Cytochemical Studies on Phytochrome-Mediated Changes of Ca2+ Localization in Etiolated Oat Coleoptile Cells

The antimonate-staining procedure and X-ray microanalysis techniquewere used to determine the pattern of Ca₂₊ localization in etiolatedoat (Avena sativa L.) coleoptile parenchyma cells. Precipitatesof calcium antimonate, indicating the presence of Ca₂₊ and confirmedby X-ray microanalysis, were found associated with the outerand inner surfaces of the plasma membrane of cells of dark-grownseedlings. After exposure of seedlings to red light, precipitatesof calcium antimonate were additionally observed in cisternaeof the endoplasmic reticulum. In the cells of oat coleoptilesexposed to red light and then followed immediately by farredlight, Ca₂₊ was observed on the outside of the plasma membrane,in cell walls and in the vacuoles. The results suggest thatphytochrome mediates the regulation of the intracellular Ca₂₊localization. Key words: Antimonate procedure, Avena sativa L., Ca²⁺ (localization), phytochrome, X-ray microanalysis     

Cytochemical Localization of Calcium and Ca2+-ATPase Activity in Plant Cells under Chilling Stress: a Comparative Study between the Chilling-Sensitive Maize and the Chilling-Insensitive Winter Wheat

Electron microscopic observations revealed that electron-denseantimonate Ca²⁺ deposits were mostly localized in the vacuoleand the intercellular space in both maize and winter wheat whentheir seedlings were grown at 25°C. The reaction products—ceriumphosphate deposits of Ca²⁺- ATPase activity were mainly seenat the cytoplasmic side of the plasma membrane. Few cerium phosphatedeposits also were observed on the nuclear envelope. In bothspecies, after 1 or 3 h 2°C chilling, antimonate Ca²⁺ depositsincreased in the cytosol and the nucleus, but cerium phosphatedeposits showed no visible difference compared to their corresponding25°C seedlings. After 12, 24, or 72 h chilling, maize seedlingsstill maintained a high level of antimonate Ca²⁺ deposits inthe cytosol and the nucleus. During these periods, maize Ca²⁺-ATPase,as indicated by the number of cerium phosphate deposits, becameless and less active as chilling proceeded. In winter wheat,the increased cytosolic and nuclear antimonate Ca²⁺ depositswere restored to a low resting level after 12, 24, or 72 h chilling,while the Ca²⁺-ATPase was maintained active, contrary to maizescenario. The transient cytosolic and nuclear Ca²⁺ increaseand the activities of Ca²⁺-ATPase during chilling are discussedin relation to plant chilling injury and cold acclimation. ³ Present address: Department of Agricultural, Food and NutritionalScience, University of Alberta, Edmonton, Canada T6G 2P5.     

桃芽休眠的自然诱导因子及钙在休眠诱导中的作用

以2年生低需冷量设施栽培适宜品种“春捷毛桃”为试材,研究桃植株进入休眠和发展抗冻性的自然诱导因子及Ca²⁺在此过程中的作用.结果表明：短日照和自然低温单个因子或其共同作用均能诱导桃植株停止生长,然后进入休眠状态和发展抗冻性,但短日照和自然低温的作用机制不同.短日照首先诱导桃植株进入休眠状态,然后诱导抗冻性发展;而自然低温则是首先诱导桃植株抗冻性发展,而后诱导其进入休眠状态;当短日照和自然低温共同作用即自然条件下时,短日照起主导作用,自然低温起辅助促进作用.短日照处理研究表明,短日照诱导桃植株停止生长、休眠诱导和抗冻性发展离不开Ca²⁺的作用,在此过程中Ca²⁺起着传递短日照信号的作用;在人工补光长日照条件下,随着温度的降低,Ca²⁺逐渐由细胞间隙、细胞壁和液泡流入细胞质和核内,同时植株生长速度变缓并最终完全停止,随后进入休眠状态并发展抗冻性.表明Ca²⁺作为传递自然低温信号的信使在自然低温诱导桃植株停止生长、抗冻性发展和休眠过程中起着重要作用.     

