A Plant-specific Cyclin-dependent Kinase Is Involved in the Control of G2/M Progression in Plants
2001; Elsevier BV; Volume: 276; Issue: 39 Linguagem: Inglês
10.1074/jbc.m011060200
ISSN1083-351X
AutoresAndrea Porceddu, Hilde Stals, Jean‐Philippe Reichheld, Gerda Segers, Lieven De Veylder, Rosa de Pinho Barrôco, Peter Casteels, Marc Van Montagu, Dirk Inzé, Vladimir Mironov,
Tópico(s)Plant tissue culture and regeneration
ResumoCyclin-dependent kinases (CDKs) control the key transitions in the eukaryotic cell cycle. All the CDKs known to control G2/M progression in yeast and animals are distinguished by the characteristic PSTAIRE motif in their cyclin-binding domain and are closely related. Higher plants contain in addition a number of more divergent non-PSTAIRE CDKs with still obscure functions. We show that a plant-specific type of non-PSTAIRE CDKs is involved in the control of the G2/M progression. In synchronized tobacco BY-2 cells, the corresponding protein, accumulated in a cell cycle-regulated fashion, peaking at the G2/M transition. The associated histone H1 kinase activity reached a maximum in mitosis and required a yet unidentified subunit to be fully active. Down-regulation of the associated kinase activity in transgenic tobacco plants using a dominant-negative mutation delayed G2/M transition. These results provide the first evidence that non-PSTAIRE CDKs are involved in the control of the G2/M progression in plants. Cyclin-dependent kinases (CDKs) control the key transitions in the eukaryotic cell cycle. All the CDKs known to control G2/M progression in yeast and animals are distinguished by the characteristic PSTAIRE motif in their cyclin-binding domain and are closely related. Higher plants contain in addition a number of more divergent non-PSTAIRE CDKs with still obscure functions. We show that a plant-specific type of non-PSTAIRE CDKs is involved in the control of the G2/M progression. In synchronized tobacco BY-2 cells, the corresponding protein, accumulated in a cell cycle-regulated fashion, peaking at the G2/M transition. The associated histone H1 kinase activity reached a maximum in mitosis and required a yet unidentified subunit to be fully active. Down-regulation of the associated kinase activity in transgenic tobacco plants using a dominant-negative mutation delayed G2/M transition. These results provide the first evidence that non-PSTAIRE CDKs are involved in the control of the G2/M progression in plants. cyclin-dependent kinase(s) polyacrylamide gel electrophoresis Progression through the major transitions of the eukaryotic cell cycle is driven by a family of serine/threonine kinases known as cyclin-dependent kinases (CDKs).1 The catalytic activity of these protein kinases is regulated by the association with their regulatory subunits, cyclins. The activity of the complexes is further controlled by a number of mechanisms including phosphorylation/dephosphorylation, interaction with inhibitory proteins, proteolysis, and intracellular trafficking (1Morgan D.O. Annu. Rev. Cell Dev. Biol. 1997; 13: 261-291Crossref PubMed Scopus (1769) Google Scholar). In yeast, a single CDK (cdc2 in Schizosaccharomyces pombe or CDC28 inSaccharomyces cerevisiae) governs both the G1/S and G2/M transitions (2Nasmyth K. Trends Genet. 1996; 12: 405-412Abstract Full Text PDF PubMed Scopus (294) Google Scholar, 3Stern B. Nurse P. Trends Genet. 1996; 12: 345-350Abstract Full Text PDF PubMed Scopus (222) Google Scholar). In animal cells, distinct CDKs that associate sequentially with different cyclins monitor the cell cycle progression (4Pines J. Biochem. Soc. Trans. 