Planta (1986)167:563-568

P l a n t a 9 Springer-Vertag 1986

Effects of abscisic acid on sequestration and exchange of Na § by barley roots R. Behl and K. Raschke Pflanzenphysiologisches Institut und Botanischer Garten, Untere Karspiile 2, D-3400 G6ttingen, Federal Republic of Germany

Abstract. Excised Na§ barley roots were suspended in solutions of Na + in combination with N O 3 - , CI-, and S 0 4 2 - , and effects of the added phytohormone, abscisic acid (ABA), to the medium were determined. Abscisic acid increased the rate of Na + (22Na+) accumulation and the amount of Na + deposited in the vacuoles. These stimulating effects of A B A were modified by anions following the sequence NO3 > C1- > SO42-. Testing whether the magnitude of the pH gradient across the plasmalemma of the cells of the root cortex affects rates of Na + accumulation and their dependence upon ABA, we observed that, in the p H range from 4 to 8, the ABA-induced stimulation was strongest at p H 5.8, and least at p H 4. Changes in p H during the experiment caused changes in the rates of Na + accumulation in agreement with experiments performed at constant p H values. Simultaneously with ABA-enhanced accumulation, loss of Na + occurred. Loss of Na + was strongest at p H 4 and was affected by anions, being greatest with S042- and following the sequence 8042- ~> C1- > N O 3 - . On the basis of the finding that initial acceleration of uptake as well as loss of Na + depended on the pH of the medium we suggest that, in barley roots, ABA stimulates an exchange of Na + for H + at the plasmalemma of the cortical cells. The results indicate that ABA-stimulated expulsion of Na § in combination with ABA-stimulated sequestration in the vacuoles, constitutes one of the mechanisms which enable barley plants to tolerate higher than normal levels of Na +. Key words: Abscisic acid - Anion - Hordeum (roots) - Plasmalemma - Sodium (accumulation, exchange) - Sodium for proton exchange (plasmalemma, p H dependence). Abbreviations: ABA = abscisic acid; FW = fresh weight

Introduction It is one of the root's tasks to absorb selectively essential ions and provide the shoot with them. Selective absorption of K § and selective exclusion of Na § provide a case in point. The potassium ion is essential as an osmoticum and for enzyme activation (L/iuchli and Pfl/iger 1978); the sodium ion is toxic at high concentrations and should be excluded from the cytoplasm (Flowers et al. 1977; Wyn Jones etal. 1979; Greenway and Munns 1980). This is accomplished by expulsion into the root's environment and by sequestration in the vacuoles (Jeschke 1984). Maintenance of a low Na + concentration in the cytoplasm contributes to salt tolerance (Wyn Jones et al. 1979; Jeschke 1984). Previous studies (recently reviewed by Jeschke 1984) have shown that preferential uptake of K + and selective exclusion of Na § are net results of the activities of at least four transport processes that operate at three locations in the root. The first is an uptake of cations into the symplast of the root. It occurs in the plasmalemma of the cortical cells. The second secretes ions from the symplast into the apoplast of the xylem tissue through the plasmalemma of the cells in the central cylinder (this process is often referred to as "transport through r o o t s " ; Anderson 1976). The third sequesters ions from the cytoplasm into the vacuoles of the cortical tissue; it operates in the tonoplast and can be opposed by a process of ion release from the vacuoles into the cytoplasm. The fourth transport process removes ions from the root into its environment; the membrane involved is again the plasmalemma of the cortical cells. Whether an ion taken up by the root will be delivered to the xylem or not will not only depend on the activity of the secretory mechanism in the central cylinder but also on the rates of sequestration into the vacuoles

