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     

ACTION OF FUSICOCCIN ON THE POTASSIUM BALANCE OF GUARD CELLS OF PHASEOLUS VULGARIS

Fusicoccin induces stomatal opening in both the light and dark. The stomatal aperture and K content of guard cells was measured to determine whether the action of fusicoccin in inducing stomatal opening is directly related to the uptake of K by the guard cells. Both detached and attached epidermis was treated with fusicoccin and the K content was determined by staining with cobalt sodium nitrite or by electron probe microanalysis. The K content of guard cells in detached epidermal strips floated on 10 μm fusicoccin in 10 mm KCl and aqueous CH₃OH (0.02%, v/v) increased in the light and dark as the stomata opened. After exposure to fusicoccin for 6 hr in the light, however, the stomata were closed and no K could be detected in the guard cells. The K content of guard cells of attached epidermis painted with fusicoccin also increased as the stomata opened, but the concentration of K in the subsidiary cells was not significantly altered by fusicoccin-stimulated opening. Moreover, painting with fusicoccin did not significantly change the Ca and P content of the guard or subsidiary cells. Stomata of epidermal strips, opened to their maximum width by fusicoccin, showed only a small and temporary closure when transferred to a solution of 10 μm abscisic acid. The use of metabolic inhibitors suggested that energy for the uptake of the K may be provided by both photophosphorylation and oxidative phosphorylation.     

Guard cells of Commelina communis L. do not respond metabolically to osmotic stress in isolated epidermis: Implications for stomatal responses to drought and humidity

We investigated the hypothesis that stomatal aperture is regulated by epidermal water status. Detached epidermal peels of Commelina communis L. or leaf disks with epidermis attached were incubated in graded solutions of mannitol (0–1.2 M) containing KCl. In isolated epidermis, guard-cell solute content of open stomata did not decrease in response to desiccation. Guard cells of closed stomata accumulated solutes to the same extent in all levels of mannitol tested. There was no evidence of stress-induced hydroactive closure nor of inhibition of hydroactive opening, even when guard cells of closed stomata were initially plasmolyzed. Hydropassive, osmometer-like, changes in stomatal aperture in the isolated epidermis were induced by addition or removal of mannitol, but these did not involve changes in guard-cell solute content. In leaf disks, stomata exhibited clear hydroactive stomatal responses. Steady-state guard-cell solute content of initially open and initially closed stomata decreased substantially with increasing mannitol. Stomata were completely closed above approx. 0.4 M mannitol, near the turgor-loss point for the bulk leaf tissue. Stomata of Commelina did not exhibit direct hydroactive responses to environmental or epidermal water status. Stomatal responses to water deficit and low humidity may be indirect, mediated by abscisic acid or other signal metabolite(s) from the mesophyll.Abbreviations ABA abscisic acid - EGTA ethyleneglycol-bis-(-aminoethyl ether)-N,N,N,N-tetraacetic acid - Mes 2-(N-morpholino)ethanesulfonic acid     

Species-dependent changes in stomatal sensitivity to abscisic acid mediated by external pH

The direct effects of pH changes and/or abscisic acid (ABA) on stomatal aperture were examined in epidermal strips of Commelina communis L. and Arabidopsis thaliana. Stomata were initially opened at pH 7 or pH 5. The stomatal closure induced by changes in external pH and/or ABA (10 microM or 10 nM) was monitored using video microscopy and quantified in terms of changes in stomatal area using image analysis software. Measurements of aperture area enabled stomatal responses and, in particular, small changes in stomatal area to be quantified reliably. Both plant species exhibited a biphasic closure response to ABA: an initial phase of rapid stomatal closure, followed by a second, more prolonged, phase during which stomata closure proceeded at a slower rate. Changes in stomatal sensitivity to ABA were also observed. Comparison of these effects between C. communis and A. thaliana demonstrate that this differential sensitivity of stomata to ABA is species-dependent, as well as being dependent on the pH of the extracellular environment.     

