Journal of Experimental Botany

This Article Abstract FREE Full Text (PDF) E-letters: Submit a response Alert me when this article is cited Alert me when E-letters are posted Alert me if a correction is posted Services Email this article to a friend Similar articles in this journal Similar articles in ISI Web of Science Similar articles in PubMed Alert me to new issues of the journal Add to My Personal Archive Download to citation manager Search for citing articles in: ISI Web of Science (43) Request Permissions Disclaimer Google Scholar Articles by Netting, A.G. Search for Related Content PubMed PubMed Citation Articles by Netting, A.G. Agricola Articles by Netting, A.G. Social Bookmarking          What's this?

Journal of Experimental Botany, Vol. 51, No. 343, pp. 147-158, February 2000
© 2000 Oxford University Press

pH, abscisic acid and the integration of metabolism in plants under stressed and non-stressed conditions: cellular responses to stress and their implication for plant water relations

A.G. Netting¹

School of Biochemistry and Molecular Genetics, University of New South Wales, Sydney, 2052, Australia

Received 25 June 1999; Accepted 26 October 1999

	Abstract

Top Abstract Introduction Guard cells Other leaf cells Root, stem and petiole... Root cortical cells References

 
A paradigm for the response of plants to stress is presented which suggests that plants move towards a state of minimal metabolic activity as a stress intensifies and remain in that state until that stress is relieved. The paradigm is based on the proposition that cells that interface with the transpiration stream employ variations on the following theme to move towards that state. Tension on the apoplastic water opens a mechanosensitive Ca²⁺ channel, a response that is augmented by apoplastic ABA. The resulting elevated cytoplasmic Ca²⁺ deactivates a plasmalemma H⁺/ATPase and also activates a K⁺-H⁺ symport. The inflow of K⁺ and H⁺ depolarizes the membrane and renders the apoplast less acidic, the protons being removed to the vacuole and the K⁺ ions being re-exported via the K⁺ outward rectifying channel. The onset of darkness in guard and mesophyll cells deactivates the plasmalemma H⁺/ATPase and then the events outlined above ensue except that these cells do not appear to utilize either Ca²⁺ or ABA during these changes. In stressed cells it is proposed that elevated cytoplasmic Ca²⁺ activates the release of an ABA precursor from a stored form. ABA is then released in the apoplast after export of the precursor if the activity of the K⁺ -H⁺ symport has brought the apoplastic pH close to 7.0. It is proposed that aquaporins in the xylem parenchyma and mesophyll cells are opened by elevated cytoplasmic Ca²⁺ when the water potential of the transpiration stream is high so that water can be stored in the ‘xylem parenchyma reservoir’. The water in this reservoir is then used to increase the water potential in the transpiration stream when the water column is under tension and to help repair embolisms by a mechanism that resembles stomatal closure.

Key words: Apoplastic pH, ABA, stress, plant water relations, H⁺/ATPase.

	Introduction

Top Abstract Introduction Guard cells Other leaf cells Root, stem and petiole... Root cortical cells References

 
If a mammal is faced with a severe biotic or abiotic stress—an emergency—it secretes adrenaline, the ‘flight or fight’ hormone, in preparation for the mobilization of large amounts of energy. But what does a plant do in this situation? It certainly cannot flee and its capacity to fight is limited essentially to chemical warfare. There seems to be only one other option: it closes down as much of its metabolic activity as possible and waits for the stress to go away; something akin to hibernation in animals. Or, to keep the ‘flight or fight’ alliteration alive, it ‘freezes’: freezes in the sense that metabolic activity is minimal. Thus, from this perspective, the closure of stomata and the reduction of growth that follows, for example, droughting or temperature extremes initiates a quiescent period of stress toleration. Such a response at the level of the whole plant also implies responses at the cellular level so that this review discusses cellular responses to stress and considers some of the implications of these responses for plant water relations.

	Guard cells

Top Abstract Introduction Guard cells Other leaf cells Root, stem and petiole... Root cortical cells References

 
Darkness
Although a good case can be made that darkness is not a stress, it is certainly an environmental stimulus that requires a response from the aerial parts of the plant. Because the effects of changes in light intensity on guard cells have probably been studied in more detail than any other stimulus on any plant cell type, this system is an excellent model for assessing the response of plant cells to external stimuli. Thus it is clear, for example, that guard cells perceive and respond to light by pumping protons into the apoplast using the light-stimulated H⁺/ATPase (Raschke and Humble, 1973; Gepstein et al., 1982; Inoue and Katoh, 1987; Fricker and Willmer, 1990). Note that the action of the H⁺/ATPase at the pHs prevalent in the cytoplasm causes one H⁺ to be extruded (Blatt et al., 1998; Palmgren, 1991). This does not necessarily imply an increase in the pH of the well-buffered cytoplasm (Blatt et al., 1998) because the proton is generated during the hydrolysis of ATP rather than by translocating a proton across the plasmalemma:

The onset of darkness causes this pump to be deactivated and perhaps activates a K⁺-H⁺ symport (see below), although no work seems to have been done on possible mechanisms. The apoplast then becomes basic within 10 min (Edwards et al., 1988) and this rise in pH inactivates the K⁺ inward rectifying channel (Czempinski et al., 1999). The imported protons seem to be transported into the vacuole (Bowling and Edwards, 1984; Blatt et al., 1998), presumably via the tonoplast H⁺/PPase (Askerlund and Sommerin, 1996) because, as with the plasmalemma H⁺/ATPase, the tonoplast H⁺/ATPase serves to energize the tonoplast according to the above equation and does not necessarily lead to a net decrease in [H⁺]_cyt. However, the equation for the pyro-phosphatase clearly implies a net transfer of protons from the cytoplasm to the vacuole with a concomitant increase in cytoplasmic pH:

Secretion of protons into the vacuole at the onset of darkness retains these protons in the guard cell for future re-incorporation into ATP, essentially by a reversal of equation 1, when light is restored. From this perspective ATP might be viewed as an energised intermediate between the store of H⁺ in the vacuole and apoplastic H⁺ that has been extruded to energize the membrane for the uptake of K⁺ and other ions. Presumably export of protons from the cytoplasm to the vacuole is responsible for an increase (0.2–0.3 pH units) in the pH of the guard cell cytoplasm (Blatt et al., 1998). This increase in cytoplasmic pH, which is thought to be independent of cytoplasmic Ca²⁺ (Blatt et al., 1998), opens the fast vacuolar channel to admit K⁺ to the cytoplasm (Allen and Sanders, 1997) and then opens the plasmalemma K⁺ outward rectifying and Cl^- outward channels (Blatt et al., 1998). These events precede the outflow of water, presumably through aquaporins, that equilibrates the apoplastic and symplastic water potentials and leads to the shrinkage of the guard cells so that stomatal closure ensues. Thus, the onset of darkness apparently induces stomatal closure without utilizing apoplastic Ca²⁺ or abscisic acid (ABA).

When light is restored this series of events is essentially reversed commencing with the activation of the H⁺/ATPase, this latter event requiring elevated cytoplasmic Ca²⁺ (Kinoshita et al., 1995). The re-acidification of the apoplast to about pH 5.1 takes some 10 min (Edwards et al., 1988) during which time protons are presumably withdrawn from the vacuole, possibly utilizing a K⁺/H⁺ antiporter, incorporated into ATP principally by oxidative phosphorylation, and then the ATP is hydrolysed to liberate protons into the apoplast. However, stomatal opening is not complete until about 80 min, by which time the apoplastic pH has returned to about 6.3, presumably because the guard cells are utilizing the electrochemical gradient to take up the appropriate ions and utilizing the osmotic pressure generated by these ions to draw water into the guard cells via aquaporins.

