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.     

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.     

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.     

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.     

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.     

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.     

Phosphorylation of cytokinin by adenosine kinase from wheat germ

Adenosine kinase was partially purified from wheat germ. This enzyme preparation, which was devoid of adenine phosphoribosyltransferase and nearly free of adenosine deaminase but contained adenylate kinase, rapidly phosphorylated adenosine and a cytokinin, N⁶-(δ²-isopentenyl)adenosine. Electrophoretic analysis indicated that only N⁶-(δ²-isopentenyl)adenosine-monophosphate was formed from the cytokinin while about 55% AMP, 45% ADP, and a trace of ATP were formed from adenosine. The biosynthesized nucleoside monophosphates were quantitatively hydrolyzed to the corresponding nucleosides by 5′-nucleotidase and the isopentenyl side chain of the phosphorylated cytokinin was not cleaved. The enzyme did not catalyze phosphorylation of inosine.     

Active cytokinins: photoaffinity labeling agents to detect binding

Four series of azidopurines have been synthesized and tested for cytokinin activity in the tobacco callus bioassay: 2- and 8-azido-N⁶-benzyladenines, -N⁶-(Δ²-isopentenyl)adenines, and -zeatins, and N⁶-(2- and 4-azidobenzyl)adenines. The compounds having 2-azido substitution on the adenine ring are as active as the corresponding parent compounds, while those with 8-azido substitution are about 10 or more times as active. The 8-azidozeatin, which is the most active cytokinin observed, exhibited higher than minimal detectable activity at 1.2 × 10⁻⁵ micromolar, the lowest concentration tested. The shape of the growth curve indicates that even a concentration as low as 5 × 10⁻⁶ micromolar would probably be effective. By comparison, the lowest active concentration ever reported for zeatin has been 5 × 10⁻⁵ micromolar, representing a sensitivity rarely attained.     

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).     

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.     

Turnip Yellow Mosaic Virus RNA as a Substrate of the Transfer RNA Nucleotidyltransferase II. Incorporation of Cytidine 5'-Monophosphate and Determination of a Short Nucleotide Sequence at the 3' End of the RNA

Turnip yellow mosaic virus (TYMV) RNA treated with snake venom phosphodiesterase accepts cytidine 5′-monophosphate and adenosine 5′-monophosphate (AMP) when it is incubated in the presence of cytidine 5′-triphosphate (CTP), adenosine 5′-triphosphate, and Escherichia coli transfer RNA nucleotidyltransferase; untreated TYMV RNA accepts only AMP. When α ³²PCTP was used for terminal labeling, the nearest neighbor analyses and the anallyses after action of various nucleases showed that the sequence of five nucleotides at the 3′ end of TYMV RNA is: pGpCpApCpC. A nuclease present in commerical preparations of snake venom phosphodiesterase leads to the fragmentation of TYMV RNA, the 3′ end of which is found in a fragment having a sedimentation constant close to 5s.     

Cytokinins of the Developing Mango Fruit : Isolation, Identification, and Changes in Levels during Maturation

The cytokinin activity has been isolated and identified from extracts of immature mango (Mangifera indica L.) seeds. The structures of zeatin, zeatin riboside, and N⁶-(Δ²-isopentenyl)adenine riboside were confirmed on the basis of their chromatographic behavior and mass spectra of trimethylsilyl derivatives. Both trans and cis isomers of zeatin and zeatin riboside were also identified by the retention times of high performance liquid chromatography. In addition, an unidentified compound appeared to be a cytokinin glucoside.

The concentration of cytokinins in the panicle and pulp of mango reached a maximum 5 to 10 days after full bloom and decreased rapidly thereafter. The cytokinin level in the seed remained high until the 28th day after full bloom. The quantity of cytokinins in pulp per fruit increased from the 10th day after full bloom, the maximum being attained around the 50th day after full bloom. Similarly, the amount of cytokinins per seed increased from the 10th day after full bloom, reaching a peak on the 40th day and decreasing gradually thereafter.

A high percentage of fruit set in mango was persistently maintained by supplying 6-benzylaminopurine (1.5 × 10³ micromolar) onto the panicle at the anthesis stage and by supplying gibberellic acid (7.2 × 10² micromolar) and naphthalene acetamide (3.1 × 10 micromolar) at the young fruit stage.

     

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.     

Functional Analyses of LONELY GUY Cytokinin-Activating Enzymes Reveal the Importance of the Direct Activation Pathway in Arabidopsis

Cytokinins play crucial roles in diverse aspects of plant growth and development. Spatiotemporal distribution of bioactive cytokinins is finely regulated by metabolic enzymes. LONELY GUY (LOG) was previously identified as a cytokinin-activating enzyme that works in the direct activation pathway in rice (Oryza sativa) shoot meristems. In this work, nine Arabidopsis thaliana LOG genes (At LOG1 to LOG9) were predicted as homologs of rice LOG. Seven At LOGs, which are localized in the cytosol and nuclei, had enzymatic activities equivalent to that of rice LOG. Conditional overexpression of At LOGs in transgenic Arabidopsis reduced the content of N⁶-(Δ²-isopentenyl)adenine (iP) riboside 5′-phosphates and increased the levels of iP and the glucosides. Multiple mutants of At LOGs showed a lower sensitivity to iP riboside in terms of lateral root formation and altered root and shoot morphology. Analyses of At LOG promoter:β-glucuronidase fusion genes revealed differential expression of LOGs in various tissues during plant development. Ectopic overexpression showed pleiotropic phenotypes, such as promotion of cell division in embryos and leaf vascular tissues, reduced apical dominance, and a delay of leaf senescence. Our results strongly suggest that the direct activation pathway via LOGs plays a pivotal role in regulating cytokinin activity during normal growth and development in Arabidopsis.     

