Planta

Planta (1983) 159:30-37

9 Springer-Verlag 1983

Cytokinin oxidase from Z e a m a y s kernels and Vinca rosea crown-gall tissue Brian A. McGaw and Roger Horgan Department of Botany and Microbiology, University College of Wales, Aberystwyth SY23 3DA, Dyfed, UK

Abstract. Cytokinin oxidase has been partially purified from mature Z e a m a y s kernels and from V i n c a r o s e a corwn-gall tissue. The molecular weights of the two enzymes, determined by gel filtration, are very different: 94,400 (+ 10%) for Z. m a y s and 25,100 (+_ 10%) for V. r o s e a . Specificity studies have been performed using a large number of synthetic and naturally occurring cytokinins. Only a small number of these compounds serve as substrates and both enzymes exhibit similar substrate specificity. In agreement with other workers, a A2 double bond in the N 6 side chain is essential for activity. The presence of glucosyl or ribosyl groups in the 7- or 9-position or an alanyl group in the 9-position of the purine moiety have little effect on their susceptibility to cytokinin oxidase, but O-glucosyl derivatives are resistant to oxidation. The relevance of these enzyme systems to studies on cytokinin metabolism and to the endogenous cytokinins is discussed. Key words: Crown gall - Cytokinin - Cytokinin oxidase (substrate specificity) - V i n c a - Z e a (cytokinin oxidase).

Introduction

A number of enzymes responsible for cytokinin metabolism have been partially purified. Using N 6AZ-isopentenyladenosine (2iP) and its derivatives Chen and co-workers have characterised a Y-nucleosidase (Chert and Kristopeit 1981a), an adenosine nucleosidase (Chert and Kristopeit 1981 b), an adenosine phosphorylase (Chen and Petschow 1978), an adenine kinase (Chen and Eckert 1977) and an adenosine phosphoribosyltransferase Abbreviations: 2iP=N6-AZ-isopentenyladenine; 2iPA=N6-A z-

isopentenyladenosine; HAP = hydroxylapatite

(Chen and Eckert 1977) from wheat germ. With all these enzymes, the presence of the N 6 side chain in the substrate slightly reduced their susceptibility relative to the adenine derivatives. Letham and coworkers have examined enzyme systems that convert' active' cytokinins into their less active or possible storage forms. A glycosyltransferase, that utilises uridine diphosphate glucose as the glucose donor and converts zeatin into its 7- and 9-glucosides, has been purified from radish cotyledons (Entsch etal. 1979a). A fi-(6-alkyl-amino-9yl)adenine synthase, which utilises 0-acetyl serine and zeatin as substrates giving 9-alanylzeatin, has been demonstrated in developing lupin seeds (Entsch et al. 1979b). Both these enzymes exhibited strong preferences for the N6-substituted purines. A number of enzymes that modify the N 6 side chain of the cytokinins have been characterised. Almond fl-glucosidase converts zeatin-0-glucoside to zeatin while zeatin-9-glucoside and zeatin-7-glucoside do not serve as substrates (Letham et al. 1975). Recently a microsomal enzyme that hydroxylates 2iP giving zeatin has been demonstrated in tobacco tissue cultures (Chen 1982). The metabolism of exogenously applied zeatin and other cytokinins (Entsch et al. 1979b; Palmer et al. 1981b) indicates the presence of other, as yet uncharacterised, enzyme systems. In virtually every case, however, the administration of labelled cytokinins to plant systems results in the irreversible destruction of cytokinin activity by side-chain cleavage. In many cases this is the major metabolic fate of exogenously supplied cytokinins. An enzyme capable of catalysing this cleavage has been partially purified from corn kernels (Whitty and Hall 1974) and cultured tobacco tissue (Paces et al. 1971). This enzyme, so-called 'cytokinin oxidase', was able to utilise 2iP, N6-A2-isopentenyladenosine (2iPA), zeatin and zeatin riboside as substrates

