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 CKXgene
Trang 1Cytokinin oxidase/dehydrogenase genes in barley and wheat
Cloning and heterologous expression
Petr Galuszka1, Jitka Fre´bortova´2, Toma´sˇ Werner3, Mamoru Yamada4, Miroslav Strnad2,
Thomas Schmu¨lling3and Ivo Fre´bort1
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;3Institute of Biology/Applied Genetics, Free University of Berlin, Germany;4Department of Biological Chemistry, Faculty of Agriculture, Yamaguchi University, Japan
The cloning of two novel genes that 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 genes 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
CKXgene family in barley and wheat The expression of
three selected barley genes was analyzed by RT-PCR and
found to 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 ribosides as substrates, which may indicate its vacuolar targeting Different substrate specifici-ties, and the pH profiles of cytokinin-degrading enzymes extracted from different barley tissues, are also presented Keywords: cereals; cloning; cytokinin oxidase/dehydro-genase; expression; gene family
Cytokinins were initially viewed as factors promoting cell
division and differentiation in plants Since then, however,
cytokinins have been shown to control other developmental
events, such as the growth of lateral buds, the release of
buds from apical dominance, leaf expansion, the delay
of senescence, the promotion of seed germination, and
chloroplast formation [1] Naturally occurring cytokinins
are mainly N6-substituted adenine derivatives that generally
contain an isoprenoid or aromatic side-chain Recently,
considerable progress has been made in elucidating the
regulation of cytokinin homeostasis during plant growth
and development New molecular biological techniques
have allowed for the identification and characterization of
genes encoding important enzymes participating in
cyto-kinin metabolic pathways Genetically engineered plants
that overexpress some of these genes were prepared as a tool
to study changes in physiological aspects caused 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 cytokinin-specific glycosylation enzymes with different substrate specificities, have been described [5–7] The principle of cytokinin catabolism has been studied for many years Enzymes capable of degrading cytokinins with unsaturated side-chains have been found in many plant tissues [8], but the details of their features and the mechanism of their action remained unknown for a long time owing to their very low content in plant tissues The ground-breaking cloning of the cytokinin oxidase maize gene ZmCKX1 [9,10] opened up the possibility for more detailed study of cytokinin degradation, both at the molecular and at the biochemical levels The recombinant maize enzyme is a glycoprotein containing a covalently bound FAD The isoprenoid side-chain of the cytokinin molecule is most efficiently cleaved in the 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 substrates [12] Studies of reaction 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, and a detailed phenotypic characterization was
Correspondence to P Galuszka, Division of Molecular Biology,
Department of Biochemistry, Faculty of Science, Palacky´ University,
Sˇlechtitelu˚ 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 oxidase/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)
Trang 2subsequently carried out All transformants displayed
reduced cytokinin content and showed distinct
develop-mental alterations in the shoot and root [15,16], most of
them in accordance with previous assumptions on
cytokinin function Two of the AtCKX proteins were
found to be targeted to the vacuoles, while another
accumulated in the reticulate structure, which may
indicate its final extracellular localization [16] One
additional CKX gene has been identified in a Dendrobium
orchid, and similar aspects of its 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 first two CKX genes of barley and
demonstrate functionality for one of them in transgenic
tobacco and 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 soil and grown in a greenhouse with
a 15 h/9 h day/night cycle at 21C
Isolation of poly(A+) RNA
All RNA was extracted from different plant tissues using
TRIZOL Reagent (Gibco BRL, Grand Island, NY, USA)
Polysaccharide contamination of the grain extract was
removed with two additional centrifugations at 14 000 g
and treatment with a high salt solution (0.8M sodium
acetate, 1.2M NaCl) before precipitation with isopropyl
alcohol/ethanol (20% isopropyl alcohol and 70% ethanol)
Poly(A+) RNA was purified from the total amount of
RNA using an Oligotex Suspension (Qiagen, Hilden,
Germany), according to the manufacturer’s instructions
Design of primers
A collection of degenerate oligonucleotide primers (CKX01,
5¢-GAYTTYGGXAAYATHAC-3¢; CKX02, 5¢-AADAT
RTCYTGXCCXGG-3¢; CKX03, 5¢-TTXARCCAXGGR
TGXGG-3¢; CKX04, 5¢-CCXCAYCCXTGGYTXAA-3¢;
and CKX05 5¢-TRXARRTARTCXGTCCA-3¢) covering
the entire assumed sequence was synthesized on the
basis of highly conserved areas between the sequences of
maize ZmCKX1 (AF044603) and Arabidopsis AtCKX2
(AC005917) genes The previously determined N-terminal
