“51’ - m. um'w . .. -...-!r.r."-~::'=‘- ..- “may MWJJ MSU .’}. . __ -‘-‘-.> A I...“ , _ RETURNING MATERIALS: Piece in book drop to nwyfymlafménwm'aq LIBRAIUES remove this checkout from "In. your record. FINES will be charged if book is returned after the date stamped be1ow. '-u.‘ :39“. “‘99; V‘ 1' ‘ V \ I 5' ,3 .3 ask! I‘" ' F TURGOR DEPENDENCE OF BIOSYNTHESIS AND METABOLISM OF ABSCISIC ACID By Margaret Lee Pierce A DISSERTATION Submitted to Michigan State University In partial fulfillment of the requirements for the degree of DOCTOR OF PHILOSOPHY Department of Botany and Plant Pathology 1981 l‘//,( (" ABSTRACT TURGOR DEPENDENCE OF BIOSYNTHESIS AND METABOLISM OF ABSCISIC ACID E" By MARGARET LEE PIERCE The relation between leaf water potential (wleaf) and steady-state level of ABA was determined for mature leaves E of Phaseolus vulgaris L., Xanthium strumarium L., and Gossypium hirsutum L. Leaf sections from any one leaf covered a range of known wleaf values, turgors (p), and osmotic pressures (n). At p=l bar, ABA content averaged A times the level found in unstressed samples. Below p=l bar, ABA content increased sharply to as much as 40 times the level found in turgid samples. Leaves from stress—condi- tioned cotton plants, having higher n, required lower wleaf in order to accumulate ABA than did leaves from previously unstressed cotton plants. Thus, turgor, rather than wleaf or n, appears to be the critical parameter of plant water relations which controls ABA production in stressed leaves. Stomatal closure occurred in leaves of Commelina CCNnmunis L. before an increase in ABA was obvious in the lfiaf as a whole, but a gradual increase, of 50—100%, in the lfiivel of ABA in the epidermis paralleled stomatal closure. Tfiie hypothesis that ABA helps cause stress—induced stomatal Margaret Lee Pierce closure is tenable, based on a comparison of these results with estimates of stomatal sensitivity to exogenous ABA. Loss of turgor in detached leaves of P. vulgaris was correlated with accumulation, not only of ABA, but also of its metabolites, predominantly phaseic acid (PA). Upon re— hydration the rate of synthesis of ABA drOpped to zero with- in about 3Eh, while conversion to PA accelerated; it reached a rate sufficient to convert almost % of the ABA present in the tissue to PA within 1 h. In contrast, the alternate route of metabolism of ABA, synthesis of conjugated ABA, was not stimulated by rehydration. Conversion of ABA to PA was accelerated at the slightest indication of a regain of turgor. Thus, the opposing processes of synthesis and metabolic removal of ABA appear to be linked to loss of turgor on the one hand and recovery of turgor on the other. In memory of Mr. Karl N. Pierce, my grandfather ii .5..-‘_.-- - - - ACKNOWLEDGEMENTS For friendship, scientific counsel and assistance, I thank especially Andrew Mort, Anita Klein, Tom Sharkey, and the members of my committee Norm Good, Ken Poff, Jan Zeevaart, and Klaus Raschke. A special thanks goes to Jan for taking over chairmanship of my committe, and to Klaus for his unflagging support. iii The research reported in this thesis was supported by the U.S. Energy Research and Development Administration and the U.S. Department of Energy under Contract EY-76- C-O2-l338. iv TABLE OF CONTENTS List of Tables List of Figures List of Abbreviations Chapter 1. General Introduction 1.1. Introductory remarks 1.2. Biosynthesis and metabolism 1.2.1. Precursors and products 1.2.2. Sites of synthesis and metabolism 1.2.3. Distribution 1.3. Factors with cause accumulation of ABA in leaves 1.3.1. Wilting 1.3.2. Interruption of transloca— tion in the phloem 1.3.3. Other factors 1.A. The possible involvement of ABA in resisting water stress 1.4.1. Regulation of stomatal aperture 1.4.2. Other possible functions 1.5. Statement of purpose Chapter 2. Correlation between loss of turgor and accumulation of abscisic acid in detached leaves V Page xi xiv (ID—1:2“) ll 11 13 15 16 16 21 23 25 2. 2. Introduction Materials and methods 2.2.1. Plants 2.2.2. Extraction and purification of abscisic acid 2.2.3. Methods of measuring leaf water status (i). Pressure—bomb method (ii). Dew-point method 2.2.4. Preparation of samples of known wleaf’ fl, and p 2.2.5. Considerations for sampling 2.2.6. Sample incubation Results 2.3.1. The relationship between wleaf’ TI, p3 and accumulation of ABA 2.3.2. The effect of elevated N2 pressure on accumulation of ABA by wilted leaves Discussion 2.4.1. Leaf w and w influence production of ABA through their effect on turgor 2.4.2. The question of a "threshold" w leaf accumulation of ABA for 2.4.3. How is accumulation of ABA related to turgor? 2.4.4. How reliable are the determinations of turgor? 2.4.5. Implications for stomata of vi Page 26 29 29 3O 32 32 33 36 37 38 45 45 53 59 59 6O 61 65 66 2.4. 2.4. 6. 7. the observed relation between accumulation of ABA and leaf turgor Implications of the results with stress-conditioned g. hirsutum "Incipient wilting" and the accumulation of ABA Chapter 3. Measurements of ABA in epidermis of Commelina communis during stomatal closure caused by water stress 3. 3. 3. l. 2. 3. Introduction Materials and methods 3.2. 3.2. Results 3.3. l. 2. 1. Plants Preparation of leaf tissue wilted in air Preparation of leaf tissue wilted in solution Analysis for ABA Estimation of guard cell potassium content Measurement of leaf water potential Abscisic acid in Commelina epidermal tissue Decline in wleaf’ accumulation of ABA, and stomatal closure Loss of potassium from guard cells and stomatal closure Accumulation of ABA in tissue which was wilted in solution vii Page 68 7O 72 73 78 78 78 79 80 82 82 83 83 86 98 103 Chapter 4. 3.4. Discussion 3.4.1. 3.4.2. 3.4.3. 3.4.4. The level of ABA prior to stress-induced accumulation Changes in stomatal aperture and ABA content related to decline in w leaf Stomatal closure and accumulation of ABA by epidermis Accumulation of ABA by tissue in solution Synthesis and metabolism of abscisic acid in detached leaves of Phaseolus vulgaris L. 4.1. 4.2. 4.3. after loss and recovery of turgor Introduction Materials and methods 4.2.1. 4.2.2. 4.2.3. Results 4.3.1. 4.3.2. 4.3.3. 4.3.4. Plants Experimental procedure Analysis for abscisic acid and its metabolites Accumulation of ABA and its metabolites as a function of leaf water deficit Identification of metabolites of ABA in bean leaves during recovery from water stress Accumulation of ABA and its metabolites as a function of time during and upon recovery from water stress Conversion of ABA to PA as a function of degree of rehydration viii Page 109 110 110 115 120 121 123 123 123 127 132 132 135 140 150 Conclusion Bibliography 4. 4. Discussion 4.4.1. Synthesis and metabolism of ABA after loss and gain of turgor 4.4.2. Stimulation of ABA synthesis after loss of turgor 4.4.3 Stimulation of ABA metabolism after recovery of turgor ix Page 161 161 161 163 166 168 LIST OF TABLES The effect of elevated N pressure on accumulation of ABA by wilted leaves. Characteristics of Commelina communis leaves. Observations during incubation of epidermal strips of Commelina leaves in osmoticum Effect of wilting and rehydration on the average rate of accumulation of PA in samples from individual, detached leaves of Phaseolus vulgaris . . . . . . . . . Page 57 78 . 108 151 hi LIST OF FIGURES Figure Page 1-1 Pathways of ABA metabolism. . . . . . . . . . . 6 2-1 A "pressure-volume curve" determined for a Gossypium hirsutum leaf . . . . . . . . . . . 35 2-2 Evidence for uniform wleaf’ n, and p for different positions in leaf blades. . . . . . 40 2-3 Time course of accumulation of abscisic acid in detached, water-stressed leaves of 3 species . . . . . . . . . . . . . . . . . . . 43 2-4 The effect of leaf water potential on abscisic acid content in single, detached leaves of Xanthium strumarium, Phaseolus vulgaris and Gossypium hirsutum . . . . . . . 47 2-5 The relationship between osmotic pressure (n) turgor (p), and leaf water potential, and the effect of wleaf on abscisic acid content in samples from a single, detached leaf of Xanthium strumarium . . . . . . . . . 50 2-6 The relationship between turgor and leaf water potential, and the effect of wleaf on abscisic acid content in two leaves of Phaseolus vulgaris. . . . . . . . . . . . . . 52 2-7 Abscisic acid content as a function of turgor in single, detached leaves of Xanthium strumarium, Phaseolus vulgaris, and Gossypium hirsutum. . . . . . . . . . . . . . 55 2-8 The effect of wleaf on ABA levels; a comparison of data redrawn from Wright (1977) and Zabadal (1974) . . . . . . . . . . 63 3-1 Increases in the level of abscisic acid in Commelina communis abaxial epidermis and the remainder of the leaf ("mesophyll") xi. Figure Page due to the wilting of intact leaves. . . . . 85 3-2 Accumulation of ABA in the lower epidermis and the rest of the leaf ("mesophyll") of Commelina communis during dehydration of intact leaf sections . . . . . . . . . . . . 88 3—3 A series of GLC—EC chromatograms indicating presence of ABA in purified extracts from Commelina communis epidermis . . . . . . . . 90 3-4 The decline in wleaf with time after removing sedfions of Commelina communis leaves from water. . . . . . . . . . . . . . 92 . —l M 3-5 The relation between -wleaf and m of original fresh weight for Commelina communis during dehydration of intact leaf sections . . 94 3-6 Stomatal closure and accumulation of abscisic acid in the lower epidermis of Commelina communis during dehydration of intact leaf sections . . . . . . . . . . . . . . . . . . 97 3-7 Stomatal aperture versus level of ABA in abaxial epidermis during dehydration of intact leaf sections of Commelina communis . 100 3-8 Correlation between stomatal aperture and relative potassium content of guard cells during stomatal response upon removal of Commelina leaf sections from their water supply . . . . . . . . . . . . . . . . . . . 102 3-9 Accumulation of ABA by mesophyll tissue of Commelina during osmotic stress. . . . . . . 105 3-10 The effect of osmotic stress on the level of ABA in Commelina leaf epidermal tissue . . . 107 4-1 Decrease in wleaf with loss in fresh weight from a leaf of Phaseolus vulgaris. . . . . . 125 4—2 The relationship between turgor ( ----- ) and wleaf’ and the effect of wleaf on the content of ABA, PA, DPA, and conjugated ABA in samples from a single, detached leaf of Phaseolus vulgaris . . . . . . . . . . . . . 134 xii Figure 4-3a-c 4-4 4-5 4—6a,b 4-7 4-8 4-9 HPLC elution profiles of radioactivity from leaf sections of Phaseolus vulgaris. Changes with time in content of ABA in sections of a single, detached leaf of Phaseolus vulgaris during water stress (——) and recovery (—--) Changes with time in content of PA (A) and DPA (0) in the same leaf sections that were analyzed for ABA content (Figure 4-4) during water stress (———) and recovery (--- . . . . . . . . . . . . . . . . . Effect of wilting (—-) and rehydration (-——) on the average rates of synthesis (A) and metabolism (0) of ABA during intervals of time after loss of turgor in samples from a single, detached leaf of Phaseolus vulgaris . . . . . . . . . . . . A comparison of duplicate determinations on the same leaf of the relationship between wleaf and fresh weight . . . . . . . . Measurements of wleaf’ which were determined with a pressure bomb, versus % of the leaf's original fresh weight compared to measurements of wleaf on sections of the same leaf, which were made by the dew- point method, versus the % to which those sections were rehydrated . The effect of degree of rehydration on the change in content of ABA, PA, and conjugated ABA in sections of a single leaf of Phaseolus vulgaris during a 3 h period of recovery from water stress xiii 143 145 148. 154 156 159 Bear-rum “a 1'. 1r ABA (:)-ABA Me-ABA DPA epi—DPA PA HEPES MES PVP-40 Bq Dw Fw MPa GLC-EC HPLC wleaf LIST OF ABBREVIATIONS Abscisic acid Racemic mixture of abscisic acid Abscisic acid methyl ester Dihydrophaseic acid epi—Dihydrophaseic acid Phaseic acid N-2—hydroxyethylepiperazine—Nl-2- ethanesulfonic acid 2—(N-morpholino)ethanesulfonic acid Polyvinylpyrrolidone Becquerel, one disintegration per second Dry weight Fresh weight Megapascal, 10 bar Gas liquid chromatography with electron capture detector High performance liquid chromatography Water potential Leaf water potential Osmotic pressure Volume-averaged turgor Volumetric modulus of elasticity xiv Chapter 1 General Introduction 2 1.1. INTRODUCTORY REMARKS Abscisic acid (ABA) has become established over the past 15 to 20 years as one of the major classes of plant growth substances (see reviews by Addicott and Lyon 1969, Milborrow 1974a, Wareing 1978, Zeevaart 1979, Walton 1980). Usually, ABA is classified as a growth inhibitor because it behaves as one in a number of bioassays, counteracting the effect of growth—promoting substances: auxins, gibberellins, and cytokinins (Wareing 1978). Numerous physiological roles have been postulated for ABA, including, but not limited to, (l) a contribution to geotropism in roots, (2) a contribu- tion to seed dormancy, especially in cases where the embryo is innately dormant (Wareing, 1978), (3) induction and maintenance of bud dormancy, (4) promotion of tuber formation, and (5) promotion of stomatal closure during water stress. Recently Nareing (1978) and Walton (1980) have critically reviewed the evidence concerning the role of ABA in these processes and concluded that we do not know enough about the effects of ABA on plant cells or the nature of the individual processes to state unequivocally in any case the degree of involvement of ABA. Possibly the most compelling evidence of a functional role for ABA comes from studies on the physiology of water-stressed plants; this evidence is summarized in Section 1.4. This theshsis an account of a further investigation of the regulation of biosynthesis and metabolism of ABA in water-stressed leaves and the regulation Ir of stomatal aperture by ABA. 4 1.2. BIOSYNTHESIS AND METABOLISM OF ABA 1.2.1. Precursors and products. The immediate precursors of ABA (I), a sesquiterpenoid, are unknown. (H' Abscisic ocid Milborrow and Robinson (1973) demonstrated the incorporation of label from mevalonate into ABA in different parts of plants, including seeds, stems, leaves, and fruits. There are basically two pathways which can account for the derivation of ABA from mevalonate: 1) direct incorporation of three isoprene units, derived from mevalonate, into ABA and 2) indirect incorporation of isoprene units into ABA via carotenoids. The bulk of the evidence, mostly from experiments on avocado fruit, has favored the first of the two pathways (Milborrow 1974a). Abscisic acid is metabolized predominantly Via 6'- hydroxymethyl-ABA, an unstable intermediate, to phaseic acid (PA). In many plant tissues, PA is further metabolized to 4'-dihydrophaseic acid (DPA), which appears to accumulate as an end product. Evidence has been presented that ABA is metabolized by this route (see Figure 1-1) in ash seeds (Sondheimer et a1. 1974), in endosperm of immature fruits of Echinocystis (Gillard and Walton 1976), in excised bean roots Figure 1-1. Pathways of ABA metabolism. The conversion of ABA to PA occurs via the unstable intermediate 6'—hydroxy- methylabscisic acid: cuppa \ .. \ \ . \ \ OH co," w o 3 0H C02“ d ”0"" OH C02H N ABSCISIC ACID PHASEIC ACID DIHYDROPHASEIC ACID (ABA) CPA) (DPA) “r n_°\;/o\c ¢° "Kai—r C+>~ABA-B—GLUCOSYL ESTER Figure 1-1 ,. .. ”dud ‘t .. ooh , . ’- ..- .-, J ... .r. . .-.. :1 r O; ,- __ .. I. 'v. ‘- .. . V4 3 ,r - 7 (Walton et a1. 