1996; 24: 15-33Crossref PubMed Scopus (34) Google Scholar). Of the five mammalian CDKs strongly implicated in cell cycle control, three (CDC2/CDK1, CDK2, and CDK3) are closely related to the prototypical yeast cdc2 and have the same characteristic PSTAIRE motif in the cyclin-binding domain (5De Bondt H.L. Rosenblatt J. Jancarik J. Jones H.D. Morgan D.O. Kim S.-H. Nature. 1993; 363: 595-602Crossref PubMed Scopus (827) Google Scholar). The other two CDKs, CDK4 and CDK6, form a distinct subfamily of CDKs in which PSTAIRE is substituted with either PISTVRE or PLSTIRE, respectively. Both CDK4 and CDK6 are known to function exclusively in the G1 phase (1Morgan D.O. Annu. Rev. Cell Dev. Biol. 1997; 13: 261-291Crossref PubMed Scopus (1769) Google Scholar). Plants, like animals, possess also an array of CDK-like kinases (referred to as CDKs hereafter), but their functions are poorly defined (6Mironov V. De Veylder L. Van Montagu M. Inzé D. Plant Cell. 1999; 11: 509-521PubMed Google Scholar). Based on sequence similarity, plant CDKs can be subdivided into a few distinct groups (7Segers G. Rouzé P. Van Montagu M. Inzé D. Bryant J.A. Chiatante D. Plant Cell Proliferation and Its Regulation in Growth and Development. John Wiley & Sons, New York1997: 1-19Google Scholar). The best characterized group (A-type) comprises plant CDKs that are most closely related to the mammalian CDC2 and CDK2 and that contain the same PSTAIRE motif. A-type CDKs can partially complement yeast cdc2/CDC28 mutations and are therefore supposed to be functional homologs of the yeast CDKs (8Colasanti J. Tyers M. Sundaresan V. Proc. Natl. Acad. Sci. U. S. A. 1991; 88: 3377-3381Crossref PubMed Scopus (163) Google Scholar, 9Ferreira P.C.G. Hemerly A.S. Villarroel R. Van Montagu M. Inzé D. Plant Cell. 1991; 3: 531-540PubMed Google Scholar, 10Hata S. FEBS Lett. 1991; 279: 149-152Crossref PubMed Scopus (61) Google Scholar, 11Imajuku Y. Hirayama T. Endoh H. Oka A. FEBS Lett. 1992; 304: 73-77Crossref PubMed Scopus (66) Google Scholar, 12Setiady Y.Y. Sekine M. Hariguchi N. Kouchi H. Shinmyo A. Plant Cell Physiol. 1996; 37: 369-376Crossref PubMed Scopus (31) Google Scholar, 13Hashimoto J. Hirabayashi T. Hayano Y. Hata S. Ohashi Y. Suzuka I. Utsugi T. Toh-E A. Kikuchi Y. Mol. Gen. Genet. 1992; 233: 10-16Crossref PubMed Scopus (65) Google Scholar). Supporting this notion, a dominant-negative mutant of an A-type CDK from Arabidopsis thaliana was found to affect negatively cell cycle progression, most probably both at the G1/S and G2/M transitions (14Hemerly A. de Almeida Engler J. Bergounioux C. Van Montagu M. Engler G. Inzé D. Ferreira P. EMBO J. 1995; 14: 3925-3936Crossref PubMed Scopus (345) Google Scholar). Currently, functions of plant CDKs other than A-type remain essentially not understood. Here, we studied the function of a plant-specific subfamily of CDKs (B-type; Ref. 7Segers G. Rouzé P. Van Montagu M. Inzé D. Bryant J.A. Chiatante D. Plant Cell Proliferation and Its Regulation in Growth and Development. John Wiley & Sons, New York1997: 1-19Google Scholar). B-type CDKs form a group of closely related kinases from diverse plant species, sharing ∼70–80% identity and comprising a distinct cluster of CDKs. Instead of PSTAIRE, all of them bear unique motifs, either PPTALRE (B1 group) or PPTTLRE (B2 group) (15Fobert P.R. Gaudin V. Lunness P. Coen E.S. Doonan J.H. Plant Cell. 1996; 8: 1465-1476PubMed Google Scholar, 16Hirayama T. Imajuku Y. Anai T. Matsui M. Oka A. Gene. 1991; 105: 159-165Crossref PubMed Scopus (129) Google Scholar, 17Kidou S.-I. Umeda M. Uchimiya H. DNA Seq. 1994; 5: 125-129Crossref PubMed Scopus (14) Google Scholar, 18Magyar Z. Mészáros T. Miskolczi P. Deák M. Fehér A. Brown S. Kondorosi E. Athanasiadis A. Pongor S. Bilgin M. Bakó L. Koncz C. Dudits D. Plant Cell. 1997; 9: 223-235Crossref PubMed Scopus (165) Google Scholar). Unlike typical CDKs, the expression of B-type CDKs is under strict cell cycle control, and attempts to complement yeast CDK-deficient mutants have been unsuccessful. We have chosen tobacco as the experimental system because of the high degree of synchronization attainable in the tobacco Bright Yellow-2 (BY-2) cell line (19Nagata T. Nemoto Y. Hasezawa S. Int. Rev. Cytol. 