Ca2+ as developmental signal in the formation of Ca-oxalate crystal spacing patterns during leaf development inCarya ovata

Changes in the spacing patterns of Ca-oxalate crystals during enlargement ofCarya ovata Mill. leaves were quantified by computerized image-analysis. Single Ca-oxalate crystals form in the vacuoles of young mesophyll cells transformed into crystal cells Crystals are very small in newly induced crystal cells and increase in size throughout leaf development. Crystal patterns thus reflect both induction and relative age of crystal cells. Shortly after the emergence of young leaves from the bud, very small crystals are formed in the mesophyll at high density. As leaves expand, these crystals grow larger and become separated by increasing distances. New small crystals appear in the gaps between the older, larger crystals. Later crystal patterns consist of widely spaced, larger crystals only. Finally, clusters of small crystals are formed again in the gaps between large crystals. No crystals were observed in young leaves expanding in a moist chamber, but large numbers of crystal cells were induced experimentally in sections of immature leaves floating on 4 mM Ca-acetate. The observations support the following mechanism of crystal-pattern formation: Ca²⁺ carried into leaves with the transpiration stream acts as the developmental signal inducing transdifferentiation of a few mesophyll cells into crystal cells when apoplastic [Ca²⁺] rises. Crystal cells precipitate absorbed Ca²⁺ as oxalate and, acting as Ca²⁺ sinks, inhibit crystal-cell induction in their vicinity by depleting apoplastic Ca²⁺. This prevents close spacing of crystal cells. New crystal cells form in the gaps between the depletion zones of older crystal cells when these move apart during leaf expansion. Later changes in crystal patterns result from increasing sink strength of crystal cells, lowered inducibility of mesophyll cells, and increased Ca²⁺ influx into leaves during intensive transpiration. Throughout leaf development, spacing of crystal cells permits rapid secretion of apoplastic Ca²⁺ as Ca-oxalate. Dedicated to Professor Erwin Bünning, University of Tübingen, Germany, who pioneered the analysis of spacing patterns     

Calcium acetate induces calcium uptake and formation of calcium-oxalate crystals in isolated leaflets of Gleditsia triacanthos L.

During treatment of isolated, peeled leaflets of Gleditsia triacanthos with 0.5–2 mM [⁴⁵Ca]acetate, saturation of the cell-wall free space with Ca²⁺ occurred within 10 min and was followed by a period of 6–10 h during which there was no significant Ca-uptake into the protoplast, but apoplastic Ca²⁺ was periodically released into the medium. Later, Ca²⁺ was absorbed for 3–4 d at rates of up to 2.2 mol Ca²⁺·h^-1·(g FW)^-1 to final concentrations of 350 mol Ca²⁺· (g FW)^-1. The distribution of absorbed Ca²⁺ between cell wall, vacuole and Ca-oxalate crystals was determined during Ca-uptake. Wheras intact, cut leaflets deposited absorbed Ca²⁺ as Ca-oxalate in the crystal cells, peeled leaflets lacking crystal cells accumulated at least 40–50 mol·(g FW)^-1 soluble Ca²⁺ before the absorbed Ca²⁺ was precipitated as Ca-oxalate. These observations indicate that the mechanisms for the continuous uptake of Ca²⁺, the synthesis of oxalate and the precipitation of Ca²⁺ as Ca-oxalate are operational in the crystal cells of intact leaflets, but not in the mesophyll cells of peeled leaflets where they must be induced by exposure to Ca²⁺. The precipitation of absorbed Ca²⁺ as Ca-oxalate by the crystal cells of isolated Gleditsia leaflets illustrates the role of these cells in the excretion of surplus Ca²⁺ which enters normal, attached leaves with the transpiration stream.In addition to acetate, only Ca-lactate and Ca-carbonate lead to Ca-uptake, but at rates well below those observed with Ca-acetate. Other small organic anions (citrate, glycolate, glyoxalate, malate) and inorganic anions (chloride, nitrate, sulfate) did not permit Ca-uptake. Acetate-¹⁴C was rapidly absorbed during Ca-uptake, but less than 20% was incorporated into Ca-oxalate; the rest remained mostly in the soluble fraction or was metabolized to CO₂. Acetate, as a permeable weak acid, may enable rapid Ca-uptake by stimulating proton extrusion at the plasmalemma and by serving as a counterion during Ca-accumulation in the vacuole, but is unlikely to function as the principal substrate for oxalate synthesis.     

