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Blue light and phytochrome-mediated stomatal opening in the npq1 and phot1 phot2 mutants of Arabidopsis.

Recent studies have shown that blue light-specific stomatal opening is reversed by green light and that far-red light can be used to probe phytochrome-dependent stomatal movements. Here, blue-green reversibility and far-red light were used to probe the stomatal responses of the npq1 mutant and the phot1 phot2 double mutant of Arabidopsis. In plants grown at 50 micromol m-2 s-1, red light (photosynthetic)-mediated opening in isolated stomata from wild type (WT) and both mutants saturated at 100 micromol m-2 s-1. Higher fluence rates caused stomatal closing, most likely due to photo-inhibition. Blue light-specific opening, probed by adding blue light (10 micromol m-2 s-1) to a 100 micromol m-2 s-1 red background, was found in WT, but not in npq1 or phot1 phot2 double mutant stomata. Under 50 micromol m-2 s-1 red light, 10 micromol m-2 s-1 blue light opened stomata in both WT and npq1 mutant stomata but not in the phot1 phot2 double mutant. In npq1, blue light-stimulated opening was reversed by far-red but not green light, indicating that npq1 has a phytochrome-mediated response and lacks a blue light-specific response. Stomata of the phot1 phot2 double mutant opened in response to 20 to 50 micromol m-2 s-1 blue light. This opening was green light reversible and far-red light insensitive, indicating that stomata of the phot1 phot2 double mutant have a detectable blue light-specific response.

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Evidence for involvement of photosynthetic processes in the stomatal response to CO2.

Stomatal conductance (gs) typically declines in response to increasing intercellular CO2 concentration (ci). However, the mechanisms underlying this response are not fully understood. Recent work suggests that stomatal responses to ci and red light (RL) are linked to photosynthetic electron transport. We investigated the role of photosynthetic electron transport in the stomatal response to ci in intact leaves of cocklebur (Xanthium strumarium) plants by examining the responses of gs and net CO2 assimilation rate to ci in light and darkness, in the presence and absence of the photosystem II inhibitor 3-(3,4-dichlorophenyl)-1,1-dimethylurea (DCMU), and at 2% and 21% ambient oxygen. Our results indicate that (1) gs and assimilation rate decline concurrently and with similar spatial patterns in response to DCMU; (2) the response of gs to ci changes slope in concert with the transition from Rubisco- to electron transport-limited photosynthesis at various irradiances and oxygen concentrations; (3) the response of gs to ci is similar in darkness and in DCMU-treated leaves, whereas the response in light in non-DCMU-treated leaves is much larger and has a different shape; (4) the response of gs to ci is insensitive to oxygen in DCMU-treated leaves or in darkness; and (5) stomata respond normally to RL when ci is held constant, indicating the RL response does not require a reduction in ci by mesophyll photosynthesis. Together, these results suggest that part of the stomatal response to ci involves the balance between photosynthetic electron transport and carbon reduction either in the mesophyll or in guard cell chloroplasts.

Carbamide Peroxide↗

Induction of Stomatal Closure by Vanadate or a Light/Dark Transition Involves Ca2+-Calmodulin-Dependent Protein Phosphorylations.

Previous studies indicate that a continual source of adenosine 5[prime]-triphosphate is required for both opening and closing of stomata. However, vanadate (Na3VO4 at 500 [mu]M) as well as a light/dark transition induced stomatal closing in epidermal peels of Commelina communis L., showing that the stoppage or even the decrease of the activity of the plasma membrane H+-adenosine 5[prime]-triphosphatase is sufficient to induce stomatal closure. Furthermore, stomatal closing in response to Na3VO4 or a light/dark transition was suppressed by inhibitors of metabolism (10 [mu]M carbonyl cyanide m-chlorophenylhydrazone) and of protein kinases (20 [mu]M 1-[5-iodonaphthalene-1-sulfonyl]-1H-hexa-hydro-1,4-diaz-epine), calmodulin antagonists (20 [mu]M N-[6-aminohexyl]-5-chloro-1-naphthalenesulfonamide), and the anion channel blocker 5-nitro-2,3-phenylpropyllamino benzoic acid (50 [mu]M). These data suggest that the slow, outward rectifying anion channel, whose opening would be related to the membrane potential, and at least one step requiring a protein phosphorylation by a Ca2+-calmodulin-dependent protein kinase of the myosin light chain kinase type might be implicated in the induction of stomatal closing by vanadate or a light/dark transition.

