Journal of Experimental Botany, Vol. 54, No. 381, pp. 303-308,
January 2, 2003
© 2003 Oxford University Press
Isolation, characterization and expression of cyclin and cyclin-dependent kinase genes in Jerusalem artichoke (Helianthus tuberosus L.)
Received 10 February 2002; Accepted 18 September 2002
Institute of Biotechnology, University of Cambridge, Tennis Court Road, Cambridge CB2 1QT, UK
1 Present address: Acambis plc, Peterhouse Technology Park, 100 Fulbourn Road, Cambridge CB1 9PT, UK.
2 Present address: Institut des Sciences de la Vie et de la Santé, Faculté des Sciences, Université de Limoges, 87100 Limoges, France.
3 To whom correspondence should be addressed. Fax: +44 (0)1223 334162. E-mail: j.murray{at}biotech.cam.ac.uk
Abbreviations: CDK, cyclin-dependent kinase; ABA, abscisic acid; ORF, open reading frame; RT-PCR, reverse-transcriptase PCR.
 |
Abstract |
|---|
 Top Abstract IntroductionMaterials and methodsResults and discussionReferences |
|---|
Tuber explants of Jerusalem artichoke (Helianthus tuberosus L.) are a model system for cell-cycle re-entry from a quiescent state, involving the activation of division of tuber parenchyma cells in response to exogenous auxin. To enable molecular studies of this system, two cyclin (Heltu;CYCD1;1 and Heltu; CYCD3;1) and two cyclin-dependent kinase (Heltu; CDKA;1 and Heltu;CDKB1;1) genes have been isolated from a Jerusalem artichoke cDNA library and their expression demonstrated during the activation of cell division. It was found that CDKA;1 transcripts are present in quiescent tubers, whereas CYCD1;1, CYCD3;1 and CDKB1;1 transcripts are induced during cell-cycle re-entry as well as during bud growth of whole tubers. Both CYCD1;1 and CYCD3;1 transcripts appear shortly before, or coincident with, the onset of S phase.
Key words: Cyclin, cyclin-dependent kinase, Helianthus tuberosus.
|
Introduction |
|---|
|
TopAbstractIntroductionMaterials and methodsResults and discussionReferences |
|---|
Co-ordination of cell division and the cell cycle is crucial for the proper growth and development of any organism. In plants, the co-ordination of the number and plane of cell divisions is particularly important, since cell migration does not occur. The identification of genes controlling cell division requires experimental systems where the division cycle of the cells is synchronized, therefore making it possible to study gene expression at particular time points in the cycle. Two such systems in plants are tobacco BY-2 cells (Nagata et al., 1992; Nagata and Kumagai, 1999) and, more recently, Arabidopsis cells (Menges and Murray, 2002). Both systems have been used in conjunction with cell-cycle blocking chemicals to identify the expression of genes during the cell cycle of rapidly dividing cells (Setiady et al., 1996; Riou-Khamlichi et al., 2000; Sorrell et al., 2001), as well as cell-cycle entry following starvation for sucrose (Menges and Murray, 2002). However, the manipulation of culture conditions required in these cases may not mimic fully the induction from a natural quiescent state, representing the exit from quiescence (G0) and entry into the active cell cycle at G1. In the past, dormant tubers of Jerusalem artichoke (Helianthus tuberosus L.) have been one of the best-studied systems for examining cell-cycle induction (Setterfield, 1963; Mitchell, 1967; Harland et al., 1973; Serafini-Fracassini et al., 1980; Bennici et al., 1982; Favali et al., 1984), although in recent times the system has been largely neglected. The attractions of these tubers as a naturally occurring reactivation model system are: (1) their tissue is extremely homogeneous, consisting of almost 100% parenchyma cells whose DNA content is unreplicated and arrested in G0, (2) as the cells are all arrested in G0 they do not need to be synchronized for induction, (3) the cells can be induced by the sole addition of the plant growth regulator auxin (Adamson, 1962; Yeoman and Mitchell, 1970; Minocha, 1979).
