Plant Growth Regulation 32: 315327, 2000. 2000 Kluwer Academic Publishers. Printed in the Netherlands.

315

Degradation of cytokinins by cytokinin oxidases in plants

Petr Galuszka*, Ivo Frbort, Marek ebela and Pavel Pec
Department of Biochemistry, Faculty of Science, Palack University, lechtitelu 11, 783 71, Olomouc, Czech Republic; *Author for correspondence (e-mail: galuszka@risc.upol.cz; phone: ++420-68-5241198; fax: ++420-68-5221332)

Key words: Cytokinin oxidase, Cytokinins, Degradation, Dehydrogenation, Function Abstract The degradation metabolism of cytokinins is an important process that controls the levels of cytokinin active forms and their distribution in plant tissues. It appears to be due, in large part, to the activity of a specic enzyme, cytokinin oxidase. This review attempts to collate the limited information available about this enzyme and introduce new facts, obtained in our laboratory, concerning the mechanism of degradation of cytokinins bearing unsaturated isoprene side chains. However, complete clarication of the effects of cytokinin oxidase on cytokinin regulation and its molecular and biochemical properties will be dependent upon the purication of the protein with cytokinin oxidase activity to homogeneity and progress in the development of requisite molecular probes. Abbreviations: AMP adenosine 5monophosphate, ckx1 gene encoding cytokinin oxidase from maize, CKOx cytokinin oxidase, DEAE diethylaminoethyl cellulose, FAD avin adenine dinucleotide, HPLC high performance liquid chromatography, iP isopentenyladenine, iPR isopentenyladenosine, ipt gene encoding isopentenyl transferase, K m Michaelis constant, NAD + nicotinamide adenine dinucleotide, Na 2EDTA sodium ethylenediamine tetraacetate, PCR polymerase chain reaction, SDS PAGE polyacrylamide gel electrophoresis in sodium dodecyl sulphate, TLC thin layer chromatography, t-Z trans-zeatin Introduction There are two main metabolic pathways leading to a complete loss in biological activity of cytokinins in plant cells: formation of N-conjugates and the cleavage of the N 6-side chain of the cytokinin molecule. All types of cytokinins can be glucosylated via the nitrogens of the purine ring and, in zeatin types, also on the hydroxy group of the N 6-side chain by specic glucosyltransferases eg. cytokinin-7-glucosyltransferase (EC 2.4.1.118) puried from radish cotyledons, zeatin O-glucosyltransferase (EC 2.4.1.203) and zeatin O-xylosyltransferase (EC 2.4.1.204) both isolated from immature bean embryos (Letham and Palni 1983; Van kov 1999). Lupinic acid, a conjugate of zeatin with alanine, which develops through the action of -(9-cytokinin)alanine synthase (EC 4.2.99.13) catalysis seems to be also biologically inactive and stable (Entsch et al. 1983). Cytokinin oxidase (CKOx) is the only known plant enzyme capable of degrading naturally occurring cytokinins. It was discovered rst in a crude extract of cultured tobacco tissue (Pac es et al. 1971) and has since been demonstrated in a number of higher and lower plants. Regardless of source, the cytokinin oxidase from higher plants specically catalyses only the cleavage of the N 6-unsaturated isoprene side chain of the cytokinin molecule. In the case of N 6-isopentenyladenine, the products of the reaction were identied as adenine (McGaw and Horgan 1983) and 3-methyl-2-butenal (Brownlee et al. 1975) originating from the oxidation of the isoprene side-chain. It is assumed that this enzymatic mechanism is the major one through which the endogenous levels and distribution of native cytokinins, i.e. zeatin, isopentenyladenine and their derivatives can be controlled in plant tissue (Hare and

