Anti-transpirant activity in xylem sap from flooded tomato (Lycopersicon esculentum Mill.) plants is not due to pH-mediated redistributions of root- or shoot-sourced ABA

In flooded soils, the rapid effects of decreasing oxygen availability on root metabolic activity are likely to generate many potential chemical signals that may impact on stomatal apertures. Detached leaf transpiration tests showed that filtered xylem sap, collected at realistic flow rates from plants flooded for 2 h and 4 h, contained one or more factors that reduced stomatal apertures. The closure could not be attributed to increased root output of the glucose ester of abscisic acid (ABA-GE), since concentrations and deliveries of ABA conjugates were unaffected by soil flooding. Although xylem sap collected from the shoot base of detopped flooded plants became more alkaline within 2 h of flooding, this rapid pH change of 0.5 units did not alter partitioning of root-sourced ABA sufficiently to prompt a transient increase in xylem ABA delivery. More shoot-sourced ABA was detected in the xylem when excised petiole sections were perfused with pH 7 buffer, compared with pH 6 buffer. Sap collected from the fifth oldest leaf of "intact" well-drained plants and plants flooded for 3 h was more alkaline, by approximately 0.4 pH units, than sap collected from the shoot base. Accordingly, xylem [ABA] was increased 2-fold in sap collected from the fifth oldest petiole compared with the shoot base of flooded plants. However, water loss from transpiring, detached leaves was not reduced when the pH of the feeding solution containing 3-h-flooded [ABA] was increased from 6.7 to 7.1 Thus, the extent of the pH-mediated, shoot-sourced ABA redistribution was not sufficient to raise xylem [ABA] to physiologically active levels. Using a detached epidermis bioassay, significant non-ABA anti-transpirant activity was also detected in xylem sap collected at intervals during the first 24 h of soil flooding.     

Manipulation of the apoplastic pH of intact plants mimics stomatal and growth responses to water availability and microclimatic variation

The apoplastic pH of intact Forsythiaxintermedia (cv. Lynwood) and tomato (Solanum lycopersicum) plants has been manipulated using buffered foliar sprays, and thereby stomatal conductance (g(s)), leaf growth rate, and plant water loss have been controlled. The more alkaline the pH of the foliar spray, the lower the g(s) and/or leaf growth rate subsequently measured. The most alkaline pH that was applied corresponds to that measured in sap extracted from shoots of tomato and Forsythia plants experiencing, respectively, soil drying or a relatively high photon flux density (PFD), vapour pressure deficit (VPD), and temperature in the leaf microclimate. The negative correlation between PFD/VPD/temperature and g(s) determined in well-watered Forsythia plants exposed to a naturally varying summer microclimate was eliminated by spraying the plants with relatively alkaline but not acidic buffers, providing evidence for a novel pH-based signalling mechanism linking the aerial microclimate with stomatal aperture. Increasing the pH of the foliar spray only reduced g(s) in plants of the abscisic acid (ABA)-deficient flacca mutant of tomato when ABA was simultaneously sprayed onto leaves or injected into stems. In well-watered Forsythia plants exposed to a naturally varying summer microclimate (variable PFD, VPD, and temperature), xylem pH and leaf ABA concentration fluctuated but were positively correlated. Manipulation of foliar apoplastic pH also affected the response of g(s) and leaf growth to ABA injected into stems of intact Forsythia plants. The techniques used here to control physiology and water use in intact growing plants could easily be applied in a horticultural context.     

Effect of leaf water status and xylem pH on metabolism of xylem-transported abscisic acid