amino acid sequence of wheat 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¢; and CKX09, 5¢-GTT
CTGCGCCTCCAGCCGCC-3¢) were designed for
ampli-fication of 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 sense: CKX11, 5¢-GCAA TGGACTTCGGCAACCTCTCTAGCTTC-3¢; CKX14, 5¢-GATTGTCATCAGAATGGAATCCCTTCGGAG-3¢; and one antisense: CKX13, 5¢-GCACCCTATCCAAGA ACTCAATGTAAGTGA-3¢) were designed to amplify fragments of HvCKX2 and HvCKX3 genes according 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 coding region of the genomic DNA fragment (AJ234763; CKX19, 5¢-GACATGCTCACGCA CCAAGACCCCGGA-3¢; CKX20, 5¢-TGCCCTGGTGA TGATGCCAAACTGGCC-3¢) showing high 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¢) and 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 transcriptase RAV-2 (Takara Shuzo Co., Shiga, Japan) and oligo(dT) primer (Promega, Madison, WI, USA) Hot-start touch-down PCR [18] was carried out using 45 cycles of amplification, with the annealing temperature of the first five cycles scaled down 1C per cycle The usual cycle consisted of melting at 94C for 30 s, annealing at 53–
49C for 30 s and extension at 72 C for 1 min The PCR mixture was prepared using a Takara Taq polymerase, as recommended by the manufacturer, 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 MarathonTM cDNA Amplification Kit (Clontech Laboratories, Palo Alto, CA, USA) The 0.5 lg of isolated poly(A+) RNA was treated exactly as advised by the manufacturer to obtain an adaptor-ligated ds cDNA library The final 3¢- and 5¢-end products of HvCKX2 and HvCKX3 genes were obtained after 35 cycles of amplification in the GeneAmp High Fidelity PCR System (Applied Biosystems, Foster City, CA, USA) using primers CKX13 and CKX14 Full-length cDNA was constructed by PCR with the template from the
ds cDNA library using specific primers from 5¢- and 3¢-product termini (HvCKX2r, 5¢-GCTGATCTTCATTG ATCTCAGTGCT-3¢; HvCKX3r, 5¢-CATATTGCTAAC CACGTGACATATG-3¢; CKX23f, 5¢-CAGTGAACCAC TACCCTGCTACACG-3¢) The annealing temperatures and the concentration of dimethylsulfoxide (4–10%) in the PCR mixture were altered to permit amplification of the cDNA ends of barley and wheat CKX genes from poly(A+) RNA treated using two other RACE-PCR kits [the
Trang 3SMARTTM RACE cDNA Amplification Kit (Clontech);
and FirstChoiceTMRLM-RACE Kit (Ambion, Austin, TX,
USA)]
Amplified fragments were excised from polyacrylamide
gels and eluted by water for 1 day at 37C DNA was
subsequently recovered by ethanol 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 blue/white screening test
Inserted DNA was completely sequenced on both strands
after amplification with internal or universal vector primers
using a BigDye-terminator Cycle sequencing kit (Applied
Biosystems) and an ABI PRISM 310 DNA sequencer
(Applied Biosystems)
Search and analysis for novel gene sequences
DNA sequences encoding putative CKX proteins in cereals
were searched using a WU-BLAST2.0 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 for wheat and
barley using theBLOSUM62 comparative matrix The search
produced gene indices that were constructed by assembling
related ESTs after filtering for possible sequence
contam-inants The 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
A 10 lL aliquot of a heat-treated (7 min, 100C)
commer-cial barley genomic library (partial Sau3AI DNA digest
cloned into the Lambda FIX II vector; Stratagene, La Jolla,
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 vector and sequenced The same primers, with
Asp718 and XbaI overhangs, were used to reamplify both
genes using PCR with Pwo DNA Polymerase (Roche
Applied Science, Mannheim, Germany) for direct sense
subcloning into a binary pBINHygTx vector downstream of
the cauliflower mosaic virus 35S promoter [21]
Full-length cDNAs were subcloned into the 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], and positive transformants were selected on the basis of
the acquired uracil autotrophy CKX activity was measured
in the media and cell lysates within 48 h of growth, or within
48 h after induction with galactose when an inducible
system was used
Plant transformation Agrobacterium tumefaciensstrain GUS3101, harboring the binary vector pBINHygTx with different transgenes, was used to transform the A thaliana ecotype Col0 via vacuum infiltration [24] A standard protocol [25], using leaf discs of 8-week-old Nicotiana tabacum L cv Samsun NN plants, was employed to generate transgenic tobacco plants The selection of all transformants was performed by adding hygromycin (15 mgÆL)1) to the selection and rooting medium
Transformed Arabidopsis plants were grown in a green-house until seed production T1 progeny seeds of Arabi-dopsistransformants were 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 after transformation, tobacco leaf discs were placed on MS-medium supplemented with selection anti-biotics and an appropriate growth regulator ratio for shoot regeneration (0.7 mgÆL)1of 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 Agrobacteriumgrowth Developing shoots were transferred
to MS-medium (without growth regulators) for root induction Young plants with several leaves were then transferred to the soil and grown in the greenhouse under the conditions described above
