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ENVIRONMENTAL STRESS AND ADAPTATION

Thermotolerant Guard Cell Protoplasts of Tree Tobacco Do Not Require Exogenous Hormones to Survive in Culture and Are Blocked from Reentering the Cell Cycle at the G1-to-S Transition¹

Department of Biology, Willamette University, 900 State Street, Salem, Oregon 97301 (N.N.G., D.H., A.K., J.U., C.F.T., S.F., S.P., L.P.M., G.T.); and Lancaster University, Department of Biological Sciences, Institute of Environmental and Natural Sciences, Bailrigg, Lancaster LA1 4YQ, United Kingdom (B.A., J.E.T.)

	ABSTRACT

TOP ABSTRACT RESULTS DISCUSSION MATERIALS AND METHODS LITERATURE CITED

 
When guard cell protoplasts (GCPs) of tree tobacco [Nicotiana glauca (Graham)] are cultured at 32°C with an auxin (1-napthaleneacetic acid) and a cytokinin (6-benzylaminopurine), they reenter the cell cycle, dedifferentiate, and divide. GCPs cultured similarly but at 38°C and with 0.1 µM ± -cis,trans-abscisic acid (ABA) remain differentiated. GCPs cultured at 38°C without ABA dedifferentiate partially but do not divide. Cell survival after 1 week is 70% to 80% under all of these conditions. In this study, we show that GCPs cultured for 12 to 24 h at 38°C accumulate heat shock protein 70 and develop a thermotolerance that, upon transfer of cells to 32°C, enhances cell survival but inhibits cell cycle reentry, dedifferentiation, and division. GCPs dedifferentiating at 32°C require both 1-napthaleneacetic acid and 6-benzylaminopurine to survive, but thermotolerant GCPs cultured at 38°C ± ABA do not require either hormone for survival. Pulse-labeling experiments using 5-bromo-2-deoxyuridine indicate that culture at 38°C ± ABA prevents dedifferentiation of GCPs by blocking cell cycle reentry at G1/S. Cell cycle reentry at 32°C is accompanied by loss of a 41-kD polypeptide that cross-reacts with antibodies to rat (Rattus norvegicus) extracellular signal-regulated kinase 1; thermotolerant GCPs retain this polypeptide. A number of polypeptides unique to thermotolerant cells have been uncovered by Boolean analysis of two-dimensional gels and are targets for further analysis. GCPs of tree tobacco can be isolated in sufficient numbers and with the purity required to study plant cell thermotolerance and its relationship to plant cell survival, growth, dedifferentiation, and division in vitro.

Cultured guard cell protoplasts (GCPs) of tree tobacco [Nicotiana glauca (Graham)] may be an excellent in vitro system for elucidating the signal transduction mechanisms that regulate plant cell thermotolerance (Roberts et al., 1995; Taylor et al., 1998). Highly purified GCP monocultures (<0.01% contamination with other cell types; Fig. 1A) are uniform and synchronous in their responses to growth regulators like 1-napthaleneacetic acid (NAA), 6-benzylaminopurine (BAP), and ± -cis,trans-abscisic acid (ABA), and to temperature (Roberts et al., 1995; Taylor et al., 1998). Parallel cultures established from the same isolate can be monitored under temperature conditions that result in full dedifferentiation and cell division (culture at 32°C; Fig. 1B), maintenance in the differentiated state (culture at 38°C in media with 0.1 µM ABA; Fig. 1C), or partial dedifferentiation without cell division (culture at 38°C; Fig. 1D; Roberts et al., 1995; Taylor et al., 1998). Under all of these conditions, GCPs survive in high percentages (70%–80%) for at least 1 week (Roberts et al., 1995), indicating that GCPs develop thermotolerance at 38°C.

View larger version (207K): [in this window] [in a new window]   Figure 1. Cultured GCPs of tree tobacco. A, FGCPs and GCPs cultured for 1 week at 32°C (B), 38°C in media containing 0.1 µM ABA (C), or 38°C (D). A to C, Magnification = 600x; B, magnification = 200x; D, magnification = 400x. Bar in A = 15 µm.

At 32°C, both NAA and BAP are required for GCPs to survive, dedifferentiate, and divide. Dedifferentiating GCPs grow 50- to 60-fold, regenerate cell walls, and undergo major cytoskeletal rearrangements (Fig. 1B; Roberts et al., 1995; Taylor et al., 1998). Their chloroplasts become chlorotic and nonfunctional (Taylor et al., 1998). The resulting dedifferentiated cells (Fig. 1B) are totipotent (Sahgal et al., 1994). Experiments with 2-aminoethoxyvinyl-Gly (AVG), an inhibitor of ethylene synthesis, suggest that dedifferentiating GCPs require endogenous ethylene production for survival (Roberts et al., 1995). ABA is lethal to GCPs cultured at 32°C (Roberts et al., 1995). At 38°C in media containing ABA, GCPs do not dedifferentiate (Fig. 1C; Roberts et al., 1995; Taylor et al., 1998). Instead, they retain the size, morphology, and many of the unique physiological characteristics of guard cells (Taylor et al., 1998). Among the functional characteristics retained are the capacity to: (a) swell when exposed to fusicoccin, (b) swell when illuminated with low fluences (15 µmol m^–2 s^–1) of blue light, (c) execute light-driven photosynthetic electron transport, and (d) accumulate zeaxanthin upon illumination (Taylor et al., 1998). At 38°C in media lacking ABA, GCPs grow (Fig. 1D) and partially dedifferentiate (Taylor et al., 1998), losing some of the functional identity of guard cells. These cells do not divide (Roberts et al., 1995; Taylor et al., 1998), indicating that elevated temperature alone is sufficient to prevent cultured GCPs from reaching M phase of the cell cycle (Fig. 1D). Whether or not media contain ABA, survival of GCPs at 38°C is not reduced by AVG treatment and, thus, does not appear to depend on endogenous ethylene production (Roberts et al., 1995). Cells cultured at 38°C ± ABA do not regenerate cell walls (Roberts et al., 1995; Taylor et al., 1998).

Despite their potential as an experimental system, limitations on the number of GCPs that can be prepared in a single isolate have made the types of routine signal transduction studies that are common in yeast and animal cell culture systems too laborious to perform with GCP. In this study, we attempted to scale procedures for isolating GCPs of tree tobacco to provide 1 to 1.5 x 10⁷ cells per isolate at a purity adequate for larger scale studies. To establish the kinetics of development of thermotolerance, GCPs were pre-incubated at 38°C for times ranging from 0 to 24 h and then cultured for an additional week at 32°C before the effects of high temperature pretreatment on cell survival and division were estimated. To evaluate whether Hsp levels increase with the same kinetics as gain of thermotolerance, at the same pre-incubation time points, proteins were extracted from GCPs cultured at 32°C or 38°C ± ABA, and levels of inducible Hsp70 and heat shock cognate (Hsc) 70 were measured by western blotting. To ascertain whether thermotolerance alters hormone requirements for cell survival, survival was estimated after 1 week of culture at 32°C or 38°C ± ABA in media containing NAA alone, BAP alone, both NAA and BAP, or neither hormone. To determine the point(s) at which high temperature and/or ABA block the cell cycle and dedifferentiation, pulse labeling with 5-bromo-2-deoxyuridine (BrdU) was employed over a 2-week period to test the hypothesis that GCPs cultured at 38°C ± ABA do not pass the restriction point between G1 and S.

MAPK similar to those involved in regulating thermotolerance in yeast (Trotter et al., 2001) and cell differentiation, division, and apoptosis in cultured animal cells (Kim et al., 2002; Yoon et al., 2002) are conserved across a number of plant species (Morris, 2001), and three putative MAPK have been identified in GCPs from pea (Pisum sativum; Burnett et al., 2000). An MAPK cascade initiated by an MAPKKK (NPK1) can suppress auxin signaling in transfected maize (Zea mays) protoplasts and transgenic tobacco (Nicotiana tabacum) plants (Kovtun et al., 1998, 2000), and several symptoms of guard cell thermotolerance are consistent with suppression of auxin signaling (e.g. failure to regenerate cell walls, senesce chloroplasts, and reenter the cell cycle; Roberts et al., 1995; Taylor et al., 1998). Thus, over a time course similar to that used for BrdU experiments, protein extracts from freshly isolated GCPs (FGCPs) or from cultures containing dedifferentiated (32°C), differentiated (38°C + ABA), or partially dedifferentiated (38°C) cells were probed for ERK1/2 homologs by western blotting with mammalian ERK1/2 antibodies.

To identify target polypeptides associated with thermotolerance for future proteomic analysis, we developed silver-stained two-dimensional gel libraries of the major polypeptides extracted from FGCPs and from GCPs cultured under each condition described. We then compared polypeptide profiles of thermotolerant cells with those of cells undergoing dedifferentiation at 32°C and with those of FGCPs by Boolean spot match analysis.

These experiments confirm that high-yield monocultures of tree tobacco GCPs can provide sufficient material to study in vitro the signal transduction pathways that regulate development of plant cell thermotolerance and its effects on cell survival, growth, differentiation, and division. They also demonstrate that protoplast yields are adequate to secure a proteomic analysis of target polypeptides associated with all of these processes.

	RESULTS

TOP ABSTRACT RESULTS DISCUSSION MATERIALS AND METHODS LITERATURE CITED

 

The Protocol for Isolating GCP Scales without Loss of Purity

We reasoned that, because a single leaf can of tree tobacco can be stripped of its epidermis in 5 to 10 min and a leaf typically yields 1.1 to 1.6 x 10⁶ GCP, it might be possible to scale the isolation protocol proportionately to give high yields of GCPs while maintaining their purity. The method used to isolate GCPs from a single leaf was scaled successfully to yield as many as 1.5 x 10⁷ GCPs from nine leaves. In the largest GCP preparations, levels of contamination with mesophyll and pavement cell protoplasts were similar to those of single leaf preparations (<0.01%; Fig. 1A; Cupples et al., 1991). As reported previously (Roberts et al., 1995), contaminating cells did not survive under any of the culture conditions employed.

Cultured GCPs Develop Thermotolerance within 12 to 24 h

GCPs cultured at 32°C begin to divide within 48 to 72 h (Cupples et al., 1991), but GCPs cultured at 38°C ± ABA do not divide. Thus, we expected that GCPs would develop thermotolerance within the first 24 to 48 h in culture and that thermotolerance should be defined by high survival but failure to divide (Roberts et al., 1995). To establish the kinetics of development of thermotolerance, GCPs were pre-incubated at 38°C for 1 to 24 h and then cultured at 32°C for another week before the effects of high-temperature pre-incubation on survival and cell division were evaluated. To establish baseline survival, GCPs were cultured at 38°C for 24 h, and the number of living and/or dead cells in cultures was estimated microscopically. After 24 h at 38°C, the mean number of dead cells was 446.3 ± 43.1, the mean number of living cells was 1,223.7 ± 79.4, and the mean percentage of survival was 73.3 ± 3.0 (mean; SE; n = 3). The mean numbers of dead cells in control cultures incubated for 1 week at 32°C or 38°C were 496 ± 34.3 and 535.3 ± 13.4, respectively. In neither of these controls was the number of dead cells significantly different from the 24-h, 38°C baseline control (Fig. 2A; ANOVA; Fisher's protected least squares difference (PLSD), P 0.05, n = 3).

