Stomatal closure is induced rather by prevailing xylem abscisic acid than by accumulated amount of xylem-derived abscisic acid

We have studied the stomatal response in relation to the xylem-derived abscisic acid (ABA) accumulation in sunflower leaves. When ABA was introduced into detached leaves of the sunflower through xylem flux, stomatal conductance was regulated, water flux was changed as a result and at the same time the xylem-derived ABA was metabolised in the leaves. We computed the xylem-derived ABA accumulation in the leaves as a function of time by taking into account the variation of ABA flux into the leaves (the product of water flux and ABA concentration) and a continuing ABA metabolism. We found that ABA accumulation was rapid during an initial lag phase, much slowed down during the decreasing phase of stomatal conductance, but still substantial when stomatal conductance reached a new stable state. The results show a poor link between the kinetics of ABA-induced stomatal closure and the xylem-derived ABA accumulation. Xylem-derived ABA was metabolised rapidly in the leaves. Tetcyclacis, as an inhibitor, substantially inhibited this process. Two hours after ABA was fed into a leaf, about 70% of the fed ABA was metabolised, but when tetcyclacis was added into the feeding solution, less than 30% of ABA was metabolised, even after 24 h of incubation. The inhibition of ABA metabolism by tetcyclacis did not lead to more stomatal closure, which was still concentration-dependent. Since the accumulation of xylem-derived ABA was enhanced substantially by the presence of tetcyclacis, these results strongly indicate that stomata mainly respond to the prevailing ABA concentration in the xylem stream, rather than to the accumulated amount of xylem-derived ABA in the leaves.     

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.     

Rapid low temperature-induced stomatal closure occurs in cold-tolerant Commelina communis leaves but not in cold-sensitive tobacco leaves, via a mechanism that involves apoplastic calcium but not abscisic acid

Commelina communis stomata closed within 1 h of transferring intact plants from 27 degrees C to 7 degrees C, whereas tobacco (Nicotiana rustica) stomata did not until the leaves wilted. Abscisic acid (ABA) did not mediate cold-induced C. communis stomatal closure: At low temperatures, bulk leaf ABA did not increase; ABA did not preferentially accumulate in the epidermis; its flux into detached leaves was lower; its release from isolated epidermis was not greater; and stomata in epidermal strips were less sensitive to exogenous ABA. Stomata of both species in epidermal strips on large volumes of cold KCl failed to close unless calcium was supplied. Therefore, the following cannot be triggers for cold-induced stomatal closure in C. communis: direct effects of temperature on guard or epidermal cells, long-distance signals, and effects of temperature on photosynthesis. Low temperature increased stomatal sensitivity to external CaCl(2) by 50% in C. communis but only by 20% in tobacco. C. communis stomata were 300- to 1,000-fold more sensitive to calcium at low temperature than tobacco stomata, but tobacco epidermis only released 13.6-fold more calcium into bathing solutions than C. communis. Stomata in C. communis epidermis incubated on ever-decreasing volumes of cold calcium-free KCl closed on the lowest volume (0.2 cm(3)) because the epidermal apoplast contained enough calcium to mediate closure if this was not over diluted. We propose that the basis of cold-induced stomatal closure exhibited by intact C. communis leaves is increased apoplastic calcium uptake by guard cells. Such responses do not occur in chill-sensitive tobacco leaves.     

Effect of the Mesophyll on Stomatal Opening in Commelina communis

The effect of a number of factors on the opening of stomatain the intact leaf and in the isolated leaf epidermis of Commelinacommunishas been investigated. Stomata in the intact leaf opened widein the light and closed rapidly on transfer to the dark. Theywere also sensitive to CO₂. In contrast, stomata in isolatedepidermis floated on an incubation solution containing 100 molm^–3KCl responded neither to light nor CO₂. They opened as widelyas those in the intact leaf when treated with fusicoccin. Stomata in isolated epidermis opened almost as wide as thosein the intact leaf when they were incubated with isolatedmesophyllcells in the light. The solution in which the mesophyll cellswere incubated was separated by centrifugation. Themedium fromcells previously incubated in the light caused the stomata inisolated epidermis to open but that from cells kept inthe darkhad no effect. A similar effect was observed when isolated chloroplastswere incubated with the isolated epidermis.However, the supernatantfrom the chloroplast suspension had no significant effect onstomatal opening. These results indicate that the mesophyll plays an importantrole in stomatal opening in the light. The mesophyll appearstoproduce in the light, but not in the dark, a soluble compoundwhich moves to the guard cells to bring about stomatal opening.Theexperiments with isolated chloroplasts suggest that this substanceis a product of photosynthesis. Key words: Commelina communis, stomata, light, mesophyll     

