quantitation of cytokinins in biological samples using antibodies against zeatin riboside

Plant Physiol. (1984) 75, 1117-11250032-0889/84/75/1117/09/$0 1.00/0

Quantitation of Cytokinins in Biological Samples UsingAntibodies Against Zeatin Riboside

Received for publication January 10, 1984 and in revised form April 16, 1984

JANE BADENOCH-JONES,' DAVID S. LETHAM, CHARLES W. PARKER, AND BARRY G. ROLFEResearch School ofBiological Sciences, Australian National University, P. 0. Box 475,Canberra City, ACT 2601, Australia

ABSTRACT

The cross-reactivity of antibodies elicited in rabbits against zeatinriboside, to a wide range of naturally occurring cytokinins, was examined.As well as to zeatin riboside, the antisera cross-reacted to a considerableextent with zeatin, lupinic acid, zeatin-9-glucoside, zeatin riboside 5'-monophosphate and to a much lesser, but measurable extent, withdihydrozeatin riboside and dihydrozeatin. Chromatographic methodswere devised which allowed separation of all these cross-reactive com-pounds. Four biological samples, extracts of immature Zea mays kernels,immature seeds of Lupinus luteus, and Datura innoxia crown gall tumortissue, and a sample of Agrobacterium tumefaciens culture supernatant,were purified by these chromatographic methods, using IHizeatin ribo-side as a recovery marker, and at each stage of the purification process,were subjected to radioimmunoassay over a range of dilutions. At eachstage of sample purification, sample dilution curves were found to beparallel to the standard curve. Sample cytokinin levels estimated byradioimmunoassay were in close agreement to those available in theliterature for similar samples assayed by alternative methods. However,in some samples, unknown cross-reacting compounds were detected.

Recently, Weiler and colleagues have developed a number ofRIAs2 for quantitating plant hormones (see 38). For cytokinins,one of the major classes of plant hormones, RIA potentially hassome considerable advantages over the techniques of bioassay(19) and mass spectrometric isotope dilution procedures (5).Bioassays are laborious, time-consuming, and although sensitive,they lack precision and are subject to interference by inhibitors.In contrast, the mass spectrometric technique has high precision,permitting unequivocal identification and accurate quantitationof cytokinins; however, it requires considerable sample purifica-tion and the sophisticated equipment required may not be readilyavailable to many investigators. Alternatively, RIA is a rapid and

'J. B.-J. was supported by a Queen Elizabeth II Fellowship.2Abbreviations: RIA, radioimmunoassay; Z, zeatin (6-[4-hydroxy-3-

methylbut-trans-2-enylamino]purine); [9R]Z, zeatin riboside (9-ft-D-ri-bofuranosylzeatin); (diH)Z, dihydrozeatin (6-[4-hydroxy-3-methylbutyl-amino]purine); (diH)[9R]Z, dihydrozeatin riboside; [9Ala]Z, lupinicacid (L-,-[6-(4-hydroxy-3-methylbut-trans-2-enylamino)-purin-9-yl]-ala-nine); (OG)Z, O-B-D-glucopyranosylzeatin; (OG)[9R]Z, O-fl-n-glucopyr-anosyl-9-j3-D-ribofuranosylzeatin; (diH OG)Z, O-fl-D-glucopyranosyldi-hydrozeatin; (diH OG)[9R]Z, O-fl-D-glucopyranosyl-9-fl-D-ribofurano-syldihydrozeatin; [7G]Z, 7-glucopyranosylzeatin; [9G]Z, 9-glucopyra-nosylzeatin; [9R]iP, 9-,f-D-ribofuranosyl-N6-4A2-isopentenyl)adenine;[9R-5'P]Z, zeatin riboside 5'-phosphate; IEC, ion exchange chromatog-raphy.

particularly sensitive method. However, when antisera are elic-ited against one of a class of phytohormones, such as cytokinins,several members of the class may cross-react, each to differingextents. An advantage of multiple cross-reactivity is the potentialability to quantitate each cross-reactive compound, but a disad-vantage is that data obtained by RIA on crude extracts containinga mixture ofthese cross-reactive hormones, are somewhat mean-ingless. Furthermore, although RIA is often regarded as a highlyspecific method (38), the technique is vulnerable to nonspecificinterference from substances in the sample which either reactwith the antiserum or alter its reaction with the compound(s)being assayed (see 6).The aim of the current investigation was therefore to assess

the reliability of RIA for quantitating cytokinins in a variety ofextracts and to develop systems that enabled the separation ofall cytokinins known to cross-react with the anti-[9R]Z-serum.

MATERIALS AND METHODS

Plant Material. Lupin plants (Lupinus luteus L. cv Weiko III)were grown from seed (source: Westralian Farmers Co-op., Perth,Australia) in a greenhouse with natural light at 15 to 25°C. Sweetcorn plants (Zea mays, Fl hybrid lochief) were grown from seed(source: Yeates Seeds) in field plots (spring, summer). Immaturelupin seeds and sweet corn kernels, which were soft and containedlittle storage material, were removed from the pods and cobs,respectively, frozen in liquid nitrogen, and,stored at -1 98°C untilrequired. Crown gall tumor tissue of Datura innoxia Mill lineB6 was a gift from Dr. L. M. S. Palni. The callus was grown at25°C in 250-ml conical flasks containing 100 ml hormone-freeB5 medium (14) solidified with 0.8% (w/v) agar and was har-vested 5 weeks after subculturing.Agrobaterium tumefaciens. Agrobacterium tumefaciens strain

C58 was a gift from Dr. P. J. J. Hooykaas, University of Leiden,the Netherlands. A 5-L culture was grown in Trifolii mediumwith yeast extract (28) in the dark at 30°C, with shaking. Theculture was harvested at mid-log phase by centrifugation (8000g,20 min, 40C).

