. mmfiistn ON THE STOMATAL RESPONSE TO ABSCISIC ACID Thesis for the Degree of Ph. D. MICHIGAN STATE UNIVERSITY WILLIAM RAYMOND CUMMINS 1971 LIBRARY Michigan Stat: University This is to certify that the thesis entitled On the Stomatal Response to Abscisic Acid presented by William Raymond Cummins has been accepted towards fulfillment of the requirements for Ph.D. Jemin Botany and Plant Pathology - Physiology 114» Luau Major professor Date W911. 0-7639 ‘ .- .; I BY E ‘k_\‘. '-sau3' 1 800K BINDERY INC. ~ LIBRARY BINDEHS I 9.0!“ .ln' .nn-.a.--.. ABSTRACT ON THE STOMATAL RESPONSE TO ABSCISIC ACID BY William Raymond Cummins Stomates of excised primary leaves of Hordeum vulgare started to close within a very short time after the introduction of abscisic acid (ABA) to the trans- 5 piration stream. Treatment with 10- M ABA initiated closure within 2.6 min. There was no response within 20 min following treatment with lO-BM ABA. The lag period between the start of treatment and the onset of the response was a function of the inverse of the concen- tration of ABA applied. A relatively simple method in- volving continuous leaf temperature measurements has been described for showing changes in transpiration rates following treatment of leaves. The closure response was specific for cis,trans- (+)-ABA. Other ABA analogs were relatively ineffective, in this short-time assay. Removal of the supply of ABA resulted in a reversal of the closing response. This reversal could not be William Raymond Cummins explained solely on the basis of catabolism of ABA since no significant breakdown of 14C-ABA occurred within the time in which reversal could be shown to have occurred. ABA treatment of isolated epidermal strips from yigia £223_leaves was effective in causing closure of stomates when the strips were floated on solutions containing low concentrations of potassium ions. The action on epidermal strips indicated that the ABA acted directly on the guard cells. Studies of changes in the levels of intercellular CO2 in leaves following treatment with inhibitors, showed that this may be a suitable method for screening for com- pounds with antitranspirant activity that do not inhibit the photosynthetic mechanism. Such studies coupled with results of experiments with flacca, a wilty mutant of tomato, demonstrated that ABA caused stomatal closure without disruption of the photosynthetic mechanism. ON THE STOMATAL RESPONSE TO ABSCISIC ACID BY William Raymond Cummins A THESIS Submitted to Michigan State University in partial fulfillment of the requirements for the degree of DOCTOR OF PHILOSOPHY Department of Botany and Plant Pathology 1971 To Eva ACKNOWLEDGMENTS The author wishes to thank the personnel of the Plant Research Laboratory for their help during the com- pletion of this thesis. The guidance of Dr. Hans Kende and Dr. Klaus Raschke and the freedom they allowed are greatly appreciated. The constructive criticism and enthusiasm of the other guidance committee members, Dr. Jan Zeevaart and Dr. Martin Bukovac is appreciated. The author wishes to thank Mr. Gene Mielke for his help with the gas-liquid chromatography. The author gratefully acknowledges a fellowship from the Allied Chemical Foundation. This work was sup- ported under Contract No. ATIll-l]—1338 by the United States Atomic Energy Commission. iii TABLE OF CONTENTS LIST OF TABLES . . . . . . . . . . . . LIST OF FIGURES. . . . . . . . . . . . ABBREVIATIONS . . . . . . . . . . . . INTRODUCTION AND LITERATURE SURVEY . . . . . ABA and Stomatal Closure . . . . . . The Effect of Other Factors on Stomatal Responses. . . . . . . . . . . Cytokinins and Hydroactive Closure . . . . The Objective of this Thesis . . . . . . MATERIALS AND METHODS. . . . . . . . . . Plant Material. . . . . . . . . . . Abscisic Acid and Analogs . . . . . . . Extraction of Leaves. . . . . . . . . Methylation of Samples . . . . . . . . Gas-Liquid Chromatography (GLC) . . . . . Thin-Layer Chromatography (TLC) . . . . . Optical Rotatory Dispersion (0RD) . . . . Ultraviolet Spectroscopy (UV). . . . . . Analysis of Gas Exchange . . . . . . . Computation of Diffusion Resistance to Water Vapor and Inter-cellular Carbon Dioxide Concentration . . . . . . . . . . Preparation of Epidermal Strips . . . . . RESULTS 0 O O O O O O O O O O O O O Transpiration Measured as Weight Loss . . . ABA-Induced Changes in Diffusion Resistance to Water Vapor . . . . . . iv Page vi vii 12 l4 14 15 15 l6 16 18 19 20 20 20 Page ABA—Induced Changes in Leaf Temperature . . 23 Estimation of the Rate of Flow of ABA Through Leaves. . . . . . . 28 Does ABA Flow in Both Directions? . . . . 34 Specificity of the Response to Abscisic Acid . . . . . . . . . . 34 Reversal of the ABA Response. . . . 42 Short-Term Metabolism of ABA in Primary Barley Leaves . . . . . . . . 48 Long-Term Metabolism of ABA . . . . . 53 Does ABA Affect Photosynthesis Directly? . 72 Experiments with a Wilty Mutant of Tomato . 77 ABA Action on Stomates in Isolated Epidermal Strips . . . . . . . . . 83 DISCUSSION AND CONCLUSIONS. . . . . . . . 88 REFERENCES . . . . . . . . . . . . . 98 Table 1. LIST OF TABLES Page Comparison of concentrations of (+) and (-) ABA preparations determined by ORD and UV . . ll Estimation of ABA flow rate determined from the lags for leaf temperature changes in a secondary barley leaf as a function of distance from the base of the leaf where ABA at 10-5 was supplied . . . . . . . . . 31 Lag for temperature change following ABA application to solution bathing cut tip of a secondary barley leaf at various distances from the point of application . . . . . . 35 Estimations of relative activities of the analogs of ABA compared to ABA in leaf temper- ature assay and embryonic axis assay. . . . 37 The lag times for leaf temperature change following treatment of barley leaves with (+) and (-) ABA preparations . . . . . . 45 Partitioning of radioactivity from leaves fed 14c-ABA . . . . . . . . . . . . 56 vi Figure BA. BB. LIST OF FIGURES Transpiration rate of excised barley leaves as a function of time before and after treat— ment with cis,trans-(RS)-abscisic acid at 10'4, 10"5 and 10'6M concentrations . . . . Leaf diffusion resistance to water vapor as a function of time . . . . . . . . . Leaf temperature changes with time upon illumination (at the time indicated by the arrow) with light from a xenon arc lamp filtered to exclude infra-red radiation and giving ca.40 mw cm'2 of photosynthetically useable light . . . . . . . . . . . Leaf temperature changes upon treatment of primary barley leaves with 10'6M or 10‘5M cis,trans-(RS)-abscisic acid at time zero . . Chemical structures of the ABA analogs used . Gas-liquid chromatography of a sample of the trans,trans-(RS)-abscisic acid following methylation . . . . . . . . . . . . Optical rotatory dispersion of the (+) and the (-) optical isomers of cis,trans- abscisic acid dissolved in 0.005N sulfuric acid in ethanol. . . . . . . . . . . GLC of preparations of the (+) and (-) Optical isomers of ABA, showing the degree of purity of the samples; 8A: (+) enantiomer; 83: (-) enantiomer . . . . . . . . . vii Page 22 25 27 30 39 41 44 47 Figure 9. 10A. 108. 11A. 118. 12. 13. 14. 15. 16. 17. 18. Changes of transpiration rates upon the addition and removal of cis,trans-(RC)- abscisic acid . . . . . . . . . . Changes in water loss, net C02 uptake and leaf temperature with time for a primary barley leaf supplied with 14C—a carbon labeled 10'5M cis,trans-(RS)—ABA at time zero . . . . . . . . . . . . . . Thin-layer chromatogram of the total extract from the leaf described in Figure 10A . . . Partial reversion of the rates of water loss, net C02 uptake and leaf temperature upon the removal of ABA supply . . . . . . . Thin-layer chromatogram of the total extract from the leaf described in Figure llA . . . Thin-layer chromatogram of an aliquot from the acidic phase extract of primary barley leaves at time zero . . . . . . . . . Thin-layer chromatogram of an aliquot from the acidic phase extraction from "light" leaves C O O O O O O O O O O O O Thin-layer chromatogram of an aliquot from the acidic phase extraction from "light" leaves which was co—chromatographed with an aliquot of a standard solution of radio- labeled 14c-ABA . . . . . . . . . . Thin-layer chromatography of an aliquot from the acidic phase extract from "dark" leaves . Thin-layer chromatogram of a methanolic solution of l4C-ABA which had been irradiated for 1 minute with UV light. . . . . . . Thin-layer chromatogram of a stock methanolic solution of l4C-ABA developed in the initial solvent system used to clear the plates of chlorophyll and other pigments. Thin-layer chromatogram of a methanolic solution of l4C-ABA developed in solvent system: benzene:ethyl acetate acetic acid, 50:5:2 v/v . . . . . . . . . . . . viii Page 50 52 52 55 55 58 61 63 65 67 69 71 Figure 19. 20. 21. 22. 