316 van Staden 1994). The presence of an enzyme capable of degrading aromatic or saturated side chain cytokinins has not yet been described in higher plants. Although CKOx has been intensively studied during the past three decades, there is still very limited knowledge about its molecular and biochemical properties and the reaction mechanism. CKOx had been mechanistically classied for many years as a copper-containing amine oxidase (Hare and van Staden 1994), but recent investigations have begun to reveal a new insight into the mechanism of cytokinin degradation through dehydrogenation. The interesting and still not entirely explained question of enhancement of CKOx activity by imidazole-copper complexes and some phenolic compounds is unresolved. Molecular characterisation of the enzyme is made difficult by very low concentrations of the CKOx protein being present in plant tissues. A better understanding of the role of CKOx in plant development may result from the recent success in cloning the CKOx gene. Recombinant protein with CKOx activity has been prepared by expression in moss protoplasts (Houba-Herin et al. 1999) and yeast (Morris et al. 1999), and its properties have been described. An enzymatic system capable of degrading endogenous and exogenously supplied cytokinins without apparent participation of the CKOx has been described in many other plant sources, such as maple (Acer pseudoplatanus) (Doree and Guern 1973), lettuce (Lactuca sativa) (Miernyk and Blaydes 1977), pea (Pisum sativum) (King and van Staden 1987), alder (Alnus glutinosa) (Henson 1978), lupine (Lupinus angustifolius) (Parker et al. 1978) and sunower (Helianthus tuberosus) (Palmer et al. 1984). A unique CKOx activity that is capable of rapidly degrading the exogenous supply of kinetin, N 6-furfuryladenine, which is not observed at higher plants, was demonstrated in protonemal cells of the moss Funaria hygrometrica (Gerhauser and Bopp 1990). A similar degradative system of cytokinins was found in the yeast Saccharomyces cerevisiae (van Kast and Laten 1987). Another organism, outside of green plants, in which CKOx activity has been identied, is the cellular slime mold Dictyostelium discoideum (Armstrong and Firtel 1989). Root apices are sites for the synthesis of cytokinins that are subsequently exported to meristematic tissues in other developing organs. Therefore it is not surprising that the levels of CKOx activity are several fold higher in roots than in the above-ground parts of plants (Jones and Schreiber 1997). The mechanism of cytokinin deactivation is different in various organisms. For example, in radish roots there is insignicant oxidative side-chain cleavage of externally applied cytokinins as compared to other plants (McGaw et al. 1985), which degrade cytokinins via CKOx. In Nicotiana and Alnus, side chain cleavage appears to be the major metabolic pathway of both endogenous and exogenously applied forms of cytokinins (Henson 1978; Singh et al. 1992). Generally, it would seem that there is a balance between oxidative and conjugative (e.g. the formation of N-glucoside and N-alanyl conjugates) metabolism. The largest quantities of CKOx are found in developing seeds. There are also differences within and between species in the compartmentation of cytokinin degradation activity within the seed. In the seed coats of lupine and soybean, the rate of endogenous cytokinin degradation is negligible and there is a large accumulation of biologically active cytokinins (Singh et al. 1988; Zhang and Letham 1990). In contrast, the degradation of labeled zeatin in bean, which may be ascribed to CKOx, was more rapid in the seed coat than in the embryo (Turner et al. 1985). In maize kernels, the amount of CKOx in the pericarp and embryo is sev-

Cytokinin oxidase sources and its compartmentation The activity of CKOx has been characterised in a number of plant species, including maize kernels (Zea mays) (Burch and Horgan 1989; McGaw and Horgan 1983; Whitty and Hall 1974) and seedlings (Burch and Horgan 1992), wheat germs (Triticum aestivum) (Burch and Horgan 1989; Frbort et al. 1999; Galuszka et al. 1998; Kamnek et al. 1994; Laloue and Fox 1989), periwinkle crown gall tumor tissue (Vinca rosea) (McGaw and Horgan 1983), oilseed rape leaves (Brassica napus) (Shukla and Sawhney 1997), sugar beet (Beta vulgaris) (Kevers et al. 1997), kidney bean (Phaseolus vulgaris and P. lunatus) (Chateld and Armstrong 1986; Kamnek and Armstrong 1990), and petunia (Petunia hybrida) (Auer et al. 1999), poplar (Populus euroamericana) (Motyka and Kamnek 1992), tobacco (Nicotiana tabacum) (Motyka and Kamnek 1992; Pac es et al. 1971; Redig et al. 1997) and soybean (Glycine max) (Jger et al. 1997) callus tissues. Recently we have found out a very high level of CKOx activity in barley seeds (Hordeum vulgare) which seem to be a very good source of CKOx for future investigations.

