Cytokinin oxidase/dehydrogenase genes in barley and wheat Cloning and heterologous expression Petr Galuszka 1 , Jitka Fre ´ bortova ´ 2 , Toma ´ s ˇ Werner 3 , Mamoru Yamada 4 , Miroslav Strnad 2 , Thomas Schmu¨ lling 3 and Ivo Fre ´ bort 1 1 Division of Molecular Biology, Department of Biochemistry, Faculty of Science, Palacky ´ Univesity, Olomouc, Czech Republic; 2 Laboratory of Growth Regulators, Palacky ´ University/Institute of Experimental Botany of the Academy of Science, Olomouc, Czech Republic; 3 Institute of Biology/Applied Genetics, Free University of Berlin, Germany; 4 Department of Biological Chemistry, Faculty of Agriculture, Yamaguchi University, Japan The cloning of two novel genes t hat encode cytokinin oxidase/dehydrogenase ( CKX) in barley is described in this work. Transformation of both genes into Arabidopsis and tobacco showed that at least one of the g enes codes for a functional enzyme, as its expression caused a cytokinin- deficient phenotype in the heterologous host plants. Addi- tional cloning of two gene fragments, and an in silico search in the public expressed sequence tag clone databases, revealed the presence of at least 13 more members of the CKX gene family in barley and wheat. The expression of three selected barley genes was analyzed by RT-PCR and found t o be organ-specific with peak expression in mature kernels. One barley CKX (HvCKX2) was characterized in detail after heterologous expression in tobacco. Interest- ingly, this enzyme shows a pH optimum at 4.5 and a pref- erence for cytokinin r ibosides as substrates, which may indicate its vacuolar targeting. Different substrate specifici- ties, a nd the pH profiles of cytokinin-degrading enzymes extracted from different barley tissues, are also presented. Keywords: cereals; cloning; cytokinin oxidase/dehydro- genase; e xpression; gene family. Cytokinins were initially viewed as factors promoting cell division and differentiation in plants. Since then, however, cytokinins have been shown t o control o ther developmental events, such a s the gr owth of lateral buds, the release o f buds from apical dominance, leaf expansion, the delay of senescence, the promotion of seed germination, and chloroplast formation [1]. Naturally occurring cytokinins are mainly N 6 -substituted adenine derivatives that g enerally contain a n isoprenoid o r aromatic s ide-ch ain. Recently, considerable progr ess has been made in elucidating the regulation of cytokinin homeostasis during plant growth and d evelopment. New molecular b iological techniques have allowed for the identification and characterization of genes encoding important enzymes p articipating in cyto- kinin metabolic pathways. Genetically engineered plants that overexpress some of these genes were p repared a s a tool to study changes in physiological aspects c aused by altered cytokinin levels. Seven genes for isopentenyltransferases – cytokinin de novo synthesizing enzymes – were identified in the Arabidopsis genome [2–4]. In addition, three novel genes, encoding cytok inin-specific glycosylation enzymes with different substrate s pecificities, have been d escribed [5–7]. The principle of cytokinin catabolism h as been studied for many years. Enzymes capable of degra ding cytokinins with unsaturated side-chains have been found in many plant tissues [8], but the details of their features a nd the mechanism of their action remained unknown f or a long time owing to their very low content in plant tissues. The ground-breaking cloning of the cytokinin oxidase maize gene ZmCKX 1 [9,10] o pened up the possibility for more detailed study of cytokinin degradation, both at the molecular and at the biochemical levels. The recombinant maize e nzyme is a glycoprotein containing a c ovalently bound FAD. The i soprenoid s ide-chain of t he cytokinin molecule is most efficiently c leaved in t he presence of an electron acceptor other than oxygen. Hence, the enzyme has been classified as a dehydrogenase with a new EC 1.5.99.12 [11]. The detailed reaction mechanism of cytokinin oxidase/ dehydrogenases (CKX) has recently been presented for the conversion of different types of cytokinin s ubstrates [12]. Studies of r eaction rates have revealed that oxygen is unlikely to be the physiological acceptor reoxidizing the FAD molecule of the enzyme in vivo. The exact character- istics of a naturally cooperating electron acceptor are still unknown, but experiments in vitro indicate that it might be p-quinone or a molecule with a similar structure [12]. The completed sequencing project of Arabidopsis and rice genomes allowed identification of the small CKX gene family of seven homologues in Arabidopsis (AtCKX1 to AtCKX7 [13]) and 11 in rice [14]. Six AtCKX genes were individually overexpressed in tobacco or Arabidopsis plants, a nd a detailed phenotypic characterization was Correspondence to P. Galuszka, Divis ion of M olecular Biol ogy, Department of Biochemistry, Faculty of Science, Palacky´ University, S ˇ lechtitelu˚ 11, 783 71 Olomouc, Czech Republic. Fax: +420 58 5634933, Tel.: +420 58 5634929, E-mail: galuszka@prfholnt.upol.cz Abbreviations: CKX, cytokinin oxidase/dehydrogenase; EST, expressed sequence tag; MS-medium, Murashige–Skoog medium; Q 0 , 2,3-dimethoxy-5-methyl-1,4-benzoquinone. Enzyme: cytokinin oxid ase/dehydrogenase (EC 1.5.99.12). Note: a web site is available at http://prfholnt.upol.cz/biochhp (Received 29 April 2004, revised 14 July 2004, accepted 16 August 2004) Eur. J. Biochem. 