Treatment with Commelina communis Extract Exerts Anti-inflammatory Effects in Murine Macrophages via Modulation of the Nuclear Factor-κB Pathway.

The incidence of severe inflammatory diseases caused by chronic inflammation has increased owing to unprecedented changes brought about by industrialization. In this study, we aimed to assess the effect of treatment of lipopolysaccharide- (LPS-) induced murine macrophages with Commelina communis Linne extract (CCE) on synthesis of nitric oxide (NO), hypersecretion of proinflammatory cytokines, intranuclear transition of the p65 subunit of nuclear factor- (NF-) κB, and degradation of the NF-κB inhibitor IκBα. Notably, CCE treatment did not affect cell viability even at a final concentration of 1.5 mg/mL. At a high concentration of CCE, the LPS-induced high levels of NO, tumor necrosis factor-α, interleukin- (IL-) 1ß, and IL-6 were decreased via downregulation of inducible NO synthase and proinflammatory cytokine mRNA expression. Furthermore, phosphorylation of IκBα was significantly decreased upon CCE treatment, and the intranuclear transition of NF-κB p65 triggered by LPS was inhibited at a high concentration of CCE. Polyphenols and flavonoids, secondary metabolites in CCE that regulate the NF-κB pathway, may be responsible for its anti-inflammatory activity. We suggest that CCE has anti-inflammatory effects related to suppression of the NF-κB pathway and can be used to treat chronic inflammation.

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.

Photoprotection of PSI by Far-Red Light Against the Fluctuating Light-Induced Photoinhibition in Arabidopsis thaliana and Field-Grown Plants.

It has been reported that PSI photoinhibition is induced even in wild-type plants of Arabidopsis thaliana, rice and other species by exposure of leaves to fluctuating light (FL) for a few hours. Because plants are exposed to FL in nature, they must possess protective mechanisms against the FL-induced photodamage. Here, using A. thaliana grown at various irradiances, we examined PSI photoprotection by far-red (FR) light at intensities comparable with those observed in nature. Dark-treated leaves were illuminated by red FL alternating high/low light at 1,200/30 µmol m-2 s-1 for 800 ms/10 s. By this FL treatment without FR light for 120 min, the level of photo-oxidizable P700 was decreased by 30% even in the plants grown at high irradiances. The addition of continuous FR light during the FL suppressed this damage almost completely. With FR light, P700 was kept in a more oxidized state in both low- and high-light phases. The protective effect of FR light was diminished more in mutants of the NADH dehydrogenase-like complex (NDH)-mediated cyclic electron flow around PSI (CEF-PSI) than in the PGR5 (proton gradient regulation 5)-mediated CEF-PSI, indicating that the NDH-mediated CEF-PSI would be a major contributor to PSI photoprotection in the presence of FR light. We also confirmed that PSI photoinhibition decreased with the increase in growth irradiance in A. thaliana and field-grown plants, and that this PSI photodamage was largely suppressed by addition of FR light. These results clearly indicate that the most effective PSI protection is realized in the presence of FR light.

Measurement of cytosolic-free Ca²âº in plant tissue.

A range of techniques have been used to measure the concentration of cytosolic-free Ca(2+) ([Ca(2+)](cyt)) in plant cells. Fluorescent Ca(2+)-sensitive indicators have been used extensively to measure plant [Ca(2+)](cyt) and a number of techniques are available for loading these into plant cells. Here we describe a method for measuring [Ca(2+)](cyt) in the guard cells of the model plant species Commelina communis by ratio photometry and imaging techniques using the ratiometric fluorescent Ca(2+)-sensitive indicator fura-2.

Initiation of the synthesis of 'stress' ABA by (+)-[2 H6 ]ABA infiltrated into leaves of Commelina communis.

The 'fettered' fraction of abscisic acid (ABA) that is held within the chloroplasts of unwilted bean and Commelina communis leaves is released when the leaves wilt and it is this 'free' ABA that is now proposed to cause the stomata to close within 2 or 3 min, well before the rise in total ABA can be detected. The large increase in 'stress' ABA begins 2-3 h later. The fettered ABA in a centrifuged homogenate is released by hyperosmotic solutions of mannitol (0.8 M) and NaCl (0.4 M). Dilute solution of halothane (10 mM) and colchicine (1 mM), the detergent sodium dodecyl sulphate (1 mM) the herbicide 2,4-d (0.1 mM) and dithiothreitol (0.01 mM) also caused ABA to be released. Zeatin (0.01 mM), cumene hydroperoxide (0.01 mM) and CaCl(2) (1 mM) had negligible effects. It was postulated that the ABA released from the chloroplasts by wilting could be the signal that initiates the synthesis of the dioxygenase and other enzymes necessary to produce the rise (up to 40-fold) in the amount of stress ABA that is seen 2-3 h later. To test this hypothesis, a solution of (+)-[(2) H(6) ]ABA was vacuum infiltrated into unwilted Commelina leaves to mimic the rise in ABA caused by wilting and gas chromatography/mass spectrometry of the ABA in the extract after 3 h showed that concentrations of (+)-[(2) H(6) ]ABA of up to 0.3 µM stimulated synthesis of endogenous [(1) H]ABA by 15-fold in the unwilted leaves. A 0.5 µM solution blocked the increase in the amount of ABA formed and also reduced the amount of ABA formed in response to a 0.8 M mannitol solution.

