Calcium- and Calmodulin-Promoted Phosphorylation of Membrane Proteins during Senescence in Apples

Sodium dodecylsulfate-polyacrylamide gel electrophoresis ofmicrosomal membrane proteins from post-climacteric apples atan early and an advanced stage of senescence showed only slightqualitative changes in the protein pattern. Though there wasa 30% reduction in the total microsomal protein content in applesat an advanced stage of senescence, a polypeptide with 18,000molecular weight increased in quantity during senescence. Invitro phosphorylation of several proteins was promoted by calciumin membranes from apples at an early stage of senescence. Phosphorylationof proteins with molecular weights of 95,000, 91,000, 53,000and 50,000 was promoted by calcium and calmodulin. Phosphorylationof these proteins increased with increasing calcium concentration.Proteins with molecular weights of 53,000 and 50,000 showedmarked promotion of phosphorylation over the calcium-promotedlevel when the amount of calmodulin in the assay mixture wasincreased. Calcium- and calmodulin-promoted phosphorylationof membrane proteins showed considerable decrease when the appleswere at an advanced stage of senescence. Moreover, increasingthe concentrations of calcium and calmodulin in the assay mixturedid not have any promoting effect on the phosphorylation ofthese proteins. Phosphoprotein phosphatase activity as measuredby the loss of label from phosphorylated proteins followingchase with cold ATP, did not differ to a great extent in membranepreparations from normal and senesced apples. Hydrolysis ofATP by senesced apple membrane preparation, however, was foundto be relatively higher. The significance of these observationsin relation to senescence is discussed. ¹ Scientific Paper No. 7084, College of Agriculture and HomeEconomics, Washington State University, Pullman, Project 0321. ² Supported in part by grants from the Washington State TreeFruit Research Commission, and National Science Foundation GrantPCM-8208408.     

Involvement of Calcium in Adventitious Bud Initiation in Torenia Stem Segments

In Torenia stem segments cultured in vitro, active meristematicdivisions are induced in the epidermis by treatment with cytokinin,resulting in the formation of adventitious buds. Applicationof the calcium ionophore A23187 [GenBank] was found to induce meristematicdivisions in the absence of cytokinin. The induction by A23187 [GenBank] was inhibited by simultaneous addition of auxin, but not byanti-cytokinin. A two hour pre-treatment with A23187 [GenBank] was alsoeffective, but only when it was applied to the explants justafter their excision from mother plants. The A23187 [GenBank] -inducedmeristematic zones developed into dome-shaped structures, butnot into complete adventitious buds. Complete elimination ofcalcium from the culture medium caused 50% inhibition of A23187 [GenBank] -and/or cytokinin-induced initiation of meristematic divisions.When the explants were preincubated with EGTA and then culturedon a Ca-free medium containing EGTA, cytokinin failed to inducebud initiation. Similar inhibition was also obtained by lanthanum,a calcium antagonist, by verapamil, a calcium channel inhibitor,and by trifluoperazine and chlorpromazine, calmodulin inhibitors.These results support the idea that adventitious bud initiationinduced by cytokinin in Torenia stem segments may be mediated,at least partially, by an increase in the level of intracellularCa²⁺. ¹Bioscience Research Center, Mitsui Petrochemical IndustriesLtd., Waki-cho, Kuga-gun, Yamaguchi 740, Japan. (Received May 9, 1985; Accepted October 5, 1985)     

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)     

Effect of Calcium and Calcium-Counteracting Drugs on the Response of Bidens pilosa L. to Wounding

We used calcium counteracting drugs known to reduce the amplitudeof wound-induced electric wave of depolarization and we showedthat in these conditions, accumulation of the calmodulin mRNA(recently found to be correlated to membrane potential) is stronglyreduced. These results bring additional evidence linking membranepotential and calmodulin mRNA accumulation. ⁵Present address: North Carolina State University, Departmentof Botany, BOX 7612, Raleigh, NC 27695, U.S.A.     

