Journal of Experimental Botany, Vol. 52, No. 361, pp. 1625-1633,
August 1, 2001
© 2001 Oxford University Press
Original Papers |
Analysis of cell division parameters and cell cycle gene expression during the cultivation of Arabidopsis thaliana cell suspensions
1 Vakgroep Moleculaire Genetica, Departement Plantengenetica, Vlaams Interuniversitair Instituut voor Biotechnologie (VIB), Universiteit Gent, K.L. Ledeganckstraat 35, B-9000 Gent, Belgium
2 Institut National de la Recherche Agronomique, Laboratoire d'Ecophysiologie des Plantes sous Stress Environnementaux, 2 Place Viala, F-34060 Montpellier, France
Received 2 January 2001; Accepted 30 March 2001
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Abstract |
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Arabidopsis thaliana cell suspension cultures were characterized for the first time in detail in terms of biomass accumulation, cell division rate and cell cycle phase durations. Subsequently, this model system was used to follow the transcription profile of key cell cycle genes during a complete cultivation cycle. According to the calculated changes in the relative division rate over time, the cell cycle genes could be classified into four groups based on their transcriptional expression pattern. These differential patterns of gene expression are discussed with respect to the putative roles of the different cell cycle genes in the division cycle. Analysis of protein levels showed that mRNA levels did not correlate with protein levels in all cases. Results obtained in other systems, such as BY-2 cell suspensions or plants, confirm that cell suspension cultures of A. thaliana are suitable for the analysis of cell cycle regulation.
Key words: Arabidopsis thaliana, cell cycle, gene expression.
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Introduction |
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The general mechanism of cell cycle control is evolutionarily conserved among eukaryotes. The progression through the cell cycle phases (G1/S/G2/M) is driven by heterodimeric Ser/Thr protein kinases consisting of a catalytic subunit, the cyclin-dependent kinase (CDK) and an activating subunit, cyclin. Some of the molecular components of the cell cycle machinery originally discovered in yeast and animals have been found in higher plants (Mironov et al., 1999
). Five types of CDK-like proteins and numerous cDNAs encoding putative cyclins have been identified in a diverse range of plant species. The cyclins have been classified in nine groups: A1, A2, A3, B1, B2, D1, D2, D3, and D4 reflecting their homologies to the mammalian cyclins A, B, and D (Renaudin et al., 1996; De Veylder et al., 1999). The complexes formed by association of CDKs and cyclins can interact with other cell cycle regulators. CKS1At, a plant homologue of p13suc1 of fission yeast and the CDK subunit (CKS) of budding yeast and vertebrates has been isolated in Arabidopsis thaliana through a two-hybrid screen using CDKA;1 as bait (De Veylder et al., 1997). CKS proteins have been proposed to act as a docking factor for positive and negative regulators of CDK activity (Bourne et al., 1996). Also CDK inhibitors (CKIs) have been isolated by the use of the two-hybrid system (Wang et al., 1997; Lui et al., 2000; De Veylder et al., 2001). In yeast and animals, CKI proteins inhibit cell cycle progression through their association with CDK complexes (Nakayama and Nakayama, 1998). More recently, plant retinoblastoma protein (Rb) and E2F homologous cDNAs have been cloned (Gutierrez, 1998). In general, transcription of all these cell cycle genes is strongly regulated, allowing the correct progression through the control points of the cell cycle situated at the late G1 phase and at the G2/M boundary. Moreover, the cellular activity of the gene products is controlled by their subcellular localization, phosphorylation status and proteolytic degradation (Mironov et al., 1999). Arabidopsis cell suspensions were used to study cell cycle regulation. The combined analyses of cell division rate and expression of cell cycle genes allowed the classification of genes into four classes depending on their different patterns of expression in the course of a cultivation cycle.
