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The role of the mesophyll in stomatal functioning in thin amphistomatous leaves was investigated by altering gas exchange for one surface and observing the effects on stomatal conductance for the other surface. Three methods of perturbing gas exchange on the adaxial surface were used. First, gas exchange for the adaxial surface was completely blocked with plastic wrap or vacuum grease. Second, leaves were inverted to induce closure of the adaxial stomata. And third, ambient humidity for the adaxial surface was reduced to induce stomatal closure on that surface. Experiments were performed at low light intensity and three different CO2 concentrations to test the idea that stomatal responses in thin amphistomatous leaves are partially controlled by a signal from the mesophyll that varies with light and CO2. In general, stomata on the abaxial surface opened when gas exchange on the adaxial surface was reduced, with the largest increases in conductance occurring at high CO2 concentration. The data are discussed with respect to role of a purported signal from the mesophyll and the partitioning of that signal between the two surfaces of the leaf. Thin amphistomatous leaves must regulate abaxial and adaxial stomatal conductances such that the total stomatal conductance for the leaf is appropriate for the prevailing environmental conditions. This manuscript investigates the possible role of a signal from the mesophyll in coordinating adaxial and abaxial stomatal conductances. [ABSTRACT FROM AUTHOR]

As plants evolved to function on land, they developed stomata for effective gas exchange, for photosynthesis and for controlling water loss. We have recently shown that sugars, as the end product of photosynthesis, close the stomata of various angiosperm species, to coordinate sugar production with water loss. In the current study, we examined the sugar responses of the stomata of phylogenetically different plant species and species that employ different photosynthetic mechanisms (i.e., C3, C4 and CAM). To examine the effect of sucrose on stomata, we treated leaves with sucrose and then measured their stomatal apertures. Sucrose reduced stomatal aperture, as compared to an osmotic control, suggesting that regulation of stomata by sugars is a trait that evolved early in evolutionary history and has been conserved across different groups of plants. [ABSTRACT FROM AUTHOR]

INDUCER OF CBF EXPRESSION 1 (ICE1) encodes a MYC-like basic helix-loop-helix (bHLH) transcription factor playing a critical role in plant responses to chilling and freezing stresses and leaf stomata development. However, no information connecting ICE1 and reproductive development has been reported. In this study, we show that ICE1 controls plant male fertility via impacting anther dehydration. The loss-of-function mutation in ICE1 gene in Arabidopsis caused anther indehiscence and decreased pollen viability as well as germination rate. Further analysis revealed that the anthers in the mutant of ICE1 (ice1-2) had the structure of stomium, though the epidermis did not shrink to dehisce. The anther indehiscence and influenced pollen viability as well as germination in ice1-2 were due to abnormal anther dehydration, for most of anthers dehisced with drought treatment and pollen grains from those dehydrated anthers had similar viability and germination rates compared with wild type. Accordingly, the sterility of ice1-2 could be rescued by ambient dehydration treatments. Likewise, the stomatal differentiation of ice1-2 anther epidermis was disrupted in a different manner compared with that in leaves. ICE1 specifically bound to MYC-recognition elements in the promoter of FAMA, a key regulator of guard cell differentiation, to activate FAMA expression. Transcriptome profiling in the anther tissues further exhibited ICE1-modulated genes associated with water transport and ion exchange in the anther. Together, this work reveals the key role of ICE1 in male fertility control and establishes a regulatory network mediated by ICE1 for stomata development and water movement in the anther. [ABSTRACT FROM AUTHOR]