Syringomycin, a bacterial phytotoxin, closes stomata

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

The Flux and Distribution of Xylem Sap Calcium to Adaxial and Abaxial Epidermal Tissue 8

Inherent differences in the responses of stomata on abaxialand adaxial epidermal surfaces of leaves of Commelina communishave previously been suggested to be due to differences in theconcentrations of apoplastic Ca. Adaxial stomata have also beenreported to be more sensitive than abaxial stomata to appliedabscisic acid (ABA). The aims of these experiments were to determinethe validity of these conclusions and to see if xylem sap Cahas a role in determining the response of stomata to ABA. It can be shown from measurements of relative stomatal resistance(determined with a viscous flow porometer) and stomatal conductancethat stomata were more open in plants grown on 8-0 mol m^–3Ca, than with those grown on 2-0 mol m^–3 Ca. When attachedleaves were fed with ABA via the transpiration stream neitherthe extent nor the rate with which conductance declined wasdependent on Ca nutrition. The extent of Ca accumulation within both epidermes was relatedto the concentration of Ca in the rhizosphere and in the xylemsap. It did not, however, appear to reflect the apparent differencesin the flux of the transpiration stream between the two epidermes.Plants growing at the lower Ca concentration accumulated proportionallymore epidermal Ca relative to Ca in xylem sap. The evidencepresented suggests that Ca movement from the xylem to the epidermiscannot be simply described by a mass flow model, and that thedistribution of Ca is not an adequate explanation of the differencesin the behaviour of adaxial and abaxial stomata. The potentialrole for changes in xylem sap Ca to act as a regulator of stomatalbehaviour are discussed. Key words: Abscisic acid, calcium, Commelina communis L., stomatal conductance     

Stomatal responses to water stress and to abscisic Acid in phosphorus-deficient cotton plants

Cotton (Gossypium hirsutum L.) plants were grown in sand culture on nutrient solution containing adequate or growth-limiting levels of P. When water was withheld from the pots, stomata of the most recently expanded leaf closed at leaf water potentials of approximately −16 and −12 bars in the normal and P-deficient plants, respectively. Pressure-volume curves showed that the stomata of P-deficient plants closed when there was still significant turgor in the leaf mesophyll. Leaves of P-deficient plants accumulated more abscisic acid (ABA) in response to water stress, but the difference was evident only at low water potentials, after initiation of stomatal closure. In leaves excised from unstressed plants, P deficiency greatly increased stomatal response to ABA applied through the transpiration stream. Kinetin blocked most of this increase in apparent sensitivity to ABA. The effect of P nutrition on stomatal behavior may be related to alterations of the balance between ABA and cytokinins.     

Stomatal Responses to Flooding of the Intercellular Air Spaces Suggest a Vapor-Phase Signal Between the Mesophyll and the Guard Cells