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A Negative Hydraulic Message from Oxygen-Deficient Roots of Tomato Plants? (Influence of Soil Flooding on Leaf Water Potential, Leaf Expansion, and Synchrony between Stomatal Conductance and Root Hydraulic Conductivity).

Four to 10 h of soil flooding delayed and suppressed the normal daily increase in root hydraulic conductance (Lp) in tomato (Lycopersicon esculentum Mill. cv Ailsa Craig) plants. The resulting short-term loss of synchrony between Lp and stomatal conductance decreased leaf water potential ([psi]L) relative to well-drained plants within 2 h. A decrease in [psi]L persisted for 8 h and was mirrored by decreased leaf thickness measured using linear displacement transducers. After 10 h of flooding, further closing of stomata and re-convergence of Lp in flooded and well-drained roots returned [psi]L to control values. In the second photoperiod, Lp in flooded plants exceeded that in well-drained plants in association with much increased Lp and decreased stomatal conductance. Pneumatic balancing pressure applied to roots of intact flooded plants to prevent temporary loss of [psi]L in the 1st d did not modify the patterns of stomatal closure or leaf expansion. Thus, the magnitude of the early negative hydraulic message was neither sufficient nor necessary to promote stomatal closure and inhibit leaf growth in flooded tomato plants. Chemical messages are presumed to be responsible for these early responses to soil flooding.

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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.

Abscisic Acid↗

Stomatal Opening in Isolated Epidermal Strips of Vicia faba. II. Responses to KCl Concentration and the Role of Potassium Absorption.

The stimulation by KCl of stomatal opening in isolated epidermal strips of Vicia faba was examined. In dark + normal air the opening response was maximal at 100 mm KCl while in light + CO(2)-free air it was maximal at about 10 mm KCl. CO(2)-free air was more influential than light in reducing the KCl concentration required for maximal opening. K(+) was essential while Cl(-) seemed to be of secondary importance in these processes.The use of (86)Rb(+) as a tracer for K(+) showed that the increase in stomatal aperture under various conditions was well correlated with K(+) uptake. The estimated amount of K(+) taken up by guard cells, along with a counter ion, was sufficient to account for the changes in solute potential associated with opening. It is suggested that the absorption of extracellular solutes, such as K(+), may be the primary mechanism of stomatal opening. Both opening and K(+) absorption are stimulated by light + CO(2)-free air.The increase in stomatal aperture was also well correlated with the decrease in stainable starch in guard cells under all conditions. It is suggested that this is a secondary change, although perhaps closely linked to K(+) absorption.

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Abnormal Stomatal Behavior and Hormonal Imbalance in flacca, a Wilty Mutant of Tomato: I. Root Effect and Kinetin-like Activity.

The wilty tomato mutant, flacca, and the normal variety, Rheinlands Ruhm, were compared for kinetin-like activity in ontogeny. The mutant wilts easily because its stomata resist closure. This stomatal resistance decreases with age. The occurrence of a root factor which induces stomatal opening was inferred from grafting experiments. It was hypothesized that the excessive stomatal openings in the mutant may result from excess of kinetin-like activity in the leaf of that plant. In addition, it was suggested that the closure of stomata in the aging mutant is due to a decrease of kinetin-like activity with age. Kinetin-like activity in the leaf was determined by incorporation of labeled leucine. The concentration of cytokinins in root exudate and leaf extract was determined by the soybean callus assay. Evidence was presented of higher kinetin-like activity in the leaves of the mutant and higher cytokinin concentration in its root exudate. Cytokinin concentration in the shoot was found to be only slightly higher in the mutant than in the normal plants. Kinetin-like activity in the leaf and cytokinin concentration of root exudate decreased with age in both mutant and normal plants. Kinetin-like activity in the leaves of mutant plants, which phenocopy the normal variety as a result of continuous application of abscisic acid, was lower than in control mutant plants. The significance of these findings per se and in connection with stomatal behavior is discussed.

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Nonsynchronized Oscillations in Stomatal Resistance among Sclerophylls of Eucalyptus umbra.

