Refilling embolized xylem conduits: Is it a matter of phloem unloading?

Plant Science 180 (2011) 604–611

Contents lists available at ScienceDirect

Plant Science journal homepage: www.elsevier.com/locate/plantsci

Review

Refilling embolized xylem conduits: Is it a matter of phloem unloading? Andrea Nardini a,∗ , Maria A. Lo Gullo b , Sebastiano Salleo a a b

Dipartimento di Scienze della Vita, Università di Trieste, Via L. Giorgieri 10, 34127 Trieste, Italy Dipartimento di Scienze della Vita, Università di Messina, Salita Sperone 31, 98166 Messina, S. Agata, Italy

a r t i c l e

i n f o

Article history: Received 13 October 2010 Received in revised form 17 December 2010 Accepted 29 December 2010 Available online 12 January 2011 Keywords: Xylem Embolism Refilling Phloem Aquaporins Sugars

a b s t r a c t Long-distance water transport in plants relies on negative pressures established in continuous water columns in xylem conduits. Water under tension is in a metastable state and is prone to cavitation and embolism, which leads to loss of hydraulic conductance, reduced productivity and eventually plant death. Experimental evidence suggests that plants can repair embolized xylem by pushing water from living vessel-associated cells into the gas-filled conduit lumina. Most surprisingly, embolism refilling is known to occur even when the bulk of still functioning xylem is under tension, a finding that is in seemingly contradiction to basic principles of thermodynamics. This review summarizes our current understanding of xylem refilling processes and speculates that embolism repair under tension can be envisioned as a particular case of phloem unloading, as suggested by several events and components of embolism repair, typically involved in phloem unloading mechanisms. Far from being a challenge to irreversible thermodynamics, embolism refilling is emerging as a finely regulated vital process essential for plant functioning under different environmental stresses. © 2011 Elsevier Ireland Ltd. All rights reserved.

Contents 1. 2. 3. 4. 5. 6.

Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Dynamics of embolism reversal . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Possible mechanisms for xylem refilling . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . A plausible synthesis: xylem refilling as a particular case of phloem unloading . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . The signal triggering xylem refilling is still unknown . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Conclusions and future perspectives . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

1. Introduction According to the cohesion-tension theory (CTT), water is transported from roots to leaves through the xylem in continuous water columns under negative pressure (= tension). Under such conditions, water is maintained in liquid phase while below its vapour pressure, i.e. water is in a metastable state [1]. Despite some vigorous challenges to its basic assumptions [2–5], the CTT has repeatedly proved to be a robust theory that can parsimoniously explain available experimental data on plant water transport [6–11]. Water in the metastable state is subject to cavitation-induced embolism, implying a sudden transition to the vapour phase. Xylem embolism is widely thought to be caused by gaseous bubbles being aspirated into xylem conduits from adjacent gas-filled com-

∗ Corresponding author. E-mail address: [email protected] (A. Nardini). 0168-9452/$ – see front matter © 2011 Elsevier Ireland Ltd. All rights reserved. doi:10.1016/j.plantsci.2010.12.011

604 605 606 606 607 609 609

partments through pit membrane pores, a process known as ‘air seeding’ [12]. This process results in conduits filled with water vapour that can eventually be substituted for air. Embolism can also be induced by freeze–thaw events causing gases to escape from solution during freezing and expanding into the conduit during thawing [13]. Gas-filled conduits are no longer conductive and hence xylem embolism causes a reduction of xylem (and plant) hydraulic conductance [14,15]. In turn, reduced hydraulic conductance of xylem may lead to a drop of leaf water potential, loss of turgor and stomatal closure, with consequent impairment of photosynthesis and productivity [16,17]. Xylem embolism is far from being a rare event, because this phenomenon has been commonly detected even in well watered plants as a consequence of dynamic daily water stress conditions [18–20]. Plants may be able to compensate for embolism-induced loss of hydraulic conductance through ion-mediated up-regulation of the hydraulic conductance of still functioning conduits [21–23]. Nonetheless, day-by-day accumulation of embolism under pro-

A. Nardini et al. / Plant Science 180 (2011) 604–611

longed drought conditions would finally lead to extensive failure of xylem water transport and plant death [24–26]. The threat that xylem embolism poses over survival can be faced by plants adopting one or more of three strategies, not alternative to one another: (a) embolism avoidance, requiring a tight control of xylem pressure; (b) embolism reversal, requiring refilling with water of gas-filled conduits; (c) production of new xylem, requiring carbon investment for growth processes. Several plants avoid xylem embolism through stomatal control of xylem pressure [15,17,27]. Reversal of xylem embolism has also been reported in several species where refilling of gas-filled conduits is driven by over-atmospheric root/stem pressures originating during the night or in specific periods of the year [28–32]. Embolized conduits can refill in absence of root pressure and with still substantially negative xylem pressures in adjacent functioning conduits [33–37], which might seem contradictory to the basic principles of irreversible thermodynamics. According to bubble dynamics theory, spontaneous dissolution of emboli in liquids should not be possible until xylem pressures rise to positive values [38]. The exact threshold pressure for embolism reversal would be dependent on sap gas content and bubble radius. The minimum xylem pressure (Px ) that must be exceeded for reversal of embolism to occur (Pxr ) can be calculated on the basis of the Young–Laplace equation: Pxr = Pgas −

 2T  r

where Pgas equals the sum of the partial pressure of gases filling the embolized conduit, T is the surface tension of water and r is the radius of the bubble (which can be roughly assumed to equal that of the conduit). Hence, the pressure required for spontaneous embolism dissolution is predicted to be higher for large conduits than for narrow ones. However, in contrast to the theory, intact plants can reverse embolism even when Px in still functioning conduits is lower than Pxr [39–42]. The term ‘novel refilling’ was proposed to indicate this process [36] as an alternative mechanism to the ‘expected’ refilling process. In the case of ‘novel refilling’, embolized conduits are thought to be pressurised independently of negative pressures (tensions) developing in neighbouring functioning conduits. This is possible only if embolized conduits remain hydraulically isolated from adjacent conduits under tension until refilling is complete, otherwise water flowing into the refilling conduit would immediately be drawn by still functioning ones at lower pressure. A mechanism was proposed of hydraulic isolation of embolized conduits, based on the chemistry of conduit walls and the geometry of interconduit pits that would permit positive pressures to develop and emboli to dissolve [43,44]. Further studies [45–47] have confirmed the importance of pit geometry for the hydraulic isolation of embolized conduits. Readers are referred to the above cited papers for detailed information on the nature and consequences of hydraulic isolation of embolized conduits. Xylem refilling under negative pressure has been observed both in Angiosperms and in Gymnosperms [48,49]. Although the basic processes of ‘novel refilling’ in both groups may be similar in many respects, this review will specifically focus on data gathered in Angiosperms and will summarize recent advances in our understanding of xylem refilling under tension, as related to: (a) the dynamics of embolism reversal; (b) the generation of the necessary driving force for refilling; (c) the role of phloem as a source of radial pressure directed to the xylem; (d) the possible role of aquaporins as modulators of the refilling process; (e) the possible signal activating embolism refilling. Future perspectives of research on xylem refilling are highlighted with special reference to the most important unanswered questions that need specific further attention.

