Distribution and Properties of Chromatin-Associated Nucleoside Triphosphate Diphosphatase Purified by a New Method

Chromatin-associated nucleoside triphosphate diphosphatase (NTDPase)activity was detected in Alaska pea plant only at the germinationstage in a study of the plant's development over 5 month fromthe dormant to fruiting stages. This enzyme activity was alsodetected in seedlings of several dicotyledonous plants includingsoybean, Usui pea, cowpea and cucumber, but almost none wasfound in monocotyledonous corn and rice plants. When the chromatin of pea epicotyl was fractionated into template-activeand -inactive forms by DNase II digestion, most of the NTDPaseactivity was found in the active chromatin. A new method to rapidly isolate and purify NTDPase from peaepicotyl chromatin was developed in which NTDPase was elutedwith a linear gradient of NaCl on a column packed with cellulosepowder and chromatin. DNA remained on the column and the elutedNTDPase was purified further by chromatography using trimethylamino-2-hydroxypropylcellulose (TMAP-cellulose) and a Sephadex G-100 column, whichgive chromatin 18-fold purer than the starting sample. This purified NTDPase was adsorbed by DNA-cellulose, which isfurther evidence that NTDPase is a kind of non-histone proteinassociated with DNA. Several cations including Ca^2$, Mg^2$, Mn^2$and Zn^2$ stimulated the enzyme activity, with the maximal eightfoldactivation by Ca^2$. NTDPase activity was clearly inhibited byKCN and pyrophosphate, but not by SH-blocking inhibitors andvarious metal chelators at 1 mM.     

Repression of Asolectin-Dependent Activation of Partially Lipid Depleted ATPase Prepared from the Plasma Membrane-Enriched Fraction of Cucumber Roots due to Ca2+ Starvation

The ATPase activity of the plasma membrane-enriched fractionwas severely inhibited by withdrawal of Ca²⁺ from the mediumfor 5 days, although the root system appeared to be unaffectedto visual inspection. Partially lipid-depleted ATPases withsimilar ratios of phospholipid to protein were prepared fromthe plasma membrane-enriched fraction of cucumber roots culturedwith control medium and one lacking Ca²⁺, and their propertieswere compared. SDS disc polyacrylamide gel electrophoresis showedthat the polypeptide components were essentially similar betweencontrol and Ca²⁺-starved roots. Partially lipid-depleted ATPasereassociated with asolectin, the lecithin from soybean, showedtypical characteristics of plasma membrane type ATPase; pH optimumat 6.5, high specificity for ATP as substrate and strong inhibitionby vanadate but not nitrate. The activity of reassociated ATPaseobtained from the control roots was apparently higher than theactivity obtained from Ca²⁺-starved roots. The amount of asolectinrequired for maximum activation of the partially lipid-depletedATPase prepared from control roots was much lower than thatprepared from Ca²⁺-starved roots. Reassociation of partiallylipid-depleted ATPase with asolectin produced higher ATPaseactivity than that with individual phospholipids. The activationof partially lipid-depleted ATPase prepared from control rootswith asolectin was not inhibited by addition of a sample preparedfrom Ca²⁺-starved roots. Thus, a decrease in the functionalassociation of ATPase with phospholipids might be one of thephysiological injuries in root cell membranes of cucumber causedby Ca²⁺ starvation. ¹Permanent address: Department of Horticulture, College of Agriculture,Chonnam National University, Chonnam 500, Korea. (Received February 23, 1988; Accepted August 18, 1988)     

Changes of the Structure of Pea Chromatin by Aluminum

Structural changes of pea chromatin due to in vitro or in vivotreatment with Al were investigated. The absorption spectrumshowed that chromatin might be condensed and/or aggregated withAl treatment. Analysis of the melting profile showed that chromatinprepared from Al-treated roots was more stable to thermal treatment. Chromatin from Al-treated roots and chromatin treated with Alin vitro were less susceptible to DNase II digestion. When thepartially digested chromatin with DNase II was chromatographedwith Bio-Gel A5m, the digestibility of chromatin obtained fromAl-treated roots was less than that from control roots. Furthermore,most of the Al was associated with a chromatin fraction whichwas minimally digested with DNase II and of large size afterthe gel chromatography. The results suggested that Al absorbedby pea roots is somehow related to alteration of the chromatinstructure, i.e. its condensation and/or aggregation. The conclusionreached was that Al toxicity results from disturbance of thenuclear activity. (Received July 22, 1987; Accepted December 7, 1987)     

