JXB Advance Access originally published online on June 4, 2004
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Journal of Experimental Botany, Vol. 55, No. 402, pp. 1499-1508, July 2004
Journal of Experimental Botany, Vol. 55, No. 402, © Society for Experimental Biology 2004; all rights reserved
RESEARCH PAPER |
Regulated gene expression by glucocorticoids in cultured Virginia pine (Pinus virginiana Mill.) cells
Department of Biology, Howell Science Complex, East Carolina University, Greenville, NC 27858-4353, USA
* To whom correspondence should be addressed. Fax: +1 252 328 4178. E-mail: tangw{at}mail.ecu.edu
Received 5 March 2004; Accepted 18 April 2004
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Abstract |
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The effects of six glucocorticoids (dexamethasone, hydrocortisone, 6-methylprednisolone, prednisolone, prednisone, and triamcinolone) on inducible gene expression, based on the chimaeric transcriptional activator GVG and carried by the binary expression vector pINDEX3-m-gfp5-ER, were evaluated in transgenic Virginia pine cell cultures. The concentration that activated GVG transcription factor activity, the level of inducible m-gfp5-ER expression, and the kinetics of inducible m-gfp5-ER expression were determined for each glucocorticoid. Transgenic cells produced green fluorescence upon blue light excitation after treatment with prednisolone, prednisone, 6-methylprednisolone, dexamethasone, triamcinolone, and hydrocortisone. Green fluorescence was observed at 6–12 h after treatment of all six glucocorticoids at concentrations of 1, 3, 5, and 10 mg l–1. Differential expression of gfp was confirmed by northern blot analysis and by quantitative fluorescence analyses of confocal images taken by a LSM 510 Laser Scanning Microscope. Fresh and dry weight increases of transgenic cell cultures were not affected by all six glucocorticoids at concentrations of 0.1, 0.5, 1, 3, and 5 mg l–1. It is shown that triamcinolone had the most potent effect on the GVG system. Different glucocorticoids can therefore be used to regulate the GVG transcriptional activator and to induce gene expression in transgenic plant cells, and this property could be useful in establishing an optimum system of transgene regulation.
Key words: Glucocorticoids, green fluorescence protein, GVG transcriptional activator, Pinus virginiana Mill
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Introduction |
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Conditional gene expression systems, based on the interaction of a chemical inducer with a specifically designed transcription factor, which results in the transactivation of a synthetic promoter upstream of the target gene, have proved to be powerful tools for basic research in molecular biology and for biotechnological applications with transgenic animals and plants (Aoyama, 1999
; Reynolds, 1999; Saez et al., 1997; Wirtz and Clayton, 1995; Zuo and Chua, 2000). Inducible gene expression systems, where the expression of target genes are tightly controlled by chemical inducers, are extremely useful for: (i) analysing biochemical and developmental processes (Gatz, 1997; Ward et al., 1993); (ii) determining temporal and spatial expression patterns of specific genes; and (iii) studying transgenes where their constitutive expression may have deleterious effects on transformed organisms (Aoyama and Chua, 1997; Ouwerkerk et al., 2001). Inducible gene expression systems may also be used to analyse gene function, site-specific DNA excision, activation tagging, conditional genetic complementation, and the restoration of male fertility (Padidam, 2003; Reynolds, 1999).
Several chemical-inducible systems based on activation and inactivation of the target gene have been developed (Gatz, 1997; Padidam, 2003; Reynolds, 1999; Shockett et al., 1995; Zuo and Chua, 2000). Artificial promoters that combine tight transcriptional control and induction by low concentrations of chemical inducers have been developed from bacterial repressor or animal steroid-binding elements (Gatz, 1997; Love et al., 2002; Zuo and Chua, 2000). These promoters include tetracycline-inducible Triple-Op (Gatz and Quail, 1988), tetracycline-repressible Top10 (Hinrichs et al., 1994; Love et al., 2002; Weinmann et al., 1994), glucocorticoid-induced GAL4-UAS (Aoyama and Chua, 1997), ethanol-inducible alcA (Caddick et al., 1998), and ecdysone-inducible GRH (Martinez et al., 1999). Chemicals that are used to regulate transgene expression include the antibiotic tetracycline, the steroids dexamethasone and estradiol, herbicide safeners, copper, ethanol, the inducer of pathogen-related proteins, benzothiadiazol, and the insecticide methoxyfenozide (Padidam, 2003; Reynolds, 1999).
