Expression of two cloned mRNA sequences during development and germination of spores of the sensitive fern,Onoclea sensibilis L.

A library of complementary DNA (cDNA) clones has been prepared from poly(A)⁺RNA of spores of the sensitive fern, Onoclea sensibilis L. By differential hybridization with labeled probes made to poly(A)⁺ RNA of spores, gametophytes and leaves, two spore-specific clones (pOSS68 and pOSS194) were selected and characterized. Northern blot analysis showed that RNA sequences homologous to the two cDNA clones first appear in the post-meiotic spore and increase in abundance during spore maturity. Both RNA sequences decay during photoinduced germination of the spores and do not reappear in the gametophytes. In spores imbibed in the dark under conditions which do not favor germination, no significant decrease in pOSS194-mRNA abundance is noted. In contrast, the decrease in pOSS68 mRNA in dark-imbibed spores parallels that observed in photoinduced spores. The predicted amino-acid sequence of pOSS194 has a striking similarity to the early light-inducible proteins expressed during the greening of etiolated pea and barley seedlings, whereas that of pOSS68 shows some homology to proteins encoded by late-embryogenesis-abundant mRNAs of angiosperm embryos.Abbreviations bp base pairs - cDNA complementary DNA - ds double-stranded - ELIP early light-inducible proteins - LEA late embryogenesis abundant - nt nucleotide - ss single stranded This work was partially supported by a NASA grant (NAGW-901) and by an allocation from the Research Challenge Investigators' Fund of the Ohio State University to V.R. Thanks are due to Mr. Clayton L. Rugh for sequencing our clones and to Dr. Paul A. Fuerst for help in the computer search of sequence alignments.     

Effects of nutrients and light pretreatment on phytochrome-mediated fern-spore germination

Spores of the ferns, Dryopteris filix-mas, D. paleacea and Polystichum minutum, sown on plain agar in quartz-distilled water, required several hours of red light in order to germinate. When, however, water agar was replaced by agar made up with a mineral nutrition medium, a single pulse of red light (about 1 min) was able fully to induce germination. Under these conditions spores became light-sensitive a few minutes after sowing. Thus, zero germination in dark controls was obtained only when all light was excluded immediately after sowing or when saturating far-red was given thereafter. The effect of the mineral medium was also obtained using low ion concentrations with an osmolality of less than 100 mol l^–1. Thus, a specific ion effect appears more probable than an unspecific osmotic effect. Species differences in light sensitivity and in dark-germination levels, as reported in the literature, might partly be the consequence of different culture media and of light acting at a very early stage after sowing, which hitherto was assumed to be still insensitive to light. On water agar as well as on mineral agar, the inducing effect of a single red pulse could be increased by the appropriate pretreatment, i.e. by preirradiation with red light for several hours, followed by a saturating pulse of far-red, the latter abolishing the direct inducing effect of the red preirradiation. The nature of both the ion-phytochrome interaction and the phytochrome-phytochrome interaction has not yet been analysed.Abbreviations FR saturating far-red light - Pfr far-red absorbin form of phytochrome - R broad-band red light, acting continuously during several hours This work was performed at the Department of Plant Physiology, University of Lund, Sweden, during a sabbatical leave     

Calcium requirement of phytochrome-mediated fern-spore germination: No direct phytochrome-calcium interaction in the phytochrome-initiated transduction chain