1992; 132: 1-30Crossref Scopus (1009) Google Scholar). The close similarity among B-type CDKs across species has allowed us to use polyclonal antibodies against CDC2bAt from A. thaliana to identify and characterize a B-type CDK in tobacco. In synchronized BY-2 cells, the accumulation of the protein and the related kinase activity is cell cycle regulated and is linked to mitosis. We further demonstrate that the specific activity is present in the form of high molecular weight complexes, whereas the fraction corresponding to the monomeric protein is inactive, which implies the presence of a yet unidentified activating subunit. To assess the function of B-type CDKs in vivo, we have expressed a dominant-negative mutant of the CDC2bAt in transgenic tobacco. These plants were found to have an increased fraction of 4C cells. Accordingly, we propose that B-type plant CDKs play a unique role in the G2/M progression. The cDNA for CDC2bAt-D161N was obtained by mutating the codon GAT (Asp-161) for AAT (Asn-161) by in vitro mutagenesis (20Porceddu A. De Veylder L. Hayles J. Van Montagu M. Inzé D. Mironov V. FEBS Lett. 1999; 446: 182-188Crossref PubMed Scopus (23) Google Scholar). Both CDC2bAt and CDC2bAt-D161N cDNAs were fused to the Triple-Op promoter, which is a derivative of the cauliflower mosaic virus 35S promoter and has an activity comparable to that of the cauliflower mosaic virus35S promoter (21Gatz C. Frohberg C. Wendenburg R. Plant J. 1992; 2: 397-404PubMed Google Scholar). The expression cassettes Triple-Op-CDC2bAt-D161N-3′Nos and Triple-Op-CDC2b-3′Nos were ligated into the pGSC1704 binary vector (22Hérouart D. Van Montagu M. Inzé D. Plant Physiol. 1994; 104: 873-880Crossref PubMed Scopus (52) Google Scholar). The resulting plasmids were transferred intoAgrobacterium tumefaciens C58C1RifR by conjugation. Tobacco plants were transformed by leaf disc transformation (23Horsch R.B. Fry J.E. Hoffmann N.L. Eichholtz D. Rogers S.G. Fraley R.T. Science. 1985; 227: 1229-1231Crossref PubMed Scopus (3717) Google Scholar) and 15 independent T0 primary transformants were analyzed by protein gel blotting. The T0 plants were self-fertilized and the segregating T1 first generation was analyzed further. The tobacco BY-2 (Nicotiana tabacum L. cv. Bright Yellow-2) suspension was maintained at a weekly dilution (1.8/100) of cells in fresh Murashige and Skoog medium modified according to Nagata et al. (19Nagata T. Nemoto Y. Hasezawa S. Int. Rev. Cytol. 1992; 132: 1-30Crossref Scopus (1009) Google Scholar) and cultured at 28 °C and 130 rpm in the dark. The cells were synchronized as described (24Reichheld J.-P. Sonobe S. Clément B. Chaubet N. Gigot C. Plant J. 1995; 7: 245-252Crossref Scopus (72) Google Scholar). A stationary culture was diluted 1/5 in fresh medium supplemented with 4 μg/ml aphidicolin (Sigma). After 24 h of culture, the drug was removed by extensive washes, and the cells were resuspended into fresh medium. DNA synthesis was determined by pulse-labeling with [3H]thymidine as described (24Reichheld J.-P. Sonobe S. Clément B. Chaubet N. Gigot C. Plant J. 1995; 7: 245-252Crossref Scopus (72) Google Scholar). Mitotic index was determined by ultraviolet light microscopic analysis of 500 cells stained with 0.1 μg/ml 4′,6-diamino-2-phenylindole (Sigma) in the presence of 0.2% Triton X-100. Tobacco BY-2 protoplasts were obtained from 5 × 104 cells by digestion with 1 ml of enzyme solution (2% cellulase Onozuka R10, 0.1% pectolyase (Kikkoman Co.), 0.66m sorbitol) for 1 h at 37 °C. After treatment, the protoplasts were pelleted by centrifugation (5 min, 1000 ×g), washed once with Murashige and Skoog medium supplemented with 4.5% mannitol. Nuclei were released from the protoplast pellet in Galbraith's extraction buffer (25Galbraith D.W. Harkins K.R. Maddox J.M. Ayres N.M. Sharma D.P. Firoozabady E. Science. 