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First published online August 17, 2007; 10.1104/pp.107.104646 Plant Physiology 145:367-377 (2007) © 2007 American Society of Plant Biologists OPEN ACCESS ARTICLE
Heat Suppresses Activation of an Auxin-Responsive Promoter in Cultured Guard Cell Protoplasts of Tree Tobacco1,[OA]Willamette University, Department of Biology, Salem, Oregon 97301 (M.A.D., J.L.B., K.C., M.Q.D., K.M., G.T.); and Japan Atomic Energy Agency, 1233 Watanuki, Takasaki 370–1292, Japan (Y.O.)
Cultured guard cell protoplasts (GCP) of tree tobacco (Nicotiana glauca) comprise a novel system for investigating the cell signaling mechanisms that lead to acquired thermotolerance and thermoinhibition. At 32°C in a medium containing an auxin (1-naphthaleneacetic acid [NAA]) and a cytokinin (6-benzylaminopurine), GCP expand, regenerate cell walls, dedifferentiate, and divide. At 38°C, GCP acquire thermotolerance within 24 h, but their expansion is limited and they neither regenerate walls nor reenter the cell cycle. These putative indicators of auxin insensitivity led us to hypothesize that heat suppresses induction of auxin-regulated genes in GCP. Protoplasts were transformed with BA-mgfp5-ER, in which the BA auxin-responsive promoter regulates transcription of mgfp5-ER encoding thermostable green fluorescent protein (GFP) or with a similar 35S-cauliflower mosaic virus constitutive promoter construct. Heat suppressed NAA-mediated activation of BA. After 21 h at 32°C in media with NAA, 49.0% ± 3.9% of BA-mgfp5-ER transformants strongly expressed GFP; expression percentages were similar to those of 35S-mgfp5-ER transformants at 32°C or 38°C. After 21 h at 38°C in media with NAA, 7.9% ± 1.6% of BA-mgfp5-ER transformants weakly expressed GFP, similar to GCP cultured at 32°C in media lacking NAA. Expression at 38°C was not increased by incubating for 48 h or increasing NAA concentrations 20-fold. After 9 to 12 h at 38°C, BA was no longer activated when cells were transferred to 32°C. Heat-stressed cells accumulate reactive oxygen species, and hydrogen peroxide (H2O2) suppresses auxin-responsive promoter activation in Arabidopsis (Arabidopsis thaliana) mesophyll protoplasts. H2O2 did not suppress BA activation at 32°C, nor did superoxide and H2O2 scavengers prevent BA suppression at 38°C.
Worldwide, the three major, interrelated abiotic plant stresses affecting plant growth and development are rising atmospheric CO2 concentration, heat, and drought (Mittler, 2006  ). Models (Scholze et al., 2006; Yesson and Culham, 2006) and experimental studies (Jump et al., 2006; Marchand et al., 2006; Qaderi et al., 2006; Sato et al., 2006; Walker et al., 2006; White et al., 2006) suggest that rising global temperatures will have important consequences both for crop production (Mittler, 2006; Qaderi et al., 2006; Sato et al., 2006; White et al., 2006) and for natural selection of noncrop plants (Marchand et al., 2006; Walker et al., 2006).
Heat can trigger cell-autonomous mechanisms that enable plants to survive extended periods of high temperature (Francis and Barlow, 1988
Cultured guard cell protoplasts (GCP) of tree tobacco (Nicotiana glauca) ‘Graham’ are a novel system for investigating the cell signaling mechanisms that lead to acquired thermotolerance and thermoinhibition (Roberts et al., 1995
GCP cultured at 38°C survive in the same high percentages as at 32°C, but neither exogenous NAA nor BAP is required for, nor do 1-aminoethoxyvinyl-glycine or ABA reduce, cell survival at 38°C (Roberts et al., 1995
Plant cell expansion, cell wall deposition, and cell division are all regulated, in part, by auxin (Kende and Zeevaart, 1997
In Arabidopsis (Arabidopsis thaliana) mesophyll protoplasts, exogenously supplied hydrogen peroxide (H2O2) activates a mitogen-activated protein (MAP) kinase, ANP1, which suppresses activation of the soybean (Glycine max) GH3 auxin-responsive promoter in a transient gene expression assay (Kovtun et al., 1998 Our results indicate that heat suppresses capacity for BA promoter activation in GCP but that neither superoxide nor H2O2 is required to suppress cellular capacity for BA activation.
