Mechanism-based inhibitors of cytokinin oxidase/dehydrogenase attack FAD cofactor

Cytokinin oxidases/dehydrogenases (CKOs) mediate catabolic regulation of cytokinin levels in plants. Several substrate analogs containing an unsaturated side chain were studied for their possible inhibitory effect on maize CKO (ZmCKO1) by use of various bioanalytical methods. Two allenic derivatives, N⁶-(buta-2,3-dienyl)adenine (HA-8) and N⁶-(penta-2,3-dienyl)adenine (HA-1), were identified as strong mechanism-based inhibitors of the enzyme. Despite exhaustive dialysis, the enzyme remained inhibited. Conversely, substrate analogs with a triple bond in the side chain were much weaker inactivators. The crystal structures of recombinant ZmCKO1 complexed with HA-1 or HA-8 were solved to 1.95 Å resolution. Together with Raman spectra of the inactivated enzyme, it was revealed that reactive imine intermediates generated by oxidation of the allenic inhibitors covalently bind to the flavin adenine dinucleotide (FAD) cofactor. The binding occurs at the C4a atom of the isoalloxazine ring of FAD, the planarity of which is consequently disrupted. All the compounds under study were also analyzed for binding to the Arabidopsis cytokinin receptors AHK3 and AHK4 in a bacterial receptor assay and for cytokinin activity in the Amaranthus bioassay. HA-1 and HA-8 were found to be good receptor ligands with a significant cytokinin activity. Nevertheless, due to their ability to inactivate CKO in the desired time intervals or developmental stages, they both represent attractive compounds for physiological studies, as the inhibition mechanism of HA-1 and HA-8 is mainly FAD dependent.     

Antagonistic Effects of Triazolo[4,5-d]pyrimidine and Pyridylurea Derivatives on Cytokinin-Induced Cytokinin Oxidase/Dehydrogenase Activity in Young Pea Plants

The effect of strong and weak cytokinin antagonists, belonging to the groups of triazolo[4,5-d]pyrimidines (TP), and pyridyl-phenylurea derivatives (PU), on cytokinin oxidase/dehydrogenase activity (CKX) in the tissues of young pea plants was studied. Tested anticytokinins, with the exception of the most efficient one – PU-1, were able to promote increased CKX activity in roots, when applied alone, but they had no significant influence on the enzymatic activity in leaves. N⁶-benzyladenine (BA) and 1-(2-chloropyridin-4-yl)-3-phenylurea (CPPU) provoked strong increase in CKX activity in roots, while in leaves considerable inhibition of enzymatic activity was observed. Different types of anticytokinins exhibited diverse preference towards taking off the action of purine and phenylurea cytokinins over CKX activity.     

Effects of cytokinin types and their concentrations on shoot proliferation and hyperhydricity in in vitro pear cultivar shoots

This study was made to clarify the effects of cytokinin type and their concentrations on shoot proliferation and hyperhydricity in in vitro Pyrus pyrifolia N. (`Hosui' and `Kosui') shoots. The shoots were subcultured in a woody plant medium supplemented with 0.5 M 3-indolyl-butyric acid, 3% (w/v) sorbitol, 0.8% (w/v) agar, and with cytokinins kinetin, 6-benzylaminopurine (BA), N-(2-chloro-4-pyridyl)-N9-phenylurea (CPPU), 1-phenyl-3-(1,2,3-thiadiazol-5-yl) urea (TDZ) added at concentrations 0.44, 4.40, 11.0 and 44.0 M. The highest number of shoots was induced by adding BA at concentration 11.0 M, then by 4.4 M BA, in both cultivars. TDZ and CPPU caused greater hyperhydricity in cultured explants than BA and kinetin. `Kosui' was more susceptible to hyperhydricity compared with `Hosui'.     