View larger version (86K): [in this window] [in a new window]   Figure 2. Effect of pre-incubating GCPs of tree tobacco at 38°C on their survival and division after an additional week of culture at 32°C. A, Mean number of dead cells after 1 week of culture at 32°C that was preceded by a 38°C pre-incubation for 0 to 24 h. B, Baseline mean number of cells surviving after 24 h at 38°C. Lines, Mean number of cells surviving after 8 d of continuous culture at 32°C or at 38°C. Values are means and SEs from three replicate experiments. a, Significantly different from baseline control (ANOVA; Fisher's protected least squares difference; P 0.05); b, significantly lower than 9-h pre-incubation (ANOVA; Fisher's protected least squares difference; P 0.05). B, GCPs cultured continuously for 8 d at 32°C (100x; bar = 100 µm). C, GCPs cultured at 38°C for 24 h and then for an additional week at 32°C. Note lack of dividing cells. Arrows, Dead cells (dc) and nondividing cells lacking cell walls.

After 1 week of culture at 32°C after 1 to 24 h of pre-incubation at 38°C, the mean number of dead cells was significantly greater than that of the baseline control at all pre-incubation times tested (Fig. 2A; ANOVA; Fisher's PLSD, P 0.05, n = 3). The number of dead cells increased significantly after 1 to 9 h of pre-incubation, peaked at 6 to 9 h, and then decreased significantly from the number of dead cells in the 9-h pre-incubation treatment after 12 to 24 h of pre-incubation (Fig. 2A; ANOVA; Fisher's PLSD, P 0.05, n = 3). After a 9-h pre-incubation at 38°C, only approximately 35% to 40% of cells survived an additional week of culture at 32°C.

Cells pre-incubated at 38°C for 9 h that survived an additional week of culture at 32°C divided in high percentages (95%; not shown). However, rates of division were low (<2%) among cells pre-incubated at 38°C for 12 h and then cultured for 1 week at 32°C (Fig. 2, B and C). A variety of cell types were observed in cultures pre-incubated for 12 h, including elongate cells (not shown) and cells without walls (Fig. 2C). GCPs cultured at 38°C for up to 24 h in media containing ABA did not survive when they were cultured subsequently at 32°C for an additional week.

GCPs Accumulate Hsp70 after 18 to 24 h at 38°C + ABA

Hsp production and activation of heat shock transcription factors (HSFs; Wu, 1995) would be expected at 38°C ± ABA and might be anticipated to affect cell survival (Queitsch et al., 2000) and capacity for cell cycle reentry (Helmbrecht et al., 2000; Kühl and Rensing, 2000). Therefore, we measured levels of inducible Hsp70 and the noninducible Hsc70 over the same time course used to measure development of thermotolerance. Mean levels of Hsp70 measured as normalized contour gel band quantities did not change significantly over the first 24 h of culture at 32°C (Fig. 3, A and C; ANOVA; Fisher's PLSD, P 0.05, n = 3), nor did levels of Hsp70 change significantly over the first 12 h of culture at 38°C or over the first 18 h of culture at 38°C in media containing ABA (Fig. 3, A and C; ANOVA; Fisher's PLSD, P 0.05, n = 3). After 18 and 24 h of culture at 38°C in media lacking ABA or 24 h of culture at 38°C in media containing ABA, Hsp70 levels were significantly greater than those of GCPs cultured at 32°C (Fig. 3, A and C; ANOVA; Fisher's PLSD, P 0.05, n = 3). Under each condition, levels of Hsc70 were unchanged over the first 24 h of culture (Fig. 3B; ANOVA; Fisher's PLSD, P 0.05, n = 3). In some instances, proteins of lower molecular mass (60–65 kD) that cross-reacted with Hsp70 and Hsc70 antibodies were detected on blots (e.g. see 38°C + ABA in Fig. 3C), but there was no consistent pattern that could be correlated with a particular treatment. Thus, only the contour quantities of predominant bands with molecular masses similar to those of control proteins were measured.

View larger version (33K): [in this window] [in a new window]   Figure 3. Levels of Hsp70 and Hsc70 in GCPs of tree tobacco cultured at 38°C (), at 38°C in media containing 0.1 µM ABA (), or at 32°C () for up to 24 h. A and C, Hsp70 levels; B, Hsc70 levels. Lanes contained equal amounts of protein extracted from cells cultured for 0, 0.5, 1, 3, 6, 9, 12, 18, or 24 h. In each experiment, band contour quantities were estimated with densitometry and normalized to standards (60 ng of human [Homo sapiens] Hsp70 or wheat [Triticum aestivum] Hsc70) before they were averaged. Values are means for three replicate experiments. Asterisk, Significantly different from corresponding 32°C (ANOVA; Fisher's PLSD; P 0.05).

Thermotolerant GCPs Do Not Require NAA or BAP to Survive

At 32°C, dividing GCPs require both NAA and BAP to survive, but it was not clear whether development of thermotolerance might induce a cell survival mechanism that would eliminate this requirement at 38°C ± ABA. At 32°C, omitting NAA, BAP, or both from culture media reduced cell survival after 1 week of culture to 0.3% to 3.9% of initial cell numbers compared with approximately 70% survival when both hormones were included in media (Table I). When GCPs were cultured at 38°C in media containing or lacking 0.1 µM ABA, omitting NAA, BAP, or both did not reduce cell survival, which ranged from 69.7% to 77.7% of initial cell numbers (Table I).

GCPs Cultured at 38°C + ABA Do Not Incorporate BrdU into DNA

In our previous studies (Roberts et al., 1995), GCPs cultured at 38° ± ABA did not reach M phase. Whether GCPs might have reached S phase or G2 was not examined. We used BrdU pulse labeling to address this question. When GCPs were cultured at 32°C, BrdU incorporation into nuclear DNA (Fig. 4, A and B) was detected within 48 to 72 h (Fig. 4C). At the end of the 48- to 72-h pulse, 18.3% ± 1.3% (mean; SE; n = 3; Fig. 4C) of nuclei were labeled with BrdU, and 1.9% ± 0.6% of cultured cells had formed cell plates. The percentage of nuclei containing BrdU-labeled DNA reached a maximum of 30.1% ± 1.7% after the 72- to 96-h pulse, ranged from 24.9% to 29% over days 5 through 7 of the experiment, and then declined steadily to <2% of nuclei examined over the remainder of the 2-week experiment (Fig. 4C). GCPs cultured at 38°C in media ± 0.1 µM ABA did not incorporate BrdU into DNA (Fig. 4C). GCPs cultured at 38° ± ABA for 2 weeks did not regenerate cell walls (Calcofluor white; not shown), but GCPs cultured at 32°C did (Fig. 1B). To determine whether failure to detect BrdU incorporation at d 10 through 14 might be due to antibody absorption by or adsorption to the fixed cell walls of GCPs cultured at 32°C for longer periods, cells from 11-d-old cultures were digested with cellulolytic enzymes, and nuclei were isolated from "reprotoplasted" cells. Only 0.2% of nuclei isolated from these protoplasts contained BrdU-labeled DNA.

View larger version (60K): [in this window] [in a new window]   Figure 4. BrdU pulse labeling of cultured GCPs of tree tobacco. A and B, Nuclei isolated from cells pulse labeled with BrdU between d 5 and 6 after cultures were established. Nuclei stained for DNA with Hoechst 33342 (A). B, Same nuclei in A stained for BrdU incorporation with a fluorescein isothiocyanate (FITC)-conjugated rabbit anti-BrdU antibody. Magnification = 600x; bar in A = 30 µm. C, Percentage of nuclei incorporating BrdU in 24-h pulse labeling experiments over a 14-d period of culture at 32°C () or at 38°C in media lacking ([trio]) or containing () 0.1 µM ABA. Each data point is the mean from three separate experiments in which 1,000 nuclei visualized initially with Hoechst staining were also scored for BrdU incorporation. Bars = SE.

Temperature-induced changes in hormone relations (Nagata et al., 2001), phosphate depletion of media (Kato et al., 1977; Sano et al., 1999), and anoxia-induced changes in cellular GA₃ contents (Sauter, 2001) could all potentially prevent cell cycle reentry. Raising hormone concentrations 20- or 50-fold, increasing phosphate concentrations to 500 mg L^–1, or including GA₃ in media at concentrations ranging from 5 to 100 µM did not trigger cell cycle reentry at 38°C (not shown). Thus, none of these factors alone appears to be responsible for failure to reenter the cell cycle at 38°C.

A 41-kD ERK1 Cross-Reacting Protein That Disappears with the Onset of S Phase Is Retained in Thermotolerant GCP

In yeast, animals, and plants, MAPKs are involved in stress responses (Kovtun et al., 2000; Morris, 2001), hormonal signaling (Kovtun et al., 1998; Burnett et al., 2000; Mockaitis and Howell, 2000), thermotolerance (Trotter et al., 2001), cell survival (Haq et al., 2002), cell cycle control (Calderini et al., 1998; Wilson et al., 1998; Haq et al., 2002; Krysan et al., 2002; Sah et al., 2002), and differentiation (Yosimichi et al., 2001; Kim et al., 2002; Yoon et al., 2002). Thus, we anticipated that regulatory patterns for some MAPK might be altered at high temperatures. Using an antibody to subdomain XI of rat (Rattus norvegicus) ERK1, three proteins were detected in extracts from FGCPs or cultured GCPs (Fig. 5, A–C; hereafter called ERK1 cross-reacting proteins [ERK1-CRPs]). No ERK1-CRPs were detected: (a) in 7-µg loads of Cellulase Onozuka RS, Pectolyase Y-23, or bovine serum albumin (BSA; not shown); (b) with a 1/1,000 (v/v) dilution of pre-immune serum in place of the primary antibody; or (c) with antibody to subdomain XI of rat ERK2. Cells cultured under all conditions contained a 47-kD ERK1-CRP that was retained at similar levels for up to 10 d (Fig. 5, A–C). At 32°C, levels of a 42-kD ERK1-CRP did not change over 10 d, but levels of a 41-kD ERK1-CRP declined rapidly with the onset of S phase (48–72 h; Fig. 5, A and D) and were undetectable by the time cultures reached stationary phase (5 d; Fig. 5, A and D). The 41-kD ERK1-CRP was retained for up to 10 d by cells cultured at 38°C ± ABA (Fig. 5, B and C), but the 42-kD ERK1-CRP found in extracts from cells cultured at 32°C (Fig. 5A) could not be clearly resolved from the 41-kD ERK1-CRP (Fig. 5, B and C).

View larger version (56K): [in this window] [in a new window]   Figure 5. Proteins that cross-react with a rat ERK1 antibody in extracts from GCPs of tree tobacco cultured for 0, 1, 2, 3, 5, 7, or 10 d at 32°C (A,) 38°C (B), or 38°C (C) in media containing 0.1 µM ABA. P–, Control from non-progesterone-treated Xenopus laevis oocytes; P+, control from progesterone-treated X. laevis oocytes; all other lanes contain 7 µg of protein extracted from GCP. D, Changes in mean contour band quantities of a 41-kD ERK cross-reacting protein as a function of days in culture at 32°C. In each experiment, band contour quantities were normalized to those of FGCPs (d 0) before they were averaged. Values are means and SEs for three replicate experiments. For purposes of illustration, BrdU incorporation for cells cultured at 32°C is repeated from Figure 4C.

