Chitosan antitranspirant activity is due to abscisic acid-dependent stomatal closure

Chitosan (CHT) is a natural compound able to activate the plant own defence machinery against pathogen attacks and to reduce both transpiration and stomatal opening when applied as foliar spray. The data here reported show that CHT-induced antitranspirant activity in bean plants is mediated by ABA, whose level raised over threefold in treated leaves, 24 h after foliar spraying. This is thought to induce partial stomatal closure via a H₂O₂-mediated process, as confirmed by scanning electron microscopy (SEM) and histo-cytochemistry, and, in turn, a decrease of stomatal conductance to water vapor (G_w) and transpiration rate (E), assessed by gas exchange measurements. The relatively high internal CO₂ concentration (C_i) values, suggest the occurrence of a slight decrease in carboxylation efficiency after CHT treatment, which however did not prevail over stomatal limitations. The intrinsic water use efficiency (WUE_i) of CHT treated plants was not statistically different from controls and the maximal photochemical efficiency (F_v/F_m) of PSII was not affected. Moreover, CHT determined a stimulation of the xanthophyll cycle towards de-epoxidation state. On the whole, these results, besides confirming the effectiveness of CHT in reducing plant transpiration, prove that the mechanism underlying this activity differs from that showed by the commercial antitranspirant Vapor Gard^® (VP). In fact, the efficacy of the latter is based on the formation of a thin antitranspirant film over the leaf and not on the reduction of stomatal opening. Finally, suggestions for possible use of the two antitranspirants in different environmental conditions are discussed.     

The effect of pH on stomatal sensitivity to abscisic acid

Abstract. The sensitivity of stomata of Commelina communis L. to abscisic acid (ABA) was evaluated by analysing the initial rates of response to the compound at different hormone concentrations. This was carried out at pH 6.8 and pH 5.5. The data were modelled and statistically analyzed by means of a computer program employing non-linear regression techniques and step-down analysis of variance. The response kinetics as quantified in terms of three sensitivity parameters were found to differ significantly between the two pH values. This finding is discussed in relation to previous research on purified ABA-binding proteins.     

Effects of abscisic acid and its derivatives on stomatal closing

Abscisic acid and its derivatives, formed with the terminalcarboxyl group replaced respectively by aldehyde, hydroxymethyland methyl groups, were examined for their effects on stomatalclosing. Only the derivative with the methyl group was inactive.The acid and the other two derivatives were very active forclosing stomata at low concentrations. (Received January 28, 1975; )     

Characterisation of abscisic acid inhibition of stomatal opening in isolated epidermal strips

Abscisic acid (ABA) inhibited the light-induced opening of stomata in isolated epidermal strips of Commelina benghalensis. It did not alter stomatal closure in the dark. The ABA-induced inhibition in light was released under conditions conducive for cyclic photophosphorylation and remarkably reversed by ATP in the presence of pyruvate. Cyclic photophosphorylation rates of isolated guard cell chloroplasts were significantly reduced by ABA. It is proposed that the direct effect of ABA on stomatal opening was mediated in two ways: (1) by inhibition of cyclic photophosphorylation activities of guard cell chloroplasts and (2) by blocking organic acid formation in guard cells.     

2-Chloroethanephosphonic acid reduces transpiration and stomatal opening in Coffea arabica L.

Summary Foliar sprays of 2-chloroethanephosphonic acid reduced transpiration rates per unit leaf area and stomatal apertures during the day in Coffea arabica L. The effect was first detected six hours after treatment and persisted for up to five days.Abbreviation CEPA 2-chloroethanephosphonic acid     

Deficient glutathione in guard cells facilitates abscisic acid-induced stomatal closure but does not affect light-induced stomatal opening

We investigated the role of glutathione (GSH) in stomatal movements using a GSH deficient mutant, chlorinal-1 (ch1-1). Guard cells of ch1-1 mutants accumulated less GSH than wild types did. Light induced stomatal opening in ch1-1 and wild-type plants. Abscisic acid (ABA) induced stomatal closure in ch1-1 mutants more than wild types without enhanced reactive oxygen species (ROS) production. Therefore, GSH functioned downstream of ROS production in the ABA signaling cascade.     