Cytokinins for Cross-Reactivity Tests and Other Chemicals.Lupinic acid ([9Ala]Z), dihydrozeatin ((diH)Z), (7-glucopyrano-sylzeatin ([7G]Z), 9-glucopyranosylzeatin ([9G]Z), O-fl-D-gluco-pyranosylzeatin ((OG)Z), O-fB-D-glucopyranosyl-9-f,-D-ribofura-nosylzeatin ((OG)[9R]Z), 0-3-D-glucopyranosyldihydrozeatin((diH OG)Z), and O-j3-D-glucopyranosyl-9-B3-D-ribofuranosyldi-hydrozeatin ((diH OG)[9R]Z)) were synthesized by publishedmethods (7-9, 20). Zeatin riboside 5'-monophosphate ([9R-5'P]Z) was synthesized by the method ofSummons et al. (34). Zeatin(Z) and zeatin riboside ([9R]Z) were purchased from Calbi-ochem. Dihydrozeatin riboside ((diH)[9R]Z) was prepared byhydrogenation of [9R]Z in methanol solution using a palladiumcatalyst at room temperature; it was purified by HPLC. Kinetin,

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BADENOCH-JONES ET AL.

6-benzylaminopurnne, adenine, adenosine, 9-fl-D-ribofuranosyl-N6-(A2-isopentenyl)adenine ([9R]iP) and BSA (fraction V) werepurchased from Sigma Chemical Company. Freund's adjuvantwas bought from Difco Laboratories. Cellulose for TLC wasbought from Serva Feinbiochemica (Heidelberg, Germany) andWoelm green fluorescent indicator (M. Woelm, Eschwerge, Ger-many) was incorporated into the cellulose at 0.8% (w/w) beforespreading the layer. Cellulose phosphate (Whatman Pl(floc)) andDEAE cellulose (DE I) were purchased from Whatman ChemicalSeparation Ltd., England. [3H][9R]Z (8.85 GBq mmolF') wasprepared as described by Summons et al. (32). [3H]Sodiumborohydride (241 GBq mmol-') and [2-3H]adenosine 5'-mono-phosphate ammonium salt ([3H]AMP) (714.1 GBq mmol-') werepurchased from Amersham International Ltd., Amersham, U.K.Sample Preparation. Plant Extracts. The tissue (fresh weights:

Z. mays kernels, 50 g; D. innoxia crown gall tumor tissue, 89.5g; L. luteus seeds, 42.5 g) was dropped into boiling 80% (v/v)ethanol (10 ml g-' fresh weight tissue). The sample was main-tained at 65C for 5 min, then rapidly cooled and homogenized.The combined extract and blender washings (80% [v/v] ethanol)were stirred overnight at room temperature, then filtered througha Buchner funnel. The pellet was resuspended in one-fifth theoriginal volume of 80% (v/v) ethanol, then filtered. [3H][9R]Z(438 Bq) was added as an internal standard for estimation ofrecovery at each stage of sample purification. The extract wasevaporated to dryness under reduced pressure at less than 40°Cand the residue was dissolved in RIA buffer and analyzed byRIA over a range of dilutions.

A. tumefaciens Culture Supernatant. [3H][9R]Z (876 Bq) wasadded to the culture supernatant which was evaporated to drynessunder reduced pressure at less than 40°C, and the residue wasdissolved in RIA buffer, and analyzed by RIA over a range ofdilutions.Sample Purification. Butanol Extraction. The concentrated A.

tumefaciens culture supernatant was adjusted to pH 8 and ex-tracted twice with an equal volume of butanol. The pooledbutanol phases were evaporated to dryness and the residuedissolved in RIA buffer and analyzed by RIA over a range ofdilutions.

Ion Exchange Chromatography (IEC) on Cellulose Phosphate.Crude plant extracts and the'butanol extract ofthe A. tumefaciensculture supernatant were applied to columns of cellulose phos-phate in the NH4' form, equilibrated to pH 3.0 by washing with0.5 M acetic acid. Cytokinin nucleotides were washed from thecolumns with 50 mm acetic acid. Cytokinin nucleosides andbases were eluted from the columns with 0.5 M NH40H till theeffluent was alkaline, followed by 2 column volumes of 0.5 MNH40H. Sample wash and eluate were evaporated to dryness,the residues disolved in RIA buffer, and aliquots analyzed byRIA over a range of dilutions.

Ion Exchange Chromatography on DEAE-Cellulose. Aqueoussolutions of evaporated nucleotide fractions from the cellulosephosphate columns were adjusted to pH 7.5 and applied tocolumns of DEAE-cellulose (HC03- form). The columns werewashed with 4 column volumes of water, and cytokinin nucleo-tides were eluted with 4 column volumes of 2M NH4HC03. [3H]AMP (873 Bq) was added to each sample prior to chromatogra-phy on DEAE-cellulose in order to estimate recovery. DEAEwash and eluate were evaporated to dryness, the residues dis-solved in RIA buffer, and aliquots analyzed by RIA over a rangeof dilutions.High Performance Liquid Chromatography. Bulk HPLC on a