Here, the first molecular study of this system is reported, including the preparation of a Jerusalem artichoke cDNA library and the isolation of two cyclin (CYCD1;1 and CYCD3;1) and two cyclin-dependent kinase (CDK) (CDKA;1 and CDKB1;1) genes. The first expression analysis of cell-cycle genes in Jerusalem artichoke has been carried out and the applicability of this system as a model for studying the control of cell-cycle induction has been demonstrated.
|
Materials and methods |
|---|
|
TopAbstractIntroductionMaterials and methodsResults and discussionReferences |
|---|
Preparation of a Jerusalem artichoke cDNA library and isolation of cyclins and CDKs
A Jerusalem artichoke cDNA library was produced by cDNA synthesis from mixed polyadenylated RNA extracted from tubers, roots and young leaves. The cDNA was ligated with Lambda ZAPII vector and cell-cycle genes were isolated by screening the resultant library with several mixtures of probes from Arabidopsis (cyclin D1;1 (CYCD1;1), cyclin D3;1 (CYCD3;1) (Soni et al., 1995)) and Antirrhinum (CYCD1, CYCD3;1, CYCD3;2, CDC2a, CDC2b, CDC2c, CDC2d (Fobert et al., 1996; Gaudin et al., 2000)). Standard methods were used for the library preparation and screening by hybridization (Sorrell et al., 1999). Four separate screens for CYCD1, CYCD2, CDKA (CDC2a and b), and CDKB (CDC2c and d) genes were carried out. Representative positive clones from each screen were sequenced on both strands. Open reading frames (ORFs) were identified in all the sequences and the translated protein sequences were aligned to other plant cyclin and CDK genes using CLUSTAL X (Thompson et al., 1997). A phylogenetic tree was constructed using protdist and the Neighbour–Joining alogorithm with the PAM protein model in PIE (Phylogeny Interface Environment, http://www.hgmp.mrc.ac.uk/Registered/Webapp/pie/).
Plant material and treatment
For storage, Jerusalem artichoke tubers were harvested in late October or November, washed in sterile water, placed in damp sand and stored for up to 5 months at 4 °C. To prepare the tubers for cell-cycle induction, they were cut into cubes to remove the outer skin, placed in a 10% (v/v) solution of bleach (Super Bleach sterilizer, Coventry Chemical Ltd., Coventry, UK; max. 5% available chlorine) for 10 min and rinsed at least three times in sterile water under low-intensity green photographic safe light. Using a 1 cm diameter cork borer, transverse cylinders were removed from the washed cubes, avoiding any xylem rays. The cylinders were sliced to 1 mm thickness using a custom-made cutting machine and had a mean weight of 58.5±12.4 mg (n=50). The slices were incubated in B&A media (Bonner and Addicott, 1937) adjusted to pH 5.5 with 1 M NaOH, and with sucrose and the auxin, 2,4-D (1 mg ml–1 in DMSO) added to give final concentrations of 4% and 2 µg ml–1, respectively. Flasks containing media and tuber slices were covered with foil to keep them in darkness and left on a shaking platform at 25 °C.
Cell-cycle monitoring and RNA analysis
[Methyl-3H]thymidine incorporation was used to detect de novo DNA synthesis and hence to monitor the progression of the cell cycle (Minocha, 1979). Nine explants were removed at each time point and incubated for 30 min with 0.37 MBq of 2.6–3.2 TBq mmol–1 [methyl-3H]thymidine (Amersham Biosciences product TRK686). Excess 3H-thymidine was removed by rinsing with methanol, followed by further sequential washes with 5% trichloroacetic acid, 0.05 M formic acid, ethanol and ether. The explants were air-dried, and nucleic acid was extracted by incubating with 0.5 M perchloric acid at 70 °C for 2 h. Activity was then measured with a scintillation counter (using three sets of three replicates for each sample).
