Characterization of membrane-bound Mg++-activated ATPase isolated from the lower epidermis of tobacco leaves

Membrane-bound Mg⁺⁺-activated ATPase was separated from thelower epidermis of tobacco leaves (Nicotiand tabacum L. SamsunNN) on stepwise sucrose density gradient centrifugation. Membrane-bound epidermal ATPase was localized in the interfaceof densities in sucrose of 1.12 to 1.16 in the sedimentary fractionbetween 1,500?g to 10,000?g from the homogenate of the lowerepidermis. The epidermal ATPase activity was activated by divalentcations (Mg⁺⁺>Mn⁺⁺Co⁺⁺>Fe⁺⁺>Zn⁺⁺>Ca⁺⁺) and furtherstimulated by KCl by ca. 20%. The pH optimum for Mg⁺⁺-activationof the epidermal ATPase was ca. 6.0. The enzyme hydrolyzed ATPmore rapidly than other nucleoside triphosphates. The optimumtemperature for activation of the epidermal ATPase activitywas ca. 40?C. 50% of the epidermal ATPase activity was lostin 18 min at 55?C and in 2.5 days at 2.5?C. The apparent Kmvalue of the epidermal ATPase was 4.7?10^–4 M and Vmaxwas 65.4 nmoles Pi/mg protein/min. The epidermal ATPase wasstrongly inhibited by N, N'-dicyclohexylcarbodiimide (DCCD)in vitro whereas oligomycin, carbonyl cyanide m-chlorophenylhydrazone(CCGP), indoleacetic acid (IAA) and abscisic acid (ABA) wereinsensitive to the epidermal ATPase activity. (Received May 23, 1978; )     

Some properties of shikimate: NADP oxidoreductase produced in sweet potato root tissue after slicing

The activity of shikimate: NADP oxidoreductase [EC 1. 1. 1.25] in sweet potato root tissue increased soon after slicing.Enzyme preparations obtained from both sliced tissue and fromfresh tissue probably contained a single enzyme component, andthey showed identical chromatographical behaviour. K_m values of the enzyme for NADP and shikimate were 1.0x10^–4Mand 1.3 x 10^–3M, respectively. Enzyme activity was potentlyinhibited by SH-inhibitors such as p-chloromercuribenzoate andoxidized glutathione. Enzyme activity was affected neither by mononucleotides suchas ATP, ADP and AMP, divalent cations, Mg⁺⁺, Ca⁺⁺ and Mn⁺⁺,nor by metabolites such as tryptophan, phenylalanine, tyrosineand t-cinnamic acid which are involved in aromatic compoundsyntheses. The enzyme rapidly lost its activity. This inactivation reactionshowed a time course consisting of two steps of the first-orderreaction. The inactivated enzyme preparation was not reactivatedby thiol compounds such as cysteine, 2-mercaptoethanol and glutathione,although these reagents, to a certain extent, protected theenzyme from inactivation. The results suggest that denaturationof the enzyme protein was involved in inactivation of the enzyme. ¹Part 74 of the phytopathological chemistry of sweet potatowith black rot and injury. ²Present address: Department of Biology, Faculty of Science,Tokyo Metropolitan University, Setagaya-ku, Tokyo. (Received August 5, 1968; )     

Morphological and physiological characterization of IAA-less-sensitive and IAA-sensitive phases in rooting of Azukia cuttings

In disbudded epicotyl cuttings taken from light grown 5-dayold Azukia angularis Phaseolus angularis) seedlings, all adventitiousrootlets appeared on the second day of incubation. No root primordiawere observed within the first 24 hr and no increase in thenumber of roots occurred after 48 hr. Puromycin (5.5?10^–5M), p-fluorophenylalanine (1?10^–3M),2-thiouracil (2.3?10^–4M) and 2,6-diaminopurine (2?10^–5M)inhibited rooting when applied to cuttings on the second day,but showed no inhibition when applied on the first day. Unlike these inhibitors, pyrithiamine (7.2?10^–5M) inhibitedrooting when it was applied to cuttings on the first day. A rooting promoting effect was observed with actinomycin D (2.4?10^–6M),2,4-dinitrophenol (3?10^–5M) and p-fluorophenylalanine(1?10^–4M) applied to the cuttings on the first day, whereasindoleacetic acid (1.7?10^–4M) showed its promoting effectmost effectively on the second day. ¹Contribution No. 17 from the Botanical Gardens, Faculty ofScience, University of Tokyo, Tokyo, Japan. (Received June 4, 1969; )     

