Metabolism of cytokinin: deribosylation of cytokinin ribonucleoside by adenosine nucleosidase from wheat germ cells

Adenosine nucleosidase (adenosine ribohydrolase, EC 3.2.2.7) which catalyzes the deribosylation of N⁶-(Δ²-isopentenyl)adenosine and adenosine to form the corresponding bases was partially purified from wheat germ. This enzyme (molecular weight 59,000 ± 3,000) deribosylates the ribonucleosides at an optimum pH of 4.7 K_m values for the cytokinin nucleoside and adenosine are 2.38 and 1.43 micromolar, respectively, in 50 millimolar Tris-citrate buffer (pH 4.7) at 30 C. The presence of adenosine and other cytokinin nucleosides inhibited the hydrolysis of N⁶-(Δ²-isopentenyl)adenosine but this reaction was insensitive to guanosine, uridine, or 3′-deoxyadenosine. It is hypothesized that an adequate level of “active cytokinin” in plant cells may be provided through the deribosylation of cytokinin riboside in concert with other cytokinin metabolic enzymes.     

Cytokinin oxidase from wheat: partial purification and general properties

As part of the study of the possible role(s) of CBF-1, a cytokinin-binding protein abundant in wheat embryo, a cytokinin oxidase was found in wheat (Triticum aestivum L.) germ and partially purified by conventional purification techniques and high performance chromatofocusing. This preparation catalyzes conversion of N⁶-(Δ²-isopentenyl)adenosine to adenosine at a V_max of 0.4 nanomol per milligram protein per minute at 30°C and pH 7.5, the K_m being 0.3 micromolar. This high affinity and the apparent molecular weight of 40,000 estimated by high performance gel permeation on a Spherogel TSK-3000 SW column indicate that this enzyme is different from other cytokinin oxidases previously reported. Oxygen is required for the reaction, as for other cytokinin oxidases already described. N⁶-(Δ²-isopentenyl)adenine and zeatin riboside are also degraded, but N⁶-(Δ²-isopentenyl)adenosine-5′-monophosphate is apparently not a substrate. Benzyladenine is degraded, but to a small extent, and it inhibits slightly the degradation of N⁶-(Δ²-isopentenyl)adenosine. The degradation of N⁶-(Δ²-isopentenyl)adenosine is strongly inhibited by diphenylurea and its highly active derivative N-(2-chloro-4-pyridyl)-N′-phenylurea.     

Kinetics of N-(Delta-Isopentenyl)Adenosine Degradation in Tobacco Cells: EVIDENCE OF A REGULATORY MECHANISM UNDER THE CONTROL OF CYTOKININS

Uptake and degradation of the cytokinin, N⁶-(Δ²-isopentenyl) adenosine, were studied in tobacco cells grown as cell suspensions. Degradation occurs by cleavage of the isopentenyl chain which gives adenylic products. Rate of N⁶(Δ²-isopentenyl)[8-¹⁴C]adenosine degradation increases several-fold after a 3- to 4-hour delay when cells have been exposed to a cytokinin. Consequently, only rates of N⁶-(Δ²-isopentenyl)adenosine degradation measured during the first 3 hours of incubation with [8-¹⁴C]-N-⁶(Δ²-isopentenyl)adenosine are representative of the intrinsic in vivo cytokinin degradative activity of tobacco cells. Within these limits, it appears that cytokinin degradative activity is high in cytokinin-autonomous tobacco cells, as indicated by the half life of the supplied N⁶(Δ² isopentenyl adenosine (about 3 hours) when it is supplied at the physiological concentration of 0.2 micromolar. This cytokinin degradative activity appears to be under the control of cytokinins themselves because N⁶-(Δ²-isopentenyl)adenosine degradative activity is increased several-fold following a 3- to 4-hour delay after these cells have been exposed to a cytokinin.     