CKX activity assay Plant samples for activity measurements were cut into pieces, powdered with liquid nitrogen using a hand mortar, and extracted with a 1.5-fold excess (v/w) of 0.2MTris/HCl buffer, pH 8.0, containing 1 mM phenylmethanesulfonyl fluoride and 1% Triton X-100 Cell debris was removed by centrifugation at 12 000 g for 10 min The extract was loaded onto a Sephadex G-25 (50· 2.5 cm) column equilibrated with 0.1M Tris/HCl, pH 8.0, to remove the low molecular mass fraction The protein 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 in a reaction mixture (total volume 0.6 mL in an Eppendorf tube) of 100 mM reaction buffer, 0.5 mM electron accep-tor [2,6-dichloroindophenol or 2,3-dimethoxy-5-methyl-1,4-benzoquinone (Q0)] and 0.5 mM substrate, for 0.5–12 h at 37C 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), and Na2HPO4/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
Trang 4and extracted in 20 mL of 70% ethanol containing
diethyldithiocarbamate (400 lgÆg)1 of tissue) for 3 h at
4C 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 C18cartridge
(Waters, Milford, MA, USA), prewashed with 80%
meth-anol to retain pigments The pass-through fraction was
collected and combined with a second fraction obtained
by elution with 8 mL of 80% methanol The resulting
sample containing cytokinins was dried on a vacuum
rotary evaporator Cytokinins were then separated by
reverse-phase HPLC, and individual HPLC fractions were
analyzed by ELISA, according to a previously described
protocol [29]
Results
Isolation ofHvCKX genes
RT-PCR with degenerate primers designed on the basis of
two conserved motifs found 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
CKXgene The gene transcript was most abundant in the
poly A+RNA fraction isolated from mature barley seeds
Thus, the putative gene was named HvCKX1 (AF362472;
Hordeum vulgare cytokinin oxidase/dehydrogenase) A
fragment of the same length was also isolated from the
poly(A+) RNA of mature wheat grains and was named
TaCKX1(AF362471; Triticum aestivum cytokinin oxidase/
dehydrogenase)
Attempts to amplify the 5¢ cDNA end sequence by
different RACE-PCR techniques did not yield any product
for either of the genes This failure may have occurred for
several reasons, 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 of the
target CKX transcripts and their rapid degradation from the
5¢-end
In addition to this PCR-based strategy, a GenBank
database search revealed several barley and wheat ESTs
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 from different barley tissues
were used as templates for amplification Two 3¢-RACE
and two 5¢-RACE reaction products of a similar size were
obtained when overlapping primers corresponding to
EST-AV835311 (a barley cDNA library fragment
gener-ated from top adult leaves) were used for amplification
Both RACE products were cloned Sequence analyses of
several clones revealed the presence of two nearly identical
gene sequences (94% homology 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 revprim-erse
primer was designed on the basis of dissimilarity at the
3¢-end of the noncoding region The new gene of the
1578 bp coding sequence, fully corresponding to the above
mentioned EST, was designated HvCKX2 (AF540382),
and its 1560 bp close homologue was named HvCKX3 (AY209184)
Wheat and barley CKX ESTs High homology between cereal gene fragments (HvCKX1 and TaCKX1 share 94% identity on the 130 amino acid fragment that includes the C-terminal region) may indicate the same evolutionary origin and possibly similar functions
of both predicted genes Both fragments show the highest degree of homology to ZmCKX1 (76%) and AtCKX2 (49%) proteins, CKX family members belonging 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 and the 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 value is shifted to 7.1 Both gene sequences contain an FAD-binding motif and other conserved regions typical of the CKX gene family An N-terminal signal peptide for targeting to the secretory pathway was predicted by the cellular localization program, TARGETP [30], for both barley genes However, predicted results were classified as medium-reliable using theIPSORT program [31], the HvCKX3 protein classified as a mito-chondrial protein Encoded CKX proteins are predicted to
be glycosylated at five potential N-glycosylation sites (calculated by NetNGly; http://www.cbs.dtu.dk/services/ NetNGlyc/) distributed along the 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 into bacteriophage k as a template Comparison of the genomic DNA sequence and the cDNA sequence showed 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 sequences were compiled in an alignment and numbered according to the homology of the
11 rice gene family members [14] For translated protein sequences of the genes and gene fragments, see Fig 1 Sequences without mutual overlapping regions showing considerable homology to only one rice template (see Table 1) were assigned the same number The compilation shown in Table 1 provided evidence for at least four additional barley (HvCKX4 to HvCKX7) and seven wheat (TaCKX2 to TaCKX8) gene homologues
Expression ofCKX 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 development, including roots, leaves, and kernels As
Trang 5Fig.