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.

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.

A confocal technique applicable to studies of cellular pH-related signaling in plants.

pH may act as a crucial signal in both animal and plant cells. It is very difficult to monitor pH signals and this has largely hindered progress in the investigation of pH signaling, particularly systematic pH signaling. Here, we report the development of a confocal technique to monitor leaf apoplastic pH in intact plants, which is particularly suitable for the studies on root to shoot signaling. A variety of different pH indicators and plant species were tested. It was found that different pH indicators, for example, 2',7'-Bis-(2-carboxyethyl)-5-(and-6)-carboxyfluoresce (BCECF), SNARF-4F 5-(and-6)-carboxylic acid (SNARF) and DM-NERF (NERF), were of different properties, and to successfully monitor pH at a sub-cellular level, the comparability between the pH indicator and plant species must be involved according to their suitable pH range and loading characteristics. The loading characteristics of different pH indicators differ with different plant species, cell types and their developing stages. No matter what methods were adopted, BCECF and SNARF could not be loaded specifically in the leaf apoplast in sunflower, tomato, and Comelina communis L. In contrast, regardless of the methods adopted, NERF could be loaded efficiently and specifically in the leaf apoplast in C. communis, but not in other plants. In C. communis, the determination coefficient for in vitro and in situ calibration of NERF was very high, which was respectively 0.9951 and 0.9916, and therefore, the adoption of NERF together with C. communis could construct an ideal experimental system that is suitable for the investigation of pH systematic signaling. Ratio image analysis demonstrated that the leaf apoplastic pH was about 5.5 in non-stressed conditions, and water deficit could trigger an increase in pH by about half a pH unit, which is the first evidence to directly indicate that pH is able to act as a systematic signal under water deficit conditions.

Photosynthesis can be enhanced by lateral CO2 diffusion inside leaves over distances of several millimeters.

This study examines the extent to which lateral gas diffusion can influence intercellular CO(2) concentrations (c(i)) and thus photosynthesis in leaf areas with closed stomata. Leaves were partly greased to close stomata artificially, and effects of laterally diffusing CO(2) into the greased areas were studied by gas-exchange measurement and chlorophyll fluorescence imaging. Effective quantum yields (Delta F/F(m)') across the greased areas were analysed with an image-processing tool and transposed into c(i) profiles, and lateral CO(2) diffusion coefficients (D(C'lat)), directly proportional to lateral conductivities (), were estimated using a one-dimensional (1D) diffusion model. Effective CO(2) diffusion distances in Vicia faba (homobaric), Commelina vulgaris (homobaric) and Phaseolus vulgaris (heterobaric) leaves clearly differed, and were dependent on D(C'lat), light intensity, [CO(2)], and [O(2)]: largest distances were approx. 7.0 mm for homobaric leaves (with high D(C'lat)) and approx. 1.9 mm for heterobaric leaves (low D(C'lat)). Modeled lateral CO(2) fluxes indicate large support of photosynthesis over submillimeter distances for leaves with low D(C'lat), whereas in leaves with large D(C'lat), photosynthesis can be stimulated over distances of several millimeters. For the plant species investigated, the surplus CO(2) assimilation rates of the greased leaf areas (A(gr)) differed clearly, depending on lateral conductivities of the respective leaves.

Phosphatidylinositol 3- and 4-phosphate modulate actin filament reorganization in guard cells of day flower.

