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Stimulus-Induced Oscillations in Guard Cell Cytosolic Free Calcium.

Ca2+ is implicated as a second messenger in the response of stomata to a range of stimuli. However, the mechanism by which stimulus-induced increases in guard cell cytosolic free Ca2+ ([Ca2+]i) are transduced into different physiological responses remains to be explained. Oscillations in [Ca2+]i may provide one way in which this can occur. We used photometric and imaging techniques to examine this hypothesis in guard cells of Commelina communis. External Ca2+ ([Ca2+]e), which causes an increase in [Ca2+]i, was used as a closing stimulus. The total increase in [Ca2+]i was directly related to the concentration of [Ca2+]e, both of which correlated closely with the degree of stomatal closure. Increases were oscillatory in nature, with the pattern of the oscillations dependent on the concentration of [Ca2+]e. At 0.1 mM, [Ca2+]e induced symmetrical oscillations. In contrast, 1.0 mM [Ca2+]e induced asymmetric oscillations. Oscillations were stimulus dependent and modulated by changing [Ca2+]e. Experiments using Ca2+ channel blockers and Mn2+-quenching studies suggested a role for Ca2+ influx during the oscillatory behavior without excluding the possible involvement of Ca2+ release from intracellular stores. These data suggest a mechanism for encoding the information required to distinguish between a number of different Ca2+-mobilizing stimuli in guard cells, using stimulus-specific patterns of oscillations in [Ca2+]i.

Journal Article↗

Immediate hypersensitivity to leaf extracts of Callisia fragrans (inch plant) in a dog.

Contact with members of the plant family Commelinaceae, which includes wandering jew (Commelina spp. formerly called Tradescantia spp.) and inch plant (Callisia fragrans), can cause cell-mediated contact dermatitis in dogs. However, reports of canine IgE-mediated hypersensitivity to these plants have not been published. The purpose of this study was to discover whether IgE antibodies specific for extractable components of C. fragrans could be identified in serum from a dog that had anaphylactic shock after exposure to the plant and after skin patch testing with the sap from a leaf of C. fragrans. Separate aqueous extracts of leaves and flowers of C. fragrans were subjected to sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and Western blotting. Serum from a dog with no history or symptoms of any allergies showed no specific IgE antibodies against the leaf extract. Serum from a dog with clinical symptoms of delayed, but not immediate hypersensitivity to leaf sap from C. fragrans, showed only minor IgE recognition of a single 65 k component in sap extracted from leaves harvested in summer but not in winter. However, IgE antibodies to a serum dilution of 1:200 specific for several components of the leaf extract were seen in serum from the dog that had anaphylactic shock after exposure to sap. The molecular weights of these molecules were in the range 51 k to 83 k. The bands on the immunoblots did not match with prominently stained protein bands in the gel, but instead identified molecules in a lightly stained area of the gel with diffuse bands. Testing for glycans indicated that the carbohydrate side chains of glycoproteins contributed significantly to the immunoreactivity of the putative allergens. All three dog sera failed to show any immunoreactivity against the extract from the flowers of C. fragrans.

Anaphylaxis↗

Diet of eared doves (Zenaida auriculata, Aves, Columbidae) in a sugar-cane colony in South-eastern Brazil.

Farmers in the Paranapanema Valley (São Paulo, Brazil) have reported problems with flocks of Eared Doves (Zenaida auriculata) eating sprouting soybeans. In this region these birds breed colonially in sugar-cane, and eat four crop seeds, using 70% of the dry weight, in the following order of importance: maize, wheat, rice, and soybeans. Three weeds (Euphorbia heterophylla, Brachiaria plantaginea, and Commelina benghalensis) were important. This information suggests that the doves adapted particularly well to the landscape created by the agricultural practices in the region, exploiting many available foods.

Animals↗

A possible role for calcium in oxidative plant stress.

