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Drought stress affects plant growth and development through several mechanisms, including the induction of oxidative stress. To cope with drought, plants have drought tolerance mechanisms at the physiological, biochemical, and molecular levels. In this study, the effects of foliar application of distilled water and methyl jasmonate (MeJA) (5 and 50 µM) on the physiological, biochemical, and molecular responses of Impatiens walleriana during two drought regimes (15 and 5% soil water content, SWC) were investigated. The results showed that plant response depended on the concentration of the elicitor and the stress intensity. The highest chlorophyll and carotenoid contents were observed at 5% SWC in plants pre-treated with 50 µM MeJA, while the MeJA did not have a significant effect on the chlorophyll a/b ratio in drought-stressed plants. Drought-induced formation of hydrogen peroxide and malondialdehyde in plants sprayed with distilled water was significantly reduced in plant leaves pretreated with MeJA. The lower total polyphenol content and antioxidant activity of secondary metabolites in MeJA-pretreated plants were observed. The foliar application of MeJA affected the proline content and antioxidant enzyme activities (superoxide dismutase, peroxidase, and catalase) in plants that suffered from drought. The expression of abscisic acid (ABA) metabolic genes (IwNCED4, IwAAO2, and IwABA8ox3) was the most affected in plants sprayed with 50 µM MeJA, while of the four analyzed aquaporin genes (IwPIP1;4, IwPIP2;2, IwPIP2;7, and IwTIP4;1), the expression of IwPIP1;4 and IwPIP2;7 was strongly induced in drought-stressed plants pre-treated with 50 µM MeJA. The study’s findings demonstrated the significance of MeJA in regulating the gene expression of the ABA metabolic pathway and aquaporins, as well as the considerable alterations in oxidative stress responses of drought-stressed I. walleriana foliar sprayed with MeJA. The results improved our understanding of this horticulture plant’s stress physiology and the field of plant hormones’ interaction network in general.

The beneficial role of fungi from the Trichoderma genus and its secondary metabolites in promoting plant growth, uptake and use efficiency of macronutrients and oligo/micro-nutrients, activation of plant secondary metabolism and plant protection from diseases makes it interesting for application in environmentally friendly agriculture. However, the literature data on the effect of Trichoderma inoculation on tomato fruit quality is scarce. Commercially used tomato cultivars were chosen in combination with indigenous Trichodrema species previously characterized on molecular and biochemical level, to investigate the effect of Trichoderma on photosynthetic characteristics and fruit quality of plants grown in organic system of production. Examined cultivars differed in the majority of examined parameters. Response of cultivar Gružanski zlatni to Trichoderma application was more significant. As a consequence of increased epidermal flavonols and decreased chlorophyll, the nitrogen balance index in leaves has decreased, indicating a shift from primary to secondary metabolism. The quality of its fruit was altered in the sense of increased total flavonoids content, decreased starch, increased Bioaccumulation Index (BI) for Fe and Cr, and decreased BI for heavy metals Ni and Pb. Higher expression of swolenin gene in tomato roots of more responsive tomato cultivar indicates better root colonization, which correlates with observed positive effects of Trichodrema.

Two tomato genotypes with constitutively different ABA level, flacca mutant and wild type of Ailsa Craig cv. (WT), were subjected to three repeated drought cycles, with the aim to reveal the role of the abscisic acid (ABA) threshold in developing drought tolerance. Differential responses to drought of two genotypes were obtained: more pronounced stomatal closure, ABA biosynthesis and proline accumulation in WT compared to the mutant were compensated by dry weight accumulation accompanied by transient redox disbalance in flacca. Fourier-transform infrared (FTIR) spectra analysis of isolated cell wall material and morphological parameter measurements on tomato leaves indicated changes in dry weight accumulation and carbon re-allocation to cell wall constituents in flacca, but not in WT. A higher proportion of cellulose, pectin and lignin in isolated cell walls from flacca leaves further increased with repeated drought cycles. Different ABA-dependent stomatal closure between drought cycles implies that acquisition of stomatal sensitivity may be a part of stress memory mechanism developed under given conditions. The regulatory role of ABA in the cell wall restructuring and growth regulation under low leaf potential was discussed with emphasis on the beneficial effects of drought priming in developing differential defense strategies against drought.

