ABA-Based Chemical Signalling: the Co-Ordination of Responses to Stress in Plants

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Blackwell Science, LtdOxford, UK PCEPlant, Cell and Environment0016-8025Blackwell Science Ltd 2002 252February 2002 824 ABA-based chemical signalling S. Wilkinson & W. J. Davies 10.1046/j.0016-8025.2001.00824.x Original ArticleBEES SGML

Plant, Cell and Environment (2002) 25, 195–210

ABA-based chemical signalling: the co-ordination of responses to stress in plants

S. WILKINSON & W. J. DAVIES

The Lancaster Environment Centre, Lancaster University, Bailrigg, Lancaster, LA1 4YQ, UK

ABSTRACT soil drying (e.g. Kramer 1969). It is now accepted, however, that many of the plant’s responses to soil drying can occur There is now strong evidence that the plant hormone absci- in the absence of changes in shoot water status, via chemical sic acid (ABA) plays an important role in the regulation of signals. In one very clever series of experiments, pressure stomatal behaviour and gas exchange of droughted plants. has been applied to roots to counteract the increasing soil This regulation involves both long-distance transport and suction that occurs as soil dries. This generated shoot water modulation of ABA concentration at the guard cells, as well relations in droughted plants that were comparable to those as differential responses of the guard cells to a given dose of of well-watered plants. Despite this, rates of gas exchange the hormone. We will describe how a plant can use the and leaf growth were still restricted compared to rates ABA signalling mechanism and other chemical signals to shown by well-watered plants (e.g. Gollan, Schurr & adjust the amount of water that it loses through its stomata Schulze 1992). in response to changes in both the rhizospheric and the Another apparent demonstration of root-sourced chem- aerial environment. The following components of the sig- ical signalling lies in the work of Gowing, Davies & Jones nalling process can play an important part in regulation: (a) (1990). Here, the roots of young apple trees were split ABA sequestration in the root; (b) ABA synthesis versus between two containers, the soil in one of which was catabolism in the root; (c) the efficiency of ABA transfer allowed to dry, while the other container was irrigated as across the root and into the xylem; (d) the exchange of normal. This treatment restricted the rate of leaf growth, ABA between the xylem lumen and the xylem parenchyma which could be restored to control rates by removing the in the shoot; (e) the amount of ABA in the leaf symplastic roots in contact with the drying soil, i.e. by removing the reservoir and the efficiency of ABA sequestration and source of the chemical signal. Blackman & Davies (1985) release from this compartment as regulated by factors such used the same technique to demonstrate that chemical sig- as root and leaf-sourced changes in pH; (f) cleavage of nals sent from drying soil could close the stomata of wheat ABA from ABA conjugates in the leaf apoplast; (g) trans- leaves. fer of ABA from the leaf into the phloem; (h) the sensitivity It is important to note, however, that several groups of the guard cells to the [ABA] that finally reaches them; have used the root pressure vessel technique to restore the and lastly (i) the possible interaction between nitrate stress shoot water relations of a range of species growing in dry and the ABA signal. soil, and have demonstrated that stomata re-open (e.g. Saliendra, Sperry & Comstock 1995; Fuchs & Livingstone Key-words: abscisic acid (ABA), apoplast, calcium, drought 1996; Comstock & Mencuccini 1998). These results indicate stress, drying soil, leaf, long-distance signalling, low that drought-induced limitations in stomatal opening in temperature, nitrate, pH, PPFD, root, stomatal guard cells, these species are mostly hydraulic, suggesting that the rel- symplast, VPD, xylem. ative importance of hydraulic versus chemical signalling may differ between species. It might be surprising if this were not the case, but we should exercise care in using the ABA AS A STRESS RESPONSE HORMONE root pressurization technique to differentiate between It has long been known that in order to conserve the water, chemical and hydraulic signalling mechanisms, as pressur- nutrients and carbohydrates required for survival, plants ization of plant parts is known to change the pH of different respond to stresses such as soil drying by reducing leaf compartments of the shoot and root (Hartung, Radin & expansion and closing stomatal pores. In addition, root Hendrix 1988). We will see below the possible significance growth rates may be maintained in order that the plant may of such a change on the modification of the signalling pro- continue to access water. Traditional explanations for cess, which raises the possibility that root pressurization can drought-induced regulation of gas exchange and leaf minimize both hydraulic and chemical limitation of sto- growth have emphasized the importance of the decline in matal behaviour simultaneously. shoot water status, which commonly accompanies severe It has long been apparent that the major plant growth regulating hormone ABA (see Addicott & Carns 1983) strongly promotes stomatal closure (Jones & Mansfield Correspondence: Sally Wilkinson. Fax: +44 01524 843854; 1970), and can often inhibit shoot growth. The synthesis of E-mail: [email protected] ABA is stimulated by the dehydration of plant cells

196 S. Wilkinson & W. J. Davies

(Wright 1977) including root cells. Leaf cells also synthesize could change the ABA concentration at the guard cell apo- ABA (Cutler & Krochko 1999) and leaf dehydration plast (2·3 µM) in the absence of bulk apoplastic changes in caused by severe soil water shortages massively increases [ABA] (185 nM), and that the apoplastic guard cell ABA bulk leaf [ABA], which often correlates well with stomatal fraction correlated with changes in stomatal aperture more closure. If we are to argue that ABA acts as a long-distance effectively than the guard cell symplastic fraction. Even chemical signal which can provide information on water more recently, ABA supplied via the xylem to non-stressed availability in the soil, it is important to be able to differ- V. faba was seen to accumulate at the guard cell apoplast entiate between such shoot-water deficit-induced increases without a concomitant increase in symplastic guard cell in leaf ABA, and those that arise due to ABA import from ABA, and this correlated with stomatal closure in the the root. Zhang & Tardieu (1996) showed that all tissues of absence of any other stress-induced signals (Zhang & Out- maize roots, regardless of their age and position, could syn- law 2001b). These data show that apoplastically-facing thesize ABA when dried. Much research has now shown guard cell ABA receptors seem to be important in the that the amount of ABA in the xylem sap can increase sub- responses to very subtle ‘stress’ signals experienced by stantially as a function of reduced soil water availability, plants. Such signalling mechanisms may not be important and that this increased delivery to shoots can increase ABA when shoot water stress develops. Harris et al. (1988) found concentrations in different compartments of the leaf (e.g. that when severe leaf water deficit develops, most ABA Loveys 1984; Zhang & Davies 1989). In order to ascribe an accumulated in the guard cell symplast, and it may be that important stress detection function to the roots we need to ABA receptors with an internal locus (Allan et al. 1994; show that the delivery of ABA by the xylem is sufficient to Schwartz et al. 1994) only operate when shoot water rela- bring about the observed degree of stomatal closure seen in tions are also affected. response to soil drying. This is only sometimes the case, but for reasons that will be explained below we can still argue that ABA plays an important regulating role in the MODIFICATION OF THE XYLEM droughted plant in response to perturbations at the root, ABA SIGNAL even when xylem ABA concentrations are not increased at all. Although we can accept that xylem ABA will modify leaf Increases in the ABA concentration in the leaf in functioning, it is clearly not just a simple matter of the response to perturbations around the root are now well [ABA] in the xylem being that perceived by the target cells, documented (see Davies & Zhang 1991). The xylem vessels given that the ABA concentration in apoplastic microsites give up their contents (including their ABA) to the leaf around the guard cells is that to which stomata respond. In apoplast (Weyers & Hillman 1979), thereby increasing the fact, there is often great variation in the apparent sensitivity concentration of the hormone in this compartment. The of leaf conductance to a given concentration of ABA in the ABA is carried with the transpiration stream inside the leaf xylem stream (e.g. Correia & Pereira 1995). The reasons for around and/or through the mesophyll cells (see below) so these findings begin to become clear in the work of Trejo that it reaches the target cells (the stomatal guard cells) in and colleagues (Trejo, Davies & Ruiz 1993; Trejo, Clephan the epidermis, which contain ABA receptors with external & Davies 1995). Their data confirmed early work by Incoll (and possibly internal) loci (see below) in their plasma & Jewer (1987a, b) that stomata in isolated epidermis were membranes (pm). Once bound, the hormone then induces often sensitive to concentrations of ABA as low as 10−9 M, an internal signal transduction cascade usually involving much lower than that found in the xylem sap even of well- increases in both externally and internally sourced cyto- watered plants. If the stomata were directly exposed to the plasmic calcium, which eventually reduces guard cell concentrations of ABA normally seen in well-watered osmotic potential via loss of K+ and Cl– to cause stomatal xylem sap (usually between 1·0 and 50 nM, e.g. Schurr, Gol- closure (for reviews see McAinsh, Brownlee & Hethering- lan & Schulze 1992) they would be permanently closed. So ton 1997; Assmann & Shimazaki 1999). Although the ABA the cells of the leaf must ‘filter out’ some of the ABA arriv- receptor has yet to be unambiguously identified in plants ing in the transpiration stream before it reaches the stomata (Assmann & Shimazaki 1999) there is much evidence in the in the epidermis. By inhibiting the catabolic degradation of literature for an apoplastic locus of ABA perception, indi- ABA in the mesophyll, Trejo et al. (1993) found that the cating that the apoplastic fraction of ABA will be the major amount of ABA reaching the epidermis was increased, and determinant of stomatal aperture (Hartung 1983; Hornberg that the concentration of ABA supplied in the xylem & Weiler 1984). Anderson, Ward & Schroeder (1994) dem- exerted an increased effect on stomatal aperture. In other onstrated a failure of direct microinjection of ABA into the words the mesophyll normally removes (by metabolism guard cells of Commelina communis L. to cause closure and sequestration) much of the ABA passing through it in (but see Allan et al. 1994 and Schwartz et al. 1994). In addi- the transpiration stream before it reaches the guard cells. tion, external ABA has been found to stimulate inward This would also explain why the same authors noted that 2+ Ca conductance and to induce elements of the IP3 signal- there was a linear relationship between the [ABA] in the ling cascade inside guard cells that leads to the release of epidermis and the stomatal response, but a very poor rela- Ca2+ from internal stores (see MacRobbie 1997). Zhang & tionship between bulk leaf ABA and stomatal aperture. Outlaw (2001a) found that stressing Vicia faba L. roots Daeter & Hartung (1995) demonstrated that the epidermal

