Rapid adjustment of guard-cell abscisic Acid levels to current leaf-water status

Detached broad bean (Vicia faba L.) leaflets were water stressed; within 15 minutes, guard-cell abscisic acid (ABA) concentration increased ninefold. This result eliminates the apparent discrepancy raised by reports of no correlation between initial water-stress effects on stomata and leaf ABA concentration. Six hours after stress relief, guard-cell ABA concentration was near the prestress value, which would seem to implicate other factors in stress aftereffects on stomata.     

Stomatal responses of Argenteum — a mutant of Pisum sativum L. with readily detachable leaf epidermis

Epidermis is easily detached from both adaxial and abaxial surfaces of leaf four of the Argenteum mutant of Pisum sativum L. The isolated epidermis has stomata with large, easily-measured pores. Hairs and glands are absent. The density of stomata is high and contamination by mesophyll cells is low. In the light and in CO₂-free air, stomata in isolated adaxial epidermis of Argenteum mutant opened maximally after 4 h incubation at 25°C. The response of stomata to light was dependent on the concentration of KCl in the incubation medium and was maximal at 50 mol m^-3 KCl. Stomata did not respond to exogenous kinetin, but apertures were reduced by incubation of epidermis on solutions containing between 10^-5 and 10^-1 mol m^-3 abscisic acid (ABA). The responses of stomata of Argenteum mutant to light, exogenous KCl, ABA and kinetin were comparable with those described previously for stomata in isolated epidermis of Commelina communis. A method for preparing viable protoplasts of guard cells from isolated epidermis of Argenteum mutant is described. The response of guard cell protoplasts to light, exogenous KCl, ABA and kinetin were similar to those of stomata in isolated epidermis except that the increase in volume of the protoplasts in response to light was maximal at a lower concentration of KCl (10 mol m^-3) and that protoplasts responded more rapidly to light than stomata in isolated epidermis. The protoplasts did not respond to exogenous kinetin, but when incubated for 1 h in the light and in CO₂-free air on a solution containing 10^-3 mol m^-3 ABA, they decreased in volume by 30%. The advantages of using epidermis from Argenteum mutant for experiments on stomatal movements are discussed.Abbreviations ABA abscisic acid - MES 2-(N-morpholino)ethanesulfonic acid     

Reversal of Abscisic Acid-Induced Stomatal Closure by trans-Cinnamic and p-Coumaric Acid

Abscisic acid (ABA)-induced increase in stomatal diffusive resistance (SDR) in excised leaves of bean (Phaseolus vulgaris L. cv Pencil Pod) and maize (Zea mays L. cv Golden Bantam) is inhibited by low concentrations of trans-cinnamic acid (TCA) (1 micromolar) and p-coumaric acid (PCA) (10 micromolar) when given together with ABA (10 micromolar) in the transpiration stream through the cut end of the petiole or leaf blade. A concentration effect is observed both in the ABA action and its reversal by phenolic acids. Leaves having attained a high diffusive resistance in ABA solution recover rapidly when transferred to water. ABA (10 micromolar) induced closure of the stomata in onion, Allium cepa L. and Vicia faba epidermal peels. This is associated with loss of K⁺ from guard cells. In the presence of TCA (10 micromolar) and PCA (10 micromolar) K⁺ is retained in the guard cells with open stomata. The dark closure of stomata is also inhibited by TCA and PCA. It is suggested that these phenolic acids may inhibit the ABA effect by competing with or acting on some ABA-specific site, probably located on the plasma membrane, regulating flux of K⁺ ions. A weak association of ABA with the plasma membrane is envisaged because of the rapid recovery obtained upon transferral of the leaves to water.     