Action of Benzoic Acid and its o-, m- and p-Hydroxy-Derivatives on the Dark- and Light-Induced Leaflet Movements in Cassia fasciculata Michx.

Benzoic acid and its o-, m- and p-hydroxy derivatives appliedon excised leaves of Cassia fasciculata modified the dark-induced(scotonasty) and light-induced (photonasty) leaflet movements.Benzoic acid inhibited the scotonastic closure in a dose-dependentmanner from 10^–4M to 10^–3M and promoted the photonasticopening at optimum concentration of 5.10^–4M. These effectswere dependent upon the position of hydroxyl group on the benzoicring, the o-derivative inducing a stronger effect than the m-and p-derivatives. Experiments showed that treatment with o-hydroxybenzoicacid had not to exceed 30–60 min and that the maximumeffect was obtained at pH 5.5. (Received September 16, 1986; Accepted June 22, 1987)     

Enzymes involved in the last steps of the biosynthesis of mannitol in brown algae

Mannitol-1-phosphate dehydrogenase (EC 1.1.1.17 [EC] ) and mannitol-1-phosphatase(EC number yet unassigned) were detected in the brown algae,Spatoglossum pacificum and Dictyota dichotoma. The enzymes wereextracted from algal fronds and their properties were investigatedusing partially purified preparations. Mannitol-1-phosphatase shows maximum activity at pH 7. The enzymehad a narrow substrate specificity. The Km value for mannitol-1-phosphateis 8.3x10^–4 M (30°C, pH 7.0). The enzyme is activatedby Mg⁺⁺ and Mn⁺⁺and is strongly inhibited by PCMB, Hg⁺⁺and NaF. Mannitol-1-phosphate dehydrogenase showed maximum activitiesat pH values 6.5 and 10.2 in reductive and oxidative reactions,respectively. The dehydrogenase also showed narrow substratespecificity; mannitol-1-phosphate and NAD or fructose-6-phosphateand NADH₂ are utilized, respectively, in oxidative and reductivereactions by the enzyme. Km values for these substrates andthe coenzymes are 2.5x10^–4 M and 7.1x10^–5 M forthe first pair and 2.8x10^–4 M and 1.3x10^–5 M forthe latter pair. This enzyme was strongly inhibited by PCMBand Hg⁺⁺, but was only slightly affected by adenosine phosphates. Possible roles of these enzymes in the biosynthesis of mannitolin brown algae are discussed. ¹ Contributions from the Shimoda Marine Biological Station ofTokyo Kyoiku University, No. 233. This work was supported inpart by a Grant-in-Aid for Co-operative Research from the Ministryof Education, Japan and in part by a grant to one of us (T.Ikawa) from the Matsunaga Science Foundation. ² Present address: Chemical and Physical Laboratory, HoechstJapan Research Laboratory, Minamidai, Kawagoe, Japan. (Received February 22, 1972; )     

Ion Fluxes Across the Transfusion Zone of Secreting Limonium Salt Glands Determined from Secretion Rates, Transfusion Zone Areas and Plasmodesmatal Frequencies