Cell and calcium oxalate crystal growth is coordinated to achieve high-capacity calcium regulation in plants

Summary Crystal idioblasts are cells which are specialized for accumulation of Ca²⁺ as a physiologically inactive, crystalline salt of oxalic acid. Using microautoradiographic, immunological, and ultrastructural techniques, the process of raphide crystal growth, and how crystal growth is coordinated with cell growth, was studied in idioblasts ofPistia stratiotes. Incorporation of⁴⁵Ca²⁺ directly demonstrated that, relative to surrounding mesophyll cells, crystal idioblasts act as high-capacity Ca²⁺ sinks, accumulating large amounts of Ca²⁺ within the vacuole as crystals. The pattern of addition of Ca²⁺ during crystal growth indicates a highly regulated process with bidirectional crystal growth. In very young idioblasts,⁴⁵Ca²⁺ is incorporated along the entire length of the needle-shaped raphide crystals, but as they mature incorporation only occurs at crystal tips in a bidirectional mode. At full maturity, the idioblast stops Ca²⁺ uptake, although the cells are still alive, demonstrating an ability to strictly regulate Ca transport processes at the plasma membrane. In situ hybridization for ribosomal RNA shows young idioblasts are extremely active cells, are more active than older idioblasts, and have higher general activity than surrounding mesophyll cells. Polarizing and scanning electron microscopy demonstrate that the crystal morphology changes as crystals develop and includes morphological polarity and an apparent nucleation point from which crystals grow bidirectionally. These results indicate a carefully regulated process of biomineralization in the vacuole. Finally, we show that the cytoskeleton is important in controlling the idioblast cell shape, but the regulation of crystal growth and morphology is under a different control mechanism.Abbreviation SEM scanning electron microscopy     

Absence of CeCl3-detectable peroxisomal glycolate-oxidase activity in developing raphide crystal idioblasts in leaves of Psychotria punctata Vatke and roots of Yucca torreyi L.

Three peroxisomal enzymes, glycolate oxidase, urate oxidase and catalase were localized cytochemically in Psychotria punctata (Rubiaceae) leaves and Yucca torreyi (Agavaceae) seedling root tips, both of which contain developing and mature calcium-oxalate raphide crystal idioblasts. Glycolate-oxidase (EC 1.1.3.1) and catalase (EC 1.11.1.6) activities were present within leaftype peroxisomes in nonidioblastic mesophyll cells in Psychotria leaves, while urate-oxidase (EC 1.7.3.3) activity could not be conclusively demonstrated in these organelles. Unspecialized peroxisomes in cortical parenchyma of Yucca roots exhibited activities of all three enzymes. Reactionproduct deposits attributable to glycolate-oxidase activity were never observed in peroxisomes of any developing or mature crystal idioblasts of Psychotria or Yucca. Catalase localization indicates that idioblast microbodies are functional peroxisomes. The apparent absence of glycolate oxidase in crystal idioblasts of Psychotria and Yucca casts serious doubt that pathways involving this enzyme are operational in the synthesis of the oxalic acid precipitated as calcium-oxalate crystals in these cells.Abbreviations AMPD 2-amino-2-methyl-1,3-propandiol - CTEM conventional transmission electron microscopy - DAB 3,3-diaminobenzidine tetrahydrochloride - HVEM high-voltage electron microscopy     

