JOHN J. BURKE*, JERRY L. HATFIELD, ROBERT R. KLEINI, AND JOHN E.MULLET. USDA Plant Stress and Water Conservation Research Unit (J.J.B.,J.L.H., ...
Plant Physiol. (1985) 78, 394-398 0032-0889/85/78/0394/05/$O 1.00/
Accumulation of Heat Shock Proteins in Field-Grown Cotton Received for publication November 20, 1984 and in revised form February 6, 1985
JOHN J. BURKE*, JERRY L. HATFIELD, ROBERT R. KLEINI, AND JOHN E. MULLET USDA Plant Stress and Water Conservation Research Unit (J.J.B., J.L.H., R.R.K), P. 0. Box 4170, Texas Tech University, Lubbock, Texas 79409; and Department of Biochemistry and Biophysics (J.E.M.), Texas A & M University, College Station, Texas 77843 ABSTRACr Cotton (Gossypinm kirsatam L.) plants grown under field water deficits exhibited an 80 to 85% reduction in leaf area index, pblt height, and dry matter accumulation compared with irrigated controls. Midday photosynthetic rates of dryland plants decreased 2-fold, and caopy temperatures increased to 40°C at 80 days after planting compared with canopy temperatures of 30°C for irripted plants. Leaves from drylbd plants which had exhibited canopy temperatures of 40°C for several weeks accumulated stainable levels of polypeptides with apparent molecular weights of 100, 94, 89, 75, 60, 58, 37, and 21 kilUltons. These polypeptides did not accumulate in leaves from irrigated plants. Addition of 135Slmethionine to leaves of growth chamber-grown cotton plants and subsequent incubation at 40°C for 3 hours radiolabeled polypeptides with molecular weights similar to those that accumulte in dryland cotton leaves. These data suggest that the proteins which accumulate in water-strssed cotton leaves at elevated temperatures (40°C) are beat shock proteins and that these proteins can accumulate to substantial levels in field-stressed plants.
Plants growing in environments with ample water supplies maintain, through transpiration, leaf temperatures at or below air temperatures. Plants exposed to drought conditions experience declining soil water levels which ultimately result in stomatal closure and reduced transpiration. Drought-induced stomatal closure limits carbon assimilation, yet may optimize the water use efficiency of the plant on a daily basis (9). As a consequence of the reduction in transpiration, leaf temperatures increase above the temperature of the surrounding air (12). The elevated leaf temperatures may limit dry matter accumulation because of increased respiration, reduced photosynthesis, and cellular damage. Living organisms have developed several endogenous protection systems which provide thermal tolerance (I 1, 13). One of these protection systems involves an acquired heat resistance mechanism associated with the synthesis and accumulation of specific proteins (HSPs2 [1, 15]). These proteins have been identified in numerous animal (20, 22, 33) and plant (7, 14, 16, 27, 29) species by following the incorporation of labeled amino acids into proteins during exposure to elevated temperatures (>37°C). Because they have only been detected by autoradiography and not by Coomassie blue or silver staining techniques, it has been suggested that heat shock proteins do not accumulate to substan-
tial quantities (8). Studies with mammalian cells (13), yeast (24), Drosophila (25), Calpodes (10), and Dictyostelium (23) have demonstrated that exposure to elevated, sublethal temperatures induces a transient thermoresistance, which protects them from a second exposure at typically lethal temperatures. A correlation between the development of thermotolerance and the synthesis of HSPs has been reported, although the mechanisms underlying the thermotolerance remain unknown (19, 21). The loss of HSPs and the decay of thermotolerance have also been shown to be related (18). The relationship between the appearance of HSPs and thermotolerance is perhaps best supported by the findings in Dictyostelium where a mutant deficient in the expression of HSPs was unable to develop thermotolerance (23). Early reports of heat shock