Ectopic over-expression of the maize -glucosidase Zm-p60.1 perturbs cytokinin homeostasis in transgenic tobacco

RESEARCH PAPER

Ectopic over-expression of the maize b-glucosidaseZm-p60.1 perturbs cytokinin homeostasis intransgenic tobacco

Nagavalli S. Kiran1,2,*, Lenka Polanska3,4,*, Radka Fohlerova1,2, Pavel Mazura1,2, Martina Valkova2,

Miloslav Smeral1, Jan Zouhar1,†, Jirı Malbeck3, Petre I. Dobrev3, Ivana Machackova3 and

Bretislav Brzobohaty1,‡

1 Institute of Biophysics AS CR, Kralovopolska 135, CZ-61265, Brno, Czech Republic2 Department of Functional Genomics and Proteomics, Faculty of Science, Masaryk University, Kamenice 5,CZ-62500 Brno, Czech Republic3 Institute of Experimental Botany AS CR, Rozvojova 135, CZ-16502 Prague, Czech Republic4 Department of Plant Physiology, Charles University, Faculty of Science, Vinicna 5, CZ-12844 Prague 2,Czech Republic

Received 26 August 2005; Accepted 2 December 2005

Abstract

The activity of the phytohormone cytokinin depends

on a complex interplay of factors such as its metab-

olism, transport, stability, and cellular/tissue local-

ization. O-glucosides of zeatin-type cytokinins are

postulated to be storage and/or transport forms, and

are readily deglucosylated. Transgenic tobacco

(Nicotiana tabacum L. cv. Petit Havana SR1) plants

were constructed over-expressing Zm-p60.1, a maize

b-glucosidase capable of releasing active cytokinins

from O- and N3-glucosides, to analyse its potential

to perturb zeatin metabolism in planta. Zm-p60.1 in

chloroplasts isolated from transgenic leaves has an

apparent Km more than 10-fold lower than the purified

enzyme in vitro. Adult transgenic plants grown in the

absence of exogenous zeatin were morphologically

indistinguishable from the wild type although differ-

ences in phytohormone levels were observed. When

grown on medium containing zeatin, inhibition of root

elongation was apparent in all seedlings 14 d after

sowing (DAS). Between 14 and 21 DAS, the transgenic

seedlings accumulated fresh weight leading later

(28–32 DAS) to ectopic growths at the base of the

hypocotyl. The development of ectopic structures

correlated with the presence of the enzyme as

demonstrated by histochemical staining. Cytokinin

quantification showed that transgenic seedlings grown

on medium containing zeatin accumulate active me-

tabolites like zeatin riboside and zeatin riboside phos-

phate and this might lead to the observed changes.

The presence of the enzyme around the base of the

hypocotyl and later, in the ectopic structures them-

selves, suggests that the development of these struc-

tures is due to the perturbance in zeatin metabolism

caused by the ectopic presence of Zm-p60.1.

Key words: b-glucosidase, chloroplast, cytokinin metabolism,

ectopic expression, hormone conjugation, zeatin-O-glucoside.

Introduction

Cytokinins (CKs) are a class of plant hormones with theability to trigger cell division in conjunction with auxinin tissue culture. They are also involved in regulating awide range of developmental processes such as chloroplastdifferentiation, nutrient assimilation and translocation,seed germination, leaf expansion, flowering, and senes-cence (for reviews see Binns, 1994; Mok, 1994; Habererand Kieber, 2002). Dozens of compounds, both natural andsynthetic, have been assigned a measure of CK activity

* These authors contributed equally to the work presented here.y Present address: Department of Botany and Plant Sciences, Center for Plant Cell Biology, University of California, Riverside, CA 92521-0124, USA.z To whom correspondence should be addressed. E-mail: [email protected]

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spanning the entire range from completely inactive to highlyactive (Letham and Palni, 1983; Mok and Mok, 2001).

Most of the studies elucidating CK action have usedthe application of exogenous hormone to various plantsand/or tissues and observing subsequent changes. Isolationof mutants with increased levels of CKs (Chaudhuryet al., 1993), expression of the Agrobacterium isopentenyl-transferase gene to increase endogenous levels of CKs(Li et al., 1992) and, recently, the over-expression of genesencoding CK oxidase/dehydrogenase to reduce endogen-ous levels (Werner et al., 2003) have been employedto study the effects of changed CK levels. While theseapproaches have allowed observation of the effects of grosschanges in hormone levels, they have not been able to shedmuch light on the regulation of many of the subtlermetabolic conversions.

Metabolic regulation of hormone levels in the plantfulfils two basic requirements: (i) the production of speedyand significant changes in active hormone (either in ab-solute concentrations or relative to other hormones) inresponse to an environmental and/or other stimulus; and(ii) maintenance of hormone homeostasis at a particularstage of development and/or in a particular part of theplant.

Conjugation, the addition of low molecular weightcompounds, represents a mechanism to regulate the cel-lular level of ‘active’ hormones by generating productswith little biological activity (for a review see Brzobohatyet al., 1994). trans-Zeatin is a major and ubiquitous CK inhigher plants, comprising an adenine with a hydroxylatedisoprenoid substituent at the N6 position. t-Zeatin canundergo conjugation on the purine ring at the N7 and N9positions as well as on the OH-group on the isoprenoidsubstituent. The N9 atom can be conjugated with ribose(with subsequent phosphorylation to form the ribotide),glucose, and amino acids such as alanine. Also known areoxidative degradation by the action of CK oxidase/de-hydrogenase, reduction to dihydrozeatin, and isomerizationto cis-zeatin (Mok and Mok, 2001). Metabolic inactivationof zeatin has long been focused on degradative reactionscatalysed by CK oxidase/dehydrogenase and conjugationto monosaccharides like glucose and xylose. Understand-ing the regulation of this complex network of metabolicconversions will contribute to a better insight into the pro-cesses leading to hormone homeostasis as well as the cor-responding physiological and developmental effects.

