Tibetan Womenâ€™s Association Central Executive Committee

Thermotolerant Guard Cell Protoplasts of Tree TobaccoDo Not Require Exogenous Hormones to Survive inCulture and Are Blocked from Reentering the CellCycle at the G1-to-S Transition1

Nathan N. Gushwa, Derek Hayashi, Andrea Kemper, Beverly Abram, Jane E. Taylor, Jason Upton,Chloe F. Tay, Sarah Fiedler, Sam Pullen, Linnsey P. Miller, and Gary Tallman*

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 ofEnvironmental and Natural Sciences, Bailrigg, Lancaster LA1 4YQ, United Kingdom (B.A., J.E.T.)

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 �m � -cis,trans-abscisic acid (ABA) remain differentiated. GCPs culturedat 38°C without ABA dedifferentiate partially but do not divide. Cell survival after 1 week is 70% to 80% under all of theseconditions. In this study, we show that GCPs cultured for 12 to 24 h at 38°C accumulate heat shock protein 70 and developa 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, butthermotolerant GCPs cultured at 38°C � ABA do not require either hormone for survival. Pulse-labeling experiments using5-bromo-2-deoxyuridine indicate that culture at 38°C � ABA prevents dedifferentiation of GCPs by blocking cell cyclereentry at G1/S. Cell cycle reentry at 32°C is accompanied by loss of a 41-kD polypeptide that cross-reacts with antibodiesto rat (Rattus norvegicus) extracellular signal-regulated kinase 1; thermotolerant GCPs retain this polypeptide. A number ofpolypeptides unique to thermotolerant cells have been uncovered by Boolean analysis of two-dimensional gels and aretargets for further analysis. GCPs of tree tobacco can be isolated in sufficient numbers and with the purity required to studyplant cell thermotolerance and its relationship to plant cell survival, growth, dedifferentiation, and division in vitro.

At high temperatures, some plant species develop athermotolerance that enables them to survive untilcooler temperatures return (for review, see Francisand Barlow, 1988). Little is known about the molec-ular mechanisms by which high temperature altersthe growth of thermotolerant plants, nor are the sig-nal transduction pathways that activate plant ther-motolerance understood. A few studies with meris-tems and seeds indicate that sublethal hightemperatures (30°C–35°C) affect cell cycle progres-sion, lengthening the cycle or certain of its phases insome tissues, but shortening them in others (Francisand Barlow, 1988). Both heat shock proteins (Hsps)and mitogen-activated protein kinases (MAPKs orextracellular signal-regulated kinases [ERKs]) havebeen implicated in development of thermotolerance.Arabidopsis plants underexpressing Hsp 101 have a

diminished capacity to develop thermotolerance,whereas those overexpressing this protein have anenhanced capacity to survive abrupt shifts to extremehigh temperatures (Queitsch et al., 2000). Evidencefor the involvement of MAPK in thermotolerancecomes mainly from studies with yeast (Saccharomycescerevisiae; Trotter et al., 2001). Still, we do not knowhow these proteins and others with which they in-teract signal the development of plant thermotoler-ance and thereby affect cell survival, growth, differ-entiation, and division. Progress in understandingplant thermotolerance has been slowed by a lack of invitro plant cell culture systems with which to studythe signal transduction mechanisms that underliethis process.

Cultured guard cell protoplasts (GCPs) of tree to-bacco [Nicotiana glauca (Graham)] may be an excellentin vitro system for elucidating the signal transductionmechanisms that regulate plant cell thermotolerance(Roberts et al., 1995; Taylor et al., 1998). Highly puri-fied GCP monocultures (�0.01% contamination withother cell types; Fig. 1A) are uniform and synchronousin their responses to growth regulators like1-napthaleneacetic acid (NAA), 6-benzylaminopurine(BAP), and � -cis,trans-abscisic acid (ABA), and to

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 (toL.P.M.).

* Corresponding author; e-mail [email protected]; fax503–375–5425.

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

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temperature (Roberts et al., 1995; Taylor et al., 1998).Parallel cultures established from the same isolate canbe monitored under temperature conditions that re-sult in full dedifferentiation and cell division (cultureat 32°C; Fig. 1B), maintenance in the differentiatedstate (culture at 38°C in media with 0.1 �m ABA; Fig.1C), or partial dedifferentiation without cell division(culture at 38°C; Fig. 1D; Roberts et al., 1995; Taylor etal., 1998). Under all of these conditions, GCPs survivein high percentages (70%–80%) for at least 1 week(Roberts et al., 1995), indicating that GCPs developthermotolerance at 38°C.

At 32°C, both NAA and BAP are required for GCPsto survive, dedifferentiate, and divide. Dedifferenti-ating GCPs grow 50- to 60-fold, regenerate cell walls,and undergo major cytoskeletal rearrangements (Fig.1B; Roberts et al., 1995; Taylor et al., 1998). Theirchloroplasts become chlorotic and nonfunctional(Taylor et al., 1998). The resulting dedifferentiatedcells (Fig. 1B) are totipotent (Sahgal et al., 1994).Experiments with 2-aminoethoxyvinyl-Gly (AVG),an inhibitor of ethylene synthesis, suggest thatdedifferentiating GCPs require endogenous ethyleneproduction for survival (Roberts et al., 1995). ABA islethal to GCPs cultured at 32°C (Roberts et al., 1995).At 38°C in media containing ABA, GCPs do notdedifferentiate (Fig. 1C; Roberts et al., 1995; Taylor etal., 1998). Instead, they retain the size, morphology,and many of the unique physiological characteristicsof guard cells (Taylor et al., 1998). Among the func-tional characteristics retained are the capacity to: (a)swell when exposed to fusicoccin, (b) swell when

illuminated with low fluences (15 �mol m�2 s�1) ofblue light, (c) execute light-driven photosyntheticelectron transport, and (d) accumulate zeaxanthinupon illumination (Taylor et al., 1998). At 38°C inmedia lacking ABA, GCPs grow (Fig. 1D) and par-tially dedifferentiate (Taylor et al., 1998), losing someof the functional identity of guard cells. These cellsdo not divide (Roberts et al., 1995; Taylor et al., 1998),indicating that elevated temperature alone is suffi-cient to prevent cultured GCPs from reaching Mphase of the cell cycle (Fig. 1D). Whether or notmedia contain ABA, survival of GCPs at 38°C is notreduced by AVG treatment and, thus, does not ap-pear to depend on endogenous ethylene production(Roberts et al., 1995). Cells cultured at 38°C � ABAdo 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 pre-pared in a single isolate have made the types ofroutine signal transduction studies that are commonin yeast and animal cell culture systems too laboriousto perform with GCP. In this study, we attempted toscale procedures for isolating GCPs of tree tobacco toprovide 1 to 1.5 � 107 cells per isolate at a purityadequate for larger scale studies. To establish thekinetics of development of thermotolerance, GCPswere pre-incubated at 38°C for times ranging from 0to 24 h and then cultured for an additional week at32°C before the effects of high temperature pretreat-ment on cell survival and division were estimated. Toevaluate whether Hsp levels increase with the samekinetics as gain of thermotolerance, at the same pre-incubation time points, proteins were extracted fromGCPs cultured at 32°C or 38°C � ABA, and levels ofinducible Hsp70 and heat shock cognate (Hsc) 70were measured by western blotting. To ascertainwhether thermotolerance alters hormone require-ments for cell survival, survival was estimated after 1week of culture at 32°C or 38°C � ABA in mediacontaining NAA alone, BAP alone, both NAA andBAP, or neither hormone. To determine the point(s)at which high temperature and/or ABA block the cellcycle and dedifferentiation, pulse labeling with5-bromo-2-deoxyuridine (BrdU) was employed overa 2-week period to test the hypothesis that GCPscultured at 38°C � ABA do not pass the restrictionpoint between G1 and S.

MAPK similar to those involved in regulating ther-motolerance in yeast (Trotter et al., 2001) and celldifferentiation, division, and apoptosis in culturedanimal cells (Kim et al., 2002; Yoon et al., 2002) areconserved across a number of plant species (Morris,2001), and three putative MAPK have been identifiedin GCPs from pea (Pisum sativum; Burnett et al.,2000). An MAPK cascade initiated by an MAPKKK(NPK1) can suppress auxin signaling in transfectedmaize (Zea mays) protoplasts and transgenic tobacco(Nicotiana tabacum) plants (Kovtun et al., 1998, 2000),

Figure 1. Cultured GCPs of tree tobacco. A, FGCPs and GCPs cul-tured for 1 week at 32°C (B), 38°C in media containing 0.1 �M ABA(C), or 38°C (D). A to C, Magnification � 600�; B, magnification �200�; D, magnification � 400�. Bar in A � 15 �m.

Gushwa et al.

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and several symptoms of guard cell thermotoleranceare consistent with suppression of auxin signaling(e.g. failure to regenerate cell walls, senesce chloro-plasts, and reenter the cell cycle; Roberts et al., 1995;Taylor et al., 1998). Thus, over a time course similarto that used for BrdU experiments, protein extractsfrom freshly isolated GCPs (FGCPs) or from culturescontaining dedifferentiated (32°C), differentiated(38°C � ABA), or partially dedifferentiated (38°C)cells were probed for ERK1/2 homologs by westernblotting with mammalian ERK1/2 antibodies.

To identify target polypeptides associated withthermotolerance for future proteomic analysis, wedeveloped silver-stained two-dimensional gel librar-ies of the major polypeptides extracted from FGCPsand from GCPs cultured under each condition de-scribed. We then compared polypeptide profiles ofthermotolerant cells with those of cells undergoingdedifferentiation at 32°C and with those of FGCPs byBoolean spot match analysis.

These experiments confirm that high-yield mo-nocultures of tree tobacco GCPs can provide suffi-cient material to study in vitro the signal transduc-tion pathways that regulate development of plant cellthermotolerance and its effects on cell survival,growth, differentiation, and division. They also dem-onstrate that protoplast yields are adequate to securea proteomic analysis of target polypeptides associ-ated with all of these processes.

