Quantitative Aspects of Leaf Acid Phosphatase Activity and the Phosphorus Status of Tomato Plants

The relation between tomato leaf acid phosphatase activity andleaf tissue P content has been examined, and a study made ofthe effects of leaf development, variation in nitrogen supply,and variation in the growing medium on this relationship. Tomatoplants were grown in sand and given various concentrations ofphosphate. Plants were also grown for an initial period in peatcontaining an adequate level of phosphate, then transferredto peat to which was added 0 or 2.3 kg superphosphate m^–3and supplied with either 50 of 300 µg N ml^–1. Expressed on a unit tissue f. wt basis, acid phosphatase activityin the control plants in sand (given 41 µg P ml^minus;1)was highest in extracts from the expanding leaves and decreasedwith leaf maturity. However, when given a reduced supply ofphosphate, the enzyme activity in the more mature leaves wasequal to, or greater than, that in the expanding leaves. Thephosphatase activity increased first in the young, fully-expandedleaves and in the mature leaves (with 4.1 µg P ml^–1),but did not increase in the expanding leaves until the supplywas restricted to 2.1 µg P ml^–1. On closer examination,the increase in enzyme activity appeared to be associated withthe P level in the leaf tissues, the activity increasing whenthe level fell below about 0.25 per cent (g P per 100 g drywt tissue). The same relation was found with the plants grownin peat, and was independent of the concentration of nitrogensupplied to the plants. The fully expanded leaves showed the best enzyme response whenthe phosphate supply was restricted and the activity reflectedclosely the local levels of tissue P. The assay of the enzymein unpurified leaf extracts is simple and rapid, and could beused in a test to detect P-deficiency in tomato plants. Lycopersicon esculentum L, tomato, acid phosphatase activity, phosphorus status     

Re-evaluation of Conditions for Plant Regeneration and Agrobacterium-Mediated Transformation from Tomato (Lycopersicon esculentum)

Conditions for plant regeneration from explants of tomato (Lycopersiconesculentum) cv. UC82B were studied for optimizing transformationprocedure. The best regeneration rate was obtained from cotyledonexplants from 8–10-d-old seedlings on a modified Murashigeand Skoog medium (1962) with 0·5 mg dm^–3 zeatinand 0·5 mg dm^–3 indolylacetic acid. Tomato cultivars(UC82B, Castone, Fl Ferline, Monalbo) and a Lycopersicon peruvkmum‘CMV sel. INRA’ were studied. The cultivarUC82Band the wild Lycopersicon species showed an efficient shootregeneration potential. Early events in the transformation of tomato cotyledons wereanalysed using an Agrobacterium tumefaciens strain carryinga binary vector with an nptII (pnos) gene and a reporter GUS-intron(p35S) chimeric gene. Two days after infection, GUS activityappeared specifically at the cut surface. Subepidermal cellswere more susceptible to transformation than epidermal cells.When selection for kanamycin resistance was applied 2 d afterinoculation, transformed cells were efficiently recovered. Preculturewith feeder cells stimulated cell transformation, but reducedregenerationcapacity from transformed cells. The optimal transformationrate was observed witha time of preculture of 1 and 2 d. Transformationevents for two tomato cultivars (UC82B and Monalbo) occurredat the same rate as 55% of the inoculated explants developedkanamycin resistant calli. However, transformed plants wereobtained at different rates of 8% and 14% for cv. Monalbo andcv. UC82B. Key words: Agrobacterium tumefaciens, ß-glucuronid, Lycopersicon esculentum, plant regeneration, transformation     

Biosynthesis of Xyloglucan in Suspension-cultured Soybean Cells. Evidence that the Enzyme System of Xyloglucan Synthesis does not Contain {beta}-1,4-Glucan 4-{beta}-D-Glucosyltransferase Activity (EC 2.4.1.12)