Metabolism and distribution of xylem-fed ABA were investigated in leaves of maize (Zea mays) and Commelina communis when water stress and xylem pH manipulation were applied. ³H-ABA was fed to excised leaves via the transpiration stream. Water stress was applied through either a previous soil-drying before leaves were excised, or a quick dehydration after leaves were fed with ABA. Xylem-delivered ABA was metabolised rapidly in the leaves (half-life 0.7 h and 1.02 h for maize and Commelina respectively), but a previous soil-drying or a post-feeding dehydration significantly extended the half-life of fed ABA in both species. In the first few hours after ABA was fed into the detached leaves, percentages of applied ABA remaining unmodified were always higher in leaves which received water stress treatments than in control leaves. However the percentage decreased to below the control levels several hours later in leaves which received a previous soil-drying treatment prior to excision, but had then been rehydrated by the xylem-feeding process itself. One possible explanation for this could be a changed pattern of compartmentalisation for xylem-carried ABA. A post-feeding dehydration treatment also changed the distribution of xylem-fed ABA within the leaves: more ABA was found in the epidermis of Commelina leaves which had been dehydrated rapidly after ABA had been fed, compared to the controls. The levels of xylem-delivered ABA remaining unmodified increased as the pH of the feeding solution increased from 5 to 8. The results support the hypothesis that water stress and a putative stress-induced xylem pH change may modify stomatal sensitivity to ABA by changing the actual ABA content of the leaf epidermis.     

Effects of iron deficiency on the composition of the leaf apoplastic fluid and xylem sap in sugar beet. Implications for iron and carbon transport

The effects of iron deficiency on the composition of the xylem sap and leaf apoplastic fluid have been characterized in sugar beet (Beta vulgaris Monohil hybrid). pH was estimated from direct measurements in apoplastic fluid and xylem sap obtained by centrifugation and by fluorescence of leaves incubated with 5-carboxyfluorescein and fluorescein isothiocyanate-dextran. Iron deficiency caused a slight decrease in the pH of the leaf apoplast (from 6.3 down to 5.9) and xylem sap (from 6.0 down to 5.7) of sugar beet. Major organic acids found in leaf apoplastic fluid and xylem sap were malate and citrate. Total organic acid concentration in control plants was 4.3 mM in apoplastic fluid and 9.4 mM in xylem sap and increased to 12.2 and 50.4 mM, respectively, in iron-deficient plants. Inorganic cation and anion concentrations also changed with iron deficiency both in apoplastic fluid and xylem sap. Iron decreased with iron deficiency from 5.5 to 2.5 microM in apoplastic fluid and xylem sap. Major predicted iron species in both compartments were [FeCitOH](-1) in the controls and [FeCit(2)](-3) in the iron-deficient plants. Data suggest the existence of an influx of organic acids from the roots to the leaves via xylem, probably associated to an anaplerotic carbon dioxide fixation by roots.     

Re-export and metabolism of xylem-delivered ABA in attached maize leaves under different transpirational fluxes and xylem ABA concentrations

By feeding radioactive³H-ABA into attached maize leaves, there-export and metabolism of xylem-delivered ABA and their relationshipswith xylem ABA transpirational fluxes and concentrations wereinvestigated. ABA entering leaves in the transpirational streamwas re-exported out of leaves slowly. Within 24 h the proportionof fed radioactivity that was re-exported was less than 45%.When different concentrations of ³H-ABA (100 nM versus 500 nM)was fed, no difference between the two concentrations was foundin their rates of re-export of the fed radioactivity duringthe first 5 h. After 5 h, very little fed radioactivity wasre-exported in leaves that were fed with 100 nM ³H-ABA, whileleaves that were fed with 500 nM ³H-ABA continued to re-exportsuch that the final proportion remaining in leaves after 24h was less as a result, suggesting a concentration-stimulatedre-export. When ³H-ABA was fed at two different transpirationrates which were induced by different air humidity, a 4-folddifference in transpirational fluxes did not produce any differencein terms of re-exportation of fed radioactivity. The rate ofcatabol-ism of xylem-fed ³H-ABA in the attached leaves was muchfaster than that of re-export. On average fed ³H-ABA had a half-lifeof 2.2 h and only 8% remained unmodified after 24 h of incubation,suggesting that re-exported radioactivity might not be the intactform of ABA at all. Using the parameters obtained from the feeding experiment, wecalculated that in a real soil-drying situation the possiblemaximum amount of xylem-delivered ABA that could accumulatein leaves during a day. It was found that the proportion ofdaily accumulated ABA was only 5% of the leaf ABA in well-wateredplants. In soil-dried plants the maximum amount of daily accumulationby xylem ABA could reach 20% of the leaf ABA at the beginningof soil drying, but it soon declined to about 5% again. Thedeclined contribution was mainly due to a reduced transpirationand an increased total leaf ABA as a result of aggravated leafwater deficit. A tight relationship between leaf conductanceand the accumulation of xylem-delivered ABA was not found. Key words: Abscisic acid, ABA, ABA export, ABA metabolism, xylem-delivered ABA, maize     