Bondapak C,8/Porasil B Column. The NH40H eluates fromcellulose phosphate columns were adjusted to pH 3.5 (aceticacid) and centrifuged (10000g, 15 min). The clear samples weresubjected to HPLC on a Bondapak C18/Porasil B column (7.8 x610 mm) (Waters Associates) equilibrated initially with 0.2 M

acetic acid. After the sample was loaded, the column was washedfor 24 min with 0.2 M acetic acid at a flow rate of 6.0 ml min',followed by a concave gradient (No. 7 on Waters programmer)run for 36 min, with increasing methanol (0-100% [v/v]) andconstant (0.2 M) acetic acid. Two fractions were collected: 1,designated bulk HPLC wash, 0 to 34 min; 2, designated bulkHPLC eluate (containing all known cytokinins), 34 to 60 min.Each fraction was evaporated and the residue dissolved in RIAbuffer and analyzed by RIA over a range of dilutions.HPLC on Zorbax C8 Column. The bulk HPLC eluate was

adjusted to pH 3.5 (with acetic acid) and made to 25% (v/v)with methanol. Aliquots were subjected to HPLC on a ZorbaxC8 column, either analytical (4.6 x 250 mm) or semipreparative(9.4 x 250 mm) (DuPont Company). For the semipreparativecolumn, the solvent used was 25% (v/v) methanol in 0.2 M aceticacid as solvent at a flow rate of 5.0 ml min-'. [9GJZ, [9Ala]Z,[9R]Z, (diH)[9R]Z, Z, and (diH)Z were collected as six individualHPLC fractions. The five fractions between these six compoundsand a further eight equal fractions, four prior to the elution of[9G]Z and four subsequent to the elution of (diH)Z, were nor-mally collected. Each fraction was evaporated and the residuedissolved in RIA buffer and analyzed by RIA. The fractionscollected at the elution times of the cross-reactive compoundswere analyzed over a range of dilutions. To estimate the recoveryof [9R]Z at this stage and to ensure that [9R]Z was eluting inthe expected fraction, an aliquot of each sample, identical to thatchromatographed for RIA, was subjected to HPLC, and fractions(5.0 ml, except for the fraction specifically containing [9R]Z)were collected, dried, and counted.HPLC on a gsBondapak Phenyl Column. To gain further

evidence for the identity of compounds showing activity in RIA,the fractions eluting at the retention times of [9RJZ, Z, and [9G]Z following HPLC on Zorbax C8 were rechromatographed on a,gBondapak Phenyl column (3.9 x 300 mm) (Waters Associates)using 25% (v/v) methanol in 0.2 M acetic acid as solvent for [9R]Z and Z, and 10% (v/v) methanol in 0.2 M acetic acid as solventfor [9G]Z at a flow rate of 1.5 ml min-'. Discrete fractions werecollected at the retention times of [9R]Z (5.7-7.1 min), Z (4.9-6.4 min) or [9G]Z (6.8-8.6 min). For each injection, anothereight fractions of approximately equal volume were collected,covering a time span of 13.4, 11.3, and 15.5 min for the threerespective compounds. The fractions were evaporated and theresidues dissolved in RIA buffer and analyzed by RIA.

Preparation of Hapten Conjugate. [9R]Z was conjugated toBSA by a modification of the method of Erlanger and Beiser(12), following the procedures outlined by Weiler (37).

Synthesis of Trtiated Zeatin Riboside Dialchol. The tracerfor the assay was synthesized according to the method of Ran-derath and Randerath (26), except that [3H]sodium borohydriderather than [3HJpotassium borohydride was used as the reducingagent. The reaction product was purified by cellulose TLC usingacetonitrile-ethyl acetate-n-butanol-isopropanol-6 M ammonia(4:3:1:2:2.7, v/v/v/v/v) as solvent. Specific activity of the tracerwas 1.01 x 102 GBq mmol-' and the trimethylsilyl derivativeexhibited the following electron impact mass spectrum (70 ev)(principal ions above m/z 280, relative intensities in parenthesis):641 (M+, 15), 626 (12), 551 (24), 538 (23), 406 (19), 350(9), 320(29), 290 (8), 201 (100). All ions could be rationalized in termsof the expected structure.Immuniation Schedule. The hapten conjugate (1.5 mg ml-')

was dissolved in 0.9% (w/v) NaCl and mixed with an equalvolume of Freund's adjuvant until a stable emulsion was formed.Rabbits (3; breed: Dutch Dwarf x New Zealand White) wereinjected at days 0, 14, 28, 52, 73, 105, 248, 262, 276, 297, and318. Freund's complete adjuvant was used on days 0, 14, 248,and 262, and Freund's incomplete adjuvant was used on theremaining days. On each day of injection, each rabbit received

1118 Plant Physiol. Vol. 75, 1984

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RADIOIMMUNOASSAY OF CYTOKININS

(b)

70F6050

403020

10n

I 0~~~~III

0\- m

., I

0.05 0.1 0.5 1.0

2 I

O _0-1 _

-2

-3-4-5

\Is

5 10 50 0.05 0.1 0.5 1.0

FIG. 1. (a), Mean standard curve for [9R]Z. (b), Logittransformation of the mean standard curve for [9R]Z.Bars indicate the SE (n = 19); B, binding of tracer toantibody in the presence of [9R]Z standards; Bo, bindingof tracer to antibody in the absence of [9R]Z.

5 10 50

[9R]Z Standard (ng)

Table I. Cross-Reactivities of Various Purines with Anti-[9RJZ-SerumResults are expressed as the inverse of the concentration, on a molar

basis, ofthe compound, at BIBo= 0.5 relative to the [9R]Z concentrationrequired to produce the same effect. Data from the literature are givenfor comparison. ND, not determined.