Effects of Calcium on Template Activity and Size of Pea Chromatin Fragments

Pea chromatin was fractionated into template active and inactivefragments by cleavage with DNase II followed by fractionationwith Bio-Gel A 5 m column chromatography. When the chromatin fragments were chromatographed in the presenceof Ca²⁺ or EGTA, the elution patterns suggested that Ca²⁺ assembledmoderately digested chromatin fractions. Moreover, total templateactivity of chromatin fractions was reduced by Ca²⁺ and theactive fraction was eluted at different positions under thechelated and non-chelated conditions. (Received February 12, 1986; Accepted May 28, 1986)     

Repression of Proton extrusion from Intact Cucumber Roots and the Proton Transport Rate of Microsomal Membrane Vesicles of the Roots due to Ca2+ Starvation

Proton extrusion from cucumber roots decreased markedly duringCa²⁺ starvation in the presence of KC1. Vesicles with ATP-dependentproton transport activity were prepared from the microsomalmembrane fraction of control and Ca²⁺-starved roots. The protontransport rate of the vesicles from Ca²⁺-starved roots was repressedto less than half of the vesicles prepared from the controlroots. K⁺-Mg²⁺-ATPase activity associated with the vesiclesprepared from Ca²⁺-starved roots was approximately one-thirdof the activity associated with those prepared from controlroots. K_m values of the proton transport rate and K⁺-Mg²⁺-ATPasefor ATP were much higher in vesicles prepared from Ca²⁺-starvedroots. The repression of proton extrusion linked with K⁺ uptake inthe Ca²⁺-starved roots could be largely caused by the reducedproton pumping activity associated with microsomal membranesin the roots. (Received May 25, 1987; Accepted October 14, 1987)     

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)     

Calmodulin-Like Activity Associated with Chromatin from Pea Buds

NAD kinase and cyclic AMP phosphodiesterase were activated bya factor prepared from pea chromatin. About 62% of the originalamount of the factor in the purified chromatin was recoveredin the reassociated chromatin. The NAD kinase- and cyclic AMP phosphodiesterase-activatingfactor was released from the chromatin by heat treatment withethylene glycol-bis(ß-aminoethyl ether)- N,N,N',N'-tetraaceticacid (EGTA) then adsorbed on an affinity gel of phenothiazineagarosederivatives in the presence of excess Ca²⁺ over EGTA, afterwhich it was eluted by a flush of EGTA. Activation of NAD kinaseand cyclic AMP phosphodiesterase by this factor depended onthe presence of Ca²⁺. The NAD kinase-activating factor and chromatin were coelutedwhen soluble chromatin was applied to a Bio-Gel A50 column.When chromatin was chromatographed on the same column afterdigestion by DNase I, the factor was eluted in association withthe digested products of the chromatin. The activation propertiesof this factor indicate that a calmodulin-like activity existsin association with pea chromatin. The activation curves of cyclic AMP phosphodiesterase with thepea bud factor and with bovine brain calmodulin were compared.The amount of the factor in the chromatin fraction that correspondedto authentic calmodulin was calculated as 5.7 µg per mgDNA. (Received August 6, 1982; Accepted February 17, 1983)     

Effect of Calcium on the Structure and Template Activity of Pea Chromatin

The absorption spectrum of native pea chromatin solubilizedunder minimal shearing conditions changed with increasing Ca²⁺concentration; the ratio of maximum to minimum absorption decreasedand the maximum absorption peak shifted to a longer wavelength.The concentration of Ca²⁺ to cause half complete sedimentationof chromatin was much lower for the solubilized native chromatin(more condensed and larger in size) than for the sonicated chromatin(less condensed and smaller in size). Solubilized native chromatinshowed a two-step melting profile in the absence of Ca²⁺. In the presence of Ca²⁺ the two T_ms disappeared and a new higherT_m appeared. Template activity of solubilized native chromatinincreased 3-fold upon dispersion and fragmentation by sonication.Addition of a small amount of ethylene glycol-bis (ß-aminoethylether)-N, N'-tetraacetic acid (EGTA) promoted the template activityof solubilized native chromatin, but not that of sonicated ordenatured DNA. The effect of EGTA was reversed by Ca²⁺. Thechromatin reconstituted in the presence of EGTA showed a lowerT_m than the chromatin reconstituted in the presence of Ca²⁺.The relationship between chromatin structure and its templateactivity is discussed in relation to Ca²⁺. (Received August 12, 1985; Accepted December 7, 1985)     