Stresses
Stresses, such as drought, appear to be transduced by a somewhat different pathway to the above adaptations to darkness as there is no clear evidence that darkness induces the influx of Ca²⁺ into the guard cell cytoplasm from the apoplast. However, the guard cell plasmalemma does contain Ca²⁺ channels (Blatt et al., 1998), such as the mechanosensitive Ca²⁺ channel (Garrill et al., 1996), which could be opened by stresses. Thus, increasing cytoplasmic Ca²⁺ could activate the Ca²⁺-dependent slow vacuolar Ca²⁺ channel (Allen and Sanders, 1997) so that sufficient Ca²⁺ was present to stimulate the phosphorylation of the H⁺/ATPase, thus deactivating it (Kinoshita et al., 1995). As with the guard cell's response to darkness, the deactivation of the H⁺/ATPase appears to activate a K⁺-H⁺ symport so that the apoplast becomes less acidic. Further, as will be discussed in detail in a future review, elevated cytoplasmic Ca²⁺ together with other factors is hypothesized to activate the release of a precursor of ABA (Netting et al., 1997) that is susceptible to hydrolysis at about pH 7.0. If this precursor is released into the apoplast it would be hydrolysed thus accounting for the increased ‘effectiveness’ of ABA in inducing stomatal closure with increasing pH (Wilkinson et al., 1998; Bacon et al., 1998): the amount of apoplastic ABA increases rather than its effectiveness. The incoming protons from the activity of the K⁺-H⁺ symport are presumably pumped into the vacuole by the PPase since the pH of the cytoplasm actually increases somewhat preceding stomatal closure (Irving et al., 1992). Elevated cytoplasmic Ca²⁺ also opens the vacuolar K⁺ channel (Allen and Sanders, 1997), closes the K⁺ inward rectifying channel (Schroeder and Hagiwara, 1989) and promotes the efflux of anions (Schroeder and Hagiwara, 1989; Hedrich et al., 1990; Schroeder and Keller, 1992) and K⁺ (Schroeder and Hagiwara, 1989) to initiate stomatal closure. Apoplastic ABA and Ca²⁺ therefore form a synergistic system which ensures that stomatal closure follows the perception of stress upstream in the transpiration stream. This conclusion is reinforced by the observations that apoplastic ABA inactivates the K⁺ inward rectifying channel (Gepstein et al., 1982) and opens a plasmalemma Ca²⁺ channel (Schroeder and Hagiwara, 1990) giving rise to increased cytoplasmic Ca²⁺ (McAinsh et al., 1990). Cytoplasmic Ca²⁺ may release the hydrolysable precursor of ABA into the cytoplasm as well as into the apoplast thus elevating cytoplasmic ABA. Cytoplasmic ABA opens the K⁺ outward rectifying channel (Schauf and Wilson, 1987) and the Cl^- outward channel (Blatt et al., 1998).

The alert reader will have noted that elevated cytoplasmic Ca²⁺ is required for both (Assmann and Shimazaki, 1999) stomatal opening and closure (Kinoshita et al., 1995). Further, both of these events are controlled by the plasmalemma H⁺/ATPase, the former requiring the activation of this pump and the latter its deactivation. So, the important question arises as to how the guard cell senses the difference between elevated cytoplasmic Ca²⁺ as a signal for opening as a opposed to a signal for closure. A possibility worth investigating is that apoplastic ABA activates a K⁺-H⁺ symport as well as opening a Ca²⁺ channel. On the imposition of stress this would increase apoplastic pH, as has been observed, and cause an influx of K⁺ and protons into the guard cell cytoplasm. Perhaps the interaction of Ca²⁺ plus K⁺ and/or H⁺ at the plasmalemma H⁺/ATPase stimulates its phosphorylation and deactivation while Ca²⁺ alone induces its activation. The increased cytoplasmic K⁺ would, of course, be removed by the K⁺ outward channel on stomatal closure. Also, if a K⁺-H⁺ symport is activated in response to apoplastic ABA, then the incoming H⁺ would have to be removed to the vacuole more rapidly than it entered from the apoplast since, at least with Paphiopedilum tonsum guard cells, the pH of the cytoplasm increased along with cytoplasmic Ca²⁺ before stomatal closure (Irving et al., 1992). Interestingly, the tonoplast PPase is activated, at least in carrot suspension cells, by cytoplasmic K⁺ (Colombo and Cerana, 1993). Certainly the influx of K⁺ and H⁺ would rapidly depolarize the membrane from at least -160 mV as observed with an active H⁺/ATPase to the -100 mV or more positive values observed with substantial anion efflux. It is of interest that Schroeder and Hagiwara suggested the Ca²⁺ channel that is opened by ABA is non-selective (Schroeder and Hagiwara, 1990), because these authors also observed the influx of K⁺. Could this be due to the activation of a K⁺-H⁺ symport?

In spite of the observations mentioned above that ABA effects stomatal closure from both the apoplastic and the cytoplasmic sides of the plasmalemma, it is not clear whether apoplastic ABA is taken into the guard cell cytoplasm. At the concentrations that induce stomatal closure, 10 nM in Commelina communis (Wilkinson and Davies, 1997) or 11.2x10^-18 mol guard cell^-1 in broad bean leaves (Harris and Outlaw, 1991), ABA may merely bind to receptors at the plasmalemma and then, together with increases in cytoplasmic Ca²⁺, induce the changes in ion fluxes just described. In any case, if guard cells release an ABA precursor in the cytoplasm in response to increased cytoplasmic Ca²⁺ the requirement for ABA uptake is eliminated.

After Ca²⁺ and/or ABA has arrived at the guard cell plasmalemma, due to the perception of a stress upstream in the transpiration stream, and instigated stomatal closure these changes in ion fluxes will need to be reversed when the stress abates. It seems likely that this is initiated by the removal of Ca²⁺ from the guard cell cytoplasm by the tonoplast Ca²⁺/H⁺ antiporter (Askerlund and Sommarin, 1996) so that protons are available for ATP synthesis in the mitochondria and perhaps the chloroplasts. When sufficient ATP is available the removal of Ca²⁺ from the cytoplasm can be completed by the plasmalemma and tonoplast Ca²⁺/ATPases. By drawing on the store of protons in the vacuole, ATP can then be synthesized, principally by oxidative phosphorylation, to provide the energy required to re-establish the electrochemical gradients at the tonoplast and plasmalemma. This will reduce the apoplastic pH and allow the uptake of K⁺ and other ions into the guard cells so that, when aquaporins are opened, water will flow into the guard cell to initiate stomatal opening.

	Other leaf cells

Top Abstract Introduction Guard cells Other leaf cells Root, stem and petiole... Root cortical cells References

 
Darkness
Perhaps the clearest evidence that mesophyll cells contain a light-activated H⁺/ATPase analogous to that found in guard cells comes from work using ion-selective vibrating microelectrodes (Shabala and Newman, 1999). These authors found that the flux of protons out of mesophyll cells was at a maximum some 7–8 min after the light was turned on. Interestingly, this was preceded by an influx of Ca²⁺ which commenced immediately the light was turned on and which reached a maximum at about 3.5 min. Similar work on the transition from light to dark has not been attempted. However, fluorescein isothiocyanate-dextran has been used to measure the pH of the apoplast in sunflower leaves by the fluorescent ratio technique (Hoffmann and Kosegarten, 1995). When the leaves were transferred from light to dark there was an increase in apoplastic pH, as measured by the integrated signal across the leaf, from about 5.7 in the light to about 5.8 in the dark with an intervening maximum of about 6.0. This is reminiscent of the pH changes in guard cells but is much less dramatic. Perhaps mesophyll cells import some protons from the apoplast via a K⁺-H⁺ symport and some of these are taken up by the chloroplast stroma where the pH drops from about 8.0 to 7.0. This is sufficient to eliminate CO₂ fixation which is negligible below pH 7.3 (Werdan et al., 1975). This provides a mechanism for co-ordinating photosynthesis with the available light and other aspects of cell metabolism which will be discussed in more detail in a future review.

Stresses in mature leaves
When excised cotton leaves were subjected to pressure-induced dehydration and the expressed sap collected and analysed, an uptake of K⁺ by the symplast was observed (Hartung et al., 1988). As there was no abrupt change in the composition of the expressed sap this was probably by both the mesophyll cells and the xylem parenchyma cells in the veins, midrib and petiole. There was also an increase in the pH of the expressed sap such that the decreasing H⁺ closely paralleled the decreasing K⁺, at least for the first 70 µl of sap. This suggests that a K⁺-H⁺ symport may have been removing both K⁺ and H⁺ from the sap. After about 100 µl of sap had been expressed the pH levelled off at about 7.6 probably under the influence of the H₂/ buffering system (Gollan et al., 1992), which has a pK of 7.2. Both the K⁺ uptake and the apoplastic pH increase were severely reduced by pretreatment with fusicoccin, implying that a plasmalemma H⁺/ATPase established an electrochemical gradient prior to the imposition of stress. In these experiments it seems likely that the pressure applied to the leaf caused shear stresses between the plasma membranes and the cell walls. As some of the mechanosensitive Ca²⁺ channels are attached to the cell walls as well as to the plasmalemma (Garrill et al., 1996) they would be opened by these shear stresses. Thus, Ca²⁺ would flow into the cytoplasm of the leaf cells and this influx, possibly supplemented by the tonoplast Ca²⁺-induced Ca²⁺ channel (Allen and Sanders, 1997), would then lead to the phosphorylation of the plasmalemma H⁺/ATPase and its deactivation (Kinoshita et al., 1995; Xing et al., 1996). As mentioned for guard cells, elevated cytosolic Ca²⁺ is believed to have the potential to release ABA in the apoplast by hydrolysis of a precursor (Netting et al., 1997), at about pH 7.0. If this is also true for mesophyll cells and for xylem parenchyma cells the imposition of pressure-induced dehydration would be predicted to increase the concentration of ABA in the apoplast and in the xylem. This is indeed what has already been observed (Hartung et al., 1988): the [ABA] started to rise when the pH of the sap was about 6.4 and about 20 µl had been expressed and peaked at above 1.0 µM when the apoplastic pH was about 7.4 and 70 µl of sap had been expressed. Further, this increase in [ABA] was completely eliminated by pretreatment with fusicoccin implying that continued proton pumping interfered with the uptake of Ca²⁺, and perhaps K⁺ plus H⁺ and, therefore, eliminated the release and hydrolysis of the ABA precursor mentioned above.