t-Zeatin-O-glucoside (ZOG) is resistant to CK oxidase/dehydrogenase-mediated breakdown and is readily con-verted into the active hormone by the action of b-glucosidases. It has also been found in xylem sap and istherefore considered to be the transport form of thehormone (Armstrong, 1994; Letham, 1994). ZOG accu-mulation when CK accumulates in tissues and its decreaseduring phases of active growth has been taken as evidenceof a storage role (Letham and Palni, 1983). For example, in

young maize seedlings, CKs are apparently not synthesizedduring germination and early seedling development. In-stead, ZOG and DHZOGwere found to be transported fromthe endosperm to the embryo, where it was activated by ab-glucosidase to supplement the developing embryo withactive CK (Smith and van Staden, 1978). It has been de-monstrated that the b-glucosidase Zm-p60.1 first isolatedfrom maize coleoptiles (Campos et al., 1992; Esen, 1992)fulfils all the requirements for a b-glucosidase involved inregulation of growth in early seedling development byreleasing Z from ZOG in maize (Brzobohaty et al., 1993). Itwas shown to be localized to plastids/chloroplasts as pre-dicted by the N-terminal signal peptide (Esen and Stetler,1993; Kristoffersen et al., 2000). In parallel, ZMGlu1 (anenzyme coded by the same genetic locus as Zm-p60.1) wasshown to hydrolyse 4-dihydroxy-7-methoxy-1,4-benzoxazin-3-one (DIMBOA)-glucoside (DIMBOA-Glc; Cicek andEsen, 1999) in a manner similar to a b-glucosidase purifiedfrom maize seedlings (Babcock and Esen, 1994). Based onthese findings, it has been suggested that ZMGlu1 isinvolved in defence against pathogens by releasing thetoxic aglycone (DIMBOA) from its storage form, DIM-BOA-Glc. Alhough this function is well supported by theanalysis of substrate specificity and substrate-enzymestructure (Czjzek et al., 2000), it does not appear to befully consistent with the Zm-p60.1 expression pattern,enzyme abundance, and immunolocalization. The transcriptis highly abundant in 3-d-old etiolated seedlings except forthe primary leaf, but is hardly detectable in the majorvegetative tissues (Brzobohaty et al., 1993) and thedistribution of both transcript and protein at the cellularlevel is highly specific (Kristoffersen et al., 2000). Further,strong accumulation of the protein in maize leaves inresponse to drought stress (Riccardi et al., 1998) indicatesother roles for the enzyme besides its involvement in pestresistance. No direct experimental evidence confirming thatZMGlu1 is actually involved in pathogen defence responsein planta has been published. The exact biological role(s) ofthe enzyme will be decided when a maize knockout linewithout any functional ZMGlu1 allele can be characterized.Plant genomes encode large families of b-glucosidases (Xuet al., 2004) with varying substrate specificities. Until now,besides Zm-p60.1, the ability to release CKs from CK-O-glucosides in vitro has been demonstrated only for aBrassica napus b-glucosidase (Falk and Rask, 1995).However, the significance of this activity was not in-vestigated in planta. Functional genomics projects areexpected to identify novel b-glucosidases involved in theregulation of CK activity by reversible conjugation.Thus, currently Zm-p60.1 represents the best character-ized enzyme available as a valuable molecular tool to under-stand the biological significance of CK O-glucosylationin planta.

ZOG1, an enzyme catalysing the O-glucosylation ofzeatin was isolated from Phaseolus lunatus (Martin et al.,

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1999). When over-expressed in tobacco, transgenic ZOG1explants required a 10-fold higher concentration of exog-enous zeatin for the initiation of callus formation (Martinet al., 2001).

Light signalling and signalling through the CK pathwayare known to interact with each other (Neff et al., 2000).Chloroplasts are photosynthetic organelles that differenti-ate from colourless proplastids in the presence of light ina process called photomorphogenesis. Exogenous CKs aswell as increased endogenous steady-state levels of CKscan partially replace the light signal leading to precociousphotomorphogenesis (Chaudhury et al., 1993; Chory et al.,1994). Despite a large body of research into the photosyn-thetic capability of chloroplasts, as well as transport ofvarious kinds of molecules into and out of them, little isknown about the occurrence and/or transport of CKsinto and out of the chloroplast. It has been shown thata wide range of CKs occurs in intact isolated chloroplasts(Benkova et al., 1999). It has recently been recognized thatthe isoprenoid precursor for the biosynthesis of t-zeatin isprimarily plastid-derived (Kasahara et al., 2004). SinceZm-p60.1 was localized in chloroplasts there was aninterest in characterizing the effects of Zm-p60.1 over-expression in transgenic tobacco and in analysing its role inperturbing CK metabolism by means of estimating the insitu enzyme activity in intact chloroplasts. It is shown herethat over-expression of Zm-p60.1 leads to a perturbance inzeatin homeostasis in intact transgenic plants, thus render-ing them hypersensitive to exogenous zeatin.

Materials and methods

Plant materials and growth conditions

Tobacco (Nicotiana tabacum, cv. Petit Havana SR1) plants harbour-ing the Zm-p60.1 cDNA under the CaMV 35S promoter wereobtained by Agrobacterium-mediated leaf-disc transformation. Twohomozygous lines (labelled T4 and T5) bearing the Zm-p60.1 cDNAincluding the N-terminal plastid-targeting signal peptide, wereselected for analysis on medium supplied with zeatin.Adult plants from an independent transgenic line were selected

with methotrexate (0.5 mg l�1) added to solidMSmedium (Murashigeand Skoog, 1962) supplemented by sucrose (15 g l�1). Transgenicplants grew slower than control plants on methotrexate-freemedium. Therefore to select plants for hormone analyses, a spectro-photometric assay was used. The enzyme activity was assayed usingthe chromogenic substrate p-nitrophenyl-b-D-glucopyranoside ac-cording to Rotrekl et al. (1999). Leaves and internodes used forquantitation of hormones and for isolation of chloroplasts for Km

determination experiments were taken from transgenic plants culti-vated in a growth chamber (16/8 h photoperiod at 150 lmol photonsm�2 s�1, 26/20 8C) for 7–8 weeks.