605

2. Dynamics of embolism reversal The physical constraints on the refilling of an embolized conduit when surrounded by functioning conduits under tension have been detailed in an elegant study [46] where the relationships between the time needed to refill an embolized conduit and the surrounding physico-chemical conditions have been carefully analyzed. Theoretical simulations suggested that the refilling time for an embolized conduit can vary between 10 min to more than 10 h, depending on conduit size, differences in the osmotic pressure of fluids in the refilling conduits and that in surrounding living cells, hydrostatic pressure in still functioning conduits, reflection coefficient and hydraulic conductivity of the refilling/living cell interface. Moreover, the dynamics of embolism reversal is probably different when single embolized conduits are refilled while surrounded by still conductive xylem or when whole embolized stems are recovered to their functional status. In any case, the knowledge of the typical refilling time of embolized conduits in planta is the key to further predict the physical and physiological conditions that must be fulfilled to meet the observed refilling times. This, in turn, would greatly help confirm or discard hypotheses relative to the origin and magnitude of necessary driving forces and water pathway(s) for refilling. Original reports of ‘novel refilling’ [33] suggested that embolism reversal under tension was a very rapid phenomenon, occurring within 20 min after inducing xylem embolism by external airpressurization of stems. This experimental procedure induces air seeding through interconduit pit membranes [12] in a similar fashion of what is expected to occur in response to critical tensions inside xylem conduits. Other studies have reported short-term changes in the percentage of embolized conduits as detected on the basis of Cryo-SEM observations of stem and root samples collected from transpiring plants [50–53]. In summary, early studies suggested that embolism reversal under tension was a rapid phenomenon, developing over a time scale of minutes to hours. Later studies doubt the shortest refilling times, and suggest that they might be biased by experimental artefacts arising either from positive pressure-driven embolism induction [36] or from freezing of water under tension in xylem conduits of transpiring plants [54]. Embolism reversal in field-grown plants of Laurus nobilis L. under xylem hydrostatic pressure between −0.5 and −1.0 MPa required about 15 h to be completed [42]. Hence, air-pressure applied to stems of plants might lead to underestimating refilling times. This non-exhaustive review of some recent studies highlights the fact that typical refilling times in planta are still far from being well characterized. Moreover, methods quantifying the refilling time on the basis of changes in percentage loss of stem hydraulic conductivity may prove to be of little help because the concurrent refilling of several conduits with different diameters does not allow the observed refilling times to be related to the putative driving forces involved. A much more promising approach to the description of embolism reversal dynamics is based on imaging techniques that monitor the status of single vessels under different physiological conditions. Both magnetic resonance imaging (MRI) and synchrotron X-ray imaging have been used to this aim. MRI was used to obtain high spatial and temporal resolution images of embolized and refilling xylem conduits [55]. In accordance with observations previously reported for Vitis vinifera [56], it was shown that refilling of embolized conduits occurred only under low-light conditions, i.e. under low transpiration. Although MRI is certainly a powerful technique to investigate refilling of xylem conduits, this experimental approach suffers from being based on induction of cavitation and stimulation of refilling by removing or adding the nutrient solution bathing the root system of young maize plants [55], or by dehydrating and then rewatering a potted plant [56]. Under these conditions,

606

A. Nardini et al. / Plant Science 180 (2011) 604–611

actual xylem hydrostatic pressure during refilling was probably quite high and raising of root pressure could not be ruled out. In fact, refilling of embolized conduits of V. vinifera was observed at leaf water potential of −0.25 MPa, and xylem pressure was probably even higher [56]. Hence, these interesting results do not solve the issue of typical refilling times under substantial negative pressures in the xylem. The potential of synchrotron 2D X-ray imaging technique for in vivo monitoring sap dynamics in xylem conduits was recently highlighted [57]. In this case, refilling of some embolized conduits was observed while adjacent ones where under relatively high hydrostatic pressure. Nonetheless, the technique provided some very interesting insights into the refilling process. As an example, sap flow dynamics in refilling conduits suggested that perforation plates of rice leaf xylem offers a substantial resistance, requiring a threshold water pressure to be exceeded before the sap can move from a refilling conduit to the next one still embolized. This observation is in accordance with the idea of the hydraulic isolation of embolized conduits [43,44]. The potential of 3D High Resolution Computed Tomography (HRCT) for in vivo monitoring of embolism repair has been recently demonstrated [58]. This technique, which is also based on a synchrotron X-ray light source, allows visualization of water droplets apparently exiting from vasicentric cells and entering embolized vessels of V. vinifera. The droplets expand over time and refill vessels while forcing the dissolution of entrapped gases. Although water droplets entering refilling conduits had been already reported in previous studies [34], convincing evidence for the role of living cells as sources of water needed for refilling was still missing. The typical refilling time was further characterized and calculated to range between about 2 and 17 h for vessels with a diameter of 20 and 150 ␮m, respectively. Moreover, embolism reversal was observed at substantially negative stem water potentials ranging between −0.45 and −0.75 MPa. We feel that high resolution imaging techniques based on MRI, synchrotron X-ray microscopy or HRCT will be fundamental to measure the typical refilling times of xylem conduits under well defined conditions. The most urgent point, in our opinion, is to carefully measure or, better, control the hydrostatic pressure of functioning conduits during the refilling process. This could be achieved by placing the root systems of the plants used for experiments in polyethylene glycol (PEG) solutions at different concentrations, an experimental set-up already used to study refilling under tension in sunflower leaf veins [41], that allows to set the maximum pressure that can be reached in xylem conduits after equilibration of plant water potential with soil/solution water potential. Such an experimental set-up, when combined with in vivo high resolution imaging techniques, would probably allow comparing the refilling times of conduits with known diameters to theoretical predictions [46], thus significantly contributing to a better understanding of the magnitude of the necessary driving forces. 3. Possible mechanisms for xylem refilling Three main hypotheses have emerged for the generation of positive pressures required to refill embolized conduits: (1) the osmotic hypothesis; (2) the reverse osmotic hypothesis; (3) the phloemdriven refilling hypothesis. The osmotic hypothesis proposes that the driving force for refilling embolized conduits is generated by enrichment of the thin sap layer remaining at the inner conduit wall with solutes. This would lower the osmotic potential in the embolized conduit and cause water to flow into the conduit under positive pressure [36,59–62]. The membranes of living cells surrounding the embolized conduit (vessel-associated cells, VAC) would represent the differentially