Cross-ReactivityCompound Present Weiler MacDonald Vold and

- Study (37) et al. (21) Leonard (36)[9R]Z 1.00 1.00 1.00 1.00Z 0.42 0.44 0.60 ND(diH)[9R]Z 0.019 ND ND ND(diH)Z 0.013 0.0172 0.33 ND[9G]Z 0.46 ND ND ND[7G]Z 0 ND ND ND[9Ala]Z 0.219 0.084 ND ND[9R]iP 0.0021 0.001 <0.005 0.0026(OG)Z 0 ND ND ND(diH OG)Z 0 ND ND ND(OG)[9R]Z 0 ND ND ND(diH OG)[9R]Z 0 ND ND NDAdenosine 0 0 <0.001 NDAdenine 0 0 ND NDKinetin 0.0008 0.0003 <0.001 ND6-Benzylaminopurine 0.0039 0.0026 ND ND[9R-5'P]Z 1.04 ND ND ND

approximately 0.75 mg of conjugate distributed as three equal0.3-ml injections: subcutaneously in the neck, subcutaneously inthe hindquarter, and intramuscularly in the thigh. Rabbits werebled by the marginal ear vein, normally 4, 6, 8, 10, and 12 dafter injection with Freund's incomplete adjuvant, and the anti-sera were screened for affinity and titer. Although all rabbitsdeveloped antibodies against [9R]Z, serum from one animal,which developed consistently superior antiserum, was selectedfor the present study. It was taken 6 d after injection on day 318.Antisera were stored at -20C, either undiluted or diluted 1:100with RIA buffer. After thawing, antisera were stored at 4°C andused within 3 d. The antisera were used in RIA without purifi-cation.

Radioimmunoassay. The assay was based on that described byWeiler (37). Standards and samples were assayed in triplicate inEppendorf centrifuge tubes (Eppendorf Geratebau Netheler andHinz GmbH, Hamburg, Germany), and all manipulations weredone at room temperature. The incubation mixture containedper tube: 250 Al RIA buffer (0.01 M sodium phosphate, 0.15 M

NaCl, pH 7.4), 50 ,l BSA (9% w/v in RIA buffer), 50 Ml(93 Bq)[3H][9R]Z dialcohol, 50 Al standard or sample and 50 ,l antise-

rum (1:1 100 dilution) (or buffer for determination of nonspecificbinding). Pipette tips used for dispensing the tracer were pre-soaked in the tracer. After the reaction mixture was mixed andincubated for 2 h, 0.6 ml 91% saturated (NH4)2S04 solution (pH7.4) was added. After mixing and incubation for 30 min, tubeswere centrifuged (10,000g, 10 min). The pellets were washed in0.5 ml 50% saturated (NH4)2SO4 solution (pH 7.4), then dis-solved in 0.1 ml water. After addition of 1 ml scintillation fluid(2) and mixing, the tubes were transferred to glass scintillationvials and counted by the procedures of Badenoch-Jones et al.(2). A computer program based on that of Brooker et al. (4) wasused for assay evaluation and computation of results.

RESULTS

The mean standard curve for [9R]Z is shown in Figure la.The curve can be linearized over the measuring range (whichextends from approximately 0.3 to 150 pmol) by logit transfor-

t 7CH2OH

NH-CH27 \CH3

N N

KN R

COMPOUND R[9RIZ ribofuranosylz H

[s9]z glucosyl

[9Ala]Z p-alanine moiety

[9R-5'P] Z ribofuranosyl-5'- phosphate

FIG. 2. Compounds cross-reactive with the anti-[9R]Z-serum (cf Ta-ble I).

100 r (a)90 i-

80h0

-110

0mm

[9R]Z Standard (ng)

v

1119

I




mation of the B/Bdilution employedabsence of unlabele(absence of antibodianalysis (29) of stancof 2.5 x 10-" mol Ivery high affinity anstudy. Antiserum sFstudies and the resultested for binding oas [9RJZ itself, fourwith the anti-[9R]-s5'P]Z, and the stnFigure 2. Two compto a much lesser, Icurves for all ofthes4were parallel with th

Several internal i

reliability. When tiinterference from odilution curve and tishould be parallel. Istages of sample pucurves were found,with slopes which dislope of the standar4(A. tumefaciens samcellulose phosphatecellulose eluate; andsample was not pasfor one of the samprecoveries of standathe purification proxFor all samples, a(

eluate from cellulos4phate step, recoveryof activity in thew,Table II. From thewash, it appearedtsample, contained alcompounds not rete

01[co

m

0

-

-1

-2

-3

-4 -

-0.