1983; 220: 1049-1051Crossref PubMed Scopus (1513) Google Scholar). After addition of 1% formaldehyde, nuclei were stored at 4 °C until analysis by flow cytometry. Before analysis, nuclei were filtered through a 10-μm nylon filter, treated with RNase A, and stained with propidium iodide (50 μg/ml). Cytometrical analyses were performed on 104nuclei with a fluorescence-activated cell sorter scan flow cytometer (Becton Dickinson). For flow cytometrical analysis of nuclear DNA content in plant tissues, cotyledons or individual calli were chopped with a razor blade in Galbraith's buffer and analyzed as described above. Polyclonal antibodies against CDC2aNt (courtesy of P. John, Australian National University, Canberra, Australia) and CDC2bAt were raised with the peptides ARNALEHEYFKDIGYVP and SAKTALDHPYFDSLDKSQF derived from the C termini of the respective proteins. The sera were purified with protein A-Sepharose (Amersham Pharmacia Biotech). Because tobacco possesses multiple nearly identical CDKs that share the same C-terminal peptides for both A-type (accession numbers L77082, L77083, D50738, and AF289467) and B1-type (accession numbers AF289465 and AF289466) CDKs, the data obtained with the sera are cumulative. Protein extracts from BY-2 cells were prepared by grinding cells with sea sand in homogenization buffer as described (26Magyar Z. Bakó L. Bögre L. Dedeoǧlu D. Kapros T. Dudits D. Plant J. 1993; 4: 151-161Crossref Scopus (69) Google Scholar). Tobacco plants were ground in liquid nitrogen. Protein concentrations were determined with the Protein Assay kit (Bio-Rad, Hercules, CA). SDS-PAGE and protein gel blots were performed according to standard procedures with primary anti-CDC2aNt and anti-CDC2bAt antibodies diluted 1/500 and 1/2500, respectively, and a secondary peroxidase-conjugated antibody (Amersham Pharmacia Biotech) diluted 1/5000. For immunoprecipitation experiments, 100–200 μg of protein extracts were preincubated for 1 h at 4 °C on a rotating platform with 25% (v/v) protein A-Sepharose (Amersham Pharmacia Biotech). After centrifugation, equal amounts of the supernatant were incubated for 4 h with purified antibodies (1/50 diluted anti-CDC2aNt or 1/50 diluted anti-CDC2bAt) and subsequently for 1 h with 25% (v/v) protein A-Sepharose beads. Beads were washed three times with homogenization buffer and a fourth time with kinase buffer (26Magyar Z. Bakó L. Bögre L. Dedeoǧlu D. Kapros T. Dudits D. Plant J. 1993; 4: 151-161Crossref Scopus (69) Google Scholar). The histone H1 kinase assay was carried out by incubating 25 μl of the packed beads with 5 μCi [γ-32P]ATP (3000 Ci/mmol) in the presence of 1 mg/ml histone H1 (Sigma), 50 mm Tris-HCl (pH 7.8), 15 mm MgCl2, 5 mm EGTA, 10 μm ATP, and 1 mm dithiothreitol, for 20 min at 30 °C in a final volume of 35 μl. Samples were analyzed on a 12% SDS-PAGE gel stained with Coomassie Brilliant Blue and autoradiographed. For competition experiments the C-terminal CDC2bAt peptide was purified to homogeneity by reverse-phase chromatography on a PRLP-S column (8 μm, 300 Å) (Polymer Laboratories) equilibrated in 0.1% trifluoroacetic acid, and eluted with a 0–70% acetonitrile gradient in 0.1% trifluoroacetic acid. The competition was achieved by preincubation of the antiserum with the purified CDC2bAt peptide at 4 °C overnight before the antiserum was used for immunoblotting and immunoprecipitation. Proliferating