Stomatal closure in response to xanthoxin and abscisic acid

Summary The stomata of detached leaves of Commelina communis L., Hordeum vulgare L., Zea mays L., Vicia faba L., Phaseolus vulgaris L. and Xanthium strumarium L. closed when xanthoxin (XAN) was added to the transpiration stream. XAN was approximately half as active as (+)-abscisic acid (ABA) at an equivalent concentration. XAN, like ABA, sensitized stomata of Xanthium strumarium to CO₂. In contrast to ABA, XAN was ineffective in closing stomata of isolated epidermal strips of C. communis or V. faba. This may be because XAN added to the transpiration stream is converted to ABA during passage from the xylem to the epidermis.Abbreviations ABA Abscisic acid - XAN xanthoxin     

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.     

Stomatal response to abscisic acid fed into the xylem of intact Helianthus annuus (L.) plants

The effect of abscisic acid on stomatal apertures of sunflower(Helianthus annuus (L.)) was investigated with a new methodfor feeding solutions into an attached leaf of an intact plant.Xylem sap was sampled with a Passioura-type pressure chamber.Then it was modified in its composition and fed back into amature leaf of the plant from which it had been collected beforethe experiment. Simultaneously, unmodified xylem sap was fedinto a control leaf at the same internode. The use of the Passioura-typepressure chamber during feeding, prevented embolisms and ensuredminimum dilution of the feeding solution. The effect of feedingwas measured by two gas exchange systems, located at the treatmentand at the control leaf. During the feeding experiments up to84% of the water volume transpired by the leaf was substitutedby the supplied feeding sap. When feeding xylem sap, to which2.5 mmol m^–3 ABA (physiological range) was added, leafconductance decreased to a similar value as in drought experiments.A log-linear relationship between the fed ABA-concentrationand leaf conductance was observed. Low stomatal con-ductancewas dependent on a continuous supply of ABA to the leaf. Whentotal ABA-influx into the leaf was large, either due to long-termfeeding of low concentrations or short-term feeding of highconcentrations (i) recovery after feeding started later and(ii) the rate of recovery was decreased. Therefore, stomatalresponses after short-term and long-term ABA-feeding were dependenton the loading of ABA into the leaf and not only on ABA-concentrations.The effectiveness of fed ABA was also dependent on the lightintensity at the fed leaf. Key words: Abscisic acid, feeding method, stomata, gas exchange, Helianthus annuus     

Stomatal response to drying soil in relation to changes in the xylem sap composition of Helianthus annuus. II. Stomatal sensitivity to abscisic acid imported from the xylem sap

Sunflower plants [Helianthus annuus L.) were subjected to soil drought. Leaf conductance declined with soil water content even when the shoot was kept turgid throughout the drying period. The concentration of abscisic acid in the xylem sap increased with decreasing soil water content. No general relation could be established between abscisic acid concentration in the xylem sap and leaf conductance due to marked differences in the sensitivity of leaf conductance of individual plants to abscisic acid from the xylem sap. The combination of these results with data from Gollan, Schurr & Schulze (1992, see pp. 551–559, this issue) reveals close connection of the effectiveness of abscisic acid as a root to shoot signal to the nutritional status of the plant.     

Nitric oxide production occurs after cytosolic alkalinization during stomatal closure induced by abscisic acid

Abscisic acid (ABA) raised the cytosolic pH and nitric oxide (NO) levels in guard cells while inducing stomatal closure in epidermis of Pisum sativum. Butyrate (a weak acid) reduced the cytosolic pH/NO production and prevented stomatal closure by ABA. Methylamine (a weak base) enhanced the cytosolic alkalinization and aggravated stomatal closure by ABA. The rise in guard cell pH because of ABA became noticeable after 6 min and peaked at 12 min, while NO production started at 9 min and peaked at 18 min. These results suggested that NO production was downstream of the rise in cytosolic pH. The ABA-induced increase in NO of guard cells and stomatal closure was prevented by 2-phenyl-4,4,5,5-tetramethyl imidazoline-1-oxyl 3-oxide (cPTIO, a NO scavenger) and partially by N-nitro-L-Arg-methyl ester (L-NAME, an inhibitor of NO synthase). In contrast, cPTIO or L-NAME had only a marginal effect on the pH rise induced by ABA. Ethylene glycol tetraacetic acid (EGTA, a calcium chelator) prevented ABA-induced stomatal closure while restricting cytosolic pH rise and NO production. We suggest that during ABA-induced stomatal closure, a rise in cytosolic pH is necessary for NO production. Calcium may act upstream of cytosolic alkalinization and NO production, besides its known function as a downstream component.     

