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Identification of cis elements involved in Commelina yellow mottle virus promoter activity.

Commelina yellow mottle virus (CoYMV) is a double-stranded DNA virus that infects a monocot host. A promoter fragment isolated from CoYMV is a strong promoter when assayed after transient introduction into monocot and dicot suspension cells and is highly active in vascular cells of flowers, leaves, stems and roots of stably transformed tobacco plants. Here it is reported that in stably transformed maize calli and transgenic tobacco leaves the CoYMV and CaMV 35S promoters exhibit similar amounts of activity. Deletion of the sequences located distal to nucleotide -230 relative to the start of transcription has no significant effect on promoter strength or tissue specificity. The region between -230 and -200 shares sequence similarity with the as-1 promoter element of the CaMV 35S promoter. Deletion of this as-1-like motif decreases promoter activity in maize suspension cells by 85%. Analysis of deletions affecting the -200 to -52 region indicates that sequences located between -159 and -84 are required for activity in vascular tissues. In addition, this region exhibits properties of a vascular tissue-specific enhancer since it confers vascular expression in an orientation-independent manner when fused to promoters that are not normally active in vascular tissues.

Base Sequence↗

Stomatal Opening Is Induced in Epidermal Peels of Commelina communis L. by GTP Analogs or Pertussis Toxin.

Pretreatment with pertussis toxin or microinjection of guanosine- 5[prime]-(3-thiotriphosphate) (GTP-[gamma]-S) into guard cells in peeled epidermis of Commelina communis L. promoted stomatal opening under subsaturating white light. Guanosine-5[prime]-(2-thiodiphosphate) (GDP-[beta]-S) and adenosine-5[prime]-(3-thiotriphosphate) (ATP-[gamma]-S) did not change stomatal aperture under identical conditions. These results indicate that G proteins may be involved in the regulation of stomatal opening.

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Evidence for an Extracellular Reception Site for Abscisic Acid in Commelina Guard Cells.

The phytohormone abscisic acid (ABA) triggers stomatal closing as a physiological response to drought stress. Several basic questions limit an understanding of the mechanism of ABA reception in guard cells. Whether primary ABA receptors are located on the extracellular side of the plasma membrane, within the intracellular space of guard cells, or both remains unknown. Furthermore, it is not clear whether ABA must be transported into guard cells to exert control over stomatal movements. In the present study, a combination of microinjection into guard cells and physiological assays of stomatal movements have been performed to determine primary sites of ABA reception in guard cells. Microinjection of ABA into guard cells of Commelina communis L. resulted in injected cytosolic concentrations of 50 to 200 [mu]M ABA and in additional experiments in lower concentrations of approximately 1 [mu]M ABA. Stomata with ABA-loaded guard cells (n > 180) showed opening similar to stomata with uninjected guard cells. The viability of guard cells following ABA injection was demonstrated by neutral red staining as well as monitoring of stomatal opening. Extracellular application of 10 [mu]M ABA inhibited stomatal opening by 98% at pH 6.15 and by 57% at pH 8.0. The pH dependence of extracellular ABA action may suggest a contribution of an intracellular ABA receptor to stomatal regulation. The findings presented here show that intracellular ABA alone does not suffice to inhibit stomatal opening under the imposed conditions. Furthermore, these data provide evidence that a reception site for ABA-mediated inhibition of stomatal opening is on the extracellular side of the plasma membrane of guard cells.

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Oligogalacturonic acid and chitosan reduce stomatal aperture by inducing the evolution of reactive oxygen species from guard cells of tomato and Commelina communis.

Stomatal opening provides access to inner leaf tissues for many plant pathogens, so narrowing stomatal apertures may be advantageous for plant defense. We investigated how guard cells respond to elicitors that can be generated from cell walls of plants or pathogens during pathogen infection. The effect of oligogalacturonic acid (OGA), a degradation product of the plant cell wall, and chitosan (beta-1,4-linked glucosamine), a component of the fungal cell wall, on stomatal movements were examined in leaf epidermis of tomato (Lycopersicon esculentum L.) and Commelina communis L. These elicitors reduced the size of the stomatal aperture. OGA not only inhibited light-induced stomatal opening, but also accelerated stomatal closing in both species; chitosan inhibited light-induced stomatal opening in tomato epidermis. The effects of OGA and chitosan were suppressed when EGTA, catalase, or ascorbic acid was present in the medium, suggesting that Ca(2+) and H(2)O(2) mediate the elicitor-induced decrease of stomatal apertures. We show that the H(2)O(2) that is involved in this process is produced by guard cells in response to elicitors. Our results suggest that guard cells infected by pathogens may close their stomata via a pathway involving H(2)O(2) production, thus interfering with the continuous invasion of pathogens through the stomatal pores.