In the case of GVG-regulated gene expression (Aoyama and Chua 1997; Ouwerkerk et al., 2001), the chimaeric transcription factor GVG consists of the yeast Gal4 binding domain, the Herpes simplex VP16 activation domain, and the rat glucocorticoid receptor. Induction is based on the property of the glucocorticoid receptor domain to localize the constitutively expressed GVG protein to the cytoplasm in the absence of the inducer (Aoyama and Chua, 1997; Ouwerkerk et al., 2001). After treatment with glucocorticoids such as dexamethasone, GVG is transferred to the nucleus and interacts with the GVG recognition site, 4UAS, cloned upstream of the targeting gene; it subsequently then activates transcription of the targeting gene (Ouwerkerk et al., 2001). The advantages of GVG-regulated gene expression in plant biotechnology include: (i) the regulatory molecules, i.e. glucocorticoid hormones, that have no naturally occurring analogues in plant cells; (ii) transgene expression is induced by the addition of glucocorticoid hormones at micromolar levels, and they have no deleterious effects on transformed tissues of some plant species at suitable concentrations, however, Kang et al. (1999) observed significant defects in transgenic Arabidopsis; and (iii) transcriptional control by the GVG transcription factor is very tight (Aoyama and Chua, 1997; Ouwerkerk et al., 2001).
Although GVG-regulated gene expression has been characterized extensively in dicotyledonous and monocotyledonous plants such as tobacco, Arabidopsis, and rice (Aoyama and Chua, 1997; McNellis et al., 1998; Ouwerkerk et al., 2001; Reynolds, 1999), it has not been tested in gymnosperms. Dexamethasone is widely used in GVG-regulated gene expression in angiosperms, but it may not be the best candidate to regulate the GVG transcription factor in gymnosperms. It was therefore investigated whether it was possible to regulate the level of transgene expression controlled by the GVG transcription factor in conifer cells more precisely, by using various glucocorticoid analogues. The effects of six readily available glucocorticoids (dexamethasone, hydrocortisone, 6-methylprednisolone, prednisone, prednisolone, triamcinolone) on GVG transcription factor-driven transgene expression in transformed Virginia pine cell cultures were tested.
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Materials and methods |
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Plasmid constructs
After the pINDEX3 binary vector, provided by Dr PBF Ouwerkerk and Dr AH Meijer (Institute of Molecular Plant Sciences, Leiden University, Clusius Laboratory, The Netherlands), was isolated and purified, the plasmid pINDEX3 was digested by SpeI and XhoI (Promega). The m-gfp5-ER fragments (Haseloff et al., 1997
; Stewart, 2001) were amplified from plasmid pBIN-m-gfp5-ER provided by Dr CN Stewart and Dr J Haseloff. The restriction enzyme SpeI and XhoI sites were introduced by PCR with Stratagene's Pfu DNA polymerase (Stratagene). For amplification of m-gfp5-ER fragments, the gfp forward primer 5'-AAACATGATGAGCTTTAAAGACTC-3' and the reverse primer 5'-CTTCATTGTTTGATCACCTTGCATCC-3' (Sigma) were used. A total of 200 ng of plasmid DNA was used as a template in a 50 µl PCR reaction mix. The PCR mixture consisted of 200 µM each of dATP, dCTP, dGTP, dTTP, 250 ng of each primer, 5 U Stratagene's Pfu DNA polymerase, 1.5 mM MgCl2, and 5 µl 10x buffer [500 mM KCl, 100 mM TRIS–HCl (pH 9.0 at 25 °C), 1% Triton X-100, 15 mM MgCl2]. The PCR conditions were 95 °C for 5 min followed by 29 cycles at 95 °C for 60 s, 57 °C for 40 s, and 72 °C for 90 s. Cycling was followed with a final incubation of 72 °C for 10 min. PCR products were purified and digested by SpeI and XhoI (Promega). Ligation of the purified insert (30 ng) and vector (90 ng) was conducted in 20 µl with 2 U T4 DNA Ligase (Roche Applied Science) at 16 °C for 16 h. One µl of ligation products was used to transform 20 µl NovaBlue competent cells (Novagen). Overnight cultures from single clones were used to isolate plasmid pINDEX3-m-gfp5-ER. The purified plasmid was introduced into Agrobacterium tumefaciens EHA105 competent cells by electroporation (Bio-Rad).