Disruption of calcium homoeostasis may be crucially important in damaging animal cells under oxidising conditions. In hepatocytes, oxidative stress causes an increase in cytosolic calcium, a process which is thought to be largely responsible for subsequent cell death. The oxidation of glutathione precedes a flux of calcium into the cytoplasm from internal and external sources. The possibility that similar events occur in plant cells has been tested using strips of epidermis taken from leaves of Commelina communis. The closure of stomata in response to many chemical or physical stimuli is thought to involve a transient rise in cytosolic calcium. Paraquat (10(-4) M) and hydrogen peroxide (10(-3) M) caused a marked reduction in stomatal aperture when included in the incubation medium of illuminated epidermal strips. The calcium channel blocker verapamil (10(-5) M) protected stomata from hydrogen peroxide-induced closure while the ion chelator EGTA (2 x 10(-3) M) protected stomata in both oxidising treatments. These results are discussed in so far as they support the hypothesis that a rise in cytosolic calcium may play a role in the pathology of oxidative stress in plant cells through a mechanism analogous to the liver cell system.

Calcium↗

High resolution imaging of photosynthetic activities of tissues, cells and chloroplasts in leaves.

Through imaging of chlorophyll fluorescence, it is possible to produce parameterized fluorescence images that estimate the operating quantum efficiency of photosystem II (PSII) photochemistry and which can be used to reveal heterogeneous patterns of photosynthetic performance within leaves. The operating quantum efficiency of PSII photochemistry is dependent upon the effective absorption cross-section of the light-harvesting system of PSII and the photochemical capacity of PSII. The effective absorption cross-section is decreased by the process of down-regulation, which is widely thought to operate within the pigment matrices of PSII and which results in non-photochemical quenching of chlorophyll fluorescence. The photochemical capacity is non-linearly related to the proportion of PSII centres in the 'open' state and results in photochemical quenching of chlorophyll fluorescence. Examples of heterogeneity of the operating quantum efficiency of PSII photochemistry during the induction of photosynthesis in maize leaves and in the chloroplast populations of stomatal guard cells of a leaf of Tradescantia albifora are presented, together with analyses of the factors determining this heterogeneity. A comparison of the operating quantum efficiency of PSII photochemistry within guard cells and adjacent mesophyll cells of Commelina communis is also made, before and after stomatal closure through a change in ambient humidity.

Chlorophyll↗

Microcosm wetlands for wastewater treatment with different hydraulic loading rates and macrophytes.

Constructed wetlands (CW) usually require large land areas for treating wastewater. This study evaluated the feasibility of applying CW with less land requirement by operating a group of microcosm wetlands at a hydraulic retention time (HRT) of less than 4 d in southern Taiwan. An artificial wastewater, simulating municipal wastewater containing 200 mg L(-1) of chemical oxygen demand (COD), 20 mg L(-1) of NH4+-N (AN), and 20 mg L(-1) of PO4(3-)-P (OP), was the inflow source. Three emergent plants [reed, Phragmites australis (Cav.) Trin. ex Steud.; water primrose, Ludwigia octovalvis (Jacq.) P.H. Raven; and dayflower, Commelina communis L.] and two floating plants [water spinach, Ipomoea aquatica Forssk.; and water lettuce, Pistia stratiotes L.] plants were tested. The planted systems showed more nutrient removal than unplanted systems; however, the type of macrophytes in CW did not make a major difference in treatment. At the HRTs of 2 to 4 d, the planted system maintained greater than 72,80, and 46% removal for COD, AN, and OP, respectively. For AN and OP removal, the highest efficiencies occurred at the HRT of 3 d, whereas maximum removal rates for AN and OP occurred at the HRT of 2 d. Both removal rates and efficiencies were reduced drastically at the HRT of 1 d. Removals of COD, OP, and AN followed first-order reactions within the HRTs of 1 to 4 d. The efficient removals of these constituents obtained with HRT between 2 and 4 d indicated the possibility of using a CW system for wastewater treatment with less land requirement.