The Role of Abscisic Acid in Acclimation of Plants Cultivated in vitro to ex vitro Conditions

The content of endogenous free abscisic acid (ABA) in the shoots of in vitro cultivated tobacco (Nicotiana tabacum L. cv. White Burley) and its changes during ex vitro acclimation of these plants to the greenhouse or growth chamber were estimated. The content of free ABA significantly increased at the 1^st and/or 2^nd day after plant transfer from in vitro to ex vitro. The ABA content of plants covered with transparent foil to maintain higher relative humidity (RH), did not significantly differ from ABA content of plants cultivated under ambient RH. Transfer to fresh medium also transiently increased the content of endogenous ABA. The ABA content in plants, which had been acclimated for 1 week to ex vitro conditions, decreased to the content found in the in vitro plants. Acclimation to ex vitro conditions affected the stomata on adaxial and abaxial sides differently: stomata on the adaxial side were less open than those on the abaxial one. The exogenous application of 5 μM ABA increased transiently its endogenous concentration in shoots of in vitro plants more than ten fold, but after 1 week the concentration in the shoots decreased. This revised version was published online in July 2006 with corrections to the Cover Date.     

Effects of patchy stomatal closure on gas exchange measurements following abscisic acid treatment

Gas exchange data and images of leaf fluorescence were collected concurrently as stomata responded to abscisic acid (ABA) application. When 10^?5kmolm^?3 ABA was applied to the transpiration stream in a short pulse, stomatal conductance (g_s), photosynthesis (A) and intercellular CO2 concentration (C_i) decreased rapidly after a short lag period and became approximately constant after 2h. There was an apparent reduction in the A versus c1 relationship as stomata closed, but the data returned to the A versus C1 curve while stomatal conductance was constant or slowly rising during the second hour after ABA treatment. Larger amounts of ABA administered during the pulse caused larger deviations from the A versus c1 relationship. When 10^?7kmolm^?3 ABA was applied continuously through the transpiration stream, gs, A and Cⁱ decreased, but there was no substantial deviation from the A versus c{ curve. Fluorescence images were patchy as stomata closed for all experiments, but became slowly more uniform during the time that gas exchange was returning to the A versus Cj curve. The distribution of con-ductance among patches was not bimodal, and larger devi-ations from the A versus ct curve had greater ranges of pixel values and more pixel values representing low values of Cj during stomatal closure than did experiments show-ing small or no deviation. Estimates of A and gs from fluo-rescence images compared favourably with measured val-ues in most cases, suggesting that the patchy distributions of fluorescence were caused by patchy distributions of stomatal conductance and that apparent reductions in the A versus ct relationship were the result of these patchy stomatai distributions and not direct effects of ABA on mesophyll functioning. The data show that stomatal patches can be temporary and that patchiness may not be reflected in gas exchange data if the range of stomatal con-ductances is not large. These observations may explain some of the discrepancies among previous studies concerning the effect of ABA on the A versus Cⁱ relationship.     

Phloem transport of abscisic acid in Ricinus communis L. seedlings

Sieve tube sap exuded from the cut hypocotyl of castor bean seedlings (Ricinus communis L.) was found to contain 0.2–0.5 mmol m^?3abscisic acid (ABA). The ABA concentration in the sieve tube sap always exceeded that in root pressure exudate under a wide range of water supply. Exudation of sieve tube sap from the cut hypocotyls caused water loss, and this induced ‘water shortage’ in the cotyledons which resulted in the ABA concentration in the cotyledons increasing by 3-fold and that in the sieve tube sap increasing by up to 50-fold within 7h. The wounded surface of the cut hypocotyl was not responsible for the ABA increase. Incubation of the cotyledons of endosperm-free seedlings in various ABA concentrations (up to 100 mmol m^?3) increased the ABA concentration in sieve tube sap. The concomitant increase in ABA, both in cotyledons and in sieve tube sap, had no effect on the phloem loading of sucrose, K⁺ and Mg²⁺ within the experimental period, i.e. up to 10h. It can be concluded that (i) the phloem is an important transport path for ABA, (ii) water stress at the phloem loading sites elevates phloem-mobile ABA, which may then serve as a water stress signal for sinks, for example stem and roots (not only for stomata), and (iii) the ABA concentration of cells next to or in the phloem is more important than the average ABA content in the whole cotyledon for determining the ABA concentration in sieve tube sap.     