B.A. McGaw and R. Horgan: Cytokinin oxidase from Zea and Vinca

giving adenine-adenosine (McGaw and Horgan 1983) and the corresponding side chain aldehyde (Brownlee et al. 1975). It was shown that sidechain saturation, A2 relocation to A 3 and the substitution of other functionalities significantly reduced susceptibility to cytokinin oxidase (Whitty and Hall 1974). There is substantial evidence that cytokinin oxidase is responsible for the catabolism of exogenously applied cytokinins. Adenine, adenosine and the adenine nucleotides have been observed as major products of zeatin (Entsch et al. 1979b), zeatin riboside (Summons et al. 1980) and 2iP (Laloue et al. 1977) catabolism, while dihydrozeatin (Palmer et al. 1981b) and benzyladenine (Parker et al. 1973), which are not substrates for the enzyme, are much more resistant to side-chain cleavage. Cytokinin oxidase is also a candidate for the control of endogenous cytokinin species and levels. So far, however~ only a limited number of naturally occurring cytokinins (2iP, 2iPA,, zeatin, zeatin riboside and dihydroisopentenyladenosine) have been tested as substrates (Whitty and Hall 1974). The purpose of this paper is to present data using a much broader spectrum of cytokinins, both naturally occurring and synthetic, with highly purified cytokinin-oxidase preparations from corn kernels. These are compared with the enzyme that has been partially purified from Vinca rosea crown-gall tissue. Materials and methods Plant material. Corn (Zea mays L. cv. Earliking) was grown under glass. Cultured crown-gall tissues of Vinca rosea L. was maintained on the medium of Miller (1974), and harvested two months after sub-culturing. Both the crown-gall tissue and the mature corn cobs were used immediately or were frozen at - 2 0 ~ C until required. Chemicals. All chemicals were from BDH Chemicals Ltd., Poole, U K unless otherwise indicated. [8-14C]-6-Chloropurine (4.33.108 Bq retool-~) was purchased from the Radiochemical Centre, Amersham, U K ; [8-14C]zeatin, dihydrozeatin, dihydrozeatin-0-glucoside, zeatin riboside, 2iP and benzyladenine were synthesised by condensing the relevant side-chain amines with the above material (Hall and Robins 1968). Dihydrozeatin riboside and zeatin riboside-0-glucoside were synthesised in the same way except unlabelled material was employed. The 2iPA and tricanthine (6-amino-3-dimethylallylpurine) were obtained commercially from Sigma (London, UK). The remaining cytokinins were synthesised cold according to their published procedures: benzyladenine-7-glucoside, benzyladenine-9-glucoside, zeatin-7-glucoside and zeatin-9-glucoside (Scott et al. 1980); benzyladenine-3-glucoside (Letham et al. 1975) ; 9-alanylzeatin and 9-alanylbenzyladenine (Duke et al. 1978); 9-alanyldihydrozeatin (Parker et al. 1978). All these compounds were purified by high-performance liquid chromatography (HPLC) prior to their use.

31

Purification of corn enzyme. The following procedures took place at approx. 4 ~ C. The method is essentially that of Whitty and Hall (1974). Corn kernels (100 g) were pulverised in 0.1 M phosphate buffer at pH 6.8 (500 cm 3) containing ascorbic acid (8.8 g) and insoluble polyvinylpyrrolidone (500 g). The slurry was allowed to stand (0.5 h) before filtering through muslin and then paper (2nd filtrate). The filtrate (280 cm 3) was centrifuged at 40,000 g (1 h). The pellet was discarded and the supernatant (crude supernantat) subjected t o ammonium-sulphate fractionation at 40% and then 60% saturation. Both were centrifuged at 40,000 g (1 h) and the precipitates re-suspended in 0.1 M phosphate buffer at pH 6.8 (35 cm 3 for the 40% and 45 cm 3 for the 60% precipitate) before dialysis against 2 1 of the same buffer (12 h). The dialysates were centrifuged at 50,000 g (1 h) and the supernatant frozen and stored at - 2 0 ~ C. During the above procedure, the various fractions were monitored for enzyme activity and protein concentration (see later sections). The 40-60% ammonium-sulphate precipitate was found to be the most active fraction and 5 cm 3 of this was applied to a G-150 super-fine Sephadex (Sigma) column (2.5 cm inner diameter, 90 cm iong) and eluted with 0.005 M phosphate buffer at pH 6.8 containing 0.02% N a N 3. The column was calibrated separately by the method of Whitty and Hall (1974). Six protein standards (Sigma), namely alkaline phosphatase (calf intestinal mucosa) MW 100,000, bovine serum albumin M W 66,200, ovalbumin MW 45,000, pepsin (hog stomach mucosa) MW 34,700, trypsinogen (bovine pancreas) MW 24,000 and lysozyme (egg white) MW 14,400, were applied to the G-150 column, previously equilibrated with 0.025 M pH 6.8 phosphate buffer. The void volume, V0, was determined with blue dextran (Sigma) and the total volume of the packed bed, Vt, was calculated geometrically. In separate experiments the 40- to 60%-precipitate preparations were run on the G-150 column using the same buffer and the etuates assayed for cytokinin-oxidase activity (Fig. 1). The molecular weight of the corn enzyme Was estimated to be 94,400 ( + 10%). It was noted that the different ionic strenghts of the elution buffers did not greatly effect column retention time of the corn enzyme. The same was also true for the Vinca enzyme. The active fractions from the elution at 0.005 M were bulked (90 cm3), and 20 cm 3 of this was applied to a spheroidalhydroxylapatite (HAP) column (1.5 cm inner diameter, 5 cm long). The first-eluted 20 cm 3 was collected as two 10-cm 3 fractions (Pre I and Pre II) before elution with 100-cm 3 aliquots of 0.005, 0.02, 0.04, 0.08 and, finally 0.2 M phosphate buffer at pH 6.8 (10-cm 3 fractions collected). The Pre II fraction, which contained most of the enzyme activity, was frozen at - 2 0 ~ C until required. This HAP step was repeated a further three times (20 cm 3 of the active G-150 eluate being used each time) and the Pre II fractions bulked together (40 cm3). Polyacrylamide disc gel electrophoresis. The fraction, Pre II (50 ~tg protein) (8 cm 3) was concentrated to 3.0 cm 3 by dialysis against a saturated solution of polyethyleneglycol ( M W = 6,000) (Koch-Light, Colnbrook, UK) and applied to two 7% polyacrylamide disc electrophoresis gels. The gels were cast with a small volume of application buffer and run at room temperature with a current of 3 mA gel- 1. The method was essentially that of Davis (1964): electrode b u f f e r - 0.025 M 2-amino-2(hydroxymethyl)-l,3-propanediol (Tris), 0.192 M glycine (pH 8.3) ; running-gel buffer - 0.375 M Tris (pH 8.8, HC1); stackinggel buffer - 0.125 M Tris (pH 6.8, HC1); application buffer - 0.125 M Tris, 20% glycerol, 0.002% bromophenol blue~(pH 6.8, HC1). The running gels were cast using the following ratios: running-gel buffer (50 cm3), N,N,N',N'-tetramethylenediamine (TEMED) (0.25 cm3), Cyanogum 41 (Sigma) (3.5 g) and am-

