Identification and Characterization of Compounds that Affect Stomatal Movements.

Regulation of stomatal aperture is essential for plant growth and survival in response to environmental stimuli. Opening of stomata induces uptake of CO2 for photosynthesis and transpiration, which enhances uptake of nutrients from roots. Light is the most important stimulus for stomatal opening. Under drought stress, the plant hormone ABA induces stomatal closure to prevent water loss. However, the molecular mechanisms of stomatal movements are not fully understood. In this study, we screened chemical libraries to identify compounds that affect stomatal movements in Commelina benghalensis and characterize the underlying molecular mechanisms. We identified nine stomatal closing compounds (SCL1-SCL9) that suppress light-induced stomatal opening by >50%, and two compounds (temsirolimus and CP-100356) that induce stomatal opening in the dark. Further investigations revealed that SCL1 and SCL2 had no effect on autophosphorylation of phototropin or the activity of the inward-rectifying plasma membrane (PM) K+ channel, KAT1, but suppressed blue light-induced phosphorylation of the penultimate residue, threonine, in PM H+-ATPase, which is a key enzyme for stomatal opening. SCL1 and SCL2 had no effect on ABA-dependent responses, including seed germination and expression of ABA-induced genes. These results suggest that SCL1 and SCL2 suppress light-induced stomatal opening at least in part by inhibiting blue light-induced activation of PM H+-ATPase, but not by the ABA signaling pathway. Interestingly, spraying leaves onto dicot and monocot plants with SCL1 suppressed wilting of leaves, indicating that inhibition of stomatal opening by these compounds confers tolerance to drought stress in plants.

Apoplastic mesophyll signals induce rapid stomatal responses to CO2 in Commelina communis.

Previous studies have suggested that the mesophyll contributes to stomatal CO(2) responses. The effects of changes in CO(2) concentration (100 or 700 ppm) on stomatal responses in red or white light were examined microscopically in a leaf segment, an epidermal strip and an epidermal strip placed on a mesophyll segment of Commelina communis, all mounted on a buffer-containing gel. In both red and white light, stomata of the leaf segment opened/closed rapidly at low/high CO(2). In red light, epidermal strip stomata barely responded to CO(2). In white light, they opened at low CO(2), but hardly closed at high CO(2). Stomata of the epidermal strip placed on the mesophyll responded in the same manner as those on the leaf segment. Insertion of a doughnut-shaped cellophane spacer (but not polyethylene spacer) between the epidermal strip and the mesophyll hardly altered these responses. Stomata in leaf segments treated with 3-(3,4-dichlorophenyl)-1,1-dimethylurea (DCMU), a photosynthesis inhibitor, did not open in red light, but opened/closed at low/high CO(2) in white light. These results indicate that the apoplast transfer of 'mesophyll signals' and the stomatal opening at low CO(2) are dependent on photosynthesis, whereas the stomatal closure at high CO(2) is independent of photosynthesis.

Differential role of ethylene and hydrogen peroxide in dark-induced stomatal closure.

Regulation of stomatal aperture is crucial in terrestrial plants for controlling water loss and gaseous exchange with environment. While much is known of signaling for stomatal opening induced by blue light and the role of hormones, little is known about the regulation of stomatal closing in darkness. The present study was aimed to verify their role in stomatal regulation in darkness. Epidermal peelings from the leaves of Commelina benghalensis were incubated in a defined medium in darkness for 1 h followed by a 1 h incubation in different test solutions [H2O2, propyl gallate, ethrel (ethylene), AgNO3, sodium orthovanadate, tetraethyl ammonium chloride, CaCl2, LaCl3, separately and in combination] before stomatal apertures were measured under the microscope. In the dark stomata remained closed under treatments with ethylene and propyl gallate but opened widely in the presence of H2O2 and AgNO3. The opening effect was largely unaffected by supplementing the treatment with Na-vanadate (PM H+ ATPase inhibitor) and tetraethyl ammonium chloride (K(+)-channel inhibitor) except that opening was significantly inhibited by the latter in presence of H2O2. On the other hand, H2O2 could not override the closing effect of ethylene at any concentrations while a marginal opening of stomata was found when Ag NO3 treatment was given together with propyl gallate. CaCl2 treatment opened stomata in the darkness while LaCl3 maintained stomata closed. A combination of LaCl3 and propyl gallate strongly promoted stomatal opening. A probable action of ethylene in closing stomata of Commelina benghalensis in dark has been proposed.