Two-Dimensional Electrophoresis Uncovers Polypeptides Unique to Thermotolerant GCP

Because proteins are the functional expression of gene regulation, polypeptides from each treatment were compared by two-dimensional gel electrophoresis to determine the number of major polypeptides unique to each culture condition. The protein extraction employed typically yielded 40 to 50 µg of protein per 10⁶ GCPs. Because 1- to 2-week-old cultures contained 20% to 30% dead cells, a control culture of 3 x 10⁶ GCPs was established in which cells were cultured at 32°C without NAA and BAP until all cells were dead. Extraction yielded 0.95 µg of protein per 10⁶ GCP.

Results of spot match analysis of two-dimensional gels are summarized in Tables II and III and are illustrated in Figure 6. In two-way comparisons among treatments in each pH range employed (Table II), major polypeptides shared between treatments ranged from 30.5% to 51.6% of total polypeptides detected.

View larger version (136K): [in this window] [in a new window]   Figure 6. Two-dimensional electrophoresis of polypeptides from GCPs of tree tobacco. A, Polypeptides from FGCPs. Polypeptides (90 µg) were extracted and separated along a pH gradient of 5 to 8 in a commercial immobilized pH gradient (IPG) strip and then separated in second dimension on an 8% to 16% (w/v) gradient gel. Gels were silver stained and scanned with a densitometer. Background was subtracted digitally. B, Enlarged view of upper left quadrant of gel shown in A. C, Polypeptides associated with guard cell function. Upper left quadrant of digital reference gel from the higher order matched set used for analysis of polypeptides unique to culture condition, physiological state, or functional capacity. Circles surround 13 polypeptides identified with Boolean operators that are held in common by, and unique to, cells with guard cell function (FGCPs and GCPs cultured at 38°C in media containing ABA).

Using combinations of Boolean operators, the two-dimensional gel library was queried for polypeptides unique either to treatment or to function (Table III). For example, in the pH range 5 to 8, 54 polypeptides associated with thermotolerance were identified by selecting for those found in both FGCPs and cells cultured at 32°C, neither of which are thermotolerant, and then removing those shared in common with cells cultured at 38°C (Table III). Accounting for overlap in pH ranges of gels by inspection, a number of polypeptides (approximately 47–70) were identified that were unique to dedifferentiated, dividing cells (Table III). Similarly, 100 to 180 polypeptides associated with ABA treatment were identified by selecting for those in the 38°C + ABA treatment that were not found in any other treatment (FGCP + 32°C + 38°C), and approximately 34 to 50 polypeptides were identified as unique to guard cell function (Table III; Fig. 6).

	DISCUSSION

TOP ABSTRACT RESULTS DISCUSSION MATERIALS AND METHODS LITERATURE CITED

 

At 38°C, Cultured GCPs Develop Thermotolerance within 24 h

The mechanism required to survive high temperatures and the mechanisms required to thwart cell cycle reentry, dedifferentiation, and division develop in parallel over the first 24 h at 38°C (Fig. 2). When cells were returned to 32°C after shorter pre-incubations (9 h) at 38°C, cell survival after a week was low (Fig. 2A), but virtually all cells that survived the transfer to a cooler temperature divided (not shown). GCPs pre-incubated at 38°C for 12 h before transfer to 32°C showed increased cell survival after a week compared with GCPs given shorter (9 h) 38°C pre-incubations (Fig. 2A), but cells given 12 h of pre-incubation at 38°C (Fig. 2C) did not divide when they were returned to 32°C. Thus, after a 24-h exposure to 38°C, cultured GCPs have developed a thermotolerance that ensures their survival, but once GCPs have developed thermotolerance they do not divide, even when NAA and BAP concentrations are raised 20- to 50-fold.

Accumulation of Hsp 70 Parallels Development of Thermotolerance

It has been proposed that, in intact plants, Hsps stabilize meristematic cells during sudden local increases in temperature so that when cooler temperatures return, the cell cycle may be resumed (Francis and Barlow, 1988). Once GCPs developed thermotolerance in culture, they did not resume division at 32°C, even after 1 week of additional culture. Increased levels of Hsp70 at 24 h were positively correlated with capacity to survive after transfer from 38°C to 32°C, but capacity for cell division among surviving cells was negatively correlated with increased Hsp70 levels. Short pre-incubations (9 h) at 38°C did not result in increased Hsp70 (Fig. 3). Under those conditions, cell survival was lower, but surviving cells divided. Hsp70 levels also increased within 24 h of culture at 38°C in ABA-containing media (Fig. 3), conditions under which GCPs also survive in high percentages but fail to divide (Roberts et al., 1995). Levels of the noninducible Hsc70 did not change over the first 24 h of culture under any condition examined (Fig. 3), suggesting that Hsp70 accumulation was probably a specific response to activation of HSFs (Wu, 1995) rather than the result of altered protein turnover.

Certain Hsps are known to be required for high temperature survival in plants. Arabidopsis plants overexpressing a gene for Hsp101 have an enhanced capacity to survive abrupt shifts to extreme high temperatures, whereas plants underexpressing the gene have a diminished survival capacity (Queitsch et al., 2000). In addition to functioning as molecular chaperones to facilitate the refolding of proteins denatured by heat shock, in mammalian cells, Hsp72, a member of the HSP70 family, can block both JNK-mediated caspase-dependent and -independent cell death pathways (Gabai and Sherman, 2002). Hsp72 can also inhibit cell survival pathways mediated through Akt and ERK (Gabai and Sherman, 2002). Whether cells survive under a particular circumstance appears to be a function of the relative effectiveness of Hsp72 in regulating the balance between these alternate pathways under any given set of conditions (Gabai and Sherman, 2002). Caspases have not been found in plants (Krishnamurthy et al., 2000), suggesting that if survival is mediated by members of the Hsp70 family in cultured GCPs, these proteins probably act by inhibiting a caspase-independent cell death mechanism.

In animal cell cultures, Hsps and HSFs (Wu, 1995) are known to regulate distribution of cultured cells among alternate fates by regulating the cell cycle (Helmbrecht et al., 2000; Kühl and Rensing, 2000), apoptosis (Gabai and Sherman, 2002), or necrosis (Gabai and Sherman, 2002). Thus, at high temperatures, Hsp could stabilize proteins to reduce death of GCPs and, at the same time, participate in blocking the G1-to-S phase transition (Fig. 4). Involvement of Hsp70 and Hsc70 in regulating early events of the cell cycle has been documented. In yeast, elevating culture temperature from 23°C to 36°C for as little as 30 min (heat shock) causes a transient arrest between G1 and S through an unknown mechanism that represses expression of the G1 cyclin genes, CLN1 and CLN2 (Rowley et al., 1993). Recent studies show that: (a) activation of HSF is required for G1 arrest, (b) titration of yeast "free" Hsp70 with misfolded proteins selectively activates HSF, and (c) activated HSF represses expression of CLN1 and CLN2 through an unknown mechanism (Trotter et al., 2001). Increased levels of Hsp70 in GCPs cultured at 38°C ± ABA probably reflect activation of HSF, but this hypothesis has not been tested. Unlike in yeast, elevated temperature does not simply extend the duration of G1 in cultured GCP; instead, GCPs do not leave G1 (Fig. 4).

Failure of GCPs cultured at 38°C to reenter the cell cycle (Fig. 4), expand fully (Fig. 1D; Roberts et al., 1995), and regenerate cell walls (Roberts et al., 1995; Taylor et al., 1998; this study) may be symptomatic of suppressed auxin signaling. Furthermore, we hypothesize that chloroplasts retain chlorophyll at 38°C (Taylor et al., 1998) because auxin is unable to trigger the ethylene production required for chloroplast senescence (Van Der Straeten et al., 1990; Yip et al., 1992; Merritt et al., 2001). We speculate that some Hsps may protect Aux/IAA proteins (Kepinski and Leyser, 2002) from auxin-induced ubiquitination and proteosomal destruction. If so, Hsps might suppress auxin signaling by preventing dimerization of auxin response factors that activate auxin response elements (Kepinski and Leyser, 2002). We envision that auxin response element activation would be required to regulate genes needed for early cell cycle events, cell expansion, wall regeneration, and ethylene synthesis.

Hsc70 could also be involved in blocking cell cycle reentry at elevated temperature. In animal cells, phosphorylation of the retinoblastoma protein, pRb, is required to activate the transcription factor E2F, which in turn activates genes for DNA replication machinery during the G1-to-S phase transition (Weinberg, 1995). Plants have a pRb equivalent (Murray, 1997), and recent studies indicate that Nicta; CycD3;3-associated kinase phosphorylates NtRb1 during the middle-G1 to early S phase boundary (Nakagami et al., 2002). In vitro, Hsc70 binds to a specific sequence in the N terminus of nonphosphorylated pRb and blocks its phosphorylation (Inoue et al., 1995). Although levels of Hsc70 did not change over the first 24 h at 32°C, Hsc70 levels have not been examined closer to cell cycle reentry after 48 to 72 h of culture, and nothing is known about how elevated temperature might affect redistribution of this protein among cellular compartments.

Despite the potential role of Hsp70 and other Hsps (e.g. Hsp101) in the processes described, Hsp70 levels increased only about 2-fold over the first 24 h at 38°C ± ABA (Fig. 3A). We have not yet determined whether increases in Hsp70 continue beyond 24 h, whether they are representative of those of other Hsp, and/or whether the increases in levels of Hsp70 would be sufficient to produce the hypothesized effects.

Thermotolerant GCPs Do Not Require NAA or BAP to Survive

Our results suggest that at 32°C, dedifferentiating and/or dividing cells require active deployment of a hormone-dependent pathway that produces factors required for cell survival. At 32°C, cultures approaching the restriction point between G1 and S or about to enter M from G2 may contain a mixture of cells, only some of which are competent or fit to complete the cell cycle. Under such circumstances, gene regulation under the control of growth regulators might allow for negative selection of cells unfit for cell cycle completion, possibly through apoptotic and/or necrotic pathways.

Both NAA and BAP were required for survival at 32°C (Table I). Our previous studies with AVG suggest that survival at 32°C may also require endogenous ethylene production (Roberts et al., 1995), which could be stimulated by auxin (Van Der Straeten et al., 1990; Yip et al., 1992; Merritt et al., 2001). It may seem odd that ethylene, which is often associated with cellular senescence and necrosis, might be required for cell survival at 32°C in this system. Exogenously applied ethylene can induce apoptosis in cultured tobacco Bright-Yellow 2 cells at G2 to M and S (Herbert et al., 2001). However, ethylene-regulated anti-apoptotic mechanisms may exist. Relatively high (27 µM) concentrations of BAP induce programmed cell death in cultured carrot (Daucus carota) and Arabidopsis cell cultures (Carimi et al., 2003). In these systems, cell death is blocked by 2,4-diphenoxyacetic acid, which could induce ethylene production (Van Der Straeten et al., 1990; Yip et al., 1992). We speculate that in GCPs, ethylene may antagonize cytokinin-induced cell death. Because both auxin and cytokinin are required for survival and division of plant cells in culture, it is possible that ethylene induced by auxin treatment modulates cytokinin signal transduction pathways to induce cell division rather than cytokinin-induced cell death. Thus, ethylene would "signal" cytokinin of the presence of the auxin required for early cell cycle events and cell growth. In the absence of cytokinin, NAA induced-ethylene would also cause cell death. Although this explanation would be consistent with requirements for NAA, BAP, and ethylene for survival of cultured GCPs at 32°C, other mechanisms are possible. The Arabidopsis ethylene-responsive element-binding protein (AtEBP) can function as a dominant suppressor of Bax-induced cell death in yeast (Pan et al., 2001). It is uncertain, however, whether plants have a system fully equivalent to Bcl-Bax (Krishnamurthy et al., 2000; Lam et al., 2001).