Sex-specific alterations in chromatin conformation of the brain of aging mouse

Chromatin conformation has been analysed in the brain cortex of adult (24±2 weeks) and old (65±4 weeks) male and female mice. Nuclei purified from different groups of mice were digested with MNase and DNase I for varying time periods (0–90 min), and with endogenous endonucleases for 1 h. MNase and DNase I digestion kinetics showed that the percentage of acid solubility of chromatin was relatively lower in old than adult and in female than male. This was further supported by electrophoretic analysis of nuclease digested DNA fragments. When the nuclei were incubated with only Ca²⁺or mg²⁺, no endonuclease digestion was observed. However, under similar conditions, the liver DNA was cleaved substantially. When divalent cations were added together, they activated endogenous endonucleases and digested the brain chromatin. The activity of Ca²⁺/Mg²⁺-dependent endogenous endonucleases was higher in male than female. Thus the accessibility of chromatin to MNase, DNase I and endogenous endonucleases was higher in male than female, and MNase as well as DNase I were more active in adult than old. Such sex- and age-dependent conformation of chromatin may attribute to differential expression of genes in the mouse brain.     

Repression of the K+ Uptake and Cation-Stimulated ATPase Activity Associated with the Plasma Membrane-Enriched Fraction of Cucumber Roots Due to Ca2+ Starvation

The uptake of K⁺ by cucumber plants decreased markedly duringCa²⁺ starvation. A plasma membrane-enriched fraction, judgedfrom the distribution of marker enzymes, was prepared from controland Ca²⁺-starved roots. The Mg²⁺- and K⁺-Mg²⁺-ATPase activitiesassociated with the plasma membrane-enriched fraction of controlroots were maxima at pH 6.5. Various monovalent cations andpotassium salts of monovalent anions stimulated Mg²⁺-ATPaseactivity. Vanadate, DES and DCCD inhibited K⁺- Mg²⁺-ATPase activity.Of the divalent cations and phosphate esters tested, Mg²⁺ andATP were most effective for the stimulation of ATPase by K⁺,whereas Ca²⁺ was ineffective in replacing Mg²⁺. Mg²⁺- and K⁺-Mg²⁺-ATPase activities associated with the plasmamembrane enriched fraction of Ca²⁺-starved roots were much lowerthan those of control roots. K_m values of K⁺-Mg²⁺-ATPase forATP were comparable for control and Ca²⁺-starved roots. The K⁺-stimulated activity of Mg²⁺-ATPase in Ca²⁺-starved rootswas approximately one fourth that of the control, whereas therate of stimulation was only slightly lower in Ca²⁺-starvedroots. (Received May 9, 1984; Accepted September 17, 1984)     

Activities of Ca2+ pump and low affinity Ca2+/H+ antiport in plasma membrane vesicles of corn roots

ATP-dependent Ca₂₊-uptake was investigated in sealed plasmamembrane vesicles isolated from corn roots (Zea mays L. cv.Hybrid-3352/Palma-Pioneer). In a chloride-containing medium,at high calcium concentrations, about 30% of the total Ca²⁺accumulation ({small tilde}4 nmol Ca²⁺ mg^–1 protein) wasshown to be protonophore-sensitive and corresponded to the fractionof Ca²⁺ not accumulated in a sulphate-containing medium. Furthermore,vesicles in the presence of nitrate, which stimulates H⁺ transport,or vesicles preloaded with H⁺, take up Ca²⁺ more rapidly, suggestingthat, at high calcium concentrations, there is a mechanism forCa²⁺ transport which depends on the magnitude of the protongradient across the membrane. The fraction of Ca²⁺ uptake shownto be sensitive to the protonophore CCCP increased by about150–200% as the Ca²⁺ concentration in the medium increasedfrom 50µM to 250µM. Under the same conditions, theCCCP-insensitive fraction of Ca²⁺ accumulated was reduced byabout 25–30% suggesting that different Ca²⁺ affinitiesexist in the two Ca²⁺ uptake processes. Although calmodulinstimulation was not observed, the sensitivity to Ca²⁺ and externalpH indicates that H⁺ gradient-independent Ca²⁺ accumulationreflects activity of the Ca²⁺–pump. These results indicatethat the plasma membrane of corn roots contain two distinctmechanisms of Ca²⁺ transport: a high Ca²⁺ affinity, proton gradient-independentCa²⁺ pump and a low Ca²⁺ affinity, proton gradient-dependentCa²⁺/H⁺ antiport, which have greatest activity at concentrationsof Ca²⁺ below and above 50+M, respectively. Key words: Ca²⁺/H+ antiport, Ca²⁺ pump, plasmalemma, roots, Zea mays L.     

