Modification of the CO2 Responses of Maize Stomata by Abscisic Acid and by Naturally-Occurring and Synthetic Cytokinins

Fragments of maize leaves were incubated at controlled temperatureand irradiance either on distilled water or on one of threeconcentrations of cytokinin (10^–1, 10^–2 and 10^–3mol m^–3). The effects of zeatin or kinetin on stomatalaperture were determined by stripping abaxial epidermis fromthe fragments after incubation and immediately measuring stomatalapertures under the microscope. At each cytokinin concentrationleaf pieces were incubated at 5 or 350 µmol mol^–1CO₂ with or without ABA (10^–1 mol m^–3). At 5.0 µmolmol^–1 CO₂ increasing the concentrations of zeatin hada negligible effect upon stomatal aperture. When air containing350 umol mol^–1 CO₂ was bubbled through the incubationsolutions, apertures of stomata incubated on water were morethan halved. Increasing cytokinin concentrations reduced theeffect of CO₂ on stomata and incubation on 10^–1 mol m^–3zeatin completely removed any CO₂ response. The addition ofABA restored the effect of CO₂, even at the highest cytokininconcentration. Key words: Maize, CO₂ response, ABA, Cytokinins     

Delay in the Response of Stomata to Abscisic Acid in CO2-free Air

Abscisic acid (10^–5 M) was fed via their petioles to leavesdetached from well watered plants of Xanthium strumartum, whilethe intercellular spaces were flushed with air of known CO₂content. A closing response to ABA occurred in the presenceor absence of CO₂, and the stomata responded to CO₂ whetheror not ABA was supplied to the leaves. A factorial experimentrevealed no interaction between CO₂ and ABA, and suggested thattheir effect on the rate of closure was purely additive. Theonly evidence of interdependence between the two corn poundswas a delay in the response to ABA in C0 air, which was moremarked in a high light intensity. A hypothesis which is consistentwith the data is that ABA induces stomatal closure by interferingwith the energy supply required for the active transport processeson which guard cell turgor depends. The inhibitory action ofABA takes longer in CO₂-free air because, in the absence ofCO₂ fixation, energy is available from chioroplasts as wellas mitochondria.     

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.     

Stomatal Responses to Water Deficits and Abscisic Acid in Leaves of Sunflower Plants (Helianthus annuus L.) Grown under Different Conditions

The decrease in diffusive conductance of a leaf exposed to waterstress or to exogenous abscisic acid (ABA) was smaller in leavesof sunflower plants (Helianthus annuus L. cv. NK285) that hadbeen grown in a phytotron in humid air than in leaves of sunflowersgrown outdoors. Stomata of the phytotron-grown plants were slowerto close after detachment of a leaf than those of the outdoorplants. When stomata closed rapidly, as they did in detachedleaves and after treatment with ABA, the extent of closure wasvaried over the leaf's surface, in particular in the case ofphytotron-grown plants, and the extent of the heterogeneitywas greater in the phytotrongrown plants than in the outdoorplants. When stomata closed gradually, for example, under conditionsof limited moisture in the soil, closure occurred uniformlyover leaves of plants of both types. The smaller decrease indiffusive conductance of leaves from phytotron-grown plantsafter treatment with ABA resulted from the presence of patcheson the surface in which stomata remained open. The smaller decreaseof diffusive conductance in the phytotron-grown plants underconditions of limited moisture in the soil resulted from theuniformly lower responsiveness of stomata on a leaf to the decreasein water potential. When estimates are made of the intercellularconcentration of CO₂ (Ci) from gas-exchange measurements, heterogeneityin stomatal closure should be monitored when stomata close rapidly,in particular in plants grown in humid air, because heterogeneousstomatal closure can lead to overestimates of Ci. (Received April 18, 1994; Accepted May 25, 1995)     

Short-Term and Long-Term Responses of Crassulacean Acid Metabolism Plants to Elevated CO(2)

For the leaf succulent Agave deserti and the stem succulent Ferocactus acanthodes, increasing the ambient CO₂ level from 350 microliters per liter to 650 microliters per liter immediately increased daytime net CO₂ uptake about 30% while leaving nighttime net CO₂ uptake of these Crassulacean acid metabolism (CAM) plants approximately unchanged. A similar enhancement of about 30% was found in dry weight gain over 1 year when the plants were grown at 650 microliters CO₂ per liter compared with 350 microliters per liter. Based on these results plus those at 500 microliters per liter, net CO₂ uptake over 24-hour periods and dry weight productivity of these two CAM succulents is predicted to increase an average of about 1% for each 10 microliters per liter rise in ambient CO₂ level up to 650 microliters per liter.     