Temperature-dependent stomatal movement in tulip petals controls water transpiration during flower opening and closing

Temperature‐dependent tulip petal opening and closing movement was previously suggested to be regulated by reversible phosphorylation of a plasma membrane aquaporin ( Azad et al., 2004a ). Stomatal apertures of petals were investigated during petal opening at 20°C and closing at 5°C. In completely open petals, the proportion of open stomata in outer and inner surfaces of the same petal was 27 ± 6% and 65 ± 3%, respectively. During the course of petal closing, stomatal apertures in both surfaces reversed, and in completely closed petals, the proportion of open stomata in outer and inner surfaces of the same petal was 74 ± 3% and 29 ± 6%, respectively, indicating an inverse relationship between stomatal aperture in outer and inner surfaces of the petal during petal opening and closing. Both petal opening and stomatal closure in the outer surface of the petal was inhibited by a Ca²⁺ channel blocker and a Ca²⁺ chelator, whereas the inner surface stomata remained unaffected. On the other hand, sodium nitroprusside, a nitric oxide donor, had no effect on stomatal aperture of the outer surface but influenced the inner surface stomatal aperture during petal opening and closing, suggesting different signalling pathways for regulation of temperature‐dependent stomatal changes in the two surfaces of tulip petals. Stomata were found to be differentially distributed in the bottom, middle and upper parts of tulip petals. During petal closing, water transpiration was observed by measuring the loss of ³H₂O. Transpiration of ³H₂O by petals was fivefold greater in the first 10 min than that found after 30 min, and the transpiration rate was shown to be associated with stomatal distribution and aperture. Thus, the stomata of outer and inner surfaces of the petal are involved in the accumulation and transpiration of water during petal opening.     

Effects of grandinol and related phloroglucinol derivatives on transpiration and stomatal closure

Grandinol, an inhibitor of seed germination and photosynthesis in Eucalyptus sp., inhibits transpiration and stomatal opening. The acylphloroglucinol structure in grandinol seemed to be essential for these activities. Enhancement of activity was achieved by the introduction of a formyl group into the molecule. Therefore, structural requirements for these activities were very similar to that for the inhibition of seed germination and photosynthesis. Other grandinol-related compounds having two electron-withdrawing groups on the phloroglucinol nuclei were also active in these assays.     

Auxin-binding-protein antibodies and peptides influence stomatal opening and alter cytoplasmic pH

Previous work has shown that stomatal opening induced by indole-3-acetic acid (IAA) in epidermal strips of the orchid Paphiopedilum tonsum L. is preceded by a reduction in cytoplasmic pH (pH_i) of the guard cells. We now report that Fab fragments of an auxin-agonist antibody (D16), directed against a putative auxin-binding domain of the auxin-binding protein ABP1, induce stomatal opening and decrease guard-cell pH_i, as monitored with the acetomethoxy ester of the ratiometric pH indicator Snarf-1. Similar activity was shown by a monoclonal antibody against the same domain. The C-terminal dodecapeptide, Pz152–163 of maize ABP1 (ABPzm1) induced guard-cell alkalinization and closed stomata, as did Fab fragments of a monoclonal antibody (MAC 256) recognising the C-terminal region of ABPzm1. By implicating, for the first time, an auxin-binding protein in mediation of an auxin-dependent physiological response, these findings strongly support an auxin-receptor role for ABP1. Received: 23 December 1997 / Accepted: 16 January 1998     

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.     

Effects of atmospheric humidity on net photosynthesis,transpiration, and stomatal resistance

Rhizomes ofHydrocotyle plants from three contrasting habitats were cloned and the ramets grown under controlled environmental conditions. Measurements of net photosynthesis, transpiration, and total leaf diffusion resistance were used to examine possible physiological adaptations to specific field environments. Increasing dryness of the growth chamber environment had large effects on gas exchange (CO₂ and water vapor) and on total diffusion resistance of plants from a pond, moderate effects on plants from a mesic forest, but plants from a coastal sand dune were unaffected by the experimentally imposed dryness. Thus the 3 Hydrocotyle types demonstrated adaptive physiological reponses to their specific field habitats. Periodic stomatal oscillations were induced in ramets from the pond by sharply increasing irradiance, but the adaptiveness of the oscillations cannot be determined with the evidence at hand.No stomatal closure could be induced by atmospheric dryness alone as long as soil and plant dessication were prevented. There were no observable differences in stomatal response to increasing atmospheric vapor pressure deficits.     

