Plant Physiol. (1971) 47, 275-281

The Metabolism and Biological Activity of a

9-Substituted Cytokinini 2Received for publication May 27, 1970

J. EUGENE Fox, CHANDER K. SOOD,' BRIAN BUCKWALTER,4 AND JAMES D. MCCHESNEYDepartment of Botany, The University of Kansas, Lawrence, Kansas 66044

ABSTRACT

In order to test the metabolic stability of 9-substitutedcytokinins, 6-benzylamino-9-methyl purine has beensynthesized and labeled with 14C in the 9-methyl carbon ordoubly labeled with 14C in the 9-methyl carbon and 3H inthe methylene moiety of the side chain. Although the 6-benzylamino-9-methylpurine is chemically stable, cyto-kinin-requiring tissues begin removing the 9-substituentin as little as 10 minutes. Among the various metabolicproducts is free benzylaminopurine. Thus, the biologicalactivity of 9-substituted cytokinins could be accountedfor by their conversion to the free base.

The widespread presence of cytokinins in the transfer RNAof animals, plants, and microorganisms is well established (seereview by Key [14]). Furthermore, in those cases where theprimary structure is known, the cytokinin is found immediatelyadjacent to the presumed anticodon (1, 5, 17). The continuingdebate as to whether or not the physiological activity of exog-enously supplied cytokinins is mediated by their presence intRNA seems to have been recently resolved by the work of Chenand Hall (4). Here it was shown that the potent cytokinin N6-(A2-isopentenyl) adenosine occurs in the tRNA of cultivatedtobacco tissue even though the tissue requires an exogenouscytokinin in order to grow. The fact that the cytokinin suppliedin the medium is not the metabolic precursor of the cytokininin the tRNA argues that the role of the former is distinct in atleast some important way from that of the latter.

This work, however, cannot be used as evidence against thenotion that exogenous cytokinins act through their observedincorporation into polynucleotides (7, 9, 10). One would needto demonstrate that cytokinins which are prevented from enter-ing into nucleotide formation nevertheless retain all of theirbiological activity. It occurred to us that the use of blockingagents at the 9 position of the purine moiety would result incompounds which might yield information concerning the rela-tion between nucleotide formation and biological activity of the

I Supported by National Science Foundation Grants GB-6010and GB-8319 and National Institutes of Health Grant GM-09902.

2 Part of this material is from a thesis presented by Chander Sood inpartial fulfillment of the requirements for the Ph.D. degree at theUniversity of Kansas.

3 Present address: Department of Biology, University of Windsor,Windsor, Ontario.

4Present address: Department of Chemistry, Indiana University,Bloomington, Indiana.

cytokinins. Accordingly, in July of 1967, we reported (11) theresults of tests with the substance 6-benzylamino-9- (3-hydroxy-propyl) purine. Although this material retains cytokinin ac-tivity, it is 10 to 100 times less effective than the correspondingfree base; it is possible that the activity of the compound is dueto its conversion to the free base in the plant tissue by removalof the substituent at the 9 position.

Earlier, Weaver et al. (22) reported that substituting benzyl-adenine at the 9 position with a tetrahydropyran ring yielded acompound with good biological activity, while Guern et al. (12)pointed out that several 9-substituted benzyladenines are activethough seemingly not hydrolyzed in vivo. More recently Kendeand Tavares (13) described studies with 6-benzylamino-9-methyl purine-benzyl-7-14C. Although radioactivity was incor-porated into the RNA of soybean callus tissue when the freebase was employed, methylation at the 9 position apparently pre-vented incorporation while in no way reducing biological ac-tivity of the cytokinin. Letham (16) and Young and Letham (23)reported that the 9-cyclohexyl analog of isopentenyladeninepossesses considerable cytokinin activity; Letham considersthat, "It is extremely unlikely that this compound with a groupnot subject to hydrolytic cleavage on the 9 position could beconverted to a ribotide and incorporated into RNA as a nucleo-tide." (16).