23. Changes in r320 and [C0211 (in ppm or ul l—l) following add1tion at time zero of water (control leaf) or of ABA (10‘7M final con- centration) to the distilled water irrigating primary barley leaves . . . . . . Changes in r320 and [C02]i (in ppm or ul 1.1) following add1tion at time zero of DCMU (lO‘SM final concentration) to the distilled water irrigating primary barley leaves . . . Changes in calculated transpiration rates in excised flacca and wild-type leaves of L. esculentum cv. Rheinlands Ruhm following changes in concentration of C02 in the air flowing over the leaves . . . . . . . . Changes in calculated transpiration rates with time for leaves excised from flacca or wild-type tomato, following addition of ABA to the distilled water irrigating the leaves . Stomatal aperature on epidermal strips after they were floated for 3 hours in the light on solutions containing various concentrations of KCl . . . . . . . . . . . ix Page 74 76 79 82 86 ABA TLC GLC DCMU Ci ORD ABBREVIATIONS Abscisic acid (unless specifically modified this means the racemic mixture of cis,trans- (RS)-abscisic acid). Thin-layer chromatography Gas-liquid chromatography Ultra-violet light 3-(3,4-dichlorophenyl)-l,l-dimethyl-urea Curie Optical rotatory dispersion Diffusion resistance to water vapor. This includes stomatal resistance and boundary layer resistance. The concentration of carbon dioxide inside the leaf (intercellular). INTRODUCTION AND LITERATURE SURVEY ABA and Stomatal Closure V/ Little and Eidt (1968) were the first to show that ABA treatment affected transpiration. Cuttings from £1223 glauca (white spruce) placed in sealed vials containing aqueous ABA solutions lost less water after six days than similar cuttings kept only in water. Mittelheuser and van Steveninck (1969) repeated this experiment and monitored water loss from graminaceous leaves. The stomates of ABA- treated leaves opened only partially. Excised wheat leaves showed a forty-fold increase in the concentration of ABA as shown by 0RD determination and by bioassay following a 4-hour period of wilting when compared to similar leaves kept well supplied with water (Wright, 1969; and Wright and Hiron, 1969). Radioactively labeled mevalonic acid was incor- porated into (+)-ABA in excised barley leaves which had been left to wilt for several hours (Milborrow and Noddle, 1970). These data suggested that water stress could stimu- late the biosynthesis of (+)-ABA in leaves, and that large increases in ABA concentration could be found within a short period after the application of the stress. No data have yet been published to show the time course of the in- crease in ABA concentration. When roots of Nicotiana tabacum plants were sub- jected to stress by increasing the osmotic strength of the solution surrounding the roots, leaves excised 4 or 48 hours after the start of such stress were shown to contain greater quantities of an ABA—like inhibitor than leaves from non-stressed plants (Mizrahi 35 31., 1970). The ABA content of such leaves remained high even after the leaves had apparently recovered full turgor. Gale 33 31. (1967) reported that the stomates of cotton plants under salinity stress were only partially open and that transpiration was considerably reduced even after the leaves regained full turgor. Stalfelt (1929) investigated the changes in stomatal aperture following the initiation of water stress. Leaves of Vicia faba were exposed to water deficits. The first #- response was an opening of the stomates. Such rapid "hydro- passive; opening, which has also been shown to occur in leaves of §32_mgy§_following the application of negative pressure to the leaf's water supply (Raschke, 1970), is caused by the reduction in water potential of the xylem resulting in a reduction in turgor of the epidermal cells and a decrease in the pressure exerted by the epidermis on the guard cells of the stomates. Willis 35 31. (1963): reported similar data. They found that the stomatal resistance started to decrease very rapidly when y, faba leaves were excised. The resistance continued to fall for at least 10 minutes after excision of the leaf. If this were the only response then the rate of water loss in stressed leaves would be accelerated by the action of the stomates. However, the transient opening of stomates as a response to water stress is followed by a second re- sponse. Starting ca. 13 HUJI after exposure of y, faba. leaves to a water deficit greater than 3% (Stalfelt, 1929) or after excision of turgid leaves (Willis et 21,, 1963) closure of stomates could be observed. Stglfelt called this “hydroactive closure" and assumed that an active closure mechanism was activated during periods of water deficit. Evidence cited above indicates that one of the . physiological roles of ABA may be to protect plants from drying out during periods of drought by mediating hydro- ' _ .. fl q...- -‘—.....,,’ r . .. _~ "h, active stomatal closure. \- I I 5 \~ The Effect of Other Factors on Stomatal Responses Stomata are capable of responding to a host of stimuli including treatment with chemicals; therefore, caution must be exercised in evaluating and understanding the responses of stomata to exogenously applied agents such as ABA. J Increased C02 concentration in the leaf leads to stomatal closure, while reduction of the C02 level causes J/stomatal opening (Heath, 1961; Linsbauer, 1916). There- fore, illumination causing photosynthetic C02 fixation leads to stomatal opening. There is possibly also a light- activated, C02-independent opening response since stomates on epidermal strips kept in C02-free air open more in the light than in the dark (Humble and Hsiao, 1969). .j The humidity of the air around the stomates also affects the extent of opening (Lange 32 31., 1971). Minshall (1960) suggested that any compound that affects photosynthesis also affects the stomatal apparatus by way of changes in intercellular C02 levels. He showed that excised leaves of Phaseolus vulgaris treated through the petiole with various inhibitors of the Hill reaction transpired less. Application of herbicides (Thorne and Minshall, 1964) and fungicides (Smith and Bucholtz, 1964) to whole plants and roots reduced transpiration. Many workers have investigated the effects of auxin on trans- piration; in some cases, auxin solutions were supplied to the roots of potted plants of Tropaeolum majus (Ferri and Lex, 1948), or by spraying auxin solutions on whole bean plants (Brown, 1946), or by dipping excised leaves of kidney beans into auxin solutions (Bradbury and Ennis, 1952). Player (1950), Kasperik (1955), and Mansfield (1967) showed that monocotyledonous plants, which are resistant to the herbicidal effects of auxins, showed no decrease in transpiration rates following auxin treatment which effec- tively closed stomata in susceptible dicotyledonous plants. J .A toxin, fusicoccin, extracted from a fungus which is a pathogen of almond and peach trees, causes opening of stomata on excised leaves of g. vulgaris and N, tabacum (Turner and Graniti, 1969). J, Treatment with various metabolic inhibitors results in a variety of stomatal responses. Treatment with 2,4- dinitrophenol and sodium azide, uncouplers of oxidative phosphorylation, inhibited Opening of stomates (Mouravieff, 1953). Stalfelt (1957), on the other hand, showed that treatment with sodium azide at 10"2 M prevented hydroactive closure suggesting that active metabolism is involved in mediating that response in excised y. faba leaves. Treating leaves with inhibitors of glycolic acid oxidase (Zelitch, 1961) or with phenyl mercuric acetate (Mansfield, 1967), an inhibitor of noncyclic photophosphorylation (Nozaki 35 31., 1961), inhibited transpiration. Cytokinins and hydroactive Closure Itai and Vaadia (1965) demonstrated a decrease in the concentration of cytokinin-like material in the root exudate collected from plants, the roots of which had been subjected to a water stress as compared to exudate from un- stressed plants. Excised barley leaves treated with a cytokinin or gibberellic acid showed greater transpiration rates than untreated leaves (Livne and Vaadia, 1965). This work was confirmed by Luke and Freeman (1967) using a 48— hour bioassay period. Meidner (1967) presented evidence that cytokinin-treated, excised mature primary barley leaves had increased rates of C02 uptake as well as greater trans- piration rates when compared to untreated leaves. O'Leary and Tarquinio Prisco (1970) gave evidence that kinetin treatment increased the stomatal diffusion resistance (i.e., closed stomates) in salt-stressed bean plants. Pallas and Box (1970) provided evidence based on psychrometric data that kihetin-treated, excised leaves of barley show de- creased turgor pressure potentials which may account for the opening of the stomata by an osmopassive response. The most rapid response of stomates to cytokinin treatment was shown by Meidner (1967) to occur 3 hours after treatment. Except for the report by O'Leary and Tarquinio Prisco (1970) on cytokinin-induced stomatal closure, it seems that reduced cytokinin supply may partially account for hydroactive closure of stomates in a period of water stress; or, in other words, a continued cytokinin supply may be needed for optimum stomatal operation. If this is true and if the root is the site of synthesis of cytokinins (Kende, 1964; Humphries and Thorne, 1964) then excised leaves placed in water should show a rapid (starting within ca.13 minutes) decrease in transpiration rate unless they are treated with cytokinin. No such data have yet been presented; the results of Livne and Vaadia (1967) definitely do not show such fast decreases of transpiration in excised water-supplied leaves. The Objective of this Thesis The evidence to date suggests that ABA may play a physiological role in the conservation of water in plants during periods of water stress. The objective of this thesis was to determine if this hypothesis is supported by experimental evidence concerning the characteristics of the stomatal response to ABA treatment. MATER IALS AND METHODS Plant Material Barley seeds (Hordeum vulgare L., cv. Himalaya) were sown in trays of vermiculite. Twice a week they were irrigated with half-strength Hoagland's solution; once a week with distilled water. The conditions under which the plants were grown were as follows: 16 hr day length; 23°C (day), and 20°C (night) temperatures; 80% relative humidity; fluorescent light supplemented with incandescent light of ca. 3000 ft candles light intensity. Primary