The UV-B represents the minor fraction of the solar spectrum, while UV-C is not contained in natural solar radiation, but both radiation types can cause damaging effects in plants. Cell walls (CWs) are one of the targets for external stressors. Juvenile P. omorika trees were treated either with 21 day-high doses UV-B or with 7 day- UV-C in open-top chambers. Using spectroscopic and biochemical techniques, it was shown that the response to UV radiation includes numerous modifications in needle CW structure: relative content of xylan, xyloglucan, lignin and cellulose decreased; cellulose crystallinity changed; yield of lignin monomers with stronger connection of C–– C in side chain with the ring increased; re-distribution of inter- and intra-polymer H-bonds occurred. The recovery was mediated by an increase in the activities and changes in isoform profiles of CW bound covalent peroxidases (POD) and polyphenol oxidases (PO) (UV–B), and ionic POD and covalent PO (UV–C). A connection between activities of specific POD/PO isoforms and phenolic species (m- and p-coumaric acid, pinoresinol and cinnamic acid derivatives) was demonstrated, and supported by changes in the sRNA profile. In vivo fluorometry showed phenolics accumulation in needle epidermal CWs. These results imply transversal connections between polymers and changed mechanical properties of needle CW as a response to UV. The CW alterations enabled maintenance of physiological functions, as indicated by the preserved chlorophyll content and/or organization. The current study provides evidence that in conifers, needle CW response to both UV-B and UV-C includes biochemical modifications and structural remodeling. Supplementary material: [http://aspace.agrif.bg.ac.rs/handle/123456789/5997]

Trichoderma species are widely known for their antagonistic properties, but plant growth promotion and abiotic stress tolerance have also been reported. Mechanisms of abiotic stress tolerance, such as drought in plants in interaction with Trichoderma spp., remain unclear. In this work, the results about the influence of Trichoderma brevicompactum on the physiological parameters and abscisic acid (ABA) content in leaves and roots of tomato, as well as the Trichodermamodulated plant response to drought are presented. Both in optimally watered plants and droughted plants treated with Trichoderma, the relative water content (RWC) was 17% higher than in untreated plants. Drought lowered RWC by 25% in both Trichoderma-untreated and -treated plants. Under drought conditions, the better water status of plants treated with Trichoderma is connected with lower stomatal conductance and closure of stomata. The lower increase of root ABA content in Trichoderma-treated plants that we observed could be an adaptive advantage in sense of smaller inhibition of plant growth induced by drought. Trichoderma modulation of the salicylic acid (SA) marker genes response to drought was observed in leaves. Our results suggest that signalling pathways of jasmonic acid (JA), SA and ABA are involved in response to both drought and Trichoderma. The mutual effect of the tested treatments leads to the modification of gene expression in comparison to the reaction to a single stress. The upregulation of SA marker genes in leaves and JA marker genes in the roots 2 weeks after the interaction with T. brevicompactum indicates that it could be promising biocontrol agent.

The aim of this study was to evaluate the content and profile of the phenolic compounds (PCs) and antioxidant properties of field-grown leaves, in vitro leaves and in vitro callus cultures of the blackberry ‘Čačanska Bestrna’ and blueberry ‘Toro’. In vitro shoots of the selected genotypes were grown either on original Murashige and Skoog (MS) medium containing 1 mg/L BA, 0.1 mg/L IBA and 0.1 mg/L GA3 (‘Čačanska Bestrna’) or on MS medium with macroelements reduced to ½, 2 mg/L zeatin and 0.2 mg/L IAA (‘Toro’). Callus cultures were induced from in vitro leaves and established on MS medium with 2 mg/L BA and 2 mg/L 2,4-D (‘Čačanska Bestrna’) or MS medium with half strength macroelements, 2 mg/L BA, 2 mg/L 2,4-D and 1 mg/L NAA (‘Toro’). Total phenolic (TPC) and flavonoid content (TFC) were the highest in blueberry leaves, whereas low TPC and TFC values were obtained in callus cultures of both cultivars. A higher content of PCs in blueberry leaves compared to blackberry leaves was determined by the UHPLC-DAD MS/MS technique. Quercetin derivatives and phenolic acids were the dominant PCs in the leaves of both berries, whereas gallocatechin was present in a significant amount in blueberry leaves. Callus cultures of both berries had a specific PC profile, with none detected in the leaves except quercetin-3-O-glucoside and quercetin-3-O-rutinoside. Blackberry leaves showed the best antioxidant properties as estimated by ferric reducing power (FRP), ABTS•+ and DPPH• scavenging activity assays. Callus cultures of both berries exhibited three to five times lower ABTS•+ and ten to seventeen times lower DPPH• scavenging activity compared to corresponding leaves. The analyzed leaves and callus cultures can be a good source of PCs with good antioxidant properties and specific phenolics, respectively, for applications in the food and pharmaceutical industries.