Ascorbic Acid↗

Rapid low temperature-induced stomatal closure occurs in cold-tolerant Commelina communis leaves but not in cold-sensitive tobacco leaves, via a mechanism that involves apoplastic calcium but not abscisic acid.

Commelina communis stomata closed within 1 h of transferring intact plants from 27 degrees C to 7 degrees C, whereas tobacco (Nicotiana rustica) stomata did not until the leaves wilted. Abscisic acid (ABA) did not mediate cold-induced C. communis stomatal closure: At low temperatures, bulk leaf ABA did not increase; ABA did not preferentially accumulate in the epidermis; its flux into detached leaves was lower; its release from isolated epidermis was not greater; and stomata in epidermal strips were less sensitive to exogenous ABA. Stomata of both species in epidermal strips on large volumes of cold KCl failed to close unless calcium was supplied. Therefore, the following cannot be triggers for cold-induced stomatal closure in C. communis: direct effects of temperature on guard or epidermal cells, long-distance signals, and effects of temperature on photosynthesis. Low temperature increased stomatal sensitivity to external CaCl(2) by 50% in C. communis but only by 20% in tobacco. C. communis stomata were 300- to 1,000-fold more sensitive to calcium at low temperature than tobacco stomata, but tobacco epidermis only released 13.6-fold more calcium into bathing solutions than C. communis. Stomata in C. communis epidermis incubated on ever-decreasing volumes of cold calcium-free KCl closed on the lowest volume (0.2 cm(3)) because the epidermal apoplast contained enough calcium to mediate closure if this was not over diluted. We propose that the basis of cold-induced stomatal closure exhibited by intact C. communis leaves is increased apoplastic calcium uptake by guard cells. Such responses do not occur in chill-sensitive tobacco leaves.

Abscisic Acid↗

Energy Supply for Stomatal Opening in Epidermal Strips of Commelina benghalensis.

The influence of light or darkness on stomatal opening in epidermal strips of Commelina benghalensis was evaluated in the presence or absence of O(2) and/or metabolic inhibitors. Opening was restricted in nitrogen and was promoted by NADH and acids of the tricarboxylic acid cycle (succinate and alpha-ketoglutarate) in CO(2)-free air in light as well as in darkness. The enhancement by light of stomatal opening was prevalent under nitrogen or in the presence of the respiratory inhibitors (sodium azide and oligomycin). Respiratory inhibitors decreased the opening in light or darkness under CO(2)-free air but exhibited no effect under nitrogen, whereas phosphorylation uncouplers were inhibitory in light or darkness under both CO(2)-free air and nitrogen. The results suggest that oxidative phosphorylation is a basic source of energy for stomatal opening, although photophosphorylation could be an energy source.

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Regulation of ADPGlucose Synthesis in Guard Cells of Commelina communis.

The activator specificity of the ADPglucose pyrophosphorylase from Commelina communis guard cells is the same as observed for the mesophyll cell enzyme. 3-Phosphoglycerate was found to be the most effective activator. Fifty per cent of maximal stimulation was observed at about 100 micromolar. Inorganic phosphate was found to be a potent inhibitor giving 50% inhibition at 0.3 millimolar. These results are discussed with respect to regulation of starch synthesis in guard cells.

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Ion-Stimulated Stomatal Opening Induced by Preillumination in Epidermal Strips of Commelina communis.

The effects of preillumination were investigated on ion-stimulated stomatal opening of epidermal strips isolated from Commelina communis L. leaves, which are dark-starved 24 hours or more. The rate and the extent of ion-stimulated stomatal openings were increased by preexposure of epidermal strips to light. The evidences are interpreted as indicating that the energy induced by preillumination can be conserved in guard cells for considerable time periods and then used for a delayed stomatal opening in the presence of higher concentration of potassium or sodium ions. Action spectrum showed two peaks, one in blue and one in the red light region. The ratio of the blue peak to the red peak is 1.2; which is the smallest reported value in action spectra of stomatal movements. 3-(4-chlorophenyl)-1,-1-Dimethylurea suppressed the ion-stimulated stomatal opening induced by the preillumination. We conclude that the photosynthetic electron transport system, containing photosystem II, in guard cell chloroplasts is a basic system of energy acquirement for stomatal opening.

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Role of Ca and EGTA on Stomatal Movements in Commelina communis L.