Phytochrome-mediated germination of fern spores of Dryopteris paleacea Sw. was initiated by a saturating red-light (R) irradiation after 20 h of imbibition. For its realization external Ca²⁺ was required, with a threshold at a submicromolar concentration, and an optimum was reached around 10^-4 M. At concentrations 10^-1 M only a reduced response was obtained, based probably on an unspecific osmotic or ionic effect. The germination response was inhibited by La³⁺, an antagonist of Ca²⁺. From these results it is concluded that Ca²⁺ influx from the medium into the spores may be an important event in phytochrome-mediated germination. In the absence of Ca²⁺ the R-stimulated system remained capable of responding to Ca²⁺, added as late as 40 h after R. Moreover, Ca²⁺ was effective even if added after the active form of phytochrome, Pfr, had been abolished by far-red (FR) 24 h after R. Thus, the primary effect of Pfr, that initiates the transduction chain, does not require calcium. Coupling of Pfr to subsequent dark reactions has been investigated by R-FR irradiations with various dark intervals. The resulting escape kinetics were characterized by a lag phase (6 h) and half-maximal escape from FR reversibility (19 h). These kinetics were not significantly changed by the presence or absence of calcium. Thus, direct interaction of Pfr and calcium is not a step in the transduction chain initiated by the active form of photochrome.Abbreviations EGTA ethyleneglycol-bis(-aminoethyl ether)-N,N,N,N-tetraacetic acid - FR far-red light - Pr red-light-absorbing form of phytochrome - Pfr far red-light-absorbing form of phytochrome - Pipes piperazine-1,4-bis(2-ethanesulfonic acid) - R red light A preliminary report of this work was presented at the XIV Int. Bot. Congr., Berlin (West), Germany, Book of Abstracts, 2-116a-5 (1987)     

Ion antagonism in phytochrome-mediated calcium-dependent germination of turions of Spirodela polyrhiza (L.) Schleiden

The light-dependent germination response of turions (resting fronds) is mediated by phytochrome and requires the presence of Ca²⁺ in the medium (K.-J. Appenroth and H. Augsten, 1990, Photochem. Photobiol. 52: 61–65). The Ca²⁺ requirement of germination is apparent only in the presence of exogenous Mg²⁺. A competitive ion antagonism was demonstrated between Ca²⁺ and Mg²⁺ in this physiological response; Mg²⁺ could also be replaced by Ba²⁺ or Sr²⁺. Without exog-enous Mg²⁺, a Ca²⁺ concentration as low as 0.9 μM fulfilled the Ca²⁺ requirement. This type of ion antagonism resembled the competitive Ca/Mg interaction reported previously for calcium-binding proteins. The physiological response was blocked by inhibitors of Ca²⁺ uptake (verapamil, La³⁺). It was concluded that uptake of Ca²⁺ from the external medium is an essential step in the phytochrome-mediated germination of turions. The results are in agreement with the assumption that the uptake of Ca²⁺ is blocked at the side of entry by other alkaline earth ions. Treatment of turions with Mg²⁺ (1 mM) for 24 h at varying times after the red light pulse in otherwise virtually Ca²⁺-free KNO₃ solution resulted in a response similar to a Ca²⁺ step-down treatment. This is in agreement with the assumption that the Ca²⁺- and the Mg²⁺-sensitive periods coincide. The ion interaction described here represents the first photophysiological example in plants of an antagonistic effect between Ca²⁺ and Mg²⁺ similar to that which occurs in vitro with calmodulin. Received: 12 June 1998 / Accepted: 28 December 1998     

Cell expansion in the filamentous gametophyte of the fernOnoclea sensibilis L.

Filamentous gametophytes of the fernO. sensibilis were exposed to paired combinations of light of different qualities, hormones and cations in the attempt to elucidate the underlying processes that regulate cell expansion. Simultaneous treatments with high-pH buffers or the auxin antagonistp-chlorophenoxyisobutyric acid abolished blue-light-mediated expansion but did not influence growth in red light. In contrast, the red-light response was preferentially altered by the ethylene absorbant KMnO₄ or the Ca²⁺ chelator ethyleneglycol-bis(-aminoethyl ether) N,N-tetraacetic acid. The Ca²⁺ ionophore A23187 caused a significant reduction in cell expansion under both blue and red irradiation. A marked promotion of expansion was mediated by high concentrations of indole-3-acetic acid, but this effect was dependent on the presence of low-pH buffers. The ethylene-generating agent 2-chloroethylphosphonic acid decreased the magnitudes of both photoresponses; this inhibition was further enhanced by high Ca²⁺ concentrations. These findings and those with other plants are interpreted in terms of two independent control mechanisms for cell expansion: 1) a blue light photoreceptor-auxin-hydrogen ion system, and 2) a phytochrome-ethylene-calcium ion system.Abbreviations APW-X artificial pond water (the associated number designates pH) - CEPA 2-chloroethylphosphonic acid - EGTA ethyleneglycol-bis(-aminoethyl ether)N,N-tetraacetic acid - IAA indole-3-acetic acid - PCIB p-chlorophenoxyisobutyric acid     