Zm-p60.1 cloning, transformation, and RT-PCR analysis

Standard procedures were used for DNA cloning and analysis(Sambrook et al., 1989). The methotrexate-resistant binary vectorpM001 (Reiss et al., 1994) was used for Agrobacterium-mediatedtransformation. The CaMV 35S::Zm-p60.1::pA cassette containing

the complete Zm-p60.1 coding region was excised frompRT101::Zm-p60.1 (Brzobohaty et al., 1993) using HindIII andinserted into pM001 cut by HindIII yielding pM001::Zm-p60.1. InpM001::Zm-p60.1, CaMV 35S::Zm-p60.1::pA was obtained in twoorientations. To minimize possible effects of genomic sequences onZm-p60.1 expression, a clone, pM001::1.3, carrying the cassetteoriented with pA close to the right border and CaMV 35S facingthe b-lactamase gene employed as an ampicillin resistance markerwithin the T-DNA was chosen for plant transformation.The binary vectors were transformed into Agrobacterium tumefa-

ciens strain GV3101::pMP90RK (Koncz and Schell, 1986) andtransgenic lines were generated in Nicotiana tabacum cv. PetitHavana SR1 by the leaf disc method. The primary transformantswere selected on 0.5 mg l�1 methotrexate. In tissue culture, plantswere grown on MS medium (Murashige and Skoog, 1962) supple-mented with 3% (w/v) sucrose and 0.8% agar. Plants were grownwith a 16/8 h light/dark regime at a daytime temperature of 24 8C.Total RNA was isolated from entire 3-week-old seedlings grown

on MS medium using Trizol (Invitrogen, Germany) according to themanufacturer’s recommendations. 5 lg of total RNA was used asa template for cDNA synthesis using SuperScript II RNase H�

reverse transcriptase (Invitrogen, Germany) and the oligo dT primerRTP3 (CGT TCG ACG GTA CCT ACG TTT TTT TTT TTT TTTTT) according to the supplier’s protocol. 4 ll of the 40 ll reactionmix were then subjected to PCR to amplify Zm-p60.1 using primersp60(+)4: (59-TCA AGG ACG AGC AGA AGG-39) and p60(-)1: (59-TCT TCT TGC TGG GCT TCT-39). Actin was amplified using theprimers Nt-Act-59ex: (59-ATT GTG (C/T)T(G/T) GA(C/T) TCTGGT GAT GGT G-39); Nt-Act-39a: (59-ATC CAG ACA CT(A/G)TAC TT(C/T) CTC TC-39) and Nt-Act-39b: (59-TCC A(A/G)A C(A/G)C TGT A(C/T)T TCC TCT C-39).

Growth on medium containing zeatin

Ten seeds each from wild-type tobacco and from transgenic lineswere sown on MS medium supplemented with 15 g l�1 sucrose and2.5 lM zeatin, solidified with 0.8% agar. The plates were incubatedvertically in a controlled-environment growth chamber (Percival)under a 8/16 h, 21/19 8C. light/dark regime with a photon fluence rateof approximately 80 lmol photons m�2 s�1. Seedlings were collectedat 14, 21, 28, and 32 d after sowing (DAS), the fresh weightdetermined in batches of five or 10 seedlings, and then processed forfurther analysis (b-glucosidase staining, CK extraction).

b-Glucosidase staining

b-Glucosidase was detected by histochemical staining using 5-bromo-4-chloro-3-indolyl-b-D-glucopyranoside (X-glc; Sigma) asa substrate. The staining was done in 0.4 M citrate-phosphate buffer(pH 5.6). The reaction contained 500 lM each of potassiumhexacyanoferrate II and III and 0.1 mg ml�1 X-glc. The seedlingswere infiltrated under vacuum for three 5 min bursts, and incubated at30 8C for 4 h. The seedlings were washed in excess bufferimmediately after incubation and decoloured before photography.

Chloroplast isolation

Intact chloroplasts were isolated and purified according to Benkovaet al. (1999). Leaves were deribbed, cut, and mixed with a homogen-ization medium (0.33 M sorbitol, 50 mM TRIS/HCl, pH 7.8,0.4 mM KCl, 0.04 mM Na2EDTA, 0.1% (w/v) bovine serumalbumin, 1% (w/v) polyvinylpyrrolidone, 5 mM isoascorbic acid)in a semi-frozen state, homogenized with a homogenizer, andfiltered through a sandwich of cotton wool between eight layers ofmuslin. The chloroplast fraction was recovered by centrifugation(1000 g; 2 min; 4 8C), washed with a resuspension medium (RM:0.33 M sorbitol, 2 mM Na2EDTA, 1 mM MgCl2, 1 mM MnCl2,50 mM HEPES, pH 7.6) and resuspended in RM. This suspension

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was layered on a Percoll density gradient (40% and 80% (v/v) Percollsolution in RM) and centrifuged for 15 min at 1000 g. Intactchloroplasts were collected at the interface of the gradient, dilutedwith RM and centrifuged for 3 min at 1000 g. The pellet wasresuspended in RM. All the procedures were done at 4 8C. Theisolated chloroplasts were used immediately.

Chlorophyll determination

Chlorophyll was extracted into 80% (v/v) acetone. The totalchlorophyll a+b content was calculated from the absorbance at 652nm of the clear extract after centrifugation (500 g, 5 min) according toArnon (1949).

Extraction and purification of IAA, ABA and CK

IAA, ABA, and CKs were extracted overnight at –20 8Cwith Bieleskisolvent (methanol:chloroform:water:acetic acid, 12:5:2:1, by vol;Bieleski, 1964) from plant tissue ground under liquid nitrogen. [3H]IAA and [3H] ABA (Sigma, USA) for 2D-HPLC analysis anddeuterium-labelled CKs ([2H5]Z, [

2H5]ZR, [2H5]Z-7G, [

2H5]Z-9G,[2H5]Z-OG, [

2H5]ZR-OG, [2H3]DZ, [

2H3]DZR, [2H6]iP, [

2H6]iPR,[2H6]iP-7G, [

2H6]iP 9G; Apex Organics, UK) for MS quantificationwere added as internal standards. After centrifugation, the extractswere purified using Sep-Pak C18 cartridges (Waters Corporation,Milford, MA, USA) and evaporated to water phase. After acidifyingwith HCOOH, hormones were trapped on an Oasis MCX mixedmode, cation exchange, reverse-phase column (150 mg, Waters)(Dobrev and Kamınek 2002). After a wash with 1 M HCOOH, IAAand ABA were eluted with 100% MeOH and evaporated to dryness.Further, CK phosphates (CK nucleotides) were eluted with 0.34 MNH4OH in water and CK bases, ribosides, and glucosides were elutedwith 0.34 M NH4OH in 60% (v/v) MeOH. The latter eluate wasevaporated to dryness. NH4OH was evaporated from the elutedfraction with CK nucleotides. 0.1 M TRIS (pH 9.6) was added tosamples and after treatment with alkaline phosphatase (30 min at37 8C), CK nucleotides were analysed as their corresponding ribo-sides. After neutralization, the solution was passed through a C18Sep-Pak cartridge. CKs were eluted with 80% (v/v) methanol andevaporated to dryness. Evaporated IAA, ABA, and CK samples werestored at –20 8C until further analysis. IAA and ABA were separatedand quantified by 2D-HPLC according to Dobrev et al. (2005).