564

R. Behl and K. Raschke: Effects of ABA on sodium movement in roots

of the root tissue and of the return to the medium by exchange at the root surface. Behl and Jeschke reported in 1981 that in barley roots the phytohormone, abscisic acid (ABA), affected the processes of bringing about selective uptake, transport, and sequestration of K § and Na +. In particular, they found that exposure to ABA caused three effects: an inhibition of alkaliion secretion into the xylem, a stimulation of the sequestration into the vacuoles, and an increase of the efflux into the medium. It was Na + which was preferentially sequestered and expelled. One can think of various ways to explain the observed phenomena. The explanation which appeared attractive to us was the possibility that ABA influenced alkali movement in the root by affecting proton translocation through the plasmalemma of the cortical cells. We decided to explore this possibility. Because barley varieties are salt tolerant and appear to achieve tolerance mainly by an exchange of Na § for H § at the plasmalemma of the cortical cells (Jeschke 1979, 1980) we restricted our experiments to a study of the accumulation and exchange of Na § by root segments of this species. Our experimental procedure was based on the following hypotheses and considerations: (1) Cortical cells excrete protons; the proton gradient thus built up is used to import monovalent cations and, by symport, monovalent anions (like C1- and NO3-, if the corresponding carriers are present). (2) If no monovalent anions are available (for instance when Na + is being offered solely in combination with SO42-) the rate of cation uptake will be determined by the rate of synthesis of organic anions in the root cells (Osmond 1976). (3) Monovalent cations, like Na +, can leave the cortical cells by being exchanged for external H § and Jacoby 1976; Jeschke 1980), and (4) cations found in the root tissue are predominantly located in the vacuoles (as concluded from volume considerations). By varying the pH of the suspension medium we modified the proton gradient across the plasmalemma, and by varying anion composition we hoped to receive indications about the importance of organic-acid synthesis in bringing about selectivity with respect to Na + movement in barley roots. A word remains to be said about the artificiality of applying ABA to roots by adding the phytohormone to the medium. Phloem sap contains ABA (Hoad 1978) and surely, in whole plants, phloem sap will reach the roots. Abscisic acid does occur in roots (Audus 1983), including roots of barley (Behl 1981), admittedly in low concentrations (1 to 100 ng per g fresh weight). After plants

have been cultivated in solutions, ABA can be found in the medium, which indicates that ABA, after having passed the endodermis, can leak from roots into the medium through the apoplast. On the strength of these observations we propose that ABA applied to the medium will reach the cells of root segments (at least those of the cortex) by one of the paths also available in the whole plant, namely through the apoplast. Distribution of ABA between apoplast and cytoplasm would depend on the distribution of pH values, as was proposed by Cowan et al. (1982). We therefore felt it to be acceptable if we applied ABA by adding it to the medium in which the root sections were suspended.

Material and methods Plants. Barley, Hordeum distichon L. cv. Aura, was obtained

from Saatzuchtwirtschaft Josef Breun, D-8522 Herzogenaurach, FRG. Caryopses were germinated for 24 h on filter paper moistened with 0.5 mmol-1 - I CaSO4. In order to obtain lowsalt roots, they were then planted on nylon gauze over aerated 0.5 mmol.1 - j CaSO4. The seedlings were cultivated in a temperature-controlled cabinet under continuous light (350 gE. m - 2 " s - ~; Pollux 069 250 Watt lamp, Norka, Hamburg, FRG). Temperatures were 22~ and 18~ from 6:00 to 19:00 h and from 19:00 to 6:00 h, respectively. Roots were harvested when they were about 8 cm long. The experiments were performed with apical segments of 20 mm length. Their Na + content was 0.7_+0.2 and their K + content 36.8_+2.8 gmol.(g FW) -~. Accumulation experiments. Apical root sections of 20 mm length were prepared and placed, 40 sections each, in plastic beakers containing 300 ml of the experimental solutions. During the experiments the solutions were aerated and stirred by a continuous stream of air. At alkaline pH the air was passed through soda lime in order to prevent an acidification of the solutions by CO2 absorption. The experiments were run in the dark and at room temperature. They were started by addition of ABA. Three hours later, sodium salts labelled with 22Na+ (308 Bqgmo1-1) were added. The anions ( N O 3 - , CI-, SO4 z-) were added as calcium (3 mmol.1-~) and sodium (1 mmol.l z) salts. In addition, all solutions contained 0.5 mmol.1- ~ MgSO4. Following Jeschke (1980) the solutions were buffered by 1 mmol1-1 succinic acid (pH4.0), l m m o l . 1 - 1 HPO4Z-/HaP04. (pH 5.8, Ca 2+ salts) or I mmol-1-1 4-(2-hydroxyethyl)-l-piperazine ethane sulfonic acid (Hepes; pH 8.0). The pH was adjusted with 0.01 tool.l-1 Ca(OH)z. At various times, samples of five roots each were removed, rinsed three times for 1 rain each with cold 0.5 retool. 1-1 CaSO4, blotted, weighed and extracted with 13% nitric acid for 24 h. Total Na+contents, as well as, contents in 22Na+ were measured by flame photometry and gamma counting, respectively. Amounts of Na + accumulated by the root sections were computed from the radioactivity of the sections, cpm. (g FW) 1, by dividing by the specific activity, cpm-gmol-1, in the incubation medium. Because the cytoplasm of barley roots contains only 5% of the total amount of Na + (Jeschke 1977) the total amounts of Na + contents found in the root samples approximate the amounts of Na+stored in the vacuoles.