Ca(2+) (0.1-1.0 millimolar) accelerated dark-induced stomatal closure and reduced stomatal apertures in the light in epidermal peels of Commelina communis L. In contrast, ethyleneglycol-bis-(beta-aminoethyl ether) N,N'tetraacetic acid (EGTA) (2 millimolar), a Ca(2+) chelator, prevented closure in the dark and accelerated opening in the light. EGTA did not promote significant opening in the dark. It is therefore concluded that EGTA does not increase ion uptake into guard cells, but rather prevents ion efflux. Addition of EGTA to incubating solutions with 10 millimolar KCl resulted in steady state apertures of 15.6 micrometers, whereas in the absence of EGTA similar apertures required 55 millimolar KCl and 150 millimolar KCl was needed in the presence of 1 millimolar CaCl(2). The results demonstrate the importance of Ca(2+) in the regulation of stomatal closure and point to a role of Ca(2+) in the regulation of K(+) efflux from stomatal guard cells.

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Localization of Carbohydrate Metabolizing Enzymes in Guard Cells of Commelina communis.

The localization of enzymes involved in the flow of carbon into and out of starch was determined in guard cells of Commelina communis. The guard cell chloroplasts were separated from the rest of the cellular components by a modification of published microfuge methods. The enzymes of interest were then assayed in the supernatant and chloroplast fractions. The chloroplast yield averaged 75% with 10% cytoplasmic contamination. The enzymes involved in starch biosynthesis, ADPglucose pyrophosphorylase, starch synthase, and branching enzyme, are located exclusively in the chloroplast fraction. The enzymes involved in starch degradation show a more complex distribution. Phosphorylase is located in both the supernatant and chloroplast fraction, 50% in each fraction. Most of the amylase and debranching enzyme activity is present in the supernatant (70%) fraction. The majority of the rest of the enzymes involved in the degradation of starch to malate and synthesis of starch from a hexose precursor were also investigated. All of the enzymes were present in the chloroplast except for hexokinase and phosphofructokinase. The inability to assay these enzymes could possibly have been due to the lack of or low activity of the enzymes or to nonoptimal assay conditions.

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Calcium Effects on Stomatal Movement in Commelina communis L. : Use of EGTA to Modulate Stomatal Response to Light, KCl and CO(2).

Stomatal movements depend on both ion influx and efflux; attainment of steady state apertures reflects modulation of either or both processes. The role of Ca(2+) in those two processes was investigated in isolated epidermal strips of Commelina communis, using the Ca(2+) chelator EGTA to reduce apoplastic [Ca(2+)]. The results suggest that a certain concentration of Ca(2+) is an absolute requirement for salt efflux and stomatal closure. EGTA (2 millimolar) increased KCl-dependent stomatal opening in darkness and completely inhibited the dark-induced closure of initially open stomata. Closure was inhibited even in a KCl-free medium. Thus, maintenance of stomata in the open state does not necessarily depend on continued K(+) influx but on the inhibition of salt efflux. Opening in the dark was stimulated by IAA in a concentration-dependent manner, up to 15.4 micrometer without reaching saturation, while the response to EGTA leveled off at 9.2 micrometer. IAA did not inhibit stomatal closure to the extent it stimulated opening. The response to IAA is thus consistent with a primary stimulation of opening, while EGTA can be considered a specific inhibitor of stomatal closing since it inhibits closure to a much larger degree than it stimulates opening. CO(2) causes concentration-dependent reduction in the steady state stomatal aperture. EGTA completely reversed CO(2)-induced closing of open stomata but only partially prevented the inhibition of opening.

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Symplastic Transfer of Fluorescent Dyes from Mesophyll to Sieve Tube in Stripped Leaf Tissue and Partly Isolated Minor Veins of Commelina benghalensis.

We have stripped small (3 x 3 mm) fields of the upper and the opposite lower epidermis of Commelina benghalensis leaves. Pectinase treatment of the resulting chlorenchyma windows produced free-lying viable minor veins with small lumps of mesophyll cells attached. These veins were still connected with the intact remainder of the leaf. Fluorescent dyes were injected into mesophyll cells or mestome sheath cells. Continuous following of the dye from the moment of injection and use of the simple vein system allowed an unhindered and precise assessment of the cell-to-cell route of dye transfer. Disodium fluorescein and Lucifer Yellow CH injected into mesophyll or mestome sheath cells readily moved to the sieve tube. This symplastic dye transfer from mesophyll to sieve tube was also observed after injection into unmacerated stripped leaf tissue. The displacement of fluorescent dyes substantiates a symplastic continuity between mesophyll and sieve tube and therefore supports the possibility of symplastic phloem loading.

