Altered development of Arabidopsis thaliana carrying the Agrobacteriumtumefaciens ipt gene is partially due to ethylene effects

Eric E. van der Graaff1,3,*, Carol A. Auer2 and Paul J.J. Hooykaas1

1Leiden University, Institute of Molecular Plant Sciences, Clusius Laboratory, Wassenaarseweg 64, 2333, ALLeiden, The Netherlands; 2University of Connecticut, Department of Plant Science, U-67, Storrs, CT 06269,USA; 3University of Zürich, Institute of Plant Biology, Zollikerstrasse 107, CH-8008, Zürich, Switzerland;*Author for correspondence (fax 41-1-6348204)

Received 1 March 2000; accepted in revised form 3 August 2000

Key words: Arabidopsis thaliana, Cytokinin, Ethylene, Ipt, Plant development, Transgenic

Abstract

Transgenic Arabidopsis thaliana plants containing the Agrobacterium tumefaciens cytokinin-biosynthesis geneipt were produced to study the effect of increased cytokinin (CK) levels on the development of this rosette plantspecies. In three independently transformed lines (ipt-156, 158 and 161), Arabidopsis plants had smaller leaves,an underdeveloped root system and decreased apical dominance in inflorescence stems. The smaller transgenicleaves were highly serrated along the margins, pale green and had pointed leaf tips. In cross section, transgenicleaves had smaller cells and irregularly shaped epidermal cells. In the ipt-161 line, leaves and hypocotyls fre-quently exhibited purple color due to anthocyanin production. The most severe phenotype was observed in tissueculture conditions, while growth in soil reduced or eliminated some phenotypic effects. Compared to C24 wildtype plants, ipt-161 plants accumulated zeatin and zeatin riboside with an approximate 10-fold increase in thetotal pool of CKs. A study of the progeny resulting from crosses between the ipt-161 transgenic line and theethylene insensitive mutants ein1, ein2 and eti5 suggested that part of the altered development exhibited by theipt transgenic plants was caused by increased ethylene levels.

Abbreviations: AGM – Arabidopsis growth medium, BAP – 6-benzyl-aminopurine, CK – cytokinin, IBA – in-dole-3-butyric acid, MS – Murashige and Skoog, NAA – �-naphthylacetic acid

Introduction

The phytohormone CK has been shown to be in-volved in many processes during plant development,including the control of cell division and chloroplastbiogenesis (Beinsberger et al. 1991), as well as to bea determinant of plant morphology by regulating theoutgrowth of axillary meristems (McGaw and Burch1995), floral development (Estruch et al. 1993;Venglat and Sawhney 1996) and senescence (Gan andAmasino 1996, 1995). Over 30 naturally-occurringCKs have been identified in plants and these com-pounds all contain an N6-substituted adenine mole-cule with a 5-carbon branched side chain (McGawand Burch 1995).

The Agrobacterium tumefaciens phytohormone-biosynthetic gene ipt, which encodes an adenos-inemonophosphate isopentenyltransferase (Hamill1993), has been expressed in many plant species tostudy the role of CK in plant development. In gen-eral, the expression of the ipt gene reduced or elimi-nated root growth, reduced apical dominance leadingto release of lateral shoots, led to formation of smallerplants with small rounded leaves, delayed leaf senes-cence, caused the expression of defense-relatedgenes, and increased tuber formation in potato plants(Binns 1994; Hamill 1993; McKenzie et al. 1998;Rupp et al. 1999). Few reports have been publishedabout the effects of ipt expression in the rosette plantArabidopsis thaliana. Medford et al. (1989) ex-

305Plant Growth Regulation 34: 305–315, 2001.© 2001 Kluwer Academic Publishers. Printed in the Netherlands.

pressed the ipt gene under the control of a maize heatshock promoter (hsp70) and produced Arabidopsisplants with reduced root growth, although shoot andinflorescence development appeared relatively nor-mal. Under short day conditions, xylem tissue in thetransgenic inflorescence stem was reduced, while ax-illary bud growth increased under long day conditions(Medford et al. 1989). More recently, Rupp et al.(1999) used the Drosophila heat shock promoter tocontrol ipt expression in Arabidopsis. A single heatshock in young plants which caused a transient in-crease in CKs did not cause a discernible change inplant development. However, daily heat shocks for 2weeks created moderate changes in plant phenotypeand anatomy.

