Effects of Xylem Ph on Transpiration from Wild-Type and Flacca Tomato Leaves1

Plant Physiol. (1998) 117: 703–709

Rapid Communication Effects of Xylem pH on Transpiration from Wild-Type and flacca Tomato Leaves1

A Vital Role for Abscisic Acid in Preventing Excessive Water Loss Even from Well-Watered Plants

Sally Wilkinson*, Janet E. Corlett, Ludovic Oger, and William J. Davies Institute of Environmental and Natural Sciences, Lancaster University, Bailrigg, Lancaster, LA1 4YQ, United Kingdom, (S.W., W.J.D.); and Horticulture Research International, Wellesbourne, Warwick, CV35 9EF, United Kingdom (J.E.C., L.O.)

flooding, diurnal or annual rhythms, and mineral nutrient The pH of xylem sap from tomato (Lycopersicon esculentum) supply (Table I) in the absence of concomitant changes in plants increased from pH 5.0 to 8.0 as the soil dried. Detached either root or shoot water status. We already know that, wild-type but not flacca leaves exhibited reduced transpiration rates like the increase in xylem ABA concentration described when the artificial xylem sap (AS) pH was increased. When a above, an increase in xylem pH can also act as a signal to well-watered concentration of abscisic acid (0.03 ␮M) was provided leaves to close their stomata (Wilkinson and Davies, 1997). in the AS, the wild-type transpirational response to pH was restored to flacca leaves. Transpiration from flacca but not from wild-type Since the conditions that affect xylem/apoplastic pH can leaves actually increased in some cases when the pH of the AS was also affect transpiration (light intensity [Cowan et al., increased from 6.75 to 7.75, demonstrating an absolute require- 1982]; soil drying [Davies and Zhang, 1991]; nitrate supply ment for abscisic acid in preventing stomatal opening and excessive [Clarkson and Touraine, 1994]; soil flooding [Else, 1996]), water loss from plants growing in many different environments. the possibility exists that the pH change that they induce could be the means by which they alter stomatal aperture. It was originally suggested that an increase in xylem sap Jones (1980) and Cowan (1982) were the first to suggest pH could putatively enhance stomatal closure by changing that plants can “measure” soil water status independently the distribution of the ABA that is present in all non- of shoot water status via the transfer of chemical informa- stressed plants at a low “background” concentration, with- tion from roots to shoots. Dehydrating roots in drying soil out requiring de novo ABA synthesis (Schurr et al., 1992; synthesize ABA more rapidly than fully turgid tissue, and Slovik and Hartung, 1992a, 1992b). This hypotheses is built resultant increases in the ABA concentration of xylem sap on the well-known fact that weak acids such as ABA accu- flowing toward the still-turgid shoot constitutes a chemical mulate in more alkaline compartments (Kaiser and Har- signal to the leaves (for review, see Davies and Zhang, tung, 1981). More recently, Wilkinson and Davies (1997) 1991): the xylem vessels give up their contents to the leaf and Thompson et al. (1997) directly demonstrated that apoplast, thereby increasing the ABA concentration in this increases in xylem sap pH reduced rates of water loss from compartment. ABA receptors on the external surface of Commelina communis and tomato (Lycopersicon esculentum) stomatal guard cells respond to the apoplastic ABA con- leaves detached from well-watered plants. This was found centration (Hartung, 1983; Anderson et al., 1994; but see to be mediated by the relatively low endogenous concen- Ϫ Schwartz et al., 1994). When bound, the receptors trans- tration of ABA (about 0.01 mmol m 3) contained in the duce a reduction in guard cell turgor, which leads to sto- xylem vessels and apoplast of these leaves, a concentration matal closure (Assmann, 1993). This maintains shoot water of ABA that did not itself affect transpiration at a well- potential despite the reduction in soil water availability. watered sap pH of 6.0. The mechanism by which the com- Another chemical change related to soil drying in the bination of high sap pH and such a low concentration of absence of a reduction in shoot water status is an increase ABA was able to increase the apoplastic ABA concentration in the pH of the xylem sap flowing from the roots (Schurr sufficiently to close stomata was also elucidated: the me- et al., 1992). The pH of the xylem and/or apoplastic sap of plants can also change dramatically in response to soil sophyll 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 1 This work was supported by the Biotechnology and Biological xylem sap (Wilkinson and Davies, 1997). Science Research Council, UK. * Corresponding author; e-mail [email protected]; fax 44–1–524–843854. Abbreviation: AS, artificial sap. 703 704 Wilkinson et al. Plant Physiol. Vol. 117, 1998