The role of calcium ions in phytochrome-controlled swelling of etiolated wheat (Triticum aestivum L.) protoplasts

Protoplasts from dark-grown wheat (Triticum aestivum L.) maintained at a constant osmotic potential at 22°C, were found to swell upon red irradiation (R) and the effect was negated by subsequent far-red light (FR), indicating phytochrome involvement. Swelling only occurred when Ca²⁺ ions were present in the surrounding medium, or were added within 10 min after R. Furthermore, Mg²⁺, Ba²⁺ or K⁺ could not replace this requirement for Ca²⁺. The presence of K⁺ did not enhance the Ca²⁺-dependent swelling response. When the Ca²⁺-ionophore A 23187 was added to the medium, protoplasts swelled in the dark to the same extent as after R. Both the Ca²⁺-channelblocker Verapamil and La³⁺ inhibited R-induced swelling. It is proposed that R causes the opening of Ca²⁺-channels in the plasma membrane. Boyle-van't Hoff analyses of protoplast volume after R and FR are consistent with the conclusion that R irradiation causes changes in membrane properties.Abbreviations EDTA ethylenediaminetetraacetic acid - FR far-red light - nov non-osmotic-volume - Pfr FR-absorbing form of phytochrome - Pr R-absorbing form of phytochrome - R red light     

The role of calcium channels in anthocyanin production in callus cultures of Daucus carota

The involvement of Ca²⁺ ATPases in anthocyanin accumulation in callus cultures of Daucus carota was investigated under the influence of calcium and calcium channel modulators. Ionophore (I) treatment enhanced callus growth and anthocyanin accumulation. Increasing the amount of calcium applied to cultures enhanced the anthocyanin level. Ionophore treatment influenced the enhancement of Ca²⁺ATPase and endogenous titres of PAs. Addition of the calcium channel blocker verapamil or the calmodulin antagonist chlorpromazine to the A23187 (ionophore) treated cells caused a reduction in anthocyanin levels. Channel blockers reduced Ca²⁺ATPase activity, which was restored by ionophore treatment, showing the importance of calcium in anthocyanin production. Higher ethylene levels were also found in treatment with ionophore or 2X calcium. Thus the influence of ionophore in anthocyanin production and its inhibition by calcium channel modulators suggests that calcium plays an important role in the production of anthocyanin by carrot callus cultures.     

Revisiting intracellular calcium signaling semantics

Cells use intracellular free calcium concentration changes for signaling. Signal encoding occurs through both spatial and temporal modulation of the free calcium concentration. The encoded message is detected by an ensemble of intracellular sensors forming the family of calcium-binding proteins (CaBPs) which must faithfully translate the message using a new syntax that is recognized by the cell. The cell is home to a significant although limited number of genes coding for proteins involved in the signal encoding and decoding processes. In a cell, only a subset of this ensemble of genes is expressed, leading to a genetic regulation of the calcium signal pathways. Calmodulin (CaM), the most ubiquitous expressed intracellular calcium-binding protein, plays a major role in calcium signal translation. Similar to a hub, it is central to a large and finely tuned network, receiving information, integrating it and dispatching the cognate response. In this review, we examine the different steps starting with an external stimulus up to a cellular response, with special emphasis on CaM and the mechanism by which it decodes calcium signals and translates it into exquisitely coordinated cellular events. By this means, we will revisit the calcium signaling semantics, hoping that we will ease communication between scientists dealing with calcium signals in different biological systems and different domains.     

Interaction of light and temperature on seed germination of Rumex obtusifolius L.

Germination of Rumex obtusifolius L. seeds (nutlets) is low in darkness at 25° C. Germination is stimulated by exposure to 10 min red light (R) and also by a 10-min elevation of temperature to 35° C. A 10-min exposure to far-red light (FR) can reverse the effect of both R (indicating phytochrome control) and 35° C treatment. Fluence-response curves for this reversal of the effect of R and 35° C treatments are quantitatively identical. Treatment for 10 min with light of wavelenght 680, 700, 710 and 730 nm, after R and 35° C treatment, demonstrates that germination induced by 35° C treatment results from increased sensitivity to a pre-existing, active, far-red-absorbing form of phytochrome (Pfr) in the seeds.Abbreviations FR far-red light - P phytochrome - Pr red-absorbing form of P - Pfr far-red-absorbing form of P - R red light     

