Regulation of Cytokinin Oxidase Activity in Callus Tissues of Phaseolus vulgaris L. cv Great Northern

The regulation of cytokinin oxidase activity in callus tissues of Phaseolus vulgaris L. cv Great Northern has been examined using an assay based on the oxidation of N⁶-(Δ²-isopentenyl)adenine-8-¹⁴C (i⁶ Ade-8-¹⁴C) to adenine. Solutions of exogenous cytokinins applied directly to the surface of the callus tissues induced relatively rapid increases in cytokinin oxidase activity. The increase in activity was detectable after 1 hour and continued for about 8 hours, reaching values two- to three-fold higher than the controls. The cytokinin-induced increase in cytokinin oxidase activity was inhibited in tissues pretreated with cordycepin or cycloheximide, suggesting that RNA and protein synthesis may be required for the response. Rifampicin and chloramphenicol, at concentrations that inhibited the growth of Great Northern callus tissues, were ineffective in inhibiting the increase in activity. All cytokinin-active compounds tested, including both substrates and nonsubstrates of cytokinin oxidase, were effective in inducing elevated levels of the enzyme in Great Northern callus tissue. The cytokinin-active urea derivative, Thidiazuron, was as effective as any adenine derivative in inducing this response. The addition of Thidiazuron to the reaction volumes used to assay cytokinin oxidase activity resulted in a marked inhibition of the degradation of the labeled i⁶ Ade-8-¹⁴C substrate. On the basis of this result, it is possible that Thidiazuron may serve as a substrate for cytokinin oxidase, but other mechanisms of inhibition have not yet been excluded.     

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     

Genotypic variation in cytokinin oxidase from phaseolus callus cultures

Genotypic variation in cytokinin oxidase has been detected in enzyme preparations from Phaseolus vulgaris L. cv Great Northern and Phaseolus lunatus L. cv Kingston callus cultures. Although cytokinin oxidase preparations from Great Northern and Kingston callus tissues appear to have very similar substrate specificities, the cytokinin oxidase activities from the two callus tissues were found to differ in a number of other properties. The cytokinin oxidase from P. vulgaris cv Great Northern callus tissue exhibited a pH optimum of 6.5 (bisTris) and had a strong affinity for the lectin concanavalin A. The cytokinin oxidase from P. lunatus cv Kingston callus tissue exhibited a pH optimum of 8.4 (Taps) and did not bind to concanavalin A. The two enzymes also differed in position of elution when chromatographed on DEAE-cellulose. Both cytokinin oxidase activities exhibited enhanced activity and lower pH optima in the presence of copper-imidazole complexes, but the optimum copper-imidazole ratio and the magnitude of enhancement differed for the two activities. In both callus tissues, transient increases in the supply of exogenous cytokinins induced increases in cytokinin oxidase activity. The differences in pH optima and in glycosylation (as evidenced by the observed difference in lectin affinity) of the cytokinin oxidases from Great Northern and Kingston callus tissues suggest that the compartmentation of cytokinin oxidase may differ in the two callus tissues. The possibility that enzyme compartmentation and isozyme variation in cytokinin oxidase may play a role in the regulation of cytokinin degradation in plant tissues is discussed in relation to known differences in the rates of cytokinin degradation in Great Northern and Kingston callus tissues.     

Effect of a cytokinin antagonist on cytokinin and light-dependent amaranthin synthesis inAmaranthus Tricolor seedlings

InAmaranthus tricolor seedlings, amaranthin synthesis can be induced under the effect either of a cytokinin or of white light. The 3-methyl-7-(n-pentylamino)pyrazolo(4,3-d)pyrimidine (PAMPP), a cytokinin analog that strongly inhibits the growth of tobacco callus, antagonizes the stimulating effect of cytokinin as well as stimulation by light. In dose-response terms, the inhibitory effect of PAMPP was described as competitive with respect to N⁶-benzyladenine (b⁶Ade) or light. The inhibition by PAMPP of the b⁶Ade amaranthin test response or the inhibition by this cytokinin analog of the light amaranthin test response were both reversed by either subsequent light or b⁶Ade treatment.     

