maintained between 23° to 24°C, Energies of illumination were as high as 8.5 X 10° erg cm-? sec’ in visible light and 6.9 xX 10° erg cm~® secin red light. At appropriate time intervals, samples were removed and plated on agar as described previously (70). The number of colonies were counted after a 7-day incubation at 26°C under 880 jJumen/m?. Euglena gracilis cells illuminated with white light under aerobic conditions demonstrate a photosensitized oxi- (17). Even after a 90-minute exposure indication that the spontaneous muta- to this intense laser beam, no cell death tion rate was less than 0.00003 percent. could be demonstrated, and MacMillan et al. (17) concluded that the cells did not contain a photosensitizer which could absorb at 632.8 nm. As shown below, however, a broader source of red light can bring about photosensitization in the absence of exogenous photosensitizers in E. gracilis. White mutant colonies developed from cells surviving either high light intensities (8.5 X 10% erg cm-? sec-!) or dation or bleaching of chlorophyll. intensities equivalent to bright sunlight ing process in air consists of an initial No such white colonies were observed when cells were exposed to the optimum light intensity for chlorophyil Under anaerobic conditions there is no loss of pigment (Fig. 1). The bleachlag period followed by a rapid disappearance of chlorophyll. This lag period can be shortened or abolished by the use of 95 percent Oz and 5 percent CO, as the gas phase. These patterns are similar to those described for Chlorella pyrenoidosa (4.5 & 10° erg cm? sec!) (Table 1). syothesis (3 X 10° erg cm-? sec"'}, an deficient bacteria, Sistrom et al. (/2) have suggested that this mechanism involves carotenoid pigments acting as “chemical buffers” to protect cells against photosensitized oxidations. In phenomenon. These white colonies could be subcultured on either agar plates or in liquid medium, giving rise to cells which are identical in appearance to the albino mutants induced by ultraviolet light, heat, or streptomycin (/8). Fluorescence microscopy did not reveal the presence of any red fluorescence, indicating not only the absence of chloroplasts containing chlorophyll but also of proplastids containing protochloro- Table 1. Mutagenic effect of visible light in Euglena gracilis var. bacillaris grown in air. White-light irradiation (11). The lag period presumably represents the time during which the normal protective mechanism is still capable of operating. Based on their studies with carotenoid- High-intensity rad light (> 610 nm) also led to the development of white mutant colonies, a strong suggestion that the endogenous chlorophyll functions as the photosensitizer for this erg cm-* sec7! Cells plated < * * x (No.) 28,000 28,000 0 6 2 5 5 60,000 27,000 27,000 60,000 150 2,000 281 127 75 15 20 63 10° 10° 10° 108 *Red light (> 610 nm) used to irradiate sample. (/4) reduced antheraxanthin to zeaxan- thin at a rate commensurate with the epoxidation of zeaxanthin to antheraxanthin by visible light and O2 (J5). Under conditions of high light intensity, this protective mechanism could no longer cope with the rate of oxidation, and various effects of aerobic photo- sensitivity, including pigment bleaching and death would occur. The duration of the lag period seen in Fig. 1 and the time needed for complete bleaching depend on light tinuous-wave gas laser (29 mw) emitting at 632.8 nm in the absence of exogenous photosensitizers, have failed ao AIR ——— a =e “a ao 100 mel 2 ERcS/cm /SEC meee 8,5 X 10° 80 —-=—=3,0x10° 60 F 407 20 in- tensity, ceil concentration, and the growth phase of the culture. Cells in the early exponential phase bleach more rapidly than do cells in the late exponential phase. Similar results have been reported for photosensitization in Rhodotorula glutinis (16). Previous attempts to photosensitize E. gracilis, with the light from a con- 8 DECEMBER 1967 120F RELATIVE CHLOROPHYLL CONCENTRATION (2) mechanism would only remain effective as long as antheraxanthin deepoxidase (No.) (No.) 6 E. gracilis, Krinsky (13) has proposed that the carotenoids zeaxanthin and antheraxanthin (5,6-epoxy-zeaxanthin) function as protective pigments, This White mutant colonies Hours 3.0 < 105 8.5 4.5 4.5 *6.9 Colonies surviving ~ 0 x i 0 1 1 rt j 2 l j 3 I L 4 I 5 i L 6 HOURS OF FLLUMINATION Fig. 1. The effect of tlumination with high-intensity visible light on the relative chlorophyll content of Euglena gracilis var. bacitlaris cells in the logarithmic phase of growth exposed to a nitrogen atmosphere (MI), air (©), or 95 percent O, and 5 percent CO, atmosphere (X). The control sample (@) was exposed to 3 x 10° erg cm™ sec* white light (the optimum light intensity for chlorophyll synthesis) in an atmosphere of air, whereas the other samples were exposed to 8.5 x 10° erg cmsec”? white light. At the indicated times, samples were removed and analyzed for chlorophyll as described by Arnon (28). The results are expressed relative to the concentration of chlorophyll present at the beginning of the experiment. q 1333