Plant Physiol. (1976) 57, 440-445
Action Spectrum between 260 and 800 Nanometers for the
Photoinduction of Carotenoid Biosynthesis in Neurospora
crassa
1
2
Received for publication June 6, 1975 and in revised form November 26, 1975
EWARD C. DE FABO, RoY W. HARDING, AND W. SHROPSHIRE, JR.
Radiation Biology Laboratory, Smithsonian Institution, Rockville, Maryland 20852
ABSTRACT
An action spectrum for light-induced carotenoid biosynthesis in Neurospora crassa was determined in 4 to 20 nm steps from 260 to 800 nm.
Four-day, dark-grown mycelial pads of N. crassa were exposed to varying amounts of monochromatic radiant energy and time. After a 48-hour
incubation period at 6 C, carotenoid content was assayed spectrophotometrically in vivo. The action spectrum has maxima at 450 and 481 nm
in the visible range and at 280 and 370 nm in the ultraviolet. A pigment
synthesized by Neurospora whose absorption spectrum resembles the
action spectrum is fl-carotene.
A model for the regulation of carotenoid biosynthesis in N. crassa is
proposed which describes a mechanism by which f8-carotene could act as
a photoregulator. This carotenoid is suggested to be both photoreceptor
for and regulator of carotenoid biosynthesis.
for carotenoid biosynthesis in Neurospora was determined for
wavelengths greater than 400 nm (32). Until now it was not
known what effect light in the UV had on carotenoid production
for Neurospora, and the precise shape of the action spectrum in
the visible was not known.
Since many other biological photoresponses such as phototropism have similar action spectra, it has been suggested that
they are controlled by the action of a single photoreceptor of
ancient origin (4). Therefore, a detailed action spectrum for
carotenoid synthesis in N. crassa may play a role in identifying
this photoreceptor.
MATERIALS AND METHODS
Sources and Dispersion Systems. Two different sources with
interference filter dispersion systems were used, one for the
visible region (400-800 nm) and one for the UV region (260400 nm). For the visible portion of the action spectrum, 1.5-kw
incandescent lamp (Sylvania No. 1500T20/100) interferencefilter monochromators of the type described by Withrow (30)
Carotenoids occur widely throughout nature in plant, animal, were used. Determination of the UV part of the action spectrum
and bacterial systems. Their exact functions are obscure. How- required the modification of a Schoeffel Universal Lamp Housever, carotenoids have been implicated in such functions as ing (Model LH 152N) utilizing a high intensity, 2.5-kw xenon
(Hanovia No. 975C-98) in conjunction with multilayered
photoprotection, photosynthesis, and the "blue light effects" arc
filters (5, 6). Second order transmission, multilayinterference
(16). Of these, protection against the photodynamic effects of
filters (5 x 5 cm) of the Fabry-Perot
light is of major significance. For example, in bacteria, carote- ered, blocked interference
filters in the 300 to 540
All
interference
of
the
were
used.
type
noids with a minimum of nine conjugated double bonds can prenm and peak transmit2
to
3
of
nm
half-band
widths
range
had
cacertain
17).
Specifically,
(16,
lethal
photosensitizations
vent
nm, the half-band
In
260
to
region
the
20%.
tances
of
10
to
rotenoids quench the excited states of singlet oxygen (9). This widths ranged from 7 to 11 nm and 10 to275
20%
peak transmitby
preventing
photosensitizations
against
quenching may protect
with these
are
associated
transmissions
secondary
tances.
Since
the formation of lipid peroxides and free radicals which could
filters, suitable blocking filters were added. For the visible relead to membrane damage (1).
(Eastman Kodak Co.)
