Thermoadaptation in Cultured Cowpea Cells - NCBI

Plant Physiol. (1987) 85, 4-7 0032-0889/87/85/0004/04/$O 1.00/0

Commnunicatio1n

Synthesis of Only Two Heat Shock Proteins Is Required for Thermoadaptation in Cultured Cowpea Cells' Received for publication March 5, 1987

KATHLEEN HEUSS-LAROSA, RANDALL R. MAYER, AND JOE H. CHERRY* Center ofPlant Environmental Stress Physiology, Horticulture Department, Purdue University, West Lafayette, Indiana 47907 ABSTRACT Cell culres of a heat sevsite geotype of cowpea (Vigva wigxkcaate) were adped to tolerate m.derate levls, of' hat by A taning cells at 32,36 38a C Owet may cell genratio. Celos adte to 32 md to W6C not py*bm the typcal heat shoc ptotetw (HSP). Cells adped Xto WC synteszed two ew pruoins, which Avow to be a sbsEtof the lSP. in mny temperatturesewgitiye orgaUms it is thought that lISt cofer thermotoenace. ld*eweerj, we hypotesze tha sVeiflc protefins are aaedat-dwitfs beat teluice is tow eter list tolerant plants ( p ctiesoch ". sorghum gad ntMft)~wag adpt cell which pVWd dwenice ace.. From lWesentdta we suest 80 two vtInsf(70 wit heat Art strongly a kilsowlton)

growing temperature. Chemical agents such as amino acid ana-

logues, arsenite, cadmium, and ethanol have also been found to induce HSP synthesis in some systems in the absence of a temperature increase (13). A varety of organisms can be rendered transiently resistant to a lethal heat dose by a prior exposure to heat shock. This phenomenon is termed thermotolerance (6, 7, 10). Using soybean seedlings, Lin et al. (17) showed that a pretreatment at a sublethal temperature (40°C) resulted in the synthesis of HSPs and an asociated development of thermotolerance, e.g. the capacity to withstand temperatures at 45°C for as long as 2 h. Although ubiquitin (2), an ATP-dependent protease (8), and enolase (11) have been shown to be induced by heat shock in tolerAnte and heat adptation organisms other than plants, the exact function and identity of the remaining HSPs remain unclear, with the possible exception of the 70 kDa protein (4, 18). This paper presents results of heat adaptation of suspension culture cells, Cal Blackeye No. 5 (CB5), maintained at 26, 32, 36, and 38C. Cells maintained at these higher temperatures (3238C) are able to grow better at high temperatures than the cells Higher plants from thermally contrasting habitats show con- maintained at 26C. We will also show that cells maintained at siderable differenes in photosynthesis, membrane fluidity, water 32, 36, and 38@C do not produce the typical HSPs at these status, stmatal responses, floral fertility, and protein synthesis. temperatures. We will present data that the 70 and 80 kDa Capacity of plant types ta make adaptations to temperature may proteins might be good candidates for heat tolerant proteins. be due to variation in key components of ceUular cnstituents which enable plans to function efficiently under the temperature MATERIALS AND METHODS regimes of their various native habitats. In this sense, one uses the concept of intrinsic hea tokrance which is applicale, for Cell suspensions of a heat sensitive cultivar (CB5) of cowpea exampl, when comparing related species from different regions. (Vigna unguiculata) were initiated from seedling hypocotyl segTo what extent te level of this heat tolerance is geletically fixed ments by the procedure of Hasegawa et al. (9). Cell suspension is not certain. We hypothesize that hea tolerance in plants is stocks were routinely maintained in 500-ml Erlenmyer flasks with specific proteins that protect cells from otherwMise containing 100 ml W38 medium (9) minus casein enzymic asocat lethal tperatures. We suggest that heat protetion, heat adap- hydrolysate. Stock cultures were inoculated at a cell density (fresh tation and heat tolerance A4 might involve different mechanisms. weight) of about 20 g L' and recultured every 6 to 9 d. Standard resentdy, the focus of research on heat tolerance is its rela- growth temperature for the cell cultures was 26°C. However, tionship to the most readily observable manifestations of heat cultures were also maintained at 32, 36, and 38°C. shock, e.g. sthesis of a set of proteins known as HSPs2 (1, 5, Two-h temperature treatments were applied to 3-ml samples 12, 14, 23). HSPs appear to represent native protective agents of cell suspension in 50-ml flasks submerged to half the flask against beat stress (or they may lead to the synthesis of protective height in water at the desired temperature. A 'Magni Whirl' water agents). In a few instnces, stress proteins have been identified bath (Blue M Co., IL) equipped with a reciprocal shaker was in plats (I6, 19, 20, 24, 25). used. For the 24°C treatment, cells were shaken using a plastic The optimal condition for HSP induction in higher plants box with a 4-cm thick layer of styrofoam at the bottom for drasi tempetature upshift to 10 to 15"C above the normal insulation and attached to the shaking mechanism. Cells were labeled during temperature treatment with L-[4,5-3H]Ieucine (50 ITbi rsearch supported by funds obtained from Purdue Unmver- Ci/mmol; Schwarz-Mann; 50 #Ci/3 ml cell suspension). At the sity cxpesiment Station, International Progrmts, Program Support end of the incubation period, 20 ml cold acetone was added per Grant. Journl PaperI ISI, of the Purdue University Agriculture flask and samples were kept at -20TC until processed further. Acetone-insoluble material was collected on Whatman No. 1 Experiment Station. filter paper, washed once with 20 ml of acetone and dried. The 2Abbreviation: HSP, heat shock protein. is a

