Isolation of a gene encoding a glycosylated cytokinin oxidase from maize

The major cytokinin oxidase in immature maize kernels was purified to homogeneity. Selected tryptic peptides were used to design degenerate oligonucleotide primers for PCR isolation of a fragment of the oxidase gene. Hybridization of the PCR fragment to a maize genomic library allowed isolation of a full-length cytokinin oxidase gene (ckx1). The gene encodes a protein of approximately 57 kDa that possesses a signal peptide, eight consensus N-glycosylation sequences and a consensus FAD binding sequence. Expression of ckx1 in Pichia caused secretion of active glycosylated cytokinin oxidase that contains a substrate-reducible FAD. The gene displays sequence homology with a putative oxidoreductase from Arabidopsis thaliana and with the fas5 gene from Rhodococcus fascians.     

Dynamics of expression and distribution of cytokinin oxidase/dehydrogenase in developing maize kernels

Maturing maize kernels are a rich source of cytokinins and cytokinin oxidase/dehydrogenase activity, but the relationship between kernel development, cytokinin levels, the induction of cytokinin oxidase/dehydrogenase and the control of cell division is not known. Using polyclonal antibodies raised against recombinant maize cytokinin oxidase/dehydrogenase, we investigated the appearance of cytokinin oxidase/dehydrogenase (ZmCKX1) in both hybrid and inbred maize kernels as a function of time after pollination. Cytokinin oxidase/dehydrogenase was detected by five days after pollination (5 DAP) in a hybrid line, but significantly later in inbred lines. The bulk of the cytokinin oxidase/dehydrogenase detected was associated with the embryo and placental/chalazal region of the kernels rather than with the endosperm. We identified additional maize sequences in the database that appear to encode cytokinin oxidase/dehydrogenase gene family members and correspond closely with a subset of the ten cytokinin oxidase/dehydrogenase genes identified in the rice genome. Gene expression of Zmckx1 was examined by RT-PCR in immature kernels and compared with that of three putative maize cytokinin oxidase/dehydrogenase homologs. We conclude that the manipulation of kernel cytokinin levels to increase endosperm cell division will require a more detailed understanding of specific expression patterns and localization of multiple cytokinin oxidase/dehydrogenases within kernels.     

Rate enhancement of cytokinin oxidase/dehydrogenase using 2,6-dichloroindophenol as an electron acceptor

Cytokinin oxidase/dehydrogenase degrades cytokinins by dehydrogenating the N6-C1 bond of cytokinins. The resulting imine is then hydrolyzed. For example, isopentenyl-adenine is cleaved into 3-methyl-2-butenal (isopentenyl-aldehyde) and adenine . The reducing equivalents from dehydrogenation are transferred to an unknown sink, in vivo. It has been hypothesized that the enzyme requires oxygen , possibly resulting in the formation of hydrogen peroxide. 2,6-dichloroindophenol (DCPIP) can function as an acceptor of reducing equivalents for in vitro cytokinin oxidase/dehydrogenase reactions. For the predominant cytokinin oxidase/dehydrogenase in maize, ZmCKX1, the addition of DCPIP to in vitro reactions increases the reaction rate to nearly 4000-fold faster than the oxygen-dependent rate. Further, the change in absorbance of DCPIP at 600 nm, as it is reduced, forms the basis for an assay suitable for following biochemical purification of cytokinin oxidase/dehydrogenases , detailed kinetic studies , and rapid measurement of cytokinin oxidase/dehydrogenase activity in large numbers of samples.     

High-level expression and characterization of Zea mays cytokinin oxidase/dehydrogenase in Yarrowia lipolytica

