Planta (1995)196:84~94 P l a n t ~

�9 Springer-Verlag 1995

The effect of auxin on cytokinin levels and metabolism in transgenic tobacco tissue expressing an ipt gene R. Zhang 1'2, X. Zhang 1, J. Wang ~, D.S. Letham 3, S.A. McKinney I, T.J.V. Higgins 2

Plant Cell Biology Group, Research School of Biological Sciences, Australian National University, PO Box 475, Canberra, Australia 2 CSIRO Division of Plant Industry, PO Box 1600, Canberra, ACT 2601, Australia 3 Cooperative Research Centre for Plant Science, Australian National University, PO Box 475, Canberra, ACT 2601, Australia

Received: 28 April 1994 / Accepted: 18 May 1994

Abstract. The ipt gene from the T - D N A of Agrobacterium tumefaciens was transferred to tobacco (Nicotiana tabacum L.) in order to study the control which auxin appears to exert over levels of cytokinin generated by expression of this gene. The transgenic tissues contained elevated levels of cytokinins, exhibited cytokinin and auxin au tonomy and grew as shooty calli on hormone- free media. Addition of 1-naphthylacetic acid to this cul- ture medium reduced the total level of cytokinins by 84% while 6-benzylaminopurine elevated the cytokinin level when added to media containing auxin. The cytokinins in the transgenic tissue were labelled with 3H and auxin was found to promote conversion of zeatin-type cytokinins to 3H-labelled adenine derivatives. When the very rapid metabolism of exogenous [3H]zeatin riboside was sup- pressed by a phenylurea derivative, a noncompetit ive in- hibitor of cytokinin oxidase, auxin promoted metabolism to adenine-type compounds. Since these results indicated that auxin promoted cytokinin oxidase activity in the t ransformed tissue, this enzyme was purified from the tobacco tissue cultures. Auxin did not increase the level of the enzyme per unit tissue protein, but did enhance the activity of the enzyme in vitro and promoted the activity of both glycosylated and non-glycosylated forms. This enhancement could contribute to the decrease in cy- tokinin level induced by auxin. Studies of cytokinin biosynthesis in the transgenic tissues indicated that trans- hydroxylat ion of isopentenyladenine-type cytokinins to yield zeatin-type cytokinins occurred principally at the nucleotide level.

Abbreviations: Ade = adenine; Ados = adenosine; BA = 6-benzy- laminopurine; C = control; Con A = concanavallin A; CP = cellulose phosphate; IPT = isopentenyl transferase; NAA = 1- naphthylacetic acid; NP=normal phase; NPPU=N-(3-nitro- phenyl)-N'-phenylurea; RIA=radioimmunoassay; RP=reversed phase. Abbreviations for natural cytokinins are listed as a footnote to Table 1. Correspondence to: D.S. Letham; FAX: 61 (6) 2475896; Tel.: 61 (6) 2492371

Key words: Agrobacterium - Auxin - Cytokinin metabolism - Cytokinin oxidase - ipt gene - Nicotiana (cytokinin)

Introduction

Crown gall is a disease of some dicotyledonous plants induced by virulent strains of Agrobacterium tumefaciens. A portion of the Ti (tumour-inducing) plasmids of these bacteria called the T-DNA, is transferred to plant cells and incorporated into nuclear DNA. Extensive insertion- al mutagenesis and transcript mapping of the T - D N A has led to the identification of all of the genes in the T-DNA. Genes 1 (iaaM), 2 (iaaH) and 4 (ipt) encode en- zymes for auxin and cytokinin biosynthesis, i.e. tryp- tophan monooxygenase, indoleacetamide hydrolase, and isopentenyl transferase, respectively (Morris 1986). Gene 3 (ocs) encodes octopine synthase, while gene 5 encodes an enzyme for the synthesis of indole-3-1actate, an auxin antagonist (Korber et al. 1991). The molecular functions of genes 6a, 6b and 7 are still not clear, but expression of gene 6b appears to reduce the activity of exogenous and endogenous cytokinins (Spanier et al. 1989) and to alter sensitivity to auxin plus cytokinin (Tinland et al. 1992).

The expression of the T - D N A in the host plant causes the "infected" cells to produce sufficient amounts of auxin and cytokinin for autonomous tumour growth. Insertion of the t ransposon Tn5 into the ipt locus results in rooty tumours containing an elevated ratio of auxin to cy- tokinin. Insertion into either the iaaM or iaaH loci yields tumours with shooty morphology and an elevated ratio of cytokinin to auxin due not only to a decrease in auxin level but also to a marked increase in cytokinin level (Akiyoshi et al. 1983; Ishikawa et al. 1988; McGaw et al. 1988). Hence, it appears that the expression of the auxin- synthesis genes iaaM and iaaH plays a role in regulating cytokinin levels in the tumour tissue.

The present paper reports a study of the effect of ex- ogenous auxin on cytokinin level in tissue expressing the

R. Zhang et al.: Effect of auxin on cytokinin metabolism 85

ipt gene. Since expression of some T - D N A genes (5, 6b, iaaM and iaaH) would greatly complicate the study, the nat ive ipt gene was separated from all other T - D N A genes and transferred to tobacco. The nat ive ipt gene, a nd the coding region of this gene unde r the cont ro l of the p romote r of the gene encoding the small subun i t of r ibu- lose- l ,5-b isphosphate carboxylase-oxygenase (Rubisco), have previously been transferred to tobacco. Rad io im- munoas say (RIA) of unpuri f ied extracts suggested that exogenous auxin reduced the cy tokin in level in the t rans- formed tissue (Beinsberger et al. 1991), but since the ex- tracts were no t purified, the results are ques t ionable and canno t be related to levels of specific cy tokin ins (see Dis- cussion). A n u m b e r of other chimeric ipt genes have also been in t roduced into plants (e.g. see Smar t et al. 1991), bu t the effect of auxin on cytokin in level and tissue devel- opmen t has no t been examined in these t ransgenic plants. In the studies now reported, the level of cytokinins was found to be greatly elevated in the t ransgenic tobacco tissue expressing the nat ive ipt gene and this level was reduced by exogenous auxin. The possibil i ty that this reduct ion was due to enhanced degradat ive metabo l i sm of cy tokin in was examined in this study, while studies of suppress ion of ipt m R N A and IPT prote in levels will be reported elsewhere.

Materials and methods

Plasmid construction and plant transformation. A 1538-bp SspI frag- ment containing the entire 723 bp of ipt coding region, 533 bp of 5' flanking region and 282 bp of 3' flanking region was excised from a cloned 12.5 kb pTiAch5 T-DNA fragment (Heidekamp et al. 1983) and subcloned into pUC18 at the SmaI site, creating plasmid pRZ1. The ipt gene was then excised with HindIII and EcoRI and inserted into the binary vector pGA492 (An 1986) to yield plasmid pRZ2. The transfer of pRZ2 from Escherichia coli. to a disarmed A. tume- faciens strain (LBA 4404) by triparental mating and then transfor- mation of tobacco (Nicotiana tabacum L. cv. Wisconsin 38) using leaf discs were performed by procedures described previously (Hig- gins et al. 1988). Transformed shoots were induced on the leaf discs by culture on MS9 medium containing cefotaxine and kanamycin (Higgins et al. 1988).

Tissue culture. For culture of the transformed tobacco tissue, four media were used frequently: (i) Murashige and Skoog (1962) medi- um without hormones (MSO); (ii) MSO with 1-naphthylacetic acid (NAA) at 5 laM (MSN); (iii) MSN plus 6-benzylaminopurine (BA) at 0.9 laM (MST), and (iv) MSO plus IAA (2.9 laM) and BA (4.5 IaM) termed MS9. All media contained agar (8 g-1-1) and kanamycin (120 mg.1-1) and cultures were maintained at 23~ under a 16-h photoperiod (20 I~mol quanta.m 2.s ~). Since control (C) tissue was transformed with the pGA492 vector only, it also expressed the kanamycin-resistance gene and could be cultured on the above media. Either shoot tips or very small callus explants were used for subculture.