RESULTS

The Protocol for Isolating GCP Scales withoutLoss of Purity

We reasoned that, because a single leaf can of treetobacco can be stripped of its epidermis in 5 to 10 minand a leaf typically yields 1.1 to 1.6 � 106 GCP, itmight be possible to scale the isolation protocol pro-portionately to give high yields of GCPs while main-taining their purity. The method used to isolate GCPsfrom a single leaf was scaled successfully to yield asmany as 1.5 � 107 GCPs from nine leaves. In thelargest GCP preparations, levels of contaminationwith mesophyll and pavement cell protoplasts weresimilar to those of single leaf preparations (�0.01%;Fig. 1A; Cupples et al., 1991). As reported previously(Roberts et al., 1995), contaminating cells did notsurvive under any of the culture conditionsemployed.

Cultured GCPs Develop Thermotolerance within12 to 24 h

GCPs cultured at 32°C begin to divide within 48 to72 h (Cupples et al., 1991), but GCPs cultured at38°C � ABA do not divide. Thus, we expected thatGCPs would develop thermotolerance within thefirst 24 to 48 h in culture and that thermotoleranceshould be defined by high survival but failure to

divide (Roberts et al., 1995). To establish the kineticsof development of thermotolerance, GCPs were pre-incubated at 38°C for 1 to 24 h and then cultured at32°C for another week before the effects of high-temperature pre-incubation on survival and cell di-vision were evaluated. To establish baseline survival,GCPs were cultured at 38°C for 24 h, and the numberof living and/or dead cells in cultures was estimatedmicroscopically. After 24 h at 38°C, the mean numberof dead cells was 446.3 � 43.1, the mean number ofliving cells was 1,223.7 � 79.4, and the mean percent-age of survival was 73.3 � 3.0 (mean; se; n � 3). Themean numbers of dead cells in control cultures incu-bated for 1 week at 32°C or 38°C were 496 � 34.3 and535.3 � 13.4, respectively. In neither of these controlswas the number of dead cells significantly differentfrom the 24-h, 38°C baseline control (Fig. 2A;ANOVA; Fisher’s protected least squares difference(PLSD), P � 0.05, n � 3).

After 1 week of culture at 32°C after 1 to 24 h ofpre-incubation at 38°C, the mean number of deadcells was significantly greater than that of the base-line control at all pre-incubation times tested (Fig.2A; ANOVA; Fisher’s PLSD, P � 0.05, n � 3). Thenumber of dead cells increased significantly after 1 to9 h of pre-incubation, peaked at 6 to 9 h, and thendecreased significantly from the number of dead cellsin the 9-h pre-incubation treatment after 12 to 24 h ofpre-incubation (Fig. 2A; ANOVA; Fisher’s PLSD, P �0.05, n � 3). After a 9-h pre-incubation at 38°C, onlyapproximately 35% to 40% of cells survived an addi-tional week of culture at 32°C.

Cells pre-incubated at 38°C for �9 h that survivedan additional week of culture at 32°C divided in highpercentages (95%; not shown). However, rates of di-vision were low (�2%) among cells pre-incubated at38°C for �12 h and then cultured for 1 week at 32°C(Fig. 2, B and C). A variety of cell types were ob-served in cultures pre-incubated for �12 h, includingelongate cells (not shown) and cells without walls(Fig. 2C). GCPs cultured at 38°C for up to 24 h inmedia containing ABA did not survive when theywere cultured subsequently at 32°C for an additionalweek.

GCPs Accumulate Hsp70 after 18 to 24 h at38°C � ABA

Hsp production and activation of heat shock tran-scription factors (HSFs; Wu, 1995) would be expectedat 38°C � ABA and might be anticipated to affect cellsurvival (Queitsch et al., 2000) and capacity for cellcycle reentry (Helmbrecht et al., 2000; Kuhl andRensing, 2000). Therefore, we measured levels of in-ducible Hsp70 and the noninducible Hsc70 over thesame time course used to measure development ofthermotolerance. Mean levels of Hsp70 measured asnormalized contour gel band quantities did notchange significantly over the first 24 h of culture at

Thermotolerance in Cultured Guard Cell Protoplasts

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32°C (Fig. 3, A and C; ANOVA; Fisher’s PLSD, P �0.05, n � 3), nor did levels of Hsp70 change signifi-cantly over the first 12 h of culture at 38°C or over thefirst 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 medialacking ABA or 24 h of culture at 38°C in mediacontaining ABA, Hsp70 levels were significantlygreater than those of GCPs cultured at 32°C (Fig. 3, Aand C; ANOVA; Fisher’s PLSD, P � 0.05, n � 3).Under each condition, levels of Hsc70 were un-changed over the first 24 h of culture (Fig. 3B;ANOVA; Fisher’s PLSD, P � 0.05, n � 3). In someinstances, proteins of lower molecular mass (60–65kD) that cross-reacted with Hsp70 and Hsc70 anti-bodies were detected on blots (e.g. see 38°C � ABA

in Fig. 3C), but there was no consistent pattern thatcould be correlated with a particular treatment. Thus,only the contour quantities of predominant bandswith molecular masses similar to those of controlproteins were measured.

Thermotolerant GCPs Do Not Require NAA orBAP to Survive

At 32°C, dividing GCPs require both NAA andBAP to survive, but it was not clear whether devel-opment of thermotolerance might induce a cell sur-vival mechanism that would eliminate this require-ment at 38°C � ABA. At 32°C, omitting NAA, BAP,or both from culture media reduced cell survivalafter 1 week of culture to 0.3% to 3.9% of initial cellnumbers compared with approximately 70% survival

Figure 2. Effect of pre-incubating GCPs of tree tobacco at 38°C ontheir 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 thatwas preceded by a 38°C pre-incubation for 0 to 24 h. B, Baselinemean number of cells surviving after 24 h at 38°C. Lines, Meannumber of cells surviving after 8 d of continuous culture at 32°C orat 38°C. Values are means and SEs from three replicate experiments.a, Significantly different from baseline control (ANOVA; Fisher’sprotected least squares difference; P � 0.05); b, significantly lowerthan 9-h pre-incubation (ANOVA; Fisher’s protected least squaresdifference; P � 0.05). B, GCPs cultured continuously for 8 d at 32°C(100�; bar � 100 �m). C, GCPs cultured at 38°C for 24 h and thenfor 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 tobaccocultured at 38°C (F), at 38°C in media containing 0.1 �M ABA (Œ), orat 32°C (f) for up to 24 h. A and C, Hsp70 levels; B, Hsc70 levels.Lanes contained equal amounts of protein extracted from cells cul-tured for 0, 0.5, 1, 3, 6, 9, 12, 18, or 24 h. In each experiment, bandcontour quantities were estimated with densitometry and normalizedto standards (60 ng of human [Homo sapiens] Hsp70 or wheat[Triticum aestivum] Hsc70) before they were averaged. Values aremeans for three replicate experiments. Asterisk, Significantly differentfrom corresponding 32°C (ANOVA; Fisher’s PLSD; P � 0.05).

Gushwa et al.



when both hormones were included in media (TableI). When GCPs were cultured at 38°C in media con-taining or lacking 0.1 �m ABA, omitting NAA, BAP,or both did not reduce cell survival, which rangedfrom 69.7% to 77.7% of initial cell numbers (Table I).

GCPs Cultured at 38°C � ABA Do Not IncorporateBrdU into DNA

In our previous studies (Roberts et al., 1995), GCPscultured at 38° � ABA did not reach M phase.Whether GCPs might have reached S phase or G2was not examined. We used BrdU pulse labeling toaddress this question. When GCPs were cultured at32°C, BrdU incorporation into nuclear DNA (Fig. 4, Aand B) was detected within 48 to 72 h (Fig. 4C). At theend of the 48- to 72-h pulse, 18.3% � 1.3% (mean; se;n � 3; Fig. 4C) of nuclei were labeled with BrdU, and1.9% � 0.6% of cultured cells had formed cell plates.The percentage of nuclei containing BrdU-labeledDNA reached a maximum of 30.1% � 1.7% after the72- to 96-h pulse, ranged from 24.9% to 29% overdays 5 through 7 of the experiment, and then de-clined steadily to �2% of nuclei examined over theremainder of the 2-week experiment (Fig. 4C). GCPscultured at 38°C in media � 0.1 �m ABA did notincorporate BrdU into DNA (Fig. 4C). GCPs culturedat 38° � ABA for 2 weeks did not regenerate cellwalls (Calcofluor white; not shown), but GCPs cul-tured at 32°C did (Fig. 1B). To determine whetherfailure to detect BrdU incorporation at d 10 through14 might be due to antibody absorption by or adsorp-tion to the fixed cell walls of GCPs cultured at 32°Cfor longer periods, cells from 11-d-old cultures weredigested with cellulolytic enzymes, and nuclei wereisolated from “reprotoplasted” cells. Only 0.2% ofnuclei isolated from these protoplasts containedBrdU-labeled DNA.

Temperature-induced changes in hormone rela-tions (Nagata et al., 2001), phosphate depletion ofmedia (Kato et al., 1977; Sano et al., 1999), andanoxia-induced changes in cellular GA3 contents(Sauter, 2001) could all potentially prevent cell cyclereentry. Raising hormone concentrations 20- or 50-

fold, increasing phosphate concentrations to 500 mgL�1, or including GA3 in media at concentrationsranging from 5 to 100 �m did not trigger cell cyclereentry at 38°C (not shown). Thus, none of thesefactors alone appears to be responsible for failure toreenter the cell cycle at 38°C.

A 41-kD ERK1 Cross-Reacting Protein ThatDisappears with the Onset of S Phase Is Retained inThermotolerant GCP

In yeast, animals, and plants, MAPKs are involvedin stress responses (Kovtun et al., 2000; Morris, 2001),hormonal signaling (Kovtun et al., 1998; Burnett etal., 2000; Mockaitis and Howell, 2000), thermotoler-ance (Trotter et al., 2001), cell survival (Haq et al.,

Table I. Percentage of GCPs of tree tobacco surviving after 1 weekof culture at 32°C or at 38°C in media containing or lacking auxin(NAA) and/or cytokinin (BAP) and/or ABA

Each value is the mean � SE of the sample mean for three culturesestablished on separate days. Survival was estimated as described in“Materials and Methods.”