Efflux of sucrose from minor veins of tobacco leaves

Mature leaves import limited amounts of nutrient when darkened for prolonged periods. We tested the hypothesis that import is restricted by the apoplast-phloem loading mechanism, ie., as sucrose exits the phloem of minor veins it is retrieved by the same tissue, thus depriving the mesophyll of nutrient. When single, attached, mature leaves of tobacco (Nicotiana tabacum L.) plants were darkened, starch disappeared from the mesophyll cells, indicating that the supply of solute to the mesophyll was limited. Starch was synthesized in mesophyll cells of darkened tissue when sucrose was applied to the apoplast at 0.1–0.3 mM concentration. Efflux from minor veins was studied by incubating leaf discs on [¹⁴C]sucrose to load the minor veins and then measuring subsequent ¹⁴C release. Efflux was rapid for the first hour and continued at a gradually decreasing rate for over 13 h. Net efflux increased when loading was inhibited by p-chloromercuribenzene-sulfonic acid, anoxia, isotope-trapping, or reduction of the pH gradient. Neither light nor potassium had a significant effect on the rate of labeled sucrose release. The site of labeled sucrose release was investigated by measuring efflux from discs in which sucrose had previously been loaded preferentially by either the minor veins or mesophyll cells. Efflux occurred primarily from minor veins.Abbreviations Mes 2(N-morpholino)ethanesulfonic acid - Mops 3(N-morpholino)propanesulfonic acid - PCMBS p-chloromercuribenzenesulfonic acid - SE-CC sieve element-companion cell complex     

Ca(2+)-dependent in vivo protein phosphorylation and encystment induction in the ciliated protozoan Colpoda cucullus

Encystment induction of Colpoda cucullus is promoted by an increase in external Ca²⁺ and overpopulation of Colpoda vegetative cells. Using phos-tag detection assays, the present study revealed that the in vivo phosphorylation level in several proteins [33 kDa, 37 kDa, 37.5 kDa, 43 kDa, 47 kDa, 49 kDa, etc.] was raised when the vegetative cells were stimulated by overpopulation to encyst in a medium containing 0.1 mM Ca²⁺ or without the addition of Ca²⁺. Both overpopulation-mediated encystment induction and protein phosphorylation were suppressed by the addition of EGTA. Ca²⁺/overpopulation-stimulated encystment induction and protein phosphorylation were also suppressed by the addition of BAPTA-AM. These results suggest that the Ca²⁺ inflow promoted by cell-to-cell stimulation due to overpopulation may activate signaling pathways involving protein phosphorylation and encystment induction. In the presence of cAMP-AM, the phosphorylation levels of 33 kDa, 37 kDa, 37.5 kDa, 43 kDa, 47 kDa and 49 kDa proteins were enhanced, and encystment induction was promoted. Enzyme immunoassays (EIAs) showed that intracellular cAMP concentration was raised prior to encystment when the cells were stimulated by overpopulation. These results suggest that cAMP/PKA-dependent protein phosphorylation, which is an event on Ca²⁺-triggered signaling pathways, may be involved in encystment induction.     

Reduced external calcium or sodium stimulates calcium influx in Pelvetia eggs

The effect of external calcium and sodium ion concentrations on the calcium fluxes on the Pelvetia fastigiata De Toni egg was measured. Decreasing external [Ca²⁺] greatly increased the permeability of the eggs to Ca²⁺; at 1 mM external Ca²⁺ this permeability was 60 times as great as it was at the normal [Ca²⁺] of 10 mM. Lowering the external [Na⁺] also increased Ca²⁺ influx; at 2 mM Na⁺, the Ca²⁺ influx was 2–3 times as great as it was at the normal [Na⁺] if choline was used as a Na⁺ substitute. Lithium was less effective as a Na⁺ substitute in increasing Ca²⁺ influx. The extra Ca²⁺ influx in low [Na⁺] seemed to be dependent on internal [Na⁺]. The Ca²⁺ efflux increased transiently and then declined in low Na⁺ media.     

Influx of extracellular Ca2+ involved in jasmonic-acid-induced elevation of [Ca2+]cyt and JR1 expression in Arabidopsis thaliana

The changes in cytosolic Ca²⁺ levels play important roles in the signal transduction pathways of many environmental and developmental stimuli in plants and animals. We demonstrated that the increase in cytosolic free Ca²⁺ concentration ([Ca²⁺]_cyt) of Arabidopsis thaliana leaf cells was induced by exogenous application of jasmonic acid (JA). The elevation of [Ca²⁺]_cyt was detected within 1 min after JA treatment by the fluorescence intensity using laser scanning confocal microscopy, and the elevated level of fluorescence was maintained during measuring time. With pretreatment of nifedipine (Nif), a nonpermeable L-type channel blocker, the fluorescence of [Ca²⁺]_cyt induced by JA was inhibited in a dose-dependent manner. In contrast, verapamil, another L-type channel blocker, had no significant effect. Furthermore, Nif repressed JA-induced gene expression of JR1 but verapamil did not. JA-induced gene expression could be mimicked by higher concentration of extracellular Ca²⁺. W-7 [N-(6-aminohexyl)-5-chloro-1-naphthalenesulfonamide], an antagonist of calmodulin (CaM), blocked the JA induction of JR1 expression while W-5 [N-(6-aminohexyl)-1-naphthalenesulfonamide], its inactive antagonist, had no apparent effect. These data provide the evidence that the influx of extracellular Ca²⁺ through Nif sensitive plasma membrane Ca²⁺ channel may be responsible for JA-induced elevation of [Ca²⁺]_cyt and downstream gene expression, CaM may be also involved in JA signaling pathway.     