Table I. pH changes that occur in plant xylem or apoplastic sap under various conditions Species Source of Sap pH Change Conditions of Change Reference Phaseolus coccineus Root xylem 6.5–7.0 Wet soil–2-d drought Hartung and Radin (1989) Anastatica hierochuntica Root xylem 6.5–7.1 Wet soil–dry soil Hartung and Radin (1989) Shoot xylem 6.5–7.6 Helianthus annuus Shoot xylem 5.8/6.6–7.1 High soil water–below 0.13 g/g Gollan et al. (1992) C. communis Shoot xylem 6.0–6.5/6.7 Wet soil–4- to 5-day drought Wilkinson and Davies (1997) L. esculentum Root xylem at 0.1 MPa 6.13–6.74 Drained soil–flooded soil Else (1996) Ricinus communis Shoot xylem 6.0–6.6 End of night–end of day Schurr and Schulze (1995) Helianthus annuus Leaf apoplast 5.7–6.4 Light–dark (ϩ2.5 mM nitrate) Hoffmann and Kosegarten (1995) Samanea pulvinus Extensor apoplast 6.2–6.7 White light–dark Lee and Satter (1989) Robinia wood Shoot xylem 6.0–5.0–5.5 Jan–April/May–Nov/Dec Fromard et al. (1995) Actinidea chinensis Shoot xylem 5.3–6.2 Spring–rest of year Ferguson et al. (1983) Betula pendula Shoot xylem 7.5/8.0–5.7 Rest of year–catkin bud break Sauter and Ambrosius (1986) Hordeum vulgare Leaf apoplast 6.6–7.3 Control–brown rust infected Tetlow and Farrar (1993) H. annuus Leaf apoplast 6.8–7.4 Low nitrate–high nitrate Dannel et al. (1995) Ϫ Ϫ H. annuus Leaf apoplast 6.0–6.5 NH4NO3 –NO3 Mengel et al. (1994) Ϫ Ϫ 6.2–7.0 NH4NO3 –HCO3

