A comparison of the chilling-stress response in two differentially tolerant cultivars of tomato (Lycopersicon esculentum)
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RANDALW. GIROUXAND W. GARYFILION J. Tuzo Wilson Research Laboratories, Department of Botany, Erindale Campus, University of Toronto, 3359 Mississauga Road North, Mississauga, Ont., Canada L5L IC6 Received June 18, 1991 GIROUX, R. W., and FILION,W. G. 1992. A comparison of the chilling-stress response in two differentially tolerant cultivars of tomato (Lycopersicon esculentum). Biochem. Cell Biol. 70: 191-198. The chilling responses of two differentially cold tolerant cultivars of tomato were monitored through in vivo labelling of polypeptides with [3S~]methionine, both during a gradual temperature decrease (2"C/day) and also during a rapid cold shock (4°C). The polypeptides were separated by one-dimensional sodium dodecyl sulfate - polyacrylamide gel electrophoresisand revealed by fluorography. Both cultivars showed changes in the polypeptide profiles resulting from either chilling treatment. During the gradual temperature decrease, there were few differences exhibited between the two cultivars. However, during cold shock both cultivars showed the altered synthesis of several unique polypeptides. Both cultivars showed the appearance of a 35-kDa polypeptide during the gradual temperature decrease and also during the cold shock. The appearance of three high relative mass polypeptides was found in both cultivars only during the gradual temperature decrease. Treatments with cycloheximide and chlorarnphenicol suggested that cold-shock polypeptides are both nuclear and organelle encoded. The cold-shock response in roots was different from the response in leaves and between cultivars. A comparison of the two cultivars showed a number of differences in polypeptide synthesis which may be related to increased cold tolerance. Key words: cold-shock protein@), tomato, chilling stress, acclimation, cold tolerance. GIROUX, R. W., et FILION,W. G. 1992. A comparison of the chilling-stress response in two differentially tolerant cultivars of tomato (Lycopersicon esculentum). Biochem. Cell Biol. 70 : 191-198. La rkponse A la rkfrigkration de deux cultivars de tomates rksistant de fa~ondiffkrente au froid est haluke par marquage in vivo de polypeptides avec la [3s~]mkthionine durant une diminution graduelle de la tempkrature (2"C/jour) et durant un choc thermique (4OC) rapide. Les polypeptides sont stparks par klectrophortse unidimensionnelle sur gel de polyacrylamide avec dodkcyl sulfate de sodium et rhklks par fluorographie. Dans les dew cultivars, les profils polypeptidiques resultant de l'un et l'autre traitement subissent des changements. Durant la diminution graduelle de tempkrature, les diffkrences entre les deux cultivars sont minimes. Cependant, dans les deux cultivars, le choc thermique modifie la synthtse de plusieurs polypeptides particuliers. Un polypeptide de 35 kDa apparaft dans les deux cultivars durant la diminution graduelle de tempkrature et aussi durant le choc thermique. Trois polypeptides de masse relative klevke apparaissent dans les deux cultivars seulement durant la diminution graduelle de tempkrature. Les traitements avec le cycloheximide et le choramphknicol suggtrent que les polypeptides de choc thermique seraient codks dans le noyau et dans les organites. La rkponse au choc thermique dans les racines est diffkrente de celle dans les feuilles et entre les cultivars. La cornparaison des deux cultivars montre un certain nombre de diffkrences dans la synthtse des polypeptides qui seraient relikes a une tolkrance accrue au froid. Mots clPs : protkine(s) de choc therrnique, tomates, stress dO au froid, acclimatation, tolkrance au froid. [Traduit par la rkdaction]
