THE EFFECT OF ABSCISIC ACID ON STOMATAL BEHAVIOUR IN FLACCA, A V\^ILTY MUTANT OF TOMATO, IN DARKNESS

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1 New Phytol (1972) 71, THE EFFECT OF ABSCISIC ACID ON STOMATAL BEHAVIOUR IN FLACCA, A V\^ILTY MUTANT OF TOMATO, IN DARKNESS BY M. T A L AND D O R O T I M B E R Division of Life Sciences, Negev Institute for Arid Zone Research, Beersheba, Israel {Received 10 June 1971) SUMMARY Young plants of the wilty tomato mutant,^acca, in which the stomata resist closure, were sprayed with ahscisic acid eight times over a period of 24 hours in light and darkness. The hormone induced closure of stomata similarly in leaves treated either in light or in darkness. Abscisic acid does not appear to induce stomatal closure in darkness by increasing the internal CO^ concentration in the leaf, since this concentration should already be ver)' high in darkness. A considerahle and rapid decrease of CO2 assimilation and increase of CO^ evolution were demonstrated in mutant as well as in normal plants transferred from light to darkness. INTRODUCTION The mode of action of plant hormones on stomatal behaviour is still an open question. One approach to the solution of this problem is to correlate the effect of hormones on stomatal behaviour with their possible effect on CO2 concentration. Meidner (1967) suggested that kinetin effects stomatal opening, at least partly, by decreasing CO2 concentration in the leaf. Similarly, Mansfield (1967) suggested that the S3'nthetic auxins i?-naphthoxyacetic acid and naphthalenacetic acid induce stomatal closure by increasing CO2 concentration in the leaf, while 2,4-dichlorophenoxyacetic acid acts directly on the guard cell. A possible relation between the effect of abscisic acid (ABA) on stomatal behaviour and the level of CO2 is studied here, using flacca {fic), a w ilty mutant of tomato. The stomata of the mutant, unlike those of the normal plant, resist closure and remain open even in the dark (Tal, 1966). This abnormal stomatal behaviour in the mutant was explained by the higher kinetin-iike activity (Tal, Imber and Itai, 1970) which resulted trom the lower concentration of substances with ABA-like activity (Tal and Imber, 1970) in ^c as compared with the control normal variety. Consequently, the mutant stomata respond more readily than the normal ones to ABA applied externally (Imber and Tal, 1970). The mutant plant seems, therefore, to be especially suitable for studying the effect of hormones with a closing effect on stomata, and especially of ABA, in the dark. Due to the absence of photosynthesis in darkness, it is expected that CO2 concentration in darkened leaves increases considerably as compared with those treated in light. If.'VBA indeed induces closure of stomata by affecting the level of CO2, it should do so by increasing the CO2 concentration. Providing the assumption of a high CO2 concentration in darkened mutant leaves is correct, it seems that any observed closing effect induced by ABA in the dark should be independent of CO2 concentration. 81

2 82 M. TAL AND DOROT IMBER MATERIALS AND METHODS The mutation flc was obtained from the normal tomato cv. Rheinlands Ruhm which is used as the control plant in the present work. Plants were grown in the greenhouse in aerated half concentration Hoagland solution up to the stage offiveto six leaves. Half of the plants were left in the greenhouse and the other half were transferred to a dark room. All plants were sprayed either with water or with ABA solution (io mg/l) eight times over 24 hours. conditions in the greenhouse during the 24-hour period were as follows: (i) from to hours light of about 1000 ft candles; (2) from to hours darkness (night); and (3) from to hours light of about 1000 ft candles. The treatment in the dark room started only i hour after the plants were transferred from the light. Assimilation of CO2 The assimilation of CO2 was determined in mutant and normal leaves in light and darkness. Detached leaves, 4th from the top, were floated on water under light of 800 ft candles for i hour before exposing tbem to ^''^C02. The leaves were exposed to **C02 for 30 minutes, one group under light of 800 ft candles and the second in darkness. The second group was transferred to darkness 15 minutes before their exposure to ^'*C02. The total assimilation of CO2 was determined by the sum of the radioactivity of different fractions such as anionic, cationic, neutral and chloroform- and HCIO4- soluble substances. The procedure followed that presented by Ben-Zioni, Vaadia and Lips (1970). The distribution of radioactivity in the various fractions was more or less equal in mutant and normal leaves. Evolution of CO2 Leaf discs, 14 mm diameter, were punched from the central region of the last leaf segment of the 4th leaf from the top. The discs were floated on water under light of 800 ft candles for i hour, blotted carefully and weighed. Samples (about 200 mg each) of these discs were then transferred to 50-ml Erlenmeyer flasks each containing 4 ml O.I M buffer phosphate (ph 6.1) and 2.5 /ici (i-'^^c) D-glucose (The Radiochemical Centre, Amersham) and infiltrated. The bottles were arranged in two shakers in water at 25 C. One group was exposed to light of 800 ft candles and the second kept in darkness. The '^^002 evolved by these discs during 30 minutes was captured by a filter paper containing 0.15 ml of 10% KOH solution. The papers were placed in 10 ml of Bray scintillation solution and radioactivity was determined. Stomatal behaviour The opening of stomata was determined directly by examining leaf impressions (Sampson, 1961; Zelitch, 1961), and indirectly by measuring the rate of transpiration from detached leaves. For these measurements, leaf petioles were inserted in water in sealed beakers for 24 hours. The transpiration of all leaves, treated in the green house and dark room, was determined in the dark room, because detached mutant leaves inserted in water wilt quickly under light (Tal et al., 1970). All operations in the dark room were performed under light of about 2-3 ft candles which is too low for effective photosynthesis (Rabinowitch, 1951). RESULTS AND DISCUSSION To verify the basic assumption that CO2 concentration in the mutant leaves increases in