Heat Suppresses NAA-Mediated Activation of the BA Early Auxin-Responsive Promoter Based on putative indicators of auxin insensitivity at high temperature, we tested the hypothesis that heat would suppress activation of an auxin-responsive promoter in cultured GCP. After 21 h at 32°C in medium with NAA, 49.0% ± 3.9% of GCP transformed with BA-mgfp5-ER expressed GFP (mean; SE; n = 3; Figs. 1 and 2, A and B ). This expression percentage was similar to those of cells from the same isolates that were transformed with 35S-mgfp5-ER and cultured for 21 h at 32°C (Figs. 1 and 2E) or at 38°C (Figs. 1 and 2F). When GCP from the same batches of BA-mgfp5-ER transformants were cultured for 21 h at 38°C in medium with NAA (Fig. 2D), the mean percentage of cells expressing GFP was 7.9% ± 1.6% (Fig. 1), similar to the 7.5% ± 0.9% of cells expressing GFP after 21 h at 32°C in medium lacking NAA (Fig. 2C). When viewed through the microscope, GFP fluorescence intensities of BA-mgfp5-ER transformants cultured in medium with NAA were visibly greater at 32°C than at 38°C (compare Fig. 2A to 2D). There was no visible expression of GFP in BA-mgfp5-ER transformants cultured at 38°C for up to 24 h in medium lacking NAA (Table I ).
We used a FACS to quantify relative GFP intensities in transformed GCP cultured at the two temperatures. In GCP transformed with BA-mgfp5-ER, after 15 h of culture at 32°C in medium containing NAA, a distinct population of GFP-expressing cells (Fig. 3A ) could be distinguished from a sham-transformed cell population from the same isolate (Fig. 3F) that was used to set the background fluorescence threshold. When GCP from the same batch of transformants were cultured at 32°C without NAA (Fig. 3B) or at 38°C with NAA (Fig. 3C), the level of GFP expression was below the threshold level required to distinguish GFP-containing cells from those of sham-treated controls. In GCP transformed with 35S-mgfp5-ER, after 15 h of culture in medium containing NAA, the median GFP intensity at 32°C (Fig. 3D) was approximately twice that of the median GFP intensity of cells from the same batch of transformants cultured at 38°C (Fig. 3E). Median GFP intensities of GCP transformed with 35S-mgfp5-ER and cultured at both temperatures were substantially greater than median GFP intensities of cells transformed with BA-mgfp5-ER and cultured at 32°C in a medium containing NAA (Figs. 2, B, E, and F, and 3, A, D, and E).
We tested the hypotheses that GCP accumulate GFP more slowly at 38°C and that higher concentrations of NAA are required to activate BA at 38°C. Extending culture times to 48 h did not increase significantly the mean percentage of GCP transformed with BA-mgfp5-ER that expressed GFP at 38°C compared to the corresponding 24-h temperature control (Table I; unpaired t test; n = 3; P = 0.22). Increasing NAA concentrations 20-fold did not increase mean GFP expression percentages over those of 1x NAA controls from the same batches of transformants either at 32°C (Table I; unpaired t test; n = 3; P = 0.57) or at 38°C (Table I; unpaired t test; n = 3; P = 0.6).
We examined whether heat-mediated suppression of BA becomes irreversible with time by preincubating GCP for various times at 38°C and then transferring them to 32°C for 18 h before examining them for GFP accumulation. After 3 or 6 h at 38°C in medium containing NAA, on average, approximately three to four times as many GCP transformed with BA-mgfp5-ER expressed GFP upon subsequent transfer to 32°C for an additional 18 h as did controls from the same batches of transformants maintained for an equal period at 38°C (Fig. 4
). After
In reciprocal experiments, culture for  12 h at 32°C before an additional 18 h of culture at 38°C reduced the mean percentages of GCP expressing GFP below those of controls from the same batches of transformants maintained at 32°C for an equal period of time (Fig. 4). After 12 h at 32°C, transfer to 38°C diminished only slightly the mean percentage of GCP expressing GFP compared to parallel transformant controls maintained at 32°C for an equal period (Fig. 4). When GCP transformed with BA-mgfp5-ER were transferred after 18 h at 32°C to 38°C for an additional 18 h, there was no visible decrease in fluorescence intensity at the end of the 36-h period (data not shown). When cells from the same batches of transformants were transferred after 18 h at 38°C to 32°C for an additional 18 h, there was no visible increase in fluorescence intensity at the end of the 36-h experiment (data not shown).