317 eral times higher than in the endosperm (Jones et al. 1992). The tissue with the most abundant CKOx activity is the pedicel/placenta-chalazal tissue, the link between the endosperm and other developing parts of plant organism (Jones et al. 1992). Thus, this tissue may function as the initial point for the metabolic control of active cytokinins. ditions when copper-imidazole complexes were present, which observation suggests that these complexes can substitute for oxygen in the reaction mechanism. Stimulating effects of other metal ions were not observed (Chateld and Armstrong 1987). Recently we substituted Cu-imidazole complexes with some articial electron acceptors such as phenazine methosulphate, 2,6-dichlorophenol-indophenol, and methylene blue in the reaction mixtures and obtained a similar or even larger enhancement of CKOx activity; tetrazolium salts showed a weaker effect. On the other hand, potassium ferricyanide and NAD + did not enhance the CKOx activity at all. 2,3-Dimethoxy5-methyl-1,4-benzoquinone, the precursor of naturally occurring quinone cofactors, increases CKOx activity at micromolar concentrations (Frbort et al. 1999; Galuszka et al. 1998). Moreover, when the incubation of the reaction mixture was performed under anaerobic conditions, reaction rate was the same as that attained with an air-saturated reaction mixture. Hence, the absence of oxygen does not affect CKOx activity. Subsequently the production of hydrogen peroxide, the hypothetical product of the oxidative degradation of isopentenyladenosine, was tested without any positive effect (Galuszka et al. 1998). Therefore we support the theory that any enzyme, which can degrade cytokinins bearing an unsaturated sidechain, will operate via dehydrogenation (Figure 1). Several years ago, Armstrong and co-workers rst described the activation of CKOx by phenolic compounds (Armstrong 1994). The most effective compounds were 2,6-dimethoxyphenol, which enhanced the activity of CKOx from bean callus tissue approximately 50-fold, and a naturally occurring acetosyringone (3,5-dimethoxy-4-hydroxy-acetophenone, Figure 2). As described above, we found that some articial electron acceptors can replace the copper-imidazole complexes in stimulating CKOx activity. It is likely that dimethoxyphenol and other similar phenolic compounds can also work as electron acceptors, drawing electrons from the N 6 to C 1 bond of the cytokinin molecule and affecting the dehydrogenation of the side chain. Other compounds similar to the 2,6-dimethoxyphenol tested by Armstrong exhibited the same effects. Caffeic acid (3,4-dihydroxy-transcinnamic acid) enhanced the CKOx activity from tissue cultures of tobacco expressing the cytokinin synthesis gene ipt two-fold in Tris-HCl buffer, but no increase was observed in an imidazole buffer containing CuCl 2 (Wang and Letham 1995). This also provides supporting evidence for the hypothesis that the

Possible mechanism of the degradative function of CKOx For some time, the mechanism of cleavage of the cytokinin N 6-side chain was considered to be similar to the oxidative deamination of biogenic amines by amine oxidases. On the basis of certain non-specic evidence, CKOx was classied as a copper-containing amine oxidase (EC 1.4.3.6) which catalyses the conversion of biogenic primary diamines to corresponding aminoaldehydes, ammonia and hydrogen peroxide through imine intermediate (McIntire and Hartmann 1992). Indeed, it has been also shown that an imine intermediate is formed during the reaction catalysed by CKOx (Laloue and Fox 1985). Cytokinin oxidases isolated from wheat and from maize were strongly inhibited by aminoacetonitrile (Burch and Horgan 1989) and cyanide (McGaw and Horgan 1983; Whitty and Hall 1974), as described for the amine oxidases. However, cytokinins are secondary amines and it is generally known that the amine oxidases do not oxidise secondary amines (McIntire and Hartmann 1992). As shown recently, naturally occurring and synthetic cytokinins are not substrates of a pea amine oxidase, on the contrary, they are weak competitive inhibitors (Galuszka et al. 1998). The activation of CKOx by cupric ions in the presence of imidazole is not a completely claried issue. This was rst observed with the enzyme isolated from Phaseolus vulgaris callus tissue (Chateld and Armstrong 1987). The addition of cupric ions in millimolar concentration to a reaction mixture containing imidazole buffer enhanced CKOx activity more than 20-fold. The effect was specic only for the copper complexed with imidazole, but not for free copper, contrary to the action of copper amine oxidases. Similar increases in the CKOx activity in vitro by cupric ions have been found in tobacco callus culture (Motyka et al. 1996). Moreover, Na 2EDTA removed the stimulating effect of cupric ions on CKOx activity and did not affect its activity in their absence. The CKOx reaction was not inhibited by anaerobic con-

318

Figure 1. Probable reaction mechanism for the degradation of isopentenyladenine by cytokinin oxidase (dehydrogenase). An electron acceptor (A) withdraws two electrons from ketimine form of the substrate via the avin cofactor in parallel with the dehydrogenation of the substrate, which is then hydrolysed to adenine and 3-methyl-2-butenal.

copper-imidazole complex works as an electron acceptor in cytokinin degradation. The total aminoacid sequence, which has recently been obtained by Morris et al. (1999), contains a consensual FAD binding domain. The presence of covalently bound avin cofactor in the CKOx molecule was conrmed by differential pulse polarography and inhibition studies with specic inhibitors of avincontaining oxidoreductases (acridine dyes, diphenyleneiodonium) (our unpublished results). It seems possible that the electrons from the substrate could be withdrawn through a avin cofactor to some naturally occurring electron acceptor (e.g. ubiquinone). The very low CKOx activity observed in the absence of electron acceptors may be due to the presence of a natural acceptor in enzyme preparations or to the existence of an oxidative degradation process. A similar enzyme is known that interacts with electron acceptors xanthine oxidase (EC 1.1.3.22). As an oxidase, xanthine oxidase converts xanthine to hypoxanthine and ureic acid with oxygen consumption. However, it can also act without the oxygen but in the presence of an electron acceptor as a dehydrogenase, depending on the reaction conditions (Bray 1975).

