Stomatal Guard Cells Research Articles

The effect of oxidative burst suppression or induction on the interaction between brown rust fungus and timopheevi wheat

The role of reactive oxygen species in the interaction between brown rust-causing pathogen Puccinia triticina Erikss. and the disease-resistant species Triticum timopheevii Zhuk. was investigated using salicylic acid treatment to amplify the oxidative burst or treatment with verapamil, an inhibitor of Ca2+-channels to suppress oxidative the burst. The levels of reactive oxygen species in control T. timopheevii plants increased during the early stages of pathogenesis due to O22← generation by stomatal guard cells contacting the appressoria and by mesophyll cells that developed a hypersensitivity reaction in response to the invasion of haustoria. Hydrogen peroxide was subsequently accumulated in the cytoplasm of cells destroyed by hypersensitivity reaction, as well as on the walls of cells contacted with fungal hyphae. The production of O22←, which results in the death of most of the inoculum on the stomata and rapid accumulation of hydrogen peroxide, were enhanced by salicylic acid pretreatment. Pretreatment of the plants with verapamil inhibited the production of reactive oxygen species and hypersensitivity reaction in disease-resistant T. timopheevii plants and thus facilitated the penetration of the fungus into the stomata and more intensive development of mycelium in plant tissues in the early stages of pathogenesis. Some colonies with single haustoria died regardless of production of reactive oxygen species by the plants.

Reconstitution of an Initial Step of Phototropin Signaling in Stomatal Guard Cells.

Phototropins are light-activated receptor kinases that mediate a wide range of blue light responses responsible for the optimization of photosynthesis. Despite the physiological importance of phototropins, it is still unclear how they transduce light signals into physiological responses. Here, we succeeded in reproducing a primary step of phototropin signaling in vitro using a physiological substrate of phototropin, the BLUS1 (BLUE LIGHT SIGNALING1) kinase of guard cells. When PHOT1 and BLUS1 were expressed in Escherichia coli and the resulting recombinant proteins were incubated with ATP, white and blue light induced phosphorylation of BLUS1 but red light and darkness did not. Site-directed mutagenesis of PHOT1 and BLUS1 revealed that the phosphorylation was catalyzed by phot1 kinase. Similar to stomatal blue light responses, the BLUS1 phosphorylation depended on the fluence rate of blue light and was inhibited by protein kinase inhibitors, K-252a and staurosporine. In contrast to the result in vivo, BLUS1 was not dephosphorylated in vitro, suggesting the involvement of a protein phosphatase in the response in vivo. phot1 with a C-terminal kinase domain but devoid of the N-terminal domain, constitutively phosphorylated BLUS1 without blue light, indicating that the N-terminal domain has an autoinhibitory action and prevents substrate phosphorylation. The results provide the first reconstitution of a primary step of phototropin signaling and a clue for understanding the molecular nature of this process.

Metabolomic Responses of Guard Cells and Mesophyll Cells to Bicarbonate.

Anthropogenic CO2 presently at 400 ppm is expected to reach 550 ppm in 2050, an increment expected to affect plant growth and productivity. Paired stomatal guard cells (GCs) are the gate-way for water, CO2, and pathogen, while mesophyll cells (MCs) represent the bulk cell-type of green leaves mainly for photosynthesis. We used the two different cell types, i.e., GCs and MCs from canola (Brassica napus) to profile metabolomic changes upon increased CO2 through supplementation with bicarbonate (HCO3 -). Two metabolomics platforms enabled quantification of 268 metabolites in a time-course study to reveal short-term responses. The HCO3 - responsive metabolomes of the cell types differed in their responsiveness. The MCs demonstrated increased amino acids, phenylpropanoids, redox metabolites, auxins and cytokinins, all of which were decreased in GCs in response to HCO3 -. In addition, the GCs showed differential increases of primary C-metabolites, N-metabolites (e.g., purines and amino acids), and defense-responsive pathways (e.g., alkaloids, phenolics, and flavonoids) as compared to the MCs, indicating differential C/N homeostasis in the cell-types. The metabolomics results provide insights into plant responses and crop productivity under future climatic changes where elevated CO2 conditions are to take center-stage.

Changes of Nitric Oxide and Its Relationship with H2O2 and Ca2+ in Defense Interactions between Wheat and Puccinia Triticina.