In order to be able to use a gene-tagging approachfor the selection of Arabidopsis mutants with an al-teration in CK levels, action or signal transduction,the role of CK in the development of this rosette plantmust be understood. Therefore, we generated trans-genic Arabidopsis thaliana lines harboring the iptgene under the control of the natural Agrobacteriumpromoter to study the effect of increased endogenousCK levels on Arabidopsis development. In this paper,we report on the phenotype, development andchanges in CK levels in our ipt-transgenic Arabidop-sis lines.

Materials and methods

Materials

The pBIN4R plant transformation vector used (kindlyprovided by JME Clement and JHC Hoge, unpub-lished results) was based on the pBin19 vector (Be-van 1984) harboring a plant kanamycin resistancemarker between the T-DNA borders and contained theipt gene from the Agrobacterium tumefaciens plasmidpTiAch5 driven by its natural full length (−283) pro-moter. The construct was maintained in Agrobacte-rium tumefaciens strain LBA 4404 (Hoekema et al.1983) for plant transformation.

Leaf-disc transformation

The Arabidopsis (ecotype C24) ipt transgenics weregenerated using a leaf transformation method as de-scribed before (van der Graaff and Hooykaas 1996,1998). Transgenic shoots were regenerated on kana-mycin selective medium (50 mg/l). During the regen-

eration of the primary ipt transformants the hormoneconcentration was altered to higher NAA (1-naphtha-lene-acetic acid) levels (1 mg/l) and lower BAP (6-benzyl-aminopurine) levels (0.2 mg/l). The primarytransformants were allowed to set seed under tissueculture conditions in either glass tubes or plastic jars.For 10 primary transformants the inflorescence stemswere separated from the shoots and subcultured onAGM medium. Such inflorescence stems were some-times able to form roots and produce a few seeds.

Growth conditions of plants

Seeds of the transgenic lines and wild type control(both ecotype C24) were either surface sterilized andgrown in tissue culture on half strength MS (Murash-ige and Skoog 1962) medium (1/2MS10) or grown insoil as previously described (van der Graaff andHooykaas 1996). For CK analysis, seedlings from thetransgenic line ipt-161 and C24 ecotype were grownon full strength MS media for 2 weeks.

PCR analysis

Genomic DNA was isolated from wild type plants andthe ipt transgenics. The presence of the T-DNA con-struct was determined using primers for the codingregion of the kanamycin gene (forward primer: 5�-TTGTCAAGACCGACCTGTCC-3� and reverseprimer: 5�-ACCGTAAAGCACGAGGAAGC-3�)producing a 574 bp fragment upon amplification. Aplasmid harboring the kanamycin plant selectionmarker was included as a positive control. As a con-trol for the presence of genomic DNA in the PCR re-actions primers for the coding region of the Arabi-dopsis glyceraldehyde 3P dehydrogenase cytosolicgene (GapC) were used (forward primer: 5�-AGCTCGTCGCTGTCAACG-3� and reverse primer:5�-GACAGCCTTGGCAGCTCCT-3�) producing a1300 bp fragment from genomic DNA. PCR amplifi-cation was performed at 5 min 95 °C followed by 35cycles of 30 s 95 °C, 45 s 62 °C and 90 s 72 °C and afinal step of 10 min 72 °C. The PCR products wereseparated on an agarose gel and the EtBr stained im-age was scanned and processed using the Adobe Pho-toshop computer program.

CK analysis

Four endogenous CKs were extracted and purifiedfrom 2-week-old seedlings of C24 and the transgenic

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line ipt-161 using an established method modified forArabidopsis tissue (Mercure 1998). Quantification ofisopentenyl adenine (iP), isopentenyl adenosine(iPAR), zeatin (Z) and zeatin riboside (ZR) in HPLC-purified CK fractions was performed by an enzymelinked immunoassay (ELISA) technique; quantifica-tion of ZR was also conducted by an internal isotopeLC/frit-FAB-mass spectrometry method.