These observations that both guard cells, and probably mesophyll cells, can respond to elevated cytoplasmic Ca²⁺ by either activating or deactivating the plasmalemma H⁺/ATPase bring to mind the conclusion (de Wit, 1995) that bacterial or fungal specific elicitors cause acidification of the apoplast while non-specific elicitors cause alkalinization. Further, both of these events are preceded by elevated cytoplasmic Ca²⁺ (Blumwald et al., 1998). In terms of the paradigm proposed in the Introduction, a quiescent period of stress tolerance is correlated with the presence of ABA and the deactivation of the plasmalemma H⁺/ATPase while active to hyperactive metabolism is correlated with the activation of the H⁺/ATPase. Or, the deactivation of the H⁺/ATPase is a prerequisite for the ‘freeze’ response and the activation of it is a prerequisite for the ‘fight’ response.

In the wild, plants are, of course, unlikely to suffer pressure-induced dehydration: they are much more likely to suffer from a water deficit which implies the imposition of tension on the water in the apoplast. On the assumption that such tensions can open mechanosensitive Ca²⁺ channels in the plasmalemma of mesophyll cells and of xylem parenchyma cells in the small veins it would be expected that the same series of events as those just described for pressure-dehydrated cotton leaves would also occur in drought-affected leaves. The plasmalemma H⁺/ATPase has been demonstrated in the pericycle, xylem parenchyma and endodermis as well as companion cells and other cell types outside the stele in oats and peas (Parets-Solar et al., 1990) and barley (Samuels et al., 1992). Further, in Commelina communis and tomatoes the pH of the sap in the stem increased when the roots were stressed (Wilkinson and Davies, 1997; Wilkinson et al., 1998) as did the pH of the root exudate when barley roots were stressed (Bacon et al., 1998). Thus, elements of the guard cell's response to stress are present in mesophyll and xylem parenchyma cells. Tension in the xylem, therefore, implies a cycle of events in the xylem parenchyma cells that proceeds up the vascular tissue leading to increasing pH, decreasing K⁺ and increasing [ABA] in the water column as the sap moves from the stem into the petiole and the leaf blade. Results recorded by others (Neumann et al., 1997) are in agreement with this scenario. They measured dramatic increases in the ABA contents of the expanding leaf zone of maize plants after 4 h of PEG treatment to the roots: 80-fold with live roots and 12.6-fold with killed roots suggesting that some, but not all, of the ABA originated in the roots. It should be noted, however, that a similar increase in [ABA] was not observed in wheat plants with live roots.

When a plant is severely stressed and the leaves wilt it implies that the mesophyll cells within a leaf have lost turgor and this loss of turgor, in turn, implies that water flows outwards across the plasmalemma, presumably through aquaporins. When aquaporins are opened by, for example, elevated cytoplasmic Ca²⁺ and phosphorylation (Johansson et al., 1996, 1998), they control the amount of water or other small neutral molecule (Tyerman et al., 1999) that flows through them. They do not control the direction of that flow (Schäffner, 1998): in the case of water, which would be the only small neutral molecule that is physiologically relevant in the present context, the direction of flow is controlled by the relative water potentials of the cytoplasm and the apoplast—or the cytoplasm and the vacuole. Thus, a loss of turgor by mesophyll cells implies that the osmolality of the leaf apoplast is higher than the cytoplasm of those mesophyll cells. Whether this is due to the export of K⁺ through an outward rectifying channel, along with accompanying anions as in guard cells, is not clear, but one suspects that the preceding events would be initiated by a mechanosensitive Ca²⁺ channel that responds to tension in the water in the apoplast. So is the aquaporin activated either directly or indirectly by this elevated cytoplasmic Ca²⁺? This is not known although a turgor-responsive gene from pea, trg-31, has been identified (Guerrero and Crossland, 1993). Transcription was induced in the leaf and particularly in the mid-rib, but not in the roots, within 30 min of the initiation of wilting. The protein from trg-31 shows strong homology with a protein from tomatoes known as TRAMP (Fray et al., 1994) which is also induced by water stress, the induction being particularly strong in wilted stems. Compared to other putative aquaporins both of these proteins have lost most of a C-terminal region that is phosphorylated by a Ca²⁺-dependent protein kinase (Fray et al., 1994), but it is not known if this is related to the opening of the aquaporin. When the roots of unstressed rice seedlings were treated with 0.5 mM HgCl₂ there was no effect on leaf growth, but when the same treatment was applied to rice seedlings that had been treated with polyethylene glycol 6000 (-0.2 MPa) for 24 h before the addition of HgCl₂ the growth rate declined by some 49% after 20 min (Lu and Neumann, 1999). The hydraulic conductance, as measured by the ‘osmotic-jump method’ (Lu and Neumann, 1999) also declined by 43% after treatment with Hg for 20 min. As Hg is a known reversible inhibitor of some aquaporins (Tyerman et al., 1999) the simplest explanation for these results is that, even under these very mild conditions of stress, aquaporins made a contribution to the maintenance of a high water potential in the xylem. If this conclusion is correct it raises some fascinating questions. How do xylem parenchyma cells and mesophyll cells monitor a change in tension corresponding to 0.2 MPa? Is movement of the cellulose cell wall relative to the plasmalemma involved and, if so, are the mechanosensitive Ca²⁺channels that form crosslinks (Garrill et al., 1996) sensitive enough to respond? If Ca²⁺ is taken up by such a mechanism is the whole suite, including the opening of an aquaporin, of responses outlined for guard cells invoked? Does this mean that K⁺ and anions are exported, perhaps just into the cell wall, so that an osmotic gradient is established to facilitate the outflow of water? If the stress is more severe is the precursor of ABA exported so that ABA can be liberated upstream of or near to the guard cells if the apoplastic pH reaches about 7.0? Whatever the answer to these questions, there are some tantalizing hints that mild water stress may open aquaporins to equilibrate water potentials in the transpiration stream and to help carry a hydrolysable ABA precursor towards the stomata.

Recovery from stresses in mature leaves
A stronger case can be made that a plasmalemma aquaporin opens when the apoplastic water potential is high so that water flows into the mesophyll cells, and particularly into cells in the vascular tissue (Johansson et al., 1998). The opening of this aquaporin, PM28A, is absolutely dependent on the presence of submicromolar concentrations of cytoplasmic Ca²⁺ (Johansson et al., 1996) which is thought to enter the cytoplasm via a stretch-activated Ca²⁺ channel. This is somewhat surprising because water is not particularly elastic: it only changes its volume by about 0.1% with a change in pressure of 1 MPa so that the changes in cross-sectional area that this implies would be minute. Perhaps, again, the plasmalemma/cell wall cross-linking Ca²⁺ channel is involved. In any case, it seems clear that both the aquaporin mentioned above (Johansson et al., 1996) and those investigated by other researchers (Guerrero and Crossland, 1993; Fray et al., 1994) respond to small pressure differences across the plasmalemma. Thus, PM28A is opened by elevated cytoplasmic Ca²⁺ which activates a membrane associated protein kinase which, in turn, phosphorylates the aquaporin, thus opening it. There are two complementary scenarios here: on the one hand elevated cytoplasmic Ca²⁺ opens an aquaporin so that water flows into the mesophyll and xylem parenchyma cells; on the other hand elevated cytoplasmic Ca²⁺ may open a presumably different aquaporin so that water flows out of these cells. This has parallels with the situation discussed above with the plasmalemma H⁺/ATPase where elevated cytoplasmic Ca²⁺ can cause either its activation or deactivation. This suggests that, at least in the cell types discussed so far, the opening of the appropriate aquaporin may be a prelude, if the stress is severe enough, to the attainment of the stress-tolerant quiescent state. Similarly, the opening of aquaporins may also be part of the process of recovering from that state.