Determination of cytokinin oxidase activity The most common in vitro assay for CKOx activity is based on measuring the rate of conversion of ringlabelled N 6-isopentenyladenine/adenosine to adenine/ adenosine. After the incubation of the enzyme preparation in a reaction mixture (usually at 37 C), the tritium-labelled adenine/adenosine formed during the reaction is determined by TLC on microcrystalline cellulose plates or HPLC. After separation, the active zones/fractions are measured using the liquid scintillation technique, or the reaction products analysed by reversed phase HPLC with a radioactivity monitor. This enzyme reaction has been carried out in various buffers over a pH range from 6.0 to 9.0 depending on plant source. This radioisotopic method is very time and labour consuming and needs specialised equipment, therefore new and easier methods were desirable. A spectrophotometric assay, based on a change in the UV absorption associated with the cleavage of the cytokinin molecule side-chain, was rst utilised in early study on maize CKOx (Whitty and Hall 1974). A new simple and rapid colorimetric assay was described recently (Libreros-Minotta and Tipton 1995).

319

Figure 2. Structures of cytokinins and related compounds.

The assay is based on the formation of a Schiff base between the enzymatic degradation product of isopentenyladenine, 3-methyl-2-butenal, and p-aminophenol, with the absorbance maximum at 352 nm under acidic conditions. The assay has a detection limit in the submicromolar range and can be used for crude plant extracts as well as for highly puried preparations. However, this discontinuous assay is specic only for cytokinins with unsaturated isoprene chains. Recently we developed a continuous and very sensitive assay based on decolourisation, measured at

600 nm, of dichlorophenol-indophenol added to the reaction mixture as an articial electron acceptor. The assay is limited to neutral or weakly alkaline conditions and can be used for all types of cytokinins as substrates (unpublished data).

Substrates and other compounds affecting cytokinin oxidase activity CKOx is highly substrate specic despite having huge variations in its molecular properties. CKOx from all

320 higher plants appears to have a denite preference for isopentenyladenine, zeatin and their ribosides as substrates. Both isomers of zeatin are degraded with almost the same velocity. Cytokinins with saturated side-chains (e.g. dihydrozeatin and isopentyladenine) or with cyclic side-chains (e.g. kinetin and benzyladenine) are not recognised by the enzymes, with the exception of the wheat enzyme which degrades benzyladenine, though 40-fold less effectively than isopentenyladenosine (Laloue and Fox 1989) and CKOx from Funaria hygrometrica, which prefers kinetin as substrate (Gerhauser and Bopp 1990). From the above facts, it is clear that the isoprene chain bearing double bond is necessary for the binding to the active site of the enzyme. Recently we discovered that the cytokinins bearing a hydrophilic group in position 2 of the purine ring are better substrates than non-substituted molecules (Frbort et al. 1999). Wheat germ CKOx degrades 9-methyl-2-(2-hydroxyethylamino)isopentenyladenine (Figure 2) approximately twice as quickly as isopentenyladenine, the best substrate described to date. Aromatic cytokinins substituted in position 2, e.g. olomoucine - 2-(2-hydroxyethylamino)-9-methyl-N 6-benzyladenine, effectively compete with isopentenyladenine as a substrate. The substitution in the position 2 evidently allows better access and linkage to the active site of the enzyme (our unpublished results). A shift in the position of the double bond from position 2 to position 3 also leads to the loss of substrate properties (Whitty and Hall 1974). N-glucosylation or other forms of ring substitution (e.g. lupinic acid-9-alanylzeatin) may decrease the affinity of CKOx for a given cytokinin, but do not necessarily eliminate its ability to act as a substrate (McGaw and Horgan 1983). On the other hand CKOx cannot accept cytokinin nucleotides as substrates, as well as with O-glucosides (Laloue and Fox 1989). Stimulation of cytokinin degradation by aromatic cytokinins or kinetin seems to be unrealistic because these compounds had little if any effect on the degradation of isopentenyladenine even when present in tenfold excess (Kamnek and Armstrong 1990). However, these cytokinins can induce accumulation of endogenous isoprene-chain bearing cytokinins in vivo which may subsequently act as substrate-inducers of CKOx (Kamnek et al. 1997; Motyka et al. 1996). Diphenylurea and phenylurea derivatives exhibit weak cytokinin activity in tissue culture systems (Letham and Palni 1983). Since they are considerably different in structure from the purine cytokinins, it has been suggested that the urea derivatives are not active per se, but are rst metabolised to active purine derivatives (Burrows and Leworty 1976). However, molecular modelling showed that diphenylurea may have high affinity to cytokinin receptors (Fox 1992). A partially puried CKOx from Triticum aestivum has been shown to be strongly inhibited by diphenylurea and N-(2-chloro-4-pyridyl)-N-phenylurea (Laloue and Fox 1989). Thidiazuron (N-phenyl-N-1,2,3-thiadizol-5-ylurea), Figure 2, inhibited the degradation of isopentenyladenine by cell-free preparations of Phaseolus vulgaris callus tissue by approximately 50% when supplied at the same concentration as the substrate (Chateld and Armstrong 1986). The inhibitory effect of phenyl urea derivatives has been conrmed in many others CKOx abundant tissues. Kinetic studies suggest that these derivatives inhibit CKOx in a noncompetitive manner (Burch and Horgan 1989). The positive of these compounds on cell division may be therefore realised via an inhibition effect on the CKOx. This hypothesis is supported by the work of Thomas and Katterman who observed an enhancement of the endogenous cytokinin level after treatment of tobacco callus cultures with thidiazuron (Thomas and Katterman 1986). Activation of CKOx by phenolic compounds was explained above in the context of a hydrogenation mechanism of degradation. However, the similarity of this stimulation to the activation of auxin degradation catalysed by indoleacetic acid oxidase and a peroxidase activity (demonstrated by similar phenolic compounds) were pointed out (Lee et al. 1982). A type of associated action of CKOx and peroxidase has also been reported. Chateld and Armstrong observed that the CKOx activity from Phaseolus vulgaris was isolated in parallel with the peroxidase activity by chromatofocusing (Chateld and Armstrong 1988). Whitty and Hall achieved similar results with the maize enzyme (Whitty and Hall 1974), but conclusive proof about the inuence of peroxidase activity on CKOx activity and a continuity of the activity enhancement by phenolic compounds has not yet been established.