Xyloglucan 4-ß-D-glucosyltransferase, an enzyme responsiblefor the formation of the xyloglucan backbone, in a particulatepreparation of soybean cells has been compared with ß-1,4-glucan4-ß-D-glucosyltransferase of the same origin. Thefollowing observations indicate that the enzyme system of xyloglucansynthesis does not contain ß-1,4-glucan 4-ß-D-glucosyltransferaseactivity, although both enzymes transfer the glucosyl residuefrom UDP-glucose to form the ß-1,4-glucosidic linkage:1. The incorporation of [¹⁴C]glucose into xyloglucan dependedon the presence of UDP-xylose in the incubation mixture. 2.No measurable amount of radioactivity was incorporated fromUDP-[¹⁴C]xylose into the cello-oligosaccharides, although theincorporation of [¹⁴C]xylose into xyloglucan depended on thepresence of UDP-glucose in the incubation mixture (Hayashi andMatsuda 1981b). 3. The activity of xyloglucan 4-ß-D-glucosyltransferasewas stimulated more strongly by Mn²⁺ than by Mg²⁺, whereas Mg²⁺was the most active stimulator for the activity of ß-1,4-glucan4-ß-D-glucosyltransferase. 4. An addition of GDP-glucose(100 µM) to the incubation mixture inhibited the activityof xyloglucan 4-ß-D-glucosyltransferase by 17%, whereasthe activity of ß-1,4-glucan 4-ß-D-glucosyltransferasewas inhibited 56% under the same conditions. 5. Irpex exo-cellulasedid not hydrolyze the xyloglucan synthesized in vitro. 6. Theß-1,4-glucan synthesized in vitro was not a branchedxyloglucan because it gave no 2,3-di-O-methyl glucose derivativeon methylation analysis. 7. Pulse-chase experiments indicatedthat the ß-1,4-glucan was not transformed into thexyloglucan. The subcellular distribution of the xyloglucan synthase, however,was similar to that of the ß-1,4-glucan synthase (Golgi-located1,4-ß-D-glucan 4-ß-D-glucosyltransferase).Thus, it appears that the latter enzyme is located at a siteclose to xyloglucan synthase and is set aside for the assemblyof these polysaccharides into the plant cell surface. (Received May 21, 1981; Accepted October 13, 1981)     

Characterization of the Hydrolytic Activity of Avocado Cellulase

The cellulase produced by ripening avocado fruits (Persea americanaMill cv. Fuerte) was isolated and purified using chromatofocusing(pH 7–4) and gel filtration on a Bio- Gel P-100 column.Characteristics of the cellulase were assessed by using, assubstrates, a range of polysaccharides containing various sugarresidues and varying types of linkages between the residues.Only those substrates containing (14)-ß-glucosyl linkageswere hydrolyzed by the purified enzyme. Two polysaccharidesthat were extensively hydrolyzed by the cellulase were carboxymethylcelluloseand (13),(14)-ß-D-glucans such as from Avena endospermcell walls. Characterization of the activity in the degradationof the mixed linked glucan of Avena and cellodextrins indicatedthat the enzyme has a limit recognition-hydrolytic site of four(l4)-ß linked glucose residues. It was also foundthat the enzyme could cleave only (14)-ß-linkagesthat were adjacent to other (l4)-ß-D-glucosyl linkages.Activity of the cellulase against isolated avocado fruit cellwalls indicated that the purified enzyme was incapable of appreciablysolubilizing the cellulosic components of these walls. ¹Supported in part by National Science Foundation Research GrantPCM 7818588. ²USDA-ARS, Dairy Forage Research Center, University of Wisconsin,Madison, WI 53706. ³Department of Vegetable Crops, University of California, Davis,CA 95616. (Received September 14, 1985; Accepted February 12, 1986)     

Fe-deficiency stress response in Fe-deficiency resistant and susceptible subterranean clover: importance of induced H+ release