Mild stresses in young leaves
The growth rate of young cereal leaves is relatively easy to measure because it is essentially confined to the longitudinal axis and has the experimental advantage that it is sensitive to minor changes in ambient conditions. For example, the elongation rate of a barley seedling leaf at high humidity was between 15 and 20 µm min^-1, but when drier air was admitted the elongation rate fell to zero in about 5 min but recovered to almost the original value after a further 15 min (Munns et al., 2000). Conversely, when the humidity was increased, the elongation rate more than doubled in about 5 min and then fell back to near its original value in a further15 min. This is a clear indication that these leaves had a graded response to changes in relative humidity so that adjustments in the internal water relations allowed a maximum growth rate to be maintained regardless, up to a point, of external conditions. So how does the barley seedling achieve this? In the light of the paradigm proposed here it is suggested that the decrease in humidity increased the tension on the apoplastic water so that a mechanosensitive Ca²⁺ channel opened, the H⁺/ATPase was deactivated, a K⁺-H⁺ symport activated, and the K⁺ outward rectifying channel and anion channels opened so that the concentration of salts in the apoplast increased. Then, when the elevated cytoplasmic Ca²⁺ opened the aquaporins, water flowed out into the apoplast. This mechanism, therefore, ascribes the initial decline in growth directly to the loss of turgor in the expanding cells. This is in agreement with the observation that a step decrease in humidity also generated a step decrease in the thickness of the leaf (R. Munns, unpublished results). At the same time the decrease in humidity and the resulting increase in tension on the apoplastic water would also tend to reduce the stomatal aperture via the mechanosensitive Ca²⁺ channels in the guard cells (Garrill et al., 1996). This again is in agreement with observation: there was an apparent slight decrease in stomatal aperture, as determined by variations in leaf temperature, over some 20 min when the humidity was reduced (R. Munns, unpublished results). Thus, the combined effect of decreasing stomatal aperture and a decrease in cell turgor would be the reduction of the tension in the apoplastic water. If, after about 5 min, the leaf cells, including the expanding cells, are able to open Ca²⁺ channels, the scenario presented above could be essentially reversed so that these cells regain turgor. This should restore the growth rate close to its original value as observed.

When the humidity was increased and the elongation rate more than doubled there was initially a decrease in tension in the apoplastic water which, it is proposed here, increased the flow of water into leaf cells by the mechanisms outlined above to give the observed increases in elongation rate and leaf thickness (R. Munns, unpublished results). If this increase in humidity and decrease in tension also tended to increase the stomatal aperture, the tension in the apoplastic water would be increased and therefore water would flow out of the expanding cells so that their elongation rate would decline to close to its original value. These suggestions are lent some credence by the observation (R. Munns, unpublished results) that when seedlings are similarly treated in an apparatus that automatically adjusts the pressure on the soil so that a cut leaf is on the point of bleeding, that is, a ‘balancing pressure’ is applied, the changes in elongation rate are virtually eliminated. This is a strong indication that the elongation rate is directly controlled by tension and, given the scenario presented here, the turgor pressure within the cells. Further, when the air was made drier the balancing pressure increased with some overshoot for the first 20 min. The stabilization of this overshoot was correlated with an apparent decrease in stomatal aperture so that, once again, the plant has made adjustments to its internal water relations so that the turgor pressure was re-equilibrated close to its original value. Thus the data summarized in this section suggest that there is a dynamic equilibrium between the magnitude of a stress and the turgor of leaf cells and the scenario developed to interpret these data imply that there will be small changes in apoplastic pH which will be directly related to changes in tension on the apoplastic water. Unfortunately, no measurements are available to substantiate these putative changes in pH.

As discussed above, changes in pH of a quite large magnitude are, however, observed under more severe conditions of stress. These, in fact, seem to reflect a graded response of a plant to stress. Thus, if the pH of the apoplast exceeds about 5.5, presumably due to the import of protons via a K⁺-H⁺ symport, expansins are deactivated (Cosgrove, 1998). These enzymes are located in the cell wall and seem to be responsible for releasing matrix glycans from cellulose microfibrils so that the cellulose and the matrix polymers slide relative to each other. Thus above about pH 5.5 growth ceases. Again, if the apoplastic pH exceeds about 6.5 to 7.0 it appears that the ABA precursor (Netting et al., 1997) is released and hydrolysed to liberate ABA in the xylem or apoplast. Thus with each increase in apoplastic pH of about one unit a new factor comes into play that, it is proposed here, transforms a plant from an active growing state to a stress-tolerant quiescent state.

In the scenarios presented here the initial response of plants to stress is proposed to be the opening, triggered by changes in tension in the apoplastic water, of mechano-sensitive Ca²⁺ channels. These mechanosensitive Ca²⁺ channels and their presumed sensitivity are, therefore, an absolutely crucial underpinning to the proposals developed here. Without them and their sensitivity these proposals fail. It is, therefore, most unfortunate that little is known of them except that they appear to be gated by tension and link the cell wall to the plasmalemma and possibly the cytoskeleton (Garrill et al., 1996).

	Root, stem and petiole xylem parenchyma cells

Top Abstract Introduction Guard cells Other leaf cells Root, stem and petiole... Root cortical cells References

 
Repair of embolisms
In the stele and vascular bundles of the root, stem and petiole there is a gradation in the diameter of the vessels and the two extremes of this range present the plant with different problems. In the narrow vessels of the leaf and petiole tensions are likely to be relatively high due to resistance to flow, a situation that was covered in the last section. However, in the older stems and particularly in tree trunks, the vessels are relatively large in diameter and the larger the vessel the more prone it is to cavitation. When gas comes out of solution to form such an embolism it poses some danger to the plant because it increases the resistance to flow in that vessel. This has the effect of increasing the tension in the water column connected to that vessel and therefore presumably activates the mechanosensitive Ca²⁺ channels, as discussed in the previous section, downstream of the embolism. Nevertheless, these embolisms need to be removed rapidly, but the mechanism by which the plant achieves this has been the subject of considerable debate. (Holbrook and Zieniecki, 1999; Tyree et al., 1999). But it does seem that the sequence of events described for guard cells could also occur in xylem parenchyma cells and that this sequence could result in the dissolution of the embolism. The plasma membrane H⁺/ATPase has been identified in 2-year-old branches of Robinia where it is particularly active in the vessel associated cells (Fromard et al., 1995), but there is little information available for other elements of the sequence in woody tissue. However, if mechanosensitive Ca²⁺ channels are present in the bordered pits it seems likely that the membrane would flex in towards the vessel lumen when an embolism was present thus activating the Ca²⁺ channel. Increased cytoplasmic Ca²⁺ would deactivate the H⁺/ATPase and activate a K⁺-H⁺ symport so that the membrane would be depolarized and the apoplastic pH would rise. K⁺ and anions would then be expected to flow out of the xylem parenchyma cells into the vessels, as has been observed, at least for K⁺ (Tyree et al., 1999): these authors observed a doubling of both xylem sap osmolality and K⁺ 15 min after pressure release in prestressed laurel twigs. But since an embolism is present there is only a film of water on the vessel wall (Tyree et al., 1999) and, in particular, within the bordered pits (Holbrook and Zwieniecki, 1999) so that the exported ions would form a particularly concentrated solution. Thus, if the influx of Ca²⁺ had also activated an aquaporin it might be expected that water would flow out of the cells containing the bordered pits so that osmotic equilibrium would be restored. This dilution of the concentrated salt solution could, therefore, commence the dissolution of the embolism: xylem parenchyma cells might, then, be characterized as the ‘guard cells of the xylem’.

It was mentioned above that elevated cytosolic Ca²⁺ was thought to release a hydrolysable precursor of ABA from a stored form. There is, therefore, a mechanism here for integrating the release of the ABA precursor and the increasing pH of the transpiration stream with the repair of embolisms so that a signal indicating excessive tension can be sent to the stomata to supplement any ABA precursor that may have been released in the narrow vessels of the young stem, petiole and leaf. Although this scenario was developed with trees in mind it raises the possibility that embolisms may occur in smaller plants also. This does, in fact, appear to be the case because it was observed that a cultivar of sugar cane lost up to 50% of its hydraulic conductivity during the day even when well-irrigated (Neufeld et al., 1992). In Vitis vinifera it was also suggested that the formation of embolisms may be common and that they could play an important role in the inhibition of shoot growth at moderate water deficits (Schultz and Matthews, 1988).

In split root experiments in which half the roots were stressed, excision of the stressed roots led to the recovery of leaf growth (Gowing et al., 1990). It follows under the present proposal that the removal of the source of a chemical inhibitor of shoot growth (as postulated by Gowing et al., 1990) was only part of the reason for recovery. Another part of the recovery would be due to the excision of the stressed roots which would relieve tension in vessels connected to those roots. Thus, when the ABA had passed through the xylem, the Ca²⁺ channels would no longer be open so that, after removal of Ca²⁺ from the xylem parenchyma cytoplasm, the H⁺/ATPases could be reactivated and the cells released from the stress-tolerant quiescent state. This proposal may also provide a resolution between those who believe that there is insufficient ABA in the xylem to cause stomatal closure (Munns and King, 1988) and those who believe that there is sufficient (Neales and McLeod, 1991). The answer depends on the presence of Ca²⁺, K⁺, an elevated apoplastic pH and the release of the ABA precursor as well as whether the xylem is sampled above or below the regions of maximum Ca²⁺ uptake.