In contrast to the hormone-dependent cell survival observed at 32°C, GCPs cultured at 38°C ± ABA did not require NAA or BAP to survive (Table I), and survival was not reduced by treatment with AVG (Roberts et al., 1995). Regardless of whether ABA is included in the culture medium, elevated temperature may block events so far upstream of the G1-to-S transition that cultured GCPs are never faced with downstream selection for life or death. It is also possible (and likely) that GCPs cultured at 38°C retain and/or develop a complement of survival factors (e.g. Hsps) that prevent their death.

Thermotolerant GCPs Do Not Make the G1-to-S Transition

Our data indicate that high temperature (38°C) prevents cultured GCPs from reentering the cell cycle by blocking the G1-to-S phase transition (Fig. 4). GCPs may be unique among plant cell types in their capacity to survive in high percentages at high temperatures in culture and, thus, may comprise a novel in vitro system for studying how temperature signal transduction mechanisms regulate the G1-to-S transition to prevent cell cycle reentry.

When cultures of GCPs of tree tobacco were established at 32°C, a temperature that in many plant species results in minimum cell cycle duration (Francis and Barlow, 1988), cells entered S phase within 48 to 72 h (Fig. 4C). Cell proliferation was exponential between days 2 and 4, but cultures reached a stationary phase at days 5 to 7 and then entered a declining phase (Fig. 4C). Nuclei isolated from cells that were reprotoplasted after 10 to 14 d of culture at 32°C did not stain with FITC-conjugated antibody to BrdU in higher percentages than those of nuclei in fixed cells that had regenerated cell walls (approximately 0.2%). Thus, failure to detect BrdU incorporation in nuclei of cells cultured at 32°C for longer periods was not due to failure of antibodies to penetrate cells with walls. The pattern of cell proliferation for cultured GCPs was similar to that reported for tobacco Bright-Yellow 2 cells cultured at 27°C, which divided rapidly with a 12- to 14-h cell cycle on days 3 to 4 before entering a stationary phase induced by exhaustion of nutrients in the medium (Nagata et al., 1992). After 72 h at 32°C, only about 10% as many cultured GCPs had developed cell plates as had incorporated BrdU into nuclear DNA, indicating that isolated GCPs were in G1 initially.

When cultures of GCPs were established at 38°C, the G1-to-S transition was blocked regardless of whether media lacked or contained ABA (Fig. 4C). Our previous studies (Taylor et al., 1998) showed that at 38°C, GCPs lose some of the functional characteristics of guard cells unless ABA is included in the culture medium. Thus, although a temperature of 38°C alone is sufficient to block the G1-to-S transition, a combination of elevated temperature and ABA is required to maintain GCPs in the differentiated state in vitro (Roberts et al., 1995; Taylor et al., 1998). ABA is known to induce the synthesis of the cyclin kinase inhibitor ICK1 in Arabidopsis (Wang et al., 1998). ICK1 interacts directly with Cdc2a and CycD3 (Wang et al., 1998), and its overexpression reduces CDK activity, cell number, and plant growth (Wang et al., 2000). Therefore, the possibility exists that the G1-to-S phase transition is blocked by a different mechanism and/or at a different point in G1 at 38°C in a medium lacking ABA than it is at 38°C in a medium containing ABA. Results of two-dimensional gel analysis (Table II) reveal a number of proteins that differ between 38°C and 38°C + ABA treatments.

Thermotolerant GCPs Retain a 41-kD ERK1 (MAPK) That Is Lost during Dedifferentiation at 32°C

It is not known whether plant MAPKs are involved in plant cell differentiation in vivo or dedifferentiation in vitro. However, plant MAPKs have been implicated in phenomena similar to those observed in this and other studies with cultured GCPs of tree tobacco. As noted above, GCPs cultured at high temperature show symptoms consistent with impaired auxin signaling, and an MAPK cascade initiated by an MAPKKK (NPK1) can suppress auxin signaling in transfected maize protoplasts and transgenic tobacco plants (Kovtun et al., 1998). MAPKs have been implicated in other similar systems and processes as well. In cultured tobacco Bright-Yellow 2 cells, a 45-kD MAPK is activated upon phosphate-induced cell cycle reentry after phosphate starvation (Wilson et al., 1998), and p43^Ntf6 appears to be required for phragmoplast formation during the cytokinetic transition from anaphase to telophase (Calderini et al., 1998). There is genetic evidence that members of the Arabidopsis MAPKKK gene family encode transcripts that are essential for regulating cytokinesis (Krysan et al., 2002). In epidermal peels of pea, an MAPK is thought to be involved in signaling guard cells during ABA-induced stomatal closure (Burnett et al., 2000), and ABA prevents growth of tree tobacco GCPs cultured at 38°C (Roberts et al., 1995). Plant MAPKs are known to be activated by auxin, cytokinin, or ethylene in a variety of plant species and tissues (Morris, 2001). All three of these growth regulators appear to be required for survival of cultured GCPs of tree tobacco during and/or after cell cycle reentry at 32°C (Roberts et al., 1995; Table I).

Similar to isolated pea guard cells (Burnett et al., 2000), GCPs of tree tobacco contained three proteins that cross-reacted with an antibody to subdomain XI of a rat ERK1 (Fig. 5). The most prominent was a 41-kD protein that disappeared from cells cultured at 32°C at the beginning of S phase and cell cycle reentry (Fig. 5, A and D). It is not known whether disappearance of the 41-kD ERK1-CRP resulted from altered protein turnover or from alteration of epitopes in subdomain XI due to protein modification or processing. At 38°C ± ABA, the protein was retained in cells for up to 10 d (Fig. 5, B and C). At the very least, disappearance of the 41-kD ERK1-CRP is diagnostic for cell cycle reentry. It is tempting to speculate that the protein is involved in maintaining GCPs in G₀ or G1 at 38°C ± ABA. In cultured human fibroblasts, constitutive activation of the stress-induced MAPK, p38^HOG, through stable expression of its activator MKK6 causes permanent cell cycle arrest at G1 (Haq et al., 2002). Still, the structure and function(s) of the 41-kD ERK1-CRP remain to be investigated.

A 42-kD ERK1-CRP was retained in cells cultured at 32°C for up to 10 d. The 42-kD ERK1-CRP was not resolvable from the 41-kD ERK1-CRP in extracts from cells cultured at 38°C ± ABA because it was obscured by the strong reaction of the antibody with the 41-kD protein even at low dilutions and film exposure times.

A 47-kD ERK1-CRP was detected in all cultures. Putative MAPKs of similar molecular masses have been reported in GCPs and isolated guard cells (Mori and Muto, 1997; Burnett et al., 2000). ABR, a 48-kD ABA-activated protein kinase capable of phosphorylating myelin basic protein, was identified in extracts from Vicia faba GCP, but it did not precipitate with an antiphosphotyrosine antibody (Mori and Muto, 1997). Two other kinases of 46 and 49 kD were also identified that precipitated with an antiphosphotyrosine antibody, but they were activated only slightly by ABA (Mori and Muto, 1997). In pea leaf epidermis, both ABA-induced stomatal closure and ABA-induced accumulation of dehydrin mRNA were shown to be inhibited by PD098059 (=PD98059), an MEK inhibitor, suggesting that MAPKs are required for ABA-induced stomatal closure (Burnett et al., 2000). In the same study, an ABA-activated 45-kD kinase, AMBPK, was identified in extracts from pea epidermal peels. AMBPK required Tyr phosphorylation for its activity and catalyzed phosphorylation of myelin basic protein (Burnett et al., 2000), but its activity was not induced by ABA in isolated guard cells. A 43-kD kinase was activated by ABA in isolated pea guard cells, but it is not clear whether this protein was an MAPK (Burnett et al., 2000). Whether the 47-kD ERK1-CRP reported here is a true guard cell MAPK similar to any of those reported also awaits investigation.

A Proteomic Analysis of Thermotolerant GCPs Is Feasible

The two-dimensional gel analysis reported here was limited to spot match analysis of the major polypeptides detected on silver-stained gels. Silver staining is sensitive, but it is not quantitative. Furthermore, although spot match analysis provides a degree of statistical certainty as to whether the same spot exists at a similar location in two different treatment gels, it does not explain why such differences in spot location exist. The presence of a polypeptide at a unique gel position may reflect changes in gene regulation for the polypeptide, changes in turnover of the polypeptide, or chemical modification of the polypeptide so that its position is shifted on the gel (hereafter, we refer to the sum of these processes as "polypeptide regulation"). Even so, two-dimensional gel comparisons of this type yield useful information. For example, the data indicate that regardless of how cells are treated, any two cell types in this system share 30% to 50% of their major polypeptides in common (allowing for overlap of pH ranges on IPG strips). This suggests that it may be possible to use such an analysis to distinguish regulation of a core of "maintenance" polypeptides involved in functions required of all cell types (metabolism, transport, etc.) from regulation of polypeptides involved in the response of cultured cells to environmental signals.

Interestingly, FGCPs and GCPs cultured at 38°C with ABA, which share guard cell function, did not share any greater proportion of their major polypeptides in common than did highly divergent cell types such as FGCPs and fully dedifferentiated, dividing cells cultured at 32°C (Table II), nor did cells given similar treatments (e.g. 38°C and 38°C + ABA) share any higher proportion of polypeptides than divergent treatments (Table II). Although the resolution of the two-dimensional system is limited to only the most abundant polypeptides, the data may suggest that polypeptide regulation can vary as radically in a guard cell that maintains its identity while responding to drastic changes in environmental signals (i.e. culture at 38°C in media containing ABA) as it does when the signals result in its dedifferentiation to a meristematic state under the influence of plant growth regulators. Thus, at the level of the proteome, dedifferentiation may be less a matter of degree of deviation from the differentiated state than the precise nature of the deviation.

Potentially, two-dimensional gel analysis can also be used to monitor temporally changes in the proteome that define dedifferentiation. Guard cell function can be highly conserved even after isolation, culture initiation, and heat shock. Conservation of guard cell function has been observed in guard cells that have been: (a) isolated from the leaf as protoplasts; (b) transferred to media enriched in nutrients and supplemented with plant growth regulators that control cell growth and division; and then (c) cultured at elevated temperature, in darkness, in media containing the antitranspirant ABA for up to 1 week (Taylor et al., 1998). It seems remarkable that any guard cell function would be retained under these conditions because any or all of these conditions might be expected to disrupt patterns of gene and protein regulation in guard cells and interfere with their normal function and/or maintenance of their genetic identity. Nevertheless, Boolean analysis of gels uncovered 34 to 50 polypeptides that were shared between and that were unique to cells with guard cell function (FGCPs and GCPs cultured at 38°C in media with ABA; Table III; Fig. 6). If the point in time can be identified at which these polypeptides are lost from GCPs dedifferentiating at 32°C, it may be possible to analyze the degree to which protein profiles can deviate from those of a guard cell before GCPs lose their unique physiological identity and are considered to be functionally "dedifferentiated."