Water deficit has a global impact on plant growth and crop yield. Climate changes are going to increase the intensity, duration and frequency of severe droughts, particularly in southern and south-eastern Europe, elevating the water scarcity issues. We aimed to assess the contribution of endogenous abscisic acid (ABA) in the protective mechanisms against water deficit, including stomatal conductance, relative water potential and the accumulation of osmoprotectants, as well as on growth parameters. To achieve that, we used a suitable model system, ABA-deficient tomato mutant, flacca and its parental line. Flacca mutant exhibited constitutively higher levels of soluble sugars (e.g., galactose, arabinose, sorbitol) and free amino acids (AAs) compared with the wild type (WT). Water deficit provoked the strong accumulation of proline in both genotypes, and total soluble sugars only in flacca. Upon re-watering, these osmolytes returned to the initial levels in both genotypes. Our results indicate that flacca compensated higher stomatal conductance with a higher constitutive level of free sugars and AAs. Additionally, we suggest that the accumulation of AAs, particularly proline and its precursors and specific branched-chain AAs in both, glucose and sucrose in flacca, and sorbitol in WT, could contribute to maintaining growth rate during water deficit and recovery in both tomato genotypes.

This study was carried out to examine the drought effect on development, physiological, biochemical and molecular parameters in Impatiens walleriana grown ex vitro. Experiment design included three treatments: Control plants&mdash, grown under optimal watering (35%&ndash, 37% of soil moisture content), drought-stressed plants&mdash, non-irrigated to reach 15% and 5% of soil moisture content and recovery plants&mdash, rehydrated for four days to reach optimal soil moisture content. Drought reduced fresh weight, total leaf area, as well as dry weight of I. walleriana shoots. Drought up-regulated expression of abscisic acid (ABA) biosynthesis genes 9-cis-epoxycarotenoid dioxygenase 4 (NCED4) and abscisic aldehyde oxidase 2 (AAO2) and catabolic gene ABA 8&prime, hydroxylase 3 (ABA8ox3) which was followed by increased ABA content in the leaves. Decrement in water potential of shoots during the drought was not accompanied with increased amino acid proline content. We detected an increase in chlorophyll, carotenoid, total polyphenols and flavonols content under drought conditions, as well as malondialdehyde, hydrogen peroxide and DPPH (1,1&prime, diphenyl-2-picrylhydrazyl) activity. Increased antioxidant enzyme activities (superoxide dismutase, peroxidase and catalase) throughout drought were also determined. Recovery treatment was significant for neutralizing drought effect on growth parameters, shoot water potential, proline content and genes expression.

In this study we exposed variegated leaves of Pelargonium zonale to strong sunlight (>1100 μmol m −2 s −1 of photosynthetically active radiation) with and without paraquat (Pq), with the aim to elucidate the mechanisms of H 2 O 2 regulation in green and white tissues with respect to the photosynthetically-dependent generation of reactive oxygen species (ROS). Sunlight induced marked accumulation of H 2 O 2 in the apoplast of vascular and (peri)vascular tissues only in green sectors. This effect was enhanced by the addition of Pq. In the presence of diphenyl iodide, an NADPH oxidase inhibitor, H 2 O 2 accumulation was abolished. Distinct light-induced responses were observed: in photosynthetic cells, sunlight rapidly provoked ascorbate (Asc) biosynthesis and an increase of glutathione reductase (GR) and catalase activities, while in non-photosynthetic cells, early up-regulation of soluble ascorbate peroxidase, dehydroascorbate reductase (DHAR) and GR activities was observed. Paraquat addition stimulated DHAR and GR activities in green sectors, while in white sectors activities of monodehydroascorbate reductase, DHAR and class III peroxidases, as well as Asc content rapidly increased. Differential antioxidative responses in the two tissues in the frame of their contrasting metabolisms, and the possible role of (peri)vascular H 2 O 2 in signaling were discussed.