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Calcium Fluxes across the Plasma Membrane of Commelina communis L. Assayed in a Cell-Free System.

The inside-out fraction of plasma membrane-rich vesicles prepared from leaves of Commelina communis L. by aqueous twophase partitioning was loaded with (45)Ca(2+) through the action of the plasma membrane Ca(2+)-ATPase. While the Ca(2+)-loaded vesicles were tightly sealed, trifluoperazine (TFP) (effective concentration giving 50% of maximum effect [EC(50)] = 70 micromolar) and W-7 (EC(50) = 100 micromolar), but to a much lesser extent, W-5 (EC(50) = 500 micromolar) led to a rapid efflux of (45)Ca(2+) from the vesicles. This efflux could be blocked efficiently with low (<1 millimolar) concentrations of La(3+), but it remained unaffected by the addition of calmodulin (CM). Further experiments with vesicles incubated in (45)Ca(2+) in the absence of ATP, as well as experiments performed with control liposomes and nonloaded as well as Ca(2+)-loaded plasma membrane vesicles using the indicator dye arsenazo III showed, that TFP and W-7 and, again to a lesser extent, W-5 mobilized a pool of membrane-bound Ca(2+) from the vesicles. No indications for a detergent effect of TFP and W-7 were obtained. The EC(50)-values of these compounds for mobilizing membrane-associated Ca(2+) (TFP = 100 micromolar, W-7 = 100 micromolar, W-5 = 500 micromolar) or for the triggering of Ca(2+) release from Ca(2+)-loaded vesicles (see above) were very similar, suggesting a common basis of antagonist action on both processes. Our results suggest the presence of a Ca(2+) channel in the plasma membrane of C. communis. The channel is obtained in a Ca(2+)-inactivated state after preparation and Ca(2+)-loading of the vesicles. The inactivation is removed by TFP or W-7, presumably due to the Ca(2+)-mobilizing effect of these compounds. The activated Ca(2+) channel is La(3+) sensitive and, in the cell, would allow for passage of Ca(2+) into the cell. The possibility that TFP or W-7 act independent of CM, or through CM tightly associated with the plasma membrane, is discussed. The system described allows a cell free analysis of Ca(2+) influx, displaying channel properties, in a higher plant.

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Functional Reconstitution of an ATP-Driven Ca-Transport System from the Plasma Membrane of Commelina communis L.

The protein(s) that constitute(s) the ATP-driven Ca(2+)-translocator of plasma membrane enriched vesicles obtained by aqueous two-phase partitioning from leaves of Commelina communis L. has/have been solubilized and reincorporated into tightly sealed liposomes. The reconstituted Ca(2+)-transport system was studied using ATP-driven (45)Ca(2+) import into the proteoliposomes as a measure of activity. The detergent, 3-[(3-cholamidopropyl) dimethylammonio]-1-propane-sulfonate proved to be the most suitable and was used at 10 millimolar concentration, i.e. just above its critical micellar concentration. The presence of additional phospholipid (2 milligrams phosphatidylcholine per milliliter) and ATP (5 millimolar) improved the solubilization and/or reconstitution. The characteristics of the reconstituted system were similar to those of the plasma membrane-bound activity, including the apparent K(m) for Ca(2+) (5.2 micromolar), inhibition by relatively high levels of vanadate (IC(50) = 500 micromolar) and lacking response to added calmodulin. The reconstituted transport system was very strongly inhibited by erythrosine B (IC(50) = 0.01 micromolar) and had a low apparent K(m) for ATP (11.4 micromolar). As in the plasma membrane vesicles, the protonophore carbonylcyanide m-chlorophenyl hydrazone did not affect Ca(2+)-transport detectably in the reconstituted system. However, low levels of the Ca(2+)-ionophore A 23187 instantaneously discharged 90% of the Ca(2+) associated with the vesicles, proving that it had been accumulated in the intravesicular volume in soluble, freely exchangeable form. Ca(2+)-transport in the reconstituted system was thus primary active, through a Ca(2+)-translocating ATPase. The system reported here may serve as a valuable tool for purifying the Ca(2+)-ATPase and for studying structural and functional aspects of the purified enzyme.

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Relationship between Respiration and Photosynthesis in Guard Cell and Mesophyll Cell Protoplasts of Commelina communis L.