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     

Protein Synthesis Linked to Respiration and Phosphorylation in Intact Euglena Mitochondria

Highly purified condensed mitochondria obtained from bleachedmutant. W₁₀BSmL of Euglena gracilis Klebs var bacillaris Coriincorporate [³⁵S]methionine into protein when fortified withmalate, ADP, Mg²⁺, phosphate and a sucrose osmoticum. Twentyto twenty-five polypeptide bands were found to be labeled inorganello when the labeled protein was subjected to sodium dodecylsulfatepolyacrylamide gel electrophoresis. Methionine incorporation,but not respiration or oxidative phosphorylation, was blockedby chloramphenicol and other 70S ribosomal translation inhibitorsbut cycloheximide and ribonuclease were without effect. Inhibitorsof electron transport and uncouplers of oxidative phosphorylationwere excellent inhibitors of protein synthesis. Thus, thesemitochondrial preparations carry out protein synthesis in organellothat is linked to respiration and oxidative phosphorylation. ¹Present address: VA Hospital Outpatient Clinic, 17 Court St.,Boston, MA 02115, U.S.A. ²Present address: Laboratories de Microbiologia e Inmunologia,Universidad Catolica de Chile, Casilla 114-D, Santiago, Chile. ³Present address: Botany Department, University of Massachusetts,Amherst, MA 01003, U.S.A. (Received June 17, 1985; Accepted October 28, 1985)     

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)     

Effects of External Ca2+ Concentration and of the lonophore A23187 on Development of Oogonia, Oospores and Gemmae in Saprolegnia diclina

In the oomycete Saprolegnia diclina, the number of oogonia formedper unit area of culture decreased as the Ca²⁺ concentrationin the culture medium was increased. The calcium ionophore A23187 [GenBank] decreased the number of oogonia formed and increased the numberof gemmae. In the presence of 0·08 µM Ca²⁺, oogoniumand oospore abortion rates were increased by A23187 [GenBank] with a peakeffect at 0·2 nM. In the presence of 10 µM Ca²⁺,abortion rates were unaffected by A23187 [GenBank] . Saprolegnia diclina, calcium, A23187, oogonia, oospores, gemmae, development, abortion     

Identification of a Phosphoprotein Expressed During Somatic Embryogenesis in Wheat Leaf Base Cultures

2,4-D mediated induction of somatic embryogenesis in wheat is enhanced in the presence of Ca⁺⁺ and its removal by EGTA reduces the response significantly. Changes that occur at the polypeptide level following 2,4-D treatment were analysed. Intense cell division activity was discernable in the leaf base explants within an hour of treatment. Changes in protein profiles were prominent in the membrane fraction as compared to the soluble fraction. The protein profile of the leaf base culture with somatic embryos was distinct from the calli induced from mature embryos on a 2,4-D containing medium. The role of Ca²⁺ in the induction of somatic embryogenesis was demonstrated by the use of EGTA (a calcium chelator), verapamil, nifedipine (calcium channel blockers), W7 (calmodulin antagonist) and Li (PI inhibitor). ^{In vitro} protein phosphorylation studies showed that 2,4-D, calcium and related treatments inhibit phosphorylation of proteins. In the membrane fraction proteins, accumulation of polypeptides at the low molecular weight range was seen in samples treated with verapamil and W7, and a 30 kO polypeptide in the samples treated with calmodulin antagonist, W7. Autoradiography of membrane fraction proteins displayed the presence of a 16 kO protein phosphorylated in samples treated with verapamil, nifedipine and W7. It thus appears that 2,4-D and Ca⁺⁺ prevent the phosphorylation of this phosphoprotein. These results thus indicate the action of 2,4-D via the Ca²⁺-CaM signaling pathway in triggering the induction of somatic embryogenesis.     

