Ion Fluxes and Abscisic Acid-Induced Proline Accumulation in Barley Leaf Segments

The increase in proline induced by ABA, a process stimulated by NaCi or KCI in barley leaves, did not occur when Na+ (or K+) was present in the external medium as the gluconate salt, namely with an anion unable to permeate the plasma membrane. However, proline increase was restored, to different extents, by the addition of various chloride salts but not by ammonium chloride. Moreover, it was shown that the stimulation of the process by NaCl (or KCI) was variously affected by the presence of different salts; all the ammonium salts (10 millimolar NH4' concentration) inhibited this stimulation almost completely. Inhibition by NH41 was accompanied by a decreased Na+ influx (-40%). Also, in the case of Na-gluconate, Na+ uptake was reduced and the addition of Clas the calcium or magnesium salt (but not as ammonium salt) restored both the ion influxes and the increase in proline typical of NaCl treatments. Both 4,4'-diisothiocyano-2,2'-disulfonic acid stilbene (DIDS), an anion transport inhibitor, and tetraethylammonium chloride (TEA), a K+ channelsblocking agent, caused, as well as with a reduction of ion influxes, an inhibition of the proline accumulation. The inhibition was practically total with 1 millimolar DIDS and about 80% with 20 millimolar TEA. A possible role of ion influxes in the process leading to the increase in proline induced by ABA is proposed. It has been shown that the increase in proline level induced by ABA in barley leaf segments practically does not occur in the absence of appropriate salts in the external medium (12) and recently it has been proposed that a protein, the synthesis of which appears to be induced by ABA, confers sensitivity to the salts (9). Sodium and potassium chlorides stimulate proline accumulation to the same extent, and stimulation by the two cations depends on the associated anions, NO3being the most effective and SO42practically ineffective. In this latter case the addition of Cl to the external medium restores the effect of Na + (or K+) chloride on the proline increase (12). The accumulation of Na+ and K+ in the tissue in the presence of Clor SO42-, evaluated over the time, is not different enough to explain the stimulation by chlorides and the lack of effect of the sulfates. Taken as a whole, these results do not exclude a possible involvement of the influx of suitable ions in the induction of proline accumulation by ABA (12). On the basis of this hypothesis, some attempt was made to elucidate the physiological role of ion fluxes in this process and the results are presented in this paper. MATERIALS AND METHODS Plant Material. Sections (5 mm long) from intermediate portions of fully expanded primary leaves of 1 week old barley seedlings (Hordeum vulgare cv Georgie; Sementi Bovo, Isola della Scala, Verona, Italy) grown in a phytotron chamber as previously described (12) were used. Proline Evaluation Experiments. The samples consisted of 300 mg fresh weight of leaf segments, prepared as previously described (12), in 20 ml 10 mM MES buffer (pH 5.5 with TRIS), 0.5 mm CaSO4 and salts at the desired concentration as specified in the individual experiments. ABA was added at the optimal 0.1 mm concentration (10); NaCl and KCI concentrations were 25 or 30 mm, concentrations that allow a good hormone-induced increase in proline (12) and at the same time a clear evaluation of inhibition kinetics. Incubation was always performed for 7 h in the dark at 25°C in a shaking bath. Proline was extracted by homogenizing the leaf fragments in a mixture of methanol:chloroform:water (12:5:1 v/v) without permutit resin and determined according to the colorimetric method of Singh et al. (16) partially modified (11). Other details were as previously described (12). All treatments were in triplicate and the data are the means (± SD) from at least two experiments. Na+, K+, and Cl Influx Experiments. The samples consisted of 100 mg fresh weight of leaf segments in 10 ml of the same buffered solution adopted for the ABA-induced proline increase experiments reported above, and incubation was performed in the dark at 25°C in a shaking bath. After 30 min preincubation in the dark the appropriate labeled solution and, when required, the compounds affecting the ion influxes were added. Each sample had a concentration of 30 mM of Na+, K+, and Clwith added radioactivity of about 0.05 ,uCi as 22Na+ or 86Rb+ or 36CI-, respectively. ABA 0.1 mm was always added in the middle of the preincubation period in all the samples. The influx experiments were stopped by vacuum withdrawal of the labeled solution followed immediately by a 2 min rinse in a corresponding ice-cold unlabeled fourfold concentrated solution. Digestion and bleaching of the leaf segments were performed with Lumasolve and benzoylperoxide treatments as previously described (11) and radioactivity was determined by liquid scintillation counting. The data are the means (± SD) from at least two experiments run in triplicate. RESULTS AND DISCUSSION Treatments with Na-gluconate (the anion of which is not able to permeate the membrane [1]) in the presence of ABA scarcely affected proline accumulation as compared with NaCl treatments (Fig. 1A). The slight effect of Na-gluconate did not change with increases of its concentration above 10 mm. As previously described for K2SO4 (12), the addition of calcium or magnesium chlorides, which have little effect on this process (see values in brackets of Table I and Ref. 12) led to an increase in proline 927 www.plantphysiol.org on October 23, 2017 Published by Downloaded from Copyright © 1988 American Society of Plant Biologists. All rights reserved. Plant Physiol. Vol. 86, 1988