Stomata, microscopic pores in leaf surfaces through which water loss and carbon dioxide uptake occur, are closed in response to drought by the phytohormone abscisic acid (ABA). This process is vital for drought tolerance and has been the topic of extensive experimental investigation in the last decades. Although a core signaling chain has been elucidated consisting of ABA binding to receptors, which alleviates negative regulation by protein phosphatases 2C (PP2Cs) of the protein kinase OPEN STOMATA 1 (OST1) and ultimately results in activation of anion channels, osmotic water loss, and stomatal closure, over 70 additional components have been identified, yet their relationships with each other and the core components are poorly elucidated. We integrated and processed hundreds of disparate observations regarding ABA signal transduction responses underlying stomatal closure into a network of 84 nodes and 156 edges and, as a result, established those relationships, including identification of a 36-node, strongly connected (feedback-rich) component as well as its in- and out-components. The network’s domination by a feedback-rich component may reflect a general feature of rapid signaling events. We developed a discrete dynamic model of this network and elucidated the effects of ABA plus knockout or constitutive activity of 79 nodes on both the outcome of the system (closure) and the status of all internal nodes. The model, with more than 1024 system states, is far from fully determined by the available data, yet model results agree with existing experiments in 82 cases and disagree in only 17 cases, a validation rate of 75%. Our results reveal nodes that could be engineered to impact stomatal closure in a controlled fashion and also provide over 140 novel predictions for which experimental data are currently lacking. Noting the paucity of wet-bench data regarding combinatorial effects of ABA and internal node activation, we experimentally confirmed several predictions of the model with regard to reactive oxygen species, cytosolic Ca2+ (Ca2+c), and heterotrimeric G-protein signaling. We analyzed dynamics-determining positive and negative feedback loops, thereby elucidating the attractor (dynamic behavior) repertoire of the system and the groups of nodes that determine each attractor. Based on this analysis, we predict the likely presence of a previously unrecognized feedback mechanism dependent on Ca2+c. This mechanism would provide model agreement with 10 additional experimental observations, for a validation rate of 85%. Our research underscores the importance of feedback regulation in generating robust and adaptable biological responses. The high validation rate of our model illustrates the advantages of discrete dynamic modeling for complex, nonlinear systems common in biology. [ABSTRACT FROM AUTHOR]

Stomatal closure is affected by various stimuli such as light, atmospheric carbon dioxide concentration, humidity and phytohormones. Our research focuses on phytohormones, specifically: abscisic acid (ABA), ethylene (ET) and methyl jasmonate (MeJA) that are responsible for the regulation of several plant processes, especially in guard cell signalling. While several studies show that these three phytohormones cause stomatal closure in plants, only two studies are notable for establishing a mathematical model of guard cell signalling involving phytohormones. Those two studies employed Boolean modelling and mechanistic ordinary differential equations modelling. In this study, we propose a new mathematical model of guard cell transduction network for stomatal closure using continuous logical modelling framework. Results showed how the different components of the network function. Furthermore, the model verified the role of antioxidants in the closure mechanism, and the diminished closure level of stomata with combined ABA-ET stimulus. The analysis was extended to ABA-ET-MeJA crosstalk. [ABSTRACT FROM AUTHOR]

Plant gas exchange is regulated by guard cells that form stomatal pores. Stomatal adjustments are crucial for plant survival; they regulate uptake of CO2 for photosynthesis, loss of water, and entrance of air pollutants such as ozone. We mapped ozone hypersensitivity, more open stomata, and stomatal CO2-insensitivity phenotypes of the Arabidopsis thaliana accession Cvi-0 to a single amino acid substitution in MITOGEN-ACTIVATED PROTEIN (MAP) KINASE 12 (MPK12). In parallel, we showed that stomatal CO2-insensitivity phenotypes of a mutant cis (CO2-insensitive) were caused by a deletion of MPK12. Lack of MPK12 impaired bicarbonate-induced activation of S-type anion channels. We demonstrated that MPK12 interacted with the protein kinase HIGH LEAF TEMPERATURE 1 (HT1)—a central node in guard cell CO2 signaling—and that MPK12 functions as an inhibitor of HT1. These data provide a new function for plant MPKs as protein kinase inhibitors and suggest a mechanism through which guard cell CO2 signaling controls plant water management. [ABSTRACT FROM AUTHOR]

There are two major methodical approaches with which changes of status in stomatal pores are addressed: indirectly by measurement of leaf transpiration, and directly by measurement of stomatal apertures. Application of the former method requires special equipment, whereas microscopic images are utilized for the direct measurements. Due to obscure visualization of cell boundaries in intact leaves, a certain degree of invasive leaf manipulation is often required. Our aim was to develop a protocol based on the minimization of leaf manipulation and the reduction of analysis completion time, while still producing consistent results. We applied rhodamine 6G staining of Arabidopsis thaliana leaves for stomata visualization, which greatly simplifies the measurement of stomatal apertures. By using this staining protocol, we successfully conducted analyses of stomatal responses in Arabidopsis leaves to both closure and opening stimuli. We performed long-term monitoring of living stomata and were able to document the same leaf before and after treatment. Moreover, we developed a protocol for rapid-fixation of epidermal peels, which enables high throughput data analysis. The described method allows analysis of stomatal apertures with minimal leaf manipulation and usage of the same leaf for sequential measurements, and will facilitate the analysis of several lines in parallel. [ABSTRACT FROM AUTHOR]