An Analysis of Osterhout and Hill's Salt Bridge Experiments in Characeae Internode

Conduction of action potentials in Chara internodal cells wasblocked at a 5%-urethane treated region. The action potentialscould be propagated beyond this region when an electric bridgewas built across it with a low enough resistance that the actioncurrent across it could depolarize the membrane at the distaljunction of the bridge up to the threshold level. After recoveryof propagation, the configuration of the action current flowingthrough the bridge changed from monophasic to diphasic. Coursesof the monophasic action current and the depolarizing potentialof the resting membrane were in parallel with the course ofaction potential, although they were slightly out of phase witheach other. The magnitudes of the current and depolarizing potentialagreed well with those estimated using a simplified equivalentcircuit of the bridge arrangement or with those observed usingan electric model circuit. ¹Present address: Department of Physiology, Tohoku UniversitySchool of Dentistry, Seiryo-machi, Sendai 980, Japan. ²Present address: Biology Laboratory, Kyoritsu Women's University,Hachioji, Tokyo 193, Japan. (Received December 18, 1985; Accepted March 26, 1986)     

Separation and Partial Characterization of Membranes from Prochloron sp.

Two differently colored membrane preparations were separatedfrom the prochlorophyte, Prochloron sp., by mechanical disintegrationof the cells followed by sucrose density gradient centrifugation.An orange-colored preparation, containing zeaxanthin as themajor constituent pigment, seemed to comprise the cytoplasmicmembrane. The other green-colored membrane preparation, containingß-carotene and chlorophyll a and b as major pigmentconstituents, was identified as the thylakoid membrane. Thetwo types of membranes were compared as to their absorptionspectra and buoyant densities. ¹ This work is one of the results of the 8th International Expeditionon Prochloron organized by Dr. R. A. Lewin, University of Californiaat San Diego. ⁵ Present address: Solar Energy Research Group, The Algatron,The Institute of Physical and Chemical Research (RIKEN), Wako-shi,Saitama 351, Japan. ⁶ Present address: National Institute for Basic Biology, Okazaki444, Japan. (Received October 19, 1984; Accepted January 7, 1985)     

Changes in H+-Pumps and a Tonoplast Intrinsic Protein of Vacuolar Membranes during the Development of Pear Fruit

Vacuolar H⁺-ATPase (V-ATPase) was purified from pear fruit andantibodies were raised against the subunits of 55 and 33 kDa.Antibodies against mung bean H⁺-pyro-phosphatase (V-PPase) andradish VM23, which is a tonoplast intrinsic protein (TIP) anda water channel, cross-reacted with the vacuolar membrane proteinsof pear fruit. To clarify the roles of these proteins in developmentof pear fruit, we determined their levels relative to the totalamount of protein by immunoblot analysis. The levels of subunitsof the V-ATPase increased with fruit development. By contrast,the level of V-PPase was particularly high at the cell-divisionstage and remained almost the same at other stages. The changesin the activities of V-ATPase and V-PPase corresponded to thosein their protein levels. The ratio of V-PPase activity to V-ATPaseactivity indicated that V-PPase is a major H⁺-pump of the vacuolarmembranes of young fruit and that the contribution of V-ATPaseincreases with fruit development, finally, V-ATPase becomesthe major H⁺-pump during the later stages of fruit development.The level of a protein analogous to VM23 (VM23P) was especiallyhigh during the active cell-expansion stage in young fruit,and VM23P might, therefore, play an important role in the rapidexpansion of cells as a vacuolar water channel. Our resultsshow that the levels of V-ATPase, V-PPase and VM23P change differentlyand reflect the roles of the respective proteins in the developmentof pear fruit. ³Research Fellow of the Japan Society for the Promotion of Science ⁴Present address: Faculty of Agriculture, Tohoku University,1-1 Tsutsumidori-Amamiyamachi, Aoba-ku, Sendai, 981 Japan     

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)     

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)     

