ORIGINAL ARTICLE

Adriana Chiappetta Æ Vania Michelotti

Marco Fambrini Æ Leonardo Bruno Æ Mariangela Salvini

Maria Petrarulo Æ Abdelkrim Azmi

Harry Van Onckelen Æ Claudio Pugliesi

Maria Beatrice Bitonti

Zeatin accumulation and misexpression of a class I knox geneare intimately linked in the epiphyllous response of the interspecifichybrid EMB-2 (Helianthus annuus · H. tuberosus)Received: 29 April 2005 / Accepted: 4 October 2005 / Published online: 6 January 2006� Springer-Verlag 2006

Abstract Epiphylly, occurring in a somaclonal variant(EMB-2) of the interspecific hybrid Helianthus annuus· H. tuberosus, was used to investigate molecular andcyto-physiological mechanisms that underlie cellular fatechange. EMB-2 plants are characterized by profuseproliferation of shoot- and embryo-like structures onsome leaves. We addressed the putative relationshipbetween cytokinins and knox genes in EMB-2 plants. Aclass I knox gene, HtKNOT1, was isolated from H. tu-berosus. A high level of HtKNOT1 transcripts was de-tected in EMB-2 epiphyllous leaves compared to non-epiphyllous (NEP) ones. In addition, epiphylly was re-lated to a localized increases in zeatin and N-glycosy-lated cytokinins. As ectopic morphogenesis proceeded,HtKNOT1 transcripts and zeatin co-localized andshowed different patterns in ectopic shoot comparedwith embryo-like structures, consistent with the differ-ential role of both cytokinin and knox genes in the twomorphogenetic events. Notably, a massive shoot/em-bryo regeneration was induced in EMB-2 NEP leaves byin vitro zeatin treatment. These results clearly indicatethat localized cytokinin accumulation and ectopicexpression of HtKNOT1 are closely linked in the epi-phylly of EMB-2 plants.

Keywords Cytokinins Æ Epiphylly Æ Helianthus ÆKnotted1-related gene Æ Meristem Æ Sunflower ÆTotipotency

Abbreviations EP: Epiphyllous leaves Æ HtKNOT1:Helianthus tuberosus knotted1-like gene Æ NEP:Non-epiphyllous leaves Æ SAM: Shoot apical meristem

Introduction

Higher plants exhibit an enormous capacity for regen-eration (D’Amato 1985). This totipotency is exemplifiedby processes of vegetative propagation in vivo (Steevesand Sussex 1989) as well as in the ability of plant cells todevelop into a complete and fertile plants by in vitrosomatic embryogenesis and/or adventitious organogen-esis (Halperin 1986). For example, structures such asembryos, shoots, leaves, and inflorescences can form onleaves. This phenomenon is epiphylly, a mechanism ofpropagation for several species (Dickinson 1978; Steevesand Sussex 1989).

Regeneration demonstrates the flexibility of plantcells that can re-acquire embryogenic or meristematiccompetence, following either developmental or envi-ronmental signals. Although widely studied, the physi-ological and genetic factors that underlie shifts in cellfate, leading to adventitious morphogenesis, are not yetfully understood. Work mainly focused on cell lineagesor mutants exhibiting high embryogenic/organogenicpotential, in Arabidopsis thaliana have begun to resolvesome genetic aspects of the regeneration processes(Mordhorst et al. 1998; Sugiyama 2000). An indetermi-nate cell state is necessary to express organogenic andembryogenic competence (Halperin 1986). Several gene‘‘markers’’ of these processes have been identified (Maet al. 1994; Teo et al. 2001; Hjortswang et al. 2002;Zhang et al. 2002; Daimon et al. 2003). In particular, afamily of knotted1-like homeobox genes (knox) are

A. Chiappetta Æ L. Bruno Æ M. Petrarulo Æ M. B. Bitonti (&)Dipartimento di Ecologia, Universita della Calabria,Arcavacata di Rende, 87030 Cosenza, ItalyE-mail: b.bitonti@unical.itTel.: +39-984-492965Fax: +39-984-492964

V. Michelotti Æ M. Fambrini Æ M. Salvini Æ C. PugliesiDipartimento di Biologia delle Piante Agrarie, Universita di Pisa,Sezione di Genetica, Via Matteotti 1B, 56124 Pisa, Italy

M. SalviniScuola Normale Superiore, Piazza dei Cavalieri 7, 56124 Pisa, Italy

A. Azmi Æ H. Van OnckelenDepartment of Biology, University of Antwerp, 2610 Antwerp,Belgium

Planta (2006) 223: 917–931DOI 10.1007/s00425-005-0150-7

important components that maintain cells in an inde-terminate state (Sinha et al. 1993; Reiser et al. 2000;Hake et al. 2004).

Based on comparative analysis of the conservedmotifs of encoded proteins, the knox family has beensubdivided in two classes, class I and class II (Reiseret al. 2000). The class I genes are mainly expressed inthe shoot apical meristem (SAM) and they may play arole in the establishment and maintenance of meri-stematic activity. However, their early repression isnecessary for leaf primordium initiation (Reiser et al.2000). In dicots as well as in monocots, knox gain offunction induces considerable alteration in leaf mor-phology and plant architecture (Hareven et al. 1996;Tamaoki et al. 1997; Frugis et al. 2001). Moreover,ectopic expression of class I knox genes can restoremorphogenetic potential in differentiated cells (Sen-toku et al. 2000; Gallois et al. 2002). Such a pheno-type was also mimicked by the mutation of genes thatnegatively regulate the knotted1-like genes (Byrne et al.2000; Daimon et al. 2003). Hence, substantial evidenceexists to link these homeobox genes to the acquisitionof meristematic competence that is such an importantfeature of regenerative responses both in vivo andin vitro.

Naturally occurring morphogenetic competence inepiphylly is well documented in many species (Dickin-son 1978; Steeves and Sussex 1989) and in someinterspecific hybrids of Nicotiana, Lycopersicon (Bayer1982) and Helianthus (Fambrini et al. 2000). Paradox-ically, epiphylly has been under exploited to addressmolecular mechanisms that regulate changes in cellularfate. The work reported here has focused on theepiphyllous (EP) response of plants (EMB-2) regener-ated in vitro from the interspecific hybrid H. annuus· H. tuberosus (Fambrini et al. 2000). EMB-2 plantsare characterized by the occurrence of numerous shootsand embryo-like structures on the adaxial surface ofleaves both in vivo and in vitro conditions, devoid ofexogenous hormones. These structures are localizedmainly in the proximal half of the oldest leaves(Fambrini et al. 2000), which raises interesting ques-tions regarding the morphogenetic competence ex-pressed by different parts of the leaf and differentleaves on the plant. Notably, the importance of cyt-okinins in promoting shoot-like structure in vitro andin vivo has been widely reported (Halperin 1986; Est-ruch et al. 1991). Moreover, a relationship has beenfound between the level and translocation of hormonesand the expression of knox genes (Tamaoki et al. 1997;Rupp et al. 1999; Hewelt et al. 2000; Sakamoto et al.2001; Frugis et al. 2001; Hamant et al. 2002; Hay et al.2002; Rosin et al. 2003).

In the present study, we demonstrate that the ectopicexpression of HtKNOT1, a class I knox gene isolatedfrom H. tuberosus, and the level of endogenous cytoki-nins are closely linked in the epiphylly exhibited byEMB-2 plants.