1976), and in the shoots or leaves of a number of species (Harrison and Walton 1975, Zeevaart 1977, Tietz et al. 1979, Sivakumaran et a1. 1980). However, the leaves of Xanthium strumarium contain only trace amounts of DPA; the metabolic fate of PA in this tissue is not yet known (Zeevaart 1980). The liquid endosperm from Echinocystis provided a cell- free system for the metabolism of ABA. After centrifugation, .ABA-hydroxylating activity was found in the particulate :fraction and PA-reducing activity was associated with the supernatant fraction (Gillard and Walton 1976). The intermediate 6'-hydroxymethy1-ABA spontaneously rearranges ‘to PA, and it is not known whether that step is enzymatic ’n vivo or not. As indicated in Figure 1-1, ABA can also be metabolized to a conjugate with glucose, abscisyl-B—D-g1ucopyranoside (Milborrow 1978), but conjugation appears normally to occur to a lesser extent than conversion to PA, at least in bean leaves (Harrison and Walton 1975, Zeevaart and Milborrow 1976). Whereas the naturally—occurring (+)-enantiomer of ABA can be converted to PA or conjugated with glucose, it appears that (-)-ABA can only be conjugated and when (i)-ABA is fed to plants, hydrolysis of the resulting glucose ester yields predominantly (—)—ABA (Milborrow 1970, Zeevaart and Milborrow 1976). It is not known whether leaves produce other conjugates of ABA (but see Weiler 1980). The conjugate B-hydroxy-B—methy1g1utary1hydroxy—abscisic acid was isolated 8 from seeds of Robina pgeudacacia (Hirai gt gt. 1978). Zeevaart and Milborrow (1976) isolated ggt—dihydrophaseic acid (ggt-DPA) and alkaline—hydrolyzable conjugates of PA, DPA, and gpt-DPA as minor metabolites of ABA when several days were allowed for the metabolism of exogenous (i)-ABA by bean shoots; PA and DPA were still the major metabolites. 1.2.2. Sites of synthesis and metabolism. Milborrow (1974b) obtained preparations of lysed chloroplasts from loean and avocado leaves and from both white and green Inertions of ripening avocado fruit, preparations which synthesized ABA from labelled mevalonate. Chloroplasts are ‘the presumed site of biosynthesis of ABA, but extrachloro— plastic synthesis of ABA has not been ruled out. In contrast, the enzymes for metabolizing ABA appear to be located outside the chloroplast. As noted in Section 1.2.1, liquid endosperm of Echinocystis has been found to metabolize ABA. Hartung gt gt. (1980), using non—aqueous isolation procedures, separated mesophyll cells of spinach into a fraction largely devoid of chloroplasts and two chloroplasts fractions, one containing intact and the other containing broken chloroplasts. Only the fraction ggt containing chloroplasts converted luC—labelled ABA into other labelled compounds. 1.2.3. Distribution. Abscisic acid is common, perhaps ubiquitous, in all families of angiosperms, gymnosperms, and ferns. It has also been isolated from horsetails, lyCOpods, and mosses, but not from liverworts, or a species 9 of blue—green alga, or several species of fungi (Weiler 1979, Dflilborrow 1978). However, Assante gt gt. (1977) found that ‘bhe fungus Cercospora rosicola produced copious amounts of leA. Abscisic acid readily moves throughout the plant via tloe xylem and phloem (Walton 1980), and it is found in all g>ajts of plants. Nevertheless, the ability to synthesize AJBA is not restricted to mature, photosynthesizing tissue; deetached roots (Walton gt gt., 1976) and young leaves that hgave just unfolded (Zeevaart 1977) can also make ABA. Buds, ffiruits, and young seeds generally have a high level of ABA (Idilborrow 1978). Young leaves have been reported to cusntain a higher concentration of ABA than mature leaves (13aschke and Zeevaart 1976). Zeevaart (1977) showed that ABA and its metabolites PA arm.DPA are translocated in the phloem of castor beans. On the other hand, conjugated ABA could not be detected in either the xylem or phloem sap of Xanthium plants, so conjugated ABA does not appear to be exported from its region of synthesis (Zeevaart 1981). Evidence that ABA readily moves from the cells which produce it comes from the finding of Hemphill and Tukey (1973) that ABA is leached from Euonymous plants by intermittent mist. Nearly all of the ABA of mesophyll cells from turgid spinach leaves was found in the chloroplasts (Loveys 1977). Heilmann gt gt. (1980) found in uptake Studies that ABA became distributed between chloroplasts and 10 the medium depending on the difference in pH between the two compartments, in accordance with the behavior of a weak acid, presuming that the chloroplast membrane is permeable to undissociated ABA but relatively impermeable to the anion. On this basis, in illuminated leaves where the chloroplast stroma would be more alkaline than the cytOplasm, it is possible to account for the presence in the chloroplasts of more than 80% of the total leaf ABA. In accordance with this View, vacuoles, with their low pH, have been found to contain relatively little ABA (Milborrow 1979, Heilmann gt Q. 1980). ov.‘ ,,.. . unu. ‘~..- ~vl- .- . ~ -. ~.. I" I‘- .- \ 11 1.3. FACTORS WHICH CAUSE ACCUMULATION OF ABA IN LEAVES 1.3.1. Wilting. Water deficit, sufficient to cause ZLeaves to wilt, generally results in increases on the order c>f 10- to 40—fold in ABA content of the leaves during a g>eriod of a few hours (see review by Wright 1978). When vvater is withheld from plants, ABA levels can increase seeveral-fold by the time early-wilting symptoms are visible (IWost 1971, Wright 1972). Little ABA accumulates in leaves a1: high wleaf; ABA appears to accumulate below a "thresh- htold wleaf" (Zabadal 1974). Section 2.1 provides further iraformation from the literature on the accumulation of ABA £18 a function of wleaf‘ Water-stressed leaves accumulate ILBA whether they are attached or detached and whether they Eire kept in light or in darkness (Wright 1978). Detached leaves can accumulate ABA due to wilting, recover pre- stress levels of ABA after rehydration, and can repeat the accumulation of ABA when wilted again on the following day (Zeevaart 1980). Leaves are not alone in their ability to respond to partial dehydration by making more ABA. Some accumulation of ABA was found in excised apices, stems, roots, immature pods, and immature seeds after partial dehydration (Wright 1978). Several theories have been proposed to account for Effects of water stress on metabolism in plants. One, Specifically proposed to account for accumulation of ABA, was outlined by Mansfield gt gt. (1978) and by Milborrow 12 (1979): If chloroplasts are the site of synthesis of ABA (Section 1.2.2) and if they retain, in turgid leaves, most of the ABA which they produce (Section 1.2.3.), then perhaps wilting increases the permeability of the chloroplast envelope to ABA, allowing ABA to leak into cytoplasm, relieving presumed feed-back inhibition by ABA on its synthesis in the chloroplasts, causing the total level of ABA in the leaf to rise. Rehydration would effectively "re-seal" the chloroplasts, restore inhibition of ABA synthesis, and allow ABA to return to its pre-stress level as it is metabolized in the cytoplasm (Section 1.2.2). More data (the identification and sub—cellular distribution of ABA-synthesizing enzymes, for example) are needed before this hypothesis can be evaluated. It is not certain that chloroplasts (plastids) are the only site of ABA synthesis, and there is only slight evidence that ABA inhibits its own synthesis (Milborrow 1978). According to the hypothesis, the level of ABA in chloroplasts of wilted leaves could be equal to or less than, but not greater than, the level in chloroplasts of turgid leaves. However, Loveys (1977) found that, while chloroplasts of wilted spinach leaves contained 15% rather than 96% of the total ABA of the leaf, the actual concentration of ABA in chloroplasts of wilted leaves was higher than in those from turgid leaves. Davies gt gt. (1980) have proposed that compounds like farnesol and fatty acids, which build up in cells during water-stress (Ogunkanmi gt g;. 1974, Willmer gt gt. 1978) and are known 13 to disrupt chknophnms (Fenton gt gt. 1976), may naturally play a role in regulating the permeability of chloroplast membranes. Exogenous farnesol induced accumulation of ABA in spinach leaves, but failed to do so in tomato or silverbeet leaves (Milborrow 1979). This hypothesis still ’leaves open the more basic question of how water stress is linked to an effect on farnesol or fatty acid metabolism. Levitt and BenZaken (1975) suggested that accumulation of ABA, decrease in C02 fixation, and other metabolic responses of wilting leaves gtggt be due to a secondary O2- deficit stress arising from reduced intercellular space during wilting. The percentage of intercellular space in wilted sunflower leaves was reduced to half or less of what it was in turgid leaves. Wilted and turgid orange leaves had the same percentage of intercellular space, but this was interpreted as evidence of the sclerophyll nature of orange leaves and of an adaptation to prevent water—stress-induced 02-deficit. Levitt and BenZaken (1975) suggested that another possible link between water stress and metabolic responses might be effects from a change in lateral compression of membrane lipids or proteins accompanying loss of turgor. This hypothesis has been amplified and examined experiment- ally by Zimmermann and colleagues (Zimmermann 1978). Section 2-4 contains more information on the subject of Sensing turgor via membrane compression. 1.3.2. Interruption of translocation in the phloem. 14 Fruit removal and stem or petiole girdling caused the level of ABA to increase in grape vine leaves (Loveys and Kriedemann 1974) or soybean leaves (Setter gt gt. 1980). The effect appears to be reproducible but variable: Setter gt gt. (1980) found that depodding of soybean plants resulted in a 2—fold increase in the level of ABA in 3 h and almost a 10— fold increase by 48 h, whereas Setter gt gt. (1981) found no increase in 3 h and less than a 2-fold increase in 24 h. The response is considerably less than that which occurs in response to wilting. Both Loveys and Kriedemann (1974) and Setter gt gt. (1980) mentioned that wleaf was as high or higher in leaves after girdling treatments than before. Setter gt gt. (1981) attributed the increased level of ABA in soybean leaves after depodding or girdling treatments to obstruction of export of ABA from the leaves rather than to enhanced synthesis. If obstruction of export of ABA from leaves can cause the level of ABA in leaves to rise, why do ‘turgid, detached leaves, ggt accumulate ABA? Detached, ‘turgid Xanthium leaves can be kept for at least 24 h without euay increase in their ABA content (Zeevaart 1980). It is Ixossible to reconcile the apparently contradictory results. Iii studies of accumulation of ABA by wilted, detached leaves, tfiie controls are normally kept turgid by preventing ‘transpiration. If, in the depodded or girdled soybean plants, the transpiration stream sweeps ABA from the mesophyll cells to the sites of tranSpiration, presuming ABA does exhibit Significant feedback inhibition of its synthesis, then ABA 15 would build up a higher steady-state level. By implica- tion, preventing transpiration from depodded soybean plants should reduce accumulation of ABA in the leaves, and allow— ing detached leaves to transpire while keeping the petiole in water should result in some build up of ABA in those leaves (see Figure 2 in Raschke and Zeevaart 1976). 1.3.3. Other factors. A variety of environmental conditions have been reported to cause accumulation of ABA in leaves: low relative humidity (2-fold increase reported by Wright 1972), long photoperiod (2—fold to 3-fold increase reported by Zeevaart 1974L flooding the soil (5-fold increase reported by Wright 1972), salinity or osmotic stress of the roots (Mizrahi 1970), mineral deprivation of the roots (5-fold increase reported by Mizrahi and Richmond 1972), and chilling (2—fold increase reported by Raschke gt gt. 1976). These environmental conditions cause consider- ably less accumulation of ABA than does withholding water until leaves wilt. In none of these cases were the leaves visibly wilted or else wilting was transient. However, (each of these conditions can be expected to either lower the lNater status of the leaves or reduce the ability of leaves tc>export materials in the phloem, or both. It is likely ‘that these conditions result in accumulation of ABA via a Secondary stress, either the approach of incipient wilting Or reduced translocation in the phloem. 16 1.4. THE POSSIBLE INVOLVEMENT OF ABA IN RESISTING WATER STRESS 1.4.1. Regulation of stomatal aperture. Stomata are viewed as the focal point of a complicated biological control system, operating to optimize CO2 uptake and water vapor loss by leaves (Raschke 1979). This section summarizes the evidence for the proposal that ABA functions in the plant as a signal in the control of water loss: appearing at the stomata when the water status of the leaves declines, pro- moting closure of the stomata, and disappearing when leaf- water status rises again. First of all, there is need for a signal. According to what is known about the mechanics of stomatal movement, a significantdecline in wleaf of 10 bar would be expected to produce an insignificant decline in stomatal aperture of about 10% due to water loss from the guard cells (Raschke 1979). In order for stomata to close further for the same lO-bar decline in wleaf’ the guard cells would have to lose :solutes. Water-stress-induced stomatal closure has been 1?eported to be paralleled by loss of K+ from the guard cells (Hsiao 1973a, Ehret and Boyer 1979). It is known that Stomata are relatively insensitive to reductions in leaf- ‘Water content until a threshold value has been reached, below which significant stomatal closure sets in (summarized in Hsiao 1973b, Turner 1974). A closing stimulus should appear at the threshold wleaf. Experiments with exogenous (i)-ABA have shown that ABA 17 would make a suitable regulator of stomatal aperture (see review by Wright 1978). ABA is a powerful antitranspirant (Little and Eidt 1968) by causing stomata of leaves to close (Mittelheuser and Van Steveninck 1969). Stomata in epi- dermal strips also close in response to ABA (Horton 1971, Tucker and Mansfield 1971). When stomatal opening is suppressed by ABA, accumulation of K+ by guard cells is prevented (Mansfield and Jones 1971, Horton and Moran 1972), and when ABA causes stomatal closure, loss of K+ from the guard cells results (Ehret and Boyer 1979). Abscisic acid, added to the water supply of detached leaves, is active in closing stomata at concentrations of lO-SM (i)-ABA or less, concentrations which are normal for the activity of phyto- hormones. Kriedemann gt gt. (1972) estimated from supplying (:)-ABA to detached leaves of bean and rose, that during stomatal closure, the leaves had taken up an amount of (+)- ABA approximately equal to their normal endogenous content. Raschke (1975a) found that as lower concentrations of ABA Ivere supplied, leaves had taken up less ABA by the time ESignificant stomatal closure had occurred. When 10—7M (i)- IEBA was fed to detached leaves of Xanthium strumarium, an Eunount of exogenous ABA equal to 1-2% of the original con- ‘tent of ABA in the leaf was enough to produce a stomatal IPesponse. The time between addition of ABA to the water Supply of leaves and observation of a stomatal response can be as short as 3 to 10 min (Cummins gt gt. 1971, Kriedemann §£_gt. 1972, Raschke 1975a). The stomatal response is Vv' . .. ...