Zinc deficiency enhanced NAD(P)H-dependent superoxider radical production in plasma membrane vesicles isolated from roots of bean plants

The production of superoxide radical (O₂^–) was studiedin plasma membrane vesicles isolated by aqueous polymer two-phasepartitioning from roots of zinc-sufficient and zinc-deficientbean (Phaseolus vulgaris L. cv. Prélude) plants. Thetwo populations of vesicles were highly enriched in plasma membraneand had similar composition as evidenced by the specific membranemarker enzymes. Vesicles from zinc-deficient roots showed higherrates of NAD(P)H oxidation compared to vesicles from zinc-sufficientplants. The NAD(P)H-dependent formation of O₂^– in plasmamembrane vesicles was also highly increased by zinc deficiency.For both activities, a higher response to zinc deficiency wasobserved when NADPH was used as electron source. Re-supply ofzinc to deficient plants for 24 h substantially decreased therates of NAD(P)H oxidation and 0₂^– production in isolatedvesicles. The NADPH-dependent O₂^– generation was stronglystimulated by FAD and showed a high pH optimum; it was scarcelyaffected by Triton X-100 or even inhibited in the presence ofFAD and was almost insensitive to Antimycin A. The results suggest the presence at the plasma membrane of beanroots of an O₂^– generating activity, preferentially utilizingNADPH, which is affected by the zinc nutritional status of theplant. This finding, together with previous observations oncytosolic and microsomal fractions prepared from zinc-deficientroots of different plants, is consistent with a role of zincin membrane stabilization by controlling the level of oxidizingO₂ species. Key words: NAD(P)H oxidase, superoxide radical, plasma membrane, zinc deficiency     

Possible interaction between peroxidase and NAD(P)H-dependent nitrate reductase activities of plasma membranes of corn roots

The relationship between the plasma membrane bound NAD(P)H-nitratereductase (NR) and a plasma membrane (PM)-bound peroxidase wasinvestigated using highly purified PM vesicles isolated fromcorn roots. The PM-bound NR activity was strongly enhanced byMnCl₂ and SHAM, which stimulated peroxidase activity. Sinceboth activities, the NAD(P)H-dependent NR and the peroxidasecompete for NAD(P)H as electron donor, we propose a model inwhich a product of peroxidation is able to offer electrons tothe nitrate reductase in a more reactive form with respect toNAD(P)H.Our hypothesis was confirmed by experiments in which the effectsof inhibitors of peroxidative reactions, catalase, superoxidedismutase, and ascorbate on the PM-bound NR were studied. Resultsindicate that the putative electron donor for nitrate reductioncould be a radicalic species, possibly NAD. Furthermore, sincecytochrome c decreased the activity of the plasma membrane-boundNAD(P)Hdependent NR, cytochrome b₅₅₇ might be the site of theenzyme accepting electrons from NAD. Our results indicate that the PM environment of the NR may beinvolved in the extent of the membrane associated nitrate reductionand that redox enzymes at the PM, the NAD(P)H-NR and a peroxidase-likeNADH-oxidase, can interact. Key words: Plasma membrane-bound nitrate reductase, peroxidase, Zea mays     

Plasma membrane isolation from freshwater and salt-tolerant species of Chara: antibody cross-reactions and phosphohydrolase activities