Introduction Many environmental stresses induce altered protein synthesis in plants and these changes in gene expression involve the synthesis of a specific set of stress proteins. Stresses such as heat (Somers et al. 1989; Mason-Apps et al. 1990)' drought (Bray 1988; Vartanian et al. 1987)' salt (Ostrem et al. 1987)' and heavy metals (Amaral et al. 1988; Edelman et al. 1988) often lead t o the synthesis of stress proteins. During cold acclimation, altered polypeptide synthesis is reported in rice (Hahn and Walbot 1989)' alfalfa (Mohaptra et al. 1987~'1987b), spinach (Guy et al. 1987)' wheat (Perras and Sarhan 1989)' and barley (Cattivelli and Bartels 1989). Some of these new polypeptides are related directly t o the ABBREVIATIONS: ddH,O, double-distilled H,O; SDS, sodium dodecyl sulfate; DTT, dithiothreitol; TCA, trichloroacetic acid; ID, one dimensional; PAGE, polyacrylamide gel electrophoresis; M,, relative mass; cpm, counts per minute; GTD, gradual temperature decrease; LHCP, light-harvesting chlorophyll a/b binding protein; LSU, large subunit of ribulose 1,s-bisphosphatecarboxylase/ oxygenase; SSU, small subunit of ribulose 1.5-bisphosphate carboxylase/oxygenase. Printed in Canada / Imprime au Canada
increase in cold tolerance during acclimation (Mohaptra et al. 1989; Guy and Haskell 1987). Increased cold tolerance can be induced by gradually lowering the temperature over a long term (1 week) (Drozdov et al. 1984) or by short exposures of plants t o lethal chilling temperatures prior to the extended chilling treatment (Graham and Patterson 1982). While changes in protein synthesis during long-term acclimation studies have been extensively examined (Perras and Sarhan 1989; Mohaptra et al. 1988; Guy et al. 1988), few studies have been directed towards the response of plants to an immediate exposure to cold temperatures or cold shock. These cold-shock studies indicate that plants rapidly respond by synthesizing a new set of polypeptides (Mesa-Basso et al. 1986; Yacoob and Filion 1986). Cold-shock polypeptides represent initial changes in polypeptide synthesis during chilling stress. While immediate chill-induced changes are not fatal to cold-sensitive tissue (Minorsky 1985), it is possible that events stemming from changes in polypeptide synthesis are ultimately responsible for chilling injury.
BIOCHEM. CELL BIOL. VOL. 70,
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Different cultivars of tomato exhibit different degrees of chilling injury when exposed t o chilling temperatures (Wolf et al. 1986; Autio and Brarnlage 1986). Dramatic differences in chilling tolerance exist between the two tomato cultivars 'Heinz 1340 VF' and 'Siberia'. 'Heinz 1340' is a chillingsensitive cultivar having a narrow growth temperature range (15-25°C) and shows no growth at 6°C (data not shown). 'Siberia' is a chilling-tolerant variety that possesses a wider growth range (4-25°C); this cultivar grows and sets fruit at chilling temperatures as low as 4°C (Amy McNulty, personal communication). An assessment of the chilling tolerance can be determined through in vivo chlorophyll fluorescence, which is a reliable method for the quantification of chillinginduced changes (Larcher and Neuner 1989). 'Siberia' showed a chilling tolerance level equal t o cold-tolerant Lycopersicon hirsutum as measured by in vivo chlorophyll fluorescence. The availability of two tomato cultivars, which vary widely in their ability to grow at low temperatures, permitted a comparison of the cold-stress response at the polypeptide level. This paper describes this comparison in an effort t o elucidate differences related t o the increased chilling tolerance.