Effect of NaCl salinity on growth,pigment and mineral element contents,and gas exchange of broad bean and pea plants

Increasing salinity of growth medium induced a reduction in growth and transpiration rate. The concentrations of chlorophylls and carotenoids were increased in most cases in broad bean leaves while in pea plants they remained more or less unchanged with the rise of salinization up to 80mM NaCl. Thereabove a significant decrease in these contents was observed. A stimulation of the net photosynthetic rate of pea was observed at the lowest levels of NaCl but at the highest levels inhibitory effect was recorded. In broad bean all salinization levels inhibited photosynthetic activity, but dark respiration of both plant species was stimulated. The content of Na⁺ in the roots and shoots of both species increased at increasing salinity. In broad bean, Ca²⁺ concentration in shoots and K⁺ and Ca²⁺ contents of roots increased at increasing salinization, while in pea plants, the content of K⁺ and Ca²⁺ was almost unaffected by salinity. Salinity induced an increase in the content of these ions in pea roots. Mg²⁺ content in shoots and roots of both broad bean and pea decreased at increasing salinity except in roots of pea, where it was generally increased.     

Inactivation and Calcium-Dependent Reactivation of Oxygen Evolution in Photosystem II Preparations Treated at pH 3.0 or with High Concentrations of NaCl

A brief treatment at pH 3.0 of Photosystem II (PS II) membranescontaining two bound Ca²⁺ from rice resulted in strong suppressionof oxygen evolution concomitant with extraction of one Ca²⁺and the lost activity was restored on addition of 50 mM Ca²⁺.However, inactivation of oxygen evolution by low pH-treatmentof oxygen-evolving PS II complexes containing only one Ca²⁺from a rice chlorophyll b-deficient mutant was not associatedwith extraction of the bound Ca²⁺, although oxygen evolutionwas markedly enhanced by the addition of Ca²⁺ to the treatedcomplexes. Thus, the acid-inactivation of oxygen evolution cannotbe related to extraction of Ca²⁺. On the other hand, low pH-treatmentwas found to share the following common features with NaCl-treatmentwhich also causes a Ca²⁺-reversible inactivation of oxygen evolution.(1) Exposure of PS II membranes to pH 3.0 resulted in solubilizationof the 23 and 17 kDa extrinsic proteins, although the releasedproteins rebound to the membranes when pH was raised to 6.5.(2) There was an apparent heterogeneity in the binding affinityof Ca²⁺ effective in restoration of the oxygen-evolving activity.(3) Low pH-treated preparations required a higher concentrationof Ca²⁺ for the maximum reactivation of oxygen evolution thandid NaCl-washed preparations. This was also the case with Sr²⁺,which stimulated oxygen evolution of both low pH-treated andNaCl-washed PS II membranes to smaller extents. When the extrinsic23 and 17 kDa proteins had been removed, however, Ca²⁺ concentrationdependence of oxygen evolution in low pH-treated membranes becamesimilar to that in NaCl-washed PS II preparations and the changeswere largely reversed by rebinding of the two proteins. Theseresults strongly suggest that low pH-treatment and NaCl-washinvolve similar mechanisms of Ca²⁺-dependent reactivation. ¹ Present address: Solar Energy Research Group, The Instituteof Physical and Chemical Research (RIKEN), Wako, Saitama, 351-01Japan (Received August 27, 1990; Accepted February 12, 1991)     

Protective Effect of External Ca2+ on Elongation and the Intracellular Concentration of K+ in Intact Mung Bean Roots under High NaCl Stress

The effects of Ca²⁺ in the external medium on intact mung beanroots under high NaCl stress were investigated. With increasingexternal concentrations of NaCl, mung bean roots showed suppressionof elongation and a decrease in the intracellular concentrationof K⁺. Addition of Ca²⁺ to the external medium alleviated theinhibition of root elongation under the high NaCl stress andmaintained a high intracellular concentration of K⁺ in the elongatingregion of the roots. This counter effect of Ca²⁺ against theNaCl stress on roots was correlated with the ratio of [Ca²⁺]/[Na⁺]²in the external medium. A value above 5.0 ? 10^–4 mM^–1resulted in almost complete recovery of root elongation undervarious high concentrations of NaCl. Root elongation for 24h under NaCl stress was correlated with the extent to whichthe intracellular concentration of K⁺ was in excess of 10 mM.Maintenance of an adequate concentration of K⁺ in root cellsis essential for root elongation under salt stress. These findingsindicate that Ca²⁺ prevents the leakage of intracellular K⁺and thereby supports the elongation of roots under salt stress. (Received November 13, 1989; Accepted June 5, 1990)     