permeable barriers allowing the osmotic pressure to build up. The proposed nature of solutes involved ranges from inorganic ions [34] to sugars derived from hydrolysis of starch contained in parenchyma cells [35,37,63], and also to large organic solutes [36] like proteins or polysaccharides [64] secreted into the xylem from neighbouring tissues. In this last case, the solutes involved would be too large to freely move across the pit membrane, which would act as the differentially permeable barrier [65] allowing positive pressures to build up in the refilling conduit. This possible mechanism is known as the ‘pit membrane osmosis hypothesis’ [36]. Attempts at estimating the actual solute concentration in refilling conduits have been performed using X-ray microanalysis techniques. Observations failed to reveal solute concentration high enough to sustain the refilling process [34,51,52]. Because analyses where focused on mineral elements, these data apparently discard the role of inorganic solutes in the refilling process, while leaving open the possibility that sugars and/or organic macromolecules act as osmotically active solutes during embolism repair. An input of solutes into the refilling conduit is not predicted by the reverse osmotic hypothesis [2,3]. According to the proposed mechanism, VAC would lower their own osmotic potential through starch-to-sugar hydrolysis. This is supposed to cause water to flow into VAC thus raising their turgor pressure. This would generate a ‘tissue pressure’ that would be contained within the stem by tissues external to VAC. Tissue pressure would squeeze water from other living cells and squeezed water would flow under positive pressure to the embolized conduits and refill them. A number of objections have been raised against this hypothesis, which does not appear realistic because parenchyma cells would have to take up water via mass flow to raise and maintain cell turgor and at the same time deliver water via mass flow to refill conduits [8,34]. The involvement of phloem in the repair of embolized xylem conduits was first suggested on the basis of the observed inhibition of xylem refilling in girdled stems [33] and later confirmed by other studies [35,66,67]. The inhibitory effect of girdling on embolism repair was suggested to derive from dissipation of phloem positive pressures [37]. In its original formulation, this hypothesis predicted that an auxin-mediated signal increased phloem loading with solutes, thus increasing phloem pressure and driving water transport to the embolized conduits. This hypothesis received further support from experiments on field-grown laurel plants [42]. Embolized stems with phloem first inactivated by girdling and then subjected to external radial pressure to simulate native conditions, recovered from embolism like intact ones. 4. A plausible synthesis: xylem refilling as a particular case of phloem unloading There is evidence for a role of wood parenchyma in xylem refilling. Experimental data support the hypothesis that embolized conduits can be restored to their functional state through inward solute transport operated by VAC [37]. This process apparently involves specific H+ /solute co-transporters [68] as energised by the plasma membrane H+ -ATPase [34,69,70]. In fact, treatments inducing inhibition of the proton pump led to inhibition of xylem refilling [37]. Altogether, these findings support the hypothesis that refilling is based on osmotic water flow driven into embolized conduits after secretion of solutes that would generate the necessary driving force for refilling (see above). Other studies have suggested that soluble sugars would be the solutes involved in the refilling process [63], as derived on depolymerisation of starch stored in xylem parenchyma [3,35,71]. There is a close, linear relationship between changes in the amount of starch in VAC and changes in percentage loss of hydraulic conductivity [37]. Starch is apparently depolymerised during xylem refilling, and treatments that inhibit refilling also block starch

A. Nardini et al. / Plant Science 180 (2011) 604–611

depolymerisation both in the lab and in field-grown plants [42]. Starch accumulation is apparently a consequence of diurnal photosynthesis and a pre-requisite for refilling to occur in drought stressed plants. Thus, it was proposed that starch depolymerisation caused sugars to be loaded into cavitated conduits, thus generating an osmotic driving force for refilling. In turn, the decrease in starch content of VAC would cause these cells to become strong sinks to phloem with consequent unloading of sugars and water from the phloem and a bulk flow directed to the refilling conduits. In this view, refilling of embolized xylem conduits should be considered as a specific case of phloem unloading where the final sink is represented by embolized xylem conduits. The proposed scenario for embolism repair is actually a mix of the osmotic hypothesis and the phloem-driven refilling hypothesis (see above) and is schematically illustrated in Fig. 1. Basically, this putative refilling mechanism is similar to other scenarios previously suggested [72], where it was shown that water moving from the phloem to the xylem can induce embolism refilling, especially if phloem unloading is accompanied by sugar entry into the embolized xylem conduits [63]. A requirement for concurrent aquaporin up-regulation in the phloem-to-xylem pathway can also be postulated, to increase the radial hydraulic conductivity up to embolized xylem conduits and significantly reduce the time required for embolism refilling. In a recently described model for embolism reversal, it has been postulated that there is no need for hydraulic isolation of embolized conduits [44], provided that the amount of water entering the refilling conduit exceeds the water volume concurrently drawn from still functioning conduits in direct contact with the refilling one [58]. Aquaporin up-regulation would probably facilitate the release of adequate water volumes to refill the conduit while keeping pace with water uptake from adjacent conduits. If refilling of embolized conduits is a specific case of phloem unloading, then useful insights into this phenomenon can be gained by a careful analysis of well known cases of phloem unloading. As an example, accumulation of sugars in the sink apoplast is mediated by a plasma membrane sugar/proton co-transport [73], which fits with the important role of proton pumps in the refilling process [37,69] and with localization of these transport proteins in VAC [74]. Also, it is known that apoplastic phloem unloading is facilitated by the expression and/or activation of aquaporins [75]. The water channel proteins (aquaporins) plays a central role in cell and plant water balance [76,77]. Molecular and microscopic studies have revealed that aquaporins are highly expressed in the plasma membranes of VAC [78,79]. An involvement of aquaporins in plant recovery after drought stress has been proposed [80,81], and two recent studies have highlighted the potential role of these water channels in xylem refilling [82,83]. In particular, high levels of expression of two PIP2 isoforms were reported in walnut VAC during the recovery from freeze-induced embolism, simultaneously with a substantial starch-to-sucrose conversion in these cells [61,82]. These results are in surprisingly good agreement with the view of xylem refilling as a particular case of phloem unloading. Even more detailed information about the involvement of aquaporins in the refilling process has been obtained in Populus trichocarpa, where PIP1 aquaporins were up-regulated under drought stress conditions with a diurnal pattern that paralleled the embolism-refilling cycle [83]. Two genes coding for aquaporins, PoptrPIP1.1 and PoptrPIP1.3 were up-regulated in response to embolism formation in xylem conduits, independently of the general plant water status, and they were down-regulated upon embolism removal. Again, this pattern is consistent with the idea that aquaporins play a role in xylem refilling and with the more general hypothesis that the ‘magic recipe’ for xylem refilling is not substantially different from the one needed for efficient phloem unloading.