NaHCO3胁迫下星星草根中Ca2+与Ca2+-ATPase 的超微细胞化学定位

利用焦锑酸盐和磷酸铅沉淀技术分别对NaHCO₃胁迫条件下星星草(Puccinellia tenuiflora)根中Ca²⁺和Ca²⁺-ATPase 进行超微细胞化学定位研究, 旨在进一步探讨Ca²⁺在NaHCO₃胁迫诱导胞内信号转导过程中的作用, 以及Ca²⁺-ATPase活性定位变化与NaHCO₃胁迫下星星草抗盐碱能力的关系。结果表明: 在正常状态下, 根毛区细胞质内Ca²⁺较少, 主要位于质膜附近和液泡中, Ca²⁺-ATPase主要定位于质膜和液泡膜, 有一定活性。在0.448%NaHCO₃胁迫下, 根毛区细胞质中Ca²⁺增多, 液泡中Ca²⁺减少, 且主要集中于液泡膜附近, 质膜和液泡膜Ca²⁺-ATPase活性明显升高。在1.054%NaHCO₃胁迫下,细胞质中分布的Ca²⁺增多, 而液泡中Ca²⁺极少, Ca²⁺-ATPase活性也降低。以上结果表明, Ca²⁺亚细胞定位和Ca²⁺-ATPase活性变化在星星草响应NaHCO₃胁迫的信号传递过程中具有重要作用。     

杨树顶芽细胞内质网与其他膜系统的结构联系及其在休眠过程中的变化

杨树（Ｐｏｐｕｌｕｓ ｄｅｌｔｏｉｄｅｓ Ｂａｒｔｒ．ｅｘ Ｍａｒｓｈ）顶芽分生组织细胞经一种改良的高锰酸钾固定法固定后,显示出一种十分清晰的内膜结构,尤其展现了内质网与其他膜系统存在一种结构上的密切联系。一些与核膜相连接的内质网伸展到细胞质中与线粒体、质体及高尔基体发生联系,可延伸到质膜。还有些内质网的一端与一个细胞的核膜相连结,其另一端穿过胞间连丝与邻近的另一个细胞的核膜相连结,在两个相邻的细     

Ca2+-homeostasis Differs Between Plant Species with Different Cold-tolerance at 4℃ Chilling

Electron microscopic observations revealed that the tissues of poplar (Populus deltoides Bartr. ex Marsh) apical bud cells, which were fixed by a modified procedure of potassium permanganate fixative, showed a distinct endomembrane organization, in particular, the structural associations of the endoplasmic reticulum (ER) with other membrane systems. The striking findings are that some ER elements were in connection with the nuclear envelopes of two adjacent cells through plasmodesmata, and many ER elements were also associated with mitochondria, plastids, Golgi bodies or the plasma membrane (PM), forming a bridge-like continuum among various endomembrane systems or between nucleus to nucleus. A great number of plasmodesmata existed between cells, indicating a perfectly integrated symplasmic structure in poplar apical bud meristem grown in a long day environment. During the short day-induced dormancy, ER contracted, leading to its disassociation between nuclei, and between the nucleus and organelles/plasmalemma in many cells. After dormancy broke and shoots growth resumed, contracted ER was no longer observed in the apical bud cells. The ER associations with other endomembrane systems and the intercellular communication channels were re-established similar to that of plants before dormancy induction. These observations suggest that ER may play an important role in linking-up between the nucleus and organelles, and between the nucleus and the nucleus (or cell-to-cell), and seemingly coordinating various physiological processes by the bridging-like associations. And the contraction of ER under short-day may result in the growth cessation and the development of dormancy in poplar.     