Materials and methods

Plant material and growth conditions

An EP plant, denominated EMB-2, was regenerated bya somatic embryo induced in vitro from a leaf explant ofthe tetraploid (2n=4x=68) interspecific hybrids H. an-nuus · H. tuberosus (A-2) (Fambrini et al. 2000). Inorder to obtain clones under sterile conditions, both theA-2 [non-epiphyllous genotype (NEP)] and the EMB-2plants (R1 generation) were multiplied by single-nodecutting on solidified (8 g l�1 Bactoagar, Oxoid, Basing-stoke, UK) MS basal medium (Murashige and Skoog1962) without growth regulators in 150- ml Erlenmeyerflasks. The cultures were incubated under a regime of25±1�C and a 16-h daily photoperiod in a growthchamber. Irradiance intensity during the light periodwas 30 lmol photons m�2 s�1, and it was provided bycool-light fluorescent lamps (Philips TLD 36W/33; Phi-lips, Eindhoven, The Netherlands). Moreover, H. tu-berosus (accession S. Pietro 1457, provided by theDepartment of Crop Plant Biology, University of Pisa,Pisa, Italy) plants were grown in growth chamber intopots containing a mixture of vermiculite, peat and soil at22±1�C under a 16-h daily photoperiod using fluores-cent tubes (Sylvania Day light F36W/56) with 200 lmolphotons m�2 s�1.

Regeneration from leaf explants of EMB-2 and A-2plants

Explants from young leaves of EMB-2 and A-2 plantswere used as starting material for in vitro experiments.Leaves of the EMB-2 clone were selected under areflecting stereoscope, before the in vitro culture, to ex-clude the presence of EP structures (NEP leaves). Leaveswere placed in Petri dishes (100·15 mm2) on basalmedium without growth regulators (MS) and on MSmedium supplemented with 1 mg l�1 zeatin (ZEA). Themedia contained 30 g l�1 sucrose and was solidified with8 g l�1 Bactoagar. Cultures were incubated in a growthchamber under the same conditions as described above.Experimental data were taken every day after placingleaves on the test media. Regeneration frequencies werepresented as the percentage of explants producingadventitious structures, from three independent experi-ments with five to six replications (Petri dishes), eachwith 8–10 explants.

Extraction, purification and analysis of cytokinins

Leaves of control (A-2) and EP (EMB-2) plants werecollected from different individuals (n=3). For EMB-2plants, basal EP leaves (EP) were also separated fromapical NEP ones. For all the samples the proximal

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halves of leaf (about 300 mg fresh weight) were dis-sected and frozen in liquid nitrogen. Samples were ex-tracted overnight in Bieleski’s solvent. Beforecentrifugation at 24,000g for 15 min at 4�C, deuteratedstandards for cytokinins (Apex International) wereadded and then the extract was purified using a com-bination of solid-phase and immunoaffinity purifica-tion, as described by Redig et al. (1996). Quantitativeanalysis of different cytokinins was performed as pre-viously described by Dewitte et al. (1999), using capil-lary column switching on a fully automatedworkstation (Famos, LC Packings, Amsterdam, TheNetherlands) coupled to a liquid chromatography set-up, consisting of an HPLC pump (model 325S, Kon-tron Instrument, Milan, Italy), an in-line UV detector(model 322, Kontron), and a triple-quadruple MS(Quattro II, Micromass UK Ltd., Cheshire, UK). Theresults were calculated according to the principles ofisotope dilution and expressed in pmol g�1 freshweight. For the calculation of detection limits and er-rors, fresh weight and recovery was taken into account.The data were treated using analysis of variance pro-cedures and means (± SE) were separated by Tukey’stest (P=0.05).

RNA isolation

The total RNA was obtained from plant tissues with asingle-step preparation, as previously described byChomczynski and Sacchi (1987). Vegetative shoot apices(HTVS), blades (HTLB) and veins (HTLV) of leaves (5-cm long), petiole (HTP) and stem internodes (HTST)were harvested from 30-day-old plants of H. tuberosusgrown in vivo. Vegetative shoot apices (VS) and leaves(L) of the A-2 interspecific hybrid (NEP clone), EP andNEP leaves of EMB-2 (EP clone) were harvested fromplants grown in vitro.

Cloning of H. tuberosus knotted1-like gene (HtKNOT1)

The total RNA (5 lg) from VS ofH. tuberosus was used,with the Superscript preamplification kit (InvitrogenS.R.L., Life Technologies, Milan, Italy), to produce thefirst-strand cDNA in conditions recommended bymanufacturer. Reverse transcription was carried outwith Superscript II retrotranscriptase in the presence ofthe adapter primer (AP, 5¢-GGCCACGCGTCGAC-TAGTACTTTTTTTTTTTTTTTTT-3¢) provided in thekit. The cDNA was employed in a PCR with the fol-lowing degenerate primers: P-1, 5¢-CCDGMDYTR-GAYCARTTCATGG-3¢ (forward) and P-4, 5¢-ATRAACCARTTGTTKATYTGYTTC-3¢ (reverse).These primers were selected in a region with highestconservation among members of the class I knox genes:KNAT1 and KNAT2 from Arabidopsis (Lincoln et al.1994), SBH1 from soybean (Ma et al. 1994) and LET6

from tomato (Chen et al. 1997). PCRs were performedin 50 ll of 1x buffer (Applied Biosystems, Applera Italia,Monza, Italy) containing 0.1 mM dNTPs, 1 lM of eachprimer, 2 mM MgCl2, 2 U AmpliTaq DNA polymerase(Applied Biosystems) and 2 ll of single-strand cDNA.Amplifications were carried out with Gene Amp� PCRSystem 2700 (Applied Biosystems) thermocycler. Afteran initial 4 min/94�C denaturation step, 40 cycles wererun each with 30 s of denaturation at 94�C, followed by30 -s annealing at 55�C and 30- s extension at 72�C.Final extension was at 72�C for 7 min. The PCR reac-tion yielded a product of about 450 bp, which wascloned into the pDrive Cloning Vector (Qiagen) andsequenced (HtK1). The RACE (rapid amplification ofcDNA ends) approach was used to isolate the 5¢ and 3¢ends of the HtKNOT1 cDNA. All reactions were per-formed with kits according to the manufacturer’sinstructions (Invitrogen). The 5¢ end of the HtKNOT1cDNA was amplified using first-strand cDNAs per-formed with Superscript II retrotranscriptase in thepresence of the HtKNOT1-specific primer P4 K (5¢-ATGAACCAATTGTTTATCTGCTTC-3¢). The termi-nal deoxynucleotidyl transferase (TdT) was used to add,at the 5¢ end of cDNA, a poly-C tail that allowed thecoupling of the 5¢ RACE abridged anchor primer (5¢-GGCCACGCGTCGACTAGTACGGGIIGGGIIGGGIIG-3¢). After C-tailing, PCR amplification was per-formed with the HtKNOT1-specific primer K5R (5¢-GAGAAAGTGATTGCCTTGAAC-3¢) and anchorprimers provided in the kit. The 3¢ end of the HtKNOT1cDNA was amplified using first-strand cDNAs madewith the AP primer. Each PCR was done with theHtKNOT1-specific primer K5 (5¢-GTTCAAGGCAATCACTTTCTC-3¢), and the abridged universal amplifi-cation primer (AUAP, 5¢-GGCCACGCGTCGACTAGTAC-3¢) provided in the kit. Specific cDNAs from 3¢and 5¢ RACE were subcloned into the pDrive CloningVector (Qiagen) and sequenced; the overlapping regionswith the first clone (HtK1) were confirmed. To isolatefull-length HtKNOT1 cDNA, sequence informationfrom the 5¢ and 3¢ RACE products was used to designthe following primers: 5¢ primer (Kstart), 5¢-TAGTTCCATTT AAAAAGTTGC-3¢ (located 29 nucleo-tides upstream the ATG); and 3¢ primer (Kend),5¢-CGTGTTT CTTGATTAGGTATTTAT-3¢ (located258 nucleotides downstream from the translational stopcodon). The primers were used for RT-PCR with thePfu-DNA polymerase with proof-reading function(Promega). PCRs were performed in 50 ll of 1x buffer(Promega) containing 0.1 mM dNTPs, 1 lM of eachprimer, 2 mM MgCl2, 2.5 U Pfu DNA polymerase and2 ll of single-strand cDNA. After an initial 4 min/95�Cdenaturation step, 35 cycles were run each with 30 s ofdenaturation at 94�C, followed by 30 s annealing at55�C and 2 min extension at 72�C. Final extension wasat 72�C for 7 min. A product of 1,378 bp were clonedinto the pDrive Cloning Vector (Qiagen) and sequenced.A clone, named HtK3, containing the entire sequencewas selected.