32

B.A. McGaw and R. Horgan: Cytokinin oxidase from Zea and Vinca

monium persulphate (0.05 g). After running, one of the gels was fixed (50% trichloroacetic acid, 24 h), stained (50% trichloroacetic acid containing 0.05% Coomassie brilliant blue, 2 h) and destained (7% acetic acid) whilst the other gel was cut into slices and assayed for cytokinin-oxidase activity (Table 1). The remaining Pre II (32 cm 3) was concentrated down (as before) to 2 cm 3 and applied to two 7% gels, as above. The active region of both gels as defined by the above gels (and containing approx. 20% of the applied protein) was cut out and pulverised in 0.5 M pH 6.0 phosphate buffer (20 cm3). The mixture was allowed to stand overnight at 4 ~ before being filtered. The filtrate was frozen and stored at - 2 0 ~ until required. This material was used to characterise the corn enzyme (Table 2).

Purification of enzyme from Vinca rosea crown gall. Vinca rosea crown-gall tissue (100 g) was extracted by the same procedure as for corn. The majority of the cytokinin-oxidase activity, however, precipitated in the fraction (L40% ammonium sulphate. This precipitate was re-suspended in 0.1 M phosphate buffer at pH 6.8 (10 cm3). Because of losses in dialysis this material (5 cm 3) was applied directly to the G-150 Sephadex column and eluted with 0.005 M pH 6.8 phosphate buffer. The oxidase activity eluted much later than for the corn preparation and the enzyme was estimated to have an MW of 25,100 (_+ 10%). The active fractions (65 cm 3) were bulked and 50 cm 3 of this were applied to a spheroidal HAP column as above. The firsteluted 50 cm 3 (HAP 1-5) was collected as 10-cm 3 fractions prior to eluting with the before-mentioned eluotrophic buffer series. The HAP 185 contained most of the enzyme activity and was bulked and frozen at - 20 ~ C until required. This material was used for characterising the Vinca enzyme.

Assay of cytokinin-oxidase activity. Side-chain cleavage has already been established as the mechanism of action of the cytokinin-oxidase enzyme from corn (McGaw and Horgan 1983). By the same procedure (ie: ultraviolet and mass spectrometry), this was also shown to be the case for the Vinca enzyme. For routine assays (i.e. for Figs. 1-3) of cytokinin-oxidase activity 8 nm of 2iP (4.33.108 Bq.mmol - t ) were incubated at 37 ~ C with 0.5 cm 3 of the sample. After 24 h, a fraction of this mixture was withdrawn and subjected to reversed-phase HPLC using a column (150 mm long, 4.5 mm inner diameter) of Hypersil ODS (Jones Chromatography, Llanbradach, UK) eluted at a rate of 2 cm 3 min-1 with acetonitrile and water pH 7 (triethylammonium bicarbonate) (18:82) (Horgan and Kramers 1979). Using this system adenine-adenosine elutes between 2 and 3 min and residual 2iP after 7 min. The oxidase reaction was monitored by liquid scintillation spectrometry. For the specificity and kinetic studies a gradient HPLC system was used. The system employed depended upon the cytokinin under investigation: 3-10% acetonitrile (10 rain) for zeatin-7-glucoside, zeatin-9-glucoside and 9-alanylzeatin; 3-12% zeatin-0-glucoside; 3-13% dihydrozeatin-0-glucoside; 3-15% zeatin and 9-alanyldihydrozeatin; 3-17% dihydrozeatin, zeatin riboside-0-glucoside, dihydrozeatin riboside-0-glucoside and zeatin riboside; 3-20% dihydrozeatin riboside; 3 30% 2iP, 2iPA, benzyladenine-3-glucoside, benzyladenine-7-glucoside, benzyladenine-9-glucoside, benzyladenine, 9-alanylbenzyladenine and tricanthine. All gradients were linear. The oxidation products of the radioactive substrates were quantified by liquid scintillation spectrometry whilst the 'cold' substrates were monitored by the disappearance of the cytokinin UVabsorbing peak. For all these experiments 0.1 cm 3 of the electrophoresis-gel extract (corn) or 0.1 cm 3 of the HAP 1 5 fraction (Vinca) were incubated at 37 ~ C for 0.5 h. The coneentra-

tions of the substrates were varied between 6.40.10 -6 and 1.28"10 -4 M. The concentrations were determined by UV absorbance based on extinction coefficients of 17,000 for all the eytokinins except zeatin-7-glucoside (14,150) (Cowley et al. 1978). Protein concentrations were measured by the method of Lowry et al. (1951). The determination of protein levels in the active region of the corn electrophoresis gels was made by gel scanning the Coomassie-stained material at 595 nm.