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Materials and methods |
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Arabidopsis thaliana cell suspension cultures
Cell suspension cultures of Arabidopsis thaliana (L.) Heynh. ecotype Col-O were grown as described previously (Glab et al., 1994
) in Gamborg's B5 medium (Sigma, St Louis, MO) containing 3% sucrose and 1 µM 
-naphthaleneacetic acid (Sigma). Cells were diluted 10-fold on a weekly basis with a precise quantity of inoculum: 5 ml of liquid culture at day 7, the equivalent of 1 g of fresh cells, was added to 45 ml fresh medium, and cultivated in a 250 ml Erlenmeyer flask.
Kinetic analysis of the cell suspension cultures
Changes in biomass over time were followed daily for 15 d after the dilution of a cell suspension culture. Relative growth rate (RGR; mg produced mg-1 and d-1) was calculated as the slope (at time j) of the relationship between the logarithm of the biomass (B) and time:
 | (1) |
Cell numbers were determined daily after dilution of the cell suspension culture. One millilitre of cell suspension was treated with 1 ml of enzymatic solution (2% cellulase, 0.1% pectinolyase in 0.66 M sorbitol) for 20 min at 37 °C. Cell density was then determined with a haemocytometer. Measurements were performed in triplicate on each of two separate samples from the same culture. Repetition on five different cultures was performed.
Dead and living cells were distinguished with fluorescein diacetate staining. Cell suspension cultures (200 µl) were incubated for 2 min in 0.1% fluorescein diacetate. The proportion of dead cells (%dead) in the cell suspension culture was estimated by counting dead and living cells under a fluorescent microscope. The number of dead cells (Ndead) in the culture was then calculated as:
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 | (3) |
Assuming that all cells divide at similar rates, the doubling time, i.e. the time required for a population of cells at time j to double in number, can be considered as a correct estimate of the duration of cell cycle (tcycle, j) (Green and Bauer, 1977; Beemster et al., 1996). From equation (3), it follows that:
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Nuclei isolation, flow cytometry and duration of cell cycle phases
Cells were first treated with an enzyme solution (2% cellulase, 0.1% pectinolyase in 0.66 M sorbitol) for 20 min at 37 °C. After two washes with B5 medium, cells were resuspended in Galbraith buffer (Galbraith et al., 1983) and filtered through a 30 µm nylon mesh. The obtained nuclei were stained with the fluorescent dye 4',6-diamidino-2-phenylindole (5 µg ml-1) and subjected to flow cytometric analysis. Fluorescence intensity of at least 10 000 nuclei was measured using a BRYTE HS flow cytometer (Biorad, Hercules, CA, USA).
Duration of cell cycle phases can be determined by combining calculation of cell doubling time with flow cytometry data (Webster and MacLeod, 1980; Tardieu and Granier, 2000). The M phase was not considered in the calculation because the nuclear envelope is absent during M phase. Because the duration of the M phase has been shown to be short both in meristems (Nougarède and Rembur, 1985) and cell suspensions (Nagata et al., 1992; Francis et al., 1995), its exclusion does not modify the conclusions on the duration of the other phases.
RNA extraction and reverse transcription PCR
Reverse transcription (RT)-PCR was used because levels of expression of most cell cycle genes are too low to be detected with classical Northern hybridization. Total RNA was extracted with Trizol (Gibco/BRL, Gaithersburg, MD, USA) according to the manufacturer's instructions.
Two independent protocols were used for cDNA preparation, the Superscript Preamplification System (Gibco/BRL) and the kit universal riboclone cDNA synthesis system (Promega, Madison, WI, USA). Gene expression obtained with these two kits was compared as an additional control. The Superscript Preamplification System (Gibco/BRL) was used for first-strand cDNA synthesis with oligo(dT) primer solution on a 3 µg RNA template. The universal riboclone cDNA synthesis system (Promega) used mRNA prepared with magnetic particules Dynabeads oligo(dT) 25 (Dynal, Oslo, Norway) from 75 µg of total RNA. First-strand synthesis of the cDNA was driven on half the volume of the mRNA obtained (approximately 0.5 µg). The quantity of cDNA obtained was estimated under UV by comparing the sample with a standard dilution of plasmid DNA labelled with ethidium bromide. The primer combinations used for the different cell cycle genes are presented in Table 1.