Materials and methods Plant material and growth of seedlings Two cultivars of Lycopersicon esculentum ('Heinz 1340 VF', Stokes Seed, St. Catharines, Ont., and 'Siberia', Siberia Seed Co., Olds, Alta.) were grown from seed at 21°C (70-80% relative humidity) in a controlled environmental chamber. Seedlings were illuminated during a 14-h photoperiod at a photosynthetic quantum flux density of 250 rnrn~l.m-~.s-'.Plants were watered on alternate days and fertilized weekly using soluble 20-20-20 fertilizer containing micronutrients (Plant Products Ltd. Brampton, Ont.) supplemented with 5 mM KNO,. All experiments were carried out using tomato seedling leaves at the third true leaf stage. Temperature treatments and labelling Gradual temperature decrease Seedlings were subjected to a gradual temperature decrease of 2"C/day from 21 to 6°C. Polypeptides were labelled in vivo during the final 2 h at each temperature. Three leaf discs (5 mm) were cut from leaves while submerged in ddH20 maintained at the specific growth temperature of the plant. Leaf discs were then placed in polyethylene test tubes containing 1 mL ddH20 and vacuum infiltrated for two 1-min intervals with the subsequent addition of 100 pL L-[35~]methionine( > 1000 Ci/mmol, 1 Ci = 37 GBq; ICN Biomedicals, St. Laurent, Que.). Cold shock For in vivo labelling, three leaf discs were cut from leaf tissue while submerged in ddH20 at 21 "C. Leaf discs were then placed in polyethylene test tubes containing 1 mL ddH20 and vacuum infiltrated for two 1-min intervals to facilitate the uptake of radiolabel. Tubes were then placed in a constant temperature water bath at the cold-shock temperature of 4°C for a total of 3 h. Following the first 30 min at the shock temperature. 100 pL of L-["~lmethioninewas added. In all experiments, leaf discs were illuminated by a 60-W incandescent bulb during the labelling period to provide illumination and increase the uptake of radiolabel (Mason-Apps et al. 1990). Root cold shock For in vivo labelling, three terminal 2-cm root segments were excised from plants while submerged in ddH20 at 21°C and the cold-shock treatment was executed as described previously. All root cold-shock experiments were performed in complete darkness.
1992
Inhibitor studies Cyclohexirnideor chloramphenicol was added to restrict translation on cytosolic (80s) ribosomes or organellar (70s) ribosomes, respectively (Galling 1982). Leaf discs were excised from plants grown at 21 "C and exposed to inhibitors 0.5 h prior to cold-shock treatment. Chloramphenicol and cycloheximide were present at 50 mg/mL. Extraction of polypeptides Following the labelling period, the leaf discs were rinsed thoroughly with ddH20 to remove excess radiolabel solution. Leaf discs were then homogenized in polyethylene microfuge tubes using polypropylene pellet pestles (BDH, Toronto, Ont.) in 0.2 mL of cold (4°C) electrophoresis buffer containing 60 mM Tris-HCI (pH 6.8), 2% (w/v) SDS, 1% (w/v) DTT, 1 mM phenylmethylsulfonyl fluoride, and 10% (w/v) glycerol (Laemmli 1970). The homogenate was centrifuged twice at 13 500 x g for 5 min at 4°C. The supernatant was stored at - 20°C until required. Protein content and incorporation of radiolabel Incorporation of radiolabel into polypeptides was determined by spotting 5 pL of sample onto a I-cm2 piece of Whatman 3 MM chromatography paper. Squares of paper were air dried and then plunged into 10% TCA for 5 rnin. Paper samples were washed three times in an excess of ddH20, 95% ethanol, and acetone, respectively. Air-dried samples were then placed in scintillationvials containing 5 mL of Aquasol cocktail (New England Nuclear, Montrtal, Que.). Radioactivityof TCA-precipitatedpolypeptides was measured using a Beckman LS-150 scintillation counter set at a 5-min counting window. Electrophoresis and fluorography The polypeptides were separated using 1D SDS-PAGE (8-20% gradient gels) containing 0.1 % (w/v) SDS and electrophoresed at 18 mA for 4 h according to the procedure of Laemmli (1970). Approximately equal counts of radioactivity (40 000 cpm) were loaded in each lane. Protein standards (phosphorylase b, 97.4 kDa; bovine serum albumin, 66.2 kDa; ovalbumin, 42.7 kDa; carbonic anhydrase, 31 kDa; soybean trypsin inhibitor, 21.5 kDa; lysozyme, 14.4 kDa; Bio-Rad Laboratories, Mississauga, Ont.) were coelectrophoresed to determine the M,s of the isolated polypeptides. Gels were impregnated with a scintillation fluor ( ~ n ~ ~ a n c New England Nuclear, Montrtal, Que.) for 1 h and subsequently dried on a slab gel drier (model 443, Bio-Rad Laboratories, Mississauga, Ont.). Fluorograms were prepared by exposing the gel to Kodak X-Omat AR film with fluorescent intensifying screen (Cronex Hi-Plus) at - 70°C for 2-5 days. Since gels were loaded with equal cpm per lane, only the relative patterns between the two cultivars could be compared.
