ABA may gain entry to plant roots in the following ways: it can be synthesized in the root (or released from conjugated forms), it can be taken up from soil water surrounding the root, or it can be delivered to the root from the shoots by the phloem. Once inside the root the ABA can be taken up by the symplast and stored or degraded, or it may be transferred from cell to cell towards the xylem vessels, or carried apoplastically with the transpiration stream towards the xylem. Alternatively, ABA may be lost from the root by diffusion into the soil water (rhizosphere). If root ABA is not degraded, stored in the symplast or lost into the soil water it is transferred into the xylem vessels for transport to the leaf. ABA loading into the xylem can either occur directly from the apoplast, or indirectly via specialized xylem parenchyma cells found along the length of the root xylem. There are several points along this root ABA uptake and transport pathway that can be influenced by the environment such that the strength of the root-sourced ABA signal in the xylem can be enhanced or reduced. These are described below.
Root cells constitutively synthesize a low ‘well-watered’ amount of ABA, and as described above this rate of synthesis is massively increased by root tissue dehydration, classically associated with soil drying. However the strength of this ‘basic’ ABA signal sent from roots to shoots can be modified in other less obvious ways. Firstly the soil around the roots may be influential, not only as regards the direct effects of its moisture or nutrient content (see last section). Hartung et al. (1996) suggested that when rhizospheric ABA is high (for example at low soil water contents) the outwardly directed concentration gradient for ABA between the root and the soil could be reduced. This makes it less likely that root ABA will diffuse out of the plant, potentially strengthening the xylem ABA signal further along the transpiration stream. In addition, different soil types are characterized by different pH values, and as described above ABA tends to accumulate in alkaline compartments as a result of the anion trapping effect (see below and Wilkinson 1999). Degenhardt et al. (2000) have shown that in alkaline soil substrates considerable portions of the ABA synthesized in the roots can be leached out into the soil solution, although species with an exodermis (e.g. Zea mays) were less susceptible to this type of stress. In this way alkaline soil could weaken the root-sourced ABA signal.
More usually though, rhizospheric ABA is transported into the root with the soil water. Water flow into and across the root (see Steudle 2000) is a result of the tension created by the transpiration stream (in which case water flows apoplastically), or of the decreased osmotic potential of the xylem sap in the absence of transpiration from the leaf (in which case water flows from cell to cell). Anything that increases water flux across the root should promote the 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). However, until recently this prediction could not have been made, as it was believed that radial transport of ABA across roots was entirely trans-cellular due to the perceived impermeability of the exo- and endodermis. This would have meant that increased flux of apoplastic water into the xylem upon increased transpiration would dilute the xylem [ABA] (e.g. Else et al. 1994). However, Freundl, Steudle & Hartung (1998, 2000) found that filtration of ABA from the apoplastic flow of water as it crossed the endodermis built its local concentration up high enough to cause ‘solvent drag’ through this potential barrier into the xylem. This is has been termed the ‘apoplastic by-pass’ for ABA.
Different species transport root water to different extents within each pathway (symplastic or apoplastic), and when the symplastic route is necessarily dominant (e.g. in heavily suberized roots), root cell plasma membranes contain large concentrations of water channels or ‘aquapoprins’ which can be actively regulated. Interestingly, ABA itself can increase the flow of water into and through the root, otherwise known as its hydraulic conductivity (Hose, Steudle & Hartung 2000). This is believed to result from ABA-induced opening of these inwardly directed water channels (Tyerman et al. 1999; Netting 2000). Soil drying and salinity have been found to increase root hydraulic conductivity, and the induction of ABA biosynthesis in the root by these stresses may effectively maximize the transport of the newly synthesized ABA within the root towards the xylem (positive feedback). As described above, water and ABA flux across the root is also regulated by the osmotic potential of the xylem sap. Again, ABA itself may reduce the xylem osmotic potential and thereby increase water and ABA flux across the root, by inducing ion loading into the xylem, e.g. Glinka (1980)– potentially another positive feedback mechanism to maximize the root-sourced ABA signal.