Seedlings of each line (2 gfw) were ground, ex-tracted in 10% perchloric acid (PCA) to prevent nu-cleotide conjugate degradation, centrifuged and thesupernatant reduced to 5 ml volume. [3H]-BA(1 × 106 dpm, 740 Mbq/�M, CEA, Gif-sur-Yvette,France) was added as an internal standard. The sam-ple was partitioned against acidified butanol (pH 3.5)and then against butanol:ammonia mixture (9:1 v/v).The organic phase was collected and dried in vacuo.The sample was taken up in 50% ethanol, passed overa SepPak C18 column (Waters, Milford, MA, USA),and prepared for HPLC. Four CK fractions were sep-arated by reverse-phase HPLC on an UltrasphereRP-18 column (octadecylsilica, 5 �m, 4.6 × 250 mm,Beckman Instruments, San Ramon, CA). Excellentseparation of endogenous CKs and the internal stan-dard occurred using an aqueous phase of 1% aceticacid (v/v) and an increasing acetonitrile gradient from5% to 100% over 32 min at 1.5 ml/min. The reten-tion time for CK standards was Z - 10.3 min, ZR -13.6 min, iP - 23.7 min, BA - 27.2 min, and iPAR -31.45 min. Fractions containing iP, iPAR, Z and ZRwere collected, dried under vacuum and resuspendedin 50 mM Tris-buffered saline (TBS) for ELISA anal-ysis. Recovery of the [3H]-BA internal standard wasbetween 48-59%, similar to that reported for iP andiPAR from another CK purification method (Astot etal. 1998).

The ELISA utilized polyclonal rabbit antibodiesproduced against ZR (cross reactivity against Z was76%, and against all other CKs tested less than 8%,titre 1:10000) and iPAR (cross reactivity against iPwas 44%, against BAR was 29%, and against all otherCKs tested was less than 5%, titre 1:10000) (anti-bodies provided by Tom Davenport, Gainesville, FL).ELISA plate wells (Corning Costar Corp., Cam-bridge, MA) were coated with goat anti-rabbit antis-era and allowed to bind for 24 h (ICN Biomedical,Costa Mesa, CA, 1 �g/well in 50 mM NaHCO3, pH9.5). The CK antiserum was allowed to bind for 20 hand non-specific binding was blocked by 50 mM TBSbuffer containing 0.1% bovine serum albumin (BSA).The reaction mixture consisted of CK standards (Sig-

ma, St. Louis, MO, USA) or HPLC-purified CK frac-tions from Arabidopsis samples. The reaction mixturewas allowed to compete for binding against alkalinephosphatase-conjugated CK tracer (1:750 dilution in50 mM TBS with 0.1% gelatin) for 3 h. A color re-action was elicited by the addition of p-nitrophenylphosphate disodium (1 mg/ml in 50 mM NaHCO3,pH 9.5) held at 37 °C for 1 h. Absorbance was re-corded at 305 nm (MR-5000 with Bio-Linx software,Dynatech Laboratories, Chantilly, VA).

A standard line was calculated based on the per-centage of tracer binding vs. the percentage of CKbinding and lower limits of detection for each CKwere 0.04–0.1 ng/100 �l. Two different tests, the testof additivity and the test of parallelism, were con-ducted to determine the accuracy of the ELISA sys-tem for Arabidopsis tissues (Grotjan and Keel 1986).Both tests showed no interference from unknowncompounds in the HPLC-purified fractions from Ara-bidopsis tissues suggesting that quantification shouldbe accurate (data not shown). Three separate tissuesamples from each Arabidopsis line were analyzed byELISA. For each purified CK fraction, three replicateELISA wells were prepared and the results averagedfor determination of CK concentration.

To support the ELISA results, a measurement ofZR was made using an established internal isotopeLC/frit-FAB-mass spectrometry method (Astot et al.1998) (conducted with the generous permission ofGoran Sandberg, Swedish University of AgriculturalSciences, Umea, Sweden). The values for ZR in C24and ipt-161 seedlings were similar to those deter-mined by the ELISA technique.

Plant crosses

To obtain ipt transgenics in the ethylene insensitivebackground of the mutants ein1, ein2 and eti5 (Guz-man and Ecker 1990; Harpham et al. 1996), pollenfrom the homozygous transgenic ipt-161 (the stron-gest CK phenotype) was transferred to the stigma ofthe ethylene insensitive mutants. The progenies ofthese crosses were screened for kanamycin resistance(marker for ipt T-DNA construct) and the occurrenceof trichomes (marker for the ecotype of the ethyleneinsensitive mutants). Plants with these traits were al-lowed to set seed and the resulting progenies (kana-mycin resistant and bearing trichomes on the rosetteleaves) were used to analyze the effect of the ethyl-ene insensitive mutations on the ipt phenotype.