Faraday, C. D., Quinton, P. M. and Thomson, W. W. 1986. Ionfluxes across the transfusion zone of secreting Limonium saltglands determined from secretion rates, transfusion zone areasand plasmodesmatal frequencies.—J. exp. Bot. 37: 482–494. The epidermal salt-secreting glands of Limonium (Plumbaginaceae)are enclosed in a cuticular envelope. Ions and metabolites enterthe glands from the mesophyll through gaps in the cuticularenvelope, the transfusion zones. Net influxes of ions acrossthe transfusion zone were calculated from measurements of secretionrates and transfusion zone areas. When leaves of L. pereziiF. T. Hubb. were treated with 300 mol m^–3 NaCl, transfusionzone influxes of Na⁺ K⁺, Ca⁺⁺ and Cl^– as high as 7?0?10^–5,1.7?10^–5, 5?8?10^–7 and 8.5?10^–5 mol m^–2s^–1 respectively, were calculated. Assuming a transmembranepathway, these fluxes would be some of the highest reportedfor ions in plant cells. Key words: Salt glands, ion fluxes, ultrastructure     

Some properties of the amine oxidase in Vicia faba seedlings

Growth of the Vicia faba seedling is accompanied by a rapid15-day increase in amine oxidase activity of the apical parts.Cotyledons and roots were found to be devoid of activity. Thepartially purified enzyme from leaves readily oxidized putrescine,cadaverine, agmatine and spermidine, while dopamine (3-hydroxytyramine)and L- and D-lysine were oxidized more slowly. The Km valueswere 1.9?10^–3 M for cadaverine, 3.7?10^–5 M for putrescine,7.8?10^–4 M for spermidine, and 5.9?10^–3 M for dopamine.Carbonyl reagents and copper-binding agents were effective inhibitorsof Vicia faba amine oxidase. The diethyldithiocarbamate-treatedenzyme could be reactivated specifically by cupric copper. (Received May 25, 1977; )     

L-Leucine Transport in Isolated Protoplasts of Vinca Suspension Culture: Characterization of Uptake

The uptake of L-leucine into Vinca protoplasts was studied undervarious conditions. The uptake was highly pH-dependent, withthe optimal pH between 3.0 and 4.0. The uptake was also energydependent, since azide, 2,4-dinitrophenol (DNP), carbonyl cyanidem-chlorophenyl hydrazone (CCCP), and iodoacetate inhibited theuptake. Oligomycin, N,N'-dicycIohexyI carbodiimide (DCCD) andvanadate, but not ouabain, inhibited the uptake, suggestingthat ATPase for H⁺ electrogenic extrusion was necessary to theuptake of L-leucine. The uptake showed stereospecificity, butwas partially inhibited by other L-amino acids. A kinetic studyof the uptake showed that the uptake was multiphasic with threesaturable phases and one unsaturable phase which occurred atconcentrations of L-leucine over 1 mM. The K_m values of thethree affinity sites were 1.4 x 10^–3 M, 1.3 x 10^–4M, 4.3 x 10^–5 M; the maximum velocity values were 3.3x 10^–8, 4.5 x 10^–9, 1.8 x 10_–9 mol/10 min/4x 10⁶ cells. (Received April 18, 1981; Accepted August 25, 1981)     

Studies on NADP-isocitrate dehydrogenase from maturing castor bean seeds

NADP-specific isocitrate dehydrogenase from the soluble fractionof maturing castor bean endosperm was partially purified (approximately180-fold) and some of its enzymatic properties were studied.Mg⁺⁺, Mn⁺⁺, Cd⁺⁺, Ba⁺⁺, Co⁺⁺, Zn⁺⁺, and Sr⁺⁺ were activatorsof the enzyme reaction at a concentration of 6.7x10 M. The optimumpH of this enzyme was about 8.5. The enzyme was stable in thenarrow range from pH 7.0 to pH 8.0. Km values for isocitrateand NADP at pH 8.5 were 3.5x10^–6 M and 3.6x10^–6M, respectively. Enzyme stability was not affected by NaCl concentrationand enzyme reaction was inhibited at 5x10^–6 M PCMB (80%inhibition). It is suggested that the condensation product ofglyoxylate and oxalacetate also inhibits the reaction. NADP-IDHin the crude extract from maturing castor bean endosperm washeat-stable but the dialyzed enzyme preparation and the partiallypurified enzyme were labile against heat treatment at 57°C.When Mg⁺⁺ was added to the partially purified enzyme in thepresence of isocitrate or NADP, the enzyme was stabilized againstheat treatment. Mn⁺⁺, Ca⁺⁺, Co⁺⁺, Sr⁺⁺ or Ba⁺⁺ could be substitutedfor Mg⁺⁺. Addition of only one of the factors, Mg⁺⁺, isocitrateor NADP, had no effect on the heat stability. Moreover, a combinationof isocitrate and NADP did not establish stabilization. A divalentcation plays a central role, while adenine nucleotide, especiallyATP, may have an important part in stabilization. (Received August 14, 1972; )     