Ribosylzeatin and zeatin in tobacco crown gall tissue

Cytokinins were extracted from two cultures of tobacco crown gall tumor tissue: an unorganized tissue and a teratoma which produced leafy shoots. On Sephadex LH-20 column chromatography, extracts of both types of tissue yielded two peaks of cytokinin activity with elution volumes similar to ribosylzeatin and zeatin. Ribosylzeatin and zeatin were detected and quantified by coupled gas chromatography — mass spectrometry selected ion monitoring (GC/MS SIM), comparable quantities being found in the two extracts. Full mass spectral evidence for the presence of ribosylzeatin in both tissues was obtained. No evidence was found for the presence of N⁶-(²-isopentenyl)adenosine (i⁶Ade) or N⁶-(²-isopentenyl)adenine (i⁶Ade) although these compounds have been reported to occur in cytokinin-habituated tobacco callus tissues.Abbreviations BAP 6-benzylaminopurine - GC gas chromatography - GC/MS coupled gas chromatography-mass spectrometry - i⁶ Ade N⁶-(²-isopentenyl)adenine - i⁶ Ado N⁶-(²-isopentenyl)adenosine - RFE rotary film evaporation - SIM selected ion monitoring - TLC thin-layer chromatography - TMS trimethylsilyl     

Hydroxylation of N-(Delta-Isopentenyl)adenine to Zeatin: Relative Activities of Organ Systems from Actinidia arguta

By incubating explants from Actinidia arguta seedlings on a nutrient medium supplemented with 20 to 30 micromolar N⁶-(Δ²-isopentenyl)adenine (i⁶Ade) and then measuring zeatin (io⁶Ade) accumulation in tissues, the distribution of i⁶Ade hydroxylase activities in whole plants could be determined. Based on analyses with three entire plants, it is estimated that, as an organ system, roots contain approximately 68% of the plant's hydroxylase, while stems and leaves account for about 26% and 6%, respectively, of the total activity. Depending on the part of the root examined, hydroxylase activities ranged from 20 to 148 nanomoles io⁶Ade accumulated per gram fresh weight per 24 hours of incubation. Stem activities ranged from 17 to 165 nanomoles per gram fresh weight per 24 hours with the lowest activities being found at the tip. Leaf activities were substantially lower (1-10 nanomoles per leaf depending on position) than either root or stem.     

Effect of a cytokinin antagonist on cytokinin and light-dependent amaranthin synthesis inAmaranthus Tricolor seedlings

InAmaranthus tricolor seedlings, amaranthin synthesis can be induced under the effect either of a cytokinin or of white light. The 3-methyl-7-(n-pentylamino)pyrazolo(4,3-d)pyrimidine (PAMPP), a cytokinin analog that strongly inhibits the growth of tobacco callus, antagonizes the stimulating effect of cytokinin as well as stimulation by light. In dose-response terms, the inhibitory effect of PAMPP was described as competitive with respect to N⁶-benzyladenine (b⁶Ade) or light. The inhibition by PAMPP of the b⁶Ade amaranthin test response or the inhibition by this cytokinin analog of the light amaranthin test response were both reversed by either subsequent light or b⁶Ade treatment.     

Cytokinin metabolism in autonomous and crown gall tissue cultures

When tissues ofCatharanthus roseus A6 crown gall were incubated on medium supplemented with 50 (μM N6-isopentenyladenine (i⁶Ade), endogenous i⁶Ade, N6-isopentenyladenosine (i⁶A) and i⁶A nucleotide (i⁶AXP) increased to. 6, 5 and 12 nmol g^-1, respectively, during 100 h. Whereas i⁶Ade and i⁶AXP increased rapidly during the initial 4 h and then remained relatively constant, the level of i⁶A continued to increase to 25 nmol g^-1 by 16 h and then decreased; Ribosylzeatin (io⁶A) and its nucleotide (io⁶AXP) remained constant at 1.5 and 1.7 nmol g^-1, respectively. Upon transfer to cytokininless medium, i⁶Ade and i⁶AXP declined rapidly but i⁶A increased to 10 nmol g^-1 after 4 h and then declined. Again, io⁶A and io⁶AXP were unchanged. Prolonged incubation of crown gall tissue on i⁶Ade completely inhibited growth. By contrast, nonrtransformed, autonomous tissue lines fromCalycanthus fertilis andActinidia chinensis Xarguta continued to proliferate on this medium. TheActinidia Une was shown to metabolize i⁶Ade to zeatin and to accumulate this cytokinin to levels in excess of 70 nmol g^-1.     

