The effect of benzo-18-crown-6, a synthetic ionophore, on stomatal opening and its interaction with abscisic acid

Abstract. Treatment of tomato seedlings with 5 mM benzo-18-crown-6, a potassium ionophore, produced a reduction in transpiration of 40%. Treatments of epidermal strips of Commelina communis with ben-zo-18-crown-6 (1-l0mM) inhibited stomatal opening, and this effect was shown to be reversible. An antagonistic interaction between abscisic acid (10⁻⁷M) and benzo-18-crown-6 (4 × 10⁻³ M) was also observed.     

Identification of jasmonic acid in Mimosa pudica and its inhibitory effect on auxin- and light-induced opening of the pulvinules

Jasmonic acid was identified from Mimosa pudica L. plants by mass spectrometry, high performance liquid chromatography and thin layer chromatography. Effects of authentic jasmonic acid on pulvinule movement and transpiration of the pinnae were compared with those of abscisic acid. Jasmonic acid and abscisic acid each at 10⁻⁵ M inhibited both auxin- and light-induced opening of the pulvinules. A closure-inducing activity of jasmonic acid at 10⁻⁴ M was greater than that of abscisic acid at 10⁻⁴ M. Pinnae transpiration was reduced by 10⁻⁵ M abscisic acid but not by 10⁻⁴ M jasmonic acid.     

Carbon dioxide induces increases in guard cell cytosolic free calcium

The hypothesis that increases in cytosolic free calcium ([Ca²⁺]_i) are a component of the CO₂ signal transduction pathway in stomatal guard cells of Commelina communis has been investigated. This hypothesis was tested using fura-2 fluorescence ratio photometry to measure changes in guard cell [Ca²⁺]_i in response to challenge with 700 µl l⁻¹ CO₂. Elevated CO₂ induced increases in guard cell [Ca²⁺]_i which were similar to those previously reported in response to abscisic acid. [Ca²⁺]_i returned to resting values following removal of the CO₂ and further application of CO₂ resulted in a second increase in [Ca²⁺]_i. This demonstrated that the CO₂-induced increases in [Ca²⁺]_i were stimulus dependent. Removal of extracellular calcium both prevented the CO₂-induced increase in [Ca²⁺]_i and inhibited the associated reduction in stomatal aperture. These data suggest that Ca²⁺ acts as a second messenger in the CO₂ signal transduction pathway and that an increase in [Ca²⁺]_i may be a requirement for the stomatal response to CO₂.     

Effects of leaf wetting and high humidity on stomatal function in leafy cuttings and intact plants of Corylus maxima

When rooted cuttings of Corylus maxima Mill. cv. Purpurea are moved from the wet and humid conditions of the rooting environment, the leaves frequently shrivel and die. Since the newly formed adventitious root system has been shown to be functional in supplying water to the shoot, stomatal behaviour in C. maxima was investigated in relation to the failure to prevent desiccation. Stomatal conductance (g_s) in expanding leaves (L3) of cuttings increased almost 10-fold over the first 14 days in the rooting environment (fog), from 70 to 650 mmol m⁻² s⁻¹. In contrast, g_s of expanded leaves (L1) changed little and was in the region of 300 mmol m⁻² s⁻¹. Midday leaf water potential was much higher in cuttings than in leaves on the mother stock-plant (−0.5 versus −1.2 MPa) even before any roots were visible. Despite this, leaf expansion of L3 was inhibited by >50% in cuttings and stomata showed a gradual reduction in their ability to close in response to abscisic acid (ABA). To determine whether the loss of stomatal function in cuttings was due to severance or to unnaturally low vapour pressure deficit and wetting in fog, intact plants were placed alongside cuttings in the rooting environment. The intact plants displayed reductions in leaf expansion and in the ability of stomata to close in response to dark, desiccation and ABA. However, in cuttings, the additional effect of severance resulted in smaller leaves than in intact plants and more severe reduction in stomatal closure, which was associated with a 2.5-fold increase in stomatal density and distinctively rounded stomatal pores. The similarities between stomatal dysfunction in C. maxima and that observed in many species propagated in vitro are discussed, as is the possible mechanism of dysfunction.     