Abscisic acid triggers whole-plant and fruit-specific mechanisms to increase fruit calcium uptake and prevent blossom end rot development in tomato fruit

Calcium (Ca) uptake into fruit and leaves is dependent on xylemic water movement, and hence presumably driven by transpiration and growth. High leaf transpiration is thought to restrict Ca movement to low-transpiring tomato fruit, which may increase fruit susceptibility to the Ca-deficiency disorder, blossom end rot (BER). The objective of this study was to analyse the effect of reduced leaf transpiration in abscisic acid (ABA)-treated plants on fruit and leaf Ca uptake and BER development. Tomato cultivars Ace 55 (Vf) and AB2 were grown in a greenhouse environment under Ca-deficit conditions and plants were treated weekly after pollination with water (control) or 500 mg l(-1) ABA. BER incidence was completely prevented in the ABA-treated plants and reached values of 30-45% in the water-treated controls. ABA-treated plants had higher stem water potential, lower leaf stomatal conductance, and lower whole-plant water loss than water-treated plants. ABA treatment increased total tissue and apoplastic water-soluble Ca concentrations in the fruit, and decreased Ca concentrations in leaves. In ABA-treated plants, fruit had a higher number of Safranin-O-stained xylem vessels at early stages of growth and development. ABA treatment reduced the phloem/xylem ratio of fruit sap uptake. The results indicate that ABA prevents BER development by increasing fruit Ca uptake, possibly by a combination of whole-plant and fruit-specific mechanisms.     

Apoplastic pH and Fe(3+) reduction in intact sunflower leaves

It has been hypothesized that under NO(3)(-) nutrition a high apoplastic pH in leaves depresses Fe(3+) reductase activity and thus the subsequent Fe(2+) transport across the plasmalemma, inducing Fe chlorosis. The apoplastic pH in young green leaves of sunflower (Helianthus annuus L.) was measured by fluorescence ratio after xylem sap infiltration. It was shown that NO(3)(-) nutrition significantly increased apoplastic pH at distinct interveinal sites (pH >/= 6.3) and was confined to about 10% of the whole interveinal leaf apoplast. These apoplastic pH increases presumably derive from NO(3)(-)/proton cotransport and are supposed to be related to growing cells of a young leaf; they were not found in the case of sole NH(4)(+) or NH(4)NO(3) nutrition. Complementary to pH measurements, the formation of Fe(2+)-ferrozine from Fe(3+)-citrate was monitored in the xylem apoplast of intact leaves in the presence of buffers at different xylem apoplastic pH by means of image analysis. This analysis revealed that Fe(3+) reduction increased with decreasing apoplastic pH, with the highest rates at around pH 5. 0. In analogy to the monitoring of Fe(3+) reduction in the leaf xylem, we suggest that under alkaline nutritional conditions at interveinal microsites of increased apoplastic pH, Fe(3+) reduction is depressed, inducing leaf chlorosis. The apoplastic pH in the xylem vessels remained low in the still-green veins of leaves with intercostal chlorosis.     

Nitrate signalling to stomata and growing leaves: interactions with soil drying, ABA, and xylem sap pH in maize