271, 3990–4002 (2004) Ó FEBS 2004 doi:10.1111/j.1432-1033.2004.04334.x subsequently carried out. All transformants displayed reduced cytokinin content a nd showed distinct develop- mental alterations in the shoot and r oot [15,16], mos t of them in accordance with previous assumptions on cytokinin f unction. Two o f the AtCKX proteins w ere found to be targeted to the vacuoles, while another accumulated i n t he reticulate structure, which may indicate its final e xtracellular l ocalization [ 16]. One additional CKX gene has been identified in a Dendrobium orchid, and similar aspects of i ts overexpression in growth and development have been described in Arabid- opsis plants [17]. In this work, we reveal the basic characterization of the CKX gene family in the cereal species Hordeum and Triticum, as well as report on the cloning of the fi rst two CKX genes of barley and demonstrate functionality for one of them in transgenic tobacco an d Arabidopsis plants. Materials and methods Plant materials Commercial barley (H. vulgare cv. Luxor) and wheat (T. aestivum L. cv. Samantha) grains were soaked in tap water for 1 day to initiate germination. The soaked grains were then transferred to s oil and grown in a greenhouse with a 15 h/9 h day/night cycle at 21 °C. Isolation of poly(A + ) RNA All RNA was extracted from different plant tissues using TRIZOL Reagent (Gibco B RL, G rand Island, NY, USA). Polysaccharide cont amination o f the grain extract was removed with two additional centrifu gations at 14 000 g and t reatment with a high s alt s olution ( 0.8 M sodium acetate, 1.2 M NaCl) before precipitation with isopropyl alcohol/ethanol (20% isopropyl alcohol and 70% ethanol). Poly(A + ) RNA was purified from the t otal amount of RNA using an Oligotex Suspension (Qiagen, Hilden, Germany), according to the manufacturer’s instructions. Design of primers A c ollection of degenerate oligonucleotide primers (CKX01, 5¢-GAYTTYGGXAAYATHAC-3¢;CKX02,5¢-AADAT RTCYTGXCCXGG-3¢; CKX03, 5 ¢-TTXARCCAXGGR TGXGG-3¢;CKX04,5¢-CCXC AYCCXTGGYTXAA-3¢; and C KX0 5 5 ¢-TRXARRTARTCXGTCCA-3¢)covering the entire assumed sequence was synthesized on the basis of h ighly conserved areas between the sequences of maize Z mCKX1 (AF 044603) and Arabidopsis AtCKX2 (AC005917) genes. The previously determined N-terminal amino acid sequence of w heat CKX [11] was not suitable for use in the primer design. Three gene-specific primers (CKX07, 5¢-CGGGGCAC GAGCACGTTGAGCCAGGGAT-3¢; CKX08, 5¢-AAG ATGTCTTGGCCCGGGGAG-3¢;andCKX09,5¢-GTT CTGCGCCTCCAGCCGCC-3¢) were designed for ampli- fication o f a 5¢-end region of barley HvCKX1,wheat TaCKX1 genes, and one antisense primer (CKX06, 5¢-ATCCCTGGCTCAACGTGCTCGTGCCCCG-3 ¢)for amplification of the 3¢-end region in RACE-PCR. Three specific primers (two s ense: CKX11, 5 ¢-GCAA TGGACTTCGGCAACCTCTCTAGCTTC-3¢;CKX14, 5¢-GATTGTCATCAGAATGGAATCCCTTCGGAG-3¢; and one antisense: CKX13, 5¢-GCACCCTATC CAAGA ACTCAATGTAAGTGA-3¢) were designed to amplify fragments of HvCKX2 and HvCKX3 genes a ccording to sequences from the barley cDNA library of top adult leaves (AV835311, AV836048) that show particular homology with the maize ZmCKX1 gene. A pair of primers was designed to amplify part of the gene predicted as HvCKX7 on the basis of the c oding region of the g enomic DNA fragment (AJ234763; CKX 19, 5¢-GACATGCTCACGCA CCAAGACCCCGGA-3¢;CKX20,5¢-TGCCCTGGTGA TGATGCCAAACTGGCC-3¢) s howing h igh homology with other CKX genes. To amplify full-length genes, and to distinguish between HvCKX2 and HvCKX3 genes, one sense pri- mer (CKX25, 5 ¢-CAGTGAACCACTACCCTGCTACA CG-3¢) a nd two antisense primers (HvCKX2 specific, CKX23, 5¢-GCTGATCTTCATTGATCTCAGTGCT-3¢; HvCKX3 specific,CKX24,5¢-CATATTGCTAACCAC GTGACATATG-3¢), covering the dissimilar region, were designed. RT-PCR The first-strand cDNA was reverse transcribed from 0.1 to 1.0 lg of poly(A + ) RNA using a reverse transcrip tase RAV-2 ( Takara Shuzo Co., Shiga, J apan) and oligo(dT) primer (Promega, M adison, WI, U SA). H ot-start touch- down PCR [18] was carried out using 45 cycles of amplification, with the annealing temperature of the fi rst five cycles scaled down 1 °C per cycle. The usual cycle consisted of melting at 94 °C for 30 s, annealing at 53– 49 °C for 30 s and extension at 72 °C for 1 min. The PCR mixture was prepared using a Takara Taq polymerase, as recommended by t he manufactu rer, with aliquots of the RT reaction, diluted 1 : 10 (v/v), as a template. RACE-PCR Different RACE-PCR techniques were used to amplify the full-length cDNA strands of barley CKX genes. Positive results were obtained by using a M arathon TM cDNA Amplification Kit (Clontech Laboratories, Palo Alto, CA, USA). The 0.5 lg of isolated poly(A + )RNAwastreated exactly as advised by the manufacturer to obtain an adaptor-ligated ds cDNA library. The final 3¢-and5¢-end products of HvCKX2 and HvCKX3 gen es were obtained after 35 cycles of a mplification in the G eneAmpÒ High Fidelity PCR System (Applied Biosystems, Foster City, CA, USA) using primers CKX13 a nd CKX14. Full-length cDNA was constructed by PCR with the template from the ds cDNA library using s pecific primers from 5¢-and 3¢-product termini (HvCKX2r, 5¢-GCTGATCTTCATTG ATCTCAGTGCT-3¢; HvCKX3r, 5¢-CATATTGCTAAC CACGTGACATATG-3¢; CKX23f, 5¢-CAGTGAACCAC TACCCTGCTACACG-3¢). The annealing t emperatures and the concentration of dimethylsulfoxide (4–10%) in the PCRmixturewerealteredtopermitamplificationofthe cDNA ends of barley and wheat CKX genes from poly(A + ) RNA treated using two other RACE-PCR kits [the Ó FEBS 2004 Cytokinin oxidase/dehydrogenase genes in cereals (Eur. J. Biochem. 271) 3991 SMART TM RACE cDNA A mplification Kit (Clontech); and F irstChoice TM RLM-RACE Kit (Ambion, Austin, TX, USA)]. Amplified fragments were e xcised from polyacrylamide gels and elute d by water for 1 day at 3 7 °C. DNA was subsequently recovered by e thanol precipitation and ligated into a pDRIVE vector (Qiagen). Transformations of Escherichia coli TOP10F¢ competent cells were made by electroporation (1.8 kV, 5 ms). Positive transformants were selected by a b-galactosidase b lue/white screen ing t est. Inserted DNA was completely sequenced on both strands after amplification with internal or universal vector primers using a BigDye-terminator C ycle sequencing kit (Applied Biosystems) and an ABI P RISM 310 DNA sequencer (Applied Biosystems). Search and analysis for novel gene sequences DNA sequences encoding putative CKX proteins in cereals were se arched using a WU - BLAST 2.0 program [19] in the expressed sequence tag (EST) clone database of the Institute for Genomic Research (TIGR: http://tigrblast.tigr.org/tgi/). All