Variation in stomatal characteristics over the lower surface of Commelina communis leaves

Abstract. The silicone rubber impression technique was used to measure stomatal apertures in 9 mm² sampling areas covering the entire lower surface of leaves of Commelina communis L. The data were analysed using a computer program which produced 'iso-aperture' contours illustrating local differences in mean stomatal aperture. Little consistency was seen in the iso-aperture patterns among sampling times, although the stomata were always relatively closed at the leaf tip and base. When stomata in the middle of the lamina were open, those near to the leaf margin tended to be relatively closed. In places, gradients of mean stomatal aperture were as high as 1 μm mm⁻¹. Measurements along a transect across the lower epidermis revealed no correlation of stomatal aperture with the presence of major veins in the mesophyll tissue. Variation in guard-cell size and stomatal frequency on the lower leaf surface was also analysed. The guard cells were smallest and the stomata more frequent near to the leaf margins. The significance of the results is discussed in relation to measurements of leaf conductance and models of stomatal function.     

Stomatal conductance and xylem sap properties of aspen (Populus tremuloides) in response to low soil temperature

The mechanisms regulating stomatal response following exposure to low (5°C) soil temperature were investigated in aspen ( Populus tremuloides Michx.) seedlings. Low soil temperature reduced stomatal conductance within 4 h, but did not alter shoot xylem pressure potential within 24 h. The xylem sap composition was altered and its pH increased from 6.5 to 7.1 within the initial 4 h of the low temperature treatment. However, the increase in abscisic acid (ABA) concentration in xylem sap was observed later, after 8 h of treatment. These changes were accompanied by a reduction in the electrical conductivity and an increase in the osmotic potential of the xylem sap. The timing of physiological responses to low soil temperature suggests that the rapid pH change of the xylem sap and accompanying changes in ion concentration were the initial factors which triggered stomatal closure in low temperature-treated seedlings, and that the role of the more slowly accumulating ABA was likely to reinforce the stomatal closure. When leaf discs were exposed to xylem sap extracted from low soil temperature-treated plants, stomatal aperture was negatively correlated with ABA and positively correlated with K⁺ concentrations of the xylem sap. The stomatal opening in the leaf discs linearly increased in response to exogenous KCl concentrations when K⁺ concentrations were in the similar range to those detected in the xylem sap. The lowest concentration of exogenous ABA to induce stomatal closure was several-fold higher compared with the concentration present in the xylem sap.     

The inhibition of photosynthesis and photorespiration in isolated mesophyll cells of Phaseolus and Lycopersicon by reduced osmotic potentials

Mesophyll cells isolated from Phaseolus vulgaris and Lycopersicon esculentum show decreasing photosynthetic rates when suspended in media containing increasing concentrations of osmoticum. The photosynthetic activity was sensitive to small changes in osmotic potential over a range of sorbitol concentrations from 0.44 M (−1.08 MPa) to 0.77 M (−1.88 MPa). Photorespiration assayed by ¹⁴CO₂ release in CO₂-free air and by ¹⁴CO₂ release from the oxidation of [1–¹⁴C] glycolate also decreased as the osmotic potential of the incubation medium was reduced. The CO₂ compensation points of the cells increased with increasing concentration of osmoticum from approximately 60 μ I⁻¹1 at −1.08 MPa to 130 μl 1⁻¹ for cells stressed at −1.88 MPa. Changes in photosynthetic and photorespiratory activities occurred at moderate osmotic potentials in these cells suggesting that in whole leaves during a reduction in water potential, non- stomatal inhibition of CO₂ assimilation and glycolate pathway metabolism occurs simultaneously with stomatal closure.     