Plants can exhibit Fe-deficiency stress response when they areexposed to Fe-deficiency conditions. The relative importanceof the individual Fe-deficiency stress-response reactions, forexample, increased release of H⁺ from roots, enhanced root plasmamembrane-bound Fe³⁺ -reductase activity, and release of reductant,in Fe-deficiency resistance is not understood. To address thisproblem, the Fe-deficiency stress response of two cultivarsof subterranean clover (subclover), Koala (Trifolium brachycalycinumKatzn. and Morley) (Fe-deficiency resistant) and Karridale (T.subterraneum L.) (Fe-deficiency susceptible), were evaluated.The plants were cultured hydroponically at 0 (–Fe) and30 (+Fe) µM Fe³⁺ EDTA conditions. After 6 d Fe treatment,the –Fe Koala and Karridale decreased the pH of the nutrientsolution by 1.83 and 0.79 units, respectively, while the +Feplants increased the pH of the nutrient solution. The H⁺ -releaserate of the –Fe Koala determined 7 d after Fe treatmentinitiation was more than three times higher than that of the–Fe Karridale. The –Fe plants had a significantlyenhanced Fe³⁺ -reduction rate compared with the +Fe plants foreach cultivar, but the resistant cultivar did not exhibit ahigher root Fe³⁺ -reduction rate than the susceptible cultivarat each Fe treatment. Reductant release from the roots of subcloverwas negligible. These results indicate that Fe-deficiency-inducedH⁺ release may be the predominant factor influencing Fe-deficiencyresistance in subclover. Key words: Fe-deficiency, Fe³⁺ reduction, H⁺ release, stress response, Trifolium     

Identification of Antheridic Acid as an Antheridiogen in Anemia rotundifolia and Anemia flexuosa

Antheridic acid was identified by retention time and full massspectra from GCMS analysis as an antheridiogen in Anemia rotundifoliaand A. flexuosa. In the dark spore germination assay, antheridicacid was active down to 10^–10 and 5 ? 10^–12g.ml^–1in A. rotundifolia and A.flexuosa, respectively. In the antheridiumformation assay, antheridic acid was active down to 10^–10g.ml^–1 in both A. rotundifolia and A.flexuosa (Received April 14, 1987; Accepted July 8, 1987)     

Acclimation of Tomato Leaves to Changes in Light Intensity; Effects on the Function of the Thylakoid Membrane

When young tomato plants grown in high light (400 µmolquanta m^–2s^–1 PAR) were transferred to low light(100 µmol quanta m^–2s^–1 PAR), non-cyclic electrontransport capacity was decreased and the rate of dark re-oxidationof Q^–, the first quinone electron acceptor of photosystemII, was decreased within 1–2 d. In contrast, the amountof coupling factor CF₁, assayed by its ATPase activity, decreasedmore gradually over several days. The total chlorophyll contentper unit leaf area remained relatively constant, although thechlorophyll a/chlorophyll b ratio declined. When young tomato plants grown in low light were transferredto high light, the ATPase activity of isolated thylakoids increasedmarkedly within 1 d of transfer. This increase occurred morerapidly than changes in chlorophyll content per leaf area. Inaddition, in vivo chlorophyll fluorescence induction curvesindicate that forward electron transfer from Q^– occurredmore readily. The functional implications of these changes arediscussed. Key words: Tomato, leaves, light intensity, thylakoid membrane     

Purification and Properties of an Extracellular Endo-1,4-r{beta}-Glucanase from Suspension-Cultured Poplar Cells

An endo-1,4-rß-glucanase (EC 3.2.1.4 [EC] ) was purifiedto apparent homogeneity from the culture medium of poplar (Populusalba L.) cells by sequential anion-exchange, hydrophobic, andgel-filtration chromatography. The preparation of extracellularrß-glucanase was homogeneous on SDS-polyacrylamidegel electrophoresis (PAGE) and native PAGE. The molecular weight,as determined by SDS-PAGE was 50,000, whereas that determinedby gel filtration was 40,000. The isoelectric point (pI) was5.5. The purified enzyme catalyzed the endohydrolysis of carboxy-methylcellulosewith a pH optimum of 6.0 and a k_m of 1.0 mg ml^–1. Theenzyme specifically cleaved the 1,4-rß-glucosyl linkagesof carboxymethylcellulose, swollen cellulose, lichenan and xyloglucan,although the last was hydrolyzed more slowly than the othertested substrates. The activity of the endo-1,4-rß-glucanaseincreased up to the early stage of the mid-logarithmic phaseof growth and then decreased rapidly, suggesting that the rß-glucanaseis induced before cell development. (Received April 28, 1993; Accepted July 19, 1993)     