Arabidopsis CKX protein sequences [14] were searched, one by one, against EST database subsets f or wheat and barley using the BLOSUM 62 comparative matrix. The search produced gene indices that were constructed b y assembling related ESTs after filtering for possible sequence contam- inants. T he resulting tentative consensus sequence was numbered and listed by relevant GenBank accession numbers representing the most overlapping sequences. Alignment of all sequences was performed with BIOEDIT software [20] using the CLUSTAL W multiple sequence alignment program. Construction of recombinant DNA for transformation and expression A10lL aliquot of a heat-treated (7 min, 100 °C) commer- cial barley genomic library (partial Sau3AI DNA digest cloned into the Lambda FIX I I v ector; Stratagene, L a J olla, CA, USA) was used as a template to amplify genomic sequences of HvCKX genes with HvCKX2r, HvCKX3r, and HvCKX23f primers. Amplified DNA was cloned into the pDRIVE v ector and s equenced. The same primers, with Asp718 and XbaI overhangs, were used to reamplify both genes u sing PCR w ith Pwo DNA P olymerase (Roche Applied S cience, Mannheim, Germany) for direct sense subcloning into a binary pBINHygTx vector downstream of the cauliflower mosaic virus 35S promoter [21]. Full-length cDNAs w ere subcloned into t he pYES2 (Invitrogen, Groningen, the Netherlands) and pDR197 binary vectors, with constitutive or inducible expression, respectively. The pDR197 plasmid was constructed from pDR195 [22] by introducing an additional cloning site (donated by D. Rentsch, ZMBP, University of Tu ¨ bingen, Tu ¨ bingen, Germany). Cells of Saccharomyces cerevisae strain 23344c ura – were transformed by electroporation [23], a nd positive transformants were selected on the b asis of the acquired u racil autotrophy. C KX activity was measured in the media and c ell lysates within 48 h of growth, or within 48 h after induction with galactose when an inducible system was used. Plant transformation Agrobacterium tumefaciens strain GUS3101, harboring the binary vector pBINHygTx with different tran sgenes, was used to transform the A. thaliana ecotype Col0 via vacuum infiltration [24]. A standard protocol [25], using leaf discs o f 8-week-old Nicotiana t abacum L. cv. Samsun NN p lants, was employed to generate transgenic tobacco plants. The selection of all transformants was performed b y adding hygromycin (15 mgÆL )1 ) to the selection and rooting medium. Transformed Arabidopsis plants were grown in a green- house until seed p roduction. T1 progeny s eeds of Arabi- dopsis transformants w ere surface sterilized and germinated on Murashige–Skoog medium (MS -medium) [26] in a controlled-environment chamber. Resistant seedlings were transferred to soil and placed in the greenhouse. Immediately a fter transfo rmation, tobacco leaf discs were placed on MS-medium supplemented with selection anti- biotics and an appropriate growth regulator ratio fo r shoot regeneration (0.7 mgÆL )1 of benzylaminopurine, 0.1 mgÆL )1 of b-naphthoxyacetic acid). After 2 days, the discs were transferred to the same medium supplemented with clafo- ram (0.5 mgÆL )1 ; Ratiopharm, Ulm, Germany), to inhibit Agrobacterium growth. Developing shoots were transferred to MS-medium (without growth regulators) for root induction. Young plants with several leaves w ere then transferred to the soil and grown in the greenhouse under the conditions described above. CKX activity assay Plant samples for a ctivity measurements were c ut into pieces, powdered with liquid nitroge n using a hand mortar, and extracted with a 1.5-fold excess ( v/w) of 0.2 M Tris/HCl buffer, pH 8.0, containing 1 m M phenylmethanesulfonyl fluoride a nd 1% Triton X-100. Cell debris was removed by centrifugation at 12 000 g for 1 0 min. The extract was loaded onto a Sephadex G-25 (50 · 2.5 cm) column equilibrated with 0.1 M Tris/HCl, pH 8.0, to r emove t he low m olecular mass fraction. The p rotein fraction was then concentrated to a minimum volume by ultrafiltration and used to assay CKX activity. The assay was performed according to a method described previously [27]. Samples were incubated i n a reaction mixture (total volume 0.6 mL in an Eppendorf tube)of100m M reaction buffer, 0.5 m M electron accep- tor [2,6-dichloroindophenol or 2,3-dimethoxy-5-methyl- 1,4-benzoquinone (Q 0 )] and 0.5 m M substrate, for 0.5–12 h at 37 °C. The following buffers (and pH ranges) were used for determining the pH profile: Tris/HCl buffer (pH 7.5–9.5), imidazole/HCl buffer (pH 6.0–7.0), Mes/ NaOH buffer (pH 5.0–5.5), a nd Na 2 HPO 4 /citric acid buffer (pH 3.0–4.5). For determination of specific activities, the protein content of the samples was assayed according to Bradford [28], with BSA as the standard. Extraction and analysis of cytokinins Two grams of frozen plant material (barley kernels, 7- and 14-day-old barley seedlings) was ground in liquid nitrogen 3992 P. Galuszka et al.(Eur. J. Biochem. 271) Ó FEBS 2004 and extracted in 20 mL of 70% ethanol containing diethyldithiocarbamate (400 lgÆg )1 of tissue) for 3 h at 4 °C. After centrifugation ( 20 min, 14 000 g), the pellet was re-extracted for 1 h in the same extraction mixture. The supernatants were combined and applied to a C 18 cartridge (Waters, Milford, MA, USA), prewashed with 80% meth- anol to retain pigments. T he pass-through f raction was collected and combined with a second fraction obtained by elution with 8 mL of 80% m ethanol. T he resulti ng sample containing cytokinins was dried on a vacuum rotary evaporator. Cytokinins were then separated by reverse-phase HPLC, and individual HPLC fractions were analyzed b y ELISA, according to a previously described protocol [29]. Results Isolation of HvCKX genes RT-PCR with degenerate primers designed on the basis o f two conserved motifs fo und among CKX proteins corres- ponding to amino acid sequences PHPWLN and PGQdIF, starting at positions 389 and 528 of the ZmCKX1 protein, revealed a 413 bp 3¢-end fragment of a potential barley CKX gene. The gene transcript was most