Monocultures of GCPs Can Be Used to Study Signal Transduction Related to Thermotolerance

There are numerous animal cell culture systems in which the signal transduction pathways governing the cell cycle and cell proliferation (e.g. Yosimichi et al., 2001; Sah et al., 2002), differentiation/dedifferentiation (Kim et al., 2002; Yosimichi et al., 2001; Yoon et al., 2002), and apoptosis (Konishi et al., 2002) can be studied. Only a few systems exist for studying these processes in plants. Zinnia elegans mesophyll protoplasts are a good example. These cells can be manipulated in vitro to expand (Lee et al., 2000) or to divide and differentiate into tracheary elements, a process which ends in programmed cell death (Groover and Jones, 1999). Similarly, a single isolate of tree tobacco GCPs can be partitioned among culture conditions that maintain GCPs in the differentiated state, result in partial dedifferentiation, end in cell death, or cause full dedifferentiation and reentry into the cell cycle. Only two signals, elevated temperature and ABA, are required to prevent dedifferentiation and cell cycle reentry, and elevated temperature alone is sufficient to prevent the latter. Furthermore, a distinct hormone-dependent cell survival mechanism employed at lower culture temperatures is not required for high survival at high temperatures. This report demonstrates that cultured GCPs of tree tobacco can be used to study the signal transduction pathways that regulate thermotolerance and its relationships to the plant cell cycle, differentiation/dedifferentiation processes, and cell survival mechanisms that are under the control of plant growth regulators (NAA and BAP), hormones (ethylene and ABA), and temperature. A number of polypeptides identified by two-dimensional profiling that are unique to these pathways are targets for future functional proteomic analysis. GCPs of Beta vulgaris have been successfully transformed and regenerated to plants that are resistant to glyphosphate herbicides (Hall et al., 1996). Ultimately, it may be possible to manipulate levels of GCP proteins identified by proteomic analysis using small interfering RNA molecules or morpholinos to target the destruction of their mRNAs.

	MATERIALS AND METHODS

TOP ABSTRACT RESULTS DISCUSSION MATERIALS AND METHODS LITERATURE CITED

 

Plants

Plants were germinated from seed at a high density on potting soil (HP Premier Pro-mix, Premier Horticulture Ltee, Rivere-du-Loup, QC, Canada) in 0.16-L plastic pots and maintained under fluorescent lights on a light/dark cycle as described (Boorse and Tallman, 1999). For 4 to 5 weeks after germination, plants were watered every other day and on alternate days were given one-half-strength modified Hoagland nutrient solution (Hoagland and Arnon, 1938; Boorse and Tallman, 1999). After 4 to 6 weeks, individual seedlings were transferred to an autoclaved mixture of 60% soil/40% sand (v/v) in 0.16-L pots and watered similarly for another 4 to 6 weeks. After they reached a height of 0.05 to 0.1 m, they were transferred to 10-L pots containing the soil/sand mix and grown to maturity as described, except that watering was for 4 min every 12 h (Boorse and Tallman, 1999).

Isolation and Culture of GCP

GCPs were isolated and cultured as described (Boorse and Tallman, 1999) with the following modifications. The procedure was scaled to process epidermis from as few as four and as many as nine leaves in a single isolation. Concentrations of ascorbic acid and polyvinylpyrrolidone 40 were doubled in solutions in which epidermis was detached from leaves (Boorse and Tallman, 1999; step 2.3.3). The same volume of enzyme solution per leaf was used in four, six, or nine-leaf preparations. Epidermis from as many as 4.5 leaves was incubated in a single 250-mL Erlenmeyer flask (Corning, Corning, NY) in a proportionate volume of enzyme solution (Boorse and Tallman, 1999; step 3.3.18). For larger isolates, two preparations of epidermis were made, and then incubation times in enzyme solutions were staggered by 4 min. At the end of the first enzyme digestion, epidermis was collected on a nylon net as described (Boorse and Tallman, 1999; step 3.3.22) and rinsed with 75 mL of solution C. Peels were transferred from the net to a 125-mL flask (Boorse and Tallman, 1999; step 3.3.24) containing 75 mL of solution C. The flask was swirled vigorously. Peels were collected again on the same nylon net and rinsed with another 50 to 100 mL of solution C before they were transferred to the second enzyme solution (solution D, step 3.3.24; Boorse and Tallman, 1999) in a clean 250-mL flask. The flask was capped and swirled vigorously. The second enzyme digestion was extended to 3.5 h at a speed of 55 rpm. During collection of protoplasts (Boorse and Tallman, 1999; step 3.3.30), flasks were swirled 10 times clockwise and then 10 times counterclockwise before contents were filtered through the fine mesh net. The cuticle remaining on the net was rinsed with an additional 5 to 10 mL of incomplete medium I (pH 6.8) before GCPs were collected and washed (Boorse and Tallman, 1999). After the second wash in incomplete medium I (pH 6.1, step 35; Boorse and Tallman, 1999), the supernatant was aspirated from each tube down to 0.5 mL, and GCPs were resuspended by rolling the tubes between the palms of the hands. The contents of all tubes were combined in one 15-mL conical centrifuge tube and were collected by centrifugation at 60g for 8 min. The supernatant was discarded down to 1 mL. GCPs were resuspended, counted with a hemocytometer, and cultured at a density of 1.25 x 10⁵ cells mL^–1 as described (Roberts et al., 1995; Boorse and Tallman, 1999). For larger preparations (eight–nine leaves), GCPs from two parallel isolates were collected separately, reduced to a single tube each, and then counted before the two batches were combined in a single tube.

Kinetics of Development of Thermotolerance

In an attempt to measure the kinetics of development of thermotolerance, we determined the length of pre-incubation at 38°C ± ABA required to produce full cell survival but inhibit cell division among cells cultured subsequently at 32°C. In three separate experiments, GCPs were first cultured at a density of 6.25 x 10⁴ cells mL^–1 in eight-well chamber slides (Roberts et al., 1995) at 38°C with or without ABA for various periods from 0 to 24 h. In each experiment, the number of dead cells in 10 fields in each of four chamber slide wells was estimated using a microscope at 200x: (a) after 24 h and 8 d of culture at 32 or 38°C, and (b) in cultures pre-incubated at 38°C for various periods from 0 to 24 h that were then cultured for another week at 32°C.

Cell Collection and Storage for Protein Extraction

FGCPs were suspended in a final volume of 1 mL as above. Cells cultured in plastic X Plate petri dishes (100 x 15 mm, Becton-Dickinson, San Jose, CA) were collected in 15-mL conical tubes by centrifugation at 60g for 10 min. After centrifugation, all but 1 mL of the supernatant was discarded. FGCPs or cultured cells were resuspended by gentle trituration and transferred to 1.5-mL cryovials. Vials were centrifuged at 60g for 7 min, and the supernatant was discarded. To maximize protein yields, cells cultured at 38°C ± ABA for more than 5 d were collected by settling instead of by centrifugation. Ninety percent of each cell suspension was transferred from petri dishes with a wide-bore pipette to a 15-mL conical centrifuge tube. Transferred suspensions and the original culture plates were returned to the incubator for 1.5 h. Supernatants from each tube were then used to rinse the remaining cells from each petri dish, and rinses were returned to their respective tubes. Tubes were returned to settle in the incubator for another 1.5 h before supernatants were discarded. Each cell pellet was stored in a cryovial in a solution made by adding 5 µL of 100 mM phenylmethanesulfonylfluoride to 0.5 mL of a solution of 50 mM Tris (pH 8.0), 1.5% (w/v)insoluble polyvinyl-polypyrrolidone, 1.8 µg of trans-epoxysuccinyl-L-leucylamido-(4-guanidino)butane, 0.2 mM 4-(2-aminoethyl)benzene-sulfonyl fluoride, 5 µg of leupeptin, and 0.02 mM ethylene glycol-bis (-aminoethyl ether) N,N,N'N'-tetra-acetic acid. The solution was added directly to cryovials containing cells collected by centrifugation. When cells were collected by settling, settled cells were resuspended in the solution, and the suspension was then transferred to cryovials. Cells to be used for Hsp/Hsc western blotting and two-dimensional gel electrophoresis were frozen immediately in liquid nitrogen and stored at –80°C.

To provide controls for ERK1/2 experiments, stage VI oocytes were isolated from female Xenopus laevis and treated with progesterone as described (Stebbins-Boaz et al., 1999).

Protein Extraction and Assay for Western Blotting

FGCPs or cultured GCPs of tree tobacco were extracted for western blotting of Hsp70 and Hsc70 with EZ extraction buffers (Martinez-Garcia et al., 1999) by adding 0.5 mL of 2x modified buffer E [250 mM Tris-HCl (pH 8.8), 2% (w/v) SDS, 20% (v/v) glycerol, 0.1 M Na₂S₂O₅ modified to contain 3% (w/v) polyvinyl-polypyrrolidone, 0.45 µg mL^–1 trans-epoxysuccinyl-L-leucylamido-(4-guanidino)butane, 0.1 mM 4-(2-aminoethyl) benzene-sulfonyl fluoride, and 1.25 µg mL^–1 leupeptin] to a cryovial containing 0.5 mL of frozen cells. Samples were suspended in microcentrifuge tubes with a pointed plastic homogenizer (GeneMate, ISC BioExpress, Kaysville, UT). Suspensions were heated at 80°C for 3 min and then centrifuged at 24°C for 4 min at 21,000g to remove insoluble material. Supernatants were transferred to tubes containing an equal volume of ice cold 20% (w/v) tricholoracetic acid (TCA), and mixtures were incubated on ice for 15 min before they were centrifuged at 4°C for 15 min at 21,000g. Supernatants were discarded, and 0.3 mL of ice cold acetone was added to each pellet. Pellets were then homogenized with a teflon microtube homogenizer until they were dispersed before an additional 0.7 mL of acetone was added. After a 20-min incubation on ice, the homogenate was centrifuged as above, and the acetone wash was repeated. After the second wash, the acetone was decanted, and the pellet was air dried for 5 to 10 min before 30 to 50 µL of buffer E (Martinez-Garcia et al., 1999) and 0.5 µL of -mercaptoethanol were added with mixing to completely dissolve the pellet.

For extraction of oocytes for ERK1/2 western-blot controls, five progesterone- or five non-progesterone-treated oocytes containing 100 to 150 µg of protein were homogenized in 10 µL of 0.1 M KCl, 1 mM MgCl₂, 50 mM Tris (pH 7.5), 1 mM dithiothreitol (DTT), and 80 mM -glycerophosphate containing 10 µg mL^–1 each of leupeptin and chymostatin. The homogenate was centrifuged at 4°C for 10 min at 21,000g. The supernatant was diluted with one volume of 2x SDS loading buffer (2% [w/v] SDS, 20% [v/v] glycerol, 5% [v/v] -mercaptoethanol, 10 µg mL^–1 bromphenol blue, and 0.1 M Tris [pH 6.8]) and heated at 80°C for 5 to 8 min.