Quantitative analysis of CKs

Purified CK samples were analysed by LC-MS system consistingof HTS PAL autosampler (CTC Analytics, Switzerland), Rheos 2000quaternary pump (FLUX, Switzerland) with Csi 6200 Series HPLCOven (Cambridge Scientific Instruments, England) and LCQ IonTrap mass spectrometer (Finnigan, USA) equipped with an electro-spray. 10 ll of sample were injected onto a C18 column (AQUA,2 mm3250 mm35 lm, Phenomenex, USA) and eluted with0.0005% acetic acid (A) and acetonitrile (B). The HPLC gradientprofile was as following: 5 min 10% B, then increasing to 17% with-in 10 min, and to 46% within further 10 min at a flow rate of0.2 ml min�1. The column temperature was kept at 30 8C. The efflu-ent was introduced in mass spectrometer being operated in the posi-tive ion, full-scan MS/MS mode. Quantification was performedusing a multilevel calibration graph with deuterated CKs as internalstandards. As standards of cis-zeatin-glucosides and cis-zeatin-9-riboside-O-glucoside were not available, the amounts of thesecompounds were estimated only from the calibration graphs of thecorresponding trans-isomers.

Determination of kinetic constant in isolated chloroplasts

The reaction mixture contained 0.64 lCi (23.5 kBq) of substrate([3H] ZOG, radioactively labelled on position 8 of the purine ring by

Dr Jan Hanus, Institute of Experimental Botany, Prague), 1.1–281.7lM unlabelled substrate ZOG and freshly isolated chloroplasts (0.9mg chlorophyll equivalent to 1.6 mg of total protein content) in RMin a final volume of 500 ll. The assays were carried out inquadruplicates at seven different substrate concentrations, incubatedat 30 8C for 10 min at 75 rpm shaking and terminated by the additionof Bieleski solution. ZOG and released zeatin were purified usingC18 SPE columns. After elution with 80% (v/v) methanol sampleswere concentrated and analysed by HPLC using column Luna C18(2) (15034.6 mm, 3 lm, Phenomenex, Torrance, CA, USA); flowrate: 0.6 ml min�1; mobile phase: A: 40 mM formic acid adjustedto pH 4.1 with ammonium hydroxide and B: acetonitrile:methanol,1:1, v/v); gradient: 0 min, 10% B; 2 min, 15% B; 11 min, 20% B;11.1 min, 34% B; 19 min, 45% B; 21 min, 100% B; 23 min, 100% B;25 min, 10% B; detection at 270 nm. The Km values were estimatedfrom a Lineweaver–Burk plot.

Determination of kinetic constant in vitro

Zeatin and glucose were assayed independently to follow enzymeactivity of purified Zm-p60.1 with ZOG as a substrate. The puri-fication scheme was essentially as described (Zouhar et al., 1999).The enzymatic reaction was started by adding the purified enzymepreparation to ZOG diluted to the individual concentrations in50 and 100 mM citrate/phosphate buffer pH 5.5, the reaction mixwas incubated at 30 8C and aliquots (50 ll) were withdrawn forglucose and zeatin determination, respectively. The reaction mixwas kept at 30 8C for not more than 10 min and 0.3 min, and aliquotswere withdrawn at 1 min and 3 s intervals, respectively, when Zand glucose were assayed as the reaction product. The shortincubation intervals when glucose was assayed were chosen toprevent any significant conversion of b- to a-glucose. The reactionbuffer was supplemented with 20% PEG as appropriate. For zeatindetermination, the reaction was stopped by mixing the aliquots with1 M gluconolactone (75 ll) and the samples were kept on ice. Thereleased zeatin was assayed using ELISA as described previously(Faiss et al., 1997). The released glucose was assayed using theglucose oxidase-peroxidase-coupled reaction (P Mazura et al., un-published results). In brief, glucose was oxidized by glucose oxidase(Fluka) which results in the generation of D-gluconolactone andhydrogen peroxide. In the presence of horseradish peroxidase(HRP Sigma), the hydrogen peroxide then reacts with AmplexUltraRed reagent (Molecular Probes) which is converted to thefluorescent resorufin. Thus, aliquots withdrawn from the reactionmix were automatically injected into detection mix (50 ll, contain-ing glucose oxidase, HRP and AmplexUltraRed) in 96-well plates.A fluorescence reader (BMG Fluostar Galaxy) was used to meas-ure resorufin fluorescence (excitation 544 nm and emission/detection 590 nm). The software package ORIGIN (OriginLabCorp., Northampton, USA) was used to determine Km values.

Results

Construction of transgenic tobacco plantsover-expressing Zm-p60.1

To analyse effects of constitutive over-expression ofZm-p60.1 on CK metabolism and action in whole plants,the transcription cassette CaMV 35S::Zm-p60.1::pA(Brzobohaty et al., 1993) was cloned into a plant trans-formation vector, and the resulting construct, pM001::Zm-p60.1 was used to generate transgenic tobacco plants. Over20 independent rooting primary transformants (T0) were



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regenerated following Agrobacterium-mediated transform-ation of tobacco leaf discs with the pM001::Zm-p60.1vector. The primary transformants were scored for thelevels of Zm-p60.1 expression by northern and western blotanalysis (data not shown). Single locus homozygous lineswere identified in T2 and propagated for several generationswithout any consistent phenotype alteration comparedwith the wild type. Leaf protoplasts derived from severalof these lines were shown to use CK-O- and N3-glucosidesto initiate cell division (Brzobohaty et al., 1993). Twoindependent single locus homozygous lines (designated T4and T5) were selected for analysis outlined below based onthe stability of transmission of b-glucosidase activitythrough several generations. Over-expression of Zm-p60.1in these lines was confirmed by RT-PCR (Fig. 1A). Adulttransgenic plants over-expressing Zm-p60.1 showed noapparent morphological deviations from the wild type

although there were differences in phytohormone levels(see below).

