Photophosphorylation after chilling in the light : effects on membrane energization and coupling factor activity

The response of in situ photophosphorylation in attached cucumber (Cucumis sativus L. cv Ashley) leaves to chilling under strong illumination was investigated. A single-beam kinetic spectrophotometer fitted with a clamp-on, whole leaf cuvette was used to measure the flash-induced electrochromic absorbance change at 518 minus 540 nanometers (ΔA_518−540) in attached leaves. The relaxation kinetics of the electric field-indicating ΔA_518−540 measures the rate of depolarization of the thylakoid membrane. Since this depolarization process is normally dominated by proton efflux through the coupling factor during ATP synthesis, this technique can be used, in conjuction with careful controls, as a monitor of in situ ATP formation competence. Whole, attached leaves were chilled at 5°C and 1000 microeinsteins per square meter per second for up to 6 hours then rewarmed in the dark at room temperature for 30 minutes and 100% relative humidity. Leaf water potential, chlorophyll content, and the effective optical pathlength for the absorption measurements were not affected by the treatment. Light- and CO₂-saturated leaf disc oxygen evolution and the quantum efficiency of photosynthesis were inhibited by approximately 50% after 3 hours of light chilling and by approximately 75% after 6 hours. Despite the large inhibition to net photosynthesis, the measurements of ΔA_518−540 relaxation kinetics showed photophosphorylation to be largely unaffected by the chilling and light exposure. The amplitude of the ΔA_518-540 measures the degree of energization of the photosynthetic membranes and was reduced significantly by chilling in the light. The cause of the decreased energization was traced to impaired turnover of photosystem II. Our measurements showed that the chilling of whole leaves in the light caused neither an uncoupling of photophosphorylation from photosynthetic electron transport nor any irreversible inhibition of the chloroplast coupling factor in situ. The sizeable inhibition in net photosynthesis observed after chilling in the light cannot, therefore, be attributed to any direct effect on photophosphorylation competence.