tobacco BY-2 cell suspension culture cells were collected three days after subculturing. Proteins were extracted in homogenization buffer as described previously (27Stals H. Bauwens S. Traas J. Van Montagu M. Engler G. Inzé D. FEBS Lett. 1997; 418: 229-234Crossref PubMed Scopus (51) Google Scholar) and bound to a Q-ceramic HyperD column (10 × 25 cm; Biosepra), equilibrated with homogenization buffer. Proteins were eluted by a one-step elution with 1 m NaCl in 0.5× homogenization buffer and further fractionated by size on a gel filtration column (Superdex 200 (Amersham Pharmacia Biotech) 1.7 × 100 cm Omnifit column). The column was equilibrated with Tris buffer (50 mm, pH 7.8) containing 15 mm MgCl2, 5 mm EGTA, 5 mm β-glycerophosphate, 1 mm NaF, 1 mm dithiothreitol, 0.1 mmNaVO4, 100 mm NaCl, and protease inhibitors (Roche Diagnostics, Brussels, Belgium). Kinase activity associated with either A- or B-type CDKs was isolated by immunoprecipitation with anti-CDC2aNt and anti-CDC2bAt antisera, respectively. The activity of 100-μl aliquots of 5-ml fractions was assayed in vitro in the presence of histone H1 (1 mg/ml), cAMP-dependent kinase inhibitor, 15 μm ATP, and 10 μCi [γ-32P]ATP (3000 Ci/mmol) in a final volume of 35 μl at 30 °C. The reactions were terminated after 20 min by heating the samples in sample buffer at 95 °C for 10 min. The proteins were separated on a 15% SDS-PAGE gel and stained with Coomassie Brilliant Blue, and the incorporated radioactivity was measured with a PhosphorImager (Amersham Pharmacia Biotech). To study the function of B-type CDKs, we first analyzed the associated kinase activity in the course of the cell cycle in the tobacco BY-2 cell suspension (19Nagata T. Nemoto Y. Hasezawa S. Int. Rev. Cytol. 1992; 132: 1-30Crossref Scopus (1009) Google Scholar). Because B-type CDKs had not been described in tobacco, we decided to use an antibody raised against the C-terminal SAKTALDHPYFDSLDKSQF peptide of CDC2bAt of Arabidopsis. This region is well conserved among the known PPTALRE kinases with 17 of 19 amino acids being identical. Thus, we expected the anti-CDC2bAt antibody to recognize tobacco orthologs. Indeed, the antibody recognized specifically a single protein in a crude protein extract of BY-2 cells fractionated by SDS-PAGE (Fig.1a). The apparent molecular mass of the protein, 34.5 kDa, is close to that of theArabidopsis CDC2b. Furthermore, the interaction was specific, as the addition of the peptide used to raise the antibody obliterated the signals corresponding to the protein (Fig.1b). We also analyzed by protein gel blotting with anti-CDC2bAt antibody immunoprecipitates obtained with either an anti-CDC2bAt or an anti-PSTAIRE antibody and found that the 34.5-kDa protein was easily detectable in the former but not the latter precipitate (data not shown). These results suggested that tobacco possesses a CDC2bAt homolog, referred to as CDC2bNt hereafter. Indeed, the sequences of two nearly identical (99.7%) tobacco B-type CDKs have been deposited into public data bases (accession numbersAF289465 and AF289466) with the identical C-terminal peptide SAKAALDHPYFDSLDKSQF, which differs from Arabidopsis CDC2b only in one position (Fig. 1c). Characteristically, the protein level of CDC2bNt fluctuated through the cell cycle (CDC2b; Fig. 2b), thus displaying the most conspicuous feature of B-type CDKs. The protein was hardly detectable in early S phase, started to accumulate in late S phase, reached the maximal level at the G2/M transition, and declined afterward. After immunoprecipitation with anti-CDC2bAt serum, the kinase activity associated with CDC2bNt was measured by in vitro phosphorylation of histone H1. In the course of the cell cycle, CDC2bNt kinase activity peaked later than the corresponding