- . ...- V.“ .. ..c -r. .... up fit ' -U- ‘U ‘ U. -h M. 18 specific for the (+)-enantiomer of ABA (Cummins and Sond- heimer 1973). There is no evidence of toxicity in the response; stomata begin to reopen within minutes of removing the supply of ABA (Cummins gt gt. 1971). The effect of ABA on stomata appears to be specific, rapid and reversible. Measurements of endogenous ABA content also support the idea that ABA is involved in regulation of water loss through stomata. As presented in Section 1.3.1, wilting induces accumulation of ABA. The response appears to be ubiquitous in vascular plants and it is particularly pronounced in mesophytes (Dbrffling gtht. 1977, Wright 1978). The wilty mutant of tomato flacca contains a low level of ABA compared to the normal variety Rheinlands Ruhm (Tal and Imber 1970). The stomata of flacca are sluggish in response to all closing stimuli (Tal 1966), so a specific role of ABA cannot be discerned, nevertheless the abnormally low level of ABA in the mutant is quite suggestive of an involvement of ABA in the normal behavior of stomata. Accumulation of ABA in response to water stress ciemonstrates a similar sensitivity to changes in wleaf as \Mas mentioned for stomatal closure: both processes are irmensitive to changes in wleaf at high wleaf’ and both Elbruptly increase in sensitivity below an apparent threshold wleaf (Zabadal 1974, Turner 1974). The threshold wleaf Values for the two processes were found to be the same in maize and sorghum (Beardsell and Cohen 1975). When ABA accumulates in response to water stress, it becomes available DA i‘d _ s.-.- - r!‘ :5 ppm ”r 19 to the rest of the plant. Increases in the concentration of ABA in the xylem sap of sunflower plants were observed in less than 1 h after wilting was induced in the leaves by osmotic stress to the roots (Road 1975). Water stress also causes the level of ABA in phloem sap to increase (Zeevaart 1977, Road 1978). Iranthe information presented on the response of stomata to exogenous ABA, it is clear that, if ABA appears in the vicinity of the guard cells during water stress, the stomata will close. From the information presented on accumulation of ABA and its movement throughout the plant during water stress, it is clear that ABA gttt appear in the vicinity of the guard cells. Abscisic acid undoubtedly acts as a natural closing stimulus to the stomata during periods of water stress. Some ambiguity remains. Hiron and Wright (1973) observed a 50% increase in the level of ABA in bean leaves within 10 min of exposing the seedlings to a stream of warm air, and increases in stomatal resistance paralleled the zincreases in level of ABA. Such a good correlation has Iiever again been reported. Considerable variation has been :Found in the coordination between onset of stomatal closure 61nd noticeable accumulation of ABA in the leaves. Section 3.1 presents details of these studies. If ABA is to trigger Stomatal closure as well as maintain the stomata closed, then the active pool of ABA must be small relative to the total ABA in the leaf.1 That is a reasonable possibility lSee Cummins (1973), Raschke and Zeevaart (1976) and Raschke gt gt. (1976) for evidence of an inactive pool of ABA in the leaf. Un~~ tr, vL- An-I 1" l , 1., ‘- ~A. {/1 ply (A: ( » 20 based on the sensitivity of stomata to exogenous ABA. Either rapid production of a small amount of ABA near the guard cells or a rapid redistribution of some of the original ABA to the vicinity of the guard cells (see Raschke (1975b) could trigger stomatal closure and account for the observation of stomatal closure prior to a measurable increase in over—all level of ABA in the leaf. A similar discrepancy in timing has been observed between reOpening the stomata and re-establishment of a low level of ABA in the leaf following rehydration. During recovery from water stress, stomatal reopening is delayed beyond the time required for recovery of pre-stress wleaf values ("aftereffect on stomata", reviewed in Hsiao 1973b). This phenomenon provided early evidence of the involvement of a stress—induced factor in the regulation of stomatal movement. Attempts have been made to discern the relationship between the aftereffect of water stress on stomata and changes in the level of endogenous ABA (Dbrffling gt gt. 1977, Bengtson gt gt. 1977, Henson 1981a). IIn.general, stomata begin to reopen when ABA is at its Inaximum level or even while the level is still rising. In cietached shoots, pre—stress levels of stomatal conductance arm.ABA are reached at about the same time, while in intact plants, complete reopening of stomata takes longer than recovery of pre—stress levels of ABA. Solution to the discrepancy, between expecting the level of ABA to drop before stomata start to reopen and the observed response, 21 may rest in how ABA is distributed between the mesophyll tissue and the epidermis, and, once again, in postulating a relatively small active pool of ABA. 1.4.2. Other possible functions. A number of responses of plants to water stress, in addition to stomatal closure, are thought to be of positive, adaptive, value to the plant in resisting drought (see Hanson and Nelsen 1980). Some of these responses may be mediated by ABA, especially the ones that concern processes which depend on H+/K+ transport, since ABA is known to affect H+/K+ exchange in causing stomatal closure (Raschke 1977). (1) Davies gt gt. (1980) have pointed out that the ability of ABA to inhibit H+/sucrose co—transport into the phloem of Rincinus leaves (Malek and Baker 1978) may provide a means by which osmotic adjustment (increasing n) in leaves takes place in response to water deficit. (2) Whereas water stress reduces the growth of leaves, the root/shoot ratio has been found to increase in water-stressed plants, and root growth may even be stimulated ciuring mild stress (Sharp and Davies 1979). Does ABA :increase the roots' sink strength for carbohydrates? (3) (Zonsiderable effort has been made to study the effect of JABA on water and ion movement in the xylem (review by Van Steveninck 1976). Tal and Imber (1971) found that exogenous .ABA increased the exudation rate of decapitated tomato plants, especially in the wilty mutant flacca. The GXperiments by Glinka (1980) with decapitated sunflower plants indicated that ABA promoted, probably independently, o 5" . . #4. b . ‘\~ ~‘M‘ 4.4 22 both sap flow and release of ions to the xylem. Transitory stimulation by ABA of guttation and ion flux from passive hydathodes of intact barley plants was measured by (Dieffenbach gt gt. 1980). If water-stress-induced accumulation of ABA by leaves of intact plants normally stimulates water uptake by the roots, that would increase the rate at which ABA-induced stomatal closure would improve the water status of the shoots. Experiments by Morgan (1980) implicated ABA as the factor responsible for reducing seed set in water-stressed wheat plants. Morgan suggested that limiting seed set could offer survival advantage for the seeds which did develop, since the amount of carbohydrate available for seed development would be reduced during water stress. Likewise, the suspected ability of ABA to inhibit germination of seeds of some species, until it has been leached from the seeds (Wareing 1978), can also be viewed as a mechanism for surviving drought. “J 23 1.5. STATEMENT OF PURPOSE Substantial evidence that the accumulation of ABA during water stress has adaptive value for the plant in resisting water stress was presented in the previous section. Such evidence warrants further attempts to increase our knowledge of the role of ABA as a phyto- hormone. Additional information would have practical value, one hopes, in defining a metabolic response that would be an appropriate criterion in breeding crop plants for adaptation to dry environments as well as helping to define appropriate irrigation practices. The study of accumulation of ABA by plants also has intrinsic value in increasing our understanding of a biological control system: how water deficit results in the appearance of ABA as a signal mediating the plant's response to an environmental stimulus and how correction of the deficit results in disappearance of the accumulated ABA. This thesis describes investigation of three specific aspects of this system. 1) Which parameter of plant water status serves as a Stimulus for inducing accumulation of ABA? 2) Based on measurements of the accumulation of ABA in epidermis during stress-induced stomatal closure, how sound is the hypothesis that ABA regulates stomatal behavior during water stress? (3) How do effects on biosynthesis and metabolism of 24 ABA contribute to the accumulation of ABA during water stress and to the disappearance of ABA after rehydration? Chapter 2 Correlation between loss of turgor and accumulation of abscisic acid in detached leaves 25 26 2.1. INTRODUCTION This investigation of water-stress-induced accumulation of ABA in leaves began with an attempt to characterize the response as a function of the parameters of leaf water status, notably leaf water potential (wleaf), osmotic pressure (n), and turgor (p).l Previous studies have, for the most part, dealt with the relationship between ABA content and wleaf' Zabadal (1974) studied accumulation of ABA in leaves of Ambrosia plants which had been placed in a desiccating environment. As the plants depleted the soil of water, ABA content increased only after wlea declined to -10 to —12 f bar. Zabadal introduced, therefore, the concept of a threshold w for the accumulation of ABA in water—stressed leaves. Similar results were obtained by Hemphill and Tukey (1975) for leaves of Euonymus alatus and by Blake and Ferrell (1977) for needles of Douglas-fir seedlings. ABA content increased steadily after wleaf reached —8 to -10 bar in maize and sorghum (Beardsell and Cohen 1975). Wright (1977) used detached leaves in order to allow the same amount of time for production of ABA at each different wleaf‘ He found that the ABA content of wheat leaves progressively increased with decreasing wleaf’ The response curve which Wright found for wheat leaves became very steep below -9.3 bar, the w of plants showing early Wilting symptoms. Beardsell and Cohen (1975) l p n is used here as a positive qmmnfity, and hence, wleaf ‘ -‘n. 27 suggested that loss of turgor might correspond to the critical wleaf at which ABA levels begin to increase. Davies and Lakso (1978) found in apple seedlings that declining turgor was better correlated with increasing ABA content than was declining wleaf' How leaf turgor was related to the pattern of accumulation of ABA during water stress became an intriguing question. Other work indicated that zero turgor might be important for ABA production. In 1974 Turner provided evidence that threshold W's which had been observed for stomatal closure in a variety of species were all associated with turgor close to zero. ABA has been strongly implicated in causing stomatal closure during water stress (Little and Eidt 1968, Mittelheuser and Vaniyxwemfimk, 1969, Wright and Hiron 1969, Hiron and Wright 1973), so one would eXpect stomatal closure to indicate that ABA was accumulating. The following experiments were designed to test the prediction that species should differ in the wleaf required for accumulation of ABA, according to the reduction of wleaf required.for elimination of turgor. Excised leaves of Egasecflus vulgaris, Xanthium strumarium, and Gossypium hirsutum were used to study the relationship between wleaf’ “: p, and abscisic acid content. Another objective was to find how gradual or abrupt the onset of accumulation of ABA was in the approach to zero turgor. Uncertainty because of variation between leaves could be eliminated by cutting a series of samples from individual leaves as they lost water. 28 Monitoring the same leaves for decreasing wleaf and fresh weight permitted calculation of w and p from a plot of -wleaf-l versus fresh weight loss (Scholander gt gt. 1965, Tyree and Hammel 1972, Talbot gt gt. 1975). 29 2.2. MATERIALS AND METHODS 2.2.1. Plants. Plants of Xanthium strumarium L. (codokbur) (from a strain collected near Chicago, 111., USA and propagated in California and later in Michigan) and plants of Phaseolus vulgaris L. cv. Mecosta (red kidney bean; seeds from Foundation Seed Co., East Lansing, Mich., USA) were grown in a potting mixture in a greenhouse. Air temperature maxima were between 23 and 29°C, and the relative humidity was generally 70—80%. The t. strumarium plants were kept pruned to the top five or six leaves and did not flower under the long—day conditions provided by extending the natural light period to 20 h/d by Sylvania (Danvers, Mass., USA) Gro-lux fhkuesmnm tubes giving an 2 irradiance of about 0.3 W m' at plant level. Fully developed leaves of t. strumarium were taken for the experiments when the plants were 10-14 weeks old. Plants of E, vulgaris were also cultivated in a growth chamber, which had a 16-h photoperiod at 85 w m’2 of light from (knaeral Electric (Cleveland, 0., USA) cool—white fluorescent Lmnps. Day temperature was 27°C; night temperature was 21°; relative humidity was 80%. The terminal leaflets of fully developed 2. vulgaris leaves were used for the experiments when the plants were 4.5-6 weeks old. Both t. strumarium and E. vulgaris plants were kept well—supplied with water. Plants of Gossypium hirsutum L. (cotton) (cv. Acala SJ-l; seeds from C. A. Beasley, University of California, 30 Riverside) were cultivated in two groups under different conditions. For 7 weeks both groups were exposed to a 13.5-h photoperiod, composed of 12 h of 60 W m_2 of light from General Electric cool—white fluorescent tubes plus 1.5 h of light from incandescent lamps only (4.5 W m-2). Day temperature was 32°C; night temperature was 22°; relative humidity was 85%. In this growth chamber the plants were watered daily. After 7 weeks one group was transferred to another growth chamber where the plants were exposed to a 13.5—h photoperiod with a peak irradiance of 2 of light from General Electric lamps H 400 DX 33-1 230 W m- (mercury vapor) and LU 400 (high temperature sodium vapor). Day temperature was 27°C; night temperature was 21°; relative humidity was 67%. Water was withheld from these plants until the mature leaves were visibly wilted. After 8 stress/recovery cycles of 2-3 days duration each, the plants were watered regularly for 3 days before an experiment was performed. The 10th and 11th oldest leaves worms used from plants 10 weeks old in both the unstressed and stress-conditioned groups of g. hirsutum. All Imxasurements of irradiance were made with an Epply pyranometer (Newport, R. 1., USA) behind a Corning (Corning, NY., USA) no. 4600 infrared-absorbing glass filter. 2.2.2. Extraction and purification of abscisic acid. Samples were cut from leaves with a razor blade to have an 2 area between 4 and 8 cm The samples were frozen and .- ~v 0d. -.. a an, .