3 Effect of ABA on flacca stomata in darkness 83 the dark as in normal tomato leaves, the assimilation of CO2 and the evolution of COj produced by respiration were studied in mutant and in normal leaves in light and darkness (Table i). The total assimilation of CO2 in both mutant and normal leaves decreased drastically within 45 minutes in the dark. The CO2 assimilated in the dark was only 4.7% in mutant and 2.6% in normal leaves of that assimilated in light. The evolution of CO2 produced by respiration, increased considerably within 30 minutes in both mutant and normal leaves in darkness. The results support the assumption made earlier that the level of CO2 in darkened mutant leaves should be much higher than in light. Table i. Assimilation {cpmx io~^/100 mg dry weight/^s minutes) and evolution {cpm xio~^/ioo mg dry weight/^o minutes) of ^*CO2 in mutant leaves, in light {800 ft candles) and darkness 1 ULai Assimilation of '^COj* Evolution of "CO^** Mutant light * Each value represents the average of io leaves. ** Each value represents the average of 3 replications. Normal %of light Abscisic acid induced similar closure of stomata in leaves treated in light and darkness, as indicated by the decrease of transpiration (Table 2). This conclusion was confirmed by a direct examination of stomata in leaf impressions. The stomata of both types of leaves, treated in light and darkness, started to react to ABA at least 6 hours after the first application of this hormone, and possibly sooner. The possibility is being investigated at present that ABA induces closure of stomata, at least partly, by affecting the extensibility of the guard cell walls. It was found (Tal, 1966) in flc that stomata remain open even when the guard cells were plasmolysed. This Table 2. Rate of transpiration {mg HiO/cm^ leaf area/24, hours) in leaves detached from control and ABA-treated mutant plants in light and dark; each value represents the average of thirty leaves, yd and ^th from top, taken from fifteen different plants Vooi % of Control ABA-treated control Control AB.^-treated control * * 75.9 * Significant diflference from the control at 95% level. phenomenon can only be interpreted as being due to a change in the degree of extensil^ility of the guard cell walls. The change may be correlated with the lower concentration of ABA-like substances demonstrated (Tal and Imber, 1970) in the/c mutant. At the time of completion of this paper, a study was published (Jones and Mansfield, 1970) which confirmed our conclusion that the effect of ABA on stomatal behaviour is independent of COj concentration.

4 84 M. TAL AND DOROT IMBER ACKNOWLEDGMENTS This research was supported in part by a research grant from the Ford Foundation (Ford-6, A-VII). The abscisic acid was a gift from F. Hoffman-La-Roche & Co. Ltd, Basle (Switzerland). REFERENCES BEN-ZIONI, A., VAADIA, Y. & LIPS, S. H. (1970). Correlation between nitrate reduction, protein synthesis and malate accumulation. Physiologia PL, 23, IMBER, D. & TAL, M. (1970). Phenotypic reversion oi flacca, a wilty mutant of tomato, by abscisic acid. Science, N.Y., 169, 592. JONES, R J. & MANSFIELD, T. A. (1970). Suppression of stomatal opening in leaves treated with abscisic acid. y. exp. Bot., 21, 714. MANSFIELD, T. A. (1967). Stomatal behaviour following treatment with auxin-like substances and phenylmercuric acetate. New Phytol., 66, 325. MEIDNER, H. (1967). The effect of kinetin on stomatal opening and rate of intake of carbon-dioxide in mature primary- leaves of barley. J. exp. Bot., 18, 556. RABINOWITCH, E. J. (1951). Photosynthesis and Related Processes. Vol. II, Part i, pp Wiley- Interscience, New York. SAMPSON, J. (1961). A method of replicating dry or moist surfaces for examination by light microscopy. Nature, Lond., 191, 932. TAL, M. (1966)..Aibnormal stomatal behavior in wilty mutants of tomato. PI. PhysioL, Lancaster, 41, TAL, M. & IMBER, D. (1970). Abnormal stomatal behavior and hormonal imbalance in fiacca, a wilty mutant of tomato. II. Auxin- and abscisic acid-like activity. PI. PhysioL, Lancaster, 46, 373. TAL, M., IMBER, D. & ITAI, C. (1970). Abnormal stomatal behavior and hormonal imbalance \n flacca, and wilty mutant of tomato. I. Root effect and kinetin-like activity. PL PhysioL, Lancaster, 46, 367. ZELITCH, I. (1961). Biochemical control of stomatal opening in leaves. Proc. natn. Acad. Sci. U.S.A., 44, 1423-

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