The Metabolism of Abscisic Acid

The light-catalysed isomerization of (+)-abscisic acid (ABA)to its trans isomer during isolation from leaves was monitoredby the addition of (±)-[2-¹⁴C]ABA to the extraction medium.(+)Trans-abscisic acid (t-ABA) was found to occur naturallyin rose (Rosa arvensis) leaves at 20µg/kg fresh weight;(+)-ABA was present at 594µg/kg. (±)-[2-¹⁴D]Trans-abscisicacid was not isomerized enzymically to ABA in tomato shoots. (±)-Abscisic acid was converted by tomato shoots to awater-soluble neutral product, ‘Metabolite B’, whichwas identified as abscisyl-ß-D-glucopyranoside. When(±)-[2-¹⁴C]trans-abscisic acid in an equimolar mixturewith (±)-[2-¹⁴C}ABA was fed to tomato shoots it was convertedto its glucose ester 10 times faster than was ABA. Trans-abscisyl-ß-D-glucopyrano8ide only was formedfrom (±)-[2-¹⁴C]t-ABA in experiments lasting up to 30h. Glucosyl abscisate was formed slowly from ABA and the freeacid fraction contained an excess of the unnatural (–).ABAas did the ABA released from abscisyl-ß-D-glucopyranosideby alkaline hydrolysis. The (+).ABA appeared to be the solesource of the acidic ‘Metabolite C" previously noted. The concentrations of endogenous (+)-, (+)-[2-¹⁴C]-, and (–)-[2-¹⁴C]ABAremaining as free acid, and also in the hydrolysate of abscisyl-ß-D-glucopyranoside,were measured by the ORD, UV absorption, and scintillation spectrometryof highly purified extracts of ABA from tomato shoots whichhad been supplied with racemic [2-^l4C]ABA.     

Effects of Nitrate Withdrawal and Resupply on the Assimilation of Nitrate in Leaves of Tomato

Nitrate reduction in leaves of tomato occurred at the same ratein plants grown in 8.0 mol m^–3 nitrate as in plants grownin 2.0 mol m^–3 nitrate, but at a much slower rate in plantsgrown in 0.1 mol m^–3 nitrate. However, the plants grownin 8.0 mol m^–3 nitrate had a larger leaf system than theplants grown in 2.0 mol m^–3 nitrate, and so the totalcapacity to assimilate nitrate was greater in the plants grownin the higher concentration. It was shown that plants grownin 8.0 mol m^–3 nitrate were better buffered against nitratewithdrawal than plants grown in 2.0 mol m^–3 nitrate asthe rate of nitrate reduction declined more slowly when plantswere transferred to 0.1 mol m^–3 nitrate from the higherconcentration than from the lower concentration. Furthermore,leaf expansion continued in the plants transferred from thehigher concentration, whereas it ceased abruptly in the plantstransferred from the lower concentration. It was concluded thatboth continuing expansion and continuing nitrate reduction wereaccompanied, and possibly caused by, a release of nitrate fromstorage pools in the lower part of the stem or the roots. Duringwithdrawal of nitrate the leaves were shown to maintain potentialactivity of the enzyme nitrate reductase although there wasno nitrate to be reduced. When nitrate was resupplied it couldbe reduced very quickly and reduction in the leaves was seento increase within 5 h of resupply. By 3 d after resupply furtherenzyme activity had been induced. Key words: Lycopersicon esculentum Mill, nitrate assimilation, nitrate reductase activity, nitrate withdrawal     

Effect of cycloheximide and cordycepin on auxin-induced elongation and {beta}-glucan degradation of noncellulosic polysaccharides of Avena coleoptile cell wall