Involvement of cellulose synthesis in actions of gibberellin and kinetin on cell expansion. Gibberellin-coumarin and kinetin-coumarin interactions on stem elongation

In azuki bean (Azukia angularis = Vignia angularis) epicotylsections, 5 ? 10^–4 M coumarin inhibited the incorporationof radioactivity from [U^–14C]glucose into the cellulosefraction by 35% in the absence of indole-3-acetic acid (IAA)and by 40% in the presence of 1 ? 10^–4 M IAA. There wasno inhibitory effect on the incorporation of radioactivity intothe other fractions. Coumarin at 5 ? 10^–4 M reversed thepromoting effect of 1 ? 10^–5 M gibberellin A₃ (GA) andthe inhibitory effect of 1 ? 10^–5 M kinetin on IAA-inducedelongation of sections with no significant effects on IAA-inducedelongation. Neither GA nor kinetin had any appreciable effectson cellulose synthesis. No inhibition of cellulose syntheiswas observed with 1 ? 10^–3 M colchichine, which has beenreported to have effects similar to those of coumarin on GA-or kinetin-affected stem elongation. Coumarin at 5 ? 10^–4M was ineffectual in breaking up wall microtubules, while adisrupting effect on wall microtubules was clearly demonstratedwith 3 ? 10^–4M colchicine. From these results, the possible involvement of cellulose synthesisin cell expansion controlled by GA or kinetin was suggested. (Received August 3, 1973; )     

Some Properties of the Arginine Decarboxylase in Vicia faba Leaves

Growth of Vicia faba seedlings is accompanied by a rapid increasein arginine decarboxylase (EC 4.1.1.19 [EC] ) in the leaves and epicotyl.Increased enzyme activity was observed under saline conditionsin the presence of NaCl and with osmotic stress by mannitol.The partially purified enzyme (about 86-fold) readily decarboxylatedL-arginine, while D-arginine, L-homoarginine, L-ornithine andL-lysine were decarboxylated very slowly, and L-citrulline andL-glutamic acid were not decarboxylated. The K_m value was 5.8?10^–4M for L-arginine. The optimal pH and temperature for activitywere 8.5 and 45?C, respectively. p-Chloromercuribenzoate andN-ethylmaleimide were effective inhibitors of the enzyme. Inhibitionby spermidine, putrescine and agmatine suggested a possiblefeed-back mechanism in the pathway of polyamine biosynthesis. (Received October 11, 1983; Accepted February 24, 1984)     

Studies on the Growth in Culture of Plant Cells: XX. UTILIZATION OF 2,4-DICHLOROPHENOXYACETIC ACID BY STEADY-STATE CELL CULTURES OF ACER PSEUDOPLATANUS L

Omission of 2,4-dichlorophenoxyacetic acid (2,4-D) from batchcultures of sycamore produced an immediate reduction in ratesof cell division and eventually in rates of biomass accumulation.The sequential responses of a chemostat and of turbidostat culturessubjected to gradual withdrawal of 2,4-P were: (i) a transientincrease in biomass accumulation, (ii) increased accumulationof p-coumaric acid, flavonoids, and lignin, (iii) increasedcell aggregation, (iv) reduced rates of cell division, and (v)death. During stepwise reduction of 2,4-D supplied to turbidostatcultures, rates of 2,4-D uptake were reduced when the spentmedium concentration fell to 3?5–1?0 ? 10^–7 M. Underthese conditions the 2,4-D concentration in soluble and insolublecell fractions declined. The growth responses were correlatedwith the spent-medium 2,4-D concentration but not with its concentrationin the intracellular fractions.     

