Effect of abscisic acid on stomatal opening in isolated epidermal strips of abi mutants of Arabidopsis thaliana

Abscisic acid-insensitive mutants of Arabidopsis thaliana L. var. Landsberg erecta were selected for their decreased sensitivity to ABA during germination. Two of these mutants, abi-1 and abi-2 , display a wilty phenotype as adult plants, indicating disturbed water relations. Experiments were undertaken to find out if this results from insensitivity of mutant stomates to ABA.
Growth conditions and methods to isolate epidermal strips were optimized to study stomatal movement. Wild type stomates required external ionic conditions comparable to those found for other species such as Commelina communis . The largest light-induced opening of A. thaliana stomates was found at an external KCl concentration of 50 m M . Stomatal apertures were increased by lowering external Ca²⁺ to 0.05 m M . The apertures of stomates incubated with 10 μ M ABA were not altered by changes in Ca²⁺ from 0.05 to 1.0 m M .
Stomates of all abi mutants showed a light-stimulated stomatal opening. The opening of wild type and abi-3 stomates was inhibited by ABA, while stomates of abi-1 and abi-2 did not respond to ABA. The insensitivity of abi-1 and abi-2 stomates to ABA may thus explain the observed disturbed water relations.     

Kinetics of maize leaf elongation IV. Effects of (+)- and (-)-abscisic acid

Abscisic acid (ABA) is involved in many of the responses of plants to environmental stress. This study focuses on the inhibitory effect of ABA on leaf expansion. In addition, the effects of (+)-ABA, the natural form of ABA, were compared to the effects of (-)-ABA. Leaf elongation rates (LER) were measured for the 3rd leaf of maize plants. ABA concentrations were measured by RIA for total ABA and an ELISA specific for (+)-ABA. ABA was added to the hydroponic solution and changes in the LER were measured over time. ABA could inhibit LER within 30 min ad reached steady-state LER within 4 h. Internal ABA concentrations in the growing zone of the leaf also reached steady-state concentrations after 4 h. This effect of ABA was reversible, because LER was fully restored upon removal of externally applied ABA, and internal concentrations of ABA in the growing zone returned to normal levels, whereas ABA concentrations remained elevated in mature tissue. Thus, steady-state LER was highly correlated with the steady-state internal ABA concentration of the growing zone. ABA inhibited leaf expansion by increasing the apparent cell wall yield threshold; no other growth parameters were affected. The (-)-enantiomer of ABA had much less effect on LER than (+)-ABA when compared upon an external concentration basis. Internal ABA concentrations rationalized the response, showing that (-)-ABA accumulation was very low, most likely due to low uptake rates. From this analysis, it was determined that LER was equally sensitive to internal concentrations of (+)- or (-)-ABA.     

Contrasting Effects of the Cotton Defoliant Thidiazuron and Aminoethoxyvinylglycine,an Inhibitor of Ethylene Formation,on Stomatal Aperture and on Ethylene Formation in Bean (Phaseolus vulgaris) Leaves

The cotton defoliant, thidiazuron, 1-phenyl-3-(1,2,3-thiadiazol-5-yl)-urea stimulated ethylene formation in primary leaves of Phaseolus vulgaris var. Favorit, three to eight days after spraying. Aminoethoxyvinylglycine (AVG), an inhibitor of ethylene formation, delayed this ethylene outburst by two to three days when sprayed together with the defoliant. Under conditions of dryness, thidiazuron inhibited the stomatal closure of bean leaves. Spraying with AVG counteracted this effect of thidiazuron on the stomates and caused a partial and reversible closure ca 1 day after spraying.     

ABA regulation of K(+)-permeable channels in maize subsidiary cells

An antiparallel-directed potassium transport between subsidiary cells and guard cells which form the graminean stomatal complex has been proposed to drive stomatal movements in maize. To gain insights into the coordinated shuttling of K(+) ions between these cell types during stomatal closure, the effect of ABA on the time-dependent K(+) uptake and K(+) release channels as well as on the instantaneously activating non-selective cation channels (MgC) was examined in subsidiary cells. Patch-clamp studies revealed that ABA did not affect the MgC channels but differentially regulated the time-dependent K(+) channels. ABA caused a pronounced rise in time-dependent outward-rectifying K(+) currents (K(out)) at alkaline pH and decreased inward-rectifying K(+) currents (K(in)) in a Ca(2+)-dependent manner. Our results show that the ABA-induced changes in time-dependent K(in) and K(out) currents from subsidiary cells are very similar to those previously described for guard cells. Thus, the direction of K(+) transport in subsidiary cells and guard cells during ABA-induced closure does not seem to be grounded solely on the cell type-specific ABA regulation of K(+) channels.     