Uptake and distribution of abscisic acid in Commelina leaf epidermis

Closure of stomata by abscisic acid (ABA) was studied by floating leaf epidermal strips of Commelina communis L. in PIPES buffer (pH 6.8) containing a range of KCl concentrations. Control apertures were greatest at high concentrations of the salt, and the effects of ABA, in terms of closure, were most pronounced below 100 mol m^-3 KCl. Stomata opened on strips floated on buffer plus 50 mol m^-3 KCl and closed within 10 min when transferred to the same medium plus 0.1 mol m^-3 ABA. [2-¹⁴C]ABA was used to study uptake and distribution of the hormone by the epidermal strips. It was calculated that no more than 6 fmol ABA were present per stomatal complex at the time of closure, although uptake continued thereafter. Microautoradiography indicated that radioactivity from [2-¹⁴C]ABA accumulated in the stomatal complex at or near the guard cells within 20 min. TLC was used to examine the state of the label after 1 h incubation. Efflux of label from preincubated tissue appeared to occur in three phases (t_1/2=7.2 s, 4.0 min, 35.2 min). Efflux was correlated with stomatal re-opening. The results confirm that ABA can accumulate in the epidermis of C. communis.Abbreviation ABA Abscisic acid     

Sensitivity of Commelina stomata to abscisic acid

Stomata of Commelina leaves pre-opened by incubation in moist air were found to close within 30 min when supplied with abscisic acid (ABA) via the transpiration stream. Radioactive ABA had similar effects, but allowed the distribution of the compound within the leaf to be measured and correlated with stomatal movements to give estimates of the sensitivity of Commelina stomata. On a whole-leaf basis, less than 163 fmol ABA per mm² leaf area were present at the time of complete stomatal closure. This was close to other published estimates. By taking epidermal ¹⁴C measurements, however, it was possible to increase the accuracy of the estimate on the assumption that only ABA present in the epidermis was physiologically active. Thus, less than 235 amol ABA for stomatal complex were present at complete closure, and statistically significant narrowing of the stomatal aperture had occurred when between 12.6 and 45.4 amol per complex were present. The distribution of ABA within the epidermal tissue after transpiration-stream application was studied using microautoradiography, and the compound appeared to have accumulated within the stomatal complex.Abbreviations ABA abscisic acid - TLC thin-layer chromatography     

The kinetics of calcium uptake by roots

Summary Barley leaves extracted 35 min after a 15-min pulse of 10^-6 M ¹⁴C-labelled cis,trans-(±)-abscisic acid (ABA) showed very little breakdown of the ABA. Leaves extracted 2 h after a 30-min pulse of 10^-5 M labelled ABA showed at least 34% degradation of the hormone. The pattern of degradation was very similar for leaves kept in either light or darkness following the labelling pulse, and the products formed resembled those described in tomato fruit and bean axes. No detectable isomerization to the physiologically inactive trans,trans-(±)-ABA isomer occurred in these leaves during the 2-h period. ABA-induced stomatal closure is partially reduced 30 min after the pulse. The reversal cannot be attributed to catabolism of the hormone and must be explained by removal of the hormone into storage sites where it cannot act on the stomata.     

The effects of two abscisic acid analogues, WL19224 and WL19377, on stomatal closure

The effect on stomatal closure by ABA and its analogues, WL19224 and WL19377 was investigated. The rate of closure showed a sigmoid curve when various concentrations of ABA were applied. A concentration of 10^-9 M ABA was the threshold for stomatal closure; maximal closure occurred at higher concentrations (from 10^-6 M to 10^-3 M). Use of the analogue WL19224 resulted in similar closure responses. However, ABA was more effective at lower concentrations. For example, at 10^-3 M of either WL19224 and ABA, stomata closed to 2.2 μm and about 3 μm, respectively. In contrast, applications of the ABA analogue WL19377 had no effect on stomatal closure. In fact, at concentrations of WL19377 higher than 10^-4 M, stomata were stimulated to open, to about 10% of their initial size. Likewise, applications of WL19377 along with ABA, inhibited ABA-induced stomatal closure. This inhibition was linearly related to the concentrations of the compounds applied. In conclusion, the structural requirements for biological activity of ABA and its analogues cannot be considered individually, but must be assessed for their roles as part of an entire functional group. Although compounds may have similar structures, their ability to control certain physiological activities may be quite different.     