Activité cytokinine de dérivés acylés,alkylés,acyl-alkylés de l'adénine et d'isost`eres azotés de la γ,γ-diméthylallyladénine

The cytokinin activities of various 6-acylaminopurines, 6-alkylaminopurines, 6-acylamino-9-benzyl-purines as well as a series of isosteric-nitrogen derivatives of N⁶-(γ, γ-dimethylallyl)adenine (I⁶Ade) have been tested using the tobacco pith and the pea bud bioassays. The interactions between active, slightly active and inactive compounds have been studied with the last assay. Acylation decreases the biological activity; e.g., N⁶-benzoyl and N⁶-furoyladenines are less active than Na⁶-benzyladenine and kinetin. Substitution at the 9-position reduces (tobacco-pith assay) or suppresses (pea-bud assay) the phytohormonal activity of otherwise active 6-acylaminopurines. In isosteric derivatives, maximum activity occurs when the side chain has the same length as in 1⁶Ade. After the analysis of interactions between more or less active compounds, it is suggested that the differences in cytokinin activity could be related to unequal affinities for a hypothetical receptor site.     

Conversion of N6-isopentenyladenine to zeatin by Actinidia tissues

Tissue cultures grown from stem explants of three $\text{Actinidia}$ species and a hybrid species rapidly converted N6-isopentenyladenine (i⁶Ade) to zeatin (io⁶Ade), a potent hydroxylated cytokinin. Within 24 h on 50 uM i⁶Ade, callus tissues of $\text{A.}\text{chinensis}\text{×}\text{arguta}$ accumulated 83 ± 6 nmol/g io⁶Ade which was purified using HPLC and identified by its characteristic UV and mass spectra. Activity converting i⁶Ade to io⁶Ade was also demonstrated in stem segments from intact plants where it was low in the tip (3 cm), highest in the region corresponding to rapid leaf growth and very low in the mature stem. Root segments converted i⁶Ade to io⁶Ade almost as rapidly as the most active region of the stem while leaf petioles produced little io⁶Ade. Fruits of $\text{A.}\text{arguta}$ and $\text{A.}\text{chinensis}$ produced little or no io⁶Ade, respectively.     

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.     

Regulation of cytokinin oxidase activity in tobacco callus expressing the T-DNA ipt gene

There are indications that the cytokinin content in transgenic tissues expressing the cytokinin biosynthetic ipt gene is under metabolic control, which prevents the accumulation of cytokinins to lethal levels. The objective of this study was to investigate the relationships between the content of endogenous cytokinins and the activity of cytokinin oxidase (which is believed to be a copper-containing amine oxidase, EC 1.4.3.6.) in ipt transgenic tobacco callus. In addition, the effect of exogenously applied N-benzyladenine (BA) on this relationship was examined. Endogenous cytokinin concentrations were measured in callus of Nicotiana tabacum L. cv. Petit Havana SRI transformed with the ipt of Agrobacterium tumefaciens under the control of a light-inducible promoter and in non-transformed tissue using LC-tandem mass spectrometry. The activity of cytokinin oxidase was estimated by measuring the conversion of [2,8-³H]N⁶-(Δ²-isopentenyl)adenine to [³H]adenine by enzyme preparations in vitro. The 14-day-old ipt-transformed callus contained a 25-fold higher amount of cytokinins as compared to the non-transformed tissue. Mainly zeatin- and dihydrozeatin-types of cytokinins (free bases, ribosides, nucleotides and O-glucosides) accumulated in the ipt transgenic tissue. The cytokinin pool of both ipt-transformed and non-transformed tissues consisted predominantly of cytokinins that are either resistant to cytokinin oxidase attack (nucleotides and O-glucosides of cytokinins and cytokinins bearing N⁶-saturated side chain) or have a low affinity for the enzyme (zeatin and its riboside). The former represented 71.6 and 74.8% and the latter 27.7 and 24.4% of the pool of endogenous cytokinins in ipt-transformed and non-transformed tissues, respectively. Enzyme preparations from ipt-transformed tissue exhibited 1.5-fold higher cytokinin oxidase activity compared with that observed in control tissues. Application of exogenous BA affected the total levels of cytokinins of the two tissue lines in different ways. The cytokinin content increased by 1.7- and 1.5-fold in ipt-transformed tissues 6 and 12 h after BA application, respectively, while it declined in the non-transformed control by 1.6- to 2.0-fold between 3 and 12 h after BA application. The increase in cytokinin content in the ipt callus is due to an increase of zeatin- and dihydrozeatin-type cytokinins (nucleotides, ribosides and free bases) leading to an enhanced accumulation of O-glucosides after 12 h. Following BA treatment, the cytokinin oxidase activity increased up to 1.8-fold in ipt-transformed and 1.6-fold in non-transformed tissues. The levels of isopentenyl-type cytokinins were near the detection limit; however, the enhancement of cytokinin oxidase activity after BA treatment in both tissue lines was correlated with the content of preferred substrate of the enzyme, N⁶-(Δ²-isopentenyl)adenosine.     