Transformation of cultured cells and glucocorticoid hormone treatments
Cell cultures of Virginia pine were prepared as described in Tang et al. (2001a). Cell cultures were maintained by weekly subculture of a 2.5 ml cell suspension into 25 ml liquid culture medium [1x TE Salt Base (Tang et al., 2001b), 30 g l–1 sucrose, 500 mg l–1 myo-inositol, 2 mg l–1 2,4-D, 0.5 mg l–1 BA, pH 5.8) and incubated at 25 °C, with orbital shaking at 150 rpm, in darkness. Cell cultures were transformed using Agrobacterium tumefaciens strain EHA105 carrying the binary vector plasmid pINDEX3-m-gfp5-ER (Fig. 1), as described in Tang et al. (2001b). Agrobacterium, grown for 24 h to an optical density (OD600 nm=0.8–1.0) of bacteria in 3 ml of YEP broth (Sambrook et al., 1989), was centrifuged and resuspended in liquid medium. Cell-suspension cultures were infected with bacterium cultures for 15 min. The infected cell-suspension cultures were harvested with 42.5 µm filter papers, transferred to 125 ml Erlenmeyer flasks, and co-cultivation was carried out for 1–2 d. Agrobacterium was removed from the cultures by washing the cell suspension cultures in 50 ml sterile tubes with 500 mg l–1 timentin (ticarcillin/clavulanic acid 3:0.1, v:v, SmithKline Beecham, Philadelphia, PA) solution for 3 min. The wash was repeated five times. Transgenic cell cultures were suspended in 25 ml liquid medium containing 5 mg l–1 hygromycin and 500 mg l–1 timetin and were incubated for 7 d at 25 °C, with orbital shaking at 150 rpm in darkness. Cells grown from these cultures were transferred to 25 ml fresh liquid medium containing 5 mg l–1 hygromycin. Further subculturing was performed weekly on media containing only hygromycin. To obtain large quantities of transformed cell cultures for further analysis, selected cell-suspension cultures were again introduced on a liquid proliferation medium. After 6 weeks, the cultures were actively producing 50–70 mg of tissue l–1 each week, and they were then used to prepare DNA for PCR and Southern blot analysis.
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Stable transgenic cell lines confirmed by PCR and Southern blot analysis were used for systemic, inducible, gene-expression experiments. For treatments with glucocorticoid hormones, transgenic cell lines were grown in liquid plant medium for 3 d, then transferred into fresh liquid medium supplemented with the different glucocorticoid hormones, prednisolone, prednisone, 6-methylprednisolone, dexamethasone, triamcinolone, and hydrocortisone at different concentrations (0.1, 0.5, 1, 3, 5, and 10 mg l–1) for 24 h (Fig. 1). Cells were washed thee times, transferred to fresh liquid medium, and were observed under the fluorescence microscope at 2, 4, 6, 12, 18, 24, 36, 48, 72, 96, 108, 120, and 144 h after application of the inducer, following the protocol described by Scott et al. (1999)
. At 24, 48, and 72 h after treatment of glucocorticoids, cell cultures were harvested for qualitative and quantitative fluorescent microscopy, and for northern blot analysis. At 21 d after treatment of glucocorticoids, cell cultures were harvested by centrifuging at 8000 rpm for 15 min for the measurement of fresh weight (Fig. 1). Cell cultures were dried at 95 °C for 3 d for the measurements of dry weight (Tang, 2000). Glucocorticoids (Sigma) were stored as 100 mM solution in dimethyl sulphoxide (DMSO) at –20 °C. Experiments were replicated at least four times.