0. 1

FIG. 3. Logit transfZ, [9GJZ, and [9R-5'F

to values (Fig. lb). The antiserum, at the ther chromatography of the cellulose phosphate wash on DEAE-(1:1100), bound 42% of the tracer in the cellulose indicated that not all of the activity present in thed [9R]Z. Nonspecific binding (binding in the cellulose phosphate wash from the D. innoxia and L. luteusy) was less than 1%. From Scatchard plot samples was due to cytokinin nucleotides. These would be foundlard curve data, a maximum affinity constant in the DEAE eluate, but particularly for the L. luteus sample,1-' was calculated, indicating the presence of considerable activity was detected in the DEAE wash. The com-tibodies in the antiserum used in the present pounds responsible for the activity detected by RIA in the DEAEecificity was determined by cross-reactivity wash are not known. Activity in the DEAE eluate may best beIts are given in Table I. The compounds were attributed to Z nucleotides, since [9R-5'P]Z cross-reactedver a range from 0.1 up to 5000 ng. As well strongly with the anti-[9R]Z-serum. The proportion of thecompounds showed marked cross-reactivity nucleoside mono-, di-, and tri-phosphates in each sample re-erum, namely, Z, [9G]Z, [9Ala]Z, and [9R- mains unknown.uctures of these compounds are shown in Chromatography on cellulose phosphate thus separated one ofounds, (diH)Z and (diH)[9R]Z, cross-reacted the cross-reactive compounds ([9R-5'P]Z) from the others. Chro-but still measurable, extent. The standard matography on the Bondapak C18/Porasil B column, which gavecompounds, following logit transformation, a 72% recovery of [3H][9R]Z, did not separate any of the cross-

ie standard curve for [9R]Z (Fig. 3). reactive cytokinins and thus in no sample was activity detectedquality checks were made to assess assay in the bulk HPLC wash. This HPLC step provided sufficienthe analysis of a sample is not subject to sample purification to enable chromatography to be carried outther compounds in the extract, the sample effectively on the Zorbax C8 column which separated all thehe standard curve (after logit transformation) remaining cross-reactive compounds ([9R]Z, Z, [9G]Z, andFor each of the four samples, at each of the [9Ala]Z, as well as (diH)Z and (diH)[9R]Z; see Fig. 5). Theirification listed below, the sample dilution Zorbax C8 analytical column was found to have insufficientby regression analysis, to be straight lines capacity, however, when a Zorbax C8 semipreparative column

id not differ significantly (P > 0.05) from the was used, the [3H][9RJZ marker in all samples eluted as a singlecurve: (a) crude extract; (b) butanol extract peak in the expected fraction at a sample injection volume of

iple only); (c) cellulose phosphate eluate; (d) 350 ;J (total volume of 500 ,ul). Recovery of the [3H][9R]Zwash; (e) bulk HPLC eluate; (f) DEAE marker off the Zorbax C8 column averaged 70%, giving a com-

(g) DEAE-cellulose wash (the A. tumefaciens bined recovery, after all stages of the purification process, aver-

sed over a DEAE-cellulose column). Details aging 47%.les (D. innoxia) are shown in Figure 4. The For each of the samples, the results of RIA on all fractionsLrd [9R]Z added to samples at any stage of collected off the Zorbax C8 column are shown in Figure 6. Forcess, were found to be satisfactory. the D. innoxia sample, RIA activity was detected in the fractionsctivity was detected in both the wash and the collected at the elution times of authentic [9G]Z,[9Ala]Z, [9R]ephosphate columns. For the cellulose phos- Z, and Z. There were also indications of activity in severalof[3H][9R]Z averaged 88%. The proportions fractions which were not at the elution times of any of theash and eluate are given for each sample in cytokinin markers, most notably, fractions 4, 7, and11. For theactivity detected in the cellulose phosphate Z. mays sample, considerable RIA activity detected in thethat the samples, particularly the L. luteus fractions at the elution times of [9G]Z, [9R]Z, and Z, and there

ppreciable amounts ofcytokinin nucleotides, appeared to be some activity in the fraction at the elution timeained by cellulose phosphate. However, fur- of[9AIa]Z. Most fractions from I to 14 showed some inhibition

of binding of tracer to antiserum compared to the zero [9R]Zstandard. For the L. luteus sample, considerable cytokinin activ-ity was detected in the fractions at the elution times of [9Ala]Z,

-*- [9R]Z Z, and (diH)[9R]Z. Since (diH)[9R]Z has a cross-reactivity of

-o-z only 1.9% with the anti-[9R]Z-serum, the inhibition of binding[9Aa]Z of tracer to antiserum by fraction1 1 must reflect a high level of[9GZ (diH)[9R]Z (see Table II). Unlike the D. innoxia and Z. mays--A-- [9G]z samples, no activity was detected in the fraction at the elution

ZN-° [9R-5'P]Z time of [9G]Z. Again, some activity was detected in fractions

that were not at the elution times of any of the marker com-

pounds, in particular, fractions 2, 4, and 17. For the A. tumefa-ciens sample, considerable activity was detected in the fraction

at the elution time of [9R]Z, but a small amount of activity wasC'ux\ also detected in the three fractions prior to and the three fractionsfollowing this fraction. For all samples, the fractions at the elution

NZN.° Dtimes of known cross-reactive compounds were assayed over a~\" N.\

range of dilutions, and in each case when activity was detected<X>x in these fractions, the logit transformation of the sample dilution\. curve was parallel with the logit transformation of the standardo curve (see Fig. 7, D. innoxia sample). RIA response, expressedI as [9R]Z equivalents, is shown for each sample, at each stage of

0. 5 1. 0 5 1 0 50 100 the purification process, in TableII. The values are corrected forrecovery of the[3H][9R]Z marker and the values for Z, [9G]Z,

Cytokinin (ng) and (diH)[9R]Z levels were calculated assuming that the recovery

rormations of standard curves for [9RJZ, Z, [9Ala] of these cytokinins was the same as the recovery of [9R]Z. These3iz. results are compared with those available in -the literature for





. [9R]Z standardx Crude extract

0coN'.co

0-J

0.1 0.2 0.5 1.0 2.0 5.0 10 20 50 100

[9R]Z standard (ng) Sample (p1)

3 - D. X Bulk HPLC eluate

4

3

2

0- 1

-2

-3

-4

R* [9R]Z standarx Cellulose pho

l.-,0 ammonia elu

\x0 X

F- x

Ird)sphatelate

0

-S

0.05 0.1 0.5 1.0 5.0 10

[9R]Z standard (ng) Sample4 r- * [9R]Z standard

2

3]

az o

N..

a-2x cn

111 ox O__ -3

-4

S I

1121

ax 1-_ xN,

a -2 - \ @0

J . -s10 0

op) [9R]Z stanar.d (ng)Samplep00 N.