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Table I. Average Growth Rates as Determined as Milligrams/25 mildfrom Inoculation to Maximum Fresh (A) or Dry (B) Weight Density CB5 cells from lines maintained for several cycles at 26, 32, 36, or 38C. Growth Temperature Maintenance Temperature 32'C 36 C 26diC 38$C OC A. Fresh weight gain 26 340 ± 70 120 ± 20 80 ± 10 370 ± 40 32 710 ± 50 180 ± 40 260 ± 20 260 ± 10 36 330± 40 800 ± 90 620 ± 90 510 ±70 38 250 ± 20 380 ± 20 360 ± 30 410 20 B. Dry weight gain 26 8.7 ± 1.2 26.6 ± 2.5 20.3 ± 3.2 4.7 0.5 32 53.2 ± 2.8 12.2 + 2.6 21.3 0.5 274 ± 3.3 36 38.1 5.2 62.2 ± 4.5 40.2 4.3 36.2 2.9 21.1 + 4.3 38 43.6 ± 2.0 32.3 ± 0.9 35.5 ± 1.4 8

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at 26°C (Table I; Fig 1), but the previous adaptation to 36°C does not enable the cells to gain fresh weight at 40°C (Fig. 1).

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(IC) FIG. 1. Fresh weight gains at various temperatures by CBS cells previously maintained at 26°C (shaded bars) and 36°C (unshaded bars). Cells were harvested after 7 d, filtered over suction, and weighed. The inoculation fresb weight of 0.5 g has not been subtracted. GROWTH TEMPERATURE

dry material was extracted with 'sample buffer' containing: 62.5 mM Tris-ICl (pH 6.8), 1 mm EDTA, 10% (v/v) glycerol, 5% (v/ v)3 f-mercaptoethanol, 0.005% (w/v) bromophenol blue, 1 mM phenylmethylsulfonyl fluoride, and 2% (w/v) SDS (0.5 ml buffer was used per 10 mg of dry material). Suspension was vortexed vigorously and incubated for 3 min in a boiling water bath. Samples were centrifuged for 8 min at 12,500g and the supernatant fluid collected.

Elctrophoresis. Samples of the protein extracts (10-4 $d; diluted, if necessary, with sample buffer) were electrophoresed as described by Laemmli (15) using 12% acrylamide in the separating gel and 3% in the stacking gel at 25 mamps (15 mamps during stacking). Equal amounts of TCA-precipitated cpm were applied to each lane. Developed gels were stained with Coomassie blue R-250, destained, and photographed. They were then prepared for fluorography as described by Bonner and Laskey (3). Kodak XAR-5 film was used for fluorography.

Other studies (data not included) show that, whether cells are maintained at 26 or 36C, maximum fresh weight gain is achieved at 7 d at 32°C and at 10-12 d at 26, 36, and 38C. As noted from the data presented from both fresh weight and dry weight measurements, cells maintained at elevated temperatures have maximum growth at 32°C in most cases. Cells maintained in culture at 26, 32, 36, and 38C were labelled at 24, 32, 36, 38, 40, 42, and 44'C (Fig. 2, A, B, and C). Cells tained at temperatures above or equal to that temperature in which they are labeled do not produce the classical array of HSP (FR 2A, lanes 1-3, 5, 6, 7, 10, and 11) except for situations in which both the maintenance and labeling temperatures exceed 36°C (Fig. 2B, lane 4). One HSP, the 80 kDa band, is visible on fluorograms in small amounts when cells are incubated at a maintenance temperature not exceeding 36TC and a band is also visible on Coomassie stained gels. Minimal elevation in temperature results in the intensifying of the 80 kDa band on fluorograms, and the appearance of a 70 kDa band (Fig. 2A, lanes 4 and 9; Fig. 2B, lane 3). Higher temperatures result in the progresive addition of other heat shock bands: first, a 94 kDa band along with bands in the 20 kD range (Fig. 2A, lane 8; Fig. 2B, lanes 2, 6, 7), followed by a 60 kDa band (Fig. 2B, lanes 1, 5, 6, 7), then a 44 kDa and a 100' kDa band (Fig. 2B, lanes 8-10; Fig. 2C, lanes 1-3). The data provided by the three PAGE (Fig. 2, A, B, C) are summafized in Table II. Cells adapted to elevated temperatures do not produce the full range of HSP at these temperatures. Cells adapted to 38°C produce only the 80 and 70 kDa heat shock

proteins at 38C.