Stomatal conductance in relation to xylem sap abscisic acid concentrations in two tropical trees, Acacia confusa and Litsea glutinosa

Two tropical trees, Acacia confusa and Litsea glutinosa, were grown under controlled conditions with their roots subjected to soil drying and soil compaction treatments. In both species, a decline in stomatal conductance resulting from soil drying took place much earlier than the decline of leaf water potential. Soil compaction treatment also resulted in a substantial decrease in stomatal conductance but had little effect on leaf water potential. A rapid and substantial increase in xylem abscisic acid (ABA) concenation ([ABA]), rather than hulk leaf ABA, was closely related to soil drying and soil compaction. A significant relationship between stomatal conductance (g_s) and xylem [ABA] was observed in both species. Artificially feeding ABA solutions to excised leaves of both species showed that the relationship bet ween g_s and [ABA] was very similar to that obtained from the whole plant, i.e. the relationship between g_s and xylem [ABA]. These results suggest that xylem ABA may act as a stress signal in the control of stomatal conductance.     

Evidence against an intermediary role of abscisic acid in stomatal closure induced by phenylmercuric acetate and faresol

The hypothesis was tested that stomatal closure induced by the antitranspirants phenylmercuric acetate and farnesol is mediated by an increase in abscisic acid content. Isolated leaves of Spinacia oleracea L. cv. Müma, Tradescantia X andersoniana Ludw. et Rohw. and Commelina communis L. were incubated in solutions of phenylmercuric acetate (10⁻³ M ) and of farnesol (2 × 10⁻³ M ). In all three species, a reduction in stomatal aperture was observed. The extent and velocity of the reaction differed between the species and also between the two compounds applied. The abscisic acid content of the leaves was determined after stomatal closure had been achieved. No significant increase in abscisic acid level was found in treated leaves of Spinacia and Commelina . In Tradescantia , on the contrary, a reduction to about 50% was observed after 120 min treatment. Visible damage of the treated leaves and membrane alterations observable by electron microscopy occurred, especially after treatment with farnesol. These changes in membrane structure suggest a connection with the reduction in stomatal aperture.     

CLE9 peptide‐induced stomatal closure is mediated by abscisic acid,hydrogen peroxide,and nitric oxide in Arabidopsis thaliana

CLE peptides have been implicated in various developmental processes of plants and mediate their responses to environmental stimuli. However, the biological relevance of most CLE genes remains to be functionally characterized. Here, we report that CLE9, which is expressed in stomata, acts as an essential regulator in the induction of stomatal closure. Exogenous application of CLE9 peptides or overexpression of CLE9 effectively led to stomatal closure and enhanced drought tolerance, whereas CLE9 loss‐of‐function mutants were sensitivity to drought stress. CLE9‐induced stomatal closure was impaired in abscisic acid (ABA)‐deficient mutants, indicating that ABA is required for CLE9‐medaited guard cell signalling. We further deciphered that two guard cell ABA‐signalling components, OST1 and SLAC1, were responsible for CLE9‐induced stomatal closure. MPK3 and MPK6 were activated by the CLE9 peptide, and CLE9 peptides failed to close stomata in mpk3 and mpk6 mutants. In addition, CLE9 peptides stimulated the induction of hydrogen peroxide (H₂O₂) and nitric oxide (NO) synthesis associated with stomatal closure, which was abolished in the NADPH oxidase‐deficient mutants or nitric reductase mutants, respectively. Collectively, our results reveal a novel ABA‐dependent function of CLE9 in the regulation of stomatal apertures, thereby suggesting a potential role of CLE9 in the stress acclimatization of plants.     

Stomatal responses of Vicia faba L. to indole acetic acid and abscisic acid

Evidence is presented that stomata in isolated epidermal peelsof Vicia faba L. open in darkness in response to the externalpresence of indole acetic acid (IAA) in the incubation medium.The effect of IAA is found to be overcome completely in thepresence of either TRIS or MES buffers. In the absence of buffer,V. faba stomata are shown to be influenced by IAA in a concentration-dependenttrend which reached a maximum at an [IAA] of 10^–3 molm^–3. Further investigations reveal that stomata in thisspecies can be shown to respond to the presence of IAA and anotherphytohormone, abscisic acid (ABA). IAA and ABA are demonstratedto be antagonistic in their effects provided the incubationconditions are suitable. The data are discussed in relationto stomatal responses of other species in different treatmentconditions. Recommendations are made with respect to standardizationof incubation media during epidermal peel experiments. Key words: Vicia faba, stomata, indole acetic acid, abscisic acid, buffers     

Early stomatal closure in waterlogged pea plants is mediated by abscisic acid in the absence of foliar water deficits

