Journal of Experimental Botany, Vol. 54, No. 393, pp. 2669-2678,
December 1, 2003
© 2003 Oxford University Press
Activation of cell proliferation by brassinolide application in tobacco BY-2 cells: effects of brassinolide on cell multiplication, cell-cycle-related gene expression, and organellar DNA contents
Received 11 August 2003; Accepted 22 September 2003
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1 Plant Functions Laboratory RIKEN, 2-1, Hirosawa, Wako, Saitama 351-0198, Japan
2 1-7-22, Suehirocho, Tsurumi-ku, Yokohama, Kanagawa, 230-0045 Japan
3 Department of Biological Sciences, Nara Women’s University, Nara 630-8506, Japan
4 Department of Integrated Biosciences, Graduate School of Frontier Sciences, University of Tokyo, Chiba 277-8562, Japan
5 Department of Biological Sciences, Graduate School of Science, University of Tokyo, Tokyo 113-0033, Japan
* To whom corresponence should be addressed. Fax: +81 48 462 4674. E-mail: yutakam{at}postman.riken.go.jp Present address: National Institute of Fruit Tree Science, Shizuoka 424-0292, Japan. Present address: Department of Life Science, Rikkyo University, Tokyo 171-8501, Japan.
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Abstract |
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Brassinosteroids (BRs) are steroidal phytohormones that are essential for many processes in plant growth and development, such as cell expansion, vascular differentiation, and responses to stress. The effects of BRs on cell division are unclear, as attested by contradictory published results. To determine the effect of BRs on cell division, the tobacco (Nicotiana tabacum) BY-2 cell line, which is a widely-used model system in plant cell biology, was used. It was found that brassinolide (BL) promoted cell division only during the early phase of culture and in the absence of auxin (2,4-D). This promotion of cell division was confirmed by RNA gel blot analyses using cell-cycle-related gene probes. At later stages in the culturing periods of BL-supplied and 2,4-D-supplied BY-2 cells, differences in cell multiplication and cell-cycle-related gene expression were observed. Moreover, the BL-treated BY-2 cells had morphological differences from the 2,4-D-treated cells. To determine whether suppressed organellar DNA replication limited this promotion of cell division during the early culture phase, this replication was examined and it was found that BL treatment had no effect on activating organellar (plastid- and mitochondrial-) DNA synthesis. As preferential organellar DNA synthesis, which is activated by 2,4-D, is necessary during successive cell divisions in BY-2 cells, these data suggest that the mechanism of the promotion of cell division by BL treatment is distinct from that regulated by the balance of auxin and cytokinin.
Key words: Brassinosteroids, BY-2 cells, cell proliferation, organellar DNAs.
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Introduction |
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Brassinosteroids (BRs) are steroid hormones that are found in various tissues of plants throughout the plant kingdom. Since the discovery of the structure of BR (Grove et al., 1979), extensive biochemical and analytical studies have come close to clarifying the BR biosynthetic pathway (Fujioka et al., 2000; Fujioka and Yokota, 2003; Noguchi et al., 2000), and the structures of more than 50 BRs have been identified. Biochemical, analytical, physiological, and genetic studies have all shown that BRs are essential for plant growth and development. Naturally occurring or exogenously applied BRs affect a wide range of physiological processes, including cell expansion, cell division, vascular differentiation, reproduction, and stress resistance (Clouse and Sasse, 1998; Bishop and Koncz, 2002; and references therein). However, the role of BRs in cell division remains unclear, as contradictory results have been reported. For example, promotion of cell division by BRs has been reported in Chinese cabbage and in petunia protoplasts in the presence of auxin and cytokinin (Nakajima et al., 1996; Oh and Clouse, 1998), whereas inhibitory effects have been reported in Agrobacterium tumefaciens-transformed hormone-autonomous tobacco cells (Roth et al., 1989). Therefore, whether BRs have a role in cell division remains an open question. Hu et al. (2000) reported that BRs promote cell division by enhancing CycD3 expression in Arabidopsis callus and suspension cells. However, because these studies used micro-callus-like large cell clusters, which often form in plant cell cultures, it was difficult to determine the extent of the effect of BRs on cell division, since BRs have been shown to affect cell expansion. Moreover, in such cells, the microenvironment surrounding each cell differs according to its position in the cell cluster, which may lead to heterogeneity in the cell population. Thus, in order to determine the effect of BRs on cell division precisely, an exceptionally homogeneous cell line is necessary.