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Dark germination

Seeds were sown on 1/2MS10 medium containingdifferent concentrations of BAP and grown for 18days in the dark. The length of the hypocotyl wasmeasured along a mm scale and the number of ex-panded leaves was recorded.

Anatomical analysis

The plant material was fixed in a 2% glutaraldehyde,0.1 M sodium cacodylate buffer (pH 7.2) for 8 h atroom temperature. The material was rinsed overnightin 0.1 M sodium cacodylate buffer (pH 7.2). Theleaves were dehydrated in 70% ethanol for 16 h, fol-lowed by 80, 90, 96 and 100% ethanol in steps of 1 heach. After dehydration the leaves were incubated inpropyleneoxide followed by an epon:propyleneoxidemixture (1:1) for 2 h each. The material was infil-trated with epon for 1 day and embedded in epon,which was allowed to polymerize at 55 °C. Sections(1 �m) were made with an ultramicrotome usingglass knives. The sections were stained with a 1%toluidine blue stain solution at 50–60 °C for 1 min,rinsed with water, dried and mounted in epon. Thesections were examined using a Leitz Diaplan micro-scope which was also used for photography (Ilford,Delta 100 film).

Results

Plant transformation and regeneration

Arabidopsis thaliana leaf discs transformed with theAgrobacterium tumefaciens ipt gene produced shootswhich frequently had a bushy appearance and vitri-fied leaves. Such shoots were unable to form rootsand subsequently failed to produce seeds. Comparedto control conditions for shoot regeneration, transfor-mation with Agrobacterium containing the ipt con-struct resulted in an increased frequency and rate ofshoot formation. Alteration of the hormone treatmentduring the shoot regeneration phase to higher NAAlevels (1 mg/l) and lower BAP levels (0.2 mg/l) al-lowed the regeneration of shoots with a more normalappearance. However, most of these shoots were un-able to form roots even when exposed to a root in-duction medium containing the auxin IBA. Only oneline (ipt-10) rooted and produced seeds under theseconditions. Surprisingly, inflorescence stems could be

separated from the primary transformants and grownin tissue culture. This treatment allowed some of theisolated inflorescence stems to root and in some casesthey produced a few seeds. Using this technique, four(ipt-156, 158, 159 and 161) out of ten primary trans-formants (ipt-156–165) produced seeds. Using PCR,the ipt transgenic lines 156, 158, 159 and 161 wereshown to contain the ipt T-DNA construct (Figure 1).

Development in tissue culture

During growth in tissue culture, the kanamycin-resis-tant progeny of two (ipt-10 and 159) of the five ipttransgenic lines which produced seeds showed no vis-ible alteration in development. The kanamycin-resis-tant progeny of three ipt transgenic lines (ipt-156, 158and 161) produced short roots and leaves that werehighly serrated, smaller in size and pointed at the tip.This latter leaf phenotype was mostly masked by thesmall size and the serration of the leaves (Fig-ure 2B,C,E). Hypocotyl length was the same as forC24 wild type plants (data not shown), but occasion-ally callus tissue formed at the base of the hypocotyl(Figure 2E). The ipt-161 line displayed the strongestCK phenotype which was characterized by the occur-rence of pale green to yellow leaves and anthocyaninproduction was occasionally observed in the shoot.Older seedlings displayed reduced apical dominanceevident by increased branching of the inflorescencestems (Figure 2G). Changes in apical dominance wereeven more striking when seedlings were grown for

Figure 1. PCR analysis of genomic DNA from C24 wild type andthe ipt transgenic lines 156, 158, 159 and 161. The kanamycinprimers detect the presence of the ipt T-DNA construct, while theGapC primers were used as a control for the presence of genomicDNA and efficiency of the PCR reaction.

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4–5 weeks in tissue culture prior to transfer to soil,resulting in the formation of very bushy plants withhighly branched inflorescences (data not shown). Thephenotype of the ipt transgenics as shown in Figure2B and C could be completely mimicked by thegrowth of C24 wild type seedlings on high levels(> 10−7 M) of applied CKs, including the productionof anthocyanin in leaves and hypocotyls (data notshown).