-4 0~1 -550 0.1 0.5 1.0 5.0 10 50

(pi) [9R]Z standard Cng) Sample (pil)N E. x DEAE eluateN OEAE wash

_

0 0N"

0,

0.1 0.5 10 5.0 10 50 100 0.1 0.5 1.0 5.0 10 50

[9R]Z standard (ng) Sample (pi) [9R]Z standard (ng) Sample (pt)FIG. 4. Logit transformation of standard curves for [9R]Z and sample dilution curves for D. innoxia extract at the following stages of sample

purification. A, Crude extract; B, ammonia eluate from cellulose phosphate column; C, wash from cellulose phosphate column; D, eluate fromBondapak C,8/Porasil B column; E, eluate and wash from DEAE column.

Table II. RIA Activity in Chromatographic Fractions Expressed as [9R]Z EquivalentsData are corrected on the basis of the recovery of [3H][9R]Z.

Cellu- Cellu- Cellulose Zorbax C8 Eluatelose lose Phosphate DEAE

Sample ECrude Phos- Phos- Eluate: Bulk HPLC Eluate DEAEExtract

phate phate Cellulose Eluate [9R]Z Z' [9G]Z8 [9Ala]Z' (diH)[9R]Zm (Z Nucleo- Wash

Elute ashPhosphate tides)Elute ash Wash

L. luteus 4282 1070 3660 0.23:0.77 1405 576 10 Possibly 1385 94 2416present (120)

Z. mays 296 155 135 0.53:0.47 145 152 30 78 20 172 0(219)

D. innoxia 311 194 70 0.73:0.27 137 81 46 282 93 78 17(100)

A. tumefaciens 2275 Butanol 1729 182 0.90:0.10 1068 718extract1873

a Recoveries of Z, [9G]Z, and (diH)[9R]Z were assumed to be the same as the recovery of [9R]Z. The cross-reactivity values in Table I were usedto calculate the concentration of Z, [9G]Z, [9Ala]Z, and (diH)[9R]Z. Data for the DEAE eluate and wash were corrected for the recovery of [3H]AMP.

similar samples, but employing alternative assay techniques (see RIA in the gBondapak Phenyl eluate in. only a single fractionTable III). which corresponded to the elution time of standard [9R]Z.The L. luteus, Z. mays, and D. innoxia samples were injected Similarly, for all these samples, when the fraction from the

onto the Zorbax C8 semipreparative column and the fractions Zorbax C8 column believed to contain Z, was reinjected ontobelieved to contain [9R]Z (according to the elution time of the gBondapak Phenyl column, activity was again detected in astandard [9R]Z) were collected and reinjected onto a gBondapak single fraction corresponding to the elution time of standard Z.Phenyl column. For all the samples, activity was detected by For the Z. mays and D. innoxia samples, when the fraction from

.A.

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2-4Nof RIA for quantitating phytohormones is in its infancy. For thecytokinins, several laboratories have pre d antisera for use inRIA, against [9RJiP (15, 17, 21, 23) or against [9R]Z (21, 24, 36,37). Weiler (37) used anti-[9R]Z-sera in RIA to assay cytokininsin crude extracts of developing tomato fruits and Weiler and

,x, Zeigler (40) assayed unpurified phloem exudate. Results from5 cr ,>, the present study, however, indicate the limitations of analyses

by RIA on crude extracts, unless the extracts contain only asingle known cytokinin that cross-reacts with the anti-[9R]Z-serum. The result obtained from analysis of a crude extractrepresents the sum, for all cross-reactive compounds, of thefollowing parameter: [(mol of cross reactive compound x per

1-4N cent molar cross-reactivity relative to [9R]Z)/ 100]. Generally,a= the amounts of each cross-reactive compound are unknown and

rJ t -hence the result on a crude extract may not even be a good'i .I indicator of the total amount of the cross-reactive cytokinins.

Furthermore, the activity of a compound in RIA is determinedS ^ \ by its structure and may bear little relation to its biological

activity. Thus, [9Ala]Z, which is only a very weakly activecytokinin in most bioassays, is more cross-reactive with anti-[9R]Z-sera than (diH)Z and (diH)[9R]Z which are very active inbioassays.

0-~ Aj The cross-reactivities of various purines to the anti-[9R]Z-seraraised in different laboratories are similar (see Table I), with the

l I510 15 20 exception of the relatively high cross-reactivity of (diH)Z to the0 5 10 15 20 antiserum of McDonald et al. (21). All workers have observed(inject)

Time (min) the effective cross-reactivity of Z with anti-[9R]Z-sera. The pu-rines examined in the present study for cross-reactivity included

he elution profile ofa standard mixture ofcompounds cross- a number ofcytokinin metabolites that are found in plant tissueshe anti-[9R]Z-serum during chromatography on a Zorbax (11), several of which have not previously been examined foris monitored by UV absorbance (X = 280 nm). cross-reactivity with anti-[9RJZ-sera because they are not readily