proteins in higher plants demonstrated HSPs in tobacco and soybean cells grown in solution culture (5), and in soybean seedling tissue (16). The HSPs of soybean consisted of 10 new bands on one-dimensional SDS gels; and a more complex pattern on two-dimensional gels (16). When the tissue was returned to 28C after 4 h at 40°C, there was a progressive decline in the synthesis of HSPs and reappearance of a normal pattern of protein synthesis by 3 to 4 h. Pitto et al. (29) demonstrated that a 38°C temperature treatment of carrot cells resulted in the gradual cessation of normal protein synthesis and an induction of a new class of peptides (HSPs). They demonstrated that carrot plants, cells grown in suspension, protoplasts, and different embryonal stages showed definite differences in the HSP pattern. Nover et al. (27) used a 39°C temperature treatment to induce HSP synthesis in tomato cell cultures and leaves. They showed that a considerable part of the dominant small HSPs with a Mr of 17,000 were structural proteins of newly forming granular aggregates in the cytoplasm (heat shock granules), whose formation strictly depended on heat shock conditions (37-40°C) and the presence or simultaneous synthesis of HSPs. Depending upon the culture conditions, it was demonstrated that HSPs also accumulated as soluble proteins without concomitant assembly of heat shock granules. Kanabus et al. (14) used tobacco cell suspensions to demonstrate that HSP synthesis was affected by the cell growth cycle. Cooper et al. (8) demonstrated on one-dimensional gels that most maize tissues synthesized a set of 10 HSPs, but the exact characteristics of the response depended upon the tissue type. They also reported that leaves constitutively synthesized low levels of HSPs and high levels could be induced at 40°C. Atkinson et aL (3) found similar results in analysis of HSP responses in quail embryos. Their data added support to the contention (2, 4, 31) that the quantity and types of HSPs expressed by a tissue or cell may be dependent upon the type of cell or tissue thermally stressed and/or upon the development or differentiative state of the stressed cells. In this report, soil water deficits which result in midday canopy
' Present address: Department of Biochemistry and Biophysics, Texas A&M University, College station, Texas 77843. 2Abbreviations: HSP, heat shock protein; DAP, days after planting; LAI, leaf area index. 394
HEAT SHOCK PROOTEINS IN COTTON temperatures of 40°C or greater for a prolonged period of time (2-3 weeks) have been used to study heat shock in field-grown cotton. The ability of cotton plants to produce and accumulate heat shock proteins in response to elevated temperatures in the field was analyzed.
MATERIALS AND METHODS A photoperiodic cotton (Gossypium hirsutum L.) strain (T 185) was planted under two soil-water regimes at Lubbock, TX. The two soil-water regimes consisted of fully irrigated and dryland tests located in the same field about 30 m apart. Dryland plots were located in a rainout shelter to prevent the addition of supplemental water by rainfall. Irrigated plots were located outside the rainout shelter. The soil type was an Acuff fine sandy loam soil (fine-loamy, mixed, thermic, Aridic Paleustolls). Three replications of each test were planted on May 17, 1983. The strain was planted in five 3-m rows with 1 m between rows. Plants were thinned after emergence to 11 plants/m. Samples were taken from the three center rows for analyses. Determination of Leaf Water Status and Gross Photosynthesis. A set of measurements was made on the same uppermost, fully expanded leaf. All data were collected between 1000 and 1600 CDT and at light levels greater than 1500 ,E m-2 s-'. Leaf water status was measured with Merrill leaf cutter psychrometers (30). Each psychrometer was calibrated with NaCl solutions at 30°C in a water bath controlled to ±0. 