CK measurements in 2-week-old seedlings showeda 10-fold increase in the total pool of Z, ZR, iP andiPAR in ipt-161 transgenic plants compared to theC24 wild type (Table 1). CK accumulation in the ipt-161 plants occurred through an 8-fold increase in Zand 22-fold increase in ZR. In contrast, iPAR showedonly a small increase in ipt-161 plants, while iP lev-els were the same in both lines. Values for ZR levelswere similar using ELISA and mass spectrometrytechniques, indicating that the ELISA method was re-liable.

Development in soil

When germinated and grown in soil, the ipt-10 and159 transgenic lines exhibited wild type development,while the transgenic lines ipt-156, 158 and 161 dis-played a clear CK phenotype. During the first fewweeks in soil, the CK phenotype was comparable tothat observed in tissue culture. However, after twoweeks of growth in soil, the CK phenotype becameless pronounced and after 4–5 weeks only the serratedleaves remained as a visibly altered phenotype (Fig-ure 2H,I). In addition, no difference was observed inthe time of flowering or in the structure of the inflo-rescence (Figure 2J,K) compared to wild type. Trans-genic Arabidopsis plants expressing a construct withthe (−184) ipt promoter and the GUS reporter geneshowed that the activity of this promoter decreasedduring growth in soil (Schouten 1999) (data notshown). Despite the loss of a strong CK phenotypeduring growth in soil, the progeny of ipt transgeniclines always exhibited the original strong CK pheno-type (tested up to the T5 generation).

An anatomical study of the leaves from 2-week-old plants grown in soil showed that, although theoverall structure was comparable between the ipttransgenics and C24 wild type, cell size was de-creased in the ipt transgenic leaves Figure 3, theshape and size of the epidermal cells were irregularand starch accumulation was increased (visualized asblack dots in the cross sections). No changes were

observed in the anatomy of the roots or inflorescencestems (data not shown).

Seedling phenotype in the dark

Because CKs are able to induce the de-etiolated phe-notype (Chory et al. 1994), the phenotype of dark-grown ipt-161 seedlings was studied (Table 2). In thedark, ipt-161 plants had shorter hypocotyls and devel-oped more expanded leaves than wild type plants. Theapplication of BAP led to a further reduction in hy-pocotyl length and an increase in the number of trueleaves formed by the ipt-161 plants. Increasing theBAP concentration reduced the difference betweenipt-161 and wild type plants and at 10−6 M BAP theipt transgenics behaved like wild type plants.

Role of ethylene

The application of high levels of CK has been re-ported to induce the production of ethylene (Cary etal. 1995; Rodrigues-Pousada et al. 1999). Hence,some of the phenotypic effects observed from the en-dogenous production of CK in ipt transgenics mightbe caused by the increased production of ethylene. Tounderstand the interaction between endogenous CKand ethylene overproduction, genetic crosses weremade between the ipt-161 line (strong CK phenotype)and the ethylene insensitive mutants ein1 (etr1), ein2and eti5 (Guzman and Ecker 1990; Harpham et al.1996). We used three different mutants to ensure areliable analysis of the influence of ethylene on theCK phenotype. The dominant ein1 (etr1) and reces-sive ein2 mutants represent different mutations in theethylene signal transduction pathway. The partiallycharacterised semi-dominant eti5 mutant differs fromein1 in that eti5 shows overproduction of ethyleneand, therefore, is likely to represent a different muta-tion than ein1. Observation of plant development inthe ‘double mutant/transgenic’ lines (ipt expression inthe ethylene insensitive mutants) showed that changesin the shoot size, leaf color and serration of the leaveswere partially caused by increased production of eth-ylene (Figure 4). The leaves of the ‘double mutant/transgenic’ were green instead of the pale green toyellow color exhibited by the original ipt-161 trans-genic. This effect was most clearly observed in tissueculture (data not shown). Leaf size was increasedcompared to the original ipt-161 transgenic, althoughstill smaller than the C24 wild type, and leaf serra-

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Figure 2. Development of ipt transgenic Arabidopsis plants in tissue culture (A-G) and in soil (H-K). (A-C) Two-week-old C24 wild type,ipt-158 and ipt-161 seedlings, respectively. (D-F) Four-week-old C24 wild type, ipt-158 and ipt-161plants, respectively. (G) Flowering ipt-161 transgenic plant. (H,I) Five-week-old C24 wild type and ipt-161 plants, respectively. The CK phenotype displayed by the transgenicsipt-156, 158 and 161 was comparable at this stage. (J,K) Flowering C24 wild type and ipt-161 transgenic. The magnification of the picturesis comparable within the series A-C, D-F, H-I and J-K.