TreatmentsCells Surviving

32°C 38°C 38°C � ABA

%

�NAA �BAP 69.3 � 5.5 71.0 � 7.5 71.2 � 15.1�NAA �BAP 0.3 � 0.3 74.7 � 3.2 77.3 � 1.2�NAA �BAP 3.9 � 3.7 77.7 � 4.0 75.3 � 2.5�NAA �BAP 2.3 � 2.3 71.3 � 6.8 69.7 � 10.1

Figure 4. BrdU pulse labeling of cultured GCPs of tree tobacco. Aand B, Nuclei isolated from cells pulse labeled with BrdU between d5 and 6 after cultures were established. Nuclei stained for DNA withHoechst 33342 (A). B, Same nuclei in A stained for BrdU incorpo-ration with a fluorescein isothiocyanate (FITC)-conjugated rabbitanti-BrdU antibody. Magnification � 600�; bar in A � 30 �m. C,Percentage of nuclei incorporating BrdU in 24-h pulse labelingexperiments over a 14-d period of culture at 32°C (F) or at 38°C inmedia lacking ([trio]) or containing (f) 0.1 �M ABA. Each data pointis the mean from three separate experiments in which 1,000 nucleivisualized initially with Hoechst staining were also scored for BrdUincorporation. Bars � SE.




2002), cell cycle control (Calderini et al., 1998; Wilsonet al., 1998; Haq et al., 2002; Krysan et al., 2002; Sah etal., 2002), and differentiation (Yosimichi et al., 2001;Kim et al., 2002; Yoon et al., 2002). Thus, we antici-pated that regulatory patterns for some MAPK mightbe altered at high temperatures. Using an antibody tosubdomain XI of rat (Rattus norvegicus) ERK1, threeproteins were detected in extracts from FGCPs orcultured GCPs (Fig. 5, A–C; hereafter called ERK1cross-reacting proteins [ERK1-CRPs]). No ERK1-CRPs were detected: (a) in 7-�g loads of CellulaseOnozuka RS, Pectolyase Y-23, or bovine serum albu-min (BSA; not shown); (b) with a 1/1,000 (v/v) dilu-tion of pre-immune serum in place of the primaryantibody; or (c) with antibody to subdomain XI of ratERK2. Cells cultured under all conditions contained a47-kD ERK1-CRP that was retained at similar levelsfor up to 10 d (Fig. 5, A–C). At 32°C, levels of a 42-kDERK1-CRP did not change over 10 d, but levels of a41-kD ERK1-CRP declined rapidly with the onset of S

phase (48–72 h; Fig. 5, A and D) and were undetect-able by the time cultures reached stationary phase (5d; Fig. 5, A and D). The 41-kD ERK1-CRP was re-tained for up to 10 d by cells cultured at 38°C � ABA(Fig. 5, B and C), but the 42-kD ERK1-CRP found inextracts from cells cultured at 32°C (Fig. 5A) couldnot be clearly resolved from the 41-kD ERK1-CRP(Fig. 5, B and C).

Two-Dimensional Electrophoresis UncoversPolypeptides Unique to Thermotolerant GCP

Because proteins are the functional expression ofgene regulation, polypeptides from each treatmentwere compared by two-dimensional gel electro-phoresis to determine the number of major polypep-tides unique to each culture condition. The proteinextraction employed typically yielded 40 to 50 �g ofprotein per 106 GCPs. Because 1- to 2-week-old cul-tures contained 20% to 30% dead cells, a controlculture of 3 � 106 GCPs was established in whichcells were cultured at 32°C without NAA and BAPuntil all cells were dead. Extraction yielded 0.95 �g ofprotein per 106 GCP.

Results of spot match analysis of two-dimensionalgels are summarized in Tables II and III and areillustrated in Figure 6. In two-way comparisonsamong treatments in each pH range employed (TableII), major polypeptides shared between treatmentsranged from 30.5% to 51.6% of total polypeptidesdetected.

Using combinations of Boolean operators, the two-dimensional gel library was queried for polypeptidesunique either to treatment or to function (Table III).For example, in the pH range 5 to 8, 54 polypeptidesassociated with thermotolerance were identified byselecting for those found in both FGCPs and cellscultured at 32°C, neither of which are thermotolerant,and then removing those shared in common withcells cultured at 38°C (Table III). Accounting for over-lap in pH ranges of gels by inspection, a number ofpolypeptides (approximately 47–70) were identifiedthat were unique to dedifferentiated, dividing cells(Table III). Similarly, 100 to 180 polypeptides associ-ated with ABA treatment were identified by selectingfor those in the 38°C � ABA treatment that werenot found in any other treatment (FGCP � 32°C �38°C), and approximately 34 to 50 polypeptides wereidentified as unique to guard cell function (Table III;Fig. 6).

DISCUSSION

At 38°C, Cultured GCPs DevelopThermotolerance within 24 h

The mechanism required to survive high tempera-tures and the mechanisms required to thwart cellcycle reentry, dedifferentiation, and division developin parallel over the first 24 h at 38°C (Fig. 2). When

Figure 5. Proteins that cross-react with a rat ERK1 antibody in ex-tracts from GCPs of tree tobacco cultured for 0, 1, 2, 3, 5, 7, or 10 dat 32°C (A,) 38°C (B), or 38°C (C) in media containing 0.1 �M ABA.P�, Control from non-progesterone-treated Xenopus laevis oocytes;P�, control from progesterone-treated X. laevis oocytes; all otherlanes contain 7 �g of protein extracted from GCP. D, Changes inmean contour band quantities of a 41-kD ERK cross-reacting proteinas a function of days in culture at 32°C. In each experiment, bandcontour quantities were normalized to those of FGCPs (d 0) beforethey were averaged. Values are means and SEs for three replicateexperiments. For purposes of illustration, BrdU incorporation forcells cultured at 32°C is repeated from Figure 4C.

Gushwa et al.



cells were returned to 32°C after shorter pre-incubations (�9 h) at 38°C, cell survival after a weekwas low (Fig. 2A), but virtually all cells that survivedthe transfer to a cooler temperature divided (notshown). GCPs pre-incubated at 38°C for �12 h beforetransfer to 32°C showed increased cell survival aftera week compared with GCPs given shorter (�9 h)38°C pre-incubations (Fig. 2A), but cells given �12 hof pre-incubation at 38°C (Fig. 2C) did not dividewhen they were returned to 32°C. Thus, after a 24-hexposure to 38°C, cultured GCPs have developed athermotolerance that ensures their survival, but onceGCPs have developed thermotolerance they do notdivide, even when NAA and BAP concentrations areraised 20- to 50-fold.

Accumulation of Hsp 70 Parallels Development ofThermotolerance

It has been proposed that, in intact plants, Hspsstabilize meristematic cells during sudden local

increases in temperature so that when cooler temper-atures return, the cell cycle may be resumed (Francisand Barlow, 1988). Once GCPs developed thermotol-erance in culture, they did not resume division at32°C, even after 1 week of additional culture. In-creased levels of Hsp70 at 24 h were positively cor-related with capacity to survive after transfer from38°C to 32°C, but capacity for cell division amongsurviving cells was negatively correlated with in-creased Hsp70 levels. Short pre-incubations (�9 h) at38°C did not result in increased Hsp70 (Fig. 3). Underthose conditions, cell survival was lower, but surviv-ing cells divided. Hsp70 levels also increased within24 h of culture at 38°C in ABA-containing media (Fig.3), conditions under which GCPs also survive in highpercentages but fail to divide (Roberts et al., 1995).Levels of the noninducible Hsc70 did not change overthe first 24 h of culture under any condition exam-ined (Fig. 3), suggesting that Hsp70 accumulationwas probably a specific response to activation of

Table II. Two-way comparisons by two-dimensional gel electrophoresis of polypeptide extracts from FGCPs of tree tobacco or those ex-tracted from guard cell protoplasts cultured for 1 week at 32°C at 38°C, or at 38°C in media containing 0.1 �M ABA

Using spot-matching software, five silver-stained two-dimensional gels of extracts from each treatment in each pH range (3–6, 5–8, and 7–10)were used to create a reference gel matched set. A higher level matched set was then created from reference gels of each matched set. Individualanalysis sets representing members of higher level matched sets were then compared using Boolean operators.

A B Total A Total B A Only B OnlyA Only �B Only �Unshared

Sharedin

Common

Shared �Unshared

% in Common � [Sharedin Common/(Shared �

Unshared) � 100]

pH 3 to 6FGCP 32°C 155 126 88 59 147 67 214 31.3FGCP 38°C 155 106 94 45 139 61 200 30.5FGCP 38°C � ABA 155 157 64 66 130 91 221 31.232°C 38°C 126 106 66 46 112 60 172 34.932°C 38°C � ABA 126 157 46 77 123 80 203 39.438°C 38°C � ABA 106 157 33 84 117 73 190 38.4



Table III. No. of polypeptides unique to certain culture treatments, differentiation states, and/or physiological functions identified by Bool-ean analysis of two-dimensional gel libraries of polypeptide extracts from FGCPs or cultured GCPs of tree tobacco

The Boolean operations by which spot match analysis sets were created are described in “Materials and Methods.”

Treatment/Function pH 3 to 6 pH 5 to 8 pH 7 to 10

Unique to thermotolerant cells 30 54 26Only found in dedifferentiated, dividing cells 31 47 15As a result of ABA treatment 32 80 83Only found in cells with guard cell function (FGCP and 38°C � ABA) 24 34 11As a result of any culture condition 121 235 73




HSFs (Wu, 1995) rather than the result of alteredprotein turnover.