Comparative Activity of Isomers of Zeatin and Ribosyl-Zeatin on Funaria hygrometrica

The activities of isomers of zeatin, ribosyl-zeatin, and 6-(γ,γ-dimethylallylamino) purine (i⁶Ade) on the moss Funaria hygrometrica are compared by measuring the ability of the cytokinins to induce callus or gametophores. The cis- and trans-ribosyl-zeatins were inactive, and therefore this kind of bioassay cannot be used as evidence for the presence or absence of a cytokinin in tests on natural products.     

Distribution of incorporated, synthetic cytokinins in ribosomal RNA preparations from tobacco callus

The distribution of incorporated synthetic cytokinins (N⁶-[8-¹⁴C]benzyladenine ([8-¹⁴C]bzl⁶Ade) and N⁶[8-¹⁴C]furfuryladenine ([8-¹⁴C]fr⁶Ade) in ribosomal RNA prepared from tobacco callus (Nicotiana tabacum L. var. Wis. No. 38) grown in the presence of one of these for 25 or 26 days has been studied. The rRNA of tissue supplied with [8-¹⁴C]bzl⁶Ade or [8-¹⁴C]fr⁶Ade was fractionated by methylated albumin-Kieselguhr column chromatography and preparative gel electrophoresis, respectively. In each case about 80% of the incorporated cytokinin was recovered as the ribonucleoside [8-¹⁴C]bzl⁶A or [8-¹⁴C]fr⁶A in the rRNA peak after the fractionations. [8-¹⁴C]fr⁶A was found associated with both the 18S and 25S rRNA components in quantities roughly proportional to their 260 nm absorbance. This pattern of apparently nonspecific association was not affected by prior denaturation of the RNA with formamide.     

Determination of plant growth substances in liquid endosperm of immature walnut (Juglans nigra) nuts by an ELISA technique

Plant growth substances (PGSs) were analysed in liquid endosperm of black walnut using HPLC and an ELISA procedure. Of all the PGSs studied, we show no GA₃, low levels of cytokinins (io⁶A, i⁶Ade, i⁶Ado) and ABA, and very high level of IAAAbbreviations ABA Abscisic acid: - Ade Adenine: - GA₃ Gibberellic acid: - IAA Indole-3-acetic acid: - i⁶Ade N⁶(²-1) adenine: - i⁶Ado N⁶(²-isopentenyl adenosine: - io⁶A Zeatin riboside:     

Kinetic comparison of cytokinin nucleosidase activity isolated from normally ripening and mutant tomato varieties

Kinetic parameters for cytokinin nucleosidase activity which catalyzes the deribosylation of N⁶(Δ²-isopentenyl)adenosine (I⁶Ado) to produce the more “active” free base N⁵(Δ²-isopenetyl)adenine (I⁶Ade) were compared for a normally ripening tomato (Lycopersicon esculentum L.) cultivar Rutgers, and two mutant tomato varieties (Nor and Rin). K_m for nucleosidase activity in Rutgers was lower (K_m = 0.1 millimolar) than that in either Nor (K_m = 0.14 millimolar) or Rin (K_m = 0.13 millimolar).     