Dark Reduction of P700+ in Photosystem I Particles by Illuminated Chlorophyllide

Illumination of chlorophyllide dissolved in a wet organic solventgenerates an unknown species of chlorophyllide which is capableof reducing P700⁺ in darkness ata considerably high rate inthe absence of ascorbate and redox mediators. The formationof this derivative species is accompanied by bleaching of boththe red and blue absorption bands of chlorophyllide concomitantwith the appearance of a new peak at around 500 nm. The generationof reducing capability is stimulated by the presence of basesbut does not require reducing agents. Some of the propertiesof this reaction are discussed in comparison with the Krasnovskiireaction. ¹Present address: Department of Environmental Biology, ResearchSchool of Biological Science, The Australian National University,Canberra City, ACT 2601, Australia. ³Present address: Department of Biology, Indiana University,Bloomington, IN 47405, U.S.A. (Received June 6, 1985; Accepted November 20, 1985)     

Mitochondrial calcium uptake stimulates nitric oxide production in mitochondria of bovine vascular endothelial cells

Although nitric oxide (NO) is a known modulator of cell respiration in vascular endothelium, the presence of a mitochondria-specific nitric oxide synthase (mtNOS) in these cells is still a controversial issue. We have used laser scanning confocal microscopy in combination with the NO-sensitive fluorescent dye DAF-2 to monitor changes in NO production by mitochondria of calf vascular endothelial (CPAE) cells. Cells were loaded with the membrane-permeant NO-sensitive dye 4,5-diaminofluorescein (DAF-2) diacetate and subsequently permeabilized with digitonin to remove cytosolic DAF-2 to allow measurements of NO production in mitochondria ([NO]_mt). Stimulation of mitochondrial Ca²⁺ uptake by exposure to different cytoplasmic Ca²⁺ concentrations (1, 2, and 5 µM) resulted in a dose-dependent increase of NO production by mitochondria. This increase of [NO]_mt was sensitive to the NOS antagonist L-N⁵-(1-iminoethyl)ornithine and the calmodulin antagonist calmidazolium (R-24571), demonstrating the endogenous origin of NO synthesis and its calmodulin dependence. Collapsing the mitochondrial membrane potential with the protonophore FCCP or blocking the mitochondrial Ca²⁺ uniporter with ruthenium red, as well as blocking the respiratory chain with antimycin A in combination with oligomycin, inhibited mitochondrial NO production. Addition of the NO donor spermine NONOate caused a profound increase in DAF-2 fluorescence that was not affected by either of these treatments. The mitochondrial origin of the DAF-2 signals was confirmed by colocalization with the mitochondrial marker MitoTracker Red and by the observation that disruption of caveolae (where cytoplasmic NOS is localized) formation with methyl--cyclodextrin did not prevent the increase of DAF-2 fluorescence. The activation of mitochondrial calcium uptake stimulates mtNOS phosphorylation (at Ser-1177) which was prevented by FCCP. The data demonstrate that stimulation of mitochondrial Ca²⁺ uptake activates NO production in mitochondria of CPAE cells. This indicates the presence of a mitochondria-specific NOS that can provide a fast local modulatory effect of NO on cell respiration, membrane potential, and apoptosis. nitric oxide; nitric oxide synthase; calcium; endothelium; mitochondria     

Intracellular Ca2+ Concentration and Cytoplasmic Streaming in Vallisneria Mesophyll Cells

Intracellular Ca²⁺ concentration regulating the cytoplasmicstreaming in Vallisneria mesophyll cells was estimated. Theleaf segment was cut open at the middle of the mesophyll celllayers and the exposed mesophyll cells were treated with testsolutions of various Ca²⁺ concentrations in the dark. This allowedA23187 [GenBank] , a calcium ionophore, to exert its full effect on thecell membrane. The streaming was induced or maintained in solutions which containedCa²⁺ at lower than 10^–6M. However, Ca²⁺ at concentrationshigher than 10^–5M had a definite, inhibitory effect. Theinduction and cessation of streaming could be repeated by alternatelychanging the solutions. (Received March 14, 1986; Accepted May 15, 1986)     

