Auxin and heat shock activation of a novel member of the calmodulin like domain protein kinase gene family in cultured alfalfa cells

A calmodulin like domain protein kinase (CPK) homologue wasidentified in alfalfa and termed MsCPK3. The full-length sequenceof cDNA encoded a 535 amino acid polypeptide with a molecularweight of 60.2 kDa. The deduced amino acid sequence showed allthe conserved motifs that define other members of this kinasefamily, such as serine-threonine kinase domain, a junction regionand four potential Ca²⁺-binding EF sites. The recombinant MsCPK3protein purified from E. coli was activated by Ca²⁺and inhibitedby calmodulin antagonist (W-7) in in vitro phosphorylation assays.The expression of MsCPK3 gene increased in the early phase ofthe 2,4-D induced alfalfa somatic embryogenesis. Heat shockalso activated this gene while kinetin, ABA and NaCl treatmentdid not result in MsCPK3 mRNA accumulation. The data presentedsuggest that the new alfalfa CPK differs in stress responsesfrom the previously described homologues and in its potentialinvolvement in hormone and stress-activated reprogramming ofdevelopmental pathways during somatic embryogenesis. Key words: Medicago sativa, CPK, stress, 2,4-D, phosphorylation, somatic embryogenesis.     

The Role of Calcium and Calmodulin in Carrot Somatic Embryogenesis

The role of Ca²⁺ and calmodulin in carrot somatic embryo formationwas examined. Embryogenic cell clumps were induced to form embryosin medium containing 0–3 mM Ca²⁺. Embryo formation wasnot affected until the concentration of Ca²⁺ was lower than200 µM and after this threshold was reached the percentof embryo formation decreased with lower Ca²⁺ concentrations.Treatment of developing embryos with either verapamil or nifedipine,Ca²⁺ -channel blockers, or the Ca²⁺ ionophore A23187 [GenBank] , inhibitedembryo formation. These results suggest that exogenous Ca²⁺or the maintenance of Ca²⁺ gradients are required for properembryo development. Analysis of membrane-associated Ca²⁺ andtotal membrane distribution using the fluorescent dyes chlorotetracyclineand N-phenyl-1-napthylamine, respectively, indicated changesin the distribution of membranes during embryogenesis withoutany significant alterations in the concentration of Ca²⁺ associatedwith the membranes. In heart- and torpedo-stage embryos, calmodulin-Ca²⁺complexes were localized in regions containing the developingmeristems of both the cotyledon tips and rhizoid regions whiletotal calmodulin protein appeared to be more uniformly distributed.Calmodulin mRNA levels increased slightly when cell clumps wereinduced to form embryos. (Received July 7, 1993; Accepted September 27, 1993)     

Effects of Calmodulin on Ca2+-Dependent Cl--Sensitive Anion Channels in the Chara Plasmalemma: a Patch-Clamp Study

The effects of calmodulin (from spinach) on the Ca²⁺-dependentCl^--sensitive anion channel in the Chara plasmalemma (Okiharaet al. 1991) were studied by the inside-out patch-clamp technique.The current of Cl^- ions, which flowed through the channel at1.0 µM Ca²⁺, tended to decrease irregularly with time.This tendency toward a decrease in the current was no longerapparent after application of calmodulin for some time. Theactivity of the channel was restored to a small extent or tendedto increase during the early stages of application of calmodulin.Such a transient action of calmodulin on the channel activitywas evident, at voltages more negative than –100 mV. (Received August 20, 1992; Accepted October 19, 1992)     

NAD Kinase in Corn: Regulation by Far Red Light is Mediated by Ca2+ and Calmodulin

Far red light irradiation of intact corn seedlings (Zea maysL.) has neither an effect on the cellular distribution nor onthe Ca²⁺, calmodulin-dependence of the NAD kinase (EC 2.7.1.23 [EC] ).The enzyme is located in the outer mitochondrial membrane andits activity is totally dependent on the presence of both Ca²⁺and calmodulin, independently of the illumination. In intactmitochondria and the presence of calmodulin the enzyme activityincreases linearly from 100 nM to 1 mM. At 100 µM Ca²⁺halfmaximal activation occurs at about 10 nM calmodulin. After solubilizationand purification by calmodulin-Sepharose chromatography theCa²⁺dependence of the enzyme changes. The activation reachesa plateau at about 100 µM Ca²⁺ and half maximal activationoccurs at about 6 µM Ca²⁺. On the other hand irradiationof intact corn seedlings as well as an increase of the cellularCa²⁺ concentration leads to an increase of NADP and a correspondingdecrease of NAD. Based on these data we suggest that the lighteffect on the NAD kinase activity is mediated by Ca²⁺ and calmodulin. (Received May 31, 1986; Accepted July 14, 1986)     