Intracellular Calcium Increase in Gerbil Taste Cell by Amino Acid Sweeteners

Gustatory transduction mechanisms for sucrose and amino acidsweeteners in gerbil taste cells were studied with Ca²⁺ imagingand whole cell recording techniques. A 100 mM sucrose stimuluswith Ca²⁺ increased the intracellular Ca²⁺ concentration ([Ca²⁺]i)in sweet-sensitive taste cells of the taste bud, but the sucrosestimulus without Ca²⁺ did not change the [Ca²⁺]i. A 10 mM D-phenylalaninesweet stimulus with or without Ca²⁺ similarly increased the[Ca²⁺]i in the taste bud. The addition of 5 µM ionomysin,a Ca²⁺-ionophore, without Ca²⁺ greatly increased the [Ca²⁺]iin the taste bud. The application of 10 mM D-phenylalanine stimuluswithout Ca²⁺ enhanced the outward K⁺ current in isolated tastecells. These results suggest that a sugar sweetener such assucrose induces a depolarization in gerbil taste cells whichactivates voltage-dependent Ca²⁺ channels and that a non-sugarsweetener such as D-phenylalanine releases Ca²⁺ from the internalstores without a depolarization. Chem. Senses 22: 83–91,1997.     

Methodological aspects of pressure loading of Fura-2 into Characean cells

Four different fura-2 compounds were tested for the applicationin Characean cells (fura-AM; fura-C₁₈; fura- K₅; fura-dextran;MW = 10 kDa). It is demonstrated that Characean cells imposespecial problems when cytosolic pCa has to be measured withfluorescent ratio dyes. Fluorescence (_ex=340 nm) from the dyewhich had diffused from the cytosol to the huge central vacuolewith milimolar Ca²⁺ concentrations overrides the signal fromthe cytosol and makes Ca²⁺ -quantification difficult. This canbe avoided by pressure injection of fura-dextran. Because ofinhomogeneities in dye concentration or in thickness of thecytoplasmic layer, cytoplasmic streaming causes high noise orpretend oscillations in pCa if data are obtained by subsequentimage grabbing. In addition, vesicles filled with high 'concentrationsof dye may sometimes be expelled into the vacuole during theloading procedure enhance this effect. These sources of inhomogeneitiescan be minimized by loading fura-dextran via the neighbouringcell. The slow loading procedure through the plasmodesmata takes1–10 h. It results in a more homogeneous distributionof the dye. The operation of the new method is illustrated bythe measurement of Ca²⁺ -transients during action potentials,the temperature dependence of the fluorescence signal in vivoand in vitro and the butyrate-induced elevation of [Ca²⁺]_c.Fura-AM was found not to be well suited for use in algal cells.Fura-C₁₈ has toxic effects and induces clotting of the cytoplasm.In addition, some aspects of the properties of dextran-derivatesare discussed. Key words: Manual pressure microinjection, fura-2, characean cells, fluorescence ratio imaging, temperature dependence     

Differential effect of Calcium and Magnesium on Mechanical Properties of Pea Stem Cell Walls

Replacement of calcium ion with magnesium ion in the cell wallof the pea epicotyl makes the wall more extensible. A possiblerole of this differential effect of Ca²⁺ and Mg^2– in regulatingcell elongation in pea epicotyl is discussed. The ratio of the content of calcium ion to that of magnesiumion (Ca²⁺/Mg²⁺) in the walls decreased markedly in the orderof the first > the second > the third internodes of thepea epicotyl. The capacity of the walls for cation exchangeincreased in the same order, whereas the calcium-magnesium ionexchange selectivity of the walls was virtually constant. Ourresults indicate that the changes in the Ca²⁺/Mg²⁺ ratio amongthe internodes is not attributable to the ion exchange propertiesof the walk per se, but is due to other physiological conditionswhich regulate the activities of free calcium and magnesiumions in the environment with which the walls are in equilibrium. ¹ Present address: Wakayama Research Laboratories, Kao SoapCo., Ltd., Wakayama 640-91, Japan ² Present address: Foold Development Laboratories, Meiji SeikaKaisha Ltd., Kawasaki 210, Japan (Received May 1, 1981; Accepted August 26, 1981)     

Structural Association of Endoplasmic Reticulum with Other Membrane Systems in Populus deltoides Apical Bud Cells and Its Alterations During the Short Day-induced Dormancy