Cultured plant cells can transiently accumulate ROS under heat stress (Vacca et al., 2004
H2O2 is a signaling molecule in ABA-induced stomatal closure (Murata et al., 2001
Heat Suppresses NAA-Mediated Activation of the BA Early Auxin-Responsive Promoter in Cultured GCP From microscopic (Figs. 1 and 2) analyses the mean percentage of BA-mgfp5-ER transformants expressing GFP after 18 to 24 h at 32°C in medium containing NAA was approximately 45% to 50% (Fig. 1). This value was not significantly lower than those of 35S-mgfp5-ER transformants from the same isolates that were cultured similarly at 32°C or 38°C (Fig. 1). In cells cultured at 32°C in medium containing NAA, GFP could be detected readily with a FACS (Fig. 3A), a CCD camera (Fig. 2B), and the eye. By contrast, <10% of GCP from the same isolates transformed with BA-mgfp5-ER expressed GFP after 18 to 24 h at 38°C in a medium containing NAA (Fig. 1). In cells cultured at 38°C in medium containing NAA, GFP fluorescence could not be detected with a FACS (Fig. 3C) or imaged effectively with a CCD camera (Fig. 2D); fluorescence levels were so low that GFP could only be detected with the eye. No GFP expression could be detected visually in BA-mgfp5-ER transformants cultured at 38°C in a medium lacking NAA at any time point (see 24-h time point, Table I; time course not shown).
In addition to suppression of promoter activation, reductions in GFP accumulation could be envisioned to result from heat-induced gene modification (e.g. methylation), global reductions in transcription rates, global reductions in translation rates, accelerated degradation of GFP mRNA, accelerated GFP degradation, or (a) combination(s) of these processes. Reductions in GFP accumulation would not be expected due to gene silencing in this system because a cryptic intron has been removed from this particular version of the GFP gene to thwart plant gene silencing mechanisms (Haseloff et al., 1997 In three separate experiments with 35S-mgfp5-ER transformants, median GFP intensities after 15 to 18 h at 38°C were approximately one-half those of cells cultured at 32°C (compare Fig. 3D to 3E). However, this decrease in intensity did not translate to a corresponding reduction in the percentage of cells that could be observed microscopically to express GFP (Figs. 1 and 2). Indeed, GFP was still expressed strongly at 38°C under the control of the constitutive 35S promoter (Figs. 2F and 3E), indicating that individually, or in combination, the effects of heat on promoter-independent processes could not account for the magnitude to which heat suppresses GFP accumulation when mgfp5-ER is under control of the auxin-responsive BA promoter. In short, if the eye can still detect GFP in 7% to 10% of cells when it can be detected neither by FACS nor a CCD camera, it would still be possible to see through the microscope GFP in the GCP shown in Figure 2B even if mean GFP intensities were reduced by 50%. At most, a heat-induced promoter-independent reduction in median GFP intensities at 38°C in BA-mgfp5-ER transformants exposed to NAA might have been expected to reduce the percentage of cells observed to express GFP from approximately 45% to 50% to approximately 20% to 25%. However, at every time point, the percentage of cells expressing GFP at 38°C in a medium with NAA was <10% (Fig. 1), and GFP intensities were low enough that this small population of cells could only be detected microscopically (Fig. 3C). Thus, we conclude that heat suppresses activation of the BA promoter.
GFP-based reporters were chosen for these experiments for a number of reasons. GFP has been shown to be a quantitative reporter of gene expression in eukaryotic cells (Soboleski et al., 2005 The percentage of BA-mgfp5-ER transformants expressing GFP at 38°C in a medium with NAA was not increased by extending incubations to 48 h (Table I), suggesting that heat-treated transformants did not simply accumulate GFP more slowly.