leaves were excised from 9-14 day old plants 6-8 hr after the beginning of the light period. Tomato seedlings (Lycopersicum esculentum Mill., cv. Rheinlands Ruhm, wild-type and flacca, a wilty mutant) were grown in vermiculite from seeds supplied by Dr. C. M. Rick of the University of California at Davis. Growth conditions were as stated above. Flacca is a recessive point mutation produced by x-ray treatment located on chromosome number 7 as indicated by Dr. Rick (see Tal, 1966) from tests of allelism and linkage. The phenotype was described by Tal (1966), and Imber and Tal (1970). Three weeks after germination the tomato seedlings were transferred to pots containing a 3:1 mixture of potting soil and sandy soil. They were irrigated daily with distilled water. In some cases the seedlings were trans- ferred to pots containing a mixture of vermiculite and soil. These were then placed into aerated half-strength Hoagland's solution. Similarly-shaped leaves were chosen from the wild-type and flacca plants, excised under water and sub- jected to analysis. Broad beans (Vicia faba L., cv. Improved Longpod) were grown from seeds in a 3:1 mixture of Bacto potting soil and sandy soil. The plants were irrigated daily with distilled water. Three to 4 weeks after germination, leaves were excised under water, and epidermal strips were taken from the abaxial leaf surface. Abscisic Acid and Analogs Cis,trans-(R,S)-ABA was obtained from Dr. J. van Overbeek of Texas A and M University and judged to be pure by thin—layer chromatography and by ultraviolet (UV) spec- troscopic examination. Trans,trans-(R,S)-ABA was kindly supplied by Dr. E. Sondheimer of Syracuse University. Gas liquid chromato- graphy (GLC) showed that the sample contained 6% of cis,trans-(R,S)-ABA (Figure 6). 10 Cis,trans-dihydroionylideneacetic acid was prepared by Dr. E. Sondheimer. Solutions of known concentration were prepared simply by dissolving weighed amounts of this isomer in distilled water. The solutions were not tested for purity. It was selected because of its chemical similarity to ABA and its relative inactivity in other biological assays. (+) and (-)—Cis,trans-ABA: the (+) and (-) optical isomers of cis,trans-ABA were supplied by Dr. E. Sondheimer. Concentrations of stock solutions of these optical isomers were determined by optical rotatory dispersion (ORD) and UV spectrosc0py. The (+) and (-) enantiomers gave mirror- image 0RD curves (Figure 7). Measurement of the concentra- tions by ORD and UV agreed very closely (Table 1) indi- cating very little, if any, contamination of one enantiomer with the other. Each sample contained contamination with trans,trans-ABA as indicated by GLC (7% in the (+) prepar- ation, 4% in the (-) preparation). Both UV and GLC results indicated considerable contamination of the (+) preparation with unknown compounds. Sharp peaks at 264 and 268 nm marked the UV Spectra of this preparation and three extra peaks were detected by GLC. The (-) preparation was free of such contaminants. Since there is uncertainty reflected in the liter- ature (see Cornforth 33 31., 1967; Burden and Taylor, 1970) as to the designation of absolute configuration of (+)—ABA 11 Table 1. Comparison of concentrations of (+) and (-) ABA preparations determined by ORD and UV. CONCENTRATION (M) PREPARAT I ON ORD UV (+) cis,trans-ABA 1.7 x 10‘4 1.7 x 10’4 (-) cis,trans-ABA 1.6 x 10'4 1.5 x 10"4 12 (i.e., either R or S), the nomenclature is herein re- stricted to the optical properties Of preparations of the two enantiomers. Radioactively labeled ABA: 14 C-cis,trans-(R,S)- abscisic acid was received from two sources. The first lot was synthesized by Dr. 0. Smith, University of Cali- fornia, Riverside and contained the label in the alpha carbon of the side chain. Its specific activity was 26 Ci mole-1. Thin-layer chromatography showed it to contain less than 5% of the trans,trans-isomer and no other contaminant. The second lot was custom-synthesized by Mallinkrodt/ Nuclear, St. Louis, MO. and contained the label in the carboxy carbon of the side chain. It was radio-chemically pure as judged by TLC. GLC showed it to contain 4% of the trans,trans-isomer. Its specific activity was 23 C1 mole-1. Extraction Of Leaves Following exposure to labeled ABA for varying periods of time, leaves were immersed into methanol which had been cooled with solid C02. The frozen leaf was finely chopped with scissors and left in methanol which was then allowed to warm to room temperature overnight. The suspension was filtered and the leaf pieces reextracted in methanol. This was repeated until the leaf extract became colorless. 13 The combined methanol filtrate was diluted with ten volumes of distilled water and evaporated under partial vacuum to remove the alcohol. The resulting aqueous solution of the total leaf extract was adjusted to pH .0 with a sat- urated solution of NaHCO thrice partitioned against diethyl 3 ether; the combined ether phases will be referred to as neutral fraction. The pH of the remaining aqueous phase was then adjusted to 3.0 with 0.1N H2804 and again partitioned three times against diethyl ether; this ether phase will be referred to as the acidic fraction of the extract. Fol- lowing hydrolysis Of the aqueous phase at 60° C and pH 11.0 for 20 min. partitioning again of diethyl ether was repeated at pH 3.0 yielding the hydrolized fraction. The ethereal phases from each Of these treatments were then dried over NaZSO4 and evaporated to small volumes under partial vacuum. Aliquots from each of these partitioned phases as well as from the remaining aqueous phase were transferred into scintillation vials to which a dioxane-based scintillation fluid was added. The vials were examined for radioactivity using a Packard Model 3375 liquid scintillation spectro- meter. Efficiency of counting was 85% as determined by external standardization. The acidic fraction of the ex- tracts were then divided: one-third was chromatographed directly by TLC. One-third was co-chromatographed with a standard solution of 14C-cis,trans-(R,S)-ABA. The final third was chromatographed after it had been methylated. 14 Alternatively, when a leaf had been exposed to low levels of radioactivity, the initial methanol extract was chromatographed directly by TLC. Methylation of Samples Methylation was carried out following the procedure of Schlenk and Gellerman (1960) as modified by Powell (1964). Since the procedure allows for complete methylation of ABA samples, the reaction products could be directly chromato- graphed (either by TLC or more generally by GLC). Gas-Liquid Chromatography (GLC) A Packard Model 7300 gas-liquid chromatograph was used throughout this investigation. This instrument was equipped with dual glass columns, flame-ionization detectors or electron-capture detectors. Gas chrome Q, 60-80 mesh, was used as the solid support, and 3% DC 200 was used as the liquid phase. Nitrogen was passed through the column at 40 ml min"1 at 40 p.s.i. The amplifier output (detector response) was recorded on a linear flat-bed recorder. Retention times were uncorrected and used only for comparative purposes. Standard ethereal solutions of methylated ABA were injected into the column to determine standard retention times. The standard ABA solution was a 1:1 mixture of cis,trans and trans,trans-isomers of the (i) synthetic ABA supplied by the Reynolds Tobacco Company, Winston-Salem, N.C. 15 Quantitative estimates Of the isomers were obtained by comparison of the relevant peak area with that of a known quantity of standard. The peak area was calculated as a function of the weight of the excised chart paper delineated by the peaks. Thin-Layer Chromatography (TLC) Thin-layer plates from Brinkman Instruments Inc., E.M. Division, Westbury, N.Y., precoated with a 0.25 mm layer of silica gel, were washed in ethanol and activated at 105°C for 10 min prior to use. The developing solutions were prepared from redistilled solvents. Since most of the solutions to be chromatographed were crude extracts, the plates were first developed three times in a hexane-ethyl acetate, 1:1 (v/v) to move a large portion of the pigments to the front. Then the plates were developed in benzene-ethyl acetate-acetic acid, 50:5:2 (v/v). This general method for whole leaf extract chromatography was described by Milborrow (1970). Optical Rotatory Dispersion (ORD) Determination Of concentration of solutions of the (+) and (-) enantiomers of ABA was carried out as described by Zeevaart (1971) using a Durrum-Jasco Spectropolarimeter model J-5 assuming equal absolute magnitudes of rotation Of the Optical isomers. l6 Ultraviolet SpectrOSCOpr(UV) Concentrations of (+), (-) and (i) preparations of cis,trans-ABA were determined after examination of UV spectra. All isomers of ABA were dissolved in ethanol con- taining 0.005N sulfuric acid and gave absorbance maxima at 261 nm. Solutions of cis,trans-(:)-ABA made up by weighing a crystalline sample supplied by Dr. J. van Overbeek showed molar extinction coefficient of 2.2 x 104 which is in agree- ment with the value published by Milborrow (1970). Analysis Of Gas Exchange Small plexiglas chambers were designed and built for analysis Of small leaves. The leaf blade under study was gently pressed between two silicone rubber gaskets. The two halves of the chamber were then pressed over the gaskets. The gaskets were wide enough to prevent leaf damage from excess pressure yet an airtight seal could be maintained around the leaf edges. The gaskets bordered an 2 on each Of the 2 surfaces Of the leaf. area of 2.0 cm Air flowed into each half of the chamber, over each sur— face of the leaf and was vented. Fixed into one gasket were fine threads