Journal of Experimental Botany, Vol. 51, No. 353, pp. 2119±2124, December 2000
SHORT COMMUNICATION
Characterization of a Mak subgroup Cdc2-like
protein kinase from sugar beet (Beta vulgaris L.)
Mark R. Fowler1, Ana I. Atanassova2, Malcolm C. Elliott, Nigel W. Scott and Adrian Slater
The Norman Borlaug Institute for Plant Science Research, De Montfort University, Scraptoft,
Leicester LE7 9SU, UK
Received 17 May 2000; Accepted 15 September 2000
Abstract
The Mak-type Cdc2-like protein kinases are, a relatively uncharacterized group of proteins. Bvcrk2
encodes a plant Mak-type kinase. Its highest levels
of expression occur in the secondary meristems of
developing sugar beet storage organs, suggesting
a role, in planta, in the regulation of cell division or
early cell differentiation.
Key words: Mak-type kinase, expression, sugar beet, cell
division, cell differentiation.
Introduction
Considerable attention has, in recent years, been focused
on the role of the Cdc2uCDKX group of protein kinases
in regulating cell division and plant development. This
effort has led to the isolation of a large number of presumptive cyclin-dependent kinase gene sequences from
various plant species (Fowler et al., 1998a). The roles
of such sequences in regulating cell division have been
elucidated in model systems such as yeast, and in higher
eukaryotes.
Despite evidence, mainly from mammalian cell studies,
that higher eukaryotes contain large numbers of Cdc2related kinases that cannot be de®ned as classical CDKs,
the functions of many of them remain unknown (Fowler
et al., 1998a). One subgroup of Cdc2-like proteins has
been termed the Mak (male-germ cell associated kinase)subgroup (after the designation of the ®rst member of this
subgroup). Members of the Mak subgroup are clearly
Cdc2-related serineuthreonine protein kinases, but they
show important structural differences (Matsushime et al.,
1990; Bladt and Birchmeier, 1993). Most obvious is the
1
2
lack of a well conserved PSTAIRE epitope in PK domain
III. Mak-type kinases also have a large (relative to CDKs)
carboxy-terminal domain, the function of which is not
well understood.
The cloning of a full length Mak-subgroup Cdc2-like
kinase from sugar beet is reported here and it is demonstrated that its expression, in planta, is correlated with cell
division activity or cell differentiation.
Materials and methods
Plant material
A leaf derived cell suspension culture of table beet (cv. Albina
Vereduna) was maintained in MS0 medium (Murashige and
Skoog, 1962). The cells exhibited a 14 d culture cycle. Sugar
beet (cv. Roberta) were grown from seed in a standard
compostuPerlite mixture under constant illumination at 25 8C.
Isolation of the full length sequence
Total RNA from cells grown in suspension culture and
from sugar beet was extracted according to Chomczynski
and Sacchi (Chomczynski and Sacchi, 1987). First strand
cDNA was synthesized from the total RNA according
to Fowler et al. (Fowler et al., 1998b). Total RNA to be used
in RACE-PCR procedures was extracted from the roots of sugar
beet (cv. Roberta) using the Wizard SV Total RNA extraction
kit according to the manufacturer's instructions (Promega). One
product from a PCR based screen for cdc2-like sequences from
sugar beet (Fowler et al., 1998c) showed considerable homology
to Mak-subgroup cdc2-like sequences. Using this original PCR
product as a starting point a full length cDNA sequence was
obtained by RACE-PCR carried out according to the manufacturer's instructions (Clontech RACE-PCR kit). The primer
dGTGTGCAAGTCCCTGAAGGACCTGAG was used for
59 RACE and the primer dGACTCAGGTCCTTCAGGGACTTGCAC was used for 39 RACE (equivalent positions are
marked on Fig. 2). The RACE-PCR products were cloned into
pGEM-T (Promega) and sequenced by an external contractor
using dye terminator chemistry.
To whom correspondence should be addressed: Fax: q44 116 2577752. E-mail: [email protected]
Present address: Vlaams Instituut voor Biotechnologie, Department of Genetics, University of Gent, B-9000, Gent, Belgium
ß Society for Experimental Biology 2000
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Fowler et al.