Metabolism of cytokinin: ribosylation of cytokinin bases by adenosine phosphorylase from wheat germ

As part of the study of cytokinin metabolic pathways, an enzyme, adenosine phosphorylase (EC 2.4.2.-), which catalyzed the ribosylation of N⁶-(Δ²-isopentenyl)adenine, N⁶-furfuryladenine, and adenine to form the corresponding nucleosides, was partially purified from wheat (Triticum aestivum) germ. The pH optimum for the ribosylation of the cytokinins and adenine was from 6.5 to 7.8; for guanine and hypoxanthine it was from 7.0 to 8.5 At pH 7.2 (63 millimolar N-2-hydroxyethyl piperazine-N′-ethanesulfonic acid) and 37 C the K_m for N⁶-(Δ²-isopentenyl)adenine was 57.1 micromolar; N⁶-furfuryladenine, 46.5 micromolar; adenine, 32.2 micromolar; and the V_max for N⁶-(Δ²-isopentenyl)adenine, N⁶-furfuryladenine, and adenine were 134.7, 137.1, and 193.1 nanomoles per milligram protein per minute, respectively. The equilibrium constants of the phosphorolysis of N⁶-(Δ²-isopentenyl)adenosine and adenosine by this enzyme indicated that the reaction strongly favored nucleoside formation. This enzyme was shown to be distinct from inosine-guanosine phosphorylase based on the differences in the Sephadex G-100 gel filtration behaviors, pH optima, and the product and p-hydroxymercuribenzoate inhibitor studies. These results suggest that adenosine phosphorylase may play a significant role in the regulation of cytokinin metabolism.     

Changes in Cytokinins before and during Early Flower Bud Differentiation in Lychee (Litchi chinensis Sonn.)

Lychee (Litchi chinensis) has been analyzed for cytokinins in buds before and after flower bud differentiation, using reversephase high performance liquid chromatography in combination with Amaranthus bioassay and gas chromatography-mass spectrometry-selected ion monitoring. Four cytokinins, zeatin, zeatin riboside, N⁶-(δ²-isopentenyl)adenine, and N⁶-(δ⁶-isopentenyl) adenine riboside, were detected in buds. There was an increase of cytokinin activity in the buds during flower bud differentiation. In dormant buds, the endogenous cytokinin content was low, and the buds did not respond to exogenous cytokinin application. Application of kinetin promotes flower bud differentiation significantly after bud dormancy. These results are interpreted as an indication that the increase in endogenous cytokinin levels during flower bud differentiation may be correlative rather than the cause of flower bud initiation.     

Cytokinins: Metabolism and Biological Activity of N-(Delta-Isopentenyl)adenosine and N-(Delta-Isopentenyl)adenine in Tobacco Cells and Callus

The cytokinin, N⁶-(Δ²-isopentenyl)adenine, is found to be at least 3.3 times as active as N⁶-(Δ²-isopentenyl)adenosine in promoting the growth of cytokinin-requiring tobacco (Nicotiana tabacum) callus. Absorption rates of N⁶-(Δ²-isopentenyl)adenine and N⁶-(Δ²-isopentenyl)adenosine by tobacco cells in liquid suspension do not differ significantly. In these cells, N⁶-(Δ²-isopentenyl)adenosine-5′-monophosphate, di-, and triphosphate are synthesized in both cases, but 7-glucosylation occurs significantly only with N⁶-(Δ²-isopentenyl)adenine, protecting thereby its N⁶-isopentenyl side chain from cleavage. Degradation by N⁶-side chain removal appears to be intense, leading to the formation of adenine, adenosine, and adenylic nucleotides. Thus, it is suggested that N⁶-(Δ²-isopentenyl)adenine-7-glucoside is a protected or storage form of the cytokinin which could account for the higher biological activity of N⁶-(Δ²-isopentenyl)adenine than of N⁶-(Δ²-isopentenyl)adenosine.     

Localization of cytokinin biosynthetic sites in pea plants and carrot roots

The biosynthesis of cytokinins was examined in pea (Pisum sativum L.) plant organs and carrot (Daucus carota L.) root tissues. When pea roots, stems, and leaves were grown separately for three weeks on a culture medium containing [8-¹⁴C]adenine without an exogenous supply of cytokinin and auxin, radioactive cytokinins were synthesized by each of these organs. Incubation of carrot root cambium and noncambium tissues for three days in a liquid culture medium containing [8-¹⁴C]adenine without cytokinin demonstrates that radioactive cytokinins were synthesized in the cambium but not in the noncambium tissue preparation. The radioactive cytokinins extracted from each of these tissues were analyzed by Sephadex LH-20 columns, reverse phase high pressure liquid chromatography, paper chromatography in various solvent systems, and paper electrophoresis. The main species of cytokinins detectable by these methods are N⁶-(Δ²-isopentyl_adenine-5′-monophosphate, 6-(4-hydroxy-3-methyl-2-butenyl-amino)-9-β-ribofuranosylpurine-5′- monophosphate, N⁶-(Δ²-isopentenyl)adenosine, 6-(4-hydroxy-3-methyl-2-butenylamino)-9-β-ribofuranosylpurine, N⁶-(Δ²-isopentenyl)adenine, and 6-(4-hydroxy-3-methyl-2-butenylamino)purine. On the basis of the amounts of cytokinin synthesized per gram fresh tissues, these results indicate that the root is the major site, but not the only site, of cytokinin biosynthesis. Furthermore, cambium and possibly all actively dividing tissues are responsible for the synthesis of this group of plant hormones.     

