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

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  • Journal of Experimental Botany, Vol. 28, No. 106, pp. 1087-1098, October 1977

    Hormones in Plants Bearing Nitrogen-fixing RootNodules: Metabolism of [8-uC]Zeatin in Root Nodulesof Alnus glutinosa (L.) Gaertn.

    I. E. HENSON AND C. T. WHEELERDepartment of Botany, University of Glasgow, Glasgow, G12 8QQ.

    Received 15 December 1976

    ABSTRACTThe metabolism of [8-14C]zeatin, supplied via micropipettes over a 24 h period to root nodulesof Alnus glutinosa (L.) Gaertn., was investigated. The major metabolites were tentativelyidentified by means of chromatographic, chemical, and enzymic treatments as adenine, adeno-sine, r

  • 1088 Henson and Wheeler-^Zeatin in Alnus Nodules

    upper part of the mature root system. Plants were selected for treatment about 8 months aftersowing. The roots were washed free of 'Peralite' and the plants were transferred to 250 mlflasks containing half-strength Crone's solution (nitrogen-free formula), and wrapped toexclude light. Except for dormant plants (which remained in an unheated glasshouse untiltreatment) the plants were transferred to a growth cabinet with a 16 h photoperiod, a 19 C dayand 15 C night temperature, and allowed to acclimatise for about 710 d before treatment.

    Application of [8-14C]zeatin[8-14C]Zeatin, specific activity 11-7 mCi mmob1, was prepared by Dr. R. Horgan, University

    College of Wales, Aberystwyth, U.K. and purified before use by paper or by cellulose phosphatepaper chromatography (see below). Radiochemical purity was checked by chromatography onTLC silica gel (solvents A and C) and on a Sephadex LH20 column. When a sample of the label,remaining as a dried residue, was collected from micropipettes at the end of a nodule feedingexperiment (see below), over 94% of the radioactivity co-eluted with unlabelled zeatin from aSephadex LH20 column.

    Application of label, at a concentration of 1 /u.Ci /il"1 in 20% aqueous ethanol, was via a micro-pipette containing 2-0 fj\, inserted into the nodule near the apex. Only a single nodule per plantwas treated. Although uptake was generally complete within 7-8 h, the micropipette was leftin position for the duration of the experiment. The treated nodules were harvested 24 h aftercommencement of feeding.

    Extraction and partial purification of metabolitesThe nodules were crushed, extracted twice in methanol:water (4:1, by vol.), the extracts

    filtered, and the combined filtrates reduced to dryness in vacuo at 30 C. The residue was thenchromatographed on Whatman 3 MM paper in solvent D (see below). After eluting a portion ofthe chromatogram to determine the distribution of radioactivity, all radioactive zones excludingthe origin were eluted from the paper with methanol:water (4:1, by vol.) and subsequentlyanalysed by partition chromatography on a Sephadex LH20 column (Armstrong, Burrows,Evans, and Skoog, 1969). The column (90 cm x 2-5 cm) was eluted with methanol: water (7:13,by vol.), a solvent which facilitates a substantial separation of dihydrozeatin from zeatin,adenine from zeatin, and adenosine from zeatin riboside (Henson and Wheeler, 19776). Thecolumn was eluted at c. 30 ml h"1 and 15 ml fractions collected. Aliquots of each fraction weretaken for determination of radioactivity and the radioactive fractions retained for furthercharacterisation.

    Chromatographic methodsPaper, cation-exchange paper, and thin layer chromatography were carried out as described

    previously (Henson and Wheeler, 19776).The following solvents (proportions by volume) were used: (A) n-butanol: ammonia: water

    (6:1:2, upper phase); (B) w-butanol:acetic acid:water (12:3:5); (C) chloroform:methanol(9:1); and (D) isopropanol:ammonia:water (10:1:1).

    Non-radioactive marker substances, co-chromatographed with extracts, were detected byu.v. absorption.

    Enzymic and chemical treatmentsIncubation of extracts with a- and j3-glucosidases and treatments with potassium perman-

    ganate were as described previously (Henson and Wheeler, 19776). Periodate oxidation ofsuspected nucleotide fractions was carried out both as described by Sondheimer and Tzou(1971) and by Parker and Letham (1973).

    Alkaline phosphatase treatment of the presumptive nucleotide fraction was conducted atpH 90 in 0-5-2-5 ml 01 M MgCl2.6H20 at 37 C for 5-5 h or longer using 0-4-2-0 mg ml-ienzyme (derived from calf intestinal mucosa, Sigma Chemical Co. Ltd.).

    RadioassayZones from paper and thin layer chromatograms were eluted directly in scintillation vials

    with 1-0 ml absolute methanol to which 10 ml scintillation cocktail (toluene containing4-0 g I"1 PPO and 0-2 g I"1 POPOP) was added. Solvent from column eluates was evaporatedand the residues remaining likewise redissolved in 1-0 ml methanol prior to adding the scintil-lation fluid.

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  • Henson and WheelerZealin in Alnus Nodules. 1089

    Samples were counted using a liquid scintillation spectrometer (Tracerlab Corumatic 200)and all counts were corrected for background and quenching using the external standard ratiomethod.