Abstract Soil waterlogging decreased leaf conductance (interpreted as stomatal closure) of vegetative pea plants (Pisuin sativum L. cv. ‘Sprite’) approximately 24 h after the start of flooding, i.e. from the beginning of the second 16 h-long photo-period. Both adaxial and abaxial surfaces of leaves of various ages and the stipules were affected. Stomatal closure was sustained for at least 3 d with no decrease in foliar hydration measured as water content per unit area, leaf water potential or leaf water saturation deficit. Instead, leaves became increasingly hydrated in association with slower transpiration. These changes in the waterlogged plants over 3 d were accompanied by up to 10-fold increases in the concentration of endogenous abscisic acid (ABA). Waterlogging also increased foliar hydration and ABA concentrations in the dark. Leaves detached from non-waterlogged plants and maintained in vials of water for up to 3 d behaved in a similar way to leaves on flooded plants, i.e. stomata closed in the absence of a water deficit but in association with increased ABA content. Applying ABA through the transpiration stream to freshly detached leaflets partially closed stomata within 15 min. The extractable concentrations of ABA associated with this closure were similar to those found in flooded plants. When an ABA-deficient ‘wilty’ mutant of pea was waterlogged, the extent of stomatal closure was less pronounced than that in ordinary non-mutant plants, and the associated increase in foliar ABA was correspondingly smaller. Similarly, waterlogging closed stomata of tomato plants within 24 h, but no such closure was seen in ‘flacca’, a corresponding ABA-deficient mutant. The results provide an example of stomatal closure brought about by stress in the root environment in the absence of water deficiency. The correlative factor operating between the roots and shoots appeared to be an inhibition of ABA transport out of the shoots of flooded plants, causing the hormone to accumulate in the leaves.     

Stomatal response to abscisic Acid is a function of current plant water status

We investigated, under laboratory and field conditions, the possibility that increasing abscisic acid (ABA) concentrations and decreasing water potentials can interact in their effects on stomata. One experiment was carried out with epidermal pieces of Commelina communis incubated in media with a variety of ABA and polyethylene glycol concentrations. In the media without ABA, incubation in solutions with water potentials between −0.3 and −1.5 megapascals had no significant effect on stomatal aperture. Conversely, the sensitivity of stomatal aperture to ABA was trebled in solutions at −1.5 megapascals compared with sensitivity at −0.3 megapascals. The effect of the change in sensitivity was more important than the absolute effect of ABA at the highest water potential. In a field experiment, sensitivity of maize stomatal conductance to the concentration of ABA in the xylem sap varied strongly with the time of the day. We consider that the most likely explanation for this is the influence of a change in leaf or epidermal water potential that accompanies an increase in irradiance and saturation deficit as the day progresses. These observations suggest that epidermal water relations may act as a modulator of the responses of stomata to ABA. We argue that such changes must be taken into account in studies or modeling of plant responses to drought stress.     

Prevention of stomatal closure by immunomodulation of endogenous abscisic acid and its reversion by abscisic acid treatment: physiological behaviour and morphological features of tobacco stomata

Transgenic tobacco ( Nicotiana tabacum L.) plants ubiquitously accumulating a single-chain variable-fragment (scFv) antibody against abscisic acid (ABA) to high concentrations in the endoplasmic reticulum (RA plants) show a wilty phenotype. High stomatal conductance and loss of CO(2) and light dependence of stomatal conductance are typical features of these plants. ABA was applied to these plants either via the petioles or by daily spraying over several weeks in order to normalise the phenotype. During the long-term experiments, scFv protein concentrations, total and (calculated) free ABA contents, and stomatal conductance and its dependence on CO(2) concentration and light intensity were monitored. The wilty phenotype of transgenic plants could not be normalised by short-term treatment with ABA via the petioles. Only a daily long-term treatment during plant development normalised the physiological behaviour completely. Scanning electron microscopy of stomata showed morphological changes in RA plants compared with wild-type plants that, for structural reasons, prevented regular stomatal movements. After long-term treatment with ABA this defect could be completely eliminated. Guard-cell-specific expression of the anti-ABA scFv did not cause any changes in physiological behaviour compared to the wild type. In addition, mesophyll-specific expression starting in leaves that were already fully differentiated resulted in normal phenotypes, too. We conclude that changes in distribution and availability of ABA in the cells of developing leaves of RA plants cause the development of structural features in stomata that prevent normal function.     