1C. Leaf samples were punched from intact leaves, immediately sealed in the psychrometer chambers, and equilibrated for 4 h prior to measuring water potential. After water potential was determined, the psychrometer chambers were placed in liquid N2 for about 1 min, allowed to equilibrate for 1 h, and osmotic potential estimated. Turgor pressure was estimated as the difference between leaf water potential and osmotic potential. Gross photosynthetic rates were determined with '4CO2 (26). The leaf discs were placed in glass liquid scintillation vials and digested in 0.2 ml of 70% HC104 and 0.2 ml of 30% H202 at Table I. Midday Water Status Measurements of the Photoperiodic Cotton Strain T185 under Irrigated and Dryland Conditions Water Status at Following DAP Treatment Potential 50 106 MPa Irrigated Water -0.67 -1.60 Osmotic -1.51 -1.73 0.84 0.12 Turgor Dryland
-0.94 -1.64 0.69
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SDS-PAGE. Leaf proteins were separated by SDS-PAGE using the discontinuous buffer system ofLaemmli (17). Electrophoresis was performed in a slab gel apparatus (32) with a continuous 12 to 16% (w/v) polyacrylamide separating gel and a 5% (w/v) stacking gel. Leaves were harvested randomly throughout the canopy. Leaf polypeptides were solubilized in 65 gM Tris-HCI sample buffer (pH 6.8) that contained 10% (v/v) glycerol, 1% (v/v) 2-mercaptoethanol, and 2% (w/v) SDS. Leaf homogenates were homogenized with a Brinkman polytron at the highest setting for 1 min. Leaf homogenates were centrifuged at 7,500g for 5 min (23°C) and the supematant boiled for 2 min prior to application to the gel sample wells. Electrophoresis was carried out at a constant current of 35 mamp. Gels were stained for protein in a solution that contained 0.2% (w/v) Coomassie blue, 50% (v/v) methanol, and 7% (v/v) glacial acetic acid for 30 min, and were destained in 20% (v/v) methanol and 7% glacial acetic acid. Mol wt determinations were accomplished by coelectrophoresis with protein standards. The protein calibration kit (Bio Rad) contained lysozyme (14,400), soybean trypsin inhibitor (21,500), carbonic anhydrase (31,000), ovalbumin (45,000), BSA (66,200), and phosphorylase b (92,500). Protein Radiolabeling and Autoradiography. T185 cotton plants were grown in a Conviron model E-1 5 growth chamber at 23°C and a 14-h photoperiod. Ten-d-old plants were excised, their stems recut under water and then transferred to tubes containing 1 mM Hepes-KOH buffer (pH 8.0) and 100 ,Ci of [35S]methionine (1098 Ci/mmol). Four plants were treated either as controls or were exposed to 40°C temperatures for 3 h. At the
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room temperature. After 24 h, 15 ml of scintillation cocktail consisting of toluene (75% by volume), Triton X-100 (25% by volume), and 6.0 g of POPOP per liter was added to each vial. Determination of Plant Growth. Plants from both irrigated and dryland plots were harvested 106 DAP and growth analyses obtained. Measurements included dry weight, height, and LAI. Determination of Canopy Temperature. Canopy temperatures were obtained 5 d each week with a hand-held IR thermometer and electronically recorded on a portable data acquisition system, Omnidata Polycorder3 model 516. The IR thermometer had a 4-degree field ot view and an 8- to 14-,um waveband filter and was held perpendicular to the row at an angle of 45 degrees from horizontal at a distance of 0.25 to 0.30 m from the row. This position allowed for canopy temperatures to be measured without interference from the soil background. Six measurements were made on each side ofthe row for a total of 36 measurements in each irrigation treatment. The measurements were made between 1400 and 1430 CDT and were completed within 15
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Plant Physiol. Vol. 78, 1985
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end of the incubation period, polypeptides were extracted and run on SDS-polyacrylamide gels as described for field grown cotton. Protein gels were analyzed by x-ray fluorography by treating with PPO (6) with subsequent drying and exposure to x-ray film.