Historically viewed as primarily functioning in the regulation of gas and water vapor exchange, it is now evident that stomata serve an important role in plant immunity. Indeed, in addition to classically defined functions related to cell architecture and movement, the actin cytoskeleton has emerged as a central component of the plant immune system, underpinning not only processes related to cell shape and movement, but also receptor activation and signaling. Using high resolution quantitative imaging techniques, the temporal and spatial changes in the actin microfilament array during diurnal cycling of stomatal guard cells has revealed a highly orchestrated transition from random arrays to ordered bundled filaments. While recent studies have demonstrated that plant stomata close in response to pathogen infection, an evaluation of stimulus-induced changes in actin cytoskeletal dynamics during immune activation in the guard cell, as well as the relationship of these changes to the function of the actin cytoskeleton and stomatal aperture, remains undefined. In the current study, we employed quantitative cell imaging and hierarchical clustering analyses to define the response of the guard cell actin cytoskeleton to pathogen infection and the elicitation of immune signaling. Using this approach, we demonstrate that stomatal-localized actin filaments respond rapidly, and specifically, to both bacterial phytopathogens and purified pathogen elicitors. Notably, we demonstrate that higher order temporal and spatial changes in the filament array show distinct patterns of organization during immune activation, and that changes in the naïve diurnal oscillations of guard cell actin filaments are perturbed by pathogens, and that these changes parallel pathogen-induced stomatal gating. The data presented herein demonstrate the application of a highly tractable and quantifiable method to assign transitions in actin filament organization to the activation of immune signaling in plants. [ABSTRACT FROM AUTHOR]

In the present study, the protective effect of nitric oxide (NO) against Cd-induced structural disturbances in pea (Pisum sativum) leaves was investigated. Cadmium treatment resulted in a decreased leaf size and thickness of the lamina, reduced intercellular spaces in the mesophyll, small pavement cells, and a high density of stomata. These abnormalities were partially or fully reversed by a simultaneous application of Cd and the NO donor, sodium nitroprusside (SNP). The concentration of 1000 µM SNP was very effective in counteracting the adverse effects of Cd and resulted in leaf structural parameters close to those of the control leaves. These findings suggest that exogenous NO can effectively facilitate structural adjustments in pea leaves under Cd stress, which could improve stress tolerance at the whole-plant level. [ABSTRACT FROM AUTHOR]

The effect of Ca2+ on programmed death of guard cells (GC) and epidermal cells (EC) determined from destruction of the cell nucleus was investigated in epidermis of pea leaves. Ca2+ at concentrations of 1–100 μM increased and at a concentration of 1 mM prevented the CN—induced destruction of the nucleus in GC, disrupting the permeability barrier of GC plasma membrane for propidium iodide (PI). Ca2+ at concentrations of 0.1–1 mM enhanced drastically the number of EC nuclei stained by PI in epidermis treated with chitosan, an inducer of programmed cell death. The internucleosomal DNA fragmentation caused by CN− was suppressed by 2 mM Ca2+ on 6 h incubation, but fragmentation was stimulated on more prolonged treatment (16 h). Presumably, the disruption of the permeability barrier of plasma membrane for PI is not a sign of necrosis in plant cells. Quinacrine and diphenylene iodonium at 50 μM concentration prevented GC death induced by CN− or CN− + 0.1 mM Ca2+ but had no influence on respiration and photosynthetic O2 evolution in pea leaf slices. The generation of reactive oxygen species determined from 2′,7′-dichlorofluorescein fluorescence was promoted by Ca2+ in epidermal peels from pea leaves. [ABSTRACT FROM AUTHOR]