ABA-based chemical signalling 197

the reservoir is full and/or catabolic enzyme activity is saturated, will increases in flux increase the [ABA] seen at the guard cells (see below). Some related work was carried out by Wilkinson, Clephan & Davies (2001). This showed that the [ABA] penetrating to the abaxial epidermis of detached C. communis leaves supplied with a concentration as high as 10−6 M via the transpiration stream, was kept uniformly low as the temperature was increased from 11 to 28 °C. As temperature, transpiration flux and ABA flux into the leaf increased, the rest of the leaf acted as a reservoir storing the extra ABA entering the leaf tissues and keeping it away from the epidermis (Fig. 1). The change in ABA flux with temperature (except between 7 and 11 °C) failed to change the epidermal [ABA]. Movements of ABA between the xylem and the leaf symplastic reservoir provide a powerful point at which the ABA stress signal response can potentially be modulated. Xylem sap/apoplastic sap pH changes are one way in which accessibility to the mesophyll (and epidermal) reservoir can be controlled. Evidence that xylem and apoplastic sap pH can increase in response to soil drying has been reviewed by Wilkinson (1999). It is important to note that sap pH Figure 1. The effect of temperature on the [ABA] in the abaxial epidermis and in the total leaf of Commelina communis L. when changes can occur even when the shoot water status of detached leaves were incubated under a PPFD of plants in drying soil is maintained using the root pressure − − 350 µmol m 2 s 1 in the presence or absence of 1 µM ABA for vessel (Schurr et al. 1992) or under partial root drying 3 h. Results are means of five separate experiments at each (PRD, Wilkinson & Davies, unpublished; Stoll, Loveys & ± temperature ( SE), and in each experiment the epidermis (and Dry 2000). Schurr et al. (1992) found that correlations the remaining tissue) from 15 leaves was bulked for ABA analysis between xylem sap ABA from droughted sunflowers and by radioimmunoassay. From Wilkinson et al. (2001). reductions in stomatal conductance were poor, but when sap pH or sap [nitrate] or sap [calcium] were also taken into account the correlation was improved (Gollan et al. 1992). symplast is an even better sink for apoplastic ABA than the The authors proposed that increasing xylem sap pH mesophyll, because it has a catabolic rate that is five-fold reduced the ability of the leaf symplastic compartment to greater. So once the ABA has reached the epidermis, there sequester ABA, so that during drought more of the ABA is still potential for ABA to be removed from its site of that enters the leaf will ultimately reach the guard cells in action (the guard cell apoplast). The importance of the con- the epidermis. This proposal was based on very detailed tributions of ABA sequestration and catabolism in the sym- information available in the literature, demonstrating that plast is made clear by the work of Kefu, Munns and King the extent of ABA uptake by all leaf cell types is reduced (1991). These authors calculated that the amount of ABA when the pH outside the cell is increased (e.g. Heilmann, carried in the transpiration stream of cotton plants each day Hartung & Gimmler 1980; Kaiser & Hartung 1981). Models is nine times in excess of the amount of ABA actually were generated by this group, based on measurements of detected in the leaves at the end of the day. ABA uptake in response to pH in isolated cells and tissues. In the past it has been argued (e.g. Jackson 1993) that as These predicted that the drought-induced pH increases the transpirational flux increases, for example with increas- detected in vivo would be enough to cause stomatal closure ing VPD or temperature, the delivery of ABA to the leaf in an intact leaf, by preferentially concentrating the ABA will increase, and that stomata will respond to the total flux that already exists in the leaf into the apoplastic as opposed of ABA into the leaf rather than to the concentration in the to the symplastic compartment (Slovik & Hartung 1992a, xylem stream. However, the fact that the leaf has a large b). It was not even necessary to incorporate an increase in reservoir (the mesophyll cells) in which to sequester and/or xylem-sourced [ABA] into the model for stomatal closure catabolize ABA entering via the xylem could explain why to occur. These predictions have since been substantiated to several lines of evidence have failed to support this theory. some extent by Wilkinson & Davies (1997) and Wilkinson For example, Trejo et al. (1995) found that increases in VPD et al. (1998) in C. communis and tomato. We were able to and temperature (19·9–36·1 °C) increased transpirational conclude that as well as potentially magnifying an effect of water loss from Phaseolus acutifolius L. leaves, but did not xylem ABA on stomatal closure during drought, a drought- affect stomatal aperture. This was despite an increased induced increase in xylem sap pH alone could constitute a delivery of ABA, as evidenced by greater than two-fold root-sourced signal to the leaf, inducing stomatal closure. In increases in bulk leaf [ABA]. Presumably the leaf symplas- essence, well-watered xylem sap has a pH around 6·0 and tic reservoir for ABA sequestration is large, and only when sap from droughted plants has a more alkaline pH of