For ERK1/2 western-blot experiments, proteins of FGCPs or cultured cells were extracted immediately after cells were collected. FGCPs or cultured GCPs were mixed with 1 volume of 20% (w/v) ice-cold TCA and incubated on ice for 10 min. Cells were centrifuged at 4°C for 10 min at 12,000g. The supernatants were removed, and 1.5 mL of 90% (v/v) ice-cold acetone was added to each tube. Tubes were incubated at –20°C for 15 min, and pellets were collected by centrifugation at 4°C for 15 min at 21,000g. The acetone wash was repeated. The acetone was aspirated, and pellets were allowed to air dry for 10 min before they were agitated for 1 h at 500 rpm in 60 µL of buffer E containing 20 mM -glycerophosphate, 1 mM Na₃VO₄, and 1 µL of -mercaptoethanol. Proteins were further solubilized by heating at 80°C for 15 min and agitating for an hour. Extracts were then clarified by centrifugation at 24°C for 10 min at 21,000g before supernatants were assayed for protein.

Protein concentrations in extracts were determined by a Non-Interfering Protein Assay (Geno Technology, St. Louis) using Protocol-1 (proprietary from supplier).

SDS-Electrophoresis and Western Blotting for Hsp70, Hsc70, ERK1, and ERK2

For western blotting Hsp70/Hsc70, 6 (trial 1), 7.5 (trial 2), or 10 (trial 3) µg of protein from GCPs cultured for 0, 0.5, 1, 3, 6, 9, 12, 18, or 24 h at 32°C, 38°C, or 38°C in media containing 0.1 µM ABA was electrophoresed. For ERK1/2 western blots, 7 µg of protein from GCPs cultured for 0, 1, 2, 3, 5, 7, or 10 d at 32°C, 38°C, or 38°C in media containing 0.1 µM ABA was electrophoresed. To ensure that ERKs detected were of cellular origin, 7 µg of Cellulase Onozuka RS, Pectolyase Y-23, or BSA was also examined by the same procedure. Proteins were separated by SDS-PAGE (Laemmli, 1970) for 15 min at a constant 80 V and then at a constant 200 V until completion on 12-well 10% (w/v) Tris-HCl Ready-Gels (catalog no. 161-0324, Bio-Rad, Hercules, CA) using a Mini-PROTEAN 3 cell (Bio-Rad). In addition to samples, either 60 ng each of purified human (Homo sapiens) Hsp70 (catalog no. SPP-755B, StressGen, Victoria, BC, Canada) or cytosolic wheat (Triticum aestivum) Hsc70 (StressGen, catalog no. SPP-791), or 5 µL of protein extracts from progesterone- or non-progesterone-treated oocytes containing 8.8 ± 0.3 µg of protein (mean; SE; n = 5) were electrophorosed as controls.

After electrophoresis, proteins were transferred to nitrocellulose membranes by western blotting as described (Stebbins-Boaz et al., 1999). Membranes were incubated at room temperature with gentle rocking at 20 rpm for 1 h in primary antibodies diluted in 5% (w/v) milk/Tris-buffered saline plus Tween 20 (TBST). Primary antibodies and their dilutions were: anti-Hsp70 (1/2,000 [v/v], StressGen catalog no. SPA-812C, rabbit anti-human polyclonal), anti-Hsc70 (1/2,000 [v/v], StressGen catalog no. SPA-795, rabbit anti-wheat polyclonal), anti-ERK1 (1/1,000 [v/v], Santa Cruz Biotechnology, Santa Cruz, CA, catalog no. SC-94, rabbit polyclonal to subdomain XI of rat ERK1), and anti-ERK2 (1/1,000 [v/v], Santa Cruz Biotechnology, catalog no. SC-153, rabbit polyclonal to subdomain XI of rat ERK2). A 1/1,000 (v/v) dilution of pre-immune serum (Santa Cruz cat No. SC-2027) was used in place of primary antibody as a control for nonspecific binding of IgG.

Membranes were washed four times for 5 min each in TBST and then incubated in secondary antibodies for 1 h. Donkey anti-rabbit IgG-horseradish peroxidase-conjugated secondary antibodies (Santa Cruz Biotechnology, catalog no. SC-2313) were diluted in 5% (w/v) milk in TBST and used to detect primary antibodies. To detect ERK antibodies, the secondary antibody was diluted 1/2,000 (v/v); Hsp70 and Hsc70 primary antibodies were detected with 1/4,000 (v/v) dilutions of secondary antibodies. Membranes were washed four times for 5 min each time in TBST. For chemiluminescent detection, equal volumes of ECL Western Blotting Detection reagents 1 and 2 (Amersham-Pharmacia Biotech, Uppsala, Sweden) were mixed and poured onto the membrane. After 1 min, membranes were drained, blotted, wrapped in plastic wrap, and exposed in darkness to 13-x 18-cm x-ray film (Kodak catalog no. 165 1496, Eastman Kodak, Rochester, NY) for 10 s to 2 min. Films were developed with an automated processor (AGFA model CP1000, Agfa-Gevaert N.V., Mortsel, Belgium). ERK molecular masses were determined against Cruz Molecular Weight Standards (Santa Cruz Biotechnology, catalog no. SC-2035) using Quantity One Software (Bio-Rad) after scanning with a Bio-Rad GS-710 densitometer. Relative amounts of protein were also analyzed by densitometry. Band contour quantities were measured and then compared among blots after normalizing to the contour quantities of standards among blots being compared.

Hormone Requirements for Survival

To ascertain whether elevated temperature (38°C) might alter auxin (NAA) and/or cytokinin (BAP) requirements for survival of GCPs, in three separate experiments the percentage of cells surviving after 1 week of culture at 32°C or at 38°C ± ABA in media containing both NAA and BAP, NAA only, BAP only, or neither hormone was estimated as described (Roberts et al., 1995).

BrdU Pulse Labeling

GCPs were cultured (Roberts et al., 1995; Boorse and Tallman, 1999) at 32°C, 38°C, or 38°C in a medium containing 0.1 µM ABA. Two-milliliter cultures were established in plastic petri dishes (3.5 x 1 cm). Immediately after isolation and at the end of each successive 24-h period of culture over a 14-d period, 5 µL of 10 µM BrdU (final concentration of 25 nM; Sigma, St. Louis) was added with gentle mixing to one dish from each treatment (32°C, 38°C, and 38°C + ABA) in a laminar flow cabinet. Dishes were returned to incubators for an additional 24 h after which cells were harvested, fixed, and stained for BrdU incorporation. Cells from each treatment were harvested with pipettes and transferred to 15-mL conical centrifuge tubes. Dishes were rinsed with 5 mL of incomplete medium I (pH 6.1; Boorse and Tallman, 1999), and the rinse was transferred to the corresponding treatment tube. Cells were collected by centrifugation at 60g for 15 min, after which all but 0.5 mL of the supernatant was discarded. To fix cells, 5 mL of ice-cold 70% (v/v) ethanol was added drop wise to each tube with mixing. Cells were incubated at room temperature for 30 min with shaking at approximately 250 excursions min^–1. Fixed cells were collected by centrifugation as above, and the supernatant was discarded to leave 0.5 mL of liquid above the pellet. To prepare nuclei and denature DNA, 1 mL of 2 N HCl and 0.5% (v/v) Triton X-100 was added drop wise with mixing, and cells were incubated at room temperature for an additional 30 min. Nuclei (38°C ± ABA) or fixed cells with intact nuclei (32°C) were collected by centrifugation for 10 min at 60g. All but 0.5 mL of the supernatant was discarded, and the remaining 0.5 mL of liquid was neutralized by addition of 1 mL of 0.1 M Na₂B₄O7 · 10 H₂O (pH 8.5). Nuclei or fixed cells were again collected by centrifugation, after which 3 mL of ice-cold 70% (v/v) ethanol was added to the remaining 0.5 mL of liquid. Isolates were stored at –20°C for no longer than 3 d before they were stained.

For direct immunofluorescent staining of BrdU-containing DNA, nuclei or fixed cells were collected by centrifugation for 10 min at 60g. The supernatant was removed to leave 0.5 mL, and nuclei or cells were resuspended with addition of 1 mL of 0.5% (v/v) Tween 20 and 1% (w/v) BSA in phosphate-buffered saline (PBS; 0.9% [w/v] NaCl in 1 mM phosphate buffer [pH 7.3]). Twenty microliters of FITC-conjugated rabbit anti-BrdU (catalog no. 347583, Becton-Dickinson) was added to each tube, and tubes were incubated for 30 min at room temperature with shaking at approximately 250 excursions min^–1. Two milliliters of Tween/BSA/PBS was added, and stained nuclei or cells were collected by centrifugation at 60g for 7 min. The supernatant was discarded to 0.5 mL, pellets were resuspended by addition of 2 mL of Tween/BSA/PBS, and nuclei or fixed cells were collected by centrifugation at 60g for 7 min. The supernatant was discarded down to 1 mL, and 0.5 µL of Hoechst 33342 (Sigma; 0.5 mg mL^–1 in incomplete medium 1 [pH 6.1]) was added with gentle mixing for 1 min at room temperature. Nuclei or cells were collected by centrifugation as above, and then the supernatant was withdrawn to leave 0.2 mL. Pellets were resuspended by trituration, transferred to glass slides with coverslips, and visualized at 400x with an inverted microscope (model IX70, Olympus, Tokyo) equipped with a 100-W mercury lamp and an epifluorescence illuminator. Hoechst-stained nuclei were viewed under UV light produced with a wide-band excitation filter (Olympus filter cube U-MWU). FITC-labeled anti-BrdU was viewed with a blue excitation filter (Olympus filter cube U-M516). For each sample in each of three separate experiments, 1,000 nuclei visualized initially with Hoechst staining were scored for BrdU incorporation.

In control experiments designed to test whether cell walls of GCPs cultured at 32°C might absorb or adsorb the FITC-conjugated antibody, cells were subjected to the second cellulolytic enzyme digestion protocol described above to isolate protoplasts from which nuclei were then prepared. GCPs cultured at 38°C ± ABA were assessed for the presence of cell walls by staining with Calcofluor white (Galbraith, 1981).

To evaluate whether FGCPs were in G1 or G2 of the cell cycle initially, in each of three separate experiments a microscope was used to score 1,000 cells for development of cell plates after 72 h of culture.

To determine whether elevated temperature might alter the concentration of NAA and BAP required to activate cell division (Nagata et al., 2001), in preliminary experiments at 38°C, concentrations of NAA and BAP were increased 20x or 50x over those required to activate cell division at 32°C but at the same ratio used at 32°C. Cell division was then estimated after 1 week as described (Roberts et al., 1995). Because phosphate depletion from media can lead to cell cycle arrest in cultured tobacco BY-2 cells (Kato et al., 1977; Sano et al., 1999), we designed experiments in which phosphate concentrations were raised to 500 mg L^–1 with KH₂PO₄ to test whether cells cultured at 38°C ± ABA might deplete phosphate in media more rapidly than at 32°C. In underwater rice (Oryza sativa), anoxia triggers synthesis of GA₃, ethylene release, and, subsequently, cell division (Sauter, 2001). Thus, in some experiments, cultures were supplemented with GA₃ in concentrations ranging from 5 to 100 µM.