香樟叶肉含晶细胞季节变化研究

为探讨香樟(Cinnamomum camphora)叶肉含晶细胞超微结构的季节变化,阐明香樟叶肉中草酸钙晶体在春夏秋冬的变化规律。该研究以多年生香樟(C. camphora)叶片为材料,分别于春夏秋冬四个季节露地取样,制作超薄切片,用透射电子显微镜(TEM)观察叶肉含晶细胞超微结构的变化。结果表明:春季时香樟叶肉中只有少数细胞有草酸钙晶体,数量较少,晶体结构多为柱状晶、方晶; 夏季时香樟叶肉细胞中随机分布于液泡的草酸钙晶体明显比春季的数量多、体积大、形态丰富,晶体多为柱状晶、方晶、针晶、簇晶; 秋季时香樟叶肉细胞草酸钙晶体和夏季的类似,数量较多,形态多样,以方晶和柱状晶针晶为主,伴有晶簇; 冬季时香樟叶肉含晶细胞晶体形态为柱状晶、方晶、针晶,数量比夏季和秋季的数量略有减少。该研究结果表明在一年四季中香樟叶肉细胞液泡中均有草酸钙晶体结构存在。     

Baicalein-Induced Apoptosis via Endoplasmic Reticulum Stress Through Elevations of Reactive Oxygen Species and Mitochondria Dependent Pathway in Mouse–Rat Hybrid Retina Ganglion Cells (N18)

Studies were designed to investigate the effects of baicalein on mouse–rat hybrid retina ganglion cells (N18) to better understand its effect on apoptosis and apoptosis-related genes in vitro. Cell viability, reactive oxygen species (ROS), cytoplasmic Ca²⁺, mitochondrial membrane potential (MMP), apoptosis induction, and caspases-3 activity were examined by flow cytometric assay. Apoptosis-associated proteins such as p53, Bax, Bcl-2, cytochrome c, and caspase-3 were examined by Western blot. We demonstrated the increase in the levels of p53, Bax, and cytochrome c and decrease in the level of Bcl-2, which are associated with the induction of apoptotic cell death after 24 h treatment with baicalein in N18 cells. Baicalein induced an increase in the cytoplasmic levels of ROS and Ca²⁺ in 1 h and reached their peak at 3 h, and thereafter a loss of MMP by flow cytometry. We also demonstrated a release of the cytochrome c from mitochondria into cytosol and an activation of caspase-3, which led to the occurrence of apoptosis in N18 cells treated with baicalein by Western blot. Pretreatment was conducted with BAPTA (intracellular calcium chelator) in baicalein-treated cells, the decline of MMP was recovered, and the increase in the level of cytoplasmic Ca²⁺ was suppressed, and the proportion of apoptosis was also markedly diminished. In conclusion, our data suggests that oxidative stress and cellular Ca²⁺ modulates the baicalein-induced cell death via a Ca²⁺-dependent mitochondrial death pathway in N18 cells.     

