PH as a stress signal

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1 Plant Growth Regulation 29: 87 99, Kluwer Academic Publishers. Printed in the Netherlands. 87 PH as a stress signal Sally Wilkinson Department of Biological Sciences, IENS, Lancaster University, Bailrigg, Lancaster LA1 4YQ, UK Accepted in revised form 23 June 1999 Key words: ABA, apoplast, drought, flacca, flooding, ph, stomatal guard cell, transpiration, xylem sap Abstract The ph of the xylem sap of plants experiencing a range of environmental conditions can increase by over a whole ph unit. This results in an increased ABA concentration in the apoplast adjacent to the stomatal guard cells in the leaf epidermis, by reducing the ability of the mesophyll and epidermal symplast to sequester ABA away from this compartment. As a result the guard cell ABA receptors become activated and the stomata close, enabling the plant to retain water. Were it not for the low concentration of ABA ubiquitous to all land plants, the increase in the ph of the apoplast adjacent to the guard cell would induce stomatal widening, and cause excessive water loss. Not only does ABA prevent this potentially harmful phenomenon, but it also converts the ph increase to a signal which can bring about plant protection. 1. Introduction Plants respond to stresses such as drought, flooding, mineral nutrient deficiency and high salinity by reducing leaf expansion and closing stomatal pores [17, 41, 60, 69]. In this way they conserve the water, nutrients and carbohydrates required for survival. Once the stress is removed and the stomata re-open, photosynthetic CO 2 fixation and the passage of water and nutrients through the transpiration stream are able to continue to provide the plant with the raw materials for further growth. The plant organ that senses the stress sends a signal to the organ or structure in which a response is required for plant protection. One such stress phenomenon is soil drying. In the past the decline in shoot water status commonly observed during severe drought was thought to be central to the control of the resultant changes in leaf conductance and growth [e.g. 46]. However more recently it has been shown that many of these responses to soil drying can occur in the absence of any detectable change in leaf water status [e.g. 26]. The plant must communicate the fact that its roots are in contact with dry soil to the shoot by some means other than a reduction in the amount of water in the shoots. Such a mechanism would have much more control over conductance than leaf water status for the simple reason that leaf water potentials can vary from minute to minute, for example under variable cloud cover, whether or not the soil around the roots is dry. Jones [43] and Cowan [8] have suggested that a measure of soil water status could be made independently from shoot water status via the transfer of chemical information from roots to shoots. Small changes in the turgor, volume, or even pressure on membranes of only a few roots may be all that is necessary to modify shoot physiology [56]. The synthesis and accumulation of the plant hormone abscisic acid (ABA) in the drying roots [87] has since been shown to be one way in which the plant measures soil moisture, and conveys this information to the shoot (see section I). A change in the ph of the xylem sap flowing from the roots in the drying soil is an alternative (or additional) method whereby this information can be conveyed to the aerial parts of the plant, and this process is reviewed here. Other types of root sourced signals include hydraulic changes [50] or action potentials [25], which may arise from stresses such as wounding, pathogens or cold [16, 58]. Below it will be shown that ph changes in root and shoot xylem sap can function as chemical signals to leaves in response to several different types of stress.

2 88 However, in order to understand how these induce the required response in the leaf tissue, namely stomatal closure, it is necessary to examine the research which has led to the discovery that ABA can sometimes, but not always, act as a root sourced signal to the leaves of stressed plants [17, but see 55]. 2. Abscisic acid as a stress signal Over the last few years work from this and other laboratories has provided much evidence for the involvement of ABA in plant protection during stress, especially during soil water deficit. In fact, it has been shown that ABA is produced in roots in contact with drying soil, that it is transported to the shoot in the xylem vessels via the transpirational stream, and that it accumulates in leaves in the vicinity of the stomata [17]. ABA binds to receptors on the external surface of the stomatal guard cell plasma membrane [30, 85; but see 68] and induces a reduction in guard cell turgor [2] such that stomatal pores close in its presence. In other words the roots sense the water deficit and send a signal to the leaves that stomatal closure is required for water conservation. ABA in the epidermis may also control leaf growth [13]. A classic demonstration of this signalling system is to split the root systems of individual plants between two containers. Withholding water from one of the containers, i.e. from half of the root system reduced stomatal conductance in maize leaves [5], and reduced the rate of leaf area development of clonal apple trees by nearly 50% over a three-week period, in the absence of any significant leaf water deficit [29]. The roots in contact with the dry soil synthesize ABA at a much greater rate than those in wet soil [87, 88], which in turn increases the ABA concentration in the xylem stream [89], and thence in the leaves [87] despite the fact that a large portion of the root system is completely unstressed. A proportion of the transpiration stream moves directly to sites of evaporation (sub-stomatal cavities) in the leaf epidermal apoplast adjacent to the guard