Because root cells may degrade or store the ABA that they synthesize or take up as it flows past them, not all the ABA that enters the root reaches the xylem vessels. Conditions that reduce the ability of root cells to degrade or take up and store ABA will increase the amounts that penetrate to the xylem. The catabolic degradation of ABA takes place constitutively in most plant cell types (see Cutler & Krochko 1999 for review), but there is some evidence that soil drying reduces the rate of this process in roots. Liang, Zhang & Wong (1997) found that the half-life of 3H-ABA fed to maize roots was extended from 1·15 to 02·27 h by drying the soil around them. Thus, more of the fed ABA could potentially penetrate to the xylem.
Soil drying also leads to changes in the pH of the various compartments of the root. That of the apoplast increases, and root dehydration causes the cell cytoplasm to acidify (Daeter, Slovik & Hartung 1993). This has multiple effects on the root ABA signal, all based on the fact that ABA always becomes concentrated in the most alkaline compartments, and all leading to an increase in the amount of ABA that finally reaches the xylem, by at least two or three-fold (Slovik, Daeter & Hartung 1995). Firstly, these pH changes inhibit ABA loss from the root to the rhizosphere. Secondly, all shoot-sourced ABA arriving at the root in the phloem is compartmentalized in the apoplast. Both Liang et al. (1997) and Neales & Mcleod (1991) have reported that root dehydration gives rise to increases in phloem-sourced ABA in the xylem sap of 25–30%, not a negligible amount. Finally, cytoplasmic acidification causes ABA synthesized in the cytoplasm or previously stored there to be released to the apoplast (see below). The fact that more root ABA becomes compartmentalized in the apoplast as a result of drought-induced pH changes means that it is less likely to be catabolized or simply stored, and a greater percentage finally reaches the xylem vessels.
To summarize, a soil drying ABA signal classically associated simply with the induction of synthesis in the root can be intensified by the simultaneous effects of dry soil to reduce ABA catabolism, to prevent rhizosphere- and phloem-sourced ABA from entering the symplast, to induce more effective efflux of the synthesized ABA to the apoplast, and potentially to increase water and therefore ABA flux towards the xylem.
Once ABA has reached the xylem vessel its transfer into this conduit can also be modulated by the environment. Xylem vessels have the ability to modify the pH of solutions passing through them (Clarkson, Williams & Hanson 1984). Fromard et al. (1995) showed that H+-pumping ATPases are much more concentrated in the plasma membranes of xylem parenchyma cells than in any other plant cell type. They showed that these pumps were involved in the control of vascular sap pH, and that this control fluctuated with season. Xylem pH values were close to neutrality in winter (when ATPase activity was low), whereas they were acidic (pH 5·5) at the beginning of spring. Hartung & Radin (1989) reported that plant water stress could reduce the activity of these H+-ATPases associated with the root xylem, and suggested that this may be one of the causes of the increased alkalinity often observed in this compartment under stress (see below for other hypotheses). Increases in xylem pH enhance ABA loading to the root xylem by increasing the capacity of the xylem sap to trap anions.
It is described above how ABA entering the leaf via the xylem does not all reach the guard cells because of sequestration into the symplastic ‘reservoir’, and that soil drying-induced changes in xylem and therefore in apoplastic sap pH modulate the rate of this process. However, several additional stress-induced phenomena occur in the leaf to intensify the root-sourced ABA signal that finally reaches the epidermis. One, or a combination of the following, could explain why the extent of stomatal closure observed in droughted plants is often greater than can be attributed alone to the concentration of ABA found in the xylem (e.g. Correia & Pereira 1995).