Stresses in shoots
In general terms then, the proposal is that a relatively severe drying down of the soil or the presence of high concentrations of osmolytes in it induces tension in the water column so that ABA release in the xylem commences in the narrow vessels in the young shoots. ABA release in or close to the leaves has the great advantage to the plant that the ABA will be produced quite close to the stomata that need to be closed. This eliminates any lag, possibly of hours in trees, between the onset of stress in the roots and a response at the stomata while a chemical message passes up the xylem. It also allows control in each leaf or even part of a leaf if tension arises in minor veins as, for example, a spot of sunlight passes over the surface.

Cohesion supplemented by the ‘xylem parenchyma reservoir’
An analogy for the proposal being put forward here is given in Fig. 1. In Fig. 1A the water potential in the transpiration stream is depicted as high by the device of drawing the water level in the stream at a higher level. This is quite artificial, of course, because the transpiration stream is continuous to the stomata and the ‘xylem parenchyma reservoir’ is essentially a closed system. Nevertheless, the diagram emphasizes that if the depicted Ca²⁺ channel opens and the aquaporin is phosphorylated and opened (as described by Johansson et al., 1996, 1998), water will ‘siphon’ from the transpiration stream into the xylem parenchyma reservoir. This scenario implies that water will be taken up by the xylem parenchyma whenever the water potential in the transpiration stream is high. This is particularly likely to be the case when the water supply is adequate but the stomata are closed; after sunset, for example. In fact, the water uptake could be sufficient, particularly if aided by the import of sugars into the xylem parenchyma cells to increase the osmolality of the cytoplasm, to apply a little pressure to the xylem. Such a mechanism could account for the diurnal rhythm in the diameter of tree trunks. For example, apple tree stems of 5–7 cm diameter increased by 1.0–1.4% in diameter at night (Link et al., 1998). Figure 1B depicts two possibilities for when the water potential in the xylem parenchyma reservoir is higher than in the transpiration stream. In the upper part, ABA opens the Ca²⁺ channel and activates a K⁺-H⁺ symport so that an aquaporin such as TRAMP (Fray et al., 1994), the protein from trg-31 (Guerrero and Crossland, 1993) or the putative Hg-inhibited aquaporin from rice seedlings (Lu and Neumann, 1999) opens and water ‘siphons’ out of the xylem parenchyma cells into the transpiration stream. This would mean that tension in the xylem would activate the outflow of water from the xylem parenchyma at the point in the transpiration stream where the uptake of Ca²⁺ is at a maximum thus equalizing the water potentials between the xylem parenchyma reservoir and the transpiration stream. It could also mean that ABA precursor would be released into the apoplast at this point so that [ABA] would increase if the apoplastic pH reached about 7.0. This scenario is proposed to take place in the narrow vessels in petioles and leaf veins. In stems, where embolisms are more likely to occur in the larger vessels, a similar series of events is envisaged, but, in this case, the opening of the aquaporin leads to the dissolution of the embolism as depicted in the lower half of Fig. 1B. Overall, then, this proposal might be named ‘cohesion supplemented by the xylem parenchyma reservoir’.

View larger version (34K): [in this window] [in a new window]   Fig. 1. An analogy for the storage of water in (A) and the utilization of water from (B) the ‘xylem parenchyma reservoir’. It is proposed that the water potentials of the two compartments, the xylem parenchyma reservoir and the transpiration stream, influence the movement of water via aquaporins in an analogous manner to the heights of water columns in the two arms of a siphon. Opening a tap between the two arms will immediately lead to an equalization in the heights of the water column in each arm. In (A) it is proposed that a mechanosensitive Ca2+ channel opens in response to the high water potential in the transpiration stream. The elevated cytoplasmic Ca2+ induces the phosphorylation of an aquaporin, thus allowing the inflow of water to equalize the water potentials (Johannsson et al., 1996, 1998). In (B) (upper, narrow xylem vessels) ABA is proposed to open a Ca2+ channel and a K+-H+ symport (see text) which together are thought to induce the phosphorylation of an aquaporin, thus opening it, and allowing the outflow of water so that the water potentials become equal. In (B) (lower, embolism repair in wide xylem vessels): similar to (A) except that the outflow of water aids in the dissolution of the embolism and also an ABA precursor is extruded to release ABA in the vessel if the remaining water approaches a pH of 7.0. This series of events is analogous to those that occur in guard cells preceding stomatal closure. Hg is depicted as inhibiting the aquaporin in (B) (an interpretation of results from Lu and Neumann, 1999). ‘P-ase’ is Phosphorylase.

 
If this proposal does approach a description of the actual relationship between the xylem parenchyma and the transpiration stream there will need to be a suite of aquaporins between the xylem parenchyma cytoplasm and the transpiration stream and also between the former and the xylem parenchyma vacuoles where the water would be stored. The study of aquaporins is in its infancy but some, perhaps most (Schäffner, 1998), of the 23 major intrinsic proteins (MIPs) identified at the DNA sequence level (Weig et al., 1997) in Arabidopsis appear to be aquaporins. Similarly, there are at least 31 MIP genes in maize (Tyerman et al., 1999). In mid-1997 seven plasmalemma intrinsic proteins (PIPs) and three tonoplast intrinsic proteins (TIPs) from Arabidopsis had been identified as aquaporins (Schäffner, 1998). Further, in situ hybridization experiments showed that the highest PIP mRNA levels were in the vasculature of Arabidopsis (Schäffner, 1998).

Thus it does seem that the available evidence, though limited, is consistent with the notion that some aquaporins could open in response to stress to attempt to maintain the water potential of the transpiration stream. It is also consistent with the idea that the maintenance of the integrity of the transpiration stream is more important than the possible loss of turgor in some xylem parenchyma cells.

	Root cortical cells

Top Abstract Introduction Guard cells Other leaf cells Root, stem and petiole... Root cortical cells References

 
Stresses in roots
For the sake of completeness aquaporins were indicated in the endodermis at the bottom of the transpiration stream in Fig. 1. There is no experimental evidence for these, but it is a necessity that, when ample water is available and the stomata are open, water be admitted to the stele. Presumably this is achieved by the opening of an aquaporin after Ca²⁺ uptake. As HgCl₂ supplied to the roots had no effect on the rate of leaf growth in unstressed rice seedlings (Lu and Neumann, 1999) it must be concluded that these putative endodermal aquaporins are not susceptible to Hg. When water is limiting these aquaporins will need to be closed, but there is no information on how this occurs. From this perspective the endodermis functions as a one-way valve admitting water into the transpiration stream and ensuring that the available water is retained within the stele, vascular bundles and mesophyll during periods of stress. The root cortical cells are, therefore, outside the main water transport and storage tissues of the plant, their principal function being the uptake of water and nutrients from the soil.

With the increasing salinization of arable land in many parts of the world there has been a good deal of interest in the mechanisms that plants use to cope with salt in the soil. Using ³¹P- and ²³Na-nuclear magnetic resonance (NMR) a small cytoplasmic alkalinization (0.1–0.2 pH units) and a larger vacuolar alkalinization (0.6 pH units) were found in maize root tips exposed to salt concentrations greater than 200 mM (Spickett et al., 1993). Their results suggest that Na⁺ enters the cytoplasm via a cation channel, as does Ca²⁺. The Ca²⁺ activates the plasmalemma H⁺/ATPase so that a plasmalemma antiporter exchanges cytoplasmic Na⁺ for apoplastic H⁺. Nevertheless, Na⁺ and H⁺ continues to build up in the cytoplasm so that the tonoplast pyrophosphatase is activated to pump protons into the vacuole. This activates a tonoplast Na⁺/H⁺ antiporter so that more Na⁺ is removed from the cytoplasm, this time to the vacuole. If Na⁺ still remains in the cytoplasm it appears that the tonoplast H⁺/ATPase is then activated, as alkalinization of the vacuole continues after the pH of the cytoplasm has stabilized, so that additional Na⁺ can be removed to the vacuole by the tonoplast Na⁺/H⁺ antiporter. In severe cases this can lead to a depletion of the energy reserves of the cell which is presumably an indication of the limits of salt toleration of this particular species. Similar results were obtained with barley using ³¹P-NMR only (Katsuhara et al., 1997). In the presence of salt (0.3 or 0.5 mM) and Ca²⁺ these root tips showed a net extrusion into the apoplast of protons of 61–64 nmol H⁺ min^-1 g^-1 fr. wt.

These examples suggest that Ca²⁺ activated the plasmalemma H⁺/ATPase, as it did with the fungal specific elicitors and with the opening of stomata. Thus, the response of maize root tip cells to salt might be characterized as an example of the ‘fight’ response: a ‘fight’ against an abiotic stress.

It seems likely that root cortical cells respond to stresses such as droughting or chilling by deactivating their plasmalemma H⁺/ATPases and synthesizing ABA, but results from experiments that cover both of these aspects together do not seem to have been published. Certainly, in beet roots it has been shown that the plasmalemma H⁺/ATPase was phosphorylated by a Ca²⁺-dependent kinase, probably a calmodulin-like domain protein kinase (Lino et al., 1998). Further, both the rates of ATP hydrolysis and of proton transport were reduced when the H⁺/ATPase was phosphorylated. Also, it has been shown that root tips synthesize ABA when the relative water content falls below 90% (Zhang and Davies, 1987). Thus, the preconditions for the control of the H⁺/ATPase in roots by ABA and Ca²⁺ are in place, but this control is yet to be demonstrated.