cells [52]. The transpiration stream can therefore provide a direct link between the roots adjacent to the dry soil and locations in the leaf where stomatal aperture (and potentially leaf growth) can be regulated. It has long been known that ABA fed directly into the xylem stream reduces leaf conductance [e.g. 49]. Quantitative evidence that ABA is indeed the xylem sourced agent inducing stomatal closure in the types of experiment described above was provided by collecting xylem sap from unwatered maize plants, and removing the ABA from this by passing it through an immunoaffinity column composed of ABA antibodies. Re-introduction of this sap to the xylem stream of fresh plants indicated that it no longer contained any anti-transpirant activity [90]. Although we now accept that ABA is a major stress signal from the roots to the leaves of plants growing in dry soil, it is important to note the great variation described in the literature in the sensitivity of leaf conductance to a given concentration of ABA in the xylem stream [e.g. 7], or even the complete lack of an effect of ABA on stomatal activity [55]. This may in part be the result of changing leaf water deficit, caused by fluctuating atmospheric evaporative demand, on stomatal sensitivity to ABA [75, 76]. However uncoupling of the correlation between xylem ABA concentration and stomatal conductance has been demonstrated to occur in sunflower plants artificially held at a constant water potential [66], so changes in leaf water potential cannot always account for this variation. Schurr and Gollan [65] analysed the composition of xylem sap from unwatered sunflower plants, and found that in addition to the expected increase in ABA concentration, there were also changes in the cation, anion and amino acid concentration of the sap, and in its ph and buffering capacity. The only factors which, when taken into account, improved the correlation between xylem ABA concentration and stomatal conductance were sap ph, and nitrate and calcium concentration [27, 66]. The authors proposed that sap ph influenced the sensitivity of stomata to the ABA entering the leaf by changing its distribution between the different leaf compartments, effectively increasing the local concentration of ABA available to the guard cell receptors. In other words the leaf itself may modify the composition of the xylem stream before it reaches the guard cells, and one of the ways in which it may do so could be controlled by xylem sap ph. This proposal was based on knowledge about the effects of ph on leaf tissue ABA distribution described by Wolfram Hartung s group among others (see section III). We now know that stress-induced change in the ph of the xylem sap (even in the absence of extra xylem sap ABA) is itself a signal to leaves to close their stomata in order to conserve water, as well as an agent which may influence the sensitivity of stomata to root sourced ABA [85].

3 89 3. Stress induced changes in xylem and apoplastic sap ph 3.1 Drought Several authors have shown that the ph of the xylem sap extracted from plants growing in drying soil becomes more alkaline. Hartung and Radin [33] found that the ph of xylem sap from water-stressed Phaseolus coccineus roots increased from control levels of 6.3, to 7.2 after two days, whilst the ABA concentration increased from 2.5 to 15.0 nm. In addition, both root and shoot xylem sap from Anastatica hierochuntica (a desert plant) increased from 6.5 (wet soil) to ph 7.1 and ph 7.6 (dry soil) respectively. Gollan et al. [27] found that the ph of shoot xylem sap removed from well-watered sunflower plants was always between 5.8 and 6.6. At soil water contents below0.13gg 1 the ph increased to 7.1 whilst the plants were artificially held at a constant water potential in a root pressure pot. In addition leaf conductance started to decrease, increases in sap ABA concentration became significant, buffering capacity was reduced, and the cation to anion ratio increased. Work from our laboratory has shown that shoot xylem sap ph increased when Commelina communis plants were subjected to 6 days of soil drying (from ca. 6.0 to when sap was collected using a pressure chamber at MPa above balancing pressure) [85]. Experiments carried out at HRI Wellesbourne showed that the ph of xylem sap from de-topped Lycopersicon esculentum plants increased dramatically from 5.0 to 8.0 as gravimetric soil water content decreased from 3.0 to 1.5 g H 2 O/g soil [84] (Figure 1). At the time of writing there is a dearth of published data on the temporal logistics of the ph signal, with respect to how quickly the ph change arises in response to soil drying, to a point where it is able to affect stomatal aperture in leaves. The data of Gollan et al. [27] for sunflower indicate that at a certain soil water content both increased sap ph and ABA concentration acted in concert to reduce conductance, although we have no information on how each signal would have acted alone. The ph of the xylem sap expressed from drying tomato plants [84] was particularly sensitive to soil water content. Results from our laboratory indicate that barley xylem sap ph is also extremely sensitive to soil water content [3]. It is to be expected that the rapidity with which the ph signal arises will vary between species: in some, ph could affect stomatal aperture prior to the accumulation of Figure 1. The effect of gravimetric soil water content (g H 2 O/g soil) on the ph of xylem sap expressed within 2.0 minutes from 3.0 cm shoot stumps of 6-7-week-old wild-type tomato plants. From Wilkinson et al. (1998). ABA in the xylem sap, and in others the reverse could be true. It is assumed that the ph changes which were measured in the roots and leaves of the plants described above are still evident when this sap reaches the leaf apoplast, due to the low buffering capacity of this compartment [34]. There is some evidence that this is indeed the