Increasing the nitrate (N) concentration in the rooting substrate above deficiency decreased stomatal conductance and leaf growth rate compared with sufficient N in maize seedlings (Zea mays L.) growing in drying substrate. Novel effects were detected when N in the non-deficient range was supplied directly to the xylem of detached shoots: concentrations above 2.0 mol m-3 KNO3 reduced transpiration, and concentrations above 12 mol m-3 KNO3 reduced leaf growth rate. Evidence is provided that the novel effects of N on transpiration and growth were mediated by pH-based ABA redistribution. ABA at 0.05 mol m-3, whilst ineffective alone, sensitized leaf growth to increases in KNO3 concentration (from 3.0 mol m-3), and the capacity of higher concentrations of ABA to reduce growth was enhanced by KNO3. Transpiration was sensitively reduced by KNO3, ABA, or buffers adjusted to pH 6.7-7.0 (compared with buffers adjusted to pH 5.0) alone. Nevertheless, a synergistic effect of KNO3 and either ABA or buffers adjusted to pH 6.7-7.0 was observed. Buffers of pH 5.6 supplied to detached shoots alleviated the depression of transpiration caused by 12 mol m-3 KNO3. Buffers adjusted to pH 6.7 increased the sensitivity of growth to KNO3. Xylem sap extracted from intact seedlings growing in drying soil exhibited an initial increase in N concentration, followed by a decrease at progressively lower soil water potentials. The importance for novel N signalling above deficiency is discussed with reference to the generality of fluctuations in soil and xylem N concentration within this range.     

Hydraulic and Chemical Responses of Citrus Seedlings to Drought and Osmotic Stress

In this work we investigated the function of abscisic acid (ABA) as a long-distance chemical signal communicating water shortage from the root to the shoot in citrus plants. Experiments indicated that stomatal conductance, transpiration rates, and leaf water potential decline progressively with drought. ABA content in roots, leaves, and xylem sap was also increased by the drought stress treatment three- to sevenfold. The addition of norflurazon, an inhibitor of ABA biosynthesis, significantly decreased the intensity of the responses and reduced ABA content in roots and xylem fluid, but not in leaves. Polyethylene glycol (PEG)-induced osmotic stress caused similar effects and, in general, was counteracted only by norflurazon at the lowest concentration (10%). Partial defoliation was able to diminish only leaf ABA content (22.5%) at the highest PEG concentration (30%), probably through a reduction of the active sites of biosynthesis. At least under moderate drought (3–6 days without irrigation), mechanisms other than leaf ABA concentration were required to explain stomatal closure in response to limited soil water supply. Measurements of xylem sap pH revealed a progressive alkalinization through the drought condition (6.4 vs. 7.1), that was not counteracted with the addition of norflurazon. Moreover, in vitro treatment of detached leaves with buffers iso-osmotically adjusted at pH 7.1 significantly decreased stomatal conductance (more than 30%) as much as 70% when supplemented with ABA. Taken together, our results suggest that increased pH generated in drought-stressed roots is transmitted by the xylem sap to the leaves, triggering reductions in shoot water loss. The parallel rise in ABA concentration may act synergistically with pH alkalinization in xylem sap, with an initial response generated from the roots and further promotion by the stressed leaves.     

The guard-cell apoplast as a site of abscisic acid accumulation in Vicia faba L.

Abscisic acid (ABA) integrates the water status of a plant and causes stomatal closure. Physiological mechanisms remain poorly understood, however, because guard cells flanking stomata are small and contain only attomol quantities of ABA. Here, pooled extracts of dissected guard cells of Vicia faba L. were immunoassayed for ABA at sub‐fmol sensitivity. A pulse of water stress was imposed by submerging the roots in a solution of PEG. The water potentials of root and leaf declined during 20 min of water stress but recovered after stress relief. During stress, the ABA concentration in the root apoplast increased, but that in the leaf apoplast remained low. The ABA concentration in the guard‐cell apoplast increased during stress, providing evidence for intra‐leaf ABA redistribution and leaf apoplastic heterogeneity. Subsequently, the ABA concentration of the leaf apoplast increased, consistent with ABA import via the xylem. Throughout, the ABA contents of the guard‐cell apoplast, but not the guard‐cell symplast, were convincingly correlated with stomatal aperture size, identifying an external locus for ABA perception under these conditions. Apparently, ABA accumulates in the guard‐cell apoplast by evaporation from the guard‐cell wall, so the ABA signal in the xylem is amplified maximally at high transpiration rates. Thus, stomata will display apparently higher sensitivity to leaf apoplastic ABA if stomata are widely open in a relatively dry atmosphere.     