Involvement of the Accumulation of Glutamine in the Initiation of Adventitious Buds in Stem Segments of Torenia

Active meristematic divisions in stem segments of Torenia culturedin vitro can be induced in the epidermis by application of cytokininor the calcium ionophore A23187 [GenBank] , resulting in the differentiationof adventitious buds. Endogenous free glutamine accumulatedat a high concentration in the epidermal tissues during theearly stages of such cultures. The accumulation of glutaminewas caused by an increase in glutamine synthetase (GS) activity,and the increase of GS activity was suppressed by the applicationof some inhibitors of GS activity, mRNA synthesis, protein synthesis,or calmodulin. Incorporation of these inhibitors into the culturemedium also inhibited initiation of adventitious buds. The inhibitoryeffect of an inhibitor of GS, methionine sulfoximine (MSX),was apparent only at the very begining of the culture, and theeffect could be overcome by the simultaneous addition of glutamine.The inhibitory action of MSX on initiation of buds seemed tobe caused by an accumulation of ammonium ions. Reduction inlevels of NH₄NO₃ in or its elimination from the culture mediumstimulated the initiation of adventitious buds. Therefore, boththe accumulation of glutamine and the reduction in levels ofammonium ions seem to play a role in the initiation of adventitiousbuds in stem., segments of Torenia. ¹Present address: Faculty of Agriculture, University of Saga,Honjo-cho, Saga, Saga, 840 Japan. (Received October 3, 1988; Accepted March 9, 1989)     

The role of calmodulin in the gravitropic response of the Arabidopsis thaliana agr-3 mutant

Calmodulin, a primary plant calcium receptor, is known to be intimately involved with gravitropic sensing and transduction. Using the calmodulin-binding inhibitors trifluoperazine, W7 and calmidazolium, gravitropic curvature of Arabidopsis thaliana (L.) Heynh, ecotype Landsberg, roots was separable into two phases. Phase I was detected at very low concentrations (0.01 μM) of trifluoperazine and calmidazolium, did not involve growth changes, accounted for about half the total curvature of the root and may represent the specific contribution of the cap to gravity sensing. Phase II commenced around 1.0 μM and involved inhibition of both growth and curvature. The agr-3 mutant exhibited a reduced gravitropic response and was found to lack phase I curvature, suggesting that the mutation alters either use or expression of calmodulin. The sequences of wild-type and agr-3 calmodulin (CaM-1) cDNAs, which are root specific were completely determined and found to be identical. Upon gravitropic stimulation, wild-type Arabidopsis seedlings increased calmodulin mRNA levels by threefold in 0.5 h. On the other hand, gravitropic stimulation of agr-3 decreased calmodulin mRNA accumulation. The possible basis of the two phases of curvature is discussed and it is concluded that agr-3 has a lesion located in a general gravity transmission sequence, present in many root cells, which involves calmodulin mRNA accumulation.     

Regulation of NADH-Glutamate Dehydrogenase Activity by Phytochrome, Calcium and Calmodulin in Zea mays

Regulation by the active form of phytochrome (P_FR) and the effectof Ca²⁺ and calmodulin was examined with glutamate dehydrogenase(GDH) of Zea mays. A brief irradiation (5 min) to 5 day oldplants with red light resulted in 5-6 fold increase in GDH activity.This effect was nullified when red light was followed immediatelyby far-red light. The photoreversibility showed that P_FR regulatesGDH activity in vivo. To the enzyme extract obtained after EGTAtreatment, when Ca²⁺ was added in vitro, GDH was activated by6 fold. The maximum response by Ca²⁺ was obtained at 80 µM.Both P_AR and Ca²⁺ effects were found to be age dependent. Theenzyme activity was inhibited by compound 48/80 in partiallypurified extracts and the effect was reversed by calmodulin.The purified GDH, however, was not activated directly by calmodulin;it required the presence of another protein factor which wasseparated by gel permeation column by HPLC. Neither anticalmodulindrugs nor addition of calmodulin had any effect on nitrate re-ductaseactivity. (Received July 13, 1988; Accepted October 31, 1988)     

Inhibition of Adventitious Root Growth in Tradescantia by Calmodulin Antagonists and Calcium Inhibitors