protein level with a maximum in the middle of mitosis (Fig. 2b). For comparison, we analyzed also the CDC2aNt protein and associated kinase activity using a specific antibody. The level of CDC2aNt protein was constant during the whole cell cycle (Fig.2b). Its activity rose in early S phase and declined during mitosis, slightly earlier than that of CDC2bNt (Fig.2b). To biochemically characterize CDC2bNt, the proteins present in a total extract of actively proliferating BY-2 suspension cells were separated in two fractions by ion exchange chromatography. The bound fraction contained most of the kinase activity (∼95 and 75% for CDC2aNt and CDC2bNt, respectively) and was further fractionated by size exclusion chromatography. The CDK-associated complexes were immunoprecipitated with either anti-CDC2aNt or anti-CDC2bAt polyclonal antibodies and assayed in an in vitro kinase assay with histone H1 as a substrate. The CDC2bNt-associated kinase activity eluted as two minor peaks of ∼250 kDa and more than 700 kDa and one major broad peak in the range of 65 to 100 kDa, referred to as the 80-kDa fraction hereafter (Fig. 3a). In contrast, CDC2aNt activity eluted essentially as a single peak of ∼200 kDa (Fig. 3a). Immunoblotting showed that most of the CDC2bNt protein was present in high molecular mass complexes with a relatively low kinase activity compared with that of the major CDC2bNt activity peak, whereas the majority of the CDC2aNt protein comigrated with the peak of activity (Fig. 3b). The fractions corresponding to monomeric CDC2aNt and CDC2bNt (fractions 20–21; Fig.3) contained detectable, albeit low, amounts of the corresponding proteins and were essentially inactive. To confirm that the kinase activity in the immunoprecipitated protein complexes was specific for CDC2bNt, we performed peptide competition assays for the bound protein fraction used in the size fractionation experiments above. As can be seen from Fig. 3c, preincubation of the serum with the peptide strongly reduced immunoprecipitated kinase activity. This corresponds well with our results obtained with a dominant-negative mutant of CDC2bAt and also indicated that at least 80% of the kinase activity immunoprecipitated with the same serum from the whole cell tobacco extracts could be attributed to CDC2bNt (see below). Additionally, it should be stressed that the peptide SAKTALDHPYFDSLDKSQF is highly specific for B-type CDKs. All the significant hits retrieved by a BLAST search with the peptide were confined to B-type CDKs, with at least 17 amino acids identical for PPTALRE kinases and only 12 for PPTTLRE kinases. Gel filtration separation of the flow-through fraction showed that the majority of the CDC2aNt protein was present in the inactive monomeric fractions and accounted for approximately half of the total CDC2aNt protein (data not shown). In contrast, only a very limited amount of the total CDC2bNt protein was found in the flow-through fraction and it eluted after gel filtration as a single peak of ∼200 kDa, co-migrating with the peak of kinase activity (data not shown). Thus, the pool of monomeric CDC2bNt is very small compared with that of CDC2aNt. Our data demonstrate that large amounts of both CDC2aNt and CDC2bNt are kept in inactive forms, albeit by different means. The residue Asp-161 ofArabidopsis CDC2b belongs to the triad of catalytic residues that are conserved in all eukaryotic protein kinases (Arg-33, Glu-51, and Asp-145 in the human CDK2) and are strictly required for the kinase activity by providing vital contacts for correct ATP orientation and Mg2+ coordination (28Jeffrey P.D. Russo A.A. Polyak K. Gibbs E. Hurwitz J. Massagué J. Pavletich N.P. Nature. 