Determination of cytoplasmic calcium concentration inDryopteris spores

Germination of Dryopteris spores is mediated by the physiologically active, far-red-absorbing form of phytochrome, Pfr, and external Ca²⁺ is necessary for the transduction of the light signal. Because knowledge about the cytoplasmic calcium ion concentration, [Ca²⁺]_i, is of great importance for understanding the role of calcium during signal transduction, this value was measured using fura-2 in fern spores undergoing the normal developmental progression into germination. Fura-2 was loaded into the spores by electroporation, which does not disrupt the normal process of germination. The intensity of the fluorescence emission of the loaded fura-2 was analysed by a microspectrophotometric assay of single spores, and successful loading could be obtained by the application of ten electrical pulses (field strength 7.5 kV · cm^–1, half-life (time constant) 230 s). Fura-2 was alternately excited by light of wavelengths 355 and 385 nm through an inverted fluorescence microscope, and the emitted fura-2 fluorescence was collected by a silicon-intensified video camera. The cytoplasmic calcium ion concentration was calculated from the ratio of the camera output obtained for both wavelengths and displayed by a pseudo-color technique. Spores responded to changes of the extracellular Ca²⁺ concentration, and this observation is considered as evidence that fura-2 is loaded into the cytoplasm. The substitution of a low external [Ca²⁺] (1 mM ethyleneglycol-bis(2-aminoethyl-ether) {ie166-01},N-tetraacetic acid (EGTA)) by 1 mM CaCl₂ caused a fast increase of [Ca²⁺]_i from approx. 50 nM to above 500 nM. In contrast, the subsequent substitution of CaCl₂ by EGTA decreased [Ca²⁺]_i again below 100 nM within 0.5 h. Furthermore, the application of ionomycin could initiate a change in [Ca2+]_i according to the Ca²⁺ gradient established between the extracellular medium and cytoplasm. In spores sown on a Ca²⁺-free medium, [Ca²⁺]_i, analysed in a buffer containing EGTA, was found to be around 50 nM during the first days of cultivation, independent of the irradiation protocol. However, if spores were grown in darkness on a Ca²⁺-containing medium and analysed in EGTA, [Ca²⁺]_i was significantly higher ( 500 nM). In red-light-irradiated spores, [Ca²⁺]_i was found to decrease with increasing time after irradiation, and was determined to be less than 100 nM when analysis was done 44 h after germination was initiated by the light treatment.Dedicated to Professor H. Mohr on the occasion of his 60th birthday     

The role of wall calcium in the extension of cell walls of soybean hypocotyls

Calcium crosslinks are load-bearing bonds in soybean (Glycine max (L.) Merr.) hypocotyl cell walls, but they are not the same load-bearing bonds that are broken during acid-mediated cell elongation. This conclusion is reached by studying the relationship between wall calcium, pH and the facilitated creep of frozenthawed soybean hypocotyl sections. Supporting data include the following observations: 1) 2-[(2-bis-[carboxy-methyl]amino-5-methylphenoxy)methyl]-6-methoxy-8-bis[carboxymethyl]aminoquinoline (Quin 2) and ethylene glycol-bis(2-aminoethyl ether)-N,N,N′,N′-tetraacetic acid (EGTA) caused only limited facilitated creep as compared with acid, despite removal of comparable or larger amounts of wall calcium; 2) the pH-response curves for calcium removal and acid-facilitated creep were different; 3) reversible acid-extension occurred even after removal of almost all wall calcium with Quin 2; and 4) growth of abraded sections did not involve a proportional loss of wall calcium. Removal of wall calcium, however, increased the capacity of the walls to undergo acid-facilitated creep. These data indicate that breakage of calcium crosslinks is not a major mechanism of cell-wall loosening in soybean hypocotyl tissues. This research was supported by Department of Energy grant DE-FG06-88ER13830 and NASA grant NAGW 1394. The authors are grateful to Dr. David Rayle (San Diego State University, Cal.) for stimulating discussions and comments during the course of this work.     