Molecular characteristics of cytokinin oxidase Due to the very low content of the enzyme in plant tissues we have only limited and insufficient knowledge about the molecular nature of CKOx. There is considerable diversity in the properties of cytokinin oxidases isolated from various plant sources. Molec-

321 ular masses of cytokinin oxidases from various plant sources have been found to be very different when estimated by gel ltration and SDS-PAGE (Table 1). The reported values range from 25.1 kDa for the enzyme from Vinca rosea to 94.4 kDa for the maize enzyme (McGaw and Horgan 1983). However these differences could be caused by inaccurate determination of molecular masses due to different levels of glycosylation or through the determination of non-homogeneous enzymes. The molecular mass of maize CKOx was recently assigned as 57.2 kDa from the total amino acid sequence (Morris et al. 1999) and our enzyme preparation from wheat shows a similar mass, in contrast to the 40 kDa determined for the wheat enzyme previously (Laloue and Fox 1989). Heterogeneity is perceptible also from the variation in pH optima over a whole physiological range, e.g., the enzymes from poplar and bean (Phaseolus lunatus) callus tissue cultures exhibit optima between 8 and 9, whereas that from maize tissue is about pH 6 (Kamnek and Armstrong 1990; McGaw and Horgan 1983; Motyka and Kamnek 1992). Isozyme variation of CKOx was demonstrated in the species Phaseolus. Two non-identical forms of the enzyme in species Phaseolus lunatus and P. vulgaris were reported which had different abilities of binding to concanavalin A (Kamnek and Armstrong 1990). This variability could be caused by different compartmentation in the plant cells. Glycosylated protein, with an optimum of ca. pH 6, could be localised in the cell wall or plasmalemma, whereas the presumably non-glycosylated enzyme has a pH optimum of 8.5 and thus may be restricted to an internal compartment which precludes access of the enzyme to endogenous cytokinins. Cell compartmentation can also explain different mechanism of N 6 bond cleavage. The enzyme in the internal structures of the cell can work as a dehydrogenase through utilisation of some membrane electron acceptor whereas classical cytokinin oxidase consuming molecular oxygen can be found free in the cytoplasm. When fractionating wheat germ enzyme preparation using a high performance chromatofocusing column, Laloue and Fox resolved two peaks of CKOx activity (Laloue and Fox 1989). The rst smaller peak was eluted with an apparent pI value of 5.0 and a second larger peak with a value of 4.75. Regarding the wheat enzyme, we also observed two activity peaks on a ceramic hydroxyapatite column. The rst isoenzyme form was eluted in the passing fraction, while the second activity peak after a weak gradient of ionic strength. Isoelectric focusing of wheat enzyme also gives several bands of CKOx activity with pI values ranging from 5.4 to 4.9 (Frbort et al. 1999; Galuszka et al. 1998). Most of the CKOx activity in tobacco callus tissue is associated with non-glycosylated enzyme (Motyka and Kamnek 1994). Cytokinin oxidase puried from transgenic tobacco tissues expressing the ipt gene yielded two activity fractions passing through a concanavalin A column (Motyka and Kamnek 1994; Zhang et al. 1995). However, the activity of the glycosylated enzyme fraction seems to be increased in transgenic tissue following derepression of the ipt gene (Motyka et al. 1996). Burch and Horgan (1989) in their work reported no indication of isozyme variation on the maize enzyme, in contrast to the identication of two active peaks after DEAE-cellulose chromatography in the latest studies on maize CKOx (Morris et al. 1999). Burch and Horgan prepared a polyclonal antibody against the Zea mays enzyme puried to near homogeneity. This antibody cross-reacted with a protein of the same molecular size from wheat seeds (Burch and Horgan 1989). Furthermore, polyclonal antibodies, raised to a 68 kDa polypeptide puried from maize seedlings, precipitated approximately 70% of the CKOx activity in the crude seedling extract and about 55-75% of the kernel extract activity (Schreiber et al. 1995). These data support the idea that multiple tissue specic CKOx isozymes appear in the same plant during development.