Effects of Cytokinin and Several Inorganic Cations on the Polyamine Content of Lettuce Cotyledons

Kinetin (4.7 x 10^–5 M) and 6-benzyladenine (2.22 x 10^–5M) were found to increase ca. 2-fold the putrescine contentin cotyledons of lettuce (Lactuca sativa L.) seedlings grownfor 3 days under fluorescent light. On the other hand, severalinorganic ions (K⁺, Na⁺, Ga⁺⁺, Mg⁺⁺) at a concentration of 3x 10^–2 M reduced the putrescine content. The combinationof kinetin with one of several inorganic ions at the same levelmarkedly increased the spermine content, but the putrescinecontent decreased; calcium and magnesium ions were less effective.The physiological significance of these findings is discussed. (Received July 4, 1982; Accepted November 6, 1982)     

Effects of indoleacetic, p-chlorophenoxyisobutyric and 2,4,6-trichlorophenoxyacetic acids on three phases of rooting in Azukia cuttings

In adventitious root formation of disbudded epicotyl cuttingstaken from light-grown, 5-day-old Azukia angularis seedlings,indoleacetic acid (IAA), 1 x 10^–4 M, applied during thefirst day showed no effect, but enhanced the effect of IAA,1 x 10^–4 M, applied during the second day. Treatment duringthe second day promoted rooting by about 70%, and a combinationof treatments for the first and second days promoted rootingsome 200%. p-Chlorophenoxyisobutyric acid (PCIB), 3 x 10^–4M, and2,4,6-trichlorophenoxyacetic acid (2,4,6-T), 2 x 10^–44M, applied the first day also enhanced the effect of IAA, 2x 10^–4 M, applied the second day. When applied the second day, PCIB, 2 x 10^–4M, increasedthe number of root primordia or clusters of small cells, butnot die number of protruded roots. Formation of the cell clusterwas inhibited by 2,4,6-T, 3 x 10^–4M, applied the secondday. Rooting processes in Azukia cuttings seem to include at leastthree phases: the first phase is induced not only by IAA butalso by PCIB or 2,4,6-T, the second phase is induced by IAAor PCIB and the diird phase depends specifically on IAA. (Received October 28, 1970; )     

Purification and Characterization of a Cytochrome P450 (trans-Cinnamic Acid 4-Hydroxylase) from Etiolated Mung Bean Seedlings

A Cyt P450 (P450_C4H) possessing trans-cinnamate 4-hydroxylase(C4H) activity was purified to apparent homogeneity from microsomesof etiolated mung bean seedlings. Upon SDS-polyacrylamide gelelectrophoresis, the purified preparation gave a single proteinband with a molecular mass of 58-kDa. Its specific P450 contentwas 12.6 nmol (mg protein)^–1. Using NADPH as electrondonor, purified P450_C4H aerobically converted trans-cinnamicacid to p-coumaric acid with a specific activity of 68 nmolmin^–1 nmol^–1 P450 in a reconstituted system containingNADPH-Cyt P450 reductase purified from the seedlings or rabbitliver microsomes, dilauroyl phosphatidylcholine, and cholate.This specific activity is by far the highest for reconstitutedC4H systems so far reported and provides direct evidence thatC4H activity is actually associated with a P450 protein. Inthe oxidized state P450_C4H showed a typical low-spin type absorptionspectrum with a Soret peak at 419 nm. A partial spectral shiftto the high spin state was observed when trans-cinnamic acidwas added to oxidized P450_C4H. By spectral titration, the dissociationconstant of the cinnamic acid-P450_C4H complex was determinedto be 2.8 µM. This value is similar to the K_m value (1.8µM) for trans-cinnamic acid determined in the reconstitutedsystem. (Received November 20, 1992; Accepted February 17, 1993)     

Dimethipin (2,3-Dihydro-5,6-dimethyl-1,4-dithiin 1,1,4,4-tetraoxide) Effects on Soybean Seedling Growth and Metabolism