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Amino acid sequence analysis

Database researches were carried out with the Blastprogram (BLAST 2.0a) at the National Center forBiotechnology Information (NCBI) (Altschul et al.1997). A data set including the HtKNOT1 and other 23aminoacidic sequences of knox genes was constructed.Homeodomain (HD) regions of ATBELL1 (BELLgroup) from A. thaliana and metazoan sequences,DMEXD, MMPBX1, HSPR1 (the EXD group), andCEW05 (the SIX2 group) were included (Bharathanet al. 1999). The sequences were aligned by ClustalW(Thompson et al. 1994), and manually adjusted.According to Bharathan et al. (1999), regions corre-sponding respectively to a putative helix–loop–helix(HLH), amphipatic helix (H3), ELK domain and HDwere used in the following steps, but Linker regions (L1and L2) were excluded (Burglin 1997). Phylogeneticanalysis was performed using programs from thePHYLIP group, PHYLogeny Inference Package, Ver-sion 3.6 (Felsenstein 1985). As support for the trees wasobtained, a bootstrap analysis, with 100 replicates, wasperformed by SEQBOOT. The search for most-parsi-monious trees was done by PROTPARS and strictconsensus trees were obtained by CONSENSE.

Construction of DIG-RNA probe

A clone K245, containing a region (244 bp), spanningfrom the HtKNOT1-specific primers, P1S: 5¢-CAG-CACTTGATCAGTTCATG-3¢, and Kint: 5¢-GAT-CCTCAAGCTGAAGATCG-3¢, was linearized withappropriate restriction enzymes and used as a templateto synthesize digoxigenin-labelled RNA sense and anti-sense probes, according to the DIG-RNA labeling Kitprotocol (Roche Diagnostics GmbH, Mannheim, Ger-many). The P1S and Kint were designed to exclude fromthe probe both the ELK and the homeobox domains.

Relative RT-PCR analyses

Extraction of total RNA and reverse transcription werecarried out as described above for the method of cDNAcloning. First-strand cDNAs derived from 5 lg of totalRNA from each sample were used for each PCR ampli-fication. PCRs were performed using gene-specific prim-ers from cDNA of HtKNOT1 (K5, 5¢-GTTCAAGGCAATCACTTTCTT-3¢ and Kint) and b-actin (ACTINA 5,5¢-GATTCCGTTGCCCA/TGAGGTC/T-3¢ and AC-TINA 3, 5¢-TC/TTCTGGA/TGGA/TGCAACCACC-3¢). Primers were designed to yield 132- bp and 243-bpfragments forHtKNOT1 and b-actin, respectively. PCRswere carried out for 32 cycles and were performed in 50 llof 1x buffer (Applied Biosystems) containing 0.1 mMdNTPs, 0.5 lM of each primer (1 primer pair for b-actinand 1 primer pair for HtKNOT1), 2 mM MgCl2, 2 UAmpliTaq DNA polymerase (Applied Biosystems) and

2 ll of single-stranded cDNA. Amplifications were car-ried out according to the following temperature profile:95�C for 4 min for denaturation, then 94�C for 30 s, 55�Cfor 30 s, 72�C for 25 s and a final extension of 7 min at72�C.

HtKNOT1-primers were designed externally to anintron of 1,318 bp (data not shown). The relativeamounts of each PCR product were readily quantifiedby direct scanning of ethidium-stained 2% TAE-agarosegels with a UVP Image Store 5000 (Ultra Violet ProductLtd, Cambridge, England) densitometer equipped withthe UVP GelBase-GelBlot TM Windows Software. Tonormalize for equal amounts of total RNA and effi-ciency of cDNA synthesis from various tissue samples,the intensities of each band were normalized with theintensity of the b-actin product within the sampleinvestigated. An arbitrary value of 100% was assignedto the b-actin product. The relative expression levels ofthe HtKNOT1 transcripts were expressed as percentagewith respect to the b-actin product. The data are means(± SD) of three independent experiments. The data,after arcsin transformation, were treated using analysisof variance procedures and means were separated byTukey’s test (P=0.05).

Zeatin immunocytolabelling

Leaves of control (A-2) and EP clone (EMB-2) werecollected from different plantlets (n=3). For EMB-2plants, basal EP leaves were separated from apical NEP-ones. For all the samples the proximal half of leaf was cutinto different portions (sectors) starting from the petiole.The leaf portions were pre-embedded, cut using the vi-bratome (Leica VT1000E, Bensheim, Germany) andincubated with primary antibody against zeatin accord-ing to the procedure described by Dewitte et al. (1999).Colloidal gold (<1 nm)-labelled secondary antibodies(1:40, Aurion, Wageningen, The Netherlands) were used.The antibodies allowed us to detect specifically only thefree cytokinin base since this fixation procedure abro-gates the ability to detect conjugate zeatin in tissues.

In situ hybridization

Excised tissues were fixed, dehydrated, embedded inparaffin, cut into 8-lm sections and hybridized (55�C) toa digoxigenin-labelled antisense K245 RNA probe aspreviously described (Construction of DIG-RNA probe)by Canas et al. (1994). For immunological detection, theslides were incubated in buffer 1 (1% [w/v] blockingsolution, 100 mM Tris, pH 7.5 and 150 mM NaCl) for45 min and then equilibrated with buffer 2 (100 mMTris, pH 7.5 and 150 mM NaCl, 1% [w/v] bovine serumalbumin, and 0.3% [w/v] Triton X-100) for 45 min.Tissue sections were then incubated with anti-digo-xygenin-alkaline-phosphatase conjugate diluted 1:100 inbuffer 2 in a humidified box for 2 h and then washed

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four times for 10 min in buffer 2. The tissue sectionswere equilibrated in buffer 3 (100 mM Tris, pH 9.5 and100 mM NaCl and 50 mM MgCl2) for 20 min and thenincubated in 3.2 lg ml�1 5-bromo-4-chloro-3-indolyl-phosphate: 6.6 lg ml�1 nitroblue tetrazolium salt inbuffer 3 in a humidified box overnight. Accumulation ofHtKNOT1 transcript is visualized as a violet/brownstain. After stopping the reaction with TE (50 mM Tris–HCl, pH 8.0, 5 mM EDTA), sections were mountedwith 50% glycerol in TE.

Accession number

The sequence data of the H. tuberosus HtKNOT1 cDNAhave been submitted to the DDBJ/EMBL/GenBankdatabases under accession No. AJ519674.

Results

Phenotypic expression of epiphylly in the EMB-2 clone

The variant clone EMB-2 derived by in vitro tissue cul-ture of the interspecific hybrid H. annuus · H. tuberosus

shows an unusual pattern of development in that itproduces, both in vitro and in vivo, EP embryo- andshoot-like structures (Fig. 1a, b) (Fambrini et al. 2000).More precisely, in addition to leaves that expandednormally, some leaves of EMB-2 plants exhibit knobsand curling or a prominent proliferation of ectopicstructures. Generally, these ectopic structures are borneon the most basal leaves. Sometimes they fill the leafadaxial surface (Fig. 1c) but usually they are arranged inclusters along veins on the proximal half of the leaf(Fig. 1d), especially at the junction between the enlargedpetiole and lamina (Fig. 1e). Isolated EP structures arealso sporadically observed near the leaf tip. The EMB-2plants were infertile; they were propagated in vitro bysingle-node cutting and in vivo by tubers that displayedtypical EP structures (Fig. 1f).