Glyphosate-induced structural variations in Commelina erecta L. (Commelinaceae).

The purpose of this study was to analyze the structural variations of Commelina erecta, a glyphosate-tolerant weed, produced by this herbicide. Regrowth shoots (vegetative cloning axes) and seedlings and two glyphosate concentrations: 900 and 1800 grams of acid equivalent per hectare (ga.e. ha⁻¹) were used. The following variations were observed: foliar shape and size changes, changes in the length of internodes, variations in the quantity of inflorescences, low seed production, and differences in the number of branches. After treatment, different phenotypic responses were observed as a result of differential sensitivity to glyphosate. Most of the vegetatively-propagated plants treated with 900 g a.e. ha⁻¹ revealed the same morphological structure as that of controls (i.e. three clearly identifiable zones: a basal or branching zone, a middle zone where branch production is inhibited, and an apical zone where only floral branches are produced). By contrast, the 900 ga.e. ha⁻¹ treated seedlings and the 1800 g a.e. ha⁻¹ treated regrowth shoots and seedlings showed two phenotypic responses, exhibiting differences in the quantity and location of branches and in the survival time of axes. The main variation is seen in branch production in the region that is usually inhibited. The different phenotypic responses and morphological changes suggest that C. erecta shows a very high intraspecific variability, which allows it to survive and thrive even in environments with intensive glyphosate application. These results underscore the need to solve the problem of tolerant weeds by means of an integrated approach including alternative management practices grounded on knowledge of the species and their behavior.

Effect of organic ligands on accumulation of copper in hyperaccumulator and nonaccumulator Commelina communis.

Better understanding of copper uptake and accumulation regulation in plants is critical to the phytoremediation of copper contaminated soil. This study employed a 30-day pot experiment to assess the relationship between organic ligands and copper accumulation in plants. Hyperaccumulator and nonaccumulator varieties of Commelina communis were used, different organic ligands were applied, and the data of copper accumulation in shoots were collected. The six organic ligands included ethylenediaminetetraacetic acid and organic acids (formic acid, citric acid, malic acid, tartaric acid, and succinic acid). The results showed that organic ligands added to culture increased the copper accumulation both varieties. The results of the copper accumulation in shoots agreed with the study of the root uptake kinetics of copper influx. The addition of organic acids could increase copper accumulation in shoots because the copper influx in roots was increased. The results also indicated that the copper influx of hyperaccumulator roots was higher than that of nonaccumulator roots. This is one of the mechanisms by which a hyperaccumulator could amass large amounts of copper in its shoots. In this accumulation process, little effect on the leaf relative water content was in the hyperaccumulator and nonaccumulator of leaves and normal physiological condition of plants.

Copper uptake and its effect on metal distribution in root growth zones of Commelina communis revealed by SRXRF.