of copper and of constantan fashioned so that they lay along 4 cm Of the abaxial leaf surface. The junction between them (one thermocouple junction) was touching this leaf surface. The reference thermocouple junction was kept in an ice 17 bath at 0°C. This arrangement allowed continuous monitor- ing Of accurate leaf temperatures. Once excised from the plant, the base of the leaf blade (or the cut surface of the petiole in the case of tomato leaves) was kept under water. When each leaf had been placed into its separate chamber, its base was im- mersed into a beaker containing 10 ml Of distilled, de- ionized water. The beaker was shaded to keep it cool and to avoid the possibility Of photo-conversion of added solutes. The leaves were vertically oriented and the light from an overhead, water-cooled Xenon arc lamp (Osram XBF 6000) was reflected Off a mirror of mylar coated aluminum film through an infra-red filter and through one side of the plexiglas chamber onto the adaxial surface of the leaf. The four chambers used in each study were positioned, using a small silica photocell so that they each received equal irradiation (ca. 40 mW cm"2 of photosynthetically useable light). Fans were positioned around the chambers to pre- vent excessive heating. Addition Of solutes to the leaf was carried out as follows: each identical beaker under each leaf was filled to capacity (10 ml); 1 ml was drawn from each and 1 m1 of experimental or control solution was then added to each. In this way precise concentrations of solutes could be administered to each leaf. Each surface studied was aerated with a constant flow (usually so 1 hr-l) of air, the dew point (usually 18 11.8: 0.05°C) and the C02 concentration (usually 300 i 2 ul 1-1) Of which were specific and constant. The compo- sition of efflux gas from each leaf surface was inde- pendently compared to the composition of influx gas using 2 differential infra-red gas analysers (URAS-2 Hartmann and Braun, Frankfurt) in series. One of them monitored changes in water vapor concentration, the other measured C02 concentration differences. The air flowing from each chamber or leaf surface was sequentially diverted to the gas analysers by solenoid valves activated by a timing mechanism. The data were recorded on analog recorders as well as through a digital data acquisition system on mag- netic tape for computation using apprOpriate programs. The gas analysis and data acquisition systems were designed and assembled by Dr. K. Raschke and will be fully described (Raschke, in preparation). Computation of Diffusion Resistance to Water Vapor and Inter-cellular Carbon Dioxide Concentration From the data recorded as described above, the diffusion resistance (including stomatal and boundary layer resistances) to water vapor (rHZO) and the inter-cellular C02 concentration [C0211 as well as the transpiration rate and the C02 assimilation rate could be computed (Raschke, in preparation) using calculations similar to those described by Moss and Rawlins (1963). 19 Preparation of Epidermal Strips Stomata on isolated epidermal strips were studied as described by Humble and Hsiao (1969). Epidermal strips were removed from the abaxial surface of bean leaves (Vicia faba L., cv. Improved Long Pod) and transferred to buffered solutions (0.5 mM tris—maleate pH 6.0) containing 1 Of Ca+2 and concentrations of KCl ranging 0.2 meq liter- from 0 to 100 meq liter-1. The solutions on which the strips were floated were then either placed in darkness, or illuminated with 8.5 chm-2 visible light (filtered through water to remove infra-red) from 2 mercury vapor lamps. These solutions were continuously flushed in a stream of humidified, C02-free air. Once the stomata were allowed to Open for 2 hours in the light, the strips were transferred to solutions containing abscisic acid. After one hour the strips were removed from the solutions, blotted dry and temporarily fixed in immersion oil. The degree of opening of the stomata was examined under a microscope fitted with an ocular micrometer. During preparation of the strips, the majority of epidermal cells were ruptured (Humble and Hsiao, 1969), but the guard cells remained intact. Intact epidermal cells could be distinguished by observing the strips under low magnification using small-angle incident light. Intact cells appeared convex and bulging. RESULTS Transpiration Measured as Weight Loss The bases Of 3 primary barley leaves were immersed into a glass vial containing water. In order to prevent evaporation, the opening of the vial around the leaf bases was sealed with parafilm. Transpiration rates were deter- mined as functions of weight loss from tared vials. Figure 1 shows the results from one such experi- ment. Transpiration rates were measured before and after abscisic acid addition to the irrigating solution. The weighing intervals were 1 hour apart,and as can be seen from the results, decreases in transpiration rates occurred within the first hour after addition Of abscisic acid. Final concentrations of ABA in the irrigating solutions of 10", 10'5 and 10'6M all caused decreases which became evi- dent as soon as could be measured by this technique. ABA-Induced Changes in Diffusion Resistance to Water Vapor Using the gas analysis technique, the lag for abscisic acid action was determined. Values for the leaf diffusion resistance to water vapor (rHZO) were calculated 20 21 Figure l.--Transpiration rate of excised barley leaves as a function of time before and after treatment with cis,trans-(RS)-abscisic acid at 10'4, 10’5 and 10'5M concentrations. Arrows indicate time of appli- cation Of the inhibitor. The horizontal bars on the first readings after treatment indicate the interval over which weight loss was determined. Transpiration rates were determined as a function of the loss of weight from vials, each containing 3 leaves. Each point is the average of 3 determinations (i.e., 9 leaves). The experiment was carried out under fluorescent lights at 20°C. The leaves were placed 50 cm below a bank of 4 Sylvania Lifeline FR40W-235 fluorescent tubes. 22 M A‘ho"4 IO'SM Io'5M \ O 2 H b b _ _ O 6 Bohr O O O 4 3 2 A .13». TE 95 28 cozogamcoek Time(hr) 23 at 10 second intervals for a barley leaf before, during, and after addition of abscisic acid to the irrigating solution. Figure 2 shows that within 7 minutes of changing the irrigating solution from pure water to a 10—7M ABA solution, the erO had started to increase and continued to increase (indicating closure) for about 30 minutes, while no change in rHZO of the untreated leaf took place. ABA-Induced Changes in Leaf Temperature Subsequent examination of temperature changes in leaves led to an easier method for determining time course of the ABA response. Figure 3 shows a typical leaf temperature versus time plot. When the light was turned on, the leaf tempera- ture rose instantaneously. As the stomates began to open, however, transpiration started to cool the leaf. The leaf temperature stabilized when transpiration rate reached a constant level. In such experiments radiation was kept constant for each leaf (approximately 40 mW'cIu"2 from a xenon arc lamp). The air flow past each surface of the leaf was also constant at 50 l h"1 and the rate of heat loss from the chambers was kept constant by fans directing air around the outside of the chambers. Very soon after ABA was added to the irrigating solution, the leaf temperature started to increase, 24 . N (80350 ¢ .m0 m0 OOOMAOMHHH mcH>Hm Ofima Ohm cocmx m mp3 condom pamflq . Is an com was coflumuucmucoo moo was .oom.HH mos Mme map mo unwomaop one .Uomm ocm ohm consumn common musumummamu moon .Hunn H om mp3 mmma masons 30am new Hobos .mmoa on» no woman cuon How mocmumammu game m mum0flocw muasmmu may Om monomusm mama nuon Ho>o mama may ocsonm omzoam Ham ucmfiflnmmxm was» OH .ucmfiummnu moumm mouscfie m on m mcfluumum oocmumfimmn ca mmmmuoca cm oozocm mama ooummnulmmm one .Hmum3 no Azhuoa mp3 coflumuucmocoo Hansel whom owmaomnmulmmvumamuu.mao umnuam museum an Ommcmso mums mm>mma Season wumeflnm Ommfloxm on» mcfiummwuua chHuOHom on» own» mfiflu pa .mfiflu mo coauocnm m on Homm> nouns ou mocmumflmon coflmOMMHo mmmqul.~ ousmflm 25 2.835: o 0 ONI 0 new... 0 O IInnvunlllmulllm Ilmvllll 0101100 1 O to .... 1m H1 e Z 0 <2 .2 we ... a... .. ...m w ..... m cos I_. 000 ls. l.\ o,- OO 0. 5F F _ _ — om o. d 26 .cofimcmEHO 80 q muflucm mnu m>onm mommusm mmma umzoa mcu suw3 uomucoo uomufio cw mHm3 momma mamsooofiumnu map on “mofim umm EO o.~ Ho Eu o.v x m.o mms ommomxm mmnm mmma may camumss omms mum3 mumnfimso HmHHmEm Anv “any: H om mo mums m um waucmocmmmocfi mmommuOm mmma anon Hm>o Omsoam Ham Amv umcH3OHHOm man How ummoxm N musmam How omumOHocH no mean may mum3 mcofluflocou .unmfla manmmms mHHMOHumnucwm Iouonm mo :50 38 o¢.mo mcH>Hm cam coflumflomu pmulmumcfl mosaoxm mu omumpaam mama mum cocmx m Eoum unwed nufl3 Azouum mnu hp omumoaocw mEHu man “my coflumcwEdHHH coma med» nufiz mmmcmno mnsumummfimu mmmqul.m musmam 27 1“ 1 l 1 J I O (D CO v N m N N N N (Go) le31 :IVB'I 3O 4O 50 TIME (min) 20 IO 28 indicating that transpiration was reduced and that stomates had started to close. Figure 4 shows such increases above leaf temperature at equilibrium. The temperature change started as early as 2 minutes after adding ABA (lo-5M final concentration) to the irrigating solution. The lag before the start of the temperature rise appeared to be a function of the inverse of the concentration of the ABA applied. Estimation of the Rate of Flow Of ABA through Leaves The rate Of flow of ABA through the leaf was esti— mated in order to determine what proportion of the lag time was due to transport of the inhibitor to its site of action. Flow rate was determined for secondary leaves of barley because their size allowed the positioning of several gas analysis chambers along the length of the leaf. Fully-grown leaves were used so that all the sto- mates along the leaf would be fully developed. The trans- piration rates per area of leaf were determined at each Of the 3 positions monitored along the leaf for comparison. The flow Of water (and presumably the dissolved ABA) through the leaf should be a function of the transpiration rate. Table 2 shows the results of one such experiment. The flow rate determined between the 3 positions was ap- proximately 10 cm min-l. The time required for abscisic 29 Figure 4.