DISCUSSION These results demonstrate that when cowpea cells are adapted to elevated temperatures (32, 36, and 38°C), the synthesis of the complete set of HSP is not required for superior growth rates. Furthermore, a much higher temperature is required to induce HSP synthesis. Based on these results and other data on pollen (26) we suggest that the majority of HSP are produced for some transient protection against stress. Even when heat stress is used RESULTS to induce the synthesis of HSPI, their synthesis lasts only for a Table I shows that CB5 cell cultures, once adapted to growth few hours (12). Therefore, it appears unlikely that the majority temperaturcs of 32 and 36°C, grow at a much faster rate at these of HSPs are involved during the adaptation of cells to heat stress, temperatures than 26C-maintained cells grow at 26, 32, or 36TC. Thermotolerance is defined (7) as the induced capacity of cells Cells maintained several cycles (at least 10 or more 74 cycles) to survive an otherwise lethal temperature after having been at 38°C also grow faster than 26"C-maintained cells. Cells survive exposed to somestressful stimulus. This type of tolerance should several days, but do not grow, at 40TC (Fig. 1). Cells adapted to not be confused with a long lasting heat tolerance such as noted 360 grow faster when switched to 38C than do cells maintained in the adaptation of cowpea cells to cope with elevated temper-

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FIG. 2. Proteins associated with heat stress in CB5 cells suspensions. Cells which have been maintained at various growth temperatures were labeled for 2 h with L-[4,5-3H]leucine at the indicated temperatures. Extracted proteins (containing the same amount of radioactivity) were separated on 12% SDS-PAGE. Arrows indicate mol wt standards.

Table II. HSP Produced Under Certain Conditions Apparent mol wt in kDa of heat shock proteins produced by CB5 cells under conditions shown, as seen in 12% SDS-PAGE fluorograms. Cells Maintained at °C Labeled at °C 38 36 32 26 None None 24 None 80 None None None 32 70, 80 80 80 36 20's, 70, 80, 94 70, 80 38 20's, 70, 80, 94 20's, 70, 80, 94 70, 80 70, 80 40 20's, 60, 70, 80, 94 20's, 60, 70, 80, 94 20's, 60, 70, 80, 94 20's, 60, 70, 80, 94 20's, 44, 60, 70, 42 20's, 45, 60, 70, 20's, 60, 70, 80, 20's, 44, 60, 70, 80, 94, 100+ 94, 100+ 80, 94, 100+ 80, 94, 100+ 20's, 44, 70, 80, 44 20's, 44, 70, 80, 94 20's, 44, 70, 80, 94, 100+ 94, 100+

PROTEINS ASSOCIATED WITH THERMOADAPTATION atures for several generations (up to 1 year). Based on the results presented in this paper we suggest that the 70 kDa and the 80

kDa HSP fit the characteristics of proteins associated with heat tolerance. In plants, it appears that some specific proteins are associated with certain stressful events. Sachs et al. (19) found that maize tissues subjected to anaerobiosis produce, among other proteins, greatly increased amounts of alcohol dehydrogenase isoforms, of which at least one (22) is necessary for maize seeds and seedlings to survive flooding. Steinback et al. (25) showed that a major 32 kDa thylakoid protein binds the herbicide, triazine, and providas plant resistance. Singh et al. (24) have identified a 26 kDa piotein that appears to be associated with salt tolerance in tobacco cell suspensions. Recently, Leland and Hanson (16) have shown that N-methyltransferase activity specific to the gramine pathway is induced in growing barley leaves by prolonged exposure to high temperature stress.