case. Van Volkenburgh and Boyer [83] measured the leaf surface ph of droughted and well-watered maize plants and found the former to be more alkaline. In 1989 Hartung and Radin concluded that drought could induce an apoplastic ph increase, from demonstrations that pressure dehydration of detached cotton leaves simultaneously increased the ph and the ABA concentration of the expressed sap [33]. Hoffman and Kosegarten [37] provided evidence that the ph change may become even more marked the further up the transpiration stream it is measured. Sunflower xylem sap was more acidic than the leaf apoplast (in response to increased nitrate), and microsites around the stomata were the most alkaline. However we still have no direct nondestructive measurements of the apoplastic ph of leaves from droughted plants. The mechanism by which drought increases xylem sap ph has yet to be elucidated. One school of thought evokes changes in proton pumping ATPase

4 90 activity at the plasma membrane of some cells as a controlling factor. It has long been known that xylem parenchyma cells surrounding the vessels can control the composition of the transpiration stream [e.g. 59]. More recently Fromard et al. [24] found that xylem vessel-associated cells from Robinia wood, which contained high concentrations of ATPases, were able to alter xylem sap ph. There is some evidence that drought-related xylem sap ph increases might arise from reduced proton pumping activity at the plasma membranes of the xylem-associated cells of these plants [33]. Another possibility is that drought-induced changes in the ionic composition of the xylem sap may result in a ph increase due to changes in the ratio between strong anions and cations, known as the strong ion difference (SID) [73]. For example Gollan et al. [27] suggested that the reduced concentration of nitrate in xylem sap from droughted sunflower plants may have increased the SID of the sap. This would result in the generation of extra negatively charged species, and an increase in sap alkalinity. However taking nitrate as a particular example, reduced concentrations of this anion in the xylem sap do not always correlate with an increase in xylem/apoplastic sap ph [see below; 14, 37, 53]. Another related way in which drought may influence xylem sap ph is that ionic exchange with the walls of the cells surrounding the xylem vessels may be affected by the changed composition of the sap [63], or by potential effects of drought on cell wall biochemistry. 3.2 Flooding Tomato plants growing in flooded soil also exhibit reduced stomatal conductances. ABA from the roots was not required for this response [40], and indeed its concentration in the xylem sap of flooded plants can be as much as 15-fold lower than in well-drained plants [e.g. 21]. Else et al. [22] proposed that an as yet unidentified anti-transpirant could be transported to the leaves from the flooded roots. This supports earlier findings that xylem sap contains something in addition to ABA that regulates stomatal aperture in droughted plants [54, 80]. Could this anti-transpirant simply be an increase in the ph of xylem sap from the droughted or the flooded plants? We already know that drought often increases the ph of sap (see above), and below it shall be described how this causes a reduction in stomatal aperture in the absence of any extra ABA in the xylem stream. Jackson et al. [42] have examined the composition of xylem sap from flooded and welldrained plants, and describe an increase in the ph of the xylem sap from the former. The ph of sap exuded from cut stumps of 24-hour flooded and well-drained tomato plants was 6.53 and 5.97 respectively before application of pressure to the root system to imitate the transpirational flow rate of sap from intact plants. At over-pressures of 0.1 MPa sap ph increased from 6.53 and 5.97 to 6.74 and 6.13 for flooded and well-drained plants respectively [20]. These ph differences may be even more marked in sap flowing through intact plants. Although Else et al. [22] detected the presence of non-aba anti-transpirant activity in the sap from the flooded plants, they did not suggest that the increase in sap ph was responsible for this activity. It is not known how soil flooding increases xylem sap ph. However, since flooded roots become anaerobic within 24 h, proton pumping from cells adjacent to the xylem may become inhibited [61], causing symplastic acidity within these cells, and increasing the alkalinity of the adjacent xylem sap. Anaerobic roots are also unable to continue to synthesize ABA. 3.3 Nitrate supply Several reports show that increasing the nitrate concentration fed to well-watered plants alkalises the leaf apoplast. Mengel et al. [53] found that the apoplastic ph of sunflower leaves increased from 6.03 to 6.3 upon addition of nitrate to the root system, and fell to ph 5.8 in the presence of ammonium nitrate. Similarly, feeding 10 mm HCO 3 to the cut petioles increased leaf apoplastic ph to 7.0. Hoffmann and Kosegarten [37] found that nitrate but not sulphate supplied to detached sunflower leaves increased the apoplastic ph from 5.7 to 6.4 within 1 hour in the dark. They found that certain micro-sites in the apoplast, especially around the stomata, became even more alkaline upon addition of nitrate (ph 7.0) than the epidermal apoplast as a whole. The anion-induced apoplastic ph increase may have been the result of a combination of processes. Co-transport of nitrate into the symplast with a proton depletes the apoplast of positive charge [82, 86]. The reduction of nitrate to ammonium in the cells of the leaf, and the subsequent synthesis of ammonium to organic material generates one excess OH anion [62]. Unless the hydroxyl ion is neutralised by the operation of the biochemical ph stat [15 which produces malic or oxalic acid from neutral precursors], it may be excreted from the cell to the apoplast, thereby increasing its ph.