Purification of cytokinin oxidase. Enzyme was extracted according to the method of Chatfield and Armstrong (1986) which involved purification with polyvinylpolypyrrolidone and removal of much unwanted protein and nucleic acid with Polymin P. Precipitation with (NH4)2SO 4 (20-60% saturation fraction) yielded a crude en- zyme fraction. Further purification utilized gel-filtration chro- matography (Sephadex G 100), followed either by chromatography on a column of concanavilin A-Sepharose [Con A-Sepharose (Phar- macia, Uppsala, Sweden); 0.1 ml per g of tissue] which was prepared

and eluted with methyl mannoside (0.1 M) according to Chatfield and Armstrong (1988), or by high-performance liquid chromatogra- phy (HPLC).

For HPLC, an Advanced Protein Purification System (Waters 650; Waters Associates, Milford, Mass., USA) consisting of a Wa- ters 600E controller and a Waters 490E programable multiwave- length detector operating at 280 nm was used. Two gel-filtration columns (Protein Pak 125 and 300 sw; Millipore Waters) were joined in series and equilibrated with 50mM Tris-HC1 buffer (pH 7.0). A flow rate of 1 ml-min 1 was used and fractions were freeze-dried.

Assay of cytokinin oxidase. The enzyme assay was based on the methods developed by Chatfield and Armstrong (1986; 1987). The reaction volumes (50 lal) contained either Tris-HCl buffer or imida- zole-HC1 buffer at a final concentration of 100 mM (pH 6.5; the latter buffer also contained 10 mM CuCI2). The substrate was usual- ly [8-3H]isopentenyladenine ([8-3H]iP; 4 p.M, 4.5 GBq.mmol- l) but other 3H-labelled cytokinins were also tested at this concentration. Reaction was normally conducted at 37~ for 30 min (imidazole- CuCl 2 buffer) or for 2 h (Tris-HC1 buffer) and was terminated by addition of ethanol-acetic acid (9:1 v/v, 100 gl) containing substrate (0.2 raM) and product [0.2 mM; adenine (Ade) or adenosine (Ados)]. In the case ofimidazole-Cu buffer, EDTA (40 mM, 20 lal) was added immediately prior to the above addition. [14C]Ade or [14C]Ados were then added as a recovery marker and the reaction solution was subjected to reversed phase thin-layer chromatography (RP TLC) on paraffin-impregnated silica gel layers using water followed by solvent C (see below) as solvents. Water eluted buffer salts to near the top of the layer prior to separation of product from substrate with the second solvent. The product spots were removed from the layer and mixed with water and scintillation fluid for determination of 3H and 14C using a model LS 3801 liquid scintillation system (Beckman Instruments, Fullerton, Calif., USA).

Chromatographic methods. For all normal phase (NP) TLC, layers were spread with Merck silica gel 60 PF254 or cellulose (Sigmacell; Sigma Chemical Co., St. Louis, Mo., USA). For RP TLC, Merck GF254 silica gel (15 Ixm) layers were impregnated with silicone fluid for studies of cytokinin metabolism or with liquid paraffin for assay of cytokinin oxidase (Singh et al. 1988; Letham et al. 1992). Solvents for TLC were as follows (proportions are by volume): A, n-bu- tanol:water:acetic acid (12:5:3); B, n-butanol:water:14 N ammonia (6:2:1, upper phase); C, 20% methanol after development with water; D, n-butanol:14 N ammonia:water:n-propanol:ethanol (6:1:3:1:1).

Analysis of cytokinins in tobacco tissues. Tissue extracts were pre- pared using methanol:water :formic acid (15:4:1, by vol.) and evap- orated as described previously (Singh et al. 1988). Three procedures were used to purify cytokinins for RIA as detailed below.

(1) Quantification of the entire cytokinin complex. The cytokinins were first separated into a basic fraction, containing cytokinin bases, ribosides and glucosides, and a cytokinin nucleotide fraction, by sequential chromatography on cellulose phosphate (CP) and diethylaminoethyl (DEAE)-cellulose columns (Badenoch-Jones et al. 1984). The basic fraction was purified on a paraffin-impregnated silica gel (PSG) column (Hall et al. 1987) and then subjected to NP TLC on silica gel (solvent A) followed by HPLC (Hall et al. 1987; Badenoch-Jones et al. 1987) to yield fractions for quantification of the cytokinins listed in Table 1 (abbreviations for all cytokinins referred to are given as a footnote). An aqueous solution of the nucleotide fraction was extracted with water-saturated n-butanol (3 times equal volume) and the extracts were discarded. The nucle- otides in the aqueous fraction were hydrolysed with alkaline phos- phatase (Hall et al. 1987) and the released ribosides were partitioned into n-butanol. After purification using a C18 column (SPE; J.T. Baker, Phillipsburg, N.J., USA) and HPLC, the ribosides were quantified by RIA. Tissue cultured on MST medium contained BA and its derivatives and these compounds cross-react with antibodies raised against N6-(2-isopentenyl)adenosine (iPA; Badenoch-Jones

86 R. Zhang et al.: Effect of auxin on cytokinin metabolism

et al. 1987). However, the HPLC procedure separated BA and its riboside from iP and iPA. In the procedure (2) below, this HPLC was not employed and iP and iPA could not be quantified.

All RIA was performed as described previously using antisera raised against zeatin riboside (ZR), dihydrozeatin riboside (DZR) and iPA (Badenoch-Jones et al. 1984; 1987). 3H-labelled recovery markers [DZ (dihydrozeatin), DZR] of high specific radioactivity (1100 and 190 GBq.mmol i respectively) were added to the extract- ing solvent (also in (2) and (3) below) to correct for cytokinin loses during purification. [14C]AMP was used to monitor nucleotide re- covery during DEAE-cellulose chromatography and [3H]DZR was added to the phosphatase hydrolysate to determine recovery during subsequent purification.

(2) Quantification of i P, zeatin ( Z ), D Z, their ribosides and nucle- otides. The basic fractions from the CP column step were purified through PSG columns using the modified procedure of Singh et al. (1992) to remove Ade, Ados, zeatin 7-glucoside (Z7G) and zeatin 9-glucoside (Z9G). Ribosides were then separated from bases using alumina columns and the riboside eluate was prepared for RIA as described previously (Singh et al. 1992). The base fraction was ex- tracted with petroleum ether (Singh et al. 1992) and further purified by NP TLC as described under (1). The iP and the Z + D Z zone eluates were chromatographed on Baker C18 columns for RIA. Nucleotides were purified as above.

(3) Simplified procedure for determining total cytokinin in base, riboside and nucleotideforms. In this method, the crude extract was hydrolysed to convert ribosides and nucleotides to bases and then the total base was determined by RIA. The usual extract was evap- orated to dryness under reduced pressure, and traces of water were then removed by addition and evaporation of the following solvents in sequence: anhydrous ethanol, ethanol-benzene (1:1, v/v) twice and dry dichloromethane. To the dried residue, the following were added per g of tissue: methanol, 1 ml; redistilled 2,2-dimethoxypro- pane, 0.2 ml; concentrated hydrochloric acid, 50 gl. The residue was dissolved in this solution and left at 25-26~ for 40 h. The solution was evaporated to dryness under reduced pressure and dry ethanol was then added and evaporated. Traces of residual acid were neu- tralised by addition of dilute ammonia and evaporation. A solution (pH 7) of the residue in water was extracted with n-butanol (three times with an equal volume). The bases in the extracts were purified for RIA using CP, triethylaminoethyl (TEAE)-cellulose (Singh et al. 1992) and Baker C18 columns in sequence. The C18 columns were eluted with 12% methanol containing 1% acetic acid (two column volumes) prior to elution of cytokinin bases with 80% ethanol con- taining acetic acid (1%). The anhydrous hydrolysis procedure de- scribed above did not hydrolyse cytokinin O- or N-glucosides ap- preciably.