A comparative study was carried out on the EM-cytochemical localization of calcium and Ca2+-ATPase activity in the suspension-cultured cells between the chilling-sensitive maize (Zea mays L. cv. Black Mexican Sweet) and chilling-insensitive Trititrigia (Triticum sect. Trititrigia mackey) at 4 ℃ chilling. When maize and Tyititrigia cells were cultured at 26 ℃, electron microscopic observations revealed that the electron-dense calcium antimonate deposits, an indication of the calcium localization, were localized mainly in the vacuoles, and few was found in the cytosol and nuclei. The electron-dense cerium phosphate deposits, an indication of Ca2+-ATPase activity, were abundantly distributed on the plasma membrane (PM). When the cells from both species were cultured at 4 ℃ for 1 and 3 h, an elevation of Ca2+ level in the cytosol and nuclei was observed, whereas the cerium phosphate deposits on the PM showed no quantitative difference from those of the 26 ℃-cultured cells, indicating that the enzymatic activities were not altered during these chilling periods. However, there was a distinct difference in the dynamics of the Ca2+ distribution and the PM Ca2+-ATPase activity between maize and Trititrigia when chilled at 4 ℃ for 12, 24 and 72 h. In maize cells, a large number of Ca2+ deposits still existed in the cytosol and nuclei, and the PM Ca2+-ATPase became less and less active, and even inactive at all. In Trititrigia cells, the increased cytosolic and nuclear Ca2+ ions decreased after 12 h chilling. By chilling up to 24 and 72 h, the intracellular Ca2+ concentration had been restored to a similar low level as those of the warm temperature-cultured cells, while the activity of the PM Ca2+-ATPase maintained high. The transient cytosolic and nuclear Ca2+ increase and the activities of PM Ca2+-ATPase during chilling are discussed in relation to plant cold hardiness.     

Sodium and calcium localization in cells and tissues by precipitation with antimonate: A quantitative study

Summary Komnick's antimonate technique, which was devised to localize Na⁺ in cells and tissues, was studied quantitatively. Some modifications, as well as its application to Ca²⁺ localization, were also investigated.We combined measurements of Na⁺ and Ca²⁺ retention in plant roots during the various procedures, electron microscopy, autoradiography, and semiquantitative X-ray microanalysis. We were able to show that (at least in barley roots) antimonate does not precipitate at all with Na⁺, irrespective of the Na⁺ content of the tissue or the method of antimonate application. (Even during precipitative freeze dissolution or after freeze drying, no Na⁺ is precipitated.) By means of Komnick's antimonate technique Ca²⁺ is trapped within the tissue, but only after serious dislocation. Perspectives for reliable localization of diffusible ions in cells and tissues, by precipitation simultaneously with conventional fixations, are bad.     

CamelliA-based simultaneous imaging of Ca2+ dynamics in subcellular compartments

As a universal second messenger, calcium (Ca²⁺) transmits specific cellular signals via a spatiotemporal signature generated from its extracellular source and internal stores. Our knowledge of the mechanisms underlying the generation of a Ca²⁺ signature is hampered by limited tools for simultaneously monitoring dynamic Ca²⁺ levels in multiple subcellular compartments. To overcome the limitation and to further improve spatiotemporal resolutions, we have assembled a molecular toolset (CamelliA lines) in Arabidopsis (Arabidopsis thaliana) that enables simultaneous and high-resolution monitoring of Ca²⁺ dynamics in multiple subcellular compartments through imaging different single-colored genetically encoded calcium indicators. We uncovered several Ca²⁺ signatures in three types of Arabidopsis cells in response to internal and external cues, including rapid oscillations of cytosolic Ca²⁺ and apical plasma membrane Ca²⁺ influx in fast-growing Arabidopsis pollen tubes, the spatiotemporal relationship of Ca²⁺ dynamics in four subcellular compartments of root epidermal cells challenged with salt, and a shockwave-like Ca²⁺ wave propagating in laser-wounded leaf epidermis. These observations serve as a testimony to the wide applicability of the CamelliA lines for elucidating the subcellular sources contributing to the Ca²⁺ signatures in plants.

A toolset for simultaneous imaging of Ca²⁺ dynamics in subcellular compartments has uncovered unrecognized Ca²⁺ signatures in Arabidopsis cells in response to developmental and external cues.     