Plasma membranes were isolated using the aqueous polymer two-phasepartition method from the algae Chara corallina and Chara longifolia,algae which differ in their ability to grow in saline environments.Enrichment of plasma membrane and depletion of tonoplast relativeto the microsomal fraction was monitored using phosphohydrolaseassays and crossreactions to antibodies raised against higherplant transporters. Antibodies to the vacuolar ATPase and pyrophosphatasecross-reacted with epitopes in the microsomal fraction, butshowed little affinity for the plasma membrane fraction. Pyrophosphataseactivity also declined in the plasma membrane fraction relativeto the microsomal fraction. The V-type H⁺ -ATPase activity,sensitive to nitrate or bafilomycin, was low in both fractions,though the cross-reaction to the antibody was reduced in theplasma membrane fraction. By contrast, the antibody recognitionof a P-type H⁺-ATPase amino acid sequence from Arabidopsis didnot occur strongly in the anticipated 90–100 kDa range.While there was enhanced recognition of a polypeptide at around140 kDa in the plasma membrane fraction, salt treatment of Charalongifolia resulted in plasma membrane fractions with reducedamounts of this epitope, but no change in vanadate-sensitiveATPase activity, suggesting that it does not represent the onlyP-type ATPase. Microsomal membranes from saltadapted C. longifoliahave higher reactivity with the antibody to the tonoplast ATPase. Key words: Chara, plasma membrane, salt tolerance, ATPase     

Comparison of Plasma Membrane and Tonoplast H+-Translocating ATPases in Phaseolus mungo L. Roots

Two membrane fractions were obtained from 16%/26% and 34%/40%interfaces following discontinuous sucrose density gradientcentrifugation of a 10,000–80,000xg pellet from mung bean(Phaseolus mungo L.) roots. The ATPases in the fractions differedfrom each other in their sensitivity toward various inhibitors,activation with salts, dependence of activity on pH, and K_mfor ATP.Mg²⁺. Judging from their sensitivity toward inhibitors,the ATPases in the low and high density membranes are consideredmainly of tonoplast and plasma membrane origin, respectively.Both ATPases were activated by gramicidin D and nigericin. ATP-inducedquenching of quinacrine fluorescence in both fractions requiredMg²⁺ and permeant anions such as Cl^– and quenching wascollapsed by carbonylcyanide p-trifluoromethoxyphenyl hydrazone.The sensitivities of quenching to the inhibitors were essentiallythe same as those of ATPase activity in the membranes. Thesefindings suggest the involvement of ATPases in H⁺-pumping acrossa plasma membrane and tonoplast. (Received April 12, 1985; Accepted October 11, 1985)     

Characterization of RNA-dependent RNA Polymerases in Healthy and Tobacco Mosaic Virus-infected Tomato Plants

Healthy tomato plants were shown to contain high levels of RNA-dependentRNA polymerase activity, mainly in a ‘soluble’ form,but also partly in a ‘ bound’ form. The ‘bound’enzyme was solublized by EDTA treatment. Both forms of enzymewere partially purified and characterized. The ion and pH optimaof the two forms were identical at all stages of purification.Both enzymes exhibited uridylyl transferase activity, whichmade up 35 per cent of total incorporation. Infection with tobacco mosaic virus (TMV) increased activityof ‘soluble’ enzyme by twofold, and of solubilized‘bound’ enzyme by less than twofold. Uridylyl transferaseactivity was also increased by infection. General propertiesof the enzymes were unaltered by infection with one exception:in the presence of TMV RNA as added template, the ‘soluble’enzyme from infected plants incorporated ³H-UTP into productswith the electrophoretic properties and RNase sensitivitiesexpected for replicative form and replicative intermediate ofTMV. ‘Soluble’ enzyme from healthy plants, and solublized‘ bound’ enzyme from either healthy or infectedplants did not synthesize these products. The ‘soluble’ and solubilized ‘bound’enzymes behaved differently on ion-exchange chromatography.Under the conditions used, ‘soluble’ enzyme didnot bind to the column, whereas solublized ‘bound’enzyme did. No differences in chromatographic behaviour werefound between enzymes from healthy or infected plants. Withboth ‘soluble’ and solublized ‘bound’enzymes, the uridylyl transferase activity co-chromatographedwith the polymerase activity. Tomato, Lycopersicon esculentum, RNA-dependent RNA polymerase, tobacco mosaic virus, tobacco mosaic virus replicase     

Inhibition of Tonoplast and Plasma Membrane H$-ATPase Activity in Rice (Oryza sativa L.) Culture Cells by Local Anesthetics

