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The protein content of clerodendrum (Clerodendrum speciosum) leaves declines following chilling (48 h, 3°C). Using western and dot blots and fluorescence immunoassays, we found that isolated leaf proteins had more conjugated ubiquitin following chilling. In contrast, the amount of free ubiquitin declined by almost 90% after chilling. The increase in ubiquitin conjugation was greater in the membrane fraction than in the soluble fraction.Plants were grown at 27/180C (day/night) under LD conditions as described before (10). Chilling was induced by exposing the plants to 30C for 48 h in a temperature-controlled chamber. Analyses of fully grown leaves were done within 1 h after the end of the treatments. Membrane PreparationChilling injury occurs in many tropical and subtropical plants following exposure Microsomal membranes were isolated from the leaves in the presence of 2 mm DTT and 2 mm PMSF using differential centrifugation as described previously (7). The membrane pellets were resuspended in 20 mm Tris-HCl at pH 7.4. To get a free ubiquitin fraction, the obtained supematant was filtered through a YM10 membrane using an ultrafiltration cell (Amicon); after this was done, the effluent was free of proteins larger than 10,000 kD. During all of the isolation steps, the temperature was kept at 0 to 40C. Immediately following isolation, protein content was determined according to Bradford (2), and the various analyses were conducted. FIA2Quantitative analysis of the ubiquitin was conducted by FIA according to a manufacturer's (Sigma) protocol: Ubiquitin-fluorescein conjugate (100 ,uL) at 20.4 pmol/mL (Sigma U-5540) was added to all test tubes except the blanks. A sample (100 ,uL of a 1 mg protein/mL solution) to be tested was added to all tubes. Finally, 100 yL of rabbit anti-ubiquitin IgG (Sigma U-5379) was added to all samples. The samples were mixed by vortexing and incubated for 60 min at room temperature in the dark. Then, 1 mL of immunoprecipitation reagent (goat anti-rabbit IgG in 0.1 M PBS) was added to each test tube, and the samples were centrifuged at 2000g for 15 min at 40C. The supematants were aspirated and discarded, and the pellets were resuspended in 3 mL of 0.1 N NaOH in 2% SDS. The samples were excited at 494 nm and the fluorescence was measured at 516 nm using a spectrofluorimeter (SLM model 4600). A decrease in the immunoprecipitated fluorescein-labeled ubiquitin resulted in a decrease in the fluorescence intensity and indicated a higher level of ubiquitin in the tested sample. The amount of the ubiquitin in the samples was calculated according to a calibration curve, which was prepared with purified ubiquitin (Sigma) (Fig. 1).

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