    Bioassay

    The soybean callus assay of Miller (1968) was used as described previously (Henson andWheeler, 1976).

    RESULTS AND DISCUSSION

    Following fractionation on Sephadex LH20 of extracts of treated nodules fromactively growing plants a number of radioactive peaks was detected (Fig. 1A). Whennodules on fully dormant plants were treated similarly under identical environ-mental conditions, although a somewhat larger proportion of certain of the meta-bolites was present, a similar distribution of radioactivity was observed (Fig. 1B).

    ~ ft -

    * 4 -

    0 300 MXI l>00

    Mi l l ion \ o l u n i c (ml )

    FIG. 1. Distribution of radioactivity ( ) following partition chromatography on a Sepha-dex LH20 column eluted with methanol: water (7:13, by vol.). Authentic 'cold' markers wereco-injected with the radioactive samples and detected by absorbance at 254 nm ( ).A. Extract of nodules from six actively growing plants co-injected with c. 200 fig each of zeatinriboside (ZR) and zeatin (Z). B. As for A but extract from nodules of dormant plants. Major

    peaks of radioactivity are labelled 1-7 in order of elution.

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  • 1090 Henson and WheelerZeatin in Alnus Nodules

    The major metaboKtes separated by the LH20 column were investigated furtherwith a view to their characterisation.

    Peak 1This was the most polar peak detected, with an elution volume of c. 270-315 ml

    on the Sephadex LH20 column. It was not extracted into re-butanol from the aqueousphase at pH 8-0 or into ethyl acetate at pH 2-5. Acidic properties were indicated by alack of retention by the cation-exchange resin Zerolit 225 (SRC 14, NH" form) andretention by the anion-exchanger Dowex 1 (formate form).

    AMP AdoZR5

  • Henson and WheelerZeatin in Alnus Nodules 1091

    by treatment with alkaline phosphatase. Although, under identical conditions, theenzyme readily cleaved AMP to yield adenosine, no hydrolysis of peak 1 could bedetected, even after prolonged (17 h) periods of incubation (Fig. 2A). The presence ofpolar metabolites of zeatin with chromatographic properties unaffected by phos-phatase, has been noted previously in radish (Parker and Letham, 1973; Gordon,Letham, and Parker, 1974). Similarly, periodate treatment (which degradesnucleoside-o'-phosphates to their bases) had little effect on peak 1, although therewas a slight increase in a minor peak of radioactivity at c. Up 0-5 (Fig. 2B). Acidhydrolysis resulted in the appearance of two peaks, at B? 0-37 and 0-52, but theJBF of the major portion of the radioactivity was again unaltered (Fig. 2c).

    In preliminary tests (Table 1) peak 1 was found to display moderate biologicalactivity in the soybean callus bioassay, equivalent to or greater than that of kinetin.This may imply the presence of an intact iV6-substituted side chain, the unsaturatednature of which was indicated by the appearance, following permanganate oxidation,of a peak co-chromatographing with adenine, the major product of KMnO4

    T A B L E 1. Biological activity of peaks 1 and 2 in the soybean callus bioassay, com-pared with kinetin.

    Peak

    1

    2

    d min-i/flask

    2972970

    297029700

    Equivalent amountof zeatin (ng/flask)

    2.525.0

    25.0250.0

    Callus yield(mg/flask)

    7701881

    53009386

    Kinetin(ng/flask)

    025

    125250625

    12502500

    Callus yield(mg/flask)

    439930

    16362206379356776952

    oxidation of zeatin itself (Fig. 2D). It is suggested therefore that at least one com-ponent of peak 1, comprising some 36-38% of the radioactivity, is a zeatin-like compound in which the side chain has become modified by the presence of anacidic group. Further investigations of this interesting metabolite are required.

    Peak laThis peak, which was not always detected, was not investigated extensively. Itselution volume on LH20 corresponded with that of xanthosine.

    Peak 2

    Peak 2 eluted from the Sephadex LH20 column around 520 ml, an elution volumeapproximating to that of a naturally occurring peak of cytokinin activity fromnodules of A. glutinosa, which contains a mixture of cytokinin glucosides (Hensonand Wheeler, 19776). In contrast to peak 1, peak 2 was retained by a cation-ex-change resin (Zerolit 225, SRC 14, NH" form) but not by an anion-exchanger(Dowex 1, formate form). Also, it could be partly recovered into ra-butanol from anaqueous solution at pH 8-0 (K = [organic phase]/[aqueous phase] = 0-72).

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  • 1092 Henson and WheelerZeatin in Alnus Nodules

    When the chromatographic properties of peak 2 were compared to those of i) theauthentic 9-glucoside of zeatin, ii) the mixed endogenous cytokinin glucoside peakfrom the nodules, and iii) the putative O-glucoside [obtained by metabolism of[8-14C]zeatin in soybean callus tissue (Horgan, 1975; Henson and Wheeler, 19776)],whereas there were marked similarities between peak 2, the endogenous glucosides,and zeatin-0-/?-D-glucoside, these substances separated clearly from the 9-gluco-side, particularly on cellulose phosphate cation-exchange paper chromatograms(Table 2.)