Ecosystem↗

Immunological characteristics of PEP carboxylase from leaves of C3-, C4- and C3-C4 intermediate species of Alternanthera--comparison with selected C3- and C4- plants.

Immunological cross-reactivity of phosphoenolpyruvate carboxylase (PEPC) in leaf extracts of C3-, C4- and C3-C4 intermediate species of Alternanthera (along with a few other C3- and C4- plants) was studied using anti-PEPC antibodies raised against PEPC of Amaranthus hypochondriacus (belonging to the same family as that of Alternanthera, namely Amaranthaceae). Antibodies were also raised in rabbits against the purified PEPC from Zea mays (C4- monocot-Poaceae) as well as Alternanthera pungens (C4- dicot-Amaranthaceae). Monospecificity of PEPC-antiserum was confirmed by immunoprecipitation. Amount of PEPC protein in leaf extracts of A. hypochondriacus could be quantified by single radial immunodiffusion. Cros- reactivity of PEPC in leaf extracts from selected C3-, C4-, and C3-C4 intermediate species (including those of Alternanthera) was examined using Ouchterlony double diffusion and Western blots. Anti-PEPC antiserum raised against A. hypochondriacus enzyme showed high cross-reactivity with PEPC in leaf extracts of A. hypochondriacus or Amaranthus viridis or Alternanthera pungens (all C4 dicots), but limited cross-reactivity with that of Zea mays, Sorghum or Pennisetum (all C4 monocots). Interestingly, PEPC in leaf extracts of Alternanthera tenella, A. ficoides, Parthenium hysterophorus (C3-C4 intermediates) exhibited stronger cross-reactivity (with anti-serum raised against PEPC from Amaranthus hypochondriacus) than that of Pisum sativum, Commelina benghalensis, Altenanthera sessilis (C3 plants). Further studies on cross-reactivities of PEPC in leaf extracts of these plants with anti-PEPC antisera raised against PEPC from leaves of Zea mays or Alternanthera pungens confirmed two points--(i) PEPC of C3-C4 intermediate is distinct from C3 species and intermediate between those of C3- and C4-species; and (ii) PEPC of C4-dicots was closer to that of C3-species or C3-C4 intermediates (dicots) than to that of C4-monocots.

Amaranthaceae↗

[Transgenic cotton plants of chitinase and glucanase genes and their performance of resistance to Verticillium dahliea].

The two disease-resistance genes chitinase and glucanase, which were respectively directed by commelina yellow mottle virus promoter (CoYMV, vascular specific) and CaMV35S promoter, were introduced into cotton genome via Agrobacterium tumefaciens. Transgenic plants were obtained from two popularly cultivated varieties Jihe321 and CRIC35. After screening by spraying kanamycin over unfolding leaves, the kanamycin resistance (KmR) plants were tested by PCR and Southern blot. The results showed that there were one or two inserts of transgenes in cotton genome. Performance test of resistance of T3 families in field and greenhouse showed that seven lines were resistant or tolerant to Verticillium dahliea. Meanwhile, the resistance at seedling stage in greenhouse was in accordance with that at the boll-setting stage in field. Among the seven lines, D9910, D9915 and D9919 had a disease resistance index of 6.5, 9.4 and 9.5, respectively in field, which showed a high resistance level. Genetics analysis of the three lines showed a classical Mendelian pattern of one pair of genes, which meant that each of the three lines contains one copy of transgene. Southern blotting also confirmed the copy number of inserts.

Blotting, Southern↗

Visualisation and measurement of the calcium message in guard cells.