607

Although the above studies have provided convincing experimental evidence for the involvement of aquaporins in embolism reversal, eventual changes in localization of these membrane proteins during xylem refilling still needs attention. In fact, according to the ‘phloem unloading model’, water would flow from the phloem through living cells down to the embolized conduits. In this case, it would be reasonable to expect membrane hydraulic conductivity to increase along the entire phloem-to-xylem pathway, as this would greatly facilitate the refilling process [72]. On the contrary, if aquaporin expression were located specifically and exclusively at the level of VAC, the water volume needed to refill the conduits would be most likely provided by these cells. This conclusion is not very plausible. As an example, woody tissues have a high modulus of elasticity, implying that volume changes of more or less 0.1% can be expected for a 1 MPa water potential drop [84]. In woody stems, vessel lumina account for about 20% of total volume [29]. Hence, if 80% of the stem volume is filled with living cells, and even if these cells lose 0.3% of their volume (corresponding to a 3 MPa change in their turgor pressure), only 2.4% of the vessel volume could be refilled [34]. It is clear that this water volume would be too small to assure the refilling of embolized conduits, beside the fact that parenchyma cells could not sustain a 3 MPa water potential drop without undergoing complete turgor loss. On the contrary, ‘Münch water’ recirculated between functioning xylem conduits and phloem would probably assure the necessary water volume needed for xylem refilling [72]. Immunolocalisation of aquaporins [85] during refilling would improve our understanding of the process, especially to elucidate the main water pathway for embolism refilling and the eventual ‘directionality’ of water flow. This might be assured not only by water potential gradients but also by ordered and polar localization of aquaporins down-stream from phloem to embolized conduits through living cells. 5. The signal triggering xylem refilling is still unknown Several lines of evidence suggest that xylem embolism can be sensed by plants and reversed independently of the general plant water status. As an example, embolism induced by air-pressurization was apparently repaired in laurel plants experiencing xylem pressure potentials ranging from 0 to −1.5 MPa [33]. Similarly, embolism reversal occurs even in well watered poplar plants induced to cavitate by air-injection [83]. The ability of plants to sense a local embolism is of physiological importance because the spatial pattern of embolism formation in the xylem can be quite random [56], so that refilling processes need to be activated only where they are really needed to repair gas-filled conduits. This implies a signal-to-response pathway including perception of a local ‘cavitation alarm’ and a down-stream cascade of events leading to embolism repair. Water cavitation in a xylem conduit is probably a critical event, implying sudden expansion of a gas phase within a volume of water under negative pressure. Such an ‘explosive’ expansion of air bubbles causes conduit walls to vibrate, thus delivering energy that can be monitored as acoustic emissions in both sonic and ultrasonic range [86,87]. Ultrasound acoustic emissions can be detected and used to monitor cavitation events in a non-destructive way [88,89]. The hypothesis that conduit wall vibrations can also represent a mechanical signal initiating the refilling process was tested. Exposing water stressed laurel twigs to ultrasounds significantly stimulated both starch depolymerisation and xylem refilling, although no effect of ultrasound on starch metabolism was observed in the case of well watered plants [90]. This suggests that the mechanical stimulus produced by vessel wall vibrations may be one of the components of the signalling pathway leading to embolism perception and repair.

608

A. Nardini et al. / Plant Science 180 (2011) 604–611

Fig. 1. Putative chain of events linking sensing of cavitation in xylem to embolism repair. The example refers to hypothetical embolism formation/reversal in a single vessel of secondary xylem connected to phloem via rays. Xylem cavitation (1) produces vessel wall vibrations (2) which are transmitted to adjacent vessel-associated cells. The mechanical stimulus induces opening of mechano-sensitive Ca2+ channels located on the plasma membrane and/or on endomembranes. Increase of intracellular [Ca2+ ] (3) activates a signal transduction cascade leading to changes in starch metabolism. Starch is depolymerised and simple sugars are loaded into the embolized conduit (4) thus lowering the osmotic potential of the thin layer of sap still adhering to the inner conduit wall. The osmotic potential inside the refilling conduit represents the first component of the driving force for embolism reversal. Vessel-associated cells become strong sinks to phloem inducing unloading of sugars and water (5). Solutes (red arrows) and water (blue arrows) are delivered from the phloem to the embolized conduit through the rays. Positive phloem pressure represents the second component of the driving force necessary to xylem refilling. Phloem-to-xylem hydraulic conductivity is eventually enhanced by activation and/or expression of aquaporins in ray cells (6). Water under positive pressure enters the embolized conduit and gradually refill it, while compressing and dissolving the entrapped gas. The refilling conduit remains isolated from adjacent functioning ones due to morphological features and biochemical composition of interconduit pits (see text for details).

Independently on vessel wall vibrations, however, cavitation events may produce important mechanical stress on the cell walls and membranes of living cells surrounding cavitating xylem conduit. When water cavitates in a conduit of a transpiring plant, the pressure inside the conduit suddenly rises from substantially negative values (e.g. −0.8 to −1.2 MPa [18]) to the saturated water vapour pressure (+2.33 kPa at 20 ◦ C referred to vacuum, or −97.7 kPa if referred to atmospheric pressure). Hence, a transient pressure change of about 1 MPa can be expected to develop in the proximity of VAC, inducing mechanical stress on cell membranes either directly because of the change of pressure in the surrounding apoplast or indirectly because of predictable rapid changes in the water potential and turgor pressure of parenchyma cells. The hypothesis that a mechanical signal produced by cavitating conduits can be perceived by VAC is somehow supported by similar cases of perception of mechanical stimuli [91,92]. As an example, mechanosensitive Ca2+ -permeable channels are involved in the fine regulation of stomatal aperture [86], as well as in perception of salinity stress in Chara cells [93]. It is well known that various extracellular stimuli are translated into specific calcium ‘signatures’, i.e. transitory changes in intracellular [Ca2+ ] that elicit specific physiological responses [94]. Most importantly, calcium signalling regulates starch metabolism in aleurone cells [95] and oat shoot pulvini [96]. Interestingly, auxins are an important component of the signalling pathway leading to regulation of starch metabolism in several physiological processes [97,98], and this puts into new light the observation that exogenous auxins significantly stimulated the extent of the refilling process [34].