R. Behl and K. Raschke: Effects of ABA on sodium movement in roots

Abscisic acid (ABA). Solutions of ABA were prepared by dissolving (__) ABA (Serva, Heidelberg, FRG) in a small volume of methanol and diluting them to a known concentration with distilled water. Aliquots were added to the experimental solutions. Controls received equal amounts of methanol; the methanol concentration of the experimental solutions was 0.0J % (v/ v).

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The first experiment was designed to test whether pH gradients between the medium and the root affected rates of Na § accumulation and the amounts of Na § deposited in the vacuoles when a steady state had been reached. The experiment was performed with N O 3 - , CI- or SO4 2- as anions accompanying Na § Initial accumulation rates of Na § were considerably lower when the pH of the medium was 4.0 than when it was 5.8 or 8.0, and this effect was virtually independent of the anion present (Fig. 1, left panels). With C1-, the initial rates were 7.7 gmol.(g F W ) - ~ - h - t at pH 8.0, 5.1 at pH 5.8, and 3.7 at pH 4.0. A change in pH during the experiment also caused a change

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566

R. Behl and K. Raschke : Effects of ABA on sodium movement in roots

Table 1. Steady-state Na + content, tlmol' (g FW) 1, of excised barley roots as modified by the external pH and the accompanying anions. The values shown are the averages • of all control treatments. In the presence of NO3 at pH 4.0 a steadystate Na + content was not reached within the duration of the

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on the accumulation o f N a § appeared also in the contents f r o m steady-state roots which were analysed 25 h after the initial exposure to 22Na§ Contents were lowest when the p H o f the m e d i u m was 4.0, and again, the quality o f the anions in the solutions had very little influence on the a m o u n t s of N a § taken up by excised roots (Fig. 1, left panels, and Table 1). The modifying influences o f the anions became a p p a r e n t when A B A was added to the incubation media. In general, the presence o f A B A led to increased rates o f accumulation o f N a § particularly at p H 5.8 and 8.0 (Fig. 1, right panels). Enhancement o f accumulation was greatest with N O 3 - , and smallest with SO42- in the solution. Considerable differences between control and A B A treatments a p p e a r e d a b o u t 10 h after the initial exposure to 22Na+. Clearly, after an initial acceleration lasting a b o u t 10 h, addition o f A B A to the m e d i u m led to a decline o f the N a § content o f the roots (Fig. 1, right panels). T h e r e were two exceptions: the decline did not occur if the A B A c o n c e n t r a t i o n had been reduced to 1 p m o l . 1 - 1 (panels for C1 and S O 4 2 - ) or if the anion was NO3 - and the p H was 5.8 or 8.0 (panel for N O 3 -). Indeed, with N O a - in the solution, steady-state N a § contents were higher in the A B A treatments than in the controls. A similar situation could be seen in the presence o f C1- and 1 I~mol' 1-1 ABA. Figure I indicates that addition o f A B A to the m e d i u m caused the d e v e l o p m e n t o f two opposing effects, an e n h a n c e m e n t o f N a + accumulation which manifested itself right f r o m the beginning and during the first 8 h o f the experiment, and an effect o f leakage o f N a + which became apparent a b o u t 10 h after the beginning. The m a g n i t u d e of the second effect d e p e n d e d on p H and on the A B A c o n c e n t r a t i o n applied. The rate o f decline in N a § content increased with decreasing p H and with increasing A B A c o n c e n t r a t i o n (Fig. 3). Figure 4 shows that in the absence o f ABA, barley