abundant in the poly A + RNA fraction isolated f rom mature barley s eeds. Thus, the putative gene was named HvCKX1 (AF362472; Hordeum vulgare cyto kinin o xidase/dehydrogenase). A fragment of the same length was also isolated from the poly(A + ) R NA of mature wheat grains and was named TaCKX1 (AF362471; Triticum aestivum cytokinin oxidase/ dehydrogenase). Attempts to am plify the 5¢ cD NA end sequence by different RACE-PCR techniques did not yield any product for e ither of the genes. This failure may have occurred for several r easons, such as decreased quality of the isolated poly(A + ) RNA owing to starch contamination, an ampli- fied GC-rich sequence, or possibly the short half-life o f the target CKX transcripts a nd their rapid degradation from the 5¢-end. In addition to this PCR-based strategy, a GenBank database search revealed several barley and wheat E STs displaying homology to the Arabidopsis CKX gene family. Sets of gene-specific primers were designed to amplify the 3¢ and 5¢ cDNA ends of potential genes using the Marathon RACE-PCR kit (Clontech Laboratories). cDNA libraries generated f rom different barley tissues were used as templates for amplification. Two 3 ¢-RACE and two 5 ¢-RACE r eaction products of a similar size were obtained when o verlapping primers c orresponding to EST-AV835311 (a barley cDNA library fra gment gener- ated from top adult leaves) were used for a mplification. Both RACE products were cloned. Sequence analyses o f several clones revealed the presence of two nearly identical gene sequences (94% ho mology between coding regions at the nucleotide level). Full-length gene sequences were recovered from independently amplified PCRs with prim- ers flanking the predicted ORF regions where the reverse primer was designed on the basis of dissimilarity at the 3¢-end of the nonco ding region. The n ew gene of the 1578 bp coding sequence, fully corresponding to the above mentioned E ST, was designated HvCKX2 (A F540382), and i ts 1560 bp close homologue was named HvCKX 3 (AY209184). Wheat and barley CKX ESTs High homology between cereal gene fragments (HvCKX1 and TaCKX1 share 94% identit y on the 130 amino acid fragment that includes t he C-terminal region) may indicate the s ame e volutionary origin and possibly similar functions of both predicted genes. Both fragments show the highest degree of homology to Z mCKX1 (76%) and AtCKX2 (49%) p roteins, CKX family members be longing to an evolutionary group with a predicted secretory pathway targeting. The HvCKX2 gene encodes a protein of 526 amino acids with a predicted molecular mass of 58.8 kDa and a predicted pI value of 6.3. There is a very high identity between the HvCKX2 an d t he HvCKX3 gene products (92% at the amino acid level, Fig. 1). The latter is shorter (58.1 kDa) with one in-frame deletion within the sequence and its predicted pI v alue is shifted t o 7 .1. Both gene sequences contain an FAD-binding motif and other conserved regions typical of t he CK X gene family. An N-terminal signal peptide for targeting to t he secretory pathway was predicted b y the cellular l ocalization program, TARGETP [30], for both barley genes. H owever, p redicted results were classified as medium-reliable using the IPSORT program [31], t he HvCKX3 protein classified as a mito- chondrial protein. Encoded CKX proteins are pred icted to be glycosylated at five potential N-glycosylation sites (calculated b y N etNGly; http://www.cbs.dtu.dk/services/ NetNGlyc/) distributed alon g t he entire amino acid sequence. The genomic structure of HvCKX2 was determined by PCR using gene-specific primers flanking the cDNA and a barley genomic library cloned i nto bacteriophage k as a template. Comparison of the gen omic DNA sequence and the cDNA sequence s howed the presence of four small introns, which corresponds well to the evolutionary conserved intron/exon pattern of most higher plant CKX genes [32]. A search for novel CKX genes in wheat and barley DNA databases revealed 24 EST clones showing significant homology to some members of the CKX gene family. Correct ORFs of partial s equences were compiled in an alignment and numbered according to the homology of the 11 ric e gene family members [14]. For translated protein sequences of the genes and gene f ragments, s ee Fig. 1. Sequences without mutual overlapping regions showing considerable homology t o only one rice template (see Table 1 ) were assigned the same number. The compilation shown in T able 1 provided e vidence f or at least four additional barley (HvCKX4 to HvCKX 7) and seven wheat (TaCK X2 to TaCKX8) gene homologues. Expression of CKX genes during barley plant development To examine the expression of CKX genes in barley plants, a series of RT-PCR experiments were carried out using poly(A + ) RNA prepared from representative plant organs during d evelopment, including roots, leaves, and kernels. As Ó FEBS 2004 Cytokinin oxidase/dehydrogenase genes in cereals (Eur. J. Biochem. 271) 3993 Fig. 1. Alignment of b arley and wheat cytokinin oxidase/dehydrogenase gene families compiled f rom TIGR EST clone databases. Amino acid residues conserved in more than half of the protein fragments a re shown in white o n a b lack b ackground. Putative con s ensus s eque nces fo r N -glyco sylation sit es o f H vCKX2 a nd HvCKX3 proteins are shaded grey. Signal peptides predicted by the TARGETP program [30] for both full-length genes are underlined. For detailed identification of gene indices see Table 1. 3994 P. Galuszka et al.(Eur. J. Biochem. 