1995; 376: 313-320Crossref PubMed Scopus (1203) Google Scholar). The mutation of the Asp residue to Asn results in loss of activity and, in the case of CDKs, has, upon overproduction, a dominant-negative effect (29Labib K. Moreno S. Nurse P. J. Cell Sci. 1995; 108: 3285-3294PubMed Google Scholar, 30van den Heuvel S. Harlow E. Science. 1993; 262: 2050-2054Crossref PubMed Scopus (969) Google Scholar) presumably because of the competition of the mutant proteins for the association with the rate-limiting interacting proteins such as cyclins. As it is well known, orthologous CDKs, even from very divergent species, are functionally interchangeable; hence, dominant-negative mutants are efficacious in heterologous systems (14Hemerly A. de Almeida Engler J. Bergounioux C. Van Montagu M. Engler G. Inzé D. Ferreira P. EMBO J. 1995; 14: 3925-3936Crossref PubMed Scopus (345) Google Scholar). To see whether kinase-negative mutants of B-type CDKs are dominant-negative as well, we generated transgenic tobacco plants that express the mutantCDC2bAt–D161N under control of a strong constitutive promoter (Fig. 4). We also produced tobacco plants expressing wild-type CDC2bAt under control of the same promoter. For 15 independent lines from each transformation, the level of the protein was analyzed in 2-week-old plantlets in the primary T0 transformants and in the self-fertilized T1 population. The level of CDC2bAt-D161N was consistently lower than that of CDC2bAt (data not shown), suggesting that a higher level of CDC2bAt-D161N is incompatible with plant regeneration and/or development. Two lines from both transformations that segregated the T-DNA as a single locus were selected for further analysis. We compared histone H1 kinase activity of protein complexes immunoprecipitated with CDC2bAt antiserum in the transgenic and parental lines (Fig. 5). Production of the CDC2bAt-D161N protein correlated with an ∼5-fold inhibition of the B-type CDK activity in both DN-1 and DN-27 lines (Fig. 5). In contrast, ectopic production of the wild-type CDC2bAt (WT-14 and WT-23; Fig. 5) did not affect the corresponding kinase activity (in this case cumulative for CDC2bNt and CDC2bAt), even when produced to a much higher level than the mutant forms (WT-23). Effectively, these results mean also that at least 80% of the kinase activity in the immunoprecipitates associates with CDC2bNt and that cross-reacting contaminants, if any, contribute to less than 20% of the total activity.Figure 5Transgene expression and histone H1 activity in transgenic tobacco. Seedlings of transgenic plants expressingCDC2bAt (lines WT14 and WT23) orCDC2bAt-D161N (lines DN-1 and DN-27) grown for 2 weeks on selective medium are compared with the SR-1 control of the same age. a, protein gel blot analysis of total proteins with anti-CDC2bAt antibodies (CDC2b) and the kinase activity in protein complexes immunoprecipitated either with anti-CDC2bAt antibodies (CDC2b activity) or with anti-CDC2aNt antibodies (CDC2a activity) (see "Experimental Procedures"). b, relative CDC2b kinase activity estimated by quantification of the radioactively phosphorylated H1 protein.View Large Image Figure ViewerDownload Hi-res image Download (PPT) To see whether CDK activities other than B-type are affected in the transgenics, we analyzed histone H1 kinase activity in protein complexes either immunoprecipitated with CDC2aNt-specific antibodies or purified with p13SUC1 affinity matrix (p13SUC1binds CDC2a but not CDC2b in tobacco). 