Fig. 3. Time course of Na+(22Na +) content, gmol.(g FW) i, in excised barley roots in the presence of 1 #mol-l-1 ABA at pH8 and with CI- as counter ion to Na +, At the time of the arrow the external ABA concentration was changed from 1 ~.mol-1-1 to 5 or l0 gmol.l 1, or the pH was lowered from pH 8 to pH 4 (while ABA remained at 1 gmol-1 1)

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r o o t segments did not lose N a +, not even if suspended in a solution free o f N a + and at a p H o f 4.0. However, when the solution contained 10 Ilmol.1 1 ABA, loss o f N a + f r o m the tissue occurred, and it was faster when the p H o f the solution was 4.0 than when it was 8.0. In other words, A B A allowed an escape o f N a + to occur, and this loss was facilitated by a high external proton concentration.

Discussion On the basis o f the hypothesis o f cation uptake and m o v e m e n t in roots which we outlined in the

R. Behl and K. Raschke: Effects of ABA on sodium movement in roots Introduction to this paper, and considering the results of the compartmental analysis by Behl and Jeschke (1981), we interpret the results of our experiments with salt-starved roots of barley in the following way. In the absence of ABA, rates of accumulation of Na § were lowest at the lowest pH used (Fig. 1, p H 4). According to Jeschke (1980), and supported by evidence produced by Rather and Jacoby (1976), this reduction in accumulation rate was not the result of a reduced rate of uptake but rather of an increased rate of exchange of N a § for H + at the plasmalemma of the cortical cells. Because rates of accumulation into the vacuoles depend on the rates of loading into the cytoplasm, changes in the vacuolar content reflect net effects of the processes occurring at the plasmalemma (Jeschke 1980), and a steady state in the Na § content of the vacuoles indicates that also the plasmalemma Na § exchange has reached a steady state. Rates of uptake and exchange of Na § through the plasmalemma and the process of sequestering of Na § into the vacuoles were independent of the kind of anion which was offered in the external solution along with Na § This was true as long as A B A was absent from the medium. In the presence of ABA, N O 3 - and C1- stimulated the initial rate of accumulation of Na + in the vacuoles whereas SO42- had virtually no influence on it (Fig. 1), in confirmation of results of Behl and Jeschke (1979). The quality of the anion in the medium also affected the rate of release of Na § that became evident 8 to 10 h after exposure of the roots to ABA. On close inspection of Fig. 1, a decline in the accumulation rate of Na § can be recognized even before 10 h had elapsed, particularly in the presence of SO~ 2- and at p H 4. We wish to discuss briefly whether this ABAinduced decline in the rate of Na § accumulation was caused by an inhibition of Na § uptake through the plasmalemma of the cortical cells or by an enhancement of Na § release. Evidence obtained by Cram and Pitman (1972) and by Behl and Jeschke (1979) from experiments with barley roots does not support the inhibition hypothesis. The decline of the Na + content we saw to occur after 10 h exposure of the barley roots to ABA (Fig. 1) rather indicates a slowly developing ability to release Na +, possibly by an ABA-induced opening of specific cation channels or, alternatively, by an ABA-stimulated N a + - H + exchange mechanism. The Na + content of the roots was lowest at pH 4 (Fig. 1), and transfer of roots from solutions of p H 8 to those of p H 4 caused a decline in Na + content (Fig. 3), whereas continuous expo-