Anoxia in roots
An interesting case study for the effects of stress in roots is that of anoxia. In the primary roots of intact maize seedlings the cytoplasmic pH was about 6.9 in the absence of O₂ and was greater than 7.3 when oxygen was not limiting (Roberts and Testa, 1988). In the absence of O₂ there was presumably little ATP synthesis, ethanol being the most likely end-product of carbohydrate metabolism (Roberts et al., 1985). Plant cells use more than 25% of their ATP to energize their plasma membranes (Lino et al., 1998) and this takes no account of the energy required to establish the electrochemical gradient across the tonoplast. As the metabolism of glucose to ethanol only produces 2 mols of ATP per mole of glucose metabolized while the respiration of glucose to CO₂ and H₂O, utilizing the malate-aspartate shuttle, produces about 32 (Stryer, 1995) there would be insufficient ATP produced to energize the cellular membranes in the absence of O₂. It is proposed here that as the amount of available oxygen declines the plasmalemma H⁺/ATPase is deactivated and a K⁺-H⁺ symport is activated so that if Ca²⁺ is also admitted to the cells the preconditions are set for the activation of the release of the ABA precursor (Netting et al., 1997) into the apoplast. If the anoxia continues the cytoplasm is eventually likely to become slightly acidic due to the action of the K⁺-H⁺ symport and the lack of energy to remove protons to the vacuole using the pyrophosphatase. Such a response should also render the apoplast less acidic, as has been described for the other cell types above, and as has been observed with the root exudate of decapitated flooded tomato plants (Else et al., 1995). If the pH of the apoplast approaches 7.0 the precursor would be hydrolysed to ABA and then the ABA would be carried towards the endodermis. Although Jackson was unable to demonstrate an increase in ABA from the very low levels in aerated roots (<5 ng g^-1) in peas after flooding for 7 d, he did observe that hydraulic resistance in the roots caused a reduction in transpiration in peas and tomatoes in as little as 8–12 h after the commencement of flooding (Jackson, 1991). After 12 h this hydraulic resistance disappeared (Jackson, 1991), but by 24 h there were substantial increases in [ABA] in the leaves from tomatoes, peas and rooted cuttings from apples. In grafted tomatoes with wild-type shoots the [ABA] increased 1.7–2.2-fold after 3 d and the stomatal conductance was reduced to about one-third; in grafted peas with wild-type shoots the [ABA] increased at least 10-fold after 7 d while the stomatal conductance decreased by one-half to one-third; in rooted apple cuttings [ABA] increased about 5-fold after 9 d (Jackson, 1991). Further, the grafting experiments just mentioned showed, using the mutants, flacca in tomato and wilty in peas, that this ABA was made in, or close to, the leaves. A possible scenario that rationalizes these results is that declining O₂ in the roots limits the energy available to the cortical cells, because oxidative phosphorylation is limited, so that the electrochemical gradient across their plasma membranes is inadequate for the uptake of ions. This in some way activates a K⁺-H⁺ symport so that the cytoplasm eventually becomes slightly acidic and the apoplast less acidic than before flooding. The activity of the K⁺-H⁺ symport together with elevated cytoplasmic Ca²⁺ leads to the release of an ABA precursor from its stored form which results in an increase in [ABA] when the apoplast becomes slightly basic, presumably in the first 12 h. The slightly basic solution in the apoplast containing ABA moves past the cortical cells and into the endodermis where, it is now proposed, it causes the closure of endodermal aquaporins. This implies that, because the leaves are still transpiring, tensions will now be generated in the xylem which will lead to the release of ABA, probably mostly in the young stems and petioles of dicotyledons. The ABA will move to the stomata and instigate stomatal closure and then it is likely that the xylem parenchyma aquaporins will open so that the shoot water potential is equilibrated. It is of interest that the application of ABA to roots inhibits the loading of K⁺ into the xylem (Tester, 1999). Perhaps K⁺ ions exported from cells in the stele in the root provide the osmotic potential to draw water through the endodermis. If this is true, ABA exported by stressed root cortical cells might close the endodermal aquaporins so that water was not drawn back from the stele into the cortex. In any case, the implementation of this scenario would bring the whole plant to a stress-tolerant quiescent state in which it could survive until oxygen tensions in the soil increased. There are many situations in which it would be advantageous to the plant to have ‘one-way valves’ in the root system; here assumed to be in the endodermis. For example, suppose that there was a mild sub-critical tension in the xylem due to a somewhat less than adequate water supply in the soil and then because, for example, the sun was setting the stomata closed. This would mean that the water in the xylem would tend to be drawn into the root cortex and could lead to a quite rapid desiccation of the shoot. However, if tensions of this type are able to close aquaporins in the endodermis, the water will be retained in the plant and the xylem parenchyma reservoir would be able to equilibrate the shoot water potentials.

Whether these scenarios have any relationship to reality is, of course, unknown but it does seem clear that if plant water relations are to be understood a means will have to be found to study events as they occur in the endodermis. In spite of this caveat it is concluded that plants respond to stress in various tissues by implementing variations on the following theme. A mechanosensitive Ca²⁺ channel is opened which admits Ca²⁺ to the cytoplasm leading to the deactivation of a plasmalemma H⁺/ATPase. A K⁺-H⁺ symport is then activated so that the apoplast becomes less acidic and the membrane is depolarized. This activates a K⁺ outward rectifying channel as well as anion channels. Elevated cytoplasmic Ca²⁺ can also lead to the release of an ABA precursor from a stored form and to the opening of aquaporins.

	Acknowledgments

 
I am grateful to Rana Munns and Barry Milborrow for critical reviews of earlier drafts of this manuscript and to Mike Blatt, Bernd Müller-Röber, Rana Munns, Michael Palmgren, and Steve Tyerman for providing copies of unpublished manuscripts. I also thank Rana Munns and the referees for pointing out weaknesses in the submitted manuscript.

	Notes

 
¹ Fax: +61 2 9385 1483. E-mail:A.Netting{at}unsw.edu.au

	References

Top Abstract Introduction Guard cells Other leaf cells Root, stem and petiole... Root cortical cells References

 
Allen GJ, Sanders D. 1997. Vacuolar ion channels of higher plants. Advances in Botanical Research 25, 217–252.

Askerlund P, Sommarin M. 1996. Calcium efflux transporters in higher plants. In: Smallwood M, Knox JP, Bowles DJ, eds. Membranes: specialized functions in plants. Oxford: Bios, 281–299.

Assmann SM, Shimazaki K-i. 1999. The multisensory guard cell. Stomatal responses to blue light and abscisic acid. Plant Physiology 119, 809–815.[Free Full Text]

Bacon MA, Wilkinson S, Davies WJ. 1998. pH-Regulated leaf cell expansion in droughted plants is abscisic acid dependent. Plant Physiology 118, 1507–1515.[Abstract/Free Full Text]

Blatt MR, Leyman B, Grabov A.1998. Cellular responses to water stress. In: Shinozaki K, ed. Cold, drought, heat and salt stress: molecular responses in higher plants. RG Landes Co. 99–124.

Blumwald E, Aharon GS, Lam BC-H.1998. Early signal transduction pathways in plant–pathogen interactions. Trends in Plant Science 3, 342–346.

Bowling DJF, Edwards A.1984. pH Gradients in the stomatal complex of Tradescantia virginiana. Journal of Experimental Botany 35, 1641–1645.[Abstract/Free Full Text]

Colombo R, Cerana R.1993. Enhanced activity of tonoplast pyrophosphatase in NaCl-grown cells of Daucus carota. Journal of Plant Physiology 142, 226–229.

Cosgrove DJ.1998. Cell wall loosening by expansins. Plant Physiology 118, 333–339.[Free Full Text]

Czempinski K, Gaedeke N, Zimmerman S, Müller-Röber B.1999. Molecular mechanisms and regulation of plant ion channels. Journal of Experimental Botany 50, 955–966.[Abstract]

de Wit PJGM.1995. Fungal avirulence genes and plant resistance genes: unravelling the molecular basis of gene-for-gene interactions. Advances in Botanical Research 21, 147–185.