2H. Stals and P. Casteels, unpublished data. In neither case, any reliable difference among the lines could be detected (Fig.5a; data not shown). To analyze whether the down-regulation of B-type CDK activity affects cell cycle progression, we compared the nuclear DNA content in segregating T1 plants grown on nonselective medium for two weeks. For each of the lines, 20 plants were chosen randomly. For each plant, half of one cotyledon was transferred to selective medium to identify the siblings that lost the transgene, while the other half was used to regenerate calli on nonselective medium. The regenerated calli and the other cotyledon were subjected to the flow cytometric analysis of nuclear DNA content. The cotyledons and calli were chosen as representatives of terminally differentiated and undifferentiated tissues, respectively. The fractions of 2C (G1 phase) and 4C (G2 phase) cells were determined for individual plants, and the siblings with and without the T-DNA were compared. As shown in Fig.6b, the expression ofCDC2bAt-D161N correlated with an ∼2- and 1.5-fold increase in the 4C/2C ratio in cotyledons and calli, respectively. The ectopic expression of CDC2bAt did not modify the G2/G1 ratio. Thus, these data showed that ectopic expression of CDC2bAt-D161N, but notCDC2bAt, affects cell cycle in transgenic tobacco. We further studied whether ectopic production of the CDC2bAt protein and its D161N mutant affects the morphology of plants. T0 seeds were germinated on nonselective medium for two weeks, and the phenotypes of the segregating populations of plants were compared. The plants ectopically expressingCDC2bAt showed no phenotypic alterations (WT-23; Fig.6a). Neither were the plants expressingCDC2bAt-D161N affected, despite a 5-fold reduction of the B-type CDK activity (Fig. 6a, DN-1). Microscopic analyses revealed no cytological modifications in any of the tissues analyzed including epidermal cells in leaves, cotyledons, and all the root cell files or particular modifications in the organization of the shoot and root meristems (the primary sources of all plant cells). Because the ploidy level and morphogenesis of seedlings is known to be under light control (31Gendreau E. Traas J. Desnos T. Grandjean O. Caboche M. Höfte H. Plant Physiol. 1997; 114: 295-305Crossref PubMed Scopus (494) Google Scholar), we compared also seedlings grown in the dark and could not find any changes either. Furthermore, we did not observe any significant differences in the rate of callus regeneration from the cotyledons (data not shown). We have attempted to elucidate the function of the CDK group distinguished by a PPT(A/T)LRE motif, which is specific for plants (referred to as B-type; Ref. 7Segers G. Rouzé P. Van Montagu M. Inzé D. Bryant J.A. Chiatante D. Plant Cell Proliferation and Its Regulation in Growth and Development. John Wiley & Sons, New York1997: 1-19Google Scholar). We identified a B-type CDK in tobacco on the basis of its similarity to the Arabidopsis CDC2b protein, the founding member of the B-type CDKs (16Hirayama T. Imajuku Y. Anai T. Matsui M. Oka A. Gene. 1991; 105: 159-165Crossref PubMed Scopus (129) Google Scholar). The single tobacco CDK characterized to date (13Hashimoto J. Hirabayashi T. Hayano Y. Hata S. Ohashi Y. Suzuka I. Utsugi T. Toh-E A. Kikuchi Y. Mol. Gen. Genet. 1992; 233: 10-16Crossref PubMed Scopus (65) Google Scholar) belongs to the A-group of plant CDKs (PSTAIRE-type). Here, we show that tobacco possesses a putative homolog of the CDC2bAt kinase, recognized by a CDC2bAt-specific antibody and displaying the expression pattern characteristic of this type of CDKs. As shown before for the alfalfa PPTALRE kinase CDC2MsD (18Magyar Z. Mészáros T. Miskolczi P. Deák M. Fehér A. Brown S. Kondorosi E. Athanasiadis A. Pongor S. Bilgin M. Bakó L. Koncz C. Dudits D. Plant Cell. 1997; 9: 223-235Crossref PubMed Scopus (165) Google Scholar), the CDC2bNt protein accumulates at the G2/M transition. Our unpublished results 3G. Segers and D. Inzé, unpublished data. with partially synchronized Arabidopsis suspension cells, albeit
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