271) Ó FEBS 2004 shown in Fig. 2, t ranscripts of HvCKX1 were found in all organs tested, such as mature kernels, roots and different developmental stages of leaves. HvCKX2 transcripts were detected in the leaves of 7-day-old seedlings, and the signal was also observed in k ernels and roots. Interestingly, the expression of HvCKX3 transcripts was only observed in mature kernels and the leaves of young seedlings. Import- antly, the presence of t he HvCKX3 gene was not detected in the commercial b arley genomic library. H owever, no signal was detected when p rimers designed for the amplification of the coding sequence o f genomic D NA fragment (AJ234763, HvCKX7) were used for RT-PCR (data not shown). An overview of cDNA lifetimes of in silico-derived genes suggests that cereal CKX enzymes a re also expressed i n additional tissues. Partially characterized novel barley genes HvCKX4 to HvCKX6 were found to be expressed in leaves, while the w heat genes w ere found in different tissues. Similarly, like HvCKX1, TaCKX1 is also expresse d in both mature grains and developed seedlings. Transcripts of four TaCKX ge nes (TaCKX2, TaCKX4, TaCKX5 and TaCKX6) were observed in an mRNA pool collected after the infection of leaves and spikes by the cereal pathogens Fusarium and Puccinia.LikeTaCKX6, TaCKX2 is also expressed in grains after pollination. Interestingly, a frag- ment of the gene c oding for t he TaCKX7 protein was present in a cDNA library generated from the mRNA of developing roots and also from spikelets at early flowering, where the fragment of TaCKX8 was also found. However, these descriptions are limited by the fact that only data presented in incomplete databases were used. Transformants overexpressing HvCKX genes To investigate whether the cloned genes code for active CKX enzymes, we overexpressed HvCKX2 and Hv CKX3 in Arabidopsis and tobacco. The cDNAs and, for HvCKX2, also the genomic clone, were placed under the control of a constitutively expressed 35S promoter. At least 30 inde- pendent tobacco transformants were regenerated for each construct. Several regenerated plants transformed with the genomic version of HvCKX2 showed a very strong pheno- type that was consistent with a cytokinin deficiency [15] Fig. 3. These plants had significantly shorter internodes, leading to a dwarf growth habit. On the contrary, the root system was noticeably e nlarged in comparison with wild- type plants, similarly to t he transgenic tobacco plants overexpressing the Arabidopsis AtCKX1 and AtCKX3 genes [15]. All of these p lants were sterile and d ied without producing seeds. Other regenerated transformants over- expressing gHvCKX2, a nd also most of the HvCKX 2 cDNA overexpressers, showed a m ilder phenotype. These plants were also characterized by shorter shoots, narrow leaves Table 1. Cytokinin oxidase/dehydrogenase (CKX) g ene families in c ereals. Sequences wit hout mutual ove rlapping regions, showin g considerable homology to only one rice template, are marked by the s ame number but with a different lowercase letter. Gene NCBI accession Closest rice homologue Homology to rice protein Tissue description HvCKX1 AF362472 OsCKX1 74% Grains BQ462284 Callus HvCKX2 AF540382 OsCKX7 84% 7-day-old leaves AV835311 Top three adult leaves HvCKX3 AY209184 OsCKX7 84% 7-day-old leaves HvCKX4a BJ479455 OsCKX4 89% Top three adult leaves HvCKX4b BJ479606 OsCKX4 94% Top three adult leaves HvCKX5a BF264028 OsCKX5 72% Seedling green leaves HvCKX5b CB877904 OsCKX5 77% Epidermis (seedlings) HvCKX6 CA031729 OsCKX6 75% Seedling apex HvCKX7 AJ234763 OsCKX3 84% Genomic DNA TaCKX1 AF362471 OsCKX1 70% Grains AL825717 Drought-stressed seedlings AL822297 Drought-stressed seedlings TaCKX2a BG905097 OsCKX6 66% Puccinia-infected leaf TaCKX2b CD932650 OsCKX6 85% Grains TaCKX3 BE404516 OsCKX6 72% Seedlings TaCKX4 BM138354 OsCKX4 89% Fusarium-infected spikes BJ306089 Spikelet at late flowering TaCKX5a BM137409 OsCKX5 87% Fusarium-infected spikes TaCKX5b BQ161648 OsCKX5 79% Fusarium-infected spikes TaCKX6a CA705202 OsCKX2 75% Developing kernels BQ903062 Fusarium-infected spikes BQ235927 Developing seeds TaCKX6b BQ238832 OsCKX2 52% Developing seeds TaCKX7a CA603337 OsCKX3 50% 7-day-old roots TaCKX7b BJ316444 OsCKX3 92% Spikelet at early flowering TaCKX8 BJ322935 OsCKX3 77% Spikelet at early flowering Ó FEBS 2004 Cytokinin oxidase/dehydrogenase genes in cereals (Eur. J. Biochem. 271) 3995 and a more branched and higher root mass than the wild type. Interestingly, T1 primary transformants overexpress- ing the HvCKX3 gene did not show any alteration of the phenotype. However, RT-PCR with specific primers f or the HvCKX3 gene revealed the presence of HvCKX3 transcripts in tobacco leaves (data not shown). Increased CKX activity was detected in the leaves of several selected transgenic plants. As expected, the activity was elevated 10- to 50-fold in gHvCKX2 transformants (Fig. 4A) with a strong phe- notype. Only a t wo- to fourfold increase was found in plants expressing the cDNA o f the same gene. In the case of HvCKX3 overexpressers, no increase in activity was found. The same three constructs of HvCKX genes in t he binary vector, pBINHygTx, were used to transform Arabidopsis plants via vacuum infiltration. Regeneration of fertile Arabidopsis transformants w as successful only from the seed progeny collected from plants transformed with constructs containing HvCKX cDNAs. In contrast, the growth of gHvCKX2 transformants was charact erized b y an enhanced root system and very slow s hoot development. All seedlings had died by t he formation of t he third pair o f rosette leaves,  3–4 weeks after germination. Thus, several Arabidopsis plants transformed with a construct carrying HvCKX2 cDNA showed sim ilar phenotypical a lterations to those recently d escribed for s trong Arabidopsis expressers of 35S:AtCKX1 and 35S:AtCKX3 [16]. Plants w ere distinctive in having delayed formation of rose tte le aves, sm aller leaf size, and delayed onset of flowering with a reduced number of flowers. After flowering, approximately half of the plants did not produce siliques, or produced only one or two siliques with a very small amount of seeds and afterwards died. CKX activity and cytokinin content during barley plant development The CKX activity was monitored in barley seedlings and young plants. The specific a ctivity was highest in the extracts of coleoptiles collected 1 day after g ermination and declined continuously thereafter, reaching about 10% of the initial activity by day 30. A t wofold increase in the enzyme activity was observed around day 9 of barley