Determination of pH and temperature optima. The pH dependence of the enzymes was investigated using 0.04 M sodium acetate (pH range 4.0 to 6.0), 0.04 M sodium phosphate (pH 5.5 to 7.5) and 0.04 M Tris-HC1 (pH range 7.0 to 9.0) (Whitty and Hall 1974). The active HAP fractions (0.1 cm 3) were used for both enzymes mixed with the relevant buffer (0.5 cm 3) and incubated at 37 ~ C with 8 nm of the radioactive 2iP. The samples were assayed after 2 h (corn) and 24 h (Vinca). Temperature optima were determined by incubating the active HAP fractions (0.1 cm 3) with 8 nm of radioactive 2iP dissolved in 0.005 M phosphate buffer (0.5 cm 3) at pH 6.0 (corn) and pH 6.8 (Vinca) which had been temperature-equilibrated at 4, 18, 37, 46 and 60 '~ C. The samples were assayed as above. In both cases the optimum was approx. 37 ~ C.

Dependence upon molecular oxygen, inhibition by cyanide and effect of magnesium ions. Radioactive 2iP (8 nm) was dissolved in 0.5 cm 3 of 0.005 M phosphate buffer at pH 6.0 (corn) and pH 6.8 (Vinca) which had been degassed and purged with helium. The HAP preparations (0.1 cm 3) were added and then incubated at 37 ~ C under an atmosphere of helium. The samples were assayed as above. The extents of side-chain cleavage were 10% (corn) and 50% (Vinea) of the control experiments where no helium was used. Traces of dissolved oxygen were obviously present in the preparations, the effects of this being more visible in the Vinea experiment where a longer incubation time was employed. Inhibition by cyanide was observed by incubating 0.1 cm 3 of the HAP preparations with 8 nm of radioactive 2iP dissolved in 0.5 cm 3 of 0.005 M phosphate buffer (pH 6.0 for the corn and pH 6.8 for the Vinca) containing 10 -5, 10 -4, 10 -3 and 1 0 - 2 M cyanide. The samples were assayed as above and at these concentrations of cyanide 5, 44, 76 and 85% (respectively) reductions of activity compared with the control (no cyanide) were observed for the corn enzyme. For the Vinca enzyme the corresponding figures were 38, 55, 80 and 88%. No magnesium dependence was observed in an identical experiment replacing the cyanide with 0.002, 0.004, 0.008 and 0.01 M magnesium chloride.

Results

The purification procedure employed was essentially that of Whitty and Hall (1974). Throughout this procedure protein concentrations and enzyme activities were monitored. These data are presented in Table 1. The cytokinin oxidase enzymes of Z e a m a y s kernels and Vinca rosea crown-gall tissue differ in two important respects. Firstly, the bulk of the oxidase activity precipitated at different ammonium-sulphate concentrations, and secondly, there was a large difference in their molecular weights. The corn enzyme was estimated to have an MW of 94,400 (_+10%), in good aggreement

B.A. McGaw and R. Horgan: Cytokinin oxidase from Z e a and Vinca

33

4.0

Z. Mays

03

~

I

'~'"-'~"'"A,,,,,%

0.2

4O

E

01

0 0

4,

o

,s

3O

60

",

->,

20

.> ~d o

,A

,10o

cO

/

V. R 0 s e a

a,

0.3

k..c 0 "o 0

40

80 >

0.2

SO

I

20

I

,"m

~0

01

10

/

],

~.--o---o" "',,g

2

4

6

8

10

Fraction

100

200

Elution

votume

300

400

(cm 3 }

Fig. t. Sephadex G-] 50 column elution profiles, using 0.025 M pH 6.8 phosphate buffer, of corn and Vinca cytokinin-oxidase activity (obtained from separate experiments)

,20

""'i ....

12

14

16

18

20

22

24

26

number

Fig. 2. Hydroxylapatite-column elution profiles of corn and Vinca showing cytokinin-oxidase activity (e e) and A28 o values (A- A) for the first 26 fractions (later fractions did not contain significant protein or oxidase activity). Fraction 2 (Pre II) was retained for the corn and fractions 1-5 (HAP 1-5) were retained for the Vinca

Table 1. Purification data for cytokinin oxidase from Zea mays kernels and Vinca rosea crown-gall tissue. The protein yield and, therefore, the total-activity data are calculated as if the entire crude supernatants were processed through each stage Preparation

Z. mays

Protein yield (rag) Crude supernatant 40% precipitate 60% precipitate G-150 HAP Polyacrylamide gel extract a

4,500 262.5 225.0 162.0 2.0 0.4

V. rosea

Total activity (nMh -1) 315 9,801 6,808 4,276

Specific activity (nMmgprotein l h - ~ ) n.d. b n.d. 1.4 60.5 3,404 10,690

Protein yield (mg) 260 80 110 45.5 6.5 -

Total activity (nMh i) 296 33 573 1,265 -

Specific activity (nMmgprotein-ih-i) n.d. 3.7 0.3 12.6 194.6 -

" These data were obtained by incubating the active gel segment from the initial electrophoresis experiment in 0.5 M phosphate buffer at pH 6.0 and not with the extract-ed material that was used in Table 2 b n.d. = activity not detected

with the 88,000 previously estimated (Whitty and Hall 1974), while the Vinca enzyme was only MW 25,100 (_+ 10%). These estimations were obtained from Sephadex G-150 column data (Fig. 1). In contrast to Whitty and Hall (1974) the crude supernatants were both inactive and subsequent steps led to substantial increases in total observed cytokinin-oxidase activity (Table 1). This may be caused by the presence of inhibitory substances.