Stomatal Responses of Variegated Leaves to CO2 Enrichment

The responses of stomatal density and stomatal index of fivespecies of ornamental plants with variegated leaves grown attwo mole fractions of atmospheric CO₂ (350 and 700 µmolmol^-1) were measured. The use of variegated leaves allowed anypotential effects of mesophyll photosynthetic capacity to beuncoupled from the responses of stomatal density to changesin atmospheric CO₂ concentration. There was a decrease in stomataldensity and stomatal index with CO₂ enrichment on both white(unpigmented) and green (pigmented) leaf areas. A similar responseof stomatal density and index was also observed on areas ofleaves with pigmentation other than green indicating that anydifferences in metabolic processes associated with colouredleaves are not influencing the responses of stomatal densityto CO₂ concentrations. Therefore the carboxylation capacityof mesophyll tissue has no direct influence on stomatal densityand index responses as suggested previously (Friend and Woodward1990 Advances in Ecological Research 20: 59-124), instead theresponses were related to leaf structure. The stomatal characteristics(density and index) of homobaric variegated leaves showed agreater sensitivity to CO₂ on green portions, whereas heterobaricleaves showed a greater sensitivity on white areas. These resultsprovide evidence that leaf structure may play an important rolein determining the magnitude of stomatal density and index responsesto CO₂ concentrations.Copyright 1995, 1999 Academic Press Leaf structure, photosynthesis, stomatal conductance, CO₂, stomatal density, stomatal index     

Humidity Pretreatment Affects the Responses of Stomata and CO2 Assimilation to Vapor Pressure Difference in C3 and C4 Plants

Experiments were carried out to investigate the long-term influenceof humidity on the short-term responses of stomata and CO₂ assimilationto vapor pressure difference in Oryza sativa (rice, C₃ species)and Panicum maximum (green panic, C₄ species). Plants were grownfor four weeks in growth chambers set at 35% and 85% relativehumidity at 25C air temperature, 38+2 Pa CO₂ partial pressureand 1,700µmol m^-2s^-1 photon flux density. Soil was saturatedwith water in both humidity treatments. Low humidity pretreatmentscaused low leaf conductance and low rates of transpiration andCO₂ assimilation in O. sativa, but small changes in stomatalresponses to humidity and in CO₂ assimilation were found inP. maximum. From the short-term gas exchange experiments, itwas noted that the responsiveness of leaf conductance to vaporpressure difference were affected by humidity pretreatmentsin O. sativa, whereas unaffected in P. maximum. In O. sativameasurements of CO₂ assimilation as a function of internal CO₂partial pressure (A-Ci curve) indicated that low humidity pretreatmentsreduced the CO₂ assimilation at high internal CO₂ partial pressure,but the initial slope of the A-Ci curve was unaffected. Furthermore,plant characteristics such as total dry weight and leaf areaof plants subjected to low umidity were lower than plants subjectedto high humidity. The reductions in O. sativa, however, werelarger than in P. maximum. Stomatal frequency from low humiditygrown plant was higher than that from high humidity grown plantsin both species although there is no significant difference.The data indicated that if the short term inhibition of netCO₂ assimilation at a high vapor pressure difference was imposedduring vegetative growth, the photosynthetic biochemistry andthe resultant plant growth were largely depressed in O. sativa,a C₃ species. (Received May 26, 1992; Accepted November 2, 1992)     

Abscisic Acid Content and Effects During Dehydration of Detached Leaves of Desiccation Tolerant Plants

Borya nitida is an angiospcrm whose detached leaves developcomplete tolerance to dehydration when they are equilibratedto air of 96% r.h. This treatment causes leaves to yellow aschlorophyll is destroyed, and abscisic acid contents increaseseveral-fold. Exogenous ABA (at 0.038–0.38 mol m^–3)promoted desiccation tolerance (a) in leaves undergoing toleranceinduction at 96% r.h., (b) only slightly during rapid dryingat rates which are normally injurious, and (c) considerablyin turgid tissue treated with ABA 48 h before rapid drying. ABA content also increased with intense water stress in Myrothamnusflabellifolia, a desiccation tolerant angiosperm which, unlikeBorya, retains most of its chlorophyll when dehydrated. Preliminaryincubation in ABA of detached leaves of this ‘resurrectionplant’ also promoted survival during rapid drying. Theability of ABA to substitute for the normal induction periodsuggests that this hormone participates in the development ofdesiccation tolerance. Key words: Abscisic acid, ABA, Drought tolerance, Resurrection plant     