growth (Fig. 2D). About 95% of the total activity in seedlings was located in the roots, while the activity in the leaves increased 7 days after germination to only slightly above the detection limit of the assay method. The content of endogenous cytokinins with unsaturated side-chains, including bases, r ibosides, nucleotides, a nd N- and O-glucosides, was measured in three developmental stages, i.e. grains, and 7- and 14-day-old barley seedlings. The measured v alues are summarized in Table 2. The total cytokinin content in the grains was approximately threefold lower than in young seedlings. The increase was mainly observed in the content of free bases and riboside types of cytokinins, which are the preferred substrates of CKX. The level o f nucleotides remained nearly constant throughout the entire period. A significant i ncrease was also visible in the content of zeatin O-glucoside during seedling Fig. 2. Expression patterns of HvCKX1, HvCKX2 and HvCKX3 genes during plant development. c DNA aliquots corresponding to 100 ng of mRNA were used as templates for PCR with gene-specific primers. Control reactions were se t up with commer cial barle y genomic and cDNA libraries to distinguish between cDNA and genomic gene fragments. To eliminate reciprocal cross-reactivity between primers, plasmids with other cloned genes were used as templates (lane cross- reactivity). No template reaction contained water instead of mRNA. (A) An HvCKX1 gene cDNA fragment with a predicted size of 332 bp. (B) HvCKX2 gene cDNA with a pre- dicted size of 1830 bp. (C) HvCKX3 gene cDNA with a predicted size of 1740 bp. (D) Time-dependence of the total specific cytokinin oxidase/dehydrogenase (CKX) activity (j) and protein content (d)inthe whole developing barley s eedlings. Inset graph shows distribution of the CKX a ctivity between shoots and roots of developing seedlings. The activity was determined with tissue extracts in imidazole/HCl buffer, pH 6.5, containing 5 m M CuCl 2 and isopentenyladenosine as a substrate. All valu es represent mean values of data obtained from two parallel extractions, e ach measured in at least two replications. 3996 P. Galuszka et al.(Eur. J. Biochem. 271) Ó FEBS 2004 development. There were no major differences in the cytokinin content of 7- and 14-day-old plants. pH optimum and substrate specificity of barley CKX The effect of pH on the activity of recombinant HvCKX2 and CKXs from grain, root and leaf extracts of barley w as measured under standard assay conditions across the p H range from 3.0 to 9.5, with Q 0 intheacidicrangeand 2,6-dichloroindophenol in the basic range as electron acceptors (Fig. 5). Overlapping pH ranges were measured in two buffer systems to exclude salt effects. These varied by only up to 5% of the total value. Protein extract from tobacco with a strong phenotype overexpressing gHvCKX2 was u sed a s a source of the recombinant protein. The same pH-dependence experiment was carried out with the extract of wild-type tobacco to eliminate the contribution of naturally present t obacco CKX t o the recombinant activity. Activity found within wild-type tobacco was more than 20-fold lower than activity found in the extract of gHvCKX2-expressing plants. Surprisin gly, the maximum value of HvCKX2 activity with isopentenyl adenosine was observed a t pH 4.5 and then the activity slowly declined through neutral to alkaline p H. A s imilar activity profile was observed when i sopentenyl adenine was used as the substrate. This behavior contrasts with the previously described pH-dependence o f C KX enzymes [8], but sup- ports new results on the subcellular t argeting of two AtCKX-green fluorescence protein fused proteins to the vacuoles [16], where the p H generally ranges from 3.0 to 5.0. This contention emphasizes the fact that one of the enzymes, AtCKX1, is the closest homologue to the HvCKX2 enzyme. At low pH, the turnover of cytokinin ribosides is significantly higher than that of free bases (Fig. 4). T his i s in agreement with the po ssible existence of vacuolar-targeted CKX [16] and the observation of glycosylated forms of cytokinins occurring in acidic content of lytic vacuoles [33]. With barley grain, root and leaf extracts, the pH profiles varied with the type of tissue from which the extract was prepared. In this case, the total activity is, however, contributed by all CKX isoenzymes expressed in the particular tissue. CKX activity from grain extract showed two m axima, one at pH 4.5 and the other, more significant one at pH 7–7.5, while the pH p rofile of leaf enzymes more or less corresponds to the HvCKX2 p rofile. T his may indicate a predominant expression of HvCKX2 or a similar type of CKX in barley leaves and the expression of other CKX forms having an optimum at pH 7.5 in g rain s and roots. These conclusions ar e i n agreement with the RT-PCR expression pattern of two evolutionarily distant HvCKX1 and HvCKX2 genes studied in this work. The study of substrate specificity agrees with previously published data [ 11]. Cytokinins with isoprenoid side-chains are the preferred substrates for all tested enzyme samples. Isopentenyl adenosine is evidently the best substrate for HvCKX2 when measured under acidic conditions and w ith Q 0 as an electron acceptor. This preference for riboside is less significant at basic pH and with 2,6-dichloroindophenol as an acceptor, while CKX e nzymes generally prefer free bases w hen t he pH of a reaction mixture is neutral o r shifted to the alkaline r egion [11,13]. Riboside forms of cytokinins were found to be degraded better under acidic conditions (Fig. 4 ). A newly described method for the detection of degrada- tion products of aromatic cytokinins [27] was used to test them as potential substrates for barley C KXs. A l ow turnover of kinetin and its riboside was detected with HvCKX2 a nd t he en zyme extract from grains. Activity w ith other aromatic cytokinins was probably under the threshold of method sensitivity for the quantities of enzyme used. Turnover of these substrates was d escribed for maize Fig. 3. Shoot and root phenotype of gHvCKX2-expressing tobacco plants. (A) Tobacco overexpressers with mild (gHvCKX2-M) and strong (gHvCKX2-S) phenotypes, and wild-type (WT) plants, a t t he flowering stage. (B) Comparison of the root systems of phenotypically mild transgenic tobacco plants w ith those of wild-type plants. Ó FEBS 2004 Cytokinin oxidase/dehydrogenase genes in cereals (Eur. J. Biochem. 