Some of the physiological effects of BRs have been shown to be related to the actions of auxin (Yopp et al., 1979, 1981; Katsumi, 1985). In addition, a comprehensive microarray analysis revealed that most of the known auxin-inducible genes and various homologues of such genes are also induced by brassinolide (BL) application (Goda et al., 2002), suggesting that a marked overlap exists between the BR and auxin signalling pathways. Again, conflicting results have been reported regarding the relationships between the effects of BR and auxin (reviewed in Clouse and Sasse, 1998). However, these observations have almost exclusively been derived from studies of cell elongation. Since auxin is a major growth factor that is required for the proliferative growth of plant cells, the relationship between the effects of BR and auxin on cell proliferation is an intriguing question.
To address the questions mentioned above, tobacco Bright Yellow-2 (BY-2) cells were used. This culture system has two advantages. First, BY-2 cells are rapidly proliferating meristematic cells with considerable homogeneity, and they have been widely used as a model system in studies of cell biology (Nagata et al., 1992; Geelen and Inzé, 2001). This feature eliminates the difficult step of preparing large quantities of homogeneous meristematic cells from plants. In liquid culture medium, BY-2 cells form small cell clusters, each composed of several cylindrical cells connected in tandem. This characteristic promotes homogeneous cultures because most of the surface area of each cell is exposed directly to the culture medium (Sakai et al., 2003). Due to this unique feature, each cell can be directly observed under the microscope, and cell proliferation can be monitored, independently of any effect of changes in cell volume. Second, the only growth factor required by BY-2 cells for proliferation is exogenous auxin (2,4-D), and the physiological and molecular biological changes caused by 2,4-D depletion from culture medium, and/or auxin application to cells grown in medium lacking 2,4-D, have already been well described (Hasezawa and Sy
no, 1983; Ishida et al., 1993; Winicur et al., 1998; Miyazawa et al., 1999, 2000, 2001, 2002b, c; Okamura et al., 2002).
The aim of this study was to examine (i) whether BL promoted cell division in a model system for cell cycle studies, and (ii) whether the effects of BL treatment and auxin treatment overlapped. The effects were examined of BL and auxin on cell numbers and on gene expression related to the cell-division cycle. As plastid and mitochondrial DNAs are preferentially synthesized during the initial phase of cell proliferation, which is thought to be a necessary process for the future proliferation of meristematic cells, (Yasuda et al., 1988; Kuroiwa et al., 1992; Suzuki et al., 1992; Fujie et al., 1993; Doener, 2000), it was also examined whether the organellar DNA content of BL-treated cells increased during the initial phase of cell proliferation.
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Cell culture
Tobacco (Nicotiana tabacum) Bright Yellow-2 (BY-2) cell suspension cultures were maintained in conventional medium (C-medium) as described in Miyazawa et al. (1999). To assess the effect of brassinolide (BL) on BY-2 cells, a suspension of stationary-phase cells grown for 8 d in conventional medium was washed with 10 vols of hormone-free medium (F-medium), transferred at a 1:20 dilution to fresh medium containing ethanol-dissolved BL at various concentrations (10–10, 10–9, 10–8, 10–7, and 10–6 M), and compared with control cultures of cells that were transferred into F-medium and C-medium with the addition of ethanol at appropriate concentrations. Other phytohormones were dissolved in distilled water at 100x concentration and added to the cultures as described in Takahashi et al. (1991).
Microscopic observations and measurement of cell number and cell length and starch content
Cells were harvested at 0, 2, and 4 d after transfer to media containing various concentrations of BL, as well as F-medium and C-medium, and observed immediately under a microscope. To measure mitotic indices, and cell numbers, cells were fixed with 1% glutaraldehyde and stained with 4',6-diamidino-2-phenylindole. Following staining, the cells were diluted and immediately counted under a fluorescence microscope. To measure cell length, randomly photographed BY-2 cells were analysed using NIH Image software. For each treatment, the lengths of approximately 200 cells were determined. Starch content was determined as described in Miyazawa et al. (2001).
DNA extraction and quantitative Southern blot analysis
Cells were collected by centrifugation, frozen in liquid nitrogen, and stored at –80 °C until used for extraction. DNA extraction and Southern blot analysis were performed as described in Miyazawa et al. (2002b).
RNA extraction and quantitative RNA gel blot analysis
RNA was extracted using Sepasol-RNA I Super (Nacalai Tesque Inc., Kyoto, Japan), according to the manufacturer’s instructions. Ten µg of total RNA were denatured with glyoxal and subjected to gel electrophoresis as described previously (Miyazawa et al., 2002a). The RNA was transferred to Hybond-XL membrane (Amersham Biosciences, UK). Cloned cDNA fragments of CYM (Ito et al., 1997), histone H4, and actin were used to detect the corresponding transcripts. A tobacco cDNA for histone H4 was amplified by PCR and cloned into the pCR-II vector (Invitrogen, Corp., Carlsbad, CA). The GenBank database was searched for similar nucleotide sequences using the BLAST algorithm. The deduced amino acid sequence of the tobacco cDNA fragment for histone H4 had significant identity to known histone H4 sequences, and so the fragment was considered suitable for use as a probe. Hybridizations and analyses were performed as described in Miyazawa et al. (2002b).