Semi-quantitative RT-PCR
Analysis of Bvcrk2, Bvcrk1 and Bvcdc2 expression during
a batch culture cycle and during the development of sugar
beet was performed according to Fowler et al. (Fowler et al.,
1998b) except that the PCR product was differentially
precipitated prior to liquid scintillation counting. The
primers used were dGGCTGTCAATAGGGAGAGTTATGAG
and dCCCGATGAAAGTATCCATTCCTATGC (Bvcrk2),
dGTGTTAAGTTCATGGCTAGAG and dCGGTGAAGGACGCCACGATT (Bvcrk1) and dGTGTGGTCTATAAGGCGCGGG and dCAGGACACGATGAGAGAGAC (Bvcdc2);
(equivalent positions are marked on Fig. 2). Primers were
designed to produce a PCR product that spanned at least one
intron and comparisons of PCR product sizes from sugar beet
genomic and cDNA con®rmed this. In order to account for any
variation in sequence composition, and therefore the amount of
radiolabel that could be incorporated into the PCR product,
results were normalized to allow comparison. Results were
normalized by taking a ratio of incorporated counts to possible
number of positions where the radiolabelled nucleotide could be
incorporated.
activity, location or binding to other proteins. The
PSTAIRE epitope of Bvcrk2, CVN-LRE, is identical to
that of the A. thaliana MHK sequence while in the Mak
subgroup sequences only the R and E residues, which are
important in determining protein kinase activity, are
conserved. Other sequence differences are also apparent.
In Pk domain I the Tyr residue (at a position equivalent
to the Tyr15 of Cdc2) present in other Mak sequences is
replaced by a cysteine residue in plant Mak-type kinases.
This may have important consequences for the regulation
of kinase activity. In CDKs Tyr15, via its phosphorylation status, is important in determining kinase activity
(Fowler et al., 1998a).
Whole mount in situ hybridization ( WISH)
WISH was performed basically according to Engler et al.
(Engler et al., 1994), but with the following modi®cations.
Sections (200 or 300 mm) were cut using a hand microtome.
Fluorescein labelled sense and antisense probes, derived from
the 59 RACE-PCR product, were generated according to the
manufacturer's instructions (Roche). The proteinase K treatment was increased to 45 min. Antibody incubation and signal
detection were performed according to the manufacturer's
instructions (Roche).
Results and discussion
The complete Bvcrk2 cDNA (accession number AJ277162)
was found to be 1979 nucleotides long, comprised of
a 474 nucleotide untranslated leader, a 1305 nucleotide
predicted open reading frame and a 200 nucleotide 39
untranslated region. The ORF encodes a protein with
a very high degree of similarity to the previously isolated
Arabidopsis thaliana MHK sequence (Moran and Walker,
1993) and belongs to the Mak subgroup of Cdc2-like
protein kinases, con®rming the presence of these
sequences in various plant species (Fig. 1). A comparison
of the derived amino acid sequences (Fig. 2) of Mak
subgroup nucleotide sequences with those of other
Cdc2-related protein kinases showed that there was considerable sequence divergence, both between Mak-type
kinases and other Cdc2-like sequences, and within the
Mak-type kinases. All Mak type sequences have
a considerable C-terminus extension relative to CDKs,
although the C-termini of the plant Mak sequences are
considerably shorter, and are much more like each other,
than to those of animal Mak sequences. It is not clear
what role, if any, the carboxy-terminus plays in Mak-type
kinases, although it may be important in determining
Fig. 1. Dendrogram (Perriere and Gouy, 1996: NJPlot-PBIL facility) of
Bvcrk2 and other Cdc2-like protein kinases produced from the MAGI
ouput ®le (Hinxton, UK). Accession numbers are: Atcdc2a ˆ P24100;
AtMAK ˆ P43294;
AtRCK ˆ AL021687;
CeMAKlike ˆ Z83731;
HsCDK7 ˆ P50613; HsCDC2 ˆ P06463; HsCDK8 ˆ P49336; HsMAK
ˆ ABO23153;
HsPCTAIRE ˆ Q00536;
HsPITSLRE ˆ U04824;
MmMAK ˆ NP_032573.1; MsCDC2 ˆ P24923; MsCDC2c ˆ P93320;
MsCDC2d ˆ P93321;
MsCDC2e ˆ P93322;
MsCDC2f ˆ P93323;
OsNRTALRE ˆ P29620; RnMAK ˆ D26178, where At ˆ Arabidopsis
thaliana; Ce ˆ Caenorhabditis elegans; Hs ˆ Homo sapiens; Mm ˆ Mus
musculus; Ms ˆ Medicago sativa; Os ˆ Oryza sativa; Rn ˆ Rattus
norvegicus.
Mak-type Cdc2-like protein kinases
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Fig. 2. Alignment of the deduced amino-acid sequences of Mak-type Cdc2-like protein kinases with Bvcrk1 and Bvcdc2. The sequences were aligned
using the BLAST programme (Hinxton, UK). Bvcrk2 (accession number AJ277162) is aligned with the Arabidopsis thaliana homologue MHK, and the
rat and mouse Mak-type kinases. Equivalent positions of primers used for RT-PCR (boxed) and RACE-PCR (underlined) are indicated. Aqfollowed
by a ®gure indicates the number of amino acid residues N-terminal of the aligned portion of the sequence. Amino acid residues conserved in all Maktype sequences are indicated by shading.