Trang 6shown in Fig 2, transcripts 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 kernels 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 the HvCKX3 gene was not detected in
the commercial barley genomic library However, no signal
was detected when primers designed for the amplification of
the coding sequence of genomic DNA 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 are also expressed in
additional tissues Partially characterized novel barley genes
HvCKX4to HvCKX6 were found to be expressed in leaves,
while the wheat genes were found in different tissues
Similarly, like HvCKX1, TaCKX1 is also expressed in both
mature grains and developed seedlings Transcripts of four
TaCKXgenes (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 Like TaCKX6, TaCKX2 is also
expressed in grains after pollination Interestingly, a
frag-ment of the gene coding for the 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 overexpressingHvCKX genes
To investigate whether the cloned genes code for active CKX enzymes, we overexpressed HvCKX2 and HvCKX3 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 enlarged in comparison with wild-type plants, similarly to the transgenic tobacco plants overexpressing the Arabidopsis AtCKX1 and AtCKX3 genes [15] All of these plants were sterile and died without producing seeds Other regenerated transformants over-expressing gHvCKX2, and also most of the HvCKX2 cDNA overexpressers, showed a milder phenotype These plants were also characterized by shorter shoots, narrow leaves
Table 1 Cytokinin oxidase/dehydrogenase (CKX) gene families in cereals Sequences without mutual overlapping regions, showing considerable homology to only one rice template, are marked by the same number but with a different lowercase letter.
Gene
NCBI accession
Closest rice homologue
Homology to rice protein Tissue description
Trang 7and 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 for the
HvCKX3gene 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 two- to fourfold increase was found in plants
expressing the cDNA of the same gene In the case of
HvCKX3overexpressers, no increase in activity was found
The same three constructs of HvCKX genes in the binary
vector, pBINHygTx, were used to transform Arabidopsis
plants via vacuum infiltration Regeneration of fertile
Arabidopsis transformants was successful only from the
seed progeny collected from plants transformed with
constructs containing HvCKX cDNAs In contrast, the
growth of gHvCKX2 transformants was characterized by an
enhanced root system and very slow shoot development All
seedlings had died by the formation of the third pair of
rosette leaves, 3–4 weeks after germination Thus, several
Arabidopsis plants transformed with a construct carrying
HvCKX2cDNA showed similar phenotypical alterations to
those recently described for strong Arabidopsis expressers of
35S:AtCKX1and 35S:AtCKX3 [16] Plants were distinctive
in having delayed formation of rosette leaves, smaller 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 activity was highest in the extracts
of coleoptiles collected 1 day after germination and declined continuously thereafter, reaching about 10% of the initial activity by day 30 A twofold 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, ribosides, nucleotides, and N- and O-glucosides, was measured in three developmental stages, i.e grains, and 7- and 14-day-old barley seedlings The measured values 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 of nucleotides remained nearly constant throughout the entire period A significant increase 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 cDNA aliquots corresponding to
100 ng of mRNA were used as templates for PCR with gene-specific primers Control reactions were set up with commercial barley 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) in the whole developing barley seedlings Inset graph shows distribution of the CKX activity 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 values represent mean values of data obtained from two parallel extractions, each measured in at least two replications.