Certain Hsps are known to be required for hightemperature survival in plants. Arabidopsis plantsoverexpressing a gene for Hsp101 have an enhancedcapacity to survive abrupt shifts to extreme hightemperatures, whereas plants underexpressing thegene have a diminished survival capacity (Queitschet al., 2000). In addition to functioning as molecularchaperones to facilitate the refolding of proteins de-natured by heat shock, in mammalian cells, Hsp72, amember of the HSP70 family, can block both JNK-mediated caspase-dependent and -independent celldeath pathways (Gabai and Sherman, 2002). Hsp72can also inhibit cell survival pathways mediatedthrough Akt and ERK (Gabai and Sherman, 2002).Whether cells survive under a particular circum-stance appears to be a function of the relative effec-tiveness of Hsp72 in regulating the balance betweenthese alternate pathways under any given set of con-ditions (Gabai and Sherman, 2002). Caspases havenot been found in plants (Krishnamurthy et al., 2000),

suggesting that if survival is mediated by membersof the Hsp70 family in cultured GCPs, these proteinsprobably act by inhibiting a caspase-independent celldeath mechanism.

In animal cell cultures, Hsps and HSFs (Wu, 1995)are known to regulate distribution of cultured cellsamong alternate fates by regulating the cell cycle(Helmbrecht et al., 2000; Kuhl and Rensing, 2000),apoptosis (Gabai and Sherman, 2002), or necrosis(Gabai and Sherman, 2002). Thus, at high tempera-tures, Hsp could stabilize proteins to reduce death ofGCPs and, at the same time, participate in blockingthe G1-to-S phase transition (Fig. 4). Involvement ofHsp70 and Hsc70 in regulating early events of thecell cycle has been documented. In yeast, elevatingculture temperature from 23°C to 36°C for as little as30 min (heat shock) causes a transient arrest betweenG1 and S through an unknown mechanism that re-presses expression of the G1 cyclin genes, CLN1 andCLN2 (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 pro-teins selectively activates HSF, and (c) activated HSFrepresses expression of CLN1 and CLN2 through anunknown mechanism (Trotter et al., 2001). Increasedlevels of Hsp70 in GCPs cultured at 38°C � ABAprobably reflect activation of HSF, but this hypothe-sis has not been tested. Unlike in yeast, elevatedtemperature does not simply extend the duration ofG1 in cultured GCP; instead, GCPs do not leave G1(Fig. 4).

Failure of GCPs cultured at 38°C to reenter the cellcycle (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 ofsuppressed auxin signaling. Furthermore, we hypoth-esize that chloroplasts retain chlorophyll at 38°C (Tay-lor et al., 1998) because auxin is unable to trigger theethylene production required for chloroplast senes-cence (Van Der Straeten et al., 1990; Yip et al., 1992;Merritt et al., 2001). We speculate that some Hsps mayprotect Aux/IAA proteins (Kepinski and Leyser, 2002)from auxin-induced ubiquitination and proteosomaldestruction. If so, Hsps might suppress auxin signal-ing by preventing dimerization of auxin response fac-tors that activate auxin response elements (Kepinskiand Leyser, 2002). We envision that auxin responseelement activation would be required to regulategenes needed for early cell cycle events, cell expan-sion, wall regeneration, and ethylene synthesis.

Hsc70 could also be involved in blocking cell cyclereentry 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 replicationmachinery during the G1-to-S phase transition(Weinberg, 1995). Plants have a pRb equivalent (Mur-ray, 1997), and recent studies indicate that Nicta;CycD3;3-associated kinase phosphorylates NtRb1

Figure 6. Two-dimensional electrophoresis of polypeptides fromGCPs of tree tobacco. A, Polypeptides from FGCPs. Polypeptides (90�g) were extracted and separated along a pH gradient of 5 to 8 in acommercial immobilized pH gradient (IPG) strip and then separatedin second dimension on an 8% to 16% (w/v) gradient gel. Gels weresilver stained and scanned with a densitometer. Background wassubtracted digitally. B, Enlarged view of upper left quadrant of gelshown in A. C, Polypeptides associated with guard cell function.Upper left quadrant of digital reference gel from the higher ordermatched set used for analysis of polypeptides unique to culturecondition, physiological state, or functional capacity. Circles sur-round 13 polypeptides identified with Boolean operators that areheld in common by, and unique to, cells with guard cell function(FGCPs and GCPs cultured at 38°C in media containing ABA).

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during the middle-G1 to early S phase boundary(Nakagami et al., 2002). In vitro, Hsc70 binds to aspecific sequence in the N terminus of non-phosphorylated pRb and blocks its phosphorylation(Inoue et al., 1995). Although levels of Hsc70 did notchange over the first 24 h at 32°C, Hsc70 levels havenot been examined closer to cell cycle reentry after 48to 72 h of culture, and nothing is known about howelevated temperature might affect redistribution ofthis protein among cellular compartments.

Despite the potential role of Hsp70 and other Hsps(e.g. Hsp101) in the processes described, Hsp70 levelsincreased only about 2-fold over the first 24 h at38°C � ABA (Fig. 3A). We have not yet determinedwhether increases in Hsp70 continue beyond 24 h,whether they are representative of those of otherHsp, and/or whether the increases in levels of Hsp70would be sufficient to produce the hypothesizedeffects.

Thermotolerant GCPs Do Not Require NAA orBAP to Survive

Our results suggest that at 32°C, dedifferentiatingand/or dividing cells require active deployment of ahormone-dependent pathway that produces factorsrequired for cell survival. At 32°C, cultures ap-proaching the restriction point between G1 and S orabout to enter M from G2 may contain a mixture ofcells, only some of which are competent or fit tocomplete the cell cycle. Under such circumstances,gene regulation under the control of growth regula-tors might allow for negative selection of cells unfitfor cell cycle completion, possibly through apoptoticand/or necrotic pathways.

Both NAA and BAP were required for survival at32°C (Table I). Our previous studies with AVG sug-gest that survival at 32°C may also require endoge-nous ethylene production (Roberts et al., 1995),which could be stimulated by auxin (Van DerStraeten et al., 1990; Yip et al., 1992; Merritt et al.,2001). It may seem odd that ethylene, which is oftenassociated with cellular senescence and necrosis,might be required for cell survival at 32°C in thissystem. Exogenously applied ethylene can induceapoptosis in cultured tobacco Bright-Yellow 2 cells atG2 to M and S (Herbert et al., 2001). However,ethylene-regulated anti-apoptotic mechanisms mayexist. Relatively high (27 �m) concentrations of BAPinduce programmed cell death in cultured carrot(Daucus carota) and Arabidopsis cell cultures (Carimiet al., 2003). In these systems, cell death is blocked by2,4-diphenoxyacetic acid, which could induce ethyl-ene production (Van Der Straeten et al., 1990; Yip etal., 1992). We speculate that in GCPs, ethylene mayantagonize cytokinin-induced cell death. Becauseboth auxin and cytokinin are required for survivaland division of plant cells in culture, it is possiblethat ethylene induced by auxin treatment modulates

cytokinin signal transduction pathways to induce celldivision rather than cytokinin-induced cell death.Thus, ethylene would “signal” cytokinin of the pres-ence of the auxin required for early cell cycle eventsand cell growth. In the absence of cytokinin, NAAinduced-ethylene would also cause cell death. Al-though this explanation would be consistent withrequirements for NAA, BAP, and ethylene for sur-vival of cultured GCPs at 32°C, other mechanismsare possible. The Arabidopsis ethylene-responsiveelement-binding protein (AtEBP) can function as adominant suppressor of Bax-induced cell death inyeast (Pan et al., 2001). It is uncertain, however,whether plants have a system fully equivalent toBcl-Bax (Krishnamurthy et al., 2000; Lam et al., 2001).

In contrast to the hormone-dependent cell survivalobserved at 32°C, GCPs cultured at 38°C � ABA didnot require NAA or BAP to survive (Table I), andsurvival was not reduced by treatment with AVG(Roberts et al., 1995). Regardless of whether ABA isincluded in the culture medium, elevated tempera-ture may block events so far upstream of the G1-to-Stransition that cultured GCPs are never faced withdownstream selection for life or death. It is alsopossible (and likely) that GCPs cultured at 38°C re-tain and/or develop a complement of survival fac-tors (e.g. Hsps) that prevent their death.

Thermotolerant GCPs Do Not Make theG1-to-S Transition

Our data indicate that high temperature (38°C)prevents cultured GCPs from reentering the cell cycleby blocking the G1-to-S phase transition (Fig. 4).GCPs may be unique among plant cell types in theircapacity to survive in high percentages at high tem-peratures in culture and, thus, may comprise a novelin vitro system for studying how temperature signaltransduction mechanisms regulate the G1-to-S tran-sition to prevent cell cycle reentry.

When cultures of GCPs of tree tobacco were estab-lished at 32°C, a temperature that in many plantspecies results in minimum cell cycle duration (Fran-cis and Barlow, 1988), cells entered S phase within 48to 72 h (Fig. 4C). Cell proliferation was exponentialbetween days 2 and 4, but cultures reached a station-ary phase at days 5 to 7 and then entered a decliningphase (Fig. 4C). Nuclei isolated from cells that werereprotoplasted after 10 to 14 d of culture at 32°C didnot stain with FITC-conjugated antibody to BrdU inhigher percentages than those of nuclei in fixed cellsthat had regenerated cell walls (approximately 0.2%).Thus, failure to detect BrdU incorporation in nucleiof cells cultured at 32°C for longer periods was notdue to failure of antibodies to penetrate cells withwalls. The pattern of cell proliferation for culturedGCPs was similar to that reported for tobacco Bright-Yellow 2 cells cultured at 27°C, which divided rap-idly with a 12- to 14-h cell cycle on days 3 to 4 before




entering a stationary phase induced by exhaustion ofnutrients in the medium (Nagata et al., 1992). After72 h at 32°C, only about 10% as many cultured GCPshad developed cell plates as had incorporated BrdUinto nuclear DNA, indicating that isolated GCPswere in G1 initially.