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tion was decreased. These effects were most clearlyobserved in the ipt*ein2 progeny (Figure 4B).

Table 1. Measurement of four CKs in 2-week-old plants of Arabidopsis C24 and the ipt-161 transgenic line. Z, ZR, iP and iPAR werequantified in 3 independent plant samples using ELISA. Mean (ng/gfw) and SE are shown. A single measurment of ZR was made for eachgenetic line using an internal isotope dilution LC/frit-FAB-MS method (values shown in parentheses).

CK concentration (ng/gfw)

Line Z ZR iP iPAR Total

C24 65.0 + 33.1 30.8 + 10.8 (36.7) 13.0 + 3.1 13.9 + 10.2 121.8 + 34.5

ipt-161 544.8 + 53.0 672.2 + 66.1 (501.9) 20.9 + 10.3 49.2 + 1.5 1287.2 + 52.1

Figure 3. Anatomy of leaves taken from 2-week-old C24 wild type plants and ipt-161 transgenic plants. Cross sections of wild type (A) andipt-161 (B). Pictures are shown at the same magnification.

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Discussion

Effect of ipt expression on Arabidopsis development

The altered development in Arabidopsis broughtabout by the expression of the ipt gene under thetranscriptional control of its natural promoter in-cluded a reduced root system, reduced leaf size, re-duced apical dominance in the inflorescence stem, in-creased serration of the leaf margins and increasedanthocyanin production in some plants. Increased ser-ration of the leaf margin was recently also observedby Rupp et al. (1999) for transgenics in which the ex-pression of the ipt gene was regulated by the Droso-phila heat shock promoter hsp70. Increased CK lev-els obtained after repeated heat shock treatments werefound to be correlated with increased expression ofthe homeobox genes STM and KNAT1 and the leafserration was suggested to be the result of either in-

creased or ectopic KNAT1 expression in the leaf pri-mordium or young leaf (Rupp et al. 1999).

CKs play a role in flower development and mayact via the control of expression of floral identitygenes (Estruch et al. 1993; Venglat and Sawhney1996). Therefore, it is likely that increased CK levelsin ipt transgenics would result in aberrant flowerstructure, decreased fertility and problems with seedproduction. Previously, the use of the CaMV 35S pro-moter to drive the expression of the ipt gene has beenshown to produce fertility problems (see review inGaudin et al. (1994)). Only few of the regenerantswhich we obtained produced seeds. Seed set undertissue culture conditions was only seen after the pri-mary transformants had rooted. Except for the ipt-10transgenic line, which showed a wild type phenotypeand, therefore, might be a non-expressor, none of theprimary transformants were able to form roots. How-ever, when inflorescence stems of the primary trans-

Table 2. Phenotype of the ipt-161 transgenic line and C24 wild type as determined by hypocotyl length and number of expanded leaves.Seedlings were grown in the dark on basal medium or in the presence of the CK BAP (0.1–50 �M). n = the number of seedlings analyzed ineach experiment.