In this research, the wheat cultivar 'Lovrin 10' and Puccinia triticina races 165 and 260 were used to constitute compatible and incompatible combinations to investigate the relationship between NO and H2O2 and between NO and calcium (Ca2+) signaling in the cell defense process by pharmacological means. The specific fluorescent probe DAF-FM DA was coupled with confocal laser scanning microscopy and used to label intracellular nitric oxide (NO) and monitoring the real-time NO dynamics during the processes of wheat defense response triggered by P. triticina infection. The results showed that at 4 h after inoculation, weak green fluorescence was observed in the stomatal guard cells at the P. triticina infection site in the incompatible combination, which indicates a small amount of NO production. Twelve hours after inoculation, the fluorescence of NO in- cell adjacent to the stomata gradually intensified, and the NO fluorescent area also expanded continuously; the green fluorescence primarily occurred in the cells undergoing a hypersensitive response (HR) at 24–72 h after inoculation. For the compatible combination, however, a small amount of green fluorescence was observed in stomata where the pathogenic contact occurred at 4 h after inoculation, and fluorescence was not observed thereafter. Injections of the NO scavenger c-PTIO prior to inoculation postponed the onset of NO production to 48 h after inoculation and suppressed HR advancement. The injection of imidazole, a NADPH oxidase inhibitor, or EGTA, an extracellular calcium chelator, in the leaves prior to inoculation, delayed the onset of NO production in the incompatible combination and suppressed HR advancement. Combined with our previous results, it could be concluded that, Ca2+ and hydrogen peroxide (H2O2) are involved in upstream of NO production to induce the HR cell death during P. triticina infection, and Ca2+, NO and H2O2 are jointly involved in the signal transduction process of HR in the interaction system.

In vitro induction of tetraploids in Vitis × Muscadinia hybrids

A protocol for in vitro induction of tetraploids of the grape rootstock 101-14 Millardet et de Grasset (Vitis riparia × V. rupestris) (101-14 Mgt) (2n = 2x = 38) × Muscadinia rotundifolia cv. Trayshed (2n = 2x = 40) hybrids was established. Different explant materials were exposed to the antimitotic chemical agents colchicine and oryzalin, at three concentrations, for 24, 48 and 72 h. Treated in vitro shoot tips, anthers and pre-embryogenic cultures were regenerated, and their ploidy levels were confirmed by chromosome counts and flow cytometry. It is generally considered that anatomical characteristics of leaves, including leaf stomatal size and number of chloroplasts in guard cells, correlate with ploidy levels of grape plants. Chromosome counts and flow cytometry are labor intensive and difficult, while the number of chloroplasts in stomatal guard cells and stomatal size are easily measured and can be stable and reliable markers. Analysis of stomata revealed clear differences between regenerated diploid and tetraploid plants, and these measurements were used as a preliminary screen, followed by chromosome counts and flow cytometry for confirmation. This study presents the results obtained by various combinations of explants, antimitotic agents, exposure time, concentration and ploidy analysis to evaluate in vitro chromosome doubling. These results provide an easy and effective method for production of tetraploid, potentially fertile Vitis × Muscadinia hybrids for utilization in rootstock breeding programs.

Genetic analysis of differences in stomatal guard cell lengths of bread wheat

Variation in stomatal guard cell length of parental cultivars and its inheritance in F 1 and F 2 hybrids have been studied after crossing between contrast genotypes of winter wheat (Triticum aestivum L.). Analysis of F 2 populations has shown the action of three non-allelic genes in control of stomatal guard cell length of parental cultivars

The guard cell metabolome: functions in stomatal movement and global food security.

Guard cells represent a unique single cell-type system for the study of cellular responses to abiotic and biotic perturbations that affect stomatal movement. Decades of effort through both classical physiological and functional genomics approaches have generated an enormous amount of information on the roles of individual metabolites in stomatal guard cell function and physiology. Recent application of metabolomics methods has produced a substantial amount of new information on metabolome control of stomatal movement. In conjunction with other “omics” approaches, the knowledge-base is growing to reach a systems-level description of this single cell-type. Here we summarize current knowledge of the guard cell metabolome and highlight critical metabolites that bear significant impact on future engineering and breeding efforts to generate plants/crops that are resistant to environmental challenges and produce high yield and quality products for food and energy security.

Arabidopsis CALCIUM-DEPENDENT PROTEIN KINASE8 and CATALASE3 Function in Abscisic Acid-Mediated Signaling and H2O2 Homeostasis in Stomatal Guard Cells under Drought Stress.