198 S. Wilkinson & W. J. Davies around 7·0 (see Wilkinson 1999). Artificial xylem sap buff- ABA is high (for example at low soil water contents) the ered to pH 7·0 supplied to whole detached C. communis or outwardly directed concentration gradient for ABA tomato leaves via the xylem stream reduced stomatal aper- between the root and the soil could be reduced. This makes ture in comparison to controls supplied with pH 6·0 buffer. it less likely that root ABA will diffuse out of the plant, The reduction of transpirational water loss by high pH in potentially strengthening the xylem ABA signal further this system was found to require absolutely the presence of along the transpiration stream. In addition, different soil a very low ‘well-watered concentration’ of ABA (c. 10−8 M) types are characterized by different pH values, and as in the xylem stream, a concentration that does not affect described above ABA tends to accumulate in alkaline com- stomatal aperture at control pH. The transpiration rate of partments as a result of the anion trapping effect (see below the ABA-deficient tomato mutant flacca could not be and Wilkinson 1999). Degenhardt et al. (2000) have shown reduced by increasing xylem sap pH up to 7·75 (highest that in alkaline soil substrates considerable portions of the tested), but the inclusion of a ‘well-watered’ concentration ABA synthesized in the roots can be leached out into the of ABA in the artificial sap buffer restored the pH response soil solution, although species with an exodermis (e.g. Zea seen in the wild-type. It was concluded that the increased mays) were less susceptible to this type of stress. In this way pH caused stomatal closure by allowing more of the ABA alkaline soil could weaken the root-sourced ABA signal. that enters the leaf to penetrate to the stomatal guard cells More usually though, rhizospheric ABA is transported as a result of a pH-induced reduction in sequestration into into the root with the soil water. Water flow into and across the symplastic reservoir as described above. the root (see Steudle 2000) is a result of the tension created We now know that soil drying-induced modulation of by the transpiration stream (in which case water flows apo- ABA sequestration into the leaf symplast via xylem- plastically), or of the decreased osmotic potential of the sourced pH changes is only one example out of many xylem sap in the absence of transpiration from the leaf (in whereby the ‘basic’ ABA signal can be influenced by the which case water flows from cell to cell). Anything that environment around the plant. Further examples are given increases water flux across the root should promote the below. transfer of ABA across this organ towards the xylem, because the uptake and radial transport of ABA occurs alongside that of water (Freundl, Steudle & Hartung 2000). Whole-plant modulation of the ABA signal However, until recently this prediction could not have been made, as it was believed that radial transport of ABA across The root roots was entirely trans-cellular due to the perceived imper- ABA may gain entry to plant roots in the following ways: it meability of the exo- and endodermis. This would have can be synthesized in the root (or released from conjugated meant that increased flux of apoplastic water into the xylem forms), it can be taken up from soil water surrounding the upon increased transpiration would dilute the xylem root, or it can be delivered to the root from the shoots by [ABA] (e.g. Else et al. 1994). However, Freundl, Steudle & the phloem. Once inside the root the ABA can be taken up Hartung (1998, 2000) found that filtration of ABA from the by the symplast and stored or degraded, or it may be trans- apoplastic flow of water as it crossed the endodermis built ferred from cell to cell towards the xylem vessels, or carried its local concentration up high enough to cause ‘solvent apoplastically with the transpiration stream towards the drag’ through this potential barrier into the xylem. This is xylem. Alternatively, ABA may be lost from the root by dif- has been termed the ‘apoplastic by-pass’ for ABA. fusion into the soil water (rhizosphere). If root ABA is not Different species transport root water to different degraded, stored in the symplast or lost into the soil water extents within each pathway (symplastic or apoplastic), and it is transferred into the xylem vessels for transport to the when the symplastic route is necessarily dominant (e.g. in leaf. ABA loading into the xylem can either occur directly heavily suberized roots), root cell plasma membranes con- from the apoplast, or indirectly via specialized xylem tain large concentrations of water channels or ‘aquapo- parenchyma cells found along the length of the root xylem. prins’ which can be actively regulated. Interestingly, ABA There are several points along this root ABA uptake and itself can increase the flow of water into and through the transport pathway that can be influenced by the environ- root, otherwise known as its hydraulic conductivity (Hose, ment such that the strength of the root-sourced ABA signal Steudle & Hartung 2000). This is believed to result from in the xylem can be enhanced or reduced. These are ABA-induced opening of these inwardly directed water described below. channels (Tyerman et al. 1999; Netting 2000). Soil drying Root cells constitutively synthesize a low ‘well-watered’ and salinity have been found to increase root hydraulic con- amount of ABA, and as described above this rate of syn- ductivity, and the induction of ABA biosynthesis in the root thesis is massively increased by root tissue dehydration, by these stresses may effectively maximize the transport of classically associated with soil drying. However the strength the newly synthesized ABA within the root towards the of this ‘basic’ ABA signal sent from roots to shoots can be xylem (positive feedback). As described above, water and modified in other less obvious ways. Firstly the soil around ABA flux across the root is also regulated by the osmotic the roots may be influential, not only as regards the direct potential of the xylem sap. Again, ABA itself may reduce effects of its moisture or nutrient content (see last section). the xylem osmotic potential and thereby increase water and Hartung et al. (1996) suggested that when rhizospheric ABA flux across the root, by inducing ion loading into the

ABA-based chemical signalling 199 xylem, e.g. Glinka (1980) – potentially another positive (1989) reported that plant water stress could reduce the feedback mechanism to maximize the root-sourced ABA activity of these H+-ATPases associated with the root signal. xylem, and suggested that this may be one of the causes of Because root cells may degrade or store the ABA that the increased alkalinity often observed in this compartment they synthesize or take up as it flows past them, not all the under stress (see below for other hypotheses). Increases in ABA that enters the root reaches the xylem vessels. Con- xylem pH enhance ABA loading to the root xylem by ditions that reduce the ability of root cells to degrade or increasing the capacity of the xylem sap to trap anions. take up and store ABA will increase the amounts that penetrate to the xylem. The catabolic degradation of ABA The xylem stream takes place constitutively in most plant cell types (see Cut- As described above, soil drying can increase the pH of the ler & Krochko 1999 for review), but there is some evidence xylem stream. Recently, work by Hartung’s group deter- that soil drying reduces the rate of this process in roots. mined that not only will this pH change maximize the initial Liang, Zhang & Wong (1997) found that the half-life of 3H- amount of ABA loaded into the xylem at the root, but also ABA fed to maize roots was extended from 1·15 to 02·27 h that ABA loading into the xylem lumen from stores found by drying the soil around them. Thus, more of the fed ABA to exist in the stem parenchyma occurred more effectively could potentially penetrate to the xylem. (Hartung, Sauter & Hose 2002). However, this only Soil drying also leads to changes in the pH of the various occurred when the initial xylem [ABA] loaded in at the root compartments of the root. That of the apoplast increases, was low, i.e. it will be an early response to soil drying. Nev- and root dehydration causes the cell cytoplasm to acidify ertheless, the possibility exists that the ABA concentration (Daeter, Slovik & Hartung 1993). This has multiple effects arriving at the leaf in the xylem stream may be greater than on the root ABA signal, all based on the fact that ABA that loaded in at the root, and this effect may be intensified always becomes concentrated in the most alkaline compart- in drying soil. ments, and all leading to an increase in the amount of ABA that finally reaches the xylem, by at least two or three-fold The leaf (Slovik, Daeter & Hartung 1995). Firstly, these pH changes inhibit ABA loss from the root to the rhizosphere. Sec- It is described above how ABA entering the leaf via the ondly, all shoot-sourced ABA arriving at the root in the xylem does not all reach the guard cells because of seques- phloem is compartmentalized in the apoplast. Both Liang et tration into the symplastic ‘reservoir’, and that soil drying- al. (1997) and Neales & Mcleod (1991) have reported that induced changes in xylem and therefore in apoplastic sap root dehydration gives rise to increases in phloem-sourced pH modulate the rate of this process. However, several ABA in the xylem sap of 25–30%, not a negligible amount. additional stress-induced phenomena occur in the leaf to Finally, cytoplasmic acidification causes ABA synthesized intensify the root-sourced ABA signal that finally reaches in the cytoplasm or previously stored there to be released to the epidermis. One, or a combination of the following, the apoplast (see below). The fact that more root ABA could explain why the extent of stomatal closure observed becomes compartmentalized in the apoplast as a result of in droughted plants is often greater than can be attributed drought-induced pH changes means that it is less likely to alone to the concentration of ABA found in the xylem (e.g. be catabolized or simply stored, and a greater percentage Correia & Pereira 1995). finally reaches the xylem vessels. To summarize, a soil drying ABA signal classically asso- A full symplastic reservoir. Under severe drought, shoot ciated simply with the induction of synthesis in the root can water potentials drop and ABA biosynthesis occurs in the be intensified by the simultaneous effects of dry soil to cells of the leaf as well as in those of the root. This means reduce ABA catabolism, to prevent rhizosphere- and that the leaf symplast contains a great deal more ABA than phloem-sourced ABA from entering the symplast, to that of a turgid leaf. This rapidly alters the ABA concentra- induce more effective efflux of the synthesized ABA to the tion at the guard cells not only as a result of the penetration apoplast, and potentially to increase water and therefore of this newly synthesized ABA, but also because ABA ABA flux towards the xylem. entering the leaf at the xylem cannot enter the symplastic Once ABA has reached the xylem vessel its transfer into reservoir and therefore ends up at the epidermis. Filling of this conduit can also be modulated by the environment. the symplastic reservoir therefore increases the sensitivity Xylem vessels have the ability to modify the pH of solutions of the stomata to the ABA coming from the xylem. For passing through them (Clarkson, Williams & Hanson 1984). example, when xylem ABA concentrations return to well- Fromard et al. (1995) showed that H+-pumping ATPases are watered values after a period of water deficit, stomatal re- much more concentrated in the plasma membranes of opening is often sluggish (e.g. Trejo et al. 1995), and this xylem parenchyma cells than in any other plant cell type. may be because the symplast is still full of ABA. Thus, even They showed that these pumps were involved in the control the low well-watered concentrations of ABA carried by the of vascular sap pH, and that this control fluctuated with sea- xylem may be capable of maintaining stomata in the closed son. Xylem pH values were close to neutrality in winter state until normal rates of symplastic sequestration are (when ATPase activity was low), whereas they were acidic restored. In related work, Heckenberger, Schurr & Schulze (pH 5·5) at the beginning of spring. Hartung & Radin (1996) found that when ABA influx to sunflower leaves was