Sensitivity of Stomata to Abscisic Acid (An Effect of the Mesophyll)

The effects of added abscisic acid (ABA) on the stomatal behavior of Commelina communis L. were tested using three different systems. ABA was applied to isolated epidermis or to leaf pieces incubated in the light in bathing solutions perfused with CO2-free air. ABA was also fed to detached leaves in a transpiration bioassay. The apparent sensitivity of stomata to ABA was highly dependent on the method used to feed ABA. Stomata of isolated epidermis were apparently most sensitive to ABA, such that a concentration of 1 [mu]M caused almost complete stomatal closure. When pieces of whole leaves were floated on solutions of ABA of the same concentration, the stomata were almost completely open. The same concentration of ABA fed through the midrib of transpiring detached leaves caused an intermediate response. These differences in stomatal sensitivity to added ABA were found to be a function of differences in the ABA concentration in the epidermes. Comparison of the three application systems suggested that, when leaf pieces were incubated in ABA or fed with ABA through the midrib, accumulation of ABA in the epidermes was limited by the presence of the mesophyll. Even bare mesophyll incubated in ABA solution did not accumulate ABA. Accumulation of radioactivity by leaf pieces floated on [3H]ABA confirmed ABA uptake in this system. Experiments with tetcyclacis, an inhibitor of phaseic acid formation, suggested that rapid metabolism of ABA in mesophyll can have a controlling influence on ABA concentration in both the mesophyll and the epidermis. Inhibition of ABA catabolism with tetcyclacis allows ABA accumulation and increases the apparent sensitivity of stomata to applied ABA. The results are discussed in the context of an important role for ABA metabolism in the regulation of stomatal behavior.     

The Influence of Prior Water Stress and Abscisic Acid Foliar Spraying on Stomatal Responses to CO2, IAA, ABA, and Calcium in Leaves of Solanum melongena

The influence of a water stress or foliar ABA spraying pretreatmenton stomatal responses to water loss, exogenous ABA, IAA, Ca²⁺,and CO₂ were studied using excised leaves of Solanum melongena.Both pretreatments increased stomatal sensitivity of water loss,in the presence and absence of CO₂, but decreased stomatal sensitivityto exogenous ABA. CO₂ greatly reduced the effect of exogenouslyapplied ABA. IAA decreased leaf diffusion resistance for controland ABA sprayed leaves, but did not influence the LDR of previouslywater-stressed leaves. CA²⁺ did not influence LDR of any leavesof any treatments. Key words: Water stress, stomatal response, pretreatments     

Osmotic Stress Temporarily Reverses the Inhibitions of Photosynthesis and Stomatal Conductance by Abscisic Acid--Evidence that Abscisic Acid Induces a Localized Closure of Stomata in Intact, Detached Leaves

Ward, D. A. and Drake, B. G. 1988. Osmotic stress temporarilyreverses the inhibitions of photosynthesis and stomatal conductanceby abscisic acid—evidence that abscisic acid induces alocalized closure of stomata in intact, detached leaves.—J.exp. Bot 39: 147–155. The influence of osmotic stress on whole leaf gas exchange wasmonitored in detached leaves of Glycine max supplied with anexogenous concentration (10^–5 mol dm^–3) of ±abscisicacid (ABA) sufficient to inhibit net photosynthesis and stomatalconductance by 60% and 70%, respectively, under a saturatingirradiance and normal air. Raising the osmotic (sorbitol) concentrationof the ABA solutions feeding leaves elicited rapid and synchronousreversals of the ABA-dependent inhibitions of net photosynthesisand conductance. These reversals reached a peak simultaneously,after which photosynthesis and conductance declined. The magnitudeof the transient stimulations at peak height was dependent uponthe sorbitol concentration of the ABA feeding solution, althoughthe time-course of the transients (half time, 4–6 min)was similar for the different osmotic concentrations applied.Irrespective of transient size the relative changes of photosynthesisand conductance were comparable; consequently the calculatedpartial pressure of CO₂ in the substomatal space (Ci) remainedrelatively constant during the transient phase. In contrastto the ABA-treated leaves, elevating the osmotic concentrationof the distilled water supply feeding control leaves stimulatedconductance to a much greater relative extent than photosynthesis.The co-stimulations of photosynthesis and conductance inducedin ABA-treated leaves by osmotic shock were not due to a restrictionin the transpirational uptake of ABA and occurred irrespectiveof the source osmoticum applied. These data are consistent with the hypothesis that the ABA-dependentinhibition of photosynthesis at constant Ci is an artifact causedby the spatially heterogeneous closure of stomata in responseto ABA. Alternative explanations for the responses are, however,considered. Key words: Abscisic acid, photosynthesis, osmotic stress, Glycine max, stomatal conductance     