Nitric oxide inhibits blue light-specific stomatal opening via abscisic acid signaling pathways in Vicia guard cells

Recent evidence suggests that nitric oxide (NO) acts as an intermediate of ABA signal transduction for stomatal closure. However, NO's effect on stomatal opening is poorly understood even though both opening and closing activities determine stomatal aperture. Here we show that NO inhibits stomatal opening specific to blue light, thereby stimulating stomatal closure. NO inhibited blue light-specific stomatal opening but not red light-induced opening. NO inhibited both blue light-induced H(+) pumping and H(+)-ATPase phosphorylation. The NO scavenger 2-carboxyphenyl-4,4,5,5-tetramethylimidazoline-1-oxyl-3-oxide (c-PTIO) restored all these inhibitory effects. ABA and hydrogen peroxide (H(2)O(2)) inhibited all of these blue light-specific responses in a manner similar to NO. c-PTIO partially restored the ABA-induced inhibition of all of these opening responses but did not restore inhibition of the responses by H(2)O(2). ABA, H(2)O(2) and NO had slight inhibitory effects on the phosphorylation of phototropins, which are blue light receptors in guard cells. NO inhibited neither fusicoccin-induced H(+) pumping in guard cells nor H(+) transport by H(+)-ATPase in the isolated membranes. From these results, we conclude that both NO and H(2)O(2) inhibit blue light-induced activation of H(+)-ATPase by inhibiting the component(s) between phototropins and H(+)-ATPase in guard cells and stimulate stomatal closure by ABA.     

Validation of a radioimmunoassay for (+)-abscisic acid in extracts of apple and sweet-pepper tissue using high-pressure liquid chromatography and combined gas chromatography-mass spectrometry

A radioimmunoassay for (+)-abscisic acid (ABA) was developed and applied to the analysis of free ABA in extracts of apple (Malus pumila Mill.) and sweet pepper (Capsicum annuum L.) leaves at various stages during extract purification. Conjugates of ABA, were quantified after alkaline hydrolysis. The validity of the radioimmunoassay was tested by comparison of immunoassay estimates of ABA at different levels of extract purity with high-pressure liquid chromatography (HPLC) and combined gas chromatography-mass spectrometry. The antiserum, raised against (+)-ABA, was almost equally sensitive to (-)-ABA. Serum cross-reactivity with the methyl ester of ABA was 160% and with the glycosyl ester of ABA was 34%. Cross-reactivity with protein-ABA conjugates was very slight for C₄-conjugated keyholelimpet haemocyanin, but about 1000% for C₁-conjugated alkaline phosphatase. Other compounds tested showed extremely low or undetectable cross-reactivities. Further evidence for the specificity of the assay came from the agreement between the results using different assay methods for both apple and pepper extracts, and from the observation that the only zone of immunoreactivity on HPLC elution profiles corresponded with authentic (+)-ABA. The use of polyvinylpyrrolidone in the assay minimised interference by other substances in plant extracts. In pepper, free ABA levels increased rapidly during water stress and recovered to pre-stress levels within two days after rewatering. Levels of ABA conjugates were significantly lowr than free ABA in unstressed plants, and also increased rapidly with stress, although not to the same extent as free ABA, and did not recover as rapidly as did free ABA. In apple, levels of free ABA and of ABA conjugates both increased more than twofold over a two-week period of water stress. In contrast to pepper, however, immunoreactivity of the conjugate fraction was increased by hydrolysis, indicating that different ABA conjugates predominate in the two species.Abbreviations ABA abscisic acid - GC-MS combined gas chromatography-mass spectrometry - HPLC high-pressure liquid chromatography - Me-ABA methyl ester of ABA - PVP polyvinylpyrrolidone - RIA radioimmunoassay     

Levels of short-chain fatty acids and of abscisic acid in water-stressed and non-stressed leaves and their effects on stomata in epidermal strips and excised leaves