Effects of chloride and sodium on gas exchange parameters and water relations of Citrus plants

Gas exchange parameters, water relations and Na⁺/Cl^- content were measured on leaves of one-year-old sweet orange ( Citrus sinensis [L.] Osbeck cv. Hamlin) seedlings grown at increasing levels of salinity. Different salts (NaCl, KCl and NaNO₃) were used to separate the effects of Cl⁻ and Na⁺ on the investigated parameters. The chloride salts reduced plant dry weight and increased defoliation. Accumulation of Cl⁻ in the leaf tissue caused a sharp reduction in photosynthesis and stomatal conductance. By contrast, these parameters were not affected by leaf Na⁺ concentrations of up to 478 m M in the tissue water. Leaf water potentials reached values near −1.8 MPa at high NaCl and KCl supplies. This reduction was offset by a decrease in the osmotic potential so that turgor was maintained at or above control values. The changes in osmotic potential were closely correlated with changes in leaf proline concentrations. Addition of Ca²⁺ (as calcium acetate) increased growth and halved defoliation of salt stressed plants. Furthermore, calcium acetate decreased the concentration of Cl⁻ and Na⁺ in the leaves, and increased photosynthesis and stomatal conductance. Calcium acetate also counteracted the reductions in leaf water and osmotic potentials induced by salinity. In addition, calcium acetate inhibited the accumulation of proline in the leaves which affected the reduction in osmotic potential. These results indicate that adverse effects of salinity in Citrus leaves are caused by accumulation of chloride.     

Flooding, gas exchange and hydraulic root conductivity of highbush blueberry

Highbush blueberry plants ( Vaccinium corymbosum L. cv. Bluecrop) growing in containers were flooded in the laboratory for various durations to determine the effect of flooding on carbon assimilation, photosynthetic response to varying CO₂ and O₂ concentrations and apparent quantum yield as measured in an open flow gas analysis system. Hydraulic conductivity of the root was also measured using a pressure chamber. Root conductivity was lower and the effect of increasing CO₂ levels on carbon assimilation less for flooded than unflooded plants after short-(i-2 days), intermediate-(10–14 days) and long-term (35–40 days) flooding. A reduction in O₂ levels surrounding the leaves from 21 to 2% for unflooded plants increased carbon assimilation by 33% and carboxylation efficiency from 0.012 to 0.021 mol CO₂ fixed (mol CO₂)⁻¹. Carboxylation efficiency of flooded plants, however, was unaffected by a decrease in percentage O₂, averaging 0.005 mol CO₂ fixed (mol CO₂)⁻¹. Apparent quantum yield decreased from 2.2 × 10⁻¹ mol of CO₂ fixed (mol light)⁻¹ for unflooded plants to 2.0 × 10⁻³ and 9.0 × 10⁻⁴ for intermediate- and long-term flooding durations, respectively. Shortterm flooding reduced carbon assimilation via a decrease in stomatal conductance, while longer flooding durations also decreased the carboxylation efficiency of the leaf.     

Phytochrome effects on K+ fluxes in guard cells of Commelina communis

The effect of phytochrome on K⁺ transport in guard cells of Commelina communis L. was studied following stomatal movement and using the K⁺−channel blockers tetraethylammonium (TEA), Cs⁺ and quinidine. TEA and quinidine prevented stomatal opening and closure in red light, but not when it was supplemented with far-red. This indicates that channels that can be blocked by TEA and quinidine are regulated by phytochrome. Evidence for a phytochrome effect on K⁺ leakage through other membranal compartments was also found. These phytochrome effects are modified by temperature. Elevated temperature decreases the involvement of channels and increases K⁺ transport through other membrane compartments, while low temperature causes channel opening and diminishes K⁺ leakage. The interaction between phytochrome effects and those of temperature is discussed.     