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Results |
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Brassinolide application induces BY-2 cell division in the absence of exogenous auxin
To investigate the effect of brassinolide (BL) application on cell multiplication, BL was added to conventional medium (C-medium) or hormone free medium (F-medium) at various concentrations, and mitotic indices were monitored at 48 h after transfer (Fig. 1). BL application to C-medium caused a slight, and not significant, increase in a dose-dependent manner. On the contrary, when BL was added to F-medium, in which cell proliferation is arrested, mitotic indices increased drastically in a dose dependent manner. This effect was further confirmed by counting cell numbers. In C-medium, BY-2 cells proliferated actively throughout the culture period (Table 1). By contrast, when BY-2 cells in the stationary phase were transferred into F-medium, the rate of cell proliferation decreased drastically. When brassinolide (BL) was added to F-medium cultures, the transferred stationary-phase cells began to proliferate in a dose-dependent manner. When BL was supplied above a concentration of 10–7 M, no statistically different change in the proliferation rate (P=0.05) was observed at 2 d after transfer, compared with cultures in C-medium. At 4 d after transfer of stationary-phase cells, the proliferation rate was greatly decreased as compared to C-medium cell cultures, even when BL was supplied at 10–6 M. Morphological differences were also observed between BL-treated cells and 2,4-D-supplied cells; on the second day of culture, BL application resulted in shorter cells and a reduced number of starch granules compared with F-medium cultured cells, and the BL-treated cells’ overall morphology was obviously different from that of 2,4-D-supplied cells, with cytoplasm-rich cells containing fewer and smaller starch granules (Fig. 2). This observation on the reduced starch granules by BL application is confirmed by measuring starch content (Table 2). As a result, starch content was significantly reduced by BL application in a dose-dependent manner and when BL was added above 10–7 M, starch content decreased to one-quarter of the usual amyloplast induces condition (F-medium). The differences between BL-treated cells and 2,4-D-supplied cells were more obvious in 4-d-old cells. The shapes of the BL-treated cells resembled those of F-medium-cultured cells, regardless of the BL concentration. These results suggest that the promotive effect of BL on cell multiplication is limited to a short period in the growth of the culture, and that BL application cannot completely compensate for a lack of 2,4-D.
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It was also examined whether other phytohormones can promote BY-2 cell division. Abscisic acid (ABA), 1-amino-cyclopropane-1-carboxylic acid (ACC), a precursor of ethylene, and gibberellic acid (GA3), respectively, were added in place of 2,4-D. The effect of cytokinin addition on cell multiplication in the absence of 2,4-D was described by Sakai et al. (1996). As shown in Table 3, none of these phytohormones promoted cell proliferation, and thus the promotive effect of BL on BY-2 cell proliferation in the absence of 2,4-D may be BL-specific. The distributions of the cell length of F-medium cultured cells, C-medium cultured cells, and BL-treated cells (Fig. 3) were also compared. BL treatment reduced the average cell length in a dose-dependent manner, and as the concentration of BL increased, similar distributions were observed. Although the promotive effect of BL on cell division was limited to a short phase in the culture period, the cell-shortening effect of BL was evident even 4 d after initiation of the culture.
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Effect of BL application on cell-cycle-related gene expression
To determine the molecular basis of BL-induced cell proliferation, RNA gel blot analysis was used to analyse whether expression of cell-cycle-related genes was also altered by BL application. CYM and histone H4 DNA fragments were used as probes, since these genes are widely accepted as marker genes for the M and S phases, respectively (Reichheld et al., 1995; Ito et al., 1997). When BL was added at or above a concentration of 10–9 M, the accumulation of CYM transcripts increased dose-dependently, and reached the same level as in 2,4-D-supplied cells grown at a BL concentration of 10–7 M after 2 d of culture (Fig. 4). Similarly, histone H4 transcripts accumulated after BL application in a dose-dependent manner, but the transcripts were far less abundant than in C-medium-cultured cells. The accumulation of actin transcripts, used as controls, showed little fluctuation. This increased accumulation of cell-cycle-related genes was consistent with the effects of BL on the cell proliferation rate. By contrast, at 4 d after transfer, the results of molecular analysis were not consistent with the cell proliferation rate. BL addition below 10–7 M resulted in decreased levels of CYM transcripts, whereas the accumulation of histone H4 transcripts was almost equal to that in C-medium cultured cells. When BL was supplied at 10–6 M, both the CYM and histone H4 transcripts accumulated at levels almost equal to or even higher than levels in C-medium cultured cells. However, in each case, the rate of cell multiplication was clearly lower than that observed in the presence of 2,4-D. These results indicate that there is another rate-limiting factor in cell multiplication, in addition to cell-cycle-related gene expression, that is distinctly different in BL-treated cells and 2,4-D-supplied cells. This additional factor could be the cause of the previously reported contradictory results.