Endogenous levels of cytokinins are altered in EP leavesof EMB-2 plants

cGiven the clear link between cytokinins and organo-genesis shown for other plants in vitro, the level ofendogenous cytokinins was measured in EP and NEP

Fig. 1 a–f Development of EPstructures in EMB-2 plants ofthe interspecific hybrid H.annuus · H. tuberosus grown invitro (a–e) or in vivo (f). aEmbryogenic structures(arrows). b Shoot-like structures(arrows). c Leaf adaxial sidefilled with ectopic somaticembryos. d Ectopic structures(arrow) developed along leafmidvein. e EP structures at thejunction between enlargedpetiole (arrow) and lamina. In dand e the leaves were partlycleared according to Candelaet al. (1999). f Ectopic embryos(arrows) on a leaf developedfrom a shoot borne on a tuberrecovered from an in vivogrown plant. Bars, 2.5 mm (a,b), 2.0 mm (c), 1.5 mm (d),3.0 mm (e), 2.7 mm (f)

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leaves of EMB-2 plants. Comparisons were then madewith a control NEP genotype (A-2). Since morphoge-netic structures preferentially formed in the proximalhalf of EP leaves, all measurements were confined to thisregion of the leaves. Total cytokinin content did notsignificantly differ in EMB-2 compared to A-2 plants asevidenced in Fig. 2a where values estimated in EP andNEP leaves are plotted together. However, a clear dif-ference in cytokinin levels was observed between differ-ent leaves of EMB-2 plants; cytokinin content was 1.8-fold higher in EP leaves compared with NEP ones(Fig. 2b). Compared with NEP leaves, zeatin-N-gly-cosylated (ZNG) was 3.2-fold higher, isopentenyl ade-nine glycosylated (iPG) was 2.4-fold higher and zeatinwas 1.8-fold higher in EP leaves (Fig. 2c). No significantdifferences were detected in the levels of other cytoki-

nins. These results indicate that total level of cytokininsper se is not necessarily an indicator of EP competence.

Ectopic shoot-like structures and increased zeatin levelsco-localize in EP leaves

We wanted to test the idea that it is localized, tissue-specific increases in, as opposed to, total cytokininlevels that are important indicators of EP competence.Hence, the spatial distribution of cytokinins wasexamined through immunocytolocalization of zeatin inboth EP and NEP leaves of EMB-2, as well in controlleaves (A-2). In cross sections of EP leaves, a strongimmunostaining was detected wherever there ectopicstructures formed, as well as at the level of the vas-cular bundles (Fig. 3a, b, f, g, i). In addition, a strongimmunoreaction marked adaxial epidermal cells, whichin some case undergo periclinal division (Fig. 3c), aswell as clusters of epidermal cells (Fig. 3d, e, h), fromwhich morphogenetic structures were likely to origi-nate. An uninterrupted immunoreaction was oftenobserved to link the vascular bundles to these cellclusters (Fig. 3d, e). As morphogenetic structuresdeveloped, a strong immunostaining was clearly de-tected at the level of the whole shoot-like structures(Fig. 3f, g). EP embryo-like structures, at differentdevelopmental stages, were also observed on theadaxial surface of EP leaves and zeatin localizationexhibited a distinct pattern, immunostaining beingconfined to a few scattered cells (Fig. 3i, j). In con-trast, a weak immunostaining was detected in NEP(Fig. 3k) and in A-2 (Fig. 3l) leaves; an immunoreac-tion was not observed in EP leaves processed withoutprimary antibody (Fig. 3m).

Zeatin treatment enhances adventitious morphogenesisin EMB-2 clone

To further investigate the relationship between zeatinand epiphylly of the EMB-2 clone, adventitious mor-phogenesis was monitored in explants of NEP leavescultured on either MS basal medium (without growthregulators) or zeatin enriched medium (ZEA medium).Leaves collected from A-2 plants and cultured under thesame conditions were used as a control.

After 3 days of culture, organized white globularstructures and buds were initiated on the adaxial surfaceof some EMB-2 leaves cultured on both media (Fig. 4a).However, a significant higher frequency of regenerationwas obtained on ZEA medium compared to MS med-ium. After 14 days the regeneration frequency of EMB-2explants increased to 74.8% and 48.7% in ZEA and MSmedium, respectively (Fig. 4a). In addition, EMB-2 ex-plants on ZEA medium showed a higher number ofadventitious structures compared to MS medium(Fig. 4b, c). By contrast, regeneration from A-2 leaveswas never detected (Fig. 4a).

Fig. 2 a–c Cytokinins’ content (pmol g�1 FW) in EP (EMB-2 EP),NEP (EMB-2 NEP) and A-2 control leaves. DHZ, dihydrozeatin;DHZR, dihydrozeatin riboside; iP, isopentenyladenine; DHZNG,dihydrozeatin-N-glucoside; iPA, isopentenyladenosine; iPG, iso-pentenyladenine-N-glucoside; ZR, zeatin riboside; ZNG, zeatin-N-glucoside. Values followed by the same letter are not significantlydifferent at the 0.05 probability level according to Tukey’s test

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Isolation of HtKNOT1, a class I knox gene

In order to provide a functional link between ectopicmorphogenesis and a cytokinin signal, it was necessaryto clone a knox gene that in other species is expressed asa prerequisite for meristematic competence. Hence, withthe aim of isolating Helianthus knotted1-like cDNAs, H.tuberosus was chosen as one of the parental species ofthe EMB-2 clone. Degenerate primers were used toamplify cDNAs derived from VS of H. tuberosus. Aproduct of the expected size of 450 bp was purified,

cloned, and sequenced. This H. tuberosus fragmentshowed a high degree of sequence similarity to theconserved positions of the consensus sequences createdfrom knox class I genes (data not shown). The recon-structed full-length cDNA sequence (1,398 bp), ob-tained from 3¢ and 5¢ RACE and confirmed from thesequence of the clone HtK3 (1,378 bp), contained a1,089 bp CDS, 54-nucleotides of 5¢-untranslated region(UTR), and 255-nucleotides of 3¢-UTR.

The predicted protein displayed 362 amino acids witha calculated molecular mass of 40.2 kDa (Fig. 5a). A

Fig. 3 a–m Zeatinimmunolocalization on crosssections of EMB-2 and A-2control plants. a–j EMB-2 EPleaves showing shoot (a–g) andembryo-like (h–j) structures. kEMB-2 NEP leaves. l A-2control leaves. m EMB-2 EPleaves treated in absence ofprimary antibody. a Section atthe level of petiole where arrowsindicate a cluster of ectopicstructures and arrow headsindicate vascular bundles. b–gSections at the distal level ofproximal portion. Arrowindicates: developing structures(b–f), periclinal division ofimmunoreactive epidermal cells(c), cluster of immunoreactivecells and continuity ofimmunoreaction betweenvascular bundle and epidermalcells (d, e), nuclear localizationof zeatin immunoreaction (g). hCluster of embryogenic cells. i, jAdvanced globular embryoswhere arrow indicates globularembryo and arrow headsindicate scattered reactive cellsalong epidermis and insideembryo. k, l Arrows indicateimmunostained vascularbundles. Bars, 120 lm (a),100 lm (b), 70 lm (c), 25 lm(d, e), 50 lm (f), 40 lm (g),20 lm (h, j), 35 lm (i), 45 lm(k), 50 lm (l), 45 lm (m)

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Fig. 4 Effects of in vitro culture on regeneration of A-2 and EMB-2leaves. a Time course of the regeneration frequency (%) in A-2 andEMB-2 leaves cultured in vitro on basal medium without hormones(MS) or on MS medium supplemented with 1 mg l�1 of zeatin(ZEA); the data are means (± SD) of three independent

experiments. b Adventitious morphogenetic structures observed inEMB-2 leaves cultured on MS medium. c ZEA-dependent increaseof morphogenetic structures in EMB-2 explants in vitro. Bars,1.2 mm (b), 2.0 mm (c)