Effect of abscisic acid on stomatal opening in isolated epidermal strips of abi mutants of Arabidopsis thaliana

Abscisic acid-insensitive mutants of Arabidopsis thaliana L. var. Landsberg erecta were selected for their decreased sensitivity to ABA during germination. Two of these mutants, abi-1 and abi-2 , display a wilty phenotype as adult plants, indicating disturbed water relations. Experiments were undertaken to find out if this results from insensitivity of mutant stomates to ABA.
Growth conditions and methods to isolate epidermal strips were optimized to study stomatal movement. Wild type stomates required external ionic conditions comparable to those found for other species such as Commelina communis . The largest light-induced opening of A. thaliana stomates was found at an external KCl concentration of 50 m M . Stomatal apertures were increased by lowering external Ca²⁺ to 0.05 m M . The apertures of stomates incubated with 10 μ M ABA were not altered by changes in Ca²⁺ from 0.05 to 1.0 m M .
Stomates of all abi mutants showed a light-stimulated stomatal opening. The opening of wild type and abi-3 stomates was inhibited by ABA, while stomates of abi-1 and abi-2 did not respond to ABA. The insensitivity of abi-1 and abi-2 stomates to ABA may thus explain the observed disturbed water relations.     

Effects of cycloheximide on abscisic Acid biosynthesis and stomatal aperture in bean leaves

Cycloheximide was shown to block abscisic acid (ABA) biosynthesis in nonstressed as well as in stressed Phaseolus vulgaris leaves. Leaf wilting caused by cycloheximide resulted from increased stomatal opening as judged by a decreased stomatal diffusion resistance. The inhibition of ABA biosynthesis by cycloheximide was at least partially responsible for the increase in stomatal opening as suggested by the cooccurrence of inhibition of ABA biosynthesis and increased stomatal opening, and the partial reversal of stomatal opening in cycloheximide-treated leaves by exogenous ABA. Dark treatment failed to close stomatal in cycloheximide-treated leaves, suggesting that stomatal closure in response to darkness may normally be mediated by ABA.     

Effects of external potassium supply on stomatal closure induced by abscisic acid

Abstract Epidermal strips of Commelina communis with ‘isolated’ stomata were incubated on Trizma-maleate buffer containing 0-500 mM KCL, with or without 10^?4 M ABA, for 2.5 h. The resulting stomatal apertures indicate that there is no absolute requirement for live epidermal and subsidiary cells for ABA-mediated closure. This implies that ABA has a direct effect on influx or efflux of K⁺ into or out of the guard cells rather than on uptake of K⁺ by the subsidiary cells. The possible in vivo role of subsidiary cells in stomatal closure is discussed.     

Effects of abscisic acid on potassium uptake and starch content of stomatal guard cells

Summary Abscisic acid (ABA) at a concentration of 100 m reduced the mean stomatal aperture on isolated epidermis of Commelina communis from 9.5 to 3.1 m. This closure resulted from a fall in osmotic pressure of the guard cells from 14.1 to 9.8 bars; the osmotic pressure of the subsidiary cells did not change significantly. Histochemical tests showed that the potassium concentration in guard cells was reduced by ABA-treatment, while the starch content of the chloroplasts increased. ABA was found to exert a significant effect on Rb⁸⁶ uptake into leaf discs, but this was relatively small in magnitude. It is concluded that ABA has a greater effect on ion uptake into guard cells than into the leaf tissues as a whole.Recent hypotheses of the stomatal mechanism are discussed in relation to these new observations, and the rejection by some writers of any major role for starch hydrolysis is challenged. Evidence from several sources suggests that starch disappearance occurs simultaneously with K⁺ entry into guard cells. Breakdown of starch may lead to formation of organic anions, with which K⁺ uptake may be associated. In this case starch breakdown would contribute as much to the increased osmotic pressure as does K⁺ uptake.     

