In the present study, an attempt was made to determine the mechanism of Pima cotton adaptation to high temperatures by analyzing heat shock proteins at different stages of growth and development. Pima cotton (Gossypium barbadense L.) cv. S-6 plants were grown from seed in a 3:1 sand, vermiculite growing medium in naturally lit, temperature and CO₂ controlled growth chambers. Plants were grown in 25/17, 30/22, 35/27, and 40/32 C 12h day/night temperature regimes and ambient (350 µmol mol^-1) and enriched (700 µmol mol^-1) CO₂ environments. The plants were irrigated with a drip irrigation system and were supplied with adequate quantities of nutrients and water during the entire period of growth. The day/night temperatures were selected to represent the temperature range of the Pima growing areas of Arizona, New Mexico, Texas, and California, USA. The day temperature was extended one hour after sunset to obtain dark respiration rates of the Pima canopy under the day temperature.

The leaf samples were collected during pre-dawn, midafternoon and 2.5 h after the shift of night temperature at 16 and 59 days after emergence. Cotyledons were collected at seedling stage (16 days). At each sampling time, a fully expanded leaf on the main stem wa@ selected. Leaf discs (5 discs/treatment) were placed in a glass tube containing 2 ml of incubation buffer (1mM KH₂PO₄ containing 1% sucrose + 0.01% Tween-20, pH 6) at each day or night time temperature in a water-bath shaker for 30 min. Following this preincubation, 25 µl of [(35)S] methionine were added and samples were incubated for an additional 90 min. Leaf discs were removed and proteins were extracted with 400 µl sample buffer (O.0625 mM Tris-HCl, pH 6.8 containing 2%, sodium-dodecyl-sulphate (SDS) 10% glycerol, 5% mercaptoethanol, 0.1% bromophenol blue and 1 mM phenyl-methyl-sulphonyl-fluoride). The protel extracts were placed in a boiling water bath for 4 min, cooled, and then centrifuged at 15,000 xg for 5 min. The supernatants were collected and samples of equal counts from (35)S labeled proteins were loaded onto polyacrylamide gels.

Protein samples were analyzed by vertical gel electrophoresis on a one-dimensional SDS-polyacrylamide linear gradient gel (10-15%). Tris-HCl buffer, pH 8.3, containing 192 mM glycine and 0.10% (w/v) SDS was used in the electrode compartments. The electrophoresis was carried out at 20 MA for 30 min, then current was increased to 40 mA until the tracking dye reached the lower end of the vertically oriented gel. The gels were stained with 0.2% Coomassie blue in 10% glacial acetic acid and 45%(v/v) methanol for 20 min at room temperature. Destaining of gels was done in 7.5% acetic acid (v/v) and 5% (v/v) methanol. The stained gels were treated with fluorographic enhancing solution (Resolution, E. M. Corporation, NY) followed by soaking in 1% glycerol. The gels were dried and exposed to Kodak AAR-5 film at -80 C. Radiolabeled gels were visualized by fluorography. Molecular weights of the leaf proteins were determined with known standards which were loaded in one of the lanes in SDS-polyacrylamide gel.

Leaves from cotton plants that had been grown in high temperature treatments (35 and 40 C) for 59 days accumulated at least seven sets of proteins that were either absent or present in modest levels in the leaves of 25/17 C grown plants. Most heat shock proteins (HSPS) are synthesized at modest levels in cells grown under normal conditions, and increased significantly to higher levels in the cells experiencing high temperature stress (Welch et al., 1983). The location of these new proteins on the gels were determined as high (75-100 kD), intermediate (37-60 kD), and low molecular weight (18-21 XD) polypeptides. Based on the position and molecular weights of the these labeled proteins on the gels, we identified these as heat shock proteins (HSPs). When sampled for varying intervals during a 24 h period it was observed that the HSPs detected in day time temperature conditions (40 C) persisted at high levels for 8 h into the dark period temperature condition (32 C).

The band intensities of existing leaf protein varied with time in both ambient and enriched CO₂ environments. However, no new major differences in protein banding patterns were observed in ambient and CO₂ environments. These results suggest the synthesis of specific heat shock proteins and perhaps their association with thermotolerance during exposure of plants to high temperature.