Aquaporins comprise a large group of transmembrane proteins responsible for water transport, which is crucial for plant survival under stress conditions. Despite the vital role of aquaporins, nothing is known about this protein family in Impatiens walleriana, a commercially important horticultural plant, which is sensitive to drought stress. In the present study, attention is given to the molecular characterization of aquaporins in I. walleriana and their expression during drought stress and recovery. We identified four I. walleriana aquaporins: IwPIP1, 4, IwPIP2, 2, IwPIP2, 7 and IwTIP4, 1. All of them had conserved NPA motifs (Asparagine-Proline-Alanine), transmembrane helices (TMh), pore characteristics, stereochemical properties and tetrameric structure of holoprotein. Drought stress and recovery treatment affected the aquaporins expression in I. walleriana leaves, which was up- or downregulated depending on stress intensity. Expression of IwPIP2, 7 was the most affected of all analyzed I. walleriana aquaporins. At 15% and 5% soil moisture and recovery from 15% and 5% soil moisture, IwPIP2, 7 expression significantly decreased and increased, respectively. Aquaporins IwPIP1, 4 and IwTIP4, 1 had lower expression in comparison to IwPIP2, 7, with moderate expression changes in response to drought and recovery, while IwPIP2, 2 expression was of significance only in recovered plants. Insight into the molecular structure of I. walleriana aquaporins expanded knowledge about plant aquaporins, while its expression during drought and recovery contributed to I. walleriana drought tolerance mechanisms and re-acclimation.

Drought is one of the main consequences of climate change that negatively affects plant growth and development, which in turn leads to a reduction in yield. The objective of this study was to investigate the effects of the physiological responses to the drought stress of maize plants (population IP3722), obtained from seeds of different ages (originating from 2012. and 2016) and different previous experience (the seeds formed in 2012. were more exposed to drought than in 2016.). Two populations responded to drought treatment by a significant decrease of shoot fresh and dry weight, this was followed with decrease leaf area and transpiration rate. Drought tre-atments enabled induction and differentiation of chemical signals of drought (abscisic acid-ABA and xylem pH) and hydraulic signals (leaf water potential), and monitoring of their activity on stomatal conductance. The data showed that hydraulic signals have the same impact on stomatal reactions in the D2012 and D2016 treatments. They also indicated that the chemical signal of ABA works toward stomatal closure, but that the effect depends on whether it originates from the leaf or the root. Leaf-originating ABA had more effect on the closure of D2012 stomata, while root-ori-ginating ABA and pH was more active in the D2016 Suša nastaje kao posledica klimatskih promena, koja negativno utiče na rast i razvoj biljaka, a to dovodi do smanjenja prinosa. Cilj ovog rada bio je da se istraži efekat suše na fiziološke reakcije biljaka kukuruza (IP3722), koje su dobijene iz semena različite starosti (regenerisano 2012. i 2016.) i različitih prethodnih iskustava (semena koja su formirana 2012. su bila u većoj meri izložena suši nego ona iz 2016.). Suša je kod oba uzorka dovela do smanjenja sveže i suve mase nadzemnog dela biljke, što je bilo praćeno opadanjem lisne površine i stomatalne provod-ljivosti. Tretmanom suše su indukovani hemijski signali (ABA i pH) i hidraulički signal (vodni potencijal), koji su uticali na provodljivost stoma. Podaci su pokazali da hidraulički signal ima podjednak udeo na zatvaranje stoma u tretmanu D2012 i D2016. Takođe, ABA kao hemijski signal dovodi do zatvaranja stoma, ali ovaj efekat zavisi od toga da li ABA potiče iz korena ili lista. ABA poreklom iz lista je imala značajniji doprinos u zatvaranju stoma kod D2012, dok ABA poreklom iz korena i pH kod tretmana D2016.