In both cases the HAP column was a very efficient 'clean-up' step. Fortunately the enzymes bound very weakly to the HAP and so the activity was separated from the bulk of the contaminating protein (Fig. 2). The HAP-purified Vinca enzyme was used for characterisation, but the corn enzyme was further purified by preparative polyacrylamide disc gel electrophoresis. These gels were either sliced and assayed for enzyme activity or stained. A typi-

34

B.A. McGaw and R. Horgan: Cytokinin oxidase from Z e a and Vinca Table 2. Kinetic constants for cytokinin oxidase from Z e a m a ys

50

kernels and Vinca rosea crown-gall tissue, derived from electrophoresis and HAP-purified material, respectively Cytokinin

Z. mays

V. rosea

~0

Km

Vm~a

Km

Vma/

2.86 1.92 3.33 3.33 3.12 5.55 3.12

2.33 2.33 2.17 2.17 1.82 1.25 1.82

2.56 2.77 2.86 3.33 4.35 4.35

0.22 0.22 0.15 0.14 0.13 0.11

( x 10-SM)

Zeatin 2iP 2iPA Zeatin riboside 9-Alanylzeatin Zeatin-7-glucoside Zeatin-9-glucoside

30 tO

"G O

20

OJ

( x 10-SM)

" Expressed in micromoles degraded per milligram of protein per hour 10

"';Z

!iili!iil;i i!!i;K

,,'d. ....,:. ...... ...,.. ..,....

2

1

Gel

3

run

4

6

~" ( c m }

Fig, 3. Polyacrytamide gel electrophoresis of corn showing cytokinin-oxidase activity and the Coomassie-staining pattern. The hatched areas represent easily discernible bands while the dotted areas show more diffuse regions of staining

cal corn gel experiment is shown in Fig. 3. The cytokinin-oxidase activity is associated with a single protein band, but at this stage it is not known whether this is pure. In subsequent electrophoresis runs the active regions of the gels were bulked and then extracted into buffer. This buffer solution was used for characterising the corn enzyme. This material had a specific activity roughly three times greater than that used by Whitty and Hall (1974). The Vinca HAP preparation was also run on polyacrylamide disc gels. Again a single region of cytokinin-oxidase activity was evident. However, recovery of activity from these gels was poor for the Vinca enzyme. The HAP material, therefore, was used for characterising the Vinca enzyme. Despite the greater purity of this corn enzyme preparation, characteristics such as pH optimum (pH 6.0), temperature optimum (37 ~ C) and the toxicity of 10 -4. M C N - (44% reduction of activity) were very similar to those previously reported (Whitty and Hall 1974). The enzyme was shown to require molecular oxygen and not magnesium. In these respects the Vinca enzyme was found to

differ in only its pH optimum which was at pH 7.0. As previously shown (Whitty and Hall 1974) some 9'-nucleosidase was observed as a contaminant of the corn enzyme. This activity was finally separated from the oxidase by the preparative-gelelectrophoresis step (this nucleosidase activity was specific for 9'-ribosyl groups). No such problem was apparent in the Vinca preparations. The specificity and kinetic data are presented in Table 2. The cytokinin 2iPA was not tested in the Vinca system. Additional cytokinins were also tested, namely: dihydrozeatin, dihydrozeatin-0glucoside, dihydrozeatin riboside, dihydrozeatin riboside-0-glucoside, zeatin-0-glucoside, zeatin riboside-0-glucoside, benzyladenine, benzyladenine-3glucoside, benzyladenine-7-glucoside, benzyladenine-9-glucoside, 9-alanyldihydrozeatin and 9alanylbenzyladenine, but none of these compounds served as substrates. The alkaloid tricanthine (6-amino-3-dimethylallylpurine) was also tested and not oxidised by the enzymes. The protein concentration of the gel-extracted material used for the corn kinetic data was calculated on the basis of total recovery from the pulverised gel slices. However, comparisons of the activities of the polyacrylamide material in the two tables indicate that losses have occurred during the extraction procedure. Discussion

Table 2 shows that the substrate specificities of the two enzymes, despite the difference in their molecular weights, are very similar. However, their kinetic data cannot be compared since they are derived from different purification procedures.