Vacuolar and cytosolic cytokinin dehydrogenases of Arabidopsis thaliana: Heterologous expression,purification and properties

The catabolism of cytokinins is a vital component of hormonal regulation, contributing to the control of active forms of cytokinins and their cellular distribution. The enzyme catalyzing the irreversible cleavage of N⁶-side chains from cytokinins is a flavoprotein classified as cytokinin dehydrogenase (CKX, EC 1.5.99.12). CKXs also show low cytokinin oxidase activity, but molecular oxygen is a comparatively poor electron acceptor. The CKX gene family of Arabidopsis thaliana comprises seven members. Four code for proteins secreted to the apoplast, the remainder are not secreted. Two are targeted to the vacuoles and one is restricted to the cytosol. This study presents the purification and characterization of each of these non-secreted CKX enzymes and substrate specificities are discussed with respect to their compartmentation. Vacuolar enzymes AtCKX1 and AtCKX3 were produced in Pichia pastoris and cytosolic enzyme AtCKX7 was expressed in Escherichia coli. The recombinant proteins were purified by column chromatography. All enzymes preferred synthetic electron acceptors over oxygen, namely potassium ferricyanide and 2,3-dimetoxy-5-methyl-1,4-benzoquinone (Q₀). In slightly acidic conditions (pH 5.0), N⁶-(2-isopentenyl)adenine 9-glucoside (iP9G) was the best substrate for AtCKX1 and AtCKX7, whereas AtCKX3 preferentially degraded N⁶-(2-isopentenyl)adenine 9-riboside-5′-monophosphate (iPMP). Moreover, vacuolar AtCKX enzymes in certain conditions degraded N⁶-(2-isopentenyl)adenine di- and triphosphates two to five times more effectively than its monophosphate.     

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.     

Metabolism of cytokinin: Phosphoribosylation of cytokinin bases by adenine phosphoribosyltransferase from wheat germ

Adenine phosphoribosyltransferase (AMP:pyrophosphate phosphoribosyltransferase EC 2.4.2.8) which catalyzes the phosphoribosylation of cytokinin bases and adenine to form the corresponding nucleotides were partially purified from the cytosol of wheat (Triticum aestivum) germ. This enzyme (molecular weight, 23,000 ± 500) phosphoribosylates the bases at an optimum Mg²⁺ concentration of 5 mm and optimum pH of 7.5 (50 mm Tris-HCl buffer). K_m values for N⁶-(Δ²-isopentenyl)adenine, N⁶-furfuryladenine, N⁶-benzyladenine, and adenine are 130, 110, 154, and 74 μm, respectively, in 50 mm Tris-HCl buffer (pH 7.5) at 37 °C. Hypoxanthine and guanine are not substrates for the enzyme. In concerting with other cytokinin metabolic enzymes, this enzyme may play a significant role in maintaining the supply of adequate levels of “active cytokinin.”     

Cytokinins from the Moss Physcomitrella patens

Gametophore-over-producing mutants of the moss, Physcomitrella patens, when grown in liquid culture export high levels of cytokinin into their culture medium. The cytokinin produced by these mutants is postulated to account for their peculiar phenotype, that of mosses treated with exogenous cytokinin. N⁶-(Δ²-isopentenyl)adenine, the major cytokinin, has been identified previously in two of these mutants (Wang, Cove, Beutelmann, Hartmann 1980 Phytochemistry 19: 1103-1105) and now in additional representatives. A second cytokinin, zeatin, has been identified by its chromatographic behavior and mass spectrum including chemical ionization mass spectrometry of its permethyl derivative.     

Streptococcal 5′-Nucleotidase A (S5nA), a Novel Streptococcus pyogenes Virulence Factor That Facilitates Immune Evasion

Streptococcus pyogenes is an important human pathogen that causes a wide range of diseases. Using bioinformatics analysis of the complete S. pyogenes strain SF370 genome, we have identified a novel S. pyogenes virulence factor, which we termed streptococcal 5′-nucleotidase A (S5nA). A recombinant form of S5nA hydrolyzed AMP and ADP, but not ATP, to generate the immunomodulatory molecule adenosine. Michaelis-Menten kinetics revealed a K_m of 169 μm and a V_max of 7550 nmol/mg/min for the substrate AMP. Furthermore, recombinant S5nA acted synergistically with S. pyogenes nuclease A to generate macrophage-toxic deoxyadenosine from DNA. The enzyme showed optimal activity between pH 5 and pH 6.5 and between 37 and 47 °C. Like other 5′-nucleotidases, S5nA requires divalent cations and was active in the presence of Mg²⁺, Ca²⁺, or Mn²⁺. However, Zn²⁺ inhibited the enzymatic activity. Structural modeling combined with mutational analysis revealed a highly conserved catalytic dyad as well as conserved substrate and cation-binding sites. Recombinant S5nA significantly increased the survival of the non-pathogenic bacterium Lactococcus lactis during a human whole blood killing assay in a dose-dependent manner, suggesting a role as an S. pyogenes virulence factor. In conclusion, we have identified a novel S. pyogenes enzyme with 5′-nucleotidase activity and immune evasion properties.