Metabolism of 3H-labelled endogenous cytokinins. Callus tissue from 15-d-old transgenic tobacco cultures (T1 line grown on MSO agar medium) was excised into smaller pieces (about 100 mg per piece). The excised tissues were incubated in 250-ml flasks with 100 ml of MSO liquid medium containing 25 nM [3H]Ade (740 GBq.mmol 1) with or without NAA (11 laM). After incubation for 12 h at 23~ with shaking (30 rpm), unlabelled Ade was added to the cultures to a final concentration of 0.3 mM which was about 104 times the [3H]Ade concentration. After another 2 h, NAA was added to half of those cultures that did not contain NAA, to a final concentration of 25 gM. The radioactivity in the medium was determined by liq- uid scintillation counting throughout the experiment, while 1-1.5 g of tissue was removed from each culture at 12, 14, 19, 38 and 62 h after incubation commenced, and was washed with sterile water (three times) before extraction.

Prior to homogenisation of tissue for extraction of 3H-labelled cytokinins derived from [3H]Ade, [14C]Z was added as an internal recovery marker. The extracts were separated into a basic fraction and a nucleotide fraction by sequential chromatography on CP and DEAE-cellulose. The basic fraction was purified on a PSG column and subjected to NP TLC on silica gel (solvent A) to yield an iP + iPA fraction and a Z + ZR + DZ + DZR fraction. By NP TLC on silica gel (solvent B) the former yielded an iP fraction and an iPA

fraction, while the latter gave a Z + DZ and a ZR + DZR fraction. Dihydrozeatin and DZR were separated from Z and ZR, respec- tively, by RP TLC (solvent C). The ribosides released from the nucleotide fraction by alkaline phosphatase hydrolysis were parti- tioned into n-butanol, purified on a PSG column and subjected to NP TLC (solvent A followed by B) and then to RP TLC as above. The radiochemical purity of the fractions obtained was confirmed by HPLC.

Metabolism of exogenous [3H]ZR. For uptake of [3H]ZR, explants of transformed tobacco tissue (T2; 15- to 20-d-old cultures) were incubated in MSO liquid medium (3 ml) in Petri dishes (diameter 8.5 cm) containing one circle of filter paper. The media contained [3H]ZR (76nM; 130GBq.mmol 1) with or without N-(3-nitro- phenyl)-N'-phenylurea (NPPU; 50 or 150 gM) and NAA (27 gM). After incubation for 24 h, the tissues were removed from the media and washed sequentially with water, ZR solution (20 gM) and water before extraction (each wash 30 s). In some experiments, the ex- plants were preineubated on MSO medium with or without NPPU and NAA for 20 h, before transfer to the above media.

In studies of the metabolism of exogenous [3H]ZR, the crude extracts plus marker metabolites were subjected directly to two-di- mensional NP TLC on silica gel (solvent B, followed by A). This enabled the radioactivity due to Ade, Ados, Z + DZ and ZR + DZR to be determined. Z-type compounds are not separated from DZ in this system and this was achieved by RP TLC (solvent C). The zone at and near the origin after TLC in the first dimension, which contained nucleotides, was eluted and hydrolysed with alkaline phosphatase; the released ribosides were separated by NP TLC on silica gel (solvent B) and by RP TLC. This enabled radioactivity due to AMP, ZNT (zeatin nucleotide) and DZNT (dihydrozeatin nude- otide) to be determined. Radioactivity due to these nucleotides was confirmed by NP TLC of the unhydrolyzed eluate on cellulose (solvent D).

Results

Transformation, growth and cytokinin levels o f tobacco tis- sues. A 1538-bp fragment from the T - D N A of pTiAch5 con ta in ing the entire ipt coding region, as well as 533 bp of Y-f lanking sequence and 282 bp of 3 ' - f lanking se- quence, was in t roduced into tobacco using A. tumefaciens and the b inary vector pGA492. This mean t that in addi- t ion to the ipt gene with its nat ive promoter , a kanamy- cin-resistance (neomycin phosphotransferase) gene modi- fied for expression in plants was also in t roduced to the same tobacco cells, thus al lowing selection of the t rans- formed cells using the antibiotic. Originally, five kanamycin- res i s tan t t ransgenic lines were ob ta ined by t rans format ion of tobacco leaf discs with the plasmid p R Z 2 ; each line was derived from one t ransgenic shoot induced on the shoot- forming med ium MS9. Two lines (T1, T2) were selected for detailed analysis. Southern b lo t t ing detected the ipt gene in all five lines which pos- sessed a similar copy n u m b e r of the gene. The ipt m R N A and a polypept ide of the molecular weight expected for the isopentenyl transferase enzyme were also detected in T1 and T2 tissues by Nor the rn and Western b lo t t ing techniques respectively (Zhang 1992).

In contras t to the control (C) shoots, t ransformed with the pGA492 vector only, which could form roots and grow into no rma l plantlets on MS O medium, the t rans- formed T1 and T2 shoots were unab le to do so and grew as shooty calli which did no t require auxin or cy tokin in dur ing subculture. On MSO medium, shoot deve lopment

R. Zhang et al.: Effect of auxin on cytokinin metabolism 87

Table 1. Cytokinin levels in control (C) and transgenic (T1, T2) tobacco tissues. The tissues were cultured on MST medium and then subcultured onto fresh MST medium for 14 d prior to analysis

Cytokinin a Cytokinin levels (pmol.(g FW) -l)

C T1 T2

Z 35.0 52.2 22.3 DZ 24.7 16.3 12.0 iP 32.7 27.4 25.7 ZR 7.6 227.9 50.8 DZR 3.8 38.5 15.0 iPA 5.7 5.9 3.9 Z9G 2.5 7.4 8.3 Z7G 5.2 8.6 8.0 DZ7G 3.4 15.0 26.4 OGZ 3.7 4.9 5.2 OGDZ 2.2 4.1 3.0 OGZR 2.1 15.6 13.6 OGDZR 0.5 6.8 10.4 iPNT 16.3 22.6 13.6 ZNT 4.4 252.0 73.1 DZNT 14.6 78.3 29.0 Total 164.4 783.5 320.3

a Z, zeatin; DZ, dihydrozeatin; iP, N6-(2-isopentenyl)adenine; ZR, zeatin riboside; DZR, dihydrozeatin riboside; iPA, N6-(2-isopen - tenyt)adenosine; Z9G, zeatin 9-glucoside; Z7G, zeatin 7-glucoside; DZ7G, dihydrozeatin 7-glueoside; OGZ, OGDZ, OGZR and OGDZR, the O-[3-glucosides of Z, DZ, ZR and DZR, respectively; iPNT, ZNT and DZNT, the 5'-phosphates of iPA, ZR and DZR, respectively (i.e. iP, Z and DZ nucleotides)

by T1 tissue was more pronounced than that by T2 tis- sue. When these shooty calli were subcultured onto M S N medium which contained N A A (5 ktM), they produced smooth-surfaced unorganized tissue, and a similar mor- phology was also observed when the tissues were cul- tured on MST medium. The fresh weight of T1 and T2 tissue cultured on M S N medium did not exceed that of tissue cultured on MSO medium at any time between 2 and 28 d after subculture, and at 25 txM, N A A did not affect growth appreciably in short-term cultures ( < 6 d), but in cultures maintained for over 12 d, this concentra- tion was supraoptimal. Calli with the tumorous mor- phology could also be induced from C tissue, when cul- tured on MST medium. However, these calli, unlike those containing the ipt gene, could not grow actively on either MSO or M S N medium, unless cytokinins were provided. While M S N medium induced C tissue to exhibit limited growth as a spongy callus, on MS9 medium and on MSO medium containing ZR (10 pM), C tissue grew actively as a shooty callus resembling T1 tissue on MSO medium.