C8 column believed to contain [9G]Z, was reinjected available. The naturally occurring cytokinins, [9G]Z, [9Ala]Z,ondapak Phenyl column, activity was detected in a and the mononucleotide of Z, were all found, as would be

on corresponding to the elution time ofstandard [9G] exPected from their structures, to cross-react to a considerableof the samples, Z. mays, these results are shown in extent with the anti-[9R]Z-serum. The di- and tri-phosphates of

[9R]Z would also, almost certainly, be cross-reactive. The 0-glucosides, which occur ubiquitously in plants, were not found

DISCUSSION to be cross-reactive, as would be expected from their structures.1st to the considerable development of RIA for the Possible disadvantages of RIA are that the properties of then of mammalian hormones (see 1), the development antisera prepared in different laboratories may be variable and

Z maysr-4

Is -CC- 01 -I

0ol I I I rI I rIn 1 1 1 1 1 1 1 1 1 o°. 1 2 3 4 5 6 7 8 9 1011 121314151617180 L. luteusC

100 100

60 s0

60-60

40 40

20-20

0 0

1 2 3 4 5 6 7 8 9 10 12131415161718

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FIG. 6. RIA activity (expressed as % bindingof tracer to antibody relative to binding oftracer to antibody in the absence of [9RJZ) for50 ju aliquots of thie final volume of fiactionseluted from the Zorbax C8 column. The frac-tions in which the authentic standards elutedare indicated by bars. Volume of fractionsvaried from approximately 2.7 to 10.3 ml.Frctions were evaporated to dryness and dis-solved in 200 ul RIA buffer.

1 2 3 4 5 6 7 8 91111213141516171819

Fraction number

0.0:

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the Zorbaxonto the iAEsingle frctiZ. For oneFigure 8.

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1122 Plant Physiol. Vol. 7 5, 1984

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the identity of the compounds measured by RIA may be uncer-tain (see 13). For anti-[9R]Z-sera, the former does not appear tobe a serious problem, since those raised in different laboratoriesare similar (see 38; Table I). The assay parameters of Weiler (37)and the present study are similar, although the assay of Weiler(37) is a little more sensitive than that of the present study,presumably due to the higher specific activity of the tracer. Also,interference with the assay from other compounds in the extracts,as indicated by nonparallelism of the standard curve and sampledilution curves, was not found to be a problem for any sample,even crude extracts, analyzed for cytokinins by RIA in the presentstudy or by Weiler (37). However, the equivocal nature of thecompounds measured may indeed be a problem, especially inlight of the complexity of the situation with cytokinins. For thesamples examined in the present study, the results from rechro-matographing the putative [9R]Z, Z, and [9G]Z fractions pro-vided reasonable evidence of the identity of these compounds.However, it would not be feasible to attempt to obtain such

evidence for every sample. Furthermore, any number of purinesnot tested for cross-reactivity, and with unknown chromato-graphic properties in the separative systems used, may cross-react with the antisera. Indeed, when all fractions from an HPLCrun were analyzed (21; the present study), some fractions, whichwere not collected at the elution times of known cross-reactivecytokinins, showed activity in RIA. Also some activity, due tounknown compounds, was detected in the DEAE-cellulose washof some samples, most notably, the L. luteus sample.

It is necessary either to use RIA in conjunction with appropri-ate systems for cytokinin separation, or to prepare antibodiesthat are specific for a single cytokinin. The latter may be possibleusing the technique of monoclonal antibody production (com-pare Refs. 35 and 41). Using conventional multispecific anti-bodies elicited in rabbits, Weiler and Spanier (39) have combinedRIA and TLC, Morris and colleagues (21, 24) have combinedRIA and HPLC, and we have combined RIA, IEC, and HPLC.Further work is required to develop optimum systems for theseparation of cytokinins prior to analysis by RIA. Immunoaffin-ity chromatography, as described by Morris et al. (24), mayindeed be a useful method for preliminary sample purification.Clearly, it is also necessary to separate all known cross-reactivecompounds, each preferably as a single fraction. The method ofMacDonald et al. (21) goes some way towards achieving thisaim, although the retention times of [9G]Z, [9Ala]Z, and [9R-5'P]Z were not indicated (21; Fig. 4) and the appropriate com-pounds were not collected as discrete peaks. The separationsystem used in the present study allows collection of each cross-reactive compound; however, HPLC systems have capacity limi-tations and the presence of cytokinins in a complex mixture ofcompounds, as in biological samples, may alter (usually, reduce)their HPLC retention times relative to authentic standards.Sample purification for RIA is reduced compared with that

required for physicochemical assays as, therefore, are the inevi-table losses which occur during the purification steps. However,appreciable losses do occur, and these need to be accounted forwhen quantitating cytokinins by RIA. Ideally, high specific activ-ity recovery markers are required for each cross-reactive cytoki-nin that one is attempting to measure, in particular, [9R]Z, Z,[9G]Z, and [9R-5'PJZ. Any possible spillover ofthe marker intoa preceding fraction could be detected and accounted for. Al-though [9Ala]Z is a cross-reactive compound, it may be difficultto quantitate because of its low levels in many samples, relativeto Z and [9R]Z, and its cross-reactivity of only approximately20%.To comprehensively investigate the cytokinins present in bio-

logical extracts, we are currently producing further antisera: anti-[9R]iP, anti4diH)[9R]Z, and anti-cis-[9R]Z-sera. Sample anal-ysis by RIA using anti-[9R]Z, anti-[9R]iP, and anti4diH)Z, in