Cloning of the CKOx gene Recently there has been much progress in our understanding of the role of CKOx in plants. Two laboratories have independently cloned the CKOx gene from maize and expressed protein with CKOx activity. Morriss group has isolated CKOx from 40 kg maize kernels by standard purication techniques including column chromatography, obtaining 100 g of relatively pure enzyme (Morris et al. 1999). The Nterminus of the protein was blocked after SDS-PAGE, nevertheless several tryptic peptides were isolated and sequenced. Degenerate oligonucleotide primers were then designed on the basis of the sequences of selected tryptic peptides and used for PCR isolation of a fragment of the CKOx gene. Hybridisation of the PCR fragment to a maize genomic library allowed isolation of a full-length CKOx gene ckx1 (NCBI GenBank accession number AF044603).

322
Table 1. Properties of cytokinin oxidases isolated from higher plants Species Source Molecular mass pH optimum (kDa) Assay method Triticum aestivum germs gel ltration 78 SDS PAGE 60 gel ltration 78 SDS PAGE 94.4 gel ltration 88 gel ltration 5560 gel ltration 57.2 calculated from Nicotiana tabacum callus tissue DNA sequence 8.59.0 iPR 6.66 b not dened germs 40 7.5 Km (M) substrate 0.3 Specic activity (pkatmg 1) Purication -fold Reference

0.01 a

625

(Laloue and Fox 1989) (Burch and Horgan 1989)

germs

6.5

55 iP

13.26 a,c,e 44 169 b 33.3 a,c 28 000 b 16.81 a 2 970 b

3330

our unpublished data

Zea mays

kernels

6.8

850

(Burch and Horgan 1989)

kernels

6.0

19.2 iP 31 iP

175

(McGaw and Horgan 1983)

kernels

5.07.0

(Whitty and Hall 1974)

seedlings

6.8

not dened

(Burch and Horgan 1992)

recombinant protein

19 t-Z 4.3

(Morris et al. 1999)

0.04 a,f

not dened

(Motyka and Kamnek 1992; Motyka and Kamnek 1994)

Phaseolus lunatus Phaseolus vulgaris

hypocotyl callus hypocotyl callus

8.4

iP

not dened

(Kamnek and Armstrong 1990) (Chateld and Armstrong 1987)

67

6.5

0.1

0.1 a,d

not dened

Vinca rosea

crown gall tissue

gel ltration 25.1 gel ltration

7.0

iP 27.7 iP 4.0 iP

1.03 a 54.05 b 0.48 a,f

53

(McGaw and Horgan 1983)

Populus euroamericana
a

callus tissue

8.09.0

not dened

(Motyka and Kamnek 1992)

The specic enzyme activity in crude extract, the specic enzyme activity in the most puried preparation. The enzyme activities were measured by the radioisotopic method and the protein content by the method of Lowry, except for cmeasured by the method of Bradford, d measured by the method of Peterson, ethe enzyme activity measured by spectrofotometric assay (Libreros-Minotta and Tipton 1995). fThe enzyme activity is expressed on a gram of callus tissue.