Tonoplast and plasma membrane vesicles were prepared from rice(Oryza sativa L. var. Yuukara) culture cells with step sucrosegradient (30% and 42.9%, w/v) and/or step dextran T-70 gradient(1% and 8%, w/w) to determine the inhibition of tonoplast andplasma membrane AT-Pases by local anesthetics. The degree towhich the anesthetics inhibited these ATPases was of the followingorder: dibucaine>lidocainetetracaine>procaineGABA. Dibucaineranging in concentration from 0.2 nui to 2 mM inhibited tonoplastATPase activity more than plasma membrane ATPase, the half inhibitionsbeing 0.8 and 1.1 mM, respectively. The K_m values of tonoplastand plasma membrane ATPases were not affected by dibucaine,but various values were noted for V_max. Dibucaine inhibitedtonoplast and plasma membrane ATPases solubilized from 0.1%DOC pellet by n-octylglucoside and zwittergent 3–14, respectively.The addition of a phospholipid mixture (asolectin) to solubilizedboth ATPases had no effect on the inhibition by dibucaine. Thus,local anesthetics may act directly on the ATPase moiety withoutlipid mediation. (Received June 15, 1987; Accepted November 13, 1987)     

A Comparison Between the ATPase and Proton Pumping Activities of Plasma Membranes Isolated from the Stele and Cortex of Zea mays Roots

Plasma membrane vesicles of high purity, determined by markerenzyme assays, were obtained by phase partitioning microsomalfractions from stelar and cortical tissues of Zea mays (cv.LG11) roots. ATP hydrolytic activities in both of the plasmamembrane fractions were inhibited by vanadate, SW26 and erythrosinB, but were insensitive to nitrate. Activity in both fractionsexhibited a marked pH optimum of 6·5 and displayed typicalMichaelis-Menten kinetics. A high substrate specificity wasapparent in both the stele and cortex plasma membrane fractions,while the lower fractions, after phase partitioning, showedlower specificity for nucleotide substrates. Specific activitiesof the stele (67·8 µmol Pi mg^–1 h^–1)and cortex (78·4 µmol Pi mg^–1 h–¹)plasma membrane H⁺ -ATPases were very similar. Proton pumping activities in microsomal membrane fractions fromstele and cortex were inhibited by nitrate and insensitive tovanadate. Homogenization of stele and cortex tissue in the presenceof 250 mol m^–3 KI resulted in microsomal fractions exhibitingvanadate-sensitive, nitrate-insensitive proton pumping activity,suggesting a plasma membrane origin for this activity. SW26was also an effective inhibitor of proton pumping activity,although results indicated an interaction between SW26 and thefluorescent probes quinacrine and acridine orange. The results are discussed in relation to models for the transportof ions into the stele and are consistent with a role for theH⁺ -ATPase activity in this process. Key words: ATPase, cortex, plasma membrane, stele, Zea mays     

Electrical Impedance Analysis in Plant Tissues8

Electrical impedance spectra (100 Hz–800 kHz) were measuredin leaves of Peperomia obtusifolia L. (a succulent) and Brassicaoleracea L. (cabbage). By measuring impedances at three or moreinter-electrode distances in a single leaf, electrode impedanceand specific tissue impedance were separated. Analysis of impedance data from B. oleracea leaves in relationto an equivalent circuit model showed that leaf developmentwas accompanied by increases in extracellular resistance, cytoplasmicresistance and vacuole interior resistance, together with decreasesin plasma membrane capacitance and tonoplast capacitance. AfterB. oleracea leaves were subjected to a –6 °C freeze-thawstress, extracellular resistance, cytoplasmic resistance andvacuole interior resistance decreased, but plasma membrane capacitanceand tonoplast capacitance did not change. These results indicatethat useful measurements of leaf parameters can be obtainedby this technique. Examination of the electrode impedance spectrum showed thatelectrode insertion produced a damaged collar, 0·4–0·5mm wide, around the electrode. This was confirmed by visualobservation of the damage in P. obtusifolia leaf. Key words: Peperomia obtusifolia L., Brassica oleracea L. (cabbage), electrical impedance, equivalent circuit, electrode polarization     