Synthesis,characterization and biological activity of ring-substituted 6-benzylamino-9-tetrahydropyran-2-yl and 9-tetrahydrofuran-2-ylpurine derivatives

In an attempt to improve specific biological functions of cytokinins routinely used in plant micropropagation, 33 6-benzylamino-9-tetrahydropyran-2-ylpurine (THPP) and 9-tetrahydrofuran-2-ylpurine (THFP) derivatives, with variously positioned hydroxy and methoxy functional groups on the benzyl ring, were prepared. The new derivatives were prepared by condensation of 6-chloropurine with 3,4-dihydro-2H-pyran or 2,3-dihydrofuran and then by the condensation of these intermediates with the corresponding benzylamines. The prepared compounds were characterized by elemental analyses, TLC, HPLC, melting point determinations, CI+ MS and ¹H NMR spectroscopy. The cytokinin activity of all the prepared derivatives was assessed in three classical cytokinin bioassays (tobacco callus, wheat leaf senescence and Amaranthus bioassay). The derivatives 6-(3-hydroxybenzylamino)-9-tetrahydropyran-2-ylpurine (3) and 6-(3-hydroxybenzylamino)-9-tetrahydrofuran-2-ylpurine (23) were selected, because of the high affinity of their parent compound meta-topolin (mT, 6-(3-hydroxybenzylamino)purine) to cytokinin receptors, as model compounds for studying their perception by the receptors CRE1/AHK4 and AHK3 in a bacterial assay. Both receptors perceived these two derivatives less well than they perceived the parent compound. Subsequently, the susceptibility of several new derivatives to enzyme degradation by cytokinin oxidase/dehydrogenase was studied. Substitution of tetrahydropyran-2-yl (THP) at the N⁹ position decreased the turnover rates of all new derivatives to some extent. To provide a practical perspective, the cytotoxicity of the prepared compounds against human diploid fibroblasts (BJ) and the human cancer cell lines K-562 and MCF-7 was also assayed in vitro. The prepared compounds showed none or marginal cytotoxicity compared to the corresponding N⁹-ribosides. Finally, the pH stability of the two model compounds was assessed in acidic and neutral water solutions (pH 3–7) by high-performance liquid chromatography (HPLC).     

The effect of auxin on cytokinin levels and metabolism in transgenic tobacco tissue expressing an ipt gene

The ipt gene from the T-DNA of Agrobacterium tumefaciens was transferred to tobacco (Nicotiana tabacum L.) in order to study the control which auxin appears to exert over levels of cytokinin generated by expression of this gene. The transgenic tissues contained elevated levels of cytokinins, exhibited cytokinin and auxin autonomy and grew as shooty calli on hormone-free media. Addition of 1-naphthylacetic acid to this culture medium reduced the total level of cytokinins by 84% while 6-benzylaminopurine elevated the cytokinin level when added to media containing auxin. The cytokinins in the transgenic tissue were labelled with ³H and auxin was found to promote conversion of zeatin-type cytokinins to ³H-labelled adenine derivatives. When the very rapid metabolism of exogenous [³H]zeatin riboside was suppressed by a phenylurea derivative, a noncompetitive inhibitor of cytokinin oxidase, auxin promoted metabolism to adenine-type compounds. Since these results indicated that auxin promoted cytokinin oxidase activity in the transformed tissue, this enzyme was purified from the tobacco tissue cultures. Auxin did not increase the level of the enzyme per unit tissue protein, but did enhance the activity of the enzyme in vitro and promoted the activity of both glycosylated and non-glycosylated forms. This enhancement could contribute to the decrease in cytokinin level induced by auxin. Studies of cytokinin biosynthesis in the transgenic tissues indicated that trans-hydroxylation of isopentenyladenine-type cytokinins to yield zeatin-type cytokinins occurred principally at the nucleotide level.Abbreviations Ade adenine - Ados adenosine - BA 6-benzylaminopurine - C control - Con A concanavallin A - CP cellulose phosphate - IPT isopentenyl transferase - NAA 1-naphthylacetic acid - NP normal phase - NPPU N-(3-nitrophenyl)-N-phenylurea - RIA radioimmunoassay - RP reversed phase We wish to thank Dr. J. Zwar for supplying phenylurea derivitives.     

The response of a cytokinin resistant mutant is highly specific and permits a new cytokinin bioassay