Effect of abscisic acid pretreatments on stomatal reopening in Vicia faba

After a pretreatment of 2 h exposure to a solution containing 2 × 10⁻⁴ M ABA, reopening of stomata occurred in epidermal strips of Vicia faba L. cv. Cavalier on an ABA-free incubation solution. After pretreatment with exogenous ABA stomatal apertures were greater when higher levels of KCl were incorporated into the solution used for reopening. Prolonged exposure to exogenous ABA (14 h) did not prevent stomatal reopening upon transfer to ABA-free solutions. However, for both ABA and ABA-free pretreatments, prolonged incubation (1 day after removal of epidermis) resulted in enhanced stomatal apertures when the epidermal strips were exposed to light. This effect was lost 2 days after removal of the epidermis and opening did not occur after 3 days. Epidermal strips containing endogenous ABA were obtained from wilted leaves. Reopening was greatly reduced by the endogenous ABA treatment, and variation of KCl concentration in the incubation solution had little effect on stomatal aperture. It is postulated that during wilting endogenous ABA becomes reversibly bound without loss of activity for a longer period than is obtained using exogenous ABA. The presence of other unidentified compounds may be involved in this process.     

How Do Stomata Read Abscisic Acid Signals?

When abscisic acid (ABA) was fed to isolated epidermis of Commelina communis L., stomata showed marked sensitivity to concentrations of ABA lower than those commonly found in the xylem sap of well-watered plants. Stomata were also sensitive to the flux of hormone molecules across the epidermal strip. Stomata in intact leaves of Phaseolus acutifolius were much less sensitive to ABA delivered through the petiole than were stomata in isolated epidermis, suggesting that mesophyll tissue and/or xylem must substantially reduce the dose or activity of ABA received by guard cells. Delivery of the hormone to the leaf was varied by changing transpiration flux and/or concentration. Varying delivery by up to 7-fold by changing transpiration rate had little effect on conductance. At a given delivery rate, variation in concentration by 1 order of magnitude significantly affected conductance at all but the highest concentration fed. The results are discussed in terms of the control of stomatal behavior in the field, where the delivery of ABA to the leaf will vary greatly as a function of both the concentration of hormone in the xylem and the transpiration rate of the plant.     

Stomatal deregulation in Plasmopara viticola-infected grapevine leaves

In grapevine, the penetration and sporulation of Plasmopara viticola occur via stomata, suggesting functional relationships between guard cells and the pathogen. This assumption was supported by our first observation that grapevine (Vitis vinifera cv. Marselan) cuttings infected by P. viticola wilted more rapidly than healthy ones when submitted to water starvation. Here, complementary approaches measuring stomatal conductance and infrared thermographic and microscopic observations were used to investigate stomatal opening/closure in response to infection. In infected leaves, stomata remained open in darkness and during water stress, leading to increased transpiration. This deregulation was restricted to the colonized area, was not systemic and occurred before the appearance of symptoms. Cytological observations indicated that stomatal lock-open was not related to mechanical forces resulting from the presence of the pathogen in the substomatal cavity. In contrast to healthy leaves, stomatal closure in excised infected leaves could not be induced by a water deficit or abscisic acid (ABA) treatment. However, ABA induced stomatal closure in epidermal peels from infected leaves, indicating that guard cells remained functional. These data indicate that the oomycete deregulates guard cell functioning, causing significant water losses. This effect could be attributed to a nonsystemic compound, produced by the oomycete or by the infected plant, which inhibits stomatal closure or induces stomatal opening; or a reduction of the back-pressure exerted by surrounding epidermal cells. Both hypotheses are under investigation.     

The relationship between guard cell water potential and the aperture of stomata in Populus

Abstract Previous work with clones of Populus trichocarpa demonstrated that the water vapour conductance of leaves from well-watered cuttings of this species does not decline with loss of turgor from the bulk leaf. In the present study, stomatal responses to water potential in Populus were examined with detached epidermal strips. Stomata in epidermal strips from well-watered plants of P. trichocarpa did not close at low water potentials which led to plasmolysis of the guard cells. In contrast, stomata of P. deltoides and a P. trichocarpa×deltoides hybrid closed when the guard cells lost turgor. A period of water stress preconditioning resulted in modified stomatal responses in P. trichocarpa such that stomata of stressed and re-watered plants nearly closed when guard cell turgor was lost.     