Cytokinin oxidase/dehydrogenase (CKO/CKX) is a flavoenzyme, which irreversibly inactivates cytokinins by severing the isoprenoid side chain from the adenine/adenosine moiety. There are several genes coding for the enzyme in maize (Zea mays). A Z. mays CKO1 cDNA was cloned in the yeast Yarrowia lipolytica to achieve heterologous protein expression. The recombinant ZmCKO1 was recovered from cultures of transformed yeasts and purified using several chromatographic steps. The enzyme was obtained as a homogeneous protein in a remarkably high-yield and its molecular and kinetic properties were characterized. The enzyme showed a molecular mass of 69 kDa, pI was 6.3. Neutral sugar content of the molecule was 22%. Absorption and fluorescence spectra were in accordance with the presence of FAD as a cofactor. Peptide mass fingerprinting using MALDI-MS correctly assigned the enzyme in MSDB protein database. The enzyme showed a relatively high degree of thermostability (T50=55 degrees C for 30 min incubation). The following pH optimum and K(m) values were determined for natural substrates (measured in the oxidase mode): pH 8.0 for isopentenyl adenine (K(m)=0.5 microM), pH 7.6 for isopentenyl adenosine (K(m)=1.9 microM), pH 7.9 for zeatin (K(m)=1.5 microM) and pH 7.3 for zeatin riboside (K(m)=2.0 microM). ZmCKO1, functioning in the oxidase mode, catalyzes the production of one molecule of H2O2 per one molecule of cytokinin substrate. This finding represents clear evidence for the existence of dual enzyme functionality (oxygen serves as a cosubstrate in the absence of better electron acceptors).     

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.     

O-glucosylation of cis-zeatin in maize. Characterization of genes,enzymes, and endogenous cytokinins

trans-Zeatin is a major and ubiquitous cytokinin in higher plants. cis-Zeatin has traditionally been viewed as an adjunct with low activity and rare occurrence. Recent reports of cis-zeatin and its derivatives as the predominant cytokinin components in some plant tissues may call for a different perspective on cis-isomers. The existence of a maize (Zea mays) gene (cisZOG1) encoding an O-glucosyltransferase specific to cis-zeatin (R.C. Martin, M.C. Mok, J.E. Habben, D.W.S. Mok [2001] Proc Natl Acad Sci USA 98: 5922-5926) lends further support to this view. Results described here include the isolation of a second maize cisZOG gene, differential expression of cisZOG1 and cisZOG2, and identification of substantial amounts of cis-isomers in maize tissues. The open reading frame of cisZOG2 has 98.3% identity to cisZOG1 at the nucleotide level and 97.8% at the amino acid level. The upstream regions contain common and unique segments. The recombinant enzymes have similar properties, K(m) values of 46 and 96 microM, respectively, for cis-zeatin and a pH optimum of 7.5. Other cytokinins, including N(6)-(delta(2)-isopentenyl)adenine, trans-zeatin, benzyladenine, kinetin, and thidiazuron inhibited the reaction. Expression of cisZOG1 was high in maize roots and kernels, whereas cisZOG2 expression was high in roots but low in kernels. cis-Zeatin, cis-zeatin riboside, and their O-glucosides were detected in all maize tissues, with immature kernels containing very high levels of the O-glucoside of cis-zeatin riboside. The results are a clear indication that O-glucosylation of cis-zeatin is a natural metabolic process in maize. Whether cis-zeatin serves as a precursor to the active trans-isomer or has any other unique function remains to be demonstrated.     

Cytokinin oxidase or dehydrogenase? Mechanism of cytokinin degradation in cereals.

An enzyme degrading cytokinins with isoprenoid side chain, previously named cytokinin oxidase, was purified to near homogeneity from wheat and barley grains. New techniques were developed for the enzyme activity assay and staining on native electrophoretic gels to identify the protein. The purified wheat enzyme is a monomer 60 kDa, its N-terminal amino-acid sequence shows similarity to hypothetical cytokinin oxidase genes from Arabidopsis thaliana, but not to the enzyme from maize. N6-isopentenyl-2-(2-hydroxyethylamino)-9-methyladenine is the best substrate from all the cytokinins tested. Interestingly, oxygen was not required and hydrogen peroxide not produced during the catalytic reaction, so the enzyme behaves as a dehydrogenase rather than an oxidase. This was confirmed by the ability of the enzyme to transfer electrons to artificial electron acceptors, such as phenazine methosulfate and 2,6-dichlorophenol-indophenol. 2,3-Dimethoxy-5-methyl-1,4-benzoquinone, a precursor of the naturally occurring electron acceptor ubiquinone, readily interacts with the enzyme in micromolar concentrations. Typical flavoenzyme inhibitors such as acriflavine and diphenyleneiodonium inhibited this enzyme activity. Presence of the flavin cofactor in the enzyme was confirmed by differential pulse polarography and by measuring the fluorescence emission spectrum. Possible existence of a second redox centre is discussed.     

