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The differentiation of sepal and petal morphologies in Commelinaceae.

The morphological transition of the first whorl of tepals into sepals occurs frequently during the diversification of angiosperms. Such transitions may play important roles in pollination modes. The B class genes, APETALA3 (AP3) and PISTILLATA (PI) in Arabidopsis thaliana and GLOBOSA (GLO) and DEFICIENS (DEF) in Antirrhinum majus, are required for the development of petals in the second whorl, and its homologs have been isolated and characterized from various plants. A recent study on tulip, a monocotyledonous plant, indicates that the morphology of petaloid tepals in the first and second whorls is consistent with the expansion of B class gene expression. Here, we report five B class genes, TRGLOA, TRGLOB, CCGLO, TRDEF and CCDEF, isolated and characterized from two commelinaceous plants, Tradescantia reflexa and Commelina communis, with distinct sepal and petal morphologies in monocots. Northern blot analysis and gene-specific reverse transcription-polymerase chain reaction (RT-PCR) studies using dissected floral organs reveal a lack or low level of DEF-like gene expression in these commelinaceous species in the first whorl, in contrast to previous results. The expression data suggest that DEF-like gene expression in Commelinaceae correlates with the production of petaloid organs in the first whorl.

Amino Acid Sequence↗

Growth-promoting effect on iron-sulfur proteins on axenic cultures of Entamoeba dispar.

A growth-promoting factor (GPF) that promotes the growth of Entamoeba dispar under axenic culture conditions was found in fractions of mitochondria (Mt), hydrogenosomes (Hg) and chloroplasts (Cp) obtained from cells of six different protozoan, mammalian and plant species. We were able to extract the GPF from the Cp-rich leaf cells of a plant (spiderwort: Commelina communis L.) in an acetone-soluble fraction as a complex of chlorophyll with low molecular weight proteins (molecular weight [MW] approximately 4,600). We also found that on treatment with 0.6% complexes of 2-mercapthoethanol (2ME), complexes of chlorophyll-a with iron-sulphur (Fe-S) proteins (e.g., ferredoxins [Fd] from spinach and Clostridium pasteurianum) and noncomplex rubredoxin (Rd) from C. posteurianum have a growth-promoting effect on E. dispar. These findings suggest that E. dispar may lack a sufficient quantity of some essential components of Fe-S proteins, such as Fe-S center.

Animals↗

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.

Abscisic Acid↗

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.

Abscisic Acid↗

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.

Abscisic Acid↗

Microtubules of guard cells are light sensitive.

Guard cells of stomata are characterized by ordered bundles of microtubules radiating from the ventral side toward the dorsal side of the cylindrical cell. It was suggested that microtubules play a role in directing the radial arrangement of the cellulose micro-fibrils of guard cells. However, the role of microtubules in daily cycles of opening and closing of stomata is not clear. The organization of microtubules in guard cells of Commelina communis leaves was studied by analysis of three-dimensional immunofluorescent images. It was found that while guard cell microtubules in the epidermis of leaves incubated in the light were organized in parallel, straight and dense bundles, in the dark they were less straight and oriented randomly near the stomatal pore. The effect of blue and red light on the organization of guard cell microtubules resembled the effects of white light and dark respectively. When stomata were induced to open in the dark with fusicoccin, microtubules remained in the dark configuration. Furthermore, when incubated in the light, guard cell microtubules were more resistant to oryzalin. Similarly, microtubules of Arabidopsis guard cells, expressing green fluorescent protein-tubulin alpha 6, were disorganized in the dark, but were organized in parallel arrays in the presence of white light. The dynamics of microtubule rearrangement upon transfer of intact leaves from dark to light was followed in single stomata, showing that an arrangement of microtubules typical for light conditions was obtained after 1 h in the light. Our data suggest that microtubule organization in guard cells is responsive to light signals.

Cell Polarity↗

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.

Calibration↗

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.

Abscisic Acid↗

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.

Animals↗

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.

Abscisic Acid↗

[Effects of tillage method and herbicide on cornfield weed community].

By the method of community ecology, this paper surveyed the weed community in a cornfield at the Shenyang Experimental Station of Ecology, CAS, and studied the effects of tillage method and herbicide on the weed composition, species diversity, and biomass at the experimental site. The results showed that the dominant weed species in the cornfield were Eriochloa villosa, Abutilon theophrasti, Bidens frondosa and Commelina communis, of which, Eriochloa villosa had the highest important value. In non-tillage field without herbicide application, the weed community had larger biomass, higher richness (S) and concentration (C), but lower species diversity (D) and species evenness (J). Herbicide could decrease weed species and inhibit biomass growth significantly in non-tillage field. It was clear that tillage method and herbicide could affect the weed composition, diversity and stability significantly.