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Five nanograms of cDNA from the kit universal riboclone cDNA synthesis system (Promega) or 1 µl of the 20 µl of cDNA product with the Superscript Preamplification System (Gibco/BRL) were submitted to the RT-PCR with 300 ng of each gene-specific primer, 160 µM of dNTP, 10 µl of PCR buffer, and 0.8 µl of Taq polymerase (Amersham Pharmacia Biotech, Little Chalfont, UK) in a total volume of 100 µl.
The PCR was performed with one denaturation cycle of 4 min at 94 °C and 15 or 20 cycles of denaturation for 45 s at 94 °C, primers annealing for 45 s at 55 °C, and elongation for 45 s at 72 °C. PCRs were done under conditions preserving linearity between the RNA input and the amount of PCR product.
Southern blot and non-radioactive hybridization
After electrophoresis, PCR products were blotted on Hybond-N+ membranes (Amersham Pharmacia Biotech) as described (Southern, 1975). Fluorescein-labelled probes specific for the different cell cycle genes were prepared with the GeneImages random prime labelling module (Amersham Pharmacia Biotech) and the GeneImages CDP-star detection module allowed the probe detection (Amersham Pharmacia Biotech). The signals were revealed by autoradiography after they were exposed for a few minutes to a Kodak film.
Western blot analysis
Protein extracts were prepared from A. thaliana cell suspensions in homogenization buffer (HB) containing 50 mM TRIS-HCl (pH 7.2), 60 mM ß-glycerophosphate, 15 mM nitrophenyl phosphate, 15 mM EGTA, 15 mM MgCl2, 2 mM dithiothreitol, 0.1 mM vanadate, 50 mM NaF, 20 µg ml-1 leupeptin, 20 µg ml-1 aprotein, 20 µg ml-1 soybean trypsin inhibitor, 100 µM benzamidine, 1 mM phenylmethylsulphonylfluoride, and 0.1% Triton X-100. Protein extract (30 µg) was denaturated for 10 min at 95 °C in SDS loading buffer. Proteins were separated on a 12.5% SDS-PAGE gel and immunoblotted on a nitrocellulose membrane (Hybond-C+; Amersham Pharmacia Biotech). Filters were blocked overnight with 2% milk powder in phosphate-buffered saline containing 0.1% Tween-20 (PBST), washed five times with PBST, and probed for 2 h with anti-CDKA;1 (1/5000), anti-CDKB1;1 (1/2000), and anti-CKS1At (1/5000) diluted in blocking solution. The blots were rinsed five times with PBST and incubated for 1 h with anti-rabbit horseradish peroxidase conjugated antibodies (Amersham Pharmacia Biotech) diluted in the blocking solution. The membranes were washed five times with PBST, and signals were developed using a chemiluminescent detection kit (NEN Life Science Products Inc., Boston, MA, USA).
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Results |
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Growth characteristics of an Arabidopsis thaliana cell suspension culture
A freshly diluted Arabidopsis culture was analysed daily for 15 d. Biomass increased linearly over time for approximately 10 d after cell dilution. Afterwards, it reached a plateau (Fig. 1a
). RGR was high during the first 2 d after cell dilution, decreased at day 3 to 0.2 mg mg-1 d-1 and stabilized (0.3 mg mg-1 d-1) between day 4 and day 6 (Fig. 1b). At day 7, RGR decreased and was approximately null from day 10 on.
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Cell number increased in the suspension from day 1 to day 7 (Fig. 2a
). Cell numbers remained high between days 7 and 11, but afterwards decreased over time. This decrease in cell numbers coincided with an increase in cell death (Fig. 2a). The percentage of cell death was low and relatively constant during the first 7 d after cell dilution (approximately 3% of total cell number), but increased linearly afterwards. On day 14, the proportion of dead cells was 38% of the total cell number. Because cell death was very low during the cell division period, RDRs calculated by considering either the total cell number or living cells only were approximately the same (Fig. 2b). Only RDRs calculated with the total number of cells will be considered hereafter. The RDR increased 2-fold between day 1 and 2 and, thereafter, decreased over time to become negligible around day 8. The shortest cell doubling time (24.5 h) was observed on day 2 (Fig. 3a ). Cell doubling time increased slowly between day 2 and day 5 and reached 53 h on day 5, after which it increased dramatically and reached 250 h on day 6.