Results Effect of temperature and inhibitors on protein synthesis and radiolabel incorporation The effect of the GTD and cold shock on the overall level of protein synthesis and incorporation of radiolabel was estimated using percent incorporation. This value represents the radioactivity in TCA-precipitable protein versus the total amount of radioactivity available t o the tissue. A significant (15 -t 2.5%) decrease in the percent incorporation was found in both cultivars during the GTD and during cold shock. A reduction in percent incorporation suggests chilling stress reduces the overall level of protein synthesis. Both cycloheximide and chloramphenicol reduced also the incorporation of radiolabel into TCA-precipitable protein owing to inhibition of protein synthesis by these chemicals.
Effect of gradual temperature decrease on polypeptide synthesis The polypeptide profiles of 'Heinz' and 'Siberia' at 21 "C
193
GIROUX AND FILION
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SIBERIA
FIG. 1. The effect of a gradual temperature decrease on polypeptide synthesis in (A) 'Heinz' and (B) 'Siberia'. Plants were subjected to a gradual decrease in temperature (2OC/day) from 21 to 6°C. During the final 3 h at each temperature, leaf discs were excised from plants and pulse labelled with L-[35~]methionine. Equal amounts (40 000 cpm) of radioactive polypeptides were separated by 1D SDSPAGE (8-20%). Numbers below the lanes indicate the temperature (OC) at which the polypeptides were pulse labelled. The MIs of protein standards are indicated on the left and the MIs of tomato polypeptides are indicated on the right.
(control temperature) were similar (Figs. 1A and 1B). After a GTD from 21 to 6"C, both cultivars exhibited a change in the polypeptide profiles (Figs. 1A and 1B). A comparison of these changes in 'Heinz' and 'Siberia' over the GTD showed 23 polypeptide bands (Mrs = 209.0, 176.2, 117.2, 104.4, 87.0, 78.8, 70.3, 67.5, 56.7, 53.9, 52.2, 50.0, 49.0, 43.0, 40.5, 36.5, 35.0, 32.0, 31.5, 30.6, 27.5, 23.0, and 21.0 kDa) with similar changes in polypeptide synthesis. Both cultivars showed the appearance of a 35-kDa band at 9°C (Figs. 1A and 1B). This band is difficult to see in the reduced proof, but is clearly evident in the original figures. A majority of the altered protein synthesis occurred at the threshold temperature of 15°C in both cultivars. We recorded (i) the increased intensity of a 50-kDa band and (ii) the decreased intensity of a 27.5-kDa band at 15°C (Figs. 1A and 1B). Although three polypeptides possessed similar Mrs of 65.1, 39.9, and 20.0 kDa in both cultivars (Figs. 1A and lB), they showed different patterns of synthesis over the GTD. These different patterns are classified as "unique patterns of synthesis." A comparison of the polypeptide profiles of the two cultivars showed the presence of two unique bands in 'Heinz' (Mrs = 160.4 and 25.3 kDa). "Unique bands" are those bands that are present in only one of the two cultivars. Effect of cold shock on polypeptide synthesis The polypeptide profiles of 'Heinz' and 'Siberia' were both altered following a 3-h cold shock at 4°C (Fig. 2). A comparison of the control profiles of the two cultivars showed few differences (Fig. 2). A majority of the bands were found in both cultivars; however, 'Heinz' showed a unique 47.5-kDa band (Fig. 2). Similar changes in the polypeptide profiles of the two cultivars during cold shock involved the disappearance of
FIG. 2. The effect of a cold shock on polypeptide synthesis. Leaf discs were cold shocked at 4°C for 3 h and pulse labelled with L-[''~lrnethionine during the final 2.5 h. Equal amounts (40 000 cpm) of radioactive polypeptides were separated by 1D SDS-PAGE (8-20%). Letters below the lanes indicate the cultivar, 'Heinz' (H) or 'Siberia' (S). Numbers at top indicate the temperature at which the leaf discs were pulse labelled. The M,.s of protein standards are indicated on the left and the M,s of tomato polypeptides are indicated on the right.