To block the explant cells in S phase, 100 mM hydroxyurea or 10 µg ml–1 aphidocolin was added after the explants had been incubated for 18 h. To block the cells in mitosis, 3.2 µg ml–1 of propyzamide or 30 µM oryzalin was added after 24 h of incubation. Total RNA was analysed by standard gel-blotting procedures, as described by Riou-Khamlichi et al. (2000), and by reverse-transcriptase PCR (RT-PCR) (Ausubel et al., 1987).
|
Results and discussion |
|---|
|
TopAbstractIntroductionMaterials and methodsResults and discussionReferences |
|---|
Synchronous S phase in the Jerusalem artichoke explant system
Most studies of dormancy and cell-cycle re-entry using Jerusalem artichoke tuber slices have concentrated on cytological and biochemical analysis (Yeoman and Mitchell, 1970; Yeoman and Davidson, 1971; Minocha, 1979; Minocha and DiBona, 1979; Bennici et al., 1982), and there has been little investigation of its control at the molecular level. For the analysis of tuber slices, progression into S phase was monitored by pulse-labelling of explants, incorporating 3H-thymidine into DNA, which indicates active DNA synthesis (Fig. 4A). Initial experiments compared tubers from two sources, after 2 weeks of storage, and confirmed the requirement for both auxin (2,4-D) and darkness for maximal induction of S phase (Fig. 1A), as previously shown (Yeoman and Davidson, 1971). Abscisic acid (ABA) has been reported to increase the proportion of cells involved in DNA replication (Minocha, 1979), but here no enhancement of the maximum rate of DNA synthesis was observed, with a possible broadening of the DNA replication peak (Fig. 1A). Subsequent experiments were carried out in darkness using only 2,4-D, and after a further week of storage of the tubers, explants from source b (Cambridge University Botanic Garden) were observed to give the sharpest peak of DNA replication at 20–25 h (Fig. 1B). Tubers from this source were used in subsequent work. Later experiments showed that after 7 weeks of storage, the S-phase peak was still pronounced, but delayed and broadened for 22–42 h (Fig. 4A), confirming the results of Minocha (1979). It was concluded that the length of S phase measured in cells allowed to continue through the cell cycle from induction varied from 7 h to12 h, and in cells temporarily blocked in early S phase by the addition of aphidicolin and then observed after the block had been removed, 3H-thymidine incorporation was observed for 10 h (data not shown). These times are in agreement with an earlier 3H-thymidine incorporation study of the Jerusalem artichoke cell cycle (Minocha, 1979).
|
|
Isolation of CDKs and cyclins from Jerusalem artichoke
To initiate molecular studies on this species, two cyclins and two CDKs from a Jerusalem artichoke cDNA library were isolated. Analysis of their DNA sequence suggests that they code for a CYCD1 with an open reading frame of 315 amino acids, named Heltu;CYCD1;1 (Accession no. AY063460); a CYCD3 with an ORF of 386 amino acids, named Heltu;CYCD3;1 (Accession no. AY063461); a CDKA with an ORF of 294 amino acids named Heltu;CDKA;1 (Accession no. AY063462) and a CDKB1 with an ORF of 304 amino acids named Heltu:CDKB1;1 (Accession no. AY063463). The cyclin proteins were named according to the conventions of Renaudin et al. (1996) and the CDK proteins according to Joubes et al. (2000). The protein sequence of both the CYCD1;1 and CYCD3;1 clones have the five key residues necessary for cyclin-CDK catalytic activity and the N-terminal LxCxE motif necessary for binding retinoblastoma protein. Similarly, the characteristic PSTAIRE and PPTALRE motifs were identified in the CDKA;1 and CDKB1;1 clones, respectively, in the regions predicted to bind to the cyclin partner (Joubes et al., 2000). The close relationships of all four genes to other plant cyclins and CDKs are illustrated in Fig. 2. To the authors’ knowledge, these sequences represent the first D-type cyclins described from the Compositae, and further illustrate the conservation of the Cyclin D group across the Angiosperms.