In this study we exposed variegated leaves of Pelargonium zonale to strong sunlight (1100μmolm

The effects of drought (partial root-zone drying-PRD and regulated deficit irrigation-RDI) and full irrigation (FI) on the expression of ABA biosynthetic genes (TAO1 and NCED), EIL1 gene and ABA content in the leaves of tomato wild-type (WT) and flacca mutant were investigated. Results confirmed differences in the expression of the investigated genes under the investigated treatments, during treatment duration as well as between investigated WT and flacca plants. The most significant differences between WT and flacca were found under PRD treatment. The similar expression pattern of all genes in the WT plants could indicate synergistic signaling pathways for ABA and ethylene. In flacca, reduced NCED and significant EIL1 expression might reflect the increase in ethylene production, which could influence the ABA signaling and production that occurred under PRD. Drought also induced an increase in ABA content that is most expressed in flacca under RDI.

The effects of drought and partial root drying (PRD) on shoot and root growth was assessed in the wild type Ailsa Craig (WT) and the flacca tomato mutant deficient in the plant hormone ABA. Our results show that drought had an inhibitory effect on shoot growth in flacca and especially in WT; the most profound effect was observed in FI (full irrigation), then PRD and the smallest in D plants. Root development in both WT and flacca was stimulated after the 3rd day of the experiment following a decrease in the soil water content. On the 11th day of the experiment, when the soil water content was reduced by about 50% of full irrigation (FI), the root density was increased in the drying part of the PDR and on both sides of the drought treatment. On the basis of these results it can be assumed that increased root density and root length represent an adaptation or root adjustment to drought conditions.

Partial root-zone drying (PRD) technique, a novel approach to watering crops, was developed on the basis of knowledge of root-to-shoot signaling in drying soil. The aim of the present paper was to investigate the effects of the PRD treatment on tomato growth and the water regime. The obtained PRD results showed significant reduction in shoot but not fruit growth in the absence of any changes in shoot water status, indicating the involvement of chemical root-to-shoot signals. Higher water use efficiency (WUE) results mean that the PRD technique can be used to reduce irrigation water without significant reduction of tomato yield. Tehnika delimičnog sušenja korenova (PRD), novi pristup u navodnjavanju biljaka, razvila se na osnovu saznanja o signalima suše na nivou koren-izdanak. Cilj rada je bio da se ispitaju efekti PRD tretmana na rastenje biljaka i vodni režim. Rezultati su pokazali da je PRD tretman izazvao značajnu redukciju rastenja izdanka, ali ne i plodova u odsustvu promene vodnog statusa izdanka što je indikator uticaja hemijskih signala na nivou koren-izdanak. Povećanje efikasnosti u korišćenju vode (WUE) je pokazalo da se primenom PRD tehnike može redukovati količina vode za navodnjavanje bez značajne redukcije prinosa paradajza.

Sunflower line with resistance for downy mildew (Ha-26R) and susceptible line (Ha-26S) were inoculated with the suspension of Plasmopara halstedii zoosporangia, race 730, in the phase of first pair of leaves. The expression of defense related genes was investigated in the time period of 2 to 96 h after treatment. Several categories of defense-related genes: signalization (EDS1 and EDR1); H2O2 producing (Hacaox and Haoxox); antioxidative response (SODc and SODp), pathogenesis related (chi, PAL, PR5) were examined. Most examined defense related genes were constitutive with higher expression in resistant line. However, in response to secondary downy mildew infection six genes were upregulated. Upregulation of HaEDS1 signaling gene 2 h after infection indicates that SA mediated response is activated. Pathogenesis related genes: chitinase and PR5, were also upregulated in the earliest time point. Other defense related genes: SODp, Caox and OxOx were sequentially upregulated from 4 to 48 h after infection in resistant line. Our results indicate that the early response of defense related genes to secondary downy mildew infection, resembles to hypersensitive-like reaction and is connected with resistance conferred by Pl6 gene. According to our results resistance to secondary infection is characterized with earlier upregulation of PR5 in comparison to primary infection.