--Leaf temperature changes upon treatment of primary barley leaves with 10" M or 10' M cis,trans- (RS)-abscisic acid at time zero. The ABA supply was removed at the time indicated by the arrow. In each case the circles represent temperature of the treated leaf; the squares indicate temperature of the control leaf. Treatment with 10’6M ABA caused initial change within 5 minutes; 10'5M ABA treatment within 3 minutes. Temperature (°C) Leaf 30 ..I 29‘- 28 27 I I -6 ABA 10 M I 32— 31(- 30" 29- ABA 165M J 0 20 Time (min) 31 Table 2. Estimation Of ABA flow rate determined from the lags for leaf temperature changes in a secondary barley leaf as a function of distance from the base of the leaf where ABA at 10‘5 was supplied. DISTANCE FROM TOTAL TRANSPIRATION FLowa BASE LAG RATE RATE (cm) (min) (g dm'2 hr'l) (cm min-1) 18.5 4.5 1.5 10.4 23.7 5.0 1.5 10.3 34.0 6.0 1.3 Total lag at 34 cm from source 6.0 min Transport lagb 3.3 min Net lagC (independent of transport) 277 min aFlow rate was determined by dividing the dif- ference in distances from the base of two monitored points by the difference in their total lags. bTransport lag was determined by dividing the distance from the ABA source by the flow rate. cNet lag is that time needed for leaf temperature change following the computed time of arrival of ABA to the center of the area of leaf that was monitored. 32 acid to reach the stomates covered by the 3 chambers (the transport lag) accounted for only a part of the total lag time. This means either that the estimate of flow rate is tOO high by a factor Of 2 or that other considerations account for the net lag time. If one considers the ABA that is added to the irrigating solution to move up through the leaf as a front from the cut surface then its rate of flow may be described according to Ohm's law as: potential difference resistance flow rate a As the front moves away from the base the potential dif- ference factor should decrease since there is a decrease in the total evaporating surface above the front. This evaporating surface is the major determining factor caus- ing the flow of water in a leaf. The resistance to flow is assumed to increase only slightly as the front moves through more and more conducting tissue. Therefore the flow rate determined over an interval of 4 cm far from the water supply should be somewhat less than the flow rate determined over a similar interval closer to the water supply. In these experiments it has been assumed that the flow rate is relatively constant along the entire leaf surface. This means that the flow rate has probably been somewhat underestimated. are: 33 Other factors which may contribute to the lag time The time required for the ABA tO diffuse along or through the evaporating surfaces to its site of action. If ABA does act on the guard cells di— rectly, then ABA must diffuse along the evapo— rating surface (presumably that layer of liquid water covering the mesohyll cells) to the epi- dermis and the guard cells. There is no way to estimate this time but it conceivably contributes to the lag of the response. A part of the lag must be due to the time required for ABA to cause closure once it has reached its site of action. For secondary leaves this time must be somewhat less than 2.7 minutes. The short— est lag for primary barley leaves was 2 minutes. In this case the distance traveled was less than 10 cm. The measurement of leaf temperature was virtually instantaneous. There was no mechanical or instru- ment lag. The time required for a rise in leaf temperature once stomata start to close, however, is very difficult to estimate. It is conceivable that stomata start to close some seconds before the change in leaf temperature. However, the lag 34 times for change in transpiration rate and change in leaf temperature are virtually the same so this time must be only a few seconds. Does ABA Flow in Both Directions? TO answer this question, secondary barley leaves were again fitted with three gas analysis chambers as before. This time the tips as well as the bases were cut and immersed under water. ABA (lo-SM final concentration) was added only to the solution bathing the tip. The pat- tern Of response was different. Table 3 shows that the leaf portion farthest from the tip source did not respond to ABA (no change in leaf temperature or transpiration rate). Leaf portions closer to the ABA supply did respond. These results indicate that ABA can move in either direction depending on the direction the water is moving. The leaf portions closer to the base draw their water from the solution bathing the base. The water flows in response to the water potential gradient established by evapo- transpiration along the path of least resistance. Specificity_of the Response to Abscisic Acid Analogs of abscisic acid which show varying degrees Of biological activity in other assay systems (Sondheimer and Walton, 1970) were tested to determine whether their relative activities in the closing response paralleled their other inhibitory activities. 35 Table 3. Lag for temperature change following ABA application to solution bathing cut tip of a secondary barley leaf at various distances from the point of application. DISTANCE FROM DISTANCE FROM TOTAL TIP SOURCEa BASE LAG (cm) (cm) (min) 6 16 3.3 11 11 16 16 6 >46 aABA lO'SM applied only to tip of leaf. 36 Table 4 Shows the lag time for leaf temperature change as a function of the concentration of synthetic abscisic acid analogs: the higher the concentration of inhibitor present, the shorter the lag. These data are compared with data published by Sondheimer and Walton (1970) which show the relative effectiveness of these compounds in inhibiting the growth of embryonic bean axes. As seen in Figure 5 the analogs are very similar to abscisic acid in chemical structure. All have a carboxylic acid terminating a conjugated side chain which is attached to a six-membered ring. The analogs demon- strated marked differences in biological activity which are paralleled in at least 2 very different types of assay systems, namely inhibition Of growth of embryonic bean axes and inhibition of transpiration. GLC of the trans,trans-ABA (Figure 6) showed it to contain about 7% of the cis,trans-isomer. This con- tamination may account for the relatively high (1-10%) activity of this sample in both assay systems since the same trans,trans-ABA preparations were used in both cases. In the very rapid leaf temperature assay, there was no activity of the other analog (dihydroionylidene- acetic acid). Such preparations contain equal amounts of both enantiomers, i.e., the (+) and the (-) optical isomers of the molecule. Only the (+) enantiomer occurs naturally in 37 Table 4. Estimations of relative activities of the analogs Of ABA compared to ABA in leaf tempera- ture assay and embryonic axis assay. % RELATIVE ACTIVITY IN TREATMENT LAG (min) BARLEY EMBRYONIC LEAVESa AXES cis,trans-(RS)-ABA 100 100 10-7M 7 10‘5M 4 10'5M 2.5 trans’trans-(RS)-ABA 1-10 6 10' M >35 10‘5M 10 10'5M 6 cis,trans-dihydro- ionylidenacicetic acid <1 2 10-7M >35 1076M >35 aBased on ability to change transpiration rates. bFrom data Of Sondheimer and Walton (1970) based on ability to inhibit elongation of bean embryonic axes. 38 Figure 5.--Chemical structures of the ABA analogs used. These were synthetic preparations and, therefore, contained equal amounts of the R and S enantiomers (i.e., they were racemic mixtures). The circled atom is the assymmetric carbon. The (+) and (-) optical isomers of cis,trans-abscisic acid (not racemic mixtures) were also tested for activity. OH\ \ o’// COZH cis, trans -(R S) - abscisic acid COZH OH\ \ <)’// trans, trans -(RS)- abscisic acid \ \ COZH cis, Irans-dihydroionylideneacetic acid 40 Figure 6.--Gas-liquid chromatography of a sample Of the trans,trans-(RS)-abscisic acid following methyl- ation. This chromatogram shows the high overall purity Of the sample and the major contamination by a peak (accounting for 6% of the total) the retention time of which corresponds to that of the cis,trans-isomer. Detector Response 41 Ir GAS uauuo CHROMATOGRAPHY or TRANS TRANS-(R31 -ABA SOLUTION CIS \ I a 3 4 6 Time(min.) 42 plants (Milborrow, 1969). Indeed the Optical activity of the (+) isomer is used to determine (+) abscisic acid concentrations from natural sources by ORD. Figure 7 shows ORD curves for the two Optical isomers. Based on ORD and UV data (see Materials and Methods),stock solutions Of known concentrations of the two isomers were prepared. Since the concentrations determined by the two methods agreed very closely (Table l), we concluded that the (-) preparation contained little, if any, (+) ABA. Table 5 shows the results Of experiments to deter- mine the relative activity Of the two Optical isomers. There was some activity of the (-) preparation at 0.8 x 10'5M but virtually none at 10'6M while the activity of the (+) preparation was comparable to or slightly greater than the activity of the racemic mixture. From such re- sults we conclude that the (+) isomer, which is the natur- ally occurring one, is considerably more active than the (-) isomer preparation. Analysis of the two isomers by GLC indicated that both preparations were more than 77% pure (Figure 8). Reversal of the ABA Response The persistence Of the ABA effect on stomata clo- sure was investigated in the following manner. The ABA supply was removed by repeatedly diluting and draining the irrigating solution. During the draining and diluting 43 Figure 7.