Morphological effects of cultivation on mediumsupplemented with exogenous zeatin

Transgenic seedlings growing on MS medium supple-mented with 2.5 lM zeatin showed a significant increasein fresh weight at 21 DAS compared with controls growingon the same plate (Fig. 1B). The difference became in-creasingly significant at the later time points (28 and 32DAS). The morphology of transgenic seedlings remainedindistinguishable from the wild type until about 21 DAS.At 28 DAS ectopic outgrowths were seen forming at thebase of the hypocotyl (Fig. 1). They resembled leaves inthat they were blade-like structures that frequently turnedgreen and developed trichomes (Fig. 1E, F). Similar

Fig. 1. RT-PCR, FW comparison, morphology and histochemical staining. (A) RT-PCR of transformed tobacco seedlings. Bands corresponding to theZm-p60.1 cDNA were detectable only in transformants (T4 and T5), but were absent from the wild type (SR1). Amplification of actin cDNA was used asa control. (B) Fresh weight comparison of seedlings growing on zeatin-containing medium. Average FW mg�1 seedling of transgenic (black) and wild-type (striped) seedlings when grown on medium containing zeatin. Bars represent SD (n=30–50). (C–G) Morphological changes in transgenic seedlingsgrown on zeatin-containing medium. (C, D) Representative photographs of wild-type (left) and transgenic (right) seedlings growing on the same plate at28 DAS (C) and 32 DAS (D). Green blade-like structure is clearly seen in (D). (E, F) Detail of the transgenic seedling in (C) and (D), respectively. (G)Detail of corresponding portion of SR1 seedling at 32 DAS. (H–L) Histochemical staining for b-glucosidase. (H, J) Representative photographs ofSR1 seedlings stained for b-glucosidase at 28 DAS (H) and 32 DAS (J). (I) Representative photograph of a transgenic seedling at 28 DAS. Note therestriction of staining to the ectopic structure. (K, L) Representative photographs of transgenic seedlings stained for b-glucosidase at 32 DAS. Note thepatchy staining pattern in the blade-like structures. Bars in (C, D) 0.5 cm; in (E–L) 1 mm.



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structures form at the base of the hypocotyl of wild-typeseedlings when the medium contains 10.0 lM zeatin(see supplementary Fig. 1 at JXB online).

Histochemical staining for b-glucosidase inseedlings grown on exogenous zeatin

An indigogenic histochemical staining procedure using5-bromo-4-chloro-3-indolyl-b-D-glucopyranoside was op-timized for use with seedlings over-expressing Zm-p60.1.The incubations were shortened to minimize false signalsfrom endogenous b-glucosidases (see Materials and meth-ods). Staining in transgenic seedlings grown on mediumcontaining zeatin was restricted to the base of the hypo-cotyl and to patches in the ectopic structures themselves(Fig. 1K, L).

Kinetic analysis of Zm-p60.1 activity

The wild type full-length Zm-p60.1 cDNA used inpM001::Zm-p60.1 encodes a nascent polypeptide includingthe N-terminal plastid-targeting signal peptide. In cellfractionation experiments performed with leaves of thetransgenic plants, Zm-p60.1 enzyme activity co-purifiedwith the chloroplast fraction (data not shown) indicatingthat Zm-p60.1 is located exclusively in chloroplasts.Kinetic analysis was performed to determine whetherZm-p60.1 is likely to act on CK-O-glucosides at physio-logically relevant levels that were determined in tobaccochloroplasts earlier (Benkova et al., 1999). Under standardin vitro assay conditions, release by purified Zm-p60.1of zeatin and glucose from ZOG, was monitored usingELISA and the glucose oxidase/peroxidase-coupled re-action, respectively. Both in vitro assays yielded compar-able, though unexpectedly high values for Km (Table 1).To assess the possible influence of the native chloroplastmicro-environment on Zm-p60.1 catalytic properties, sub-sequent kinetic analysis was performed using a highlypurified fraction of freshly isolated, intact chloroplastsfrom fully expanded leaves of transgenic plants. 3H-labelled ZOG was taken up rapidly into and converted inthe chloroplasts (see supplementary Results and supple-mentary Table 2 at JXB online). The ZOG conversion wasalmost completed after a 4 h incubation period in chloro-plasts isolated from the transgenic plants while more than90% of ZOG remained unconverted in wild-type chloro-plasts. These results enabled us to establish an assay todetermine the apparent Km of Zm-p60.1 in isolated chloro-plasts with 3H-labelled ZOG as a substrate.

The apparent Km in these chloroplasts using 3H-labelledZOG as a substrate was more than ten times lower com-pared with the apparent Km from the in vitromeasurements.Thus, the Km value in chloroplasts is within the physio-logical range for CK-O-glucoside levels. The inclusionof an inert hydrophilic polymer (polyethylene glycol,PEG) in the standard in vitro assay buffer resulted in

a more than 3-fold decrease in Km with ZOG as a substrate(Table 1), suggesting a strong dependence of Zm-p60.1catalytic properties on water content in the reactionenvironment. Intact chloroplasts isolated from wild-typeand transgenic tobacco were incubated with ZOG and itsuptake and conversion followed. Only 1% of ZOG re-mained unconverted at the end of a 4 h incubation periodwith chloroplasts isolated from the transgenic plants.When chloroplasts isolated from wild-type were used,91% of initial ZOG remained unconverted under the sameincubation conditions. ZOG uptake into chloroplasts wastemperature dependent (not shown). However, the extentof uptake (a maximum of 2.5% of total radioactivity) doesnot indicate ZOG enrichment in the chloroplasts.

Hormone quantification in adult transgenic plants inthe absence of exogenous zeatin

CK quantification from plants grown in the absence ofexogenous zeatin revealed a higher level of active CKs(free bases and ribosides) in the top portion (leaf numbers

Table 1. Michaelis constants of Zm-p60.1 with ZOG as sub-strate in various environments

Assaya Zm-p60.1 in Km (lM)

GOX-HP Bufferb 697.0641.3ELISA-Z Buffer 6006105ELISA-Z Buffer+20%PEG 177679HPLC-scintillation Chloroplasts 62.961.0

a GOX-HP, glucose oxidase/peroxidase-coupled reaction to assayreleased glucose; ELISA-Z, ELISA to determine released zeatin;HPLC-scintillation, released 3H-labelled zeatin determined by a scintilla-tion detector following HPLC separation.

b Buffer – see Materials and methods.

Table 2. Endogenous contents of CKs (bases+ribosides), IAAand ABA in internodes of adult control (SR1) and transgenic(CaMV 35S-Zm-p60.1) tobacco

Values represent the mean of two replicates (individual measurements areshown in parentheses).