We have previously isolated six independent cytokinin-resistant mutants of Nicotiana plumbaginifolia which define three complementation groups, zeal, zea2 and zea3. We report here the characterization of the phenotypic response to cytokinin treatment of the mutant 1–64, belonging to the zeal group, and the result of the study of the specificity of this response. The phenotype of this mutant grown in the presence of cytokinin concentrations higher than 0.1 M is characterized by a hypertrophy of the cotyledons and hypocotyl which results in an increase of plantlet fresh weight. This hypertrophy is correlated to cytokinin concentration in a range between 0.01 to 10 M. The specificity of this response has been verified by using adenine and urea type cytokinins, as well as enantiomers of methylzeatin and methylbenzyladenine which differ widely in their cytokinin activities. We show that the high specificity of the hypertrophic response to cytokinins can be used as a convenient bioassay to screen the cytokinin activity of adenine or urea type molecules.Abbreviations zeatin [6-(4-hydroxy-3-methylbut-trans-2-enylamino)purine] - iP isopentenyladenine [6-(3-methylbut-2-enylamino)purine] - BA benzyladenine [6-(benzylamino)purine] - (R)-(+)-MeZea [(R)--methylzeatin] - (S)-(–)-MeZea [(S)--methylzeatin] - (R)-(+)-MeBA [(R)--methylbenzyladenine] - (S)-(–)-MeBA [(S)--methylbenzyladenine] - CPPU N-(2-chloro-4-pyridyl)-N-phenylurea - thidiazuron N-(1,2,3-thiadiazol-5-pyridyl)-N-phenylurea The authors dedicate this paper to the memory of Jean-Pierre Bourgin, Director of the Laboratoire de Biologie Cellulaire, who died suddenly on October 29, 1994.     

Effect of cytokinins on shoot regeneration from cotyledon and leaf segment of stem mustard (<Emphasis Type="Italic">Brassica juncea</Emphasis> var. <Emphasis Type="Italic">tsatsai</Emphasis>)

Cotyledon and leaf segments of stem mustard (Brassica juncea var. tsatsai) were cultured on Murashige and Skoog medium supplemented with various concentrations of different cytokinins [6-benzyladenine (BA), N-(2-chloro-4-pyridyl)-n-phenylurea (CPPU), 6-furfurylaminopurine (KT) and thidiazuron (TDZ)] in combinations with different levels of α-naphthalene acetic acid (NAA). The shoot regeneration frequency of cotyledon and leaf segment was dependent on the kinds and concentrations of cytokinins used in the medium, while in most cases cotyledon gave high regeneration frequency than leaf segment. TDZ proved to be the best cytokinin to induce shoot from both cotyledon and leaf segments compared to BA, KT and CPPU. The highest frequency of shoot regeneration was 61.3–67.9 % in cotyledon and 40.7–52.4% in leaf segment respectively when 2.27 or 4.54 μM TDZ was combined with 5.37 μM NAA. Next to TDZ, CPPU was also very suitable to induce shoot formation both in cotyledon and leaf segment. When 1.61 μM CPPU was combined with 2.69 μM NAA, shoot regeneration frequency was 45.0% in cotyledon and 36.4% in leaf segment, respectively. It was also shown that KT and BA affected shoot regeneration from cotyledon and leaf segment, the shoot regeneration was greatly increased when NAA was added together with cytokinins. The efficient and reliable shoot regeneration system was developed in both cotyledon and leaf segments. This regeneration protocol may be applicable to the improvement of this crop by genetic engineering in the future.     

Biochemical characteristics and ligand-binding properties of Arabidopsis cytokinin receptor AHK3 compared to CRE1/AHK4 as revealed by a direct binding assay

The cytokinin receptor AHK3 of Arabidopsis thaliana plays a predominant role in shoot development. A study of the hormone-binding characteristics of AHK3 compared with the mainly root-confined receptor CRE1/AHK4 has been accomplished using a live-cell binding assay on transgenic bacteria expressing individual receptor proteins. Both receptors bound trans-zeatin (tZ) with high affinity. Scatchard analysis showed a linear function corresponding to an apparent K(D) of 1-2 nM for the AHK3 receptor-hormone complex, which is close to the K(D) (2-4 nM) for the CRE1/AHK4 receptor-hormone complex. The specific binding of tZ to both receptors was pH dependent, AHK3 being more sensitive to pH changes than CRE1/AHK4. Hormone binding was reversible, at least for the bulk of (3)H-zeatin, and influenced by monovalent cations, while divalent cations (Ca(2+), Mg(2+), Mn(2+)) at physiological concentrations had no significant effect. AHK3 differed significantly from CRE1/AHK4 in relative affinity to some cytokinins. AHK3 had an approximately 10-fold lower affinity to isopentenyladenine (iP) and its riboside, but a higher affinity to dihydrozeatin than CRE1/AHK4. For AHK3, cytokinin ribosides (tZR, iPR) and cis-zeatin had true binding activity, although lower than that of tZ. The phenylurea-derived cytokinin thidiazuron was a strong competitor and bound to the same site as did adenine-derived cytokinins. The inhibitor of cytokinin action butan-1-ol had little effect on cytokinin-receptor complex formation. The revealed properties of AHK3 suggest its specific function in root-to-shoot communication.     