Straight-chain saturated fatty acids (C6-C11) and abscisic acid (ABA) accumulate in the leaves of Phaseolus vulgaris L. and Hordeum vulgare L. under water stress. ABA and certain of the fatty acids, particularly decanoic and undecanoic acid, can inhibit stomatal opening and cause stomatal closure in epidermal strips of Commelina communis L. depending on the incubating medium used. 10^-4 M (±)-ABA inhibits opening in media containing either high or relatively low concentrations of KCl but causes closure only in the latter medium. The fatty acids (at 10^-4 M) prevent opening in both media while significant closure of open stomata was caused only by undecanoic acid in both media and, additionally, by decanoic acid in the low-KCl medium. 10^-4 M formic acid also caused stomatal closure and prevented opening to significant extents in the low-KCl medium (it was not tested in the high-KCl medium). The efficacy of undecanoic acid in causing 50% inhibition of opening is about three orders of magnitude lower than that of ABA. At a concentration of 10^-3 M, nonanoic, decanoic and particularly undecanoic acid and all-trans-farnesol cause increased cell leakage in Beta vulgaris L. root tissue. Undecanoic acid (10^-4 M) also causes some loss of guard cell integrity in C. communis within 1.5 h of treatment. ABA (10^-4 M) reduces transpiration rates in barley and C. communis leaves when applied via the transpiration stream but decanoic and undecanoic acids did not have this effect. Transpiration was not affected when ABA or the fatty acids were applied to the leaf surfaces.Abbreviations ABA abscisic acid - RWC relative water content - SCFA short-chain fatty acids Deceased May 1977     

Expression of an abscisic acid-binding single-chain antibody influences the subcellular distribution of abscisic acid and leads to developmental changes in transgenic potato plants

Potato (Solanum tuberosum L. cv. Désirée) plants were transformed to express a single-chain variable-fragment antibody against abscisic acid (ABA), and present in the endoplasmic reticulum at to up to 0.24% of the soluble leaf protein. The resulting transgenic plants were only able to grow normally at 95% humidity and moderate light. Four-week-old plants accumulated ABA to high extent, were retarded in growth and their leaves were smaller than those of control plants. Leaf stomatal conductivity was increased due to larger stomates. The subcellular concentrations of ABA in the chloroplast, cytoplasm and vacuole, and the apoplastic space of leaves were determined. In the 4-week-old transgenic plants the concentration of ABA not bound to the antibody was identical to that of control plants and the stomates were able to close in response to lower humidity of the atmosphere. A detailed analysis of age-dependent changes in plant metabolism showed that leaves of young transformed plants developed in ABA deficiency and leaves of older plants in ABA excess. Phenotypic changes developed in ABA deficiency partly disappeared in older plants.     

Stomatal density and responsiveness of banana fruit stomates

Determination of stomatal densities of the banana peel (Musa acuminata L. var Hort. Valery) by microscopic observations showed 30 times fewer stomates on fruit epidermis than found on the banana leaf. Observations also showed that peel stomates were not laid down in a linear pattern as on the leaf.

It was demonstrated that stomatal responses occurred in banana fruit. Specific conditions of high humidity and light were necessary for stomatal opening: low humidity and darkness were necessary for closure. Responsiveness of the stomates continued for a considerable length of time after the fruit had been severed from the host.

     

Involvement of phospholipase C-independent calcium-mediated abscisic acid signalling during <Emphasis Type="Italic">Arabidopsis</Emphasis> response to drought

The present study investigated whether Ca²⁺ mobilization independent of phosphoinositide-specific phospholipase C (PI-PLC) would delay wilting in Arabidopsis thaliana (L.) Heynh. cv. Columbia through mediating stomatal closure at abscisic acid (ABA) concentrations rising beyond a drought-specific threshold value. In wild type (WT) epidermis, the PI-PLC inhibitor (U73122) affected the stomatal response to 20 μM ABA but not to 30 μM ABA. Disruption in GTP-binding protein ά subunit 1 (GPA1) affected the stomatal response to 30 μM ABA, but not to 20 μM ABA. In the gpa1-4 mutant, the inhibitory effects of the Ca²⁺ buffer, 1,2-bis(0-aminophenoxy)ethane-N,N,N′,N′-tetraacetic acid (BAPTA), the inactive mastoparan analogue, mas17 and the antagonist of cyclic ADP-ribose synthesis, nicotinamide, were differentially attenuated on 30 μM ABA-induced stomatal closure. By contrast, the NADPH oxidase atrbohD/F double mutation fully suppressed inhibition of 20 μM ABA-induced stomatal closure by BAPTA or U73122 as well as inhibition of 30 μM ABA-induced stomatal closure by BAPTA, mas17 or nicotinamide. On the contrary, The Al resistant alr-104 mutation modulated ABA-induced stomatal closure by a stimulatory effect of U73122 and an increased sensitivity to mas17, nicotinamide and BAPTA. Compared to WT, the atrbohD/F double mutant was more hypersensitive than the gpa1-4 mutant to wilting under the tested water stress conditions, whereas wilting was delayed in the alr-104 mutant. Since the atrbohD/F mutation breaks down ABA-induced Ca²⁺ signalling through fully preventing apoplastic Ca²⁺ to enter into the guard cells, these results showed that a putative guard cell GPA1-dependent ADP-ribosyl cyclase activity should contribute to drought tolerance within PI-PLC-independent-Ca²⁺-mediated ABA signalling.     