SR1 Zm-p60.1

CKs (B+R) (pmol g�1 FW)Apex 23.6 (40.5; 6.8) 55.5 (64.6; 46.3)Internode 1 27.7 (45.7; 9.6) 49.1 (51.6; 46.6)Internode 2 29.0 (47.3; 10.7) 33.8 (32.8; 34.9)Internode 3 25.1 (40.6; 9.6) 32.6 (34.9; 30.4)Internode 4 15.2 (22.8; 7.6) 32.9 (28.5; 37.3)

IAA (pmol g�1 FW)Apex 535 (559; 512) 407 (362; 452)Internode 1 776 (761; 791) 564 (522; 607)Internode 2 981 (939; 1022) 646 (760; 532)

ABA (pmol g�1 FW)Internode 5 616 (776; 456) 729 (641; 818)Internode 6 536 (666; 406) 728 (716; 741)Internode 7 476 (521; 430) 702 (672; 731)Internode 8 497 (483; 510) 656 (638; 674)



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1–4 and internode numbers 1–4) of plants over-expressingZm-p60.1 than in the corresponding parts of wild-typeplants (Table 2; Fig. 2A). However, no consistent trendwas observed in levels of storage and inactivated forms ofCKs (see supplementary Table 1 at JXB online). Auxin(free IAA) measurements revealed that transgenic tobaccoover-expressing Zm-p60.1 has lower levels of IAA in theapex and the first two internodes than in the correspondingcontrols (Table 2). The gradient in leaf IAA levels fromhigh to low from the apex downward was steeper than

wild-type in plants over-expressing Zm-p60.1 (Fig. 2B).ABA levels were measured in plants over-expressingZm-p60.1. In older leaves (numbers 5–10 from the apex)as well as in older internodes (numbers 5–8), ABA levelswere higher than the wild type in plants over-expressingZm-p60.1 (Table 2; Fig. 2C).

CK quantification analyses of seedlings grown inthe presence of exogenous zeatin

CK quantification was carried out on seedlings grown onmedium containing zeatin. The changes in CK levels wererestricted to the zeatin-type and the iP-type metaboliteswere not affected. The most significant pattern was seenin the levels of active zeatin metabolites. The transgenicseedlings accumulated Z, ZR, and ZRP to levels higherthan the wild type (Fig. 3A). Seedlings over-expressingZm-p60.1 were able to accumulate the substrate ZOG tolevels higher than their wild-type counterparts in a time-dependent manner (Fig. 3B). The same was also true, to agreater extent, of Z7G, a terminal inactivation conjugate(Fig. 3C).

Presentation of the CK quantification results fromseedlings grown on exogenous zeatin

Fresh weight is most commonly used to normalize CKquantification data. However, in this case, transgenicseedlings were significantly heavier than the wild type(Fig. 1B) and so the quantification data were normalizedto number of seedlings. This also has the advantage ofproviding an average picture in whole seedlings of steady-state levels of individual CK metabolites. The y-axis inFig. 3 represents the number obtained when the valuefor transgenic seedlings was subtracted from the cor-responding wild-type value. In addition, a table showingthe CK accumulations in transgenic seedlings as percen-tages of wild-type values is presented (Table 3).

Discussion

Transgenic tobacco plants over-expressing Zm-p60.1,a maize b-glucosidase capable of releasing active CKfrom O- and N3- glucosides, were analysed in order tounderstand the regulation of zeatin metabolism in planta.Although the plants showed no apparent phenotype alter-ations, they had substantial differences in phytohormonelevels. The apparent Km value for Zm-p60.1 in isolatedchloroplasts with ZOG as a substrate was close to physi-ological levels of CK-O-glucosides. Unexpectedly, thevalue was 10-fold lower than the apparent Km valuedetermined in vitro for the purified enzyme suggestinga higher affinity of the enzyme for ZOG in its native com-partment. When grown on exogenous zeatin, the trans-genic seedlings displayed a hypersensitivity to zeatin. The

Fig. 2. CK, IAA and ABA levels in leaves of adult plants. Graphshowing the gradients in CK (bases+ribosides) (A), IAA (B), and ABA(C) levels in leaves of transgenic (black) and wild-type (striped) plants. Asteeper fall in IAA levels in leaves of transgenic plants is apparent.Graphs represent two independent measurements.



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study revealed a perturbation in zeatin homeostasis, andthat the chloroplast is not the storage organelle for zeatin-O-glucoside. It is proposed that Zm-p60.1 can be used asa molecular tool in investigating the biological role(s) ofCK-O-glucosylation.

Morphological changes in seedlings over-expressingZm-p60.1 grown on exogenous zeatin

Juvenile hypocotyls are known to be competent for invitro bud formation in woody species ‘recalcitrant’ tobud regeneration like Eucalyptus globulus (Azmi et al.,1997a). Shoot formation in callus with numerous shootswas correlated with high levels of Z and ZR in shooty tu-mours compared with non-shooty tumours (Azmi et al.,1997b). In addition, the shooty capacity of the tumourwas not associated with an overall increase in CK levelsbut only with the presence of small transformed areashighly provided with CKs, while the buds themselveswere provided with a moderate CK signal. The restrictionof histochemical staining for Zm-p60.1 to the ectopicstructures suggests that the enzyme is involved in in-creasing the local availability of active CKs. In thisrespect these results recall the pattern of CK localizationin E. globulus calli (Azmi et al., 2001). The hypocotyl–rootjunction is one of the sites of auxin accumulation (Ni et al.,2001; Dr Alena Kuderova, personal communication), andthe contemporaneous availability of active CKs mightlead to an additive effect (Rashotte et al., 2005) on itsdevelopment. This might be a reason why the ectopicstructures arise in this region. Zm-p60.1 expression isdriven by the CaMV 35S promoter that is known to beactive to higher levels in vascular tissues. The ectopicstructures contain a high proportion of vasculature andthus are enriched in Zm-p60.1. Increased levels of Zm-p60.1 in these structures might cause a kind of autocatalyticeffect (where the high proportion of vasculature allowsfurther accumulation of Zm-p60.1) with respect to perturb-ance of zeatin metabolism.