Increasing NAA concentrations 20-fold did not increase the percentage of BA-mgfp5-ER transformants that expressed GFP at 38°C (Table I). These data suggest that GCP harbor at least one heat-sensitive auxin-mediated promoter activation pathway that is fully suppressed at high temperature rather than just reduced in its sensitivity to NAA. This observation is consistent with our previous result showing that neither 20- nor 50-fold increases in NAA concentration enabled cultured GCP to reenter the cell cycle at 38°C (Gushwa et al., 2003
When BA-mgfp5-ER transformants were preincubated at 38°C in an NAA-containing medium for <9 h prior to being cultured for 18 h at 32°C, substantial percentages (20%–40%) of cells still accumulated GFP (Fig. 4). When BA-mgfp5-ER transformants were preincubated at 38°C for 9 to 18 h prior to being cultured at 32°C, cells did not accumulate GFP in percentages greater than those of controls maintained at a constant 38°C (approximately 7%–8%; Fig. 4). Reciprocal controls preincubated at 32°C for 9 to 12 h before transfer to 38°C did accumulate GFP. These data indicate that 9 to 12 h are required to irreversibly suppress GCP capacity for NAA-mediated BA activation. This period is much longer than those typical of heat shock experiments (Vacca et al., 2004
In previous experiments, returning GCP to the lower culture temperature of 32°C after 9 to 12 h at 38°C resulted in a high percentage of cell death, but GCP incubated for >12 h survived in higher percentages upon transfer to 32°C (Gushwa et al., 2003
Unlike GCP cultured at 32°C, GCP cultured at 38°C are thermoinhibited. Their expansion is limited and they fail to regenerate cell walls and make the G1-to-S phase transition required for cell cycle reentry. While the data presented here do not establish definitively a causal linkage between development of auxin insensitivity and thermoinhibition, all of the processes above have been linked to auxin signaling for gene expression (Meyer et al., 1984
At 32°C, a small percentage (approximately 5%–10%) of GCP accumulate GFP in medium without auxin. GFP accumulation in the absence of auxin could be due to promoter leakiness, to the action of endogenous auxin, or both. In no experiment did culturing BA-mgfp5-ER transformants at 38°C in media containing NAA completely suppress BA-mediated GFP expression. Even when GCP were preincubated at 38°C for 18 h before they were cultured at 32°C in a medium containing NAA, nearly 10% of cells expressed GFP (Fig. 4). These results could be due to leakiness in the promoter, a heat-insensitive promoter activation pathway, or both, but the observation that GFP does not accumulate in BA-mgfp5-ER transformants at 38°C in a medium lacking NAA indicates that heat-insensitive GFP accumulation at 38°C is NAA dependent.
The percentage of BA-mgfp5-ER transformants expressing GFP at 38°C in a medium with NAA was similar to that of BA-mgfp5-ER transformants cultured at 32°C in a medium lacking NAA. However, GCP do not survive at 32°C in a medium lacking NAA (Gushwa et al., 2003
In mesophyll protoplasts isolated from Arabidopsis leaves, exogenous H2O2 activates a MAP kinase, ANP1, which suppresses activation of the soybean GH3 auxin-responsive promoter (Kovtun et al., 1998
H2O2 is a component of a guard cell ABA signaling pathway(s) that prevents stomatal opening/triggers stomatal closure (Murata et al., 2001
Within 9 to 12 h of exposure of cultured GCP of tree tobacco to high temperature (38°C), heat irreversibly suppresses their capacity for NAA-mediated activation of the BA early auxin-responsive promoter. Previous studies show that when GCP are returned after 9 to 12 h at 38°C to 32°C, a large percentage die (Gushwa et al., 2003 ), probably because at the lower temperature they still require auxin, cytokinin, and endogenously produced ethylene and therefore must be auxin responsive to survive (Gushwa et al., 2003). If cells are left at 38°C for 24 h, their survival no longer depends on exogenous auxin or cytokinin or endogenously produced ethylene at either temperature (Gushwa et al., 2003). However, as they become thermotolerant they also become thermoinhibited. Cell expansion is limited and GCP fail to regenerate cell walls and make the G1-to-S cell cycle transition as they do at lower culture temperatures (Gushwa et al., 2003). While these experiments do not establish a causal linkage between heat-induced suppression of NAA-mediated auxin-responsive promoter activation and thermoinhibition, cell expansion, cell wall regeneration, and/or the G1-to-S transition would all be expected to require auxin-mediated gene activity. Taken together, our time course data suggest that at high temperature, development of thermotolerance is subsequent to development of auxin insensitivity and that auxin insensitivity could be responsible, at least in part, for thermoinhibition of cell expansion, cell wall regeneration, and cell cycle reentry. Whether signaling for development of thermotolerance is coordinately and/or obligatorily linked to loss of auxin sensitivity at high temperature awaits investigation.