Cytokinin binding proteins from mammalian sera

A cytokinin-binding protein fraction was isolated from normal rabbit sera by affinity chromatography. The protein fraction bound tritium labelled N⁶- (δ2-risopentenyl) adenosine and the order of inhibition of this binding by competing non-radioactive compounds was, N⁶-(δ²-isopentenyl) adenosine < N⁶-benzyIadenosine < zeatin-riboside > N⁶-(δ²-isopentenyl) adenine < kinetin riboside > adenosine. The protein fraction showed broad specificity, the prefered cytokinin being N⁶-(δ²-isopentenyl) adenosine. This is the first report of the isolation of cytokinin binding proteins from mammalian sources.     

Cytokinin structure-activity relationships and the metabolism of N-(delta-isopentenyl)adenosine-8-C in phaseolus callus tissues

The activities of the free base and ribonucleoside forms of cytokinins bearing saturated and unsaturated N⁶-isoprenoid side chains have been examined in callus cultures derived from Phaseolus vulgaris cv. Great Northern, P. lunatus cv. Kingston, and the interspecific hybrid Great Northern × Kingston. In callus of cv. Great Northern, cytokinins bearing saturated side chains (N⁶-isopentyladenine, N⁶-isopentyladenosine, dihydrozeatin, and ribosyldihydrozeatin) were always more active than the corresponding unsaturated analogs (N⁶-[Δ²-isopentenyl]adenine, N⁶-[Δ²-isopentenyl]adenosine, zeatin, and ribosylzeatin). In callus of cv. Kinston, the cytokinins bearing unsaturated side chains were either more active or equally as active as the saturated compounds. These differences in cytokinin structure-activity relationships were correlated with differences in the metabolism of ¹⁴C-N⁶-(Δ²-isopentenyl)adenosine. In Great Northern tissues, this cytokinin was rapidly degraded to adenosine; in Kingston tissues, the major metabolite was the corresponding nucleotide. The growth responses of callus of the interspecific hybrid were intermediate between the parental tissues, and the metabolism of ¹⁴C-N⁶-(Δ²-isopentenyl)adenosine by the hybrid callus exhibited characteristics of both parental tissues. The results are consistent with the hypothesis that the weak activity of cytokinins with unsaturated side chains in promoting the growth of Great Northern callus is due to the rapid conversion of these cytokinins to inactive metabolites.     

Cytokinin Biosynthesis in Mutants of the Moss Physcomitrella patens

Three cytokinin-over-producing mutants of the moss, Physcomitrella patens, have been shown to convert [8-¹⁴C]adenine to N⁶-[¹⁴C](Δ²-isopentenyl)adenine, the presence of which was confirmed by thin layer chromatography, high performance liquid chromatography, and recrystallization to constant specific radioactivity. The labeled cytokinin was detected in the culture medium within 6 hours and the tissue itself appears to contain both labeled N⁶-(Δ²-isopentenyl)adenine and N⁶-(Δ²-isopentenyl)adenosine monophosphate.     

