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

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

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

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-

)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

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
–

)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

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.

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

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

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

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

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

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

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

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

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

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

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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