It is known that exogenously applied CKs (specificallyzeatin at micromolar concentrations) can induce D-typecyclins in responsive cells (Riou-Khamlichi et al., 1999).Further, the over-expression of a D-type cyclin acceleratescell-cycle progression from G-phase to S-phase andNicta;CycD3;2 controls the G1/S transition in tobaccocells (Nakagami et al., 2002). Boucheron et al. (2002)have shown CK involvement in the redifferentiation of

Table 3. Accumulation of CK metabolites in transgenic seedlings grown on medium containing 2.5 lM zeatin

Values are percentages of wild-type levels and are averages of two independent measurements. The individual percentages are given in parentheses.

Z7G ZOG Z+ZR+ZRP

14 DAS 101.88 (105.27; 98.48) 107.45 (117.98; 96.93) 110.74 (113.89; 107.58)21 DAS 129.00 (115.55; 142.45) 126.18 (108.79; 143.57) 223.76 (246.12; 201.39)28 DAS 145.58 (147.85; 143.31) 157.80 (177.48; 138.12) 239.95 (266.68; 213.22)32 DAS 221.33 (173.42; 269.23) 229.87 (239.87; 220.61) 225.11 (238.99; 211.23)

Fig. 3. CK levels in seedlings grown on zeatin-containing medium.Graphs representing the deviation from wild-type of CK levels intransgenic seedlings. The y-axis represents the number obtained when thevalue for transgenic seedlings was subtracted from the correspondingwild-type value (see text). (A) Deviations from wild-type of levels ofactive CKs; (B) deviations from wild-type of ZOG levels; (C) deviationsfrom wild-type of Z7G levels.



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xylem and phloem tissues in tobacco explants growingon medium containing exogenous CKs. These resultsstrongly suggest that the increased availability of activeCK metabolites like ZR and ZRP leads to the formation ofthe ectopic structures at the base of the hypocotyl.

Enzymatic activity of Zm-p60.1 in isolated chloroplasts

The apparent Km value for Zm-p60.1 in isolated chloro-plasts was 10-fold lower against ZOG than the apparentKm determined in vitro for the purified enzyme againstthe same substrate. This value is more than 2-fold lowerthan against 4-methylumbelliferyl-b-D-glucopyranoside(140 lM) and 10-fold lower than against p-nitrophenyl-b-D-glucopyranoside (640 lM) both of which were obtainedusing purified enzyme in vitro (Zouhar et al., 2001).

The chloroplast is an organelle that presents a consider-ably different environment for proteins and it is generallybelieved that the water content is significantly lower thanthat of the cytoplasm. Given this it can be expected thatchloroplast enzymes may behave very differently in vitro.Interestingly, a more than 3-fold decrease in Zm-p60.1 Km

with ZOG was observed when the standard in vitro assaybuffer was supplemented with 20% PEG. This representsthe first direct experimental evidence suggesting thata decrease in water content might result in increasedcatalytic efficiency of a plastid/chloroplast enzyme. Besidesthis, other, more specific, mechanisms might be involvedin modulation of chloroplast enzyme activity. Active trans-port of CK-O-glucosides into chloroplasts might contributeto the observed low value of the apparent Km. However,when wild-type chloroplasts were incubated with ZOG,significant ZOG accumulation in the chloroplasts was notobserved (data not shown).

A system of reductive activation of disulphide-bond-containing enzymes has been well-characterized in chloro-plasts. It involves the reduction and reformation ofdisulphide bridges mediated by chloroplast thioredoxinwhich, in turn, is activated by ferredoxin and hence bylight (Buchanan et al., 2002). Zm-p60.1 has five cysteineresidues. Formation of an intramolecular disulphidebridge between two of them (C205 and C211) was shownto be essential for enzyme activity through stabilizationof a loop forming a part of an aglycone binding site andacquisition of the competence to assemble a catalysis-competent homodimer (Rotrekl et al., 1999; Czjzek et al.,2000; Zouhar et al., 2001). It has previously been reportedthat CK-O-glucosides transiently occur in chloroplasts atthe end of the dark phase (Benkova et al., 1999). The light-activation of glucoside cleavage could thus involve theaction of a similar glucosidase.

Hormone quantification in adult transgenic plants inthe absence of exogenous zeatin

CK-mediated reduction in auxin levels has been docu-mented in plants over-expressing the CK biosynthesis gene

ipt from Agrobacterium (Eklof et al., 2000). A recent studyusing plants over-expressing ipt has shown that elevatedCK levels lead to a long-term reduction in auxin biosyn-thesis rates and pool sizes. However, the authors concludeit to be an indirect effect, possibly mediated by develop-mental changes (Nordstrom et al., 2004). It was found thatCK effects are partly mediated by elevation of ethylenebiosynthesis (Genkov et al., 2003). Elevated ethylenebiosynthesis in its turn can stimulate the oxidative decar-boxylation of IAA (Winer et al., 2000). Thus, the elevatedlevels of active CK metabolites in plants over-expressingZm-p60.1 could lead to lower levels of free IAA.

Since Zm-p60.1 was identified in drought-stressedmaize plants (Riccardi et al., 1998), ABA levels weredetermined in plants over-expressing Zm-p60.1. It wasfound that ABA tends to accumulate in older leaves aswell as in older internodes of transgenic plants comparedwith the wild type. Correlations between CKs and ABAlevels have been reported. Increased levels of ABA wereobserved in potato plants with a high CK content carryingthe Agrobacterium ipt gene (Machackova et al., 1997).ABA levels increased in response to benzyladenine treat-ment in micropropagated explants of Actinidia deliciosa(Moncalean et al., 2003). A correlated increase in free ABAand ZOG was observed during germination of plantscarrying the etr1-2 mutation (Chiwocha et al., 2005). Bycontrast, delayed corolla senescence in petunia flowersoverproducing CKs due to the action of PSAG12-driven iptis correlated with lower accumulation of ABA (Changet al., 2003). Pretreatment with benzyladenine of beanand maize resulted in lower ABA accumulation duringsubsequent water stress (Pospısilova et al., 2005). Simi-larly, a contrasting role for ABA and CK in the senescenceprocess is well documented (Panavas et al., 1998; Ganand Amasino, 1996). On the other hand, an increase inABA levels can cause a decrease in CK levels. ABAaccumulates in kernels of drought-stressed maize and isaccompanied by a decrease in zeatin-type CKs (Setteret al., 2001). It is also known that ABA can induce theexpression of CK oxidase/dehydrogenase. Thus, in stressedand/or senescing tissue ABA-mediated CK oxidase/dehydrogenase induction can lead to lowered CK levels.However, recent evidence has raised the possibility thatthe ABA-induced increase in CK oxidase/dehydrogenaseactivity may be important in regulating levels of CKstransiting the vascular system, but that endogenous CKsthat are not transported away from their site of synthesisby vascular system may be compartmentalized away fromthis CK oxidase/dehydrogenase activity (Brugiere et al.,2003; Yang et al., 2002).