Likewise, Shaw et al. (21) reported that 9-methylzeatin gavebiological activity of the same order as zeatin and concludedthat, "The results imply that the mechanism of cytokinin ac-tivity in substituted adenines does not require prior formationof nucleotide derivatives."

Clearly, however, such conclusions are premature unless it isdemonstrated directly that the substituent at the 9 position isnot removed by the plant tissue and thus constitutes a truly ef-fective block to ribonucleotide synthesis. In this paper the syn-thesis and biological activity of 9-methyl benzyladenine is de-scribed. In addition, evidence is presented which indicates thatsoybean and tobacco tissues remove substituents at the 9 posi-tion within a few minutes of incubation.

MATERIALS AND METHODS

Biological Tests. The origin of the soybean and tobacco tissuesused here and their growth on various cytokinin and auxinlevels have previously been described (8). The basal mediumand culture methods have been reported (6). In some shortterm metabolic studies with labeled compounds, liquid mediawere used and the flasks were shaken. Compounds tested forcytokinin activity were sterilized at room temperature by filtra-tion.

Synthesis and Characterization of 6-Benzylamino-9-methylPurine. The 6-benzylaminopurine used in this and followingsynthetic procedures was prepared as previously described (9).The methylated derivative was synthesized by alkylation of the

275

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Plant Physiol. Vol. 47, 1971

100

50

0c

3.0

-0

20

a

10

10

is

15II.

H I-C*

NCKN

N HCH,;

45

51

50 100II ..I ll. u.1ll

a?

.I .1. 1,150

(M+)I

200 240

meFiG. 1. Mass spectrum of 6-benzylamino-9-methyl purine determined on a Finnigan 1015 quadrapole mass spectrophotometer by the direct

inlet technique.

2I

53~~ ~ ~ ~ ~~

II

50 100

Hl-CH24_

N1-CH

H

7,0L

,1.11,it1

,.I- 1,1.1i0

FIG. 2. Mass spectrum of 6-benzylaminopurine (see legend to Fig. 1).

potassium salt of BA.5 This procedure is similar in many respectsto that of Carraway et al. (3) who report an increase in the selec-tivity of the alkylation which yields an increased ratio of the9- to the 3-isomer as compared with earlier methods. In a typicalsynthesis 5.8 x 10-4 moles (130.2 mg) of BA were dissolved in15 ml of anhydrous tetrahydrofuran to which were added 7.0 X10- moles (27.3 mg) of metallic potassium. This mixture was

stirred at room temperature under nitrogen for 12 hr after which6.4 X 10-3 moles (912 mg) of methyl iodide was added, and thewhole mixture was stirred for an additional 12 hr. The reactionflask was fitted to a condenser through which water at 0 C wascirculated to prevent the escape of gaseous methyl iodide. Thereaction mixture was then taken to dryness at 30 C under re-

5 Abbreviations: BA: 6-benzylaminopurine; MBA: 6-benzylamino-9-methylpurine.

duced pressure in a rotary evaporator. The dried material wasdissolved in a small amount of ethyl acetate and streaked on 1-mmlayers of aluminum oxide G (Merck) on glass plates activated at60 C for 24 hr. The thin layer chromatogram was developed in a

mixture of chloroform and benzene (9:1). In this system ultra-violet-absorbing material presumed to be 6-benzylamino-9-methyl purine migrated to an RF of 0.25 while the unreactedpotassium salt of BA remained at the base line. In addition, atleast two other ultraviolet-absorbing compounds could oftenbe detected at RF 0.55 and at the solvent front; these were pre-sumed to be other methylated derivatives although they were notfurther characterized. The ultraviolet quenching band at RF-0.25was eluted from the alumina with ethanol and taken to drynessin vacuo. In the run described here 38 mg of product were re-

covered, which on the basis of the expected compound MBA,constitutes a 27% yield.For further characterization and tests of biological activity,

100

0S

0

- 30

*> 20l010

(m*I2.5

10

25

200 300

276 FOX ET AL.

39

1 -1 II

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METABOLISM OF A CYTOKININ

Table I. Signal Assignments of the Paramagnetic ResonanceSpectrum of 6-Benzylamino-9-methyl Purinel