Calcium distribution in fertilized and unfertilized ovules and embryo sacs of Nicotiana tabacum L.

Potassium antimonate was used to localize Ca²⁺ in tobacco ovules from 0 to 7 d after anthesis in pollinated and emasculated flowers. Antimonate binds “loosely bound” Ca²⁺ into calcium antimonate; less-soluble forms are unavailable and free calcium usually escapes. Ovules are immature at anthesis. Abundant calcium precipitates in nucellar cells surrounding the micropylar canal. A difference between calcium in the two synergids emerges at 1 d, which is enhanced in pollinated flowers. The future receptive synergid accumulates more precipitates in the nucleus, cytoplasm and cell walls. After fertilization, micropyle precipitates diminish, and the ovule is unreceptive to further tube entry. In emasculated flowers 6 d after anthesis, ovular precipitates essentially disappear; however, flowers pollinated at 4–5 d and collected 2 d later largely restore their prior concentration of precipitates. Ovular precipitates occur initially in the nucellus, then the embryo sac, and finally the synergid and micropylar filiform apparatus. Possibility, calcium is released from the embryo sac, although no structural evidence of exudate formation was observed. Calcium precipitates in the ovule correlate with the ability of the ovule to be fertilized, suggesting that successful pollen tube entry and later development may require calcium of the class precipitated by antimonate. Received: 14 August 1996 / Accepted: 9 October 1996     

Calcium antagonists and calmodulin inhibitors block cytokinin-induced bud formation in Funaria

The plant hormone cytokinin stimulates nuclear migration followed by an asymmetric cell division in target cells of the protonema of the moss Funaria hygrometrica, leading to bud formation. The role of calcium in this developmental event was investigated by examining the effects of various calcium antagonists on the cytokinin-induced division. Calcium-free medium (buffered with EGTA), the extracellular Ca²⁺ antagonist La³⁺ (lanthanum), and the Ca²⁺ channel inhibitors D 600 and verapamil all block bud formation. These inhibitions are partially reversed by washing the cells or by raising the extracellular [Ca²⁺]. The Ca²⁺ ionophore A23187 partially reversed the effects of D 600 and verapamil. Bud formation is also inhibited by the intracellular Ca²⁺ antagonist TMB-8 (8-diethylamino)ocytl 3,4,5-trimethoxybenzoate HCl), and this inhibition is partially reversed by washing or raising the extracellular [Ca²⁺]. The cross walls of both the filaments and bud initial cells formed during TMB-8 exposure exhibit a distorted morphology. High concentrations of TMB-8 block nuclear migration. The calmodulin inhibitor trifluoperazine stops cytokinin-induced budding more effectively than the related compound chlorpromazine. Low concentrations of these two compounds do not affect nuclear migration; however, the target cell does not enter mitosis. These results support the hypothesis that a rise in intracellular calcium mediates cytokinin-induced bud formation in Funaria. It is concluded that the proposed cytokinin-induced rise in intracellular calcium may be effected in part by the activation of calmodulin. The essential source of Ca²⁺ appears to be extracellular, because blocking Ca²⁺ uptake with Ca²⁺ transport inhibitors can block both nuclear migration and subsequent division.     

A probable role of dihydropyridine receptors in repression of Ca2+ sparks demonstrated in cultured mammalian muscle