Oscillations in stomatal resistance to viscous air flow have been recorded in the juvenile and adult sclerophylls of Eucalyptus umbra R. T. Baker, in both controlled and natural environments. Under constant illumination, temperature, and humidity, irregular oscillations in stomatal resistance with a periodicity of approximately 20 to 30 minutes occurred spontaneously, when the soil water potential was approximately -0.2 bar. In the natural summer environment, similar stomatal oscillations developed with periods ranging from 60 to 100 minutes only after imposition of soil moisture stress equivalent to -2 to -3 bars water potential. These cycles in stomatal resistance were not synchronized among the leaves of a given plant in any of the experiments.

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Evidence for Two Photoreactions and Possible Involvement of Phytochrome in Light-dependent Stomatal Opening.

Leaves of the xantha mutant of Helianthus annuus have a higher rate of transpiration and a lower diffusive resistance in the light than in the dark. Stomates of this nonphotosynthetic mutant open in the light and close in the dark.Comparative studies of tobacco, xantha mutant, and wild-type sunflower stomatal opening over a range of light intensities in isolated portions of the spectrum reveal two patterns of response: (a) a low intensity opening in the green and far red characterized by partial opening, absence of a threshold, and saturation of the response at low light intensities; (b) a high intensity response in the blue characterized by a threshold (intensities greater than 100 microwatts per square centimeter needed for opening) and a linear opening response at higher incident light intensities. In xantha mutant stomates only the low intensity system appears to be operational, while both low and high intensity systems are present in the wild-type sunflower and tobacco.Red light has an inhibiting effect on stomatal opening in both mutant and wild-type sunflowers. They require prior exposure to far red for opening to occur in red light. This redfar red antagonism suggests the involvement of phytochrome.

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Stomatal Response to Light of Solanum pennellii, Lycopersicon esculentum, and a Graft-induced Chimera.

To learn how species differences in stomatal behavior are regulated, the response of epidermal and leaf diffusive resistance to light was investigated in Lycopersicon esculentum Mill., Solanum pennellii Corr., and a periclinal chimera having an S. pennellii epidermis and an L. esculentum mesophyll that was produced from a graft of the two species. S. pennellii has about 23% fewer stomata per square millimeter than does L. esculentum, and the two species have contrasting stomatal sensitivities to light. The abaxial stomata of L. esculentum open in dimmer light and to a greater extent than the adaxial stomata. The abaxial and adaxial stomata of S. pennellii respond similarly to light incident on the adaxial epidermis and are less open at all quantum flux densities than comparable stomata of L. esculentum. The patterns of response to light of the abaxial and adaxial stomata of the chimera were practically identical to those of L. esculentum, and quite unlike those of S. pennellii. Thus, the pattern of stomatal light response in the chimera was regulated by the L. esculentum mesophyll. The reduction in stomatal frequency of the chimera, which was regulated by the epidermis of S. pennellii, contributed to the 40% difference in leaf diffusive resistance between the plants in moderate light.

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Relationships between Stomatal Behavior and Internal Carbon Dioxide Concentration in Crassulacean Acid Metabolism Plants.

Measurements of internal gas phase CO(2) concentration, stomatal resistance, and acid content were made in Crassulacean acid metabolism plants growing under natural conditions. High CO(2) concentrations, sometimes in excess of 2%, were observed during the day in a range of taxonomically widely separated plants (Opuntia ficus-indica L., Opuntia basilaris Engelm. and Bigel., Agave desertii Engelm., Yucca schidigera Roezl. ex Ortiges, Ananas comosus [L.] Merr., Aloe vera L., Cattleya sp. and Phalanopsis sp.) and below ambient air concentrations were observed at night.Stomatal resistance was always high when CO(2) concentration was high and experiments in which attempts were made to manipulate internal CO(2) concentrations gave data consistent with stomatal behavior in Crassulacean acid metabolism being controlled by internal CO(2) concentration. Exogenous CO(2) applied in darkness at a concentration similar to those observed in the light caused stomatal resistance to increase.In pads of Opuntia basilaris Engelm. and Bigel. subjected to severe water stress internal gas phase CO(2) concentrations exhibited fluctuations opposite in phase to fluctuations in acid content. Stomatal resistance remained high and the opening response to low CO(2) concentration was almost entirely eliminated.