B.A. McGaw and R. Horgan: Cytokinin oxidase from Zea and Vinca

It is clear from these invstigations (Table 1) that there is considerably more cytokinin-oxidase activity in the corn kernels than in the same weight of Vinca crown-gall tissue. Both these tissues contain large quantities of cytokinins (Summons et al. 1979; Scott et al. 1982a), and it is possible that the cytokinin oxidase is important in the regulation of cytokinin activity in the tissues. However, in comparison with 'normal' V. rosea callus tissue, which is cytokinin requiring, there is little difference in cytokinin oxidation ability (Scott et al. 1982b), and this tissue contains extremely low levels of cytokinins (personal communication by I.M. Scott, Department of Botany and Microbiology, U.C.W., Aberystwyth). Dihydrozeatin is not a substrate, confirming that the presence of the A2 double bond in the side chain is essential for cytokinin-oxidase activity. In previous work (Whitty and Hall 1974) dihydroisopentenyladenosine was shown to be resistant to this enzyme. In further agreement with this work the results show that benzyladenine is not a substrate. Zeatin and 2iP are almost equally easily oxidised which indicates that the terminal functionality is of little consequence. Whitty and Hall (1974) have shown that the substitution of a chlorine atom for a terminal methyl group of 2iPA, to give N6-3-chlorobut-2-enyladenine, still permits considerable enzyme activity. Interestingly, however, our results show that the presence of a bulky 0-glucosyl group in this position (eg: zeatin-0-glucoside) renders the cytokinin completely resistant to cytokinin oxidase. The substitution of a ribosyl, glucosyl or alanyl residue at the 9-position of the purine ring causes a slight reduction in the Vmax values. There is little difference between the effects of these different substituents. Substitution of a glucosyl group at the 7-position (ie: zeatin-7-glucoside) also reduced Vmax values, but again there was little difference between these figures and those obtained for the most easily oxidised cytokinins. The indifference of cytokinin oxidase towards 9- and 7-glucosyl or 9-alanyl substitution is in contrast to the many metabolic studies performed by Letham and co-workers which indicate that these compounds are metabolically stable (Parker and Letham 1973; Parker etal. 1978; Entsch etal. 1979b; Summons et al. 1980; Letham et al. 1982). However, none of these 'stable' compounds have been fed to tissues where cytokinin-oxidase-type metabolism is the predominant fate of exogenously supplied zeatin (i.e. derooted sweet-corn seedlings or kernels, V. rosea crown-gall and Phaseolus vulgaris tissue). Gawer et al. (1977) have fed labelled

35

benzyladenine-7-glucoside to tobacco cell suspensions and shown it to be metabolically very stable. This tissue is known to actively catabolise 2iP to adenylic products (Terrine and Laloue 1980). However, we know that benzyladenine is not a substrate for cytokinin oxidase and the stability of benzyladenine-7-glucoside may be a consequence of the benzyl rather than the 7-glucosyl group. On the other hand, zeatin-7-glucoside is the major metabolite of zeatin feeds to derooted radish seedlings (Parker and Letham 1973) and radish roots (Gordon et al. 1974), and it was thought that the 7-glucosyl group may render the cytokinins insensitive to cytokinin oxidase. The results presented in Table 2 show that this is not the case. The apparent metabolic stability of zeatin-7-glucoside in radish tissue may be due to compartmentation or, perhaps, to the absence of or low levels of cytokinin oxidase. Evidence for the latter is the very iow level of cytokinin-oxidase-type catabolism when zeatin (Parker and Letham 1973; Gordon etal. 1974) and zeatin-7-glucoside (Letham et al. 1982) were exogenously applied to radish tissue. In sweet corn the situation is different. A number of metabolic studies where zeatin has been fed to corn kernels (Entsch et al. 1979b; Summons et al. 1980) or roots and derooted seedlings of corn (Parker and Letham 1974) all give adenine, adenosine and adenine nucleotides as the major metabolites, indicating that cytokinin oxidase is playing an important role. In detached P. vulgaris leaves (Palmer et al. 1981 b) and V. rosea crown gall (Horgan et al. 1981), side-chain cleavage is also the fate of the majority of exogenously applied zeatin. Lupin, on the other hand, presents a more complex picture. Following feeds with zeatin, this tissue accumulated quantities of 9-alanylzeatin, dihydrozeatin riboside, zeatin and dihydrozeatin nucleotides and 0-glucosides as well as oxidising zeatin to adenine and adenosine (Parker etal. 1978; Entsch et al. 1979b). The 0-glucosides are also thought to be metabolically stable forms (Parker et al. 1978). With the exception of radish tissue the 0-glucosides have been identified as metabolites and, in some tissues, as major metabolites, of zeatin feeds (Entsch et al. 1979b). On the other hand, in recent work by Scott (personal communication) equal quantities of zeatin-0-glucoside and zeatin-7glucoside were found in radish leaves following feeds with zeatin. The 0-glucosides are therefore unique among the proposed 'stable forms' in that they are of universal occurrence as metabolites of zeatin and they are resistant to cytokinin oxidase. The 0-glucosides are strong candidates as storage forms of the cytokinins. Their accumulation and