Fig. 5 Sequence analysis of HtKNOT1 gene of H. tuberosus. aDeduced amino acid sequence of the HtKNOT1 gene. Theasparagine stretch in N-terminus are dotted underlined, the helix–loop–helix region (HLH) is underlined, the anphipatic helix (H3) isshaded while the complete MEINOX domain is delimited byopposed arrows. The GSE domain is bold, the ELK region is doubleunderlined and the homeodomain (HD) is represented with dashedunderline. b Diagrammatic representation of the organization ofcoding region of HtKNOT1 gene and deduced amino acid sequenceof the HD of theHtKNOT1 gene. The Pro-Tyr-Pro (PYP) sequenceis double underlined and the placement of the helix–turn–helix motifis shown above the sequence. The helices of HD are depicted as inVollbrecht et al. (1991). In the HD, the highly conserved amino

acid residues in the loop are bolded. Conserved position for theSBH1 subclass is shaded. The HD of HtKNOT1 and selected class Iplant homeobox proteins were aligned. The deduced amino acidsequences used for the comparison and their correspondingaccession numbers are: H. annuus HAKN1 (AY096802) andHAKN2 (AY096803), Nicotiana tabacum NTH15 (T01735), Gly-cine max SBH1 (L13663), Lycopersicon esculentum LET6(AF000141) and LETKN1 (U32247), Arabidopsis thaliana KNAT1(U14174) and KNAT2 (U14175), Zea mays ZMLG3 (AF100455),KN1 (CAA43065) and RS1 (T03946), Hordeum vulgareHVKNOX3 (X83518), Oryza sativa OSH1 (D16507), and Piceaabies HBK1 (AF063248). Asterisks indicate conserved amino acidresidues among these homeodomains. M, methionine

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BLAST search against the protein database of the Na-tional Center for Biotechnology Information (NCBI)indicated that the encoded protein shared high sequenceidentity with members of the class I knox subfamily.Thus the gene product was designated as H. tuberosusKNOTTED1 (HtKNOT1).

To compare the H. tuberosus gene with knox genesfrom other species, a phylogenetic analysis employingprotein sequences from several monocots and dicotstogether with sequences of metazoan HD proteins wasconstructed. All the analyses yielded results largelyconsistent with a previously published dendrogram(Bharathan et al. 1999). The KNOX proteins are wellsupported as monophyletic and there is strong con-firmation for both KNOX class I and class II clades(100 bootstrap values). Based on our phylogeneticanalysis (Fig. 6) and the detailed comparison of theHDs of 15 class I knox genes from various species(Fig. 5b), HtKNOT1 can be assigned as a class I ofKNOX HD protein. Furthermore, our results alsoindicated that HtKNOT1 along with HAKN1, NTH15,LET6, SBH1 and STM, among others, constitute adistinguishable subgroup (SBH1 subclass) of the classI knox genes.

HtKNOT1 is highly expressed in shoot meristemsand stem internodes of H. tuberosus and in EP leavesof the EMB-2 clone

Relative levels of HtKNOT1 transcripts in vegetativeorgans of in vivo grown plants of H. tuberosus wereestimated by using RT-PCR. Expression of HtKNOT1was detected in vegetative shoot apices and stem inter-nodes, while leaves (blades and veins) and petioles didnot accumulate any detectable HtKNOT1 transcripts(Fig. 7).

In EMB-2 plants, epiphylly is manifested by in vivogrown plants but the development of ectopic structuresis far more evident in in vitro conditions (Fambrini et al.2000). Thus, the expression of HtKNOT1 in interspecifichybrids was examined in in vitro grown plants. Firstly,we examined shoot meristems for the presence ofHtKNOT1 transcripts by using in situ hybridization.Sections of VS from H. annuus · H. tuberosus plants(A-2) were hybridized to an antisense HtKNOT1-specificprobe (K245). In longitudinal sections, HtKNOT1transcripts were strongly detected in the meristematicdome (Fig. 8a, b). Weak presence of transcripts wasdetected also in incipient leaf primordia (Fig. 8b) andyoung leaves (Fig. 8a). No transcription was detectedwith the sense RNA probe (Fig. 8c).

Relative RT-PCR showed that HtKNOT1 was highlyexpressed in vegetative shoots of A-2 plants (Fig. 8d, e).A comparatively low levels of HtKNOT1 transcripts wasdetected in both leaves of A-2 and NEP leaves of EMB-2plants (Fig. 8d, e). By contrast, the HtKNOT1 expres-sion in EP leaves was significantly higher with respect toNEP ones.

In EP leaves of the EMB-2 clone, HtKNOT1 expressionprecedes the ectopic morphogenetic processes

To determine the spatial pattern of HtKNOT1 expres-sion during the initiation and development of EPstructures, in situ hybridization with digoxigenin-la-belled RNA probes was performed (Fig. 9).

It was evident that in the absence of morphogeneticstructures (Fig. 9a), HtKNOT1 transcripts were con-fined to discrete zones along the leaf lamina (Fig. 9b) atthe level of the palisade cell layer (Fig. 9c). Thereafter,transcripts accumulated in developing morphogeneticstructures, but the signal did not spread to epidermalcells (Fig. 9d). However, as development of shoot-likestructures proceeded, transcript signal was also spreadthroughout epidermal cells (Fig. 9e, f). Afterwards, thelevel of HtKNOT1 transcripts decreased and mainlyaccumulated in the external cell layers of ectopic struc-tures (Fig. 9e, f). In contrast, in the case of putativeembryo-like structures, HtKNOT1 transcripts werestrictly confined to the basal portion of the ectopicstructure (Fig. 9g), whereas in the advanced globularembryos, the signal was restricted to a few scattered cellsinside the structure (Fig. 9h). A clear signal was alsoobserved at the level of vascular bundles (Fig. 9h).HtKNOT1 transcript accumulation was not detected ineither A-2 or NEP expanded leaves (data not shown).No transcription was detected with the HtKNOT1 senseRNA probe (Fig. 9i).

Notably, before ectopic structures became evident,HtKNOT1 expression and zeatin signal in EP leaveswere localized in distinct histological domains fromwhich ectopic shoots/embryos would arise (mesophylltissue vs. epidermis) (compare Fig. 9b, c with Fig. 3e, h,respectively). However, as morphogenetic processes oc-curred, the accumulation of HtKNOT1 transcripts andzeatin clearly overlapped (compare Fig. 9e, h withFig. 3g, i, respectively).

Discussion

Isolated through a PCR approach from vegetative shootcDNA of H. tuberosus, HtKNOT1 is a homeobox genebelonging to the knox gene family. Several lines of evi-dence presented here support the notion that HtKNOT1is phylogenetically related to the class I knox genes.First, sequence analyses revealed that HtKNOT1 issimilar to this class of genes, especially in the regions ofencoding functional domains of KNOX proteins. Sec-ond, HtKNOT1 is expressed in apical meristems and isdown-regulated in leaves of H. tuberosus as usually re-ported for class I knox genes (Hake et al. 2004).