Shifts Splitting Pattern No. of Assignment

ppm

3.81 Singlet 3 IP-methyl4.94 Doublet 2 -CH2-phenyl

(J about 6 Hertz)6.65 Multiplet 1 N6-hydrogen7.38 Multiplet 5 phenyl-7.63 Singlet 1 purine C88.46 Singlet 1 purine C2

Refer to Figure 3 for spectrum.

the product was dissolved in ethanol and precipitated as thehydrochloride. The precipitate was dissolved in water and re-converted to the free base by titration with sodium bicarbonate.MBA was then extracted from the mixture with diethyl ether andrecovered in crystalline form as colorless needles by evaporatingthe solvent. Final purification was achieved by sublimation ofcrystalline material. The sublimed material melted at 138 to138.5 C. Elemental analysis yielded the following results: C:65.27; H: 5.85; N: 29.09; calculated for CuHuN5: C: 65.26; H:5.48; N: 29.27.A mass spectrum of the compound (Fig. 1) shows a molecular

ion at mle 239 which is consistent with a methyl-substitutedbenzyladenine. In addition, the fragmentation pattern of thecompound is not unlike that of BA (Fig. 2) with strong peaks inboth at mle 91 and mle 106 identifiable respectively as the tro-pylium ion (C7H7+) and a species (C6H5CH2N+H) formed by theloss of the purine ring system (2).

Paramagnetic resonance spectra (Table 1, Fig. 3) indicate thatthe purine 2 and 8 positions are unsubstituted and support a9-substituted structure assignment. That the substance is a 6-,9-disubstituted adenine is likewise indicated by the ultravioletabsorption spectrum (Fig. 4). Features of the spectrum which

permit this conclusion are the shoulder on the high wavelengthside of the maximum under alkaline conditions, and maxma inthe region of 270 with little shift of variation in extinction co-efficient with change in pH (15).

Further evidence that the compound is substituted at the 9position is provided by the infrared absorption spectrum (Fig.

.9

.8

.7

u

zc .4

0co<

5

5

4

3

2

.1

I

615

I110

3131

220 240 260 280WAVELENGTH (m1u)

300 320

FIG. 4. Ultraviolet absorption spectrum of 6-benzylamino-9-methlypurine at a concentration of 2.1 X 10-6 M in water determined on aBausch and Lomb 505 recording spectrophotometer. )max, pH 2.0(HCI) 271.5, ?max, pH 6.0 (H20) 270, Xmax, pH 11.0 (NH4OH)271.5.

FIG. 3. Proton magnetic resonance spectrum of 6-benzylamino-9-methyl purine in deuterated chloroform determined on a Varian A 60A PMRspectrophotometer.

Plant Physiol. Vol. 47, 1971 277

..I

.

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Plant Physiol. Vol. 47, 1971

I,. . I I I I II IIII

- .-r (.1

_;H

+ r-n

- -s pt+ + + .

,,,8 t

-; -- t-t-I. , ! i;, |+ _! 1 k-fi

.,' -& e - Lt

.

A

,. *. n is n .0

11-1111 1I@,

I~~~~I"X *4C 100 *00 10

FIG. 5. Infrared spectra taken in KBr pellets with a Beckman IR 10 Infrared spectrophotometer of A: 6-benzylaminopurine and, B: 6-benzyl-amino-9-methyl purine.

5). The infrared spectrum of BA exhibits typical weak absorptionbetween 2800 to 2400 cm-l due to the acidic hydrogen on the 9nitrogen (2) whereas the corresponding spectrum of the com-pound in question lacks this feature. Finally, the compound wasnot precipitated with AgNO3 under acidic conditions (0.05 Nacetic acid) although BA precipitated immediately with thistreatment.MBA was also synthesized by a second method which pro-

vided still more evidence for its structure. In a typical experiment81.5 mg of 6-chloro-9-methyl purine (Cyclo Chemical Co.)