Nitrate does not result in iron inactivation in the apoplast of sunflower leaves

It has been hypothesized that nitrate (NO(3)(-)) nutrition might induce iron (Fe) deficiency chlorosis by inactivation of Fe in the leaf apoplast (H.U. Kosegarten, B. Hoffmann, K. Mengel [1999] Plant Physiol 121: 1069-1079). To test this hypothesis, sunflower (Helianthus annuus L. cv Farnkasol) plants were grown in nutrient solutions supplied with various nitrogen (N) forms (NO(3)(-), NH(4)(+) and NH(4)NO(3)), with or without pH control by using pH buffers [2-(N-morpholino)ethanesulfonic acid or 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid]. It was shown that high pH in the nutrient solution restricted uptake and shoot translocation of Fe independently of N form and, therefore, induced Fe deficiency chlorosis at low Fe supply [1 micro M ferric ethylenediaminedi(O-hydroxyphenylacetic acid)]. Root NO(3)(-) supply (up to 40 mM) did not affect the relative distribution of Fe between leaf apoplast and symplast at constant low external pH of the root medium. Although perfusion of high pH-buffered solution (7.0) into the leaf apoplast restricted (59)Fe uptake rate as compared with low apoplastic solution pH (5.0 and 6.0, respectively), loading of NO(3)(-) (6 mM) showed no effect on (59)Fe uptake by the symplast of leaf cells. However, high light intensity strongly increased (59)Fe uptake, independently of apoplastic pH or of the presence of NO(3)(-) in the apoplastic solution. Finally, there are no indications in the present study that NO(3)(-) supply to roots results in the postulated inactivation of Fe in the leaf apoplast. It is concluded that NO(3)(-) nutrition results in Fe deficiency chlorosis exclusively by inhibited Fe acquisition by roots due to high pH at the root surface.     

Rapid low temperature-induced stomatal closure occurs in cold-tolerant Commelina communis leaves but not in cold-sensitive tobacco leaves, via a mechanism that involves apoplastic calcium but not abscisic acid

Commelina communis stomata closed within 1 h of transferring intact plants from 27 degrees C to 7 degrees C, whereas tobacco (Nicotiana rustica) stomata did not until the leaves wilted. Abscisic acid (ABA) did not mediate cold-induced C. communis stomatal closure: At low temperatures, bulk leaf ABA did not increase; ABA did not preferentially accumulate in the epidermis; its flux into detached leaves was lower; its release from isolated epidermis was not greater; and stomata in epidermal strips were less sensitive to exogenous ABA. Stomata of both species in epidermal strips on large volumes of cold KCl failed to close unless calcium was supplied. Therefore, the following cannot be triggers for cold-induced stomatal closure in C. communis: direct effects of temperature on guard or epidermal cells, long-distance signals, and effects of temperature on photosynthesis. Low temperature increased stomatal sensitivity to external CaCl(2) by 50% in C. communis but only by 20% in tobacco. C. communis stomata were 300- to 1,000-fold more sensitive to calcium at low temperature than tobacco stomata, but tobacco epidermis only released 13.6-fold more calcium into bathing solutions than C. communis. Stomata in C. communis epidermis incubated on ever-decreasing volumes of cold calcium-free KCl closed on the lowest volume (0.2 cm(3)) because the epidermal apoplast contained enough calcium to mediate closure if this was not over diluted. We propose that the basis of cold-induced stomatal closure exhibited by intact C. communis leaves is increased apoplastic calcium uptake by guard cells. Such responses do not occur in chill-sensitive tobacco leaves.     

Effects of Xylem pH on Transpiration from Wild-Type and flacca Tomato Leaves : A Vital Role for Abscisic Acid in Preventing Excessive Water Loss Even from Well-Watered Plants

