Abscisic Acid-Induced Stomatal Closure in Vicia faba Epidermal Strips. Excretion of Solutes from Guard Cells and Increase in Elastic Modulus of Guard Cell Wall

The effects of abscisic acid (ABA) on the size of the apertureof stomata on epidermal strips of Vicia faba were studied inincubation media with different pH values. The osmotic potentialof guard cells, as determined by the limiting plasmolysis method,was higher at pH 4.0 than at pH 6.0, although the size of thestomatal apertures was almost identical at both pH values. AtpH 4.0, ABA effectively caused stomatal closure but had onlya small effect on the osmotic potential, whereas, at pH 6.0,ABA significantly increased the osmotic potential. ABA promotedthe efflux of Cl^– and malate from epidermal strips intothe incubation medium, an effect which was more marked at pH6.0, with a concomitant efflux of K⁺ to balance the charge onthe exported anions. From these results, it is suggested thatABA may cause an increase in the elastic modulus of the cellwalls of guard cells. ³ Present address: Nagano Prefectural Vegetable and OrnamentalCrops Experimental Station, 2206 Oomuro, Matsusiro-machi, Nagano381-12, Japan (Received September 30, 1986; Accepted January 9, 1987)     

Characterisation of Non-Uniform Photosynthesis Induced by Abscisic Acid in Leaves Having Different Mesophyll Anatomies

The effects of abscisic acid (ABA) on photosynthesis in leavesof Helianthus annuus L. were compared with those in leaves ofVicia faba L. After the ABA treatment, the response of photosyntheticCO₂ assimilation rate, A, to calculated intercellular partialpressure of CO₂, P_i, (A(p_i) relationship) was markedly depressedin H. annuus. A less marked depression was also observed inV.faba. However, when the abaxial epidermes were removed fromthese leaves, neither the maximum rate nor the CO₂ responseof photosynthetic oxygen evolution was affected by the applicationof ABA. Starch-iodine tests revealed that photosynthesis was not uniformover the leaves of H. annuus treated with ABA. The starch contentwas diffferent in each bundle sheath extension compartment (thesmallest subdivision of mesophyll by veins with bundle sheathextensions, having an area of ca. 0.25 mm² and ca. 50 stomata).In some compartments, no starch was detected. The distributionof open stomata, examined using the silicone rubber impressiontechniques, was similar to the pattern of starch accumulation.In V.faba leaves, which lack bundle sheath extensions, distributionof starch was more homogeneous. These results indicate that the apparent non-stomatal inhibitionof photosynthesis by ABA deduced from the depression of A(pⁱ)relationship is an artifact which can be attributed to the non-uniformdistribution of transpiration and photosynthesis over the leaf.Intercellular gaseous environment in the ABA-treated leavesis discussed in relation to mesophyll anatomy. ¹ Present address: Department of Botany, Duke University, Durham,NC 27706, U.S.A. (Received September 30, 1987; Accepted January 13, 1988)     

Age-related Changes in Stomatal Response to Cytokinins and Abscisic Acid

Kinetin and zeatin(100 mmol m^–3)reversald the ABA-mediated(100mmol ^m-2)closure of stomata of young maize leaves but did notaffect stomatal apertures of these leaves when applied alone.As leaves aged, kinetin or zeatin alone promoted increased stomatalapertures, while abscisic acid (ABA) applied alone had a reducedeffect on stomata. Even with older leaves, cytokinins reversadthe effect of ABA on stomata. Maize, stomata, abscisic acid, kineusc, zeatin, Zea mays     

Effect of abscisic acid on membrane-bound epidermal ATPase from tobacco leaves

Membrane-bound Mg⁺⁺-activated ATPase activity in epidermal stripsfrom tobacco leaves (Nicotiana tabacum L. Samsun NN) was stimulatedby abscisic acid (ABA) when the strips were floated on ABA solutionin light or in darkness. The optimum ABA concentrations in lightand in darkness were 10^–5 M and 10^–6 M, respectively.Carbonyl cyanide m-chlorophenylhydrazone (CCCP) and N, N'-dicyclohexylcarbodiimide(DCCD) completely blocked the basal level membrane-bound epidermalATPase activity. ABAinduced membrane-bound epidermal ATPaseactivity was completely inhibited by CCCP, but only partly byDCCD. H⁺-influx into epidermal strips on a solution in light was lowerthan that in darkness. ABA stimulated H⁺-influx into epidermalstrips in light and in darkness. CCCP suppressed basal levelH⁺-influx, whereas DCCD did not. CCCP also suppressed ABA-inducedH⁺-influx, whereas DCCD did not. Interaction between H⁺-influxand membranebound epidermal ATPase activity is discussed. (Received May 23, 1978; )     