Table II. Effect of BA and MBA on the Growth ofSoybean CallusTissue' 2

BA AIBA

Concn of CytokininFresh Dry Fresh Dry

wt/flask wt/flask wt/flask wt/flask

X g g

0 0.16 0.01 0.16 0.011 X 10'9 0.17 0.01 0.17 0.01X 10-8 0.66 0.04 0.17 0.01x 107 1.63 0.10 0.77 0.06

1 X 1-- 1.42 0.08 1.36 0.10x 10-' 1.19 0.07 1.38 0.09

1 X 104 0.20 0.01 0.24 0.02

lTissues were cultured for 22 days under standard conditions(see text).

2Each figure is an average of 4 flasks with 2 pieces of tissue ineach.

were refluxed in 20 ml of water with 0.1 ml of benzylamine for 6hr. The reaction mixture was taken to dryness in a rotary evapo-rator and washed with a small amount of water at 0 C to removeresidual benzylamine. The product was converted to the HCl saltand purified as described above. Crystalline material was re-covered in a 68% yield and proved identical with the materialsynthesized by method 1.

Synthesis of 6-Benzylamino-9-methyl Purine-methylJ4C. Thepreparation of the '4C-labeled compound was performed as inthe first method described above except that the synthesis wascarried out in a sealed reaction bomb. In this procedure 7.2 X10-' moles of the potassium salt of BA were reacted with 0.5mc of 14CH.I (Amersham Searle, batch 95) sp. act. 41.2 mc/mmole, i.e., 12.1 ,umoles. After purification, 0.62 mg (2.6 X 10'moles) of the desired product were recovered. The specific radio-activity of the product as estimated with the aid of a Packardmodel 3375 liquid scintillation spectrometer was 43.0 mc/mM.

Synthesis of 6-Benzylamino-9-methyl Purine-methylJ4C-methy-1ene-3H. The tritium, carbon 14 doubly labeled compound wassynthesized by reducing N'-benzoylaminopurine (18) with lithiumaluminum tritiide (New England Nuclear Corp., specific radio-activity 125 mc/mM). The reactants were refluxed in N-methylmorpholine overnight, taken to a small volume in vacuo, andsubjected to chromatography on thin layers of silica gel in chloro-form-methanol-i N NH40H (5:2:1), a procedure which con-veniently separates benzyladenine from unreacted benzoyladenine.The tritiated BA then became the starting compound for methyl-ation at the 9 position with 14CH3I as described above.From this procedure 0.5 mg of doubly labeled MBA was

recovered with specific radioactivities of ('4C) 10 mc/mM and(3H) 37 mc/mm.

113NT -FceN

sl *s z Z5 loll .'

278 FOX ET AL.

It

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METABOLISM OF A CYTOKININ

RESULTS AND DISCUSSION

Biological Activity. The ability of MBA to support growth incytokinin-requiring tissues approached but never equaled thatexhibited by BA itself. The latter exhibited an optimum at 1 x107 M for both soybean and tobacco cultures, while the 9-substituted derivative was most active at 1 X 10' M in tobaccotissue and perhaps at slightly higher concentration in soybean(Table II, Fig. 6). This relative difference has been a consistentfeature in repeated tests.

In addition, the activity ofMBA has been contrasted with thatof BA in the assay for cytokinins depending upon the initiationof deoxyisoflavone synthesis in soybean cultures described byMiller (19). This test, which was performed by Carlos Miller inBloomington, Indiana, indicated that the presence of a methylgroup at the 9 position greatly interfered with the ability of BAto promote synthesis of deoxyisoflavones in soybean tissuesincubated 45 hr in liquid medium (Fig. 7). Some activity wasnoticed, however, at concentrations of MBA 10 to 100 timeshigher than the unsubstituted compound.

Metabolic Stability of the 9-Substituent. In order to test formetabolic cleavage of the 9-methyl moiety, soybean and tobaccotissues were incubated for varying periods on liquid or solidmedia containing MBA labeled with '4C in the 9-methyl carbon.The tissues employed in these studies were in the log phase ofgrowth but starved for 72 hr prior to use on a basal medium,complete except for the cytokinin. At the end of the incubationperiod, the gases present in the flask were passed through a

BA

- LOG CONC.