5 91 It is important to note however, that the tissue location of nitrate assimilation differs between species. In sunflower plants this occurs both in the leaves and in the roots, but in other species this process takes place solely in the root (Vicia and Lupinus) orsolelyinthe shoot (Xanthium) [1]. Plants which reduce nitrate in the root lose the resultant hydroxyl ions to the soil, such that the nitrate supply to the plant should have no effect on xylem sap ph. Not all authors record a correlation between high nitrate and increased xylem/apoplastic sap ph however. Schurr et al. [66 sunflower] and Jackson et al. [42 tomato] noted that drought- or floodinginduced reductions in xylem sap nitrate concentration were associated with an increase in the ph of the sap. This seems strange, particularly in the case of sunflower, in the light of the results described above for the same species [14, 37, 53]. Perhaps the difference between these two studies lies in the condition of the sunflower plants used. There is some evidence that removal of nitrate from the xylem stream affects its strong ion difference, which in turn increases its alkalinity (see above). This process, rather than a change in nitrate reductase activity, may be the factor which controls the xylem sap ph of droughted and flooded plants. Further study is required to discover whether we can predict the effect that nitrate supply might have on xylem or leaf apoplastic sap ph, by taking into account the position and extent of nitrate reductase activity in each species, and the water status of the individual plant. There is some evidence in the literature that reduced nitrate supply can enhance that sensitivity of stomata to ABA in cotton plants [60], but as yet there is no information as to whether nitrate-induced changes in sap ph are the basis of any change in stomatal aperture or its sensitivity to ABA. 3.4 Fungal infection One instance of fungal infection-induced alkalisation of leaf apoplastic sap exists in the literature. Tetlow and Farrar [77] found that brown rust increased Hordeum vulgare leaf apoplastic ph from 6.6 to 7.3. They suggested that the fungus affected the sugar composition of the leaf apoplast, which may have affected the ph through changes in sugar uptake into the phloem, which involves ATPase activity [18]. No data were presented concerning the effect of brown rust on stomatal aperture. 3.5 Conditions not classified as stresses also induce xylem/apoplastic sap ph changes Diurnal and seasonal periodicity are not classified as stresses to a plant, yet xylem sap ph changes occur over the day/night period, or over the course of a year, as do stomatal apertures. Schurr and Schulze [67] found that Ricinus communis xylem sap ph consistently rose from 6.0 at the end of the dark period, to 6.6 at the end of the day. This contrasts with other findings that darkness induces an increase in apoplastic ph, which correlates with stomatal closure: Hoffmann and Kosegarten [37] found that the ph of sunflower leaf apoplastic sap increased transiently from 5.7 to 6.4 in darkness in the absence of nitrate, and permanently in its presence. Lee and Satter [47] found that the ph of the apoplast of the Samanea pulvinus extensor increased from 6.2 to 6.7 upon transfer to darkness. Several authors have observed that the ph of xylem sap from several species is more acidic in the spring than it is for the rest of the year [23 Actinidea chinensis,24 Robinia wood, 64 Betula pendula]. Irradiance changes both diurnally and annually, as does temperature. Both of these factors affect proton pumping activity [51, 70], which in turn affects ph. The sugar composition of the apoplastic sap also changes both annually and diurnally, and this also affects ATPase activity in this compartment. One or all of these mechanisms could be responsible for the observed ph changes. 4. The mechanism of high ph-induced stomatal closure 4.1 Is ABA required? Direct evidence that the increased ph of sap from droughted plants could function as an early signal to the leaf to reduce stomatal aperture was first provided by Wilkinson and Davies [85]. Well-watered sap from Commelina shoots had a ph of 6.0, whilst that from droughted plants had increased to ca Applications of artificial sap buffered to ph 7.0 to whole, detached Commelina leaves via the xylem stream reduced stomatal aperture in comparison to controls supplied with ph 6.0 buffer, thus reducing abaxial and adaxial leaf conductance and traspirational water loss. Subsequently, this was also demonstrated in tomato leaves (Figure 2) [35, 78, 84], the tomato xylem sap ph having been shown to range between 5.0 and 6.0 in well-watered plants, and between 6.75 and 8.0 in