Responses of individual stomata in Ipomoea pes-caprae to various CO2 concentrations

The responses of individual stomata to CO₂ concentrations ranging from 0 to 900 μmol mol⁻¹ air were analysed in Ipomoea pes-caprae L. Sweet (Convolvulaceae). The stomata were directly observed using a measurement system that permitted continuous observation of stomatal movement under controlled light and CO₂ conditions. A CO₂ concentration of 350 μmol mol⁻¹ or higher induced stomatal closure, whereas concentrations below 350 μmol mol⁻¹ did not. The time lag before stomatal closure decreased with increasing CO₂ concentration, as did the steady-state aperture of the stomata after a change in CO₂ concentration. However, the rate of stomatal closure increased with increasing CO₂ concentration. Therefore, not only the stomatal closure rate but also the time from the CO₂ concentration change to the beginning of stomatal closure changed with increasing CO₂ concentration. These results suggest that atmospheric CO₂ may be the stimulus for the closure of guard cells. No significant differences were observed between adaxial and abaxial stomata in terms of their responses to CO₂. However, heterogeneous responses were detected between neighbouring stomata on each leaf surface.     

Light activates H2 15O flow in rice: Detailed monitoring using a positron-emitting tracer imaging system (PETIS)

Water (H₂ ¹⁵O) translocation from the roots to the top of rice plants ( Oryza saliva L. cv. Nipponbare) was visualized over time by a positron-emitting tracer imaging system (PETIS). H₂ ¹⁵O flow was activated 8 min after plants were exposed to bright light (1 500 μmol m⁻² s⁻¹). When the light was subsequently removed, the flow gradually slowed and completely stopped after 12 min. In plants exposed to low light (500 μmol m⁻² s⁻¹), H₂ ¹⁵O flow was activated more slowly, and a higher translocation rate of H₂ ¹⁵O was observed in the same low light at the end of the next dark period. NaCl (80 m M ) and methylmercury (1 m M ) directly suppressed absorption of H₂ ¹⁵O by the roots, while methionine sulfoximine (1 m M ), abscisic acid (10 μ M ) and carbonyl cyanide m -chlorophenylhydrazone (10 m M ) were transported to the leaves and enhanced stomatal closure, reducing H₂ ¹⁵O translocation.     

Phosphate transport in potato cuttings: Effect of gibberellic acid and abscisic acid

Apical cuttings of Solanum tuberosum L. cv. Sirtema were used al different stages of development to study long-distance transport of phosphate. The effects of two hormones, gibberellic acid (GA₃) and abscisic acid (ABA), on this process were also investigated. Before tuberization, phosphate (³²P) supplied to a single leaf was transported preferentially in the young and growing parts of the plant: apical bud, young leaves and roots. After tuberization, the tuber became the principal site of phosphate accumulation. GA³ treatment (10⁻⁴ M) of the tuber as well as of the leaves led to reduced transport of ³²P into the tuber. By contrast, treatment of the tuber with ABA (10⁻⁴M) did not change the ³²P distribution within the plant, while foliar spray with ABA greatly increased the transport into the tuber. The opposite effects of the two hormones on phosphate accumulation by tubers are discussed with regard to their opposite effects on the tuberization process.     

The stomatal response to CO2 is linked to changes in guard cell zeaxanthin*

The mechanisms mediating CO₂ sensing and light–CO₂ interactions in guard cells are unknown. In growth chamber-grown Vicia faba leaves kept under constant light (500 μ mol m^–2 s^–1) and temperature, guard cell zeaxanthin content tracked ambient [CO₂] and stomatal apertures. Increases in [CO₂] from 400 to 1200 cm³ m^–3 decreased zeaxanthin content from 180 to 80 mmol mol^–1 Chl and decreased stomatal apertures by 7·0 μ m. Changes in zeaxanthin and aperture were reversed when [CO₂] was lowered. Guard cell zeaxanthin content was linearly correlated with stomatal apertures. In the dark, the CO₂-induced changes in stomatal aperture were much smaller, and guard cell zeaxanthin content did not change with chamber [CO₂]. Guard cell zeaxanthin also tracked [CO₂] and stomatal aperture in illuminated stomata from epidermal peels. Dithiothreitol (DTT), an inhibitor of zeaxanthin formation, eliminated CO₂-induced zeaxanthin changes in guard cells from illuminated epidermal peels and reduced the stomatal CO₂ response to the level observed in the dark. These data suggest that CO₂-dependent changes in the zeaxanthin content of guard cells could modulate CO₂-dependent changes of stomatal apertures in the light while a zeaxanthin-independent CO₂ sensing mechanism would modulate the CO₂ response in the dark.     