|
Expression analysis
The RNA expression of CYCD3;1 and CDKA;1 was compared in dormant tubers and their buds (Fig. 3). CYCD3;1 expression was not evident in the dormant tuber, but was found in the buds, whereas CDKA;1 was expressed in both the dormant tuber (at a low level) and the buds (at a high level). Expression of a CDKA;1 gene has also been observed in dormant potato tubers, and similarly, a higher level of expression was observed in actively growing shoot tips and a log-phase suspension (Campbell et al., 1996). Moreover, CDKA is expressed rather widely in Arabidopsis plants in all cells with the capacity to respond to signals promoting division (Hemerly et al., 1993). The particularly strong expression of CDKA;1 after dormancy break in the Jerusalem artichoke could indicate that it is linked to re-entry into the cell cycle, consistent with CDKA being the CDK partner of the D-type cyclins CYCD2 and CYCD3;1 in Arabidopsis (Healy et al., 2001)
|
Tuber explants were prepared and placed in media, as described above and in the Materials and methods, to induce cell-cycle entry. A clear peak of S phase was observed (Fig. 4A), and RNA samples were prepared from tuber explants at the times indicated. The RNA expression of CYCD1;1, CYCD3;1 and CDKA;1 genes was examined by Northern blotting (Fig. 4B). CDKA;1 appears to be expressed both in unstimulated tuber slices, confirming the result for dormant tubers (Fig. 3) and, subsequently, throughout the time-course examined. In contrast, CYCD3;1 and CYCD1;1 RNA expression was detected from the G1/S boundary at about 23 h and continued at a relatively constant level until the end of the experiment at 44 h. Interestingly, CYCD1;1 expression was detected in tuber explants with similar expression timing to CYCD3;1. Whilst CYCD1 expression has been detected in Antirrhinum seedlings, it was not detected during cell-cycle re-entry of Arabidopsis suspension-cultured cells (Riou-Khamlichi et al., 2000). CYCD3;1 expression is activated in late G1 shortly before S phase entry in tobacco BY-2 and Arabidopsis cells (Sorrell et al., 1999; Riou-Khamlichi et al., 2000), and the same timing was observed in cell-cycle re-entry from quiescent tuber explants.
The accumulation of transcripts in response to various inhibitors of cell-cycle progression (Planchais et al., 2000) was also examined (Fig. 4C). Aphidicolin and hydroxyurea are inhibitors of DNA polymerase 
and ribonucleotide reductase, respectively, and block progression at the G1/S boundary or in early S phase. Propyzamide and oryzalin are herbicides with anti-microtubule effects that block mitosis. RT-PCR analysis showed that CDKB1;1 transcripts are present in aphidicolin and hydroxyurea-treated cells, but are more abundant in cells blocked in mitosis. This is consistent with results observed in synchronized tobacco BY-2 cells (Sorrell et al., 2001) and Arabidopsis cells (Menges and Murray, 2002), which showed CDKB1;1 expression at a low level from early S phase and reaching a relatively higher level in early mitosis. CYCD1;1 and CYCD3;1 RNA levels were relatively high in mitotic-blocked cells, consistent with the continued expression of these genes throughout the cell cycle found in Arabidopsis. Indeed these results may suggest a higher level of expression in mitotic cells, as previously observed for CYCD2;1 and CYCD3;1 in tobacco BY-2 cells (Sorrell et al., 1999), or may reflect further cells in the explants entering the cell cycle during the period of the block. Both CYCD1;1 and CYCD3;1 were observed in hydroxyurea-blocked cells, whereas in aphidicolin-treated cells, CYCD1;1 transcripts were present, but CYCD3;1 was barely detected. Since aphidicolin and hydroxyurea block at similar positions in the cell cycle, this may suggest a specific role for CYCD3 in responding to the disruption of DNA synthesis, which has also been observed for similarly treated Arabidopsis cells (Menges and Murray, 2002).