Drought is a major threat to plant growth and crop productivity. Calcium-dependent protein kinases (CDPKs, CPKs) are believed to play important roles in plant responses to drought stress. Here, we report that Arabidopsis thaliana CPK8 functions in abscisic acid (ABA)- and Ca(2+)-mediated plant responses to drought stress. The cpk8 mutant was more sensitive to drought stress than wild-type plants, while the transgenic plants overexpressing CPK8 showed enhanced tolerance to drought stress compared with wild-type plants. ABA-, H2O2-, and Ca(2+)-induced stomatal closing were impaired in cpk8 mutants. Arabidopsis CATALASE3 (CAT3) was identified as a CPK8-interacting protein, confirmed by yeast two-hybrid, coimmunoprecipitation, and bimolecular fluorescence complementation assays. CPK8 can phosphorylate CAT3 at Ser-261 and regulate its activity. Both cpk8 and cat3 plants showed lower catalase activity and higher accumulation of H2O2 compared with wild-type plants. The cat3 mutant displayed a similar drought stress-sensitive phenotype as cpk8 mutant. Moreover, ABA and Ca(2+) inhibition of inward K(+) currents were diminished in guard cells of cpk8 and cat3 mutants. Together, these results demonstrated that CPK8 functions in ABA-mediated stomatal regulation in responses to drought stress through regulation of CAT3 activity.

Stomatal Guard Cells Co-opted an Ancient ABA-Dependent Desiccation Survival System to Regulate Stomatal Closure

During the transition from water to land, plants had to cope with the loss of water through transpiration, the inevitable result of photosynthetic CO2 fixation on land [1, 2]. Control of transpiration became possible through the development of a new cell type: guard cells, which form stomata. In vascular plants, stomatal regulation is mediated by the stress hormone ABA, which triggers the opening of the SnR kinase OST1-activated anion channel SLAC1 [3, 4]. To understand the evolution of this regulatory circuit, we cloned both ABA-signaling elements, SLAC1 and OST1, from a charophyte alga, a liverwort, and a moss, and functionally analyzed the channel-kinase interactions. We were able to show that the emergence of stomata in the last common ancestor of mosses and vascular plants coincided with the origin of SLAC1-type channels capable of using the ancient ABA drought signaling kinase OST1 for regulation of stomatal closure.

Industrial dust sulphate and its effects on biochemical and morphological characteristics of Morus (Morus alba) plant in NCR Delhi.

Abundance of CaCO3 rich soil dust is a typical feature of atmospheric environment in the Indian region. During prevailing dry weather conditions, dustfall is deposited onto the foliar surfaces of plant affecting their morphology, stomata and the levels of biochemical constituents. This study reports the chemical characteristics of dustfall, its effect on foliar morphology and biochemical constituents of a medicinal plant (Morus alba) at two sites which are differentiated on the basis of landuse pattern, viz., (i) residential, Jawaharlal Nehru University (JNU), and (ii) industrial, Sahibabad (SB), located in the National Capital Region (NCR) of Delhi. Dustfall was characterized for major anions (F(-), Cl(-), NO3 (-) and SO4 (--)) and cations (Na(+), NH4 (+), K(+), Mg(++) and Ca(++)). Biochemical parameters such as chlorophyll a, chlorophyll b, total chlorophyll, carotenoid, proline and ascorbic acid were determined in foliar samples. The results showed that the dustfall fluxes of all the major ions were found to be higher at the industrial site (SB) as compared to the residential site (JNU). Foliar analysis revealed that the levels of biochemical parameters were more affected at SB site due to higher levels of dust SO4 (--) contributed by various anthropogenic sources resulting in more stressful conditions affecting the biochemistry of the plant. The possible entry pathways for dust SO4 (--) into foliar cells are also discussed in the paper. It was noticed that the deposition of urban dust was responsible for the damage of trichome, epidermis, cuticle and stomatal guard cells significantly affecting foliar morphology. SB exhibited more damage to these morphological parts suggesting that industrial dust is harmful to the plants.