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The percentage of cells in G1 was always higher than in G2 or S (Fig. 3b
). The percentage of cells in G1 and S increased over time, whereas that in G2 decreased. On the first day after dilution, durations of the S and G1 phases were approximately the same (11.4 h and 10.9 h, respectively) and that of G2 was close to 26 h at this time. Durations of G2 and S decreased between days 1 and 2 and reached their lowest value on day 2 (10 h and 2.5 h, respectively). Duration of all the phases increased with time after day 2. The increase in cell cycle duration observed between days 2 to 5 was essentially due to the more pronounced increase in duration of the G1 phase.
Transcriptional regulation of cell cycle genes
The expression of various cell cycle genes was analysed at four time points after initial dilutions of the culture: day 1 (characterized by a high RDR), day 5 (specific for the exponential accumulation of biomass and decrease in cell division rate), day 8 (entry into stationary phase, total cessation of cell division), and day 12 (stationary phase, linear increase in cell death).
Expression of all studied cyclin genes and CDKB1;1 decreased with time during the culture cultivation while expression of CDKA;1 remained constant
The cyclin genes CYCA2;1, CYCB1;1, CYCB2;1, and CDKB1;1 displayed decreasing levels of mRNA during a culture cycle (Fig. 4). CYCB1;1 and CYCB2;1 show a very close expression pattern and their corresponding mRNA levels decreased rapidly compared to CYCA2;1. In contrast to A- and B-type cyclins, the steady-state levels of the D-type cyclins (CYCD2;1, CYCD3;1, and CYCD4;1) decreased in a shorter time interval from day 1 to day 5. The mRNA levels of the CDKA;1 gene remained constant during the cultivation period, whereas those of CDKB1;1 decreased but to a minor extent compared to cyclins. From day 1 to day 12 it was reduced only by a factor of 2.
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A close pattern of expression for all the studied CKI and CKS genes
The maximum of mRNA levels for the CKI genes (ICK1, ICK2, KRP3, and KRP4) was reached during the exponential accumulation in biomass, while the cells actively divided (Fig. 4). The mRNA levels of ICK1 and KRP3 were up to 6-fold higher on day 5 than on day 8, while cell division rate was close to 0. However, ICK2 and KRP4 were still expressed in the early stationary phase. CKS gene expression increased with time to reach a maximum value around day 5, and decreased afterwards (Figs 4, 5).
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E2Fb was expressed only at the beginning and end of the culture
The E2Fb gene presented a particular pattern of mRNA accumulation. Expression was detected at the beginning of the cultivation at day 1. A 7-fold decrease from day 1 to day 5 was recorded. Remarkably, expression could again be observed when cells were in late stationary phase (day 12) (Fig. 4).
Translational regulation of cell cycle gene expression
Currently, only a few specific antibodies against plant cell cycle proteins are available. However, the results obtained demonstrate that mRNA levels and protein levels did not correlate in all cases.
The levels of CDKA;1 mRNA and proteins remained constant during the whole cultivation (Fig. 5). For CDKB1;1, mRNA and protein levels showed a similar decreasing accumulation pattern. The above correlations between amounts of transcripts and proteins were not observed for CKS1At. The CKS1At mRNA levels increased until days 5–6 and remained high until late stationary phase. In contrast, on Western blotting a constant CKS1At signal was observed from day 1 to day 4, then the CKS1At protein amounts decreased to become undetectable after day 7.
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Discussion |
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Growth characteristics of Arabidopsis cell suspension culture
Three days after dilution, the relative increase in biomass of the cell suspension culture is stabilized indicating that with time changes in biomass follow an exponential progression. After this phase, RGR decreases with time. As in plant leaves (Granier and Tardieu, 1998
), in cell suspensions, cell division and increase in biomass occur first simultaneously and then cell division stops a few days before the arrest of biomass increase.