The pH of xylem sap from tomato (Lycopersicon esculentum) plants increased from pH 5.0 to 8.0 as the soil dried. Detached wild-type but not flacca leaves exhibited reduced transpiration rates when the artificial xylem sap (AS) pH was increased. When a well-watered concentration of abscisic acid (0.03 μm) was provided in the AS, the wild-type transpirational response to pH was restored to flacca leaves. Transpiration from flacca but not from wild-type leaves actually increased in some cases when the pH of the AS was increased from 6.75 to 7.75, demonstrating an absolute requirement for abscisic acid in preventing stomatal opening and excessive water loss from plants growing in many different environments.Jones (1980) and Cowan (1982) were the first to suggest that plants can “measure” soil water status independently of shoot water status via the transfer of chemical information from roots to shoots. Dehydrating roots in drying soil synthesize ABA more rapidly than fully turgid tissue, and resultant increases in the ABA concentration of xylem sap flowing toward the still-turgid shoot constitutes a chemical signal to the leaves (for review, see Davies and Zhang, 1991): the xylem vessels give up their contents to the leaf apoplast, thereby increasing the ABA concentration in this compartment. ABA receptors on the external surface of stomatal guard cells respond to the apoplastic ABA concentration (Hartung, 1983; Anderson et al., 1994; but see Schwartz et al., 1994). When bound, the receptors transduce a reduction in guard cell turgor, which leads to stomatal closure (Assmann, 1993). This maintains shoot water potential despite the reduction in soil water availability.Another chemical change related to soil drying in the absence of a reduction in shoot water status is an increase in the pH of the xylem sap flowing from the roots (Schurr et al., 1992). The pH of the xylem and/or apoplastic sap of plants can also change dramatically in response to soil flooding, diurnal or annual rhythms, and mineral nutrient supply (Table ​(TableI)I) in the absence of concomitant changes in either root or shoot water status. We already know that, like the increase in xylem ABA concentration described above, an increase in xylem pH can also act as a signal to leaves to close their stomata (Wilkinson and Davies, 1997). Since the conditions that affect xylem/apoplastic pH can also affect transpiration (light intensity [Cowan et al., 1982]; soil drying [Davies and Zhang, 1991]; nitrate supply [Clarkson and Touraine, 1994]; soil flooding [Else, 1996]), the possibility exists that the pH change that they induce could be the means by which they alter stomatal aperture. Table IpH changes that occur in plant xylem or apoplastic sap under various conditions It was originally suggested that an increase in xylem sap pH could putatively enhance stomatal closure by changing the distribution of the ABA that is present in all nonstressed plants at a low “background” concentration, without requiring de novo ABA synthesis (Schurr et al., 1992; Slovik and Hartung, 1992a, 1992b). This hypotheses is built on the well-known fact that weak acids such as ABA accumulate in more alkaline compartments (Kaiser and Hartung, 1981). More recently, Wilkinson and Davies (1997) and Thompson et al. (1997) directly demonstrated that increases in xylem sap pH reduced rates of water loss from Commelina communis and tomato (Lycopersicon esculentum) leaves detached from well-watered plants. This was found to be mediated by the relatively low endogenous concentration of ABA (about 0.01 mmol m⁻³) contained in the xylem vessels and apoplast of these leaves, a concentration of ABA that did not itself affect transpiration at a well-watered sap pH of 6.0. The mechanism by which the combination of high sap pH and such a low concentration of ABA was able to increase the apoplastic ABA concentration sufficiently to close stomata was also elucidated: the mesophyll and epidermis cells of these leaves had a greatly reduced ability to sequester ABA away from the apoplast when the pH of the latter was increased by the incoming xylem sap (Wilkinson and Davies, 1997).In contrast to the indirect ABA-mediated effect of pH on stomata, it was also demonstrated that increasing the pH of the external solution (from 5.0 to 7.0) bathing isolated abaxial epidermis tissue peeled from well-watered C. communis leaves actually increased stomatal aperture (Wilkinson and Davies, 1997). Mechanisms for this direct effect of pH on guard cells have been speculated on by Thompson et al. (1997). If this process were to occur in vivo, environments that increase xylem sap pH could potentially induce excessive water loss from the plants experiencing them, over and above rates of transpiration occurring in unstressed plants. The latter may contain stomata with apertures smaller than the maximum that is possible, even under favorable local conditions. It was assumed that high-pH-induced apoplastic ABA accumulation in C. communis in vivo was sufficient to override the direct stomatal opening effect seen in the isolated tissue (Wilkinson and Davies, 1997). To test these possibilities, effects of pH on transpiration rates from leaves of the flacca mutant of tomato were investigated. flacca does not synthesize ABA as efficiently as wild-type tomato (Parry et al., 1988; Taylor et al., 1988). It contains a very low endogenous ABA concentration (Tal and Nevo, 1973), although it retains the ability to respond to an application of this hormone (Imber and Tal, 1970). The results demonstrate not only that ABA mediates high xylem sap pH-induced stomatal closure but also that it is necessary to prevent high xylem sap pH-induced stomatal opening and dangerously excessive water loss.     