A mass spectrometric method combining (16)O/(18)O and (12)C/(13)C isotopes was used to quantify the unidirectional fluxes of O(2) and CO(2) during a dark to light transition for guard cell protoplasts and mesophyll cell protoplasts of Commelina communis L. In darkness, O(2) uptake and CO(2) evolution were similar on a protein basis. Under light, guard cell protoplasts evolved O(2) (61 micromoles of O(2) per milligram of chlorophyll per hour) almost at the same rate as mesophyll cell protoplasts (73 micromoles of O(2) per milligram of chlorophyll per hour). However, carbon assimilation was totally different. In contrast with mesophyll cell protoplasts, guard cell protoplasts were able to fix CO(2) in darkness at a rate of 27 micromoles of CO(2) per milligram of chlorophyll per hour, which was increased by 50% in light. At the onset of light, a delay observed for guard cell protoplasts between O(2) evolution and CO(2) fixation and a time lag before the rate of saturation suggested a carbon metabolism based on phosphoenolpyruvate carboxylase activity. Under light, CO(2) evolution by guard cell protoplasts was sharply decreased (37%), while O(2) uptake was slowly inhibited (14%). A control of mitochondrial activity by guard cell chloroplasts under light via redox equivalents and ATP transfer in the cytosol is discussed. From this study on protoplasts, we conclude that the energy produced at the chloroplast level under light is not totally used for CO(2) assimilation and may be dissipated for other purposes such as ion uptake.

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Redox Processes in the Blue Light Response of Guard Cell Protoplasts of Commelina communis L.

Guard cell protoplasts from Commelina communis L. illuminated with red light responded to a blue light pulse by an H(+) extrusion which lasted for about 10 minutes. This proton extrusion was accompanied by an O(2) uptake with a 4H(+) to O(2) ratio. The response to blue light was nil in darkness without a preillumination period of red light and increased with the duration of the red light illumination until about 40 minutes. However, acidification in response to a pulse of blue light was obtained in darkness when external NADH (1 millimolar) was added to the incubation medium, suggesting that redox equivalents necessary for the expression of the response to blue light in darkness may be supplied via red light. In accordance with this hypothesis, the photosystem II inhibitor 3-(3,4-dichlorophenyl)-1, 1-dimethylurea (10 micromolar) decreased the acidification in response to blue light more efficiently when it was added before red light illumination than before the blue light pulse. In the presence of hexacyanoferrate, the acidification in response to a blue light pulse was partly inhibited (53% of control), suggesting a competition for reducing power between ferricyanide reduction and the response to blue light.

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Role of Calcium in Signal Transduction of Commelina Guard Cells.

The role of cytosolic Ca2+ in signal transduction in stomatal guard cells of Commelina communis was investigated using fluorescence ratio imaging and photometry. By changing extracellular K+, extracellular Ca2+, or treatment with Br-A23187, substantive increases in cytosolic Ca2+ to over 1 micromolar accompanied stomatal closure. The increase in Ca2+ was highest in the cytoplasm around the vacuole and the nucleus. Similar increases were observed when the cells were pretreated with ethyleneglycol-bis-(o-aminoethyl)tetraacetic acid or the channel blocker La3+, together with the closing stimuli. This suggests that a second messenger system operates between the plasma membrane and Ca2+-sequestering organelle(s). The endogenous growth regulator abscisic acid elevated cytosolic Ca2+ levels in a minority of cells investigated, even though stomatal closure always occurred. Ca2+-dependent and Ca2+-independent transduction pathways linking abscisic acid perception to stomatal closure are thus indicated.

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Characterization and purification of the fusicoccin-binding complex from plasma membranes of Commelina communis.

The fungal phytotoxin fusicoccin binds with high affinity to plasma membranes of the monocotyledonous plant, Commelina communis L. The sites bind the toxin with an apparent Kd of 5.2 nM and a pH optimum of 6.0. They occur at a level of approximately 6-8 pmol/mg plasma membrane protein. Photoaffinity labeling with the biologically active fusicoccin derivative 9'-nor-8'-(4-azido[3,5-3H]benzoyl) diaminoethylfusicoccin identified a polypeptide of 31.5 kDa on SDS/PAGE which was strongly labeled. A second 32.5-kDa band was also consistently labeled, although not to the same extent. The binding sites were solubilized in functional form and a purification scheme was developed based on affinity and ion-exchange procedures. The purified fraction contains two polypeptides of apparent molecular masses of 30.5 kDa and 31.6 kDa. A detailed molecular analysis of the fusicoccin-binding complex is now possible.

Affinity Labels↗