A Comparison of the Effects of Chilling on Thylakoid Electron Transfer in Pea ( Pisum sativum L .

Experiments comparing the photosynthetic responses of a chillingresistant species (Pisum sativum L. cv Alaska) and a chiHling-sensitive species (Cucumis sativus L. cv Ashley) have shown that cucumber photosynthesis is adversely affected by chilling temperatures in the light, while pea photosynthesis is not inhibited by chilling in the light. To further investigate the site ofthe differential response of these two species to chilling stress, thylakoid membranes were isolated under various conditions and rates of photosynthetic electron transfer were determined. Preliminary experiments revealed that the integrity of cucumber thylakoids from 25C-grown plants was affected by the isolation temperature; cucumber thylakoids isolated at 50C in 400 millimolar NaCI were uncoupled, while thylakoids isolated at room temperature in 400 millimolar NaCl were coupled, as determined by addition of gramicidin. The concentration of NaCl in the homogenization buffer was found to be a critical factor in the uncoupling of cucumber thylakoids at 5°C. In contrast, pea thylakoid membranes were not influenced by isolation temperatures or NaCl concentrations. In a second set of experiments, thylakoid membranes were isolated from pea and cucumber plants at successive intervals during a whole-plant light period chilling stress (5°C). Drin wholeplant chilling, thylakoids isolated from cucumber plants chilled in the light were uncoupled even when the membranes were isolated at warm temperatures. Pea thylakoids were not uncoupled by the whole-plant chilling treatment. The difference in integrity of thylakoid membrane coupling following chilling in the light demonstrates a fundamental difference in photosynthetic function between these two species that may have some bearing on why pea is a chilling-resistant plant and cucumber is a chilling-sensitive plant. Photosynthesis has been shown to be very sensitive to environmental stresses such as heat, drought, and chilling (0-15°C) (12). Several workers have found that photosynthetic activity is inhibited by long periods of chilling in the dark (4, 8). Photosynthesis is inhibited more rapidly, however, by chilling in the light, and especially at high irradiances (10). The inhibition of photosynthesis caused by chilling temperatures during illumination has been attributed to photooxidative processes (12, 15). However, the mechanism of the photooxidative process has not yet been elucidated. In an effort to determine the effects of whole plant chilling in the light on photosynthetic ' Supported by National Science Foundation grants PCM-840491 1 to A. W. N. and BSR 83-14925 to the Duke University Phytotron. 2Current address: Department ofBotany, University ofTexas, Austin, TX 78713. electron transfer, rates of electron transfer were assayed in thylakoid membranes isolated at different times during the course ofa chilling treatment. Chilling-sensitive cucumber and chillingresistant pea were the two species compared in this series of experiments. The results show that chilling in the light does not immediately inhibit the potential for electron transfer in either ofthe species tested. Rates of electron transfer in cucumber were even more rapid than normal; these increased rates were generated because chilling in the light uncoupled electron transfer from photophosphorylation in the thylakoid membranes. MATERIALS AND METHODS Pea (Pisum sativum L. cv Alaska) and cucumber (Cucumis sativus L. cv Ashley) were grown in the Duke University Phytotron. Control conditions were 25°C day/l 8C night, 16 hr photoperiod, and 350 umol m 2sirradiance. For chilling treatment, two hours after the beginning of the light period the chamber temperature was dropped to 5C day/ night. Just fully expanded leaves of pea and cucumber were removed at various times before and during the chilling treatment and used immediately for thylakoid isolation. Approximately 3 g of leaves were sectioned into small pieces with a razor blade and placed in 200 ml of homogenization buffer which contained 0.4 M NaCl, 1.0 mm EDTA, 2.0 mg/ml BSA and 20 mm HEPES, pH 7.5. The tissue was homogenized using a Polytron grinder at high speed for 5 s. The resulting slurry was poured through four layers of cheesecloth, and the filtrate was centrifuged at 2000g to remove cellular debris. The length oftime for this centrifugation was kept as short as possible by turning offthe centrifuge timer as soon as 2000g was reached. Thylakoid membranes were then pelleted from the supernatant by centrifugation at 5000g for 4 min. The pellet was resuspended and washed once in a buffer containing 0.15 M NaCl and 20 mM HEPES, pH 7.5. The final pellet was resuspended to a concentration of approximately 1 mg chlorophyll/ml of resuspension buffer. The temperature for the thylakoid isolation procedure was 4°C, except for the initial homogenization step which was performed at 25°C, as described in "Results." Electron transfer rates from H20 to MV3 were determined by measuring O2 uptake using a Clark-type oxygen electrode. The chamber volume of the water-jacketed electrode was 2.0 ml, and temperature was maintained at 25°C. The assay buffer contained 0.1 M sorbitol, 50 mm KCI, 5.0 mM MgCl2, 50 mm HEPES (pH 7.8), 30 ,AM MV, and thylakoids containing 25 to 50 Mg of Chl. Gramicidin (0.2 Mm) was used to uncouple electron transfer from the generation of a pH gradient. A projector lamp was used to 'Abbreviations; MV, methyl viologen; DCCD, dicyclohexylcarbodiimide; CF, chlorophyll coupling factor. 147 www.plantphysiol.org on July 22, 2017 Published by Downloaded from Copyright © 1988 American Society of Plant Biologists. All rights reserved.