Potassium Chloride as Stomatal Osmoticum in Allium cepa L., a Species Devoid of Starch in Guard Cells

K⁺ and Cl⁻ contents of guard cells and of ordinary epidermal cells were determined in epidermal samples of Allium cepa L. by electron probe microanalysis; malate contents of the same samples were determined by enzymic oxidation. KCl was, in general, the major osmoticum in guard cells, irrespective of whether stomata had opened on leaves or in epidermal strips floating on solutions. The solute requirement varied between 50 and 110 femtomoles KCl per micrometer increase in aperture per pair of guard cells. Stomata did not open on solutions of K iminodiacetate, presumably because its anion could not be taken up. Stomata opened if KCl or KBr was provided. Taken together, the results indicate that the absence of starch from guard cells deprived them of the ability to produce malate in amounts of osmotic consequence and that the presence of absorbable Cl⁻ (or Br⁻) was necessary for stomatal opening.     

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.     

Differential stomatal closure by abscisic acid in epidermal strips of green and pigmented leaves or leaf parts

Response of stomata in epidermal strips from green leaves ofTradescantia sillamontana and anthocyanin-rich purple leaves ofT. virginiana and from green and pigmented regions ofPedilanthus tithymaloides leaves, to ABA have been compared. Stomata from anthocyanin-rich leaves or leaf parts appeared to be relatively insensitive to ABA as compared to those from green leaves or leaf parts. Observations indicate the possibility of the involvement of endogenous anthocyanins in antagonising ABA in preventing the stomatal opening.     

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.     

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     

Effect of Potassium Levels on the Stomatal Behavior of the Hemi-Parasite Striga hermonthica

The hemi-parasite Striga hermonthica, exhibits an anomalous pattern of stomatal response, stomata remaining open in darkness and when subjected to water stress. This suggests irregularity in stomatal response due to malfunction of the stomatal mechanism. To test this suggestion guard cells were isolated from the effects of surrounding cells, by incubating epidermal strips at low pH. These stomata responded rapidly to low CO₂ concentrations, darkness, and ABA. Thus, a paradox exists between stomatal behavior observed in whole leaves and that in isolated guard cells. However, when incubated in the presence of high potassium concentrations (>200 millimolar KCl) stomatal responses in epidermal strips resembled those found in whole leaves, with enhanced opening and reduced closing responses. It is suggested that the anomalous behavior of stomata in Striga and other leafy hemiparasites can be explained by the modulatory effects of high potassium concentrations which accumulate in the leaves as a consequence of high transpiration rates and the lack of a retranslocation system.     

Stomatal movement and potassium transport in epidermal strips of Zea mays: The effect of CO2

Summary The correlation between stomatal action and potassium movement in the epidermis of Zea mays was examined in isolated epidermal strips floated on distilled water. Stomatal opening in the isolated epidermis is reversible in response to alternate periods of light or darkness, and is always correlated with a shift in the potassium content of the guard cells. K accumulates in guard cells during stomatal opening, and moves from the guard cells into the subsidiary cells during rapid stomatal closure. When epidermal strips are illuminated in normal air, as against CO₂-free air, the stomata do not open and there is a virtually complete depletion of K from the stomatal apparatus. In darkness CO₂-containing air inhibits stomatal opening and K accumulation in guard cells, but does not lead to a depletion of K from the stomata as observed in the light.     

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.     

Stomata of Micropropagated DelphiniumPlants Respond to ABA, CO2, Light and Water Potential, but Fail to Close Fully

Morphological and physiological characteristics of micropropagatedplants of Delphinium cv. Princess Caroline were studied. Leavesproduced in vitro showed poor control of water loss which appearsto result from restricted responses by stomata and not frompoor cuticular development. Stomata of leaves produced in vitrowere larger and more frequent than those produced during acclimatization.Despite the fact that stomata from isolated epidermis of leavesproduced in vitro reduced their apertures when exposed to turgor-reducingtreatments, they did not close fully. This, together with highstomatal frequencies might explain the poor control of waterloss shown by intact leaves produced in culture when exposedto dry air. While leaves from acclimatized plants showed almostcomplete closure with ABA, low water potentials, darkness andCO₂, stomata from leaves produced in vitro reduced their apertureswhen exposed to those factors, but only to a limit. Therefore,stomata from leaves cultured in vitro seem to be partially functional,but some physiological or anatomical alteration prevents themfrom closing fully. Stomata from leaves produced in vitro wereparticularly insensitive to ABA which appears to be partly associatedwith the high cytokinin concentration in the culture medium.In the long-term, this stomatal insensitivity to ABA might contributeto plant losses when micropropagated plantlets are transferredto soil. Key words: Micropropagation, stomatal physiology, dehydration, PEG, ABA, BAP, darkness, CO₂, Delphinium     