Protein Extraction for Two-Dimensional Electrophoresis

Proteins were extracted by a modification of the method of Shimazaki and Kinoshita (1995). Frozen samples were thawed in cryovials, ice-cold 20% (w/v) TCA (0.5 mL) was added with mixing, and samples were incubated on ice for 10 min. Samples were centrifuged at 21,000g for 10 min at 4°C, and the supernatant was discarded. The precipitate was resuspended in 200 µL of ice-cold deionized water and homogenized for approximately 10 s at 4.5 to 8 x 10³ rpm with a teflon microtube homogenizer attached to a rotary tool (Craftsman model no. 572.610530, Sears Roebuck, Hoffman Estates, IL). One milliliter of acetone (–20°C) was added with mixing followed by incubation at –20°C for 20 min. The precipitate was collected by centrifugation as described and gently resuspended by hand homogenization with the teflon homogenizer in 300 µL of 4% (w/v) SDS, 2% (v/v) 2-mercaptoethanol, 20% (v/v) glycerol, and 0.1 M Tris (pH 8.5). Samples were heated at 80°C for 3 min and centrifuged at 24°C for 10 min at 21,000g. The supernatant was collected, and 1.2 mL of acetone (–20°C) was added with mixing followed by incubation for 1 h at –20°C. The precipitate was collected by centrifugation at 4°C for 15 min at 21,000g. The water/acetone wash was repeated, but the pellet was hand-homogenized in water before acetone was added. The supernatant was discarded, the pellet was solubilized by hand homogenization in 10 mM Tris (pH 7.4) and 5 mM MgCl₂. Protease-free RNAse A (4.5 units in 0.8 µL; catalog no. LS002131, Worthington Biochemical, Lakewood, NJ), and 3.1 units (0.5 µL) of protease-free DNAse I (catalog no. LS006333, Worthington Biochemical) were added, and samples were incubated on ice for 15 min. After another acetone precipitation as described above, pellets were air dried for 5 min and solubilized with shaking for 3 to 4 h in an appropriate volume of 5 M urea, 2 M thiourea, 2% (w/v) CHAPS (Sigma), 2% (w/v) SB3-10 (Sigma), 0.2% (w/v) BioLytes 3/10 (Bio-Rad; Rabilloud et al., 1997), and 2 mM tributylphosphine (TBP; Rabilloud et al., 1997). Protein content of samples was assayed by the method of Bradford (1976; Bio-Rad) using bovine gamma globulin (Bio-Rad) as a standard. To evaluate the potential contribution of protein from dead cells to the pool of extracted protein, debris was collected and proteins were extracted from 3 x 10⁶ GCPs that had died after 10 d of culture in a medium lacking NAA and BAP. Samples were diluted to give approximately 100 µg of protein in 185 µL of isoelectric focusing (IEF) solution, and TBP was added to a final concentration of 2 mM. For estimation of polypeptide molecular masses, two-dimensional electrophoresis standards (2.5 µL; catalog no. 161-0320, BioRad) were electrophoresed in a well separate from the IPG strip well.

Two-Dimensional Electrophoresis

IEF was performed using the Bio-Rad Protein II IEF cell. To increase resolution, 11-cm IPG strips were rehydrated with the samples present (Rabilloud et al., 1994). IPG strips (pH 3–10 and 5–8) were placed over liquid samples in the focusing tray and allowed to rehydrate for 1 h before they were overlayed with mineral oil. Gels were allowed to continue to rehydrate for an additional 13 h at 22°C. IEF strips of pH 3 to 6 were rehydrated passively in disposable trays, and two wicks dampened with deionized water were placed over each electrode before focusing. IEF strips of pH 7 to 10 were rehydrated in disposable trays with 185 µL of IEF solution containing 2 mM TBP, and then samples were loaded cathodically in 30 µL before electrophoresis. A temperature of 22°C and a current limit of 50 µA per strip was maintained for all subsequent IEF steps. Immediately after rehydration, gels were electrophoresed sequentially and continuously at 200 V for 1 h (rapid ramping), ramped linearly from 200 to 5,000 V over 3 h, and then ramped rapidly to their limits (top limit = 8,000 V) and electrophoresed for 25,000 V h^–1. A 500-V hold was included at the end of the program to prevent protein diffusion. Immediately after IEF, gels were either wrapped in plastic wrap and stored at – 80°C or prepared for SDS-PAGE.

Before separation in the second dimension, strips were placed in individual screw top culture tubes and rocked at 35 rpm for 10 min in 5 mL of 6 M urea, 2% (w/v) SDS, 0.375 M Tris (pH 8.8), 20% (v/v) glycerol, and 130 mM DTT. Gels were then transferred to a similar solution containing 135 mM iodoacetamide instead of DTT and equilibrated with rocking for another 10 min.

For SDS-PAGE, strips were mounted on top of 8% to 16% (w/v) acrylamide gradient Tris-HCl gels with 4% (w/v) stacking gels (Criterion Gels, Bio-Rad) and cemented with 1% (w/v) low-melt agarose (Sigma) in Tris-Gly-SDS electrode buffer (Laemmli, 1970) at approximately 30°C. Cold packs were inserted between buffer tanks to prevent heating during electrophoresis. Gels were electrophoresed at a constant 200 V for 65 to 70 min.

Gels were silver-stained (Bio-Rad Silver Stain Plus) by the manufacturer's instructions. All glassware was cleaned with 50% (v/v) nitric acid and rinsed with deionized water before staining. After staining, gels were cleaned with cotton-tipped applicators to remove any precipitate, soaked with gentle agitation (40 rpm) in deionized water for 5 min, and then transferred to a gel-drying solution (Bio-Rad, catalog no. 161-0752) and gently agitated for 30 min. Gels were dried between cellophane sheets in a Bio-Rad GelAir Dryer with forced air for 3 h and then for an additional 24 h in the drying frames.

Dried gels were scanned with a GS-710 Calibrated Imaging Densitometer (Bio-Rad) and analyzed with PD-Quest software (Bio-Rad). Scanning resolution was 63.5 x 63.5 µm and images were cropped to 1,902 x 1,201 pixels and filtered using a contramean filter with a 5-x 5-pixel filter size. Spot centers were resolved and marked using automatic parameters and were then edited manually.

Five gels per pH range per treatment were used to create a matched set with a standard image representing all proteins for that cell/treatment type. The percentage of spots matched initially by manual landmarking ranged among the four experimental treatments as follows: pH 3 to 6, 19.1% to 29.6%; pH 5 to 8, 23.1% to 37.2%; and pH 7 to 10, 52% to 65.9%. The resulting digital standard image included all spots appearing in at least two matched set members. Digital standard gels from each treatment were used to create a higher order matched set with a composite reference image representing all proteins appearing in all treatments. The percentage of spots matched by manual landmarking were: pH 3 to 6, 44.4%; pH 5 to 8, 34.9%; and pH 7 to 10, 40.1%. Analysis sets were created by two-way comparisons among the treatments of the higher order matched set. Further analyses were performed to identify polypeptides unique to a culture condition or physiological or functional state with analysis sets created from composite and standard images using a combination of Boolean operators as follows:

Unique to thermotolerant GCPs = Polypeptides IN [38°C] NOT IN [FGCPs

OR 32°C]

Only found in dedifferentiated, dividing cells= Polypeptides IN

[32°C] NOT IN [[FGCPs OR 38°C] OR 38°C + ABA]

As a result of ABA treatment = Polypeptides IN [38°C + ABA] NOT

IN [FGCPs OR 32°C] OR 38°C]

Only found in cells with guard cell function = Polypeptides IN [FGCPs

AND 38°C + ABA] NOT IN [32°C OR 38°C]

As a result of culture = Polypeptides IN [32°C OR 38°C] OR 38°C+

ABA] NOT IN [FGCP]

Distribution of Materials

Upon request, all novel materials described in this publication will be made available in a timely manner for noncommercial research purposes.

	ACKNOWLEDGMENTS

 
We thank Ian H. Street, Barbara Stebbins-Boaz, and Eduardo Zeiger for reading an earlier version of the manuscript, and E. Vierling for an early gift of Hsp 70 antibodies.

Received March 23, 2003; returned for revision April 22, 2003; accepted May 5, 2003.

	FOOTNOTES

 
Article, publication date, and citation information can be found at www.plantphysiol.org/cgi/doi/10.1104/pp.103.024067.

¹ This work was supported by the National Science Foundation (grant no. 9900525), by the M.J. Murdock Charitable Trust (to S.F.), and by an Arthur A. Wilson Research Scholarship Award (to L.P.M.).

^* Corresponding author; e-mail gtallman{at}willamette.edu; fax 503–375–5425.

	LITERATURE CITED

TOP ABSTRACT RESULTS DISCUSSION MATERIALS AND METHODS LITERATURE CITED

 
Boorse G, Tallman G (1999) Guard cell protoplasts: isolation, culture, and regeneration of plants. In RD Hall, ed, Methods in Molecular Biology: Plant Cell Culture Protocols, Vol. 111. Humana Press, Totawa, NJ, pp 243–257

Bradford MM (1976) A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Anal Biochem 72: 248–254[CrossRef][Web of Science][Medline]

Burnett EC, Desikan R, Moser RC, Neill SJ (2000) ABA activation of an MBP kinase in Pisum sativum epidermal peels correlates with stomatal responses to ABA. J Exp Bot 51: 197–205[Abstract/Free Full Text]

Calderini O, Bögre L, Vicente O, Binarova P, Heberle-Bors E, Wilson C (1998) A cell cycle regulated MAP kinase with a possible role in cytokinesis. J Cell Sci 111: 3091–3100[Abstract]

Carimi F, Zottini M, Formentin E, Terzi M, Schiavo F (2003) Cytokinins: new apoptotic inducers in plants. Planta 216: 413–421[Web of Science][Medline]

Cupples W, Lee J, Tallman G (1991) Division of guard cell protoplasts of Nicotiana glauca (Graham) in liquid cultures. Plant Cell Environ 14: 691–697

Francis D, Barlow PW (1988) Temperature and the cell cycle. In SP Long, FI Woodward, eds, Plants and Temperature, 42nd Symposium of the Society for Experimental Biology. Company of Biologists, Cambridge, UK, pp 181–201

Gabai VL, Sherman MY (2002) Invited review: interplay between molecular chaperones and signaling pathways in survival of heat shock. J Appl Physiol 92: 1743–1748[Abstract/Free Full Text]

Galbraith DW (1981) Microfluorimetric quantitation of cellulose biosynthesis by plant protoplasts using Calcofluor white. Physiol Plant 53: 111–116

Groover A, Jones AM (1999) Tracheary element differentiation uses a novel mechanism coordinating programmed cell death and secondary cell wall synthesis. Plant Physiol 119: 375–384[Abstract/Free Full Text]

Hall RD, Riksen-Bruinsma T, Weyens GJ, Rosquin IJ, Denys PN, Evans IJ, Lathouwers JE, Lefebvre MP, Dunwell JM, van Tunen A et al. (1996) A high efficiency technique for the generation of transgenic sugar beets from stomatal guard cells. Nat Biotechnol 14: 1133–1138[Web of Science][Medline]

Haq R, Brenton JD, Takahashi M, Finan D, Rottapel R, Zanke B (2002) Constitutive p38HOG mitogen-activated protein kinase activation induces permanent cell cycle arrest and senescence. Can Res 62: 5076–5082[Abstract/Free Full Text]

Herbert RJ, Vilhar B, Evett C, Orchard CB, Rogers HJ, Davies MS, Francis D (2001) Ethylene induces cell death at particular phases of the cell cycle in the tobacco TBY-2 cell line. J Exp Bot 52: 1615–1623[Abstract/Free Full Text]