Involvement of ATP,increase of intracellular calcium and the early expression of <Emphasis Type="Italic">c-fos</Emphasis> in the repair of rat fetal articular cartilage

To compare the potential of adult and fetal animals to repair articular cartilage, we investigated the early process after creating superficial defects in the femoral knee cartilage in rat models. In fetuses at 19 days of gestation, both chondrocytes and the extracellular matrix responded notably by 48 h after artificial injury. Staining patterns with safranin O revealed that, by 1 h after injury, some components of the extracellular matrix around the wound were modified, and the change spread from the limited region to the entire knee cartilage within 24 h. The chondrocytes in the area surrounding the wound transiently expressed increased level of c-fos from 1 h to 6 h. The wound remained 1 day after birth, i.e., 72 h after injury, but was completely repaired 10 days after birth. In contrast, neither visible responses nor transient c-fos expression was observed in 12-week-old adult articular cartilage 48 h after injury. We also examined the relationships between the intracellular Ca²⁺ concentration ([Ca²⁺]_i) and the induction of c-fos expression in the cartilage. Applications of ATP or Ca²⁺ ionophore A23187, both of which increase [Ca²⁺]_i, induced immediate expression of c-fos in primary cultured chondrocytes: 1 M ATP elicited an increase of [Ca²⁺]_i in chondrocytes in fetal cartilage slices, but 1 mM was required in adult cartilage slices. Our findings show the presence of a signaling pathway that is apparently active in the repair of fetal but not adult articular cartilage and that involves the intercellular transfer of ATP, increase of [Ca²⁺]_i, and expression of c-fos in cartilage.This study was supported in part by Health Sciences Research Grants for Research on Human Genome, Tissue Engineering and Food Biotechnology to M.O. from the Japanese Ministry of Health, Labor and Welfare     

Turgor regulation and cytoplasmic free Ca2+ in the algaLamprothamnium

Summary In internodal cells ofLamprothamnium succinctum, turgor regulation in response to hypotonie treatment is inhibited by lowering external Ca²⁺ concentration ([Ca²⁺]_e) from 3.9 (normal) to 0.01 (low) mM. In order to clarify whether a change in the cytoplasmic free Ca²⁺ concentration ([Ca²⁺]_c) is involved in turgor regulation, the Ca²⁺ sensitive protein aequorin was injected into the cytoplasm of internodal cells. A large transient light emission was observed upon hypotonic treatment under normal [Ca²⁺]_e but not under low [Ca²⁺]_e. Thus hypotonic treatment induces a transient increase in [Ca²⁺]_c under normal [Ca²⁺]_e but not under low [Ca²⁺]_e.Abbreviations ASW artificial sea water - _i cellular osmotic pressure - [Ca²⁺]_c cytoplasmic free Ca²⁺ concentration - EDTA ethylenediamine-tetraacetic acid - EGTA ethylenglycol-bis(-aminoethyl ether(N,N-tetraacetic acid - [Ca²⁺]_e external Ca²⁺ concentration - _e external osmotic pressure - GM glass micropipette - GP glass plate - HEPES N-2-hydroxyethylpiperazine-N-2-ethansulfonic acid - MS microscope stage - OL objective lens - PIPES piperazine-N-N-bis(2-ethanesulfonic acid) - W Weight     

Ajmalicine production in methyl jasmonate-induced Catharanthus roseus cell cultures depends on Ca2+ level

Cytosolic Ca²⁺ and jasmonate mediate signals that induce defense responses in plants. In this study, the interaction between Ca²⁺ and methyl jasmonate (MJ) in modulating defense responses was investigated by monitoring ajmalicine production in Catharanthus roseus suspension cultures. C. roseus suspensions were treated with nine combinations of CaCl₂ (3, 23, and 43 mM) and MJ (0, 10, and 100 μM) on day 6 of growth. Increased Ca²⁺ influx through the addition of extracellular CaCl₂ suppressed ajmalicine production in MJ-induced cultures. The highest ajmalicine production (4.75 mg/l) was observed when cells were treated with a low level of calcium (3 mM) combined with a high level of MJ (100 μM). In the presence of 3 mM CaCl₂ in the medium, the addition of Ca²⁺ chelator EGTA (1, 2.5, and 5 mM) or Ca²⁺ channel blocker verapamil (1, 10, and 50 μM) to MJ-induced (100 μM) cultures on day 6 also inhibited ajmalicine production at higher levels of the Ca²⁺ inhibitors. Hence, ajmalicine production in MJ-induced C. roseus cultures depended on the intracellular Ca²⁺ concentration and a low extracellular Ca²⁺ concentration (3 mM) enhanced MJ-induced ajmalicine production.     