By a combinat ion of chromatography and radioim- munoassay, cytokinin levels were assayed in the ipt- t ransformed tobacco tissues (T1, T2), and in C tissue. The levels of 16 cytokinins in 14-d-old tissue cultured on MST medium are presented in Table 1 (a footnote to this Table lists all abbreviations used for natural cytokinins). MST medium, which contains BA and NAA, was chosen because C and the ipt-transformed tissues all grew on this medium with a very similar morphology, namely, undif- ferentiated callus. The level of certain cytokinins was

greatly increased in the ipt-gene-transformed tissues, compared to the C tissue. The greatest increase, among the 16 cytokinins measured, was found in the level of Z N T which increased 57-fold in T1 tissue, and 17-fold in T2 tissue. The levels of ZR, DZR, D Z N T and the glu- cosides were also markedly increased. However, no in- creases were found in iP-type cytokinins, which include the nucleotide iPNT, the product of isopentyl transferase (IPT) action, thus indicating that iPNT was very rapidly converted to hydroxylated (Z-type) cytokinins which then accumulated in the tissues.

The effect of auxin and BA on cytokinin levels in trans- formed tobacco. The auxin-induced change in the mor- phology of the transgenic tissues from shooty to smooth callus was correlated with a marked decline in levels of cytokinins in the tissues. Table 2 shows the cytokinin lev- els in the transformed T1 and T2 tobacco tissues (12 d old) cultured on MSO medium without hormones (tis- sues beginning to develop shoots), on M S N (smooth cal- lus), and on MST medium (smooth callus). The total lev- els of cytokinins Z, DZ, ZR, DZR, Z N T and D Z N T in these tissues grown on M S N were about 17% of those in tissues cultured on MSO and auxin reduced the level of each cytokinin considerably. The relatively low cytokinin levels in C tissues were further reduced by auxin (Table 2). While N A A reduced levels of Z and D Z cy- tokinins considerably in T1 and T2 tissues, the levels of iP-type cytokinins did not change appreciably. It is note- worthy that exogenous BA elevated the cytokinin levels in the T1 and T2 tissues cultured on auxin-containing media but there was no similar elevation in the case of C tissue (Table 2).

A reduction in cytokinin level caused by exogenous auxin was also evident in further experiments using T1 and T2 tissue previously cultured on either MST or MSO media, and in these experiments, cytokinin ribosides and nucleotides were hydrolyzed to bases and the total level of each base was then determined. With callus tissue derived from culture on MST medium, N A A reduced the levels of Z plus D Z for T1 and T2 tissue by 90% and 81%, respectively, after culture for 15 d; the correspond- ing values for tissue derived by culture on MSO media (explants consisted of callus plus shoots) were 50 and 73%, respectively. The cytokinin level of T1 and T2 tissue was also reduced by N A A after culture for only 4 d, well before the auxin could have induced any change in mor- phology. Thus when T2 explants grown on MSO media were subcultured onto MSO, M S N and MST media for 4 d, the Z and D Z levels (latter in parentheses) after hy- drolysis were 354 (186), 171 (106) and 298 (180) pmol.(g FW) -~, respectively. Hence the ability of BA to elevate the cytokinin level in the presence of auxin was also evident after only 4 d.

The effect of auxin on metabolism of endogenous 3H-la- belled cytokinins in 77 tissue. In this study, the cytokinins in T1 tissue were first labelled with [3H]Ade. While the objective was to examine the effect of exogenous auxin on their subsequent metabolism, some comments on the in-

88 R. Zhang et al. : Effect of auxin on cytokinin metabolism

Table 2. The levels of cytokinin bases, ribosides and nucleotides in control (C) and transgenic (T1, T2) tobacco tissues cultured on MSO, MSN and MST media for 12d. MSO medium does not contain hormones; MSN medium is MSO with NAA (5 ~tM) while MST medium contains both NAA (5 gM) and BA (0.9 gM). The C tissue for the experiment was grown on MST medium and consisted

of callus while the T1 and T2 tissues were grown on MSO medium and callus tissue was used for the experiment. The iP-type cy- tokinins were not determined (ND) in tissues cultured on MST medium due to interference from BA and its derivatives which cross-react with anti-iPA antibodies

Cytokinin Cytokinin levels (pmol.(g FW) ~)

C T1 T2

MSO MSN MST MSO MSN MST MSO MSN MST

iP ND ND ND 24.8 20.6 ND 29.8 21.9 ND Z 50.0 18.2 17.9 248.9 36.0 290.1 66.5 13.7 25.5 DZ 25.5 6.9 2.8 73.8 15.5 53.0 20.3 6.6 11.0 iPA ND ND ND 18.8 18.5 ND 9.3 10.3 ND ZR 11.4 5.2 19.4 412.5 43.9 235.0 117.1 14.0 55.3 DZR 17.0 5.4 10.8 165.0 28.0 128.6 40.0 17.9 27.8 iPNT ND ND ND 5.7 7.5 ND 6.0 4.9 ND ZNT 30.7 8.6 6.9 327.1 63.5 182.7 278.3 18.6 44.6 DZNT 20.2 6.7 9.7 104.2 22.4 96.3 42.1 24.8 11.8 Total a 154.6 51.0 67.5 1331.5 208.3 985.7 564.3 95.6 176.0

a For Z- and DZ-type cytokinins

corporat ion of labelled Ade into cytokinins, i.e. their biosynthesis and interconversion, are merited.

After uptake of [3H]Ade for 12 h, the incorporat ion of radioactivity into the following cytokinins was deter- mined: iPNT, ZNT, DZNT, iPA, ZR, DZR, iP, Z and DZ. Results from these studies showed that: (i) overall radioactivity associated with the cytokinins in the tissues cultured on MSO medium was 3050 Bq'(g FW) 1, which was 1.54% of the soluble 3H recovered from the tissues after incubation for 12 h; (ii) the radioactivity was mainly associated with Z- and DZ-type cytokinins, while only a small amount of radioactivity (50 Bq-(g FW) -~) was de- tected in iPNT but none was present in either iPA or iP; (iii) of the radioactivity attr ibutable to cytokinins, 84% was associated with cytokinin nucleotides (iPNT, 2%; ZNT, 56%; DZNT, 26%) and 16% with bases plus ri- bosides (Z, 3% ; DZ, 9%; ZR, 2%; DZR, 2%). When this experiment was repeated with C tissue after transfer to MSO medium, negligible incorporat ion of 3H into cy- tokinins was found.

The addition of excess unlabetled Ade to the cultures after a 12-h incubation, resulted in an immediate inhibi- tion in [3H]Ade uptake by the tissues. The concentration of Ade supplied should have flooded the pool of Ade and related compounds in the cells and, consequently, the incorporat ion of [3H]Ade into cytokinins would be rapid- ly terminated, which was confirmed by a declining level of 3H-labelled cytokinins found in the tissues 2 h later. Hence, it is reasonable to assume that metabolism would become the major factor which influenced the levels of 3H-labelled cytokinins in tissues after addition of excess unlabelled Ade.

Although cytokinin nucleotides were the dominant form of 3H-labelled cytokinins in the tissue at the time of addition of unlabelled Ade, the level of this nucleotide radioactivity declined rapidly after the addition. At 0, 2, 7 and 26 h after supply of unlabelled adenine, the total

radioactivity due to cytokinin nucleotides (iPNT, ZNT, DZNT) was 2495, 1812, 847, and 368 Bq.(g FW) 1 respec- tively, which represented 83.6, 64.9, 52.1 and 45.6% of total radioactivity associated with cytokinins analysed at each time point. Concomitant with the initial decline in level of radioactive nucleotides, the level of 3H-labelled ribosides, and bases increased. The total radioactivity of cytokinin ribosides at 0, 2, 7 and 26 h after unlabelled Ade supply was 132, 135, 250 and 223 Bq.(g FW) 1 re- spectively, which represented 4.0, 4.9, 15.9 and 29.7% of total radioactivity associated with cytokinins analysed. The corresponding percentages for cytokinin bases were 12.4, 29.5, 30.4 and 21.4%, respectively. No 3H-labelled iP or iPA was detected. After addition of excess Ade to T1 cultures on MSO medium to terminate incorporat ion of label into cytokinins, the level of total Z-type cytokinins (half-life about 5 h), which initially exceeded the level of DZ-type compounds, declined much more rapidly than the latter, and after 24 h, the DZ-type compounds were dominant (Fig. 1, cf. A and B).