When cultures of GCPs were established at 38°C,the G1-to-S transition was blocked regardless ofwhether media lacked or contained ABA (Fig. 4C).Our previous studies (Taylor et al., 1998) showed thatat 38°C, GCPs lose some of the functional character-istics of guard cells unless ABA is included in theculture medium. Thus, although a temperature of38°C alone is sufficient to block the G1-to-S transi-tion, a combination of elevated temperature and ABAis required to maintain GCPs in the differentiatedstate in vitro (Roberts et al., 1995; Taylor et al., 1998).ABA is known to induce the synthesis of the cyclinkinase inhibitor ICK1 in Arabidopsis (Wang et al.,1998). ICK1 interacts directly with Cdc2a and CycD3(Wang et al., 1998), and its overexpression reducesCDK activity, cell number, and plant growth (Wanget al., 2000). Therefore, the possibility exists that theG1-to-S phase transition is blocked by a differentmechanism and/or at a different point in G1 at 38°Cin a medium lacking ABA than it is at 38°C in amedium containing ABA. Results of two-dimensionalgel analysis (Table II) reveal a number of proteins thatdiffer 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 involvedin plant cell differentiation in vivo or dedifferentia-tion in vitro. However, plant MAPKs have been im-plicated in phenomena similar to those observed inthis and other studies with cultured GCPs of treetobacco. As noted above, GCPs cultured at high tem-perature show symptoms consistent with impairedauxin signaling, and an MAPK cascade initiated byan MAPKKK (NPK1) can suppress auxin signaling intransfected maize protoplasts and transgenic tobaccoplants (Kovtun et al., 1998). MAPKs have been im-plicated in other similar systems and processes aswell. In cultured tobacco Bright-Yellow 2 cells, a45-kD MAPK is activated upon phosphate-inducedcell cycle reentry after phosphate starvation (Wilsonet al., 1998), and p43Ntf6 appears to be required forphragmoplast formation during the cytokinetic tran-sition from anaphase to telophase (Calderini et al.,1998). There is genetic evidence that members of theArabidopsis MAPKKK gene family encode tran-scripts that are essential for regulating cytokinesis(Krysan et al., 2002). In epidermal peels of pea, anMAPK is thought to be involved in signaling guardcells during ABA-induced stomatal closure (Burnettet al., 2000), and ABA prevents growth of tree to-bacco 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 speciesand tissues (Morris, 2001). All three of these growthregulators appear to be required for survival of cul-tured GCPs of tree tobacco during and/or after cellcycle 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 proteinsthat cross-reacted with an antibody to subdomain XIof a rat ERK1 (Fig. 5). The most prominent was a41-kD protein that disappeared from cells cultured at32°C at the beginning of S phase and cell cycle reen-try (Fig. 5, A and D). It is not known whetherdisappearance of the 41-kD ERK1-CRP resulted fromaltered protein turnover or from alteration ofepitopes in subdomain XI due to protein modifica-tion or processing. At 38°C � ABA, the protein wasretained in cells for up to 10 d (Fig. 5, B and C). At thevery least, disappearance of the 41-kD ERK1-CRP isdiagnostic for cell cycle reentry. It is tempting tospeculate that the protein is involved in maintainingGCPs in G0 or G1 at 38°C � ABA. In cultured humanfibroblasts, constitutive activation of the stress-induced MAPK, p38HOG, through stable expressionof its activator MKK6 causes permanent cell cyclearrest at G1 (Haq et al., 2002). Still, the structure andfunction(s) of the 41-kD ERK1-CRP remain to beinvestigated.

A 42-kD ERK1-CRP was retained in cells culturedat 32°C for up to 10 d. The 42-kD ERK1-CRP was notresolvable from the 41-kD ERK1-CRP in extracts fromcells cultured at 38°C � ABA because it was obscuredby the strong reaction of the antibody with the 41-kDprotein even at low dilutions and film exposuretimes.

A 47-kD ERK1-CRP was detected in all cultures.Putative MAPKs of similar molecular masses havebeen reported in GCPs and isolated guard cells (Moriand Muto, 1997; Burnett et al., 2000). ABR, a 48-kDABA-activated protein kinase capable of phosphory-lating myelin basic protein, was identified in extractsfrom Vicia faba GCP, but it did not precipitate with anantiphosphotyrosine antibody (Mori and Muto,1997). Two other kinases of 46 and 49 kD were alsoidentified that precipitated with an antiphosphoty-rosine antibody, but they were activated only slightlyby ABA (Mori and Muto, 1997). In pea leaf epidermis,both ABA-induced stomatal closure and ABA-induced accumulation of dehydrin mRNA wereshown to be inhibited by PD098059 (�PD98059), anMEK inhibitor, suggesting that MAPKs are requiredfor ABA-induced stomatal closure (Burnett et al.,2000). In the same study, an ABA-activated 45-kDkinase, AMBPK, was identified in extracts from peaepidermal peels. AMBPK required Tyr phosphoryla-tion for its activity and catalyzed phosphorylation ofmyelin basic protein (Burnett et al., 2000), but itsactivity was not induced by ABA in isolated guardcells. A 43-kD kinase was activated by ABA in iso-

Gushwa et al.



lated pea guard cells, but it is not clear whether thisprotein was an MAPK (Burnett et al., 2000). Whetherthe 47-kD ERK1-CRP reported here is a true guardcell MAPK similar to any of those reported alsoawaits investigation.

A Proteomic Analysis of Thermotolerant GCPsIs Feasible

The two-dimensional gel analysis reported herewas limited to spot match analysis of the majorpolypeptides detected on silver-stained gels. Silverstaining is sensitive, but it is not quantitative. Fur-thermore, although spot match analysis provides adegree of statistical certainty as to whether the samespot exists at a similar location in two different treat-ment gels, it does not explain why such differences inspot location exist. The presence of a polypeptide ata unique gel position may reflect changes in generegulation for the polypeptide, changes in turnoverof the polypeptide, or chemical modification of thepolypeptide so that its position is shifted on the gel(hereafter, we refer to the sum of these processes as“polypeptide regulation”). Even so, two-dimensionalgel comparisons of this type yield useful information.For example, the data indicate that regardless of howcells are treated, any two cell types in this systemshare 30% to 50% of their major polypeptides incommon (allowing for overlap of pH ranges on IPGstrips). This suggests that it may be possible to usesuch an analysis to distinguish regulation of a core of“maintenance” polypeptides involved in functionsrequired of all cell types (metabolism, transport, etc.)from regulation of polypeptides involved in the re-sponse of cultured cells to environmental signals.

Interestingly, FGCPs and GCPs cultured at 38°Cwith ABA, which share guard cell function, did notshare any greater proportion of their major polypep-tides in common than did highly divergent cell typessuch as FGCPs and fully dedifferentiated, dividingcells cultured at 32°C (Table II), nor did cells givensimilar treatments (e.g. 38°C and 38°C � ABA) shareany higher proportion of polypeptides than diver-gent treatments (Table II). Although the resolution ofthe two-dimensional system is limited to only themost abundant polypeptides, the data may suggestthat polypeptide regulation can vary as radically in aguard cell that maintains its identity while respond-ing to drastic changes in environmental signals (i.e.culture at 38°C in media containing ABA) as it doeswhen the signals result in its dedifferentiation to ameristematic state under the influence of plantgrowth regulators. Thus, at the level of the proteome,dedifferentiation may be less a matter of degree ofdeviation from the differentiated state than the pre-cise nature of the deviation.

Potentially, two-dimensional gel analysis can alsobe used to monitor temporally changes in the pro-teome that define dedifferentiation. Guard cell func-

tion can be highly conserved even after isolation,culture initiation, and heat shock. Conservation ofguard cell function has been observed in guard cellsthat have been: (a) isolated from the leaf as proto-plasts; (b) transferred to media enriched in nutrientsand supplemented with plant growth regulators thatcontrol cell growth and division; and then (c) cul-tured at elevated temperature, in darkness, in mediacontaining the antitranspirant ABA for up to 1 week(Taylor et al., 1998). It seems remarkable that anyguard cell function would be retained under theseconditions because any or all of these conditionsmight be expected to disrupt patterns of gene andprotein regulation in guard cells and interfere withtheir normal function and/or maintenance of theirgenetic identity. Nevertheless, Boolean analysis ofgels uncovered 34 to 50 polypeptides that wereshared between and that were unique to cells withguard cell function (FGCPs and GCPs cultured at38°C in media with ABA; Table III; Fig. 6). If the pointin time can be identified at which these polypeptidesare lost from GCPs dedifferentiating at 32°C, it maybe possible to analyze the degree to which proteinprofiles can deviate from those of a guard cell beforeGCPs lose their unique physiological identity and areconsidered to be functionally “dedifferentiated.”

Monocultures of GCPs Can Be Used to Study SignalTransduction Related to Thermotolerance

There are numerous animal cell culture systems inwhich the signal transduction pathways governingthe cell cycle and cell proliferation (e.g. Yosimichi etal., 2001; Sah et al., 2002), differentiation/dedifferen-tiation (Kim et al., 2002; Yosimichi et al., 2001; Yoonet al., 2002), and apoptosis (Konishi et al., 2002) canbe studied. Only a few systems exist for studyingthese processes in plants. Zinnia elegans mesophyllprotoplasts are a good example. These cells can bemanipulated in vitro to expand (Lee et al., 2000) or todivide and differentiate into tracheary elements, aprocess which ends in programmed cell death(Groover and Jones, 1999). Similarly, a single isolateof tree tobacco GCPs can be partitioned among cul-ture conditions that maintain GCPs in the differenti-ated state, result in partial dedifferentiation, end incell death, or cause full dedifferentiation and reentryinto the cell cycle. Only two signals, elevated tem-perature and ABA, are required to prevent dediffer-entiation and cell cycle reentry, and elevated temper-ature alone is sufficient to prevent the latter.Furthermore, a distinct hormone-dependent cell sur-vival mechanism employed at lower culture temper-atures is not required for high survival at high tem-peratures. This report demonstrates that culturedGCPs of tree tobacco can be used to study the signaltransduction pathways that regulate thermotoleranceand its relationships to the plant cell cycle, differen-tiation/dedifferentiation processes, and cell survival




mechanisms that are under the control of plantgrowth regulators (NAA and BAP), hormones (eth-ylene and ABA), and temperature. A number ofpolypeptides identified by two-dimensional profilingthat are unique to these pathways are targets forfuture functional proteomic analysis. GCPs of Betavulgaris have been successfully transformed and re-generated to plants that are resistant to glyphosphateherbicides (Hall et al., 1996). Ultimately, it may bepossible to manipulate levels of GCP proteins iden-tified by proteomic analysis using small interferingRNA molecules or morpholinos to target the destruc-tion of their mRNAs.