Spatial and temporal patterns of intracellular calcium in colonic smooth muscle

Summary Intracellular calcium [Ca²⁺] _i measurements in cell suspension of gastrointestinal myocytes have suggested a single [Ca²⁺] _i transient followed by a steady-state increase as the characteristic [Ca²⁺] _i response of these cells. In the present study, we used digital video imaging techniques in freshly dispersed myocytes from the rabbit colon, to characterize the spatiotemporal pattern of the [Ca²⁺] _i signal in single cells. The distribution of [Ca²⁺] _i in resting and stimulated cells was nonhomogeneous, with gradients of high [Ca²⁺] _i present in the subplasmalemmal space and in one cell pole. [Ca²⁺] _i gradients within these regions were not constant but showed temporal changes in the form of [Ca²⁺] _i oscillations and spatial changes in the form of [Ca²⁺] _i waves. [Ca²⁺] _i oscillations in unstimulated cells (n = 60) were independent of extracellular [Ca²⁺] and had a mean frequency of 12.6 +1.1 oscillations per min. The baseline [Ca²⁺], was 171 ± 13 nm and the mean oscillation amplitude was 194 ± 12 nm. Generation of [Ca²⁺] _i waves was also independent of influx of extracellular Ca²⁺. [Ca²⁺] _i waves originated in one cell pole and were visualized as propagation mostly along the subplasmalemmal space or occasionally throughout the cytoplasm. The mean velocity was 23 +3 m per sec (n = 6). Increases of [Ca²⁺] _i induced by different agonists were encoded into changes of baseline [Ca²⁺] _i and the amplitude of oscillations, but not into their frequency. The observed spatiotemporal pattern of [Ca²⁺] _i regulation may be the underlying mechanism for slow wave generation and propagation in this tissue. These findings are consistent with a [Ca²⁺] _i regulation whereby cell regulators modulate the spatiotemporal pattern of intracellularly generated [Ca²⁺] _i oscillations.The authors thank Debbie Anderson for excellent technical assistance with the electron microscopy and Dr. M. Regoli for providing the NK-1 agonist [Sar⁹,Met(O₂)¹¹]-SP. This work was supported by National Institutes of Health Grants DK 40919 and DK 40675 and Veterans Administration Grant SMI.     

Differential Mechanisms of Glutamate-Stimulated Perturbations in the Kinetics of c-fos mRNA Induction Are Associated with Maturation of Cerebellar Granule Cells in Primary Culture

In further exploring proposals for the measurement of early gene (c-fos mRNA) levels as a predictive index for in vitro excitotoxicity, this study, using immature (2 days in vitro) cultures of mouse cerebellar granule cells as an experimental model system, was undertaken to determine the effect of glutamate (Glu) i) in stimulating increases in intracellular free-calcium ([Ca²⁺]_i), ii) on cell viability and iii) on induction of steady-state c-fos mRNA levels. In parallel experiments the action of agents (viz. 55 mM KCl and the calcium ionophore, A23187) that mediate Ca²⁺ entry into cells via different routes was also evaluated. Glu was unable to induce excitotoxicity in granule cells at this stage of development in culture, but did stimulate a concentration-dependent and marked increase in [Ca²⁺]_i levels while also mediating a dramatic concentration-dependent perturbation in the kinetics of c-fos mRNA induction that appeared to arise solely from NMDA receptor-mediated Ca²⁺ influx. The results are presented in comparison to the actions of KCl and A23187 and considered in relation to earlier studies undertaken using mature (7 days in vitro) cultures of cerebellar granule cells.     

Effects of salt on growth,ion accumulation,photosynthesis and leaf anatomy of the mangrove,<Emphasis Type="Italic"> Bruguiera parviflora</Emphasis>