- b 31 1yophilized. They ranged in dry weight from 10 to 50 mg. For these small samples the extraction procedure of Zeevaart (1974) was simplified. Each lyophilized sample was homogenized at room temperature in 15 ml methanol. The methanol extract was separated from the debris by vacuum filtration and the debris was re-extracted by shaking overnight in another 15 ml of methanol. The second methanol fraction was combined with the first. A 250 Bq-aliquot of (i)-[3H]ABA (hereafter designated [3HJABA) was added to the methanol extract for monitoring recovery. In some experiments chlorophyll concentration was determined in the methanol solution according to Holden (1965). Five ml H2O were added to the methanol extract, and the methanol was evaporated under reduced pressure. Material insoluble in H2O was removed by filtration through a Millipore (Bedford, Mass., USA) AP prefilter. The aqueous phase was acidified to pH 2.5 with HCl and extracted 3 times with equal volumes of ethyl acetate. ABA in the ethyl acetate extract was further purified by TLC and methylated as described by Zeevaart (1977). Recovery of ABA from the original methanol extract averaged 70—75%. The amounts of Me-ABA in the samples were determined with a Varian (Palo Alto, Cal., USA) 3700 gas chromatograph equipped with a 63Ni electron capture detector. A 1.8 m glass column, 2 mm internal diameter, packed with 3% SE-30 on 80/100 Supelcoport (Supelco, Bellefonte, Pa., USA) was used. Carrier gas was N2, flowing at a rate of 20 ml min"1 32 The column oven temperature was 200°C; the detector temperature was 300°C. Measurements of ABA by GLC-EC was not difficult even in the leaf sample which contained the least amount of ABA (2.7 ng). In that case, when one fiftieth of the sample was injected, the detector response at the retention time for Me—ABA produced a peak 30 mm high and well-resolved from any other peaks. Calibration curves were prepared by injecting known amounts of standards. The concentration of (i)—Me-ABA in a stock solution was measured by spectrophotometry in methanol using an extinction l -l coefficient of 20900 M- cm at Amax = 265 nm (Milborrow and Robinson 1973). 3 . . 14 -l The [ HJABA, speCific actiV1ty 8.3 - 10 Bq mol , was purchased from Amersham, Arlington Heights, 111., prepared according to Walton gt gt. (1977). The starting material for the synthesis of [BHJABA, l—hydroxy—4—keto-d-ionone, was a gift from R. J. Reynolds Tobacco Company, Winston—Salem, N.C., USA. 2.2.3. Methods of measuring leaf water status. (i) Pressure-bomb method. The pressure-bomb technique developed by Dixon (1914) and by Scholander gt gt. (1965) was used to determine wleaf' The chamber (PMS Instrument, Corvallis, Ore., USA models 600L and 1000) was lined with moist filter paper to reduce water loss from the leaf during measurements. Chamber pressure was increased by adding compressed nitrogen l at a rate approximately 0.05 bar 8. until xylem sap appeared at the cut surface of the petiole. At this point the chamber 33 ressure is equal in magnitude but Opposite in sign to the xylem tension and is called the "balancing pressure" (pbomb). When cell and apoplastic water potential are in equilibrium, wleaf = - pbomb-nylem' The “xylem was determined to be negligible for the leaves used in our experiments: all tests of turgid and wilted leaves gave n readings of less xylem r than 0.5 bar in a dew point hygrometer (Wescor, Logan, Ut., USA; model HR—33(T) Dew Point Microvoltmeter with a C-52 sample chamber) used as a psychrometer. Therefore, balancing pressures were taken as measurements of -w1eaf' Leaf osmotic and turgor pressures were determined from "pressure-volume curves" (Scholander gtht. 1965). These were prepared by plotting inverse balancing pressure (= -w1eaf-l) versus decreasing leaf fresh weight (Talbot gt gt. 1975) as in Figure 2-1. The theory relating balancing pressures as a function of tissue volume has been developed by Tyree and Hammel (1972) and by Tyree gt gt. (1973). The reciprocal bulk leaf osmotic pressure can be read from the linear portion of the curve or its extrapolation to the ordinate. Leaf turgor can be derived from the curve as the difference between osmotic and balancing pressures. (ii) Dew-point method. The theoretical basis of the Wescor dew—point microvoltmeter for measurements of w has been described by Campbell gt gt. (1973). Samples consisted of l-cm leaf discs. Repeating the readings after freezing and thawing the leaf discs three times provided measurements of osmotic pressure. 34 Figure 2-1. A "pressure—volume curve" determined for a Gossypium hirsutum leaf. The original fresh weight of the leaf was 3.41 g; the leaf weighed 89% of its weight at p = 0. The same leaf provided the ABA analysis, the results of which are shown g. hirsutum, watered daily. (l/irO = inverse osmotic pressure.) original fresh samples for in Figure 2-4, original bulk /nverse 50/0/76th Pressure {bar 7/ 35 I 0.4 0.3 - 0.2 r- O.I Gossyp/Um h/ksu/um _. ‘ ‘uri’l"'.“1-——-'H..- W (D .2 .4 Leaf Fresh Weight Loss (9) Figure 2—1 36 2.2.4. Preparation of samples of known wleaf’ n, and 3. Plants were watered and placed in a dark CEBIHet in the evening before an experiment was performed. A mature leaf was out under water and allowed to take up water for about 1 h still in darkness. Such turgid g. hirsutum leaves were obtained by applying 0.5-l bar positive pressure for 10 min to the water in which the cut petiole was placed. To enable pressure-bomb work with E. vulgaris the groove in the petiole was filled with silicone rubber (not acetate- cured) (Wacker—Chemi, Munich, Germany) several hours before the leaf was cut. The dehydration procedure consisted of the following sequence. The leaf was weighed. A balancing pressure was determined. The bomb pressure was released at less than 0.1 bar s-l. Two more minutes were allowed for w equilibration within the leaf. Then the leaf was taken from the chamber, and a sample was quickly excised and wrapped in pre-weighed foil. Sample plus foil were weighed. The rest of the leaf was left on the laboratory bench to lose water, and its weight was monitored until the leaf was judged to be at appropriate water content for a new measurement of wleaf with the pressure bomb. The cycle was repeated until enough samples had been taken to span the range from full turgor to several bars beyond the wilting point. At the beginning of the experiment samples had to be taken one after another as quickly as possible, but the rate of water loss dropped and ) the water loss requirement (per bar decrease in wleaf 37 increased as the leaves wilted. To speed up the sampling rate, when necessary, toward the end of the experiment, the pressure bomb was used to force water from the cut end of the petiole (as in the original description by Scholander gt gt. 1965, for preparing pressure—volume curves). Cutting samples from the leaf meant that the running total of fresh-weight loss could not be used directly in plotting -wleaf-l versus fresh weight. The weight of the samples and the fact that the amount of leaf left to lose water was continuously decreasing had to be taken into account. The weight losses between sampling were calculated according to the following formula: (wt before sampling - sample wt) - wt after water loss fraction of leaf remaining The fraction of leaf which a sample represented was estimated from dry-weight measurements. Curves prepared as described were indistinguishable from those of control leaves from which no pieces were cut. Therefore, it was concluded that no significant error was introduced, either by unavoidable cutting of vascular tissue during sampling or by calculating whole-leaf- equivalent weight loss. Cutting major veins during sampling was avoided. Twelve experiments were performed which provided samples for analysis of ABA as a function of wleaf’ w, and p in individual leaves. Six examples are presented here. 2.2.5. Considerations for sampling. In the procedure 38 described above, samples for analysis of ABA were out from random positions around the leaf. Variability would be introduced into the results if there were gradients of w or n along the leaf. The positional destribution of w and n was checked on control leaves (no samples cut for analysis of ABA) which were close to wilting. The dew—point method was used to measure w and w in discs cut from various positions on the leaf blades. Figure 2—2 shows the range of values obtained. In no case was there a discernable pattern in the variability in terms of the location on the leaf from which a sample was taken. It was concluded that taking all samples for analysis of ABA from one leaf was justified and superior to using different leaves for each point. 2.2.6. Sample incubation. When samples for analysis of ABA were excised from a leaf they were at known w's. After weighing, the foil covered samples were placed in Petri dishes lined with wet filter paper for incubation to allow ABA to accumulate or not. After the incubation period the samples were unwrapped, reweighed, then frozen with liquid nitrogen. Samples were lyophilized for dry weight determination. An unavoidable but small loss of weight occurred during sample incubation. In general the change in weight was around 1%, and samples generally weighed between 100 and 200 mg. Respiration during 10 h of incubation could account for roughly 1/3 of the weight loss, and perhaps another 1/3 of the loss happened during the post—incubation 39 Figure 2—2. Evidence for uniform wleaf’ n, and p for different positions in leaf blades. Measurements of leaf water potential (wleaf’ upper number of each pair) and osmotic pressure (n, lower number) were made with a dew- point microvoltmeter on discs out from leaves of Xanthium strumarium, Phaseolus vulgaris, and Gossypium hirsutum. Each value is the mean from measurements made on three leaves of similar wleaf‘ 40 Phaseo/us vulgar/'5 Gossyp/Um Mrs/rum stress-conditioned Xanthium s/rumar/Um Cosmo/21m h/i'svfum 13.5 watered doily Figure 2—2 41 weighing. For fully turgid samples, loss in weight no matter how small represents a significant change in and such u)leaf turgid samples may have declined in w during the incubation by as much as 1 bar. For most of the samples, however, the water status was nearly constant during the incubation period. Choice of the sample incubation period was based on the time course of accumulation of ABA for the three species (Figure 2-3). The time courses were prepared as follows. After cutting a turgid control sample, a leaf was dehydrated until turgor was zero. The leaf was removed from the bomb and divided into about 10 pieces, which were wrapped individually in foil and incubated. At various times a piece was frozen and later extracted for ABA. The control sample was incubated until the last wilted sample was frozen (Figure 2-3, at the left), affirming that excision and long iiicubation by themselves do not result in accumulation of IUBAJ In Figure 2—3 ABA content was plotted versus time from vnien zero turgor was reached. Considerable ABA had acusumulated by 6 h, and there was a tendency for the rate of acnsumulation to decrease after that time; and at least 8 h vwere required for a new steady-state level to be reached. On the basis of the time courses all samples for a dehydration series were incubated until 8.5 h had elapsed from the time zero turgor was reached. It took about 1.5 h to reach zero turgor and a total of about 4 or 5 h to take a leaf through the entire dehydration procedure. By . . whoa llllllll mpcoEoLSmmoE one pom pom: who; Atop : man u 9v hawmo commons oLSpHSOIIESPSmLHQ Edwoxmmoo pom .Asmo z.ml u mmoasv opzpazo omsoocomnwllmflgowao> msHoommLm .Atmo H.Hfi| mmoae mamas no .hmo H.0HI n Nov ESHLmEBABm Sofinpomx mo mo>moq .moflooom m mo mo>moa oommohpmlpopmz noooompoo CH oaom owmflownm mo soapmasezoom mo omosoo oEflB .m1m otsmflm 43 mum whom; E .092. oemN 5:4 mEF mmanm m m s m h e m m _ o 292 o _ q a a _ _ 3 \S\l?lfi.lql II] \« +\+.+.+ n + i N o\/ \oe\ /+ Aw \\ -e w ESxSmSQ + \ 7 ESEXmmSw + 1 w W o. S o\ <\ 5 to S; 1 m os\ot\\8§ + h . (W \+ Ill. we w. \EStSESxxm 1 O_ +/\ \+ ESSEX + 44 freezing samples all at the same time the bias of having samples with lower wleaf also having experienced water stress for a longer time (as occurs in soil-drying experiments) was eliminated. The samples should all have had close to a new steady-state level of ABA for each respective wleaf’ 2.3.1. The relationship between wleaflw’ p and accumulation of ABA. These experiments all followed the same procedure. Individual leaves were allowed to wilt slowly. Samples for ABA analysis were cut from a leaf as it lost known amounts of water and incubated at constant water content to allow accumulation of ABA to a new steady— state level. Just before a sample was cut, wleaf was determined with a pressure bomb. Curves were prepared from the wleaf and weight-loss measurements (e.g. Figure 2-1). These were used to determine n and p. The results of leaf—dehydration series are shown in .Figure 2-4 for leaves of Xanthium strumarium, Phaseolus 'vulgaris, Gossypium hirsutum, and g. hirsutum which had tween forced into osmotic adjustment by periodic withholding of? water. Values for ABA content were plotted as ng ABA hug—l dry weight. Lines of the same shape were obtained vdien ABA content was expressed on the basis of chlorophyll ccnutent. The curves of ABA content versus wleaf all show ea region of high wleaf (right side of Figure 2-4) over which little ABA accumulated, followed by gradual transition to a region of wleaf over which ABA content rose steeply. The Curves differed in the wleaf at which the steepest slope occurred. The responses became very steep below -8 bar for these examples of t. strumarium and E. vulgaris, below —13.5 bar for a g. hirsutum leaf, and below -18.5 bar for a leaf A6 Figure 2_u. The effect of leaf water potential on abscisic acid content in single, detached leaves of Xanthium strumarium, Phaseolus vulgaris, and Gossypium hirsutum. ng ABA/mg dry M “7 O Xanthium sflwMMMm 0 20» + § 0 '5 ' Gossypium 'rsurum . . 0\ stress‘condmoned P$$%1:5/\/\ 0 IO- .tfiwspmm him/Mm \ watered daily 0 5. o 4' o\oao ‘\‘ + \O+o_o\o ‘\‘\/ ‘ ‘ O 1 i “Hie—{Md -25 -20 -l5 -l0 --‘.5L 0 Leaf Water Potential (bars) Figure 2-A A CD from a stress—conditioned g. hirsutum plant. The variation appeared to artxafrom the leaves having different t's and, hence, different u's at which loss of turgor occurred. At full hydration (wleaf = 0) these leaves had bulk osmotic pressures of 8.3 bar for E. vulgaris, 8.6 bar for X. strumarium, 11.6 bar for g. hirsutum and 17.2 bar for stress- f -n) the leaves had H's of 9.6 bar for B. vulgaris, 9.7 bar conditioned g. hirsutum. At zero turgor (when wlea reached for g. strumarium, 13.7 bar for g. hirsutum, and 21.5 bar for stress-conditioned g. hirsutum. Figure 2—u shows that leaves with similar original bulk osmotic pressures had almost overlapping curves of accumulation of ABA in response to decreasing wleaf' Leaves with different N's, even of the same species, differed with respect to the wleaf (and n) at which accumulation of ABA took place. Data for n and p were added to a graph of ABA content versus wlea for another g. strumarium leaf in Figure 2-5. f Figure 2-5 illustrates the correspondence between loss of turgor and accumulation of ABA. Samples with low but still positive turgor developed levels of ABA which were up to three times the level of ABA in fully turgid samples. Zero turgor strongly affected the accumulation of ABA; the steepest increase in ABA content occurred between the first two samples having zero turgor. Greenhouse and growth chamber conditions produced 3. vulgaris plants with slightly different fl'S. Figure 2-6 shows that the small difference in w was reflected in an 149 Figure 2-5. The relationship between osmotic pressure (w), turgor (p), and leaf water potential, and the effect of wleaf on abscisic acid content in samples from a single, detached leaf of Xanthium strumarium. 5O .