Key's group has studied the expression of the 15 to 18 kDa HSP of soybean (14, 17, 21). The low molecular weight HSPs are of interest in the study of thermotolerance because of the intensity of their mRNA accumulation and of their uniqueness to plants (21). We believe that the small number of proteins (70 and 80 kDa protein) produced in cowpea cells during adaptation and maintenance near maximum growth temperatures provides the opportunity to study the role of specific proteins during adaptation to high temperature. In the future we plan to determine the role of the 70 and 80 kDa proteins, in the adaptation of cells to elevated temperatures, and whether the proteins are involved in heat tolerance of cowpea plants under field conditions. LITERATURE CITED 1. BARNETT, T, M ALTSHULER, CN McDANIEL, JP MASCARENHAS 1980 Heat shock induced proteins in plant cells. Dev Genet 1: 331-340 2. BOND U, MJ SCHLESINGER 1985 Ubiquitin is a heat shock protein in chicken embryo fibroblasts. Mol Cell Biol 5: 949-956 3. BONNER WM, RA LASKEY 1974 A film detection method for tritium-labelled proteins and nucleic acids in polyacrylamide gels. Eur J Biochem 46: 83-88 4. CHAPPELL TG, WJ WELCH, DM SCHLOSSMAN, KB PALTER, MJ SCHLESINGER, JE ROTHMAN 1986 Uncoating ATPase is a member of the 70 kilodalton family of stress proteins. Cell 45: 3-13 5. COOPER P, THD Ho 1983 Heat shock proteins in maize. Plant Physiol 71: 215-222

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6. GERNER EW, DH RUSSELL 1977 The relationship between polyamine accumulation and DNA replications in synchronized Chinese hamster ovary cells after heat shock. Cancer Res 37: 482-489 7. GERNER EW, WJ SCHNEIDER 1975 Induced thermal resistance in HeLa cells. Nature 256: 500-502 8. GOFF SA, LP CASSON, AL GOLDBERG 1984 Heat shock regulatory gene htpR influences rates of protein degradation and expression of the Ion gene in Escherichia coli. Proc Natl Acad Sci USA 81: 6647-6651 9. HASEGAWA PM, RA BRESSAN, AK HANDA 1980 Growth characteristics of NaCl selected and nonselected cells of Nicotiana tabacum L. Plant Cell Physiol 21: 1347-1355 10. HENLE KJ, DB LEEPER 1976 Interaction of hyperthermia and radiation in CHO cells: recovery kinetics. Radiat Res 66: 505-518 11. IIDA H, I YAHARA 1985 Yeast heat-shock protein of Mr 48000 is an isoprotein of enolase. Nature 315: 688-690 12. KANABUS J, CS PIKAARD, JH CHERRY 1984 Heat Shock proteins in tobacco cell suspension during growth cycle. Plant Physiol 75: 639-644 13. KELLEY PM, MJ SCHLESINGER 1978 The effect of amino acid analogues and heat shock on gene expression in chicken embryo fibroblasts. Cell 15: 12771286 14. KEY JL, CY LIN, YM CHEM 1981 Heat shock proteins of higher plants. Proc Natl Acad Sci USA 78: 3526-3530 15. LAEMMLI UK 1970 Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature 227: 680-685 16. LELAND TJ, AD HANSON 1985 Induction of a specific N-Methyltransferase enzyme by long-term heat stress during barley leaf growth. Plant Physiol 79: 451-457 17. LIN CY, JK ROBERTS, JL KEY 1984 Acquisition of thermotolerance in soybean seedlings. Plant Physiol 74: 152-160 18. PALTER KB, M WATANABE, L STINSON, AP MAHOWALD, EA CRAIG 1986 Expression and localization of Drosophila melanogaster hsp 70 cognate proteins. Mol Cell Biol 6: 1187-1203 19. SACHS MM, M FREELING, R OKIMOTO 1980 Selective synthesis of alcohol dehydrogenase during anaerobic treatment of maize. Cell 20: 761-767 20. SACHS MM, THD Ho 1986 Alteration of gene expression during environmental stress in plants. Annu Rev Plant Physiol 37: 363-376 21. SCHOFFL F, JL KEY 1982 An analysis of mRNAs for a group of heat shock proteins of soybean using cloned cDNAs. J Mol Appl Genet 1: 301-314 22. SCHWARTZ D 1969 An example of gene fixation resulting from selective advantage in suboptimal conditions. Am Nat 103: 479-481 23. SHAH DM, DE ROCHESTER, GG KRIVI, CM HIRONAKA, TJ MOZER, RT FRALEY, DC TIEMEIR 1985 Structure and expression of maize HSP 70 gene. In JL Key, T Kosuge, eds, Cellular and Molecular Biology of Plant Stress. Alan R Liss Inc., New York, pp 181-200 24. SINGH NK, AK HANDA, PM HASEGAWA, RA BRESSAN 1985 Proteins associated with adaptation of cultured tobacco cells to NaCl. Plant Physiol 79: 126137 25. STEINBACK KE, L MCINTOSH, L BOGORAD, CJ ARNTZEN 1981 Identification ofthe triazine receptor protein as a chloroplast gene product. Proc Natl Acad Sci USA 78: 7463-7467 26. XIAO CM, JP MASCARENHAS 1985 High temperature-induced thermotolerance in pollen tubes of Tradescantia and heat-shock proteins. Plant Physiol 78: 887-890