The effects of a range of salinity (0, 100, 200 and 400 mM NaCl) on growth, ion accumulation, photosynthesis and anatomical changes of leaves were studied in the mangrove, Bruguiera parviflora of the family Rhizophoraceae under hydroponically cultured conditions. The growth rates measured in terms of plant height, fresh and dry weight and leaf area were maximal in culture treated with 100 mM NaCl and decreased at higher concentrations. A significant increase of Na⁺ content of leaves from 46.01 mmol m^-2 in the absence of NaCl to 140.55 mmol m^-2 in plants treated with 400 mM NaCl was recorded. The corresponding Cl^- contents were 26.92 mmol m^-2 and 97.89 mmol m^-2. There was no significant alteration of the endogenous level of K⁺ and Fe²⁺ in leaves. A drop of Ca²⁺ and Mg²⁺ content of leaves upon salt accumulation suggests increasing membrane stability and decreased chlorophyll content respectively. Total chlorophyll content decreased from 83.44 g cm^-2 in untreated plants to 46.56 g cm^-2 in plants treated with 400 mM NaCl, suggesting that NaCl has a limiting effect on photochemistry that ultimately affects photosynthesis by inhibiting chlorophyll synthesis (ca. 50% loss in chlorophyll). Light-saturated rates of photosynthesis decreased by 22% in plants treated with 400 mM NaCl compared with untreated plants. Both mesophyll and stomatal conductance by CO₂ diffusion decreased linearly in leaves with increasing salt concentration. Stomatal and mesophyll conductance decreased by 49% and 52% respectively after 45 days in 400 mM NaCl compared with conductance in the absence of NaCl. Scanning electron microscope study revealed a decreased stomatal pore area (63%) in plants treated with 400 mM NaCl compared with untreated plants, which might be responsible for decreased stomatal conductance. Epidermal and mesophyll thickness and intercellular spaces decreased significantly in leaves after treatment with 400 mM NaCl compared with untreated leaves. These changes in mesophyll anatomy might have accounted for the decreased mesophyll conductance. We conclude that high salinity reduces photosynthesis in leaves of B. parviflora, primarily by reducing diffusion of CO₂ to the chloroplast, both by stomatal closure and by changes in mesophyll structure, which decreased the conductance to CO₂ within the leaf, as well as by affecting the photochemistry of the leaves.     

Effects of membrane potential on calcium fluxes of Pelvetia eggs

⁴⁵Ca²⁺ fluxes across the plasma membrane of zygotes of the fucoid alga, Pelvetia fastagiata (J. Ag.) De Toni, were studied in artificial sea waters of various potassium concentrations. Except for two cases, hyperpolarization of the cell membrane (with low [K⁺]) increases, and depolarization (with high [K⁺]) decreases the influx of Ca²⁺ over the range of [K⁺] studied (1–100 mM). The fractional increases of influx during hyperpolarization are close to the fractional increases in membrane potential but the decreases during depolarization are much smaller than those in membrane potential. In two anomalous cases, the influxes of ⁴⁵Ca²⁺ at a potassium concentration of 30 mM were about 20% higher than the control value instead of being 10% lower.The effluxes of ⁴⁵Ca²⁺ are increased by both hyperpolarization and by depolarization. On balance (and excepting the two anomalous cases) the net result of hyperpolarization should be to increase and that of depolarization to decrease intracellular [Ca²⁺].     

External bioenergy-induced increases in intracellular free calcium concentrations are mediated by Na+/Ca2+ exchanger and L-type calcium channel

External bioenergy (EBE, energy emitted from a human body) has been shown to increase intracellular calcium concentration ([Ca²⁺]_i, an important factor in signal transduction) and regulate the cellular response to heat stress in cultured human lymphoid Jurkat T cells. In this study, we wanted to elucidate the underlying mechanisms. A bioenergy specialist emitted bioenergy sequentially toward tubes of cultured Jurkat T cells for one 15-minute period in buffers containing different ion compositions or different concentrations of inhibitors. [Ca²⁺]_i was measured spectrofluorometrically using the fluorescent probe fura-2. The resting [Ca²⁺]_i in Jurkat T cells was 70 ± 3 nM (n = 130) in the normal buffer. Removal of external calcium decreased the resting [Ca²⁺]_i to 52 ± 2 nM (n = 23), indicating that [Ca²⁺] entry from the external source is important for maintaining the basal level of [Ca²⁺]_i. Treatment of Jurkat T cells with EBE for 15 min increased [Ca²⁺]_i by 30 ± 5% (P 0.05, Student t-test). The distance between the bioenergy specialist and Jurkat T cells and repetitive treatments of EBE did not attenuate [Ca²⁺]_i responsiveness to EBE. Removal of external Ca²⁺ or Na⁺, but not Mg²⁺, inhibited the EBE-induced increase in [Ca²⁺]_i. Dichlorobenzamil, an inhibitor of Na⁺/Ca²⁺ exchangers, also inhibited the EBE-induced increase in [Ca²⁺]_i in a concentration-dependent manner with an IC50 of 0.11 ± 0.02 nM. When external [K⁺] was increased from 4.5 mM to 25 mM, EBE decreased [Ca²⁺]_i. The EBE-induced increase was also blocked by verapamil, an L-type voltage-gated Ca²⁺ channel blocker. These results suggest that the EBE-induced [Ca²⁺]_i increase may serve as an objective means for assessing and validating bioenergy effects and those specialists claiming bioenergy capability. The increase in [Ca²⁺]_i is mediated by activation of Na⁺/Ca²⁺ exchangers and opening of L-type voltage-gated Ca²⁺ channels. (Mol Cell Biochem 271: 51–59, 2005)     