4 5 6

(M)

7 8 Y

MBA

r FIG. 6. Effect of BA and MBA on the growth of soybean callus tis-sue. The tissues were grown for 3 weeks on a basal medium (see text)to which was added the appropriate concentration of the cytokininsterilized at room temperature by filtration.

E

(CC'4

(5

CC

1.8

1.7

1.6

1.5

1.4: 1.3

) 1.2

; 1.1

) 1.0

0.90.8

0.7

0.6C 9 8 7 6

- LOG CONC. (M)

FIG. 7. Influence of BA and MBA on the synthesis of deoxyiso-flavones in soybean tissue; 0.6 g of finely chopped tissue was incubatedfor 45 hr at 26 C in 20 ml of medium and then extracted by the addi-tion of 80 ml of 95% ethanol. The increase in absorbance at 260 nmof the extract is due largely to the synthesis of deoxyisoflavones stimu-lated by active cytokinins (19). Each point is the average of threecultures. See Miller (19) for details of procedure.

Table III. 14C02 Evolution from Tobacco and Soybean TissuesIncubated on a Culture Medium Containing MBA-methyl-'4C

Tissue', 2,I Incubation Period CPM-background

Liquid mediumTobacco 10 min 283Control 10 min 19Tobacco 60 min 1680Control 60 min 2Tobacco 360 min 3550Control 360 min 8Tobacco 48 hr 4469Soybean 48 hr 352Control 48 hr 6

Solid mediumTobacco 48 hr 4633Soybean 48 hr 792Control 48 hr 7

1 See text for details of trapping and radioactivity estimationof released C02.

2 Experimental manipulations of the control flask were identicalwith the test flask except that the tissue was omitted.

3 Tissue (approximately 3 gm fresh weight/flask) was incubatedwith 10 ml of a standard culture medium containing 1 mg/I, i.e.,4.15 X l0-8 moles/10 ml, 6-benzylamino-9-methylpurine-methyl14C sp. ac. 43 mc/mmole, i.e., 3.5 X 106 cpm/10 ml. Tissues werestarved on a medium complete except for a cytokinin for 72 hrprior to use.

column containing finely divided filter paper soaked in a con-centrated solution of barium hydroxide in order to trap CO2.The filter paper was air-dried and suspended in a standard fluidfor liquid scintillation spectrometry. In order to rule out the pos-

BA

/ ~~~MB

v~~~~~~~~~~~~~0,0..~ *

Plant Physiol. Vol. 47, 1971 279

)v

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Plant Physiol. Vol. 47, 1971

C' 2

5- _ A

4- 6 hr n96 hrl(.)5BA ~ ~ MB

0 Q5 1.0 0.5 1.0

Rf

FIG. 8. Tobacco tissue metabolites of MBA. Tissues which had been starved on a basal medium without cytokinin for 3 days were incubatedin 5 ml of a standard liquid culture medium containing 1 mg/l of MBA-methyl-14C, methylene-3H. Extracts (see text for preparation) were chro-matographed on alumina thin layers (see text) in chloroform-ethanol (97:3, v/v). Radioactivity of the fractions estimated as described in thetext.

sibility of microbial contamination during the manipulationsrequired by the experiment, a control flask complete except forthe tissue was subjected to the same operations for each experi-ment.The results (Table III) indicate that both soybean and tobacco

tissue on either liquid or solid media metabolize MBA in such away that at least some portion of the methyl moiety is releasedas CO2. Furthermore, the amount of 14CO2 released is a functionof time and the process begins to occur within at least 10 min.Only a tiny fraction of the counts supplied to the tissue as