Flooding the intercellular air spaces of leaves with water was shown to cause rapid closure of stomata in Tradescantia pallida, Lactuca serriola, Helianthus annuus, and Oenothera caespitosa. The response occurred when water was injected into the intercellular spaces, vacuum infiltrated into the intercellular spaces, or forced into the intercellular spaces by pressurizing the xylem. Injecting 50 mm KCl or silicone oil into the intercellular spaces also caused stomata to close, but the response was slower than with distilled water. Epidermis-mesophyll grafts for T. pallida were created by placing the epidermis of one leaf onto the exposed mesophyll of another leaf. Stomata in these grafts opened under light but closed rapidly when water was allowed to wick between epidermis and the mesophyll. When epidermis-mesophyll grafts were constructed with a thin hydrophobic filter between the mesophyll and epidermis stomata responded normally to light and CO₂. These data, when taken together, suggest that the effect of water on stomata is caused partly by dilution of K⁺ in the guard cell and partly by the existence of a vapor-phase signal that originates in the mesophyll and causes stomata to open in the light.Stomatal responses to the environment have been studied in leaves for well over 100 years. More recently, the mechanisms for these responses have been investigated using isolated epidermes or isolated guard cell protoplasts. Despite the combination of these two approaches, the mechanisms by which stomata respond to environmental signals are not well understood. Since stomata control CO₂ uptake and water loss from leaves, the responses of stomata to environmental factors are important determinants of terrestrial productivity and water use. It is therefore critical that we understand the mechanisms by which stomata respond to the environment if we are to accurately predict the effects of future climates on productivity and water cycles (Randall et al., 1996).There are two assumptions about stomata that are implicit in much of the recent literature: (1) that stomatal responses result from sensory mechanisms that reside within the guard cells, and (2) that stomata in isolated epidermes respond similarly to those in a leaf. The exception to this generalization is the stomatal response to humidity, which has been suggested to be the result of changes in guard cell water potential (Dewar, 1995, 2002) or of signaling from other cells in the leaf to the guard cells (Buckley et al., 2003). The assumption that guard cells directly sense CO₂ and light is largely based on data from isolated epidermes that show effects of light and CO₂ on stomatal apertures. As pointed out by Mott (2009), however, stomatal responses to light and CO₂ in isolated epidermes are generally much different from those observed in leaves; e.g. responses in isolated epidermes are generally smaller than those in leaves, opening in response to light is slower, and closing in darkness is rarely observed. These observations were used to suggest that the mesophyll is somehow involved in stomatal responses to red light and CO₂. This idea is supported by several recent studies that suggest that guard cells do not respond directly to red light. In the first of these studies it was shown that guard cells in an intact leaf do not show hyperpolarization of the plasma membrane in response to red light if the red light is applied to only the guard cell (Roelfsema et al., 2002). In contrast, blue light applied only to the guard cell does cause hyperpolarization, and red light does cause hyperpolarization if applied to the guard cell and the underlying mesophyll. The second study showed that stomata in albino areas of a leaf do not respond to red light, although they contain chloroplasts and do respond to blue light (Roelfsema et al., 2006). Finally, a third study has shown that isolated epidermes are much more sensitive to light and CO₂ when placed in close contact with an exposed mesophyll from a leaf from the same or a different species (Mott et al., 2008). These epidermis-mesophyll grafts showed stomatal responses to light and CO₂ that were indistinguishable from those in an intact leaf—a sharp contrast to the behavior of stomata in isolated epidermes that are floating on buffer solutions. In that study, illumination of a single stoma in a leaf using a small-diameter fiber optic did not produce stomatal opening, but opening did occur if several stomata and the underlying mesophyll were illuminated. Furthermore, this treatment actually caused opening of adjacent, but unilluminated, stomata (Mott et al., 2008).In constructing the epidermis-mesophyll grafts in the study described above (Mott et al., 2008), it was noticed that functional grafts could be produced only if both the mesophyll and the epidermis were blotted completely dry of any free water before placing them together. Although the tissues were apparently still fully hydrated, there was very little free water present (i.e. water not contained within the walls of the leaf cells), and both the mesophyll and epidermis felt and looked dry prior to assembly. In addition, even when free water was blotted away initially, stomata did not open in grafts that ended up with visible water on the epidermis or mesophyll that was caused by condensation during the experiment. These observations suggest that the presence of free water somehow prevented the stomata in the grafts from opening. Assuming that the mechanisms operating in the grafts were similar to those in an intact leaf, this result also suggests that free water may have an effect on stomata in leaves as well. In addition, it seems possible that the effect of free water on stomata could be related to the disruption of the signal from the mesophyll that was proposed in an earlier study (Mott et al., 2008). We hypothesize that disruption of this signal could be caused by (1) dilution of some solute that is necessary for opening (such as K⁺) in the guard cell walls, (2) dilution of an apoplastic, liquid-phase opening signal from the mesophyll to the guard cells, and (3) blockage of a vapor-phase opening signal from the mesophyll to the guard cells. This study was initiated to test these three hypotheses by examining the effect of free water and other liquids on stomatal functioning.     

Responses of stomata of senescing and nonsenescing leaves of Nicotians glauca to changes in intercellular concentrations of leaf carbon dioxide