A possible stress physiological role of abscisic acid conjugates in root-to-shoot signalling

Abscisic acid (ABA) conjugates, predominantly their glucose esters, have recently been shown to occur in the xylem sap of different plants. Under stress conditions, their concentration can rise substantially to levels that are higher than the concentration of free ABA. External ABA conjugates cannot penetrate apoplastic barriers in the root. They have to be hydrolysed by apoplastic enzymes in the root cortex. Liberated free ABA can then be redistributed to the root symplast and dragged directly across the endodermis to the stele. Endogenous ABA conjugates are formed in the cytosol of root cells, transported symplastically to the xylem parenchyma cells and released to the xylem vessels. The mechanism of release is unknown; it may include the action of ABC-transporters. Because of its extremely hydrophilic properties, ABA-GE is translocated in the xylem of the stem without any loss to the surrounding parenchyma. After arrival in the leaf apoplast, transporters for ABA-GE in the plasmalemma have to be postulated to redistribute the conjugates to the mesophyll cells. Additionally, apoplastic esterases can cleave the conjugate and release free ABA to the target cells and tissues. The activity of these esterases is increased when barley plants are subjected to salt stress.     

Stress-dependent redistribution of abscisic acid (ABA) in Hordeum vulgare L leaves: the role of epidermal ABA metabolism, tonoplastic transport and the cuticle

When ¹⁴C-labelled abscisic acid ([¹⁴C]ABA) was supplied to isolated protoplasts of the barley leaf at pH 6, initial rates of metabolism were about five times higher in epidermal cell protoplasts than in mesophyll cell protoplasts if equal cytosolic volumes were considered. In spite of the fact that epidermal cells make up only about 35% of the total water space in barley leaves, and despite the small cytosolic volume of these cells, in intact leaves all epidermal cells would thus metabolize half as much ABA per unit time as the mesophyll cells (0–27 and 0–51 mmol h^?1 m^?3 leaf water). Therefore, under these conditions epidermal cells seem to be a stronger sink than mesophyll cells for ABA that arrives via the transpiration stream. However, at an apoplastic pH of 7–25, which occurs in stressed leaves, the proportion of total metabolized ABA would be much smaller in epidermal than in mesophyll cells (0–029 and 0–204 mmolh^?l m^?3 leaf water). Our results indicate that under conditions of slightly alkaline apoplastic pH the epidermis may serve as the main source for fast stress-dependent ABA redistribution into the guard cell apoplast. This is partly the result of ABA transport across the epidermal tonoplast, which is dependent on the apoplastic pH and possibly on the cytosolic calcium concentration. The cuticle seems to be of no particular importance in stress-induced apoplastic ABA shifts and cannot be regarded as a significant sink for high ABA concentrations under stress.     

Abscisic acid in apoplastic sap can account for the restriction in leaf conductance of white lupins during moderate soil drying and after rewatering

We studied the effects of drought on leaf conductance (g) and on the concentration of abscisic acid (ABA) in the apoplastic sap of Lupinus albus L. leaves. Withholding watering for 5d resulted in complete stomatal closure and in severe leaf water deficit. Leaf water potential fully recovered immediately after rewatering, but the aftereffect of drought on stomata persisted for 2d. ABA and sucrose were quantified in pressurized leaf xylem extrudates. We assumed that the xylem sucrose concentration is negligible and hence that the presence of sucrose in leaf extrudates indicated that they were contaminated by phloem. To eliminate this interference, the concentration of ABA in leaf apoplast was estimated by extrapolation to zero sucrose concentration, using the regression between ABA and sucrose concentrations. The estimated apoplastic ABA concentration increased by 100-fold with soil drying and did not return to pre-stress values immediately following rewatering. g was closely related to the concentration of ABA in leaf apoplast. Furthermore, the feeding of exogenous ABA to leaves detached from well-watered plants brought about the same degree of depression in g as resulted from the drought-induced increase in ABA concentration. We therefore conclude that the observed changes in the concentration of ABA in leaf apoplast were quantitatively adequate to explain drought-induced stomatal closure and the delay in stomatal reopening following rewatering.     