Second-messenger-induced signalling events in pollen tubes of Papaver rhoeas

A role for cytosolic free Ca²⁺ (Ca²⁺_i) in the regulation of growth of Papaver rhoeas pollen tubes during the self-incompatibility response has recently been demonstrated [Franklin-Tong et al. Plant J. 4:163–177 (1993); Franklin-Tong et al. Plant J. 8:299–307 (1995); Franklin-Tong et al. submitted to Plant J.]. We have investigated the possibility that Ca²⁺_i is more generally involved in the regulation of pollen tube growth using confocal laser scanning microscopy (CLSM). Data obtained using Ca²⁺ imaging, in conjunction with photolytic release of caged inositol 1,4,5-trisphosphate [Ins(1,4,5)P₃], point to a central role of the phosphoinositide signal transduction pathway in the control of Ca²⁺ fluxes and control of pollen tube growth. These experiments further revealed that increases in cytosolic levels of Ins(1,4,5)P₃ resulted in the formation of distinct Ca²⁺ waves. Experiments using the pharmacological agents heparin, neomycin and mastoparan further indicated that Ca²⁺ waves are propagated, at least in part, by Ins(1,4,5)P₃-induced Ca²⁺ release rather than by simple diffusion or by “classic” Ca²⁺-induced Ca²⁺ release mechanisms. We also have data which suggest that Ca²⁺ waves and oscillations may be induced by photolytic release of caged Ca²⁺. Ratio-imaging has enabled us to identify an apical oscillating Ca²⁺ gradient in growing pollen tubes, which may regulate normal pollen tube growth. We also present evidence for the involvement of Ca²⁺ waves in mediating the self-incompatibility response. Our data suggest that changes in Ca²⁺_i and alterations in growth rate/patterns are likely to be closely correlated and may be causally linked to events such as Ca²⁺-induced, or Ins(1,4,5)P₃-induced wave formation and apical Ca²⁺ oscillations.Presented at the 1997 SEB Annual Meeting: Interactive MultiMedia Biology - Experimental Biology Online Symposium, Canterbury, 7-11 April     

Ca2+ and Mg2+ ions counteract the reduction by fosetyl-Al (aluminum tris[ethyl phosphonate]) of peroxidase activity from suspension-cultured grapevine cells

Grapevine (Vitis vinifera cv. Monastrell) cell suspension cultures were treated with 1.5 mM fosetyl-Al, a frequently used systemic fungicide for grapevine diseases caused by oomycetes. These cells showed a reduction in the level of peroxidase activity secreted into the culture media when compared to non-treated cells, the effect being mainly related to a decrease in the level of the basic B₁ peroxidase isozyme. The effect of fosetyl-Al on peroxidase was analogous to that observed with the Ca²⁺-channel blockers Co²⁺, Cd²⁺ and La³⁺, and was counteracted by Ca²⁺ ions, but was not reversed when the Ca²⁺-ionophore A23187 was added to the culture media. Moreover, the effect of fosetyl-Al on peroxidase activity and peroxidase isozymes was also partially reversed by Mg²⁺ ions but not by Sr²⁺, and was accentuated by Ba²⁺ ions. These results suggested that Ca²⁺ and Mg²⁺ ions specifically overcome the inhibitory effect of fosetyl-Al on peroxidase. In this context, an apoplastic Ca²⁺/Mg²⁺-displacement hypothesis is proposed for the mechanism of action of fosetyl-Al on peroxidase from grapevine cells.