative effects of Hg on Lhr are much greater in WS/R than in IRR plants. In particular, and in contrast to Kss, Lhr in Hg-treated plants is even lower than in nontreated plants at the end of water stress. If water stress had reduced Lhr via root embolism formation, also in the root, metabolic perturbations induced by Hg would have hindered embolism removal. Embolisms in roots have been detected much more rarely than in shoots, but in all studies that compared root and shoot embolisms, roots of both hardwoods (Sperry & Saliendra, 1994; Hacke & Sauter, 1996; Matzner et al., 2001) and conifers (Sperry & Ikeda, 1997; Oliveras et al., 2003) have been shown to be more vulnerable than stems to xylem cavitation. This is reasonable, as smaller safety margins from cavitation in roots may be beneficial in limiting water use during mild drought, and in protecting the stem from low xylem tensions during extreme drought (Sperry & Ikeda, 1997). The alternative hypothesis, which provides an HgCl2 effect at the shoot level, is supported by the observation that negative pressures were present in all plant organs, both after drought stress and after recovery. This means that after rehydration the stem-to-root-stalk tension gradient was approx. −0.05 MPa and the leaf-to-shoot tension gradient was approx. −0.2 MPa. Although these measurements were not made in Hg-treated plants, the observed decrease in root conductance (which is 80–90% composed of radial conductance: Martre et al., 2001; North et al., 2004) makes it unlikely that, after Hg treatment, shoots may have experienced higher water potentials. This hypothesis also requires that Hg administered to the intact root should be able to move to the shoot, as happened during the 2-h period of Hg treatment. Penetration of the Hg ion within the plant has been little studied, and it has been reported for onion that most of the Hg given to root systems is blocked at the root surface, provided roots have a suberized exodermis (Barrowclough et al., 2000). However, several plants growing on Hg-polluted soils show the presence of significant Hg concentrations in their aerial parts (Zarcinas et al., 2004; Moreno et al., 2005). Also, grapevine roots in our experiments blocked most of the Hg independently of the drought stress treatment, but approx. one-fiftieth of the mercuric content measured at the root level was present at the

shoot level. In our experiment, a further element supporting an effect of HgCl2 on shoot aquaporins is that the effects of the Hg treatment on Kss diminished when the shoot itself was directly treated with 60 mM β-mercaptoethanol (recovery option D). This suggests that Hg-sensitive metabolic processes on a short-term time scale in the shoot are basic requirements for the recovery of hydraulic conductivity during plant rehydration. In irrigated control plants, HgCl2 reduced shoot hydraulic conductivity significantly by approx. 20%. This may suggest that Hg-inhibited water transport in grapevine shoots may, in principle, contribute to 20% of overall water transport (irrigated control plants did not show any significant extent of embolization). More precisely, only about half of that 20% of water transport showed reversibility after β-ME treatment and can be explained by blocking of Hg-sensitive processes affecting water transport, while the remaining 10% has to be attributed to the general, irreversible toxicity of the Hg treatment. However, it is possible to speculate that, in Hg-treated irrigated plants, the 20% reduction of Kss recovered after flushing. Thus Kss reduction induced by Hg is not easily attributable to an impairment of overall water transport, but rather to an Hg-induced perturbation of short-time tension– pressure imbalances causing cycles of embolism and their partial refilling (McCully, 1999; Bucci et al., 2003), also subsisting under irrigated conditions.

Acknowledgements We thank S. Bragagnolo, S.F. Martinez, T. Strano and S. Tramontini for help in executing hydraulic measurements, and P. Doria and F. Fracchia for chemical analysis. We thank P.M. Neumann, S. Salleo, H.R. Schultz and J.J. Zwiazek for critical observations on preliminary results, and three anonymous reviewers for helpful suggestions about this manuscript.

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