Three-day-old dark-grown soybean [Glycine max (L.) Merr.] seedlingswere transferred to 2 mM CaSO₄ or 10^–5 M dimethipin in2 nM CaSO₄ and root-fed via liquid culture. Plants were placedin continuous darkness or in continuous white light (200 µE.m^–2?s^–11,PAR) at 25?C. Dimethipin inhibited root and shoot elongationin dark-grown plants after 24 h and 48 h, respectively. In thelight, root elongation was inhibited also after 24 h, but hypocotylelongation was not significantly affected. Extractable phenylalanineammonia-lyase (PAL) activity per axis in dimethipin-treateddark-grown axes was not generally affected but, in the lightdimethipin caused a significant decrease in PAL activity (24to 96 h). Total soluble hydroxyphenolics in axes were not affectedby dimethipin in light- or dark-grown plants. Anthocyanin andchlorophyll levels were lowered in hypocotyls of dimethipin-treatedplants after 48 to 96 h. Soluble protein in hypocotyls of light-or dark-grown seedlings was not substantially affected by dimethipin.Nitrate reductase (NR) activity (per organ) was generally notaffected by dimethipin in light-grown cotyledons, but in theroots of these seedlings, NR activity was significantly decreased.Proteolytic enzyme activity using three substrates (leucine-p-nitroanilide,LPNA; proline-p-nitroanilide, PPNA; and benzoylarginine-p-nitroanilide,BAPA) indicated little effect on enzyme activities per organin roots and hypocotyls. These data suggest that dimethipinat low concentrations can cause significant growth inhibitionin soybean seedlings grown in either light or darkness and thatfurthermore, extractable activities of some enzymes associatedwith nitrogen metabolism and secondary metabolism are alteredby this chemical. Light also plays a role in the activity ofthis chemical. (Received November 29, 1983; Accepted January 25, 1984)     

Preliminary characterization of 1-aminocyclopropane-1-carboxylate oxidase properties from embryonic axes of chick-pea (Cicer arietinum L.) seeds

For a deeper understanding of the germination of chick–pea(Cicer arietinum) seeds, which is dependent upon ethylene synthesis,a crude extract containing authentic ACC oxidase (ACCO) activitywas isolated in soluble form from the embryonic axes of seedsgerminated for 24 h. Under our optimal assay conditions (200mM HEPES at pH 7.0, 4µM FeS0₄, 6 mM Na–ascorbate,1 mM ACC, 20% 0₂, 3% CO₂ , and 10%glycerol) this enzyme was5–fold more active than under the conditions we used initiallyin the present work. The enzyme has the following K_m: 28 µMfor ACC (approximately 4–fold less than in vivo), 1.2%for O₂ (in the presence of an optimal CO₂ concentration of 3%),and 1% for CO₂ in the presence of O₂ (20%). The enzyme is inhibitedby phenanthroline (PNT) (specific chelating agent of ferrousion), and competitively inhibited (K₁, =0.5 mM) by 2–aminoisobutyricacid (AIB), and the enzymatic activity was not detectable inthe absence of CO₂. Under optimal assay conditions, the enzymehas two optimum temperatures (28 C and 35 C) and is inhibitedby divalent metal cations (Zn²⁺> CO²⁺>Ni²⁺>Cu²⁺>Mn²⁺>Mg²⁺) and by salicylic acid, propylgallate, carbonyl cyanidem–chlorophenyl hydrazone (CCCP), dinitrophenol (DNP),and Na–benzoate. The in vitro ACCO activity which we recoveredin soluble form is equivalent to approximately 80–85%of the apparent activity evaluated in vivo. Key words: ACC oxidase, Cicer arietinum, ethylene, germination, seeds     

Carrier-Mediated ABA Uptake by Suspension-Cultured Phaseolus coccineus L. Cells: Stereospecificity and Inhibition by Ionones and ABA Esters