+ ('71 l ng ABA/mg dry wt C5 ABA Xanthium sfrumarirm l O ~16 -€3 -é «3 -'2 Leaf Water Potential (bars) Figure 2—5 0 Turgor ‘ 0r Osmotic Pressure {bors/ 51 Figure 2—6. The relationship between turgor and leaf water potential, and the effect of wleaf on abscisic acid content in two leaves of Phaseolus vulgaris. The leaves were detached from plants cultivated in either a growth chamber or a greenhouse. (Turgor: """" ABA: A,A.) I5 ("'5 ng ABA/mg dry wt 52 Lfi A/ A~.‘ Growth Chamber ’,.\ / Phaseo/us vulgar/s ’ \ A (AGreen house - /€\;‘r‘ as o Turgor Pressure {bars} I CD q 45 DO 1 O -Ié -lO -8 -6 -4 -2 Leaf Water Potential (bars) Figure 2—6 0 equivalent displacement between the curves of increasing ABA content. 3. vulgaris samples with a turgor of 1 bar developed levels of ABA which were four to eight times the level of ABA in fully turgid samples. Below p = 1 bar, ABA content increased sharply to as much as forty times the level found in unstressed samples. As in Figure 2-5, both examples in Fig. 2-6 show that the steepest slope in the relation between ABA content and wleaf occurred at zero turgor. Some of the curves of ABA content versus wleaf in Figures 2-U, 2-5, and 2-6 indicate a tendency for the response to level off within several bar of the point of zero turgor. Saturation of the response is particularly obvious in Figure 2-6. The dependence of ABA production on loss of turgor is summarized in Figure 2-7. ABA content progressively increased as turgor approached zero. Accumulation of ABA in the example from g. hirsutum plants which were well— supplied with water was exceptionally abrupt; all of the increase in ABA above unstressed levels took place below p = 0.5 bar. For the other leaves, more than 80% of the increase in ABA above unstressed levels took place at less than 1 bar turgor. 2.3.2. The effect of elevated N2 pressure an accumulation of ABA by wilted leaves. Elevated atmospheric pressure increases w by the amount of the added pressure. That allows the possibility of having a wilted leaf (p = O) with a high a = O). This experiment was designed to (wleaf 5“ Figure 2—7. Abscisic acid content as a function of turgor in single, detached leaves of Xanthium strumarium, Phaseolus vulgaris, and Gossypium hirsutum. ABA content is plotted as the percent of the maximum ABA content accumulated by the same leaves at any wleaf tested. The relationship between ABA content and wleaf for these leaves is shown in Figures Z—U, 2-5, and 2-6. A 84 Come/7f /% ofmax/mum/ I00 55 70- 60 - 30- 20- I IO Phaseolus vu/gari'su, A Xanthium strumar/l/m - +, x Gossyp/Um hirsutum -' 0 watered daily 0 stress-conditioned \ / + \A/ \ "‘O— "‘10—"1 o—Cb—joxo—o 6 8 no I2 Ii; Turgor Pressure (bars) Figure 2-7 / 50 test whether a high wleaf in a wilted leaf could prevent the normal water-stress induced accumulation of ABA. Mature leaves of Xanthium strumarium were used. After a sample was cut from a turgid leaf, the remainder of the leaf was allowed to wilt. The wilted part was divided into two samples, one of which was put in a pressure chamber. All three samples were wrapped in foil to prevent water loss during a subsequent U-h incubation. Nitrogen was added to the pressure chamber until the pressure in the chamber balanced the wilted leaf n raising wleaf to zero. The results are presented in Table 2-1. The turgid samples contained the amount of ABA that is typical of unstressed Xanthium leaves (e.g. Figure 2-3). The wilted samples which were maintained at elevated N2 pressure consistently tripled their content of ABA in the u-h period, but the wilted samples that experienced normal atmospheric pressure increased their content of ABA 7 to lO-fold. The role of wleaf per se in affecting accumulation of ABA was not clarified by these results. If the leaf samples had been brought to wleaf = O by immediate rehydration rather than by applied pressure, one would expect that they would contain no more ABA than unstressed leaves. (This was the case in other experiments.) Pressure—induced high wleaf’ on the other hand, did not completely prevent accumulation of ABA. It is unlikely that lack of 02 was reSponsible for attenuating accumulation of ABA by samples in the pressure chamber. During the U-h period, 57 Table 2-1. The effect of elevated N2 pressure on accumulation of ABA by wilted leaves. The wleaf of the turgid samples ranged between -l and -3 bar. Wilting occurs in Xanthium leaves near wleaf = —lO bar; water loss was allowed to continue until the wilted samples reached wleaf = -11 bar. Eleven bar N2 pressure was added to the pressure chamber. All samples were incubated in darkness at constant weight for A h, and were frozen and lyophilized for subsequent extraction of ABA. Wilted Sample, Wilted Sample, Leaf Turgid Bench Pressure Chamber Number Sample Incubation Incubation ng ABA mg‘l Dw 1 1.9 13.3 5.2 2 1.8 13.9 5.6 3 0.8 8.1 2.4 ABA Relative to Unstressed Level 1 1.0 7.2 2.8 R) 2 1.0 7.9 3. 3 1.0 10.“ 3.0 “to v a. 5' 58 respiration by the tissue would have consumed less than 2% of the available 02. The effect of the increased concentration of dissolved N2 in and around the compressed cells is not known. It is thought that the most likely explanation, of how pressure could reduce the accumulation of ABA by wilted leaves, would be the possibility of applied pressure mimicking turgor in the cells. (See Section 2.3.3.) to )1 2.“. DISCUSSION 2.U.l. Leaf u and n influence production of ABA through their effect on turgor. ABA content in water- stressed leaves increased at different water potentials for different leaves (Figures 2-U, 2—5, and 2-6), but in each case the capacity for accumulation of ABA rose sharply as turgor approached zero (Figure 2-7). These results are interpreted to mean that wleaf per s: does not control ABA content. It is unlikely that bulk leaf N directly influences production of ABA either for the following reasons: (1) Increases in ABA content were not uniquely related to n just as they were not uniquely related to wleaf' (2) In most leaves the entire change in ABA content occurred over a range of w of about 2—A bar, for instance from 7 to 9 bar in E. vulgaris or from 1” to 18 bar in g. hirsutum (e.g. Figure 2—5). Changes in n of this magnitude can normally occur in leaves during the course of a day and may be attributed to solute accumulation rather than dehydration, as Acevedo et a1. (1979) reported for leaves of maize and sorghum. Even larger diurnal changes in n were observed in cotton leaves (Cutler et a1. 1977) when some dehydration accompanied solute accumulation during the day, but even so the stomata did not close, suggesting ABA levels did not rise in this case. The changes in bulk leaf N which were observed in the present study were almost certainly not responsible for the 10 to UO—fold accumulation of ABA that 60 occurred. The 2-A bar change in n represents a 30% increase in solute concentration; a change of that size, for any component of n, is unlikely to trigger accumulation of ABA. (3) It is probably concidental that a nearly linear relationship appears if ABA content for an individual leaf is plotted versus w. The linearity results from the fact that with decreasing wleaf only small changes in leaf n and ABA occur so long as turgor is positive, but that these changes increase after turgor has been lost (e.g. Figure 2—5). It is not possible to assign significance to the linearity because the relation holds only for a narrow (2-4 bar) range of n. (A) Not all, but at least half, of the leaves showed saturation of the response within the range of water potentials tested (see Figures 2—U and 2—6); increases in solute concentration above about 30% were less effective or not at all effective in promoting accumulation of ABA. Thus, accumulation of ABA in water- stressed leaves is most probably a turgor-dependent process rather than a w or N—dependent one. leaf- 2.“.2. The question of a "threshold" for wleaf accumulation of ABA. Single mature leaves were used in these experiments in the hope of getting a clearer answer as to whether ABA production in response to water stress exhibits a threshold phenomenon or not. In one case——that of g. hirsutum well—supplied with water——the response curve versus wleaf in Figure 2—U showed an abrupt change of slope coincident with the point of zero turgor. In the rest of the examples in Figure 2—A, 2—5, and 2-6, the curves were more sigmoid in shape, just as Wright (1977) found for 61 detached wheat leaves, showing no clear threshold. Although Zabadal's (l97fl) data produced the appearance of a threshold wleaf for the accumulation of the ABA, the data of this study and that of Wright, in fact, showed much steeper responses. The data of Zabadal and Wright have been replotted in Figure 2—8 for comparison. Soil and plant u were falling throughout Zabadal's experiment. Presumably he would have obtained results more like those of this study and Wright's study if time had been allowed at each wleaf for full expression of the effect on accumulation of ABA. 2.4.3. How is accumulation of ABA related to turgor? The results, when plotted against turgor (Figure 2—7), showed a lack of respOnse at high turgor and a progressive increase in accumulation of ABA by the leaf samples as they approached zero turgor. This pattern of response may reflect the nature of turgor sensing by the individual cells of the leaf. The curves are of the same shape as that found for the turgor-dependence of K+ influx into cells of Valonia utricularis (Zimmermann and Steudle 1977). According to the electrochemical model of turgor sensing by plant cell membranes (Coster et a1. 1977, Coster and Zimmermann l976, Zimmermann 1978), turgor is sensed by membrane compression. Both mechanical forces (pressure) and the electric field in a membrane can affect membrane thickness. These authors have presented evidence that the elastic, compressive modulus for plant cell membranes is 62 Figure 2-8. The effect of wlea on ABA levels; a f comparison of data redrawn from Wright (1977) and Zabadal (1974). ABA in excised wheat leaves: I - I right-hand 9 scale, (Wright 1977). ABA in leaves of Ambrosia artemisiifolia: O — - 0, left—hand scale, and Ambrosia trifida: A - A, left-hand scale, during a period of progressive dehydration of intact plants (Zabadal 197A). 63 - ISO :5... -o 9: <3 0 p m l 1 -l6 -l2 wleaf (bar) -20 I -24 :50 roe 9: m meow one mcfi>m£ mpcfloo MCOHpoom mama mamcflm a co psoEoLSmmoE m mpcommpoop pCAOQ memo comm .Lopwz Eonm wo>moa mficzEEoo mcHHoEEoo mo m:oapoom mca>oEmp Lopgm mafia gpflz mamas cm ocflaooo one .zlm opswfim 92 llfirl’l mmmno EduauoEmo CH mo>wofi wcHHoEEoo mo .mlm oHQmB 109 3.4. DISCUSSION 3.4.1. The level of ABA prior to stress-induced accumulation. The level of ABA in epidermal tissue from unstressed leaves of Commelina was reported by Singh gt gt. (1979) and by DBrffling gt gt. (1980) to be, on the average, between 0.1 and 0.2 ng mg.l dry weight. The results presented here agree with that estimate. Vicia faba epidermis from unstressed leaves appears to contain much more ABA than that, 0.8 nm Ing-l dry weight (Loveys 1977). A comparison of the same reports indicates that Vtgtg mesophyll also contains a higher basal level of ABA than Commelina mesophyll does. It is puzzling that the absolute level of ABA in epidermis, prior to accumulation as a result of experimentally—imposed water stress, was found to vary considerably from one group of plants to another: from 0.05 (Figure 3-7) to 0.25 ng mg.l dry weight (Figure 3—2). To some extent the variation of ABA level in the epidermis paralleled a variation in ABA level in the mesophyll. We already know from experiments with Xanthium strumarium (Raschke gt gt. 1976) that the basal level of ABA in a leaf, as a whole, can vary several-fold without an apparent effect on the stomata. Now it seems that, possibly, ABA can be somewhere even within the epidermis without affecting the stomata. In some groups of leaves (Figures 3—6 and 3—7) the stomata were closed at a level of ABA which, in another 110 group of leaves (Figure 3-2) was well below the initial value. Understanding the compartmentation of ABA within the epidermis may prove to be critical for an evaluation of the role which ABA plays in regulating stomatal behavior. 3.4.2. Changes in ABA content and stomatal aperture related to decline in wleaf' An increase in ABA in the epidermis was apparent 6y_36 min after removing the leaf- halves from water (Figure 3—6), which was within 13 min of when leaf turgor fell to zero (Figure 3—4). If ABA content from Figure 3-6 were plotted against wleaf from Figure 3-4, the wleaf at which leaf turgor equalled zero would appear as a threshold for increasing ABA content in the epidermis. It is not possible, however, to conclude what the exact relation was between leaf turgor and epidermal ABA content; since wleaf was falling throughout the experiment, any wleaf above the apparent threshold could have initiated the increase in ABA level. A comparison of Figure 3—4 and 3-6 indicated, as one might expect, that the peak of transient stomatal opening occurred as turgor in the leaf sections reached zero. 3.4.3. Stomatal closure and accumulation of ABA by gpidermis. Increases in ABA in response to water stress were evident sooner in the abaxial epidermis than in the remainder of the leaf. The level of ABA in the epidermis gradually increased as the stomata closed. These observations support the idea that the stomatal closure could have been caused by ABA. 0n the other hand, the 111 changes in the amount of ABA in the epidermis were small, less than a doubling by the time the stomata were closed. Loss of potassium from the guard cells occurred in parallel with decreasesin stomatal aperture (Figure 3—8). Had the accumulation of ABA by the epidermis begun by the time stomata had started to close? The accumulation of ABA certainly did not start before stomatal closure; the two F processes became apparent at about the same time (Figure 3—6). The data in Figures 3-6 and 3—7 indicate that Commelina stomata closed to 50% of the original aperture with about a 10-20% increase in the level of ABA in the epidermis, and complete closure was associated with a 50-100% increase in level of ABA. How does that result compare to the amount of exogenous ABA that is required to close stomata in Commelina abaxial epidermis?l Raschke (1975a) showed that the amount of ABA, which detached leaves take up in the transpiration stream by the time stomatal closure is initiated, depends on the concentration in which the exogenous ABA is supplied. The amount of ABA increased with a power of about 0.67 of the concentration of ABA in the transpiration stream. The concentration in which endogenous ABA appears in the epidermis when leaves wilt is not known. A reasonable estimate may be that concentration of exogenous L 1The comparison will be affected by any variation in stomatal sensitivity to ABA. Stomatal sensitivity to ABA is affected by factors such as potassium (Cummins 1971), 002 supply (Raschke 1975a), or previous exposure to water stress (Davies 1978). 