Polymerase chain reaction and Southern blot analyses of transgenic cultures
Genomic DNA was extracted from 500 mg cell cultures of control and putative transgenic cell lines, using a Genomic DNA Isolation Kit (Sigma) following the manufacturer's protocol. The PCR reactions were performed with a PTC-100TM Programmable Thermal Controller (MJ Research) using DNA extracted from putative transformed and control cell cultures, respectively. The primers used for PCR reactions are the gfp forward primer (gfpf) 5'-AAACATGATGAGCTTTAAAGACTC-3' and the reverse primer (gfpr) 5'-CTTCATTGTTATATCACCTTGCAT-3'. A total of 200 ng of genomic DNA was used as a template in a 50 µl PCR reaction mix. The PCR reaction was conducted as previously described (Tang et al., 2001b). PCR products were separated by electrophoresis on 1.0% agarose gels in 1x TAE buffer (Sambrook et al., 1989) and were detected by fluorescence under UV light (302 nm) after staining with 0.1% ethidium bromide. A molecular marker of 1 kb (Gibco-BRL, Gaithersburg, MD, USA) was used. Southern blot analysis was conducted as previously described (Tang et al., 2001b).
RNA isolation and northern blot analysis
Total RNA was isolated from 1.5 g transgenic cell cultures harvested though 42.5 µm filter papers and ground in liquid nitrogen using the RNeasy Plant Mini Kit (QIAGEN Inc.) following the manufacturer's protocol. Ten µg RNA was separated by agarose-gel electrophoresis. Electrophoresis and northern blotting of RNAs were performed as described by Tang and Tian (2003). Baked blots were prehybridized in 1 M NaCl, 1% SDS, 10% dextran sulphate, and 50 µg ml–1 denatured herring sperm DNA at 64 °C, washed with 0.1x SSPE (1x SSPE is 180 mM NaCl, 10 mM NaH2PO4, 1 mM EDTA, pH 6.5), 0.5% SDS at 45 °C, and autoradiographed. Probes (816 pb fragment of m-gfp5-ER DNA) were labelled by Digoxigenin (DIG) (Roche Diagnostics Corporation). Equal loading of RNA samples was verified on the control of 25S rRNA.
Qualitative and quantitative analysis of inducible GFP expression
To determine the effect of different concentrations of dexamethasone and different time of treatments on GFP expression, transformed cell cultures were observed at various times after treatments. GFP expression in transgenic cell cultures was observed with a stereo dissecting microscope equipped with a fluorescence module consisting of a 100 W mercury lamp and GFP Plus excitation and emission filters (Leica, Heerbrugg, Switzerland). This system (excitation filter 480 nm; dichoic mirror 505 nm LP; barrier filter 510 nm LP) permits visualization of GFP following excitation by blue light. For quantitative fluorescence determinations of m-gfp5-ER activity, samples were examined with a LSM 510 Laser Scanning Microscope (Carl Zeiss, Inc., Thornwood, NY, USA) using excitation with the 488 nm Argon laser line and detection of emitted light between 500 nm and 520 nm. The confocal images of m-gfp5-ER expression cells were created in the Expert Mode. Fluorescence intensities of different samples were calculated from confocal images with the Zeiss LSM Image Examiner software. The fluorescence level was quantified separately for the whole cell by circumscribing the respective area as a region of interest. Thirty to fifty cells were used for each sample.
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Results |
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Transgenic cell lines
To generate transgenic cell lines, 476 cell cultures derived from single embryos were infected with an Agrobacterium tumefaciens strain containing pINDEX3-m-gfp5-ER (Fig. 1) carrying a modified hygromycin phosphotransferase gene, a chimaeric transcription factor GVG, and a modified GFP protein with an endoplasmic reticulum targeting sequence. Forty-four independent Virginia pine cell lines that were transformed with the pINDEX3-m-gfp5-ER plasmid (Ouwerkerk et al., 2001
) and were resistant to hygromycin, were generated. After the T-DNA insert was confirmed by both PCR (Fig. 2) and Southern blot analyses (data not shown), six transgenic cell lines (P2, P5, P12, P16, P25, and P32) with 1–2 copies of the transgene were selected as candidates for inducible gene-expression experiments. A summary of inducible GFP expression in all six transgenic cell lines was demonstrated in Table 1. After measurement of growth and cell survival rates at the end of the subculture period (7 d), one transgenic cell line (P32) with high growth and cell survival rates and containing one copy of the T-DNA insert, was selected for inducible gene expression. This transgenic cell line was transferred weekly into fresh proliferation medium for 10 weeks to produce more cells. No background GFP expression was observed in non-transformed control cell lines and all transgenic cell lines in the absence of inducer.