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     

Structural Requirements of Abscisic Acid (ABA) and its Impact on Water Flow During Radial Transport of ABA Analogues Through Maize Roots

The effect of (+) (ABA) and (?)-abscisic acid and nine ABA metabolites, precursors or derivatives on radial water movement through maize roots, was investigated using a suction technique (Freundl and others 1998). (+)-ABA, (+)- and (?)-abscisyl aldehyde, (+)-8?-hydroxymethyl ABA, (+)-8?-methylene, and (+)-8?-acetylene ABA stimulated radial water transport. (?)-ABA, phaseic acid, and (+)-8?-acetylene methyl ABA were ineffective. ELISA analysis for ABA detected and apparent increase of free ABAxyl in xylem sap of excised root systems that were perfused with either (+)-abscisyl aldehyde, (+)-8?-methylene, (+)8?-acetylene-ABA, or ABA-glucose ester. The analogues (+)-8?-hydroxymethyl ABA and (?)-abscisyl aldehyde passed the cortex of maize roots without changing the ABAxyl. The data from this study permit conclusions about the structural requirements for hormonal regulation of hydraulic conductivity.     

ABA metabolites induce group 3 LEA mRNA and inhibit germination in wheat

In plants, metabolism of the plant hormone (+)- S -abscisic acid (ABA) occurs by oxidation at the 8'-carbon on the ABA ring, producing (+)-8'-hydroxy-ABA (8'OH-ABA), which undergoes ring closure to phaseic acid (PA), or at the 7'-carbon producing 7'-hydroxy-ABA (7'OH-ABA). New methods for synthesixing and stabilizing 8'OH-ABA have enabled us for the first time to compare the biological activities of the ABA metabolites. 8'OH-ABA. PA and 7'OH-ABA. Activities were determined in wheat ( Triticum aestivum , cvs Brevor and Clarks Cream) using optically pure natural enantiomers of ABA. 8'OH-ABA, PA, and 7'OH-ABA. Comparison of wheat embryo germination inhibitory activity showed that only the metabolite 7'OH-ABA had significant activity (10 to 40% of effective ABA activity) as a germination inhibitor of wheat embryos. The metabolites had differential effects on ABA-responsive gene expression. Both 8'OH-ABA and PA were effective inducers of an ABA-responsive LEA (late embryogenesis abundant) gene, wheat group 3 LEA , in seedling roots, suggesting that ABA metabolites can maintain and prolong LEA gene expression. The metabolites had only a slight effect on accumulation of an ABA-responsive protein kinase ( PKABA1 ) mRNA or pMA1951 . These results are consistent with the idea that there are several control points in the ABA metabolic pathway and indicate that assessment of ABA signalling responses should include consideration of the biological activities of ABA metabolites.     

Structure-activity relationship of uniconazole, a potent inhibitor of ABA 8'-hydroxylase, with a focus on hydrophilic functional groups and conformation

The plant growth retardant S-(+)-uniconazole (UNI-OH) is a strong inhibitor of abscisic acid (ABA) 8'-hydroxylase, a key enzyme in the catabolism of ABA, a plant hormone involved in stress tolerance, stomatal closure, flowering, seed dormancy, and other physiological events. In the present study, we focused on the two polar sites of UNI-OH and synthesized 3- and 2'-modified analogs. Conformational analysis and an in vitro enzyme inhibition assay yielded new findings on the structure-activity relationship of UNI-OH: (1) by substituting imidazole for triazole, which increases affinity to heme iron, we identified a more potent compound, IMI-OH; (2) the polar group at the 3-position increases affinity for the active site by electrostatic or hydrogen-bonding interactions; (3) the conformer preference for a polar environment partially contributes to affinity for the active site. These findings should be useful for designing potent azole-containing specific inhibitors of ABA 8'-hydroxylase.     