The effect of cycloheximide (10^–5 M) and cordycepin (10^–4M) used as protein and RNA synthesis inhibitors, respectively,on auxin action in noncellulosic ß-glucan degradationof Avena coleoptile cell wall was investigated. Both depressedauxin-induced ßglucan degradation of the cell wallas well as auxin-induced elongation and cell wall loosening,suggesting that the process of ß-glucan degradationof the cell wall is closely associated with cell wall looseningand that auxin enhances the activity of an enzyme for ß-glucandegradation through de novo synthesis of RNA and protein butnot through activation of the enzyme in situ. Kinetic studywith the inhibitors showed that RNA metabolism involved in ß-glucandegradation was stimulated by auxin treatment of only 15 minwhile a longer lag phase (about 1 hr) existed for the synthesisof the enzyme. (Received December 16, 1978; )     

Effectiveness of Lotus Root Nodules: II. RELATIONSHIP BETWEEN ROOT NODULE EFFECTIVENESS AND 'IN VITRO' SENSITIVITY OF FAST-GROWING LOTUS RHIZOBIA TO FLAVOLANS

An extract from the roots of Lotus pedunculatus plants was foundto contain a compound toxic towards fast-growing Lotus rhizobia.This compound was identified as a flavolan, which has a prodeiphinidin:procyanidin ratio of 75:25. A fast-growing strain of Rhizobium(NZP2213) which forms ineffective root nodules on L. pedunculatuswas four times more sensitive to this flavolan (ED₅₀ = 25 ?gml^–1) than another strain (NZP2037, ED₅₀ = 100 ?g ml^–1)which forms effective root nodules on this species. The rootsof another Lotus species, L. tenuis, on which both strains ofRhizobium form effective root nodules, also contained a flavolan( 95% procyanidin) but both strains were relatively insensitiveto this flavolan (EDED₅₀ = 350 to 500 ?g ml^–1) L. pedunculatusplants bearing ineffective root nodules contained two to threetimes more flavolan in their roots (5–7 mg g^–1 fr.wt.)than uninoculated control plants. Experiments with seven otherLotus species and with hybrid plants developed between L. pedunculatusand L. tenuis showed a relationship between the prodeiphinidin:procyanidin ratio of the flavolan in their roots and the effectivenessof root nodules formed on these plants by NZP2213. Quantitativebinding studies of the flavolan from L. pedunculatus to NZP2037and NZP2213 indicated that, while the affinity constants forbinding were similar for both strains, the surface of strainNZP2037 contained four times more binding sites than NZP2213,possibly correlating with this strain's ability to toleratehigher concentrations of this flavolan. It is suggested thatthe differential sensitivity of these two strains of Rhizobiumto flavolans is related to their ability to form effective rootnodules on Lotus species.     

Purification and Properties of a Wall-Bound Endo-1,4-{beta}-Glucanase from Suspension-Cultured Poplar Cells

A wall-bound endo-1,4-ß-glucanase (EC 3.2.1.4 [EC] ) wasobtained from a preparation of the cell walls of suspension-culturedpoplar cells and purified to electrophoretic homogeneity bycation-exchange, hydrophobic, and gel-filtration chromatography.The molecular mass was estimated to be 47 kDa by SDS-PAGE and48 kDa by gel filtration on Superdex 200 pg. The isoelectricpoint (pI) was 5.6. The purified enzyme catalyzed the endo-hydrolysisof carboxymethylcellulose with an optimal pH of 6.5, a K_m of1.2 mg ml^-1, and a V_max of 280 units. The purified enzyme specificallyhydrolyzed the 1,4-ß-glucosyl linkages of carboxymethylcellulose,phospho-swollen cellulose, lichenan, xylan and xyloglucan. Theactivity of the enzyme was strongly stimulated by cysteine-HCl.The N-terminal sequence of the enzyme was similar to that ofan extracellular endo-1,4-ß-glucanase found in suspensioncultures of poplar cells and some homology was recognized toavocado fruit-ripening and bean abscission endo-1,4-ß-glucanases. ¹This work was supported in part by a grant from the Toray ScienceFoundation, Japan, and by a Grant-in-Aid from the Ministry ofEducation, Science and Culture of Japan.     