Syringomycin, a bacterial phytotoxin, closes stomata

The effects of the bacterial phytotoxin, syringomycin, on stomata were investigated using detached leaves of Xanthium strumarium and isolated epidermes of Vicia faba. Syringomycin is known to cause K⁺ efflux in fungal and higher plant cells. Doses of syringomycin as low as 0.3 unit per square centimeter (about 0.88 pmole per square centimeter) resulted in measurable stomatal closure when applied through the transpiration stream of detached leaves; higher doses produced larger reductions in stomatal conductance. Stomatal apertures of isolated epidermes were also reduced by low concentrations (3.2 units per milliliter; 10⁻⁸ molar) of syringomycin. The effects of syringomycin were similar to those of ABA. Both compounds closed stomata at a similar rate and at similar concentrations. In addition, neither compound significantly affected the relationship between photosynthesis and intercellular CO₂ based on data taken after stomatal conductance had stabilized following the treatment. It is possible that syringomycin and ABA activate the same K⁺ export system in guard cells, and syringomycin may be a valuable tool for studying the molecular basis of ABA effects on guard cells.     

Compartmental distribution and redistribution of abscisic acid in intact leaves

Using a computer model written for whole leaves (Slovik et al. 1992, Planta 187, 14–25) we present in this paper calculations of abscisic acid (ABA) redistribution among different leaf tissues and their compartments in relation to stomatal regulation under drought stress. The model calculations are based on experimental data and biophysical laws. They yield the following results and postulates: (i) Under stress, compartmental pH-shifts come about as a consequence of the inhibition of the pH component of proton-motive forces at the plasmalemma. There is a decrease of net proton fluxes by about 8.6 nmol · s^–1 · m^–2. (ii) Using stress-induced pH-shifts we demonstrate how stress intensities can be quantified on a molecular basis. (iii) As the weak acid ABA is the only phytohormone which behaves in vivo and in vitro ideally according to the Henderson-Hasselbalch equation, pH-shifts induce a complicated redistribution amongst compartments in the model leaf. (iv) The final accumulation of ABA in guard-cell walls is intensive: up to 16.1-fold compared with only up to 3.4-fold in the guard-cell cytosol. We propose that the binding site of the guard-cell ABA receptor faces the apoplasm. (v) A twoto three-fold ABA accumulation in guard-cell walls is sufficient to induce closure of stomata. (vi) The minimum time lag until stomata start to close is 1–5 min; it depends on the stress intensity and on the guard-cell sensitivity to ABA: the more moderate the stress is, the later stomata start to close or they do not close at all. (vii) In the short term, there is almost no influence of the velocity of pH-shifts on the velocity of the ABA redistribution, (viii) Six hours after the termination of stress there is still an ABA concentration 1.4-fold the initial level in the guard-cell cytosol (delay of ABA relaxation, aftereffect), (ix) The observed induction of net ABA synthesis after onset of stress may be explained by a decrease in cytosolic ABA degradation. About 1 h after onset of stress the model leaf would start to synthesise ABA (and its conjugates) automatically, (x) This ABA net synthesis serves to inform roots via an increased ABA concentration in the phloem sap. The stress-induced ABA redistribution is per se not sufficient to feed the ploem sap with ABA. (xi) The primary target membrane of stress is the plasmalemma, not thylakoids. (xii) The effective stress sensor, which induces the proposed signal chain finally leading to stomatal closure, is located in epidermal cells. Mesophyll cells are not capable of creating a significant ABA signal to guard cells if the epidermal plasmalemma conductance to undissociated molecular species of ABA (HABA) is indeed higher than the plasmalemma conductance of the mesophyll (plasmodesmata open), (xiii) All model conclusions which can be compared with independent experimental data quantitatively fit to them. We conclude that the basic experimental data of the model are consistent. A stress-induced ABA redistribution in the leaf lamina elicits stomatal closure.Abbreviations ABA abscisic acid - CON vacuolar ABA conjugates We are grateful to Prof. U. Heber (Lehrstuhl Botanik I, University of Würzburg, FRG) for stimulating discussions. This work has been performed within the research program of the Sonderforschungsbereich 251 (TP 3 and 4) of the University of Würzburg. It has been also supported by the Fonds der Chemischen Industrie.     