323 Laloues group used a photolabelling CKOx inhibitor, 1-(2-azido-6-chloropyrid-4-yl)-3-(4[ 3H])phenylurea, and obtained about 6 g of pure enzyme protein after denaturing 2D-electrophoresis (Houba-Herin et al. 1999). Peptides obtained after endoproteolytic cleavage were then sequenced and used to design primers for RT-PCR performed on mRNA puried from maize cobs. Corresponding cDNA was then cloned and sequenced (NCBI GenBank accession number Y18377). Both clones encode the same protein of 57.2 kDa with 18 amino acid-signal peptide, 8 consensus Nglycosylation sequences and a consensus FAD binding sequence. By screening gene libraries a hypothetical CKOx gene Arabidopsis thaliana (NCBI GenBank accession number AC002510) was found showing over 40% identity (Morris et al. 1999). It is interesting that this hypothetical protein contains far less N-glycosylation sequences than the maize CKOx. This indicates considerable variability in the glycosylation levels between cytokinin oxidases. A very interesting and curious nding is the 34% homology at the peptide level between the gene ckx1 and a gene fas5 from the bacterium Rhodococcus fascians (NCBI GenBank accession number P46377) which causes shoot hypertrophy disease in dicotyledonous plants (Crespi et al. 1994). However, no connection between plant pathogen invasion and the production of CKOx has been demonstrated so far. The Fas5 protein has been tentatively described as an electron carrier (Goethals et al. 1995). The question arises as to whether this homologous sequence could have some function in the electron transfer by cytokinin dehydrogenase? By screening gene libraries we have found another hypothetical CKOx gene from Arabidopsis thaliana (NCBI GenBank accession number AL079344) that shows 49% identity and 64% similarity (Figure 3). Attempts to express the CKOx gene in E. coli were not successful, but when the intronless ckx1 gene was inserted into the yeast Pichia pastoris cytoplasmic expression vector under the control of the alcohol oxygenase promoter, large amounts of CKOx activity were detected in the culture media (Morris et al. 1999). Preliminary kinetic studies indicate that the recombinant enzyme shows the same substrate specicity and K m values as the maize enzyme characterised previously. The other group used moss protoplasts of Physcomitrella patens, an expression system that should be appropriate for the production of correctly folded glycosylated plant protein and has the advantage that there is no requirement for exogenous auxins and cytokinins to promote growth and division in the medium. The CKOx activity was detected, but the enzyme was not recovered from moss cell homogenates, which may indicate liberation of recombinant enzyme by cell lysis or its excretion (HoubaHerin et al. 1999).

Physiological signicance of cytokinin oxidase Generally, CKOx plays major role in controlling the endogenous levels of cytokinins. Exogenous application of cytokinins induces a signicant but transient increase in degradation of iP as was rst demonstrated in cell suspension cultures of tobacco (Terrine and Laloue 1980) and then in other plants. Both substrates and non-substrates (benzyladenine, kinetin) of CKOx are effective in inducing this increase. For example, cytokinins applied to the surface of bean callus tissue caused a detectable increase in CKOx during the rst hour following the application and reaching a 3-fold higher maximum during the next 8 h (Chateld and Armstrong 1986). The enhancement of enzyme activity promoted by supplying cytokinin is greater than the enhancement in total protein content (Motyka and Kamnek 1992) and is sensitive to RNA and protein synthesis inhibitors (cordycepin and cycloheximide) suggesting that any increase in the amount of enzyme is under transcriptional or translational regulation (Chateld and Armstrong 1986). In developing seeds, CKOx is the tool for altering physiologically active cytokinin levels. Zeatin and its riboside, two principal substrates of the enzyme, are the main cytokinins that accumulate in developing seeds. The levels of these cytokinins increase as much as 400-fold during the early phases of maize seed development after pollination pursuant to maximal cell division (Cheikh and Jones 1994). However, high levels during this period are transient and decline rapidly to initial levels over the following 3 to 5 days (Summons et al. 1980). In maize kernels, the CKOx activity increases after pollination, by as much as 3to 9-fold, coincident with the increase in kernel cytokinin levels (Dietrich et al. 1995; Jones et al. 1992). The exogenous supply of non-substrate cytokinins to maize kernels changes grain weight, kernel numbers and heat tolerance. A deeper understanding of the metabolic control of CKOx by endogenous substrate supply has been achieved by exploitation of the Agrobacterium tumefaciens Ti-plasmid T-DNA ipt gene. Although a com-