Specific photoaffinity labelling of a thylakoid membrane protein with an azido-cytokinin agonist

A cytokinin-binding peptide (CBP) of 46 kDa (Thy46) has been identified in thylakoid membranes of pea chloroplasts, by photoaffinity labelling with tritiated 1-(2-azido-6-chloropyrid-4-yl)-3-phenylurea ([³H]azidoCPPU), a urea-type cytokinin agonist. The labelled peptide is also detected in Nicotiana plumbaginifolia, Nicotiana tabacum and spinach thylakoid membranes, but is absent in thylakoid membranes of Chlamydomonas reinhardtii. A pharmacological study of the interaction of this peptide with different cytokinin agonist molecules has been achieved. Urea derivatives are the most efficient competitors of photolabelling, and this efficiency is in good agreement with the cytokinin activity of these compounds. A quantitative analysis of the displacement of the photoaffinity labelling of the peptide by increasing concentrations of CPPU indicates an apparent dissociation constant of 1 M for this ligand. Purine-type cytokinins are weaker competitors than urea-type molecules, but the efficiency of the competition is also correlated to their respective cytokinin activity. A partial purification of Thy46 by a protocol involving ion exchange chromatography and 2D-gel electrophoresis is described.     

Cytokinin receptor antagonists derived from 6-benzylaminopurine

Recently we reported 6-(2-hydroxy-3-methylbenzylamino)purine (PI-55) as the first molecule to antagonize cytokinin activity at the receptor level. Here we report the synthesis and in vitro biological testing of eleven BAP derivatives substituted in the benzyl ring and in the C2, N7 and N9 positions of the purine moiety. The ability of the compounds to interact with Arabidopsis cytokinin receptors AHK3 and CRE1/AHK4 was tested in bacterial receptor and in live-cell binding assays, and in an Arabidopsis ARR5:GUS (Arabidopsis response regulator 5) reporter gene assay. Cytokinin activity of the compounds was determined in classical cytokinin biotests (tobacco callus, wheat leaf senescence and Amaranthus bioassays). 6-(2,5-Dihydroxybenzylamino)purine (LGR-991) was identified as a cytokinin receptor antagonist. At the molecular level LGR-991 blocks the cytokinin receptor CRE1/AHK4 with the same potency as PI-55. Moreover, LGR-991 acts as a competitive inhibitor of AHK3, and importantly shows reduced agonistic effects in comparison to PI-55 in the ARR5:GUS reporter gene assay and in cytokinin bioassays. LGR-991 causes more rapid germination of Arabidopsis seeds and increases hypocotyl length of dark-grown seedlings, which are characteristics of plants with a reduced cytokinin status. LGR-991 exhibits a structural motive that might lead to preparation of cytokinin antagonists with a broader specificity and reduced agonistic properties.     

Cytokinins regulate a bidirectional phosphorelay network in Arabidopsis

The cytokinin class of plant hormones plays key roles in regulating diverse developmental and physiological processes. Arabidopsis perceives cytokinins with three related and partially redundant receptor histidine kinases (HKs): CRE1 (the same protein as WOL and AHK4), AHK2, and AHK3 (CRE-family receptors). It is suggested that binding of cytokinins induces autophosphorylation of these HKs and subsequent transfer of the phosphoryl group to a histidine phosphotransfer protein (HPt) and then to a response regulator (RR), ultimately regulating downstream signaling events. Here we demonstrate that, in vitro and in a yeast system, CRE1 is not only a kinase that phosphorylates HPts in the presence of cytokinin but is also a phosphatase that dephosphorylates HPts in the absence of cytokinin. To explore the roles of these activities in planta, we replaced CRE1 with mutant versions of the gene or with AHK2. Replacing CRE1 with CRE1(T278I), which lacks cytokinin binding activity and is locked in the phosphatase form, decreased cytokinin sensitivity. Conversely, replacing CRE1 with AHK2, which favors kinase activity, increased cytokinin sensitivity. These results indicate that in the presence of cytokinins, cytokinin receptors feed phosphate to phosphorelay-integrating HPt proteins. In the absence of cytokinins, CRE1 removes phosphate from HPt proteins, decreasing the system phosphoload.     