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Inducible gfp expression in transgenic cells
To assess the glucocorticoid, hormone-inducible, gene-expression system, the selected transgenic cell line was used to test inducible gene expression by six different glucocorticoid hormones (prednisolone, prednisone, 6-methylprednisolone, dexamethasone, triamcinolone, and hydrocortisone) at different concentrations (0.1, 0.5, 1, 3, 5, and 10 mg l–1). After the transgenic cell line was treated with glucocorticoid hormones for 24 h, cells were washed thee times and transferred to fresh liquid medium. Transgenic cells were observed with the aid of a fluorescence microscope at 2, 4, 6, 12, 18, 24, 36, 48, 72, 96, 108, 120, and 144 h after the application of the inducer.
Inducible gfp activity was observed at 4 h after treatment with dexamethasone and triamcinolone at concentrations of 1, 3, 5, and 10 mg l–1, and at 6 h after treatment of glucocorticoid hormones dexamethasone and triamcinolone at concentrations of 0.1 and 0.5 mg l–1. Green fluorescence was observed at 6 h after treatment with prednisolone, prednisone, 6-methylprednisolone, and hydrocortisone at concentrations of 1, 3, 5, and 10 mg l–1, and at 12 h after treatment with prednisolone, prednisone, 6-methylprednisolone, and hydrocortisone at concentrations of 0.1 and 0.5 mg l–1. Transgenic cells produced green fluorescence upon blue light excitation 24 h after treatment with prednisolone, prednisone, 6-methylprednisolone, dexamethasone, triamcinolone, and hydrocortisone at all six concentrations tested. Inducible expression of m-gfp5-ER was visible in cultures incubated in all inducer concentrations. A sample of inducible m-gfp5-ER expression 24 h after treatment with prednisolone, prednisone, 6-methylprednisolone, dexamethasone, and triamcinolone at concentrations of 0.5, 1, 3, 5, and 10 mg l–1, is shown in Fig. 3A–Y. Fluorescent images, 24 h after treatment with prednisolone, prednisone, 6-methylprednisolone, dexamethasone, and triamcinolone at concentrations of 0.1 mg l–1 and from hydrocortisone at different concentrations (0.1, 0.5, 1, 3, 5, and 10 mg l–1) were not included because of the dimness of the fluorescence. Inducible gene expression was also analysed by northern blot analysis of transgenic cell cultures treated with glucocorticoid hormones at a concentration of 1 mg l–1. The results demonstrate that a high constitutive level of GVG gene expression was conferred by the CaMV35S promoter, and that m-gfp5-ER mRNA only appeared after glucocorticoid treatment (data not shown). Among samples taken 24, 48, and 72 h after application of 1 mg l–1 glucocorticoids, higher transcripts were observed from treated transgenic cell cultures at 48 h (Fig. 4).
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To evaluate the effects of glucocorticoid-inducible gene expression on cell growth and development, transgenic cell cultures were treated with concentrations of 0.1, 0.5, 1, 3, 5, and 10 mg l–1 for 24 h, and then subcultured weekly on liquid medium without inducer. Transgenic cell cultures were harvested at 21 d after glucocorticoid treatment for the measurement of fresh and dry weights. There were no significant differences in fresh and dry weight increases when transgenic cell cultures were treated with all six glucocorticoids at concentrations of 0, 0.1, 0.5, 1, 3, and 5 mg l–1. These results have demonstrated that cell growth and development were not inhibited by the addition of these compounds at suitable concentrations (Tables 2, 3). However, these results have demonstrated that fresh and dry weight increases of transgenic cell cultures were reduced by prednisolone, prednisone, 6-methylprednisolone, dexamethasone at concentration of 10 mg l–1, but growth was not inhibited by triamcinolone and hydrocortisone at a concentration of 10 mg l–1 (Tables 2, 3). The reduction in growth caused by the high levels of glucocorticoids was probably due to potentially toxic effects on cell cultures.