The above data stimulate exciting speculation about the possible chain of events linking xylem cavitation perception to refilling, as summarized in Fig. 1. According to our hypothesis, water cavitation would produce a mechanical stimulus either directly (vessel wall vibrations) or indirectly (turgor changes in VAC). The mechanical stimulus would be translated into opening of mechanosensitive Ca2+ channels located on the plasma membrane or endomembranes of VAC. Changes in cytosolic [Ca2+ ], would trigger a signal transduction cascade finally leading to starch depolymerisation into soluble sugars. The sugars would be loaded into the cavitated conduit with participation of proton pumps and sugar/H+ co-transporters. Sugar accumulation in the thin layer of sap remaining in the cavitated conduit would in turn lower the osmotic potential inside the conduit, sufficiently to generate the first component of the necessary driving force for refilling. Sugar export from VAC would cause these cells to become strong sinks, thus favoring phloem unloading of solutes and water toward the VAC and embolized conduits. Positive phloem pressures would therefore represent the second component of the driving force for refilling. Water flow from the phloem to the embolized conduit would be facilitated by expression and/or activation of aquaporins along the phloem-to-xylem pathway. Water under pressure would gradually refill the embolized conduit and hydraulic isolation of the refilling conduit from adjacent functioning ones (under negative pressure) would be assured by intervessel pit geometry and chemical composition [44,58]. Although several of the proposed steps of the refilling process have not yet been experimentally demonstrated and, hence, the scenario depicted above is purely speculative, we feel that it might

A. Nardini et al. / Plant Science 180 (2011) 604–611

represent an interesting working scheme to design future experiments addressed at elucidating the several question marks still limiting our understanding of xylem refilling under tension. During the final revision of this manuscript, we became aware of a study [99] testing the hypothesis for a different mechanism for embolism sensing and initiation of refilling processes, initially proposed by [100]. In this recent study, it is argued that cavitation events in xylem conduits are unlikely to represent critical events producing acoustic waves of sufficient energy to generate a mechanical signal. Moreover, while acoustic signals might well act as signals initiating the refilling process, it is objected that this mechanism would not provide plants with any ‘off’ signal ending the refilling process. According to the alternative model proposed, xylem parenchyma cells would constantly release small amounts of sucrose into xylem conduits. Sucrose would be constantly drawn away along the transpiration stream in functioning conduits. Following conduit embolism, living cells would continue to release sugars into the thin sap layer remaining at the wall level in the gas-filled conduit, but in this case sucrose could not be removed by the transpiration stream and high sugar concentration would build up. Increased apoplastic sugar concentration would act as a signal initiating the chain of events finally leading to embolism reversal. The progressive dilution of sugars once the conduit is again connected to the transpiration stream would also provide a signal for switching off the refilling mechanism. We feel that the ideas presented in [99], as partially confirmed by experimental data, deserve great attention and future research efforts aimed at measuring sucrose concentration in functioning, embolized and refilling conduits while simultaneously observing the initiation and ending of key processes in the refilling mechanism like starch depolymerisation and aquaporin expression. 6. Conclusions and future perspectives Our understanding of embolism repair occurring while the bulk of xylem is under tension, has significantly improved during the last decade. Experimental observations and theoretical modeling have revealed that embolism repair under tension does not imply any violation of the laws of thermodynamics. Simply, this is a physiological process requiring a fine coordination of (a) biochemical and morphological features of interconduit pits and perforation plates to assure hydraulic isolation of refilling conduits, (b) changes in VAC sugar metabolism to provide the necessary driving force for delivering water into embolized conduits, and (c) source-to-sink solute and water transport from phloem to the refilling conduit. While several pieces of this intriguing puzzle are in place, a comprehensive picture detailing the sequence of events starting from xylem cavitation to complete refilling is still lacking. In this review, we have higlighted some of the still obscure facts regarding embolism refilling. Hopefully, this and other recent ‘thought’ papers [100] will stimulate further research on: • typical refilling times for embolized conduits with different diameter and under different water potential values in still functioning conduits and surrounding living cells; • perception of cavitation events and down-stream signal transduction cascades leading to embolism sensing and activation of refilling processes; • sugar metabolism in vessel-associated cells during embolism refilling; • changes in composition of the fluids in refilling conduits with special reference to concentration of sugars and other organic and/or inorganic solutes; • changes in the phloem pressure and activity in proximity to the refilling xylem;