To activate skeletal muscle contraction, action potentials must be sensed by dihydropyridine receptors (DHPRs) in the T tubule, which signal the Ca²⁺ release channels or ryanodine receptors (RyRs) in the sarcoplasmic reticulum (SR) to open. We demonstrate here an inhibitory effect of the T tubule on the production of sparks of Ca²⁺ release. Murine primary cultures were confocally imaged for Ca²⁺ detection and T tubule visualization. After 72 h of differentiation, T tubules extended from the periphery for less than one-third of the myotube radius. Spontaneous Ca²⁺ sparks were found away from the region of cells where tubules were found. Immunostaining showed RyR1 and RyR3 isoforms in all areas, implying inhibition of both isoforms by a T tubule component. To test for a role of DHPRs in this inhibition, we imaged myotubes from dysgenic mice (mdg) that lack DHPRs. These exhibited T tubule development similar to that of normal myotubes, but produced few sparks, even in regions where tubules were absent. To increase spark frequency, a high-Ca²⁺ saline with 1 mM caffeine was used. Wild-type cells in this saline plus 50 µM nifedipine retained the topographic suppression pattern of sparks, but dysgenic cells in high-Ca²⁺ saline did not. Shifted excitation and emission ratios of indo-1 in the cytosol or mag-indo-1 in the SR were used to image [Ca²⁺] in these compartments. Under the conditions of interest, wild-type and mdg cells had similar levels of free [Ca²⁺] in cytosol and SR. These data suggest that DHPRs play a critical role in reducing the rate of spontaneous opening of Ca²⁺ release channels and/or their susceptibility to Ca²⁺-induced activation, thereby suppressing the production of Ca²⁺ sparks. excitation-contraction coupling; sarcoplasmic reticulum; ryanodine receptors; Ca²⁺ imaging     

Functional expression of heterologous proteins in yeast: insights into Ca2+ signaling and Ca2+-transporting ATPases

The baker's yeast Saccharomyces cerevisiae is a well-developed, versatile, and widely used model organism. It offers a compact and fully sequenced genome, tractable genetics, simple and inexpensive culturing conditions, and, importantly, a conservation of basic cellular machinery and signal transducing pathways with higher eukaryotes. In this review, we describe recent technical advances in the heterologous expression of proteins in yeast and illustrate their application to the study of the Ca²⁺ homeostasis machinery, with particular emphasis on Ca²⁺-transporting ATPases. Putative Ca²⁺-ATPases in the newly sequenced genomes of organisms such as parasites, plants, and vertebrates have been investigated by functional complementation of an engineered yeast strain lacking endogenous Ca²⁺ pumps. High-throughput screens of mutant phenotypes to identify side chains critical for ion transport and selectivity have facilitated structure-function analysis, and genomewide approaches may be used to dissect cellular pathways involved in Ca²⁺ transport and trafficking. The utility of the yeast system is demonstrated by rapid advances in the study of the emerging family of Golgi/secretory pathway Ca²⁺,Mn²⁺-ATPases (SPCA). Functional expression of human SPCA1 in yeast has provided insight into the physiology, novel biochemical characteristics, and subcellular localization of this pump. Haploinsufficiency of SPCA1 leads to Hailey-Hailey disease (HDD), a debilitating blistering disorder of the skin. Missense mutations, identified in patients with HHD, may be conveniently assessed in yeast for loss-of-function phenotypes associated with the disease. Saccharomyces cerevisiae; calcium ion; transporters; functional complementation     

Tastants evoke cAMP signal in taste buds that is independent of calcium signaling

We previously showed that rat taste buds express several adenylyl cyclases (ACs) of which only AC8 is known to be stimulated by Ca²⁺. Here we demonstrate by direct measurements of cAMP levels that AC activity in taste buds is stimulated by treatments that elevate intracellular Ca²⁺. Specifically, 5 µM thapsigargin or 3 µM A-23187 (calcium ionophore), both of which increase intracellular Ca²⁺ concentration ([Ca²⁺]_i), lead to a significant elevation of cAMP levels. This calcium stimulation of AC activity requires extracellular Ca²⁺, suggesting that it is dependent on Ca²⁺ entry rather than release from stores. With immunofluorescence microscopy, we show that the calcium-stimulated AC8 is principally expressed in taste cells that also express phospholipase C₂ (i.e., cells that elevate [Ca²⁺]_i in response to sweet, bitter, or umami stimuli). Taste transduction for sucrose is known to result in an elevation of both cAMP and calcium in taste buds. Thus we tested whether the cAMP increase in response to sucrose is a downstream consequence of calcium elevation. Even under conditions of depletion of stored and extracellular calcium, the cAMP response to sucrose stimulation persists in taste cells. The cAMP signal in response to monosodium glutamate stimulation is similarly unperturbed by calcium depletion. Our results suggest that tastant-evoked cAMP signals are not simply a secondary consequence of calcium modulation. Instead, cAMP and released Ca²⁺ may represent independent second messenger signals downstream of taste receptors. calcium-sensitive adenylyl cyclase; capacitative entry; cross talk; taste transduction     