112 ABA which causes stomatal closure with a time course similar to the one which was observed in this study. According to Figure 3-6, significant stomatal closure occurred within about 30-40 min after the leaves were removed from their supply of water. In the experiments of Weyers and Hillman (1979), when (i)-[2—luC]ABA was fed via the transpiration stream to detached Commelina leaves, a concentration between 1 and 5 - 10-6M was required to obtain significant stomatal closure in 40 min. These authors used the 1MC activity to estimate how much ABA the leaves took up in 40 min and what percent of that was partitioned to the epidermis. Using the dry weight/area ratio in Table 3-1, the data of Weyers and Hillman (from their Table 2) were converted to give the estimate that significant stomatal closure would occur in 40 min by the addition of less than 0.12 ng (+)-ABA,2 but probably more than 0.03 ng (+)-ABA mg.l dry weight of epidermis. Whether this estimate amounts to a significant increase in epidermal ABA depends, of course, on how much ABA was in the epidermis to begin with, and, as mentioned, that level was variable. Using an average from all sources (this work, Singh gt gt. 1979, Ddrffling gt gt. 1980) of 0.1 ng mg-1 dry weight, epidermal ABA would be expected to increase by 30—120% as stomatal closure occurred. This projection and the results that were actually obtained (Figures 3-6 and 3-7) are not widely 2The estimate by Weyers and Hillman was divided by 2 and expressed as (+)-ABA because Cummins and Sondheimer (1973) demonstrated that the (-)-enantiomer has little or no effect on stomata. 113 disparate. It is still conceivable that ABA plays a major role in closing stomata during water stress. Would a doubling of the level of ABA in the abaxial epidermis represent a significant inmxmse in the level of ABA in the whole leaf, for instance an addition of 0.1 ng mg-l dry weight of epidermis (= 25 pg cm-2)? One cm2 of leaf would contain (at 0.235 ng mg‘l dry weight) 800 pg ABA. Thus, 25 pg would represent 3% of the total ABA in a cm2 section of an unstressed Commelina leaf. Such a small change could hardly be detected. Another estimate can be made, which, like the others, highlights the smallness of the active pool of ABA and, in doing so, makes a major role of ABA in regulating stomata seem credible. This is an estimate of how much the concentration of ABA might change around the guard cells if the level of ABA in the epidermis, as a whole, doubled. The ABA in the epidermis of an unstressed leaf may be rather uniformly distributed; since the basal level of ABA can vary by several—fold, presumably a large portion is not in contact with the guard cells. tt additional ABA arrives from the mesophyll during stress, there is reason to suspect preferential deposition of that ABA in the vicinity of the guard cells. Certainly ABA which enters the apoplast and moves with the transpiration stream ends up mostly near the guard cells (Weyers and Hillman 1979; Raschke 1979). The implication is clear, then, that what may be a modest increase in terms of the whole epidermis, may really be a 114 very large increase in the concentration of ABA near the guard cells. Commelina epidermis weighs roughly 4 mg fresh weight cm-2, and thus has a volume of about 4 ul cm_2. At 6000 stomata cm—2 and roughly 20 pl per guard cell pair (including cell walls) (Raschke 1979), the guard cells make up about 0.12 ul of the 4 ul cm"2 of epidermis, or 3% of the volume. If 30% of the additional ABA were to go to the guard cells, during a doubling of the level of ABA in the whole epidermis, then the concentration of ABA at the stomata would have increased lO-fold. In conclusion, the small and gradual increase in ABA in the epidermis, which paralleled the decrease in stomatal aperture in leaves of Commelina (Figures 3-6 and 3-7), is sufficient for us to still maintain the hypothesis that ABA is a major factor in closing stomata during water stress. The preceding calculations point out the difficulty we will have in proving whether or not the hypothesis is correct. The difficulty which is imposed by a requirement for only a very small active pool of ABA is compounded by the knowledge that stomata are sensitive to environmental and physiological parameters other than ABA that also pertain to plant water economy. As pointed out by Raschke (1979), biological control systems appear to depend on signals from more than one source. Efforts to evaluate the role of ABA would be greatly helped by the discovery of an inhibitor of endogenous pro- duction of ABA. Meanwhile, the following experiment might yield interesting results. We know that guard cells, but not ordinary epidermal cells, can retain their viability after 115 epidermal strips have been sonicated (Ogawa gt gt. 1978). We also know that compounds can be transferred from the mesophyll to the epidermis even if the epidermis has been removed and then replaced (Dittrich and Raschke 1977). If sonication removes the large background pool of ABA in the epidermis, then it should be easier to see a subsequent stress—induced accumulation of ABA after replacing the epidermis on the mesophyll and subsequently wilting the leaf sections. 3.4.4. Accumulation of ABA by tissue in solution. One of the purposes of this set of experiments was to check whether conditions, which induced the rest of the leaf to accumulate ABA, would also induce its accumulation in isolated epidermis. The results were negative (Figure 3-10). Loveys (1977) and Darffling gt gt. (1980) also did not detect any increase in ABA in stressed, isolated epidermis. The negative result was expected, since production of ABA by the epidermis would diminish the effectiveness of such a signal of water-stress coming from the mesophyll. The absence of accumulation of ABA by isolated epidermis certainly does not prove, however, that the mesophyll provides ABA to the epidermis during water stress. Several interpretations are possible, especially since mannitol treatment caused stomatal closure and epidermal cell breakage (Table 3—2). The main purpose of the experiments with solutions of mannitol was to develop a system in which mesophyll tissue, 116 in solution, would produce ABA just as well as dehydrated leaves do. This purpose was achieved. Incubation of mesophyll tissue (leaf minus lower epidermis) in basal medium plus 0.75M mannitol induced accumulation of ABA in 4 h to a greater degree than was observed in dehydrated leaves in 4 h (Figure 3-9 vs. Figure 3—2). Loveys (1977) reported higher levels of ABA in mesophyll tissue that was infiltrated with a 0.88M solution of mannitol rather than buffer, but the total ABA in that tissue plus incubation medium was, for unknown reasons, at a lower level than the content of ABA which Loveys reported for unstressed leaves. Recently Mawson gt gt. (1981) found enhanced production of ABA by leaf slices of Phaseolus vulgaris that were incubated in medium which contained 0.77M sorbitol. Others have found that addition of mannitol to suspension cultures caused enhanced production of ABA by grape pericarp tissue (Loveys gt gt. 1975) and tobacco cells (Wong and Sussex 1980). The results in Figure 3-9 are preliminary to extending the study that was presented in Chapter 2. There it was reported that leaves were slightly stimulated to accumulate ABA if turgor fell to within a few bars of being zero. The stimulation progressively increased as turgor approached zero. There was evidence of saturation of the response as wleaf was further reduced. The consequences of reducing turgor by osmotic stress should provide further insight on the relationship between loss of turgor and accumulation of ABA. The results in Figure 3-9 are, in fact, curious in 117 this respect. Commelina communis leaves are wilted at wleaf = —6.8 bar (Figure 3-5), and yet there was a large difference in the accumulation of ABA between Commelina tissue incubated in solution at w = -l3.6 bar versus -20.9 bar, even though both sets of samples were expected to have been without turgor. The phenomenon requires investigation; the possibility exists that the tissue at w = -l3.6 bar ‘ regained turgor part way through the incubation by taking up solute from the medium.3 That suggestion is supported by the following observation. As the leaf-halves wilted upon being immersed in either solution of mannitol, they curled '5! with the long axis along the circumference of the incubation tube, whereas the leaf-halves in buffer were turgid and hung vertically in the tube. By the end of the incubation period, the sections at w = -20.9 bar were still curled, but the sections at w = -l3.6 bar had straightened. An tg ttttg system of osmotic stress greatly increases the options for studying the induction of accumulation of ABA. Some examples follow. 1) An tg vitro system will allow direct measurements 3Adding the equivalent of 0.15 M KCl to the cell sap would have re-established turgor in the tissue that was incubated at w = 413.6 bar. If that had happened in 2 h, the required rate of K+ uptake would have been 75 umole g"1 fresh weight h-l. This rate is half that calculated for tgg mays guard cells in response to light (Raschke and Fellows 1971) and 3.6 times the rate reported for barley roots in 25 mM KCl (Epstein et a1. 1963). The required rate of K+ uptake would be reduced—by—the extent to which mannitol was taken up. It may be worth noting that K+ influx into cells of Valonia utricularis is stimulated by loss of turgor (Zimmermann and Steudle 1977). 118 of cell turgor with a pressure probe (Zimmermann and Steudle 1974). 2) Assuming that changes in turgor are monitored; it should be possible to decrease wleaf beyond the wilting point in order to find out if accumulation of ABA saturates once all cells in a sample have lost turgor. Attempts to decrease wleaf much beyond the wilting point, by using a pressure bomb to force water out of a leaf, were thwarted by breakage of cells. 3) Populations of cells can be uniformly exposed to osmotic stress so that one may be able to distinguish whether the progressive induction of accumulation of ABA that was observed in leaves was due to a progressive induction within the individual cells or to differences in the water relations among the cells in a leaf. 4) Turgor-dependent processes may depend on the exact position of the plasmalemma relative to the cell wall. Can protoplasts accumulate ABA? How does accumulation of ABA compare in response to osmotica which affect cells in different ways physically? Polyethylene glycol 6000 causes protoplast and cell wall to shrivel; mannitol causes cells to plasmolyze; ethylene glycol causes only a temporary loss of turgor since it penetrates cells rapidly. Ethylene glycol has been used to distinguish turgor-dependent processes since it affects wlea and n without affecting p f (Greenway gt gt. 1972). 5) An tg vitro system would facilitate testing the effect of potentially inhibitory or stimulatory compounds and testing hypotheses for turgor- transduction such as membrane compression (Coster gt gt. 1977) . 119 Chapter 4 Synthesis and metabolism of abscisic acid in detached leaves of Phaseolus vulgaris L. after loss and recovery of turgor 120 121 4.1. INTRODUCTION As demonstrated by the results presented in Chapters 2 and 3, loss of turgor causes the amount of ABA in a leaf to increase many—fold. Upon rehydration of a leaf, its ABA content returns to a level typical of leaves of unstressed plants (for review, see Wright 1978). The level of ABA will depend on the relative rates of synthesis and metabolic removal of this hormone. Knowledge of the factors that affect accumulation and disappearance of ABA will contribute to an understanding of how plants cope with water stress and recover from it. Harrison and Walton (1975) and Zeevaart and Milborrow (1976) showed that, in leaves of Phaseolus vulgaris, ABA is metabolized either by hydroxylation then rearrangement to phaseic acid (PA) followed by oxidation of PA to dihydrophaseic acid (DPA), or by formation of alkaline- hydrolyzable conjugated ABA. Of these pathways, the former appears to be the predominant one. Harrison and Walton (1975) fed (+)-[2-1uC]—ABA to leaves of t. vulgaris and found that metabolism of ABA continues during water stress. This chapter examines how the pool sizes of the important metabolites of ABA change with time during and after water stress, in order to compare the rate of synthesis with the rate of metabolic removal of ABA and to estimate how much each of the two pathways for the metabolism of ABA contributes to the reduction of 122 the ABA level in leaves during recovery from water stress. Recently, Zeevaart (1980) presented data on this subject obtained with detached leaves of Xanthium strumarium. Among his results was the finding that much more ABA is converted to PA than is being conjugated, both in wilted and rehydrated leaves of Xanthium. Chapter 2 (t.g. Pierce and Raschke 1980) provided evidence that turgor, rather than wlea or w, is the f critical parameter of leaf water status that affects accumulation of ABA. This chapter reports on the influence of turgor on the metabolism of ABA. Effects of changes in turgor were examined both with increasing severity of water stress and with increasing relief from water stress. This work is presented in Pierce and Raschke (1981). .fl- 9' 123 4.2. MATERIALS AND METHODS 4.2.1. Plants. Plants of Phaseolus vulgaris L. cv. Mecosta (red kidney bean) were cultivated as described in Section 2.2.1. The terminal leaflets of fully developed leaves were used for the experiments when the plants were 4.5-6 weeks old. For simplicity's sake, "leaf" is used throughout instead of "leaflet". 4.2.2. Experimental procedure. On the evening before an experiment was performed, a leaf was excised under water. I» The leaf was left to hydrate overnight by standing it in a beaker of water in a darkened cabinet (relative humidity close to 100% at 25°C). After hydration, the wleaf was always 3 —2 bar (1 bar = 0.1 MPa). The fresh weight of the leaf which corresponded to a wleaf of -2 bar was taken as the leaf's original fresh weight, and will hereafter be referred to as such. The measurements of wleaf were made with a pressure chamber as described in Section 2.2.3. In some experiments measurements of wleaf were made periodically on the same leaf as it became more and more dehydrated. Then, in order to determine turgor and n, inverse balancing pressures (= _w1eaf71) were plotted versus decreasing leaf fresh weight, as in Figure 4—1. In some experiments measurements of wleaf were also made by the dew-point method, as also described in Section 2.2.3. 124 Figure 4—1. Decrease in wleaf with loss in fresh weight for a leaf of Phaseolus vulgaris. (Inverse balancing —1 -wleaf ’ pressure = l/n = inverse bulk osmotic pressure.) 125 I I I I 0.4. Phaseolus vulgaris .. {s E \ S. I» § O'BP. J 8 \ ti o o. E 0.2- \o " 5 \. s \K ‘1’ _ .:o~ 4 S I I I I IOO 95 90 85 80 75 ‘70 of Original Fresh Weight Figure 4—1 126 The experiments were of three general types: (1) Accumulation of ABA and its metabolites in leaves of t. vulgaris was studied as a function of leaf water deficit. This type of experiment was performed as described in Sections 2.2.4, 2.2.5, and 2.2.6. (2) ABA synthesis and metabolism were studied as a function of time during and upon recovery from water stress. Individual, detached leaves were removed from their water supply and allowed to transpire until they reached 82% of their original fresh weight, the degree of water deficit which produced the greatest accumulation of ABA in the first type of experiment. They were then wrapped in foil and maintained at constant weight for up to about 8 h from the time the leaf lost turgor. Part of the leaf was removed from foil after usually 5 h, and fully rehydrated by floating on water in a covered dish for up to 0.5 h. This part of the leaf was kept further in the rehydrated condition. Sections of wilted and rehydrated parts of the leaf were frozen at various times for subsequent analysis of the levels of ABA and its metabolites. (3) Metabolism of ABA was studied as a function of degree of rehydration. Curves for inverse balancing pressure versus fresh weight were determined for leaves until they reached 82% of their original fresh weight. These curves were used to determine by what percentage the fresh weight of a wilted leaf section would have to be increased in order to re—establish a desired degree of turgor. After allowing 127 a wilted leaf to accumulate ABA for a period of about 5 h, leaf sections were cut. These sections were frozen immediately, left wilted for 3 h longer, or else rehydrated to varying degrees and kept for a further 3 h in the rehydrated state. Leaf sections were rehydrated by floating them on water in a covered dish until they reached the desired, pre-determined weight. 