To explore the copper uptake mechanisms by the Cu-tolerant plant Commelina communis, the contents of Cu and other metals (including Fe, Zn, and Mn) in roots were detected using atomic absorption spectrometer under transporter inhibitors, partial element deficiency, or Cu excess treatments, while distribution characters of Cu and other metals in root growth zones were investigated by synchrotron radiation X-ray fluorescence spectroscopy (SRXRF). Cu uptake was inhibited by the uncoupler DNP and P-type ATPase inhibitor Na(3)VO(4), not by the Ca(2+) ion channel inhibitor LaCl(3), suggesting that Cu could probably be assimilated actively by root and be related with P-type ATPase, but not through Ca(2+) ion channel. Fe or Zn deficiency could enhance Cu uptake, while 100 µM Cu inhibited Fe, Zn, and Mn accumulation in roots significantly. Metal distribution under 100 µM Cu treatment was investigated by SRXRF. High level of Cu was found in the root meristem, and higher Cu concentrations were observed in the vascular cylinder than those in the endodermis, further demonstrating the initiative Cu transport in the root of C. communis. Under excess Cu stress, most Fe was located in the epidermis, and Fe concentrations in the endodermis were higher than those in the vascular cylinder, suggesting Cu and Fe competition not only in the epidermal cells but also for the intercellular and intracellular transport in roots. Zn was present in the meristem and the vascular cylinder similar to Cu. Cu and Zn showed a similar pattern. Mn behaves as Zn does, but not like Fe.

Effects of fusicoccin on ion fluxes in guard cells.

The pharmacology has been further investigated of the two transport systems mediating potassium (rubidium) (K(+)(Rb(+))) release from the guard cell vacuole, responsible, respectively, for the resting efflux and abscisic acid (ABA)-induced transient stimulation of efflux, and for the transient stimulation induced by hypotonic treatment. Here, the effects of fusicoccin and of butyrate-induced cytoplasmic acidification on (86)Rb efflux were measured in isolated guard cells of Commelina communis. Fusicoccin (10 microM) inhibited the resting efflux at the tonoplast and the ABA-induced transient, but had no effect on the hypotonic transient. All three processes were inhibited by cytoplasmic acidification. Fusicoccin did not inhibit efflux at the plasmalemma. As the hypotonic response is inhibited by cytoplasmic acidification but not by fusicoccin, the effect of fusicoccin on the resting efflux and ABA response must be direct, and not the result of fusicoccin-induced cytoplasmic acidification. The collected tonoplast efflux properties resemble those of TPC1 (two-pore channel) rather than TPK1 (two-pore K channel). The flux and TPC1 are both activated by Ca(2+), but inhibited by phenylarsine oxide and by cytoplasmic acidification. The flux is inhibited by fusicoccin. TPC1 is inhibited by 14-3-3 proteins and has the C-terminal sequence STSDT, a type III binding site for 14-3-3 proteins, of the kind involved in fusicoccin binding.

Modification of leaf apoplastic pH in relation to stomatal sensitivity to root-sourced abscisic acid signals.

The confocal microscope was used to determine the pH of the leaf apoplast and the pH of microvolumes of xylem sap. We quantified variation in leaf apoplast and sap pH in relation to changes in edaphic and atmospheric conditions that impacted on stomatal sensitivity to a root-sourced abscisic acid signal. Several plant species showed significant changes in the pH of both xylem sap and the apoplast of the shoot in response to environmental perturbation. Xylem sap leaving the root was generally more acidic than sap in the midrib and the apoplast of the leaf. Increasing the transpiration rate of both intact plants and detached plant parts resulted in more acidic leaf apoplast pHs. Experiments with inhibitors suggested that protons are removed from xylem sap as it moves up the plant, thereby alkalinizing the sap. The more rapid the transpiration rate and the shorter the time that the sap resided in the xylem/apoplastic pathway, the smaller the impact of proton removal on sap pH. Sap pH of sunflower (Helianthus annuus) and Commelina communis did not change significantly as soil dried, while pH of tomato (Lycopersicon esculentum) sap increased as water availability in the soil declined. Increasing the availability of nitrate to roots also significantly alkalinized the xylem sap of tomato plants. This nitrogen treatment had the effect of enhancing the sensitivity of the stomatal response to soil drying. These responses were interpreted as an effect of nitrate addition on sap pH and closure of stomata via an abscisic acid-based mechanism.

Species-dependent changes in stomatal sensitivity to abscisic acid mediated by external pH.