Developmental Responses to Drought and Abscisic Acid in Sunflower Roots: 2. MITOTIC ACTIVITY

Mitotic activity was studied in the root apices of aeroponicallygrown sunflower seedlings (Helianthus annum L. var. RussianGiant) which were draughted or treated with abscisic acid (ABA)over a 7 d period. Labelling index (LI) and mitotic index (MI)were scored from autoradiographs of median longitudinal sectionsof [³H] methyl-thymidine treated root apices. Both drought stressand ABA-treatment (at a concentration of 10^–2 mol m^–3inhibited DNA synthesis and mitosis within the first 6 h oftreatment. The depression of mitotic activity was first evidentin the proximal regions of the meristem (1000–1500 µmfrom the cap junction). This was followed by a general depressionof mitotic activity throughout the meristem which was, in turn,followed by a partial recovery of mitotic activity in the distalregions of the meristem. The beginning of this partial recoverywas concurrent with the activation of the quiescent centre (QC).Treatment with lower concentrations of ABA (10^–3 mol m^–3and 10^–4 mol m^–3) also inhibited mitotic activity.Exogenous supplements of sucrose to the plant did not alleviatethe inhibition of mitotic activity by drought or ABA. Thesefindings support the hypothesis that ABA mediates drought-inducedchanges in the primary development of sunflower roots. Key words: Abscisic acid, drought, mitotic activity     

Internal CO(2) Measured Directly in Leaves : Abscisic Acid and Low Leaf Water Potential Cause Opposing Effects

Observations of nonuniform photosynthesis across leaves cast doubt on internal CO₂ partial pressures (p_i) calculated on the assumption of uniformity and can lead to incorrect conclusions about the stomatal control of photosynthesis. The problem can be avoided by measuring p_i directly because the assumptions of uniformity are not necessary. We therefore developed a method that allowed p_i to be measured continuously in situ for days at a time under growth conditions and used it to investigate intact leaves of sunflower (Helianthus annuus L.), soybean (Glycine max L. Merr.), and bush bean (Phaseolus vulgaris L.) subjected to high or low leaf water potentials (ψ_w) or high concentrations of abscisic acid (ABA). The leaves maintained a relatively constant differential (Δp) between ambient CO₂ and measured p_i throughout the light period when water was supplied. When water was withheld, ψ_w decreased and the stomata began to close, but measured p_i increased until the leaf reached a ψ_w of −1.76 (bush bean), −2.12 (sunflower) or −3.10 (soybean) megapascals, at which point Δp = 0. The increasing p_i indicated that stomata did not inhibit CO₂ uptake and a Δp of zero indicated that CO₂ uptake became zero despite the high availability of CO₂ inside the leaf. In contrast, when sunflower leaves at high ψ_w were treated with ABA, p_i did not increase and instead decreased rapidly and steadily for up to 8 hours even as ψ_w increased, as expected if ABA treatment primarily affected stomatal conductance. The accumulating CO₂ at low ψ_w and contrasting response to ABA indicates that photosynthetic biochemistry limited photosynthesis at low ψ_w but not at high ABA.     

Abscisic Acid Content, Transpiration, and Stomatal Conductance As Related to Leaf Age in Plants of Xanthium strumarium L

Among the four uppermost leaves of greenhouse-grown plants of Xanthium strumarium L. the content of abscisic acid per unit fresh or dry weight was highest in the youngest leaf and decreased gradually with increasing age of the leaves. Expressed per leaf, the second youngest leaf was richest in ABA; the amount of ABA per leaf declined only slightly as the leaves expanded. Transpiration and stomatal conductance were negatively correlated with the ABA concentration in the leaves; the youngest leaf lost the least amount of water. This correlation was always very good if the youngest leaf was compared with the older leaves but not always good among the older leaves. Since stomatal sensitivity to exogenous (±)-ABA was the same in leaves of all four age groups ABA may be in at least two compartments in the leaf, one of which is isolated from the guard cells.     

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.     