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Effect of BL application on organellar DNA replication
Because several lines of evidence have indicated that organellar DNAs are preferentially synthesized during the initial phase of cell proliferation, when plant cells undergo multiple successive cell and organellar divisions (Yasuda et al., 1988; Kuroiwa et al., 1992; Suzuki et al., 1992; Fujie et al., 1993; Doener, 2000), the organellar DNA content of BL-treated BY-2 cells was measured. As the organellar DNA content per cell is highest 1–2 d after renewal of the medium (Okamura et al., 2002), the total cellular DNA was extracted from 2-d-old BY-2 cells for quantitative DNA gel blot analysis. Samples of total cellular DNA were digested with HindIII and standardized to contain equal amounts of nuclear DNA, using preliminary hybridizations with 26S and 18S rDNA probes. The standardized samples were then hybridized with probes specific for plastid rbcL and mitochondrial coxI to determine the amounts of plastid and mitochondrial DNAs, respectively. Unexpectedly, BL application increased neither plastid nor mitochondrial DNA content, irrespective of the final concentration of the added BL (Fig. 5), whereas increases in both plastid and mitochondrial DNA content (2.0-fold for plastid DNA and 2.2-fold for mitochondrial DNA) were observed in C-medium cultured cells. Considered in conjunction with the finding that BL addition did not induce preferential organellar DNA synthesis, BL addition, therefore, cannot replace the effect of 2,4-D in the initial culture phase, although increases in cell numbers and cell-cycle-related transcript levels occur at similar rates as in 2,4-D-supplied cells throughout the period. These results indicate that exogenously-supplied BL promotes BY-2 cell proliferation by a mechanism that is distinct from that induced by application of 2,4-D, which is already known to be an indispensable phytohormone for cell division.
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Discussion |
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Promotion of BY-2 cell proliferation by exogenously-supplied brassinolide
BRs are steroidal phytohormones that are essential for many aspects of plant growth and development, including cell expansion, cell division, reproductive and vascular development, and stress responses (reviewed in Clouse and Sasse, 1998; Bishop and Koncz, 2002). However, BRs have been reported to have both promotive and inhibitory effects on cell division in different plant species and cultured cell lines (Roth et al., 1989; Kauschmann et al., 1996; Nakajima et al., 1996; Oh and Clouse, 1998; Hu et al., 2000). In this study, tobacco BY-2 cell cultures were used, which do not have the limitations of other systems that may have led to contradictory results. It was demonstrated in three different ways that BL has a promotive effect on cell division in BY-2 cells. First, mitotic indices of BL-treated cells were counted and it was found that BL application increases mitotic indices in a dose-dependent manner. Second, the numbers and lengths of BL-treated cells were monitored using a microscope and the proliferation rates and cell length distributions were determined when 2,4-D was replaced by BL at various concentrations. Third, RNA gel blot analysis was performed using two gene probes related to cell cycle progression: B-type cyclin (CYM) and histone H4, whose expression corresponds to the G2 to M phase and the S phase, respectively. When BY-2 cells were cultured in F-medium, the accumulation of both transcripts was significantly lower than in cells cultured in C-medium. These results are in solid agreement with the finding that cells at the G1 phase accumulate in F-medium cultured cells (Miyazawa et al., 1999). When BL was added to F-medium cultures, the proliferation rate of the cells began to increase in a dose-dependent manner, possibly related to the enhanced accumulation of cell-cycle-related transcripts (Table 1; Figs 1, 4); when BL was supplied at 10–7 M, the proliferation rate and the levels of the CYM transcript on the second day of culture showed no statistically significant differences from those of cells grown in C-medium. However, microscopic observation revealed that the BL-treated cells were morphologically quite different from C-medium cultured cells (Fig. 2). Although adding increasing concentrations of BL led to proportionally higher proliferation rates, the BL-treated cells somewhat retained the characteristics of auxin-deprived BY-2 cells. These results indicated that application of BL could not fully substitute for the effect of auxin. This inability of BL to substitute for auxin became more obvious by the fourth day of culture, when the cell proliferation rates of BL-treated cultures were significantly lower than those of 4-d C-medium cultures, although CYM and histone H4 transcript levels were relatively high even in BL-treated cultures.