The direct effects of pH changes and/or abscisic acid (ABA) on stomatal aperture were examined in epidermal strips of Commelina communis L. and Arabidopsis thaliana. Stomata were initially opened at pH 7 or pH 5. The stomatal closure induced by changes in external pH and/or ABA (10 microM or 10 nM) was monitored using video microscopy and quantified in terms of changes in stomatal area using image analysis software. Measurements of aperture area enabled stomatal responses and, in particular, small changes in stomatal area to be quantified reliably. Both plant species exhibited a biphasic closure response to ABA: an initial phase of rapid stomatal closure, followed by a second, more prolonged, phase during which stomata closure proceeded at a slower rate. Changes in stomatal sensitivity to ABA were also observed. Comparison of these effects between C. communis and A. thaliana demonstrate that this differential sensitivity of stomata to ABA is species-dependent, as well as being dependent on the pH of the extracellular environment.

The mutual effect of extracellular Ca2+, abscisic acid, and pH on the rate of stomatal closure.

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.

Isolation and stomatal opening activity of two oxylipins from Ipomoea tricolor.

Bioassay-guided fractionation of a 50% MeOH extract of Ipomoea tricolor enabled the isolation of two oxylipins, cis-12-oxophytodienoic acid (OPDA, 1) and a novel monogalactosylmonoacylglyceride (2) containing OPDA, that acted as inducers of stomatal opening. These oxylipins enhanced stomatal opening of Commelina communis in darkness at micromolar concentrations.

Chemical interactions of Brachiaria plantaginea with Commelina bengalensis and Acanthospermum hispidum in soybean cropping systems.

Previous results obtained in soybean-wheat rotations under no-tillage conditions showed reductions in the seedbank of the weed species Commelina benghalensis, but no alteration in the seedbank of Acanthospermum hispidum in areas infested with Brachiaria plantaginea. Analyses of the soluble fraction of B. plantaginea indicated the predominance of aconitic acid (AA) among the aliphatic acids and ferulic acid (FA) among the phenolic acids. Laboratory bioassays using C. benghalensis and A. hispidum were carried out to evaluate phytotoxic effects of pure organic acid solutions and dilute extracts of B. plantaginea on seed germination, root development, and fungal germination. Solutions of AA and FA were prepared at 0.25, 0.50, and 1.0 mM. Extracts of B. plantaginea were diluted to obtain concentrations of AA similar to those in the prepared solutions. Seeds were sown on 0.5% agar (containing AA, FA, or diluted extract) in plastic-covered receptacles and maintained in a germination chamber for 10 days. AA and FA solutions and the B. plantaginea extract reduced germination and root length, mainly of C. benghalensis. AA also stimulated the development of endophytic fungi (Fusarium solani), which had complementary adverse effects on C. benghalensis germination. FA and AA may play important roles in reducing the seedbank of some weed species, acting directly on germination and development and, indirectly, by stimulating endophytic fungi that alter germination.

The effects of manipulating phospholipase C on guard cell ABA-signalling.

Studies using stably transformed tobacco plants containing very low levels of PI-PLC in their guard cells show that this enzyme plays a role in the events associated with the inhibition of stomatal opening by ABA, but not in the cellular reactions that are responsible for ABA-induced stomatal closure. However, Commelina communis guard cells microinjected with the InsP3 antagonist, heparin, fail to close on addition of ABA. There are three possible explanations for this apparent data mismatch. The differences may be indicative of species-specific signalling pathways, the presence of a PI-PLC isoform(s) that is not down-regulated in these transgenic lines and/or they may reflect differences between short-term (acute) administration of an inhibitor and long-term (chronic) effects of gene manipulation. It is possible that the guard cell is a robust signalling system that is able to adapt or compensate for the chronic loss of PI-PLC, but which is unable to adjust quickly to acute loss of this component. It would be interesting to investigate this possibility further using either transient manipulation of gene expression or through the use of an inducible promoter.