MATERIALS AND METHODS

Plants

Plants were germinated from seed at a high density on potting soil (HPPremier 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 aftergermination, plants were watered every other day and on alternate dayswere given one-half-strength modified Hoagland nutrient solution (Hoag-land 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 6weeks. After they reached a height of 0.05 to 0.1 m, they were transferred to10-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 processepidermis from as few as four and as many as nine leaves in a singleisolation. Concentrations of ascorbic acid and polyvinylpyrrolidone 40 weredoubled in solutions in which epidermis was detached from leaves (Boorseand Tallman, 1999; step 2.3.3). The same volume of enzyme solution per leafwas used in four, six, or nine-leaf preparations. Epidermis from as many as4.5 leaves was incubated in a single 250-mL Erlenmeyer flask (Corning,Corning, NY) in a proportionate volume of enzyme solution (Boorse andTallman, 1999; step 3.3.18). For larger isolates, two preparations of epider-mis were made, and then incubation times in enzyme solutions were stag-gered by 4 min. At the end of the first enzyme digestion, epidermis wascollected 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 toa 125-mL flask (Boorse and Tallman, 1999; step 3.3.24) containing 75 mL ofsolution C. The flask was swirled vigorously. Peels were collected again onthe same nylon net and rinsed with another 50 to 100 mL of solution Cbefore they were transferred to the second enzyme solution (solution D, step3.3.24; Boorse and Tallman, 1999) in a clean 250-mL flask. The flask wascapped and swirled vigorously. The second enzyme digestion was extendedto 3.5 h at a speed of 55 rpm. During collection of protoplasts (Boorse andTallman, 1999; step 3.3.30), flasks were swirled 10 times clockwise and then10 times counterclockwise before contents were filtered through the finemesh net. The cuticle remaining on the net was rinsed with an additional 5to 10 mL of incomplete medium I (pH 6.8) before GCPs were collected andwashed (Boorse and Tallman, 1999). After the second wash in incompletemedium I (pH 6.1, step 35; Boorse and Tallman, 1999), the supernatant wasaspirated from each tube down to 0.5 mL, and GCPs were resuspended byrolling the tubes between the palms of the hands. The contents of all tubeswere combined in one 15-mL conical centrifuge tube and were collected bycentrifugation at 60g for 8 min. The supernatant was discarded down to 1mL. GCPs were resuspended, counted with a hemocytometer, and culturedat a density of 1.25 � 105 cells mL�1 as described (Roberts et al., 1995; Boorseand Tallman, 1999). For larger preparations (eight–nine leaves), GCPs fromtwo 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 toproduce full cell survival but inhibit cell division among cells culturedsubsequently at 32°C. In three separate experiments, GCPs were first cul-tured at a density of 6.25 � 104 cells mL�1 in eight-well chamber slides(Roberts et al., 1995) at 38°C with or without ABA for various periods from0 to 24 h. In each experiment, the number of dead cells in 10 fields in eachof four chamber slide wells was estimated using a microscope at 200�: (a)after 24 h and 8 d of culture at 32 or 38°C, and (b) in cultures pre-incubatedat 38°C for various periods from 0 to 24 h that were then cultured foranother week at 32°C.

Cell Collection and Storage for Protein Extraction

FGCPs were suspended in a final volume of 1 mL as above. Cells culturedin plastic X Plate petri dishes (100 � 15 mm, Becton-Dickinson, San Jose,CA) were collected in 15-mL conical tubes by centrifugation at 60g for 10min. After centrifugation, all but 1 mL of the supernatant was discarded.FGCPs or cultured cells were resuspended by gentle trituration and trans-ferred to 1.5-mL cryovials. Vials were centrifuged at 60g for 7 min, and thesupernatant was discarded. To maximize protein yields, cells cultured at38°C � ABA for more than 5 d were collected by settling instead of bycentrifugation. Ninety percent of each cell suspension was transferred frompetri dishes with a wide-bore pipette to a 15-mL conical centrifuge tube.Transferred suspensions and the original culture plates were returned to theincubator for 1.5 h. Supernatants from each tube were then used to rinse theremaining cells from each petri dish, and rinses were returned to theirrespective tubes. Tubes were returned to settle in the incubator for another1.5 h before supernatants were discarded. Each cell pellet was stored in acryovial in a solution made by adding 5 �L of 100 mm phenylmethane-sulfonylfluoride to 0.5 mL of a solution of 50 mm Tris (pH 8.0), 1.5%(w/v)insoluble polyvinyl-polypyrrolidone, 1.8 �g of trans-epoxysuccinyl-l-leucylamido-(4-guanidino)butane, 0.2 mm 4-(2-aminoethyl)benzene-sulfonylfluoride, 5 �g of leupeptin, and 0.02 mm ethylene glycol-bis (�-aminoethylether) N,N,N�N�-tetra-acetic acid. The solution was added directly tocryovials containing cells collected by centrifugation. When cells were col-lected by settling, settled cells were resuspended in the solution, and thesuspension was then transferred to cryovials. Cells to be used for Hsp/Hscwestern blotting and two-dimensional gel electrophoresis were frozen im-mediately in liquid nitrogen and stored at �80°C.

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

Protein Extraction and Assay for Western Blotting

FGCPs or cultured GCPs of tree tobacco were extracted for westernblotting of Hsp70 and Hsc70 with EZ extraction buffers (Martinez-Garcia etal., 1999) by adding 0.5 mL of 2� modified buffer E [250 mm Tris-HCl (pH8.8), 2% (w/v) SDS, 20% (v/v) glycerol, 0.1 m Na2S2O5 modified to contain3% (w/v) polyvinyl-polypyrrolidone, 0.45 �g mL�1 trans-epoxysuccinyl-l-leucylamido-(4-guanidino)butane, 0.1 mm 4-(2-aminoethyl) benzene-sulfonyl fluoride, and 1.25 �g mL�1 leupeptin] to a cryovial containing 0.5mL of frozen cells. Samples were suspended in microcentrifuge tubes witha pointed plastic homogenizer (GeneMate, ISC BioExpress, Kaysville, UT).Suspensions were heated at 80°C for 3 min and then centrifuged at 24°C for4 min at 21,000g to remove insoluble material. Supernatants were trans-ferred to tubes containing an equal volume of ice cold 20% (w/v) tri-choloracetic acid (TCA), and mixtures were incubated on ice for 15 minbefore they were centrifuged at 4°C for 15 min at 21,000g. Supernatants werediscarded, and 0.3 mL of ice cold acetone was added to each pellet. Pelletswere then homogenized with a teflon microtube homogenizer until theywere dispersed before an additional 0.7 mL of acetone was added. After a20-min incubation on ice, the homogenate was centrifuged as above, and theacetone wash was repeated. After the second wash, the acetone was de-canted, and the pellet was air dried for 5 to 10 min before 30 to 50 �L ofbuffer E (Martinez-Garcia et al., 1999) and 0.5 �L of �-mercaptoethanol wereadded with mixing to completely dissolve the pellet.

For extraction of oocytes for ERK1/2 western-blot controls, fiveprogesterone- or five non-progesterone-treated oocytes containing 100 to

Gushwa et al.



150 �g of protein were homogenized in 10 �L of 0.1 m KCl, 1 mm MgCl2, 50mm Tris (pH 7.5), 1 mm dithiothreitol (DTT), and 80 mm �-glycerophosphatecontaining 10 �g mL�1 each of leupeptin and chymostatin. The homogenatewas centrifuged at 4°C for 10 min at 21,000g. The supernatant was dilutedwith one volume of 2� SDS loading buffer (2% [w/v] SDS, 20% [v/v]glycerol, 5% [v/v] �-mercaptoethanol, 10 �g mL�1 bromphenol blue, and0.1 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 culturedcells were extracted immediately after cells were collected. FGCPs or cul-tured GCPs were mixed with 1 volume of 20% (w/v) ice-cold TCA andincubated on ice for 10 min. Cells were centrifuged at 4°C for 10 min at12,000g. The supernatants were removed, and 1.5 mL of 90% (v/v) ice-coldacetone 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. Theacetone wash was repeated. The acetone was aspirated, and pellets wereallowed to air dry for 10 min before they were agitated for 1 h at 500 rpmin 60 �L of buffer E containing 20 mm �-glycerophosphate, 1 mm Na3VO4,and 1 �L of �-mercaptoethanol. Proteins were further solubilized by heatingat 80°C for 15 min and agitating for an hour. Extracts were then clarified bycentrifugation at 24°C for 10 min at 21,000g before supernatants wereassayed for protein.

Protein concentrations in extracts were determined by a Non-InterferingProtein Assay (Geno Technology, St. Louis) using Protocol-1 (proprietaryfrom 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)�g 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 �m ABA was electrophoresed. ForERK1/2 western blots, 7 �g 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 �m ABA waselectrophoresed. To ensure that ERKs detected were of cellular origin, 7 �gof Cellulase Onozuka RS, Pectolyase Y-23, or BSA was also examined by thesame procedure. Proteins were separated by SDS-PAGE (Laemmli, 1970) for15 min at a constant 80 V and then at a constant 200 V until completion on12-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 tosamples, either 60 ng each of purified human (Homo sapiens) Hsp70 (catalogno. SPP-755B, StressGen, Victoria, BC, Canada) or cytosolic wheat (Triticumaestivum) Hsc70 (StressGen, catalog no. SPP-791), or 5 �L of protein extractsfrom progesterone- or non-progesterone-treated oocytes containing 8.8 �0.3 �g of protein (mean; se; n � 5) were electrophorosed as controls.