The effect of calcium chelators on microsomal pyridine nucleotide-linked dehydrogenases of sugarbeet cells

Cell suspension cultures of Beta vulgaris L., treated with calciumchelators or untreated, were used to characterize pyndine nucleotide-dependentdiaphorases of microsomes. The microsomal activity of NADH-dependentduroquinone reductase from cultures treated with 10 mM Na₂EGTAfor 24 h increased by a factor of 1.8 with respect to controlmicrosomes, and was mainly associated with particles of d=1.17gml^–1. NADPH-duroquinone reductase and NADH-ferricyanidereductase activities showed smaller increases. Bacterial protein-lipopolysaccharidecomplexes (prLPS) also promoted the increase of microsomal diaphorases;CaEGTA was Ineffective. EGTA effects on enzymes of supernatantand mitochondria were negligible, although Na₂EGTA treatmentinduced cell aggregation and strong acidification of the medium. When microsomes from control cultures were solubilized with1% LPC and fractionated in high-efficiency gel permeation columns(FPLC) the diaphorase activities were found associated to threemajor proteins: (i) NADH-specific quinone reductase (NADH-QR)of 340 kDa; (ii) pyndine nucleotide-nonspecific quinone reductase(NAD(P)H-QR) of 85 kDa also having ferricyanide reductase activity;(iii) NADH-specific ferricyanide reductase (NADH-FCR) of 38kDa. The microsomes from EGTA-treated cells also showed a highlyactive NADH-QR having a larger molecular mass (440 kDa) thanin control cells. NAD(P)H-QR also showed increased activity.We conclude that external Ca²⁺ chelation induces changes indehydrogenase components in microsomes. Furthermore, prLPS probablyexert part of their effect on plants through Ca²⁺ chelation. Key words: Beta vulgaris, cell cultures, calcium chelators, diaphorase, NAD(P)H-dehydrogenase, lipopolysaccharide, EGTA, quinone reductase     

Regulation of Mo-cofactor, NADH- and NAD(P)H-specific nitrate reductase activities in the wild type and two nar-mutant lines of barley (Hordeum vulgare L.)

Seedlings of three genotypes of barley, Hordeum vulgare L.,cv. Winer, were grown in nutrient solutions for 12 d: (a) Wt,the wild type; (b) Chlo19 and (c) Chlo29, two nitrate reductase(NR) deficient nar-mutants. Nar-mutant plants grown in nitratedeveloped about 5–24% of NADH-NR (EC 1.6.6.1 [EC] .) activitylevel characteristic of the Wt. The NR in vitro assays in whichNADH or NADPH were used as electron donors showed that the twomutant lines contained a mixture of NADH-specific and NAD(P)H-bispecific(EC 1.6.6.2 [EC] .) NRs. Chlo19 had a very low level of MoCo activityas compared to Chlo29 and Wt. Chlo19 appeared to be mutatedin a MoCo gene rather than in the genes coding for the nitrateNR apoenzyme. NAD(P)H-NR was found in the shoots and roots of both mutantsbut only in the roots of Wt. Several aspects of the regulationof NADH and NAD(P)H specific NRs in plants of the barley cv.Winer genotypes are discussed. MoCo was a strong limiting factorfor NR biosynthesis in nitrate-fed plants of Chlo19, but lesslimited in N-starved and ammonium-fed plants. Biomass productionby the three genotypes was similar during first 12 d after germination,regardless of the level of NR detected in vitro. Mutant plantsmay be able to supply the nitrogen required for growth withonly 5–24% of the NR level of the WT. Key words: Hordeum vulgare, mutants, nitrate, nitrate reductase, molybdenum cofactor     

In vitro binding of IAA to plasma membrane-rich fractions containing Mg++-activated ATPase from mung bean hypocotyls