Metabolism of Cytokinin : DEPHOSPHORYLATION OF CYTOKININ RIBONUCLEOTIDE BY 5'-NUCLEOTIDASES FROM WHEAT GERM CYTOSOL

Two forms (F-I and F-II) of 5′-nucleotidases (5′-ribonucleotide phosphohydrolase, EC 3.1.3.5) which catalyze the dephosphorylation of N⁶-(Δ²-isopentenyl)adenosine 5′-monophosphate and AMP to form the corresponding nucleosides were partially purified from the cytosol of wheat (Triticum aestivum) germ. Both the F-I (molecular weight, 57,000) and F-II (molecular weight, 110,000) 5′-nucleotidases dephosphorylate the ribonucleotides at an optimum pH of 7. The K_m values for the cytokinin nucleotide are 3.5 micromolar (F-I enzyme) and 12.8 micromolar (F-II enzyme) in 100 millimolar Tris-maleate buffer (pH 7) at 37 C. The F-I enzyme is less rapidly inactivated by heating than is the F-II enzyme. Both nucleotidases hydrolyze purine ribonucleoside 5′-phosphates, AMP being the preferred substrate. N⁶-(Δ²-isopentenyl)Adenosine 5′-monophosphate is hydrolyzed at a rate 72 and 86% that of AMP by the F-I and F-II nucleotides, respectively. Phenylphosphate and 3′-AMP are not substrates for the enzymes. It is proposed that dephosphorylation of cytokinin nucleotide by cytosol 5′-nucleotidases may play an important role in regulating levels of “active cytokinin” in plant cells.     

Effects of Thidiazuron on Cytokinin Autonomy and the Metabolism of N-(Delta-Isopentenyl)[8-C]Adenosine in Callus Tissues of Phaseolus lunatus L

The effects of a highly cytokinin-active urea derivative, N-phenyl-N′-1,2,3-thiadiazol-5-ylurea (Thidiazuron), and zeatin on cytokinin-autonomous growth and the metabolism of N⁶-(Δ²-isopentenyl)[8-¹⁴C]adenosine ([¹⁴C]i⁶ Ado) were examined in callus tissues of two Phaseolus lunatus genotypes, cv Jackson Wonder and P.I. 260415. Tissues of cv Jackson Wonder maintained on any concentration of Thidiazuron became cytokinin autonomous, whereas only tissues exposed to suboptimal concentrations of zeatin displayed cytokinin-autonomous growth. Tissues of P.I. 260415 remained cytokinin dependent under all these conditions. The metabolism of [¹⁴C]i⁶ Ado was similar for the two genotypes, but differed with the medium used. [¹⁴C]i⁶ Ado was rapidly converted to N⁶-(Δ²-isopentenyl)[8-¹⁴C]adenosine 5′-P ([¹⁴C]i⁶ AMP) by tissues grown on zeatin-containing medium, whereas only traces of the nucleotide were formed in tissues grown on medium with Thidiazuron. Incubation with [¹⁴C] i⁶ AMP of tissues grown in the presence of Thidiazuron resulted in rapid conversion to [¹⁴C]i⁶ Ado, while [¹⁴C]i⁶ AMP persisted in tissues maintained on zeatin. Thus, Thidiazuron appears to stimulate enzyme activity converting the ribonucleotide to ribonucleoside. Although the cytokininactive phenylureas and adenine derivatives differ in their effects on cytokinin autonomy as well as nucleotide formation, the two types of effects do not seem to be related.     

Isolation and Estimation of Cytokinins and Cytokinin-Containing Transfer RNAs from Cucumis sativus L. var. Guntur Seedlings

N⁶-(Δ²-Isopentenyl) adenosine antibodies were used for the isolation of free cytokinins and cytokinin-containing tRNAs from parts of Cucumis sativus L. var. Guntur seedlings and for the estimation of cytokinins in them. Immobilized N⁶-(Δ²-isopentenyl) adenosine antibodies retained tRNAs containing N⁶-(Δ²-isopentenyl) adenosine and N⁶-(4-hydroxy-3-methylbut-2-enyl) adenosine with equal efficiencies. There were at least five cytokinins in the free form in cucumber seedlings. N⁶-(4-Hydroxy-3-methylbut-2-enyl) adenosine, N⁶-(Δ²-isopentenyl) adenosine, and N⁶-(Δ²-isopentenyl) adenine were present at least to the extent of 80, 23, and 9 nanograms, respectively, in the cotyledons and 40, 6, and 3 nanograms, respectively, in the decotyledonated seedlings per gram of tissue. Only two cytokinins were found in the tRNAs of cucumber cotyledons, namely N⁶-(Δ²-isopentenyl) adenosine and N⁶-(4-hydroxy-3-methylbut-2-enyl) adenosine in amounts of 12 and 318 nanograms, respectively, per gram of tissue. Immunoaffinity chromatographic analysis of radiolabeled aminoacyl tRNAs from cucumber cotyledons showed that tRNA^Phe and tRNA^Tyr contained cytokinins whereas tRNA^Ala did not.     