The mechanisms by which stomata respond to red light and CO2 are unknown, but much of the current literature assumes that these mechanisms reside wholly within the guard cells. However, responses of guard cells in isolated epidermes are typically much smaller than those in leaves, and there are several lines of evidence in the literature suggesting that the mesophyll is necessary for these responses in leaves. This paper advances the opinion that although guard cells may have small direct responses to red light and CO2, most of the stomatal response to these factors in leaves is caused by an unknown signal that originates in the mesophyll. [ABSTRACT FROM AUTHOR]

Stomatal responses to light and CO2 were investigated using isolated epidermes of Tradescantia pallida, Vicia faba and Pisum sativum. Stomata in leaves of T. pallida and P. sativum responded to light and CO2, but those from V. faba did not. Stomata in isolated epidermes of all three species could be opened on KCl solutions, but they showed no response to light or CO2. However, when isolated epidermes of T. pallida and P. sativum were placed on an exposed mesophyll from a leaf of the same species or a different species, they regained responsiveness to light and CO2. Stomatal responses in these epidermes were similar to those in leaves in that they responded rapidly and reversibly to changes in light and CO2. Epidermes from V. faba did not respond to light or CO2 when placed on mesophyll from any of the three species. Experiments with single optic fibres suggest that stomata were being regulated via signals from the mesophyll produced in response to light and CO2 rather than being sensitized to light and CO2 by the mesophyll. The data suggest that most of the stomatal response to CO2 and light occurs in response to a signal generated by the mesophyll. [ABSTRACT FROM AUTHOR]

Stomatal responses to light and CO2 were investigated using isolated epidermes of Tradescantia pallida, Vicia faba and Pisum sativum. Stomata in leaves of T. pallida and P. sativum responded to light and CO2, but those from V. faba did not. Stomata in isolated epidermes of all three species could be opened on KCl solutions, but they showed no response to light or CO2. However, when isolated epidermes of T. pallida and P. sativum were placed on an exposed mesophyll from a leaf of the same species or a different species, they regained responsiveness to light and CO2. Stomatal responses in these epidermes were similar to those in leaves in that they responded rapidly and reversibly to changes in light and CO2. Epidermes from V. faba did not respond to light or CO2 when placed on mesophyll from any of the three species. Experiments with single optic fibres suggest that stomata were being regulated via signals from the mesophyll produced in response to light and CO2 rather than being sensitized to light and CO2 by the mesophyll. The data suggest that most of the stomatal response to CO2 and light occurs in response to a signal generated by the mesophyll. [ABSTRACT FROM AUTHOR]

Stomatal pores, each surrounded by a pair of guard cells, regulate CO2 uptake and water loss from leaves. Stomatal opening is driven by the accumulation of K+ salts and sugars in guard cells, which is mediated by electrogenic proton pumps in the plasma membrane and/or metabolic activity. Opening responses are achieved by coordination of light signaling, light-energy conversion, membrane ion transport, and metabolic activity in guard cells. In this review, we focus on recent progress in blue- and red-light-dependent stomatal opening. Because the blue-light response of stomata appears to be strongly affected by red light, we discuss underlying mechanisms in the interaction between blue-light signaling and guard cell chloroplasts. [ABSTRACT FROM AUTHOR]

In Mimosa pudica L., heat stimulation triggers leaflet folding in local, neighbouring and distant leaves. Stomatal movements were observed microscopically during this folding reaction and electrical potentials, chlorophyll fluorescence, and leaf CO2/H2O-gas exchange were measured simultaneously. Upon heat stimulation of a neighbouring pinna, epidermal cells depolarized and the stomata began a rapid and pronounced transient opening response, leading to an approximately 2-fold increase of stomatal aperture within 60 s. At the same time, net CO2 exchange showed a pronounced transient decrease, which was followed by a similar drop in photochemical quantum yield at photosystem (PS) II. Subsequently, CO2-gas exchange and photochemical quantum yield recovered and stomata closed partly or completely. The transient and fast stomatal opening response is interpreted as a hydropassive stomatal movement caused by a sudden loss of epidermal turgor. Thus, epidermal cells appear to respond in a similar manner to heat-induced signals as the pulvinar extensor cells. The subsequent closing of the stomata confirms earlier reports that stomatal movements can be induced by electrical signals. The substantial delay (several minutes) of guard cell turgor loss compared with the immediate response of the extensor and epidermal cells suggests a different, less direct mechanism for transmission of the propagating signal to the guard cells. [ABSTRACT FROM AUTHOR]

