Photooxidation by Photosystem II of Tris . washed Chloroplasts ' Takashi

Irradiation of tris-washed chloroplasts with moderate intensities of red light caused a partial bleaching of chloroplast pigments and an inhibition of the hydroquinonesupported photoreduction of NADP. The presence of an electron donor for photosystem 2 (PS2) during the irradiation prevented the bleaching and inhibition. It is concluded that the strong oxidant produced by PS2 accumulates in tris-washed chloroplasts during irradiation and an electron donor for PS2 protects against the photooxidation reactions. \Ve have shown previously (4, 5, 6) that washing chloroplasts with tris buffer inhibits electron transport between water and PS2 and that a number of reduced compounds, e.g., PD2 and HQ can restore plhotosyntlhetic electron transport by donating electrons to PS2. It was noted in experiments in which ferricyanide was the electron acceptor for tris-washed chloroplasts (6) that irradiation in the absence of an electron donor resulted in an initial decrease in the absorbance ratio. A420/A480 used to monitor ferricyanide photoreduction. Subsequent investigation revealed that this absorbance change was due to a bleaching of the chloroplast pigments. The work reported in the present paper continues from that observation and shows that irradiation of tris-washed chloroplasts by moderate intensities of red actinic light in the absence of an electron donor svstem caused bleaching of chlorophyll and carotinoid pigmeents, an inhibition of NADP photoreduction in the presence of PS2-specific electron donor and a loss of the fluorescence of variable yield. These destructive effects of actinlic irradiation were all prevented if an electron donorsystem was present during the irradiationi. It is concluded that the destructive effects are due to the strong oxidant produced by PS2 which tends to accumulate during irradiation in the absence of ani electron donor. 'This work \vas supported by USPHS, National Institutes of Health Grant GM-15048 and a Charles F. Kettering Research Award. 2 Abbreviations: PD, p-phenylenediamine; HQ, phydroquinione; PSI and PS2, photosystem I and photosystem II: DCMU, 3(3,4-(ichlorop)lhenyl) -1,1-dimethylurea: DCPIP, 2,6-dichlorophenol-in(lophenol. Experimental Procedures Chloroplasts were prepared by grinding 50 g of spinach leaves in a Warinig Blendor for 20 sec in 150 ml of solution containing 0.4 M stucrose, 0.05 M tris-HCl (pH /.8), and 0.01 M NaCl (abbreviated as STN solution). The stupernatant from a 1-min centrifugation at 300g was centrifuged again at 600g for 7 min in 4 centrifuge tubes. The pellet from 2 tubes was suspended at 0° in 20 nil of 0.8 M tris-TICI (pH 8.0) to about 0.1 nig clil/mil (tris-waslhed chloroplasts). The pellet from the otlher 2 tubes was suspended in 20 ml of STN solution (intact cliloroplasts). After standing for 10 min, lheavy particles of botlh batches of chloroplasts were removed by centriffugation at 3OOg for 1 min. The remaining supernatants were centrifuiged at 15OOg for 7 min and the precipitates were resuspended in 2 ml of STN solution. Measurenments were made with chloroplasts suspended in a standard reaction medium consistinig of 15 mM tris-HCl, 4 m-i KPO4, 1 mM ADP, 4 maI MgCl.,, and 20 imM NaCl, pH 7.8. Chlorophyll concentration was determined by the method of MacKinney (2). Ferredoxin was purified from spinach leaves; DCMU donated by Dr. P. G. Heytler was used as a methanolic solution. ADP and NADP wvere obtained from Calbioclhem Company. p-Hydroquinone was obtained from Aldrich Chemical Company; DCPIP, from tlle La Motte Chemical Products Company. NADP photoreduction was measured under aerobic conditions withl an Aminco-Clhance dual wavelength spectrophotometer witlh monitoring wavelengths of 340 and 370 nm; ferricyanide pliotoreduction was measured vith monitoring wavelengtlhs of 420 and 480 nm; carotenoid photobleaclhing with wavelenigths of 490 and 530 nm. The EMI 9524 1342 www.plantphysiol.org on October 23, 2017 Published by Downloaded from Copyright © 1969 American Society of Plant Biologists. All rights reserved. YAMASHITA AND BUTLER-PHOTOOXIDATION BY PHOTOSYSTEMI II phototuibe was blocked with Corning filters 5840 and 5970 for the NADP measurement and with 2 Corning 9788 filters and an Optics Technology 600 nm short pass cut-off filter for the ferricyanide and carotenoid measurements. The fluorescence-yield measurements were the same as those described previously (5). The actinic light was obtained with a Unitron LKR microscope illuminator and Baird Atomic interference filters (approximately 10 nm lhalf band width), with additional infrared blocking filters. Photobleaching and photoinhibition experiments were carried out by irradiating chloroplasts (10 ug chl/ml suspenided in the standard reaction medium) in 1-cm cuvettes with 680 nm actinic light at 2 X 104 ergs cm-2 sec'1 except where other intensities are noted. 500 600 WAVELENGTH nm FIG. 1. Difference spectra (A through D) of irradiated vs. non-irradiated chloroplasts (10 Ag chl/ml). 10 wain irradiation with 680 nm light 2 X 104 ergs cm-2 sec-1 of A) tris-washed chloroplasts; B) tris-washed chloroplasts in the presence of 200 AM HQ and 330 4AM ascarbate; C) tris-washed chloroplasts in the presence of 1 AM I)CMU; D) intact chloroplasts; E) difference spectrunm of intact chloroplasts treated with 10-4 M K,lrCl. in the dark vs. nontreated. *00 .04 Tris-Washed (Suspension) .00 Intact (Acetone Ext.