200 S. Wilkinson & W. J. Davies large, either due to long-term feeding of low concentrations chemical changes may occur either in addition to or instead or short-term feeding of high concentrations via the xylem, of the root-sourced increases in xylem sap pH, which stomatal opening after the cessation of feeding took longer increase the penetration of ABA to the guard cells by to occur. ABA flux into the leaf may therefore be an impor- reducing its symplastic uptake, even in leaves that are still tant determinant of stomatal aperture under severe but not fully turgid. incipient drought stress, when the capacity of the symplastic reservoir to remove ABA from the apoplast is mini- Evidence for chemical signals originating in leaves that mized by a reduction of the inwardly-directed pm may control ABA penetration to guard cells. Recently concentration gradient for ABA. it has been found that the leaf may be able to affect the penetration of ABA to the guard cells in the absence of leaf Loss of ABA from the symplast. The symplastic ABA water deficits and/or root-sourced changes in xylem pH. In reservoir in the leaf may also lose its ability to hold on to other words, chemical changes originating in leaves that are the ABA that it contains. The occurrence of ABA release not obviously hydraulically induced may influence the from root cells has already been described above. There is ABA signal. This was first indicated by the finding that plenty of evidence in the literature that leaf cells are also changes in VPD (and mild soil drying episodes) reduced capable of releasing ABA to the surrounding medium, and leaf growth and stomatal aperture in some species without Hartung and co-workers have suggested that drought- affecting water potential, apparently by sensitising growth induced increases in xylem sap pH cause leaf cells to release receptors and guard cells to xylem ABA (Tadieu & Davies ABA to the apoplast. This could cause stomatal closure in 1992, 1993). This is an entirely different response to that the absence of any increase in bulk leaf ABA concentra- described above, in which large soil water deficits (or tion, as has often been described (references in Wilkinson increases in VPD) induce obvious hydraulic signals that & Davies 1997). However, Wilkinson & Davies (1997) reduce leaf water status prior to stomatal closure. The could find no evidence for ABA efflux from tissue isolated authors coined the term ‘isohydric’ to describe the resultant from turgid leaves in response to an increase in pHext. On stability of leaf water status that they described in the field, the other hand, when leaves become dehydrated, the cell in the face of fluctuating VPD and soil water content. They cytoplasm is acidified. Hartung, Kaiser & Burschka (1983) proposed that mild stresses induce small hydraulic changes, showed that wilting induced the efflux of 14C-ABA from the possibly undetectable in bulk shoot water measurements, mesophyll of preloaded Valerianella locusta leaf tissue so that can directly sensitize guard cells to ABA so that sto- that it was able to penetrate to the lower epidermis. Har- mata close and maintain leaf water status. We have pro- tung & Radin (1989) showed that cotton leaf dehydration posed here and elsewhere that soil drying-induced pH reduced the activity of outwardly rectifying pm H+ATPases, changes can often be responsible for that heightened sen- which would acidify the symplast. This mechanism was sim- sitivity of leaf physiological responses to xylem ABA some- ilar to, but separate from that involved in the alkalinization times observed, such as those described by Tardieu & of the xylem sap of plants in drying soil referred to above. Davies (1992, 1993). It is also possible that leaf-sourced Kaiser & Hartung (1981) demonstrated that the efflux changes in pH can occur in response to variable VPDs in of ABA from the cytoplasm of isolated mesophyll cells of the absence of VPD-induce leaf water deficits, possibly

Papaver somniferum increased with pHext in the presence of alongside the VPD-induced hydraulic inﬂuence proposed

KNO2, which acidified the cytoplasm. However, they were by Tardieu & Davies (1992, 1993). Some evidence that this unable to detect an effect of pHext, from 5 to 8, on ABA does indeed occur is provided below; however, it is by no release in the absence of this salt. ABA is usually present in means a universal phenomenon. For example, in sunflower its dissociated form (ABA–) in the normally high pH of the ABA and plant water status do not interact at all even when cytoplasm. This form cannot cross the plasma membrane leaf water deficits are clearly detectable (Tardieu, Lafarge and it is therefore trapped in the symplast. ABAH formed & Simonneau 1996) and plant water status is variable and upon cytoplasmic acidification is lipophilic, and can diffuse unregulated (anisohydric). out of the symplast such that ABA accumulates in the apo- Recent work at Lancaster University has shown that the plast if this is alkaline enough. aerial environment can change leaf sap pH in both For- Thus, episodes of soil (or air) drying that are severe sythia × intermedia cv Lynwood and Hydrangea macro- enough to induce reductions in shoot water potential may phylla cv Bluewave. Increases in sap pH correlated with increase the penetration of ABA to the guard cells in one of reductions in stomatal conductance in the absence of mea- three ways. Firstly, the up-regulation of ABA biosynthesis surable changes in leaf relative water content in F. interme- in leaves means that bulk leaf ABA concentrations dia. Aerial conditions that increase transpiration (high increase, but it also means that ABA arriving at the leaf via VPD, PPFD and leaf surface temperature) increased the the xylem is more easily able to penetrate to the epidermis pH of sap expressed under pressure from the upper parts of as the symplastic reservoir will be full. Finally, leaf dehy- F. intermedia and H. macrophylla shoots, and the stomata dration-induced cytoplasmic acidification induces more were more closed (see Fig. 2A and B for data from H. mac- effective release of symplastic ABA to the apoplast, rophylla, see Davies, Wilkinson & Loveys 2002 for data increasing its likelihood of penetrating to the guard cells in from F. intermedia). The same conditions that caused sto- the epidermis. These leaf-sourced hydraulically induced matal closure in the afternoon did not do so in the morning