Phototropins (phot1 and phot2) are suggested to be multifunctional blue‐light (BL) receptors mediating phototropism, chloroplast movement, stomatal opening, and leaf expansion. The Arabidpsis phot1 phot2 double mutant lacks all of these responses. To confirm the requirement of phototropins in BL responses, the Arabidopsis phot1 phot2 double mutant was transformed with PHOT1 cDNA and the phenotypic restoration was analysed in the transformants. It was found that all BL responses were restored, although differentially, by the transformation of the Arabidopsis phot1 phot2 double mutant with PHOT1 cDNA. The results showed that phot1 was an essential component for all these BL responses in planta, and that the cellular level of phot1 might determine the individual BL responses. [ABSTRACT FROM PUBLISHER]

A rapid stomatal bioassay to test abscisic acid (ABA)-like inhibition of stomatal opening was elaborated. The assay was sensitive to low concentrations of ABA (>50% inhibition of opening at 0.02 μM (+)-S-ABA) and was insensitive to 20 μM (±)-S-ABA-1-(methyl ester), which is consistent with previous work. With the original intent of developing anti-idiotypic antibodies, we made conjugates of ABA at the 4'carbonyl because of its accessibility and known requirements for other positions of the ABA molecule in stomatal signal transduction. We found that these conjugates lacked ABA-like activity and did not interfere with (+)-S-ABA in the stomatal bioassay, implying the importance of the 4' carbonyl. These observations cast doubt on the feasibility of using anti-idiotypic antibodies produced through the use of 4'-substituted ABA for the isolation of a guard-cell ABA receptor. [ABSTRACT FROM AUTHOR]

Maize plants (Zea mays L. hybrid INRA 508) were placed under controlled conditions of light and CO2 partial pressure. The K+, Cl− and P contents were then determined by X‐ray microanalysis in the bulbous end of guard cells and in the center of subsidiary cells. The results were interpreted in connection with the stomatal conductance at the time of sampling. In normal air, the K+ and Cl− contents in guard cells only rose from a light threshold of about 300 μmol m−2 s−1 at which stomata were already largely open. At 600 μmol m−2 s−1, the K+ and Cl− levels in guard cells attained values that were 3‐ and 8‐fold greater, respectively, than the values observed in darkness. The K+ and Cl− contents in the subsidiary cells remained quite constant irrespective of the light conditions. CO2‐free air in darkness induced a significant K+ influx towards guard and subsidiary cells. Under light and in CO2‐free air, the K+ and Cl− contents dramatically increased in the guard cells, but slightly decreased in the subsidiary cells. Thus, when subjected to strong light in CO2‐free air, the K+ and Cl− contents in the subsidiary cells were approximately equal to those measured in normal air conditions. In the guard cells, stomatal opening was associated with a marked shift of the Cl−/K+ ratio – from 0.3 for closed stomata to ca 1 for fully open stomata. This could imply a slow change in the nature of the principal counterion accompanying K+ during stomatal opening. The content of P in guard cells appeared, in contrast to that of K+ and Cl−, to be practically independent of stomatal aperture.

The fluorescent probes 6-carboxyfluorescein and Lucifer yellow CH which do not pass the plasmalemma have been used to examine cell-to-cell communication in the leaf of Commelina cyanea, Dye movement from cell-to-cell occurs in epidermal, spongy and palisade mesophyll, and vascular cells. Dye movement between these tissues was also found. Hence, the epidermis, spongy and palisade mesophyll cells, and vascular tissue are all linked in a continuous symplast. However, dye injected into the epidermal cells rarely moves into guard cells, epidermal cells rarely moves into guard cells in Vicia faba and the C4 grass Anthephora pubescens also appeared to be isolated from epidermal cells. Thus guard cell isolation from cell-to-cell communication appears to be a common phenomenon. Hence, the ion fluxes required for guard cell function must occur via the apoplast. [ABSTRACT FROM AUTHOR]