271) 3997 recombinant CKX as being 200- to 1000-fold lower than that of isopentenyl adenine [12]. A newly estimated value of molar absorption coefficient for 4-(-4-hydroxyphenyl- imino)-3-methyl-2-buten-1-ol [26], the conjugated d egrada- tion product of zeatin-type cytokinins, results in a 4.5-fold increase in detected activities for these cytokinins than was previously assumed f or purified CK X from b arley grains [11]. Cleavage of cis-zeatin seems to be catalyzed only by some forms of CKX. Relatively high turnover rates were detected only with enzymes present in grains and roots at pH 7.5. This zeatin isomer does not serve as a substrate for HvCKX2 (Fig. 4 ). Discussion In recent years, genomics and re verse genetics have devel- oped t ools and techniques that are crucial f or a better understanding of the a ctivity a nd function of cytokinins. Complete sequencing of t he Arabidopsis genome revealed the p resence o f a small g ene family encoding CKX. Detailed characterization o f six out of the seven AtCKX gene family members demonstrated differential subcellular compart- mentalization and their expression predominantly in mer- istematic t issues where t he main pool of cytokinins is located [16]. Characterization of the CKX gene families in o ther species seems to be m ore d ifficult to assess, especially in monocot crop plants with large genomes in which complete sequences are unlikely to b e obtained i n the near future. Large genomes of cereals, with a high content of repetitive DNA sequences and their polyploid nature, make t he study of gene or ganization m ore d ifficult. To date, one gene encoding a functional CKX enzyme has been described i n maize [9,10], and two othe r full-length homologues have recently been deposited in the gene database. In this work, we p resent the cloning of the first CKX genes of barley and their f unctional expression in tobacco Fig. 4. Substrate specificity of c ytokinin oxidase/ dehydrogenase (CKX) enzymes. ActivitywasmeasuredinanNa 2 HPO 4 /citric acid buffer, pH 4.5, with 2,3-dimethoxy-5-methyl-1,4-benzoquinone (Q 0 ) as the electron acceptor (dark b ars) and in Tris/HCl buffer, pH 7.5, with 2,6-dichloroindo- phenol as the electron acceptor (light bars). (A) Activity of the H vCKX2 enzyme extracted from transgenic tobacco leaves. (B) CKX activity extracted from mature barley grains. (C) CKX activity extracted from 7-day-old barley roots. (D) CKX activity extracted from 7-day-old barley leaves. Table 2. Endogenous isopr enoid cytokinin levels in Hordeum vulgare during early development. Values are expressed as pmol of cytokinin- equivalents per gram of fresh weight (FW). All values represent the mean of two independent measurements. Standard e rrors were in the range of 4–20%. Cytokinin compound Cytokinin content (pmolÆg )1 FW) Grain 7-day seedling 14-day seedling Isopentenyladenine 1.33 4.82 5.03 Isopentenyladenosine 3.41 4.66 4.22 Isopentenyladenosine monophosphate 0.11 0.15 0.09 Isopentenyladenine-9-glucoside 0.08 1.01 2.73 Zeatin 0.19 0.64 1.17 Zeatin riboside 0.59 2.13 2.65 Zeatin ribotide 0.21 0.27 0.29 Zeatin-9-glucoside 0.32 0.98 1.14 Zeatin O-glucoside 0.68 5.04 5.78 Zeatin riboside O-glucoside 0.43 0.31 0.83 Total 7.35 20.01 23.93 3998 P. Galuszka et al.(Eur. J. Biochem. 271) Ó FEBS 2004 and Arabidopsis plants. We describe two novel members of the CKX gene family with a typical FAD-binding domain and p redicted glycosylation sites. Surprisingly, Hv CKX cDNAs share 89% homology at the nucleotide level that leads to 37 changes in the protein sequence, and sequences noticeably differ only in the length of a 3¢-end noncoding sequence. This high homology may indicate a rather recent evolutionary duplication of the HvCKX2 and HvCKX3 genes. A similar duplication event probably took place in the r ice genome, w here two paralogs of the CKX gene with 88% homology lie on neighboring loci on chromosome 2 (AP004996) [14]. T hree recently annotated Zea mays mRNAs for CK X also show features of a r ecent duplication event. While two almost-identical isolated mRNAs (Z mCKX2: AJ606943, AJ606944) are obviously allelic versions of the same g ene, the sequence annotated as ZmCKX3 (AJ606942) is probably their close paralog (sharing 93% homology at the amino acid level). Prelim- inary comparative mapping of selected gene regions in barley, wheat and maize has shown that gene duplication plays a significant role in the evolution of gene families within large cereal genomes [34]. However, it is still questionable whether all of these paralogs encode functional proteins. Whereas transformation of the HvCKX2 gene into the tobacco genome unambiguously elevates the level of the endogenous CKX activity and causes phenotypic altera- tions typical f or cytokinin-deficient plants, no enhancement of the CKX level and no cytokinin-deficiency syndrome were found when the HvCKX3 paralog was overexpressed. Following heterologous expression of the HvCKX3 gene in the yeast S. cerevisiae, active CKX was not demonstrably present either in yeast media or in the cell extract (data not shown). E ffectiveness in expression of the genomic and cDNA versions of the HvCKX2 transgene, respectively, in tobacco and Arabidopsis plants was significantly different. While transformation of model p lants by the genomic version of the transgene led to strong cytokinin-deficiency phenotypes, the cDNA overexpresser showed just mild phenotypic alterations with only a moderately increased CKX l evel. A similar phenomenon was observed w hen expressing genomic and cDNA versions of ZmCKX1 gene in tobacco (K. Bilyeu, personal communication). It has been demonstrated many times that