Plants
Tree tobacco (Nicotiana glauca) ‘Graham’ was grown from seed (Garden Makers) as described (Tallman, 2005
Escherichia coli (DH5-
Tree tobacco GCP were isolated and cultured as described (Tallman, 2005
GCP were transformed with either BA-mgfp5-ER containing a truncated version of the early auxin BA promoter from Pisum sativum or with 35S-mgfp5-ER containing the 35S CaMV constitutive promoter. The GFP gene in these reporter constructs, mgfp5-ER, has been modified to yield a thermostable protein (Siemering et al., 1996
Arabidopsis (Arabidopsis thaliana) mesophyll protoplast transformation protocols (Chiu et al., 1996
Because tree tobacco GCP had not been transformed previously, neither the time required for maximum GFP accumulation nor the transformation percentages to be expected were known. Therefore, the effects of culture at 32°C versus 38°C on GFP accumulation regulated by the BA promoter (Ballas et al., 1995 Because heat greatly reduced GFP expression in GCP transformed with BA-mgfp5-ER (above), some experiments were extended to 48 h to determine whether GFP was simply accumulating more slowly or with different kinetics at 38°C than at 32°C. In other attempts to increase GFP accumulation at 38°C, NAA concentrations were increased 20-fold to 16.2 µM and GCP were cultured for 18 h. To initiate experiments, 0.3 mL of S&T medium, pH 6.1, containing 4 x 104 transformed GCP was pipetted into each well of an 8-well chamber slide. NAA (0.012 g) and/or BAP (0.003 g) was dissolved in 10 mL of ethanol. NAA and/or BAP in ethanol (12.5 µL) was added to 25 mL of S&T medium at pH 6.1, and 0.1 mL of the appropriate hormone-containing medium was added to each well to final concentrations of 0.81 µM NAA and/or 0.166 µM BAP. For 20x stocks, 0.24 g NAA and/or 0.06 g BAP was dissolved in 10 mL of ethanol. In each experiment, cells in some wells were treated with a combination of NAA and BAP while GCP in other wells were treated with BAP alone. Chamber slides were incubated at 32°C or at 38°C in water-jacketed incubators.
The number of GCP expressing GFP was estimated microscopically every 3 h for 24 h using an Olympus inverted microscope (Gushwa et al., 2003
To confirm that the manual counting methods described above accurately reflected GFP accumulation levels in GCP transformed with BA-mgfp5-ER or 35S-mgfp5-ER, a FACS was used to measure the fraction of cells in each treatment expressing GFP and the intensity distribution of GFP fluorescence in cell populations. GCP transformed with either BA-mgfp5-ER or 35S-mgfp5-ER were incubated for 15 h at 32°C or 38°C in media containing BAP with or without NAA. Thirty-thousand events were then machine scored using a FACS Aria (Becton-Dickinson) at low pressure setting and a flow rate of 6 to 7. Voltage settings were: 150 forward scatter, 320 side scatter, 500 fluorescein isothiocyanate (GFP). Because they exhibit intense red chloroplast autofluorescence, GCP subjected to transformation protocols without plasmid DNA (sham controls) were used to establish conservative threshold levels of autofluorescence beyond which only GCP that were unequivocally expressing GFP were scored by the machine.
In all experiments with BA-mgfp5-ER, fluorescence intensities were substantially lower in cells cultured at 38°C than in those cultured at 32°C (above). Reciprocal temperature transfer experiments were conducted to determine: (1) whether GCP preincubated at 38°C would develop a higher fluorescence intensity upon transfer to 32°C; (2) whether the higher fluorescence intensity observed in cells cultured initially at 32°C would be diminished upon transfer to 38°C; and (3) the approximate time period at 38°C beyond which cellular capacity for BA promoter activation was irreversible. Nine chamber slides with BA-mgfp5-ER-transformed cells in media containing NAA and BAP (above) were incubated at 32°C or at 38°C. The percentage of cells expressing GFP was estimated (above) in one slide from each temperature treatment after 0, 3, 6, 9, 12, 15, or 18 h. Each culture was then incubated at the alternate temperature for an additional 18 h before the percentage of GCP expressing GFP was reestimated. As a control, at every time point the percentage of cells expressing GFP was also estimated in BA-mgfp5-ER-transformed GCP incubated continuously at 32°C or at 38°C for the same accumulated time periods as treatments.