MBA-methyl-'4C are represented by '4CO2. Extracts of the tissuechromatographed in a variety of systems have made it clear,however, that MBA persists for only a very short time and isquickly metabolized by the tissue. The most instructive experi-ments were done with tobacco tissue incubated with MBAdoubly labeled with '4C and 'H. The tissue was extracted withsufficient 95% ethanol at room temperature to achieve a finalconcentration of 70% by grinding with a mortar and pestle.Insoluble material was separated by centrifugation and re-extracted twice more. The combined supematants were reducedin vacuo to a small volume and spotted on precoated sheets ofalumina for thin layer chromatography (Eastman-Chromato-gram sheet 6062). The chromatogram was developed in chloro-form-ethanol (97:3, v/v), a solvent system chosen for its abilityto separate widely BA (RF 0.15) and MBA (RF 0.75). The de-veloped chromatogram was then air dried, cut into small strips,and eluted at 50 C with 50% ethanol for 1 hr. The eluate wasdried at 30 C in counting vials to which was then added 15 mlof a standard solution for liquid scintillation spectrometry. Thevials were assayed for both 14C and 'H.The results (Fig. 8) indicate that even within 1 hour, less than

10% of the 14C is associated with MBA; the bulk of the radio-activity is distributed across several fractions. By 4 days there islittle, if any, intact MBA in tobacco tissue. The compound isapparently much more stable in soybean tissue however, so that,although other metabolites appear early, as much as 40% of theMBA taken up by the tissue on a liquid medium may persist upto 7 days. In a control run in which manipulations were identicalwith the experiment except that no tissue was placed in the flask,over 990% of the radioactivity recovered from the medium after 4days was coincident with MBA in three chromatographic systems.

A question crucial to this study is whether or not free BAappears during the metabolism of MBA. One ought to be able todetect this compound easily since it would be tritium labeledbut devoid of "IC. However, Figure 8 shows that "4C is presentin substantial amounts even at the RF of BA. Presumably, though,the 14C from the methyl group of MBA enters into the methylpool and subsequently into a number of metabolites, some ofwhich migrate into the same general area of the chromatogramas BA. In order to test for free BA, portions of the chromatogramhaving the proper RF were eluted in ethanol and the eluate co-crystallized with authentic, unlabeled BA from mixtures ofethanol and water. The product was sublimed and the resultingcrystals were scraped from the walls of the sublimation tube di-rectly into counting vials. Similar studies were performed on theincubation medium after tissue had been growing for either 6hr or 4 days. In all cases the product was radioactive and con-tained largely tritium although a small amount of "4C was ob-served as well, probably due to a tenaciously bound contaminant.Thus, in one instance a sample which contained 3600 dpm tritiumalso registered 178 dpm of 14C. As much as 10% of the tritiumcounts in the liquid medium after 6 hours incubation with tobaccotissue was identified with BA.The biological activity of MBA, which in our hands is at least

10 times less active than BA, is therefore probably best explainedby its conversion in the tissue to the free base. The methyl groupat the 9 position does not constitute an effective block as has beenassumed by previous workers but is in fact rather readily metabol-ized by plant tissues. This finding makes suspect the presumedstability of other 9-substituted cytokinins. In the absence ofinformation regarding their metabolic stability, one cannot,therefore, use the biological activity of 9-substituted cytokininsas evidence against the possibility that cytokinin activity dependsupon nucleotide formation.

LITERATURE CMD

1. BIEMANN, R., S. TSUNAKAWA, J. FELDMAN, D. DUTTING, AND H. ZAcHAu. 1966.Struktur eines ungewohnlichen Nucleosides aus Serine-spezifischer transferRibonucleinsaure. Angew. Chem. 78: 600.

2. BUDZIKIEWICZ, H., C. DiERASSI, AND D. WILLIAMS. 1967. Mass Spectrometryof Organic Compounds. Holden-Day Inc., San Francisco. pp. 593-594.

3. CARRAWAY, K., P. C. HUANG, AND T. Scorr. 1968. 9-Benzyladenine, 9-(w-Chloro-alkyl) adenines, and 3,9-(Oligomethylene) adeninium chloride. In: W. Zorbach

280 FOX ET AL.

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METABOLISM OF

and R. Tipton, eds., Synthetic Procedures in Nucleic Acid Chemistry, Vol. 1,John Wiley-Interscience Publishers, Inc. pp. 3-5.