RNA-dependent RNA Polymerase Activities in Tomato Plants Susceptible or Resistant to Tobacco Mosaic Virus

RNA-dependent RNA polymerase activities were measured in healthyand tobacco mosaic virus (TMV)-infected tomato plants, to investigatethe possibility that altered activity might be involved in theoperation of the Tm-I gene for resistance to TMV. Healthy, susceptibleand resistant plants had similar levels of enzyme activity.Infection with TMV strain 0, which is inhibited by Tm-I, causeda 2-fold increase in activity in susceptible plants but no increasein Tm-I plants. Infection with a number of strain 1 isolates,which overcome Tm-I resistance, led to a 2 to 4-fold increasein enzyme activity in resistant plants. RNA-dependent RNA polymerase, Tm-I resistance gene, tobacco mosaic virus, tomato, Lycopersicon esculentum     

Hormones in Plants Bearing Nitrogen-fixing Root Nodules: Metabolism of [8-14C]Zeatin in Root Nodules of Alnus glutinosa (L.) Gaertn

The metabolism of [8-¹⁴C]zeatin, supplied via micropipettesover a 24 h period to root nodules of Alnus gliutinosa (L.)Gaertn., was investigated. The major metabolites were tentativelyidentified by means of chromatographic, chemical, and enzymictreatments as adenine, adenosine, trans-zeatin riboside, dihydrozeatin,trans-zeatin-O-ß-D-glucoside, and the O-ß-D-glucosideof dihydrozeatin. In addition, a prominent water-soluble peakof radioactivity was present. This did not appear to be a ribosidebut was biologically active in the soybean callus test. The number and nature of the metabolites formed in the noduleswas similar in both dormant and non-dormant plants.     

Ethylene and 1 -Aminocyclopropane-1-Carboxylic Acid Production in flacca, a Wilty Mutant of Tomato, Subjected to Water Deficiency and Pre-treatment with Abscisic Acid

Neill, S. J., McGaw, B. A. and Horgan, R. 1986. Ethylene and1-aminocyclopropane-l-carboxylic acid production in flacca,a wilty mutant of tomato, subjected to water deficiency andpretreatment with abscisic acid —J. exp. Bot. 37: 535–541. Plants of Lycoperstcon esculentum Mill. cv. Ailsa Craig wildtype and flacca (flc) were sprayed daily with H²O or 2?10^–2mol m^–3 abscisic acid (ABA). ABA treatment effected apartial phenotypic reversion of flc shoots; leaf areas wereincreased and transpiration rates decreased. Leaf expansionof wild type shoots was inhibited by ABA. Indoleacetic acid (IAA), ABA and l-aminocyclopropane-l-carboxylicacid (ACC) concentrations were determined by combined gas chromatography-massspectrometry using deuterium-labelled internal standards ABAtreatment for 30 d resulted in greatly elevated internal ABAlevels, increasing from 1?0 to 4?3 and from 0?45 to 4?9 nmolg^–1 fr. wt. in wild type and flc leaves respectively.Endogenous IAA and ACC concentrations were much lower than thoseof ABA. IAA content ranged from 0?05 to 0?1 nmol g^–1 andACC content from 0?07 to 0?24 nmol g^–1 Ethylene emanationrates were similar for wild type and flc shoots. Wilting of detached leaves induced a substantial increase inethylene and ACC accumulation in all plants, regardless of treatmentor type. Ethylene and ACC levels were no greater in flc leavescompared to the wild type. ABA pretreatment did not preventthe wilting-induced increase in ACC and ethylene synthesis. Key words: ABA, ACC, ethylene, wilting, wilty mutants     

Responses of Nine Trifolium repens L. Populations to Ultraviolet-B Radiation: Differential Flavonol Glycoside Accumulation and Biomass Production