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Figure 3. Comparison of deduced amino acid sequence of CKOx from Zea mays and the hypothetical CKOx protein from Arabidopsis thaliana. The same amino acid sequences of CKOx from maize obtained by the Morriss group (Morris et al. 1999) (NCBI GenBank accession number AF044603) and Laloues group (Houba-Herin et al. 1999) (NCBI GenBank accession number Y18377) were aligned to that of the hypothetical protein (NCBI GenBank accession number AL079344) from A. thaliana by the Blast2 programme on a NCBI server, using blosum90 comparison matrix. Shaded area shows the consensus FAD binding sequence, consensus N-glycosylation sites are underlined, and the + mark indicates similarity between amino acids.

parable gene has not been isolated from plants, the tmr region of T-DNA encodes an enzyme, isopentenyl transferase, which synthesises the nucleotide of iP from AMP and isopentenyl pyrophosphate, the rst step in the biosynthesis of cytokinins. No signicant difference in the CKOx activity was observed in transformed plants despite the levels of zeatin and its derivatives being 2- to 12-fold higher than in wild type plants (Eklf et al. 1996). On the other hand the derepression of the tetracycline-dependent ipt gene transcript in a transgenic tobacco calli resulted in a 100- to 300-fold increase in the level of cytokinins degradable by CKOx and about 10-fold higher CKOx activity (Motyka et al. 1996). This increase in CKOx level was also observed in detached leaves and roots.

Endogenous cytokinin levels are modulated by other phytohormones, the most notable in this regard being the auxins. Palni found that the rate of the degradation of zeatin riboside in tobacco pith explants could be doubled by increasing the auxin concentration in the medium (Palni et al. 1988) and similar results have been obtained from studies with other plant systems. Treatment of soybean leaves with auxins increased the side chain cleavage of externally supplied benzyladenine (Zhang et al. 1987), presumably by inhibiting an enzyme that forms alanine conjugates of cytokinins (Parker et al. 1986). A stimulatory effect of auxins on CKOx activity is supported indirectly by the nding that tobacco crown gall tissues carrying an insertionally inactivated auxin tms gene exhibit ex-

325 tremely elevated levels of cytokinins, for example 7-glucosylzeatin is often found in 100-fold higher concentrations than in normal transformed tissues (Akiyoshi et al. 1983; McGaw et al. 1988). However, application of three different auxins on to the surface of tobacco callus did not affect activity of extracted CKOx (Motyka and Kamnek 1992). The recent nding of Zhang that auxin decreased ipt mRNA as well as the ipt level in ipt-transgenic tobacco tissue indicates that auxin affects cytokinin biosynthesis (Zhang et al. 1995). Recent data on cytokinin- and auxinoverproducing transgenic tobacco plants demonstrate that cytokinin or auxin overproduction decreases the content of the other hormone, apparently by decreasing its rate of synthesis and/or transport, rather than by increasing rates of turnover or conjugation (Eklf et al. 1997). Some effects of abscisic acid on the regulation of cytokinin degradation have been also noted. These include the suppression of exogenously applied kinetin degradation and the inhibition of zeatin conversion to dihydrozeatin followed abscisic acid treatment in lettuce seeds (Miernyk 1979) and embryonic axes of Phaseolus vulgaris (Sondheimer and Tzou 1971), respectively. We have noted with great interest that cytokinins and their synthetic derivatives have been shown to be powerful inhibitors of cyclin-dependent protein kinases, the conserved regulators of the eukaryotic cell division cycle (Vesel et al. 1994). The fact that exogenous application of these compounds to various tumour cell lines could stop their division has triggered extensive research in this area. Thus, with possible future use of cytokinin-derived compounds as cancerostatics, there is also a need for monitoring such compounds when administered exogenously. This could be conveniently achieved by using CKOx either as a reagent or even an active component of a biosensor.

Acknowledgements The work was supported by the grants 522/00/0121 and 204/96/K235 from the Grant Agency Czech Republic and VS96154 from the ministry of Education, Czech Republic.

References Conclusions and possible future directions of research Our knowledge about the regulation of cytokinin levels in plants and the mechanisms by which enzymes degrade cytokinins with an unsaturated side chain is still limited. Such enzymes are likely to play a key role in the cytokinin regulatory process. Understanding the developmental regulation, compartmentation and tissue specicity of CKOx in plants should help to determine the mechanisms by which cytokinins regulate plant development. It is hoped that current and future studies involving expression and manipulation of the gene encoding CKOx will introduce more possibilities of utilising recombinant enzyme techniques in physiological experiments. In our laboratory we have just obtained rst results in cloning CKOx genes from wheat and barley. We think this will be a useful contribution and it should be fascinating to compare our results with those from maize CKOx cloning. Furthermore, identication of the enzyme system capable of cleaving benzyl and furfuryl side chains will be necessary, as these cytokinins are frequently used in studies of plant growth regulation and have also been shown to occur in plant tissues.
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