In general, little is known about the expression and
function of Mak-type sequences (this is particularly true
for the plant sequences). The mammalian sequences are
known to be expressed in a variety of tissues and have
been implicated in processes such as meiosis and spermatogenesis and sensory signal transduction (Matsushime
et al., 1990; Bladt and Birchmeier, 1993; Jinno et al.,
1993). In plants, a limited Northern blot analysis indicated that expression was found in Arabidopsis thaliana
roots and rosettes (Moran and Walker, 1993). Semiquantitative RT-PCR was used to analyse the expression
of Bvcrk2 (Fig. 3). Analysis of Bvcrk2 expression in Beta
vulgaris cells during a batch suspension culture cycle
indicated that expression was not correlated with any
particular growth state of the cells, it was expressed
throughout the batch culture cycle. Comparison of the
expression pattern with that of Bvcdc2 and Bvcrk1
(Fowler et al., 1998c) showed that, despite belonging to
the Cdc2-like family of protein kinases, the expression
pattern did not match that of either a bona ®de CDK
(e.g. Bvcdc2) or a different Beta vulgaris Cdc2-like
sequence (Bvcrk1). The expression of Bvcrk2 was also
determined at key points during the development of
sugar beet and compared with the results obtained for
Bvcdc2 and Bvcrk1 expression (Fig. 4). Bvcrk2 expression was, like that of Bvcrk1 and Bvcdc2, low in
young and old leaves, the highest level of expression
being found in developing storage organs. In this
respect the pattern of Bvcrk2 expression most closely
resembled that of Bvcrk1. Bvcdc2 expression was relatively stable throughout development of the storage
organ.
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Fowler et al.
Fig. 3. Expression of Bvcrk2 during the batch culture cycle of Beta
vulgaris cells grown in suspension culture. Semi-quantitative RT-PCR
was used to determine expression levels of Bvcrk2, Bvcrk1 and Bvcdc2 in
stationary phase cells (0 h after subculture) and at various times after
subculture until the cells were dividing exponentially (96 h after
subculture). Results are the meanqSD of three observations.
In order to determine, more precisely, in which cells
Bvcrk2 was expressed, a WISH analysis was carried
out during the development of sugar beet (Fig. 5).
Hybridization signals were only detected in tissues
of sugar beet plants that were undergoing cambial
initiation and the early stages of development, which
correlated with the maximal period of expression as
measured by RT-PCR analysis. The hybridization signals
were localized in the secondary cambia, thus implicating
Bvcrk2 in either cell division control or the early events
of differentiation. The relatively high levels of expression
observed during the early stages of storage organ
development by RT-PCR can be explained by the
fact that sugar beet initiates cambial activity early in
development. Thus young storage organs are comprised
of a relatively high proportion of cambial cells which
are initiating cell division. Later in development, parenchymatous storage cells predominate, and cambial
Fig. 4. Expression of Bvcrk2, Bvcrk1 and Bvcdc2 during the development of the sugar beet storage organ as determined by RT-PCR.
Samples 1, 2, 3, and 4 correspond to seedling cotyledon, hypocotyl, root,
and root-tip, respectively. The position of samples 5±15 is indicated on
the diagram of the developing storage organ below. Sample 16 is from
young leaf and sample 17 from old leaf. Results are the meanqSD. of
three observations.
activity is restricted to the periphery and lower parts of
the storage organ. Bvcrk2 expression is much lower than
that of Bvcdc2 (as determined by RT-PCR) which
probably re¯ects the fact that Bvcdc2 is expressed in the
majority of cells in the developing storage organ (results
not shown) whereas Bvcrk2 expression is limited to a
distinct subset of cells. No hybridization signals were
detected in older sugar beet or in seedling tissues (although
the latter may be due to technical dif®culties with very
young material).
Mak-type Cdc2-like protein kinases
2123
Fig. 5. Whole mount in situ hybridization analysis of Bvcrk2 expression in young sugar beet storage organs. (A) Transverse section through a storage
organ using control sense probe. Bar represents 1 cm. (B) Transverse section through a storage organ probed with anti-sense probe. Hybridization
signals (rings of blue colour) are seen in regions corresponding to cambia (secondary meristems). Bar represents 1 cm. (C, D) Higher magni®cation
portions of (B) (bar represents 0.1 cm). The numbers to the right are the number of the cambia (from the middle of the storage organ). Hybridization
signals can clearly be seen in areas corresponding to cambia, running through the middle of the developing vascular tissue. Xylem vessels appear black.
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