ABA-based chemical signalling 201 in F. intermedia (and vice versa in H. macrophylla), suggest- the guard cells of environmental information received by ing that the sensitivity of guard cells to pH (and ABA) both the roots and the shoot. Changes in sap pH may be a change over the course of the day. unifying influence on guard cells through which both classes In F. intermedia, high VPD/PPFD, increased pH and of information may act. reduced stomatal conductance correlated with increases in It is as yet unknown which aerial factor caused the bulk leaf (but not xylem sap) ABA, and we suggest that changes in pH and stomatal aperture described above. High ABA removal by the phloem may have been inhibited (see VPD may have reduced leaf cell pm H+-ATPase activity by below). In H. macrophylla the same set of conditions had causing slight (localised) changes in water relations (see no effect on bulk leaf [ABA], and it can only be hypothe- above, Hartnung & Radin 1989). Alternatively (or addi- sized that the increased pH increased the penetration of tionally) at high PPFD, increased removal of CO2 from the xylem-sourced ABA to the guard cells and/or induced its apoplast for photosynthesis may alkalize this compartment. release from the symplast as a result of the types of pro- Stahlberg, Van Volkenburgh & Cleland (2001) found that cesses described above. That the climatically induced photosynthetic removal of CO2 from Coleus leaves induced changes in sap pH originated from changes within the leaf an increase in apoplastic pH that could be propagated over apoplast rather than from the incoming xylem sap itself a distance of 2 cm. A high PPFD- and/or temperature- were indicated by effects of soil drying on the response. induced increase in nitrate uptake by leaf cells could have Unusually (but see below), soil drying acidified the xylem the same effect (see below). sap of F. intermedia plants. In plants in drying soil the cor- The multiple effects of pH (in the root, stem and leaf) on relation between apoplastic sap pH and both the changes in the ABA signalling process raise an interesting possibility aerial conditions and changes in stomatal conductance were that species that exhibit a very low stomatal sensitivity to poor (Davies et al. 2002). This was hypothesized to result ABA may do so because the xylem/apoplastic pH has not from the combination of the sap from the two different become more alkaline with soil drying/climatic changes. sources (root and leaf, see also Mühling & Lauchli 2001), Schurr & Schulze (1995, 1996) have shown that there are which would partially override the climate-induced pH marked differences between species in the effects of soil change. There is plenty of evidence in the literature that drying on hormone and nutrient fluxes to shoots. In our such gradients in pH exist between the leaf apoplast and the experience, this is also true for changes in pH (soil drying xylem, with pH often being higher in the apoplast (Hoff- increases sap pH in tomato, C. communis and barley; mann & Kosegarten 1995; Mühling & Lauchli 2000). Reg- decreases pH in F. intermedia, and has no effect on sap pH ulation of gas exchange will be the result of a processing by from H. macrophylla and Cotinus coggyria cv Royal Pur- )

1 (a)

– (b) s 2 – Xylem sap pH Stomatal conductance (mmol m

PPFD (mmol m–2 s–1) PPFD (mmol m–2 s–1)

Figure 2. Ambient PPFD (n = 6 ± SE) incident on the first fully expanded leaf of well-watered Hydrangea macrophylla cv Bluewave plants of approx. 50 cm in height (three or four branches) growing in 3 L pots in polythene tunnels in July–September 2000, plotted against (a) the stomatal conductance of the leaves (n = 6 ± SE) and (b) the pH of xylem sap expressed immediately afterwards from the top 10 cm of a dominant shoot under pressure (n = 3 ± SE). Each point represents measurements taken approximately every other day (a) or every 5– 6 d (b). Second-order regressions are shown, along with 95% confidence intervals (dotted lines) and r2 curve coefficients, as calculated on Sigmaplot version 1·02.

© 2002 Blackwell Science Ltd, Plant, Cell and Environment, 25, 195–210 202 S. Wilkinson & W. J. Davies ple). These results suggest that the differences in signalling apoplastic pH on loading of ABA into the phloem comes responses to drought are especially significant between from work by Peuke, Jeschke & Hartung (1994), who woody and herbaceous species (see below). showed that ABA flux in the phloem from the leaves of ammonium-treated plants is extremely large. As will be Release of ABA from inactive transport forms within explained below, ammonium nutrition reduces the apoplas- the leaf. Another way in which the leaf may modify the tic pH of leaves. ABA signal has been described by Dietz et al. (2000). ABA Other work has shown that the ABA concentration actu- conjugates, which are themselves unable to affect stomatal ally increases in the phloem in response to stress (Wolf, aperture or plant growth, have been detected in the xylem Jeschke & Hartung 1990). This presumably occurs only sap of stressed plants (Netting, Willows & Milborrow 1992). under more severe and/or prolonged stress in which [ABA] ABA-GE (ABA-glucose ester) seems to be the most com- will be high in all tissues due to both root and shoot bio- mon of these. Glycosylation of ABA is thought to be a synthesis. mechanism to allow for the export of ABA from cells independently of the prevailing transmembrane proton gradient. In addition, its release from root stelar cells and THE SENSITIVITY OF GUARD CELLS TO A liberation to the xylem seems to be greatest under condi- PERCEIVED CONCENTRATION OF ABA tions of inhibited ABA metabolism (Sauter & Hartung 2000). ABA-GE transport may therefore be important as a As described above, plants control the amounts of ABA root-sourced stress signal during soil flooding when ABA that reach the guard cells in the leaf epidermis via processes synthesis becomes inhibited. ABA-GE in barley xylem sap that originate in the root, the stem or the leaf. However, has also been shown to increase under salinity, whereas its they also control the sensitivity with which guard cells concentration in the apoplast of primary leaves was respond to the ABA concentration that finally reaches unchanged (Dietz et al. 2000). This is because the barley them. Changes in guard cell sensitivity to ABA occur over leaf apoplastic sap contained the enzyme β-glucosidase, the day/night cycles and as a plant matures, often in which rapidly cleaved ABA from the inactive ABA-GE response to changes in electrical and osmotic membrane pool arriving at the leaf apoplast via the xylem. The data potentials generated by guard cells themselves (for reviews support the hypothesis that ABA-GE is a long-distance see Assmann & Shimazaki 1999; Blatt 2000). transport form of ABA. Anything that affects the activity of β-glucosidase in the leaf will therefore affect the [ABA] in the leaf apoplast, presumably thereby affecting stomatal Effects of apoplastic composition on guard cell behaviour. There is also some evidence that ABA can be sensitivity to ABA released from its conjugated form in the root before transport in the xylem (Sauter & Hartung 2000). Calcium Calcium ions are known to enter guard cells and promote Removal of ABA from leaves by the phloem. Finally, stomatal closure and inhibit opening when applied to iso- leaves may alter the ABA signal by influencing the rate at lated epidermis, and apoplastic calcium sensitizes guard which ABA is removed from them via the phloem. If less cells to ABA (De Silva, Hetherington & Mansfield 1985). ABA exits the leaf then presumably there is an increased This is because ABA-induced stomatal closure involves likelihood that the remaining ABA will penetrate to the increases in cytosolic-free Ca 2+ in guard cells as part of the guard cells. Jia & Zhang (1997) detected a large reduction of signal transduction chain leading to loss of turgor ABA transport out of detached maize leaves in the phloem (McAinsh, Brownlee & Hetherington 1990). The addition when buffers at a ‘droughted’ pH of 7·4 as opposed to a ‘well of 8 mM calcium to the transpiration stream of detached watered’ pH of 5·5 were injected into the xylem. Wilkinson leaves initiates rapid reductions in stomatal conductance & Davies (1997) found that detached C. communis leaves (Ruiz, Atkinson & Mansfield 1993). Variations in calcium µ taking up 3 M ABA from the external medium had a and ABA concentration in the xylem with soil drying greater bulk leaf [ABA] 3 h later when this was fed at a (Schurr et al. 1992) may interact to limit stomatal opening. ‘droughted pH’ of 7 as opposed to pH 6. These data suggest To add further complexity, guard cell sensitivity to external that an alkaline apoplastic pH prevents ABA movement out calcium can also change under some circumstances (see of the leaf via the phloem. This could either result from the below). decreased pH gradient between the apoplast and the phloem, trapping the ABA in the leaf apoplast (if apoplastic Potassium ABA transfer is important), or from a decrease in the amount of ABA present in the sieve element companion Potassium is centrally involved in the up-regulation of tur- cell (if symplastic phloem transfer is predominant). Phloem gor-driven stomatal opening, and the effectiveness of ABA sieve tubes are living cells with an internal pH of 7·5 (Baier at closing stomata may be reduced when its application to & Hartung 1991), so that the phloem’s efficiency as an ABA the medium bathing epidermal strips exceeds a certain con- reservoir (and export conduit) will depend on the same centration (Wilson, Ogunkanmi & Mansfield 1978; see types of processes described above. Support for an effect of below). The significant reductions in xylem sap [K+]