Ethylene and 1 -Aminocyclopropane-1-Carboxylic Acid Production in flacca, a Wilty Mutant of Tomato, Subjected to Water Deficiency and Pre-treatment with Abscisic Acid

Neill, S. J., McGaw, B. A. and Horgan, R. 1986. Ethylene and1-aminocyclopropane-l-carboxylic acid production in flacca,a wilty mutant of tomato, subjected to water deficiency andpretreatment with abscisic acid —J. exp. Bot. 37: 535–541. Plants of Lycoperstcon esculentum Mill. cv. Ailsa Craig wildtype and flacca (flc) were sprayed daily with H²O or 2?10^–2mol m^–3 abscisic acid (ABA). ABA treatment effected apartial phenotypic reversion of flc shoots; leaf areas wereincreased and transpiration rates decreased. Leaf expansionof wild type shoots was inhibited by ABA. Indoleacetic acid (IAA), ABA and l-aminocyclopropane-l-carboxylicacid (ACC) concentrations were determined by combined gas chromatography-massspectrometry using deuterium-labelled internal standards ABAtreatment for 30 d resulted in greatly elevated internal ABAlevels, increasing from 1?0 to 4?3 and from 0?45 to 4?9 nmolg^–1 fr. wt. in wild type and flc leaves respectively.Endogenous IAA and ACC concentrations were much lower than thoseof ABA. IAA content ranged from 0?05 to 0?1 nmol g^–1 andACC content from 0?07 to 0?24 nmol g^–1 Ethylene emanationrates were similar for wild type and flc shoots. Wilting of detached leaves induced a substantial increase inethylene and ACC accumulation in all plants, regardless of treatmentor type. Ethylene and ACC levels were no greater in flc leavescompared to the wild type. ABA pretreatment did not preventthe wilting-induced increase in ACC and ethylene synthesis. Key words: ABA, ACC, ethylene, wilting, wilty mutants     

Abscisic Acid and Apical Dominance in Phaseolus coccineus L. The Role of Tissue Age

Abscisic acid (ABA) applied to the decapitated second internodeof runner bean plants enhanced outgrowth of lateral buds onlywhen internode stumps were no longer elongating. Applied toelongating internodes of slightly younger plants, ABA causesinhibition of bud outgrowth. Together with 10^–4 M indol-3-ylacetic acid (IAA), ABA stimulated internode elongation and interactedadditively in the inhibition of bud outgrowth. A mixture of10^–5 M ABA and 10^–6 M gibberellic acid (GA₃ ) causedsimilar effects on internode growth as IAA + ABA, but was mutuallyantagonistic in effect on growth of the lateral buds. Abscisic acid, apical dominance, gibberellic acid, indol-3yl acetic acid, Phaseolus coccineus, bean     

Characterization of a wilty sunflower (Helianthus annuus L.) mutant: I. Abscisic acid content, light-dark changes in the stomatal conductance and genetic analysis

The present study was conducted to characterize geneticallyand physiologically a spontaneous mutation in sunflower whichconfers a wilty phenotype. The wilting condition of the mutantis due to abnormal stomatal behaviour. The mutant stomata resistclosure in darkness. This abnormality is associated with lowlevels of endogenous abscisic acid (ABA). By artificially elevatingthe ABA content of the mutant plants by spray treatments with10^– and 10^– M solutions it proved possible to effecta phenotypic reversion of the mutant. It has, therefore, beenproposed that the primary effect of this spontaneous mutationis to reduce the level of ABA. The genetic analysis has shownthat the willy phenotype is due to recessive nuclear mutationat a single locus. Key words: ABA, Helianthus annuus L, stomatal conductance, wilty mutant     