-—Optical rotatory dispersion of the (+) and the (-) Optical isomers Of cis,trans-abscisic acid dissolved in 0.005N sulfuric acid in ethanol. The (+) enantiomer shows a positive rotation peak at 289 nm, zero rotation at 269 nm and a negative rotation peak at 246 nm. The curve for the (-) enantiomer shows a mirror image except that the point of zero rotation is slightly shifted to a lower wave length. There is no reasonable explanation for this small shift although it could indicate Slight contamination of this enan- tiomer by an optically-active species containing a UV- sensitive chromOphore. +25 A U) m 0 h a m E : O E v C o '4: O 4— o h -25 44 (I l 1 l 250 300 350 wavelength (nm) 45 Table 5. The lag times for leaf temperature change following treatment of barley leaves with (+) and (-) ABA preparations. LEAF TEMPERATURE ABA TOTAL INITIAL CONCENTRATION LAG a TRANSPIRATION APPLIED (min) INIEIAL FI§AL (9 dm-2 hr-l) 1.0 x 10‘6b (t) 10 24.2 25.2 1.35 (i) 10 24.5 25.4 1.53 (+) 7 24.5 25.5 1.68 (-) >30 24.8 24.6 1.53 0.8 x 10'5b (+) <7 - 26.7 1.35 (-) 12 25.8 26.0 1.35 (+) 5.5 25.4 26.9 1.28 (-) 12 25.8 26.0 1.35 1.6 x 10’6C (+) 5 27.4 28.4 1.20 (-) >20 27.9 27.9 1.35 (:)d 7 27.9 28.6 1.35 aLeaf temperature 30 HOT: after treatment started. bExperiment carried out in COZ-free air. -1 cExperiment carried out in air containing 300u1 1 C0 20 d(i) applied to leaf which failed to respond to (-) preparation. 46 .HmEOHyamam any ”mm nymEOHycmam A+v "4m “mmamfimm may mo Myflusm mo mmymmo may mcwzoam .mmm mo mHmEOmw Hmoyymo any can A+v may mo mCOHymHmmmym yo UAUII.mm Cam «m mmysmfim 47 ... a: I— —1 h- -( II-——_=: =51 asuodsaa 10:33:30 - < P -I --_=: ‘T {,2 asuodsaa 10:33:30 3 Time (min) 2 3 2 Time (min.) 48 procedure, the base of the leaf was kept submerged so that the transpiration stream was not broken. Removal of the ABA supply caused a reversal Of the ABA effect, i.e., the transpiration rates started to increase again (Figure 9). Those leaves to which ABA supply had been maintained showed continued decline of transpiration rates. Such reversion of the response can most simply be explained in the following 2 ways. Active ABA must be removed from its site of action either by being sequestered into a cellular compartment which is different from its site Of action, or else by being chemically altered to an inactive form. To determine which of the two possibilities is most likely, efforts were directed at following the metabolism of the applied ABA. Short-Term Metabolism of ABA in Primary Barley Leaves Primary barley leaves were fitted into gas analysis chambers where leaf temperature, C02 exchange and H20 ex- change could be monitored. After the leaves were illumi- nated for 2 hr, radioactively labeled ABA was added to the irrigating solution. After a labeling period of 15 min, when the stomates closed as a response to ABA treatment (Figure 10), one leaf was removed and immediately ex- tracted. At the same time the ABA supply to the other leaf was removed as described above. Thirty-two minutes after removal of the ABA supply when reversal of the ABA 49 .N musmym How omayyommo mmoay Oy Hmoyycmow mums maOHyHOCOU .wammsm «m4 may yo Hm>osmy Hmymm mmyscye oa .mo mmmmyoafl am Omsoam mymy aowymywmmamyy mas .aoHysHom 4mm may CH omaHmEmy mama Omymmuy Hmayo maB .mamasm 4mm may mo Hm>oemy coma mama may mo mymu aoflymyymmamuy may mymoyoaw mmamcmwyy ammo maa .mmmaymyo awayymm cam aOHyOHflO Omymmmmy ma aOnyHOm mayymmyyyy may Eoym om>OEmy mm3 «ma may mBOyHm may ha omymoyoay mEHy may yd .Hoamaym wmoo.o maaaymyaoo aomm Ammaoyyo ammov Hmym3 omaayymwo HO Ammaoyyo ommOHOV «mm Shuoa Hmayym ayys Oymu mEyy ya omymmyy mym3 mm>mma amayma mHmEyum ommyoxm mo mmomaa may mawymmyyuy aoyysaom may .Cyom OymyomamnammvlmCMHy.mHO mo Hm>OEmH mam aoyywoom may coma mmymy aowymuymmamuy MO mmmamaorr.m mysmym 50 ([14 3111p 03:45) uonmgdsuml “to .13 .E. om .. “”5 i-"-' . 0 ° -—0 —.lfi 1 l m N .— 51 Figure 10A.--Changes in water loss, net C0 uptake and leaf temperature with time for a primarg barley leaf supplied with 14C-a carbon labeled 10‘ M cis,trans-(RS)-AEA at time zero. At the time indicated by the arrow the leaf was removed and ex- tracted. AH 0 is the difference in water vapor concentrations in the air flow into and out of the chamber. ACO is the difference in C02 concentra- tion between hese two air flows. The units for the two are arbitrary and not related. TL is the leaf temperature. Conditions identical to those described for Figure 3. Figure lOB.--Thin-layer chromatogram of the total extract from the leaf described in Figure 10A. 52 UL mkaHOm may ymay mmymuymCOEmo mHaB .amdumamyy.mHo mo ammm HmmymH oumoamym may no ommam mmmymsm Aamdlmamuy.mcmyy Oy monommmuuoo aOHa3 mo mOHm> mm mayv ammm 3m: m .yamHH >D mo GHE H cho Hmywm .mH musmHm OH omaHHOmmo EmymMm may aH cmay .hH mysmHm CH omaHHOmmo Emymmm ycm>Hom may aH ymHHu wHHmHyamsomm ommOHm>mo mm3.fimymoymaouao mas .80 H m0 mocmymHo m ym AMHCHOMHHmU .HmHHamu cam ..OGH .myuseoee ymHoH>umuyaev mamH HH.m>: yemHHmumeHz “mOHOOm yamHH .yamHH >5 ast mysaHE H you omymHomu :yH amma oma aOHa3 amdnoeH mo aOHyDHOm OHHoamaymE a mo Emymoymfionao Hm>MHIaHaBII.mH musmHm 67 0.5 to v :0 N ,-o|xwdo (.0 68 Figure l7.--Thin-layer chromatogram of a stock methanolic solution of 14C-ABA developed in the initial solvent system used to clear the plates Of chlorophyll and other pigments. Solvent system used was hexane-ethyl acetate, 1:1 v/v. cpm x (0'2 (5 I0 69 V I I V I T T I '4c-ABA STANDARD 70 Figure 18.--Thin-layer chromatogram of a methanolic solution of 14C—ABA developed in solvent system: benzene:ethyl acetate acetic acid, 50:5:2 v/v. cpm 2000 I500 500 71 '4c-ASA STANDARD :- -I ('1 t . 1 d I J . - A .1 I I , I - 0 5 IO 72 capacity to sequester ABA into an inactive compartment. This is the simplest hypothesis which can account for the reversion of the short-term ABA response upon removal of the ABA supply. The Observed low rate of ABA metabolism cannot entirely be responsible for the reopening Of the stomates. Does ABA Affect Photosynthesis Directly? Figure 19 shows the change in computed [C0211 and r320 after cis,trans-(R,S)-ABA treatment. [C0211 decreased as the stomata closed. This indicates that the photo- synthetic C02 fixation mechanism remained functional after the stomata started to close. That is, an effective sink for CO2 remained. On the other hand when DCMU, an in- hibitor Of photosynthesis,was added to the irrigating solution (Figure 20) the stomata also closed with a com- parably short lag. In this case, however, the [C02]i increased during closure. Since it is known that increased external C02 concentrations will cause stomatal closure, (Linsbauer, 1916) and since DCMU does not cause closure of stomata on isolated epidermal strips of y, £323 leaves, in C0 -free air (Humble and Hsiao, 1970), one may conclude 2 that DCMU probably causes closure in the whole leaf through changes in internal C02 concentration. Since ABA treatment at 10-7 M did not cause [C0211 to rise (it actually fell) one may conclude that ABA prob- ably does not effectively inhibit photosynthetic C02 73 Figure l9.--Changes in rHZO and [C02]i (in ppm or ul 1'1) following addition at time zero of water (control leaf) or of ABA (10‘7M final con- centration) to the distilled water irrigating primary barley leaves. E 245 Foali.._._o_.e_._n_n—-o—o-1— “'235» - I § 4 .rHZO ._._._._._._._o—'—4-O— 250~ ' ' l A 240_ $021” W’O 3.2.230“ \ 220» I?“ 2"; ABA TREATED LEAF g I IO'7 M gr: 7 I. ... E 5‘ / 0 O 5 b g 4 " r J H 0 0 3 2 . . . . 74 -l5 0 IS 30 TIME (min) 75 Figure 20.--Changes in rH 0 and [C0211 (in ppm or ul 1‘1) following addition at time zero of DCMU (10'5M final concentration) to the distilled water irrigating primary barley leaves. Both the DCMU and the so-called H 0 additions contained equal concentrations Of the so vent used to dissolve the DCMU. 77 fixation (at least in meSOphyll cells) and therefore ABA most probably acts more directly on the stomatal mechanism. Since ABA does cause closure it is only by limiting the supply Of C0 to the photosynthetic tissues that it ef- 2 fectively decreases C02 fixation. It is interesting to note (Table 5) that ABA treat- ment caused decreased transpiration with comparable effec- tiveness in leaves exposed to C02-free air as in leaves exposed to air containing 300 ul 1'1 C02. Experiments with a Wilty Mutant Of Tomato In an effort to further demonstrate that the ABA- induced closure is independent of changes in C02 concen- tration, several experiments were carried out using flacca, a wilty mutant of tomato. The stomates of flacca do not respond to many stimuli which cause either closure or opening in the wild-type. Neither darkness, wilting, guard cell plasmolysis, nor treatment with phenyl mercuric acetate cause closure Of flacca stomates (Tal, 1966). On the other hand, ABA is reported (Imber and Tal, 1970) to cause stomatal closure in excised flacca leaves. Figure 21 shows the changes in transpiration rates when the C02 concentration of the air flowing over the leaves was first increased from 270 to 470 ul 1.1 and then decreased to 0 ul 1'1. Only the wild-type leaves responded. The very slight and abrupt changes in the apparent 78 Figure 21.