BIOCHEM. CELL BIOL. VOL. 70, 1992
A Mrx10-3 97.4 66.2 -
-
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-
HEINZ 1340 21'C 4'C
SIBERIA 21'C 4'C
I Mrx10-3
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-. # h
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- iE;
- 56.7
- 50.0
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RANDALW. GIROUXAND W. GARYFILION J. Tuzo Wilson Research Laboratories, Department of Botany, Erindale Campus, University of Toronto, 3359 Mississauga Road North, Mississauga, Ont., Canada L5L IC6 Received June 18, 1991 GIROUX, R. W., and FILION,W. G. 1992. A comparison of the chilling-stress response in two differentially tolerant cultivars of tomato (Lycopersicon esculentum). Biochem. Cell Biol. 70: 191-198. The chilling responses of two differentially cold tolerant cultivars of tomato were monitored through in vivo labelling of polypeptides with [3S~]methionine, both during a gradual temperature decrease (2"C/day) and also during a rapid cold shock (4°C). The polypeptides were separated by one-dimensional sodium dodecyl sulfate - polyacrylamide gel electrophoresisand revealed by fluorography. Both cultivars showed changes in the polypeptide profiles resulting from either chilling treatment. During the gradual temperature decrease, there were few differences exhibited between the two cultivars. However, during cold shock both cultivars showed the altered synthesis of several unique polypeptides. Both cultivars showed the appearance of a 35-kDa polypeptide during the gradual temperature decrease and also during the cold shock. The appearance of three high relative mass polypeptides was found in both cultivars only during the gradual temperature decrease. Treatments with cycloheximide and chlorarnphenicol suggested that cold-shock polypeptides are both nuclear and organelle encoded. The cold-shock response in roots was different from the response in leaves and between cultivars. A comparison of the two cultivars showed a number of differences in polypeptide synthesis which may be related to increased cold tolerance. Key words: cold-shock protein@), tomato, chilling stress, acclimation, cold tolerance. GIROUX, R. W., et FILION,W. G. 1992. A comparison of the chilling-stress response in two differentially tolerant cultivars of tomato (Lycopersicon esculentum). Biochem. Cell Biol. 70 : 191-198. La rkponse A la rkfrigkration de deux cultivars de tomates rksistant de fa~ondiffkrente au froid est haluke par marquage in vivo de polypeptides avec la [3s~]mkthionine durant une diminution graduelle de la tempkrature (2"C/jour) et durant un choc thermique (4OC) rapide. Les polypeptides sont stparks par klectrophortse unidimensionnelle sur gel de polyacrylamide avec dodkcyl sulfate de sodium et rhklks par fluorographie. Dans les dew cultivars, les profils polypeptidiques resultant de l'un et l'autre traitement subissent des changements. Durant la diminution graduelle de tempkrature, les diffkrences entre les deux cultivars sont minimes. Cependant, dans les deux cultivars, le choc thermique modifie la synthtse de plusieurs polypeptides particuliers. Un polypeptide de 35 kDa apparaft dans les deux cultivars durant la diminution graduelle de tempkrature et aussi durant le choc thermique. Trois polypeptides de masse relative klevke apparaissent dans les deux cultivars seulement durant la diminution graduelle de tempkrature. Les traitements avec le cycloheximide et le choramphknicol suggtrent que les polypeptides de choc thermique seraient codks dans le noyau et dans les organites. La rkponse au choc thermique dans les racines est diffkrente de celle dans les feuilles et entre les cultivars. La cornparaison des deux cultivars montre un certain nombre de diffkrences dans la synthtse des polypeptides qui seraient relikes a une tolkrance accrue au froid. Mots clPs : protkine(s) de choc therrnique, tomates, stress dO au froid, acclimatation, tolkrance au froid. [Traduit par la rkdaction]
Introduction Many environmental stresses induce altered protein synthesis in plants and these changes in gene expression involve the synthesis of a specific set of stress proteins. Stresses such as heat (Somers et al. 1989; Mason-Apps et al. 1990)' drought (Bray 1988; Vartanian et al. 1987)' salt (Ostrem et al. 1987)' and heavy metals (Amaral et al. 1988; Edelman et al. 1988) often lead t o the synthesis of stress proteins. During cold acclimation, altered polypeptide synthesis is reported in rice (Hahn and Walbot 1989)' alfalfa (Mohaptra et al. 1987~'1987b), spinach (Guy et al. 1987)' wheat (Perras and Sarhan 1989)' and barley (Cattivelli and Bartels 1989). Some of these new polypeptides are related directly t o the ABBREVIATIONS: ddH,O, double-distilled H,O; SDS, sodium dodecyl sulfate; DTT, dithiothreitol; TCA, trichloroacetic acid; ID, one dimensional; PAGE, polyacrylamide gel electrophoresis; M,, relative mass; cpm, counts per minute; GTD, gradual temperature decrease; LHCP, light-harvesting chlorophyll a/b binding protein; LSU, large subunit of ribulose 1,s-bisphosphatecarboxylase/ oxygenase; SSU, small subunit of ribulose 1.5-bisphosphate carboxylase/oxygenase. Printed in Canada / Imprime au Canada