Hypocotyl length (mm) Number of expanded leaves per plant

BAP (�M) C24 n ipt-161 n C24 n ipt-161 n

0 18.4 ± 1.3 46 12.6 ± 1.6 46 0 46 1.5 ± 0.8 46

0.1 14.2 ± 1.4 40 11.3 ± 1.9 48 0 40 1.2 ± 0.7 48

0.5 11.6 ± 1.8 47 9.6 ± 2.7 47 0.9 ± 0.7 47 1.7 ± 1.2 47

1 11.1 ± 1.7 49 10.4 ± 2.1 45 1.4 ± 1.0 49 1.5 ± 0.9 45

5 10.2 ± 1.9 50 9.6 ± 1.9 44 2.1 ± 1.0 50 2.2 ± 1.2 44

10 9.5 ± 1.6 53 9.3 ± 1.7 51 2.5 ± 1.2 53 2.5 ± 1.1 51

20 9.0 ± 1.1 49 7.0 ± 1.8 48 3.4 ± 1.4 49 2.7 ± 1.2 48

30 7.5 ± 1.2 47 7.2 ± 1.2 42 4.0 ± 1.3 47 3.0 ± 0.9 42

50 7.0 ± 0.8 43 6.1 ± 1.1 40 3.6 ± 1.3 43 3.0 ± 0.9 40

Figure 4. Influence of ethylene insensitive mutations on the development of ipt transgenics. The progenies resulted from crosses made be-tween the ipt-161 transgenic line and ethylene insensitive mutants. Progeny were grown in soil for three weeks. (A) ipt-161 transgenic inein1 background. (B) ipt-161 transgenic in ein2 background. (C) ipt-161 transgenic in eti5 background. (D) ipt-161 transgenic. Pictures areshown at the same magnification.

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formants were subcultured, most inflorescence stemsrooted and four lines produced seeds. This suggeststhat the difficulty in obtaining rooted primary trans-formants was responsible for the low number of re-generants producing seeds.

In 2-week-old seedlings of Arabidopsis line ipt-161, the phenotypic changes described above corre-lated with an approximately 10-fold increase in en-dogenous CK. CK accumulation in ipt-161 occurredprimarily through large increases in Z and ZR, whileiP and iPAR remained constant. This result was con-sistent with previous reports from ipt-transgenic to-bacco and Arabidopsis. Four studies in tobacco usingdifferent ipt constructs showed large increases in Zand ZR, with little or no increase in iP and iPAR(McKenzie et al. 1998; Motyka et al. 1996; Redig etal. 1997; Zhang et al. 1995). In ipt-transgenic tobaccotissues, other CKs also accumulated, such as dihy-drozeatin-type compounds, O-glucoside conjugatesand nucleotide conjugates (McKenzie et al. 1998;Motyka et al. 1996; Redig et al. 1997; Zhang et al.1995). In a recent study, Arabidopsis plants express-ing ipt linked to a heat-shock promoter showed a tran-sient increase in Z, ZR and zeatin nucleotides 8 h af-ter heat shock; iP-type CKs showed little or no in-crease after heat shock. After 72 h, only the zeatin-9-glucoside conjugate was increased. A detailed studyof Arabidopsis detected 10 natural endogenous CKcompounds including Z, ZR, zeatin-O-glucoside(ZOG), zeatinriboside-O-glucoside (ZROG) andzeatin nucleotides; dihydrozeatin-type CKs were notdetected in Arabidopsis (Astot et al. 1998). Therefore,the ipt-161 plants may be accumulating Z-type con-jugates besides Z and ZR, although they could not bequantified in our study.

Activity of the ipt promoter

Gene expression patterns conferred by two forms ofthe natural ipt-promoter (−184 and −283) have beenstudied in potato (Dymock et al. 1991), tobacco (Neu-teboom et al. 1993) and Arabidopsis (Schouten 1999).In potato and tobacco, the full length (−283) ipt-pro-moter conferred expression in most cell types inleaves, stem and roots, with the strongest expressionin roots and vascular tissue (Dymock et al. 1991;Neuteboom et al. 1993). The shorter (−184) promoterhad the same activity as the full length (−283) ipt-promoter in tobacco (Neuteboom et al. 1993) andshowed similar activity in Arabidopsis (Schouten1999). In tobacco, ipt-promoter activity gradually in-

creased during plant development with low activityjust after germination and higher activity after 3weeks, similar to the constitutive CaMV 35S-promot-er (Neuteboom et al. 1993). In potato and Arabidop-sis, ipt-promoter activity has not been studied duringplant development. However, our Arabidopsis ipt-transgenics exhibited a clear CK phenotype after ger-mination and during continued growth in tissue cul-ture, suggesting that ipt-promoter activity in Arabi-dopsis was not affected by plant development.

In potato and Arabidopsis, a decrease in ipt-pro-moter activity was observed in soil grown plants com-pared to plants grown in tissue culture (Dymock etal. 1991; Schouten 1999). Our ipt transgenics onlydisplayed the CK phenotype during the first 2–3weeks of growth in soil. After 3 weeks of growth insoil, the CK phenotype disappeared and 4–5 weeksafter germination the ipt transgenics resembled wildtype plants except for the increased serration of theleaf margins indicating that loss of ipt-promoter ac-tivity occurred in our transgenic lines under thesegrowth conditions. However, a loss of ipt-promoteractivity was not observed in tobacco plants (Neute-boom et al. 1993), suggesting that the effect of growthconditions on ipt-promoter activity is species depen-dent.