Cytokinin Oxidase from Phaseolus vulgaris Callus Tissues : Enhanced in Vitro Activity of the Enzyme in the Presence of Copper-Imidazole Complexes

The effects of metal ions on cytokinin oxidase activity extracted from callus tissues of Phaseolus vulgaris L. cv Great Northern have been examined using an assay based on the oxidation of N⁶-(Δ²-isopentenyl)-adenine-2,8-³H (i⁶ Ade) to adenine (Ade). The addition of cupric ions to reaction mixtures containing imidazole buffer markedly enhanced cytokinin oxidase activity. In the presence of optimal concentrations of copper and imidazole, cytokinin oxidase activity was stimulated more than 20-fold. The effect was enzyme dependent, specific for copper, and observed only in the presence of imidazole. The substrate specificity of the copper-imidazole enhanced reaction, as judged by substrate competition tests, was the same as that observed in the absence of copper and imidazole. Similarly, in tests involving DEAE-cellulose chromatography, elution profiles of cytokinin oxidase activity determined using a copper-imidazole enhanced assay were identical to those obtained using an assay without copper and imidazole. On the basis of these results, the addition of copper and imidazole to reaction mixtures used to assay for cytokinin oxidase activity is judged to provide a reliable and specific assay of greatly enhanced sensitivity for the enzyme. The mechanism by which copper and imidazole enhance cytokinin oxidase activity is not certain, but the reaction catalyzed by the enzyme was not inhibited by anaerobic conditions when these reagents were present. This observation suggests that copper-imidazole complexes are substituting for oxygen in the reaction mechanism by which cytokinin oxidase effects cleavage of the N⁶-side chain of i⁶Ade.     

Cytokinin Utilization by Adenine-Requiring Mutants of the Yeast Saccharomyces cerevisiae

By monitoring the growth of several adenine auxotrophs of the yeast Saccharomyces cerevisiae on cytokinin-supplemented media, we have demonstrated that this organism can utilize some of these derivatives as a source of adenine. Growth of a mutant lacking adenylosuccinate synthetase suggests that the conversion of cytokinins to adenine does not involve a hypoxanthine intermediate and may be catalyzed by an enzyme analogous to cytokinin oxidase.     

Cytokinin oxidase: Biochemical features and physiological significance

The catabolism of cytokinin in plant tissues appears to be due, in large part, to the activity of a specific enzyme, cytokinin oxidase. This enzyme catalyses the oxidation of cytokinin substrates bearing unsaturated isoprenoid side chains, using molecular oxygen as the oxidant. In general, substrate specificity is highly conserved and cytokinin substrates bearing saturated or cyclic side chains do not serve as substrates for most cytokinin oxidases tested to date. Despite variation in molecular properties of the enzyme from a number of higher plants, oxygen is always required for the reaction. Cytokinin oxidases from several sources have been shown to be glycosylated. Cytokinin oxidase activity appears to be universally inhibited by cytokinin-active urea derivatives. Auxin has been reported to act as an allosteric regulator which increases activity of the enzyme.
Cytokinin oxidase activity is subject to tight regulation. Levels of the enzyme are controlled by a mechanism sensitive to cytokinin supply. The up-regulation of cytokinin oxidase expression in response to exogenous application of cytokinin suggests that the metabolic fate of exogenously applied cytokinins may not accurately mimic that of the endogenous compounds.
Cytokinin oxidase is believed to be a copper-containing amine oxidase (EC 1.4.3.6). Considerable evidence strongly supports a common mechanism for amine oxidases. It is possible that advances in understanding of other amine oxidases could be extrapolated to increase our understanding of cytokinin oxidase at the molecular level. This is discussed with reference to what is currently known about the catalytic mechanism of the enzyme. The possibility of pyrroloquinoline quinone, or a closely related compound, as a redox cofactor of cytokinin oxidase is considered, as are the implications of the glycosylated nature of the enzyme for its regulation and compartmentalisation within the cell.     

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.     