Trang 8development 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 was
measured under standard assay conditions across the pH
range from 3.0 to 9.5, with Q in the acidic range and
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 used as 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 tobacco CKX to 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 Surprisingly, the maximum value of HvCKX2 activity with isopentenyl adenosine was observed at pH 4.5 and then the activity slowly declined through neutral to alkaline pH A similar activity profile was observed when isopentenyl adenine was used as the substrate This behavior contrasts with the previously described pH-dependence of CKX enzymes [8], but sup-ports new results on the subcellular targeting of two AtCKX-green fluorescence protein fused proteins to the vacuoles [16], where the pH 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) This is in agreement with the possible 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 maxima, one at pH 4.5 and the other, more significant one at pH 7–7.5, while the pH profile of leaf enzymes more
or less corresponds to the HvCKX2 profile This 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 grains and roots These conclusions are in 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 with
Q0as an electron acceptor This preference for riboside is less significant at basic pH and with 2,6-dichloroindophenol
as an acceptor, while CKX enzymes generally prefer free bases when the pH of a reaction mixture is neutral or shifted
to the alkaline region [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 CKXs A low turnover of kinetin and its riboside was detected with HvCKX2 and the enzyme extract from grains Activity with other aromatic cytokinins was probably under the threshold
of method sensitivity for the quantities of enzyme used Turnover of these substrates was described 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, at the
flowering stage (B) Comparison of the root systems of phenotypically
mild transgenic tobacco plants with those of wild-type plants.
Trang 9recombinant 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
degrada-tion product of zeatin-type cytokinins, results in a 4.5-fold
increase in detected activities for these cytokinins than was
previously assumed for purified CKX from barley 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 reverse genetics have devel-oped tools and techniques that are crucial for a better understanding of the activity and function of cytokinins Complete sequencing of the Arabidopsis genome revealed the presence of a small gene family encoding CKX Detailed characterization of six out of the seven AtCKX gene family members demonstrated differential subcellular compart-mentalization and their expression predominantly in mer-istematic tissues where the main pool of cytokinins is located [16] Characterization of the CKX gene families in other species seems to be more difficult to assess, especially in monocot crop plants with large genomes in which complete sequences are unlikely to be obtained in the near future Large genomes of cereals, with a high content of repetitive DNA sequences and their polyploid nature, make the study
of gene organization more difficult To date, one gene encoding a functional CKX enzyme has been described in maize [9,10], and two other full-length homologues have recently been deposited in the gene database
In this work, we present the cloning of the first CKX genes of barley and their functional expression in tobacco
Fig 4 Substrate specificity of cytokinin oxidase/dehydrogenase (CKX) enzymes Activity was measured in an Na 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 bars) and in Tris/HCl buffer, pH 7.5, with 2,6-dichloroindo-phenol as the electron acceptor (light bars) (A) Activity of the HvCKX2 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 isoprenoid 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 errors were in the
range of 4–20%.
Cytokinin compound
Cytokinin content (pmolÆg)1FW)
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 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
Trang 10and Arabidopsis plants We describe two novel members of
the CKX gene family with a typical FAD-binding domain
and predicted glycosylation sites Surprisingly, HvCKX
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 rice genome, where two paralogs of the CKX gene with
88% homology lie on neighboring loci on chromosome 2
(AP004996) [14] Three recently annotated Zea mays
mRNAs for CKX also show features of a recent duplication
event While two almost-identical isolated mRNAs
(ZmCKX2: AJ606943, AJ606944) are obviously allelic
versions of the same gene, 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 for 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) Effectiveness 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 plants 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 level A similar phenomenon was observed when expressing genomic and cDNA versions of ZmCKX1 gene
in tobacco (K Bilyeu, personal communication) It has been demonstrated many times that incorporating introns into transgenes has an enhancing effect on gene expression This phenomenon was observed predominantly in GC-rich monocot genomes [35], but the mechanisms underlying the enhancement of gene expression are not entirely clear, especially when introducing monocot introns into dicot plants [36]
The great number of ESTs in public databases helped us
to assemble at least a partial picture of CKX gene families in Hordeumand Triticum species Gene indices were construc-ted for a minimum of seven barley and eight wheat CKX genes, respectively However, background noise was observed within the constructed consensus sequences, which 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 addition, wheat is a hexaploid organism in which sequence diversity could be attributed to the origin of genomes inherited from different ancestors Therefore, a partial sequence of several highly homologous ESTs did not
Fig 5 pH dependence of HvCKX activity with 2,3-dimethoxy-5-methyl-1,4-benzoquinone (Q 0 ) (j) and 2,6-dichloroindophenol (d) as the electron 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 barley leaves See Materials and methods for details on the buffer and reaction mixture composition.