To study the effects of auxin on metabolism of endoge- nous cytokinins, the levels of 3H-labelled cytokinins (Z, DZ, ZR, DZR, Z N T and DZNT) were compared in T1 tissue during short-term culture on one of three media as follows (excess unlabelled Ade was added to all cultures after 12 h): (i) on MSO medium plus [3H]Ade (MSO or control cultures); (ii) MSO plus [3H]Ade plus N A A added at start of culture period (MSO + N A A (1) cultures); (iii) MSO + [3H]Ade, with N A A added 2 h after the addition of unlabelled Ade (MSO + N A A (2) cultures). Tissue was usually sampled at 12, 14, 19, 38 and 62 h after com- mencement of incubation with [3H]Ade.

Addition of N A A to the medium resulted in lower total levels of 3H-labelled cytokinins in the tissues. The levels of 3H-labelled cytokinins in MSO + N A A (2) cul- tures were 76.9, 61.6 and 66.3% of the levels found in controls (MSO cultures) at 5, 24 and 48 h after N A A

R. Zhang et al. : Effect of auxin on cytokinin metabolism 89

x

7-,

I00 - '.,,<>.

"".. "0 . . . . . . . . <) 10-

1.0-

0.3- i i i i ,

c

10 , ~

1.0 - ' , , "'O

0.3- ........... + c

B

..... +---:---:----2

D 9 . . . . . . *-.

;~ 2'4 a; ,o 10 72

Time (h)

Fig. IA-D. The effect of exogenous auxin on the levels of endoge- nous 3H-labelled cytokinins in transgenic tobacco (TI) tissue. The time specified is from the start of the culture period when [3H]Ade was supplied to the cultures. Excess unlabelled adenine was added to each culture at 12 h and while N A A was added to M S O + N A A (2) cultures at 14 h, NAA was supplied to M S O + N A A (1) cultures throughout their growth. A 3H-Labelled Z-type cytokinins (Z + ZR + ZNT); B 3H-labelled DZ-type cytokinins (DZ + DZR + DZNT); C 3H-labelled ZR; D 3H-labelled DZR <>---O Control cultures (MSO medium); O - O, MSO + N A A (1) cultures; + - - - + , M S O + N A A (2) cultures

addition respectively. The total levels of 3H-labelled cy- tokinins in MSO+NAA (1) cultures were 40.8 and 41.1% of that in MSO cultures at 19 and 38 h after incu- bation started, i.e. 7 and 26 h after addition of unlabelled Ade to prevent further incorporation of 3H into cy- tokinins. The effect of NAA in promoting degradation of Z-type cytokinins was markedly greater than the effect on DZ-type cytokinins (Fig. 1, cf. A and B). The levels of 3H-labelled Z- and DZ-type cytokinins in MSO + N A A (2) cultures were 14 and 82%, respectively, of the levels found in the controls after NAA application for 48 h. The Z-type cytokinin that was affected most markedly by NAA on a percentage basis was [3H]ZR which was unde- tectable in MSO + NAA (2) cultures 24 h after NAA ad- dition (Fig. 1C). Reduction of [3H]DZR level was also promoted by NAA but to a much lesser degree (Fig. 1D). Auxin supplied to MSO + NAA (2) cultures markedly re- duced the level of [3H]ZNT (40% reduction in 5 h), but [3H]DZNT was not affected.

The above rapid NAA-induced degradation of Z-type cytokinins evident at 5 h and subsequently can be at- tributed to cytokinin-oxidase action. However, N A A is now known to reduce ipt mRNA levels by about 50% in 4 d (Zhang 1992), but it is unlikely that changes in cy- tokinin pool size and specific activity could have an ap- preciable influence on the rapid metabolism results re- ported above. Two observations support this view: prein- cubation of T1 tissue with NAA prior to addition of unlabelled Ade (as in MSO+NAA (1) cultures) did not enhance auxin-induced cytokinin metabolism measured

subsequently; preincubation with NAA also did not en- hance the metabolism of exogenous ZR caused by simul- taneous supply of NAA.

The 3H-labelled cytokinin levels in MSO+NAA (1) cultures, which received auxin throughout the incubation period, were consistently lower than the levels in MSO + NAA (2) cultures (Fig. 1). Only 7 h after addition of unlabelled Ade to eliminate further 3H incorporation into cytokinin, the level of 3H-labelled Z-type cytokinins in MSO + NAA (1) cultures was only 27% of the control level in MSO cultures (Fig. 1A).

Effect of auxin on metabolism of exogenous ZR in the transformed tobacco tissues. When [3HIZR or [3H]iP (2 gM) was supplied to the transformed tobacco tissues (T1 and T2) for 22 h, less than 3% of the radioactivity then extracted was due to the unmetabolized cytokinin. When [3H]ZR was supplied, less than 1.5% of recovered 3H could be attributed to Z and the principal metabolites were Ados and AMP. The main metabolites of iP were iP-7-glucoside (identity established by HPLC, and NP and RP TLC), Ade and Ados. Supply of [3H]ZR to the control tobacco tissues resulted in similarly rapid metabolism. Because of this rapidity of the cytokinin metabolism, it was not possible to demonstrate any pro- motion of metabolism by auxin. Formation of Ade-type compounds from ZR as dominant metabolites is indica- tive of active degradation by cytokinin oxidase. Suppres- sion of this oxidation might allow detection of auxin-in- duced metabolism of ZR. Since two substituted ureas, N, N'-diphenylurea and N-(2-chloro-4-pyridyl)-N'-pheny- lurea, are known to inhibit purified cytokinin oxidase prepared from wheat germ (Laloue and Fox 1989), urea derivatives available to us were tested for ability to sup- press formation of Ade-type compounds from exogenous [3HIZR in the tobacco cultures T1 and T2. When tissue was transferred to MSO medium containing [3H]ZR, ad- dition of the following ureas at 150 pM did not suppress formation of 3H-labelled adenine compounds: N-benzyl- N'-(3-chlorophenyl)urea, N,N'-diphenylurea, N-benzyl- N'-(3,4-dichlorophenyl)urea, and N-benzyl-N'-pheny- lurea. However, N-(3-nitrophenyl)-N'-phenylurea (NP- PU) at 150 gM reduced conversion to these compounds by about 50%, and the 3H due to total Z-type com- pounds was elevated threefold (Table 3). The radioactivi- ty associated with Z, ZR and ZNT were all increased by NPPU and that due to DZ-type compounds was also elevated but to a much lesser degree. Addition of NAA to the NPPU medium partially reversed the effect of NPPU and promoted formation of Ade-type metabolites from the [3H]ZR. The effects of NPPU at 50 and 150 gM were similar.