Edwards MC, Smith GN, Bowling DJF.1988. Guard cells extrude protons prior to stomatal opening—a study using fluorescence microscopy and pH micro-electrodes. Journal of Experimental Botany 39, 1541–1547.[Abstract/Free Full Text]

Else MA, Hall KC, Arnold GM, Davies WJ, Jackson MB.1995. Export of abscisic acid, 1-aminocyclopropane-1-carboxylic acid, phosphate, and nitrate from roots to shoots of flooded tomato plants. Accounting for effects of xylem sap flow rate on concentration and delivery. Plant Physiology 107, 377–384.[Abstract]

Fray RG, Wallace A, Grierson D, Lycett GW.1994. Nucleotide sequence and expression of a ripening and water-stress related cDNA from tomato with homology to the MIP class of membrane channel proteins. Plant Molecular Biology 24, 539–543.[Web of Science][Medline]

Fricker MD, Wilmer CM.1990. Some properties of proton pumping ATPases at the plasma membrane and tonoplast of guard cells. Biochemie und Physiologie der Pflanzen 186, 301–308.

Fromard L, Babin V, Fleurat-Lessard P, Fromont J-C, Serrano R.19955. Control of vascular sap pH by the vessel-associated cells in woody species. Plant Physiology 108, 913–918.[Abstract]

Garrill A, Findlay GP, Tyerman SD.1996. Mechanosensitive ion channels. In: Smallwood M, Knox JP, Bowles DJ, eds. Membranes: specialised functions in plants. Oxford: Bios, 247–260.

Gepstein S, Jacobs M, Taiz L.1982/3. Inhibition of stomatal opening in Vicia faba epidermal tissue by vanadate and abscisic acid. Plant Science Letters 28, 63–72.

Gollan T, Schurr U, Schulze E-D.1992. Stomatal response to drying soil in relation to changes in the xylem sap composition of Helianthus annuus. I. The concentration of cations, anions amino acids in, and pH of, the xylem sap. Plant, Cell and Environment 15, 551–559.

Gowing DJG, Davies WJ, Jones HG.1990. A positive root-sourced signal as an indicator of soil drying in apple, Malusxdomestica Borkh. Journal of Experimental Botany 41, 1535–1541.[Abstract/Free Full Text]

Guerrero FD, Crossland L.19. Tissue-specific expression of a plant turgor-responsive gene with amino acid sequence homology to transport-facilitating proteins. Plant Molecular Biology 21, 929–935.

Harris MJ, Outlaw Jr WH.1991. Rapid adjustment of guard-cell abscisic acid levels to current leaf-water status. Plant Physiology 95, 171–173.[Abstract/Free Full Text]

Hartung W, Radin JW, Hendrix DL.1988. Abscisic acid movement into the apoplastic solution of water-stressed cotton leaves. Plant Physiology 86, 908–913.[Abstract/Free Full Text]

Hedrich R, Busch H, Raschke K.1990. Ca²⁺ and nucleotide-dependent regulation of voltage-dependent anion channels in the plasma membrane of guard cells. The EMBO Journal 9, 3889–3892.[Web of Science][Medline]

Hoffmann B, Kosegarten H.1995. FITC-dextran for measuring apoplast pH and apoplastic pH gradients between various cell types in sunflower leaves. Physiologia Plantarum 95, 327–335.

Holbrook NM, Zwieniecki MA.1999. Embolism repair and xylem tension: do we need a miracle? Plant Physiology 120, 7–10.[Free Full Text]

Inoue H, Katoh Y.1987. Calcium inhibits ion-stimulated stomatal opening in epidermal strips of Commelina communis L. Journal of Experimental Botany 38, 142–149.[Abstract/Free Full Text]

Irving HR, Gehring CA, Parish RW.1992. Changes in cytosolic pH and calcium of guard cells precede stomatal movements. Proceedings of the National Academy of Sciences, USA 89, 1790–1794.[Abstract/Free Full Text]

Jackson MB.1991. Regulation of water relationships in flooded plants by ABA from leaves, roots and xylem sap. In: Davies WJ, Jones HJ, eds. Abscisic acid, physiology and biochemistry. Oxford: Bios, 217–226.

Johansson I, Karlsson M, Shukla VK, Chrispeels, MJ, Larsson C, Kjellbom P.1998. Water transport activity of the plasma membrane aquaporin PM28A is regulated by phosphorylation. The Plant Cell 10, 451–459.[Abstract/Free Full Text]

Johansson I, Larsson C, Ek B, Kjellbom P.1996. The major integral proteins of spinach leaf plasma membranes are putative aquaporins and are phosphorylated in response to Ca²⁺ and apoplastic water potential. The Plant Cell 8, 1181–1191.[Abstract]

Katsuhara M, Yazaki Y, Sakano K, Kawasaki T.1997. Intracellular pH and proton-transport in barley root cells under salt stress: in vivo ³¹P-NMR study. Plant Cell Physiology 38, 155–160.[Abstract/Free Full Text]

Kinoshita T, Nishimura M, Shimazaki K.1995. Cytosolic concentration of Ca²⁺ regulates the plasma membrane H⁺-ATPase in guard cells of fava bean. The Plant Cell 7, 1333–1342.[Abstract]

Link SO, Thiede ME, van Bavel MG. 1998. An improved strain gauge device for continuous field measurement of stem and fruit diameter. Journal of Experimental Botany 49, 1583–1587.[Abstract/Free Full Text]

Lino B, Baizabel-Aguirre VM, Gonzalez de la Vara LE.1998. The plasma-membrane H⁺-ATPase from beet root is inhibited by a calcium-dependent phosphorylation. Planta 204, 352–359.[Web of Science][Medline]

Lu Z, Neumann PM.1999. Water stress inhibits hydraulic conductance and leaf growth in rice seedlings, but not the transport of water via mercury-sensitive water channels in the root. Plant Physiology 120, 143–151.[Abstract/Free Full Text]

McAinsh MR, Brownlee C, Hetherington AM.1990. Abscisic acid-induced elevation of guard cell cytosolic Ca²⁺ precedes stomatal closure. Nature 343, 186–188.[Web of Science]

Munns R, King RW.1988. Abscisic acid is not the only stomatal inhibitor in the transpiration stream of wheat plants. Plant Physiology 88, 703–708.[Abstract/Free Full Text]

Munns R, Passioura JB, Guo J, Chazen O, Cramer GR. 2000. Cell turgor and leaf expansion: importance of time scale. Journal of Experimental Botany 51, (in press).

Neales TF, McLeod AL.1991 Do leaves contribute to the abscisic acid present in the xylem sap of ‘droughted’ sunflower plants? Plant, Cell and Environment 14, 979–986.

Netting AG, Windsor ML, Milborrow BV.1997. Endogenous biosynthetic precursors of (+)-abscisic acid. III. Incorporation of ²H₂O from ¹⁸O₂ into precursors. Australian Journal of Plant Physiology 24, 175–184.

Neufeld HS, Grantz DA, Meinzer FC, Goldstein G, Crisoto GM, Crisoto C.19. Genotypic variability in vulnerability of leaf xylem to cavitation in water-stressed and well-irrigated sugar cane. Plant Physiology 1992, 1020–1028.

Neumann P, Chazen O, Bogoslavsky L, Hartung W.1997. Role of root derived ABA in regulating early leaf growth responses to water deficits. In: Altman, Waisel, eds. Biology of root formation and development. New York: Plenum Press, 147–154.

Parets-Solar A, Pardo JM, Serrano R.1990. Immunocytolocalization of plasma membrane H⁺-ATPase. Plant Physiology 93, 1654–1658.[Abstract/Free Full Text]

Palmgren MG.19. Regulation of plant plasma membrane H⁺-ATPase activity. Physiologia Plantarum 95, 327–335.

Raschke K, Humble GD.1973. No uptake of anions required by opening stomata of Vicia faba: guard cells release hydrogen ions. Planta (Berl.) 115, 47–57.[Web of Science]

Roberts JKM, Lane AN, Clark RA, Nieman RH.1985. Relationships between the rate of synthesis of ATP and the concentrations of reactants and products of ATP hydrolysis in maize root tips, determined by ³¹P nuclear magnetic resonance. Archives of Biochemistry and Biophysics 240, 712–722.[Web of Science][Medline]

Roberts JKM, Testa MP.1988. ³¹P-NMR spectroscopy of roots of intact corn seedlings. Plant Physiology 86, 1127–1130.[Abstract/Free Full Text]

Samuels AL, Fernando M, Glass ADM. 19. Immunofluorescent localisation of plasma membrane H⁺-ATPase in barley roots and effects of K nutrition. Plant Physiology 99, 1509–1514.[Abstract/Free Full Text]

Schäffner AR.1998. Aquaporin function, structure and expression: are there more surprises to surface in water relations? Planta 204, 131–139.[Web of Science][Medline]

Schauf CL, Wilson KJ.1987. Effects of abscisic acid on K⁺ channels in Vicia faba guard cell protoplasts. Biochemical and Biophysical Research Communications 145, 284–290.[Web of Science][Medline]

Schroeder JI, Hagiwara S.1989. Cytosolic calcium regulates ion channels in the plasma membrane of Vicia faba guard cells. Nature 338, 427–430.[Web of Science]

Schroeder JI, Hagiwara S.1990. Repetitive increases in cytosolic Ca²⁺ of guard cells by abscisic acid activation of non-selective Ca²⁺ permeable channels. Proceedings of the National Academy of Sciences, USA 87, 9305–9309.[Abstract/Free Full Text]

Schroeder JI, Keller, BU.1992. Two types of anion channel currents in guard cells with distinct voltage regulation. Proceedings of the National Academy of Sciences, USA 89, 5025–5029.[Abstract/Free Full Text]

Schultz HR, Matthews MA.1988. Resistance to water transport in shoots of Vitis vinifera L. Relation to growth at low water potential. Plant Physiology 88, 718–724.[Abstract/Free Full Text]

Shabala S, Newman I. 1999. Light-induced changes in hydrogen, calcium, potassium, and chloride ion fluxes and concentrations from the mesophyll and epidermal tissues of bean leaves. Understanding the ionic basis of light-induced bioelectrogenesis. Plant Physiology 119, 1115–1124.[Abstract/Free Full Text]

Spickett CM, Smirnoff N, Ratcliffe RG.1993. An in vivo nuclear magnetic resonance investigation of ion transport in maize (Zea mays) and Spartina anglica roots during exposure to high salt concentrations. Plant Physiology 102, 629–638.[Abstract]

Stryer L.1995. Biochemistry, 4th edn. New York: W.H. Freeman & Co., 552.