Cytokinins of sycamore spring sap

Summary The cytokinins present in the spring sap of Acer pseudoplatanus L. were investigated. Ribosyl-trans-zeatin, trans-zeatin and dihydrozeatin were isolated and identified by combined gas chromatography-mass spectrometry (GC-MS). A number of other cytokinin active fractions were observed. One of these was less polar than zeatin and did not behave as any known cytokinin. Two other fractions were more polar than ribosylzeatin and were unstable. A decomposition product of one of these was identified as ribosyl-trans-zeatin by GC-MS. The possible nature of the unstable compounds is discussed. Data on the changes in cytokinin activity of the various fractions during spring 1973 are presented and discussed.Abbreviations GLC gas-liquid chromatography - GG-MS combined gas chromatography-mass spectrometry - KE kinetin equivalents - TLC thin-layer chromatography - TMS trimethylsilyl - tRNA transfer RNA - i⁶ Ade 6-(3-methylbut-2-enylamino)-purine - i⁶ Ado 6-(3-methylbut-2-enylamino)-9--D-ribofuranosyl-purine     

Cytokinin oxidase activity in the cellular slime mold, Dictyostelium discoideum

The cytokinin N6-(delta 2-isopentenyl)adenine (i6Ade) is produced during the development of the cellular slime mold, Dictyostelium discoideum, and functions in this organism as the immediate precursor of the spore germination inhibitor, discadenine. The metabolism of i6Ade in axenic cultures of D. discoideum Ax-3 amoebae has been investigated in the present study. An enzyme activity that specifically catalyzes the degradation of i6Ade has been detected in Ax-3 amoebae. This enzyme is similar to the cytokinin oxidases present in higher plant systems and cleaves the N6-side chain of i6Ade to form adenine. Discadenine synthase activity was also detected in axenically cultured Ax-3 amoebae. The cytokinin oxidase activity detected in Dictyostelium decreased during aggregation and development of Ax-3 amoebae and in starving Ax-3 amoebae maintained under either fast-shake (230 rpm) or slow-shake (70 rpm) conditions. In the latter case, the fall in enzyme activity was accelerated by treatment with cyclic AMP. In contrast to these results, discadenine synthase activity in Ax-3 amoebae rose sharply during the culmination phase of development, exhibited little change in starving Ax-3 amoebae maintained under fast-shake conditions, and fell under slow-shake conditions unless the amoebae were treated with cyclic AMP. Possible functions of the Dictyostelium cytokinin oxidase and the significance of the i6Ade metabolism observed in vegetative Dictyostelium amoebae are discussed.     

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.     

Protein Folding: Grand Challenge of Nature

Abstract

Conformational preferences of the hypermodified nucleic acid bases N6-(Δ² -cis-hydroxyisopentenyl)adenine, cis-io⁶Ade also known as cis-zeatin, and N⁶-(Δ² -trans-hydroxyisopentenyl)adenine, trans-io⁶ade or trans-zeatin, and 2-methylthio derivatives of these cis-ms²io⁶Ade or cis-ms²zeatin, and trans-ms²io⁶Ade or trans-ms²zeatin have been investigated theoretically by the quantum chemical Perturbative Configuration Interaction with Localized Orbitals (PCILO) method. Automated geometry optimization using quantum chemical MNDO, AMI and PM3 methods has also been made to compare the salient features. The predicted most stable conformation of cis-io⁶Ade, trans-io⁶Ade, cis-ms²io⁶Ade and trans-ms²io⁶Ade are such that in each of these molecules the isopentenyl substituent spreads away (has “dista” conformation) from the five membered ring imidazole moiety of the adenine. The atoms N(6), C(10) and C(11) remain coplanar with the adenine ring in the predicted preferred conformation for each of these molecules. In cis-io⁶Ade as well as cis- ms²io⁶Ade the hydroxyl oxygen may participate in intramolecular hydrogen bonding with the H-C(10)-H group. In trans-io⁶Ade the hydroxyl group is oriented towards the H-C(2) instead. This orientation is retained in trans-ms²io⁶Ade, possible O-H…S hydrogen bonding may be a stabilizing factor. In all these four modified adenines C(11)-H is favourably placed to participate in intramolecular hydrogen bonding with N(1). In cis-ms²io⁶Ade as well as trans-ms²io⁶Ade the 2-methylthio group preferentially orients on the same side as C(2)-N(3) bond, due to this nonobstrusive placing, orientation of the hydroxyisopentenyl substituent remains unaffected by 2-methylthiolation. Thus the N(1) site remains shielded irrespective of the 2-methylthiolation status in these various cis-and trans-zeatin analogs alike. Firmly held orientation of hydroxyisopentenyl substituent in zeatin isomers and derivatives, in contrast to adaptable orientation of isopentenyl substituent in i⁶Ade and ms²i⁶Ade, may account for the increased efficiency of suppressor tRNA and reduced codon context sensitivity accompanied with the occurrence of ms²-zeatin (ms²io⁶Ade) modification.