4.2.3. Analysis for ABA and its metabolites. Each lyOphilized sample was homogenized at room temperature in 15 ml methanol containing 1% (v/v) glacial acetic acid and 10 mg l-1 of antioxidant 2,6-di-tgtt-butyl-4-methylphenol (Milborrow and Mallaby 1975). The methanol extract was separated from the debris by vacuum filtration, and the debris was re-extracted by shaking overnight in another 15 ml of methanol. The second methanol fraction was combined with the first. Tritiated (i)-ABA, [3HJPA, [3HJDPA, and conjugated [3H]ABA (250 Bq each) were added to the methanol extract for monitoring recovery. Hereafter, (i)-[3H]ABA is referred to simply as [BHJABA. Five ml H20 were added to the methanol extract, and the methanol was evaporated under reduced pressure. Material insoluble in H20 was removed by filtration through a Millipore (Bedford, Mass., USA) AP prefilter. The filtered aqueous solution was loaded on a SEP-PAK 018 cartridge (Waters Associates, Milford, Mass., USA) and followed by a wash with 5 ml 1% aqueous acetic acid. This method of extraction and preliminary purification was highly 128 efficient. A single extraction of the tissue with methanol and subsequent purification of the treated extract on a SEP- PAK cartridge recovered more than 95% of the radioactivity added through the application of a known amount of 3[HJABA to leaf sections just prior to freezing. Ethanol and 1% acetic acid were combined in a ratio 40:60 (v/v), and ABA, PA, DPA, and conjugated ABA were all removed from the cartridge with 7 ml of this solvent mixture. A sample was evaporated to a smaller volume under reduced pressure, filtered through a 0.45-um Millipore filter, and evaporated to dryness under a stream of N2. Further purification was achieved with a high- performance liquid chromatography (HPLC) system (Model SP 8000; Spectra—Physics, Santa Clara, Cal., USA) using a procedure modified from the one developed by Zeevaart (1980). The two solvents used for the first HPLC column were re- distilled 95% ethanol (A) and 1% (v/v) aqueous acetic acid (B). A sample was dissolved in 0.5 ml of 10% A: 90% B and injected on a 0.5-ml sample loop connected to a guard column filled with ODS pellicular packing (Whatman, Clifton, N.J., USA) followed by an analytical column packed with 10 um Spherisorb ODS (Spectra-Physics). The sample was eluted with a linear gradient of 10 - 30% A in B in l h following 10 min at initial conditions. Solvent flow rate was 1 ml min.1 at 2 7 MPa (1000 pounds per square inch, psi). The collection times for ABA, PA, DPA, and conjugated ABA were based on retention times of standards, which were 129 detected in the HPLC eluant by absorption of light at 254 nm. Abscisic acid (racemic mixture) was purchased from Calbiochem, La Jolla, Cal., USA. Phaseic acid and DPA were a gift from T.D. Sharkey (see Sharkey and Raschke 1980). The conjugated ABA (B-D-glucopyranoside ester of (i)—ABA was a gift from J.A.D. Zeevaart (see Zeevaart 1980). The retention time of alkaline-hydrolyzable conjugated ABA in extracts of P. vulgaris leaves was originally determined from HPLC trials in which fractions throughout the gradient were subjected to mild alkaline hydrolysis and re—run, collecting at the retention time for ABA. Subsequently it was found that the retention time originally determined for alkaline-hydrolyzable conjugated ABA of E. vulgaris corresponded to the retention time of synthetic glucosyl ester of (i)—ABA and to the retention time of a major radioactive metabolite when [BHJABA was supplied to leaves of E. vulgaris. Conjugated ABA which was collected from the reverse- phase HPLC column was hydrolyzed at pH 13 (adjusted with 5 N NaOH) at 60°C for 30 min (Milborrow and Mallaby 1975). After hydrolysis, the pH was adjusted to 2.5 with phosphoric acid, and ABA was extracted from the solution by partitioning three times with an equal volume of ethyl acetate. The ABA in ethyl acetate was further purified using the same procedure as for the free-ABA fraction collected from the reverse-phase HPLC column. The fractions containing ABA, PA, or DPA were taken to dryness under a stream of N2 and methylated with diazomethane 130 in ethyl acetate. The methylated fractions were filtered through a 0.5-um Millipore FHLP filter and taken to dryness. A sample was dissolved in 0.5 ml of a mixture of ethyl acetate and hexane and injected on a 0.5-ml sample loop connected to a guard column filled with PAC pellicular packing (Whatman) followed by a uBondapak-NH2 analytical column (Waters Associates). A sample was injected and eluted with solvent of constant composition. A mixture of 40% ethyl acetate in hexane was used for ABA; 50% ethyl acetate in hexane was used for PA; 70% ethyl acetate in hexane was used for DPA. Solvent flow rate was 1 ml min-1 at g 2 MPa (300 psi). Retention times were determined with standards. The extracts of the individual leaf sections contained amounts of ABA and its metabolites too small to be analyzed quantitatively by absorption of light at 254 nm. Quantitative analysis was therefore performed by GLC-EC as described in Section 2.2.2. The concentrations of standards for calibration were determined by ultraviolet spectro- photometry in methanol using published extinction co- efficients (Milborrow and Robinson 1973, Harrison and Walton 1975). Samples were transferred to scintillation vials and taken to dryness. Overall recovery was estimated by determining the radioactivity in samples using a Packard (Downer's Grove, 111., USA) model 3255 Tri-Carb Liquid Scintillation Spectrometer. Recovery of ABA from the 131 original methanol extract was generally between 60 and 70%; recovery of the metabolites of ABA was generally between 40 and 60%. The [3HJABA, specific activity 8.3 - 10114 mol-l, was purchased from Amersham Corp., Arlington Heights, Bq 111., USA, prepared according to Walton gt gt. (1977). The [3HJPA, [3HJDPA, and conjugated [BHJABA were extracted and purified from t. vulgaris leaves which had been fed [3HJABA through the transpiration stream and incubated for 24 h (see Harrison and Walton 1975). The specific activities of the radioactive metabolites varied from one preparation to another. In no case did the amount of radioactive metabolite, which was added to a sample for monitoring recovery, equal more than 10% of the endogenous metabolite, and in most cases it was less than 1%. When warranted, the results were corrected for the quantity of radioactive material that was added to the samples. All results were corrected for degree of recovery and expressed as pmol mg-1 dry weight. 132 4.3. RESULTS 4.3.1. Accumulation of ABA and its metabolites as a function of leaf water deficit. Leaf samples were prepared according to the first type of experiment outlined above in Section 4.2.2: As a leaf last known amounts of water its wleaf was monitored with a pressure bomb and sections were fer cut and wrapped for incubation. A sample graph of the water relations parameters is shown in Figure 4-1. After about 8 h of incubation, a new steady-state level of ABA for each in wleaf had been reached in the samples (see Figure 2-3). e- The result of the experiment, shown in Figure 4—2,illustrates again what was found before for ABA (Figure 2-6): Eighty percent or more of the maximum increase in ABA content, that was developed above the unstressed level, occurred at a turgor of less than 1 bar (= 0.1 MPa). Contents of PA, conjugated ABA, and free ABA were nearly the same in samples having a high water potential (>—5 bar). Unstressed leaf sections contained about three times as much DPA as ABA. The DPA content was not measurably affected by decreasing water potential (<—5 bar) within the time of the experiment (3 10 h). Samples with a low wleaf contained twice as much conjugated ABA as samples with a high wleaf' In contrast, with loss of leaf turgor, PA content increased in parallel with increasing ABA content to more than 10 times the basal level. The sum of the contents of ABA and PA more accurately reflects how much ABA was synthesized during the incubation 133 Figure 4-2. The relationship between turgor ( °°°° ) and wleaf and the effect of wleaf on the content of ABA, PA, DPA, and conjugated ABA in samples from a single, detached leaf of Phaseolus vulgaris. 134 E a TcmlOOt' I I I I I I I I E . / 8 ' 7' \ 3 80 ABA I‘ T + 5 PA LI.J - _ I— P 6 60- o o l TURGORj' ‘6 (<6 ’ PAOK‘X A \ ... O 01 O N O 145 IO— / _. =_=_==._ I7“. ""' ' DPA I I I 0 2 4 6 IO I 8 HOURS FROM TURGOR = 0 Figure 4—5 146 PA to DPA. During stress and during recovery the formation of conjugated ABA occurred to a much smaller extent than did the formation of PA, and rehydration did not accelerate the conjugation of ABA. Unstressed bean leaves were found to contain about 3.8 pmol of conjugated ABA mg.1 dry weight. In the time-course experiment illustrated in Figures 4—4 and 4-5, the content of conjugated ABA increased to 11 pmol mng dry weight in the sample which was kept wilted 1 for 9.5 h and to 5 pmol mg_ dry weight in the sample which had been wilted but then was allowed to rehydrate for 4.5 h. H In another experiment, the corresponding wilted sample also contained more than twice as much conjugated ABA as the rehydrated sample did. Rates of synthesis and metabolism of ABA were estimated from the data of the time—course experiments. This was possible because detached leaves were used; translocation of ABA or its metabolites from the leaf did not contribute to the changes that were observed. The rate of synthesis of ABA equals the rate of change in the content of ABA plus the rates of change in the contents of its metabolites. The rate of metabolism of ABA equals the sum of the rates of change in the levels of just the metabolites of ABA. The maximum rate of synthesis of ABA averaged 15 pmol mg.1 dry weight h.1 in 5 experiments, with a range from 6 to 23 pmol mg-1 dry weight h-l. The calculations for the results shown in Figures 4-4 and 4-5 are presented in Figure 4-6. After wilting occurred, 147 Figure 4—6a,b. Effect of wilting (————) and rehydration (—---) on the average rate of synthesis (A) and metabolism (0) of ABA during intervals of time after loss of turgor in samples from a single, detached leaf of Phaseolus vulgaris. Panel (a): continued wilting. Panel (b): rehydration after 5 h. The data points are placed at the midpoints of the intervals. pmol ABA mg" aw h" 148 I I I I I I I 1 20b 0 “'- b . _I REHYDRATION /\ t It I I I5” .4.- I ‘ _ SYNTHESIS ‘ , \ ‘/ A ‘/ \l \ .0- -. I I _ II \ c ' ‘\ I / I METABOLISM o I 5_ O/ -- O/ \\ \ -I / / \ O . \‘ \. l l l l l I \\l a 2 4 s 8 2 4 5 8. I0 HOURS FROM TURGOR =0 Figure 4—6a,b 147 Figure 4-6a,b. Effect of wilting (————) and rehydration (----) on the average rate of synthesis (A) and metabolism (0) of ABA during intervals of time after loss of turgor in samples from a single, detached leaf of Phaseolus vulgaris. Panel (a): continued wilting. Panel (b): rehydration after 5 h. The data points are placed at the midpoints of the intervals. 148 I I I fl I 20" O J- b g _ REHYDRATION /\ I II I I 3; I5— 4 I I - 3 SYNTHESIS ‘ , \ ‘ 0 Q \‘ §‘|' ‘ To. A/ ‘/ \{ ‘ E IO- _- ‘ I 1 < ’H I m \ < t I " / I \ g 5_ METABatIsyo .. ./ \ I _ a / / \ I \ - . I l. ‘\ I I I I I l I \l a 2 4 6 8 2 4 5 8. HOURS FROM TURGOR =0 Figure 4—6a,b IO 149 the rate of synthesis of ABA jumped to a maximum that averaged 13 pmol mg_l dry weight h.l between 2.5 and 5 h. Thereafter, the rate of synthesis of ABA subsided to an average of 7 pmol mg.l dry weight h-l. In wilted tissue the rate of metabolism of ABA climbed stamfily until it matched the rate of synthesis between 7.5 and 9.5 h. Upon rehydration of the leaf tissue, the synthesis of ABA did not cease abruptly. The pool size of the metabolites of ABA increased by 13 pmol mg_1 dry weight, and the level of ABA fell by 3 pmol mg”l dry weight during the first hour after rehydration, which meant that ABA was being synthesized at an average rate of 10 pmol mg"l dry weight h-l, down from the maximum rate found just prior to rehydration. Synthesis of ABA fell rapidly after that to zero within about 3 h after rehydration. If the loss of stimulation of ABA synthesis had been the only effect of rehydration, ABA would have 1 ’ disappeared at a rate of 6 or 7 pmol mg-l dry weight h- which was the average rate of metabolism of ABA in wilted tissue. However, after 1 h of recovery, ABA disappeared at a rate of more than 19 pmol mg-1 dry weight h-l, which was possible only if rehydration actually enhanced the metabolic removal of ABA. Later, as synthesis of ABA slowed down and the conversion to PA proceeded rapidly, the supply of ABA was depleted and turnover of ABA slackened in the last period of recovery. wwwz M 150 The rate of accumulation of PA during the period of rehydration was compared with the rate in tissue that was left wilted. Results of five experiments are shown in Table 4—1. Individual leaves varied considerably in their response. In three experiments, rehydration caused increased production of PA; in two experiments no large change occurred (Table 4-1, left column). In spite of the declining size of the precursor pool, accumulation of PA in rehydrated bean leaves was equal to or greater than it was in wilted tissue. An acceleration of the conversion of ABA to PA becomes strikingly apparent if the rates of PA formation are related to the sizes of the ABA pools and coefficients of conversion are obtained (Table 4—1, right column). The same data also indicate that the relative rates of ABA conversion to PA in rehydrated tissue were similar in all experiments (between 0.4 and 0.5 pmol ABA converted pmol.1 ABA present h-l). What appears to have varied from one experiment to the next is the rate of conversion of ABA to PA in the wilted tissue. The cause of this variation is not known at present. 