An unusual effect of the far-red absorbing form of phytochrome: Photoinhibition of seed germination inBromus sterilis L.

Seeds ofBromus sterilis L. germinated between 80–100% in darkness at 15° C but were inhibited by exposure to white or red light for 8 h per day. Exposure to far-red light resulted in germination similar to, or less than, that of seeds maintained in darkness. Germination is not permanently inhibited by light as seeds attain maximal germination when transferred back to darkness. Germination can be markedly delayed by exposure to a single pulse of red light following 4 h inhibition in darkness. The effect of the red light can be reversed by a single pulse of far-red light indicating that the photoreversible pigment phytochrome is involved in the response. The response ofB. sterilis seeds to light appears to be unique; the far-red-absorbing form of phytochrome (Pfr) actually inhibiting germination.Abbreviations Pr red absorbing form of phytochrome - Pfr far-red absorbing form of phytochrome     

Interaction of light and temperature on the germination of Rumex obtusifolius L.

Seeds (nutlets) of Rumex obtusifolius L. fail to germinate in darkness at 25° C, but are stimulated by short exposure to red light (R) the effectiveness of which can be negated by a subsequent short exposure to far red light (F) indicating phytochrome control. Short periods of elevated temperature treatment (e.g. 5 min at 35° C) can induce complete germination in darkness. Although short F cannot revert the effect of 35° C treatment, cycling the phytochrome pool by exposure to short R before short F results in reversion of at least 50% of the population. Prolonged or intermittent F can also revert the germination induced by 35° C treatment. The effect of elevated temperature treatment is interpreted on the basis of two possible models; (i) that it increases the sensitivity of the seeds to a low level of pre-existing active form of phytochrome (Pfr) (ii) that it induces the appearance of Pfr in the dark. In both cases it is envisaged that elevated temperature treatment and Pfr control germination at a common point in the series of reactions that lead to germination.Abbreviations D Dark - F far red light - P phytochrome - Pr red absorbing form of P - Pfr far red absorbing form of P - R red light     

The effect of exogenous Ca2+ ions on pollen grain germination and pollen tube growth

Summary The involvement of exogenous calcium ions in the regulation of pollen tube formation has been investigated in Haemanthus albiflos L. and Oenothera biennis L. by following the changes that occur in pollen germination, tube growth, and ⁴⁵⁺Ca²⁺ uptake and distribution upon application of Verapamil (an inhibitor of calcium channels), lanthanum (a Ca²⁺ substitute), and ruthenium red (believed to raise the intracellular calcium level). It was found that exogenous Ca²⁺ takes part in the formation of the calcium gradient present in germinating pollen grains and growing pollen tubes. Ca²⁺ ions enter the cells through calcium channels. Raising or reducing ⁴⁵Ca²⁺ uptake causes disturbances in the germination of the pollen grains and in the growth of the pollen tubes.     

Effects of long-term storage on phytochrome-mediated germination in lettuce seeds

The effects of long-term seed storage on the physiological properties of phytochrome-mediated germination including water uptake, the temperature and light flunnce dependencies of germination and dark germination were studied. The fluenceresponse relationships of the brief irradiation with monochromatic red (660 nm, 7.5 W m⁻²) and far-red (750 nm, 6.6 W m⁻²) light at various times after sowing were also studied. The samples used consisted of three lots of seeds ofLactuca sativa L. cv. MSU-16, which had been harvested in 1976, 1979 and 1985 and stored dry for 9, 6 and 0 years, respectively, in darkness at 23±2 C until the experiments were carried out in July–August, 1985. Seeds with the longer storage periods showed the higher ability to germinate in both continuous darkness and continuous white fluorescent light at 20–30 C. In the seeds stored for 6 or 9 years, red light irradiation for 20 sec given at 15 min or more after sowing at 25 C induced as high a percent germination (85–95%) as those under continuous white fluorescent light. In the freshly harvested seeds, however, germination under continuous white fluorescent light (46%) was considerably lower than the germination induced by the red pulse (97%). Germination of the seeds decreased when the intervals between sowing and a far-red irradiation for 20 sec increased up to 100 min (or 30 min in the freshly harvested seeds). The far-red pulse given later than 100 min (or 6 hr in the freshly harvested seeds) after sowing resulted in an increased germination up to the dark-germination levels with increasing intervals between sowing and the pulse irradiation. Before or at 3 min after sowing, the seeds stored for 6 or 9 years were responsive to the far-red pulse although they were not or hardly responsive to the red pulse, while the freshly harvested seeds were responsive to both the far-red and the red pulses. These data indicate that normal functions of phytochrome completely survived in the dry seeds during storage at 25 C for as long as 6 or 9 years and that these functions are restored into full operation by means of imbibition. The differences in the dependence of germination on the time and fluence of a single pulse of red or far-red light seems to be related to the smaller water content throughout the imbibition in the seeds with the longer storage periods. The greater ability to germinate in the dark indicates the greater amounts of P_FR or the greater responsivity to P_FR, in the seeds with the longer storage periods.     

