Kinetic Behavior of Electron Paramagnetic Resonance Signal

The electron paramagnetic resonance (EPR) characteristics of wild type Chiamydomonas reinhardtii are compared with these of a mutant strain (ac-206) which lacks cytochrome 553. The steady-state signals I and II are similar but differ in their responses to light of long and short wavelengths, reflecting the fact that the electron transport chain linking photosystems I and II is interrupted. The kinetic behavior of signal I is simpler in the mutant, which lacks induction effects prominent in the wild type. The deoay of the signal when light ceases is not dependent on the length or intensity of illumination in the mutant, ,whereas it is in the wild type. These data can be interpreted in terms of signal I being a reflection of cyclic flow in a pathway which does not involve cytochrome 553 in the mutant, whereas in the wild type there is also a contribution of electrons from photosystem II. Preceding papers (7, 8) have described induction effects in the formation of electron paramagnetic resonance (EPR) signal I in intact algae of 3 species. This signal is attributed to the oxidized form of the photoreac'tive center for system I (P700); its kinetic behav'ior provides a sensitive tool for the observation of the state of P700. It was concluded that the dependence of the rate of rise of the signal on the length of dark time preceding the onset of illumination (induction effect) was liargely a property of photosystem I. This conclusion was, based on the observation that adding 3-(3,4-dichloropheenyl)-1, 1-dimethyl urea (DCMU) to whole cells had little effect on the induction curves, and that mutants of both Scenesdesmus and Chlamydomonas which lacked a functioning photosystem II still displayed prominent induction effects. Vredenberg and Amesz (6) had also found that the rate of bleaching of P700 in intact algal cells was strongly dependent on the length of the preceding dark period and was unaffected by DCMU. This correspondence was one of the bases for the contention that formation of EPR signal I and bleaching of P700 were the same process. Levine and Gorman (2, 4) have reported a mutant of Chlamydomonas reinhardtii (ac-206) which lacks optically or functionally detectable cytochrome 553, which appears to be identical to cytochrome f found in higher plants, and they have kindly made this culture available to us. They have demonstrated that cytochrome 553 is an integral part of the intermediate electron transport chain, but that 1 Paper I of this series is E. C. Weaver, Photochem. Photobiol. 7: 93-100 (1968). it is not essential to NADP photoreduction when the source of electrons is reduced 2,6-dichlorophenolindophenol. However, the absence of cytochrome 553 isolates the 2 photosystems from each other and we have demonstrated how this fact is reflected in the steady-state signal I and in its kinetic behavior. Materials and Methods Wild type (Sager's 21 gr) and mutant ac-206 strains were grown in shake culture on mineral mediumii supplemented with 0.5 g per liter sodium acetate and 0.25 g per liter yeast extract. The temperature was 250 and light intensity 7000 lux. The cells were harvested after 5 or 6 days of growth and suspended in tris-HCl, 0.05 M, pH 7.2. Photosynthetic competence was tested with a Yellow Springs Instruiment Company (YSI) Biological Oxygen Analyzer, Model 53. Chlo,rophyll was determined in a Cary 14 spectrophotometer in an 80 % acetone extract, using the method of Arnon (1). EPR techniques were the same as those described previously (6), although a new instrument (Varian 4500) with a 9-inch magnet was employed at X-band. A modulation amplitude of 4.45 gauss was routinely employed. The response time used was 1 sec for field scans, which were made at a 'rate of 20 gauss per min, and 0.1 to 0.01 sec for kinetic traces. During the latter, a larger modulation amplitude (10.5 gauss) was occasionally used to enhance signal-to-noise ratio. Light was provided by a system consisting of 2 separate lamps, described in detail elsewhere (5), whose output could be superimposed. Baird-Atomic interference filters (type B-1) with 10 nm width at half transmission height isolated desired spectral bands; 703 nm was 1538 www.plantphysiol.org on January 30, 2018 Published by Downloaded from Copyright © 1969 American Society of Plant Biologists. All rights reserved. KINETICS OF EPR SIGNAL I IN Chlamydomonas used to preferentially excite photosystem I, 633 nm for photosystem II. For saturation studies, a wider interference filter (type B-3) was used with 30 nm half width centered about 680 nm. Calibrated neutral density filters (Special Optics) attenuated the light intensity, which was measured in the EPR cavity with a Yellow Springs Instrument Company Radiometer, model 65, fit-ted with a special probe designed to fit in the cavity. Solenoid-activated shutters were either controlled by an automatic timing mechanism or manually switched. No computer averaging was employed.