Tài liệu Báo cáo khoa học: Cytokinin oxidase/dehydrogenase genes in barley and wheat docx

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:
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 />(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: />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; />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
allow us to distinguish whether they belonged to allelic
variations or to two independent genes. Owing to the
relationship of cereal plants, we were able to assign the
closest rice CKX orthologs t o a ll predicted Hv CKX and
TaCKX genes (50–94% homology). H ence, the r ice genome
revealed 11 CK X homologues, some of which had more
than two corresponding copies in barley or wheat. Only
detection a nd characterization of full-length genes, and their
localization on cereal genomes, can give us a better
understanding of the character and evolutionary relation-
ships within this multiple gene family.
The expression patterns of the four CKX genes charac-
terized during b arley p lant development s uggest that the
expression of particular genes is organ-specific. Three of the
four genes studied are expressed in mature kernels, two of
them in the roots and leaves of young seedlings, whereas
expression of the fourth gene was not detected at all.
Expression studies of maize CKX homologues have resulted
in similar c onclusions. The ZmCKX1 gene was found to be
expressed in the vascular bundles of developing kernels,
roots, and coleoptiles [37]. Transcripts of the other two
maize genes were detected at two different time-points
during kernel development, but they were not tested for in
other organs [38]. These results indicate that d ifferent CKX
genes a re active mainly during k ernel d evelopment. Fur-
thermore, wide variations in the p H optimum of CKXs
suggest that subcellular compartmentalization can be also a
specific parameter a mong particular members of CKX gene

families. Localization of two AtCKX proteins in the
vacuoles and one in the endoplasmic reticulum, or possibly
in the extracellular space, has been recently described [16].
The rapid loss of CKX activity during the first few days
of plant growth, to the residual levels i n t he roots, is in
agreement w ith the Western blot a nalysis presented fo r
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 develop-
ment. This i ndicates t hat t he CKXs detected in barley
regulate the active cyto kinin level principally in developing
grains, and that the biologically active cytokinins are
probably deactivated by other m etabolic pathways, such
as glycosylation, in the later stages of development when
increased levels of both O- and N
9
-glucosides are observed.
The addition of the detergent Triton X-100 to the
extraction buffer noticeably increased the total extracted
CKX activity in all tested tissues. A relatively high
concentration (1%) of detergent effectively destroys c ellular
membranes, probably allowing all forms of CKX proteins
to be released without their substantial denaturation .
Detailed c haracterization of the CKX reaction mechanism,
carried out with recombinant ZmCKX1 protein, also
helped us to determine conditions for the assay of CKX
activities in vitro [12]. The mechanism of cytokinin cleavage
involving a ternary enzyme–substrate–electron acceptor
complex formation indicates that previously used concen-
trations of substrate and electron acceptors in the reaction

mixture may limit the assays used for determination of the
total activity [39]. T he high stability of C KX proteins
allowed us t o prolong incubation times f or the a ctivity
assay. A linear increase of product formation w as deter-
mined w ithin 7 h o f i ncubation. H ence, the upgraded
method utilizing p-aminophenol seems t o be both sensitive
enough and more effective than t he previously and more
commonly used radioisotope method.
Differences in CKX activity over t he whole physiological
range of p H were p resented for partially purified enzymes
from different sources [8]. Variations are also significant in
one plant s pecies. This heterogeneity may r eflect differences
in subcellular l ocalization and/or glycosylation of enzymes.
Indeed, differently glycosylated forms of C KX detected in
conditionally cytokinin-overproducing tobacco cell suspen-
sions have recently been studied [40]. While a glycosylated
CKX with a pH optimum of 6.0 is secreted to the culture
medium, the nonglycosylated form remains in the cells and
shows a pH optimum of 8.5. On the contrary, an extract
from barley grains, where the HvCKX1 form is predom-
inant, shows a pH optimum of 7.0–7.5. In accordance, the
two extracellular CKXs – AtCKX2 and ZmCKX1 –
possibly the closest orthologs t o HvCKX1, were also found
to be most active under mildly alkaline conditions [13,27].
The surprisingly low pH optimum of the recombinant
HvCKX2 indicates its potential targeting t o t he plant
vacuole, despite the computer prediction showing possible
secretion out of the cell. This form of enzyme is probably the
main one in the leaf CKX pool. Preliminary characteriza-
tion of Arabidopsis CKX proteins shows that e nzymes

with enhanced activity at low pH might also exist in other
species [16].
The b arley CKXs studied here have broad substrate
specificities. Until recently, kinetin and aromatic cytokinins
were not thought to be substrates for N
6
side-chain cleavage
by C KX. It was demonstrated recently, with the recombin-
ant m aize enzyme, t hat these compounds can a lso be
irreversibly converted to adenine and a corr esponding
aldehyde by CKX. However, the degradation of aromatic
cytokinins is not significantly affected by the cooperation of
the enzyme with an e lectron acceptor. The reaction rates of
these c ytokinins 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 conversion seems to b e nonspecific for this
particular isoenzyme. In contrast, the conversion of cis-
zeatin was detectable only in grain and root extracts at
pH 7.5. This zeatin isomer is obviously preferably cleaved
by only a specific isoform of CKX that is apparently visible
from a substrate preference study of the r ecombinant maize
enzyme [13]. Cleavage of cis-zeatin by this isoenzyme was
detected only with a large q u antity of enzyme, an d was
determined to have a 3 0-fold higher K
m
and two or ders
lower k
cat
/K

m
values for cis-zeatin than that estimated for
isopentenyladenine. This indicates that cis- zeatin h as 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], h as promoted
interest in the further investigation of barley and wheat
CKX genes. Characterization of direct HvCKX barley
overexpressers, and especially the possibility of preparing
knockout mutants, will help us to elucidate t he precise
function of cytokinin-degrading e nzymes in these c rop
plants. G enetic manipulation o f CKX activity also holds the
promise o f improving agricultural traits, such as yield
attributes or adaptation to environmental stress, in barley
and other cereals.
4000 P. Galuszka et al.(Eur. J. Biochem. 271) Ó FEBS 2004
Acknowledgements
This work was supported by grants 204/03/P103 and 522/03/0979 from
the Grant Agency and M SM 153100008 and KO NTAKT CZE01/023
from the Ministry of E ducat ion, Youth a nd Physical Educ ation , Czech
Republic and Bundesministerium fu
¨
r Bildung un d Forschung,
Germany.
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