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Quantitative analysis of inducible gfp expression
To analyse glucocorticoid-inducible gene expression in further detail, m-gfp5-ER activities were determined quantitatively. The effects of prednisolone, prednisone, 6-methylprednisolone, dexamethasone, triamcinolone, and hydrocortisone (at concentrations of 0.1, 0.5, 1, 3, 5, and 10 mg l–1) on m-gfp5-ER activities of transgenic cell cultures, were investigated. Transgenic cell cultures were subcultured on liquid medium with different concentrations for 24 h, and green fluorescence of transgenic cells was determined quantitatively. Confocal images were taken at different times after treatment with dexamethasone (Fig. 5A–J). Inducible gene expression turns off completely 144 h after the application of inducer (Fig. 5A, J). As shown in Fig. 6, inducible m-gfp5-ER activities ranged between 63 and 80 (at 0.1 mg l–1), 66 and 92 (at 0.5 mg l–1), 79 and 127 (at 1 mg l–1), 103 and 190 (at 3 mg l–1), 105 and 192 (at 5 mg l–1), and 106 and 193 (at 10 mg l–1) units of relative fluorescence 24 h after treatment of prednisolone, prednisone, 6-methylprednisolone, dexamethasone, triamcinolone, and hydrocortisone. The highest level of green fluorescence was from transgenic cell cultures treated with 3–10 mg l–1 triamcinolone for 24 h (Fig. 6). In the absence of inducer, no m-gfp5-ER activity could be detected either qualitatively or quantitatively.
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Glucocorticoid-inducible gene expression was also quantitatively determined in transgenic cell cultures treated with prednisolone, prednisone, 6-methylprednisolone, dexamethasone, triamcinolone, and hydrocortisone, for different intervals (24, 48, and 72 h) after treatment, respectively. No GFP fluorescence was detected in transgenic cell cultures that were not treated with glucocorticoids. However, background fluorescence, not from GFP expression but from the reflection of the light source, was observed. The same amount of background fluorescence (20–28 arbitrary units in intensity; Table 1; Figs 5, 6, 7) was observed in both non-transgenic cells and transgenic cells before the application of the inducer. The differences in inducible gfp activity were observed among different glucocorticoids and at different times after treatment (Fig. 7). GFP fluorescence was initially undetectable and increased from 0 h to 48 h after application of inducer. In agreement with the northern blot analyses data (Fig. 4), transgenic cell cultures from 24–48 h after treatment at 1 mg l–1 glucocorticoids showed a greater increase of fluorescence compared with the increase from 0–24 h and from 48–72 h. In general, high levels of green fluorescence were observed with transgenic cell cultures treated with dexamethasone and triamcinolone. Triamcinolone appeared to be the best inducer for the transgenic cell line investigated here. However, the dose and time required to saturate the system will be largely dependent on the growth stage and transgenic cell lines in different plant species used.
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Discussion |
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Chemically inducible systems that activate or inactivate gene expression have many potential applications in plant biotechnology, particularly in the determination of gene function (Padidam, 2003
). The precise timing and control of gene expression are important aspects of chemically inducible systems. Several systems-based chemical inducers have been developed and used. Chemicals that are used to regulate transgene expression include the antibiotic tetracycline (Gatz, 1997, 1999; Bohner et al., 1999), the steroids dexamethasone and estradiol (Schena et al., 1991; Aoyama and Chua, 1997), copper (McKenzie et al., 1998; Reynolds, 1999), ethanol (Caddick et al., 1998; Salter et al., 1998), ecdysone (Martinez et al., 1999), the inducer of pathogen-related proteins benzothiadiazol, herbicide safeners, and the insecticide methoxyfenozide (Padidam, 2003). Transgenic technology involving the introduction of one or more transgenes can turn desired traits on or off in animals and plants (Gatz, 1997; Gossen et al., 1995; Uknes et al., 1993). Although constitutive promoters are used to transcribe a gene of interest, a major limitation of constitutive promoters is that they cannot be used to investigate genes whose constant over- or under-expression has deleterious effects on the animals and plants where expression of a sense or anti-sense transgene in transformed cells may be toxic (Friedrich et al., 1996; Gatz and Lenk, 1998; Gatz, 1999; Picard et al., 1990; Reynolds, 1999).