6 92 Figure 2. The effect of buffer ph on the transpiration rate of detached wild-type tomato leaflets in the light. The transpiration rate was calculated for each leaf every 30 minutes, and exprssed as a percentage of the initial rate (after 30 min). Results are means (n = 8) ± SE. droughted plants. Prior evidence for this phenomenon consisted of correlations between sap ph and stomatal aperture [e.g. 27], or conclusions drawn from models that used data from ph measurements and ABA fluxes from individual cells and tissues (see sections III and IV) to predict how ph changes might affect stomatal aperture in an entire leaf [71, 72]. An increase in xylem sap ph could cause stomatal closure by a variety of mechanisms: 1) It could increase bulk leaf ABA concentration by a) increasing de novo ABA synthesis, b) reducing catabolic degradation or conjugation of endogenous ABA, or c) reducing transport of ABA away from the leaf in the phloem. 2) It could change the water status of the leaf, which might a) directly affect guard cell turgor pressure or b) increase guard cell sensitivity to an unchanged local ABA concentration [75, 76]. 3) It could have a direct effect on guard cell activity and stomatal aperture, perhaps by changing membrane ion fluxes. 4) It could change the distribution of endogenous ABA within the leaf to increase the concentration in the vicinity of the guard cells by a) changing the distribution of ABA between the different leaf tissues, perhaps by inducing movement from the mesophyll to the epidermis; by b) causing the release of ABA from symplastic stores (mesophyll cells, epidermis cells, or even guard cells themselves), as predicted in the model described by SlovikandHartung[72 seebelow];orbyc) causing an accumulation of ABA in the apoplast through inhibition of the normal modes of sequestration into the symplast, bearing in mind that ABA will still arrive from the veins of the leaf. No effects of ph on shoot water potential, bulk leaf ABA concentration, or on the relative concentrations of ABA in the separated leaf tissues (mesophyll, midrib, abaxial and adaxial epidermes) were detected in Commelina [85]; and the reduction of transpirational water loss by ph 7.0 buffer was found to require absolutely the presence of a very low ABA concentration in the leaves. These findings lead us to the conclusion that either mechanism 4b) or 4c) (see above) was responsible for the high ph-induced reduction in stomatal aperture observed in the intact leaves. The absolute requirement for ABA in the response of Commelina leaf transpiration to xylem ph was demonstrated by pre-incubating the leaves in distilled water overnight. This treatment, and an initial transpiration period of 1 2 hours in the same solution, rendered the leaves unresponsive to subsequent transfer to solutions buffered to ph 7.0, i.e. transpiration was no longer reduced by increasing xylem sap ph [85]. However if the pre-incubation in distilled water was carried out in the presence of a low concentration of ABA (10 8 M), which was not great enough alone to affect transpiration at a control ph of 6.0, the subsequent response to ph 7.0 was restored. It was assumed that pre-incubation in water removed most, if not all of the endogenous ABA from the apoplast and the xylem vessels throughout the leaf. In other words the low background concentration of ABA present in the veins and the apoplast of a well-watered leaf [ca M 65] is all that is required for the leaf to be able to respond to an increase in the ph of its xylem stream by closing its stomata. The presence of a well-watered concentration of ABA was also shown to be necessary for the reduction of transpiration by high ph in tomato leaves [84], by investigating the effects of ph on transpiration from leaves of the wilty flacca mutant of tomato. flacca does not synthesize ABA as efficiently as wild-type tomato [e.g. 57]. It contains a very low endogenous ABA concentration [74], although it retains the ability to respond to an external application of this hormone [39]. We were able to show [84] that flacca leaf transpiration rates could not be reduced by phs of

7 93 up to 7.75, whilst leaves from the wild-type plants responded to a ph as low as The stomata of the flacca leaves were not stuck open for some reason, as they closed as normal in response to decreased PAR (photosynthetically active radiation). When a low concentration of ABA (0.03 µm equivalent to that from well-watered plants) was supplied in the artificial xylem sap, which did not itself reduce transpiration at ph 6.25, the high ph was able to mimic the effect it normally had in wild-type leaves, i.e. its ability to reduce the rate of transpirational water loss was restored. This is direct evidence that ABA is an absolute requirement for the reduction of transpirational water loss from detached tomato leaves by increased xylem sap ph. More recently we have also demonstrated that ABA is required for a high ph induced reduction in barley leaf growth [3]. 4.2 ABA and high ph act in concert The basis of the mechanism by which a low wellwatered concentration of ABA and high ph act to close stomata lies in the fact that the symplastic uptake of ABA in the leaf is a ph regulated phenomenon. ABA, like all weak acids, is present in its undissociated form (ABAH) in the relatively acidic milieu of well-watered leaf apoplastic sap (ca. ph ). ABAH is lipophilic, and is thus able to diffuse easily over the lipid plasma membrane of the cells which it encounters as the transpiration stream traverses the leaf (first mesophyll then epidermal cells). However it can only be taken up by the cells if its concentration gradient over the membrane is high. The high concentration gradient for ABAH over the plasma membrane of the cells of the leaf is partially maintained by virtue of the well-buffered alkalinity of the cytoplasm (ph ), which in turn is maintained by the constant removal of protons from this compartment by plasma membrane ATPases. When ABAH enters the alkaline cytoplasm, it dissociates to ABA +H +, thereby maintaining an inward concentration gradient for further ABAH accumulation. ABA is a lipophobic molecule which cannot freely diffuse over the plasma membrane and it is