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

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<ms-zeatin riboside, dihydrozeatin, irans-zeatin-O-jS-D-glucoside, and the O-/J-D-glucoside of dihydrozeatin. In addition, a prominent water-soluble peak of radioactivity waspresent. This did not appear to be a riboside but was biologically active in the soybean callustest.

The number and nature of the metabolites formed in the nodules was similar in both dormantand non-dormant plants.

INTRODUCTIONAlthough the metabolism of zeatin has been studied in several species, includingseedlings of Baphanus sativus (Parker and Letham, 1973; Gordon, Letham, andParker, 1974), Zeamays (Parker and Letham, 1974), Lupinus angustifolius (Parker,Letham, Wilson, Jenkins, MacLeod, and Summons, 1975), and Phaseolus vulgaris(Sondheimer and Tzou, 1971; Wareing, Horgan, Henson, and Davis, 1977),embryos of Fraxinus americana (Tzou, Galson, and Sondheimer, 1973), and callustissue of Glycine max (soybean) (Horgan, 1975), there have been only a few attemptsto relate such metabolism to the cytokinins found naturally in the plant (e.g. Vonk,1974, 1976; Wareing el al., 1977).

The root nodules of Alnus glutinosa (L.) Gaertn. are a particularly.rich source ofcytokinins (Henson and Wheeler, 1977a), the main types of which appear to besimilar to or identical with zeatin, zeatin riboside, zeatin-0-j9-D-glucoside, dihydro-zeatin-O-jS-D-glucoside, and a /?-D-glucoside of zeatin riboside (Henson and Wheeler,19776). We now report on the short-term metabolism of [8-l4C]zeatin by the rootnodules of both dormant and non-dormant plants of A. glutinosa.

MATERIALS AND METHODSPlant materialPlants of Alnus glutinosa were raised from seed and grown in a heated glasshouse in 'Peralite'-filled pots as described previously (Wheeler, 1969). Treatment of the seedlings at the two-leafstage with a crushed nodule inoculum resulted in the formation of numerous nodules on the

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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 7—10 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 Wheeler—Zealin 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 Wheeler—Zeatin 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<)r " - 1 - 1

B

ZR

r-3

c Adc Z Adc Z

00 0-5 Ml 0-5 Ml

FIG. 2. The distribution of radioactivity of samples of peak 1 following TLC on silica gel insolvent A. A. Control ( ) and alkaline phosphatase-treated ( ) samples. B. Control( ) and sodium periodate/cyclohexylamine-treated ( ) samples, c. Sample subject toacid hydrolysis (0-2 N HC1 for 1 h at 100 °C). D. Control ( ) and potassium permanganate-treated ( ) samples. 0-l% aqueous KMnO4 was applied as a spray following loading butbefore development with chromatographic solvent. Marker compounds, indicated by hori-zontal bars, were: adenine (Ade), adenosine (Ado), adenosine-5'-monophosphate (AMP),

zeatin (Z), and zeatin riboside (ZR).

Peak 1 co-chromatographed on Sephadex LH20, on paper chromatograms(solvent D), and on TLC (solvent A), with adenosine-5'-monophosphate (AMP).However, it was more mobile than AMP on TLC in solvent B (JKF of AMP = 0-16-0-21; peak 1 = 0-25—0-35). That peak 1 was not adenylosuccinic acid was shown bychromatography on Sephadex LH20.

Peak 1 was run on TLC in solvent A following various chemical treatments (Fig.2). The possibility that peak 1 contained a ribonucleotide(s) of zeatin was explored

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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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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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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 Wheeler—Zeatin 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 itself should be produced following oxidation. Treatment ofpeak 2 by KMnO4 gave two main peaks, one of which co-chromatographed withadenine and was presumably derived from the breakdown of the major zeatin-likeglucoside (Fig. 5A) . The nature of the other oxidation product at c. B-p 0- 3 is unknown.No such products were detected following identical treatment of zeatin-9-jS-D-glucoside with KMnO4 but, by contrast, a sample of the putative O-glucoside ofzeatin was almost completely converted by KMnC>4 to an adenine-like peak (Fig.OB).

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A further finding relevant to the question of the position of substitution is thebiological activity of the glucosides. Preliminary tests suggested that in the soybeancallus bioassay the glucosides of peak 2 were some 10 times more active than kine-tin, and hence probably equally as active as zeatin itself (Table 1). This again isconsistent with attachment of glucose via the side chain as it is reported that zeatin-O-glucoside exhibits activity comparable to zeatin (Parker et al., 1975), while the 7-and 9-glucosides of zeatin are much less active than the parent molecule (Parker andLetham, 1973; Parker et al., 1975; Henson, unpublished results).

