Journal of Experimental Botany, Vol. 51, No. 353, pp. 2119-2124,
December 2000
© 2000 Oxford University Press
Short Communication |
Characterization of a Mak subgroup Cdc2-like protein kinase from sugar beet (Beta vulgaris L.)
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 Cdc2/CDKX 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 Cdc2-related kinases that cannot be defined 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 first member of this subgroup). Members of the Mak subgroup are clearly Cdc2-related serine/threonine protein kinases, but they show important structural differences (Matsushime et al., 1990; Bladt and Birchmeier, 1993). Most obvious is the 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 compost/Perlite mixture under constant illumination at 25 °C.
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 5' RACE and the primer dGACTCAGGTCCTTCAGGGAC-TTGCAC was used for 3' 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.
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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 confirmed 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.
Whole mount in situ hybridization (WISH)
WISH was performed basically according to Engler et al. (Engler et al., 1994), but with the following modifications. Sections (200 or 300 µm) were cut using a hand microtome. Fluorescein labelled sense and antisense probes, derived from the 5' 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 3' 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, confirming 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 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).
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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). Semi-quantitative 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 fide 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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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 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 reflects 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 difficulties with very young material).
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Notes
1 To whom correspondence should be addressed: Fax: +44 116 2577752. E-mail: mrfowler{at}dmu.ac.uk 
2 Present address: Vlaams Instituut voor Biotechnologie, Department of Genetics, University of Gent, B-9000, Gent, Belgium
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