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In xylem tissues of plants, water flows under negative pressure along the pathway from root tips to stomata (Pockman et al., 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 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 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.
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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 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 vine-like 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). 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 mol 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 mol 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 mmβ-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 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 mmβ-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.