© 2002 Blackwell Science Ltd, Plant, Cell and Environment, 25, 195–210 ABA-based chemical signalling 203 observed at high bulk soil density and in dry soil (Mulhol- tivity to local ABA, otherwise we would find that high land et al. 1999; Roberts & Snowman 2000) may have max- xylem sap pH opens stomata, and this is often clearly not imized a potential reduction by ABA of stomatal the case (e.g. Patonnier, Peltier & Marigo 1999, Wilkinson conductance. It is also interesting to note that ABA in the 1999). root inhibits potassium loading into the xylem by xylem parenchyma cells (Roberts & Snowman 2000), indicating Sugars that ABA in the root could potentially sensitize stomata to ABA in the leaf by reducing K+ availability as a guard cell There is some evidence in the literature that apoplastic sug- osmoticum. ars could potentially increase guard cell sensitivity to ABA. As the water potential in soil around ash seedlings decreased, there was a significant increase in the concen- Protons trations of malate and mannitol in the xylem and a concom- Protons can also directly affect stomatal aperture and/or its itant decrease in maximum stomatal conductance sensitivity to ABA. There may be an optimum apoplastic (Patonnier et al. 1999). A transpiration bioassay using pH for stomatal opening related to the activity of guard cell detached leaves from control plants found that mannitol pm H+-ATPases and other pm ion channels that directly had no effect on stomata, whereas malate between 0·5 and control turgor. Wilkinson & Davies (1997) and Wilkinson et 3 mM (the range found in xylem sap from the droughted al. (1998) have provided evidence that the direct effect of plants) prevented stomatal opening. Other structural pH on stomatal aperture in isolated epidermis can be very malate analogues (citrate, aspartate or shikimate) had the different to that seen when buffers of differing pH are sup- same effect, whereas neutral amino acids did not. The plied to intact leaves via the xylem. Increasing the pH of the authors suggested that the effect of malate on stomata was medium on which isolated epidermal strips of C. communis to specifically increase the activity of the anion efflux chan- were floating actually opened stomata and reduced their nel at the pm of the guard cells (GCAC1), as described by sensitivity to ABA (see also Schwartz et al. 1994). Feeding Hedrich & Marten (1993) for V. faba. As ABA also induces artificial sap of pH 7 to intact detached leaves of the ABA- anion loss and therefore reduces turgor in guard cells, it deficient tomato mutant flacca increased stomatal aperture may be supposed that malate and ABA act synergistically and transpirational water loss compared to when the fed at GCAC1 to close stomata. Alternatively, because of the sap was buffered to pH 6, but the response to pH was non-specificity of the effect in ash, Patonnier et al. (1999) reversed when ABA was supplied in the fed sap. Several suggest that the effects of the inorganic acids on stomatal other studies have shown that, whereas pH per se had no aperture were directly dependent on the negative charge of effect on stomata in isolated epidermis, reduced pH did the molecules. However, given that under some circum- sensitize stomata to ABA (e.g. Anderson et al. 1994), as stances increasing the local apoplastic pH at the guard cell described above for C. communis. These authors proposed induces stomatal opening and/or reduced guard cell sensi- that because guard cells take up ABA more efficiently at tivity to ABA as described above, it is possible instead that more acidic pH, its contact with internally located receptors the negative charge of the inorganic acids exerted an indi- would be increased, thereby inducing more sensitive sto- rect effect on stomata. Namely, the increased pH that they matal closure. However, given the evidence that guard cell induce (see below) may cause preferential accumulation of symplastic ABA is not always necessary for stomatal clo- ABA in the apoplast, rather than directly affecting guard sure, it may instead be the case that an acidic external pH cell sensitivity to a given ABA concentration. directly modifies the ionic balance and thereby the turgor High CO2 in the substomatal cavity (see below) also of the guard cell, as suggested above, to predispose it reduces stomatal aperture and may enhance the response toward stomatal closure. For example, there is already evi- of guard cells to ABA. Hedrich & Marten (1993) have sug- dence that under some circumstances (but see below) gested that high CO2 induces photosynthetically derived reduced external pH can directly reduce K+ influx and malate to be released from mesophyll cells adjacent to increase K+ efflux at the guard cell plasma membrane guard cells, which, as described above may directly induce (Roelfsema & Prins 1998). The reason for the fact that in loss of guard cell turgor. Mesophyll-derived apoplastic some cases (Wilkinson & Davies 1997) but not others sucrose is another CO2-sensing candidate which can reduce (Anderson et al. 1994) acidic pH closes stomata in isolated stomatal aperture, by virtue of its osmotic effect to draw out epidermis in the absence of ABA could be related to the guard cell water, but this will presumably occur only in spe- ionic composition of the bathing medium used. Thompson cies which load sucrose into the phloem apoplastically (Lu et al. (1997) describe how in the presence of 75–100 mM KCl et al. 1997). Apoplastic malate or sucrose may signal the stomatal aperture no longer varied with pHext as it did in the plant that mesophyll-derived photosynthetic products are presence of 50 mM KCl, whereas the sensitivity of aperture high and stomata can therefore close to limit water loss. to ABA varied with pH as usual in both media. It must be However, Schmidt & Schroeder (1994) have suggested that supposed, however, that the more potent effect of pH in the the efflux of osmotically active malate from guard cells intact plant is on the distribution of ABA between the com- themselves (which also contain photosynthetic apparatus) partments of the leaf. This must normally override the via anion channels may provide a positive feedback mech- opposing direct effect of pH to modulate guard cell sensi- anism for ‘plentiful photosynthate’-induced stomatal clo-

© 2002 Blackwell Science Ltd, Plant, Cell and Environment, 25, 195–210 204 S. Wilkinson & W. J. Davies sure. As described above, it may be supposed that ABA and malate or sucrose therefore act synergistically to reduce guard cell turgor, and this may be the basis of a high CO2- induced sensitization of stomata to ABA. Alternatively, the apoplastic mode of sucrose loading into the phloem could + remove H from the apoplast and increase its pH (see Tet- ) M low & Farrar 1993), which in an intact leaf would cause m ABA to compartmentalize in the apoplast. However, Zeiger & Zhu (1998) provide evidence that in the light high