Cell homogenates of dark-grown mung bean hypocotyls were fractionatedinto six fractions (L-0, L-l to L-5) by stepwise sucrose density-gradientcentrifugation. The majority (ca. 84%) of Mg⁺⁺-activated ATPase activity ofthe 10,000 x g pellet was localized in the L-0 (1.03 d 1.14)and L-l (1.14 d 1.16) fractions. Over 40% of the vesicularmembrane in the L-0 fraction and 60% of the L-l fraction couldbe stained with phosphotungstic acid (PTA)-chromic acid, a selectivestaining for the plant plasma membrane. In vitro binding of ¹⁴C-IAA to the fraction components was thegreatest in the L-l fraction among the six. The binding of ¹⁴C-IAAto the L-l fraction in vitro was markedly interfered with bythe presence of a high concentration of cold IAA (2 x 10^–4M).However, it was not affected by the IAA analogues IPA, IBA andIAN. This indicates that IAA highly specifically binds to theL-l fraction. In vitro specific binding of ¹⁴C-IAA to L-l andL-0 was decreased with an increasing acidity from pH 8.0 to5.0. In vitro binding of ¹⁴C-IAA to L-l and L-5 was furtherenhanced when these fractions were isolated from sections pretreatedwith 10^–5M cold IAA for 60 min ¹Present address: Institute for Plant Virus Research, 959 Aobacho,Chiba 280, Japan. (Received August 14, 1975; )     

NAD-dependent malate dehydrogenase and glyceraldehyde 3-phosphate dehydrogenase isoenzymes play an important role in dark metabolism of various plastid types

Chloroplasts isolated from spinach (Spinacia oleracea L.) leaves and green sweet-pepper (Capsicum annuum L. var. grossum (L.) Sendt.) fruits contain NADP-dependent malate dehydrogenase (MDH; EC 1.1.1.82) and the bispecific NAD(P)-glyceraldehyde 3-phosphate dehydrogenase (GAPDH; EC 1.2.1.13). The NADP-dependent MDH and GAPDH are activated in the light, and inactive in the dark. We found that chloroplasts possess additional NAD-dependent MDH activity which is, like the NAD-dependent GAPDH activity, not influenced by light. In heterotrophic chromoplasts from red sweet-pepper fruits, the NADP-dependent MDH and the NAD(P)-GAPDH isoenzymes disappear during the developmental transition and only NAD-specific isoforms are found. Spinach chloroplasts contain both NAD/H and NADP/H at significant concentrations. Measurements of the pyridine dinucleotide redox states, performed under dark and various light conditions, indicate that NAD(H) is not involved in electron flow in the light. To analyze the contribution of NAD(H)-dependent reactions during dark metabolism, plastids from spinach leaves or green and red sweet-pepper fruits were incubated with dihydroxyacetone phosphate (DHAP). Exogenously added DHAP was oxidized into 3-phosphoglycerate by all types of plastids only in the presence of oxaloacetate, but not with nitrite or in the absence of added electron acceptors. We conclude that the NAD-dependent activity of GAPDH is essential in the dark to produce the ATP required for starch metabolism; excess electrons produced during triose-phosphate oxidation can selectively be used by NAD-MDH to form malate. Thus NADPH produced independently in the oxidative pentose-phosphate pathway will remain available for reductive processes inside the plastids. Received: 2 July 1997 / Accepted: 20 October 1997     

Identification of a magnesium-dependent NAD(P)(H)-binding domain in the nicotinoprotein methanol dehydrogenase from Bacillus methanolicus