Thus the principal components of peak 2 are tentatively designated as trans-zeatin-O-/3-D-glucoside and dihydrozeatin-0-/8-D-glucoside, both of which alsooccur naturally in the nodule (Henson and Wheeler, 19776). Zeatin-O-/?-D-gluco-side has previously been found as a metabolite of zeatin in seedlings of Lupimisangustifolius (Parker et al., 1975), in soybean callus tissues (Horgan, 1975) and inseedlings of Phaseolus vulgaris (Wareing et al., 1977), while dihydrozeatin-0-)8-D-glucoside has been identified as a naturally occurring cytokinin in Phaseolus (Wang,Thompson, and Horgan, 1977). The presence of a third aglycone in trace amounts,which co-chromatographed with adenine, was also indicated, but no evidence wasobtained for the occurrence of a glucoside of zeatin riboside or dihydrozeatin ribo-side, despite the natural occurrence of a compound of this nature in the nodules(Henson and Wheeler, 19776).

Peak 2a

This peak was present only in minor amounts. It was not extensively investigatedbut was unaffected by both a- and /9-glucosidases and yielded two peaks on bothpaper (solvent D) (at R$ 0-13 and 0-70) and on cellulose phosphate paper (R$ 0-45and 0-65) chromatograms.

PeakS

The elution volume on Sephadex LH20 of peak 3 corresponded to that of adeno-sine. It co-chromatographed with adenosine and adenine on paper chromatograms(solvent D) and with adenosine on TLC (solvent A).

Peak 4

The elution volume of peak 4 on LH20 corresponded to that of zeatin riboside(Fig. 1A, B). The peak co-chromatographed with zeatin riboside on paper chromato-grams (solvent D) (R-p 0-63), on TLC in solvents A (J?F 0-37) andB (iJF 0-57), and oncellulose phosphate paper chromatograms in distilled water (R$ 0-80). Followingpotassium permanganate treatment, the peak of radioactivity shifted to the RF ofadenosine (0-30 on TLC in solvent A) with less than 13-0% of the radioactivity stillco-chromatographing with the riboside, indicating little, if any, contribution fromdihydrozeatin riboside. Following TLC in solvent C, peak 4 co-chromatographedwith the trans isomer of zeatin riboside. Finally, when peak 4 was subject to treat-ment with sodium periodate and cyclohexylamine there was a shift, consistent withthe loss of the ribosyl moiety, to the position of zeatin on both TLC (solvent A)

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and cellulose phosphate paper chromatograms. Thus peak 4 appears to be trans-zeatin riboside.

Peak 5

This minor peak had an i?p of 0-65 on paper chromatograms (solvent D) and of0-38 on TLC (solvent A); its possible identity is unknown.

Peak 6

The position of elution of this peak on Sephadex LH20 corresponded to that ofadenine and dihydrozeatin, which have identical elution volumes on this column.Evidence for the presence of both substances was obtained following re-chromato-graphy when two peaks were resolved, on paper in solvent D (B$ 0-28 and 0-70) andon TLC in solvent A (J?p O43 and 0-53), corresponding in both cases to adenine andzeatin/dihydrozeatin respectively. On TLC in solvent B peak 6 ran as a broadlyspread peak encompassing the JSF of both adenine (R$ 0-50) and zeatin/dihydro-zeatin (R? 0-57).

The peak corresponding on paper chromatograms to adenine (termed 6i) co-chromatographed with adenine on i) TLC in solvent A (R? 0-45) and ii) on cellulosephosphate paper (Rp 0-13). Similarly the second, dihydrozeatin-like peak from thepaper chromatograms (6ii) co-chromatographed with zeatin/dihydrozeatin whenrun on TLC in solvent A (R? 0-57).

Prior to permanganate treatment the dihydrozeatin-like peak was eluted frompaper chromatograms and checked for the presence of contaminating [8-14C]-zeatin (e.g. from peak 7, see below) by rechromatography on Sephadex LH20together with authentic dihydrozeatin as a marker. The symmetry of the peakobtained, which co-eluted with the marker, indicated the absence of detectablezeatin. No degradation to less mobile compounds (on TLC, solvent A) was observedsubsequently on treatment of an aliquot with KMnO-4.

Thus peak 6 contains substances identical or closely similar to both adenine anddihydrozeatin, the latter constituting 50—55% of the total radioactivity of the peak.Dihydrozeatin appears to have been reported previously as a zeatin metabolite onlyin Phaseolus vulgaris (Sondheimer and Tzou, 1971).