--Changes in calculated trans- piration rates in excised flacca and wild-type leaves of L. esculentum cv. Rheinlands Ruhm fol- lowing chafiges in concentration of C0 in the air flowing over the leaves. At 21 min, first arrow) the C02 concintration was increased from 270 ul 1'1 to 470 ul 1’ . At 36 min, (second arrow) the C02 supply was stopped and COZ-free air flowed over the leaves. Both leaves were of nearly identical size and shape taken from analogous positions on 80-day- Old plants. No correction was made for air flow rate changes resulting from changing rate of in- jection of C02 into the COZ-free air supply. 79 I I I I l400h M - TE flacca ‘1’ g IOSO- 4 O N I E 700- - c .9 ‘2' "' 350- 8’ COz-free air + c E 3. O l l l I 0 (0 20 30 40 60 Time (min) 80 transpiration rate of the flacca leaves arose as an arti- fact due tO the changes in rate of air flow past the leaves when C02 containing air was added tO or withdrawn from the system. These slight changes in air flow were not taken into account when the transpiration rates were calculated. The lack of response to C02 of the flacca leaves is understandable since flacca stomates respond to neither light nor darkness (Tal, 1966). Having established that CO2 did not affect stomata of flacca, I then tried to demonstrate a response to ABA treatment. As shown in Figure 22, only the wild-type stomates responded to ABA treatment. The flacca leaves showed no change in transpiration within 40 minutes of treatment with either 10"6 or lO-SM ABA. The reported (Imber and Tal, 1970) closure of stomates in excised flacca leaves was Observed only in darkened leaves whereas in experiments reported here both flacca and wild-type leaves were illuminated equally. Imber and Tal do not report on experiments to show stomatal closure in flacca leaves in the light, nor on ABA responses that occur earlier than 24 hours after start of the treat- ment. It seems that the reported closure of flacca leaves following long-term ABA treatment is a phenomenon quite different from the rapid responses Observed with the wild- type leaves. 81 Figure 22.--Changes in calculated transpiration rates with time for leaves excised from flacca or wild- type tomato, following addition of ABA to the distilled water irrigating the leaves. At 18 min ABA was injected into irrigating water to give a final concentration of 10'6M. At 43 min the ABA concentration was raised to 10'5M. The air supply flowing over each leaf contained 295 ul‘l C02. Comparable-sized leaves were excised from analogous positions on 80-day-old plants. Trans- piration rates were calculated for lower leaf surface only. Transpiration (mgm H20 dm"2 hr") 82 T T f I I 2|OO~ " flacca moo , . . . . .. wild-type1 . ' . IO‘5M ’ 700'. ABA It, q IO M ABA O 1 l l l 1 0 IO 20 30 4O 50 60 Time (min) 83 These experiments suggest that flacca leaves would be an excellent system to study rapid physiological re- sponses in leaves which are uncoupled from known stomatal controls. Since the guard cells do not respond to plas- molysis (Tal, 1966), flacca may be a morphological mutant with relatively inflexible guard cell walls. One other very interesting Observation was made during these investigations Of flacca. ABA treatment at 10'6M or lO-SM did not cause any change in the rate of C02 uptake in excised flacca leaves. If the mutation just renders the stomates non-functional, this is quite good proof that ABA does not inhibit photosynthetic C02 fixation: and that ABA normally causes stomatal closure by acting directly upon the guard cells. ABA Action on Stomates in Isolated Epidermal Strips The response of stomates in epidermal strips from V. fgpg leaves was investigated in an effort to demonstrate more directly an effect of ABA on the stomatal system. Strips were isolated from the leaves as described in the methods section; they were floated under illumination on buffered solutions containing CaClz, various concentrations Of KCl and were aerated with humidified C02-free air. After two hours, when the stomates had Opened, some strips were transferred to solutions identical to those on which they had been floated before but containing 10'6M ABA. 84 Figure 23 shows the stomatal aperture on such strips after the strips had floated for one hour on ABA solutions. Apparently stomates that Opened over solutions of high K+ concentration did not close as a result of ABA treatment. Those that Opened over solutions of low K+ concentration (1—10 meq liter'l) did close in response to ABA. Since the strips were prepared in such a manner that at least 80% of the non-stomatal epidermal cells were ruptured, it is reasonable to conclude that ABA causes closure by a direct action on the guard cells and not on the epidermal cells. E, §3p3_leaves were chosen as a source for epidermal strips because they are easily pre- pared and the system is well described (Humble and Hsiao, 1970). Attempts were made at treating epidermal strips from corn and barley in the same manner. However, the stomates from such strips did not stay open in either ABA- treated or control strips for long enough periods to show any differences in aperture due to ABA treatment. The 2, £222 stomatal system is quite different from that Of H. vulgare. The former does not have the well defined subsidiary cells of the latter, but does have larger and slower-moving guard cells which are apparently more resistant to the destruction that occurs when the strips are peeled from the leaf. With these points in mind, one cannot immediately transfer conclusions arising 85 .ooHymm yamHH may yo ysoa ymmH may mcHyso «ma ZOIOH oy ammomxm mums aOHaB mmHuym co mmymEoym mo myayummm mmymoHoaH maHH amaoya mas .amd ysoasz chHyOHOm co omymOHm mmHyym no musyymmm mmyMOHoaH maHH OHHOm mas .HUM mo maoHymyyamoaOO mOOHHm> maHaHmycoo mGOHyOHOm ao yamHH may cH mnsoa m you omyMOHm mums hmay Hmywm mmHHym HmEHmOHmm co musyummm HmymEOymrl.m~ mHOmHh 86 r... amp: .ucOu+¥ oo— o— — o jli I'll—III J j .T m T e ““‘ F \ L a M\\\( :th— F b amusdv (suongw) 87 from epidermal strip studies in V, fepa_to responses found in intact excised leaves of barley. Nonetheless the anal— ogy is presented. Stomates on strips floated on solutions of high K+ concentration Open even in the dark (Humble and Hsiao, 1970). Light facilitates opening predominantly at lower + concentrations. K It seems that ABA may reduce the aperture to the same degree that light increases aperture. If this proves to be correct then ABA may be inhibiting the same process which causes opening when activated by light. DISCUSSION AND CONCLUSIONS The results have shown that ABA applied to barley leaves causes very rapidly stomatal closure, and that the rapidity of the response is a function of the inverse of the concentration of ABA applied. This is the most rapid response to ABA that is known. Mittelheuser and van Steveninck (1971) have recently shown closure to occur within 10 minutes of application Of 3.8 x 10-6M ABA to barley leaves. Jones and Mansfield (1970) reported closure within 30 minutes after treatment of excised tobacco leaves with 10’4M ABA. Warner and Leopold (1971) have recently shown that treatment of individual pea plants SM ABA resulted in a decrease in the growth rate with 10' which started 5.1 min after treatment. Loveys et 31. (1971) have reported results regard- ing the rapidity and specificity Of the ABA-induced stoma- tal closure in leaves from several species. They also determined that the ABA content needed for closure was Of the same order as the endogenous levels. Such rapid reSponses to ABA treatment indicate that ABA may be involved in the inhibition of discrete 88 89 enzyme systems which are involved in maintaining stomatal aperture. Gene repression by ABA seems to be unlikely since the lag for the ABA—induced response is shorter than 2 min (after transport lag is deducted), and since the typical time for assembly of a complete protein molecule in higher organisms is estimated to be 5 min (Goodwin, 1963; also see discussion by Evans and Ray, 1969). The lag time might be shortened even more. Since there is a concentration dependence of the lag time, a diffusion process may be involved in the involvement of ABA to its site of action. Possibly this diffusion step could be reduced if leaves were immersed in a solution and the stomates continuously examined microscopically after the addition of ABA to the solution. Since the response is so rapid, ABA is probably not acting by a general enhancement of senescence such as enhanced degradation of cellular constituents. There is probably no irreversible damage done to the stomatal system by the ABA treatment since removing the supply of ABA results in a reOpening of the stomates. If ABA is the agent responsible for hydroactive closure, this is very significant since leaves do recover from hydroactive closure with time. The reversal of the closing response upon removal of the ABA supply cannot readily be explained on the basis of metabolism of ABA. In leaves where reversal of 90 l4C-ABA from the response was observed after withdrawal of the medium, at least 90% of the radioactivity was still associated with unchanged ABA. Some other mechanism, such as compartmentation of ABA must then account for the reversal. The closure may be reversed by an adaptation of the guard cells to the presence of low concentrations of ABA. The Opening may also reflect an action of ABA on the other epidermal cells. If ABA treatment also caused a loss of turgor of the epidermal cells the stomates might partially open by a hydro-passive response. This is not likely, however, since continuing the supply of ABA to the transpiration stream did not result in reOpening. The long-term closure caused by water stress probably reflects a continued production of ABA by