The Effect of pH on the Binding of Calcium to Pea Epicotyl Cell Walls and its Implications for the Control of Cell Extension

Cell walls were prepared from the epicotyls of dark-grown pea(Pisum sativum L.) seedlings. The walls were found to bind externally-added⁴⁵Ca²⁺, with a binding constant of 4 ? 10^–4 mol dm^–3and a maximum capacity of 1.5 ? 10^–8 g-ions of Ca²⁺ perg fresh weight of epicotyl. The binding capacity decreased asthe pH of the medium was decreased below 6.0, suggesting thatthe calcium was bound by an anionic group with an apparent pKof 4.7. More than half the calcium binding was due to polygalacturonicacid in the wall, since up to 60% of the calcium binding capacitywas removed by pre-incubation of the cell walls with polygalacturonase(E.C.3.2.1.15). Only small decreases in calcium binding wereseen following pre-incubation with protease, nucleases, phospholipaseand hemicellulase. These results indicate that calcium willbe displaced from the cell wall at hydrogen ion concentrationswhich are known to occur in the wall during wall extension.They are consistent with a mechanism by which calcium inhibitswall extension by forming ionic bridges between polygalacturonicacid molecules, and also with the hypothesis that calcium andhydrogen ions exert opposing influences on cell wall extensionby competing for the same binding sites on the polygalacturonicacid. Key words: Pea epicotyl, Cell wall, Calcium, pH     

Localization of membrane-associated calcium following cytokinin treatment in Funaria using chlorotetracycline

We have investigated the changes in membrane-associated calcium that occur during cytokinin induced bud formation in Funaria hygrometrica Hedw. using the fluorescent Ca²⁺-chelate probe chlorotetracycline (CTC). In the target caulonema cells a localization of CTC fluorescent material becomes evident at the presumptive bud site 12 h after cytokinin treatment. By the time of the initial asymmetric division this region is four times as fluorescent as the entire caulonema cell. Bright CTC fluorescence remains localized in the dividing cells of the bud. To relate the changes in CTC fluorescence to changes in Ca²⁺ as opposed to membrane-density changes we employed the general membrane marker N-phenyl-1-naphthylamine (NPN). NPN fluorescence increases only 1.5 times in the initial bud cell. We conclude that the relative amount of Ca²⁺ per quantity of membrane increases in this localized area and is maintained throughout bud formation. We suggest that these increases in membrane-associated Ca²⁺ indicate a localized rise in intracellular free Ca²⁺ concentration brought about by cytokinin action.Abbreviations BA 6-benzyladenine - CTC chlorotetracycline - ER endoplasmic reticulum - NPN N-phenyl-1-naphthylamine     

Calneuron I inhibits Ca channel activity in bovine chromaffin cells

Calneuron I (CalnI) is a calmodulin-like protein that contains two functional EF-hand motifs at the N-terminal and a hydrophobic segment at the C-terminal. CalnI was cloned from the adult rat cortex and fused with GFP at its N-terminal. When expressed in bovine chromaffin cells, wild-type CalnI was localized at the plasma membrane. However, a mutant that lacked the hydrophobic segment was localized in the cytosol and nucleus, while a Ca²⁺-binding-deficient mutant was found in the cytosol and at the plasma membrane. Evaluation using the whole-cell patch-clamp technique revealed that Ca²⁺ currents were inhibited by both wild-type CalnI and the Ca²⁺-binding-deficient mutant. When the bovine N-type Ca²⁺ channel was expressed in 293T cells, Ca²⁺ currents were mostly inhibited by co-expression of CalnI, but not by the mutant without the hydrophobic tail. These results suggest that CalnI attenuates Ca²⁺ channel activity and that its subcellular localization is important for this effect.