In contrast to the indirect ABA-mediated effect of pH on daily. At this stage flacca plants were no longer sprayed stomata, it was also demonstrated that increasing the pH of with ABA to prevent contamination of subsequent experi- the external solution (from 5.0 to 7.0) bathing isolated ments. Although humidity was not controlled, the in- abaxial epidermis tissue peeled from well-watered C. com- creased water supply prevented leaf wilt, but leaf surface munis leaves actually increased stomatal aperture (Wilkin- area eventually became smaller than that of wild-type son and Davies, 1997). Mechanisms for this direct effect of leaves. When plants were 6 to 9 weeks old, single leaflets pH on guard cells have been speculated on by Thompson et from the two or three youngest fully expanded wild-type al. (1997). If this process were to occur in vivo, environ- leaves were used as a source of experimental material. In ments that increase xylem sap pH could potentially induce the case of flacca, the three most apical leaflets from appro- excessive water loss from the plants experiencing them, priate leaves were detached together and used as a single over and above rates of transpiration occurring in un- experimental unit to standardize leaf surface area between stressed plants. The latter may contain stomata with aper- the two cultivars. tures smaller than the maximum that is possible, even under favorable local conditions. It was assumed that high- Chemicals pH-induced apoplastic ABA accumulation in C. communis in vivo was sufficient to override the direct stomatal open- Synthetic racemic (Ϯ)-ABA was obtained from Lancaster ing effect seen in the isolated tissue (Wilkinson and Davies, Synthesis Ltd. (Morecambe, Lancashire, UK). General re- 1997). To test these possibilities, effects of pH on transpi- agents used in experiments were all BDH Analar grade ration rates from leaves of the flacca mutant of tomato were from Sigma. investigated. flacca does not synthesize ABA as efficiently as wild-type tomato (Parry et al., 1988; Taylor et al., 1988). Measurement of Xylem Sap pH, Shoot Water Potential, It contains a very low endogenous ABA concentration (Tal and Soil Water Content and Nevo, 1973), although it retains the ability to respond to an application of this hormone (Imber and Tal, 1970). Fifty-two wild-type plants were watered as described The results demonstrate not only that ABA mediates high above, whereas another group of 52 plants was left unwa- xylem sap pH-induced stomatal closure but also that it is tered for 1 to 2 d. Several measurements were taken from necessary to prevent high xylem sap pH-induced stomatal these plants during daylight during the 48-h period, and opening and dangerously excessive water loss. correlations between them were investigated. We found no evidence for a diurnal effect on xylem sap pH when soil moisture content was kept high (results not shown). MATERIALS AND METHODS The stem was cut 30 mm above the soil surface, and the Seeds of tomato (Lycopersicon esculentum L. cv Ailsa remaining stump was blotted to remove any contaminants Craig) (wild-type) or flacca were sown in Levington F2 from cut cells. The pH of xylem sap exuded from the stump compost (Fisons, Ipswich, UK). Seedlings were trans- was measured within 2 min by immersing an Orion com- planted to 0.125- ϫ 0.125-m pots in a greenhouse with a bination needle pH electrode (Orme Scientific Ltd., day/night temperature of 22/15°C. They were watered Manchester, UK) into the droplet. Exudation rates from daily with tap water and weekly with full-strength Hoag- de-topped roots are slow and the sap may become more land nutrient solution (Epstein, 1972). flacca plants were acidic than that in an intact transpiring plant (by up to 0.5 Ϫ sprayed twice weekly with 0.2 mmol m 3 (ϩ)-ABA to keep pH unit after 9 h [Schurr and Schulze, 1995]); however, the growth conditions between the two cultivars as similar as reduction in flow rate is proportional between stressed and possible. Plants 4 to 5 weeks of age were watered twice unstressed plants (Else, 1996). To artificially increase sap Xylem pH Changes Affect Tomato Leaf Transpiration 705 flow rates by pressurizing the root system can dehydrate that they must be signals arising from an effect of de- the tissue (Hartung and Radin, 1989), and this may be creased soil water on the root. disproportionate between stressed and unstressed plants. Several theories exist to explain how the environment To measure water potential, the shoot from the same might control xylem sap pH. It has long been known that plant was quickly sealed into a pressure bomb (Soil Mois- xylem parenchyma cells surrounding the vessels can con- ture Equipment, Santa Barbara, CA). The chamber was trol the composition of the transpiration stream (Pitman et gradually pressurized until the meniscus of the xylem sap al., 1977). More recently, Fromard et al. (1995) found that became visible at the cut tissue surface, and the pressure xylem vessel-associated cells from Robinia pseudoacacia was noted. wood, which contain high concentrations of plasma mem- The soil in which the plant had been growing was sep- brane proton-pumping ATPases, were able to influence arated from the roots and weighed, dried in an oven for xylem sap pH. Presumably this also occurs in roots. There 2 d, and reweighed to calculate gravimetric soil water is some evidence that drought-related and PAR-related pH content. changes in xylem/apoplastic sap might result from differ- ential proton-pumping activity in the cells of these plants (Hartung and Radin, 1989; Marre´ et al., 1989; Hoffmann Transpiration Bioassay and Kosegarten, 1995). It is not known, however, whether Each experiment described below was carried out on a proton-pumping rates are directly affected by tissue dehy- separate batch of plants of an equivalent age. Leaflets were removed from plants that had been kept in the dark for 1 h, and the petiole was recut under degassed distilled water to avoid embolism. They were immediately transferred to Ϫ Ϫ plastic vials of 7.0 ϫ 10 6 m 3 volume that were covered with aluminum foil secured with Parafilm (American Na- tional Can, Greenwich, CT) to reduce evaporation. A “V”- shaped slit was cut into the foil so that the leaf could have access to the contents of the vial. Each of these contained Ϫ Ϫ 5.0 ϫ 10 6 m 3 of AS (unless otherwise stated): 1.0 mol Ϫ3 Ϫ3 Ϫ3 m KH2PO4, 1.0 mol m K2HPO4, 1.0 mol m CaCl2, 0.1 Ϫ3 Ϫ3 Ϫ3 mol m MgSO4, 3.0 mol m KNO3, and 0.1 mol m Ϫ1 MnSO4 buffered to specified pHs with 1.0 mol L HCl or Ϫ KOH, with or without 0.03 mmol m 3 (ϩ)-ABA (the ap- proximate concentration present in the xylem sap of well- watered, well-drained tomato plants [Else, 1996]). The vials Ϫ were randomized in a growth cabinet (PPFD 500 ␮mol m 2 Ϫ s 1) and weighed every 30 min for 3 to 4 h, after which time the leaf area above the foil was measured in a planimeter (Paton Industries, Pty. Ltd., South Australia). The transpirational rate of water loss was calculated per unit leaf area (single surface) every 30 min.