The Effects of Temperature and ABA on Stomata of Zea mays L.

Epidermal fragments were removed from maize leaves by tearingparallel to the veins. These were incubated at a number of differenttemperatures in several concentrations of ABA. The sensitivityof stomata to temperature was dependent upon the technique usedto incubate epidermis. Generally, the widest apertures wererecorded at around 25°C. In all experiments, stomata incubatedat low (10°C) temperatures on 5.6 x 10^–4 M ABA showedwider apertures than those incubated on distilled water. ThisABA-stimulated stomatal opening was accompanied by an increasein the intensity of potassium staining in the guard cells. At25 °C, epidermis incubated on several concentrations ofABA showed some stomatal closure, decreased potassium stainingin the guard cells and increased potassium staining in the subsidiarycells.     

Guard cell photosynthesis is critical for stomatal turgor production,yet does not directly mediate CO2‐ and ABA‐induced stomatal closing

Stomata mediate gas exchange between the inter‐cellular spaces of leaves and the atmosphere. CO₂ levels in leaves (Ci) are determined by respiration, photosynthesis, stomatal conductance and atmospheric [CO₂]. [CO₂] in leaves mediates stomatal movements. The role of guard cell photosynthesis in stomatal conductance responses is a matter of debate, and genetic approaches are needed. We have generated transgenic Arabidopsis plants that are chlorophyll‐deficient in guard cells only, expressing a constitutively active chlorophyllase in a guard cell specific enhancer trap line. Our data show that more than 90% of guard cells were chlorophyll‐deficient. Interestingly, approximately 45% of stomata had an unusual, previously not‐described, morphology of thin‐shaped chlorophyll‐less stomata. Nevertheless, stomatal size, stomatal index, plant morphology, and whole‐leaf photosynthetic parameters (PSII, qP, qN, F_V′/F_M′) were comparable with wild‐type plants. Time‐resolved intact leaf gas‐exchange analyses showed a reduction in stomatal conductance and CO₂‐assimilation rates of the transgenic plants. Normalization of CO₂ responses showed that stomata of transgenic plants respond to [CO₂] shifts. Detailed stomatal aperture measurements of normal kidney‐shaped stomata, which lack chlorophyll, showed stomatal closing responses to [CO₂] elevation and abscisic acid (ABA), while thin‐shaped stomata were continuously closed. Our present findings show that stomatal movement responses to [CO₂] and ABA are functional in guard cells that lack chlorophyll. These data suggest that guard cell CO₂ and ABA signal transduction are not directly modulated by guard cell photosynthesis/electron transport. Moreover, the finding that chlorophyll‐less stomata cause a ‘deflated’ thin‐shaped phenotype, suggests that photosynthesis in guard cells is critical for energization and guard cell turgor production.     

叶子花表皮特征的研究及意义

对叶子花（Bougainvillea spectabilis）正常叶和变态叶上气孔密度、气孔指数和保卫细胞大小进行了研究。结果表明：正常叶上表皮的表皮细胞为多边形,垂周壁平直;下表皮的表皮细胞为不规则型,垂周壁浅波状;气孔类型为不规则型。变态叶上表皮没有发现气孔,变态叶下表皮的表皮细胞垂周壁则由浅波形逐渐变为深波形,气孔类型为不规则型和轮列型。随着变态叶的发育,变态叶下表皮的气孔密度降低,气孔指数升高;变态叶保卫细胞的长增大,宽减小。变态叶的平均气孔密度和平均气孔指数明显低于正常叶。正常叶和变态叶的保卫细胞均呈肾形。