Induction of Fluorescence in Quinone Poisoned Chlorella Cells.

Abstract

The efficiency of fluorescence quenchers has been shown to be dependent on the concentration of the fluorescing dye. This effect was attributed to the occurrence of the migration of electronic excitation (6). In view of the special importance of this problem in the theory of the photochemical aspect of photosynthesis, it was found desirable to look at a possible difference between the efficiencies of fluores-cence quenching of chlorophyll by quinone in vitro and in vivo. However, our attention was soon diverted toward a very striking change in the induction of fluorescence upon poisoning of Chlorella cells witlh quinone. Contrary to the impression given by one published observation (9), we found that quinone at moderate concentration did not suppress the induction of fluorescence completely. On the other hand under suitable conditions, the mean fluorescence level is depressed and a new induction course appears, the main feature of it being a rapid rise of fluorescence followed by a slower decay to a steadly state. The general significance of the Hill reaction and related processes is now well established: mechanical separation (isolated chloroplasts) or chemical inhibition (quinone reaction with Chlorella) uncouples the water-photolysis nmechainisnm from the key hydrogen acceptor of the C02 reduction cycle. This must bring considerable simplification in the kinetic picture and might support the hope of untangling some of its components more easily. The present report will try to illustrate this point of view. MATERIAL AND METHODS Algae (CChlorella pyrenoidos) were grown in Knopp solution at 23° C with 2 % C02-air mixture bubbling at the rate of 1.5 1 per minute. Light was supplied by 2 banks of 3 fluorescent tubes (16-watt), 1 bank on each side of the culture flasks. For use, cells were centrifuged and resuspended in phosphate buffer 0.1 M, pH 6.4 plus KCl 0.05 M. A stock solution of purified quinone was added to the suspension giving a quinone concentration of 3 X 10-4M (unless otherwise stated) and chlorophyll concentrations from 0.03 to 0.05 mg per ml. The mixture was incubated for ca. 10 minutes before the measurements were made. The fluorescence was excited with monochromatic light at 670 m,u (half-band width: 13 mnL) from a Bausch and Lomb grating monochromator operated Received March 23, 1959. 2 Present address: Laboratoire de Photosynthese du C.N.R.S. Gif-sur-Yvette (Seine et Oise), France. with a tungsten ribbon lamp. The fluorescent light was passed through a 2nd monochromator of the same type to the detector, an infra-red sensitive photomultiplier tube (Dumont, type K 1292). This 2nd monochromator was set at 725 myA with a half-band of 26 mn. Fluorescence curves were automatically recorded with a galvanometer and a Photodyne recorder (Sefram) ; in several instances, oscillographic recording has been used. The cell suspension was introduced in a narrow vertical glass tube 1.5 mm inside diameter. A 10 mm section of this tube was illumi-natedl with the exciting light which had a maximum incident intensity of ca. 3500 erg/sec cm2 which will be referred to as I = 100. Constant temperature was maintained with a water jacket surrounding the glass tube. The fluorescence measurements were corrected for stray light using a correction factor estimated from measurements of the light scattered by light bleached cells. RESULTS AND DISCUSSION Figure 1 is a typical fluorescence curve, recorded under high exciting light intensity. Letters 0, P and S have been used throughout this report as representing the initial, maximum and stationary levels of fluorescence; they divide the fluorescence curve into 2 main phases: O-P and P-S. We will also make extensive use of the quantities PM, P and s, as defined on figure 1. Several basic observations are to be noted first: 1.) Level S lies only slightly above level 0, and conditions can be chosen that will maximize the ratio pm,/(light intensity) : low temperature and high light intensity. 2.) The illumination can be interrupted for short periods of time during the P-S phase without significantly disturbing the time course of p. 3.) The time curve of p is best described by the equation: 1//p = A + Bt (1) where A and B are constants and t is time. However, the validity of equation 1 is restricted to the end of the P-S phase, the 1st points lying definitely above the straight line (fig 2). 4.) The whole fluores-cence curve can be repeated several times if the preparation is allowed to stay in darkness for a minimum period of time. The minimal interval depended on the temperature. Recording the fluorescence peaks at shorter periods of dark rest gives intermediate PM values, which fall on a smooth curve when plotted as a function of dark time (fig 3). This process will be referred to as the recovery phase.