RESULTS AND DISCUSSION Changes in Leaf Water Status and Gross Photosynthesis. Midday leaf water status was monitored at 50 and 106 DAP (Table I). Little difference in the water status of leaves from dryland and irrigated plants was observed at 50 DAP. The water potential of dryland leaves at 106 DAP, however, had decreased to nearly -2.4 MPa compared with a water potential of -1.6 MPa in the leaves from the irrigated plots. Turgor of the dryland and irrigated leaves was similar at both sampling dates. Analysis of gross photosynthesis at 50 and 106 DAP also illustrated the severity ofthe water stress. At 50 DAP, the irrigated and dryland treatments had photosynthetic rates of 0.72 to 0.81 mg CO2 fixed/m2/s. Similar measurements at 106 DAP showed photosynthetic rates in the irrigated treatment between 0.78 and 0.97 mg CO2 fixed/m2/s. The dryland treatments, however, had photosynthetic rates between 0.36 and 0.50 mg CO2 fixed/m2/s at 106 DAP. Leaf conductance levels decreased from 2.67 cm/s in irrigated leaves to 1.04 cm/s in the dryland treatments. These results indicate that the stress was severe enough to result in a 50% decline in gross photosynthesis and a 60% decline in leaf conductances. Stress-Induced Changes in Plant Morphology. The second parameter used to identify the severity of the soil water deficit was analysis of changes in the growth parameters of the photoperiodic cotton strain at 106 DAP. All of the parameters exhibited dramatic declines under the stress conditions (Fig. 1). An 80 to 85% reduction in plant height, LAI, and plant dry weight was observed between irrigated and dryland treatments. Measurements of Midday Canopy Temperatures. Midday canopy temperatures of dryland and irrigated plants are shown in Figure 2. Leaf temperatures of irrigated plants remained in the
FIG. 3. SDS-polyacrylamide slab gel of polypeptide banding patterns of dryland and irrigated cotton leaves. A, Mol wt standards; B, irrigated cotton leaf polypeptides; C, dryland cotton leaf polypeptides. Arrows and associated numbers in the right margin indicate the location and estimated mol wt of polypeptides unique to dryland leaves. Arrows and associated numbers in the left margin indicate mol wt of standards. range of 26 to 32C throughout the growing season. Between 55 and 70 DAP canopy temperatures of irrigated plants declined because of a reduction in the warm soil background component on the canopy temperature. This effect has been noted by several researchers (12) and is present even though the readings were collected at an angle to the foliage. After 70 DAP there was a trend for increased canopy temperatures to 30'C because of increasing canopy foliage density and a reduction in windspeed in the crop and its impact on mixing within the canopy (28). In contrast to temperatures of irrigated plants, midday canopy temperatures of dryland plants increased to greater than 38°C by 70 DAP. Midday canopy temperatures remained above 38C until irrigation was applied to the dryland plots. Addition of irrigation water to the dryland plots resulted in a rapid drop in midday leaf temperatures. Induction of Heat Shock Proteins in Dryland Cotton Leaves. The reported induction temperatures for HSP synthesis in laboratory grown plants range from 38C to 410C (16). In the present study, midday canopy temperatures of dryland plants reached 380C by 70 DAP and remained above the reported induction
HEAT SHOCK PROTEINS IN COTTON
leaves analyzed. The apparent mol wt of the polypeptides were 100, 94,89, 75, 60, 58, 37, and 21 kD. No accumulation of these The polypeptides was detected in leaves of irrigated plants. polybanding pattern and apparent mol wt of the accumulated from peptides closely resemble the reported mol wt for HSPs other plant species (1, 5, 6, 16). Because leaves were harvested randomly through the canopy, the effect of leaf position could