RESULTS AND DISCUSSION Correlations between Xylem Sap pH, Soil Water Content, and Shoot Water Potential The pH of xylem sap from wild-type tomato plants grown as described above was negatively correlated with gravimetric soil water content (Fig. 1A), which only affected shoot water potential within a range of 0.05 MPa (Fig. 1B). Sap pH increased over a range of 3 units as soil water content decreased, although most readings were between pH 5.5 and 7.5. These results are comparable to those in which xylem sap pH was measured over a drying period in other species (Table I) except in castor bean, in which sap became more acidic with drought (Schurr and Schulze, 1996), and the range of pH covered here is similar to that Figure 1. Effect of gravimetric soil water content on the pH (A) of detected in B. pendula shoot xylem sap by Sauter and xylem sap expressed within 2 min from 30-mm shoot stumps and on Ambrosius (1986). As expected (Davies and Zhang, 1991), the water potential (B) of shoots cut from 6- to 9-week-old wild-type these changes were detected in plants that exhibited only tomato plants. Linear regressions with 95% confidence limits are very small variations in shoot water potential, indicating shown. 706 Wilkinson et al. Plant Physiol. Vol. 117, 1998 dration, i.e. whether the pH changes are confined to the vided (Wilkinson and Davies, 1997) by preincubating root or whether they are indirectly influenced by alter- leaves overnight in distilled water with or without a low ations in ion concentrations in the sap flowing from roots concentration of ABA, equivalent to that found in xylem Ϫ adjacent to drying soil. If the effect of drought on ATPase sap from well-watered plants (0.01 mmol m 3). Only when activity is indirect, reduced proton pumping from shoot ABA had been present were the leaves able to respond to xylem parenchyma cells could enhance increases in the pH subsequent treatments with high- pH buffers. It was as- of the sap flowing upward from the root. Some evidence sumed that the solution in which the leaves were preincu- exists for the latter, since increases in stress-induced sap bated replaced the contents of the xylem vessels and the pH tend to become more marked when measured farther apoplast of the fresh leaf. High-pH-induced ABA accumu- up the plant stem (Hartung and Radin, 1989; Hoffmann lation in the apoplast adjacent to the guard cells was no and Kosegarten, 1995). These findings could also be ex- longer possible in leaves treated in water alone and, con- plained by the possibility that xylem/apoplastic pH might sequently, stomata remained open. be controlled by ionic exchange between sap constituents and the walls of adjacent cells (Ryan et al., 1992). Since the High pH Only Reduces Transpiration from flacca Leaves ionic composition of sap flowing from the roots is changed in Combination with a Low Concentration of ABA by the proximity of drying soil (Gollan et al., 1992), the pattern of exchange with wall-bound ions along the length Figure 3 illustrates an example of several experiments of the transpiration stream could be altered, thereby chang- that showed that flacca leaf transpiration rates were not ing its pH. Some authors have detected a correlation between the pH and the sugar composition of the leaf apoplast (Sauter and Ambrosius, 1986; Tetlow and Farrar, 1993). Since sugar uptake by the phloem involves ATPase activity (Delrot and Bonnemain, 1981), apoplastic sugar content may also influence apoplastic pH and could explain some of the changes in sap pH with time of day or year. An alternative proposal is that nitrate levels in xylem sap may somehow control its pH (Raven and Smith, 1976; Allen et al., 1988). Increasing the nitrate concentration supplied to roots of sunflower plants has recently been demonstrated to increase the apoplastic pH of leaves when sap was extracted by centrifugation (Dannel et al., 1995) or investigated with fluorescent dyes (Mengel et al., 1994; Hoffmann and Kosegarten, 1995). It is thought that proton- nitrate cotransport over the plasma membrane of leaf cells Ϫ and localized production of OH after nitrate reduction in the cytoplasm are responsible for increased apoplastic pH (Ullrich, 1992). However, increased nitrate levels in xylem sap are not always associated with increases in pH, and in fact, the reverse can be true (Gollan et al., 1992; Jackson et al., 1996). The fact that some plant species reduce nitrate in the roots and others perform this vital function in shoots (Andrews, 1986) could explain some of the variability in xylem/apoplastic pH between species and in the effects of environmental conditions on their physiology.