Gamma-irradiated plant callus tissue: Cytokinins from transfer ribonucleic acid

Summary Callus tissue ofHaworthia mirabilis Haw. was irradiated with⁶⁰Co gamma rays. tRNA was isolated, hydrolyzed enzymatically, and cytokinin-active ribonucleosides were separated by Sephadex LH-20 column chromatography and assayed with the tobaccocallus cytokinin bioassay. Three cytokinins were detected in tRNA from irradiated tissue, two of which chromatographed with zeatin riboside and N⁶-(Δ²-isopentenyl)adenosine. The third cytokinin-active ribonucleoside was retained longer than the above compounds on the Sephadex column and may be 2-methylthio-N⁶-(Δ²-isopentenyl)adenosine. Two cytokinins were detected in tRNA from nonirradiated tissue—those chromatographed with zeatin riboside and N⁶-(Δ²-isopentenyl)adenosine. Relationships between cytokinins from tRNA and free cytokinins found in tissue earlier are discussed. This is paper 78-10-124 of the Kentucky Agricultural Experiment Staton and is published with approval of the Director.     

Cytokinin Secretion by Frankia sp. HFP ArI3 in Defined Medium

Frankia sp. HFP ArI3 (host plant Alnus rubra Bong.) was grown in defined medium and the culture solution was analyzed for the presence of various cytokinins and related compounds. N⁶- (Δ²-isopentenyl) adenosine was the only cytokinin detected by both high performance liquid chromatography and gas chromatography-mass spectrometry, at levels of approximately 1 ng/ml culture medium.     

Cytokinins: A Rapid Extraction and Purification Method

A method is described for the isolation, purification and quantitation of free cytokinin bases and ribosides using ethyl acetate at pH 7.7 for the extraction. The extraction is almost complete (97.7%) as determined by using N⁶-(Δ²-isopentenyl)adenine-8-¹⁴C. The subsequent fast purification by chromatography on a standardized silica gel column in chloroform-methanol (7:3 v/v) is followed by thin layer chromatography (silica gel 60 F²⁵⁴) in chloroform-acetic acid (8:2 v/v). The recovery of N⁶-(Δ²-isopentenyl)adenine-8-¹⁴C after this two step purification was 78–82%. The efficiency of the method was determined by applying this procedure to N⁶-(Δ²-isopentenyl)adenine and N⁶(Δ²-isopentenyl)adenosine. Using gas liquid chromatography the recovery for N⁶-(Δ²-isopentenyl)adenosine was determined to be 61% and compared to 43% for N⁶-(Δ²-isopentenyl)adenosine, showing the suitability of the described method for gas liquid chromatography.     

Cytokinin oxidase from auxin- and cytokinin-dependent callus cultures of tobacco (Nicotiana tabacum L.)

Cytokinin oxidase was extracted and partially purified from auxin- and cytokinin-dependent callus tissue of tobacco (Nicotiana tabacum L. cv. Wisconsin 38). The activity of the enzyme preparation was examined using an assay based on the conversion of tritiated N⁶-(²-isopentenyl)adenine ([2,8-³H]iP) to adenine. Cytokinin oxidase exhibited a temperature optimum at 45–50°C and a relatively high pH optimum (8.5–9.0). The apparent K_m value of the enzyme was 4.3 M for iP. On the basis of the substrate competition assays, iP was determined to be the preferred substrate of the enzyme. Substrate competition was also observed with zeatin and the cytokinin-active urea derivative Thidiazuron. Cytokinins bearing saturated isoprenoid side chains or cyclic side chain structures, as well as auxins and abscisic acid, had no effect on the conversion of [2,8-³H]iP. The cytokinin oxidase exhibited increased activity in the presence of copper-imidazole complex in the reaction mixture. Under optimal concentrations of copper (15 mM CuCl₂) and imidazole (100 mM), the enzyme activity was enhanced ca. 40-fold. Under these conditions the pH optimum was lowered to pH 6.0, whereas the temperature optimum, the apparent K_m value, and the substrate specificity were not altered. Most of the enzyme moiety did not bind to the lectin concanavalin A. The characteristics of cytokinin oxidase presented here suggest that a novel molecular form of the enzyme, previously identified and characterized in Phaseolus lunatus callus cultures (Kamínek and Armstrong (1990) Plant Physiol 93:1530), also occurs in cultured tobacco tissue.Abbreviations Ade adenine - iP N⁶-(²-isopentenyl)adenine - [2,8-³H]iP [2,8-³H]-N⁶-(²-isopentenyl)adenine - [9R]iP N⁶-(²-isopentenyl)adenosine - (diH)iP N⁶-isopentyladenine - (diH)Z dihydrozeatin - BAP N⁶-benzyladenine - ( _o OH)[9R]BAP N⁶-(o-hydroxybenzyl)adenosine - (mOH)[9R]BAP N⁶-(m-hydroxybenzyl)adenosine - IAA indole-3-acetic acid - IBA indole-3-butyric acid - NAA naphthalene-1-acetic acid - ABA abscisic acid - Con A concanavalin A     