Abstract. Gas exchange measurements were performed to test the hypothesis that failure of stomata to open in senescing leaves of Nicotiana glauca is caused by elevated concentrations of carbon dioxide in the intercellular spaces of leaf mesophyll tissue (c_i). Senescing leaves selected for experiments were completely chlorotic and lacked positive rates of photosynthesis. When stomata in detached epidermis from senescing leaves were illuminated in CO2-free air, they opened to similar apertures as those in detached epidermis from nonsenescing leaves. To compare the effects of changes in c_i on stomatal responses of the two leaf types, leaf 'flags' of either nonsenescing or senescing leaves were illuminated at a photosynthetic photon flux density of 500 μmol m⁻² s⁻¹ in a gas exchange cuvette. Leaf temperatures were maintained at 23.5 ± 0.5°C, and vapour pressure differences between leaves and the air were maintained between 0.70 and 0.75kPa. C_i was adjusted by changing external concentrations of carbon dioxide in air circulating through the cuvette. Conductances and photosynthetic rates of nonsenescing leaves changed in response to changes in c_i, but neither the conductances nor the photosynthetic rates of senescing leaves were affected significantly by changes in q. We conclude that guard cells of senescing leaves of Nicotiana glauca do not lose the capacity to respond to changes in carbon dioxide concentration and that increases in c_i resulting from declining rates of mesophyll photosynthesis are not the sole cause of maintenance of stomatal closure during leaf senescence. The data suggest that factors external to guard cells may prevent them from responding to changes in carbon dioxide concentrations in intact senescing leaves.     

The Effects of Cytokinins and ABA on Stomatal Behaviour of Maize and Commelina

Epidermal strips and leaf fragments of Commelina and leaf fragmentsof maize were incubated on solutions containing naturally-occurringor synthetic cytokinins and/or ABA. The effects of these treatmentson stomatal behaviour were assessed. Cytokinins alone did notpromote stomatal opening in either species but concentrationsof both zeatin and kinetin from 10^–3 to 10^–1 molm^–3 caused some reversal of ABA-stimulated closure ofmaize stomata. The reversal of the ABA effect increased withincreasing cytokinin concentration. Cytokinins had no effecton ABA-stimulated closure of Commelina stomata. When appliedalone, at high concentration (10^–1 mol m^–3), toCommelina epidermis or leaf pieces both zeatin and kinetin restrictedstomatal opening. Key words: ABA, Cytokinins, Stomata, Maize, Commelina     

ABA xylem concentrations determine maximum daily leaf conductance of field-grown Vitis vinifera L. plants

Differences in maximum leaf conductance in grapevine plants growing in soils with contrasting water availabilities during mid-summer in Portugal could be accounted for by differences in the concentration of ABA in xylem sap. This conclusion is reinforced by the observation that the relationship between leaf conductance and endogenous ABA concentration can be mimicked by the application of exogenous ABA to leaves detached from irrigated plants. During the day, leaf conductance decreased after a morning peak, even when the leaves remained in a constant environment at a moderate temperature and leaf-to-air vapour pressure difference. This decline in leaf conductance was not a consequence of an increase in the xylem ABA concentration or the rate of delivery of this compound by the transpiratory stream. The afternoon depression in leaf conductance was associated with an apparent limitation in stomatal opening potential, which persisted even when detached leaves were fed with water and rehydrated. The reason for this inhibition has still to be identified.     

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

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

Kinetic characterization of reduced pyridine nucleotide dehydrogenases (duroquinone-dependent) in cucurbita microsomes

Stomatal conductances of normally oriented and inverted leaves were measured as light levels (photosynthetic photon flux densities) were increased to determine whether abaxial stomata of Vicia faba leaves were more sensitive to light than adaxial stomata. Light levels were increased over uniform populations of leaves of plants grown in an environmental chamber. Adaxial stomata of inverted leaves reached maximum water vapor conductances at a light level of 60 micromoles per square meter per second, the same light level at which abaxial stomata of normally oriented leaves reached maximum conductances. Abaxial stomata of inverted leaves reached maximum conductances at a light level of 500 micromoles per square meter per second, the same light level at which adaxial stomata of normally oriented leaves reached maximum conductances. Maximum conductances in both normally oriented and inverted leaves were about 200 millimoles per square meter per second for adaxial stomata and 330 millimoles per square meter per second for abaxial stomata. Regardless of whether leaves were normally oriented or inverted, when light levels were increased to values high enough that upper leaf surfaces reached maximum conductances (about 500 micromoles per square meter per second), light levels incident on lower, shaded leaf surfaces were just sufficient (about 60 micromoles per square meter per second) for stomata of those surfaces to reach maximum conductances. This `coordinated' stomatal opening on the separate epidermes resulted in total leaf conductances for normally oriented and inverted leaves that were the same at any given light level. We conclude that stomata in abaxial epidermes of intact Vicia leaves are not more sensitive to light than those in adaxial epidermes, and that stomata in leaves of this plant do not respond to light alone. Additional factors in bulk leaf tissue probably produce coordinated stomatal opening on upper and lower leaf epidermes to optimally meet photosynthetic requirements of the whole leaf for CO₂.     