4.3.4. Conversion of ABA to PA as a function of degree of rehydration. In leaf samples differing in degree of water deficit, synthesis of ABA was stimulated by loss of turgor (Figure 4-2 and results in Chapter 2). Is regulation of the metabolism of ABA to PA also related to turgor? The following experiments were designed to test whether enhanced 1 m:.o 0.0H ooumnomCoC C m + oopHHz C m sm.o s.m mcHoHHs eoscHocoo m mm.o s.m ocomnosson C m + oooHHz C m mm.o :.oH mCHpHHz ooCCHpCoo : ms.o m.om eoomwoscop a mm.m + eooHHz C ms.m mH.o m.m mCHpHHz UCCCHpCoo m m” ms.o m.mH eoomnosgow C m.m + oooHHs c m Tl No.0 m.: mCHpHHz oosCHpCoo m Hm.o m.s eoomnessoh c m.m + eooHHs s m.m mo.o m.H wCHpHHB ooCCHpCoo H AHICV CQHmCo>Coo Co oCmHonCooo HIC 30 HIme am Hose wCHpHHz mo C m .xonddm wCHBOHHom COHpod wCszo < pCoEpmmCB .pdxm "COHmCo>Cod .pCommCo Coo oH CH mmCmCo 0C9 .ooumnozCoC mm: mmoH Como mo puma COHCB mmpmm .C m .xondom Com CopHHz who; mo>moq .mHmeHC> mCHommmCm mo mo>moH ooCowpoo .HmCoH>HCCH Eonz moHdEmm CH m CC» Co COHumCozCon oCo wCHpHHz mo poommm .HI: mHQmB 152 conversion of ABA to PA correlates with re-establishment of turgor. The experimental procedure was of the third type described in Section 4.2.2. In general, leaves were wilted and left to accumulate ABA for 5 h. Then leaf sections were rehydrated to varying degrees and 3 h later examined for their production of PA. A graph of Twleaf-l versus percent of original fresh weight (g.g. Figure 4—1), which was determined during dehydration of a leaf, was used as a calibration curve for calculating turgor when sections from the same leaf were subsequently rehydrated to different percentages of their original fresh weights. It was important to determine whether the original relation between wlea and fresh weight f could be used to calculate the turgor of the rehydrated samples. Figure 4-7 shows the results of one kind of test. A bean leaf was dehydrated, yielding one set of wleaf and fresh weight values. When the same leaf was rehydrated and then dehydrated a second time the second set of wleaf and fresh weight values yielded a curve which closely matched the original relationship. The two curves fell within about 0.25 bar of each other. In another kind of test (Figure 4-8) a relation between wleaf and fresh weight was determined using a pressure bomb, then the leaf was cut into sections, and these were rehydrated to varying degrees. The wlea values of the f rehydrated samples were determined by the dew-point method. Once again the agreement was good between the two sets of 153 Figure 4—7. A comparison of duplicate determinations on the same leaf of the relationship between wleaf and fresh weight. After the first set of measurements, the leaf was floated on water until it had rehydrated. Leaf Water Potential (bar) 154 O T I I I -l _ ' .1 ' Phaseolus vulgaris -21 ' Pressure-bomb Measurements 1 . 0 ISI Ran - I 2nd un 3r- I . _ I -4_ . _. I ’5— . _. O -6... —I II -7_ I . _ I I ’8’- I . _ I .. _9__ ' a . ._ I .. I I I l IOS I00 95 90 ,85 80 % of Original Fresh Weight Figure 4-7 I ‘1 “at. 155 Figure “—8. Measurements of wleaf’ which were determined with a pressure bomb, versus % of the leaf's original fresh weight compared to measurements of wleaf on sections of the same leaf, which were made by the dew-point method, versus the % to which those sections were rehydrated. Leaf Wafer Potential (bar) 156 O I I r -l _ _‘ Phaseolus vulgaris '2?— 0 Pressure - bomb Method - ' I Dew- point Method T"... -3 _ I — _ 'i .- ..4 _ _ . I -5 t— l d g N I -6 _ _ O ;7__ " q. - .— . O -8— ' ' ' . ~ I I - O '5?“ .1.- ‘_ I I I I00 95 9O 85 80 % of Original Fresh Weight Figure “-8 157 measurements. Figure “—8 shows that wle values of af individual leaf pieces, as determined by the dew-point method, mgy deviate by as much as 1 bar from wleaf values as originally determined by the pressure—bomb method for the entire leaf. Calculations from the original relation between wleaf and fresh weight were reasonable estimates of wleaf and turgor for the rehydrated samples. Bean leaf samples were prepared for analysis of the content of ABA and its metabolites. Tissue was frozen to assess the contents after 5 h of wilting. A number of leaf sections were rehydrated and incubated for another 3 h, after which they were also frozen. The results of this experiment are shown in Figure u—9. The changes in ABA, PA, or conjugated ABA from the levels after 5 h of wilting, that occurred during the 3 h recovery period, were plotted versus the wleaf which a sample had during the recovery period. The relation between wleaf and turgor for these samples is also indicated. The sample having the lowest wleaf {-10.9 bar) and no turgor had not been rehydrated at all. It can be compared to the samples in the time course experiments (Figures A-A and “-5) which were kept wilted throughout the experiment. The sample having the highest wleaf (-2.3 bar) and the highest turgor (5.6 bar) had been rehydrated to 100% of its original fresh weight and can be compared to the samples which were rehydrated in the time course experiments. Rehydration triggered the disappearance of ABA and l58 Figure A-9. The effect of degree of rehydration on the change in content of ABA, PA, and conjugated ABA in sections of a single leaf of Phaseolus vulgaris during a 3 h period of recovery from water stress. Degree of rehydration is indicated by (i) water potential and (ii) turgor which the leaf sections had during the period of recovery. I I A N N «b m (I) O O O O O O O CHANGE IN CONTENT DURING 3h OF RECOVERY (pmol mg" DW) .0: OOO -|O 159 TURGOR (bar) .‘ I \_,-/- ABA l l l l l l l l 7T — A : /A\‘/A \ ‘A . _ CONJUGATED ABA _ -l2 -IO -8 -é -4 -2 0 WATER POTENTIAL (bar) Figure “-9 160 increased the production of PA (Figure “-9). Samples having turgor accumulated more than twice as much PA during the recovery period than did the sample having the lowest wleaf' In all samples, the magnitude of the decrease in ABA was less than the rise in PA, which indicates that synthesis of ABA must have continued during at least part of the recovery period. These results are consistent with those presented in Figures “-4 and A—5. Increased synthesis of PA and disappearance of ABA was not a linear function of the degree of rehydration. Re-establishment of the slightest turgor was sufficient to elicit maximum enhancement of the conversion of ABA to PA. We cannot tell whether the response anticipated re- establishment of turgor in the leaf or not. The position of zero turgor on the wleaf scale in Figure “-9 could be incorrect by about i 1 bar (refer to Figure 4-8). Re- establishment of turgor did not increase the production of conjugated ABA (Figure A-9). , lbl 4.“. DISCUSSION A.A.l. Synthesis and metabolism of ABA after loss and gain of turgor. The results of these experiments demonstrate that leaf turgor affects both the metabolic system which catalyzes the synthesis of ABA, and that which catalyzes the conversion of ABA to PA. The systems respond to changes in turgor in opposite ways. Synthesis of ABA is more rapid in wilted than in turgid leaves; metabolism of ABA to PA is more rapid in leaves that have regained turgor 1.1....11' than in wilted leaves. The results are a net accumulation of ABA in wilted tissue, and a decline of the ABA content to a low level in tissue to which turgor has returned. A.A.2. Stimulation of ABA synthesis after loss of turgor. The conclusion that turgor loss causes an increase in the rate of synthesis of ABA is based on the elimination of other possible causes of ABA accumulation, namely (1) inhibiton of the metabolism of ABA to PA, (2) release of ABA from a conjugated form, or (3) inhibition of the formation of ABA conjugates. With respect to the first possibility, Harrison and Walton (1975) were the first to showtimt metabolism of ABA continued in leaves under water stress. The extents to which [luCJABA disappeared and labeled products appeared in the tissue were the same in both stressed and unstressed leaves. The content of PA and DPA actually built up in wilted bean leaves. As shown by Harrison and Walton (1975) and indicated by Figures A-2 and .I'm- 162 A-S, inhibition of the metabolism of ABA through the PA - DPA pathway was not responsible for the accumulation of ABA that had occurred. Hydrolysis of ABA conjugates could not have been the source of free ABA either. As demonstrated by Figure 4-2, bean leaves which had not experienced water stress did not contain sufficient alkaline-labile conjugated ABA to yield the amounts of ABA which appeared in the wilted 1‘- tissue (see Zeevaart 1980 and review by Wright 1978). Furthermore, Milborrow and Noddle (1970) obtained more conversion of labeled mevalonate to ABA in wilted than in turgid wheat leaves. Turning to the third possibility, we I see from Figure “-2 that the level of conjugated ABA was ‘ slightly higher in wilted than in turgid leaves. Inhibition of the metabolism of ABA to conjugated ABA cannot have been the cause of the observed accumulation of ABA. As the pathways via PA and alkaline-labile conjugated ABA account for the bulk of the metabolism of ABA in bean leaves (g.g. Figure “-3), it follows that the principal cause of the accumulation of ABA in water—stressed bean leaves was the stimulation of the synthesis of ABA. Simultaneous with the accumulation of ABA in wilted leaves occurred an accumulation of PA and, to a much smaller degree, of conjugated ABA (Fignes 4—2 and “—5). Since ABA and PA (and DPA) increased in parallel fashion with respect to changes in wleaf (Figure A-Z), and with respect to time (Fighxfi A-A and A-S), the accumulation of the metabolites of ABA in wilted tissue can be explained as an effect of mass 163 action of ABA. With confidence that determinations of changes in the levels of PA, DPA, and alkaline-labile conjugated ABA would account for the metabolism of ABA during a short-term wilt- recovery cycle in bean leaves (see Figure “-3 and Harrison and Walton 1975), rates of synthesis and metabolism of ABA were calculated. Seven h after turgor was lost, accumulation of ABA leveled off, then production and metabolic removal of ABA balanced each other. At this time, the rate of synthesis or metabolism was77pmol mg-l dry weight b.1 (Figure “—6). This is about two times the rate Harrison and Walton (1975) determined for a steady-state level of ABA in wilted primary leaves of two-week old bean plants (at 15% dry weight, 0.15 ug ABA g-1 fresh weight h-l correspond to 3.8 pmol mg-1 1). dry weight h‘ Harrison and Walton felt that twice the rate they calculated was closer to being accurate. Wilted Xanthium leaves appear to possess a capacity to synthesize and metabolize ABA at rates very similar to those of wilted bean leaves. The data of Zeevaart (1980, Figures 3 and A, time interval 5-l2 h) indicate a rate of 8 pmol mg.l dry 1 weight h- for the steady state. A.A.3. Stimulation of ABA metabolism after recovery of turgor. After the return of turgor to the tissue, conversion of ABA to PA accelerated (Figure A-S, A-9, Table A-l). Since this stimulation of the metabolism of ABA occurred at a time of declining ABA content (Figures A—A and A-9), recovery of turgor must have caused an increase in Twas 16A the amount or the activity of the enzyme(s) involved in the .conversion of ABA to PA; or, possibly, recovery of turgor increased the accessibility of critical substrates to the enzyme(s). A stimulation of the conversion of ABA to PA after rehydration was also discovered by Zeevaart (1980) in leaves of Xanthium strumarium. Previously, it was possible to consider that the concentration of ABA falls when plants are re-watered because of diminished capacity for synthesis in combination with, as Milborrow (1979) wrote, "a constant, large degradative capacity for ABA...". The evidence indicates that the capacity of bean leaves to remove ABA by metabolism is not constant but increases when turgor returns to a wilted leaf (Table A-l, Figure A—9). This acceleration becomes particularly evident when the rate of ABA conversion is expressed relative to the amount of ABA present in the tissue (yielding a coefficient of conversion; second column in Table “-1). In rehydrated tissue, almost one half of the amount of ABA present was converted to PA.per h (O.AA : l 0.06 h'l, as compared to 0.16 i 0.11 h' in wilted tissue). An increase in the rate of conversion appears also in Zeevaart's data on Xanthium leaves. Calculating from Figures 3 and A of Zeevaart, during periods when PA was increasing linearly with time, the coefficient of conversion averaged l O.AO h- for leaves rehydrated after four hours of wilting (time interval: O-A h after rehydration), and it averaged l 0.12 h" for wilted leaves (time interval: A-IZ h of wilting). 165 The stimulation of the metabolic removal of ABA after rehydration was restricted to the PA pathway. Conjugation of ABA continued at a low rate, if at all (Figure A-9). An early report by Hiron and Wright (1973) indicated some conversion of ABA to conjugated ABA during recovery of bean seedlings from water stress. But neither this work with bean leaves, nor that of D8rffling _e_t a_l_. (1974), who tested F pea seedlings, nor that of Zeevaart (1980), who analyzed Xanthium leaves, showed the formation of conjugated ABA to be of importance during short-term stress and recovery from E it. Prolonged or repeated stress may be a different matter: w leaves from cotton plants which had experienced 8 wilt- recovery cycles over the course of two weeks contained 20 to 30 times more conjugated ABA than did comparable samples from unstressed plants. (See also Hiron and Wright 1973, Zeevaart 1980.) CONCLUSION Abscisic acid has come to be considered a plant "stress hormone" (Jones 1978). In the biological control system by which plants regulate CO2 and water vapor exchange,Raschke (1979) positioned ABA as a signal in a feed— back loop controlling plant water status. The results of this Umsis strengthen the candidacy of ABA as an important signal. Accumulation of ABA and disappearance of ABA from leaf tissue are tightly linked to loss and recovery of turgor (Chapters 2 and A). Plants have the ability to adapt to stress conditions by adjustment of their solute content (Turner and Jones 1980). Linking the control of ABA level to turgor automatically takes into account the plant's success in making an osmotic adjustment, when ABA is produced as a signal of water stress. Abscisic acid appears to play an important role in the way plants cope with water stress. In wilting Commelina leaves, an increase in ABA was obvious in the epidermis before it was obvious in the mesophyll, and the increase paralleled stomatal closure (Chapter 3). At least in this Species, ABA is likely to be an important factor in causing stomatal closure during water stress. Stomatal closure in response to ABA is probably only one among several 166 167 ameliorative reactions. For instance, ion transport as well as water permeability of roots have been reported to be enhanced by ABA (Glinka 1980). Changes in ABA are net changes from effects on both synthesis and metabolism of ABA (Chapter A and Zeevaart 1980). Conversion of ABA to PA continues when ABA is being synthesized in wilted tissue, and, coupled with slackening of ABA synthesis, leads to a steady state. Synthesis of ABA continues for about an hour after turgor has been regained and conversion of ABA to PA has been accelerated. Thereafter, synthesis of ABA declines. Accumulation of ABA sets in before turgor of the bulk of the leaf has become zero; conversion of ABA to PA is accelerated at the slightest indication of a regain of turgor (Chapters 2 and A). Anticipatory production of removal of ABA could be caused by deviations from bulk properties in the solute content or the cell—wall elasticity of individual cells in the tissue, but turgor sensing may also be a progressive response in individual cells (Zimmermann (1978) . Of the questions arising from this work, what the turgor sensor is and how it causes changes in the opposing mechanisms of synthesis and metabolic removal of ABA appear particularly important and challenging. BIBLIOGRAPHY BIBLIOGRAPHY Acevedo, E., Fereres, E., Hsiao, T.C., Henderson, D.W. (1979) Diurnal growth trends, water potential, and osmotic adjustment of maize and sorghum leaves in the field. Plant Physiol. 83, A76-A80 Addicott, F. T., Lyon, J.L. (1969) Physiology of abscisic acid and related substances. Ann. Rev. Plant Physiol. 29, 139-164 Assante, G., Merlini, L., Nasini, G. (1977) (+)-Abscisic acid, a metabolite of the fungus Cercospora rosicola. Experientia 33, 1556—1557 If 1.! 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