Kinetic and chemical analyses of the cytokinin dehydrogenase-catalysed reaction: correlations with the crystal structure

CKX (cytokinin dehydrogenase) is a flavoprotein that cleaves cytokinins to adenine and the corresponding side-chain aldehyde using a quinone-type electron acceptor. In the present study, reactions of maize (Zea mays) CKX with five different substrates (N6-isopentenyladenine, trans-zeatin, kinetin, p-topolin and N-methyl-isopentenyladenine) were studied. By using stopped-flow analysis of the reductive half-reaction, spectral intermediates were observed indicative of the transient formation of a binary enzyme-product complex between the cytokinin imine and the reduced enzyme. The reduction rate was high for isoprenoid cytokinins that showed formation of a charge-transfer complex of reduced enzyme with bound cytokinin imine. For the other cytokinins, flavin reduction was slow and no charge-transfer intermediates were observed. The binary complex of reduced enzyme and imine product intermediate decays relatively slowly to form an unbound product, cytokinin imine, which accumulates in the reaction mixture. The imine product only very slowly hydrolyses to adenine and an aldehyde derived from the cytokinin N6 side-chain. Mixing of the substrate-reduced enzyme with Cu2+/imidazole as an electron acceptor to monitor the oxidative half-reaction revealed a high rate of electron transfer for this type of electron acceptor when using N6-isopentenyladenine. The stability of the cytokinin imine products allowed their fragmentation analysis and structure assessment by Q-TOF (quadrupole-time-of-flight) MS/MS. Correlations of the kinetic data with the known crystal structure are discussed for reactions with different cytokinins.     

Ligand-binding properties and subcellular localization of maize cytokinin receptors

The ligand-binding properties of the maize (Zea mays L.) cytokinin receptors ZmHK1, ZmHK2, and ZmHK3a have been characterized using cytokinin binding assays with living cells or membrane fractions. According to affinity measurements, ZmHK1 preferred N(6)-(Δ(2)-isopentenyl)adenine (iP) and had nearly equal affinities to trans-zeatin (tZ) and cis-zeatin (cZ). ZmHK2 preferred tZ and iP to cZ, while ZmHK3a preferred iP. Only ZmHK2 had a high affinity to dihydrozeatin (DZ). Analysis of subcellular fractions from leaves and roots of maize seedlings revealed specific binding of tZ in the microsome fraction but not in chloroplasts or mitochondria. In competitive binding assays with microsomes, tZ and iP were potent competitors of [(3)H]tZ while cZ demonstrated significantly lower affinity; adenine was almost ineffective. The binding specificities of microsomes from leaf and root cells for cytokinins were consistent with the expression pattern of the ZmHKs and our results on individual receptor properties. Aqueous two-phase partitioning and sucrose density-gradient centrifugation followed by immunological detection with monoclonal antibody showed that ZmHK1 was associated with the endoplasmic reticulum (ER). This was corroborated by observations of the subcellular localization of ZmHK1 fusions with green fluorescent protein in maize protoplasts. All these data strongly suggest that at least a part of cytokinin perception occurs in the ER.     

Heterologous expression and characterization of mouse spermine oxidase

Polyamine oxidases are key enzymes responsible of the polyamine interconversion metabolism in animal cells. Recently, a novel enzyme belonging to this class of enzymes has been characterized for its capability to oxidize preferentially spermine and designated as spermine oxidase. This is a flavin adenine dinucleotide-containing enzyme, and it has been expressed both in vitro and in vivo systems. The primary structure of mouse spermine oxidase (mSMO) was deduced from a cDNA clone (Image Clone 264769) recovered by a data base search utilizing the human counterpart of polyamine oxidases, PAOh1. The open reading frame predicts a 555-amino acid protein with a calculated M(r) of 61,852.30, which shows a 95.1% identity with PAOh1. To understand the biochemical properties of mSMO and its structure/function relationship, the mSMO cDNA has been subcloned and expressed in secreted and secreted-tagged forms into Escherichia coli BL21 DE3 cells. The recombinant enzyme shows an optimal pH value of 8.0 and is able to oxidize rapidly spermine to spermidine and 3-aminopropanal and fails to act upon spermidine and N(1)-acetylpolyamines. The purified recombinant-tagged form enzyme (M(r) approximately 68,000) has K(m) and k(cat) values of 90 microm and 4.5 s(-1), respectively, using spermine as substrate at pH 8.0. Molecular modeling of mSMO protein based on maize polyamine oxidase three-dimensional structure suggests that the general features of maize polyamine oxidase active site are conserved in mSMO.     

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.     

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.