Astle, M. and Rubery, P. 1987. Carrier-mediated ABA uptake bysuspension-cultured Phaseolus coccineus L. cells: Stereospecificityand inhibition by ionones and ABA esters.—J. exp. Bot.38: 150–163. The substrate for the abscisic acid (ABA) carrier in Phaseoluscoccineus L. suspension-cultured cells is shown to be the (S)ABAenantiomer, K_m = 1?0 mmol m^–3. The methyl (MeABA) andphenyl (PheABA) esters of ABA inhibit carrier-mediated uptakeof ABA with half-maximal inhibition achieved at about 7?0 mmolm^–3 and 10 mmol m^–3 respectively: with (S)MeABAthis value is decreased to about 2?0 mmol m^–3. There isno demethylation of radioactive MeABA by the cells during 5min incubations. Although MeABA reversibly inhibits the ABAcarrier, it is not a transport substrate: association of radioactiveMeABA with living cells is unaffected by non-radioactive MeABAor ABA and, by comparison with frozen-and-thawed cells, it isshown that the radioactivity remains extracellular. It is proposedthat MeABA binds to the carrier to form an abortive complexthat is not translocated. The terpenoid ABA analogue LAB 144143also inhibits carrier-mediated ABA uptake. At concentrationsup to about 20 mmol m^–3 - and ß-ionone specificallyinhibit the ABA carrier with the half-maximal effect at about0?6 mmol m^–3 ß-ionone. However, at higher iononeconcentrations, the uptake of ABA, indol-3-yl acetic acid andof 5,5-dimethyloxazolidine-2,4-dione (DMO) are all stimulated:this may reflect general permeabilization of the membrane toweak acids by ionone. Key words: Uptake carrier, abscisic acid, methyl and phenyl esters of ABA, ionone, Phaseolus coccineus L. suspension culture     

Effect of abscisic acid on membrane-bound epidermal ATPase from tobacco leaves

Membrane-bound Mg⁺⁺-activated ATPase activity in epidermal stripsfrom tobacco leaves (Nicotiana tabacum L. Samsun NN) was stimulatedby abscisic acid (ABA) when the strips were floated on ABA solutionin light or in darkness. The optimum ABA concentrations in lightand in darkness were 10^–5 M and 10^–6 M, respectively.Carbonyl cyanide m-chlorophenylhydrazone (CCCP) and N, N'-dicyclohexylcarbodiimide(DCCD) completely blocked the basal level membrane-bound epidermalATPase activity. ABAinduced membrane-bound epidermal ATPaseactivity was completely inhibited by CCCP, but only partly byDCCD. H⁺-influx into epidermal strips on a solution in light was lowerthan that in darkness. ABA stimulated H⁺-influx into epidermalstrips in light and in darkness. CCCP suppressed basal levelH⁺-influx, whereas DCCD did not. CCCP also suppressed ABA-inducedH⁺-influx, whereas DCCD did not. Interaction between H⁺-influxand membranebound epidermal ATPase activity is discussed. (Received May 23, 1978; )     

Determination of Volume of Dunaliella Cells by Lithium Dilution Measurements and Derivation of Internal Solute Concentrations

A method has been developed to measure the cell volume of theunicellular green alga Dunaliella parva 19/9 using Li⁺ measurementsonly. Concentrations of internal solutes can also be calculatedif they are assayed in the same samples as Li⁺. We found thatD. parva cells grown in 0.4 kmol m^–3 NaCl have an averageaqueous cell volume of 65.1 ?2.9 µm³, a K⁺ concentrationof 126?6 mol m^–3, a Na⁺ concentration of 11?11 mol m^–3and a glycerol concentration of 615?27 mol m–3 (n= 12).Algae grown in 1.5 kmol m^–3 NaCl have an average aqueouscell volume of 131 ?7.5 µm³, a K⁺ concentration of 109?4mol m^–3, a Na⁺ concentration of 10?39 mol m^–3 anda glycerol concentration of 1 425?59 mol m^–3 (n = 12).These results indicate that D. parva cells adapted to high salinitieshave larger cell volumes than those adapted to lower salinities.However, there is no evidence for a significant difference ininternal Na⁺ concentration, despite the almost 4-fold differencein the concentration of external NaCl. The intracellular glycerolconcentration alone accounts for 65% and 54%, respectively,of the osmotic balance in low and high salt grown cells. Key words: Dunaliella, cell volume, intracellular solutes