incorporating i ntrons into transgenes has an enhancing effect on gene expression. This phenomenon was observed predominantly in GC-rich monocot genomes [ 35], but the m echanisms underlying the enhancement o f gene expression are not entirely c lear, especially when introducing monocot in trons into dicot plants [36]. The great number of ESTs in public databases helped us to assemble at least a partial picture of CK X gene families in Hordeum and Triticum species. Gene indices were construc- ted for a minimum of seven barley and eight wheat CKX genes, respectively. However, background noise was observed w ithin t he constructed consensus s equences, w hich could be attributed to the limited fidelity of the reverse transcription step of cDNA library construction and sequence artifacts caused by the biochemistry of sequencing reactions. In a ddition, wheat i s a hexaploid organism in which sequence diversity could be attributed to t he origin of genomes inherited from different ancestors. Therefore, a partial sequence o f s everal h ighly homologous ESTs did not Fig. 5. pH d epend ence of HvCKX ac tivity with 2,3-dimethoxy-5-methyl-1,4-benzoquinone (Q 0 )(j) and 2,6-dichloroindophenol (d)astheelectron acceptor . (A) Activity of HvCKX2 enzyme extracted from transgenic tobacco leaves. (B) Cytokinin oxidase/dehydrogenase (CKX) activity extracted from mature barley grains. (C) CKX activity extracted from 7-day-old barley roots. (D) CKX activity extracted from 7-day-old b arley leaves. See Materials and methods for details on the buffer and reaction mixtu re comp osition. Ó FEBS 2004 Cytokinin oxidase/dehydrogenase genes in cereals (Eur. J. Biochem. 271) 3999 [...]... 30-fold higher Km and two orders lower kcat/Km values for cis-zeatin than that estimated for isopentenyladenine This indicates that cis-zeatin has a much lower affinity for this enzyme than other isoprenoid cytokinins Progress made during recent years in the transformation of monocot plants, including barley [41], has promoted interest in the further investigation of barley and wheat CKX genes Characterization... Identification of genes encoding adenylate isopentenyltransferase, a cytokinin biosynthesis enzyme, in Arabidopsis thaliana J Biol Chem 276, 26405–26410 5 Martin, R.C., Mok, M.C., Habben, J.E & Mok, D.W.S (2001) A maize cytokinin gene encoding an O-glucosyltransferase specific to cis-zeatin Proc Natl Acad Sci USA 98, 5922–5926 6 Martin, R.C., Mok, M.C & Mok, D.W.S (1999) A gene encoding cytokinin enzyme zeatin O-xylosyltransferase... (2001) Molecular and biochemical characterization of a cytokinin oxidase from maize Plant Physiol 125, 378–386 ´ ´ ´ 14 Popelkova, H., Galuszka, P., Frebortova, J., Bilyeu, K.D & ´ Frebort, I (2004) Cytokinin dehydrogenase: characterization and structure homology modeling of the flavoprotein catabolizing plant hormones cytokinins In Recent Research Developments in Proteins, vol 2 (Pandalai, S.G., ed.),... HvCKX barley overexpressers, and especially the possibility of preparing knockout mutants, will help us to elucidate the precise function of cytokinin- degrading enzymes in these crop plants Genetic manipulation of CKX activity also holds the promise of improving agricultural traits, such as yield attributes or adaptation to environmental stress, in barley and other cereals Ó FEBS 2004 Cytokinin oxidase/dehydrogenase. .. Kerala, India 15 Werner, T., Motyka, V., Strnad, M & Schmulling, T (2001) ¨ Regulation of plant growth by cytokinin Proc Natl Acad Sci USA 98, 10487–10492 16 Werner, T., Motyka, V., Laucou, V., Smets, R., van Onckelen, H & Schmulling, T (2003) Cytokinin- deficient transgenic Arabi¨ dopsis plants show multiple developmental alterations indicating opposite functions of cytokinins in the regulation of shoot and. .. Different effects of intron nucleotide composition and secondary structure on premessenger-RNA splicing in monocot and dicot plants EMBO J 10, 2635–2644 37 Brugiere, N., Jiao, S.P., Hantke, S., Zinselmeier, C., Roessler, J.A., Niu, X.M., Jones, R.J & Habben, J.E (2003) Cytokinin oxidase gene expression in maize is localized to the vasculature, and is induced by cytokinins, abscisic acid, and abiotic stress... during the first few days of plant growth, to the residual levels in the roots, is in agreement with the Western blot analysis presented for maize tissues [13] The levels of cytokinins that are good substrates of CKX increase antagonistically with the enzyme level during the early days of the plant’s development This indicates that the CKXs detected in barley regulate the active cytokinin level principally... Kakimoto, T (2001) Identification of plant cytokinin biosynthetic enzymes as dimethylallyl diphosphate: ATP/ADP isopentenyltransferases Plant Cell Physiol 42, 677–685 3 Miyawaki, K., Matsumoto-Kitano, M & Kakimoto, T (2004) Expression of cytokinin biosynthetic isopentenyltransferase genes in Arabidopsis: tissue specificity and regulation by auxin, cytokinin, and nitrate Plant J 37, 128–138 4 Takei, K.,... converted to adenine and a corresponding aldehyde by CKX However, the degradation of aromatic cytokinins is not significantly affected by the cooperation of the enzyme with an electron acceptor The reaction rates of these cytokinins are still several hundred fold lower then those of isoprenoid ones [12] The barley enzymes studied also convert kinetin and its riboside with two-order lower velocity, and this... P., Werner, T., Schmulling, T & ¨ ˇ Pec, P (2002) Cytokinin oxidase/dehydrogenase assay: optimized procedures and applications Anal Biochem 306, 1–7 28 Bradford, M.M (1976) A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding Anal Biochem 72, 248– 254 ´ 29 Faiss, M., Zalubilova, J., Strnad, M & Schmulling, T (1997) ¨ Conditional . he CKXs detected in barley regulate the active cyto kinin level principally in developing grains, and that the biologically active cytokinins are probably. S chmu ¨ lling, T. (2003) Cytokinin- induced upregulation of cytokinin oxidase activity in tob acco includes changes in enzyme glycosylation and secretion.