the leaf. It is not known when ABA synthesis ceases after a water stress has been relieved. After 2 hr and 25 min exposure to ABA there was significant metabolism of ABA as indicated by partitioning followed by TLC. Thirteen percent of the radioactivity did not partition into the acidic fraction. Most of this radioactivity was found in the hydrolysate fraction indi- cating possible esterification of ABA. Of the radio- activity which partitioned into the acidic phase 75% co- chromatographed with ABA. The remainder moved to a Rf characteristic of the hydroxylated ABA (a precursor of 91 phaseic acid as described by Milborrow, 1970). As judged by partitioning and TLC, approximately 66% of the radio- activity was still present in the form of cis,trans-(RS)- ABA. Esterification of an aliquot of the acidic phase followed by TLC again showed the majority of the radio- activity to move as cis,trans-(RS)-ABA methyl ester. This is quite good evidence that no further metabolism had occurred. No attempt was made to differentiate be— tween the amounts of the (+) and (-)-isomers metabolized. Milborrow (1970) showed that in tomato the (-)—isomer is metabolized more rapidly than the (+)-isomer. The demonstrable metabolism of ABA within 2.5 hr after its application, suggests that ABA may not be useful as an antitranspirant. It may be metabolized too quickly. However, the use of repeated applications of ABA or of analogs of ABA which show activity but are not as easily catabolized may prove practical. 43 This work has also shown that the stomatal appara- tus responds specifically to cis,trans-(+)-ABA. The re- sponse to trans,trans-(RS)—ABA is significantly lower and may even be entirely accounted for by contamination of the preparation with the cis,trans-isomer. This latter point could be demonstrated better if samples with fewer impurities were available. However, it is very difficult to separate the two isomers with greater efficiency except by preparative GLC. It is also possible that some 92 isomerization occurs in the leaf after treatment with trans,trans-ABA to yield cis,trans-ABA. However, this appears less probable for two reasons: 1. ABA absorbs very little light energy at wave lengths above 300 nm (see UV spectral curve pub- lished by Milborrow, 1970). The absorption peak is at 240-260 nm, depending on the degree to which ABA is dissociated. In the experiments described here, the leaves were always shielded with glass filters which blocked the transmission of short wave-length irradiation. 2. In eXperiments using radioactive cis,trans-(RS)- ABA there was no detectable isomerization after eXposing the leaves for 2 hr and 25 min to the labeled preparation either in the light or in darkness, whereas such isomerization was detected after exposing methanolic solutions of the labeled hormone to UV light for 1 minute (Figure 18). The optical isomer of the naturally occurring cis,trans-(+)-ABA showed very little activity in closing 5 stomates. Only at 0.8 x 10- M did it cause a slight change in leaf temperature which started 12 min after treatment. It had no activity at lO-6M. This result was surprising since Milborrow (1968) claimed that both optical isomers were biologically active. It is not clear from 93 Milborrow's report, however, in which assay system and at which concentration the activity of the cis,trans- (—)-ABA was tested. Sondheimer et 31. (1971) claim that the activity of the (-)-isomer was either equal to or less than that of the naturally occurring (+)— form in inhibiting the germination of barley seeds and the growth of roots and shoots. These assays involved 56 hr treatments of the seeds with the (+) and (—)— ABA preparations. It appears then that the short-time inhibition of transpiration is very specific for the (+)-isomer and that the (-)-isomer is much less effec— tive in initiating this fast response. The (-)-isomer possibly acts only during long periods of incubation. The relatively low effectiveness of the ABA analogs in causing stomatal closure indicates that the response is very specific and not caused by a trivial mechanism such as a lowering of the pH of the solution in the trans- piration stream. The high degree of specificity of the response to cis,trans-(+)-ABA also means that the re- sponse is very likely of physiological significance and not a pharmacological phenomenon. This is especially significant since it is the cis,trans—(+)—ABA which occurs naturally in plants and which is synthesized in leaves in reSponse to water stress. 94 There must be highly specific receptor sites, probably in (or on) the guard cells which recognize ABA and are involved in translation of the ABA stimulus to give the observed response. The results also indicate two novel methods for differentiating between direct effects of "anti-trans- pirant" preparations either on the stomatal apparatus or on the photosynthetic mechanism. The first method in- volves the determination of changes in the [C02]i after treatment of excised leaves. ABA treatment caused de- creased levels of [C02]i with stomatal closure, whereas DCMU treatment caused an increase in [C02]i while the stomates closed. This has been interpreted as meaning that ABA treatment caused the stomates to close without affecting the C0 -fixing mechanism of the leaf, while 2 DCMU treatment caused closure because it inhibited photo- synthetic CO -fixation and thereby allowed [C02]i to 2 increase sufficiently to effect closure. The second method involves the use of the wilty mutant flacca, the stomates of which do not respond to increased CO levels nor to ABA during short-time experi- 2 ments. By monitoring the C02 assimilation rate in the mutant after treatment with a potential "anti-transpirant ll preparation, it is possible to monitor effects on the ability of the leaf to assimilate CO in the absence of 2 stomatal movement. ABA treatment did not change the 95 assimilation rate at all in this mutant. It would be interesting to investigate the effect of photosynthetic inhibitors on flacca. It would also be important to understand why ABA does not cause stomatal closure in flacca in short-term experiments, yet apparently does during extended exposure to ABA (Imber and Tal, 1971). Mittelheuser and van Steveninck (1970) monitored rH20 and photosynthetic CO2 fixation rates in leaves as a function of time following application of ABA. C02 fixation was measured by supplying the leaves with air containing 14C02 and determining the amount of radio- activity assimilated. They concluded that closure of stomates in response to ABA treatment preceded a decrease in the rate of CO2 fixation. These results support the ones described in this thesis insofar as they show that ABA treatment causes closure without inhibiting the photo- synthetic mechanism. However, it is very difficult to understand how the results were obtained since closing of the stomates restricts the supply of C02 to the photo- synthetic mechanism. This should, therefore, cause de- creases in the net rate of C02 uptake. The interpretation of these results is made difficult by the fact that the determinations of stomatal aperture and C02 fixation were carried out on different sets of leaves. Even in the leaves which were not supplied with ABA, the rate of CO2 fixation varied considerably. It is concluded that the 96 gas analysis method coupled with [C02]i determinations yields more conclusive evidence concerning the lack of a direct effect of ABA treatment on the photosynthetic apparatus. The results obtained after ABA treatment of isolated epidermal strips also suggest that ABA acts directly on the guard cells. Horton (1971) and Tucker and Mansfield (1971) have reported similar results show- ing that ABA inhibits stomatal opening in epidermal strips. Horton (1971) also showed that stomates of ABA-treated strips would Open when the strips were transferred to solutions which did not contain ABA. Therefore, the response in strips is also reversible. Since the epi- dermal cells on such strips are disrupted, it can be con— cluded that the site of ABA action is localized at the guard cells. Humble and Hsiao (1970) have shown that light activates the influx of potassium into guard cells as the stomates open. Humble and Raschke (1971) demon— strated that the guard cells of open stomates contain a twenty-fold higher content of potassium than guard cells of closed stomates. They also postulated that potassium is the predominant cation involved in the maintenance of turgor pressure of open stomates. Since the ABA response is so fast, it is reasonable to postulate that ABA may be involved in the regulation of potassium transport in guard cells. ABA could cause closure by one of three mechanisms: 97 l. Inhibiting a potassium pump. 2. Rendering the guard cell membrane(s) more leaky to potassium (or ions in general). 3. Interfering with anion production in the guard cell. The movement of leaflets in Albizzia have many features in common with stomatal closure. Such movements result from the differential turgor changes in dorsal and ventral pulvinule cells which are attributable to potassium ion fluxes. ABA treatment promotes the closure of excised pulvinules (Satter and Galston, 1971). ‘ ABA treatment (Glinka and Rheinhold, 1971) apparently increases the permeability of carrot root cells to water. This is the only reported direct effect of ABA on membranes. It is hard to see how increased water permeability of guard cell membranes would cause closure, unless this observation reflects a general increase in permeability of the membrane which allowed increased efflux of potassium ions as well as water from the guard cell. 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