Although leaves from adult transgenic plants over-expressing Zm-p60.1 show deviations from wild-typelevels of phytohormones, the plants show no obviousmorphological alterations unless they are challengedwith exogenous zeatin. In tobacco over-expressing the



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zeatin-specific O-glucosyltrasferase ZOG1, CK-dependent explants differed significantly from the wildtype in their capacity to differentiate on medium supple-mented with zeatin, but few substantial changes in adulthabit were observed under normal growth conditions(Martin et al., 2001).

CK quantification in seedlings grown onexogenous zeatin

It is generally accepted that N7- and N9-glucosylation isirreversible while O-glucosylation is reversible and thatit plays a role in storage and/or transport of zeatin. Inline with this, enzymes from plants have been identifiedfor both glucosylation (ZOG1 from Phaseolus lunatus;Martin et al., 1999) and deglucosylation (Zm-p60.1 fromZea mays; Brzobohaty et al., 1993) at the hydroxyl oxygenwhile no enzyme has yet been identified that deglucosylatesN7- and/or N9-glucosides. It has been shown that over-expression of the glucosyltransferase ZOG1 leads toa specific increase in the levels of O-glucosides and thechanges are largely restricted to O-glucosylation (Martinet al., 2001). It is shown here that the over-expression ofan enzyme catalysing the reverse reaction increasesthroughput through a larger set of zeatin conversions(conversion to N-glucosides, ribosides, and ribotides)compared with controls and, further, that seedlings over-expressing this activity are hypersensitive to exogenouszeatin. The exact tissue and temporal distribution of thesemetabolites is still not clear. The presence of the enzymein and around the ectopic structures indicates that a localmaximum of active CKs leads to the formation of thosestructures. The observation that wild-type seedlings formsimilar ectopic structures when incubated with higherconcentrations of zeatin leads us to conclude that theseseedlings are indeed hypersensitive to exogenous zeatin.It is known that moderate increases in steady-state CKlevels result in a moderate increase in zeatin pool-sizes, buta large increase in the content of Z7G (Eklof et al., 1996),as observed in the case of Zm-p60.1 transgenic seedlingsgrown on zeatin. iP-type CK metabolites were not affected,in line with previous observations (Lexa et al., 2003).

Both shoots and roots are now recognized to be sites ofCK biosynthesis. In tobacco roots, iPA nucleotides arethe major CKs detected, and the low levels of ZR andZMP is in good agreement with results from Arabidopsis,where root-derived biosynthesis of CKs occurs mainlyvia the iPMP-dependent pathway (Nordstrom et al., 2004).The pattern of expression of AtIPT3 and AtIPT7 in roottissue is consistent with their possible involvement in suchsynthesis (Miyawaki et al., 2004).

Subcellular compartmentation is expected to play asignificant role in CK homeostasis. The hydroxylatedisoprenoid side-chain substituent for t-Z biosynthesis isderived largely from the plastid-localized methylerythritol

phosphate (MEP) pathway, and four Arabidopsis IPTproteins (AtIPT1, AtIPT3, AtIPT5, and AtIPT8) are locatedin the chloroplast (Kasahara et al., 2004). Further, it hasbeen suggested that the presence of chloroplasts might bea prerequisite for the iPMP-independent pathway, whichmay explain the shoot-localization of t-Z biosynthesis(Nordstrom et al., 2004).

The ability of seedlings over-expressing Zm-p60.1 toaccumulate ZOG was unexpected. This observation intransgenic seedlings supplied with exogenous zeatinstrongly implies that the terminal subcellular destinationof ZOG is not the chloroplast. It has been shown thatCK-O-glucosides accumulate preferentially in the vacuole(Fubeder and Ziegler, 1988). Previous results have shownthat ZOG accumulates transiently in chloroplasts (Benkovaet al., 1999) where it may fulfill specific biologicalfunction(s). Thus, in the transgenic plants over-producingZm-p60.1 in chloroplasts, it was possible to affect only thispart of the total pool of intracellular ZOG. This relativelysmall perturbance is probably overcome during the normalcourse of plant development and, therefore, the adult plantis morphologically indistinguishable from the wild type.However, this perturbance is sufficient for the phenotypemanifestation on medium containing zeatin, as seen by theability of the transgenic seedlings to accumulate active CKmetabolites to a larger extent than they accumulate ZOG.Experiments are in progress in this laboratory using arecombinant Zm-p60.1 to probe the subcellular compart-mentation of zeatin metabolism further.

Taken together, it is proposed that over-expression ofZm-p60.1 can be used as a powerful tool for understandingthe regulation of zeatin metabolism in general and itssubcellular compartmentation in particular.

Supplementary data

The supplementary data available at JXB online are: (i)Table 1 listing the individual measurements of CKs inleaves and internodes from adult plants grown in theabsence of zeatin; (ii) a figure showing the phenotype ofwild-type and transgenic seedlings grown on concentra-tions of zeatin in the range 1.0 to 10.0 lM; and (iii) resultspresenting uptake (Table 2) and conversion of ZOG inchloroplasts together with relevant materials and methods.

Acknowledgements

We thank Professor Miroslav Strnad for his generous help duringthe initial phases of the ELISA assays and for providing us withthe polyclonal anti-zeatin-riboside antibodies. We thank ProfessorAsim Esen for stimulating discussions on the effects of chloroplastmicro-environment on Zm-p60.1 catalytic properties, and Dr AlenaKuderova for sharing unpublished information. This work wassupported by grant nos LN00A081, MSM143100008, andMSM0021622415 (Ministry of Education of the Czech Republic),



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AVOZ50040507 and AV0Z50380511 (Academy of Sciences of theCzech Republic), IAA600380507 (Grant Agency of the Academy ofSciences of the Czech Republic), and 206/03/0369 (Grant Agency ofthe Czech Republic).

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Ectopic over-expression of the maize -glucosidase Zm-p60.1 perturbs cytokinin homeostasis in transgenic tobacco

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