PH as a stress signal

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.     

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

There is now strong evidence that the plant hormone abscisic acid (ABA) plays an important role in the regulation of stomatal behaviour and gas exchange of droughted plants. This regulation involves both long-distance transport and modulation of ABA concentration at the guard cells, as well as differential responses of the guard cells to a given dose of the hormone. We will describe how a plant can use the ABA signalling mechanism and other chemical signals to adjust the amount of water that it loses through its stomata in response to changes in both the rhizospheric and the aerial environment. The following components of the signalling process can play an important part in regulation: (a) ABA sequestration in the root; (b) ABA synthesis versus catabolism in the root; (c) the efficiency of ABA transfer across the root and into the xylem; (d) the exchange of ABA between the xylem lumen and the xylem parenchyma in the shoot; (e) the amount of ABA in the leaf symplastic reservoir and the efficiency of ABA sequestration and release from this compartment as regulated by factors such as root and leaf-sourced changes in pH; (f) cleavage of ABA from ABA conjugates in the leaf apoplast; (g) transfer of ABA from the leaf into the phloem; (h) the sensitivity of the guard cells to the [ABA] that finally reaches them; and lastly (i) the possible interaction between nitrate stress and the ABA signal.     

Modulation of Root Signals in Relation to Stomatal Sensitivity to Root-sourced Abscisic Acid in Drought-affected Plants

Stomatal sensitivity to root signals induced by soil drying may vary between environments and plant species. This is likely central role in root to shoot signaling. pH and hydraulic signals may interact with ABA signals and thus, jointly regulate stomatal responses to changed soil water status. pH itself can be modified by several factors, among which the chemical compositions In the xylem stream and the live cells surrounding the vessels play crucial roles. In addition to the xylem pH,more attention should be paid to the direct modulation of leaf apoplastic pH, because many chemical compositions might strongly modify the leaf apoplastlc pH while having no significant effect on the xylem pH. The direct modulation of the ABA signal intensity may be more important for the regulation of stomatal responses to soil drying than the ABA signal per se.The ABA signal is also regulated by the ABA catabolism and the supply of precursors to the roots If a sustained root to shoot communication of soil drying operates at the whole plant level. More importantly, ABA catabolism could play crucial roles In the determination of the fate of the ABA signal and thereby control the stomatal behavior of the root-sourced ABA signal.     

Modulation of Root Signals in Relation to Stomatal Sensitivity to Root-sourced Abscisic Acid in Drought-affected Plants

Stomatal sensitivity to root signals induced by soil drying may vary between environments and plant species. This is likely to be a result of the interactions and modulations ámong root signals. As a stress signal, abscisic acid （ABA） plays a central role in root to shoot signaling, pH and hydraulic signals may interact with ABA signals and thus, jointly regulate stomatal responses to changed soil water status, pH itself can be modified by several factors, among which the chemical compositions in the xylem stream and the live cells surrounding the vessels play crucial roles. In addition to the xylem pH, more attention should be paid to the direct modulation of leaf apoplastic pH, because many chemical compositions might strongly modify the leaf apoplastic pH while having no significant effect on the xylem pH. The direct modulation of the ABA signal intensity may be more important for the regulation of stomatal responses to soil drying than the ABA signal per se. The ABA signal is also regulated by the ABA catabolism and the supply of precursors to the roots if a sustained root to shoot communication of soil drying operates at the whole plant level. More importantly, ABA catabolism could play crucial roles in the determination of the fate of the ABA signal and thereby control the stomatal behavior of the root-sourced ABA signal.