36

B.A. McGaw and R. Horgan: Cytokinin oxidase from Zea and Vinca

rapid utilisation at different stages of plant development (Palmer et al. 1981a) and their apparent metabolic stability (Parker etal. 1978; Palmer et al. 1981b) provide good evidence for this hypothesis. The 0-glucosides are also found to be major endogenous cytokinins in P. vulgaris (Wareing et al. 1977; Palmer et al. 1981a), corn kernels (Summons et al. 1980) and in V. rosea crown-gall tissue (Scott et al. 1982 a). On the other hand, previous work in this laboratory (Horgan et al. 1981), showed that zeatin-0-glucoside was rapidly metabolised to adenine and adenosine in Vinca crown gall. This probably reflects high levels offl-glucosidase activity, but it also stresses the difficulty in extrapolating the fate of exogenously applied cytokinins to the endogenous situation and vice versa. The same could be said of the apparent high biological activity of the 0-glucoside in bioassays. Perhaps this is also a consequence of/~-glucosidase activity. The stability of the endogenous 0-glucosides in certain plant tissues indicates that they are not coming into contact with fl-glucosidases. Like other tissues, V. rosea crown gall accumulates 0glucosides following feeds with zeatin. This contrasts with the rapid oxidation of zeatin-0-glucoside when exogenously supplied (Horgan et al. 1981). The oxidation presumably goes via zeatin. 0-Glucosides, therefore, are readily accumulated and easily metabolised; both important criteria for a putative storage form. Conversely, the apparently stable zeatin-7-glucoside, zeatin-9-glucoside and 9-alanylzeatin may be detoxification products incapable of further contribution to the active cytokinin pool in the tissues where they accumulate. There are therefore several options open to the plant when cytokinins are applied exogenously. In restoring a level of cytokinin activity, cytokinin oxidase appears to be of central importance to bean, corn and V. rosea crown-gall tissue. In other tissues, like radish or poplar leaves (Duke et al. 1979), alternative means of reducing levels of cytokinin activity are probably more important (i.e. the formation of 7-glucosides or 0-glucosides). In lupins, all methods of'inactivation' and oxidation appear to be operating. It is much more difficult to assign a role for cytokinin oxidase in the control of endogenous levels of cytokinins. In Vinca crown-gall tissue and corn kernels the most abundant cytokinins are zeatin riboside and zeatin (Scott et al. 1982a; Summons et al. 1980), both of which are readily oxidised by cytokinin oxidase. A faster rate of biosynthesis than degradation is not very likely when the high cytokinin-oxidation potential in these tissues is considered. It is probable that compartmenta-

tion prevents the cytokinins coming into contact with the oxidase system. As with the metabolic studies, cytokinin oxidase must be considered as potentially important in the control of cytokinin levels and species, but it is certainly not the only factor involved. We are grateful to Mr. Phil Williams for technical assistance. This work forms part of a project financed by the Agricultural Research Council.

References Brownlee, B.G., Hall, R.H., Whitty, C.D. (1975) 3-Methyl-2butenal: an enzymatic product of the cytokinin, N6-(A 2isopentenyl)adenine. Can. J. Biochem. 53, 47-41 Chert, C.-M., Eckert, R.L. (1977)Phosphorylation of cytokinin by adenosine kinase from wheat germ. Plant Physiol. 59, 443-447 Chen, C.-M., Petschow, B. (1978) Metabolism of cytokinin: ribosylation of cytokinin bases by adenosine phosphorylase from wheat germ. Plant Physiol. 62, 871-874 Chen, C.-M., Kristopeit, S.M. (1981 a) Metabolism of cytokinin: dephosphorylation of cytokinin ribonucleotide by 5'nucleotidases from wheat germ cytosol. Plant Physiol. 67, 494-498 Chen, C.-M., Kristopeit, S.M. (1981 b) Metabolism of cytokinin: deribosylation of cytokinin ribonucleoside by adenosine nucleosidase from wheat germ cells. Plant Physiol. 68, 1020-1023 Chen, C.-M. (1982) Cytokinin biosynthesis in cell-free systems. In: Plant growth substances 1982, pp. 155-163, Wareing, P.F., ed. Academic Press, London New York Cowley, D.E., Duke, C.C., Liepa, A.J., MacLeod, J.K., Letham, D.S. (1978) The structure and synthesis of cytokinin metabolites. I. The 7- and 9-fl-D-glucofuranosides and pyranosides of zeatin and 6-benzylaminopurine. Aust. J. Chem. 31, 1095-1111 Davis, B.J. (1964) Disc electrophoresis. II. Method and application to human serum proteins. Ann. N.Y. Acad. Sci. 121, 404-427 Duke, C.C., Letham, D.S., Parker, C.W., MacLeod, J.K., Summons, R.E. (1979) The complex of O-glucosylzeatin derivatives formed in Papulus species. Phytochemistry 18, 819-824 Duke, C.C., MacLeod, J.K., Summons, R.E., Letham, D.S., Parker, C.W. (1978) The structure and synthesis of cytokinin metabolites. II. Lupinic acid and O-fl-D-glucopyranosylzeatin from Lupinus angustifolius. Aust. J. Chem. 31, 1291-1301 Entsch, B., Parker, C.W., Letham, D.S., Summons, R.E. (1979a) Preparation and characterisation, using high-performance liquid chromatography, of an enzyme forming glucosides of cytokinins. Biochim. Biophys. Acta 570, 124-139 Entsch, B., Letham, D.S., Parker, C.W., Summons, R.E., Gollnow, B.I. (1979b) Metabolites of cytokinins. In: Plant growth substances 1979, pp. 109-118, Skoog, F., ed. Springer, Berlin Heidelberg New York Gawer, M., Laloue, M., Terrine, C., Guern, J. (1977) Metabolism and biological significance of benzyladenine-7-glucoside. Plant Sci. Lett. 8, 267 274 Gordon, M.E., Letham, D.S., Parker, C.W. (1974) The metabolism and translocation of zeatin in intact radish seedlings. Ann. Bot. 38, 809-825