Extracellular ATP mediates H2S-regulated stomatal movements and guard cell K+ current in a H2O2-dependent manner in Arabidopsis

Hydrogen sulfide (H2S) is a newly discovered gaseous signaling molecule and involved in ethylene and ABA-induced stomatal closure. As an important factor, extracellular ATP (eATP) was believed to participate in regulation of stomatal closing. However, the mechanism by which eATP mediates H2S-regulated stomatal closure remains unclear. Here, we employed Arabidopsis wild-type and mutant lines of ATP-binding cassette transporters (Atmrp4, Atmrp5 and their double mutant Atmrp4/5) to study the function of eATP in H2S-regulated stomatal movement. Our results indicated that H2S affected stomatal closing through stimulating guard cell outward K+ current. Moreover, we found that H2S induced eATP generation by regulating the activity of an ABC transporter. The inhibitor of ABC transporters, glibenclamide (Gli), could impair H2S-regulated stomatal closure and reduce H2S-dependent eATP accumulation in Atmrp4 and Atmrp5 mutants. In addition, the promotion effect of H2S on outward K+ currents was diminished in Atmrp4/5 double mutant. Our data suggested that hydrogen peroxide (H2O2) is required for H2S-induced stomatal closure, and the production of H2O2 is regulated by eATP via NADPH oxidase. Based on this work, we conclude that H2S-induced stomatal closure requires ABC transporter-dependent eATP production and subsequent NADPH oxidase-dependent H2O2 accumulation.

A molecular pathway for CO₂ response in Arabidopsis guard cells.

Increasing carbon dioxide (CO₂) levels in the atmosphere have caused global metabolic changes in diverse plant species. CO₂ is not only a carbon donor for photosynthesis but also an environmental signal that regulates stomatal movements and thereby controls plant-water relationships and carbon metabolism. However, the mechanism underlying CO₂ sensing in stomatal guard cells remains unclear. Here we report characterization of Arabidopsis RESISTANT TO HIGH CO₂ (RHC1), a MATE-type transporter that links elevated CO₂ concentration to repression of HT1, a protein kinase that negatively regulates CO₂-induced stomatal closing. We also show that HT1 phosphorylates and inactivates OST1, a kinase which is essential for the activation of the SLAC1 anion channel and stomatal closing. Combining genetic, biochemical and electrophysiological evidence, we reconstituted the molecular relay from CO₂ to SLAC1 activation, thus establishing a core pathway for CO₂ signalling in plant guard cells.

Decreased photosynthesis in the erect panicle 3 (ep3) mutant of rice is associated with reduced stomatal conductance and attenuated guard cell development.

The ERECT PANICLE 3 gene of rice encodes a peptide that exhibits more than 50% sequence identity with the Arabidopsis F-box protein HAWAIIAN SKIRT (HWS). Ectopic expression of the Os02g15950 coding sequence, driven by the HWS (At3g61950) promoter, rescued the hws-1 flower phenotype in Arabidopsis confirming that EP3 is a functional orthologue of HWS. In addition to displaying an erect inflorescence phenotype, loss-of-function mutants of Os02g15950 exhibited a decrease in leaf photosynthetic capacity and stomatal conductance. Analysis of a range of physiological and anatomical features related to leaf photosynthesis revealed no alteration in Rubisco content and no notable changes in mesophyll size or arrangement. However, both ep3 mutant plants and transgenic lines that have a T-DNA insertion within the Os02g15950 (EP3) gene exhibit smaller stomatal guard cells compared with their wild-type controls. This anatomical characteristic may account for the observed decrease in leaf photosynthesis and provides evidence that EP3 plays a role in regulating stomatal guard cell development.

A Protocol to Measure the Cytoplasmic Calcium in Arabidopsis Guard Cells

Cytoplasmic calcium ([Ca2+]cyt) acts as a stimulus-induced second messenger in multiple signal transduction cascades (Allen et al., 1999). In plant cells, a dramatic and readily assayed response to stimulus is the change of stomatal aperture. Changes in [Ca2+]cyt of stomatal guard cells were involved in stomatal movement in response to various stimuli and cellular processes. In general, there are two available ways to measure [Ca2+]cyt in guard cells, i.e., loading of calcium-sensitive fluorescence dyes such as fluo-3 AM and fura-2 or expressing genetically encoded calcium indicators such as yellow cameleon (Krebs et al., 2012). In this protocol, we aim at describing the experimental procedure to record [Ca2+]cyt fluctuation in guard cells with loading of fluo-3 AM upon ABA or PA treatment combining with fluorescence imaging performed with confocal laser scanning microscope.

Arabidopsis reduces growth under osmotic stress by decreasing SPEECHLESS protein.