effectively trapped inside the cell [e.g. 36, 44]. For this reason alkaline compartments have come to be known as anion traps. In this way the cells of the leaf are able to remove a great deal of the ABA from the apoplastic sap that flows by them as it is pulled along by the transpiration stream. By the time the sap of well-watered plants reaches the stomata, it does not contain a high enough concentration of ABA to induce their closure. A second mechanism operates alongside this anion trapping phenomenon, to enhance the ability of the leaf cells to sequestre ABA away from the apoplast. ABA trapped in the cell cytoplasm is rapidly catabolised, in both mesophyll cells [28, 81], and even more rapidly in epidermal cells [12]. Published half-lives of ABA range from 36 minutes in cherry to 3.2 hours in Hordeum vulgare [28]. In this way the concentration gradient for further ABA uptake into the cells is supplemented. The mesophyll is able to reduce the concentration of ABA in the sap that would otherwise reach the epidermis [79, 81]. Trejo et al. [81] demonstrated that the concentration of ABA found in the xylem stream of well-watered Commelina shoots before it enters the leaf (ca M) was actually able to induce stomatal closure in isolated epidermal strips, even though it had no effect on the apertures of these in the intact leaf. This is because filtration of ABA from the solution moving towards the epidermis was no longer occurring. It is the concentration of ABA immediately adjacent to the guard cell plasma membrane, which contains the ABA receptors, that directly controls stomatal aperture. In well-watered plants, ABA entering the leaf via the transpiration stream is kept away from this location by the methods described above (Figure 3). However the situation can change dramatically in stressed plants. In stressed plants the apoplastic ph is increased by the xylem sap which enters it from the vascular bundles (see above), which in turn reduces the ph gradient over the plasma membrane of both the mesophyll and the epidermal cells. It has long been understood that the diffusive uptake of weak acids into alkaline compartments is reduced when the external ph is increased. The increased external ph causes undissociated weak acids to dissociate to the anionic form (and a proton). This has two consequences on symplastic acid accumulation. Firstly there is no longer a concentration gradient to drive the passive diffusion of the remaining undissociated acid into the cells, and secondly the dissociated anion is lipophobic and becomes trapped in the external solution. This process was discovered to be relevant to the uptake of ABA by all leaf cell types [36]. A classic demonstration of this phenomenon is to show that the rate of symplastic uptake of 3 H-ABA by both isolated mesophyll and abaxial epidermal tissue is faster when the external medium is buffered to ph 6.0 than to ph 7.0 [e.g. 85 Commelina]. In an intact leaf

8 94 Figure 3. A schematic representation of the cells of a leaf from a well-watered plant (A) removing ABA from the apoplast after it has been delivered there by the xylem stream. ABA does not accumulate at the stomatal pores in the epidermis, so these remain open and lose water. B represents the cells of a droughted plant. Here the ABA is no longer efficiently removed from the apoplast, so it accumulates to a high enough concentration to stimulate the ABA receptors on the external face of the guard cells to close the stomata. this process would cause ABA to accumulate in the apoplast when the ph of this compartment is increased by the incoming alkaline xylem sap. The leaf cells would no longer be able to remove the ABA from the sap as they do in the well-watered plant, and the concentration of ABA in the sap which finally reaches the guard cells in the epidermis is great enough to cause stomatal closure (Figure 3). We were able to show that ABA filtration from the apoplast by the epidermal cells was a more complex process than originally believed [85]. We detected the existence of a saturable uptake component in the epidermis but not in the mesophyll tissue from wellwatered Commelina plants. Adding a high concentration of unlabelled ABA to the external medium bathing the epidermal strips reduced the ability of the tissue to accumulate radiolabelled ABA ( 3 H-ABA) from the same medium, when this was buffered to ph 6.0. This information tells us that the two types of ABA molecule were competing for a limited number of binding sites on the plasma membrane, the binding of ABA to which resulted in facilitated transport to the cell interior. We were able to conclude that both carrier-mediated and diffusive uptake contribute to the efficiency of ABA sequestration by the epidermis at a ph equivalent to that of well-watered sap. When the same experiment was carried out at ph 7.0, the saturable component of ABA uptake disappeared, as did the unsaturable diffusive component. This probably reflects the fact that the ph optimum for ABA binding to the carrier/channel protein in the epidermal plasma membrane was closer to ph 6.0 than to ph 7.0. This process will contribute to the inability of the epidermis to filter ABA from the apoplast, when this becomes perfused with the alkaline sap from the stressed plant, and thus it has a role in high ph-induced stomatal closure. Daeter and Hartung [11] also found evidence for an uptake carrier in epidermal protoplasts from barley, however in contrast to some reports of ABA carrier activity in plant tissues [see 85], its operation was maximal at ph 7.25, and was reduced at ph 6.0. Its action would tend to oppose high ph-induced apoplastic ABA accumulation. However, it may act to prevent excessive ABA build-up in the epidermis. This may prevent barley guard cells from becoming de-sensitised to ABA. 4.3 Apoplastic ph might also influence ABA efflux from symplastic stores The mechanism for the high ph-induced accumulation of ABA in the apoplast described above adequately explains the action of the ph signal on stomatal aperture. However there are data in the literature which provide support for an additional mechanism whereby high apoplastic ph might bring about further ABA accumulation in this compartment: the efflux of ABA from the symplasm. There is some evidence