567

sure to p H 8 or transfer from p H 4 to pH 8 permitted further net uptake of Na + (Fig. 2). These observations would be consistent with the notion of a N a + - H + antiport. In our experiments, the presence of ABA in the medium turned out to be required for a net release of Na +. A high external H + concentration alone was insufficient to cause a net efflux of Na +, even if the roots were suspended in a N a § medium (Fig. 4, pH 4). The ABA-stimulated efflux of Na § was strongest in the presence of SO42 - and declined following the series SO42- > C1- > N O 3 - . The causes for this sequence are very likely the plant's differing abilities to absorb these ions and its ability to metabolize N O 3 - . Sulfate is taken up slowly, necessitating the synthesis of organic acids during Na + uptake, C1- can be absorbed, and N O 3 - will be absorbed and some of it removed by reduction. Jeschke (1980) suggested that a gradual acidification of the cytoplasm of Na+-loaded barley roots caused a release of Na + from the vacuoles. If this is true, acidification during exposure to N a z S O 4 would ultimately stimulate an efflux of N a +. No net efflux of Na + occurred in the ABA treatments when N O 3 - was present at pH 5.8 and 8. It appears that ABA stimulated a Na + effiux mechanism that was pH dependent. Alternatively or in combination, ABA itself could have caused an acidification of the cytoplasm which then was enhanced by organic-acid synthesis (as was suggested to occur in guard cells; Raschke 1975), and least when N O 3 - was offered as an anion that could be removed by metabolism. The two effects of ABA on Na § movement in barley roots differed in their dependences on ABA concentration. The enhancement of net accumulation of Na § during the first 8 or 10 h exposure to ABA was already fully expressed at a concentration of i gmol. 1-1. The enhancement of net release of Na + during the subsequent time required 5 g m o l ' l - 1 (Fig. 1). An increase of the ABA concentration to 10 gmol.1-1 led to an acceleration of Na + efflux (Fig. 3). Enhancement by ABA of Na + sequestration in the vacuoles and the observed stimulation of the N a + - H + exchange at the plasmalemma of the cells of the root cortex can be considered to constitute a combined mechanism which keeps the Na + content of the cytoplasm at a low level. This view receives support from the compartmental analyses of barley roots (Behl and Jeschke 1981) and of tissue from the storage roots of red beet (Francois et al. 1982). In both investigations, the Na + content of the cytoplasm of ABAtreated tissue was found to have been lower than that of control tissue. We suggest that the phyto-

568

R. Behl and K. Raschke: Effects of ABA on sodium movement in roots

hormone ABA could be involved in mechanisms that enable barley plants to tolerate higher-thannormal levels of Na +. Further tests would require inclusion of competition experiments with simultaneous exposure to Na § and K § In this context it is of particular interest that Cornish and Zeevaart (1985) reported that wilted or osmotically stressed roots (admittedly of J(anthium strumarium and Lycopersicum esculentum) were able to produce ABA. We gratefully acknowledge the support of the "Zentrales Isotopenlabor der Universitfit G6ttingen". These studies were supported by the DFG (Ra 122/11 1).