A full symplastic reservoir. Under severe drought, shoot water potentials drop and ABA biosynthesis occurs in the cells of the leaf as well as in those of the root. This means that the leaf symplast contains a great deal more ABA than that of a turgid leaf. This rapidly alters the ABA concentration at the guard cells not only as a result of the penetration of this newly synthesized ABA, but also because ABA entering the leaf at the xylem cannot enter the symplastic reservoir and therefore ends up at the epidermis. Filling of the symplastic reservoir therefore increases the sensitivity of the stomata to the ABA coming from the xylem. For example, when xylem ABA concentrations return to well-watered values after a period of water deficit, stomatal re-opening is often sluggish (e.g. Trejo et al. 1995), and this may be because the symplast is still full of ABA. Thus, even the low well-watered concentrations of ABA carried by the xylem may be capable of maintaining stomata in the closed state until normal rates of symplastic sequestration are restored. In related work, Heckenberger, Schurr & Schulze (1996) found that when ABA influx to sunflower leaves was large, either due to long-term feeding of low concentrations or short-term feeding of high concentrations via the xylem, stomatal opening after the cessation of feeding took longer to occur. ABA flux into the leaf may therefore be an important determinant of stomatal aperture under severe but not incipient drought stress, when the capacity of the symplastic reservoir to remove ABA from the apoplast is minimized by a reduction of the inwardly-directed pm concentration gradient for ABA.
Loss of ABA from the symplast. The symplastic ABA reservoir in the leaf may also lose its ability to hold on to the ABA that it contains. The occurrence of ABA release from root cells has already been described above. There is plenty of evidence in the literature that leaf cells are also capable of releasing ABA to the surrounding medium, and Hartung and co-workers have suggested that drought-induced increases in xylem sap pH cause leaf cells to release ABA to the apoplast. This could cause stomatal closure in the absence of any increase in bulk leaf ABA concentration, as has often been described (references in Wilkinson & Davies 1997). However, Wilkinson & Davies (1997) could find no evidence for ABA efflux from tissue isolated from turgid leaves in response to an increase in pHext. On the other hand, when leaves become dehydrated, the cell cytoplasm is acidified. Hartung, Kaiser & Burschka (1983) showed that wilting induced the efflux of 14C-ABA from the mesophyll of preloaded Valerianella locusta leaf tissue so that it was able to penetrate to the lower epidermis. Hartung & Radin (1989) showed that cotton leaf dehydration reduced the activity of outwardly rectifying pm H+ATPases, which would acidify the symplast. This mechanism was similar to, but separate from that involved in the alkalinization of the xylem sap of plants in drying soil referred to above. Kaiser & Hartung (1981) demonstrated that the efflux of ABA from the cytoplasm of isolated mesophyll cells of Papaver somniferum increased with pHext in the presence of KNO2, which acidified the cytoplasm. However, they were unable to detect an effect of pHext, from 5 to 8, on ABA release in the absence of this salt. ABA is usually present in its dissociated form (ABA–) in the normally high pH of the cytoplasm. This form cannot cross the plasma membrane and it is therefore trapped in the symplast. ABAH formed upon cytoplasmic acidification is lipophilic, and can diffuse out of the symplast such that ABA accumulates in the apoplast if this is alkaline enough.
Thus, episodes of soil (or air) drying that are severe enough to induce reductions in shoot water potential may increase the penetration of ABA to the guard cells in one of three ways. Firstly, the up-regulation of ABA biosynthesis in leaves means that bulk leaf ABA concentrations increase, but it also means that ABA arriving at the leaf via the xylem is more easily able to penetrate to the epidermis as the symplastic reservoir will be full. Finally, leaf dehydration-induced cytoplasmic acidification induces more effective release of symplastic ABA to the apoplast, increasing its likelihood of penetrating to the guard cells in the epidermis. These leaf-sourced hydraulically induced chemical changes may occur either in addition to or instead of the root-sourced increases in xylem sap pH, which increase the penetration of ABA to the guard cells by reducing its symplastic uptake, even in leaves that are still fully turgid.