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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.Previous nutrient supply of the plants determined whether the charges of K(+) in guard cells were completely balanced by Cl(-) or only partially. Addition of K(2)SO(4) to the nutrient solution reduced the participation of Cl(-) in stomatal ion transfer, even if epidermal strips of these plants were later exposed to KCl solution. The anion supplying the charge complement in these cases is not known.Although malate appeared not to participate in stomatal ion transfer in onion, epidermal samples of this species did contain malate. Malate accumulated in the epidermis of leaves put into the light but disappeared from illuminated epidermal strips floating on solutions. In whole leaves, epidermal malate content was positively correlated with stomatal opening; in epidermal strips floating on solutions, the correlation was negative or absent.

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Response of leaf water potential, stomatal resistance, and leaf rolling to water stress.

Numerous studies have associated increased stomatal resistance with response to water deficit in cereals. However, consideration of change in leaf form seems to have been neglected. The response of adaxial and abaxial stomatal resistance and leaf rolling in rice to decreasing leaf water potential was investigated. Two rice cultivars were subjected to control and water stress treatments in a deep (1-meter) aerobic soil. Concurrent measurements of leaf water potential, stomatal resistance, and degree of leaf rolling were made through a 29-day period after cessation of irrigation. Kinandang Patong, an upland adapted cultivar, maintained higher dawn and midday leaf water potential than IR28, a hybrid selected in irrigated conditions. This was not explained by differences in leaf diffusive resistance or leaf rolling, and is assumed to result from a difference in root system extent.Stomatal resistance increased more on the abaxial than the adaxial leaf surface in both cultivars. This was associated with a change in leaf form or rolling inward of the upper leaf surface. Both responses, increased stomatal resistance and leaf rolling, were initiated in a similar leaf water potential range (-8 to -12 bars). Leaves of IR28 became fully rolled at leaf water potential of about -22 bars; however, total leaf diffusive resistance was only about 4 to 5 seconds per centimeter (conductance 0.25 to 0.2 centimeter per second) at that stage. Leaf diffusive resistance and degree of leaf rolling were linearly related to leaf water potential. Thus, leaf rolling in rice may be used as an estimate of the other two less obvious effects of water deficit.

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Stomatal closure and photosynthetic inhibition in soybean leaves induced by petiole girdling and pod removal.

The presence of strong sinks of photoassimilates is thought to stimulate photosynthesis by minimizing photosynthetic end product accumulation in leaves. This hypothesis was examined in soybeans (Glycine max [L] Merr.) with treatments designed to alter the phloem translocation of photoassimilates out of source leaves. Pod removal and petiole girdling resulted in 70 and 90% reductions, respectively, in leaf CO(2) exchange rate. Reductions of similar magnitude also were observed in stomatal diffusive conductivity.Time course data showed that CO(2) exchange rates as well as stomatal conductivities were significantly reduced 0.5 hour after girdling and 5 hours after pod removal, thus suggesting that stomatal closure was the cause of the reduced rates of photosynthesis.Mesophyll conductivities to CO(2), calculated from gas exchange data, were not affected by the treatments. Radiocarbon assimilation rates by leaf slices floated in KH(14)CO(3) solutions were not reduced by pod removal and were reduced only 10% by girdling. It was concluded that treatments which block or slow translocation from source leaves reduce their photosynthetic rate by inducing stomatal closure.

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Effect of obstructed translocation on leaf abscisic Acid, and associated stomatal closure and photosynthesis decline.

Pod removal or petiole girdling, which causes obstruction of translocation, was found in our previous study to cause reduced rates of photosynthesis in soybean leaves due to stomatal closure. The purpose of this research was to determine the involvement of photoassimilate accumulation and leaf abscisic acid (ABA) levels in the mechanism of stomatal closure induced by such treatments.Leaf glucose and sucrose levels increased during the initial 12-hour period after depodding or petiole girdling. Starch, which represents a much larger pool of leaf carbohydrate, was not perceptibly increased above control levels during the 12-hour posttreatment period.When leaflets were exposed to nonphotosynthetic environments (shading or CO(2)-free air) for a 24-hour period after the translocation-obstructing treatments were applied and then returned to normal light or CO(2) concentration, stomatal diffusive conductivity was reduced 65% and 85% with depodding and girdling, respectively. These reductions were comparable to those previously observed without an intervening nonphotosynthetic exposure, thus indicating that photosynthate accumulations were not necessary for the observed response.Free and bound ABA (released on alkaline hydrolysis) were determined by gas liquid chromatography with electron capture detection following preparative high performance liquid chromatography. Free ABA in monitored leaves increased almost 10-fold 48 hours after complete depodding and 25-fold 24 hours after petiole girdling of such leaves. By 3 hours after treatment, in a time course study, free ABA had increased 2-fold above control values in depodded and 5-fold in girdled leaves. Leaf concentrations of bound ABA did not appear to be related to the treatment effects on stomata.Thus, the translocation-obstructing treatments cause an increased level of ABA by a mechanism not involving accumulation of photoassimilate. Increased leaf ABA levels, which were independent of water stress or leaf water potential, appear to be involved in the stomatal closure response. It is suggested that the mechanism of increased leaf ABA levels following translocation-obstruction may be due to an interference with normal translocation of ABA out of leaves.