Xylem Sap pH Increase: A Drought Signal Received at the Apoplastic Face of the Guard Cell That Involves the Suppression of Saturable Abscisic Acid Uptake by the Epidermal Symplast

Drought increased the pH of Commelina communis xylem sap from 6.1 to 6.7. Conductances of transpiring leaves were 50% lower in pH 7.0 than in pH 6.0 buffers, but bulk leaf abscisic acid (ABA) concentration and shoot water status were unaffected by pH. Stomatal apertures of isolated abaxial epidermis incubated on simple buffers increased with external pH, so in vivo this must be overridden by alternative pH effects. Reductions in leaf transpiration rate at pH 7.0 were dependent on the presence of 10-8 mol dm-3 ABA in the xylem stream. We inferred that at pH 7.0 leaf apoplastic ABA concentrations increased: pH did not affect distributions of ABA among leaf tissues, but isolated epidermis and mesophyll tissue took up more 3H-ABA from pH 6.0 than from pH 7.0 buffers. The apoplastic ABA increase at pH 7.0 may result from reduced symplastic sequestration. A portion of 3H-ABA uptake by the epidermis was saturable at pH 6.0 but not at pH 7.0. An ABA uptake carrier may contribute to ABA sequestration by the leaf symplast of well-watered plants, and its inactivity at pH 7.0 may favor apoplastic ABA accumulation in draughted plants. Effects of external pH on stomatal apertures in the isolated epidermis indicate that published data supporting a role for internal guard cell ABA receptors should be reassessed.     

An Investigation into the Stomatal Behaviour of a Wilty Mutant of Pisum sativum

The water relations and stomatal behaviour of a wilty line ofpea (JI 1069) were investigated and compared with those of severalnon-wilty lines (JI 1180, JI 1194, and JI 74). The leaves ofthe wilty line were found to have a lower percent water content,water potential and diffusive resistance and the dimensionsof the stomatal cells were larger than those of the non-wiltytypes. The aperture of stomata on epidermal samples taken from plantsin the light or dark period of a diurnal rhythm was consistentlylarger for the wilty pea than for the non-wilty lines, however,their stomatal responses on detached epidermis to light, CO₂and KC1 concentration were similar. There was no differencein response to ABA of stomata on detached epidermis of wiltyor non-wilty types of pea. Key words: Pisum sativum, Wilty mutant, Water relations, Stomatal behaviour     

Is the signal from the mesophyll to the guard cells a vapour‐phase ion?

Previous studies have suggested that the red light and CO₂ responses of stomata are caused by a signal from the mesophyll to the guard cells. Experiments were conducted to test the idea that this signal is a vapour‐phase ion. Stomata in isolated epidermes of Tradescantia pallida were found to respond to air ions created by an electrode that was positioned under the epidermes. Anthocyanins in the epidermes of this species were observed to change colour in response to these air ions, and this change in colour was attributed to changes in pH. A similar change in lower epidermal colour was observed in intact leaves upon illumination and with changes in CO₂ concentration. Based on the change in epidermal colour, the pH of the epidermis was estimated to be approximately 7.0 in darkness and 6.5 in the light. Stomata in isolated epidermes responded to pH when suspended over (but not in contact with) solutions of different pH. We speculate that stomatal responses to CO₂ and light are caused by vapour‐phase ions, possibly hydronium ions that change the pH of the epidermis.     

Hormonal Interactions and Stomatal Responses

Both environmental and hormonal factors and their interactions affect stomatal behavior. Methodologies for identifying hormonal interactions affecting stomatal function are reviewed. Although there is abundant evidence that abscisic acid (ABA) closes stomata, evidence that the other classical plant hormones (auxins, cytokinins, ethylene, gibberellins) in isolation alter stomatal response often comes from exogenous applications to detached epidermes and leaves, rather than correlation of endogenous concentrations with stomatal conductance (g_s). Evidence for hormonal interactions comes from isolated tissues with exogenous hormones supplied at nonphysiological concentrations, or from variation in stomatal response to xylem ABA concentration in planta. The roles of hormonal changes in causing stomatal closure following changes in soil environment are considered. Although soil drying induces multiple changes in xylem sap composition, analysis of stomatal responses suggests a dominant role for increased endogenous ABA concentrations and relatively little evidence of roles for other hormones. A similar picture emerges from studies of soil compaction. Although soil flooding decreases ABA export from the root system, there is some evidence that apoplastic ABA accumulation elicits stomatal closure. Stomatal closure following nitrogen deprivation does not appear to involve ABA and may provide a suitable experimental system to investigate roles for other hormones. The availability of mutant or transgenic lines with altered hormone homeostasis or sensitivity provides opportunities to screen for altered stomatal behavior in response to different environments, and may provide new evidence that hormonal interactions are important in the control of stomatal behavior.     