Phylogenetic analyses of the homeodomains fromangiosperm class I knox genes showed a high degree ofsequence conservation throughout plant evolution,demonstrating that dicot and monocot genes occur inthis family without very clear demarcations by subfam-ilies, in spite of evolutionary distances between some

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genes (Bharathan et al. 1999; Reiser et al. 2000). How-ever, the results presented here clearly demonstrated thatHtKNOT1 is clustered within a group comprising onlydicot genes such as HAKN1, STM, SBH1, NTH15,

MTKNOX, and LET6. The homology of this cluster ofgenes is well supported by sequence data but theirrelationship based on expression studies is ambiguous(Ma et al. 1994; Long et al. 1996; Chen et al. 1997;

Fig. 6 Bootstrap consensus tree based on maximum parsimony,generated using Protpars program (Phylip package 3.572 version).The bootstrap replicates were 100 (values are given at the nodes);the deduced amino acid sequence of the HtKNOT1 of H. tuberosuswas compared to representative sequences of the knox homeobox

genes. Homeodomain regions of ATBELL1 from A. thaliana andmetazoan sequences DMEXD, MMPBX1, HSPR1 were included.The amino acid sequence CEW05 from C. elegans was used asoutgroup (Bharathan et al. 1999). The GenBank accession numbersof the sequences are indicated in brackets

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Fig. 7 Expression analysis of HtKNOT1. Amplification ofHtKNOT1 and b-actin transcripts performed using 5 lg of totalRNA isolated from vegetative shoot apices (HTVS), blades

(HTLB), veins (HTLV) and petioles (HTP) of 5-cm-long leaves,stem internodes (HTST) of H. tuberosus; the PCR products wereresolved on a TAE 2.0% agarose gel. M, marker

Fig. 8 a–e Expression analysis of HtKNOT1. a Distribution ofHtKNOT1 transcripts in vegetative shoot apex of in vitro grownplantlets of the interspecific hybridH. annuus · H. tuberosus (A-2);longitudinal section through the vegetative shoot apex hybridizedwith an antisense probe (K245) labelled with digoxigenin-UTP; thetranscript-specific hybridization signal is visible as purple staining;arrow indicates the shoot apical meristem. b Magnification of a;arrows indicate the leaf primordia. c Control experiment performedwith dig-labelled HtKNOT1 sense probe. d Amplification ofHtKNOT1 and b-actin transcripts performed using 5 lg of total

RNA isolated from vegetative shoot apex (VS) and leaves (L) ofcontrol A-2 plants, from NEP and EP leaves of the EMB-2 clone;the PCR products were resolved on a TAE 2.0% agarose gel. e Therelative expression levels of the HtKNOT1 transcripts, expressed aspercentage with respect to b-actin product levels, were estimated bymeasuring the ethidium bromide staining of the PCR productsresolved by gel electrophoresis in d; the data are means (± SD) ofthree independent experiments; values followed by the same letterare not significantly different at the 0.05 probability level accordingto Tukey’s test. M, marker. Bars, 250 lm (a), 70 lm (b, c)

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Tamaoki et al. 1997; Bharathan et al. 1999; Tioni et al.2003).

In particular, STM and NTH15, as most of class Iknox genes, are typically expressed in the SAM andsubtending stem and excluded from lateral organs (Longet al. 1996; Tamaoki et al. 1997). By contrast, LET6transcripts were detected in leaf primordia of tomato(Parnis et al. 1997). Transcripts of the HAKN1 gene, forwhich HtKNOT1 showed the highest identity, wereaccumulated in several organs of H. annuus, includingpetioles, leaves, and leaf homologues (Tioni et al. 2003).HtKNOT1 expression was not widespread as that ofHAKN1. In fact, HtKNOT1 mRNAs was detectedin vegetative shoot apices and stem internodes ofH. tuberosus, but not in petioles and leaves. However, a

low level of HtKNOT1 transcripts was detected in leafprimordia and developing leaves of the interspecifichybrid H. annuus · H. tuberosus grown in vitro. Thesefindings indicate that, in some species, the transcriptionaldown-regulation of class I knox genes is not a prerequisitefor lateral organ initiation (Parnis et al. 1997; Bellaouiet al. 2001; Muller et al. 2001; Chen et al. 2003).

Our data showed that HtKNOT1 is strongly ex-pressed in EP leaves of EMB-2 plants as compared toNEP ones. Note that contrasting expression of the mu-tant phenotype within a single plant (i.e. NEP and EPleaves) is a somewhat common trait of the class I knoxmutants. For example, in both knotted1 of mais (Freel-ing and Hake 1985) and hooded of barley (Muller et al.1995), the mutant phenotype, also in relation to genetic

Fig. 9 a–h Localization of HtKNOT1 transcripts (b–h) on crosssections of EP leaves of EMB-2 plants. a Morphological features ofEP leaves showing sectors with (A) or without (B) ectopicstructures. b–c Cross sections at the level of the B sector: arrows(b) indicate scattered labelled cells along leaf lamina. d–i Crosssections at the level of the A sector: initial stage of ectopic

structures (d), ectopic shoot-like structures (e, f), early globularembryo (g), advanced globular embryo (h). i Control experimentperformed with dig-labelled HtKNOT1 sense probe. Bars, 3.0 mm(a), 18 lm (b), 20 lm (c), 35 lm (d, e), 40 lm (f), 75 lm (g, i),45 lm (h)

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background, is strongly associated with ectopic geneexpression but only in some leaves and awns, respec-tively. Thus, it is likely that the differential HtKNOT1expression in EP leaves compared with lack of expres-sion in NEP leaves is also regulated by other factors thatare expressed in a spatially or temporally differentialpattern during leaf development.

Using in situ hybridization we demonstrated that inEP leaves localized HtKNOT1 overexpression precedesthe development of ectopic structures. Indeed, beforeepiphylly becomes evident, transcript presence wasselectively detected at the level of mesophyll and vas-cular bundles whereas epidermal cells never exhibitedHtKNOT1 expression. Notably, in leaves of Arabidopsisoverexpressing KN1 under the control of tissue specificpromoters, the gene product was shown to move fromthe inner layers to epidermis, but not in the oppositedirection (Kim et al. 2003). However, when KN1 wasexpressed in the epidermis, the plants did not exhibitectopic morphogenetic structures but transgenic plantsoverexpressing KN1 in the mesophyll cells producedectopic shoots on the adaxial leaf side (Kim et al. 2003).These results fully agree with the ectopic HtKNOT1expression that we detected in the mesophyll cells ofEMB-2 EP leaves.

An intriguing feature of the EMB-2 variant is that itproduces not only ectopic shoots but also ectopic em-bryo-like structures, which have been never describedeither in transgenic or mutant plants with alteredexpression of class I knox genes (Lincoln et al. 1994;Sentoku et al. 2000; Gallois et al. 2002). However,members of a knox gene family mark the embryogenicpotential of cell lines of Abies alba, being a key factor forin vitro development of somatic embryos (Hjortswanget al. 2002). Thus, to our knowledge, the epiphylly ofEMB-2 represents a unique example of an ectopicembryogenic process related to misexpression of a knoxgene. In this context, it is important to recall that theEMB-2 clone is derived from a single somatic embryoinduced in an enriched cytokinin medium (Fambriniet al. 2000), which is an unusual condition to restoreembryogenic competence in determinate cells (Halperin1986).

The pattern of HtKNOT1 expression was clearlydifferent in embryo- compared to shoot-like structures.In somatic embryos, HtKNOT1 transcripts were con-fined to the basal region and became scattered in a fewembryogenic cells as development proceeded. Similarly,in somatic embryos of maize at the globular stage, KN1was expressed in a small group of cells (Zhang et al.2002), but these cells were clearly located in a morespecific region (e.g. initial stage of SAM organization)than those of EMB-2 EP embryos. Perhaps in the latter,some cells can themselves become new embryogenicgrowth centres rather than participate in the organiza-tion of a functional SAM. The abnormal cellular pro-liferation, which frequently characterizes thedevelopment of ectopic EMB-2 embryos, strongly sup-ports this suggestion (Fambrini et al. 2000). In contrast,

shoot-like structures were heavily labelled at the begin-ning of their formation, while in more developed struc-tures, HtKNOT1 transcripts were progressively confinedto the external layers, which perhaps represent the his-tological domains of SAM establishment. An analogouspattern of expression was observed for BROSTM, aclass I knox gene of Brassica oleracea, during thedevelopment of adventitious shoots induced in vitrofrom stem segments (Teo et al. 2001).