This study aimed to quantify and identify flavonoids involvedin the response of nine populations of white clover (Trifoliumrepens L.) to ultraviolet-B radiation (UV-B). Plants were grownfor 12 weeks in controlled environment rooms with or withoutsupplemental UV-B radiation of 13.3 kJ m^-2d^-1. Methanol–waterextractable flavonoids were quantified using high performanceliquid chromatography (HPLC). Two major peaks showed significantenhancement in the HPLC chromatogram in response to supplementalUV-B. The structures of the compounds responsible were identifiedby¹H and¹³C nuclear magnetic resonance (NMR) spectroscopy tobe the flavonols quercetin-3-O-ß- D -xylopyranosyl-(1 2)-ß- D -galactopyranoside and kaempferol-3-O-ß-D -xylopyranosyl-(1 2)-ß- D -galactopyranoside. Withsupplemental UV-B, quercetin glycoside levels increased on averageby 200% while the kaempferol glycoside response was much smaller.Significant differences in flavonol accumulation were foundamong T. repens populations, both constitutively and in responseto UV-B. Stress-adapted populations displayed particularly highflavonol levels under UV-B. There was an inverse correlationbetween plant productivity and quercetin accumulation. Furthermore,higher quercetin accumulation under UV-B was correlated withtolerance against UV-B-induced growth reduction. In conclusion,within-species comparisons in T. repens lend support to a distinctrole for ortho -dihydroxylated flavonoids in the adaptationto UV-B stress and suggest particular advantages in this UV-B-inducedbiochemical adaptation for populations characterized by lowhabitat and plant productivity. Copyright 2000 Annals of BotanyCompany Ultraviolet-B, Trifolium repens, white clover, HPLC, NMR, flavonoids, flavonols, quercetin, kaempferol, biomass, genetic variation, intraspecific     

Types, Formation, and Metabolism of Cytokinins in Leaves of Alnus Glutinosa (L.) Gaertn.

The nature of the cytokinins present in mature, expanded leavesof alder (Alnus glutinosa (L.) Gaertn.) was investigated usinga chromatographic system capable of partially resolving zeatinfrom (?)-dihydrozeatin. A zeatin-like cytokinin, present asboth a ‘free’ form and as a polar conjugate, postulatedto the the O-ß-D-glucoside of zeatin, accounted forthe bulk of the cytokinin activity (detected by the soybeancallus assay). Studies on the fate of [8-¹⁴C]zeatin supplied to detached alderleaves indicated this cytokinin to undergo extensive metabolism.An appreciable proportion of the radioactivity was incorporatedinto 80% methanol-insoluble and soluble acidic/neutral fractions,while adenosine- and adenine-like peaks were prominent metabolitesin the basic n-butanol-soluble fraction. Small amounts of glucosides,with the properties of both zeatin-O-ß-D-glucosideand dihydrozeatin-O-ß-D-glucoside were formed, thelatter becoming the most prominent with time. The ability of alder leaves to form glucoside directly from(?)-dihydrozeatin was assessed using the soybean callus assay.(?)-Dihydrozeatin was subject to a relatively rapid, continuousand substantial conversion to its putative O-ß-D-glucosideand cytokinin activity was consequently conserved while, incontrast, leaves supplied with zeatin exhibited a progressiveloss of cytokinin activity and, in agreement with the radioactivityexperiments, produced only a small amount of activity attributableto the putative O-glucosides. The significance of the observed cytokinin metabolism is discussedin relation to the endogenous cytokinin status of the leaf.     

Purification and some properties of L-tyrosine carboxy-lyase from barley roots

L-Tyrosine carboxy-lyase (E. C. 4. 1. 1. 25) was extracted fromthe roots of barley seedlings and purified approximately 25fold. Optimum pH for the enzyme activity was found to be 7.3.The Km value for L-tyrosine was calulated as 4.5?10^–4M.D-Isomer did not react with the enzyme. L-DOPA, m-tyrosine ando-tyrosine were decarboxylated to some extent. Pyridoxal phosphateactivated the enzyme 4 fold. Caffeic acid and p-coumaric acidare competitive inhibitors. Ki values were 4.5?10^–5M forcaffeic acid and 1.6?10^–4M for p-coumaric acid. L-DOPAand m-tyrosine had an inhibitory effect on the decarboxylationof L-tyrosine. Hydroxylamine, semicarbazide, p-CMB, Fe⁺⁺, Cu⁺⁺,and Hg⁺⁺ inhibited the decarboxylation of tyrosine. Enzyme activitywas also found in extracts from Triticum aestivum, Zea maysand Cytisus scoparius. (Received November 30, 1973; )     

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; )