The role of abscisic acid in disturbed stomatal response characteristics of Tradescantia virginiana during growth at high relative air humidity

In this study, the role of abscisic acid (ABA) in altered stomatal responses of Tradescantia virginiana leaves grown at high relative air humidity (RH) was investigated. A lower ABA concentration was found in leaves grown at high RH compared with leaves grown at moderate RH. As a result of a daily application of 20 microM ABA to leaves for 3 weeks during growth at high RH, the stomata of ABA-treated leaves grown at high RH showed the same behaviour as did the stomata of leaves grown at moderate RH. For example, they closed rapidly when exposed to desiccation. Providing a high RH around a single leaf of a plant during growth at moderate RH changed the stomatal responses of this leaf. The stomata in this leaf grown at high RH did not close completely in response to desiccation in contrast to the stomata of the other leaves from the same plant. The ABA concentration on a fresh weight basis, though not on a dry weight basis, of this leaf was significantly lower than that of the others. Moreover, less closure of stomata was found in the older leaves of plants grown at high RH in response to desiccation compared with younger leaves. This was correlated with a lower ABA concentration in these leaves on a fresh weight basis, though not on a dry weight basis. Stomata of leaves grown at moderate RH closed in response to short-term application of ABA or sodium nitroprusside (SNP), while for leaves grown at high RH there was a clear difference in stomatal responses between the leaf margins and main-vein areas. The stomatal aperture in response to short-term application of ABA or SNP at the leaf margins of leaves grown at high RH remained significantly wider than in the main-vein areas. It was concluded that: (i) a long-term low ABA concentration in well-watered plants during growth at high RH could be a reason for less or no stomatal closure under conditions of drought stress; and (ii) the long-term ABA concentration on a fresh weight basis rather than on a dry weight basis is likely to be responsible for structural or physiological changes in stomata during leaf growth.     

Abscisic acid in apoplastic sap can account for the restriction in leaf conductance of white lupins during moderate soil drying and after rewatering

We studied the effects of drought on leaf conductance (g) and on the concentration of abscisic acid (ABA) in the apoplastic sap of Lupinus albus L. leaves. Withholding watering for 5d resulted in complete stomatal closure and in severe leaf water deficit. Leaf water potential fully recovered immediately after rewatering, but the aftereffect of drought on stomata persisted for 2d. ABA and sucrose were quantified in pressurized leaf xylem extrudates. We assumed that the xylem sucrose concentration is negligible and hence that the presence of sucrose in leaf extrudates indicated that they were contaminated by phloem. To eliminate this interference, the concentration of ABA in leaf apoplast was estimated by extrapolation to zero sucrose concentration, using the regression between ABA and sucrose concentrations. The estimated apoplastic ABA concentration increased by 100-fold with soil drying and did not return to pre-stress values immediately following rewatering. g was closely related to the concentration of ABA in leaf apoplast. Furthermore, the feeding of exogenous ABA to leaves detached from well-watered plants brought about the same degree of depression in g as resulted from the drought-induced increase in ABA concentration. We therefore conclude that the observed changes in the concentration of ABA in leaf apoplast were quantitatively adequate to explain drought-induced stomatal closure and the delay in stomatal reopening following rewatering.     

The role of the mesophyll in stomatal responses to light and CO2

Stomatal responses to light and CO₂ were investigated using isolated epidermes of Tradescantia pallida , Vicia faba and Pisum sativum . Stomata in leaves of T. pallida and P. sativum responded to light and CO₂, but those from V. faba did not. Stomata in isolated epidermes of all three species could be opened on KCl solutions, but they showed no response to light or CO₂. However, when isolated epidermes of T. pallida and P. sativum were placed on an exposed mesophyll from a leaf of the same species or a different species, they regained responsiveness to light and CO₂. Stomatal responses in these epidermes were similar to those in leaves in that they responded rapidly and reversibly to changes in light and CO₂. Epidermes from V. faba did not respond to light or CO₂ when placed on mesophyll from any of the three species. Experiments with single optic fibres suggest that stomata were being regulated via signals from the mesophyll produced in response to light and CO₂ rather than being sensitized to light and CO₂ by the mesophyll. The data suggest that most of the stomatal response to CO₂ and light occurs in response to a signal generated by the mesophyll.