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not be determined. Because of the variability in the reported mol wt of HSPs from different species (1, 5, 6, 16), two sets of cotton plants were heat shocked in the presence of[35S]methionine and radiolabeled polypeptides were compared with the Coomassie blue-stainedof polypeptides of the field-stressed plants (Fig. 4). Comparison the polypeptide patterns of the control and heat-shocked cotton leaves showed a reduction in normal protein synthesis and increased synthesis of HSPs. At least eleven newly synthesized polypeptides were detectable in the heat-shocked sample.to Eight of the eleven HSPs of cotton leaves had mol wt similar those leaves. In this polypeptides that accumulate in dryland cotton from study, we cannot definitively separate water stress responses thermal stress responses; however, the 3 weeks of leaf temperatures above the induction temperature of heat shock proteins and the marked similarities in the electrophoretic mobilities of the accumulated proteins with the heat shock proteins suggests that the polypeptides which accumulate in dryland cotton leaves at elevated canopy temperatures are heat shock proteins. We have demonstrated that HSPs accumulate to substantial levels in leaves from temperature-stressed cotton. Our findings are supported by a similar study by Kimpel and Key (personal communication) on field-stressed soybeans. Their study showed the presence of HSP RNA in nonirrigated soybean plants and the presence of accumulated HSPs on SDS-polyacrylamide gels. Thus, in arid and semi-arid regions, dryland crops may synthesize HSPs in response to elevated leaf temperatures and these proteins can accumulate to substantial levels. LITERATURE CITD 1. ALTSCHULER M, JP MASCARENHAS 1982 Heat shock proteins and effects of heat shock in plants. Plant Mol Biol 1: 103-115 2. ATKINSON BG 1981 Synthesis of heat-shock proteins by cells undergoing myogenesis. J Cell Biol 89: 666-673 3. ATKINSON BG, T CUNNINGHAM, RL DEAN, M SOMERVILLE 1983 Comparison of the effects of heat shock and metal-ion stress on gene expression in cells undergoing myogenesis. Can J Biochem Cell Biol 61: 404-413 4. ATKINSON BG, M POLLOCK 1982 Effect of heat shock on gene expression on human epidermal carcinoma cells (strain KB) and in primary cultures of mammalia and avian cells. Can J Biochem 60: 316-327
5. BARNETT T, M ALTSCHULER, CN McDANIEL, JP MASCARENHAS 1980 Heat shock induced proteins in plant cells. Dev Gen 1: 331-340 6. BONNER WM, RA LASKY 1974 A film detection method for tritium-labeled proteins and nucleic acids in polyacrylamide gels. Eur J Biochem 46: 83-88
21000
FIG. 4. A fluorogram of radiolabeled polypeptides from control (C) and heat-shocked cotton plants (40'C). The arrows and mol wt in the right margin illustrate the location of the heat shock proteins that migrated with mol wt similar to the Coomassie-stained polypeptides that
accumulated in the dryland cotton leaves.
temperatures for HSPs for 36 d. Leaves from dryland and irri-
gated cotton were harvested at 106 DAP and leaf proteins were separated on one-dimensional SDS-polyacylamide slab gels (Fig. 3). Comparison of polypeptide patterns of dryland and irrigated cotton leaves revealed that at least eight new polypeptides accumulated to stainable levels in approximately 50% of the dryland