609

• precise localization of aquaporins activated and/or expressed during xylem refilling. Further research in this area, as combined with promising findings emerging from studies on the ion-mediated regulation of xylem hydraulics [21–23], will likely change our current view of xylem as an inert, vulnerable pipeline into a new paradigm where xylem is seen as a finely regulated water transport system closely depending on vital processes for optimal functioning and maintenance, especially under environmental stress conditions. References [1] E. Steudle, The cohesion–tension mechanism and the acquisition of water by plant roots, Annu. Rev. Plant Physiol. Plant Mol. Biol. 52 (2001) 847–875. [2] M.J. Canny, Vessel contents during transpiration – embolisms and refilling, Am. J. Bot. 84 (1997) 1223–1230. [3] M.J. Canny, Applications of the compensating pressure theory of water transport, Am. J. Bot. 85 (1998) 897–909. [4] U. Zimmermann, H. Schneider, L.H. Wegner, A. Haase, Water ascent in tall trees: does evolution of land plants rely on a highly metastable state? New Phytol. 162 (2004) 575–615. [5] M. Westhoff, D. Zimmermann, H. Schneider, et al., Evidence for discontinuous water columns in the xylem conduit of tall birch trees, Plant Biol. 11 (2009) 307–327. [6] J.A. Milburn, Sap ascent in vascular plants: challengers to the cohesion theory ignore the significance of immature xylem and the recycling of Münch water, Ann. Bot. 78 (1996) 399–407. [7] M.T. Tyree, The cohesion–tension theory of sap ascent: current controversies, J. Exp. Bot. 48 (1997) 1753–1765. [8] J.P. Comstock, Why Canny’s theory doesn’t hold water, Am. J. Bot. 86 (1999) 1077–1081. [9] V. Stiller, J.S. Sperry, Canny’s compensating pressure theory fails a test, Am. J. Bot. 86 (1999) 1082–1086. [10] C. Wei, M.T. Tyree, E. Steudle, Direct measurement of xylem pressure in leaves of intact maize plants. A test of the cohesion-tension theory taking hydraulic architecture into consideration, Plant Physiol. 121 (1999) 1191–1205. [11] H. Richter, The cohesion theory debate continues: the pitfalls of cryobiology, Trends Plant Sci. 6 (2001) 456–457. [12] M.T. Tyree, M.H. Zimmermann, Xylem Structure and the Ascent of Sap, Springer, New York, 2002. [13] S. Mayr, U. Hacke, P. Schmid, et al., Frost drought in conifers at the alpine timberline: xylem dysfunction and adaptations, Ecology 87 (2006) 3175–3185. [14] M.A. Zwieniecki, N.M. Holbrook, Diurnal variation in xylem hydraulic conductivity in white ash (Fraxinus americana L.) red maple (Acer rubrum L.) and red spruce (Picea rubens Sarg.), Plant Cell Environ. 21 (1998) 1173–1180. [15] S. Salleo, A. Nardini, F. Pitt, M.A. Lo Gullo, Xylem cavitation and hydraulic control of stomatal conductance in Laurel (Laurus nobilis L.), Plant Cell Environ. 23 (2000) 71–79. [16] J.S. Sperry, W.T. Pockman, Limitation of transpiration by hydraulic conductance and xylem cavitation in Betula occidentalis, Plant Cell Environ. 16 (1993) 279–287. [17] J.S. Sperry, N.N. Alder, S.E. Eastlack, The effect of reduced hydraulic conductance on stomatal conductance and xylem cavitation, J. Exp. Bot. 44 (1993) 1075–1082. [18] A. Nardini, S. Salleo, Limitation of stomatal conductance by hydraulic traits: sensing or preventing xylem cavitation? Trees 15 (2000) 14–24. [19] T.J. Brodribb, N.M. Holbrook, Diurnal depression of leaf hydraulic conductance in a tropical tree species, Plant Cell Environ. 27 (2004) 820–827. [20] B. Choat, E.C. Lahr, P.J. Melcher, et al., The spatial pattern of air seeding threshold in mature sugar maple trees, Plant Cell Environ. 28 (2005) 1082–1089. [21] M.A. Zwieniecki, P.J. Melcher, N.M. Holbrook, Hydrogel control of xylem hydraulic resistance in plants, Science 291 (2001) 1059–1062. [22] A. Gascò, A. Nardini, E. Gortan, S. Salleo, Ion-mediated increase in the hydraulic conductivity of laurel stems: role of pits and consequences for the impact of cavitation on water transport, Plant Cell Environ. 29 (2006) 1946–1955. [23] P. Trifilò, M.A. Lo Gullo, S. Salleo, et al., Xylem embolism alleviated by ionmediated increase in hydraulic conductivity of functional xylem: insights from field measurements, Tree Physiol. 28 (2008) 1505–1512. [24] S.D. Davis, F.W. Ewers, J.S. Sperry, et al., Shoot dieback during prolonged drought in Ceanothus (Rhamnaceae) chaparral of California: a possible case of hydraulic failure, Am. J. Bot. 89 (2002) 820–828. [25] M.T. Tyree, G. Vargas, B.M.J. Engelbrecht, T.A. Kursar, Drought until death do us part: a case study of the desiccation tolerance of a tropical moist forest seedling-tree Licania platypus (Hemsl.) Fritsch, J. Exp. Bot. 53 (2002) 2239–2247. [26] T.J. Brodribb, H. Cochard, Hydraulic failure defines the recovery and point of death in water-stressed conifers, Plant Physiol. 149 (2009) 575–584. [27] H.G. Jones, R.A. Sutherland, Stomatal control of xylem embolism, Plant Cell Environ. 49 (1991) 607–612. [28] J.S. Sperry, N.M. Holbrook, M.H. Zimmermann, M.T. Tyree, Spring filling of xylem vessels in wild grapevine, Plant Physiol. 83 (1987) 414–417.