9 95 that symplastic ABA efflux to the apoplast can occur in detached leaves dehydrating in air [6, 19, 48]. Hartung et al. [32] and Hartung and Radin [33] dehydrated detached leaves under pressure so that they could simultaneously collect the expelled liquid. They assumed that this represented uncontaminated apoplastic fluid, and detected increased concentrations of ABA in the exudate from the leaves placed under the highest pressures, along with a concomitant increase in its ph. When sections of leaves or individual leaf tissues were pre-loaded with radioactive ABA, subsequent dehydration in hyperosmotic media caused the ABA to be effluxed into the external solution [e.g. 10]. All the leaf tissue used in these experiments was subject to a very severe reduction in water potential, whereas the leaves of the stressed plants and the experimental material referred to in the previous sections of this report were fully turgid. It is to be expected that the tissues of turgid and dehydrated leaves will behave very differently from each other, and plants growing in the field, even under stressful conditions, rarely experience such low water potentials in their shoots. It seems that hyperosmotic treatment reduces the ctyoplasmic ph of leaf tissues, and that this phenomenon, rather than a reduced membrane ph gradient per se correlates with symplastic ABA efflux [31, 33]. Kaiser and Hartung [44] were unable to detect any effect of external ph, from 5.0 to 8.0, on ABA release from isolated mesophyll cells of Papaver somniferum, unless an agent was also present which acidified the cytoplasm (in this case KNO 2 ). Similarly we found no effect of increasing the external ph on the rate of 3 H-ABA efflux from either mesophyll or epidermis tissue isolated from well-watered Commelina plants [85]. It would seem therefore, that there is no evidence that increasing the external ph increases the rate of efflux of ABA from the leaf tissues of turgid plants. A contribution to the apoplastic ABA concentration may be made by its efflux from the symplast only under conditions of drought severe enough to cause leaf dehydration and cytoplasmic acidification. Alternatively, fluctuating evaporative demand may locally reduce the water potential of some of the leaves, or even part of a leaf, of an otherwise turgid plant. This phenomenon has already been shown to affect stomatal sensitivity to the xylem ABA concentration of field-grown maize [75, 76], although a role for changes in leaf cell ph gradients was not implied. It is possible that local water-deficit-induced cytoplasmic acidification could lead to a reduction in the ph gradient over the plasma membrane of the leaf cells, which in turn might cause symplastic ABA efflux, and control the aperture of the stomata in this leaf. 5. Potentially harmful effects of a xylem sap ph increase We have provided evidence that external ph can also affect stomatal aperture directly, if ABA accumulation in the vicinity of the guard cells is prevented [85]. We floated isolated abaxial epidermal strips peeled from Commelina leaves on solutions of KCL buffered to a range of phs with either K 2 HPO 4 /KH 2 PO 4 or Mes. In contrast to the effects of ph described above, increasing the ph of the buffer solution increased the aperture of the stomata in the strip. Alkaline ph can be an opening signal in isolated epidermis, although it is a closing signal in the intact leaf. This phenomenon was probably a result of ph-stimulated modifications in guard cell ionic balance. For example Kondo and Maruta [45] found the osmotic potential of Vicia faba guard cells to be more negative at ph 6.0 than at ph 4.0, although in this case apertures were unaffected by ph. Final concentrations of K + and malate in the epidermal strips containing these guard cells were much higher at ph 6.0. It is not clear how high external ph decreases guard cell osmotic potential or increases stomatal aperture, but given that several other authors have shown that it induces K + efflux from guard cells [4, 38], the accumulation of some other solute such as malate may be involved. Alkaline ph-induced malate accumulation may be brought about by the operation of the ph stat mechanism described above, where the production of malic acid occurs to neutralise OH [15]. This normally occurs in response to a potential alkalinisation of the cytosol. However, it is possible that an increased external ph may, for example, promote loss of positively charged species from the guard cell cytosol [as has indeed been shown to occur already 4, 38], which in turn could cause cytosolic alkalinisation were it not for the operation of the biochemical ph stat. Increased ph does not induce stomatal opening in vivo, and this must be a result of the overriding effect that it has on the apoplastic accumulation of ABA in the vicinity of the guard cells. We tested this possibility by comparing the effects of artificial xylem sap ph on transpiration from wild-type and flacca tomato plants. As described above flacca is a mutant which is unable to synthesize its own ABA. Flacca leaf