CO2 detection occurs at the blue light photoreceptor in the guard cell chloroplast. It is therefore possible that CO2 and ABA could interact directly at the guard cell pm H+- ATPase, as its down-regulation comprises part of the signal

response chain of both ABA and CO2 perception in guard Stomatal aperture ( cells. Apoplastic sugars may only be involved in the stomatal response to CO2 when its concentration is high due to high light-saturated photosynthesis, whereas the direct effect of CO2 to close stomata may occur when its concentration is high because photosynthesis is limiting at low light intensities (which include the blue light wavelengths). However, because stresses such as drought increase the [ABA] (M) concentration of sugars in the xylem as described above, they may still exert an important influence on stomatal sen- Figure 3. The effect of [ABA] on stomatal aperture in detached sitivity to ABA. abaxial epidermal strips of Commelina communis L. at two different temperatures. Strips (approx 35–40) were pre-incubated 3 Other hormones on 25 cm 70 mM KCl at room temperature for 3 h, then transferred to treatment solutions, pre-chilled where necessary, of Cytokinins in the xylem have been shown to decrease sto- 70 mM KCl (4·0 cm3) ± ABA for 1·5 h. All leaves received a PPFD −2 −1 matal sensitivity to xylem ABA in cotton (Radin, Parker & of 350 µmol m s at all times. Stomatal aperture was measured Guinn 1982), and their presence at high concentrations in under the microscope. Each point represents the mean of 25 apertures in each of three strips per treatment (n = 75 ± SE). From the transpiration stream can act directly on stomata to Wilkinson et al. (2001). increase their aperture (Incoll & Jewer 1987a, b). Blackman & Davies (1985) showed that these effects became larger as leaves aged. Fusseder et al. (1992) report data from field studies of almond trees under desert conditions that suggest ABA biosynthesis, it seems counterintuitive that stomata an ameliorating influence of increasing xylem cytokinin become less responsive to this hormone. This finding may concentration on ABA’s effect on stomatal aperture. More explain why some research has shown that low tempera- recently, Stoll et al. (2000) showed that under partial root tures can actually open stomata in vivo in chill-sensitive drying there was an increase in xylem sap and leaf ABA in species. Although we found that stomata remained open grape vines which correlated with reduced stomatal con- when C. communis epidermis was isolated from the rest of ductance. Concomitant increases in xylem sap pH and the leaf and floated on pre-chilled media, the closing reductions in cytokinins were observed, both of which response to low temperatures could be restored (Wilkinson could have enhanced stomatal sensitivity to ABA, the latter et al. 2001) by supplying the epidermal strips with the cal- via direct sensitization. cium concentration estimated to exist in the leaf apoplast (0·01–0·05 mM, De Silva, Honor & Mansfield 1996). Sto- mata in strips floating on the same calcium concentration remained open at room temperature. Thus, chill-induced Effects of the aerial microclimate around the leaf stomatal closure occurs via a sensitization of guard cells to on guard cell sensitivity to ABA calcium in the apoplast, even though stomatal sensitivity to ABA is reduced (Wilkinson et al. 2001). In the context of Temperature this review, it might be more pertinent to state that increas- We have shown recently that chilling temperatures applied ing temperature increases the sensitivity of guard cells to to epidermal strips reduce the sensitivity of C. communis ABA (Fig. 3), presumably by increasing ABA binding to its stomata to ABA in the bathing medium (Fig. 3). This is receptors in the guard cell pm, and/or by increasing the despite the fact that the same conditions induce stomatal activity of the ion transporters involved in the ABA signal closure in the intact leaves of this chill-tolerant species to response chain. counteract chill-induced reductions in water uptake at the Although early responses to chilling temperatures root (Wilkinson et al. 2001). Given much evidence in the lit- directly reduce guard cell sensitivity to ABA, there is evi- erature that chilling temperatures can eventually induce dence in the literature that a previous period of chilling

© 2002 Blackwell Science Ltd, Plant, Cell and Environment, 25, 195–210 ABA-based chemical signalling 205 stress followed by recovery can sensitize guard cells to close chain, will already be less active when light is limiting. How- in response to a second stress such as drought (Wilson 1976) ever, work by Clephan in our laboratory could only detect – ‘chill-hardening’. Allan et al. (1994) found that guard cells evidence for a sensitization of C. communis stomata to in epidermis isolated from cold-acclimated C. communis ABA at low light intensities when leaves were intact, and leaves did not require cytoplasmic increases in calcium for not in isolated epidermis (personal communication). This ABA to cause stomatal closure. Our work (Wilkinson et al. suggests that only indirect effects of light (e.g. on the mes-

2001) demonstrates that chilling temperatures may replen- ophyll-determined [CO2]) affect the response of stomata to ish internal stores of calcium in guard cells. In effect, cells ABA. The same series of experiments revealed that more are ‘primed’ by cold pre-treatment for closure in response to ABA reached the epidermis of intact leaves at lower light a subsequent exposure to ABA, as internal calcium is cen- intensities (despite decreased ABA fluxes into them). It trally involved in the ABA signal transduction chain. was suggested that light-induced alkalinization of the mesophyll chloroplast stroma and therefore ABA retention in VPD chloroplasts (Kaiser & Hartung 1981) may have inhibited ABA penetration to the epidermis in saturating light, such There is much evidence in the literature that increasing that stomata were less sensitive to the [ABA] supplied. VPD closes stomata and increases their sensitivity to ABA, These responses occur over very low light intensities, although again the underlying cellular mechanisms for and will be very different to those suggested to result from these responses remain under debate. We have proposed stressfully high PPFD and/or VPD described earlier, which some explanations for this response in the sections above correlate with apoplastic alkalinization and stomatal (see also Zhang & Outlaw 2001a), based on ABA redistri- closure. bution. However, Assmann, Snyder & Lee (2000) found that increasing VPD closed stomata in both ABA-deficient and ABA-insensitive mutants of Arabidopsis thaliana, showing that, at least in some species, it is necessary to NITRATE STRESS AND ABA SIGNALS MAY invoke the more traditional view that dry air has a more INTERACT AS SOIL DRIES direct effect on guard cell and/or epidermal cell turgor. Plants growing in dry soil frequently show a sensitive Like responses to soil drying, responses to VPD may differ reduction in the delivery of nitrate to shoots and this signal between species, and could be hydraulic or chemical or a can regulate leaf processes independently from a variation combination of both. Saliendra et al. (1995) found that in in plant water relations (Shaner & Boyer 1976). When western water birch,restoration of shoot water potential nitrate supply is limiting (regardless of soil water availabil- using a root pressure vessel fully reversed stomatal closure ity), stomata close, leaves grow more slowly, root growth is in response to both a soil water deficit and increased VPD. maintained, and is often characterized by greater lateral By contrast, Fuchs & Livingstone (1996) found that sto- root proliferation. All of these are also symptoms of water matal responses to VPD in Douglas fir and alder seedlings stress. It may be that the soil drying response that is often were only partially reversed by restoration of shoot water characterized is at least in part a response to a limitation in relations. N supply. One way in which limiting nitrate could reduce leaf Light growth would simply be the result of a decrease in the sup- It is well established that indirect responses of stomata to ply of N-derived amino acids and structural proteins. How- light involve photosynthetic effects on intercellular CO2 ever, there is much evidence that responses to N- concentrations (see above). Stomata are also directly deprivation are governed instead by fast root–shoot (or responsive to light (for reviews see Assmann & Shimazaki shoot–root) chemical signals. Krauss (1978) found marked 1999 and Zeiger & Zhu 1998): photosynthetically active increases in ABA in the xylem sap of rooted sprouting radiation (400–700 nm) induces guard cell photosynthesis potato tubers deprived of N, and increases in shoot and leaf and opens stomata via the production of osmotically active ABA in N-deprived plants have since been observed in sev- solutes such as malate. In addition, blue light (425–475 nm) eral species (see Radin et al. 1982; Clarkson & Touraine induces stomatal opening by transposing a signal cascade in 1994). However Peuke et al. (1994) found that ABA in low guard cells that includes a stimulation of H+ efflux to the nitrate-grown Ricinus communis was only increased in the apoplast via pm H+-ATPases. This provides the driving xylem and not in the leaves. Similar data were reported by force for uptake of K+ and Cl– and therefore increases Brewitz, Larsson & Larsson (1995) for barley. Some studies guard cell turgor (depending on the prevailing membrane detected no long-term effect of reduced nitrate supply on potential, see Roelfsema & Prins 1998). Stomata may be tissue ABA concentrations at all (Chapin et al. 1988, barley more sensitive to ABA under conditions of low light and tomato). When Peuke et al. (1994) grew R. communis because: (a) the intercellular [CO2] will be higher (less pho- on ammonium as a substitute N source, rather than on low tosynthetic removal); (b) reduced guard cell photosynthesis N, they were able to demonstrate large increases in leaf and means lower guard cell [malate]; and/or (c) the blue-light- xylem ABA, and also large increases in phloem ABA trans- dependent H+-ATPase in the guard cell pm, which is port, but the stomata in the leaves did not close in response directly down-regulated as part of the ABA signal response to this increase in ABA flux around the plant.