610

A. Nardini et al. / Plant Science 180 (2011) 604–611

[29] M.T. Tyree, S. Yang, Hydraulic conductivity recovery versus water pressure in xylem of Acer saccharum, Plant Physiol. 100 (1992) 669–676. [30] H. Cochard, F.W. Ewers, M.T. Tyree, Water relations of a tropical vine-like bamboo (Rhipidocladum racemiflorum): root pressures, vulnerability to cavitation and seasonal changes in embolism, J. Exp. Bot. 45 (1994) 1085–1089. [31] U. Hacke, J.J. Sauter, Xylem dysfunction during winter and recovery of hydraulic conductivity in diffuse-porous and ring-porous trees, Oecologia 105 (1996) 435–439. [32] M. Westhoff, H. Schneider, D. Zimmermann, et al., The mechanisms of refilling of xylem conduits and bleeding of tall birch during spring, Plant Biol. 10 (2008) 604–623. [33] S. Salleo, M.A. Lo Gullo, D. De Paoli, M. Zippo, Xylem recovery from cavitationinduced embolism in young plants of Laurus nobilis: a possibile mechanism, New Phytol. 132 (1996) 47–56. [34] M.T. Tyree, S. Salleo, A. Nardini, et al., Refilling of embolized vessels in young stems of Laurel. Do we need a new paradigm? Plant Physiol. 120 (1999) 11–21. [35] S.J. Bucci, F.G. Scholz, G. Goldstein, et al., Dynamic changes in hydraulic conductivity in petioles of two savanna tree species: factors and mechanisms contributing to the refilling of embolized vessels, Plant Cell Environ. 26 (2003) 1633–1645. [36] U. Hacke, J.S. Sperry, Limits to xylem refilling under negative pressure in Laurus nobilis and Acer negundo, Plant Cell Environ. 26 (2003) 303–311. [37] S. Salleo, M.A. Lo Gullo, P. Trifilò, A. Nardini, New evidence for a role of vesselassociated cells and phloem in the rapid xylem refilling of cavitated stems of Laurus nobilis L., Plant Cell Environ. 27 (2004) 1065–1076. [38] S. Yang, M.T. Tyree, A theoretical model of hydraulic conductivity recovery from embolism with comparison to experimental data on Acer saccharum, Plant Cell Environ. 15 (1992) 633–643. [39] S. Salleo, M.A. Lo Gullo, Different aspects of cavitation resistance in Ceratonia siliqua, a drought avoiding Mediterranean tree, Ann. Bot. 64 (1989) 325–336. [40] V. Stiller, J.S. Sperry, H.R. Lafitte, Embolized conduits of rice (Oryza sativa L., Poaceae) refill despite negative xylem pressure, Am. J. Bot. 92 (2005) 1970–1974. [41] A. Nardini, M. Ramani, E. Gortan, S. Salleo, Vein recovery from embolism occurs under negative pressure in leaves of sunflower (Helianthus annuus), Physiol. Plant. 133 (2008) 755–764. [42] S. Salleo, P. Trifilò, S. Esposito, et al., Starch-to-sugar conversion in wood parenchyma of field-growing Laurus nobilis plants: a component of the signal pathway for embolism repair? Funct. Plant Biol. 36 (2009) 815–825. [43] N.M. Holbrook, M.A. Zwieniecki, Embolism repair and xylem tension: do we need a miracle? Plant Physiol. 120 (1999) 7–10. [44] M.A. Zwieniecki, N.M. Holbrook, Bordered pit structure and vessel wall surface properties. Implications for embolism repair, Plant Physiol. 123 (2000) 1015–1020. [45] W. Konrad, A. Roth-Nebelsick, The dynamics of gas bubbles in conduits of vascular plants and implications for embolism repair, J. Theor. Biol. 224 (2003) 43–61. [46] T. Vesala, T. Hölttä, M. Perämäki, E. Nikinmaa, Refilling of a hydraulically isolated embolized xylem vessel: model calculations, Ann. Bot. 91 (2003) 419–428. [47] M.J. Canny, J.P. Sparks, C.X. Huang, M.L. Roderick, Hypothesis: air embolism exsolving in the transpiration water – the effect of constrictions in the xylem pipes, Funct. Plant Biol. 34 (2007) 95–111. [48] J.S. Sperry, K.L. Nichols, J.E.M. Sullivan, S.E. Eastlack, Xylem embolism in ringporous, diffuse-porous, and coniferous trees of northern Utah and interior Alaska, Ecology 75 (1994) 1736–1752. [49] S Mayr, F. Schwienbacher, H. Bauer, Winter at the alpine timberline: why does embolism occur in Norway spruce but not in stone pine? Plant Physiol. 131 (2003) 780–792. [50] M.R. Facette, M.E. McCully, M.W. Shane, M.J. Canny, Measurements of the time to refill embolized vessels, Plant Physiol. Biochem. 39 (2001) 59–66. [51] M.E. McCully, C.X. Huang, L.E. Ling, Daily embolism and refilling of xylem vessels in the roots of field-grown maize, New Phytol. 138 (1998) 327–342. [52] M.E. McCully, Root xylem embolism and refilling. Relation to water potentials of soil, roots and leaves, and osmotic potentials of root xylem sap, Plant Physiol. 119 (1999) 1001–1008. [53] P.J. Melcher, G. Goldstein, F.C. Meinzer, et al., Water relations of coastal and estuarine Rhizophora mangle: xylem pressure potential and dynamics of embolism formation and repair, Oecologia 126 (2001) 182–192. [54] H. Cochard, C. Bodet, T. Améglio, P. Cruiziat, Cryo-scanning electron microscopy observations of vessel contents during transpiration in walnut petioles. Facts or artefacts? Plant Physiol. 124 (2000) 1191–1202. [55] I. Kaufmann, T. Schulze-Till, H.U. Schneider, et al., Functional repair of embolized vessels in maize roots after temporal drought stress, as demonstrated by magnetic resonance imaging, New Phytol. 184 (2009) 245–256. [56] N.M Holbrook, E.T. Ahrens, M.J. Burns, M.A. Zwieniecki, In vivo observation of cavitation and embolism repair using magnetic resonance imaging, Plant Physiol. 126 (2001) 27–31. [57] H.K. Kim, S.J. Lee, Synchrotron X-ray imaging for nondestructive monitoring of sap flow dynamics through xylem vessel elements in rice leaves, New Phytol. 188 (2010) 1085–1098. [58] C.R. Brodersen, A.J. McElrone, B. Choat, et al., The dynamics of embolism repair in xylem: in vivo visualizations using High Resolution Computed Tomography, Plant Physiol. 154 (2010) 1088–1095.