After electrophoresis, proteins were transferred to nitrocellulose mem-branes by western blotting as described (Stebbins-Boaz et al., 1999). Mem-branes were incubated at room temperature with gentle rocking at 20 rpmfor 1 h in primary antibodies diluted in 5% (w/v) milk/Tris-buffered salineplus Tween 20 (TBST). Primary antibodies and their dilutions were: anti-Hsp70 (1/2,000 [v/v], StressGen catalog no. SPA-812C, rabbit anti-humanpolyclonal), anti-Hsc70 (1/2,000 [v/v], StressGen catalog no. SPA-795, rabbitanti-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 ratERK1), 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 placeof primary antibody as a control for nonspecific binding of IgG.

Membranes were washed four times for 5 min each in TBST and thenincubated in secondary antibodies for 1 h. Donkey anti-rabbit IgG-horseradish peroxidase-conjugated secondary antibodies (Santa Cruz Bio-technology, catalog no. SC-2313) were diluted in 5% (w/v) milk in TBST andused to detect primary antibodies. To detect ERK antibodies, the secondaryantibody was diluted 1/2,000 (v/v); Hsp70 and Hsc70 primary antibodieswere detected with 1/4,000 (v/v) dilutions of secondary antibodies. Mem-branes were washed four times for 5 min each time in TBST. For chemilu-minescent detection, equal volumes of ECL Western Blotting Detectionreagents 1 and 2 (Amersham-Pharmacia Biotech, Uppsala, Sweden) weremixed and poured onto the membrane. After 1 min, membranes weredrained, blotted, wrapped in plastic wrap, and exposed in darkness to 13- �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 molec-

ular masses were determined against Cruz Molecular Weight Standards(Santa Cruz Biotechnology, catalog no. SC-2035) using Quantity One Soft-ware (Bio-Rad) after scanning with a Bio-Rad GS-710 densitometer. Relativeamounts of protein were also analyzed by densitometry. Band contourquantities were measured and then compared among blots after normaliz-ing 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 threeseparate experiments the percentage of cells surviving after 1 week ofculture 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) at32°C, 38°C, or 38°C in a medium containing 0.1 �m ABA. Two-millilitercultures were established in plastic petri dishes (3.5 � 1 cm). Immediatelyafter isolation and at the end of each successive 24-h period of culture overa 14-d period, 5 �L of 10 �m 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 toincubators for an additional 24 h after which cells were harvested, fixed, andstained for BrdU incorporation. Cells from each treatment were harvestedwith pipettes and transferred to 15-mL conical centrifuge tubes. Dishes wererinsed 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 but0.5 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 wereincubated at room temperature for 30 min with shaking at approximately250 excursions min�1. Fixed cells were collected by centrifugation as above,and the supernatant was discarded to leave 0.5 mL of liquid above thepellet. 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 wereincubated at room temperature for an additional 30 min. Nuclei (38°C �ABA) or fixed cells with intact nuclei (32°C) were collected by centrifugationfor 10 min at 60g. All but 0.5 mL of the supernatant was discarded, and theremaining 0.5 mL of liquid was neutralized by addition of 1 mL of 0.1 mNa2B4O7 � 10 H2O (pH 8.5). Nuclei or fixed cells were again collected bycentrifugation, after which 3 mL of ice-cold 70% (v/v) ethanol was added tothe remaining 0.5 mL of liquid. Isolates were stored at �20°C for no longerthan 3 d before they were stained.

For direct immunofluorescent staining of BrdU-containing DNA, nucleior fixed cells were collected by centrifugation for 10 min at 60g. Thesupernatant was removed to leave 0.5 mL, and nuclei or cells were resus-pended with addition of 1 mL of 0.5% (v/v) Tween 20 and 1% (w/v) BSAin phosphate-buffered saline (PBS; 0.9% [w/v] NaCl in 1 mm phosphatebuffer [pH 7.3]). Twenty microliters of FITC-conjugated rabbit anti-BrdU(catalog no. 347583, Becton-Dickinson) was added to each tube, and tubeswere incubated for 30 min at room temperature with shaking at approxi-mately 250 excursions min�1. Two milliliters of Tween/BSA/PBS wasadded, and stained nuclei or cells were collected by centrifugation at 60g for7 min. The supernatant was discarded to 0.5 mL, pellets were resuspendedby addition of 2 mL of Tween/BSA/PBS, and nuclei or fixed cells werecollected by centrifugation at 60g for 7 min. The supernatant was discardeddown to 1 mL, and 0.5 �L of Hoechst 33342 (Sigma; 0.5 mg mL�1 inincomplete medium 1 [pH 6.1]) was added with gentle mixing for 1 min atroom temperature. Nuclei or cells were collected by centrifugation as above,and then the supernatant was withdrawn to leave 0.2 mL. Pellets wereresuspended by trituration, transferred to glass slides with coverslips, andvisualized at 400� with an inverted microscope (model IX70, Olympus,Tokyo) equipped with a 100-W mercury lamp and an epifluorescence illu-minator. Hoechst-stained nuclei were viewed under UV light produced witha wide-band excitation filter (Olympus filter cube U-MWU). FITC-labeledanti-BrdU was viewed with a blue excitation filter (Olympus filter cubeU-M516). For each sample in each of three separate experiments, 1,000nuclei visualized initially with Hoechst staining were scored for BrdUincorporation.




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

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

To determine whether elevated temperature might alter the concentra-tion of NAA and BAP required to activate cell division (Nagata et al., 2001),in preliminary experiments at 38°C, concentrations of NAA and BAP wereincreased 20� or 50� over those required to activate cell division at 32°Cbut at the same ratio used at 32°C. Cell division was then estimated after 1week as described (Roberts et al., 1995). Because phosphate depletion frommedia 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 phosphateconcentrations were raised to 500 mg L�1 with KH2PO4 to test whether cellscultured at 38°C � ABA might deplete phosphate in media more rapidlythan at 32°C. In underwater rice (Oryza sativa), anoxia triggers synthesis ofGA3, ethylene release, and, subsequently, cell division (Sauter, 2001). Thus,in some experiments, cultures were supplemented with GA3 in concentra-tions ranging from 5 to 100 �m.

Protein Extraction for Two-Dimensional Electrophoresis

Proteins were extracted by a modification of the method of Shimazakiand Kinoshita (1995). Frozen samples were thawed in cryovials, ice-cold20% (w/v) TCA (0.5 mL) was added with mixing, and samples wereincubated on ice for 10 min. Samples were centrifuged at 21,000g for 10 minat 4°C, and the supernatant was discarded. The precipitate was resuspendedin 200 �L of ice-cold deionized water and homogenized for approximately10 s at 4.5 to 8 � 103 rpm with a teflon microtube homogenizer attached toa rotary tool (Craftsman model no. 572.610530, Sears Roebuck, HoffmanEstates, IL). One milliliter of acetone (�20°C) was added with mixingfollowed by incubation at �20°C for 20 min. The precipitate was collectedby centrifugation as described and gently resuspended by hand homogeni-zation with the teflon homogenizer in 300 �L of 4% (w/v) SDS, 2% (v/v)2-mercaptoethanol, 20% (v/v) glycerol, and 0.1 m Tris (pH 8.5). Sampleswere 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 addedwith mixing followed by incubation for 1 h at �20°C. The precipitate wascollected by centrifugation at 4°C for 15 min at 21,000g. The water/acetonewash was repeated, but the pellet was hand-homogenized in water beforeacetone was added. The supernatant was discarded, the pellet was solubi-lized by hand homogenization in 10 mm Tris (pH 7.4) and 5 mm MgCl2.Protease-free RNAse A (4.5 units in 0.8 �L; catalog no. LS002131, Worth-ington Biochemical, Lakewood, NJ), and 3.1 units (0.5 �L) of protease-freeDNAse I (catalog no. LS006333, Worthington Biochemical) were added, andsamples were incubated on ice for 15 min. After another acetone precipita-tion as described above, pellets were air dried for 5 min and solubilized withshaking 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; Rabilloudet al., 1997). Protein content of samples was assayed by the method ofBradford (1976; Bio-Rad) using bovine gamma globulin (Bio-Rad) as a stan-dard. To evaluate the potential contribution of protein from dead cells to thepool of extracted protein, debris was collected and proteins were extractedfrom 3 � 106 GCPs that had died after 10 d of culture in a medium lackingNAA and BAP. Samples were diluted to give approximately 100 �g of proteinin 185 �L of isoelectric focusing (IEF) solution, and TBP was added to a finalconcentration of 2 mm. For estimation of polypeptide molecular masses,two-dimensional electrophoresis standards (2.5 �L; catalog no. 161-0320, Bio-Rad) 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 increaseresolution, 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 liquidsamples in the focusing tray and allowed to rehydrate for 1 h before they

were overlayed with mineral oil. Gels were allowed to continue to rehydratefor an additional 13 h at 22°C. IEF strips of pH 3 to 6 were rehydratedpassively in disposable trays, and two wicks dampened with deionizedwater were placed over each electrode before focusing. IEF strips of pH 7 to10 were rehydrated in disposable trays with 185 �L of IEF solution contain-ing 2 mm TBP, and then samples were loaded cathodically in �30 �L beforeelectrophoresis. A temperature of 22°C and a current limit of 50 �A per stripwas 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 thenramped rapidly to their limits (top limit � 8,000 V) and electrophoresed for25,000 V h�1. A 500-V hold was included at the end of the program toprevent protein diffusion. Immediately after IEF, gels were either wrappedin plastic wrap and stored at � 80°C or prepared for SDS-PAGE.

Before separation in the second dimension, strips were placed in indi-vidual screw top culture tubes and rocked at 35 rpm for 10 min in 5 mL of6 m urea, 2% (w/v) SDS, 0.375 m Tris (pH 8.8), 20% (v/v) glycerol, and 130mm DTT. Gels were then transferred to a similar solution containing 135 mmiodoacetamide instead of DTT and equilibrated with rocking for another 10min.

