TWO PHOTOREACTIONS IN PHOTOSYNTHESIS: EVIDENCE FROM THE DELAYED LIGHT EMISSION OF CHLORELLA.

Following Emerson's1' 2 finding that the efficiency of photosynthesis drops at long wavelengths and that supplementary illumination at short wavelengths eliminates this long wavelength drop, a number of authors have presented evidence that two photoreactions are involved in photosynthesis.3-'0 Since 1951, it has been known that for several minutes after illumination photosynthetic organisms emit light from chlorophyll. Although the physical mechanism of this delayed light production is still uncertain,11-'3 the emission would appear to allow direct measurement of energy conversion processes within the photosynthetic apparatus. The present experiments on living green plants indicate that the delayed light is emitted by functionally active chlorophyll and that the presence of two pigment systems may be inferred from the wavelength dependence of the decay curves. Materials and Methods. The time course of delayed light emission was measured by a pumping method similar to that of Strehler and Arnold.1' Three hundred cc of Knop's medium containing Chlorella was illuminated by 6,500 A light (Farrand model No. 300 visible monochromator with 1,000-watt tungsten source, half band width of 350 A) and by far-red light containing wavelengths longer than 7,000 A (500-watt tungsten source with Corning filters 2030 and 9836). Illumination of the cells could be performed by the two light sources simultaneously or by each source individually. The cells were pumped with a Maisch metering pump from the illumination chamber, through black rubber tubing, and through glass tubing in front of a DuMont K 1292 photomultiplier, which was cooled to the temperature of dry ice. The signal from the photomultiplier was amplified on an electrometeramplifier and recorded on a Brown recorder. Black tubing channeled the cells from the photomultiplier to a stainless steel coil in a temperature control bath, then through the pump, and finally back to the illumination chamber. Decay curves for delayed light could be constructed by measuring the intensity of delayed light at various pump speeds, since the time in dark (from illumination chamber to photomultiplier) depended on the speed at which cells were pumped. The volume of unilluminated cells in the pumping system was less than 5 per cent of those in the illumination chamber. Chlorella pyrenoidosa Chick, strain 252, was obtained from the Culture Collection of Algae at Indiana University. The cells were grown in Knop's solution and aerated with 5 per cent C02-95 per cent air at 18'C under continuous red illumination from neon tubes. Before being placed in the delayed light apparatus, exponential phase cultures were diluted to a standard concentration. This was done by first determining the volumetric concentration of cells in a culture by a hematocrit method in which 10 cc of a culture was centrifuged at 500 X g for 5 min. The volume of cells in a culture wvas read directly from the hematocrit tube, and the culture diluted to a volumetric concentration of 20 mm3/100 cc Knop's solution. This cell concentration absorbed less than 20 per cent of the incident light.