The specificity of cytokinin signalling in Arabidopsis thaliana is mediated by differing ligand affinities and expression profiles of the receptors

Arabidopsis thaliana has three membrane‐located cytokinin receptors (AHK2, AHK3 and CRE1/AHK4), which are sensor histidine kinases containing a ligand‐binding CHASE domain. Despite their structural similarity the role of these receptors differs in planta. Here we have explored which parameters contribute to signal specification. In a bacterial assay, the CHASE domain of AHK2 has a similar ligand binding spectrum as CRE1/AHK4. It shows the highest affinity for isopentenyladenine (iP) and trans‐zeatin (tZ) with an apparent K_D of 1.4 and 4.0 nm , respectively. Real‐time PCR analysis of cytokinin primary response genes in double mutants retaining only single receptors revealed that all receptors are activated in planta by cytokinin concentrations in the low nanomolar range. However, there are differences in sensitivity towards the principal cytokinins iP and tZ. The activation of the cytokinin‐sensitive P_ARR5:GUS reporter gene in three different double mutants shows specific, but also overlapping, spatial domains of activity, which were for all receptors predominantly in the shoot apical meristems and root cap columella. AHK2 and AHK3 signal specifically in leaf parenchyma cells, AHK3 in stomata cells, and CRE1/AHK4 in the root vasculature. Promoter‐swap experiments demonstrate that CRE1/AHK4 can functionally replace AHK2 but not AHK3. However, the cytoplasmic AHK3 histidine kinase (Hk) domain can be replaced by the CRE1/AHK4 Hk domain, which suggests that functionality is mediated in this case by the extracytosolic domain. Together, the data show that both differential gene expression and ligand preference contribute to specify the receptor activity.     

Plant growth promotion by Bacillus megaterium involves cytokinin signaling

Accumulating evidence indicates that plant growth promoting rhizobacteria (PGPR) influence plant growth and development by the production of phytohormones such as auxins, gibberellins, and cytokinins. Little is known on the genetic basis and signal transduction components that mediate the beneficial effects of PGPRs in plants. We recently reported the identification of a Bacillus megaterium strain that promoted growth of A. thaliana and P. vulgaris seedlings. In this addendum, the role of cytokinin signaling in mediating the plant responses to bacterial inoculation was investigated using A. thaliana mutants lacking one, two or three of the putative cytokinin receptors CRE1, AHK2 and AHK3, and RPN12 a gene involved in cytokinin signaling. We show that plant growth promotion by B. megaterium is reduced in AHK2-2 single and double mutant combinations and in RPN12. Furthermore, the triple cytokinin-receptor CRE1-12/AHK2-2/AHK3-3 knockout was insensitive to inoculation in terms of growth promotion and root developmental responses. Our results indicate that cytokinin receptors play a complimentary role in plant growth promotion by B. megaterium.Key words: Arabidopsis, plant growth stimulation, root development, rhizobacteria     

Urea derivatives on the move: cytokinin-like activity and adventitious rooting enhancement depend on chemical structure

Urea derivatives are synthetic compounds, some of which have proved to be positive regulators of cell division and differentiation. N -phenyl- N '-(2-chloro-4-pyridyl)urea (forchlorofenuron, CPPU) and N -phenyl- N '-(1,2,3-thiadiazol-5-yl)urea (thidiazuron, TDZ), well known urea cytokinin representatives, are extensively used in in vitro plant morphogenesis studies, as they show cytokinin-like activity often exceeding that of adenine compounds. In recent years, renewed interest in structure–activity relationship studies allowed identification of new urea cytokinins and other urea derivatives that specifically enhance adventitious root formation. In this review, we report the research history of urea derivatives, new insights into their biological activity, and recent progress on their mode of action.     