Stomatal responses to vapour pressure deficit are regulated by high speed gene expression in angiosperms

Plants dynamically regulate water use by the movement of stomata on the surface of leaves. Stomatal responses to changes in vapour pressure deficit (VPD) are the principal regulator of daytime transpiration and water use efficiency in land plants. In angiosperms, stomatal responses to VPD appear to be regulated by the phytohormone abscisic acid (ABA), yet the origin of this ABA is controversial. After a 20 min exposure of plants, from three diverse angiosperm species, to a doubling in VPD, stomata closed, foliar ABA levels increased and the expression of the gene encoding the key, rate‐limiting carotenoid cleavage enzyme (9‐cis‐epoxycarotenoid dioxygenase, NCED) in the ABA biosynthetic pathway was significantly up‐regulated. The NCED gene was the only gene in the ABA biosynthetic pathway to be up‐regulated over the short time scale corresponding to the response of stomata. The closure of stomata and rapid increase in foliar ABA levels could not be explained by the release of ABA from internal stores in the leaf or the hydrolysis of the conjugate ABA‐glucose ester. These results implicate an extremely rapid de novo biosynthesis of ABA, mediated by a single gene, as the means by which angiosperm stomata respond to natural changes in VPD.     

Abscisic Acid Regulation of DC8, A Carrot Embryonic Gene

DC8 encodes a hydrophylic 66 kilodalton protein located in the cytoplasm and cell walls of carrot (Daucus carota) embryo and endosperm. During somatic embryogenesis, the levels of DC8 mRNA and protein begin to increase 5 days after removal of auxin. To study the role of abscisic acid (ABA) in the regulation of DC8 gene, fluridone, 1-methyl-3-phenyl,-5(3-trifluoro-methyl-phenyl)-4(1H)-pyridinone, was used to inhibit the endogenous ABA content of the embryos. Fluridone, 50 micrograms per milliliter, effectively inhibits the accumulation of ABA in globular-tage enbryos. Western and Northern analysis show that when fluridone is added to the culture medium DC8 protein and mRNA decrease to very low levels. ABA added to fluridone supplemented culture media restores the DC8 protein and mRNA to control levels. Globular-stage embryos contain 0.9 to 1.4 × 10⁻⁷ molar ABA while 10⁻⁶ molar exogenously supplied ABA is the optimal concentration for restoration of DC8 protein accumulation in fluridone-treated embryos. The mRNA level is increased after 15 minutes of ABA addition and reaches maximal levels by 60 minutes. Evidence is presented that, unlike other ABA-regulated genes, DC8 is not induced in nonembryonic tissues via desiccation nor addition of ABA.     

Action of proline on stomata differs from that of abscisic Acid, g-substances, or methyl jasmonate

Methyl jasmonate (MJ) and a mixture of G₁, G₂, and G₃ (G-substances) inhibited stomatal opening in abaxial epidermis of Commelina benghalensis and complete closure occurred at 10⁻⁶ molar MJ, or 10⁻³ molar G-substances compared to 10⁻⁵ molar abscisic acid (ABA). Proline, even at 10⁻³ molar caused only a partial stomatal closure. Apart from ABA, other endogenous plant growth regulators do regulate stomata. Reduction in the stimulation by fusicoccin and complete stomatal closure, at 30 millimolar KCl or less, were affected by ABA, MJ, or G-substances, but not by proline. The action of MJ or G-substances was similar to ABA in decreasing proton efflux and the levels of potassium, malate, or reducing sugars. Proline, however, interfered with starch-sugar interconversion but had no effect on proton efflux or potassium content of epidermis.     