7. COOPER P. T-HD Ho 1983 Heat shock proteins in maize. Plant Physiol 71: 215-222 8. COOPER P. T-HD Ho, RM HAUPTMANN 1984 The specificity of the heat shock response in maize. Plant Physiol 75: 431-441 9. COWAN IR, GD FARQUHAR 1977 Stomatal junction in relation to leaf metabolism and environment. Symp Soc Exp Biol 31: 471-505 10. DEAN RL, BG ATKINSON 1983 The acquisition of thermal tolerance in larvae of Calpodes ethelius (Lepidoptera) and the in situ and in vitro synthesis of heat shock proteins. Can J Biochem Cell Biol 61: 472-479 11. HAHN GM, GC Li 1982 Thermotolerance and heat shock proteins in mammalian cells. Radiat Res 92: 452-457 12. HATFIELD JL 1979 Canopy temperatures. The usefulness and reliability of remote measurements. Agron J 71: 889-892 13. HENLE KJ, LA KETHLEFSEN 1978 Heat fractionation and thermotolerance: A review. Cancer Res 38: 1843-1851 14. KANAKUS J, CS PIKAARD, JH CHERRY 1984 Heat shock proteins in tobacco cell suspension during growth cycle. Plant Physiol 75: 639-644 15. KEY JL, C-Y LIN, E CEGLARZ, F SCHOFFL 1982 The heat shock response in M Ashburner, A plants: physiological considerations. In ML Schlesinger, Cold Spring Harbor Tissieres, eds, Heat Shock from Bacteria to Man. Laboratory. Cold Spring Harbor, New York, pp 329-355 CY Sci YM 78: 1981 Heat shock proteins of higher plants. Proc 16. KEY CHEN LIN,USA 3526-3530 NatlJL,Acad
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17. LAEMMLI UK 1970 Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature 227: 680685 18. LANDRY J, D BERNIER, P CHRETIEN, LM NICOLE, RM TANGUAY, N MARCEAU 1982 Synthesis and degradation of heat shock proteins during development and decay of thermotolerance. Cancer Res 42: 2457-2461 19. LANDRY J, P CHRETIEN, D BERNIER, LM NICOLE, N MARCEAU, RM TANGUAY 1982 Thermotolerance and heat shock proteins induced by hyperthermia in rat liver cells. Int J Radiat Oncol Biol Phys 8: 59-62 20. Li GC 1983 Induction of thermotolerance and enhanced heat shock protein synthesis in Chinese hamster fibroblasts by sodium arsenite and by ethanol. J Cell Physiol 115: 116-122 21. Li G, NS PETERSEN, HK MITCHELL 1982 Induced thermal tolerance and heat shock protein synthesis in Chinese hamster ovary cells. Int J Radiat Oncol Biol Phys 8: 63-67 22. Li GC, DC SHRIEVE 1982 Thermal tolerance and specific protein synthesis in Chinese hamster fibroblasts exposed to prolonged hypoxia. Exp Cell 142: 464-468 23. LooMis WF, SA WHEELER 1982 Chromatin-associated heat shock proteins of Dictyostelium. Dev Biol 90: 412-418 24. McALISTER L, DB FINKELSTEIN 1980 Heat shock proteins and thermal resistance in yeast. Biochem Biophys Res Commun 93: 819-824
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25. MITCHELL HK, G MOLLER, NS PETERsEN, L LIPPS-SARMIENTO 1979 Specific protection from phenocopy induction by heat shock. Dev Genet 1: 181-192 26. NAYLOR DS, ID TEARE 1975 An improved rapid field method to measure photosynthesis with 14CO2. Agron J 67: 404-406 27. NOVER L, K-D SCHARF, D NEUMANN 1983 Formation of cytoplasmic heat shock granules in tomato cell cultures and leaves. Mol Celi Biol 3: 16481655 28. O'TooLE JC, JL HATFIELD 1984 Effect of wind on the crop water stress index derived by infrared thermometry. Agron J 75: 811-815 29. PTrro L, F LOSCHIAVO, G GIOLIANO, T TERZI 1983 Analysis of the heat-shock protein pattern during somatic embryogenesis of carrot. Plant Mol Biol 2: 231-237 30. QUISENBERRY JE, BG CARTWRIGHT, BL MCMICHAEL 1984 Genetic relationships between turgor maintenance and growth in cotton germplasm. Crop Sci 24: 479-482 31. ROCCHERI MC, MG Di BERNARDO, G GiuDICE 1981 Synthesis of heat-shock proteins in developing sea urchins. Dev Biol 83: 173-177 32. STUDIER FW 1973 Analysis of bacteriophage T7 early RNA's and proteins on slab gels. J Mol Biol 79: 237-248 33. SUBJECK JR, J SCIANDRA, E REPASKY, RJ JOHNSON 1981 Induction of protein synthesis foliowing hyperthermia at 45C or 41IC in CHO. Radiat Res 87: 390