Due to the above-mentioned characteristics and well-documented relationship between knox genes and cyto-kinin signalling (Rupp et al. 1999; Hewelt et al. 2000;Frugis et al. 2001; Hamant et al. 2002), the level andlocation of this hormone class in EMB-2 leaves was alsoinvestigated. Clear differences in cytokinin levels wereobserved between NEP and EP leaves. However, ourdiscovery that EMB-2 plants are not cytokinin over-producers is not surprising. In fact, although EMB-2 ischaracterized by EP shoots, like ipt overexpressingplants (Estruch et al. 1991), other features of cytokininoverproduction were not observed (i.e. reduced inter-node length, reduced leaf surface, retarded leaf senes-cence). Indeed, our results indicate that epiphylly ofEMB-2 plants is tightly related to a localized increase ofcytokinins (ZNG, iPG and zeatin) in EP leaves ascompared to NEP ones. Zeatin-induced stimulation ofshoot and embryo development on NEP leaves culturedin vitro further supports this link between cytokininavailability and adventitious morphogenesis in EMB-2plants. However, EP structures were not formed inzeatin-treated A-2 leaves suggesting that an intrinsic cellcompetence to regeneration is required. It is likely thatin EMB-2 plants such competence is switched by specificinductors (i.e. cytokinins and knox misexpression).

The involvement of cytokinins in ectopic morphoge-netic events of EMB-2 plants is strengthened by immu-nolocalization results. The presence of active zeatin,revealed by immunostaining, marks the development ofadventitious shoot-like structures, from the first divisionof epidermal cells to SAM establishment (data notshown). However, in the ectopic embryogenesis, thescattered distribution of zeatin, from the initial cluster ofembryogenic cells until the globular stage, suggests thatcytokinin action is limited to the initial switch of epi-dermal cell fate. Accordingly, in vitro somatic embryodevelopment does not occur in the presence of a con-tinuous/high cytokinin treatment (Halperin 1986).

A characteristic of zeatin distribution in EP leaveswas the massive localization at the level of vascularbundles, a feature that was also observed in lettuceplants overexpressing KNAT1 (Frugis et al. 2001). Inparticular, ectopic shoot-like structures formed whereverzeatin was found in vascular bundles through to theepidermis. These distinct histological traits and thepreferential accumulation of cytokinins in EP leavesstrongly suggest that EP leaves could acts as a sink forcytokinins that could be related to the conspicuouspresence of N-glycosylated forms. Although the preciserole of each cytokinin is not yet fully understood,

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N-glucoside conjugates are usually regarded as irre-versible inactive forms (Mok and Mok 2001). Hence, theexcessive amount of cytokinins loaded in EP leavescould be inactivated through the formation ofN-glucosyl conjugate. Clearly, hormonal activities are afunction of not only their endogenous concentrationsbut also on their interactions with other growthregulators. For example, in addition to the associationbetween class I knox genes and cytokinins, several otherstudies have shown that knox expression is also linked toother hormonal activities, including gibberellic acid(Sakamoto et al. 2001; Hay et al. 2002), ethylene(Hamant et al. 2002) and polar auxin transport (PAT)(Tsiantis et al. 1999; Scanlon 2003). In general, ectopicKNOX accumulation is correlated with a defective PAT,which in turn could influence cytokinin level and/ortranslocation (Mok and Mok 2001).

In conclusion, we have isolated a HtKNOT1, ahomeobox gene which belongs to the knox gene familyand both sequence analyses and expression patternsupport the notion that HtKNOT1 is phylogeneticallyrelated to the class I knox genes. In EP leaves of theEMB-2 clone, the ectopic proliferation of shoot- andembryo-like structures appears to be associated withboth a local accumulation of zeatin and HtKNOT1misexpression. On this basis we propose that both eventsare intimately linked to the epiphylly of EMB-2 plants.

Acknowledgements This work was supported by grants from PRIN2004 and from Scuola Normale Superiore to M. Salvini.

References

Altschul SF, Madden TL, Schaffer AA, Zhang J, Zhang Z, MillerW, Lipman DJ (1997) Gapped BLAST and PSI-BLAST: a newgeneration of protein database search programmes. NucleicAcids Res 25:3389–3402

Bayer MH (1982) Genetic tumors: physiological aspects of tumorformation in interspecific hybrids. In: Kahl G, Schell JS (eds)Molecular biology of plant tumors. Academic Press, New York,pp 33–67

Bellaoui M, Pidkowich MS, Samach A, Kushalappa K, Kohalmi S,Modrusan Z, Crosby WL, Haughn GW (2001) The ArabidopsisBELL1 and KNOX TALE homeodomain proteins interactthrough a domain conserved between plants and animals. PlantCell 13:2455–2470

Bharathan G, Janssen BJ, Kellog EA, Sinha NR (1999) Phyloge-netic relationship and evolution of the KNOTTED class ofplant homeodomain proteins. Mol Biol Evol 16:553–563

Burglin TR (1997) Analysis of TALE superclass homeobox genes(MEIS, PBC, KNOX, Iroquois, TGIF) reveals a novel domainconserved between plants and animals. Nucleic Acids Res25:4173–4180

Byrne ME, Barley R, Curtis M, Arroyo JM, Dunham M, HudsonA, Martienssen RA (2000) Asymmetric leaves1 mediates leafpatterning and stem cell function in Arabidopsis. Nature408:967–971

Canas LA, Busscher M, Angenent GC, Beltran JP, van Tunen AJ(1994) Nuclear localization of petunia MADS box proteinFBP1. Plant J 6:597–604

Candela H, Martinez-Laborda A, Micol JL (1999) Venation pat-tern formation in Arabidopsis thaliana vegetative leaves. DevBiol 205:202–216

Chen JJ, Janssen BJ, Williams A, Sinha N (1997) A gene fusion at ahomeobox locus: alterations in leaf shape and implications formorphological evolution. Plant Cell 9:1289–1304

Chen H, Rosin FM, Prat S, Hannapel DJ (2003) Interacting tran-scription factors from three-amino acid loop extension super-class regulate tuber formation. Plant Physiol 132:1391–1404

Chomczynski P, Sacchi N (1987) Single-step method of RNA iso-lation by acid guanidinium thiocyanate-phenol-chloroformextraction. Anal Biochem 162:156–159

Daimon Y, Takabe K, Tasaka M (2003) The CUP-SHAPEDCOTYLEDON genes promote adventitious shoot formation oncalli. Plant Cell Physiol 44:113–121

D’Amato F (1985) Cytogenetics of plant cell and tissue culturesand their regenerates. Crit Rev Plant Sci 3:73–112

Dewitte W, Chiappetta A, Azmi A, Witters E, Strnad M, RemburJ, Noin M, Chriqui D, Van Onckelen H (1999) Dynamics ofcytokinins in apical shoot meristem of a day-neutral tobaccoduring floral transition and flower formation. Plant Physiol119:111–122

Dickinson TA (1978) Epiphylly in angiosperms. Bot Rev 44:181–232

Estruch JJ, Prinsen E, Van Onckelen H, Schell J, Spena A (1991)Viviparous leaves produced by somatic activation of inactivecytokinin-synthesizing gene. Science 254:1364–1367

Fambrini M, Cionini G, Bianchi R, Pugliesi C (2000) Epiphylly in avariant of Helianthus annuus · H. tuberosus induced by in vitrotissue culture. Int J Plant Sci 161:13–22

Felsenstein J (1985) Confidence limits on phylogenies: an approachusing the bootstrap. Evolution 39:783–791

Freeling M, Hake S (1985) Developmental genetics of mutants thatspecify knotted leaves in maize. Genetics 111:617–634