Stomatal closure in response to xanthoxin and abscisic acid

Summary The stomata of detached leaves of Commelina communis L., Hordeum vulgare L., Zea mays L., Vicia faba L., Phaseolus vulgaris L. and Xanthium strumarium L. closed when xanthoxin (XAN) was added to the transpiration stream. XAN was approximately half as active as (+)-abscisic acid (ABA) at an equivalent concentration. XAN, like ABA, sensitized stomata of Xanthium strumarium to CO₂. In contrast to ABA, XAN was ineffective in closing stomata of isolated epidermal strips of C. communis or V. faba. This may be because XAN added to the transpiration stream is converted to ABA during passage from the xylem to the epidermis.Abbreviations ABA Abscisic acid - XAN xanthoxin     

Circadian Rhythm of Stomatal Movements in Epidermal Strips

Measurements were made of changes in stomatal pore widths inepidermal strips of leaves ofVicia faba and Commelina communis.Strips were incubated in dilute KCI solutions (1 and 10 molm^–3) flowing through a perfusion chamber on the stageof a microscope and kept for 4 d in continuous light. Circadianrhythms of stomatal apertures were detected in both species.Although the amplitude was small it was statistically significant.It is concluded that at least partof the mechanism for the stomatalrhythm resides in the epidermis, probably in the guard cells. Key words: Cireadian rhythm, epidermal strips, stomata     

Acid-Induced Stomatal Opening in Commelina communis and Vicia faba

The effects of H^$ and fusicoccin (FC) on stomatal opening inthe dark were investigated using epidermal strips of Commelinacommunis and Vicia faba cv. Ryosai Issun. Citrate-phosphatebuffer induced maximal opening of stomata at pH 3.0 when testedover the range of 2.7 to 5.0. HCl at 1 mM also induced stomatalopening without appreciable accumulation of K^$ in the guardcells. After 4 hr treatment with 10 µM FC, stomata openedwith concomitant accumulation of K^$ in the guard cells, although1–2 hr treatment caused opening without concomitant K^$increase. These results suggest that stomatal opening can be caused bysalt accumulation and/or changes of the physicochemical conditionsin the cell wall of the guard cells due to high acidity. ¹ Present address: Biological Laboratory, Faculty of Education,Nagasaki University, Nagasski 852, Japan. (Received April 30, 1982; Accepted July 17, 1982)     

Effect of CO2 on Volume Change of Guard Cell Protoplast from Vicia faba L

Guard cell protoplasts (GCP) were isolated from epidermal stripsof Vicia faba L. by enzymatic digestion. The presence of non-osmoticvolume in the protoplast was suggested by the relationship betweenprotoplast volume and the mannitol concentration of the suspendingmedium. Light illumination caused swelling of GCP only whenKCl was present in the suspending medium. Dark treatment causedshrinking of GCP irrespective of the presence of 10 mM KCl.In the presence of 10 µM abscisic acid (ABA), GCP shrank.Light-induced swelling was suppressed at concentrations of ambientCO₂ higher than that in normal air. Promotion of swelling wasnot always observed at lower CO₂ concentration. These volumechange responses to light, ABA and CO₂ suggest that GCP retainsits physiological activity as a guard cell. The osmotic contributionof K⁺ to volume increase was lower than expected. Ambient CO₂seems to have some effect on the contribution of K⁺ to osmoregulationof GCP. (Received January 30, 1982; Accepted June 25, 1982)     

Effect of Abscisic Acid on Glucose Metabolism in Guard Cells of Vicia faba L.

The effects of abscisic acid (ABA) on the formation of osmoticallyactive solutes and on cell wall synthesis in guard cells wereexamined using sonicated abaxial epidermal strips of Vicia fabaL. incubated with ¹⁴C-glucose at pH 4 and 6. Radioactivity wasincorporated mainly into malate,sucrose, starch and cell-wallfractions. ¹⁴C- Glucose uptake by the guard cells was reducedwhen 1 µm ABA was added. Malate formation, which was moreactive at pH 6 than at 4, was inhibited by ABA at pH 6, butnot at pH 4. Conversion of ¹⁴C-glucose into ¹⁴C-sucrose wasstimulated by ABA at both pH values. Release of radio activesolutes (composed mainly of glucose and malate)from the guardcells into the medium was more active at pH 6 than at pH 4.ABA stimulated there lease at both pH values. Turnover of starchwas more remarkable when the pH value was 6. ABA inhibited thesynthesis of starch, but did not affect its degradation. Cell-wallsynthesis inthe guard cells was also inhibited by ABA, the inhibitionrate being greater at pH 4 than at pH 6.These results suggestthat ABA may have two different actions on stomatal movement:to changethe metabolic activities in the guard cells so as tolower the concentration of osmotically active solutes, and tochange the mechanical properties of cell walls by modulatingcell-wall metabolism. (Received September 7, 1987; Accepted November 30, 1987)     

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.     