Effects of pH on Transpiration from Wild-Type Leaves The transpiration rates of freshly detached wild-type tomato leaflets taking up AS buffered to a well-watered pH of 6.0 were comparable to those of leaves taking up distilled water only, indicating that AS itself had no effect on stomatal aperture (Fig. 2A). Transpiration was reduced from control (well-watered) levels within 90 min when the pH of the AS supplied to the leaves was adjusted to the pH of xylem sap extracted from plants growing in drying soils (Fig. 2A, pH 6.75; Fig. 2B, pH 7.75). These findings support Figure 2. Comparison of the effect of water (A) and AS pH (A and B) the hypothesis that increased sap pH closes stomata, but on the transpiration rate of detached wild-type tomato leaflets in the they do not provide evidence for the involvement of ABA light. The transpiration rate was calculated for each leaf every 30 min in this process. For C. communis such evidence was pro- and expressed as a mean (n ϭ 8) Ϯ SE. Xylem pH Changes Affect Tomato Leaf Transpiration 707 reduced by pH of up to 7.75 (within the range detected in droughted tomato plants), although the stomata were able to close in response to decreased PAR. This is in direct contrast to the situation in wild-type leaves (Fig. 2). How- ever, when a low, well-watered concentration of (ϩ)-ABA Ϫ (0.03 mmol m 3) was supplied in the AS, which did not reduce transpiration at pH 6.25, the high pH mimicked its normal effect in wild-type leaves, i.e. it reduced the rate of transpirational water loss (Fig. 4). 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.

