Activation of the chloroplast ATP-ase measured by the electrochromic change in intact leaves


by

David M. Kramer and Antony R. Crofts, Department of Physiology and Biophysics, University of Illinois, 407 S. Goodwin, Urbana, IL 61801

Summary

The activation of the ATP-ase in intact cucumber leaves has been studied, using a novel instrument developed for the measurement of absorbance changes in intact leaves. The flux through the ATP-ase was measured by using the decay of the electrochromic shift at 515 nm as an indicator of the flux of protons across the thylakoid membrane. Plots of the rate of decay of the electrochromic shift against its amplitude were used to determine the threshold for activation of the ATP-ase. A rapid phase of decay of the electrochromic shift was found above a certain threshold amplitude in control leaves. This rapid phase was eliminated after treatment of the leaves with dicyclohexylcarbodiimide (DCCD), indicating that it was associated with flux through the active ATP-ase. The threshold amplitude of the electrochromic shift was lower in light-adapted leaves than in dark-adapted leaves indicating a lower threshold amplitude for the activation of the ATP-ase. The lowering of the threshold amplitude by light-adaption was eliminated by treatment of the leaves with methyl viologen, which blocks electron flow to the thioredoxin system. These results are interpreted in terms of a model presented by Junesch and Graber (Biochim. Biophys. Acta. 893, 275-288) to explain results from isolated systems. The reoxidation kinetics of the -subunit of the ATP-ase were followed by observing the extent of the slow decay phase of electrochromic shift. These kinetics showed a lag time which was dependent on the amount of light adaptation, and a recovery which did not appear to be dependent on the amount of light adaptation. The kinetics are interpreted in terms of a model consisting of a large redox buffering pool with a midpoint potential somewhat more negative than that of the sulfhydryl groups of the -subunit of the ATP-ase, in equilibrium with a smaller pool equipotential with the -subunit sulfhydryls. Some possible characteristics of this pool are discussed. Our results suggest that the activation of the chloroplast ATP-ase through the thioredoxin system occurs at levels of illumination less than 0.2% of normal actinic intensities, and that this process is never limiting for photosynthesis.