Abscisic Acid Biosynthesis in Leaves and Roots of Xanthium strumarium

Research on the biosynthesis of abscisic acid (ABA) has focused primarily on two pathways: (a) the direct pathway from farnesyl pyrophosphate, and (b) the indirect pathway involving a carotenoid precursor. We have investigated which biosynthetic pathway is operating in turgid and stressed Xanthium leaves, and in stressed Xanthium roots using long-term incubations in ¹⁸O₂. It was found that in stressed leaves three atoms of ¹⁸O from ¹⁸O₂ are incorporated into the ABA molecule, and that the amount of ¹⁸O incorporated increases with time. One ¹⁸O atom is incorporated rapidly into the carboxyl group of ABA, whereas the other two atoms are very slowly incorporated into the ring oxygens. The fourth oxygen atom in the carboxyl group of ABA is derived from water. ABA from stressed roots of Xanthium incubated in ¹⁸O₂ shows a labeling pattern similar to that of ABA in stressed leaves, but with incorporation of more ¹⁸O into the tertiary hydroxyl group at C-1′ after 6 and 12 hours than found in ABA from stressed leaves. It is proposed that the precursors to stress-induced ABA are xanthophylls, and that a xanthophyll lacking an oxygen function at C-6 (carotenoid numbering scheme) plays a crucial role in ABA biosynthesis in Xanthium roots. In turgid Xanthium leaves, ¹⁸O is incorporated into ABA to a much lesser extent than it is in stressed leaves, whereas exogenously applied ¹⁴C-ABA is completely catabolized within 48 hours. This suggests that ABA in turgid leaves is either (a) made via a biosynthetic pathway which is different from the one in stressed leaves, or (b) has a half-life on the order of days as compared with a half-life of 15.5 hours in water-stressed Xanthium leaves. Phaseic acid showed a labeling pattern similar to that of ABA, but with an additional ¹⁸O incorporated during 8′-hydroxylation of ABA to phaseic acid.     

Leaf Senescence and Abscisic Acid in Leaves of Field-grown Soybean

Leaf senescence in field-grown soybean (Merrill) as defined by the period after full expansion, was studied by measuring abscisic acid (ABA), total soluble protein, and chlorophyll in leaves through the later part of the growing season. ABA concentrations increased significantly at the end of the season when leaves had started to turn yellow, well after total soluble protein and chlorophyll had started to decline. The results indicate that events occurring before leaf yellowing are more significant in evaluating leaf senescence since the yellowing condition and rise in ABA are effects of changes in physiological activity beginning when leaves are still green.     

长期喷施ABA对云杉幼苗生长和生理特性的影响

采用单因素盆栽实验, 通过叶面喷施5、10、15和20 mg.L-1 4个浓度的ABA溶液, 研究了长期外源ABA处理对云杉(Picea asperata)幼苗生长及生理特性的影响。5年的研究结果表明: 长期不同浓度ABA处理显著影响了云杉幼苗的多种生长及生理生化指标。当ABA浓度为5、10和15 mg.L-1时有利于云杉幼苗根重、茎重和总生物量的积累, 并且提高了叶片中可溶性蛋白和脯氨酸的含量, 降低了MDA含量; 20 mg.L-1ABA处理使幼苗的叶重、总生物量、脯氨酸及可溶性糖含量显著下降,明显增加了叶片中MDA含量。此外, 各浓度ABA处理均显著降低了云杉幼苗的株高、叶绿素含量以及SOD和APX活性。本研究结果显示, 长期ABA处理对云杉幼苗生长和生理特性的影响与所喷施的ABA浓度有关, 长期高浓度ABA(20 mg.L-1)处理不利于云杉幼苗生长。     

长期喷施ABA对云杉幼苗生长和生理特性的影响

采用单因素盆栽实验,通过叶面喷施5、10、15和20mg·L^-1 4个浓度的ABA溶液,研究了长期外源ABA处理对云杉（Piceaasperata）幼苗生长及生理特性的影响。5年的研究结果表明：长期不同浓度ABA处理显著影响了云杉幼苗的多种生长及生理生化指标。当ABA浓度为5、10和15mg·L^-1叫时有利于云杉幼苗根重、茎重和总生物量的积累,并且提高了叶片中可溶性蛋白和脯氨酸的含量,降低了MDA含量：20mg·L^-1 ABA处理使幼苗的叶重、总生物量、脯氨酸及可溶性糖含量显著下降,明显增加了叶片中MDA含量。此外,各浓度ABA处理均显著降低了云杉幼苗的株高、叶绿素含量以及SOD和APX活性。本研究结果显示,长期ABA处理对云杉幼苗生长和生理特性的影响与所喷施的ABA浓度有关,长期高浓度ABA（20mg·L^-1）处理不利于云杉幼苗生长。