Demonstration of a portable double-flash kinetic spectrophotometer for measurement of electron transfer reactions in intact plants


by

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

Abstract

A highly sensitive, portable spectrophotometer for use in measuring flash-induced absorbance changes in intact leaves is demonstrated. The design of the instrument is modified for whole plant use from that suggested by Joliot and Joliot (Biochim. Biophys. Acta 765, 210-218). The spectrophotometer uses trifurcated light guides to deliver measuring and actinic beams to two comparable areas of the leaf. The measuring beam is provided by a series of short, relatively intense light pulses from a xenon flashlamp in place of the constant weak measuring beam used in conventional machines. The use of a flash measuring beam and differential detection allows for a high signal-to-noise ratio (noise levels of 10-5A) without significant actinic effects. The time resolution of the instrument is 2 usec and the noise level is independant of the experimental time range. The instrument is battery or mains powered, computer operated, and has a liquid crystal display for computer-user interface and dialogue, and to show the kinetic traces graphically. Wavelength selection is provided by interchangable interference filters. The instrument can communicate with a laboratory-based computer, recieving programming information and sending experimental data to be processed and plotted. The instrument is demonstrated by following the kinetics of the electrochromic shift, and of the change in redox states of cytochrome f and the b cytochromes in an intact cucumber leaf, and in the same leaf after infiltration with DCMU.