The Bacillus methanolicus methanol dehydrogenase (MDH) is a decameric nicotinoprotein alcohol dehydrogenase (family III) with one Zn(2+) ion, one or two Mg(2+) ions, and a tightly bound cofactor NAD(H) per subunit. The Mg(2+) ions are essential for binding of cofactor NAD(H) in MDH. A B. methanolicus activator protein strongly stimulates the relatively low coenzyme NAD(+)-dependent MDH activity, involving hydrolytic removal of the NMN(H) moiety of cofactor NAD(H) (Kloosterman, H., Vrijbloed, J. W., and Dijkhuizen, L. (2002) J. Biol. Chem. 277, 34785-34792). Members of family III of NAD(P)-dependent alcohol dehydrogenases contain three unique, conserved sequence motifs (domains A, B, and C). Domain C is thought to be involved in metal binding, whereas the functions of domains A and B are still unknown. This paper provides evidence that domain A constitutes (part of) a new magnesium-dependent NAD(P)(H)-binding domain. Site-directed mutants D100N and K103R lacked (most of the) bound cofactor NAD(H) and had lost all coenzyme NAD(+)-dependent MDH activity. Also mutants G95A and S97G were both impaired in cofactor NAD(H) binding but retained coenzyme NAD(+)-dependent MDH activity. Mutant G95A displayed a rather low MDH activity, whereas mutant S97G was insensitive to activator protein but displayed "fully activated" MDH reaction rates. The various roles of these amino acid residues in coenzyme and/or cofactor NAD(H) binding in MDH are discussed.     

Characterization of ATPases Associated with Various Cellular Membranes Isolated from Etiolated Hypocotyls of Vigna radiata (L.) Wilczek

Cellular membrane fractions, including endoplasmic reticum (ER),Golgi-enriched membrane, plasma membrane and tonoplasts, wereisolated from Vigna radiata seedlings. Each of these membranefractions was associated with specific ATPases which were highlydependent on Mg²⁺. ATPases of ER, Golgi-enriched membrane andplasma membrane were sensitive to vanadate but the tonoplastATPase was not. ATPases were mostly dependent on Cl¹, but aslight stimulation by K⁺ was observed in the case of ATPasesof Golgi-enriched membrane and plasma membrane. KNO₃ inhibitedtonoplast ATPase but stimulated the other ATPases. ER ATPasecan be distinguished from other ATPases by the following characteristics:specific inhibition by KNO₂ and Triton X-100, stimulation bylow concentrations of diethylstilbestrol and 4,4'-diisothiocyanostilbene-2,2'-disulfonicacid, and high sensitivity to heat. The ATPases showed typicalMichaelis-Menten kinetics and had K_m values of 0.5 to 0.6 ITIMMg²⁺-ATP for ER, Golgienriched-membrane and tonoplast ATPases,and 2.27 msi Mg²⁺-ATP for plasma membrane ATPase. ATPases ofGolgi-enriched membranes and plasma membranes had similar properties,but they were still distinguishable by the differences in theirK_m values and their responses to Triton X-100. Based on theseresults, it is postulated that each cellular membrane is associatedwith a specific ATPase in cells of V. radiata. ¹Contribution No. 3171 from the Institute of Low TemperatureScience. (Received April 22, 1988; Accepted September 28, 1988)     

Expression of the Plastid ndhF Gene Product in Photosynthetic and Non-Photosynthetic Tissues of Developing Barley Seedlings

A fragment of the NDH-F subunit of the plastid NAD(P)H dehydrogenasecomplex (NAD(P)H-plastoquinone-oxidoreductase) from barley wasexpressed as a fusion protein in Escherichia coli and an antibodyto the fusion protein was prepared. Western blot analysis usingthe anti-NDH-F antibody showed specificity towards a plastidpolypeptide of approximately 70 kDa present in both photosyntheticand non-photosynthetic barley tissue. The polypeptide was foundin thylakoid membranes of green leaves whereas in etiolatedleaves it was shown to be associated with the membrane fractionof etioplasts. NDH-F levels were higher in roots and etiolatedtissue than in greening or young leaves. During leaf ontogeny,NDH-F levels decreased from young to mature tissue but increasedduring senescence. The accumulation of NDH-F in thylakoids ofyoung leaves was stimulated by photooxidative treatment. Theresults indicate a high degree of expression of plastid ndhgenes (which encode NAD(P)H dehydrogenase sub-units) in non-photosyntheticplastids and under conditions which impair the photosyntheticactivity of chloroplasts. In addition to its putative implicationin photosynthetic electron transport, a non-photosynthetic role,such as chloro-respiration, is proposed for the plastid NAD(P)Hdehydrogenase complex. (Received May 20, 1997; Accepted October 8, 1997)     