The human L-pipecolic acid oxidase is similar to bacterial monomeric sarcosine oxidases rather than D-amino acid oxidases

L-Pipecolic acid oxidase activity is deficient in patients with peroxisome biogenesis disorders (PBDs). Because its role, if any, in these disorders is unknown, the authors cloned the human gene to order to further study its functions. BLAST search of the translated sequence showed greatest homology to Bacillus sp. NS-129 monomeric sarcosine oxidase. The purified enzyme could use either L-pipecolic acid or sarcosine as a substrate. No homology was found to the peroxisomal D-amino acid oxidases. A further comparison of L-pipecolic acid oxidase to the two D-amino acid oxidases in peroxisomes showed that the proteins differed in many ways. First, both D-amino acid oxidase and L-pipecolic acid oxidase showed no enzyme activity in liver from Zell-weger syndrome patients; D-aspartate oxidase activity was unchanged from control levels. Although all were targeted to peroxisomes, their targeting signals differed. No L-pipecolic acid oxidase was found in brain or other tissues outside of liver and kidney. The D-amino acid oxidases were similarly and more widely distributed. Finally, although D-amino acid degradation is limited to peroxisomes in mammals, L-pipecolic acid can be oxidized in either mitochondria or peroxisomes, or both.     

Effect of leaf water status and xylem pH on metabolism of xylem-transported abscisic acid

Metabolism and distribution of xylem-fed ABA were investigated in leaves of maize (Zea mays) and Commelina communis when water stress and xylem pH manipulation were applied. ³H-ABA was fed to excised leaves via the transpiration stream. Water stress was applied through either a previous soil-drying before leaves were excised, or a quick dehydration after leaves were fed with ABA. Xylem-delivered ABA was metabolised rapidly in the leaves (half-life 0.7 h and 1.02 h for maize and Commelina respectively), but a previous soil-drying or a post-feeding dehydration significantly extended the half-life of fed ABA in both species. In the first few hours after ABA was fed into the detached leaves, percentages of applied ABA remaining unmodified were always higher in leaves which received water stress treatments than in control leaves. However the percentage decreased to below the control levels several hours later in leaves which received a previous soil-drying treatment prior to excision, but had then been rehydrated by the xylem-feeding process itself. One possible explanation for this could be a changed pattern of compartmentalisation for xylem-carried ABA. A post-feeding dehydration treatment also changed the distribution of xylem-fed ABA within the leaves: more ABA was found in the epidermis of Commelina leaves which had been dehydrated rapidly after ABA had been fed, compared to the controls. The levels of xylem-delivered ABA remaining unmodified increased as the pH of the feeding solution increased from 5 to 8. The results support the hypothesis that water stress and a putative stress-induced xylem pH change may modify stomatal sensitivity to ABA by changing the actual ABA content of the leaf epidermis.     

Stomatal behaviour and water movement through roots of wheat plants treated with abscisic acid

Abstract Experiments with isolated roots of wheat plants suggested that when water uptake rates are low, low concentrations of abscisic acid (ABA) may increase the flux of water into roots. This increase was recorded despite an ABA-stimulated reduction in the hydraulic conductance of the whole root system. Hydraulic conductances were measured under steady-state conditions. A system is described where the stomatal behaviour and water movement through roots of a single intact plant may be concurrently monitored. Experiments with intact plants confirmed that application of ABA could increase the rate of water movement into roots when uptake rates were low. No such increase was observed at high flux rates. Application of ABA to roots caused partial stomatal closure and caused conductance to oscillate around a reduced mean value. An ABA-stimulated increase in the turgor sensitivity of stomata is postulated and the significance of this effect is discussed.     

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 CO₂ exchange rate. Reductions of similar magnitude also were observed in stomatal diffusive conductivity.     

Changes of free and conjugated abscisic acid and phaseic acid in xylem sap of drought-stressed sunflower plants

Abscisic acid (ABA), conjugated abscisic acid, phaseic acid (PA), and conjugated phaseic acid were determined by enzyme-linked immunosorbent assay (ELISA) and gas chromatography (GC) in xylem sap of well-watered and drought-stressed sunflower plants. Conjugated ABA and conjugated PA were determined indirectly after chemical or enzymatic hydrolysis. Conjugated ABA was found to be the predominant ABA metabolite in xylem sap. In xylem sap from well-watered plants at least five, and in sap from drought-stressed plants at least six alkaline hydrolysable ABA conjugates were found. One of them corresponds chromatographically (HPLC) with abscisic acid glucose ester (ABAGE). Under drought conditions the concentrations of ABA, alkaline hydrolysable ABA conjugates, -glucosidase hydrolysable ABA conjugates, PA, and conjugated PA increased. After rewatering the drought-stressed plants, the ABA and the conjugated ABA content decreased. The possible function of the ABA conjugates in the xylem sap as a source of free ABA is discussed.