The role of ethylene in the establishment of the CKphenotype

The altered development of the Arabidopsis ipt trans-genics in this study resembled that of ipt transgenicsin other plant species. However, the formation of palegreen leaves has not been described. Increased endog-enous CK levels have been reported to cause an in-crease in mRNA levels for chloroplast proteins whichcorrelate with the formation of greener leaves (Beins-berger et al. 1991; Yusibov et al. 1991). The palegreen leaves of the our ipt transgenics contradict pre-vious reports and resemble the pale green leaf pheno-type reported for several CK-resistant mutants (Deik-man and Ulrich 1995; Su and Howell 1992). Crossesof the ipt-161 transgenic line with three ethylene in-sensitive mutants showed that the pale green color ofthe original ipt-161 transgenic was not displayed inthe ethylene insensitive mutant background. Further-more, these ‘double mutant/transgenic’ plants werelarger in size than ipt-161 and the serration of theleaves was less pronounced. This indicates that partof the CK phenotype displayed by the ipt transgenicswas caused by increased ethylene levels.

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The role of CK in de-etiolation

CK has been shown to be involved in many develop-mental processes including those controlled by light.The growth of seedlings in the absence of light re-sults in an etiolated phenotype including long hypo-cotyls and small unopened cotyledons which containetioplasts. In the light, seedlings have short hypocot-yls and open, expanded cotyledons (von Arnim andDeng 1996). It has been shown that CKs can mimicthe effects of light because dark-grown seedlings ex-posed to CK have short hypocotyls and expanded cot-yledons (de-etiolated phenotype). Both CK and lighthave an independent effect on hypocotyl elongationin Arabidopsis and these effects have been shown tobe additive (Su and Howell 1995). Both CK and lightcan completely saturate the inhibition of hypocotylelongation explaining our observeration that hypo-cotyl length for the ipt transgenics was comparable towild type plants in light. In dark grown seedlings, thede-etiolation response was observed in det and copmutants (Mayer et al. 1996) and the CK overproduc-ing mutant amp1 (Chin-Atkins et al. 1996), whichwas shown to be allelic to cop2 (Rupp et al. 1999).Our ipt transgenics only showed a mild de-etiolationphenotype when grown in the dark, which could befurther enhanced by the exogenous application of CK.

Comparison with other CK overproducingArabidopsis lines

Although a small number of CK-overproducing Ara-bidopsis lines have been reported, our ipt transgeniclines differed in development from both the heat-shock ipt transgenics and the CK-overproducing mu-tants cri1 and amp1. The ipt transgenics reported hereexhibited no alteration in xylem formation as ob-served in the maize heat shock promoter-ipt trans-genic line (Medford et al. 1989). Despite the fact thatour ipt lines shared decreased apical dominance andincreased leaf margin serration with the Drosophilaheat shock promoter-ipt lines, we did not observe in-creased leaf size, leaf thickness or hypocotyl diameteras was reported by Rupp et al. (1999). Unlike theamp1 CK-overproducing mutant, polycotyledony(Chaudhury et al. 1993) was not detected in our ipttransgenics and our lines showed only a mild de-eti-olation phenotype in dark grown seedling. The vitri-fication described for the cri1 mutant (Santoni et al.1997) was only observed for some primary transfor-mants and the cri1 mutant did not show reduced api-

cal dominance in tissue culture. Considering the factthat the increased CK levels in our ipt transgenicswere comparable to other CK-overproducing lines(6–10 fold wild type CK levels), the phenotypic dif-ferences could be due to differences in the mechanismof CK production, CK localization, or the spatial andtemporal distribution of CK within the plants as de-termined by the ipt promoter. The Arabidopsis ipt-161line described in this article has been deposited in theNASC stock centre and is available to the researchcommunity under accession number N117.

Acknowledgements

We would like to thank JCM. Clement and JHC.Hoge for providing the pBIN4R construct, J.Schouten for providing us with the transgenic linesharboring the ipt promoter GUS fusion and M. Hall(Aberystwyth) for the eti5 mutant. We would also liketo thank Eric W. Mercure and Qun-Hua Zhao for theirassistance with the ELISA analysis. This work wassponsored through the EU INCO program ERBIC15CT9609/4.

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