Effect of sulphate deficiency on the plasma membrane polypeptide composition of Brassica napus

Highly purified oilseed rape (Brassica napus) root plasma membranefractions were prepared and their polypeptide patterns analysedby two-dimensional gel electrophoresis. Sulphur starvation enhancesthe sulphate uptake capacity of B. napus roots. The relativeabundance of several polypeptides increased significantly andspecifically after sulphur starvation. Several of them (37,38, 60, and 65 kDa), found in sulphur-starved plants, were moreabundant in a phase-partitioned membrane fraction treated withTriton X-100/KBr, indicating that they are intrinsic polypeptides.One polypeptide (47 kDa) was identified in the in vitro translationproducts of the roots mRNAs as specific for S-starved plants.It was also present among the intrinsic polypeptides specificfor —S plants. These plasma membrane polypeptides mightbe involved in sulphate uptake. Key words: Sulphate, sulphur-starvation, plasma membrane, polypeptides, root, transport     

Effects of 4-Chloro-2-methylphenoxypropionate (an Auxin Analogue) on Plasma Membrane ATPase Activity in Herbicide-Resistant and Herbicide-Susceptible Biotypes of Stellaria media L.

ATPase activity was examined in plasma membrane (PM) fractionsprepared from mecoprop-resistant and -susceptible biotypes ofStellaria media L. (chickweed). Treatment with the herbicidecaused an 18% increase in ATP hydrolysis, but this was not significantlydifferent from control plants and was similar for both biotypes.However, there was an overall significant biotype effect, herbicide-resistantplants having greater enzyme activity than susceptible ones.Proton-pumping was readily demonstrated in PM fractions obtainedfrom both biotypes using the fluorescent probe amino-chloro-methoxyacridine(ACMA), indicating a relatively large proportion of 'inside-out'vesicles. Proton-pumping was significantly greater in PM preparationsobtained from the resistant compared with susceptible plants.The differences in ATPase activity between the two biotypescould not be attributed to differences in the main sterol orphospholipid components of the PM. There were no effects ofthe herbicide on ATP hydrolysis in vitro, but proton-pumpingwas affected in a herbicide concentration-dependent manner.At 1·0 mol m mecoprop caused an increase in the rateof proton-pumping, whereas at 10 and 100 mol m^–6, an inhibitionin this rate was observed. Both biotypes behaved similarly,irrespective of mecoprop concentration. These data indicatethat mecoprop resistance in chickweed is unlikely to be dueto a direct effect of the herbicide on PM H+-ATPase activity. Key words: Stellaria media, mecoprop, ATPase, plasma membrane, herbicide resistance     

Tryptophan Oxidizing Enzyme and Basic Peroxidase Isoenzymes in Arabidopsis thaliana (L.) Heynh.: Are They Identical?