To explain these rather variable responses, it has been its symplastic sequestration and increasing the efficiency of suggested that rather than inducing increases in [ABA] per its transfer into the phloem and out of the leaf. Similarly, + se, low nitrate availability may modulate responses in the Allen & Raven (1984) found that R. communis with NH4 shoot via a sensitization of stomata/leaf growth receptors to as the N source transpired more water per g dry matter syn- – ABA. Radin et al. (1982) have shown that both N and P thesized than when NO3 was the N source. Raven, Griffiths deficiency can enhance stomatal sensitivity to an ABA sig- & Allen (1984) went a step further and showed that nal. Schurr et al. (1992) found that the concentration of ammonium-grown plants actually had increased stomatal nitrate in the xylem sap of droughted sunflower plants reg- apertures. ulated stomatal sensitivity to ABA. It is possible that ABA So far, the data described above would tend to suggest and nitrate can interact to influence stomata via nitrate- that a reduction in nitrate availability, by whatever means induced changes in the pH relations of the leaf (Raven & (soil nutrient deficiency, soil moisture deficiency, soil com- Smith 1976). paction or soil flooding), would tend to decrease the leaf There is now much evidence available for effects of N- apoplastic pH, as nitrate nutrition alkalizes this compart- containing compounds on plant pH balance. Mengel, ment. This is counterintuitive because, as discussed above, Planker & Hoffmann (1994) found that young sunflower acidic apoplastic sap pH reduces the accessibility of ABA to – plants growing on a sole NO3 (or bicarbonate) medium stomata, and would tend to oppose stomatal closure (as in had chlorotic pale leaves, but those on ammonium nitrate ammonium nutrition). However, all of these stresses are – did not. On NO3 (or bicarbonate), the pH of the leaf apo- characterized by stomatal closure and increased sensitivity plast was increased in comparison to that of plants on of stomata to ABA. Some of them are characterized by ammonium, and this reduced leaf chlorophyll content by increases in xylem sap pH. How can we explain these dis- inhibiting iron reduction in the apoplast (this process has an crepancies? acidic pH optimum). Tagliavini et al. (1994) reported simi- The answer lies in the fact that N availability influences lar findings in field-grown Kiwi fruit: many of the plants dis- the species of N-containing molecule that is carried in the played chlorotic pale leaves, and these were found to xylem. Different N-containing molecules have different contain xylem sap with a higher pH than those with healthy abilities to influence the xylem and/or apoplastic pH. When green leaves. The plants were growing on calcareous soils, nitrate is plentiful, nitrate is transported in the xylem to the – – which contain high levels of HCO3 and NO3 . When the leaf, where it is reduced in the mesophyll. When nitrate is leaves were sprayed with citric acid or H2SO4, to counteract limiting, either due to low soil availability or to soil the (presumably) nitrate-derived alkalinization, re-green- drought/flooding/compaction, then nitrate reduction is ing occurred. More recently, Kosegarten, Hoffmann & switched from the shoot to the root (see Lips 1997). Root Mengel (1999) found that nitrate nutrition (and nitrate plus nitrate reductase activity produces hydroxyl ions, which are – – HCO3 ) gave rise to a high apoplastic pH in immature but then converted to COO (often as malate). This is carried in not mature sunflower leaves, especially at distinct inter- the xylem to the leaf. Malate and related compounds alka- + veinal sites. This did not occur in sole NH4 or NH4/NO3 lize apoplastic sap to an even greater extent than nitrate nutrition. Mühling & Lauchli (2001) also found that nitrate can. It is well known that organic anion salts such as malate nutrition gave rise to more alkaline leaf apoplastic sap than have higher pH values than mineral anion salts such as ammonium nutrition, in both Phaseolus vulgaris and sun- nitrates (Wallace, Wood & Soufi 1976). Kirkby & Arm- flower, but not in Vicia faba or Zea mays. Mengel et al. strong (1980) showed that under nitrate nutrition, organic – (1994) hypothesize that NO3 may alkalize apoplastic sap acids were present in only trace amounts in the xylem sap of – because it is co-transported into the leaf cytoplasm with a castor oil plants, which had a pH of 5·6. Under NO3 stress proton (Ullrich 1992), and this presumably occurs to a the pH of the sap rose to 7·3, and considerable amounts of greater extent in young leaves because these are the site of organic acid anions were present. Patonnier et al. (1999) – + greatest N reduction. HCO3 may simply neutralize H describe drought-induced increases in xylem sap pH and in + pumped into the xylem sap by ATPases. Soil-sourced NH4 the organic acid fraction of the xylem (that presumably is reduced in roots, and the neutral amino acids which are result from a drought-induced switch of nitrate reduction to produced are transported in the xylem to the leaf instead of the root) that could reduce stomatal aperture in ash. Neu- – NO3 (see below). Amino acids are already protonated at tral amino acids (the product of ammonium nutrition that is apoplastic pH values, so uptake into the mesophyll will transported in the xylem) fed into the xylem of detached – + remove fewer protons from the apoplast than NO3 /H co- leaves had no affect on stomatal aperture. These authors transport, presumably giving rise to a more acidic apoplast. suggested that the organic acids had a direct effect on guard These results could help to explain the findings of Peuke et cells (see above), but it is just as likely that their effect to al. (1994) described above, that even though ammonium- increase xylem sap pH allowed ABA more access to the grown R. communis plants contain more root-sourced ABA guard cells by reducing symplastic sequestration. than nitrate-grown plants, they have open stomata and Rather than having a direct affect on xylem and there- poor water use efficiencies. Ammonium nutrition may have fore leaf apoplastic pH, organic acids could increase apo- given rise to a more acidic leaf apoplast than nitrate nutri- plastic sap pH by virtue of their di-anionic form: upon tion, as it did in sunflower, which as described above can transfer of these to the symplast, the uptake of two protons reduce the accessibility of ABA to guard cells by increasing and therefore their removal from the apoplast will be

© 2002 Blackwell Science Ltd, Plant, Cell and Environment, 25, 195–210 ABA-based chemical signalling 207 required. Whether they directly increase xylem sap pH, or guard cells. This technology will allow us to test some of the increase apoplastic sap pH by removing protons during hypotheses proposed in this review. symplastic sequestration, the result will be the same: greater penetration of ABA to its site of action at the guard cells. This could explain why much research shows ACKNOWLEDGMENTS increased stomatal sensitivity to ABA in response to N- deficiency, whereas N-deficiency does not always lead to The authors would like to thank the members of MAFF increases in leaf [ABA]. It could also be one explanation Horticulture Link project HL0132LHN/HNS 97 for for the effect of soil drying to increase xylem sap pH. financial support towards the work with ornamental To summarize, the literature shows that xylem/apoplas- species. We are grateful to the American Society of Plant tic sap seems to be most alkaline under N deficiency when Biologists for permission to reprint Fig. 1 (represented as nitrate reduction in the root produces inorganic anions and Fig. 2 at source) from: Wilkinson, Clephan & Davies (2001). loads them to the xylem. It is of intermediate alkalinity This work is copyrighted by the American Society of Plant when nitrate is carried to leaves in the xylem stream, and Biologists. most acidic when ammonium is reduced in roots and the neutral amino acids produced are loaded to the xylem stream. REFERENCES An explanation for differences between species in the effects of rhizospheric stresses on xylem sap pH could lie in Addicott F.T. & Carns H.R. (1983) History and introduction. In the inherent species response to low nitrate availability. Abscisic Acid (ed. F. T. Addicott), pp. 1–21. Praeger, New York. 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