Evidence for chemical signals originating in leaves that may control ABA penetration to guard cells. Recently 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 water deficits and/or root-sourced changes in xylem pH. In other words, chemical changes originating in leaves that are not obviously hydraulically induced may influence the ABA signal. This was first indicated by the finding that changes in VPD (and mild soil drying episodes) reduced leaf growth and stomatal aperture in some species without affecting water potential, apparently by sensitising growth receptors and guard cells to xylem ABA (Tadieu & Davies 1992, 1993). This is an entirely different response to that described above, in which large soil water deficits (or increases in VPD) induce obvious hydraulic signals that reduce leaf water status prior to stomatal closure. The authors coined the term ‘isohydric’ to describe the resultant stability of leaf water status that they described in the field, in the face of fluctuating VPD and soil water content. They proposed that mild stresses induce small hydraulic changes, possibly undetectable in bulk shoot water measurements, that can directly sensitize guard cells to ABA so that stomata close and maintain leaf water status. We have proposed here and elsewhere that soil drying-induced pH changes can often be responsible for that heightened sensitivity of leaf physiological responses to xylem ABA sometimes observed, such as those described by Tardieu & Davies (1992, 1993). It is also possible that leaf-sourced changes in pH can occur in response to variable VPDs in the absence of VPD-induce leaf water deficits, possibly alongside the VPD-induced hydraulic influence proposed by Tardieu & Davies (1992, 1993). Some evidence that this does indeed occur is provided below; however, it is by no means a universal phenomenon. For example, in sunflower ABA and plant water status do not interact at all even when leaf water deficits are clearly detectable (Tardieu, Lafarge & Simonneau 1996) and plant water status is variable and unregulated (anisohydric).
Recent work at Lancaster University has shown that the aerial environment can change leaf sap pH in both Forsythia×intermedia cv Lynwood and Hydrangea macrophylla cv Bluewave. Increases in sap pH correlated with reductions in stomatal conductance in the absence of measurable changes in leaf relative water content in F.intermedia. Aerial conditions that increase transpiration (high VPD, PPFD and leaf surface temperature) increased the pH of sap expressed under pressure from the upper parts of F. intermedia and H. macrophylla shoots, and the stomata were more closed (see Fig. 2A and B for data from H. macrophylla, see Davies, Wilkinson & Loveys 2002 for data from F. intermedia). The same conditions that caused stomatal closure in the afternoon did not do so in the morning in F. intermedia (and vice versa in H. macrophylla), suggesting that the sensitivity of guard cells to pH (and ABA) change over the course of the day.
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.
In F. intermedia, high VPD/PPFD, increased pH and reduced stomatal conductance correlated with increases in bulk leaf (but not xylem sap) ABA, and we suggest that ABA removal by the phloem may have been inhibited (see below). In H. macrophylla the same set of conditions had no effect on bulk leaf [ABA], and it can only be hypothesized that the increased pH increased the penetration of xylem-sourced ABA to the guard cells and/or induced its release from the symplast as a result of the types of processes described above. That the climatically induced changes in sap pH originated from changes within the leaf apoplast rather than from the incoming xylem sap itself were indicated by effects of soil drying on the response. Unusually (but see below), soil drying acidified the xylem sap of F. intermedia plants. In plants in drying soil the correlation between apoplastic sap pH and both the changes in aerial conditions and changes in stomatal conductance were poor (Davies et al. 2002). This was hypothesized to result from the combination of the sap from the two different sources (root and leaf, see also Mühling & Lauchli 2001), which would partially override the climate-induced pH change. There is plenty of evidence in the literature that such gradients in pH exist between the leaf apoplast and the xylem, with pH often being higher in the apoplast (Hoffmann & Kosegarten 1995; Mühling & Lauchli 2000). Regulation of gas exchange will be the result of a processing by the guard cells of environmental information received by both the roots and the shoot. Changes in sap pH may be a unifying influence on guard cells through which both classes of information may act.
It is as yet unknown which aerial factor caused the changes in pH and stomatal aperture described above. High VPD may have reduced leaf cell pm H+-ATPase activity by causing slight (localised) changes in water relations (see above, Hartnung & Radin 1989). Alternatively (or additionally) at high PPFD, increased removal of CO2 from the apoplast for photosynthesis may alkalize this compartment. Stahlberg, Van Volkenburgh & Cleland (2001) found that photosynthetic removal of CO2 from Coleus leaves induced an increase in apoplastic pH that could be propagated over a distance of 2 cm. A high PPFD- and/or temperature-induced increase in nitrate uptake by leaf cells could have the same effect (see below).