RDR is never constant, indicating that temporal changes in cell numbers are never exponential. After day 2, the cell cycle duration increases with time as documented in BY-2 cell suspension (Francis et al., 1995). The peak of RDR observed at day 2 correlates perfectly with the measured temporal changes in CDK activity of proliferating Arabidopsis cells suspensions grown under similar conditions (Stals et al., 2000).
As noticed for the tobacco BY-2 cell line (Francis et al., 1995), the calculations lead to the conclusion that, with time, the increase in cell cycle duration is due to a progressive lengthening of the G1 phase without any significant changes on the duration of S and G2. Lengthening of the G1 phase has also been reported in plants under different situations: spatial gradient in RDR in dicotyledonous leaves (Granier and Tardieu, 1998), effects of temperature (Wimber, 1966), sucrose starvation (Van't Hof et al., 1973), toxic metals on roots (Powell et al., 1986), and water deficit or reduction in incident light on leaves (Granier and Tardieu, 1999a, b).
Different behaviours of CDKA;1 and CDKB1;1: evidence for a post-translational regulation of CDKA;1
Expression of CDKA;1 and CDKB1;1 genes correlates with their protein level. Whereas the CDKA;1 mRNA and protein levels remain constant with time, the CDKB1;1 protein and mRNA levels gradually decrease in parallel with a reduction in cell division rate. Transcript and protein levels of CDKA;1 do not correlate with cell division rate or with the CDKA;1 kinase activities as quantified by the affinity of this protein to p9CKShs1 beads and in vitro by phosphorylation of histone H1 (Stals et al., 2000). The differences in activity and in abundance of CDKA;1 indicate a post-translational regulation of this kinase, as shown in wheat (Schuppler et al., 1998) and maize leaves (Granier et al., 2000).
Levels of cyclin gene expression correlate with cell division rate
The expression of cyclin genes decreases with time, following the reduction in cell division rate. However, two different patterns emerge: A- and B-type cyclins are weakly produced when RDR is negligible (on day 8). In contrast, D-type cyclins are not expressed while the cell division rate is null. In whole plants, CYCA2;1 is expressed in dividing cells, but also in specific non-actively dividing cells, such as parenchyma and root pericycle (Burssens et al., 2000). CYCB1;1 expression is linked to active cell division because CYCB1;1 transcripts are exclusively detected in newly forming organs and tissues (Ferreira et al., 1994). Such differential behaviour of A- and B-type cyclins is not visible in this model system.
In plants, D-type cyclins are involved in a direct response to nutritional signals and plant hormones (Soni et al., 1995; Fuerst et al., 1996; De Veylder et al., 1999). In Arabidopsis cell suspensions, the expression level of CYCD2;1 and CYCD4;1 is significantly reduced after growth in media depleted of hormones and sucrose and, after re-addition of sucrose, the initial transcript levels are rapidly reached. CYCD3;1 transcript levels depend strongly on cytokinin and are induced at the G1-to-S transition after this phytohormone is added again. These observations imply a function for the D-type cyclins as part of the cellular machinery that integrates diverse signals impinging on the cell cycle (Fuerst et al., 1996). The absence, or very low abundance, of D-type cyclins after day 5 of subculturing suggests that, at this stage, essential mitogenic signals are depleted in the medium.
Unfortunately, no specific antibodies against cyclins of A. thaliana are available to follow the cyclin evolution at the protein level. Nevertheless, the amount of CYCB2;1 that is co-purified with CDKA;1 on p9CKShs1 beads is low 4 d after subculturing, while the associated kinase activity decreases by 50% (Stals et al., 2000). The decreasing amount of CYCB2;1 proteins correlate with decreasing transcript levels. Although preliminary, this finding suggests that in plants, as in yeast, cyclin transcript levels reflect the levels of the corresponding proteins (Nasmyth, 1993).