increase in cold tolerance during acclimation (Mohaptra et al. 1989; Guy and Haskell 1987). Increased cold tolerance can be induced by gradually lowering the temperature over a long term (1 week) (Drozdov et al. 1984) or by short exposures of plants t o lethal chilling temperatures prior to the extended chilling treatment (Graham and Patterson 1982). While changes in protein synthesis during long-term acclimation studies have been extensively examined (Perras and Sarhan 1989; Mohaptra et al. 1988; Guy et al. 1988), few studies have been directed towards the response of plants to an immediate exposure to cold temperatures or cold shock. These cold-shock studies indicate that plants rapidly respond by synthesizing a new set of polypeptides (Mesa-Basso et al. 1986; Yacoob and Filion 1986). Cold-shock polypeptides represent initial changes in polypeptide synthesis during chilling stress. While immediate chill-induced changes are not fatal to cold-sensitive tissue (Minorsky 1985), it is possible that events stemming from changes in polypeptide synthesis are ultimately responsible for chilling injury.
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Different cultivars of tomato exhibit different degrees of chilling injury when exposed t o chilling temperatures (Wolf et al. 1986; Autio and Brarnlage 1986). Dramatic differences in chilling tolerance exist between the two tomato cultivars 'Heinz 1340 VF' and 'Siberia'. 'Heinz 1340' is a chillingsensitive cultivar having a narrow growth temperature range (15-25°C) and shows no growth at 6°C (data not shown). 'Siberia' is a chilling-tolerant variety that possesses a wider growth range (4-25°C); this cultivar grows and sets fruit at chilling temperatures as low as 4°C (Amy McNulty, personal communication). An assessment of the chilling tolerance can be determined through in vivo chlorophyll fluorescence, which is a reliable method for the quantification of chillinginduced changes (Larcher and Neuner 1989). 'Siberia' showed a chilling tolerance level equal t o cold-tolerant Lycopersicon hirsutum as measured by in vivo chlorophyll fluorescence. The availability of two tomato cultivars, which vary widely in their ability to grow at low temperatures, permitted a comparison of the cold-stress response at the polypeptide level. This paper describes this comparison in an effort t o elucidate differences related t o the increased chilling tolerance.
Materials and methods Plant material and growth of seedlings Two cultivars of Lycopersicon esculentum ('Heinz 1340 VF', Stokes Seed, St. Catharines, Ont., and 'Siberia', Siberia Seed Co., Olds, Alta.) were grown from seed at 21°C (70-80% relative humidity) in a controlled environmental chamber. Seedlings were illuminated during a 14-h photoperiod at a photosynthetic quantum flux density of 250 rnrn~l.m-~.s-'.Plants were watered on alternate days and fertilized weekly using soluble 20-20-20 fertilizer containing micronutrients (Plant Products Ltd. Brampton, Ont.) supplemented with 5 mM KNO,. All experiments were carried out using tomato seedling leaves at the third true leaf stage. Temperature treatments and labelling Gradual temperature decrease Seedlings were subjected to a gradual temperature decrease of 2"C/day from 21 to 6°C. Polypeptides were labelled in vivo during the final 2 h at each temperature. Three leaf discs (5 mm) were cut from leaves while submerged in ddH20 maintained at the specific growth temperature of the plant. Leaf discs were then placed in polyethylene test tubes containing 1 mL ddH20 and vacuum infiltrated for two 1-min intervals with the subsequent addition of 100 pL L-[35~]methionine( > 1000 Ci/mmol, 1 Ci = 37 GBq; ICN Biomedicals, St. Laurent, Que.). Cold shock For in vivo labelling, three leaf discs were cut from leaf tissue while submerged in ddH20 at 21 "C. Leaf discs were then placed in polyethylene test tubes containing 1 mL ddH20 and vacuum infiltrated for two 1-min intervals to facilitate the uptake of radiolabel. Tubes were then placed in a constant temperature water bath at the cold-shock temperature of 4°C for a total of 3 h. Following the first 30 min at the shock temperature. 100 pL of L-["~lmethioninewas added. In all experiments, leaf discs were illuminated by a 60-W incandescent bulb during the labelling period to provide illumination and increase the uptake of radiolabel (Mason-Apps et al. 1990). Root cold shock For in vivo labelling, three terminal 2-cm root segments were excised from plants while submerged in ddH20 at 21°C and the cold-shock treatment was executed as described previously. All root cold-shock experiments were performed in complete darkness.