[59] J.J. Sauter, W. Iten, M.H. Zimmermann, Studies on the release of sugar into the vessels of sugar maple (Acer saccharum), Can. J. Bot. 51 (1973) 1–8. [60] J.J. Sauter, Seasonal variation of sucrose content in the xylem sap of Salix, Zeit. Pflanzenphysiol. 98 (1980) 377–391. [61] T. Améglio, F.W. Ewers, H. Cochard, et al., Winter stem pressures in walnut trees: effects of carbohydrates, cooling and freezing, Tree Physiol. 21 (2001) 384–394. [62] F.W Ewers, T. Améglio, H. Cochard, et al., Seasonal variation in xylem pressure of walnut trees: root and stem pressures, Tree Physiol. 21 (2001) 1123–1132. [63] T. Améglio, M. Decourteix, G. Alves, et al., Temperature effects on xylem sap osmolarity in walnut trees: evidence for a vitalistic model of winter embolism repair, Tree Physiol. 24 (2004) 785–793. [64] P.R. Neumann, R. Weissman, G. Stefano, S. Mancuso, Accumulation of xylem transported protein at pit membranes and associated reductions in hydraulic conductance, J. Exp. Bot. 61 (2010) 1711–1717. [65] R.W. Johnson, M.T. Tyree, Effect of stem water content on sap flow from dormant maple and butternut stems: induction of sap flow in butternut, Plant Physiol. 100 (1992) 853–858. [66] M.A. Zwieniecki, L. Hutyra, M.V. Thompson, N.M. Holbrook, Dynamic changes in petiole specific conductivity in red maple (Acer rubrum L.) tulip tree (Liriodendron tulipifera L.) and northern fox grape (Vitis labrusca L.), Plant Cell Environ. 23 (2000) 407–414. [67] S. Salleo, P. Trifilò, M.A. Lo Gullo, Phloem as a possibile major determinant of rapid cavitation reversal in stems of Laurus nobilis (laurel), Funct. Plant Biol. 33 (2006) 1063–1074. [68] M. Decourteix, G. Alves, N. Brunel, et al., JrSUT1, a putative xylem sucrose transporter, could mediate sucrose influx into xylem parenchyma cells and be up-regulated by freeze–thaw cycles over the autumn-winter period in walnut tree (Juglans regia L.), Plant Cell Environ. 29 (2006) 36–47. [69] G Alves, J.J. Sauter, J.L. Julien, et al., Plasma membrane H+ -ATPase, succinate and isocitrate dehydrogenases activities of vessel associated cells in walnut trees, J. Plant Physiol. 158 (2001) 1263–1271. [70] A.H. De Boer, V. Volkov, Logistics of water and salt transport through the plant: structure and functioning of the xylem, Plant Cell Environ. 26 (2003) 87–101. [71] J.J. Sauter, M. Wisniewski, W. Witt, Interrelationships between ultrastructure, sugar levels, and frost hardiness of ray parenchyma cells during frost acclimation and deacclimation in poplar (Populus canadensis Moench ‘robusta’) wood, J. Plant Physiol. 149 (1996) 451–461. [72] T. Hölttä, T. Vesala, M. Perämäki, E. Nikinmaa, Refilling of embolised conduits as a consequence of ‘Münch water’ circulation, Funct. Plant Biol. 33 (2006) 949–959. [73] J.W. Patrick, Phloem unloading: sieve element unloading and post-sieve element transport, Annu. Rev. Plant Physiol. Plant Mol. Biol. 48 (1997) 191–222. [74] L. Fromard, V. Babin, P. Fleurat-Lessard, et al., Control of vascular sap pH by the vessel-associated cells in woody species: physiological and immunological studies, Plant Physiol. 108 (1995) 913–918. [75] Y. Zhou, N. Setz, C. Niemietz, et al., Aquaporins and unloading of phloemimported water in coats of developing bean seeds, Plant Cell Environ. 30 (2007) 1566–1577. [76] R. Kaldenhoff, M. Ribas-Carbo, J. Flexas, et al., Aquaporins and plant water balance, Plant Cell Environ. 31 (2008) 658–666. [77] N. Sade, M. Gebretsadik, R. Seligmann, et al., The role of tobacco Aquaporin1 in improving water use efficiency, hydraulic conductivity, and yield production under salt stress, Plant Physiol. 152 (2010) 245–254. [78] F Barrieu, F. Chaumont, M.J. Chrispeels, High expression of the tonoplast aquaporin ZmTIP1 in epidermal and conducting tissues of maize, Plant Physiol. 117 (1998) 1153–1163. [79] H.H. Kirch, R. Vera-Estrella, D. Golldack, et al., Expression of water channels proteins in Mesembryanthemum crystallinum, Plant Physiol. (2000) 111–124. [80] P. Martre, R. Morillon, F. Barrieu, et al., Plasma membrane aquaporins play a significant role during recovery from water deficit, Plant Physiol. 130 (2002) 2101–2110. [81] C. Lovisolo, A. Schubert, Mercury hinders recovery of shoot hydraulic conductivity during grapevine rehydration: evidence from a whole-plant approach, New Phytol. 172 (2006) 658–666. [82] S. Sakr, G. Alves, R. Morillon, et al., Plasma membrane aquaporins are involved in winter embolism recovery in walnut tree, Plant Physiol. 133 (2003) 630–641. [83] F. Secchi, M.A. Zwieniecki, Patterns of PIP gene expression in Populus trichocarpa during recovery from xylem embolism suggest a major role for the PIP1 aquaporin subfamily as moderators of refilling process, Plant Cell Environ. 33 (2010) 1285–1297. [84] J. Irvine, J. Grace, Continuous measurements of water tensions in the xylem of trees based on the elastic properties of wood, Planta 202 (1997) 455–461. [85] B. Otto, R. Kaldenhoff, Cell-specific expression of the mercury-insensitive plasma-membrane aquaporin NtAQP1 from Nicotiana tabacum, Planta 211 (2000) 167–172. [86] M.T. Tyree, M.A. Dixon, Cavitation events in Thuja occidentalis L.? Ultrasonic acoustic emissions from the sapwood can be measured, Plant Physiol. 72 (1983) 1094–1099. [87] A.P. Sandford, J. Grace, The measurement and interpretation of ultrasound from woody stems, J. Exp. Bot. 36 (1985) 298–311. [88] M.T. Tyree, J.S. Sperry, Characterization and propagation of acoustic emission signals in woody plants: towards an improved acoustic emission counter, Plant Cell Environ. 12 (1989) 371–382.

A. Nardini et al. / Plant Science 180 (2011) 604–611 [89] M.A. Lo Gullo, S. Salleo, Three different methods for measuring xylem cavitation and embolism: a comparison, Ann. Bot. 67 (1991) 417– 424. [90] S. Salleo, P. Trifilò, M.A. Lo Gullo, Vessel wall vibrations: trigger for embolism repair? Funct. Plant Biol. 35 (2008) 289–297. [91] H.G.L. Coster, E. Steudle, U. Zimmermann, Turgor pressure sensing in plant cell membranes, Plant Physiol. 58 (1977) 636–643. [92] T. Furuichi, H. Tatsumi, M. Sokabe, Mechano-sensitive channels regulate the stomatal aperture in Vicia faba, Biochem. Biophys. Res. Commun. 366 (2008) 758–762. [93] V.A. Shepherd, M.J. Beilby, S.A.S. Al Khazaaly, T. Shimmen, Mechanoperception in Chara cells: the influence of salinity and calcium on touch-activated receptor potentials, action potential and ion transport, Plant Cell Environ. 31 (2008) 1575–1591. [94] T. Yang, B.W. Poovaiah, Calcium/calmodulin-mediated signal network in plants, Trends Plant Sci. 8 (2003) 505–512.

611

[95] S. Gilroy, Signal transduction in barley aleurone protoplasts is calcium dependent and independent, Plant Cell 8 (1996) 2193–2209. [96] S.C. Chang, M.H. Cho, B.G. Kang, P.B. Kaufman, Changes in starch content in oat (Avena sativa) shoot pulvini during the gravitropic response, J. Exp. Bot. 52 (2001) 1029–1040. [97] E. Purgatto, J.R.O. do Nascimento, F.M. Lajolo, B.R. Cordenunsi, The onset of starch degradation during banana ripening is concomitant to changes in the content of free and conjugated forms of indole-3-acetic acid, J. Plant Physiol. 159 (2002) 1105–1111. [98] E.P. Chu, A.R. Tavares, S. Kanashiro, et al., Effects of auxin on soluble carbohydrates, starch and soluble protein content in Aechmea blanchetiana (Bromeliaceae) cultured in vitro, Sci. Hortic. 125 (2010) 451–455. [99] F. Secchi, M.A. Zwieniecki, Sensing embolism in xylem vessels: the role of sucrose as a trigger for refilling, Plant Cell Environ., in press. [100] M.A. Zwieniecki, N.M. Holbrook, Confronting Maxwell’s demon: biophysics of xylem embolism repair, Trends Plant Sci. 14 (2009) 530–534.