New fava bean guard cell signaling mutant impaired in ABA-induced stomatal closure

We isolated a mutant from Vicia faba L. cv. House Ryousai. Itwilts easily under strong light and high temperature conditions,suggesting that its stomatal movement may be disturbed. We determinedresponses of mutant guard cells to some environmental stimuli.Mutant guard cells demonstrated an impaired ability to respondto ABA in 0.1 mM CaCl₂ and stomata did not close in thepresence of up to 1 mM ABA, whereas wild-type stomata closedwhen exposed to 10 µM ABA. Elevating external Ca²⁺caused a similar degree of stomatal closure in the wild typeand the mutant. A high concentration of CO₂ (700 µlliter^–1) induced stomatal closure in the wild type, butnot in the mutant. On the basis of these results, we proposethe working hypothesis that the mutation occurs in the regiondownstream of CO₂ and ABA sensing and in the region upstreamof Ca²⁺ elevation. The mutant is named fia (fava bean impairedin ABA-induced stomatal closure). ³ Corresponding author: E-mail, smoiwai{at}agri.kagoshima-u.ac.jp;Fax, +81-99-285-8556.     

The Anisotropy of the Mesophyll and CO2 Capture Sites in Vicia faba L. Leaves at Low Light Intensities

Experiments are reported on the spatial distributions of isotopiccarbon within the mesophyll of detached leaves of the C₃ plantVicia faba L. fed ¹⁴CO₂ at different light intensities. Eachleaf was isolated in a cuvette and ten artificial stomata providedspatial continuity between the ambient atmosphere (0.03–0.05%v/v CO₂) and the mesophyll from the abaxial leaf side. Paradermalleaf layers exhibited spatial profiles of radioactivity whichvaried with the intensity of incident light in 2 min exposures.At low light, when biochemical kinetics should limit CO₂ uptake,sections through palisade cells contained most radioactivity.As the light intensity was increased to approximately 20% offull sunlight, peak radioactivity was observed in the spongycells near the geometric mid-plane of the mesophyll. The resultsindicate that diffusion of carbon dioxide within the mesophyllregulated the relative photosynthetic activity of the palisadeand spongy cells at incident photosynthetically active lightintensities as little as 110 µE m^–2 s^–1 whenCO₂ entered only through the lower leaf surface. Key words: CO₂ capture sites, Vicia faba L., Artificial stomata     

Stomatal Functioning of In Vitro and Greenhouse Apple Leaves in Darkness, Mannitol, ABA, and CO2

Leaves from in vitro and greenhouse cultured plants of Malusdomestica (Borkh.) cv. Mark were subjected to 4 h of darkness;4 h of 1 M mannitol induced water stress; 1 h of 10^–4M to 10^–7 M cis-trans abscisic acid (ABA) treatment; 1h of 0.12% atmospheric CO₂. Stomatal closure was determinedby microscopic examination of leaf imprints. In all treatments,less than 5% of the stomata from leaves of in vitro culturedplants were closed. The diameter of open stomata on leaves fromin vitro culture remained at 8 µm. In contrast, an averageof 96% of the stomata on leaves of greenhouse grown plants wereclosed after 4 h in darkness; 56% after 4 h of mannitol inducedwater stress; 90% after 1 h of 10^–4 M ABA treatment; 61%after 1 h in an atmosphere of 0.12% CO₂. Stomata of in vitroapple leaves did not seem to have a closure mechanism, but acquiredone during acclimatization to the greenhouse environment. Thelack of stomatal closure in in vitro plants was the main causeof rapid water loss during transfer to low relative humidity.     