The multiple effects of pH (in the root, stem and leaf) on the ABA signalling process raise an interesting possibility that species that exhibit a very low stomatal sensitivity to ABA may do so because the xylem/apoplastic pH has not become more alkaline with soil drying/climatic changes. Schurr & Schulze (1995, 1996) have shown that there are marked differences between species in the effects of soil drying on hormone and nutrient fluxes to shoots. In our experience, this is also true for changes in pH (soil drying increases sap pH in tomato, C. communis and barley; decreases pH in F. intermedia, and has no effect on sap pH from H. macrophylla and Cotinus coggyria cv Royal Purple). These results suggest that the differences in signalling responses to drought are especially significant between woody and herbaceous species (see below).
Release of ABA from inactive transport forms within the leaf. Another way in which the leaf may modify the ABA signal has been described by Dietz et al. (2000). ABA conjugates, which are themselves unable to affect stomatal aperture or plant growth, have been detected in the xylem sap of stressed plants (Netting, Willows & Milborrow 1992). ABA-GE (ABA-glucose ester) seems to be the most common of these. Glycosylation of ABA is thought to be a 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 liberation to the xylem seems to be greatest under conditions of inhibited ABA metabolism (Sauter & Hartung 2000). ABA-GE transport may therefore be important as a root-sourced stress signal during soil flooding when ABA synthesis becomes inhibited. ABA-GE in barley xylem sap has also been shown to increase under salinity, whereas its concentration in the apoplast of primary leaves was unchanged (Dietz et al. 2000). This is because the barley leaf apoplastic sap contained the enzyme β-glucosidase, which rapidly cleaved ABA from the inactive ABA-GE pool arriving at the leaf apoplast via the xylem. The data support the hypothesis that ABA-GE is a long-distance 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 behaviour. There is also some evidence that ABA can be released from its conjugated form in the root before transport in the xylem (Sauter & Hartung 2000).
Removal of ABA from leaves by the phloem. Finally, leaves may alter the ABA signal by influencing the rate at which ABA is removed from them via the phloem. If less ABA exits the leaf then presumably there is an increased likelihood that the remaining ABA will penetrate to the guard cells. Jia & Zhang (1997) detected a large reduction of ABA transport out of detached maize leaves in the phloem when buffers at a ‘droughted’ pH of 7·4 as opposed to a ‘well watered’ pH of 5·5 were injected into the xylem. Wilkinson & Davies (1997) found that detached C. communis leaves taking up 3 µm ABA from the external medium had a greater bulk leaf [ABA] 3 h later when this was fed at a ‘droughted pH’ of 7 as opposed to pH 6. These data suggest that an alkaline apoplastic pH prevents ABA movement out of the leaf via the phloem. This could either result from the decreased pH gradient between the apoplast and the phloem, trapping the ABA in the leaf apoplast (if apoplastic ABA transfer is important), or from a decrease in the amount of ABA present in the sieve element companion cell (if symplastic phloem transfer is predominant). Phloem sieve tubes are living cells with an internal pH of 7·5 (Baier & Hartung 1991), so that the phloem’s efficiency as an ABA reservoir (and export conduit) will depend on the same types of processes described above. Support for an effect of apoplastic pH on loading of ABA into the phloem comes from work by Peuke, Jeschke & Hartung (1994), who showed that ABA flux in the phloem from the leaves of ammonium-treated plants is extremely large. As will be explained below, ammonium nutrition reduces the apoplastic pH of leaves.
Other work has shown that the ABA concentration actually increases in the phloem in response to stress (Wolf, Jeschke & Hartung 1990). This presumably occurs only under more severe and/or prolonged stress in which [ABA] will be high in all tissues due to both root and shoot biosynthesis.