Preincubation of tissue with NPPU prior to transfer to the medium containing NPPU and [3H]ZR did not en- hance the suppression of Ade-type metabolite formation. However, the preincubated tissue showed a more pro- nounced NAA-induced conversion of Z-type compounds to Ade-type compounds compared with tissue trans- ferred directly to [3H]ZR medium containing NPPU (Table 3). NAA reduced the radioactivity due to both ZR and ZNT markedly (70% reduction) in NPPU-preincu-

90 R. Zhang et al.: Effect of auxin on cytokinin metabolism

Table 3. Radioactivity due to metabolites in extracts of transgenic tobacco (T2) tissue supplied with [3H]ZR in the presence and ab- sence of NPPU and NAA. The NPPU and NAA were supplied at 150 gM and 27 p.M, respectively. In experiment A, the tissue previ- ously cultured on MSO medium was transferred directly to media containing [3H]ZR for 24 h. In experiment B, the tissue was first

preincubated on MSO medium with or without NPPU for 20 h. The metabolites specified below accounted for 60-78% of the extracted radioactivity; the remainder was due to 7-glucosides of Z and DZ and to degradation products of Ade-type compounds. Auxin did not affect the percentage of radioactivity in these forms

Medium

For preincubation For [3H]ZR uptake and metabolism

Radioactivity (% of total extracted)

Ados Ade AMP ZR DZR ZNT DZNT Z DZ

Expt. A MSO 50.9 5.2 MSO+NPPU 21.6 1.2 MSO+NPPU + NAA 34.7 1.9

Expt. B MSO MSO 28.4 1.7 MSO + NPPU MSO + NPPU 14.7 0.25 MSO + NPPU MSO + NPPU + NAA 28.6 0.75

9.6 3.2 1.5 3.9 1.7 0.9 0.7 8.9 7.5 2.3 13.0 3.4 4.5 1.2 8.9 4.3 1.7 7.0 2.8 4.2 2.1

7.0 1.7 0.9 7.8 4.5 0.4 0.2 4.5 8.7 2.0 20.3 4.8 4.6 0.6 6.3 2.6 1.9 6.6 4.8 4.2 3.2

Table 4. Effect of NAA on tobacco cytokinin oxidase activity in two different buffer systems. The enzyme used for these assays was puri- fied by HPLC. Both buffers were at pH 6.5 and the imidazole buffer contained CuC12. The Ade formed from [3H]iP is expressed on a

picomolar basis and as a percentage of the control value without NAA. The values presented are the mean for two experiments which showed very similar trends

NAA concn. Enzyme activity (p~M)

Tris-HC1 buffer Imidazole buffer

(pmol.h- 1-(~tg protein)- i) (% of control) (pmol.h- 1.(~tg protein)- 1) (% of control)

0 1.10 100 8.31 100 5 1.36 124 8.06 97

10 1.79 163 7.95 96 25 1.69 154 5.57 67 50 2.09 190 6.53 79

100 1.74 158 6.75 81

b a t e d t issue bu t tha t due to Z was not d iminished. The inh ib i t i on of Z R m e t a b o l i s m by N P P U and its counte r - ac t ion by N A A occur red in b o t h shoo t s and cal lus tissue when these were excised a n d cu l tu red separa te ly .

In a fur ther exper iment , T2 tissue was p r e i n c u b a t e d with bo th N P P U a lone and wi th N P P U + N A A for 12 h p r io r to u p t a k e of [3H]ZR in the presence of these com- pounds . P r e i n c u b a t i o n with N A A did no t enhance the d e g r a d a t i o n of [3H]ZR to A d e - t y p e c o m p o u n d s relat ive to tha t i nduced by N A A co- supp l i ed with the 3H-label led cy tokin in . A l t h o u g h B A e leva ted cy tok in in levels of T1 and T2 t issue in cul ture , it d id no t suppress d e g r a d a t i o n of the supp l i ed [3H]ZR. In all the a b o v e s tudies involv ing N P P U , when N A A p r o m o t e d d e g r a d a t i o n of Z- type cy- tok in ins to A d e - t y p e c o m p o u n d s , it d id no t reduce the p r o p o r t i o n of r ad ioac t i v i t y due to D Z - t y p e cytokinins . The effect of N A A was therefore specific for Z - type cy- tokin ins .

Cytokinin oxidase activity in tobacco tissue. Since N A A p r o m o t e d m e t a b o l i s m of e n d o g e n o u s 3H-label led Z- type cy tok in ins , a n d conve r s ion of exogenous Z R to A d e - t y p e c o m p o u n d s , the effect of N A A on the ac t iv i ty of t obacco c y t o k i n i n ox idase in vi t ro a n d on the t issue levels of this ac t iv i ty were assessed. C y t o k i n i n ox idase was p r e p a r e d f rom t o b a c c o cul tures T1 a n d T2 and was p rec ip i t a t ed by

a m m o n i u m su lpha te to yield crude enzyme which was fur ther purif ied by gel f i l t ra t ion and o the r methods . The subs t ra te specificity of the purif ied enzyme was deter- mined in two buffer systems: Tris-HC1 buffer and imida- zole-HC1 buffer with CuC12. As r epo r t ed for ox idase f rom Phaseolus vulgaris (Chatf ield and A r m s t r o n g 1987), the reac t ion rate in the la t te r buffer was much h igher than tha t in the former. In bo th buffers, iP was the prefer red subs t ra te of those tested. Whi le Z and cis-Z were sub- strates, the co r r e spond ing r ibos ides and the s a tu ra t ed c o m p o u n d 6 - (3 -methy lbu ty lamino)pur ine and its ri- bos ide were no t d e g r a d e d apprec iab ly . Charac te r i s t i cs of the t obacco oxidase purif ied by H P L C were as fol lows: Vma x and K m (subst ra te iP in Tr is-HC1 buffer), 3 .4pmol .h - l - (gg prote in) -1 and 4 . 9 g M , respect ively; mo lecu l a r weight based on H P L C gel f r ac t iona t ion and gel e lec t rophores is , 85 000; p H o p t i m u m 7.0-8.0. In Tris- HC1 buffer, with enzyme pur i f ied by H P L C , the ra tes of convers ion of iP, Z and cis-Z to Ade (pmol .h 1.0ag prote in) 1 protein)-1 were 1.39, 0.48 and 0.29 respect ively; in imidazo le buffer with CuCI2, the rates for iP and Z were 14.4, and 3.2 respectively.

D e p e n d i n g on the buffer used, N A A ei ther p r o m o t e d or inh ib i ted the ac t iv i ty of H P L C - p u r i f i e d oxidase. In imidazo le buffer with CuCI 2, an inh ib i t ion was a lways obse rved which was m a x i m a l at 25 laM (33% inhibi t ion) .

R. Zhang et al.: Effect of auxin on cytokinin metabolism 91

140O0

12OOO

,oooo

2

~ o o

=,

,.= 4ooo

2o0o

o , , . . . . . . , , , , , , , , , . . . . . , , , , , . . . . . . . , , , , , . . . . . . . .

5 15 25 35 45 Fraction number

55

o.~= g

o.1

i 0 .0~

=

0.06 ]

Fig. 2. Concanavalin-A column chromatography of cytokinin oxi- dase from transgenic tobacco tissue. The UV absorption is indicat- ed by �9 and cytokinin oxidase activity is indicated by the his- togram. In fractions 1-9, 25-45 and 55-60, no appreciate enzyme activity was detected. Fractions 12-20 were combined as Con-A column fraction B and fractions 48-52 were termed fraction A. Elution with methyl mannoside was commenced when fraction 40 had been collected. The enzyme activity shown is that found in a 30-1al aliquot of each fraction (2.5 ml)

In Tris-HC1 buffer, however, oxidase activity was pro- moted and the maximum promotion (90%) was observed at a f f ' N A A concentration of 50 laM (Table 4). The effect of N A A on the activity of cytokinin oxidase purified in other ways was also determined. Thus, enzyme purified by chromatography on a Sephadex G 100 column was then passed through a Con A-Sepharose column which yielded two cytokinin oxidase fractions (Fig. 2) - one held on the column (presumably glycosylated) and eluted with methyl mannoside (fraction A, about 22% of the total enzyme activity) and the other not retained (fraction B, about 78% of the total enzyme activity). The activity of both cytokinin oxidase fractions was promoted by N A A in Tris-HC1 buffer (Table 5), but not in imidazole buffer. Although N A A also enhanced the activity of crude to- bacco cytokinin oxidase prepared by precipitation with ammonium sulphate only (see Materials and methods), the effects evoked were less marked (10-30% promotions at 25-50 IxM) than those found with HPLC-purified en- zyme and tended to be more variable. Enzyme which had been stored and was of low activity was also less respon- sive to N A A than freshly prepared oxidase. Cytokinin oxidase activity per unit protein was not affected by incu- bation of T1 and T2 tissue with N A A for 24 h. However, cytokinin oxidase activity was elevated fivefold in tissues expressing the ipt gene, relative to C tissue.