1992
Inhibitor studies Cyclohexirnideor chloramphenicol was added to restrict translation on cytosolic (80s) ribosomes or organellar (70s) ribosomes, respectively (Galling 1982). Leaf discs were excised from plants grown at 21 "C and exposed to inhibitors 0.5 h prior to cold-shock treatment. Chloramphenicol and cycloheximide were present at 50 mg/mL. Extraction of polypeptides Following the labelling period, the leaf discs were rinsed thoroughly with ddH20 to remove excess radiolabel solution. Leaf discs were then homogenized in polyethylene microfuge tubes using polypropylene pellet pestles (BDH, Toronto, Ont.) in 0.2 mL of cold (4°C) electrophoresis buffer containing 60 mM Tris-HCI (pH 6.8), 2% (w/v) SDS, 1% (w/v) DTT, 1 mM phenylmethylsulfonyl fluoride, and 10% (w/v) glycerol (Laemmli 1970). The homogenate was centrifuged twice at 13 500 x g for 5 min at 4°C. The supernatant was stored at - 20°C until required. Protein content and incorporation of radiolabel Incorporation of radiolabel into polypeptides was determined by spotting 5 pL of sample onto a I-cm2 piece of Whatman 3 MM chromatography paper. Squares of paper were air dried and then plunged into 10% TCA for 5 rnin. Paper samples were washed three times in an excess of ddH20, 95% ethanol, and acetone, respectively. Air-dried samples were then placed in scintillationvials containing 5 mL of Aquasol cocktail (New England Nuclear, Montrtal, Que.). Radioactivityof TCA-precipitatedpolypeptides was measured using a Beckman LS-150 scintillation counter set at a 5-min counting window. Electrophoresis and fluorography The polypeptides were separated using 1D SDS-PAGE (8-20% gradient gels) containing 0.1 % (w/v) SDS and electrophoresed at 18 mA for 4 h according to the procedure of Laemmli (1970). Approximately equal counts of radioactivity (40 000 cpm) were loaded in each lane. Protein standards (phosphorylase b, 97.4 kDa; bovine serum albumin, 66.2 kDa; ovalbumin, 42.7 kDa; carbonic anhydrase, 31 kDa; soybean trypsin inhibitor, 21.5 kDa; lysozyme, 14.4 kDa; Bio-Rad Laboratories, Mississauga, Ont.) were coelectrophoresed to determine the M,s of the isolated polypeptides. Gels were impregnated with a scintillation fluor ( ~ n ~ ~ a n c New England Nuclear, Montrtal, Que.) for 1 h and subsequently dried on a slab gel drier (model 443, Bio-Rad Laboratories, Mississauga, Ont.). Fluorograms were prepared by exposing the gel to Kodak X-Omat AR film with fluorescent intensifying screen (Cronex Hi-Plus) at - 70°C for 2-5 days. Since gels were loaded with equal cpm per lane, only the relative patterns between the two cultivars could be compared.