The in vitro conversion of [³H]tryptophan by a plasma membraneenriched fraction from Arabidopsis thaliana (L.) Heynh. seedlings,grown in liquid culture, revealed indole-3-acetaldoxime (IAOX)as the only detectable reaction product. The pH optimum of thereaction was at pH 8, the K_m value for tryptophan 12 µM.The formation of IAOX was stimulated about 10-fold by H₂O₂ Incubationexperiments with solubilized proteins and membrane vesiclesshowed that the investigated enzyme(s) were bound covalent tothe plasma membrane. Tryptophan oxidizing enzyme (TrpOxE) andperoxidase activity were not only found in the plasma membrane,but also in the culture medium. Specific IAOX forming activitywas 74-fold and 6-fold higher compared to the crude extractand the plasma membrane fraction, respectively. After isoelectricfocusing of solubilized plasma membrane and precipitated mediumproteins, TrpOxE activity co-migrated with two prominent highpI peroxidase bands stained with benzidine-guaiacol. The zonesof the IEF gel with peroxidase and TrpOxE activity were analyzedby SDS PAGE and revealed in all fractions a main protein bandof ca. 55 kDa. TrpOxE activity and peroxidase activity wereboth inhibited by antisera directed against tobacco and horseradishperoxidase. TrpOxE activity and peroxidase activity were determinedduring plant development. TrpOxE activity peaked after 8 and42 days, whereas peroxidase activity was consistently presentduring the whole life cycle. The inhibitory effects of indolederivatives, especially indole-3-glyoxylic acid, on (i) seedlingdevelopment and (ii) on TrpOxE and peroxidase activity werealso compared. (Received November 1, 1991; Accepted September 2, 1992)     

Alicyclic acid metabolism in plants 10. Partial purification and some properties of 3-dehydroquinate synthase from Phaseolus mungo seedlings

Dehydroquinate synthase from Phaseolus mungo seedlings was purified120-fold by DE-23, hydroxylapatite and Sephadex G-100 columnchromatography. The final preparation was free of dehydroquinatehydro-lyase and NAD(P)H₂ oxidase. The dehydroquinate synthaserequired Co²⁺ and NAD as cofactors. Co²⁺ could be replaced byCu²⁺ at 0.1 mM, but Cu²⁺ at higher levels was inhibitory. Noneof the other metal ions tested activated the enzyme. Some activitywas observed in the absence of added Co²⁺ and this activitywas inhibited by EDTA but not by diethyldithiocarbamate, NaN₃or NaCN. Heavy metal ions, such as Ag⁺ and Hg²⁺, and p-chloromercuribenzoatestrongly inhibited the enzyme activity. Of the pyridine nucleotidestested only NAD was required for the maximum activity of theenzyme. In the absence of NAD, the enzyme retained 30 to 40%of the activity obtained with added NAD. The apparent Km valuefor DAHP at pH 7.4 was about 23 µM. The enzyme activityappeared to be maximum at about pH 8.5. However, the characteristicsof the enzyme were studied at pH 7.4, because of the labilityof the enzyme under alkaline conditions. An Arrhenius plot ofthe enzyme reaction showed a break at about 21?C, and belowthis critical temperature the activation energy increased. (Received March 4, 1977; )     

The Permeability of the Guard Cell Plasma Membrane and Tonoplast

Uptake experiments and efflux compartmental analysis of planthormones, osmotica and toxins using ‘isolated’ guardcells of Valerianella locusta and guard cell protoplasts (GCP)of Vicia faba were performed in order to study the permeabilityproperties of guard cell plasma membrane and tonoplast. Theplasma membrane of guard cells exhibits a higher permeabilitythan plasma membranes of mesophyll cells for most solutes investigated.The permeability coefficients (P_s calculated for the guard cellplasma membranes are also significantly higher than the P_s valuesfor the guard cell tonoplast. This applies also for protonatedABA. We suppose that the high permeability for ABAH could bepart of the target cell properties. A Collander analysis demonstratesa linear correlation between P_s, values and the ratio K_r/M_r^1,5for both plasma membrane (r = 0.87) and for the tonoplast (r=0.93). Because of deviations from the observed correlations,the permeation of some solutes (ABA, GA, IAA through the tonoplast;methylamine through the plasma membrane) seems to be facilitatedby an additional transport mechanism. The Collander analysisof the plasma membrane of GCP shows very similar results tothe analysis of the plasma membrane of ‘isolated’guard cells, indicating that isolation of protoplasts does notalter the permeability of the guard cell plasma membrane. Key words: Permeability coefficient, guard cells, plasma membrane, tonoplast