Frugis G, Giannino D, Mele G, Nicolodi C, Chiappetta A, BitontiMB, Innocenti AM, Dewitte W, Van Onckelen H, Mariotti D(2001) Overexpression of KNAT1 in lettuce shifts leaf deter-minate growth to a shoot-like indeterminate growth associatedwith an accumulation of isopentenyl-type cytokinins. PlantPhysiol 126:1370–1380

Gallois JL,WoodwardC,ReddyGV, SablowskiR (2002)CombinedSHOOT MERISTEMLESS and WUSCHEL trigger ectopicorganogenesis in Arabidopsis. Development 129:3207–3217

Hake S, Smith HMS, Holtan H, Magnani E, Mele G, Ramirez J(2004) The role of knox genes in plant development. Annu RevCell Dev Biol 20:125–151

Halperin W (1986) Attainment and retention of morphogeneticcapacity in vitro. In: Vasil IK (ed) Plant regeneration and ge-netic variability, vol 3. Academic Press, London, pp 3–47

HamantO,Nogue F, Belles-Boix E, JublotD,GrandjeanO, Traas J,Pautot V (2002) The KNAT2 homeodomain protein interactswith ethylene and cytokinin signaling. Plant Physiol 130:657–665

Hareven D, Gutfinger T, Parnis A, Eshed Y, Lifschitz E (1996) Themaking of a compound leaf: genetic manipulation of leafarchitecture in tomato. Cell 84:735–744

Hay A, Kaur H, Phillips A, Hedden P, Hake S, Tsiantis M (2002)The gibberellin pathway mediates KNOTTED1-type homeo-box function in plants with different body plans. Curr Biol12:1557–1565

Hewelt A, Prinsen E, Thomas M, Van Onckelen H, Meins FJ(2000) Ectopic expression of maize knotted1 results in thecytokinin-autotrophic growth of cultured tobacco tissues.Planta 210:884–889

Hjortswang HI, Sundas Larsson A, Bharathan G, Bozhkov PV,Von Arnold S, Vahala T (2002) KNOTTED1-like homeoboxgenes of gymnosperm, Norway spruce, expressed during so-matic embryogenesis. Plant Physiol Biochem 40:837–843

Kim JY, Yuan Z, Jackson D (2003) Developmental regulation andsignificance of KNOX protein trafficking in Arabidopsis.Development 130:4351–4362

Lincoln C, Long J, Yamaguchi J, Serikawa K, Hake S (1994) Aknotted1-like homeobox gene in Arabidopsis is expressed in thevegetative meristem and dramatically alters leaf morphologywhen overexpressed in transgenic plants. Plant Cell 6:1859–1876

930

Long JA, Moan EI, Medford JI, Barton MK (1996) A member ofthe KNOTTED class of homeodomain proteins encoded by theSTM gene of Arabidopsis. Nature 378:66–69

Ma H, McMullen MD, Finer JJ (1994) Identification of ahomeobox-containing gene with enhanced expression duringsoybean (Glycine max L.) somatic embryo development. PlantMol Biol 24:465–473

Mok DWS, Mok MC (2001) Cytokinin metabolism and action.Annu Rev Plant Physiol Plant Mol Biol 52:89–118

Mordhorst AP, Voerman KJ, Hartog MV, Meijer EA, Van Wen J,Koornneef M, De Vries SC (1998) Somatic embryogenesis inArabidopsis thaliana is facilitated by mutations in genesrepressing meristematic cell divisions. Genetics 149:549–563

Muller J, Wang Y, Franzen R, Santi L, Salamini F, Rohde W(2001) In vitro interactions between barley TALE homeodo-main protein suggest a role for protein–protein associations inthe regulation of Knox gene function. Plant J 27:13–23

Muller KJ, Romano N, Gerstner O, Garcia-Maroto F, Pozzi C,Salamini F, Rohde W (1995) The barley Hooded mutation iscaused by a duplication in a homeobox gene intron. Nature374:727–730

Murashige T, Skoog F (1962) A revised medium for rapid growthand bioassays with tobacco tissue cultures. Physiol Plant15:473–497

Parnis A, Cohen O, Gutfinger T, Hareven D, Zamir D, Lifschitz E(1997) The dominant developmental mutant of tomato Mouse-ear and Curl, are associated with distinct modes of abnormaltranscriptional regulation of a Knotted gene. Plant Cell 9:2143–2158

Redig P, Schmulling T, Van Onckelen H (1996) Analysis of cyto-kinin metabolism in ipt transgenic tobacco by liquid chroma-tography-tandem mass spectrometry. Plant Physiol 112:141–148

Reiser L, Sanchez-Baracaldo P, Hake S (2000) Knots in the familytree: evolutionary relationships and functions of knox homeo-box genes. Plant Mol Biol 42:151–166

Rosin FM, Hart Jk, Horner HT, Davies PJ, Hannapel DJ (2003)Overexpression of a Knotted-like homeobox gene of potato al-ters vegetative development by decreasing gibberellin accumu-lation. Plant Physiol 132:106–117

Rupp HM, Frank M, Werner T, Strand M, Schmulling T (1999)Increased steady state mRNA levels of the STM and KNAT1homeobox genes in cytokinin overproducing Arabidopsis thali-ana indicate a role for cytokinins in the shoot apical meristem.Plant J 18:557–563

Sakamoto T, Kamiya N, Ueguchi-Tanaka M, Iwahori S, Mats-uoka M (2001) KNOX homeodomain protein directly sup-presses the expression of a gibberellin biosynthetic gene in thetobacco shoot apical meristem. Genes Dev 15:581–590

Scanlon MJ (2003) The polar auxin transport inhibitor N-1-naphthylphthalamic acid disrupts leaf initiation, KNOX pro-tein regulation, and formation of leaf margins in maize. PlantPhysiol 133:597–605

Sentoku N, Sato Y, Matsuoka M (2000) Overexpression of riceOSH genes induces ectopic shoots on leaf sheaths of transgenicrice plants. Dev Biol 220:358–364

Sinha NR, Williams Jr RE, Hake S (1993) Overexpression of themaize homeo box gene, KNOTTED-1, causes a switch fromdeterminate to indeterminate cell fate. Genes Dev 7:787–795

Steeves TA, Sussex IA (1989) Patterns in plant development, 2ndedn. Cambridge University Press, Cambridge

Sugiyama M (2000) Genetic analysis of plant morphogenesis invitro. Int Rev Cytol 196:67–84

Tamaoki M, Kusaba S, Kano-Murakami Y, Matsuoka M (1997)Ectopic expression of a tobacco homeobox gene, NTH15,dramatically alters leaf morphology and hormone levels intransgenic tobacco. Plant Cell Physiol 38:917–927

Teo WL, Kumar P, Goh CJ, Swarup S (2001) The expression ofBrostm, a KNOTTED1-like gene, marks the cell type and timingof in vitro shoot induction in Brassica oleracea. Plant Mol Biol46:567–580

Thompson JD, Higgins DG, Gibson TJ (1994) CLUSTALW:improving the sensitivity of progressive multiple sequencealignment through sequence weighting, position-specific gappenalties and weight matrix choice. Nucleic Acids Res 22:4673–4680

Tioni MF, Gonzalez DH, Chan RL (2003) Knotted1-like genes arestrongly expressed in differentiated cell types in sunflower. JExp Bot 54:681–690

Tsiantis M, Brown MIN, Skibinski G, Langdale JA (1999) Dis-ruptions of auxin transport is associated with aberrant leafdevelopment in maize. Plant Physiol 121:1163–1168

Vollbrecht E, Veit B, Sinha N, Hake S (1991) The developmentalgene Knotted-1 is a member of a maize homeobox gene family.Nature 350:241–243

Zhang S, Wong L, Meng L, Lemaux PG (2002) Similarity ofexpression patterns of knotted1 and ZmLEC1 during somaticand zygotic embryogenesis in maize (Zea mays L.). Planta215:191–194

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