Control by cytokinins of the cellular behavior in the plate meristem of zucchini cotyledons

The temporal and spatial effects of exogenous cytokinins on both cell expansion and division activity in the plate meristem of cultured zucchini cotyledons were studied. N ⁶-benzylaminopurine (1–100 μM) and N-(2-chloro-4pyridyl)-N′-phenylurea (4PU-30) (0.1–100 μM) greatly stimulated the cell growth and division. They provoked multiple cell cycles, formation of larger clusters of daughter cells and an increase of the final number of cells. Both cytokinins led to earlier achievement of final cotyledon size and shortened the cell doubling time. By contrast to the purine cytokinin, phenylurea cytokinin 4PU-30 enlarged the cotyledon predominantly in length. Zeatin and kinetin were less effective, particularly in stimulating cell expansion. In low concentrations, all cytokinins were more effective in stimulating division activity rather than expansion. The cells in the cotyledon margins displayed a higher division activity, especially when treated with exogenous cytokinins. The final cotyledon and cluster areas were not of the strict proportional dependence upon the number of their cells. These results provide a novel example where stimulated cell division fails to evoke a respective increase in the final organ size.     

Cytokinin activity of N-phenyl-N′-(4-pyridyl)urea derivatives

We have synthesized 35 N-phenyl-N′-(4-pyridyl)urea derivatives and tested their cytokinin activity in the tobacco callus bioassay. Among them, N-phenyl-N′- (2-chloro-4-pyridyl)urea is highly active, the optimum concentration of which is lower than 4 × 10^?9 M (0.001 ppm), 3 compounds, i.e. N-(2-methylphenyl)-N′-(2-chloro-4-pyridyl)urea, N-(3-methylphenyl)-N′-(2-chloro-4-pyridyl)urea and N-(3-chlorophenyl)-N′-(2-chloro-4-pyridyl) urea are as active as N⁶-benzyladenine (concentration for optimum yield: 4.4 × 10^?8 M or 0.01 ppm), and N-phenyl-N′-(2-methyl-4-pyridyl)urea and N-(2-chlorophenyl)-N′-(2-chloro-4-pyridyl)urea are as active as N-phenyl-N′-(4-pyridyl)urea (concentration for optimum yield: 4.7 × 10^?7 M or 0.1 ppm), while the activity of the other 29 compounds are not so remarkable and 11 of them are almost or completely inactive.     

Regulation of cytokinin oxidase activity as a factor affecting the content of cytokinins

In the shoots of 7-day-old seedlings of wheat (Triticum durum Desf., cv. Bezenchukskaya 139), ion deficiency in Hoagland-Arnon nutrient solution induced a decrease in the cytokinin content by the end of the first day, whereas the excision of four out of five primary roots brought about an opposite response (accumulation of cytokinins) as early as during the first hour. It was assumed that changes in the content of cytokinins might depend on the activity of cytokinin oxidase (CKO) and the expression of gene encoding this enzyme. It was shown that tenfold dilution of the nutrient solution activated CKO and raised the level of CKO gene expression, whereas excision of some roots brought about a quick decrease in enzyme activity and gene expression. The role of ABA and arrival of cytokinins from the roots to the shoot as factors affecting CKO activity in the shoot is discussed; arguments for the priority of hormonal signal over the influx of nitrates from the roots are offered. It was concluded that the regulation of CKO activity might be one of the important mechanisms determining plant response to treatments via the changes in the cytokinin concentration.     

New aromatic 6-substituted 2′-deoxy-9-(β)-d-ribofuranosylpurine derivatives as potential plant growth regulators

Cytokinins are naturally occurring substances that act as plant growth regulators promoting plant growth and development, including shoot initiation and branching, and also affecting apical dominance and leaf senescence. Aromatic cytokinin 6-benzylaminopurine (BAP) has been widely used in micropropagation systems and biotechnology. However, its 9-glucoside (BAP9G) accumulates in explants, causing root inhibition and growth heterogenity. To overcome BAP disadvantages, a series of ring-substituted 2′-deoxy-9-(β)-d-ribofuranosylpurine derivatives was prepared and examined in different classical cytokinin bioassays. Amaranthus, senescence and tobacco callus bioassays were employed to provide details of cytokinin activity of 2′-deoxy-9-(β)-d-ribosides compared to their respective free bases and ribosides. The prepared derivatives were also tested for their recognition by cytokinin receptors of Arabidopsis thaliana AHK3 and CRE1/AHK4. The ability of aromatic N⁶-substituted adenine-2′-deoxy-9-(β)-d-ribosides to promote plant growth and delay senescence was increased considerably and, in contrast to BAP, no loss of cytokinin activity at higher concentrations was observed. The presence of a 2′-deoxyribosyl moiety at the N9-position led to an increase in cytokinin activities in comparison to the free bases and ribosides. The antioxidant capacity, cytotoxicity and effect on the MHV-68 gammaherpesvirus strain were also examined.