When cuttings of Tradescantia fluminensis stem were incubatedin distilled water, the buds located at the node grew into adventitiousroots. The root growth could be inhibited by calmodulin antagonists,trifluoperazine, chlorpromazine, compound 48/80 and calmidazolium,in a concentration-dependent manner. The divalent cation chelatorethyleneglycol-bis-(ß-aminoethyl ether)-N, N, N, N-tetraaceticacid had no effect, however, the intracellular chelator TMB-8completely inhibited root growth. The growth was also inhibitedby calcium ionophore A23187 [GenBank] , lanthanum, a competitive inhibitorof Ca²⁺ uptake and verapamil, a calcium channel inhibitor. AWestern blot of the adventitious root extract followed by immunostainingwith an anti-spinach calmodulin antibody clearly showed thepresence of calmodulin in this tissue. These results stronglysuggest the involvement of calmodulin and calcium in the growthof Tradescantia advenitious roots. ¹A part of this work has been published in abstract form in"Molecular and Cellular Aspects of Calcium in Plant Development"(Editedby A.J.Trewavas, Plenum Publishing Co. 1986. ³Present address: Plant Laboratory , Kirin Brewerry Co. Ltd.,Kitsuregawa-cho, Tochigi-ken 329-14 ,Japan (Received May 2, 1987; Accepted September 17, 1987)     

Inducible Accumulation of mRNA for NADH-Dependent Glutamate Synthase in Rice Roots in Response to Ammonium Ions

After growth for 26 d in water, rice seedlings were transferredto a medium containing 1 mM NH⁴Cl. The mRNA for NADH-glutamatesynthase was markedly increased in the roots within 3 h. Methioninesulfoximine completely inhibited this accumulation, suggestingthat NH⁴⁺ is not a direct inducer of this process. (Received July 11, 1997; Accepted September 10, 1997)     

Computer-assisted Video Image Analysis of Spatial Variations in Membrane-associated Ca2+ and Calmodulin during Pollen Hydration, Germination and Tip Growth in Nicotiana tabacum L.

Video images of the distributional pattern of membrane-associatedcalcium (Ca²⁺) and calmodulin (CaM) have been documented andanalysed during pollen hydration, germination and tip growthin Nicotiana tabacum. Digitization of fluorescence microscopeimages of chlorotetracycline (CTC) and fluphenazine (FPZ)-fluorescenceemissions reveal that there is a maximum concentration of membrane-associatedCa²⁺ and also CaM in the vicinity of germination apertures ofhydrated pollen. With the onset of germination relatively higheramounts of Ca²⁺ and CaM were found to regionalize towards theaperture through which the pollen tube would emerge Both shortand long growing pollen tubes manifest tip-to-base Ca²⁺ andCaM gradients which are disturbed in non-growing tubes. Tubegrowth and the Ca²⁺-gradient were significantly affected byvanadate and verapamil suggesting that both a vanadate-sensitiveCa²⁺-transport system and verapamil-sensitive Ca²⁺ channelsare involved in maintaining Ca²⁺ homeostasis during pollen germinationand tube growth. The possible interactions of Ca²⁺ and CaM withdifferent cytoskeletal proteins modulating organelle movementare also briefly discussed. Image analysis, calcium, calmodulin, Nicotiana tabacum L., pollen germination, pollen tube, tip growth, Ca²⁺-channels, Ca²⁺ transport ATPase     

Volume-dependent taurine release from cultured astrocytes requires permissive [Ca2+]i and calmodulin

Cell swelling results in regulatory activation of multipleconductive anion pathways permeable toward a broad spectrum of intracellular organic osmolytes. Here, we explore the involvement ofextracellular and intracellularCa²⁺ in volume-dependent[³H]taurine effluxfrom primary cultured astrocytes and compare theCa²⁺ sensitivity of this efflux inslow (high K⁺ medium induced) andfast (hyposmotic medium induced) cell swelling. NeitherCa²⁺-free medium norCa²⁺-channel blockers prevented thevolume-dependent[³H]taurine release.In contrast, loading cells with the membrane-permeable Ca²⁺ chelator1,2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid (BAPTA)-AM suppressed[³H]taurine efflux by65-70% and 25-30% underhigh-K⁺ and hyposmotic conditions,respectively. Fura 2 measurements confirmed that BAPTA-AM, but notCa²⁺-free media, significantlyreduced resting intracellular Ca²⁺concentration([Ca²⁺]_i).The calmodulin antagonists trifluoperazine and fluphenazine reversiblyand irreversibly, respectively, inhibited thehigh-K⁺-induced[³H]taurine release,consistent with their known actions on calmodulin. In hyposmoticconditions, the effects were less pronounced. These data suggest thatvolume-dependent taurine release requires minimal basal[Ca²⁺]_iand involves calmodulin-dependent step(s). Quantitative differences inCa²⁺/calmodulin sensitivity ofhigh-K⁺-induced and hyposmoticmedium-induced taurine efflux are due to both the effects of theinhibitors on high-K⁺-induced cellswelling and their effects on transport systems and/or signalingmechanisms determining taurine efflux.