We have applied several novel technologies to investigate the role of cytosolic free calcium [Ca2+]i in signal transduction in guard cells of Commelina communis L. Fluorescence ratio imaging and photometry together with the fluorescent Ca2+ indicator Indo-1 were used to directly visualise and measure dynamic spatial and temporal changes in [Ca2+]i in response to various exogenous stimuli. More subtle manipulation of the Ca2+ signal transduction pathway was achieved through the use of photoactivateable, caged Ca2+ and caged inositol-1,4,5-triphosphate (InsP3) released directly into the cytoplasm of the guard cell after microinjection. In these experiments, changes in [Ca2+]i were simultaneously monitored with the fluorescent Ca2+ indicator, Fluo-3. Resting levels of [Ca2+]i (100-200 nM) increased in response to elevated [Ca2+]e, lowering [K+]e, application of the ionophore A-23187 or cytosolic release of either Ca2+ or InsP3 from their caged forms. Stomatal closure was triggered if [Ca2+]i increased above a threshold of about 600 nM. Abscisic acid (ABA) had little effect on [Ca2+]i in the majority of cells studied, being elevated in only a minority of cells investigated. However, stomatal closure occurred in all cases after ABA application. This suggests that ABA acts through both Ca(2+)-independent and Ca(2+)-dependent pathways. The imaging data revealed a substantial heterogeneity in [Ca2+]i within the guard cell. Cytoplasmic regions, particularly near the nucleus, often showed marked elevations and sometimes oscillations. The origin and kinetics of the Ca2+ fluxes leading to the dynamic spatial patterns is discussed along with several new approaches directed towards identification of the source of the Ca2+. These methods include optical sectioning and 3-D reconstruction of both the endomembrane system and [Ca2+]i in living guard cells using confocal microscopy. Overall, our data is consistent with multiple sources for [Ca2+]i, including uptake across the plasma membrane and InsP3- or Ca(2+)-induced Ca2+ release from internal stores.

Calcium↗

Voltage-gated Ca2+ release channels in vacuolar membranes from beet storage roots and guard cells.

The ubiquity and wide-ranging roles of Ca(2+)-mediated stimulus-response coupling in plant cells is likely to demand flexibility in the duration and amplitude of the cytosolic Ca2+ signal. The presence of both ligand- and voltage-gated Ca2+ release channels at the vacuolar membrane suggests that this flexibility might be achieved through differential regulation of these discrete channel classes. The properties and distribution of voltage-gated vacuolar Ca2+ channels is described in this chapter. In beet storage roots, the channels display a single channel conductance of 12 pS (5-20 mM Ca2+ on the lumenal side), Ca2+:K+ selectivity = 15:1 and a unitary current which saturates as a function of (physiological) negative voltages at about 0.5 pA. Channel opening is enhanced by negative voltages and by intravacuolar (lumenal) Ca2+. Variation of lumenal Ca2+ concentration has enabled the binding sites both for ionic selectivity and for Ca2+ gating to be mapped with respect to their fractional electrical distance across the membrane. Voltage-gated Ca2+ channels with similar permeation properties also reside in the vacuolar membranes of Vicia faba and Commelina communis guard cells. Although these channels exhibit somewhat higher unitary conductances and voltage-saturating currents than the beet channel, they are similar in many other respects, including sensitivity to Gd3+. We conclude that the widespread distribution of an emerging class of voltage-gated Ca2+ channel in the vacuolar membrane of plants points towards a fundamental role of these channels in plant cell biology.

Calcium↗

Control of volume and turgor in stomatal guard cells.

Water loss from plants is determined by the aperture of stomatal pores in the leaf epidermis, set by the level of vacuolar accumulation of potassium salt, and hence volume and turgor, of a pair of guard cells. Regulation of ion fluxes across the tonoplast, the key to regulation of stomatal aperture, can only be studied by tracer flux measurements. There are two transport systems in the tonoplast. The first is a Ca(2+)-activated channel, inhibited by phenylarsine oxide (PAO), responsible for the release of vacuolar K(+)(Rb(+)) in response to the "drought" hormone, abscisic acid (ABA). This channel is sensitive to pressure, down-regulated at low turgor and up-regulated at high turgor, providing a system for turgor regulation. ABA induces a transient stimulation of vacuolar ion efflux, during which the flux tracks the ion content (volume, turgor), suggesting ABA reduces the set-point of a control system. The second system, which is PAO-insensitive, is responsible for an ion flux from vacuole to cytoplasm associated with inward water flow following a hypo-osmotic transfer. It is suggested that this involves an aquaporin as sensor, and perhaps also as responder; deformation of the aquaporin may render it ion-permeable, or, alternatively, the deformed aquaporin may signal to an associated ion channel, activating it. Treatment with inhibitors of aquaporins, HgCl(2) or silver sulfadiazine, produces a large transient increase in ion release from the vacuole, also PAO-insensitive. It is suggested that this involves the same aquaporin, either rendered directly ion-permeable, or signalling to activate an associated ion channel.