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Dark Stomatal Movement in Sunflowers in Response to Illumination under Nitrogen.

Experiments were performed on intact sunflowers (Helianthus annuus) placed in a specially designed experimental chamber which allows instantaneous modifications of the atmospheric composition without changing any other conditions. After one night in normal conditions, the plant was illuminated under pure nitrogen atmosphere; the opening stomatal movement, measured as a transpiration rate variation, was inhibited. After an anoxia time period not exceeding one hour, the light was turned off and normal air restored. The stomatal movement was no longer inhibited, and a transient increase in the transpiration rate, referred to here as the postillumination transpiration peak (PITP), was observed.The quantity of transpired water during the PITP can be related to the total incident light energy supplied during the light-nitrogen period. Furthermore, the addition of a dark-nitrogen period between the light-nitrogen and dark-air periods caused the PITP to decrease. The PITP is almost suppressed after a 20-minute dark-nitrogen period.It is shown that the PITP does not result from a hydropassive mechanism but is metabolically controlled. Moreover, it seems that the PITP is not due to a CO(2)-suppression effect during the light-nitrogen period.The results are interpreted in terms of stomatal mechanism. The metabolites leading to PITP originate from the reducing equivalents created during the light-nitrogen period. They could be synthesized at the beginning of the PITP period (darkness under normal air) or during the dark-nitrogen plus CO(2) period between the light-nitrogen and PITP periods. The results obtained are related to the first steps of classical photoactive stomatal opening.

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Gas Exchange, Stomatal Behavior, and deltaC Values of the flacca Tomato Mutant in Relation to Abscisic Acid.

The relationship between stomatal conductance and capacity for assimilation was investigated in flacca, a mutant of tomato (Lycopersicon esculentum Mill.) that has abnormal stomatal behavior and low abscisic acid (ABA) content. The assimilation capacity, determined by measuring assimilation rate as a function of intercellular CO(2) pressure, did not differ in leaves of flacca and its parent variety, Rheinlands Ruhm (RR). On the other hand, stomatal conductance of flacca leaves was greater than that of RR, and could be phenotypically reverted by spraying with 30 micromolar ABA. Stomatal conductance of flacca leaves was also reduced by increasing CO(2) pressure, increasing leaf to air vapor pressure difference, and decreasing quantum flux, irrespective of ABA treatment.The high conductance of flacca leaves resulted in a high intercellular CO(2) pressure. This allowed greater discrimination against (13)CO(2), as evidenced by more negative delta (13)C values for flacca as compared to RR. The delta (13)C values of both flacca and RR plants as influenced by ABA treatment were consistent with predictions based on gas exchange measurements, using a recent model of discrimination.

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Role of Ca and EGTA on Stomatal Movements in Commelina communis L.

Ca(2+) (0.1-1.0 millimolar) accelerated dark-induced stomatal closure and reduced stomatal apertures in the light in epidermal peels of Commelina communis L. In contrast, ethyleneglycol-bis-(beta-aminoethyl ether) N,N'tetraacetic acid (EGTA) (2 millimolar), a Ca(2+) chelator, prevented closure in the dark and accelerated opening in the light. EGTA did not promote significant opening in the dark. It is therefore concluded that EGTA does not increase ion uptake into guard cells, but rather prevents ion efflux. Addition of EGTA to incubating solutions with 10 millimolar KCl resulted in steady state apertures of 15.6 micrometers, whereas in the absence of EGTA similar apertures required 55 millimolar KCl and 150 millimolar KCl was needed in the presence of 1 millimolar CaCl(2). The results demonstrate the importance of Ca(2+) in the regulation of stomatal closure and point to a role of Ca(2+) in the regulation of K(+) efflux from stomatal guard cells.

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