     

The Level of mRNA Transcribed from psaL, Which Encodes a Subunit of Photosystem I, Is Increased by Cytokinin in Darkness in Etiolated Cotyledons of Cucumber

A cDNA clone for an mRNA whose level increased within 2 h ofthe start of treatment with N⁶-benzyladenine in etiolated cotyledonsof cucumber was isolated by differential hybridization. ThecDNA was homologous to psaL, which encodes subunit XI (PSI-L)of photosystem I. The accumulation of psaL mRNA was specificallyinduced by cytokinins or light. (Received April 10, 1996; Accepted July 31, 1996)     

Calcium, Calmodulin and the Control of Respiration in Protoplasts Isolated from Meristematic Tissues8

Owen, J. H., Hetherington, A. M. and Wellburn, A. R. 1987. Calcium,calmodulin and the control of respiration in protoplasts isolatedfrom meristematic tissues by abscisic acid.—J. exp. Bot.38: 1356–1361. A study was made of the possible involvement of calcium channelsand calmodulin during the calcium-dependent inhibition of mitochondrialrespiration by abscisic acid (ABA) in meristematic protoplastsobtained from light-grown barley (Hordeum vulgare L. cv. Patty)seedlings. The calcium channel blockers lanthanum, verapamiland nifedipine were all found to reduce the Ca²⁺-dependent inhibitionof protoplast respiration by ABA. The ionophore A23187 [GenBank] itselfcaused an inhibition of protoplast respiration, possibly becauseit mimicked the action of ABA by increasing plasmalemma permeabilityto extracellular calcium. By contrast, calmodulin antagoniststrifluoperazine and compound 48/80 both caused a partial decreasein the Ca²⁺-dependent inhibition of protoplast respiration byABA. In contrast to the action of ABA, gibberellic acid markedlyincreased the rates of protoplast respiration but this did notappear to require the presence of extracellular calcium ions.These results support the hypothesis that ABA increases plasmalemmapermeability to extracellular calcium which might then directlyor indirectly act as a second messenger, possibly in conjunctionwith calmodulin, to regulate mitochondrial dark respirationwhich is an important part of early meristematic cell development. Key words: Abscisic acid, calcium, calmodulin, meristematic respiration     

Activation of ATPase Activity in the Chromatin Fraction of Pea Nuclei by Calcium and Calmodulin

ATPase, especially the Ca^2$-dependent enzyme, in the chromatinfraction isolated from nuclei of pea seedlings was activatedby bovine brain calmodulin and by protein that was judged tobe calmodulin from its Ca^2$-dependent activation of NAD kinase.This calmodulin-dependent ATPase activity was blocked by calmodulin-specificinhibitors. (Received July 11, 1983; Accepted October 24, 1983)     

Visualization of Membrane Calcium and Calmodulin in Embryo Sacs in situ and Isolated from Petunia hybrida L. and Nicotiana tabacum L.

We have used chlorotetracycline (CTC) and fluphenazine (FPZ)as fluorescent probes to visualize the distributional patternsof membrane calcium (mCa²⁺) and the Ca²⁺-receptor protein calmodulin(CaM) in various cell types of unfixed living isolated and unisolatedembryo sacs of Petunia hydrida L. and Nicotiana tabacum L. Ourresults indicate that in the young embryo sacs of Petunia, bothsynergids and the central cell sequester relatively higher amountsof mCa²⁺ and CaM than the egg cell and the antipodals. Much of the mCa²⁺ in the synergids is polarized in its distributionin that the mCa²⁺ is higher towards the micropylar end of thesynergids. Interestingly, in the mature embryo sacs of Petuniaonly one of the two synergids and the egg cell proper manifesta higher level of mCa²⁺. In vivo only one of the synergids inthe young as well as in the mature embryo sacs if Nicotianaconsistently show higher mCa²⁺. In the mature embryo sacs of Petunia the level of CaM is almostuniform in all the cell types except that one of the synergidsand the three antipodal cells show a slightly higher level ofCaM. The possible implications of these findings in the late eventsof vectorial orientation of pollen tube tip, pollen-tube-synergidinteractions and sperm delivery mechanism are discussed.Copyright1993, 1999 Academic Press Membrane-Ca²⁺, calmodulin, living embryo sacs, Petunia, Nicotiana, pollen tube-synergid interaction     