4. CHEN, C., AND R. HALL 1969. Biosynthesis of N6 (A2-isopentenyl) adenosinein the transfer ribonucleic acid of cultured tobacco pith tissue. Phytochemistry.8: 1687-1695.

5. DOCTOR, B., J. LoEBEL, M. SODD, AND D. WINTER. 1969. Nucleotide sequenceof Escherichia coli tyrosine transfer RNA. Science 163: 693-695.

6. Fox, J. E. 1963. Growth factor requirements and chromosome number in tobaccotissue cultures. Physiol. Plant. 16: 793-803.

7. Fox, J. E. 1964. Incorporation of Idnins into the RNA of plant tissue cultures.Plant Physiol. 39: xxxi.

8. Fox, J. E. 1964. Indoleacetic acid-kinetin antagonism in certain tissue culturesystems. Plant Cell Physiol. 5: 251-254.

9. Fox, J. E. 1966. Incorporation of a kinin, N, 6-benzyladenine, into soluble RNA.Plant Physiol. 41: 75-82.

10. Fox, J. E. AND C. CHEN. 1967. Characterization of labeled ribonucleic acid fromtissue grown on 14C-containing cytokinins. J. Biol. Chem. 242: 4490-4494.

11. Fox, J. E. AND C. CHEN. 1968. Cytokinin incorporation into RNA and its pos-sible role in plant growth. In: F. Wightman and G. Setterfield, eds., Biochem-istry and Physiology of Plant Growth Substances. Runge Press, Ottawa. pp.777-789.

12. GuERN, J., M. DoREE, AND P. SORDAGE. 1968. Transport, metabolism and bio-logical activity of some cytokinins. In: F. Wightman and G. Setterfield, eds.,Biochemistry and Physiology of Plant Growth Substances. Runge Press,Ottawa. pp. 1155-1167.

A CYTOKNIN 281

13. KENDE, H. AND J. E. TAVARES. 1968. On the significance of cytokinin incorpora-tion into RNA. Plant Physiol. 43: 1244-1248.

14. KEY, J. 1969. Hormones and nucleic acid metabolism. Annu. Rev. Plant Physiol.20: 449-474.

15. LEONARD, N. J., K. CARRAWAY, AND J. HELGESON. 1965. Characterization ofNx, Ny-disubstituted adenines by ultraviolet absorption spectra. J. HeterocyclicChem. 2: 291-297.

16. LErTEw, D. S. 1969. Cytokinins and their relation to other phytohormones.Bioscience 19: 309.316.

17. MADISON, J., G. EvERE-r, AND H. HUNG. 1966. Nucleotide structure of a yeasttransfer RNA. Science 153: 531-534.

18. MARTIN, J. 1968. A study of cytokinin metabolism. Ph.D. thesis. University ofKansas, Lawrence.

19. MILIER, C. 0. 1969. Control of deoxyisoflavone synthesis in soybean tissue.Planta 87: 26-35.

20. MONTGOMERY, J., AND L. HOLUM. 1957. Synthesis of potential anticancer agentsXl, N2. 6-alkyl derivatives of 2,6-diaminopurine. J. Amer. Chem. Soc. 80:404-408.

21. SHAw, G., B. M. SMALLwooD, AND F. C. STEwARD. 1968. Synthesis and cyto-kinin activity of the 3-, 7- and 9-methyl derivatives of zeatin. Experientia 24:1089-1090.

22. WEAVER, R. J., J. VAN OvERsEr, AND R. M. PooL- 1965. Induction of fruit setin Vitis vinifera L. by a kinin. Nature 206: 952-953.

23. YOUNG, H. ANm D. LEmAm. 1969. 6-[Substituted amino] purines: synthesis bya new method and cytokinin activity. Phytochemistry 8: 1199-1203.

PlantfPhysiol. Vol. 47, 1971

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