Phosphatidylinositol 3-kinases (PtdIns 3-kinases) that produce phosphatidylinositol (3,4,5) triphosphate (PtdIns(3,4,5)P(3)) are considered to be important regulators of actin dynamics in animal cells. In plants, neither PtdIns(3,4,5)P(3) nor the enzyme that produces this lipid has been reported. However, a PtdIns 3-kinase that produces phosphatidylinositol 3-phosphate (PtdIns3P) has been identified, suggesting that PtdIns3P, instead of PtdIns(3,4,5)P(3), regulates actin dynamics in plant cells. Phosphatidylinositol 4-kinase (PtdIns 4-kinase) is closely associated with the actin cytoskeleton in plant cells, suggesting a role for this lipid kinase and its product phosphatidylinositol 4-phosphate (PtdIns4P) in actin-related processes. Here, we investigated whether or not PtdIns3P or PtdIns4P plays a role in actin reorganization induced by a plant hormone abscisic acid (ABA) in guard cells of day flower (Commelina communis). ABA-induced changes in actin filaments were inhibited by LY294002 (LY) and wortmannin (WM), inhibitors of PtdIns3P and PtdIns4P synthesis. Expression of PtdIns3P- and PtdIns4P-binding domains also inhibited ABA-induced actin reorganization in a manner similar to LY and WM. These results suggest that PtdIns3P and PtdIns4P regulate actin dynamics in guard cells. Furthermore, we demonstrate that PtdIns3P exerts its effect on actin dynamics, at least in part, via generation of reactive oxygen species (ROS) in response to ABA.

Function and stability of abscisic acid acyl hydrazone conjugates by LC-MS2 of ex vivo samples.

We prepare a biotinylated conjugate of the ubiquitous plant hormone (S)-(+)-abscisic acid via an acyl hydrazone linkage at the C4' position and demonstrate in vivo cleavage of the otherwise stable acyl hydrazone linkage using LC-MS2. As part of a wider chemical genomic study, biological activity of the conjugate was assessed using standard epidermal peel and gravimetric transpiration assays, showing significant activity but at a level lower than the unconjugated hormone. When deuterated samples of the conjugate were fed to the plant, however, it was apparent by LC-MS2 experiments that significant levels of hydrolysis of the acyl hydrazone had taken place, contrary to in vitro stability assays in artificial sap. We conclude that abscisic acid is liberated in sufficient quantities to account for the observed physiological response and that LC-MS2 monitoring of conjugates is a simple and practical method by which such events may be assessed, whether in plants or other organisms.

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.

Biolistic delivery of Ca2+ dyes into plant and algal cells.

In eukaryotes, changes in cytosolic Ca2+ concentrations ([Ca2+]cyt) are associated with a number of environmental and developmental stimuli. However, measuring [Ca2+]cyt changes in single plant or algal cells is often problematic. Although a wide range of Ca2+-sensitive fluorescent dyes is available, they are often difficult to introduce into plant cells. Micro-injection is the most robust method for dye loading, but is time-consuming, technically demanding, and unsuitable in many cell types. To overcome these problems, we have adapted biolistic techniques to load Ca2+-sensitive dyes into guard cells of the flowering plant, Commelina communis, cells of the green alga Chlamydomonas reinhardtii, and zygotes of the brown alga, Fucus serratus. Using this approach, we have been able to monitor [Ca2+]cyt changes in response to various stimuli, including a novel [Ca2+]cyt response in C. reinhardtii. The method allows the use of free acid and dextran-conjugated dyes. Biolistic loading of differentiated plant cells is easier, quicker, and more widely applicable than micro-injection, and should broaden the study of plant signal transduction.

Stomatal movement in response to long distance-communicated signals initiated by heat shock in partial roots of Commelina communis L.

The systematic or long-distance signal transmission plays crucial roles in animal lives. Compared with animals, however, much less is known about the roles of long-distance signal communication in plant lives. Using the model plant Commelina communis L., we have probed the root to shoot communication mediated by heat-shock signals. The results showed that a heat shock of 5 min at 40 degrees C in partial roots, i.e. half or even 1/4 root system, could lead to a significant decrease in stomatal conductance. The regulation capability depends on both heat shock temperature and the amount of root system, i.e. with higher temperature and more roots stressed, the leaf conductance would decrease more significantly. Interestingly, the stomatal regulation by heat shock signal is in a manner of oscillation: when stomata conductance decreased to the lowest level within about 30 min, it would increase rapidly and sometimes even exceed the initial level, and after several cycles the stomata conductance would be finally stabilized at a lower level. Feeding xylem sap collected from heat-shocked plants could lead to a decrease in stomata conductance, suggesting that the heat shock-initiated signal is basically a positive signal. Further studies showed that heat shock was not able to affect ABA content in xylem sap, and also, not able to lead to a decrease in leaf water status, which suggested that the stomatal regulation was neither mediated by ABA nor by a hydraulic signal. Heat shock could lead to an increase in xylem sap H2O2 content, and moreover, the removal of H2O2 by catalase could partially recover the stomatal inhibition by xylem sap collected from heat-shocked plants, suggesting that H2O2 might be able to act as one of the root signals to control the stomatal movement. Due to the fact that heat-shock and drought are usually two concomitant stresses, the stomatal regulation by heat-shock signal should be of significance for plant response to stresses. The observation for the stomatal regulation in an oscillation manner by presently identified new signals should contribute to further understanding of the mystery for the pant systematic signaling in response to stresses.