References Anderson, W.P. (1976) Transport through roots. In: Encyclopedia of plant physiology, N.S., vol. 2 B : Transport in plants II, pp. 129-156, Liittge, U., Pitman, M.G., eds. Springer, Berlin Heidelberg New York Audus, L.J. (1983) Abscisic acid in root growth and geotropism. In: Abscisic acid, pp. 421-477, Addicott, F.T., ed. Praeger Publishers, New York Behl, R. (198l) Wirkung von Abscisins/iure auf die Fluxe und die intrazellulfire Kompartimentierung von Kalium und Natrium in isolierten Gerstenwurzeln. Ph.D. thesis, UniversitS.t Wfirzburg Behl, R., Jescbke, W D . (1979) On the action of abscisic acid on transport, accumulation, and uptake of K + and Na + in excised barley roots ; effect of the accompanying anions. Z. Pflanzenphysiol. 95, 335-353 Behl, R., Jeschke, W.D. (1981) Influence of abscisic acid on unidirectional fluxes and intracellular compartmentation of K + and Na + in excised barley root segments. Physiol. PIant. 53, 95 100 Cornish, K., Zeevaart, J.A.D. (1985) Stress-induced abscisic acid accumulation in Xanthium and tomato roots. (Abstr.) Plant Physiol. 77, Suppl. 117 Cowan, I.R., Raven, J.A., Hartung, W., Farquhar, G.D. (1982) A possible role for abscisic acid in coupling stomatal conductance and photosynthetic carbon metabolism in leaves. Aust. J. Plant Physiol. 9, 489-498 Cram, W.J., Pitman, M.G. (1972) The action of abscisic acid on ion uptake and water flow in plant roots. Aust. J. Biol. Sci. 25, 1125-1132 Flowers, T.J., Troke, P.F., Yeo, A.R. (1977) The mechanism

of salt tolerance in halophytes. Annu. Rev. Plant Physiol. 28, 89-121 Francois, G., Bogemans, J_, Neirinckx, L. (1982) Unidrectional sodium fluxes in red beet storage tissue (Beta vulgaris L. vc. Platronde Egyptische) : effects of the phytohormones indol-3yl-acetic acid and abscisic acid. Plant Cell Environ. 5, 5-8 Greenway, H., Munns, R. (1980) Mechanisms of salt tolerance in nonhalophytes. Annu. Rev. Plant Physiol. 31,149-190 Hoad, G.V. (1978) Effect of water stress on abscisic acid levels in white lupin (Lupinus albus L.) fruit, leaves and phloem exudate. Planta 142, 28%290 Jeschke, W.D. (1977) K +-Na + exchange and selectivity in barley root cells: effect of Na + on the Na + fluxes. J. Exp. Bot. 28, 128%1305 Jeschke, W.D. (1979) Univalent cation selectivity and compartmentation in cereals. In: Recent advances in the biochemistry of cereals, pp. 37-61, Laidman, D.L., Wyn Jones, R.G., eds. Academic Press, London New York San Francisco Jeschke, W.D. (1980) Involvement of proton fluxes in the K +Na + selectivity at the plasmalemma; K +-dependent net extrusion of sodium in barley roots and the effect of anions and pH on sodium fluxes. Z. Pflanzenphysiol. 98, 155 175 Jeschke, W.D. (1984) K + - N a + exchange at cellular membranes, intracellular compartmentation of cations, and salt tolerance. In: Salinity tolerance in plants: strategies for crop improvement, pp. 37-66, Staples, R.C., ed. John Wiley and Sons, New York L/iuchli, A., Pfltiger, R. (1978) Potassium transport through plant cell membranes and metabolic rote of potassium in plants. In: Potassium Research - Review and Trends, pp. I l l , Proc. l l t h Congr. Int. Potash Inst., Gething, P.A., Peter, A. von, eds. Bern Osmond, C.B. (1976) Ion absorption and carbon metabolism in cells of higher plants. In : Encyclopedia of plant physiology, N.S., vol. 2A: Transport in plants II, pp. 347 372, Lfittge, U., Pitman, M.G., eds. Springer, Berlin Heidelberg New York Raschke, K. (1975) Stomatal action. Annu. Rev. Plant Physiol. 26, 309-340 Rather, A., Jacoby, B. (1976) Effect of K*, its counteranion and pH on sodium efflux from barley root tips_ J. Exp. Bot. 27, 843-852 Wyn Jones, R.G., Brady, C.J., Speirs, J. (1979) Ionic and osmotic relations in plant cells. In: Recent advances in the biochemistry of cereals, pp. 63 103, Laidman, D.L., Wyn Jones, R.G. eds. Academic Press, London New York San Francisco Received 12 August; accepted 28 November 1985