Several aspects of current research into the role of calcium in plant physiology

Calcium plays a variety of significant roles in the life cycle of plants. This review describes a brief summary of several examples of such roles in an attempt to provide some common ground relevant to the roles of calcium, with emphasis on the coupling between various stimuli and their respective responses. The selected topics include the regulation of turgor pressure, tropic responses, the cell cycle, and cell motility.     

The role of asymmetric cell division in pteridophyte cell differentiation. I. Localized metal accumulation and differentiation inVittaria gemmae andOnoclea prothallia

Summary In gemmae ofVittaria graminifolia and prothallia ofOnoclea sensibilis, cell differentiation is initiated by nuclear migration and geometrically asymmetric cell division. The small daughter cells inVittaria develop into antheridia in the presence of gibberellic acid or into rhizoids or new prothallia in its absence. Antheridial differentiation from asymmetric division is induced inOnoclea byPteridium antheridiogen, whereas rhizoid or vegetative cell formation occurs in its absence. Although asymmetric cytokinesis initiates differentiation, it does not in itself determine the developmental fate of the smaller cell. Several histochemical techniques demonstrate that prior to nuclear migration and cell division, Ca²⁺ accumulates in the cytoplasm and wall of the cell at the site where asymmetric division will occur, regardless of the developmental fate of the small cell. The cytoplasmic localization of Ca²⁺ appears to reflect a mobilization of Ca²⁺ from within the cell that eventually moves into the cell wall. We propose that this internal accumulation of Ca²⁺ leads to a localized decrease in cytosolic [Ca²⁺] which in turn may regulate developmental events such as nuclear migration.Publishing prior to 1984 as Alix R. Bassel.     

Activation of the human red cell calcium ATPase by calcium pretreatment

Some kinetic parameters of the human red cell Ca²⁺-ATPase were studied on calmodulin-free membrane fragments following preincubation at 37°C. After 30 min treatment with EGTA(1 mm) plus dithioerythritol (1 mm), a V _max of about 0.4 μmol P_i/mg × hr and a K _s of 0.3 μm Ca²⁺ were found. When Mg²⁺ (10 mm) or Ca²⁺(10 μm) were also added during preincubation, V _maxbut not Kwas altered. Ca²⁺ was more effective than Mg²⁺, thus increasing V _max to about 1.3 μmol P_i/mg × hr. The presence of both Ca²⁺ and Mg²⁺ during pretreatment decreasedKto 0.15 μm, while having no apparent effect on V _max. Conversely, addition of ATP (2 mm) with either Ca²⁺ or Ca²⁺ plus Mg²⁺increased V_max without affecting K. Preincubation with Ca²⁺ for periods longer than 30 min further increased V_maxand reduced Kto levels as low as found with calmodulin treatment. The Ca²⁺ activation was not prevented by adding proteinase inhibitors (iodoacetamide, 10 mm; leupeptin, 200 μm; pepstatinA, 100 μm; phenylmethanesulfonyl fluoride, 100 μm). The electrophoretic pattern of membranes preincubated with or without Mg²⁺, Ca²⁺ or Ca²⁺ plus Mg²⁺ did not differ significantly from each other. Moreover, immunodetection of Ca²⁺-ATPase by means of polyclonal antibodiesrevealed no mobility change after the various treatments. The above stimulation was not altered by neomycin (200 μm), washing with EGTA (5 mm) or by both incubating and washing with delipidized serum albumin (1 mg/ml), or omitting dithioerythritol from the preincubation medium. On the other hand, the activation elicited by Ca²⁺ plus ATP in the presence of Mg²⁺ was reduced 25–30% by acridine orange (100 μm), compound 48/80 (100 μm) or leupeptin (200 μm) but not by dithio-bis-nitrobenzoic acid (1 mm). The fluorescence depolarization of 1,6-diphenyl-and l-(4-trimethylammonium phenyl)-6-phenyl 1,3,5-hexatriene incorporated into membrane fragments was not affected after preincubating under the different conditions. The results show that proteolysis, fatty acid production, an increased phospholipid metabolism or alteration of membrane fluidity are not involved in the Ca²⁺ effect. Ca²⁺ preincubation may stimulate the Ca²⁺-ATPase activity by stabilizing or promoting the E₁ conformation.     