Considering the overlapping effects of auxin and BRs on cell expansion, two alternative effects of BL on the auxin-related response can be assumed: activation of auxin biosynthesis and activation of auxin signal transduction pathways. BY-2 cells can synthesize and retain indole-3-acetic acid (IAA) at trace levels throughout the cell cycle (Redig et al., 1996), despite their inability to divide without exogenously supplied 2,4-D. If the application of BL activates either IAA biosynthesis or the auxin signalling pathway, then BL-treated cells should have a proliferation rate that is similar to that of C-medium cultured cells. In fact, measurements of cell length revealed that as increased BL concentrations were added, the distributions of cell length resembled those of 2,4-D-treated cells, even at 4 d of culture (Fig. 3). However, the proliferation rates of these culture types were never comparable at the fourth day of culture, irrespective of the BL concentration. From these results, it was hypothesized that there is an alternative signalling pathway in BY-2 cells, which leads to cell division that is independent of auxin. Similar observations of a promotive effect of BRs on cell division have been reported by Hu et al. (2000), who showed that epibrassinolide can substitute for the effects of cytokinin in Arabidopsis cultured cells. The authors reported that both cytokinin and BR could induce cycD3 gene expression, resulting in the promotion of cell division. Although they did not compare the effects of BR and auxin, the induction of cycD3 gene expression by BR also differs from the expression in Arabidopsis cells under standard culture conditions in which auxin and cytokinin are supplied.
It was also shown that BL promotes cell division only during the early culture phase (Table 1), suggesting that there is a rate-limiting factor in BL-induced cell division. In the search for this factor, it was found that both plastid and mitochondrial DNAs did not replicate during the early phase of the culture (Fig. 5). Several lines of evidence have indicated that organellar DNAs are preferentially synthesized during the initial phase of the proliferation of plant meristematic cells, such as root and shoot apical meristems, and egg cells preparing for fertilization and subsequent embryogenesis (Kuroiwa et al., 1992; Suzuki et al., 1992; Fujie et al., 1993; Doerner, 2000). In BY-2 cells as well, active replication of organellar DNAs occurs during the initial phase of cell proliferation (Yasuda et al., 1988; Suzuki et al., 1992). Recently, Okamura et al. (2002) reported that both auxin and sucrose have indispensable roles in this process. In BL-treated BY-2 cells, the organellar DNA content per cell did not increase, whereas an increase in organellar DNA content was observed in C-medium cultured cells. The cessation of cell propagation in BL-treated cells coincides well with a previous observation in BY-2 cells treated with nalidixic acid, an inhibitor of prokaryotic DNA gyrase. The organellar genomes of these cells failed to replicate and, as a result, the cells did not proliferate (Suzuki et al., 1996). No obvious increase or decrease in organellar DNA content per cell was observed in BL-treated BY-2 cells, even when the cells multiplied, indicating that the organellar DNA replication maintained a consistent amount of organellar DNA in each cell. This retention of organellar DNA was also observed in cells cultured in F-medium (Fig. 5). A recent analysis of plastid and mitochondrial DNA synthesis in BY-2 cells using isolated organelles and nuclei showed that both plastid and mitochondrial nuclei are capable of DNA synthesis at the stationary phase, and that this process was promptly activated by replacement of the medium, which contained auxin and sucrose (Okamura et al., 2002). Taking this indispensable role of organellar DNA synthesis into account, these present data show that BL treatment could not replace the function of 2,4-D in a process closely related to cell division.
Based on these considerations, together with the result that other phytohormones could not induce cell division (Table 3), it is concluded that (i) BL quickly promotes cell division mediated by the enhanced accumulation of cell-cycle-related genes, and (ii) the mechanism that supports this short-term promotion of cell division by BL is unique and probably distinct from that regulated by the balance of auxin and cytokinin levels.
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Acknowledgements |
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The authors would like to thank Dr Masaki Ito (Nagoya University) for providing the NtCYM cDNA, Dr Yukihisa Shimada (Plant Science Center, RIKEN) for useful discussions, and Mr Makoto Kobayashi (Plant Science Center, RIKEN) for technical assistance. This work was supported by a Grant-in-Aid from the Special Postdoctoral Researchers Program of RIKEN to YM, and also by a grant from the Research Project for the Study of Biological Cross-Talk Functions from the Ministry of Education, Culture, Sports, Science and Technology, Japan.
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