CKIs and CKS1At, a close pattern of regulation
In cell suspension cultures, the CDK inhibitors, ICK1 and KRP3, are strongly expressed during the exponential growth phase in which cells divide actively, indicating a role at the G1 and G2 checkpoints. Transcriptional activation of CKIs could be necessary to control the correct progression through the different phases of the cell cycle. Recently, animal CKIs were shown to play a role as adaptor proteins that help to assemble CDK/cyclin complexes (LaBaer et al., 1997; Cheng et al., 1999). A similar function might be true for ICK1 and KRP3. In contrast, ICK2 and KRP4 are expressed when cell division rate is high, but also when division stops and when cell weight increases due to cell expansion only. In animals, CKIs have been postulated to play a distinct role in differentiation. Cell suspensions are not really appropriate to investigate the role of CKIs in such a mechanism. In whole plants, CKI genes are involved in the exit of mitosis and are strongly expressed in differentiated tissues that have stopped dividing (L De Veylder, unpublished data).
Like for ICK2 and KRP4, transcription of CKS1At is maintained at high levels even after the cessation of cell division. However, no correlation between protein and mRNA levels has been recorded. An increase of the transcript level until days 5–6 is noticed while the amount of protein decreases over the same period, suggesting that an active degradation mechanism is switched on that suppresses the accumulation of the CKS1At protein. Alternatively, the translation of the mRNA could be repressed. Because the CKS proteins are essential for cell division, down-regulation of the CKS1At protein level may be one of the mechanisms that arrests the cell cycle under unfavourable conditions.
E2Fb, a particular pattern of expression
The E2Fb transcript accumulation pattern can be correlated with an expression in non-cycling cells or a G1 phase-specific expression, as shown by flow cytometry. Indeed after day 6, the population of nuclei in G1 continues to increase to reach 80% at day 12 (data not shown). These hypotheses are also consistent with a dual role of the animal E2F in S phase entry and apoptosis (Lavia and Jansen-Dürr, 1999). Further studies need to be done to analyse in detail these possibilities. However, the pattern observed for this gene is different from that of D-type cyclins, also involved in G1-to-S transition. This difference could be explained by a different sensitivity to mitogens present in the culture medium. Hormones or sucrose do not clearly affect E2Fb expression, in contrast to D-type cyclins that are sensitive to mitogens (Soni et al., 1995; Fuerst et al., 1996; De Veylder et al., 1999; Riou-Khamlichi et al., 1999; C Richard, unpublished data).
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Conclusions |
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TopAbstractIntroductionMaterials and methodsResultsDiscussionConclusionsReferences |
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An A. thaliana cell suspension culture, of which the main cell cycle parameters were determined, was used as a model system for studying the transcriptional regulation of key cell cycle genes during a cultivation cycle. The genes examined can be classified into four groups according to their transcript profile: (i) a gene with a constant level of expression independently of variations in cell division rate (CDKA;1); (ii) genes with a decreasing expression parallel to decreasing cell division rate during cultivation (A-, B-, and D-type cyclins; CDKB1;1); (iii) genes with a peak in transcript level when the relative division rate is maximum (ICK1, ICK2, KRP3, KRP4, CKS1At); and (iv) a gene expressed only at the start and the end of the cultivation cycle, when cell division was high and arrested, respectively (E2Fb). Although protein levels do not always correlate with the transcript profile, as demonstrated here for the CKS1At gene, the observed differences in temporal expression of the examined cell cycle genes could correspond with differential functions of their gene products in the cell cycle. Thus, the A. thaliana culture system is useful for cell cycle studies.
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Acknowledgments |
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The authors wish to thank Dr Isabelle Landrieu and Dr James Dat for critical reading of the manuscript, Martine De Cock for help in preparing it, and Rebecca Verbanck and Stijn Debruyne for illustrations. This work was supported by grants from Interuniversity Poles of Attraction Programme (Belgian State, Prime Minister's Office—Federal Office for Scientific, Technical and Cultural Affairs; P4/15). CR and CG are indebted to the Biopôle végétal d'Amiens (France) and the Ministère des Affaires Etrangères (France) for a PhD fellowship and a Lavoisier post-doctoral fellowship, respectively.
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3 To whom correspondence should be addressed. Fax: +32 9 264 5349. E-mail: diinz{at}gengenp.rug.ac.be
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