For SDS-PAGE, strips were mounted on top of 8% to 16% (w/v) acryl-amide 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. Coldpacks were inserted between buffer tanks to prevent heating during elec-trophoresis. 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’sinstructions. All glassware was cleaned with 50% (v/v) nitric acid andrinsed with deionized water before staining. After staining, gels werecleaned with cotton-tipped applicators to remove any precipitate, soakedwith gentle agitation (40 rpm) in deionized water for 5 min, and thentransferred to a gel-drying solution (Bio-Rad, catalog no. 161-0752) andgently agitated for 30 min. Gels were dried between cellophane sheets in aBio-Rad GelAir Dryer with forced air for 3 h and then for an additional 24 hin 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 reso-lution was 63.5 � 63.5 �m and images were cropped to 1,902 � 1,201 pixelsand filtered using a contramean filter with a 5- � 5-pixel filter size. Spotcenters were resolved and marked using automatic parameters and werethen edited manually.

Five gels per pH range per treatment were used to create a matched setwith a standard image representing all proteins for that cell/treatment type.The percentage of spots matched initially by manual landmarking rangedamong the four experimental treatments as follows: pH 3 to 6, 19.1% to29.6%; pH 5 to 8, 23.1% to 37.2%; and pH 7 to 10, 52% to 65.9%. The resultingdigital standard image included all spots appearing in at least two matchedset members. Digital standard gels from each treatment were used to createa higher order matched set with a composite reference image representingall proteins appearing in all treatments. The percentage of spots matched bymanual landmarking were: pH 3 to 6, 44.4%; pH 5 to 8, 34.9%; and pH 7 to10, 40.1%. Analysis sets were created by two-way comparisons among thetreatments of the higher order matched set. Further analyses were per-formed to identify polypeptides unique to a culture condition or physiolog-ical or functional state with analysis sets created from composite andstandard 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]

Gushwa et al.



Distribution of Materials

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

ACKNOWLEDGMENTS

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

Received March 23, 2003; returned for revision April 22, 2003; accepted May5, 2003.

LITERATURE CITED

Boorse G, Tallman G (1999) Guard cell protoplasts: isolation, culture, andregeneration of plants. In RD Hall, ed, Methods in Molecular Biology:Plant Cell Culture Protocols, Vol. 111. Humana Press, Totawa, NJ, pp243–257

Bradford MM (1976) A rapid and sensitive method for the quantitation ofmicrogram quantities of protein utilizing the principle of protein-dyebinding. Anal Biochem 72: 248–254

Burnett EC, Desikan R, Moser RC, Neill SJ (2000) ABA activation of anMBP kinase in Pisum sativum epidermal peels correlates with stomatalresponses to ABA. J Exp Bot 51: 197–205

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

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

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

Francis D, Barlow PW (1988) Temperature and the cell cycle. In SP Long, FIWoodward, eds, Plants and Temperature, 42nd Symposium of the Soci-ety for Experimental Biology. Company of Biologists, Cambridge, UK, pp181–201

Gabai VL, Sherman MY (2002) Invited review: interplay between molecularchaperones and signaling pathways in survival of heat shock. J ApplPhysiol 92: 1743–1748

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

Groover A, Jones AM (1999) Tracheary element differentiation uses a novelmechanism coordinating programmed cell death and secondary cell wallsynthesis. Plant Physiol 119: 375–384

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) Ahigh efficiency technique for the generation of transgenic sugar beetsfrom stomatal guard cells. Nat Biotechnol 14: 1133–1138

Haq R, Brenton JD, Takahashi M, Finan D, Rottapel R, Zanke B (2002)Constitutive p38HOG mitogen-activated protein kinase activation in-duces permanent cell cycle arrest and senescence. Can Res 62: 5076–5082

Herbert RJ, Vilhar B, Evett C, Orchard CB, Rogers HJ, Davies MS, FrancisD (2001) Ethylene induces cell death at particular phases of the cell cyclein the tobacco TBY-2 cell line. J Exp Bot 52: 1615–1623

Helmbrecht K, Zeise E, Rensing L (2000) Chaperones in cell cycle regula-tion and mitogenic signal transduction: a review. Cell Prolif 33: 341–365

Hoagland DR, Arnon DI (1938) The water culture method for growingplants without soil. Circular of the California Agricultural ExperimentStation, 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 cognateprotein interacts directly with the N-terminal region of the retinoblas-toma protein pRb. J Biol Chem 270: 22571–22576

Kato K, Fukasawa S, Shimizu Y, Soh Y, Nagai S (1977) Requirements ofPO4

3�, NO3�, SO4

2�, K�, and Ca2� for the growth of tobacco cells insuspension culture. J Fermentation Technol 55: 207–212

Kepinski S, Leyser O (2002) Ubiquitination and auxin signaling: a degrad-ing 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 differentia-tion in LNCaP cells independently of cell cycle withdrawal and STAT3phosphorylation. Can Res 62: 1549–1554

Konishi Y, Lehninen M, Donovan N, Bonni A (2002) Cdc2 phosphorylationof BAD links the cell cycle to the cell death machinery. Mol Cell 9:1005–1016

Kovtun Y, Chiu W-L, Tena G, Sheen J (2000) Functional analysis of oxida-tive stress-activated mitogen-activated protein kinase cascade in plants.Proc Natl Acad Sci USA 97: 2940–2945

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

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 Arabidopsismitogen-activated protein kinase kinase kinase gene family encodes es-sential positive regulators of cytokinesis. Plant Cell 14: 1109–1120

Kuhl NM, Rensing L (2000) Heat shock effects on cell cycle progression.Cell Mol Life Sci 57: 450–463

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

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

Lee S, Woffenden BJ, Beers EP, Roberts AW (2000) Expansion of culturedZinnia mesophyll cells in response to hormones and light. Physiol Plant108: 216–222

Martinez-Garcia JF, Monte E, Quail PH (1999) A simple, rapid and quan-titative method for preparing Arabidopsis extracts for immunoblot anal-ysis. Plant J 20: 251–257

Merritt F, Kemper A, Tallman G (2001) Inhibitors of ethylene synthesisinhibit auxin-induced stomatal opening in epidermis detached fromleaves of Vicia faba L. Plant Cell Physiol 42: 223–230

Mockaitis K, Howell SH (2000) Auxin induces mitogenic activated proteinkinase (MAPK) activation in roots of Arabidopsis seedlings. Plant J 24:785–796

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

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

Murray JA (1997) The retinoblastoma protein is in plants! Trends Plant Sci2: 82–84

Nagata T, Kumagai F, Sano T (2001) The regulation of the cell cycle incultured 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

Nakagami H, Kawamura K, Sugisaka K, Sekine M, Shinmyo A (2002)Phosphorylation of retinoblastoma-related protein by the cyclinD/cyclin-dependent kinase complex is activated at the G1/S phase tran-sition in tobacco. Plant Cell 14: 1847–1857

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

Queitsch C, Hong S-W, Vierling E, Lindquist S (2000) Heat shock protein101 plays a crucial role in thermotolerance in Arabidopsis. Plant Cell 12:479–492

Rabilloud T, Adessi C, Giraudel A, Lunardi J (1997) Improvement of thesolubilization of proteins in two-dimensional electrophoresis with immo-bilized pH gradients. Electrophoresis 18: 307–316

Rabilloud T, Valette C, Lawrence JJ (1994) Sample application by in-gelrehydration improves the resolution of two-dimensional electrophoresiswith immobilized pH gradients in the first dimension. Electrophoresis 15:1552–1558

Roberts C, Sahgal P, Merritt F, Perlman B, Tallman G (1995) Temperatureand abscisic acid can be used to regulate survival, growth, and differen-tiation of cultured guard cell protoplasts of tree tobacco. Plant Physiol109: 1411–1420

Rowley A, Johnston GC, Butler B, Werner-Washburne M, Singer RA(1993) Heat shock-mediated cell cycle blockage and G1 cyclin expressionin the yeast Saccharomyces cerevisiae. Mol Cell Biol 13: 1034–1041




Sah JF, Eckert RL, Roshantha AS, Chandraratna AS, Rorke EA (2002)Retinoids suppress epidermal growth-factor associated cell proliferationby inhibiting epidermal growth factor receptor-dependent ERK1/2 acti-vation. J Biol Chem 277: 9728–9735

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

Sano T, Kuraya Y, Amino S, Nagata T (1999) Phosphate as a limiting factorfor the cell division of tobacco BY-2 cells. Plant Cell Physiol 40: 1–8

Sauter M (2001) Gibberellic acid-mediated regulation of the cell cycle dur-ing 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 instomatal guard cells. In DW Galbraith, HJ Bohnert, DP Bourque, PTMatsudaira, eds, Methods in Cell Biology, Vol 49. CRC Press, Boca Raton,FL, pp 501–513

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

Taylor JE, Abram B, Boorse G, Tallman G (1998) Approaches to evaluatingthe extent to which guard cell protoplasts of Nicotiana glauca (tree to-bacco) retain their characteristics when cultured under conditions thataffect their survival, growth, and differentiation. J Exp Bot 49: 377–386

Trotter EW, Berenfeld L, Krause SA, Petsko GA, Gray JV (2001) Proteinmisfolding and temperature up-shift cause G1 arrest via a commonmechanism dependent on heat shock factor in Saccharomyces cerevisiae.Proc Natl Acad Sci USA 98: 7313–7318

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 AcadSci USA 87: 4859–4863

Wang H, Qi Q, Schorr P, Cutler AJ, Crosby WL, Fowke LC (1998) ICK1, acyclin-dependent protein kinase inhibitor from Arabidopsis thaliana inter-acts both with Cdc2a and CycD3, and its expression is induced byabscisic acid. Plant J 15: 501–510

Wang H, Zhou Y, Gilmer S, Whitwill S, Fowke LC (2000) Expression of theplant cyclin-dependent kinase inhibitor ICK1 affects cell division, plantgrowth, and morphology. Plant J 24: 613–623

Weinberg RA (1995) The retinoblastoma protein and cell cycle control. Cell81: 323–330

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 cellcycle 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

Yip W-K, Moore T, Yang SF (1992) Differential accumulation of transcriptsof four tomato 1-aminocyclopropane-1-carboxylate synthase homologsunder various conditions. Proc Natl Acad Sci USA 89: 2475–2479

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 chondrocytesby protein kinase C and extracellular signal-regulated protein kinase.J Biol Chem 277: 8412–8420

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

Gushwa et al.



Tibetan Womenâ€™s Association Central Executive Committee

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