Chemical-inducible systems for regulated gene expression offer a more general and flexible solution because chemical-inducible systems are quiescent in the presence or absence of inducers, and therefore will not inhibit physiological activities (Aoyama, 1999; Gatz et al., 1992; Thompson and Myatt, 1997). In addition, the use of an appropriate promoter to express the chemical-responsive transcription factor can further restrict the target transgene expression to specific organs, tissues, or even cell types (Faiss et al., 1996; Gossen et al., 1993). Chemical-inducible systems for regulated gene expression are extremely useful for basic biology research and biotechnology applications (Schena et al., 1991; Williams et al., 1992). In E.coli, an example of a promoter that provides such fidelity is the isopropyl ß-D-thiogalactoside (IPTG) inducible lac promoter, which is routinely used whenever expression of the recombinant protein is going to interfere with growth (Beckwith and Zipser, 1970; Benhamou, 1996). Similarly, if a foreign gene product expressed in animals and plants is going to interfere with regeneration, growth or reproduction, an inducible promoter is required (Gatz, 1997). With such a tool, plants can be regenerated while the promoter is inactive. Further analysis can then be performed after activating expression of the transgene (Zuo and Chua, 2000; Reynolds, 1999).
For the application of the GVG system in transgenic Virginia pine cell cultures, the m-gfp5-ER reporter gene was inserted into a well-defined vector, pINDEX3, to test the system. It was reported that plant growth could be affected due to the nuclear localization of GVG after dexamethasone treatment (Kang et al., 1999). In this study, it was possible to distinguish transgenic Virginia pine cell cultures that showed limited growth retardation. Of the six glucocorticoids investigated in this study, prednisolone, prednisone, 6-methylprednisolone, and dexamethasone did not inhibit cell growth at 0.1–5 mg l–1, but did inhibit cell growth at 10 mg l–1 (Table 2, 3). Triamcinolone and hydrocortisone had no inhibitory effect on Virginia pine cell growth at concentrations of 0.1–10 mg l–1. Quantitative analysis showed that triamcinolone treatment could induce m-gfp5-ER activities in pINDEX3-m-gfp5-ER transgenic cell cultures to the highest levels (Fig. 6). No m-gfp5-ER activities were detected qualitatively or quantitatively in the absence of triamcinolone. However, hydrocortisone only induced a low expression level of target gene. A similar effect was observed in tobacco (Aoyama and Chua, 1997). Therefore, triamcinolone appeared to be the best choice for inducible gene expression in transgenic Virginia pine cells. These results demonstrated that the GVG driven by the CaMV 35S promoter worked in transgenic Virginia pine cell cultures, because inducible gene expression was observed in all six glucocorticoids selected. The system will be particularly useful when the pINDEX system is applied to study the functions of genes, for example, genes involved in cell division and cell wall formation, because these processes can be addressed in cell cultures.
The 4UAS sequence upstream of the target gene is not recognized by Virginia pine transcriptional activators in the nuclei in the absence of inducer. This is in agreement with the results of others for Arabidopsis, where GVG-mediated control of target genes also demonstrated that the system is silent without inducer (Aoyama and Chua, 1997; McNellis et al., 1998; Kunkel et al., 1999). Although it has been reported that methylation of UAS sites can interfere with reporter-gene activation sites in transgenic tobacco plants (Gälweiler et al., 2000), these results demonstrated that the use of the GVG system was not hampered by UAS methylation problems.
Transgenic Virginia pine cells are particularly suitable to investigate inducible gene expression and regulation in plant cells, as a large number of independently transformed lines can be produced. Moreover, cells are maintained in liquid culture, allowing maximum exposure between the cells and the dissolved chemicals. However, it is therefore essential to select a sufficient number of independent transformants in order to obtain a satisfactory number of individuals or cell lines possessing the desired GVG-regulated phenotype (Aoyama and Chua, 1997; Ouwerkerk et al., 2001). Among six glucocorticoids, prednisolone, prednisone, 6-methylprednisolone, dexamethasone, triamcinolone, and hydrocortisone, triamcinolone is the most potent activator of GVG activity in Virginia pine cells. However, the effective concentration remains extremely low for these compounds. The level of GVG-controlled m-gfp5-ER expression is different after treatment with different concentrations of glucocorticoids. Considerable differences in the level of induction can be obtained though manipulation of concentration and inducer compound. This property allows the control of transgene expression at levels intermediate between the on and off states, as well as comparisons of different transgene expression levels in the same transgenic line.
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Acknowledgements |
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The authors are grateful to Dr PBF Ouwerkerk and Dr AH Meijer for the gift of the vector pINDEX1-4, and to Dr CN Stewart and Dr J Haseloff for providing us with the m-gfp5-ER constructs, and to Dr D Weidner for technical assistance with confocal microscopy for imaging and quantitative analysis of green fluorescence.
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