Agriculture↗

Herbicide-resistant crops and weed resistance to herbicides.

The adoption of genetically modified (GM) crops has increased dramatically during the last 3 years, and currently over 52 million hectares of GM crops are planted world-wide. Approximately 41 million hectares of GM crops planted are herbicide-resistant crops, which includes an estimated 33.3 million hectares of herbicide-resistant soybean. Herbicide-resistant maize, canola, cotton and soybean accounted for 77% of the GM crop hectares in 2001. However, sugarbeet, wheat, and as many as 14 other crops have transgenic herbicide-resistant cultivars that may be commercially available in the near future. There are many risks associated with the production of GM and herbicide-resistant crops, including problems with grain contamination, segregation and introgression of herbicide-resistant traits, marketplace acceptance and an increased reliance on herbicides for weed control. The latter issue is represented in the occurrence of weed population shifts, the evolution of herbicide-resistant weed populations and herbicide-resistant crops becoming volunteer weeds. Another issue is the ecological impact that simple weed management programs based on herbicide-resistant crops have on weed communities. Asiatic dayflower (Commelina cumminus L) common lambsquarters (Chenopodium album L) and wild buckwheat (Polygonum convolvulus L) are reported to be increasing in prominence in some agroecosystems due to the simple and significant selection pressure brought to bear by herbicide-resistant crops and the concomitant use of the herbicide. Finally, evolution of herbicide-resistant weed populations attributable to the herbicide-resistant crop/herbicide program has been observed. Examples of herbicide-resistant weeds include populations of horseweed (Conyza canadensis (L) Cronq) resistant to N-(phosphonomethyl)glycine (glyphosate). An important question is whether or not these problems represent significant economic issues for future agriculture.

Adaptation, Physiological↗

Pharmacological properties of slow anion currents in intact guard cells of Arabidopsis. Application of the discontinuous single-electrode voltage-clamp to different species.

More electrophysiological studies have been carried out on guard cells than on any other cell type of vascular plants. The characterization of their ion channels has been achieved using mainly the whole-cell patch-clamp technique applied to guard-cell protoplasts. The aim of this study was to obtain recordings of ion channel currents in intact guard cells and especially of slow anion channels of Arabidopsis thaliana, a species of fundamental genetic interest. Application of the discontinuous single-electrode voltage-clamp technique enabled the first characterization of K+ currents in Commelina communis and of slow anion currents in C. communis and A. thaliana in intact guard cells to be made. Inward K+ channels from A. thaliana were inhibited by external application of tetraethylammonium (TEA) or Ca2+. In the presence of K+ channel blockers, slow anion channel currents were elicited in almost all guard cells tested and were confirmed by the application of anion channel blockers. In A. thaliana, only anthracene-9 carboxylic acid was able to inhibit slow anion currents, to promote stomatal opening in the dark and to reverse the effect of 25 microM abscisic acid under light. Use of a single microelectrode and preservation of cell integrity make this technique well suited for the study of ion channel regulation in species that have guard cell protoplasts with which it is difficult to form good seals.

Anions↗

Cortical actin filaments in guard cells respond differently to abscisic acid in wild-type and abi1-1 mutant Arabidopsis.

Cortical actin filaments in guard cells of Commelina communis L. show signal-specific organization during stomatal movements [S.-O. Eun and Y. Lee (1997) Plant Physiol 115: 1491-1498; S.-O. Eun and Y. Lee (2000) Planta 210: 1014-1017]. To study the roles of actin in signal transduction, it is advantageous to use Arabidopsis thaliana (L.) Heynh., an excellent model plant with numerous well-characterized mutants. Using an immunolocalization technique, we found that actin deployments in guard cells of A. thaliana were basically identical to those in C. communis: actin proteins were assembled into radial filaments under illumination, and were disassembled by ABA. In addition, we examined actin organization in an ABA-insensitive mutant (abi1-1) to test the involvement of protein phosphatase 2C (PP2C) in the control of actin structure. A clear difference was observed after ABA treatment, namely, neither stomatal closing nor depolymerization of actin filaments was observed in guard cells of the mutant. Our results indicate that PP2C participates in ABA-induced actin changes in guard cells.

Abscisic Acid↗

Stomatal opening by fusicoccin is accompanied by depolymerization of actin filaments in guard cells.