ATP-Dependent Ca2+ Transport in Tonoplast Vesicles from Apple Fruit

Protoplasts and vacuoles were isolated from immature apple fruit(Malus pumila Mill. cv. Golden Delicious). ATP-stimulated Ca²⁺uptake was identified in both protoplast vesicles and tonoplastvesicles. The apparent K_m for Ca²⁺ of the tonoplast transportsystem was 43.4 µM. The pH optima were 7.2 and 6.7 forCa²⁺ transport by protoplast and tonoplast vesicles, respectively.Ca²⁺ transport in tonoplast vesicles was strongly inhibitedby the calmodulin antagonists fluphenazine and N-(6-aminohexyl)-5-chloro-l-naphthalensulfonamidehydrochloride (W-7), while N-aminohexyl)-l-naphthalensulfonamidehydrochloride (W-5) was relatively ineffective. Addition ofexogenous calmodulin stimulated transport by 35%. Ca²⁺ uptakewas inhibited by vanadate, but not by the ionophores carbonylcyanidem-chlorophenyl hydrazone (CCCP) or valinomycin. The resultsindicate that apple tonoplasts have a Ca²⁺ transport systemthat is driven by the direct hydrolysis of ATP, and may be calmodulindependent. ¹Present address: Morioka Branch, Fruit Tree Research Station,Ministry of Agriculture, Forestry and Fisheries, Shimokuriyagawa,Morioka 020-01, Japan. To whom reprint requests should be addressed. (Received October 18, 1985; Accepted January 29, 1986)     

Effect of pH on Pb2+ accumulation in Saccharomyces cerevisiae and Aureobasidium pullulans

The optimum pH conditions of Pb²⁺ accumulation in Saccharomyces cerevisiae and Aureobasidium pullulans were 4~5 and 6~7, respectively. The initial Pb²⁺ accumulation rates according to the increase of initial Pb²⁺ concentration and pH were increased both in S. cerevisie and A. pullulans. And the initial Pb²⁺ accumulation rate of A. pullulans was much higher than that of S. cerevisiae because of the difference of Pb²⁺ accumulation mechanism. The Pb²⁺ accumulation isotherm of S. cerevisae obeyed a fully competitive inhibition, whereas that of A. pullulans showed a mixed inhibition of competition and non-competition associated with the proton (H⁺) as an accumulation inhibitor.     

Orthovanadate Suppresses Accumulation of Phenylalanine Ammonia-Lyase mRNA and Chalcone Synthase mRNA in Pea Epicotyls Induced by Elicitor from Mycosphaerella pinodes

Orthovanadate delayed accumulation of mRNAs encoding phenylalanineammonia-lyase and chalcone synthase in pea epicotyls inducedby an elicitor from Mycosphaerella pinodes. However, accumulationof mRNA for a putative P-type ATPase was not affected. The relationshipbetweenthe ATPase and defense responses is discussed. ³Present address: Plant Pathology Laboratory, School of Agriculture,Nagoya University, Chikusa, Nagoya, 464-01 Japan.     

Stage-Specific Changes in Calcium-Regulated Protein Phosphorylation in Developing Tomato Fruits

The role of calcium and calmodulin in the in vitro phosphorylationof soluble and membrane proteins was studied in relation togrowth and development of tomato fruits. Calcium at micromolarconcentrations promoted the phosphorylation of both solubleand membrane proteins. The calmodulin antagonists, chlorpromazineand trifluoperazine, inhibited the phosphorylation of severalproteins. Qualitative changes were observed in the pattern ofprotein phosphorylation at different developmental stages. Therewas a general decrease in protein phosphorylation towards ripening.These results indicated that calcium may be involved in theregulation of phosphorylation of different proteins at differentstages of fruit development. ¹ Scientific Paper No. 7149, College of Agriculture and HomeEconomics, Washington State University, Pullman, Project 0321. ² Supported in part by a grant from the National Science Foundation.DCB-8502215. (Received May 14, 1985; Accepted September 12, 1985)     

The Effect of Salinity on Germination and Its Correlation with K+, Na+, Cl- Accumulation in Germinating Seeds of Triticum aestivum L. cv. Akbar

NaCl salinity stress consistantly decreased the rate of germinationof wheat. GA alone or in combination with kinetin alleviatedthe inhibitory effect of salinity on germination. However, kinetinfurther decreased the rate of germination under NaCl salinitystress. NaCl salinity increased accumulation of Na⁺ and Cl^–while it decreased K⁺ accumulation in germinating seeds. GAcaused an increase in K⁺ accumulation and a decrease in Cl^–accumulation in the germinating seeds while kinetin increasedCl^– accumulation in salinity stressed plants. The co-relationbetween the effect of salinity on germination and that on accumulationof ions is discussed. (Received February 12, 1992; Accepted August 4, 1992)