Plants, which are sessile unlike most animals, have evolved a system to reduce growth under stress; however, the molecular mechanisms of this stress response are not well known. During programmed development, a fraction of the leaf epidermal precursor cells become meristemoid mother cells (MMCs), which are stem cells that produce both stomatal guard cells and epidermal pavement cells. Here we report that Arabidopsis plants, in response to osmotic stress, post-transcriptionally decrease the protein level of SPEECHLESS, the transcription factor promoting MMC identity, through the action of a mitogen-activated protein kinase (MAPK) cascade. The growth reduction under osmotic stress was lessened by inhibition of the MAPK cascade or by a mutation that disrupted the MAPK target amino acids in SPEECHLESS, indicating that Arabidopsis reduces growth under stress by integrating the osmotic stress signal into the MAPK-SPEECHLESS core developmental pathway.

Tomato-Pseudomonas syringae interactions under elevated CO₂ concentration: the role of stomata.

Increasing atmospheric CO₂ concentrations ([CO₂]) in agricultural and natural ecosystems is known to reduce plant stomatal opening, but it is unclear whether these CO₂-induced stomatal alterations are associated with foliar pathogen infections. In this study, tomato plants were grown under ambient and elevated [CO₂] and inoculated with Pseudomonas syringae pv. tomato strain DC3000, a strain that is virulent on tomato plants. We found that elevated [CO₂] enhanced tomato defence against P. syringae. Scanning electron microscopy analysis revealed that stomatal aperture of elevated [CO₂] plants was considerably smaller than their ambient counterparts, which affected the behaviour of P. syringae bacteria on the upper surface of epidermal peels. Pharmacological experiments revealed that nitric oxide (NO) played a role in elevated [CO₂]-induced stomatal closure. Silencing key genes involved in NO generation and stomatal closing, nitrate reductase (NR) and guard cell slow-type anion channel 1 (SLAC1), blocked elevated [CO₂]-induced stomatal closure and resulted in significant increases in P. syringae infection. However, the SLAC1-silenced plants, but not the NR-silenced plants, still had significantly higher defence under elevated [CO₂] compared with plants treated with ambient [CO₂]. Similar results were obtained when the stomata-limiting factor for P. syringae entry was excluded by syringe infiltration inoculation. These results indicate that elevated [CO₂] induces defence against P. syringae in tomato plants, not only by reducing the stomata-mediated entry of P. syringae but also by invoking a stomata-independent pathway to counteract P. syringae. This information is valuable for designing proper strategies against bacterial pathogens under changing agricultural and natural ecosystems.

A RELATIONSHIP BETWEEN PLOIDY LEVEL AND THE NUMBER OF CHLOROPLASTS IN STOMATAL GUARD CELLS IN DIPLOID AND AMPHIDIPLOID Brassica SPECIES

Doubled haploid lines production through isolated anthers and isolated microspore cultures has been used widely for genetic studies and plant breeding of Brassica crops. The ploidy level of microspore derived plants varies, and normally haploid, diploid and mixoploid plants could be obtained in vitro. The determination of ploidy level is essential in doubled haploid pure line production. The determination of ploidy level by counting the number of chloroplast in stomatal guard cells (NCSGC) is less time consuming, laborious and expensive comparing to chromosome counting in root tip cells or mother pollen cells and flow cytometry methods. Several studies have been reported concerning relationship between ploidy level and number of chloroplast in stomatal guard cells of Brassica rapa, B. napus and B. oleracea species, however a small number of genotypes had been analyzed. In our study, the NCSGCs of haploid (n) and diploid (2n) Chinese cabbage (B. rapa ssp. pekinensis), winter oilseed rape (B. napus var. napus) and haploid, di- and tetraploid white cabbage (B. oleracea var. capitata) microspore derived plants were estimated, and also the influence of plant growth temperature (6±2 °N e 24±2 °N) and development stage (vegetative or generative) was investigated. High correlation between the ploidy level of microspore-derived plants and NCSGC is found for white cabbage (Brassica oleracea, r = 0.94), Chinese cabbage (B. rapa, r = 0.90) and oilseed rape (B. napus, r = 0.94). The chloroplast average number in stomatal guard cells was very similar among the same ploidy genotypes of Chinese cabbage as well as rapeseed, while the variation of chloroplast number in diploid and tetraploid white cabbage plants was significant. In a range of early, middle and late maturing diploid white cabbage inbred lines there was established the tendency to form more chloroplasts in the early lines (Pl, Sus and others) and less in the late lines (AM2, Sa1, Ges2 and others), with the difference up to 1.7 times, that is comparable to the difference between haploid and diploid plants of Chinese cabbage or rapeseed. The chloroplast number in stomatal guard cells is 4.2-7.8 and 7.9-13.6 for Chinese cabbage (B. rapa) haploids and diploids, respectively, 7.5-12.4 and 14.1-20.3 for rapeseed (B. napus) amphihaploids and amphidiploid, respectively, and 7.7-9.9, 11.7-17.9 and 18.0-26.5 for white cabbage (B. oleracea) haploids, diploids and tetraploids, respectively. No significant influence of vegetative or generative stage of plant development or growth temperature on NCSGC was found.