In vivo Treatments That Modulate PPi-Dependent H+ Transport Activity of Tonoplast-Enriched Membrane Vesicles from Barley Roots

The PP_i-dependent H⁺ transport activity of tonoplast-enrichedmembrane vesicles prepared from barley roots was greatly reducedwhen the plants were grown for 4 or 5 days with an additional3 raM KC1 in growth medium that contained only 0.1 mM CaCl₂in water. To characterize the mechanism of this reduction inactivity, we attempted to treat barley roots with K⁺ ions, Cl^-ions(or acetate), and A23187 [GenBank] (with or without Ca²⁺ ions), whichmight be expected to cause alkalization, acidification and mobilizationof Ca²⁺ ions in the cytoplasm, respectively. One-day treatmentof barley roots with K⁺ ions significantly decreased PP_i--dependentH⁺ transport activity of prepared tonoplast-enriched membranevesicles, while treatment with Cl^- ions or acetate significantlyincreased the activity. A similar increase in the activity alsooccurred by treatment with Ca²⁺ ions alone or in combinationwith A23187 [GenBank] . Determination of the PP_i-hydrolyzing activity ofmembrane vesicles showed that changes in this activity by thevarious treatments were similar to those in the PP_i-dependentH⁺ transport activity. The changes in ATP-dependent H⁺ transportactivity of membrane vesicles caused by these treatments weresmall. These results indicate that the in vivo treatments hadsignificant effects on the H⁺ transport activity of H⁺-PP_i-ase,one of the two active vacuolar H⁺-pumps (H⁺-PP_iase and H⁺-ATPase).In addition, these results suggest the possibility that changesin levels of cytoplasmic H⁺ or Ca²⁺ ions may be involved inmodulation of the H⁺ transport activity of the vacuolar H⁺-PP_iaseduring plant growth. (Received September 14, 1992; Accepted March 1, 1993)     

Specific Increase in Phosphatase Isozymes in Cucumber Roots Caused by Calcium Deficiency

Phosphatases in cucumber roots, whose production was inducedby Ca^2$ deficiency, were characterized chromatographically usingATP, 2'(3')-AMP and p-nitrophenyl-phosphate (PNPP) as substrates.Ca^2$ deficiency stimulated greater than 10-fold increases inthe activities with these substrates of the non-adsorbed fractionfrom a DEAE-cellulose column. Several fractions associated withthese phosphatase activities were eluted from the column withNaCl solution; their levels increased less with Ca^2$ starvation.When the non-adsorbed fraction from Ca^2$-straved roots was appliedto a Sephadex G-200 column, fractions associated with 2'(3')-AMPase(phosphatase I) and with both ATPase and PNPPase (phosphataseII) were separated. In the control roots, very weak activitiesof phosphatases I and II were observed at the same positionon the gel filtration. The phosphatase I isolated from boththe control and Ca^2$-starved roots was extremely specific tonucleoside 2'(3')-monophosphates, whereas phosphatase II fromboth types of roots had a relatively broad substrate specificity.When phosphatase I from Ca^2$-starved roots was stained with2'(3')-AMP in CaCl₂ after polyacrylamide gel electrophoresis,a single band was obtained. Phosphatase I from control rootsalso showed a single band, with the same Rf value. PhosphataseII from both types of roots contained two isozyme bands whenthe activities were stained with either ATP or PNPP. These resultsindicate that Ca^2$ starvation causes specific increases in thelevel of phosphatases I and II in cucumber roots. (Received October 28, 1981; Accepted January 19, 1982)     

Unidirectional Ca2+ Fluxes in Roots of Rye (Secale cereale L). A Comparison of Excised Roots with Roots of Intact Plants

The unidirectional Ca²⁺ fluxes across the plasma membrane andtonoplast were determined in both excised roots and roots ofintact seedlings of rye (Secale cereale L. cv. Rheidol). Theunidirectional Ca²⁺ fluxes across the plasma membrane and tonoplastmeasured in excised roots were of a similar order of magnitudeto those determined in roots of intact plants. Influx and effluxof Ca²⁺ across the root plasma membrane were similar (estimatedto be between 0·7 and 3·4 µmol g     