Metal contamination represents a strong selective pressure favoring tolerant genotypes and leading to differentiation between plant populations. We investigated the adaptive capacity of early-colonizer species of Verbascum recently exposed to Zn- and Cu-contaminated soils (10-20 years). Two Verbascum thapsus L. populations from uncontaminated sites (NMET1, NMET2), one V. thapsus from a zinc-contaminated site (MET1), and a Verbascum lychnitis population from an open-cast copper mine (MET2) were exposed to elevated Zn or Cu in hydroponic culture under glasshouse conditions. MET populations showed considerably higher tolerance to both Zn and Cu than NMET populations as assessed by measurements of growth and net photosynthesis, yet they accumulated higher tissue Zn concentrations in the shoot. Abscisic acid (ABA) concentration increased with Zn and Cu treatment in the NMET populations, which was correlated to stomatal closure, decrease of net photosynthesis, and nutritional imbalance, indicative of interference with xylem loading and divalent-cation homeostasis. At the cellular level, the sensitivity of NMET2 to Zn and Cu was reflected in significant metal-induced ROS accumulation and ion leakage from roots as well as strong induction of peroxidase activity (POD, EC 1.11.1.7), while Zn had no significant effect on ABA concentration and POD activity in MET1. Interestingly, MET2 had constitutively higher root ABA concentration and POD activity. We propose that ABA distribution between shoots and roots could represent an adaptive mechanism for maintaining low ABA levels and unaffected stomatal conductance. The results show that metal tolerance can occur in Verbascum populations after relatively short time of exposure to metal-contaminated soil, indicating their potential use for phytostabilization.

Mullein (Verbascum) plants have extensive distribution and can grow in variable environmental conditions. Seed was collected from mullein plants grown at 4 locations, two metals contaminated and two metal uncontaminated areas. Genetic variability of progeny was examined. Populations collected from unpolluted areas were genetically more similar than those collected from polluted areas as revealed by RAPD and SSR markers and UPGMA analysis. The results indicate that there is genetic differentiation between examined populations and therefore they represent suitable material for further investigation of plant adaptation mechanisms to increased metal content. [Projekat Ministarstva nauke Republike Srbije, br. III43010]

Metal contamination represents a strong selective pressure favoring tolerant genotypes and leading to differentiation between plant populations. We investigated the adaptive capacity of early-colonizer species of Verbascum recently exposed to Zn- and Cu-contaminated soils (10-20 years). Two Verbascum thapsus L. populations from uncontaminated sites (NMET1, NMET2), one V. thapsus from a zinc-contaminated site (MET1), and a Verbascum lychnitis population from an open-cast copper mine (MET2) were exposed to elevated Zn or Cu in hydroponic culture under glasshouse conditions. MET populations showed considerably higher tolerance to both Zn and Cu than NMET populations as assessed by measurements of growth and net photosynthesis, yet they accumulated higher tissue Zn concentrations in the shoot. Abscisic acid (ABA) concentration increased with Zn and Cu treatment in the NMET populations, which was correlated to stomatal closure, decrease of net photosynthesis, and nutritional imbalance, indicative of interference with xylem loading and divalent-cation homeostasis. At the cellular level, the sensitivity of NMET2 to Zn and Cu was reflected in significant metal-induced ROS accumulation and ion leakage from roots as well as strong induction of peroxidase activity (POD, EC 1.11.1.7), while Zn had no significant effect on ABA concentration and POD activity in MET1. Interestingly, MET2 had constitutively higher root ABA concentration and POD activity. We propose that ABA distribution between shoots and roots could represent an adaptive mechanism for maintaining low ABA levels and unaffected stomatal conductance. The results show that metal tolerance can occur in Verbascum populations after relatively short time of exposure to metal-contaminated soil, indicating their potential use for phytostabilization.

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.

An analysis of the rate of stomatal closure in epidermal strips of Commelina communis L. induced by different calcium concentrations (10 nM to 1 mM) demonstrated a biphasic dependency, the initial process of closure being more calcium dependent. This dependency was more evident at pH 7 than pH 5 of the bathing medium. Addition of low concentrations of abscisic acid (10 nM) induced a broadening of the Ca(2) dependency, while 10 microM abscisic acid increased the rate of stomatal closure in the whole Ca(2+) range and to a large extent abolished the calcium-induced effects. Such results indicate an interaction of external protons, Ca(2+), and abscisic acid, and possible competition for the membrane associated binding sites of the mechanism(s) responsible for the regulation of stomatal closure.