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.     

Responses of stomata of barley and maize to phenylmercuric acetate

A reduction of stomatal aperture in light was found in leaves of maize after they had been treated with 10“3-5 m phenylmercuric acetate (PMA). Complete closure of the stomata in darkness was prevented, whilst there was total closure in the controls. Higher PMA concentrations had bigger effects. The relative water content (RWC) of barley tissues was slightly reduced 12 hours after treatment with PMA. The transpiration rate observed on PMA-treated barley plants was lower in light and higher in darkness than in untreated plants. Water saturation deficit (WSD) was higher by about 5%, and water holding capacity (WHC) lower (25%) than in untreated plants. The results suggest that the concentration of PMA normally applied as an antitranspirant is unfavourable for healthy growth of maize and barley.     

Early effects of excess cadmium uptake in Phaseolus vulgaris

Abstract. Seedlings of Phaseolus vulgaris were exposed to solutions containing Cd²⁺ in the range 0 to 1 molm⁻³. Ethylene formation started following 3 h of exposure to 10⁻², 10⁻¹ and 1 mol m⁻³ Cd²⁺, peaked at 18 h and returned to a relatively low rate after 24 h. Cadmium-induced ethylene formation depended on the formation of 1-aminocyclopropane-1-carboxylic acid (ACC). Aminoethoxyvinylglycine (AVG, 0.1 mol m⁻³) inhibited ACC accumulation and ethylene production during exposure to 0.2 mol m⁻³ Cd²⁺.
Activity of soluble and ionically-bound peroxidase increased after 18 h of exposure to Cd²⁺ concentrations above 10⁻³ mol m⁻³ due to an increase in activity of cathodic isoperoxidases. Stimulation of soluble and ionically-bound peroxidase by 0.2 mol m⁻³ Cd²⁺ was reduced in the presence of 0.1 mol m⁻³ AVG.
Accumulation of soluble and insoluble ('ligninlike') phenolics was found in plants exposed to Cd²⁺ (10⁻² mol m⁻³ or above) in the presence or absence of AVG. Deposition of insoluble (autofluorescing) material occurred in cell walls around vessels and was associated with reduced expansion and water content of leaves.     

Silencing of NtMPK4 impairs CO-induced stomatal closure, activation of anion channels and cytosolic Casignals in Nicotiana tabacum guard cells

Light-induced stomatal opening in C3 and C4 plants is mediated by two signalling pathways. One pathway is specific for blue light and involves phototropins, while the second pathway depends on photosyntheticaly active radiation (PAR). Here, the role of Nt MPK4 in light-induced stomatal opening was studied, as silencing of this MAP kinase stimulates stomatal opening. Stomata of Nt MPK4-silenced plants do not close in elevated atmospheric CO₂, and show a reduced response to PAR. However, stomatal closure can still be induced by abscisic acid. Measurements using multi-barrelled intracellular micro-electrodes showed that CO₂ activates plasma membrane anion channels in wild-type Nicotiana tabacum guard cells, but not in Nt MPK4-silenced cells. Anion channels were also activated in wild-type guard cells after switching off PAR. In approximately half of these cells, activation of anion channels was accompanied by an increase in the cytosolic free Ca²⁺ concentration. The activity of anion channels was higher in cells showing a parallel increase in cytosolic Ca²⁺ than in those with steady Ca²⁺ levels. Both the darkness-induced anion channel activation and Ca²⁺ signals were repressed in Nt MPK4-silenced guard cells. These data show that CO₂ and darkness can activate anion channels in a Ca²⁺-independent manner, but the anion channel activity is enhanced by parallel increases in the cytosolic Ca²⁺ concentration. Nt MPK4 plays an essential role in CO₂- and darkness-induced activation of guard-cell anion channels, through Ca²⁺-independent as well as Ca²⁺-dependent signalling pathways.     