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     

Transient expression of <Emphasis Type="Italic">AtNCED3</Emphasis> and <Emphasis Type="Italic">AAO3</Emphasis> genes in guard cells causes stomatal closure in <Emphasis Type="Italic">Vicia faba</Emphasis>

Abscisic acid (ABA) regulates stomatal closure in response to water loss. Here, we examined the competence of guard cells to synthesize ABA, using two Arabidopsis ABA biosynthetic enzymes. 35S pro::AtNCED3-GFP and AAO3-GFP were introduced into guard cells of broad bean leaves. AtNCED3-GFP expression was detected at the chloroplasts, whereas green fluorescent protein (GFP) and AAO3-GFP were in the cytosol. The stomatal aperture was decreased in AtNCED3-GFP- and AAO3-GFP-transformed guard cells. This indicated that ABA biosynthesis is stimulated by heterologous expression of AtNCED3 and Arabidopsis aldehyde oxidase 3 (AAO3) proteins, which both seem to be regulatory enzymes for ABA biosynthesis in these cells. Furthermore, stomatal closure by the expression of AtNCED3 and AAO3 suggested that the substrates of the enzymes are present and native ABA-biosynthesis enzymes are active in guard cells. Electronic supplementary material The online version of this article (doi:) contains supplementary material, which is available to authorized users. V. Melhorn and K. Matsumi contributed equally to this work.     

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.     

Abscisic Acid and Desiccation Tolerance in Mosses

In higher plants the phytohormone ABA is involved in processes that are connected to water deficit, like stomatal closure or desiccation tolerance. In bryophytes, also containing ABA in their tissues, physiological functions remained uncertain for a long time. Quite recently, several papers have shown different effects of exogenously applied ABA: stomatal closure in Anthoceros, drought hardening in Funaria and production of the landform in Riccia. In all these cases the relevant conditions (water deficit) enhance the endogenous ABA level significantly. For induced desiccation tolerance, ABA serves as a mediator to induce specific proteins (dehydrins) strongly connected with this tolerance. Therefore, it can be concluded that in bryophytes ABA has the same function as in higher plants. It acts as a mediator in stress conditions.     

Effects of external potassium supply on stomatal closure induced by abscisic acid

Abstract Epidermal strips of Commelina communis with ‘isolated’ stomata were incubated on Trizma-maleate buffer containing 0-500 mM KCL, with or without 10^?4 M ABA, for 2.5 h. The resulting stomatal apertures indicate that there is no absolute requirement for live epidermal and subsidiary cells for ABA-mediated closure. This implies that ABA has a direct effect on influx or efflux of K⁺ into or out of the guard cells rather than on uptake of K⁺ by the subsidiary cells. The possible in vivo role of subsidiary cells in stomatal closure is discussed.     

Effects of Abscisic Acid Metabolites and Analogs on Freezing Tolerance and Gene Expression in Bromegrass (Bromus inermis Leyss) Cell Cultures

Optical isomers and racemic mixtures of abscisic acid (ABA) and the ABA metabolites abscisyl alcohol (ABA alc), abscisyl aldehyde (ABA ald), phaseic acid (PA), and 7[prime]hydroxyABA (7[prime]OHABA) were studied to determine their effects on freezing tolerance and gene expression in bromegrass (Bromus inermis Leyss) cell-suspension cultures. A dihydroABA analog (DHABA) series that cannot be converted to PA was also investigated. Racemic ABA, (+)-ABA, ([plus or minus])-DHABA, and (+)-DHABA were the most active in inducing freezing tolerance, (-)-ABA, ([plus or minus])-7[prime]OHBA, (-)-DHABA, ([plus or minus])-ABA ald, and ([plus or minus])-ABA alc had a moderate effect, and PA was inactive. If the relative cellular water content decreased below 82%, dehydrin gene expression increased. Except for (-)-ABA, increased expression of dehydrin genes and increased accumulation of responsive to ABA (RAB) proteins were linked to increased levels of frost tolerance. PA had no effect on the induction of RAB proteins; however, ([plus or minus])- and (+)-DHABA were both active, which suggests that PA is not involved in freezing tolerance. Both (+)-ABA and (-)-ABA induced dehydrin genes and the accumulation of RAB proteins to similar levels, but (-)-ABA was less effective than (+)-ABA at increasing freezing tolerance. The (-)-DHABA analog was inactive, implying that the ring double bond is necessary in the (-) isomers for activating an ABA response.