<i>In Vitro</i> Induction and Identification of Tetraploid in Watermelon [<i>Citrullus lanatus</i> (Thunb.) Matsum and Nakai

This research induced tetraploid watermelon through tissue culture. The cotyledons of a diploid mini-watermelon (A7) were treated with different concentrations of colchicine on medium for different time. The autotetraploid plants were identified basing on morphology, determination of the number of chloroplasts in stomatal guard cells and flow cytometry analysis. A stable autotetraploid material was observed. The results showed that tetraploid watermelons could be obtained under different treatments, and the highest tetraploid induction rate was up to 25 %. The most effective way was cutting the proximal cotyledons at the 7th day after sowing, then explants were cultivated on MS medium with 0.1 %(w/v) colchicine for 72 h, the adventitious shoot induction rate was 62.5 %, and multiplication coefficient was 3.6.

Hydrogen sulfide generated by L-cysteine desulfhydrase acts upstream of nitric oxide to modulate abscisic acid-dependent stomatal closure.

Abscisic acid (ABA) is a well-studied regulator of stomatal movement. Hydrogen sulfide (H2S), a small signaling gas molecule involved in key physiological processes in mammals, has been recently reported as a new component of the ABA signaling network in stomatal guard cells. In Arabidopsis (Arabidopsis thaliana), H2S is enzymatically produced in the cytosol through the activity of l-cysteine desulfhydrase (DES1). In this work, we used DES1 knockout Arabidopsis mutant plants (des1) to study the participation of DES1 in the cross talk between H2S and nitric oxide (NO) in the ABA-dependent signaling network in guard cells. The results show that ABA did not close the stomata in isolated epidermal strips of des1 mutants, an effect that was restored by the application of exogenous H2S. Quantitative reverse transcription polymerase chain reaction analysis demonstrated that ABA induces DES1 expression in guard cell-enriched RNA extracts from wild-type Arabidopsis plants. Furthermore, stomata from isolated epidermal strips of Arabidopsis ABA receptor mutant pyrabactin-resistant1 (pyr1)/pyrabactin-like1 (pyl1)/pyl2/pyl4 close in response to exogenous H2S, suggesting that this gasotransmitter is acting downstream, although acting independently of the ABA receptor cannot be ruled out with this data. However, the Arabidopsis clade-A PROTEIN PHOSPHATASE2C mutant abscisic acid-insensitive1 (abi1-1) does not close the stomata when epidermal strips were treated with H2S, suggesting that H2S required a functional ABI1. Further studies to unravel the cross talk between H2S and NO indicate that (1) H2S promotes NO production, (2) DES1 is required for ABA-dependent NO production, and (3) NO is downstream of H2S in ABA-induced stomatal closure. Altogether, data indicate that DES1 is a unique component of ABA signaling in guard cells.

Cytosolic calcium signals elicited by the pathogen-associated molecular pattern flg22 in stomatal guard cells are of an oscillatory nature.

Changes in cytosolic free calcium ([Ca(2+)]cyt) are an early and essential element of signalling networks activated by the perception of pathogen-associated molecular patterns (PAMPs), such as flg22. The flg22-induced calcium signal has been described on whole-plant, but not on single-cell scale so far. Also, the Ca(2+) sources and channels contributing to its generation are still obscure. Ratiometric fluorescence imaging employing the calcium reporter Yellow Cameleon 3.6 was performed to analyse the flg22-induced calcium signature in single guard cells of Arabidopsis thaliana. Calcium stores and channel types involved in its generation were determined by a pharmacological approach. In contrast to the calcium signal determined on whole-plant level, the signature on single-cell level is not characterized by one sustained response, but by oscillations in [Ca(2+)]cyt. These oscillations were abolished by EGTA and lanthanum, as well as by U73122, neomycin and TMB-8, but only partially or not at all affected by inhibitors of glutamate receptor-like channels and cyclic nucleotide-gated channels. Our analyses suggest that the response observed on whole-plant level is the summary of oscillations occurring in single cells. Parallel to external calcium, influx via channels located at internal stores contributes to the signal.