High pH Can Directly Increase Transpiration from flacca Leaves Figure 4A also shows that in the absence of ABA the transpiration rate of flacca leaves at pH 7.75 was initially higher than at pH 6.25. This effect was present to different degrees in different experiments and was sometimes com- pletely absent (Fig. 3). Figure 4B illustrates an example of a group of experiments in which, in the absence of exoge- nous ABA, the transpiration rate at pH 7.75 was steadily much higher than at pH 6.75. This is a novel finding in whole leaves, although as described above there is evidence that increasing the external pH opens stomata in isolated abaxial epidermal peels of C. communis (Wilkinson and Davies, 1997; see also Schwartz et al., 1994). That increased transpirational water loss from flacca leaves was induced by high xylem sap pH to different degrees in different experiments could have been ac- counted for by a variability in the endogenous, although

Figure 4. Effect of AS pH on the transpiration rate of detached flacca leaves in the light in the presence and absence of 0.03 ␮M (ϩ)-ABA. Leaves were preincubated in AS for1hinthedark. It is assumed that the endogenous ABA content of the leaves used in A was higher than that in the leaves used in experiment B. Transpiration rates were calculated and expressed as in Figure 3 (n ϭ 4–6).

very low, ABA concentration between individual flacca plants or between the batches of plants used for each experiment due, for example, to day-to-day fluctuations in greenhouse temperature, light intensity, and/or humidity. Preliminary measurements of bulk leaf ABA by radioim- munoassay (for protocol, see Wilkinson and Davies, 1997) revealed that flacca leaves picked on different days con- Ϫ tained between 0.1 and 1.5 nmol g 1 dry weight ABA, whereas wild-type tomato leaves contained between 2.0 Ϫ and 6.0 nmol g 1 dry weight ABA. Bulk leaf ABA is not Figure 3. Effect of AS pH and PAR (PPFD 500 ␮mol mϪ2 sϪ1 and always a good measure of xylem ABA, and cross-reactive darkness) on the transpiration rate of detached flacca leaves. Leaves substances in tomato that interfere with the radioimmuno- were preincubated in AS for1hinthedark. The transpiration rate assay can result in considerable overestimations of the was calculated for each leaf every 30 min and expressed as a mean ABA concentration actually present (Else et al., 1996). (n ϭ 4–8) Ϯ SE. However, these results demonstrate the potential for the 708 Wilkinson et al. Plant Physiol. Vol. 117, 1998 endogenous ABA concentration of an individual flacca of nitrogen supply on apoplasmic pH. J Plant Physiol 146: plant to vary on a daily basis. The plants used in the 273–278 experiment illustrated in Figure 3 may have contained Davies WJ, Zhang J (1991) Root signals and the regulation of Ϫ1 growth and development of plants in drying soil. Annu Rev closer to 1.5 nmol g dry weight bulk leaf ABA, whereas Plant Physiol Plant Mol Biol 42: 55–76 those used in Figure 4B may have had a 15-fold lower Delrot S, Bonnemain JL (1981) Involvement of protons as a sub- concentration, with those in Figure 4A containing an inter- strate for the sucrose carrier during phloem loading in Vicia faba mediate amount. leaves. Plant Physiol 67: 560–564 That such small concentrations of ABA in the AS (up to Else MA (1996) Xylem-borne messages in the regulation of shoot Ϫ 0.03 mmol m 3) were all that was required to inhibit the responses to soil flooding. PhD thesis. Lancaster Univer- sity, UK pH-induced stomatal opening effect seen in some experi- Else MA, Tiekstra AE, Croker SJ, Davies WJ, Jackson MB (1996) ments (Fig. 4B) demonstrates the potency of this hormone Stomatal closure in flooded tomato plants involves abscisic acid and its great capacity for plant protection, at least in tomato. and a chemically unidentified anti-transpirant in xylem sap. Plant Physiol 112: 239–247 Epstein E (1972) The media of plant nutrition. In Mineral Nutrition CONCLUSIONS of Plants: Principles and Perspectives. John Wiley & Sons, New York, pp 29–49 These findings show that the increases in xylem sap pH Ferguson AR, Eiseman JA, Leonard JA (1983) Xylem sap from described in Table I have the potential to widen the sto- Actinidia chinensis: seasonal changes in composition. 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