An enzyme mediating the conversion of zeatin to dihydrozeatin in phaseolus embryos

A reductase catalyzing the conversion of zeatin to dihydrozeatin was detected in soluble fractions of immature Phaseolus vulgaris embryos. The enzyme was partially purified by ammonium sulfate fractionation and affinity, gel filtration, and anion exchange chromatography. NADPH was the only cofactor required for enzyme activity, and the pH optimum was 7.5 to 8.0. The enzyme did not recognize compounds closely related to zeatin, such as ribosylzeatin, cls-zeatin, O-xylosylzeatin, N⁶-(Δ²-isopentenyl)adenine, or N⁶-(Δ²-isopentenyl)adenosine. No conversion of dihydrozeatin to zeatin by the enzyme was observed. Two forms of the reductase could be separated by either gel filtration or anion exchange high performance liquid chromatography. The high molecular weight isozyme (M_r 55,000 ± 5,000) eluted as the second peak from the anion exchange column, while the low molecular weight isozyme (M_r 25,000± 5000) was less negatively charged. The results suggest that side chain reduction occurs at the free base level. In addition, Phaseolus embryos are useful for the detection of zeatin-specific metabolic enzymes.     

Phosphorylated α(1→4)Glucans as Substrate for Potato Starch-Branching Enzyme I

The possible involvement of potato (Solanum tuberosum L.) starch-branching enzyme I (PSBE-I) in the in vivo synthesis of phosphorylated amylopectin was investigated in in vitro experiments with isolated PSBE-I using ³³P-labeled phosphorylated and ³H end-labeled nonphosphorylated α(1→4)glucans as the substrates. From these radiolabeled substrates PSBE-I was shown to catalyze the formation of dual-labeled (³H/³³P) phosphorylated branched polysaccharides with an average degree of polymerization of 80 to 85. The relatively high molecular mass indicated that the product was the result of multiple chain-transfer reactions. The presence of α(1→6) branch points was documented by isoamylase treatment and anion-exchange chromatography. Although the initial steps of the in vivo mechanism responsible for phosphorylation of potato starch remains elusive, the present study demonstrates that the enzyme machinery available in potato has the ability to incorporate phosphorylated α(1→4)glucans into neutral polysaccharides in an interchain catalytic reaction. Potato mini tubers synthesized phosphorylated starch from exogenously supplied ³³PO₄³⁻ and [U-¹⁴C]Glc at rates 4 times higher than those previously obtained using tubers from fully grown potato plants. This system was more reproducible compared with soil-grown tubers and was therefore used for preparation of ³³P-labeled phosphorylated α(1→4)glucan chains.     

Differential cytokinin structure-activity relationships in phaseolus

The activities of eight cytokinins in promoting callus growth were tested in two Phaseolus genotypes, P. vulgaris L. var. Great Northern, and P. lunatus L. var. Kingston. The structural feature which contributes to the major genotypic difference in cytokinin structure-activity relationships is the presence or absence of a double bond at the 2,3-position of the isoprenoid N⁶ side chain. In Kingston, trans-zeatin was 3-fold more active than dihydrozeatin and 30-fold more active than cis-zeatin. The activities of N⁶-(Δ²-isopentenyl)adenine and N⁶-isopentyladenine were nearly the same. In Great Northern, however, dihydrozeatin was at least 30-fold more active than both trans-zeatin and cis-zeatin, and N⁶-isopentyladenine was 100-fold more active than N⁶-(Δ²-isopentenyl)adenine. The results suggest the possibility of employing cytokinin structure-activity relationships in distinguishing genotypic differences in cytokinin function and metabolism.