Swelling of Etiolated Oat Protoplasts Induced by cAMP and Red Light

Etiolated oat protoplasts were treated with dibutyryl cAMP tostudy possible function of cAMP in the development by measuringthe protoplast swelling. The mean diameter of protoplasts inthe absence of any chemical treatment was 33.58±1.26(SE) µm, which increased to 36.96±0.86 µmin the presence of 100 µM dibutyryl cAMP. Prostacyclin,a potent activator of adenyl cyclase, also showed a significantswelling effect (diameter 38.01±0.98 µm). Red lightalso elicited the swelling of protoplasts (40.26±0.8µm). ¹Present address: Department of Biology, Pusan National University,Pusan 607, Korea. ²Present address: Department of Horticulture, Cheju NationalUniversity, Cheju 590, Korea. ³Present address: Department of Biological Sciences, Texas TechUniversity, Lubbock, TX 79409, U.S.A. (Received June 29, 1985; Accepted November 18, 1985)     

Alterations in Protein Synthesis in vivo in Chilling Sensitive Mung Bean Hypocotyls Caused by Chilling Stress

The effects of chilling on protein synthesis in vivo in etiolatedhypocotyls of Vigna radiata L. were investigated. After exposureof the tissues to 0?C for various periods of time, proteinswere labeled with [³⁵S]-methionine at 26?C. The total amountof ³⁵S incorporated into soluble and membrane proteins was reversiblyreduced by chilling for 24 h, during which time the tissuessuffered no injury. Further prolonged chilling produced an irreversibledecline both in the incorporation of radioactivity and in cellviability as assessed by the extent of leakage of electrolyte.The ³⁵S-labeled proteins in the soluble and the total membranefractions were analyzed quantitatively. Chilling of etiolatedhypocotyls for one or two days induced the syntheses of twosoluble proteins (82 and 74 kDa) and one membrane protein (80kDa). Moreover, three heat-shock proteins (HSPs) that were inducedby heat stress (41 ?C, 4h) had the same electrophoretic mobilitiesas those of the proteins induced directly or indirectly by thechilling treatment. ¹Contribution No. 3170 from the Institute of Low TemperatureScience. (Received April 22, 1988; Accepted September 30, 1988)     

Changes of Some Membrane-Associated Enzyme Activities Degradation of Membrane Phospholipids in Cucumber Roots Due to Ca2+ Starvation

Deprivation of Ca²⁺ from a complete culture medium affectedthe enzyme activities associated with five membrane fractionsof cucmber roots obtained by discontinuous sucrose density gradientcentrifugation. The total activity of K⁺-ATPase, Cyt. c oxidaseand NADPH-Cyt. c reductase of Ca²⁺-deficient roots, starvedfor only 4 days, had decreased to 14, 38 and 60% of the activityof the control roots. In general, loss of enzyme activitieswas accompanied by a shift of activity distribution from theheavier density fractions to lighter ones. The amounts of Ca²⁺ associated with membranes from Ca²⁺-starvedroots decreased to 50–60% of those of the control roots.Both phospholipid and neutral lipid contents in the membranesdecreased markedly while the protein content was not changedby Ca²⁺ deficiency. Phospholipid analysis indicated a drasticdrop in the percent composition of phosphatidylinositol butan increase of phosphatidic acid. Also, phospholipase D activityincreased remarkably during Ca²⁺ starvation, paralleling theappearance of Ca²⁺-deficiency symptoms. Thus, the major effects of Ca²⁺ deficiency appear to be to stimulatephospholipase D activity and a reduction in membrane bound Ca²⁺.These effect may be involved in disorganization of the membranestructure and the changes of enzyme activities associated withthe altered membranes. ¹Rubber Research Institute of Sri Lanka, Dartonfield, Agalwatta,Sri Lanka. (Received July 15, 1985; Accepted November 21, 1985)