Actin in guard cells is assembled in a radial pattern when stomata are induced to open under light, but the filaments are disassembled when stomata are closed under darkness or by abscisic acid (S.-O. Eun and Y. Lee, 1997, Plant Physiol. 115: 1491-1498). To test if signals that open stomata commonly generate the polymerized form of actin in guard cells, leaves of Commelina communis L. were treated with a potent stomatal opening agent, fusicoccin, and the actin organization examined by immunolocalization techniques. When stomata were induced to open by fusicoccin, hardly any of the filamentous form of actin was detected; instead, the actin resembled that present in guard cells that had been treated with an antagonist to actin filaments, cytochalasin D, and showed a sharp contrast to the long filaments developed in illuminated guard cells. Furthermore, treatment of illuminated leaves with fusicoccin disintegrated actin filaments that had already been formed in the guard cells. Preincubation of leaves with phalloidin, which interferes with fusicoccin-induced actin depolymerization, delayed fusicoccin-induced opening during the early phase. These observations suggest that the prevention of actin filament formation and/or depolymerization of actin filaments may accelerate the stomatal opening process in response to fusicoccin.

Actins↗

The fusicoccin receptor of plants is a member of the 14-3-3 superfamily of eukaryotic regulatory proteins.

The receptor for the wilt-inducing phytotoxin fusicoccin was purified to homogeneity from plasma membranes of Commelina communis as a complex with the radioligand [3H]9'-nor-8'-hydroxyfusicoccin. The preparation consisted of two polypeptides with apparent molecular masses of 30.5 kDa and 31.5 kDa and with isoelectric points of around pH 5.2 and 5.3, respectively. The proteins were N-terminally blocked. Internal amino acid sequences were obtained for both polypeptides of the fusicoccin-binding complex. Sequence information, as well as subsequent immunological analysis, proved that both polypeptides are members of the eukaryotic 14-3-3 family, which comprises structurally conserved regulatory proteins of widespread occurrence and a wide range of functions. 14-3-3 isoform(s) constituting the fusicoccin receptor are distinguishable from other cellular 14-3-3 proteins by their tight association with the plasma membrane. Applying temperature-induced Triton X-114 phase separation experiments, they, as well as the target enzyme of fusicoccin action, the H(+)-ATPase, partitioned into the phospholipid-rich fraction which contains the most hydrophobic proteins. The results discussed herein provide a basis for the elucidation of the molecular mechanism of fusicoccin action.

Adenosine Triphosphatases↗

In vitro tests and ethnopharmacological investigations: wound healing as an example.

In vitro tests are now widely employed in ethnopharmacological research because of ethical reasons and their usefulness in bioactive-guided fractionation and determination of active compounds. For many disease conditions, a variety of in vitro tests can now be employed as the biochemical mechanisms underlying disease and healing processes are understood. Approaches to the in vitro investigations of wound healing processes are exemplified by studies on extracts of Buddleja species and three Ghanaian species Spathodea campanulata, Commelina diffusa and Secamone afzelii. Most studies have been carried out on Buddleja officinalis or Buddleja globosa. The extracts have been shown to have anti-inflammatory and antioxidant properties due to flavonoids, triterpenoids, diterpenoids and caffeic acid derivatives. There appears to a slight effect on proliferation of fibroblasts at lower concentrations, but this was not significant, and higher concentrations appeared to be cytotoxic. Novel findings are the ability of Buddleja globosa leaf extracts to induce differentiation in keratinocytes and to alter the profile of proteins produced by cultured fibroblasts. Extracts also had some effect on lattice contraction. The three Ghanaian species examined show a mixture of antimicrobial and antioxidant activities. The evolution over recent years of tests for wound healing, from in vivo tests to cell-based systems and chemical reactions and on to investigations into effects on secondary messengers and protein expression, is described.

Animals↗

Identification of GTP-binding proteins in the plasma membrane of higher plants.

Antisera raised against a highly conserved amino acid sequence (G alpha-common peptide) of animal Gs alpha, Gi alpha, Go alpha and Gt alpha recognize, in plasma membranes of several higher plants, sets of proteins of Mr = 37 and 31 kDa (Vicia faba), 36 and 31 kDa (Arabidopsis thaliana) and 38 and 34 kDa (Commelina communis). The A. thaliana proteins were solubilized and partially purified. They bind [35S]GTP gamma S with high affinity (apparent Kd approximately 10 nM) and, with lower affinity, GTP but not the other nucleotides tested (ATP, CTP, ITP, UTP).

Blotting, Western↗