    T A B L E 2. A comparison of the chromatographic properties of i) peak 2, ii) anendogenous peak of cytokinin activity from alder nodules ('peak a')a, iii) zeatin-9-jS-D-glucoside (Z-9-G), iv) putative [8-14C]zeatin-O-fi-T>-glucoside extracted from soybeancallus11 (Z-O-G), and v) zeatin.

    Peakorcompound

    Peak 2Peak aZ-9-GZ-O-GZeatin

    Chromatographic system

    Sephadex LH20eluted withmethanol: water(7:13, by vol.)

    Peak elutionvolume (ml)

    520500500500820

    Silica gel TLC

    SolventA

    J?P

    0.170.150.230.150.65

    SolventB

    BF

    0.360.370.44

    0.57

    Cellulose paperchromatography(solvent D)

    -RF

    0.280.300.510.270.68

    Cellulose phosphatepaperchromatography(distilled water)

    EF

    0.450.45 and 0.8"0.850.430.28

    a see Henson and Wheeler (19776).

    Glucosidase treatment of peak 2Whereas treatment with a-glucosidase had no effect, incubation with j8-gluco-

    sidase generally led to the complete conversion of peak 2 to less polar products. OnTLC (solvent A), on paper (solvent D), and on cellulose phosphate paper chromato-grams (distilled water), the main aglycone peak obtained co-chromatographed withzeatin (and thus with dihydrozeatin). In addition, following ^-glucosidase treat-ment, a small amount of the radioactivity, from 7 to 16%, was located at By 0-3-0-40on paper chromatograms (solvent D). This peak co-chromatographed with adenineon TLC in solvent A (i?p 0-44) and may account for a small 'shoulder' on the mainzeatin-like peak of radioactivity (c. R? 0-50) sometimes present following TLC of thetotal products of hydrolysis.

    When either all the products of j3-glucosidase treatment or only the zeatin-likeaglycone(s) (comprising a peak at i?p 0-68 on paper chromatograms in solvent D)were passed through a Sephadex LH20 column, two major peaks of radioactivity,termed 2i and 2ii, were resolved (Fig. 3). Peak 2i corresponded in its elution volumeto adenine and dihydrozeatin (which do not separate on this system) while peak 2ii

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  • Henson and WheelerZeatin in Alnus Nodules 1093

    corresponded to zeatin. The latter was always the major peak and generally rep-resented some 70% or more of the combined radioactivity of the two peaks.

    Peak 2ii co-chromatographed with trans-zea,tin on TLC in solvent C, a systemwhich separates the cis and trans isomers (Fig. 4A). The peak was degraded by

    1 0 r

    300 60(1

    Llution wilume (ml)900 1200

    FIG. 3. Distribution of radioactivity following separation on a Sephadex LH20 column of aa sample of peak 2 treated with j8-glucosidase. The column was eluted with methanol: water

    (7:13, by vol.). Z = zeatin.

    Z+KMnOj

    Ado DHZ.Z

    - :o

    f l

    (I L

    80 -

    0 0 0 5 10 0-0 0 5 M)

    FIG. 4. Distribution of radioactivity on silica gel TLC of the major aglycone of peak 2 (peak2ii from the Sephadex LH20 column, see Fig. 3). A. Sample run in solvent C together with cisand trans isomers of zeatin. B. Sample run in solvent A. Effect of permanganate oxidation;

    , control; , treated with 0.1 % KMnC>4. Marker compounds, indicated by horizontalbars, were adenine (Ade), zeatin (Z), and dihydrozeatin (DHZ). Positions of u.v.-absorbing

    oxidation products resulting from permanganate treatment of zeatin marker are shown.

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  • 1094 Henson and WheelerZeatin in Ahras Nodules

    potassium permanganate, with the distribution of degradation products on TLCclosely similar to that produced following identical treatment of authentic zeatin(Fig. 4B). Hence peak 2ii appears to be zeatin.

    Permanganate treatment of peak 2

    The position of attachment of the glucose moiety in the glucosides is unknown,although the 9-position was excluded on chromatographic grounds (Table 2).Permanganate oxidation of zeatin results in a substantial loss of the side chain, withadenine a major product. Similarly, adenosine arises as an oxidation product of theriboside. It can be surmised therefore that, with glucose attached to the purinering,

    Z + KMn()4

    /(^Ado75 r KMnO,

    Ado /

    f l

    (HI (ill 0-5 I d

    FIG. 5. The effect of potassium permanganate oxidation on the distribution of radioactivityand of u.v.-absorbing compounds on silica gel TLC developed in solvent A. A. Peak 2 fromnodules, B. Putative [8-14C]zeatin-O-/?-D-glucoside from soybean callus. .control; ,treated (0-1% KM11O4 spray). Marker compounds, indicated by horizontal bars were : adenine

    (Ade), adenosine (Ado), zeatin (Z), and zeatin-9-/J--D-glucoside (ZG).

    permanganate oxidation of a zeatin glucoside should produce the correspondingadenine glucoside, whereas, with a glucose attached via the hydroxyl group of theside chain, adenine it...

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