To determine whether endogenously produced superoxide and/or H2O2 might be required to suppress activation of the BA promoter at high temperature, GCP were cultured at 32°C or at 38°C in a medium containing both the superoxide scavenger, 4,5,dihydroxy-1,3-benzenedisulfonic acid (Tiron; 50 µM), and N-acetyl-L-Cys (50 µM), a scavenger of H2O2. To achieve final concentrations, 5 µL of a 4-mM stock solution containing both compounds prepared in cell culture medium were added to 400-µL chamber-slide cultures. To determine whether H2O2 accumulating in the culture medium might be required to suppress BA activation at high temperature, bovine liver catalase (EC 1.11.1.6; 0.03 mg mL–1, Sigma; Lee et al., 1999 The endogenous capacity of GCP to metabolize and/or scavenge H2O2 was evaluated using cell extracts prepared by freezing 2 x 105 GCP at –80°C for 5 min in 1 mL of cell culture medium, thawing, centrifuging for 2 min at top speed in a microcentrifuge to clear insoluble debris, and transferring supernatants to a fresh tube. Extracts were stored on ice while their effects on H2O2 metabolism and/or scavenging were determined. In preparation for H2O2 assays, extracts were diluted with cell culture medium, pH 6.1, over the range of 0.04% to 20% of their full concentration, and an H2O2 solution was prepared by diluting a 1 x 104 ng mL–1 stock 1:9 with culture medium, pH 6.1, lacking hormones (final concentration 1 x 103 ng mL–1). To evaluate the capacity of extracts to metabolize and/or scavenge H2O2, 50 µL of medium (control) or each diluted or undiluted extract was added to 200 µL of diluted H2O2 (above) so that the final H2O2 concentration was 800 ng mL–1 (23.5 µM). After exactly 5 min at room temperature, 50 µL of the mixture was transferred to a luminometer tube and H2O2 remaining was measured with a chemiluminescent assay (catalog no. 907–102; Assay Designs). The luminometer (Zylux Femtomaster FB15, Zylux) was programmed to inject 20 µL of chemiluminescent substrate and then 20 µL of trigger after a 2-s delay. After injection of the trigger, samples were counted for 10 s. To determine whether loss of H2O2 was due to enzymatic or nonenzymatic processes, assays were repeated with undiluted extracts in media containing the peroxidase inhibitors sodium azide (final concentration 10 mM), KCN (10 mM), or 3-amino-1,2,4-triazole (10 mM), or the NADPH oxidase inhibitor, diphenylene iodonium chloride (final concentration 0.2 mM from a 32-mM dimethyl sulfoxide stock). Assays containing inhibitors but with cell culture medium in place of extracts were used as controls. Experiments were repeated three times on separate days.
We thank J. Oost and B. Druker for assistance with FACS, and E. Meyerowitz, J. Sheen, and A.S. Raghavendra for helpful discussions. Received June 26, 2007; accepted August 13, 2007; published August 17, 2007.
1 This work was supported by a grant from the M.J. Murdock Charitable Trust (to G.T.). 
2 Present address: Department of Energy Plant Research Laboratory and Department of Plant Biology, Michigan State University, East Lansing, MI 48824.
3 Present address: University of Kentucky College of Medicine, Box 40, Chandler Medical Center, 800 Rose Street, Lexington, KY 40536–0298. The author responsible for distribution of materials integral to the findings presented in this article in accordance with the policy described in the Instructions for Authors (www.plantphysiol.org) is: Gary Tallman (gtallman{at}willamette.edu).
[OA] Open Access articles can be viewed online without a subscription. www.plantphysiol.org/cgi/doi/10.1104/pp.107.104646 * Corresponding author; e-mail gtallman{at}willamette.edu.
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ABSTRACT
RESULTS
) generally increases when it acquires thermotolerance (Suzuki and Mittler, 2006
, 
) or by the 35S CaMV promoter (
) over 24 h at 32°C (
9 h at 38°C, the mean percentages of cells expressing GFP after transfer to 32°C for an additional 18 h were similar to those of parallel transformant controls (approximately 7%–8%) maintained for an equal period at 38°C (
) or from protoplasts cultured for 12 h at 32°C (
) or at 38°C (
) by freezing 2 x 105 cells in culture medium at –80°C, thawing, and centrifuging. Extract dilutions are expressed as cell equivalents in the fixed-volume assay mixture. Inset, Expanded version of region between 0 and 2,500 cell equivalents. Values are means and SE from three replicate experiments on separate days. The mean initial RLU s–1 of untreated solutions in all treatments was 3.2 ± 0.4 x 106 (n = 8); the mean value after 5-min exposure to culture medium containing no cells was 3.2 ± 0.4 x 106 RLU s–1 (n = 8).
) harboring BA-mgfp5-ER/pPZP121 (Aspuria et al., 2002