Table III. A Comparison ofthe Cytokinin Concentrations as Determined by RIA in L. luteus Seeds, Z. maysKernels, and D. innoxia Tissue, with the Concentrations Determined on Similar Tissues by Alternative

TechniquesL. luteus L. luteus Z. mays Z. mays D. innoxia D. innoxia'

Cytokinin (RIA, Present (MS, Summons (RIA, Present (MS, Summons (RIA, Present (MS, PalniStudy) et al., 33) Study) et al., 31) Study) et al., 25)

ng g' fresh wt tissue[9R]Z 576 393 152 530 81 78Z 10 18.5 30 220 46 27(diH)[9R]Z 1385 666 NAb ND NA 7[9G]Z n.d. n.d. 78 15 282 250[9R-5'P]Z 120 ND 212 ND 82 47' These values represent minimal levels, since some losses may have occurred prior to the addition to the

deuterium-labeled internal standards which were added following chromatography on Sephadex LH.20.b NA, not possible to quantitate accurately; ND, not determined; n.d., not detected.

3-NI-

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80

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Fraction numberFIG. 8. RIA activity in fractions eluting from the jsBondapak Phenyl

column following injection of the fractions from the Zorbax C8 columnbelieved to contain (A) [9R]Z, (B) Z, and (C) [9G]Z, for the Z. mays

sample. The fractions inwhich the authentic standards eluted are indi-cated by bars.

conjunction with appropriate recovery markers, should providequantitative data on the levels of Z, (diH)Z, [9R]Z, (diH)[9R]Z,[9G]Z, (diH)[9G]Z, iP, [9R]iP, and possibly [9Ala]Z and(diH)[9Ala]Z, as well as the nucleotides of Z, (diH)Z, andiP. Insome samples, cis-Z and cis-[9R]Z may occur (see 19), and theanti-cis-[9R]Z-serum would be employed for their analysis. Itmay be possible to individually quantitate the O-glucosides ifthey were separated from other cytokinins (for example, by TLC,see Badenoch-Jones et al. [3]), hydrolyzed with,8-glucosidase,and the Zorbax C8 eluate of the hydrolyzed sample was analyzedby RIA employing, separately, the anti-[9R]Z- and anti(diH)[9R]Z-sera. The major problem would be synthesizing high specificactivity recovery markers for each of theO-glucosides.A comparison of the cytokinin concentrations determined by

RIA of the various tissues examined in the present study withthe concentrations previously determined for the same tissues byMS (Table III) shows encouraging similarities. The tissue samplesused in the present and previous studies would be very similarin the cases of the L. luteus seeds and D. innoxia tissue. It issignificant that the levels of [9R]Z, Z, and (diH)[9R]Z deter-mined by RIA in L. luteus and the levels of [9R]Z, Z, [9GJZ,and [9R-5'P]Z determined by RIA in D. innoxia were similar tothe values found by MS (TableIII). It should be noted that thevalues found by Palni et al. (25) for the latter will be a slightunderestimate since the2H standards for MS were added afterthe initial extraction had been made. Summons et al. (33) were

not able to detect [9Ala]Z in lupin seed by MS. Results fromRIA suggested the possible presence of [9Ala]Z in lupin seed,but the low levels, combined with the low cross-reactivity of[9Ala]Z with the anti-[9R]Z-serum, preclude an accurate quan-

titation of [9Ala]Z in this tissue. Appreciable quantities of Znucleotides were detected in the L. luteus samples by RIA (Table

III), but data on the level ofcytokinin nucleotides is not availablefrom the study of Summons et al. (33).The two sets of data for Z. mays kernels (Table III) are not as

consistent as those for the other two tissues. This might beexpected since the Z. mays sample in the present study was notcarefully matched in terms of maturity or cultivar with thesample assayed by Summons et al. (31). However, according toboth RIA and MS quantification, [9RJZ was more abundantthan Z. [9G]Z was first identified and quantitated in plant tissuesonly recently, in MS studies with Z. mays kernels (31, 32) andcrown gall tumor tissue (30); the present RIA results confirmthese findings. The cytokinin nucleotides were not determinedby Summons et al. (31, 32) in Z. mays kernels but a previousstudy (18) had unambiguously identified [9R-5'P] as a cytokininin sweet corn kernels. Based on the weight isolated in crystallineform, it appeared to be the major cytokinin present. Similarly,in the present study, the level of [9R-5'PJZ exceeded that of Zand [9R]Z.The estimated [9R]Z level of 0.72 jig 1' in A. tumefaciens

culture supernatant is close to the values of 0.44 and 0.62observed by Einset (10) and of 0.31 observed by McCloskey etal. (22) for the same or similar strains. cis[9R]Z and cisZ havealso been detected in culture supernatants of A. tumefaciens (16,22), but anti-[9RJZ serum has very low cross-reactivity to theseisomers (21, 37) and is therefore not useful for detecting them insamples. A. tumefaciens has been found to produce Z (10, 16),but this cytokinin was not detected in the volume of sampleassayed in the present study.The identification and quantitation of cytokinins in biological

extracts is a prerequisite for many physiological studies of plantdevelopment. Current analytical techniques remain problematic(5, 27) and there is a need for a method that combines simplicityand rapidity with accuracy and sensitivity. Results from thepresent study suggest that RIA has the potential to fulfil theserequirements, but for this potential to be reached, further devel-opment of the technique per se and of the associated separativeprocedures, is required.

Acknowledgment-The authors wish to thank C. M. Bates for technical assist-ance.

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quantitation of cytokinins in biological samples using antibodies against zeatin riboside

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