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 transition

Abstract

When guard cell protoplasts (GCPs) of tree tobacco [Nicotiana glauca (Graham)] are cultured at 32 degrees 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 degrees C and with 0.1 micro M +/- -cis,trans-abscisic acid (ABA) remain differentiated. GCPs cultured at 38 degrees 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 degrees C accumulate heat shock protein 70 and develop a thermotolerance that, upon transfer of cells to 32 degrees C, enhances cell survival but inhibits cell cycle reentry, dedifferentiation, and division. GCPs dedifferentiating at 32 degrees C require both 1-napthaleneacetic acid and 6-benzylaminopurine to survive, but thermotolerant GCPs cultured at 38 degrees C +/- ABA do not require either hormone for survival. Pulse-labeling experiments using 5-bromo-2-deoxyuridine indicate that culture at 38 degrees C +/- ABA prevents dedifferentiation of GCPs by blocking cell cycle reentry at G1/S. Cell cycle reentry at 32 degrees 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.

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 = 600×; B, magnification = 200×; D, magnification = 400×. Bar in A = 15 μm.

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 (100×; 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.

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).

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 = 600×; 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.

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.

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).