N-(3-nitrophenyl)-N'-phenylurea, which suppressed conversion of [3H]ZR to Ade-type metabolites in vivo,

x

0 0 .07 0 .2 0 .7 2 8 17 50

N P P U ( uM)

Fig. 3. Inhibition of cytokinin oxidase from transgenic tobacco tis- sue by NPPU. Crude enzyme was incubated with [3H]iP in imida- zole-CuC12 buffer (pH 6.5) containing increasing concentrations of NPPU. Oxidase activity is expressed as pmol Ade.h-l.(~tg protein)

2 0 / / 1 .8

1 .6

1 ,4

1 .2

~ 1 .0 § §

0 .8

0 .6

0 .4

0 .2 /

[ ~ I I I I I

- 0 .1 0 .0 0 .1 0 .2 0 .3 0 .4 0 .5

1 / IS ]

Fig. 4. Double reciprocal plots of substrate ([3H]iP) concentration and reaction velocity determined in the presence of varying concen- trations of NPPU. Assays were performed in imidazole-CuCl 2 buffer (pH 6.5) with cytokinin oxidase purified on a Sephadex G 100 column. The Ki for NPPU was 0.7 gM. The concentrations of NP- PU were zero (O-O) , 0.7 p.M (A--A), 1.41~M ( x - x) and 2.1 ixM (+ - - +)

was found to be a potent inhibitor of cytokinin oxidase in vitro (Fig. 3) and it acted in a non-competitive manner (Fig. 4). A concentration of 0.7 ~tM caused 50% inhibi- tion of the tobacco enzyme in the standard assay in imi- dazole buffer. In Tris-HCl buffer a similar level of inhibi- tion occurred. However, N A A (5, 10, 20, 25 and 50 laM) did not suppress the activity of N P P U in the in-vitro enzyme assay. The effects of N A A on cytokinin metabolism in the presence of N P P U are therefore not due to a simple interaction between the two compounds at the level of cytokinin oxidase.

Table 5. Effect of NAA on the activity of tobacco cytokinin oxidase purified by chromatography on Con A Sepharose. As- says were performed in Tris-HC1 buffer. Fraction A was retained on the column and eluted with methyl mannoside; frac- tion B was not retained

Con A fractions NAA concn. Enzyme activity (p.M)

(pmol.h 1.([.tg protein) i) % of control

Fraction A 0 0.32 100 50 0.48 150

Fraction B 0 0.98 100 50 1.44 147

92 R. Zhang et al. : Effect of auxin on cytokinin metabolism

Discussion

Inactivation of the auxin-synthesis genes (iaaM and iaaH) results in a marked increase in the level of cy- tokinins in the transformed tissues. This observation, coupled with the report that exogenous auxin inhibits ZR accumulation in one of three lines of tobacco tissue transformed with a wild-type strain of Agrobacterium (Hansen et al. 1987), implies a regulatory role for auxin in the control of cytokinin biosynthesis and/or metabolism in tissues transformed with Ti plasmids. However, this speculation was not supported by the observation that exogenous auxin did not affect cytokinin level in cultures of tobacco tissue transformed with a mutant Ti plasmid defective in auxin biosynthesis (Rudelsheim et al. 1987).

Because of these conflicting results regarding the effect of exogenous auxin on the cytokinin level in transgenic tissues, and the possibility that other T -DNA genes (such as 5 and 6b) may also be involved in regulation of auxin and cytokinin action, the ipt gene from the Ti plasmid pTiAch5 was separated from all other genes and trans- ferred to tobacco using a binary vector pGA492 via a fully disarmed A. tumefaciens strain. Exogenous auxin significantly reduced the cytokinin level, and also sup- pressed shoot formation in the transgenic T1 and T2 tis- sues. Furthermore, auxin was shown to promote degra- dation of cytokinins by cleavage of the isoprenoid sidechain to yield adenine derivatives. However, in con- trolling cytokinin level in the tobacco tissues, auxin ap- peared to have a bifunctional role. Auxin has been found to suppress ipt gene expression (Zhang 1992) and this will be reported in detail elsewhere.

The transgenic tissues and their endogenous cytokinins. The transgenic T1 and T2 tissues expressing the ipt gene showed autonomy for both cytokinin and auxin, and such autonomy has been associated previously with ex- pression of this gene (Binns et al. 1987). In the present studies, shoots were initiated 4 8 d after subculture of T1 and T2 tissues on MSO medium. This early formation of shoots which then produce auxin could account for the auxin autonomy. The apparent auxin autonomy of C tissue when cultured on MSO medium containing cy- tokinin may have a similar basis. In other tissues, auxin production has been associated with shoot development (Binns et al. 1987; van Slogteren et al. 1983).

Elevated cytokinin levels have been reported previ- ously in tissues transformed with the ipt gene (e.g. Smart et al. 1991; Beinsberger et al. 1991). However, in most of these studies only a few cytokinins were measured, often by procedures based on RIA or related antibody meth- ods after inadequate or no extract purification. As a re- sult, several cytokinins with differing antibody affinities could be detected in a particular assay. Apparent changes in cytokinin level might only be due to change in the ratios of the cytokinins measured. Non-cytokinins could also interfere in the assays (see, e.g. Neuman and Smit 1990). Furthermore, in the studies mentioned above, there was usually no information on the levels of DZ- type compounds or cytokinin glucosides.

In the present study, all major, naturally-occurring, cytokinins have been assayed in the control and trans-

formed tissues. The extraction procedure used is known to minimize hydrolysis of nucleotides by phosphatase ac- tion. The total level of 16 cytokinins measured in T1 tissues was 5 times higher than the levels in the control tissues. Shoot development in T1 tissue exceeded that in T2 tissue and this was positively correlated with cy- tokinin levels (Table 1). The Z and DZ types of cy- tokinins, in particular ZR, ZNT, DZR and DZNT, were markedly elevated in both transgenic tissues T1 and T2, while the iP-type cytokinins, including iPNT, the product of the IPT enzyme, were not. The levels of cytokinin glu- cosides (Z9G, Z7G, DZ7G, OGZ, OGDZ, O G Z R and OGDZR) were also elevated, although the absolute levels of these compounds were much lower than those of the bases, ribosides and nucleotides.

The above data support the view that iPNT, the product of the IPT enzyme, serves as a short-lived inter- mediate compound in cytokinin biosynthesis. The tissues appeared to maintain a threshold level of iP, iPA and iPNT, and when this was exceeded, rapid trans-hydroxy- lation occurred to yield Z-type cytokinins. Although mi- crosomal enzymes have been found to cause trans-hy- droxylation of iP and iPA (Chen and Leisner 1984), the metabolite level (i.e. base, riboside or nucleotide) at which trans-hydroxylation of iP compounds occurs in plant tis- sue is obscure (see Letham and Palni 1983). The results of the present study indicate that, in the transgenic tobacco cultures, hydroxylation occurs principally at the nucle- otide level. Thus, after uptake of [3H]Ade for 12 h, 2, 56 and 26% of the 3H due to cytokinins was in the form of iPNT, ZNT and DZNT, respectively, while ZR, DZR, Z and DZ accounted for approximately 2, 2, 3 and 10% respectively; no radioactivity was found as iP or iPA. After addition of excess unlabelled Ade to terminate fur- ther incorporation of 3H into cytokinins, the radioactivi- ty due to ZNT declined and that in Z and DZ bases and ribosides increased. These results indicated that iPNT is rapidly hydroxylated to yield ZNT which is then metabo- lized to bases and ribosides. The Z-type cytokinins were readily converted to the DZ-type derivatives in the to- bacco tissues. An enzyme responsible for such conversion has been found in immature Phaseolus vulgaris embryos (Martin et al. 1989), and zeatin but not its riboside was found to be the substrate of the enzyme. However the high level of [3H]DZNT found in this study indicates that such conversion may have occurred mainly at the nucle- otide level in the tobacco tissues.