Abscisic Acid↗

Tobacco ZFT1, a transcriptional repressor with a Cys2/His2 type zinc finger motif that functions in spermine-signaling pathway.

We previously proposed that a spermine (Spm)-mediated signal transduction pathway is involved in the hypersensitive response induced by Tobacco mosaic virus (TMV) in tobacco plants. To identify regulatory component(s) of this pathway, we surveyed a tobacco cDNA library and found that the ZFT1 gene, which encodes a Cys2/His2 type zinc-finger protein, is Spm-responsive. ZFT1 was not induced by two other polyamines, putrescine and spermidine, or by salicylic acid (SA), jasmonic acid or ethylene. Furthermore, ZFT1 was upregulated in TMV- inoculated tobacco plants in an N gene-dependent manner. Notably, induction of ZFT1 by Spm and by TMV infection was unimpaired in NahG-transgenic tobacco plants, indicating that cross-talk with an SA signaling pathway is not involved in this response. Within the Spm-signaling pathway, we found that ZFT1 functioned downstream of both mitochondrial dysfunction and mitogen-activated protein kinase activation. The ZFT1 protein has two zinc finger motifs and shows a high degree of similarity to ZPT2-3 in petunia and SCOF1 in soybean. However, unlike the latter two proteins, ZFT1 binds to the EP1S sequence and functions as a transcription repressor. Moreover, interestingly, ZFT1 overexpression rendered tobacco plants more tolerant to TMV. Based on the results presented here, we propose that ZFT1 functions as a transcription repressor in a Spm signaling pathway, thereby accelerating necrotic local region formation in tobacco leaves.

Amino Acid Sequence↗

Direct current electrical field effects on intact plant organs.

Intact plant tissues (of hypocotyls, radicles, cotyledons and leaves) were contracted by applying a low DC electrical field through them. Stomatal opening as a result of the electrical treatment of leaves was observed, presumably due to the differential influence of the electrical treatment on guard cell turgor pressure versus turgor pressure of the surrounding epidermal cells. In addition, leakage of minerals from the treated leaves was detected (higher contents of potassium, sodium, calcium and sulfur), as was leakage of betanin from electrically treated red beet roots (higher OD value of the immersion solution with increasing time of applied electrical field). Application of such a treatment can be used for initial drying or as part of another more drastic drying method. The advantages of this method lie in its nonthermal character and its potential to increase the quality of processed foods by maintaining their "like-fresh" quality, e.g., fruits and vegetables that are less damaged by browning. An understanding of the mechanism involved in this nonthermal application can help in controlling the process and predicting its outcome.

Betacyanins↗

Cell wall arabinan is essential for guard cell function.

Stomatal guard cells play a key role in the ability of plants to survive on dry land, because their movements regulate the exchange of gases and water vapor between the external environment and the interior of the plant. The walls of these cells are exceptionally strong and must undergo large and reversible deformation during stomatal opening and closing. The molecular basis of the unique strength and flexibility of guard cell walls is unknown. We show that degradation of cell wall arabinan prevents either stomatal opening or closing. This locking of guard cell wall movements can be reversed if homogalacturonan is subsequently removed from the wall. We suggest that arabinans maintain flexibility in the cell wall by preventing homogalacturonan polymers from forming tight associations.

Cell Wall↗