Tester M.1999. The control of long-distance K⁺ transport by ABA. Trends in Plant Science 4, 5–6.

Tyree MT, Salleo S, Nardini A, Lo Gullo MA, Mosca R.1999. Refilling of embolized vessels in young stems of laurel. Do we need a new paradigm? Plant Physiology 120, 11–21.[Abstract/Free Full Text]

Tyerman SD, Bohnert HJ, Maurel C, Steudle E, Smith JAC.1999. Plant aquaporins: their molecular biology, biophysics and significance for plant water relations. Journal of Experimental Botany 50, 1055–1071.[Abstract]

Weig A, Deswarte C, Chrispeels MJ.1997. The major intrinsic protein family of Arabidopsis has 23 members that form three distinct groups with functional aquaporins in each group. Plant Physiology 114, 1347–1357.[Abstract]

Werdan K, Heldt HW, Milovancev M.1975. The role of pH in the regulation of carbon fixation in the chloroplast stroma. Studies on CO₂ fixation in the light and dark. Biochimica et Biophysica Acta 396, 276–292.[Medline]

Wilkinson S, Corlett JE, Oger L, Davies WJ.1998. Effects of xylem pH on transpiration from wild-type and flacca tomato leaves: a vital role for abscisic acid in preventing excessive water loss even from well-watered plants. Plant Physiology 117, 703–709.[Abstract/Free Full Text]

Wilkinson S, Davies WJ.1997. Xylem sap pH: a drought signal received at the apoplastic face of the guard cell that involves the suppression of saturable abscisic acid uptake by the epidermal symplast. Plant Physiology 113, 559–573.[Abstract]

Xing T, Higgins VJ, Blumwald E.1996. Regulation of plant defense response to fungal pathogens: two types of protein kinases in the reversible phosphorylation of the host plasma membrane H⁺-ATPase. The Plant Cell 8, 555–564.[Abstract]

Zhang J, Davies WJ.1987. Increased synthesis of ABA in partially dehydrated root tips and ABA transport from roots to leaves. Journal of Experimental Botany 38, 2015–2023.[Abstract/Free Full Text]

CiteULike    Connotea    Del.icio.us    What's this?

This article has been cited by other articles:

				A. G. Netting Limitations within "The Limits to Tree Height"1 Am. J. Botany, February 1, 2009; 96(2): 542 - 544. [Abstract] [Full Text] [PDF]

				G. W. Koch and S. C. Sillett A response to: Limitations within "The Limits to Tree Height" Am. J. Botany, February 1, 2009; 96(2): 545 - 547. [Abstract] [Full Text] [PDF]

				R. Aroca, P. Vernieri, and J. M. Ruiz-Lozano Mycorrhizal and non-mycorrhizal Lactuca sativa plants exhibit contrasting responses to exogenous ABA during drought stress and recovery J. Exp. Bot., May 9, 2008; (2008) ern057v1. [Abstract] [Full Text] [PDF]

				K. P. Lee, C. Kim, F. Landgraf, and K. Apel EXECUTER1- and EXECUTER2-dependent transfer of stress-related signals from the plastid to the nucleus of Arabidopsis thaliana PNAS, June 12, 2007; 104(24): 10270 - 10275. [Abstract] [Full Text] [PDF]

				M. A. Else, J. M. Taylor, and C. J. Atkinson Anti-transpirant activity in xylem sap from flooded tomato (Lycopersicon esculentum Mill.) plants is not due to pH-mediated redistributions of root- or shoot-sourced ABA J. Exp. Bot., September 1, 2006; 57(12): 3349 - 3357. [Abstract] [Full Text] [PDF]

				R. G. L. op den Camp, D. Przybyla, C. Ochsenbein, C. Laloi, C. Kim, A. Danon, D. Wagner, E. Hideg, C. Gobel, I. Feussner, et al. Rapid Induction of Distinct Stress Responses after the Release of Singlet Oxygen in Arabidopsis PLANT CELL, October 1, 2003; 15(10): 2320 - 2332. [Abstract] [Full Text] [PDF]

				I. C. Dodd, L. P. Tan, and J. He Do increases in xylem sap pH and/or ABA concentration mediate stomatal closure following nitrate deprivation? J. Exp. Bot., April 1, 2003; 54(385): 1281 - 1288. [Abstract] [Full Text] [PDF]

				R. TUBEROSA, S. SALVI, M. C. SANGUINETI, P. LANDI, M. MACCAFERRI, and S. CONTI Mapping QTLs Regulating Morpho-physiological Traits and Yield: Case Studies, Shortcomings and Perspectives in Drought-stressed Maize Ann. Bot., June 15, 2002; 89(7): 941 - 963. [Abstract] [Full Text] [PDF]

				N. M. Holbrook, V.R. Shashidhar, R. A. James, and R. Munns Stomatal control in tomato with ABA-deficient roots: response of grafted plants to soil drying J. Exp. Bot., June 1, 2002; 53(373): 1503 - 1514. [Abstract] [Full Text] [PDF]

				A. Bahrun, C. R. Jensen, F. Asch, and V. O. Mogensen Drought-induced changes in xylem pH, ionic composition, and ABA concentration act as early signals in field-grown maize (Zea mays L.) J. Exp. Bot., February 1, 2002; 53(367): 251 - 263. [Abstract] [Full Text] [PDF]

				W. Van Ieperen, J. Nijsse, C.J. Keijzer, and U. Van Meeteren Induction of air embolism in xylem conduits of pre-defined diameter J. Exp. Bot., May 1, 2001; 52(358): 981 - 991. [Abstract] [Full Text] [PDF]

				C.L. V. Santos, A. Campos, H. Azevedo, and G. Caldeira In situ and in vitro senescence induced by KCl stress: nutritional imbalance, lipid peroxidation and antioxidant metabolism J. Exp. Bot., February 1, 2001; 52(355): 351 - 360. [Abstract] [Full Text] [PDF]

				W. J. Davies, M. A. Bacon, D. Stuart Thompson, W. Sobeih, and L. Gonzalez Rodriguez Regulation of leaf and fruit growth in plants growing in drying soil: exploitation of the plants' chemical signalling system and hydraulic architecture to increase the efficiency of water use in agriculture J. Exp. Bot., September 1, 2000; 51(350): 1617 - 1626. [Abstract] [Full Text] [PDF]

This Article Abstract FREE Full Text (PDF) E-letters: Submit a response Alert me when this article is cited Alert me when E-letters are posted Alert me if a correction is posted Services Email this article to a friend Similar articles in this journal Similar articles in ISI Web of Science Similar articles in PubMed Alert me to new issues of the journal Add to My Personal Archive Download to citation manager Search for citing articles in: ISI Web of Science (43) Request Permissions Disclaimer Google Scholar Articles by Netting, A.G. Search for Related Content PubMed PubMed Citation Articles by Netting, A.G. Agricola Articles by Netting, A.G. Social Bookmarking          What's this?

Online ISSN 1460-2431 - Print ISSN 0022-0957

Copyright © 2005 Society for Experimental Biology

Oxford Journals Oxford University Press

• Site Map
• Privacy Policy
• Frequently Asked Questions

Other Oxford University Press sites: Oxford University Press Oxford Journals China Oxford Journals Japan American National Biography Booksellers' Information Service Children's Fiction and Poetry Children's Reference Corporate & Special Sales Dictionaries Dictionary of National Biography Digital Reference English Language Teaching Higher Education Textbooks Humanities International Education Unit Law Medicine Music Online Products Oxford English Dictionary Reference Rights and Permissions Science School Books Social Sciences Very Short Introductions World's Classics