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