Osmotic effects on vacuolar ion release in guard cells.

Tracer flux experiments in isolated guard cells of Commelina communis L. suggest that the vacuolar ion content is regulated and is reset to a reduced fixed point by abscisic acid (ABA) with no significant change in cytoplasmic content. The effects of changes in external osmotic pressure were investigated by adding and removing mannitol from the bathing solution. Two effects were distinguished. In the new steady state of volume and turgor, the vacuolar ion efflux was sensitive to turgor: efflux increased at high turgor and reduced at lower turgor after the addition of mannitol. These changes were inhibited by phenylarsine oxide and are likely to involve the same channel that is involved in the response to ABA. After a hypoosmotic transfer, there was an additional effect: a fast transient stimulation of vacuolar efflux during the period of water flow into the cell; the size of this hypopeak increased with the size of the hypoosmotic shock, with increased water flow. No corresponding transient in reduced vacuolar efflux was observed upon hyperosmotic transfer. The fast hypopeak was not inhibited by phenylarsine oxide and appears to involve a different ion channel from that involved in the resting efflux, the response to ABA, or the turgor sensitivity. Thus, the tonoplast can sense an osmotic gradient and respond to water flow into the vacuole by increased vacuolar ion efflux, thereby minimizing cytoplasmic dilution. An aquaporin is the most likely sensor and may also be involved in the signal transduction chain.

Lateral diffusion of CO2 in leaves is not sufficient to support photosynthesis.

Lateral diffusion of CO(2) was investigated in photosynthesizing leaves with different anatomy by gas exchange and chlorophyll a fluorescence imaging using grease to block stomata. When one-half of the leaf surface of the heterobaric species Helianthus annuus was covered by 4-mm-diameter patches of grease, the response of net CO(2) assimilation rate (A) to intercellular CO(2) concentration (C(i)) indicated that higher ambient CO(2) concentrations (C(a)) caused only limited lateral diffusion into the greased areas. When single 4-mm patches were applied to leaves of heterobaric Phaseolus vulgaris and homobaric Commelina communis, chlorophyll a fluorescence images showed dramatic declines in the quantum efficiency of photosystem II electron transport (measured as F(q)'/F(m)') across the patch, demonstrating that lateral CO(2) diffusion could not support A. The F(q)'/F(m)' values were used to compute images of C(i) across patches, and their dependence on C(a) was assessed. At high C(a), the patch effect was less in C. communis than P. vulgaris. A finite-volume porous-medium model for assimilation rate and lateral CO(2) diffusion was developed to analyze the patch images. The model estimated that the effective lateral CO(2) diffusion coefficients inside C. communis and P. vulgaris leaves were 22% and 12% of that for free air, respectively. We conclude that, in the light, lateral CO(2) diffusion cannot support appreciable photosynthesis over distances of more than approximately 0.3 mm in normal leaves, irrespective of the presence or absence of bundle sheath extensions, because of the CO(2) assimilation by cells along the diffusion pathway.

Measurement of cytosolic-free Ca²+ in plant tissue.

Several techniques have been used to measure the concentration of cytosolic-free Ca(2+) ([Ca(2+)](cyt)) in plants. These include Ca(2+)-sensitive microelectrodes, luminescent photoproteins, cameleons, and fluorescent Ca2(+) indicators. Ca(2+)-sensitive microelectrodes can be used only in cells that are able to withstand impalement with two electrodes or a double-barrelled electrode. In addition, microelectrodes suffer from slow response times and difficulties with calibration. These problems are particularly acute in plant cells in which the high turgor often results in partial displacement of the sensor, and the subsequent loss of sensitivity, following impalement. Consequently, the use of Ca(2+)-sensitive electrodes has been limited to only a few studies in plants and algae.

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.

Biosynthesis of 1-deoxynojirimycin in Commelina communis: a difference between the microorganisms and plants.

1-Deoxynojirimycin is a glycosidase-inhibitory alkaloid obtained from several plants and microorganisms. Administration experiments using [1-(13C)] glucose in the higher plant Commelina communis and 13C NMR spectroscopic analyses of products suggested that 1-deoxynojirimycin was biosynthesized through a different route compared with that in Streptomyces and Bacilli microorganisms.