Helmbrecht K, Zeise E, Rensing L (2000) Chaperones in cell cycle regulation and mitogenic signal transduction: a review. Cell Prolif 33: 341–365[CrossRef][Web of Science][Medline]

Hoagland DR, Arnon DI (1938) The water culture method for growing plants without soil. Circular of the California Agricultural Experiment Station, No. 347. California Agricultural Experiment Station, Berkeley, CA, pp 39

Inoue A, Torigoe T, Sogahata K, Kamiguchi K, Takahashi S, Sawada Y, Saijo M, Taya Y, Ishii S, Sato N et al. (1995) 70-kDa heat shock cognate protein interacts directly with the N-terminal region of the retinoblastoma protein pRb. J Biol Chem 270: 22571–22576[Abstract/Free Full Text]

Kato K, Fukasawa S, Shimizu Y, Soh Y, Nagai S (1977) Requirements of PO₄^3–, NO₃^–, SO₄^2–, K⁺, and Ca²⁺ for the growth of tobacco cells in suspension culture. J Fermentation Technol 55: 207–212

Kepinski S, Leyser O (2002) Ubiquitination and auxin signaling: a degrading story. Plant Cell Suppl 2002: S81–S95

Kim J, Adam RM, Freeman MR (2002) Activation of the Erk mitogen-activated protein kinase pathway stimulates neuroendocrine differentiation in LNCaP cells independently of cell cycle withdrawal and STAT3 phosphorylation. Can Res 62: 1549–1554[Abstract/Free Full Text]

Konishi Y, Lehninen M, Donovan N, Bonni A (2002) Cdc2 phosphorylation of BAD links the cell cycle to the cell death machinery. Mol Cell 9: 1005–1016[CrossRef][Web of Science][Medline]

Kovtun Y, Chiu W-L, Tena G, Sheen J (2000) Functional analysis of oxidative stress-activated mitogen-activated protein kinase cascade in plants. Proc Natl Acad Sci USA 97: 2940–2945[Abstract/Free Full Text]

Kovtun Y, Chiu W-L, Zeng W, Sheen J (1998) Suppression of auxin signal transduction by a MAPK cascade in higher plants. Nature 395: 716–720[CrossRef][Medline]

Krishnamurthy KV, Krishnaraj R, Chozhavendan R, Christopher FS (2000) The programme of cell death in plants and animals: a comparison. Curr Sci India 79: 1169–1181

Krysan PJ, Jester PJ, Gottwald JR, Sussman MR (2002) An Arabidopsis mitogen-activated protein kinase kinase kinase gene family encodes essential positive regulators of cytokinesis. Plant Cell 14: 1109–1120[Abstract/Free Full Text]

Kühl NM, Rensing L (2000) Heat shock effects on cell cycle progression. Cell Mol Life Sci 57: 450–463[CrossRef][Web of Science][Medline]

Laemmli UK (1970) Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature 227: 680–685[CrossRef][Medline]

Lam E, Kato N, Lawton M (2001) Programmed cell death, mitochondria and the plant hypersensitive response. Nature 41: 848–853

Lee S, Woffenden BJ, Beers EP, Roberts AW (2000) Expansion of cultured Zinnia mesophyll cells in response to hormones and light. Physiol Plant 108: 216–222

Martinez-Garcia JF, Monte E, Quail PH (1999) A simple, rapid and quantitative method for preparing Arabidopsis extracts for immunoblot analysis. Plant J 20: 251–257[CrossRef][Web of Science][Medline]

Merritt F, Kemper A, Tallman G (2001) Inhibitors of ethylene synthesis inhibit auxin-induced stomatal opening in epidermis detached from leaves of Vicia faba L. Plant Cell Physiol 42: 223–230[Abstract/Free Full Text]

Mockaitis K, Howell SH (2000) Auxin induces mitogenic activated protein kinase (MAPK) activation in roots of Arabidopsis seedlings. Plant J 24: 785–796[CrossRef][Web of Science][Medline]

Mori IC, Muto S (1997) Abscisic acid activates a 48-kilodalton protein kinase in guard cell protoplasts. Plant Physiol 113: 833–839[Abstract]

Morris PC (2001) MAP kinase signal transduction pathways in plants. New Phytol 151: 67–89

Murray JA (1997) The retinoblastoma protein is in plants! Trends Plant Sci 2: 82–84

Nagata T, Kumagai F, Sano T (2001) The regulation of the cell cycle in cultured cells. In D Francis, ed, The Plant Cell Cycle and its Interfaces. CRC Press, Boca Raton, FL, pp 74–86

Nagata T, Nemoto Y, Hasezewa S (1992) Tobacco BY-2 cell line as the "HeLa" cell line in the cell biology of higher plants. Int Rev Cytol 132: 1–30[Web of Science]

Nakagami H, Kawamura K, Sugisaka K, Sekine M, Shinmyo A (2002) Phosphorylation of retinoblastoma-related protein by the cyclin D/cyclin-dependent kinase complex is activated at the G1/S phase transition in tobacco. Plant Cell 14: 1847–1857[Abstract/Free Full Text]

Pan L, Kawai M, Yu L-H, Kim K-M, Hirata A, Umeda M, Uchimiya H (2001) The Arabidopsis thaliana ethylene-responsive element binding protein (AtEBP) can function as a dominant suppressor of Bax-induced cell death of yeast. FEBS Lett 508: 375–378[Medline]

Queitsch C, Hong S-W, Vierling E, Lindquist S (2000) Heat shock protein 101 plays a crucial role in thermotolerance in Arabidopsis. Plant Cell 12: 479–492[Abstract/Free Full Text]

Rabilloud T, Adessi C, Giraudel A, Lunardi J (1997) Improvement of the solubilization of proteins in two-dimensional electrophoresis with immobilized pH gradients. Electrophoresis 18: 307–316[CrossRef][Web of Science][Medline]

Rabilloud T, Valette C, Lawrence JJ (1994) Sample application by in-gel rehydration improves the resolution of two-dimensional electrophoresis with immobilized pH gradients in the first dimension. Electrophoresis 15: 1552–1558[CrossRef][Web of Science][Medline]

Roberts C, Sahgal P, Merritt F, Perlman B, Tallman G (1995) Temperature and abscisic acid can be used to regulate survival, growth, and differentiation of cultured guard cell protoplasts of tree tobacco. Plant Physiol 109: 1411–1420[Abstract]

Rowley A, Johnston GC, Butler B, Werner-Washburne M, Singer RA (1993) Heat shock-mediated cell cycle blockage and G1 cyclin expression in the yeast Saccharomyces cerevisiae. Mol Cell Biol 13: 1034–1041[Abstract/Free Full Text]

Sah JF, Eckert RL, Roshantha AS, Chandraratna AS, Rorke EA (2002) Retinoids suppress epidermal growth-factor associated cell proliferation by inhibiting epidermal growth factor receptor-dependent ERK1/2 activation. J Biol Chem 277: 9728–9735[Abstract/Free Full Text]

Sahgal P, Martinez G, Roberts C, Tallman G (1994) Regeneration of plants from cultured guard cell protoplasts of Nicotiana glauca (Graham). Plant Sci 97: 199–208

Sano T, Kuraya Y, Amino S, Nagata T (1999) Phosphate as a limiting factor for the cell division of tobacco BY-2 cells. Plant Cell Physiol 40: 1–8[Abstract/Free Full Text]

Sauter M (2001) Gibberellic acid-mediated regulation of the cell cycle during stem growth. In D Francis, ed, The Plant Cell Cycle and its Interfaces. CRC Press, Boca Raton, FL, pp 57–73

Shimazaki K, Kinoshita T (1995) Analysis of the light signaling pathway in stomatal guard cells. In DW Galbraith, HJ Bohnert, DP Bourque, PT Matsudaira, eds, Methods in Cell Biology, Vol 49. CRC Press, Boca Raton, FL, pp 501–513[Medline]

Stebbins-Boaz B, Cao Q, de Moor CH, Mendez R, Richter JD (1999) Maskin is a CPEB associated factor that regulates maternal mRNA translation by transiently interacting with eIF-4E. Mol Cell Biol 4: 1017–1027

Taylor JE, Abram B, Boorse G, Tallman G (1998) Approaches to evaluating the extent to which guard cell protoplasts of Nicotiana glauca (tree tobacco) retain their characteristics when cultured under conditions that affect their survival, growth, and differentiation. J Exp Bot 49: 377–386[Abstract]

Trotter EW, Berenfeld L, Krause SA, Petsko GA, Gray JV (2001) Protein misfolding and temperature up-shift cause G1 arrest via a common mechanism dependent on heat shock factor in Saccharomyces cerevisiae. Proc Natl Acad Sci USA 98: 7313–7318[Abstract/Free Full Text]

Van Der Straeten D, Van Wiemeersch L, Goodman HM, Van Montagu M (1990) Cloning and sequence of two different cDNAs encoding 1-aminocyclo-propane-1-carboxylate synthase in tomato. Proc Natl Acad Sci USA 87: 4859–4863[Abstract/Free Full Text]

Wang H, Qi Q, Schorr P, Cutler AJ, Crosby WL, Fowke LC (1998) ICK1, a cyclin-dependent protein kinase inhibitor from Arabidopsis thaliana interacts both with Cdc2a and CycD3, and its expression is induced by abscisic acid. Plant J 15: 501–510[CrossRef][Web of Science][Medline]

Wang H, Zhou Y, Gilmer S, Whitwill S, Fowke LC (2000) Expression of the plant cyclin-dependent kinase inhibitor ICK1 affects cell division, plant growth, and morphology. Plant J 24: 613–623[CrossRef][Web of Science][Medline]

Weinberg RA (1995) The retinoblastoma protein and cell cycle control. Cell 81: 323–330[CrossRef][Web of Science][Medline]

Wilson C, Pfosser M, Jonak C, Hirt H, Heberle-Bors E, Vicente O (1998) Evidence for the activation of a MAP kinase upon phosphate-induced cell cycle re-entry in tobacco cell. Physiol Plant 102: 532–538

Wu C (1995) Heat shock transcription factors: structure and regulation. Annu Rev Cell Dev Biol 11: 441–469[CrossRef][Web of Science][Medline]

Yip W-K, Moore T, Yang SF (1992) Differential accumulation of transcripts of four tomato 1-aminocyclopropane-1-carboxylate synthase homologs under various conditions. Proc Natl Acad Sci USA 89: 2475–2479[Abstract/Free Full Text]

Yoon Y-M, Kim S-J, Oh C-D, Ju J-W, Song WK, Yoo YJ, Huh T-L, Chun J-S (2002) Maintenance of differentiated phenotype of articular chondrocytes by protein kinase C and extracellular signal-regulated protein kinase. J Biol Chem 277: 8412–8420[Abstract/Free Full Text]

Yosimichi G, Nakanishi T, Nishida T, Hattori T, Takano-Yamamoto T, Takigawa M (2001) CTGF/Hcs24 induces chondrocyte differentiation through a p38 mitogen-activated protein kinase (p38MAPK), and proliferation through a p44/p42 MAKP/extracellular-signal regulated kinase (ERK). Eur J Biochem 268: 6058–6065[Web of Science][Medline]

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