Calcium and phytochrome control of leaf unrolling in dark-grown barley seedlings

The red light-stimulated component of unrolling in sections from 7-d-old dark-grown barley (Hordeum vulgare L.) leaves is inhibited by ethyleneglycol-bis-(-aminoethyl ether)-N,N,N,N-tetracetic acid (EGTA). A free-Ca²⁺ activity of less than 40 M restores the ability to respond to red light, but only if supplied within 1 h of red light. Magnesium ions are an ineffective substitute. At least two processes in unrolling appear to be Ca²⁺-sensitive.Fluence-response measurements indicate that the levels of the far-red-absorbing from of phytochrome (Pfr) still present 4 h after red-light treatment should be above saturation for the unrolling response; consequently, loss of Pfr does not explain the loss in effectiveness of Ca²⁺ during prolonged EGTA treatment. However, if a further red-light treatment is given simultaneously with Ca²⁺ addition 4 h after the initial light stimulus, then full unrolling occurs in EGTA-treated sections. These data indicate that, under normal circumstances, a functional change in the properties of Pfr must occur, uncoupling it from the transduction chain.Abbreviations EGTA ethyleneglycol-bis-(-aminoethylether)-N,N,N,N,-tetracetic acid - FR far-red light - Mes 2-(N-morpholino)ethanesulphonic, acid - Pfr far-red absorbing form of phytochrome - Pr red-absorbing form of phytochrome - R red light     

Voltage- and calcium-dependent inactivation of calcium channels in Lymnaea neurons.

Ca(2+) channel inactivation in the neurons of the freshwater snail, Lymnaea stagnalis, was studied using patch-clamp techniques. In the presence of a high concentration of intracellular Ca(2+) buffer (5 mM EGTA), the inactivation of these Ca(2+) channels is entirely voltage dependent; it is not influenced by the identity of the permeant divalent ions or the amount of extracellular Ca(2+) influx, or reduced by higher levels of intracellular Ca(2+) buffering. Inactivation measured under these conditions, despite being independent of Ca(2+) influx, has a bell-shaped voltage dependence, which has often been considered a hallmark of Ca(2+)-dependent inactivation. Ca(2+)-dependent inactivation does occur in Lymnaea neurons, when the concentration of the intracellular Ca(2+) buffer is lowered to 0.1 mM EGTA. However, the magnitude of Ca(2+)-dependent inactivation does not increase linearly with Ca(2+) influx, but saturates for relatively small amounts of Ca(2+) influx. Recovery from inactivation at negative potentials is biexponential and has the same time constants in the presence of different intracellular concentrations of EGTA. However, the amplitude of the slow component is selectively enhanced by a decrease in intracellular EGTA, thus slowing the overall rate of recovery. The ability of 5 mM EGTA to completely suppress Ca(2+)-dependent inactivation suggests that the Ca(2+) binding site is at some distance from the channel protein itself. No evidence was found of a role for serine/threonine phosphorylation in Ca(2+) channel inactivation. Cytochalasin B, a microfilament disrupter, was found to greatly enhance the amount of Ca(2+) channel inactivation, but the involvement of actin filaments in this effect of cytochalasin B on Ca(2+) channel inactivation could not be verified using other pharmacological compounds. Thus, the mechanism of Ca(2+)-dependent inactivation in these neurons remains unknown, but appears to differ from those proposed for mammalian L-type Ca(2+) channels.