Carrier-Mediated ABA Uptake by Suspension-Cultured Phaseolus coccineus L. Cells: Stereospecificity and Inhibition by Ionones and ABA Esters

Astle, M. and Rubery, P. 1987. Carrier-mediated ABA uptake bysuspension-cultured Phaseolus coccineus L. cells: Stereospecificityand inhibition by ionones and ABA esters.—J. exp. Bot.38: 150–163. The substrate for the abscisic acid (ABA) carrier in Phaseoluscoccineus L. suspension-cultured cells is shown to be the (S)ABAenantiomer, K_m = 1?0 mmol m^–3. The methyl (MeABA) andphenyl (PheABA) esters of ABA inhibit carrier-mediated uptakeof ABA with half-maximal inhibition achieved at about 7?0 mmolm^–3 and 10 mmol m^–3 respectively: with (S)MeABAthis value is decreased to about 2?0 mmol m^–3. There isno demethylation of radioactive MeABA by the cells during 5min incubations. Although MeABA reversibly inhibits the ABAcarrier, it is not a transport substrate: association of radioactiveMeABA with living cells is unaffected by non-radioactive MeABAor ABA and, by comparison with frozen-and-thawed cells, it isshown that the radioactivity remains extracellular. It is proposedthat MeABA binds to the carrier to form an abortive complexthat is not translocated. The terpenoid ABA analogue LAB 144143also inhibits carrier-mediated ABA uptake. At concentrationsup to about 20 mmol m^–3 - and ß-ionone specificallyinhibit the ABA carrier with the half-maximal effect at about0?6 mmol m^–3 ß-ionone. However, at higher iononeconcentrations, the uptake of ABA, indol-3-yl acetic acid andof 5,5-dimethyloxazolidine-2,4-dione (DMO) are all stimulated:this may reflect general permeabilization of the membrane toweak acids by ionone. Key words: Uptake carrier, abscisic acid, methyl and phenyl esters of ABA, ionone, Phaseolus coccineus L. suspension culture     

Control of the Co2 Responses of Stomata by Indol-3ylacetic Acid and Abscisic Acid

Previous studies of stomatal behaviour on detached epidermisof Commelina communis L. have suggested that abscisic acid (ABA)and C02 act independently to cause stomatal closure. Evidenceis presented here that if indol-3ylacetic acid (IAA) is addedto the medium used for incubating the epidermis, an interactionbetween ABA and Co₂ becomes apparent. Increasing concentrationsof IAA reduce the ability of the stomata to respond to CO₂,and ABA appears to antagonize this effect. Recognition of therole of IAA enables us to reconcile earlier conflicting reportsconcerning the interdependence of effects of ABA and Co₂on stomata.     

Some Effects of Abscisic Acid and Water Stress on Stomata of Vicia faba L.

Vicia faba seedlings grown under a plastic tent in the laboratorywere either watered well throughout their growth period or weresubjected to a water stress treatment for several days priorto an experimental treatment. The effects of a further waterstress treatment or an application of an aqueous solution ofabscisic acid (ABA) on the stomata of these plants were determined.Stomata of previously water-stressed plants proved to be moresensitive than stomata of well watered plants to ABA appliedthrough the petiole via the transpiration stream and sprayedonto leaf surfaces. Stomata of previously water-stressed plantsclosed more rapidly and to a greater degree than stomata ofwell watered plants. The hormone had only a small effect whenapplied directly to epidermal fragments removed from both groupsof plants. Stomata of plants which had received a water stresspretreatment were less sensitive to a subsequent period of waterstress than were stomata of previously well watered plants.It is proposed that stomatal adaptation to water stress maybe related to changes in the hormonal balance of the plant.     

Responses of Commelina communis L. Guard Cell Protoplasts to Abscisic Acid

Fitzsimons, P. J. and Weyers, J. D. B. 1987. Responses of Commelinacommunis L. guard cell protoplasts to abscisic acid.—J.exp. Bot. 38: 992–1001. Guard cell protoplasts (GCPs) isolated from the leaf epidermisof Commelina communis L. responded to abscisic acid (ABA) ina manner which was qualitatively and quantitatively similarto that of intact stomata. ABA inhibited swelling of GCPs underlow-CO₂ conditions and swollen GCPs responded to the hormoneby shrinking. Both the absolute volume decrease and the initialrate of shrinking were commensurate with the extent and ratesof solute loss computed for ABA-treated intact, open stomata.This indicates that GCPs represent a suitable experimental systemfor studies of ABA-mediated solute fluxes. A radiotracer equilibrationmethod was developed for the rapid estimation of GCP osmoticvolume changes. Using this technique it was found that, on average,82% of the reduction in solute content caused by ABA treatmentwas due to the loss of K⁺. It is envisaged that electroneutralitymight be maintained during ABA-induced shrinkage of GCPs bynet inward proton movement leading to acidification of the vacuole. Key words: Abscisic acid, Commelina communis L., guard cells, protoplasts