In conclusion, the foundations for studies of cell-cycle induction in the Jerusalem artichoke system have been laid. Several of the genes likely to be involved in the control of this stage in the cell cycle have been identified. Further functional studies, including the isolation of other cyclin and CDK genes and identifying their interacting partners, will allow a greater understanding of this important juncture in the cell cycle.
|
Acknowledgements |
|---|
We thank David Hanke for help with the Jerusalem artichoke tuber system, and John Doonan, Cambridge University Botanic Garden and Oswyn Murray for gifts of tubers. We also thank Graham Armstrong, our Publications Manager, for his contribution to the manuscript and figures. This work was supported by BBSRC grant PO1552.
|
References |
|---|
|
TopAbstractIntroductionMaterials and methodsResults and discussionReferences |
|---|
Adamson D. 1962. Expansion and division in auxin-treated plant cells. Canadian Journal of Botany 40, 719–744.
Ausubel FM, Brent R, Kingston RE, Moore DM, Seidman JG, Smith JA, Struhl K. 1987. Current protocols in molecular biology. New York: John Wiley & Sons.
Bennici A, Cionini PG, Gennai D, Cionini G. 1982. Cell cycle in Helianthus tuberosus tuber tissue in relation to dormancy. Protoplasma 112, 133–137.[CrossRef]
Bonner J, Addicott F. 1937. Cultivation in vitro of excised pea roots. Botanical Gazette 99, 144–170.[CrossRef]
Campbell MA, Suttle JC, Sell TW. 1996. Changes in cell cycle status and expression of p34 (cdc2) kinase during potato tuber meristem dormancy. Physiologia Plantarum 98, 743–752.[CrossRef]
Favali MA, Serafini-Fracassini D, Sartorato P. 1984. Ultrastructure and autoradiography of dormant and activated parenchyma of Helianthus tuberosus. Protoplasma 123, 192–202.[CrossRef]
Fobert PR, Gaudin V, Lunness P, Coen ES, Doonan JH. 1996. Distinct classes of cdc2-related genes are differentially expressed during the cell division cycle in plants. The Plant Cell 8, 1465–1476.[Abstract]
Gaudin V, Lunness PA, Fobert PR, Towers M, Riou-Khamlichi C, Murray JA, Coen E, Doonan JH. 2000. The expression of D-cyclin genes defines distinct developmental zones in snapdragon apical meristems and is locally regulated by the Cycloidea gene. Plant Physiology 122, 1137–1148.
Harland J, Jackson JF, Yeoman, MM. 1973. Changes in some enzymes involved in DNA biosynthesis following induction of division in cultured plant cells. Journal of Cell Science 13, 121–138.
Healy JM, Menges M, Doonan JH, Murray JAH. 2001. The Arabidopsis D-type cyclins CycD2 and CycD3 both interact in vivo with the PSTAIRE cyclin-dependent kinase Cdc2a but are differentially controlled. Journal of Biological Chemistry 276, 7041–7047.
Hemerly AS, Ferreira P, de Almeida Engler J, Van Montagu M, Engler G, Inzé D. 1993. cdc2a expression in Arabidopsis is linked with competence for cell division. The Plant Cell 5, 1711–1723.[Abstract]
Joubes J, Chevalier C, Dudits D, Heberle-Bors E, Inzé D, Umeda M, Renaudin JP. 2000. CDK-related protein kinases in plants. Plant Molecular Biology 43, 607–620.[CrossRef][Web of Science][Medline]
Menges M, Murray JAH. 2002. Synchronous Arabidopsis suspension cultures for analysis of cell cyle gene activity. The Plant Journal 30, 203–212.[CrossRef][Web of Science][Medline]
Minocha SC. 1979. Abscisic acid promotion of cell division and DNA synthesis in Jerusalem artichoke tuber tissue cultured in vitro. Zeitschrift für Pflanzenphysiologie 92, 327–339.
Minocha SC, DiBona S. 1979. Effect of auxin and abscisic acid on RNA and protein synthesis prior to the first cell division in Jerusalem artichoke tuber tissue cultured in vitro. Zeitschrift für Pflanzenphysiologie 92, 367–374.