B.A. McGaw and R. Horgan: Cytokinin oxidase from Z e a and Vinca Hall, R.H., Robbins, M.J. (1968) In: Synthetic procedures in nucleic acid chemistry, vol. i, p. 11, Zorback, W.W., Tipson, R.S., eds. Wiley, New York Horgan, R., Kramers, M.R. (1979) High-peformance liquid chromatography of cytokinins. J. Chromatogr. 173, 263-270 Horgan, R., Palni, L.M.S., Scott, I.M., McGaw, B.A. (1981) Cytokinin biosynthesis and metabolism in Vinca rosea crown gall tissue. In: Metabolism and molecular activities of cytokinins, pp. 56-65, Guern, J., Peaud-Lenoal, C., eds. Springer, Berlin Heidelberg New York Laloue, M., Terrine, C., Guern, J. (1977) Cytokinins: metabolism and biological activity of N6-(A2-isopentenyl) adenosine and N6-(A2-isopentenyl) adenine in tobacco cells and callus. Plant Physiol. 59, 478-483 Letham, D.S., Wilson, M.M., parker, C.W., Jenkins, I.D., Macleod, J.K., Summons, R.E. (1975) Regulators of cell division in plant tissues. XXIII. The identity of an unusual metabolite of 6-benzylaminopurine. Biochim. Biophys. Acta 399, 61-70 Letham, D.S., Tao, G.Q., Parker, C.W. (1982) An overview of cytokinin metabolism. In:Plant growth substances 1982, pp. 143-153, Wareing, P.F., ed. Academic Press, London New York Lowry, O.H., Rosebrough, N.J., Farr, A.L., Randall, R.J. (1951) Protein estimation with the folin phenol reagent. J. Biol. Chem. 193, 265-275 McGaw, B.A., Horgan, R. (1983) Cytokinin catabolism and cytokinin oxidase. Phytochemistry 22, 1103-1105 Miller, C.O. (1974) Ribosyl-trans-zeatin, a major cytokinin produced by crown gall tumour tissue. Prec. Natl. Acad. Sci. USA 71, 334-338 Paces, V., Werstiuk, E., Hall, R.H. (1971) Conversion of N 6(A2-isopentenyl) adenosine to adenosine by enzyme activity in tobacco tissue. Plant Physiol. 48, 775-778 Palmer, M.V., Horgan, R., Wareing, P.F. (1981a) Cytokinin metabolism in Phaseolus vulgaris L. I. Variation in cytokinin levels in leaves of decapitated plants in relation to lateral bud outgrowth. J. Exp. Bet. 32, 1231 1241 Palmer, M.V., Scott, I.M., Horgan, R. (1981b) Cytokinin metabolism in Phaseolus vuIgaris L. II. Comparative metabolism of exogenous cytokinins by detached leaves. Plant Sci. Lett. 22, 187-195 Parker, C.W., Letham, D.S. (1973) Regulators of cell division in plant tissues. XVI. Metabolism of zeatin by radish cotyledons and hypocotyls. Planta 114, 199-218

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Parker, C.W., Letham, D.S. (1974) ReguIators of cell division in plant tissues. XVIII. Metabolism of zeatin in Z e a m a y s seedlings. Planta 115, 337-344 Parker, C.W., Letham, D.S., Gollnow, B.I., Summons, R.E., Duke, C.C., MacLeod, J.K. (1978) Regulators of cell division in plant tissues. XXV. Metabolism of zeatin by lupin seedlings. Planta 142, 239 251 Parker, C.W., Wilson, M.M., Letham, D.S., Cowley, D.E., MacLeod, J.K. (1973) The glucosylation of cytokinins. Biochem. Biophys. Res. Commun. 55, 1370-1376 Scott, I.M., Horgan, R., McGaw, B.A. (1980) Zeatin-9-glucoside, a major endogenous cytokinin of Vinca rosea L. crown gall tissue. Planta 149, 472-475 Scott, I.M., Martin, G.C., Horgan, R., Heatd, J.K. (1982a) Mass spectrometric measurement of zeatin glycoside levels in Vinca rosea L. crown gall tissue. Planta 154, 273-276 Scott, I.M., McGaw, B.A., Horgan, R., Williams, P.E. (1982b) Biochemical studies on cytokinins in Vinca rosea crown gall tissue. In: Plant growth substances 1982, pp. 165-174, Wareing, P.F., ed. Academic Press, London New York Summons, R.E., Duke, C.C., Eichholzer, J.V., Entsch, B., Letham, D.S., MacLeod, J.K., Parker, C.W. (1979) Mass spectrometric analysis of cytokinins in plant tissues. II. Quantitation of cytokinins in Z e a m a y s kernels using deuterium labelled standards. Biomed. Mass Spectrom. 6, 407 413 Summons, R.E., Entsch, B., Letham, D.S., Gollnow, B.I., MacLeod, J.K. (1980) Regulators of cell division in plant tissues. XXVIII. Metabolites of zeatin in sweet-corn kernels: purification and identification using high-performance liquid chromatography and chemical-ionization mass spectrometry. Planta 147, 422-434 Terrine, C., Laloue, M. (1980) Kinetics of N6-(A2-isopentenyl) adenosine degradation in tobacco cells. Plant Physiol. 65, 1090-1095 Wareing, P.F., Horgan, R., Henson, I.E., Davies, W. (1977) Cytokinin relations in the whole plant. In: Plant growth regulation, pp. 147-153, Pilet, P.E., ed. Springer, Berlin Heidelberg New York Whitty, C.D., Hall, R.H. (t974) A cytokinin oxidase in Z e a mays. Can. J. Biochem. 52, 781-799

Received 24 February; accepted 5 May 1983

Cytokinin oxidase fromZea mays kernels andVinca rosea crown-gall tissue.

Cytokinin oxidase has been partially purified from matureZea mays kernels and fromVinca rosea corwn-gall tissue. The molecular weights of the two enzy...
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