Effects of patchy stomatal closure on gas exchange measurements following abscisic acid treatment

Gas exchange data and images of leaf fluorescence were collected concurrently as stomata responded to abscisic acid (ABA) application. When 10^?5kmolm^?3 ABA was applied to the transpiration stream in a short pulse, stomatal conductance (g_s), photosynthesis (A) and intercellular CO2 concentration (C_i) decreased rapidly after a short lag period and became approximately constant after 2h. There was an apparent reduction in the A versus c1 relationship as stomata closed, but the data returned to the A versus C1 curve while stomatal conductance was constant or slowly rising during the second hour after ABA treatment. Larger amounts of ABA administered during the pulse caused larger deviations from the A versus c1 relationship. When 10^?7kmolm^?3 ABA was applied continuously through the transpiration stream, gs, A and Cⁱ decreased, but there was no substantial deviation from the A versus c{ curve. Fluorescence images were patchy as stomata closed for all experiments, but became slowly more uniform during the time that gas exchange was returning to the A versus Cj curve. The distribution of con-ductance among patches was not bimodal, and larger devi-ations from the A versus ct curve had greater ranges of pixel values and more pixel values representing low values of Cj during stomatal closure than did experiments show-ing small or no deviation. Estimates of A and gs from fluo-rescence images compared favourably with measured val-ues in most cases, suggesting that the patchy distributions of fluorescence were caused by patchy distributions of stomatal conductance and that apparent reductions in the A versus ct relationship were the result of these patchy stomatai distributions and not direct effects of ABA on mesophyll functioning. The data show that stomatal patches can be temporary and that patchiness may not be reflected in gas exchange data if the range of stomatal con-ductances is not large. These observations may explain some of the discrepancies among previous studies concerning the effect of ABA on the A versus Cⁱ relationship.     

Nitric oxide inhibits blue light-specific stomatal opening via abscisic acid signaling pathways in Vicia guard cells

Recent evidence suggests that nitric oxide (NO) acts as an intermediate of ABA signal transduction for stomatal closure. However, NO's effect on stomatal opening is poorly understood even though both opening and closing activities determine stomatal aperture. Here we show that NO inhibits stomatal opening specific to blue light, thereby stimulating stomatal closure. NO inhibited blue light-specific stomatal opening but not red light-induced opening. NO inhibited both blue light-induced H(+) pumping and H(+)-ATPase phosphorylation. The NO scavenger 2-carboxyphenyl-4,4,5,5-tetramethylimidazoline-1-oxyl-3-oxide (c-PTIO) restored all these inhibitory effects. ABA and hydrogen peroxide (H(2)O(2)) inhibited all of these blue light-specific responses in a manner similar to NO. c-PTIO partially restored the ABA-induced inhibition of all of these opening responses but did not restore inhibition of the responses by H(2)O(2). ABA, H(2)O(2) and NO had slight inhibitory effects on the phosphorylation of phototropins, which are blue light receptors in guard cells. NO inhibited neither fusicoccin-induced H(+) pumping in guard cells nor H(+) transport by H(+)-ATPase in the isolated membranes. From these results, we conclude that both NO and H(2)O(2) inhibit blue light-induced activation of H(+)-ATPase by inhibiting the component(s) between phototropins and H(+)-ATPase in guard cells and stimulate stomatal closure by ABA.     

Influence of stomatal distribution on transpiration in low-wind environments

Abstract According to computer energy balance simulations of horizontal thin leaves, the quantitative effects of stomatal distribution patterns (top vs. bottom surfaces) on transpiration (E) were maximal for sunlit leaves with high stomatal conductances (g_s) and experiencing low windspeeds (free or mixed convection regimes). E of these leaves decreased at windspeeds > 50 cm s^?1, despite increases in the leaf-to-air vapour density deficit. At 50 cm s^?1 wind-speed, rapidly transpiring leaves had greater E when one-half of the stomata were on each leaf surface (amphistomaty; 10.16 mmol H₂O m^?2 s^?1) than when all stomata were on either the top (hyperstomaty; 9.34 mmol m^?2s^?1) or bottom (hypostomaty; 7.02 mmol m^?2s^?1) surface because water loss occurred in parallel from both surfaces. Hyperstomatous leaves had larger E than hypostomatous leaves because free convection was greater on the top than on the bottom surface. Transpiration of leaves with large g, was greatest at windspeeds near zero when ～60–75% of the stomata were on the top surface, while at high windspeeds E was greatest with, 50% of the stomata on top. For leaves with low g_s, stomatal distribution exerted little influence on simulated E values. Laboratory measurements of water loss from simulated hypo-, hyper-, and amphistomatous leaf models qualitatively supported these predictions.