Peak!This peak, which co-eluted with zeatin, was the latest-eluting zone of radio-

activity observed in the extracts. It was retained by a cation-exchange resin(Zerolit 225, SRC 14, NH1[ form) and was recovered into w-butanol from an aqueoussolution at pH 8-0 (K = 5-79). Peak 7 co-chromatographed with zeatin on paperchromatograms (solvent D) (JSJ 0-65) and on cellulose phosphate paper (Rp 0-35)and with the trans isomer of zeatin on TLC in solvent C (J?p 0-10). It was com-pletely degraded following permanganate oxidation, the major product co-chro-matographing with adenine. These findings suggest therefore that peak 7 consistsof unmetabolized [8-14C]zeatin.

The major metabolites of [8-14C]zeatin in the root nodules of Alnus glutinosaappeared to be more polar than zeatin itself as they eluted from the Sephadex LH20

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column prior to zeatin. These metabolites were present in extracts of nodules fromboth dormant and non-dormant plants, although only in the latter would activefixation of nitrogen and import of photosynthate by the nodules be occurring.Differences in metabolism of zeatin by dormant and non-dormant plants wereprobably not significant as quite large variations were found, in preliminaryexperiments, in the extent of metabolism by individual nodules. These variations,which were most probably due to differences in the rate of uptake of the appliedradioactive solutions into individual nodules from the micropipettes, rendereddifficult an examination of the rates of metabolism and the time of appearance of theindividual metabolites. However, preliminary experiments indicated that the re-lative proportion of the polar metabolites (peaks 1 and 2) tended to increase betweenthe first and third day after treatment.

The extent to which the observed metabolism reflects normal processes in thenodule remains for the moment uncertain. However, the similarity of many of themetabolites to the naturally occurring cytokinins in the nodule does suggest thatthe observed transformations have some physiological significance. Nevertheless itmust be borne in mind that the amounts of cytokinin applied in these experimentsgreatly exceed normal endogenous levels. The nodule tissues apparently possess ahigh glucosylating capacity, as in one experiment it was calculated that an averageof some 137 ng of labelled glucosides was present per nodule 24 h after the start ofzeatin uptake, whereas the normal endogenous levels were estimated to be around0-1 ng per nodule. (This result may be of significance in view of suggestions (e.g.Parker and Letham, 1973) that cytokinin glucosides may be of importance in thestorage and regulation of cytokinin levels in plant tissues.) The extension of thesestudies awaits the availability of 3H-labelled cytokinins of high specific activity sothat the effect of isotope dosage on metabolism can be ascertained.

ACKNOWLEDGEMENTSWe are grateful to Dr R. Horgan for a sample of zeatin- 9-/?-D-glucoside and forsynthesis of the [8-14C]zeatin used in the experiments. The work was supported byGrant No. B/RG/71340 from the Science Research Council.

LITERATURE CITEDARMSTRONG, D. J., BURROWS, W. J., EVANS, P. K., and SKOOG, F. 1969. Biochem. biophys. Res.

Commun. 37, 451-6.GORDON, M. E., LETHAM, D. S., and PARKER, C. W., 1974. Ann. Bot. 38, 809-25.HENSON, I. E., and WHEELER, C. T., 1976. New Phytol. 76, 433-9.

1977a, J. exp. Bot. 28, 205-14.19776. Ibid. 28, 1076-86.

HORGAN, R., 1975. Biochem. biophys. Res. Commun. 65, 358-63.MULER, C. O., 1968. In Biochemistry and physiology of plant growth substances. Eds. F. Wight-

man and G. Setterfield. Runge Press, Ottawa. Pp. 33-45.PARKER, C. W., and LETHAM, D. S., 1973. Planta, 114, 199-218.

1974. Ibid. 115, 337-44.WILSON, M. M., JENKINS, I. D., MaoLEOD, .J. K., and SUMMONS, R. E., 1975. Ann.

Bot. 39, 375-6.SONDHEIMER, E., and Tzou, D., 1971. PI. Physiol., Baltimore, 47, 516-19.

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Tzou, D., GALSON, E. C, and SONDHEIMER, E., 1973. Ibid. 51, 894-7.VONK, C. R., 1974. Acta bot. neerl. 23, 541-8.

1976. Ibid., 25, 153-66.WANG, T. L., THOMPSON, A. G., and HOBGAN, R., 1977. Planta, 135, 285-88.WABEING, P. F., HOBGAN, R., HENSON, I. E., and DAVIS, W., 1977. In Plant growth regulation.

Proc. 9th int. Conf. on Plant Growth Substances. Ed. P. E. Pilet, Springer-Verlag, Berlin.Pp. 147-53.

WHEELER, C. T., 1969. New Phytol. 68, 675-82.

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Documents

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