Results Effect of temperature and inhibitors on protein synthesis and radiolabel incorporation The effect of the GTD and cold shock on the overall level of protein synthesis and incorporation of radiolabel was estimated using percent incorporation. This value represents the radioactivity in TCA-precipitable protein versus the total amount of radioactivity available t o the tissue. A significant (15 -t 2.5%) decrease in the percent incorporation was found in both cultivars during the GTD and during cold shock. A reduction in percent incorporation suggests chilling stress reduces the overall level of protein synthesis. Both cycloheximide and chloramphenicol reduced also the incorporation of radiolabel into TCA-precipitable protein owing to inhibition of protein synthesis by these chemicals.
Effect of gradual temperature decrease on polypeptide synthesis The polypeptide profiles of 'Heinz' and 'Siberia' at 21 "C
193
GIROUX AND FILION
Biochem. Cell Biol. Downloaded from www.nrcresearchpress.com by YORK UNIV on 11/12/14 For personal use only.
SIBERIA
FIG. 1. The effect of a gradual temperature decrease on polypeptide synthesis in (A) 'Heinz' and (B) 'Siberia'. Plants were subjected to a gradual decrease in temperature (2OC/day) from 21 to 6°C. During the final 3 h at each temperature, leaf discs were excised from plants and pulse labelled with L-[35~]methionine. Equal amounts (40 000 cpm) of radioactive polypeptides were separated by 1D SDSPAGE (8-20%). Numbers below the lanes indicate the temperature (OC) at which the polypeptides were pulse labelled. The MIs of protein standards are indicated on the left and the MIs of tomato polypeptides are indicated on the right.
(control temperature) were similar (Figs. 1A and 1B). After a GTD from 21 to 6"C, both cultivars exhibited a change in the polypeptide profiles (Figs. 1A and 1B). A comparison of these changes in 'Heinz' and 'Siberia' over the GTD showed 23 polypeptide bands (Mrs = 209.0, 176.2, 117.2, 104.4, 87.0, 78.8, 70.3, 67.5, 56.7, 53.9, 52.2, 50.0, 49.0, 43.0, 40.5, 36.5, 35.0, 32.0, 31.5, 30.6, 27.5, 23.0, and 21.0 kDa) with similar changes in polypeptide synthesis. Both cultivars showed the appearance of a 35-kDa band at 9°C (Figs. 1A and 1B). This band is difficult to see in the reduced proof, but is clearly evident in the original figures. A majority of the altered protein synthesis occurred at the threshold temperature of 15°C in both cultivars. We recorded (i) the increased intensity of a 50-kDa band and (ii) the decreased intensity of a 27.5-kDa band at 15°C (Figs. 1A and 1B). Although three polypeptides possessed similar Mrs of 65.1, 39.9, and 20.0 kDa in both cultivars (Figs. 1A and lB), they showed different patterns of synthesis over the GTD. These different patterns are classified as "unique patterns of synthesis." A comparison of the polypeptide profiles of the two cultivars showed the presence of two unique bands in 'Heinz' (Mrs = 160.4 and 25.3 kDa). "Unique bands" are those bands that are present in only one of the two cultivars. Effect of cold shock on polypeptide synthesis The polypeptide profiles of 'Heinz' and 'Siberia' were both altered following a 3-h cold shock at 4°C (Fig. 2). A comparison of the control profiles of the two cultivars showed few differences (Fig. 2). A majority of the bands were found in both cultivars; however, 'Heinz' showed a unique 47.5-kDa band (Fig. 2). Similar changes in the polypeptide profiles of the two cultivars during cold shock involved the disappearance of
FIG. 2. The effect of a cold shock on polypeptide synthesis. Leaf discs were cold shocked at 4°C for 3 h and pulse labelled with L-[''~lrnethionine during the final 2.5 h. Equal amounts (40 000 cpm) of radioactive polypeptides were separated by 1D SDS-PAGE (8-20%). Letters below the lanes indicate the cultivar, 'Heinz' (H) or 'Siberia' (S). Numbers at top indicate the temperature at which the leaf discs were pulse labelled. The M,.s of protein standards are indicated on the left and the M,s of tomato polypeptides are indicated on the right.
BIOCHEM. CELL BIOL. VOL. 70, 1992
A Mrx10-3 97.4 66.2 -
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Biochem. Cell Biol. Downloaded from www.nrcresearchpress.com by YORK UNIV on 11/12/14 For personal use only.
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