Ineffectiveness of Abscisic Acid in Stomatal Closure of Yellow Lupin, Lupinus luteus var. Weiko III

Stomata of yellow lupin leaves are remarkably insensitive toabscisic acid (ABA). Stomatal resistance was monitored usingboth a viscous now porometer and a diffusion porometer. Resultswere confirmed with scanning electron microscopy. When exogenousABA solutions were supplied via petioles, 10^–6 M solutionshad no effect on stomatal resistance. Upper (adaxial) stomatawere not affected by 10^–5 M ABA but lower stomata showed3-fold more resistance after 2 h. Stomata of both surfaces closedafter 30 min in 10^–4 M ABA. Isolated epidermal peels of lupin leaves were floated on ABAsolutions yet upper surface peels showed no stomatal closingin 10^–4 M ABA, while lower surface stomata closed to abarely significant extent. Stomata of intact leaves were not very sensitive to darkness,showing at most a doubling in resistance after 6 h darkness.Complete stomatal closure, however, was readily produced bywilting leaves. Hence, lupin stomata are physically capableof closing. Endogenous ABA levels of water-stressed leaves increased approximately10-fold, which corresponds to concentrations below 10 µMABA. It is concluded that ABA is unlikely to play a role incontrolling short-term stomatal response of lupins.     

Stomatal responses in isolated epidermis of the crassulacean acid metabolism plant Kalanchoe daigremontiana Hamet et Perr.

The optimal conditions for opening of stomata in detached epidermis of the Crassulacean Acid Metabolism (CAM) plant Kalanchoe daigremontiana were determined. Stomatal opening in CO₂–free air was unaffected by light so subsequently all epidermal strips were incubated in the dark and in CO₂–free air. Apertures were maximal after 3 h incubation and were significantly greater at 15° C than 25° C. Thus stomata in isolated epidermis of this species can respond directly to temperature. Stomatal opening was greatest when the incubating buffer contained 17.6 mol m^–3 K⁺, but decreased linearly with increasing K⁺ concentrations between 17.6 and 300 mol m^–3; the decrease in aperture was shown to be associated with increasing osmotic potentials of the solutions. Reasons for this behaviour, which differs from that of many C₃ and C₄ species, are discussed. Stomatal apertures declined linearly upon incubation of epidermis on buffer solutions containing between 10^–11 and 10^–5 mol m^–3 abscisic acid (ABA). Hence stomata on isolated epidermis of K. daigremontiana respond to lower concentrations of ABA than those of any species reported previously.     

Stomatal Closure in Flooded Tomato Plants Involves Abscisic Acid and a Chemically Unidentified Anti-Transpirant in Xylem Sap

We address the question of how soil flooding closes stomata of tomato (Lycopersicon esculentum Mill. cv Ailsa Craig) plants within a few hours in the absence of leaf water deficits. Three hypotheses to explain this were tested, namely that (a) flooding increases abscisic acid (ABA) export in xylem sap from roots, (b) flooding increases ABA synthesis and export from older to younger leaves, and (c) flooding promotes accumulation of ABA within foliage because of reduced export. Hypothesis a was rejected because delivery of ABA from flooded roots in xylem sap decreased. Hypothesis b was rejected because older leaves neither supplied younger leaves with ABA nor influenced their stomata. Limited support was obtained for hypothesis c. Heat girdling of petioles inhibited phloem export and mimicked flooding by decreasing export of [14C]sucrose, increasing bulk ABA, and closing stomata without leaf water deficits. However, in flooded plants bulk leaf ABA did not increase until after stomata began to close. Later, ABA declined, even though stomata remained closed. Commelina communis L. epidermal strip bioassays showed that xylem sap from roots of flooded tomato plants contained an unknown factor that promoted stomatal closure, but it was not ABA. This may be a root-sourced positive message that closes stomata in flooded tomato plants.