Incorporation of [3H]Ade into cytokinins could not be demonstrated in C tissue, but expression of the ipt gene resulted in a level of incorporation similar to that ob- served in Vinca rosea crown gall tissue (Palni et al. 1983). Although iPNT was labelled, iP and iPA were not and this suggested that these two cytokinins were not pro- duced as a result of cytokinin synthesis due to the ipt gene. However, iP and iPA were present in T1 and T2 tissue and also in C tissue which has a low endogenous cytokinin level (Table 1) maintained by synthesis due to enzymes normal to plants. This normal synthesis, which presumably also occurs in the transgenic tissues T1 and T2, concomitantly with that due to ipt gene expression, could account for the low level of iP and iPA in these tissues.

R. Zhang et al.: Effect of auxin on cytokinin metabolism 93

Cytokinin biosynthesis has been critically studied in one other tissue expressing the ipt gene, namely Vinca rosea crown gall tissue which has been transformed with the native Ti plasmid (see Discussion and references in Letham and Palni 1983). In these tissues, radiolabelled iPNT, iPA and iP were not detected at any time, and Z N T was the major labelled product of biosynthesis after incubation with [14C]Ade for up to 24 h. These studies and those with tobacco tissue discussed above are there- fore in accord regarding the importance of ZNT as a biosynthetic product.

The effect of auxin and BA on cytokinin metabolism. This was first studied by comparing the stability of endoge- nously synthesized cytokinins (3H-labelled) in the ipt- transformed tissues cultured with or without exogenous- ly supplied NAA. The greatly elevated capacity for ey- tokinin biosynthesis in these tissues made it possible to label endogenous cytokinins by supplying radioactive Ade as the substrate; incorporation of label could then be terminated by supply of excess unlabelled Ade and the degradative metabolism of the labelled cytokinins could be followed.

The labelled cytokinins were metabolized more rapid- ly in the tissues treated with NAA than in the controls (Fig. 1) and this was evident 5 h after NAA addition. This indicated that exogenous auxin may reduce cytokinin levels in the cultured tobacco tissues by directly stimulat- ing cytokinin degradation. Furthermore, NAA markedly stimulated the metabolism of Z-type cytokinins, but not of DZ-type compounds. On a percentage basis, the 3H-la- belled Z-type cytokinin which was affected most marked- ly by NAA was ZR. It is relevant to note that the cy- tokinin which exhibited the greatest NAA-induced re- duction in level after culture for 12 d on agar medium was also ZR (Table 2).

The conversion of exogenously supplied [3H]ZR to Ade-type compounds in the transformed tobacco tissues was very rapid and only a negligible amount remained in the tissues after continuous uptake for 24 h. This rapid metabolism is attributable to conversion of ZR to Z (which was noted in Table 3); the latter is rapidly degrad- ed by tobacco cytokinin oxidase in vitro, while the for- mer is not. The rapidity of ZR metabolism made it im- possible to perform experiments with treatments (such as NAA) which may enhance the metabolism further. How- ever, the urea derivative NPPU was found to reduce the metabolism of exogenous [3H]ZR considerably by in- hibiting cytokinin oxidase noncompetitively. Recently, three other urea derivatives have been found to inhibit partially purified cytokinin oxidase (Chatfield and Arm- strong 1986; Laloue and Fox 1989; Burch and Horgan 1989). These three compounds, N,N'-diphenylurea, N-(2- chloro-4-pyridyl)-N'-phenyl-urea and thidiazuron, are all active as cytokinins and the same applies to N P P U (Bruce and Zwar 1966). However, N,N'-diphenylurea did not suppress formation of Ade-type compounds from ZR in the transgenic tobacco tissue and neither did three other ureas which were tested (see Results) and which lack cytokinin activity (Bruce and Zwar 1966). Thus, NP- PU may be the first compound known to markedly in- hibit cytokinin oxidase in vivo. Using transformed tissue

supplied with NPPU, it was possible to study the effects of auxin on metabolism of exogenous [3H]ZR, and such studies indicated that auxin promoted metabolism of Z- type cytokinins to Ade derivatives while radioactivity due to DZ-type compounds derived from ZR was not affected.

Two observations indicate that auxin-induced cy- tokinin metabolism to Ade derivatives may occur gener- ally in normal (untransformed) plant tissues. Firstly, in artichoke tuber explants, Palmer et al. (1984) found that IAA rapidly induced conversion of [3H]ZR to Ados (160- fold increase) and AMP, and markedly suppressed the accumulation of ZNT, the dominant metabolite in con- trol tissue. Secondly, N A A promoted conversion of ZR to Ade, Ados and AMP in excised tobacco pith tissue (Palni et al. 1988). It is important to identify the basis of the above effects of auxin on cytokinin metabolism which are mediated by cytokinin oxidase. Although auxins were reported to have no effect on the activity of crude cy- tokinin oxidase from callus tissue of tobacco and poplar (Motyka and Kaminek 1992), Palni et al. (1988) reported that N A A increased by 20-30% the activity of partially purified cytokinin oxidase from Zea mays kernels. Using oxidase from these kernels purified by retention on, and elution from, a Con A-Sepharose column, this auxin-in- duced stimulation was confirmed when Tris-HC1 buffer was used. However, when crude corn enzyme was tested, N A A evoked little effect. Also, NAA was found to en- hance the activity of cytokinin oxidase prepared from tobacco T1 and T2 cultures when Tris-HC1 buffer was used for the assay. This applied to crude enzyme and to enzyme purified by gel filtration (Sephadex G 100 and HPLC), and Con A-Sepharose chromatography. In imi- dazole buffer, which promoted oxidase activity about 10- fold and may constitute an unnatural system with a high level of CuC12 (10 raM), N A A did not enhance oxidase activity. The imidazole-CuC12 buffer and auxin possibly enhance the oxidase activity by equivalent mechanisms. The results presented show that N A A can directly pro- mote the activity of cytokinin oxidase in vitro but the effect observed varies depending on the degree of purifi- cation of the enzyme and the buffer used.

In the present studies of the effect of auxin on purified cytokinin oxidase activity, iP was used as substrate. Since Z-type compounds were the dominant cytokinins in the ipt-transformed tissues, a study of the effect of auxin on cytokinin oxidase activity using such compounds as sub- strate is desirable. However, since oxidation of Z-type cytokinins was promoted by auxin in vivo, promotive effects in vitro seem likely and would be in accord with the results of Palni et al (1988) who noted that NAA and other synthetic auxins promoted the oxidation of both ZR and iP by corn kernel cytokinin oxidase.

An unexpected finding in the present studies was the observation that BA elevated the endogenous cytokinin level in transgenic tobacco tissues T1 and T2 cultured on auxin-containing media. It is relevant that kinetin has been found to elevate the endogenous cytokinin level in both cytokinin-requiring and cytokinin-autonomous (crown gall) tobacco tissues (Hansen et al. 1987). Further- more, exposure of immobilized cytokinin-dependent cells to BA increased secretion of both Z (threefold increase)

94 R. Zhang et al.: Effect of auxin on cytokinin metabolism

and ZR (fivefold increase) (Vankova et al. 1987). The above effects on cytokinin level probably reflect a promo- tion of cytokinin synthesis and enhancement of ipt gene expression in T1 tissue (MSN medium) in response to BA has been detected by Northern and Western blotting methods (Zhang 1992).

Plant development is controlled by a complexity of hormonal interactions which can be synergistic or antag- onistic. However, there is another component to plant hormonal interaction: one hormone can influence the level of another hormone and this type of interaction has been discussed previously (Leopold and Nood~n 1984). Studies based on tissues transformed with the ipt gene have now contributed to the recognition of a further, and probably very important, interaction of this type, namely, auxin-induced down-regulation of cytokinin level.

We wish to thank Dr. J. Zwar for supplying phenylurea derivitives.

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