10 96 transpiration rates were actually increased by increasing xylem sap ph (Figure 4), which was assumed to be the result of direct high ph-induced stomatal opening [84]. Transpiration rates of leaves from the wild-type plant were reduced, aswehavecometo expect and understand (see above), and this effect could be micmicked in the flacca plants by providing them with a low well-watered concentration of ABA (0.03 µm) which was itself not high enough to reduce stomatal aperture at a well-watered ph (Figure 4). In conclusion, increasing the apoplastic ph immediately under the guard cells can directly increase stomatal aperture in vivo, provided that ABA is not present to accumulate to an apoplastic concentration sufficient to induce stomatal closure. The latter effect seems to have the greater control over guard cell osmotic potential. These findings tell us that the increases in xylem sap ph described in section II have the potential to widen the stomata in the leaves of these plants, were it not for the ubiquitous presence of a low background concentration of ABA. In other words the ph signal is potentially harmful, and since it seems to be such a common response to changes in the external environment, which are not necessarily stressful, plants may require ABA to function normally, even when they are not experiencing a water deficit in any of their tissues. The plant has used a potentially harmful chemical change induced by several different factors to actually improve its water use efficiency, and therefore its chances of survival. 6. Conclusions There are a number of gaps in our knowledge about the ph signal, not least of these being the mechanism(s) by which it arises (see section II). One could be tempted to label a ph increase as a negative chemical signal, as it is classically associated with a reduction in proton concentration. However given that we do not yet know how water deficit, let alone the other environmental factors mentioned above change the ph, it is impossible to categorise this signal. For instance drought-induced changes in strong ion concentrations of the sap can result in a ph change without reducing sap proton concentration [73]. We are also uncertain of the extent of the influence of the ph signal on stomatal aperture or leaf growth in vivo during the development of the environmental change which induces it. For example we Figure 4. The effect of buffer ph on the transpiration rate of detached flacca leaves in the light, in the presence and absence of 0.03 µm ABA. Leaves were pre-incubated in artificial xylem sap for 1 h in the dark. The transpiration rate was calculated for each leaf every 30 min and expressed as a mean ± SE (n = 4 6). From Wilkinson et al. (1998). know that during a soil drying cycle the roots also synthesize extra ABA, and the increased ABA concentration itself constitutes a signal to the leaf to close its stomata. The ph signal could either enhance the effect of this new high ABA concentration on stomatal guard cells if it arises later in the drying cycle, or it could initiate stomatal closure before a more severe drought induces the increase in xylem ABA concentration. In the case of a flooded plant, there is no extra ABA signal to the leaves [40], so it would appear that the plant may rely on the ph signal for stomatal closure, although the presence of some other unknown antitranspirant has been proposed to occur in response to flooding [22]. Although we know that nitrate supply affects the xylem and apoplastic sap ph of some plant species under some circumstances, there are no data concerning effects of nitrate supply on stomatal aperture other than via a potential increase in xylem sap ABA concentration. Finally dark and winter-induced stomatal closure are well-known phenomena, and there are several reports that both of these conditions affect at least apoplastic sap ph, but it is not since an early report by Cowan et al. [9] that the two phenomena have been suggested to be causally linked. It seems that the ph signal is a much more widespread phenomenon than originally thought. Most

11 97 research concerns its function as a drought-induced signal. The ph signal could be of very great significance to the ability of plants to survive in the field under a variety of conditions, or at the very least it could greatly affect the efficiency with which a plant uses the resources available to it. Acknowledgements The author is grateful to Professor Bill Davies for providing helpful advice during the preparation of this manuscript. Figures 1 and 4 are copyrighted by the American Society of Plant Physiologists and are reprinted with their kind permission. References 1. Andrews M (1986) The partitioning of nitrate assimilation between root and shoot of higher plants. Plant Cell Environ 9: Assmann SM (1993) Signal transduction in guard cells. Annu Rev Cell Biol 9: Bacon MA, Wilkinson S and Davies WJ (1998) ph-regulated leaf cell expansion in droughted plants is abscisic acid dependent. 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