Mitchell JP. 1967. DNA synthesis during the early division cycles of Jerusalem artichoke callus cultures. Annals of Botany 31, 427–435.
Nagata T, Kumagai F. 1999. Plant cell biology through the window of the highly synchronized tobacco BY-2 cell line. Methods in Cell Science 21, 123–127.[CrossRef][Medline]
Nagata T, Nemoto Y, Hasezawa S. 1992. Tobacco BY-2 cell line as the ‘HeLa’ cell in the cell biology of higher plants. International Review of Cytology 132, 1–30.
Planchais S, Glab N, Inzé D, Bergounioux C. 2000. Chemical inhibitors: a tool for plant cell cycle studies. FEBS Letters 476, 78–83.[CrossRef][Web of Science][Medline]
Renaudin JP, Doonan JH, Freeman D, et al. 1996. Plant cyclins: a unified nomenclature for plant A-, B- and D-type cyclins based on sequence organization. Plant Molecular Biology 32, 1003–1018.[CrossRef][Web of Science][Medline]
Riou-Khamlichi C, Menges M, Healy JM, Murray JA. 2000. Sugar control of the plant cell cycle: differential regulation of Arabidopsis D-type cyclin gene expression. Molecular Cell Biology 20, 4513–4521.
Serafini-Fracassini D, Bagni N, Cionini PG, Bennici A. 1980. Polyamines and nucleic acids during the first cell cycle of Helianthus tuberosus tissue after the dormancy break. Planta 148, 332–337.[CrossRef]
Setiady YY, Sekine M, Hariguchi N, Kouchi H, Shinmyo A. 1996. Molecular-cloning and characterization of a cDNA clone that encodes a cdc2 homolog from Nicotiana tabacum. Plant and Cell Physiology 37, 369–376.
Setterfield G. 1963. Growth regulation in excised slices of Jerusalem artichoke tuber tissue. Symposia of the Society for Experimental Biology 17, 98–126.[Medline]
Soni R, Carmichael JP, Shah ZH, Murray JAH. 1995. A family of cyclin D homologs from plants differentially controlled by growth regulators and containing the conserved retinoblastoma protein interaction motif. The Plant Cell 7, 85–103.[Abstract]
Sorrell DA, Combettes B, Chaubet-Gigot N, Gigot C, Murray JA. 1999. Distinct cyclin D genes show mitotic accumulation or constant levels of transcripts in tobacco bright yellow-2 cells. Plant Physiology 119, 343–352.
Sorrell DA, Menges M, Healy JM, et al. 2001. Cell cycle regulation of cyclin-dependent kinases in tobacco cultivar bright yellow-2 cells. Plant Physiology 126, 1214–1223.
Thompson JD, Gibson TJ, Plewniak F, Jeanmougin F, Higgins DG. 1997. The CLUSTAL X windows interface: flexible strategies for multiple sequence alignment aided by quality analysis tools. Nucleic Acids Research 25, 4876–4882.
Yeoman MM, Davidson AW. 1971. Effect of light on cell division in developing callus cultures. Annals of Botany 35, 1085–1100.
Yeoman MM, Mitchell JP. 1970. Changes accompanying the addition of 2,4-D to excised Jerusalem artichoke tuber tissue. Annals of Botany 34, 799–810.
CiteULike 
Connotea 
Del.icio.us What's this?
This article has been cited by other articles:
|
|
 |
|
  O. A. Koroleva, M. Tomlinson, P. Parinyapong, L. Sakvarelidze, D. Leader, P. Shaw, and J. H. Doonan CycD1, a Putative G1 Cyclin from Antirrhinum majus, Accelerates the Cell Cycle in Cultured Tobacco BY-2 Cells by Enhancing Both G1/S Entry and Progression through S and G2 Phases PLANT CELL, September 1, 2004; 16(9): 2364 - 2379. [Abstract] [Full Text] [PDF]
|
|
| ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||