Reactivation of Cytoplasmic Streaming in a Tonoplast-Permeabilized Cell Model of Acetabularia

To find whether cytoplasmic streaming in Acetabularia is controlledby Ca²⁺, a tonoplast-permeabilized cell model was prepared usinga vacuolar perfusion technique. The cytoplasmic streaming remainedalmost normal after perfusion with EGTA medium (10 mM EGTA,40 mM PIPES, 5mM MgCl₂ and 800 mM sorbitol, pH 6.9), but stoppedwithin 10 min when saponin medium (EGTA medium plus 50 µg/mlsaponin, 50 µg/ml hexokinase and 5 mM glucose) was perfused.This model system was reactivated with a solution containing0.5 mM ATP and different concentrations of Ca²⁺ (reactivationmedium). With the reactivation medium at pCa 6–5, theresumed streaming lasted for about 10 min before the cytoplasmaggregated. At pCa 4–3, the streaming was observed onlyfor a few minutes because the cytoplasm aggregated quickly.At pCa 7, no reactivated movement was observed. Reactivationwas not induced in an ATP- or Mg²⁺-deficient medium even inthe presence of an adequate concentration of Ca²⁺, and was inhibitedby 50 µg/ml cytochalasin B or 1 mM N-ethylmaleimide. We concluded from these observations that the cytoplasmic streamingin Acetabularia is very likely to be driven by the actomyosinsystem in the presence of Mg-ATP and Ca²⁺ at pCa 6–5. (Received October 31, 1984; Accepted April 1, 1985)     

Effects of Ca2+ and Cd2+ on the Carbohydrate Metabolism in Sugar Beet (Beta vulgaris)

Sugar beet (Beta vulgaris L. cv. Monohill) were cultivated ina nutrient solution with different combinations of Ca²⁺ (36,180, 720 or 3560µM) and Cd²⁺ (0, 1, 5 or 20µM).The dry and fresh weights, the content of Ca²⁺ and Cd²⁺ , sucrose,fructose, glucose and starch in 5-week-old plants was analysedas well as the rate of [¹⁴C]-sucrose uptake in discs from 3-month-oldstorage roots. The carbohydrate metabolism was indirectly affectedby the presence of calcium or cadmium. Cadmium caused a diminisheddry weight and carbohydrate concentration. The dry weight wasunaffected by the Ca²⁺ level but the carbohydrate distributionbetween storage and growth processes was affected; at low Ca²⁺in the tissue, the growth was retarded and the level of storagecarbohydrate increased, while at high Ca²⁺ the opposite wasfound. The [¹⁴C]-sucrose uptake decreased in tap roots cultivatedat low Ca²⁺ . Long term exposure to Cd²⁺ also decreased thesucrose uptake in tap roots. Direct Cd²⁺ addition to the assaymedium, however, increased the sucrose uptake, probably at thetonoplast, while Ca²⁺ had no transient effect on the uptake.Cadmium increased the Ca²⁺ concentration in the plant, but Ca²⁺did not affect the net-uptake of Cd²⁺. Key words: Sugar beet, cadmium uptake, calcium uptake, carbohydrate formation, growth     

Chromatin structure variation in successful and unsuccessful arbuscular mycorrhizas of pea

Summary Lincoln and Frisson varieties of endomycorrhiza-forming pea plants and isogenic mycorrhiza-resistant Frisson mutant (P2) plants were inoculated withGlomus mosseae. Nuclei released from inoculated and non-inoculated (control) roots were analysed for chromatin structure and activity using flow cytometric techniques. Chromatin accessibility to the specific DNA fluorochrome DAPI at saturating and non-saturating concentrations was measured. DNA fluorescence of nuclei of mycorrhizal Lincoln and wild genotype Frisson plants was significantly increased, compared to the controls, at saturating and, more strongly, at non-saturating DAPI concentrations. In contrast, the nuclei of inoculated P2 mutant roots showed a much lower increase in fluorescence, compared to uninoculated controls. Nuclei released from mycorrhiza-infected Lincoln roots were more sensitive to DNase I than those of uninfected ones. These results indicate a dramatic increase in that portion of the genome which can be transcribed in response to AM infection.Abbreviations AM arbuscular mycorrhizas - CRBCs chicken red blood cells - CV coefficient of variation - DAPI 4 6-diamidino-2-phenylindole - DNase I deoxyribonuclease I - EDTA ethylenediamine tetraacetic acid - FCM flow cytometry - TMN Tris MgCl₂ NaCl buffer