Effect of 2,5-norbornadiene on abscission and ethylene production in citrus leaf explants

Citrus ( Citrus sinensis L. Osbeck) leaf explants completely abscise within 48 h when exposed to saturating amounts of ethylene at 25°C. When 2,5-norbornadiene was added, 2000 μl 1⁻¹ reduced abscission of explants also exposed to 2 μl 1⁻¹ of ethylene to the level of the control, and 8000 μl 1⁻¹ reduced abscission in explants exposed to 10 μl 1⁻¹ of ethylene to the level of the control, but abscission was complete when 1 000 μl 1⁻¹ of ethylene was used in the presence of 8 000 μl 1⁻¹ of 2,5-norbornadiene. When explants were exposed to 2 μl 1⁻¹ of ethylene, 2000 μl 1⁻¹ of 2,5-norbornadiene prevented abscission if applied up to 10 h after exposure to ethylene. After 18 h, applied 2,5-norbornadiene had little effect on abscission at 48 h. A Lineweaver-Burk plot gave a 1/2 maximum value of 0.12 μl 1⁻¹ for ethylene on abscission, 2,5-Norbornadiene gave competitive kinetics with respect to ethylene with a K₁ value of approximately 120 μl 1⁻¹ of 2,5-norbornadiene. The presence of 2,5norbornadiene stimulated ethylene production, which progressively increased as the 2,5-norbornadiene concentration was increased from 250 to 8 000 μl 1⁻¹ 2,5-Norbornadiene also suppressed the induction of cellulase and polygalacturonase by ethylene. Together, 2,5-norbornadiene and 2,4-dichlorophenoxyacetic acid were more effective than either alone in reducing abscission. 2,5-Norbornadiene also was effective in preventing the reduction of indole-3-acetic acid transport induced by ethylene.     

Stomatal and photosynthetic responses to shade in sorghum, soybean and eastern gamagrass

We studied photosynthetic and stomatal responses of grain sorghum ( Sorghum bicolor [L.] Moench cv. Pioneer 8500), soybean ( Glycine max L. cv. Flyer) and eastern gamagrass ( Tripsacum dactyloides L.) during experimental sun and shade periods simulating summer cloud cover. Leaf gas exchange measurements of field plants showed that short-term (5 min) shading of leaves to 300–400 μmol m⁻² s⁻¹ photosynthetic photon flux density reduced photosynthesis, leaf temperature, stomatal conductance, transpiration and water use efficiency and increased intercellular CO₂ partial pressure. In all species, photosynthetic recovery was delayed when leaves were reilluminated, apparently by stomatal closure. The strongest stomatal response was in soybean. Photosynthetic recovery was studied further with soybeans grown indoors (maximum photosynthetic photon flux density 1 200 μmol m⁻² s⁻¹). Plants grown indoors had responses to shade similar to those of field plants, except for brief nonstomatal limitation immediately after reillumination. These responses indicated the importance of the light environment during leaf development on assimilation responses to variable light, and suggested different limitations on carbon assimilation in different parts of the soybean canopy. Photosynthetic oxygen evolution recovered immediately upon reillumination, indicating that the light reactions did not limit soybean photosynthetic recovery. While shade periods caused stomatal closure and reduced carbon gain and water loss in all species, the consequences for carbon gain/water loss were greatest in soybean. The occurrence of stomatal closure in all three species may arise from their shared phenologies and herbaceous growth forms.