Such a photoprotective mechanism may also be at work in gion, blocking filters of the Wratten type
a solution of copper sulfate (30) were used. For the UV
Neurospora. For example, it is known that in dark, aerobically or
region, the reflectance type blocking factor was built into the
grown mycelia of N. crassa, low levels of certain carotenoids can filter
itself and transmitted on the order of less than 10-3 of the
be detected (32). However, light and 02 are needed to trigger
incident
energy outside the bandpass.
the major synthesis of these and other carotenoids, especially
Systems. The general principles of the optical system
those with a greater degree of unsaturation (31). Therefore, in Optical
used for the visible portion have been
those
monochromators
for
when
the
conditions
occurs
since carotenoid biosynthesis
the UV, the optical system indescribed
(30).
previously
photodynamic sensitivity are present, the existence of a well cluded a double quartz lens For
in a lever-operated focusing
system
is
for
carotenoid
mechanism
biosynthesis
controlled, regulatory
It is 7.5 cm in diameter, with an effective aperture of 7.2
sleeve.
postulated.
and a continuously variable arc image magnification
An action spectrum for light-induced carotenoid biosynthesis cm, f/1.1
-1
to
from
infinity. In addition, a filter of circulating chilled
was determined in 4 to 20 nm steps from 260 to 800 nm by an in
to remove most of the IR. The beam from
was
H20
(4
C)
vivo spectrophotometric assay. Previously, an action spectrum the xenon arc, used
after passing through the lens system was reflected by a front surface mirror (20 x 25 cm) into a cabinet
1 This work was supported in part by a predoctoral research fellowship identical to those used in the Withrow system. The exposure area
(beam cross section) for both systems was greater than 15 cm
to E.C.D. from the office of Academic Studies, Smithsonian Institution.
2 This research was presented by E.C.D. in partial fulfillment of Ph.D.
diameter and large enough to irradiate three mycelial pads
D.C.
throughout the entire spectral region used.
simultaneously
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University, from
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Copyright © 1976 American Society of
440Plant Biologists. All rights reserved.
Plant Physiol. Vol. 57, 1976
CAROTENOID BIOSYNTHESIS IN NEUROSPORA
Cultures. Conidia of a seven day stock culture of N. crassa
(wild type, Emerson 5297a) were suspended in sterile, distilled
H20 at a concentration of approximately 107 spores-ml-'. For
each experiment, a 0.1-ml aliquot of this suspension was inoculated into each of 35 to 50 Erlenmeyer flasks (125 ml) containing 20 ml of sterile 2% Vogel's minimal medium (28). The
inoculated flasks were incubated in the dark at 20 C for 4 days.
At the end of the incubation period, three mycelial pads were
poured into the bottom half of a Petri dish (15 cm diameter).
This procedure was carried out under red light previously determined to be noninductive. The three pads were placed in each
plate with the same "top/bottom" orientation as grown and
transported in light-tight containers to the monochromators.
Two or three dark control plates were included for each experiment in order to measure the carotenoid content of noninduced
pads as well as to ensure against any light leaks.
Irradiance Measurements and Exposure Procedure. A typical
experiment consisted of dark controls, two to three sensitivity
controls, and three to nine test plates. The test plates were
subjected to different time exposures at some constant irradiance for each wavelength.
Irradiance was measured with a Hewlett-Packard radiant flux
meter. This meter has a sensitivity of 0.1 ,uv. Iw- Icm-2. It has a
flat spectral response over the wavelength range used and was
calibrated, usually weekly, against a standard lamp (Bureau of
Standards EPI 1060). Incident energy was adjusted to some
constant value of irradiance (0.2 w.m-2 for the visible and 0.05
to 0.2 w-m-2 for the UV) at the center of the plate. Field
uniformity of irradiance in both visible and UV varied 10 to
15%.
Incubation and Assay. Immediately following exposure, the
pads were incubated in the dark at 6 C (11) for 48 hr. Preliminary experiments showed that at least 30 hr at this temperature
were needed for maximal carotenoid formation (about 0.6 absorbance) when measured spectrophotometrically, and this value
remained constant between 30 and 72 hr (5). At the conclusion
of the incubation period, the in vivo absorbance was measured
(Shimadzu MPS-SOL). Three mycelial pads were pressed between two stacks of paper toweling to remove most of the liquid
and permit easy manipulation. The three press-dried pads were
then superimposed upon each other, enclosed between two glass
slides (7.5 x 5 x 0.1 cm) and placed in the spectrophotometer.
Carotenoid content was determined by measuring the absorbance at 480 nm corrected for scatter at 580 nm. This procedure
was repeated at two additional areas of the same pad. These
three absorbance readings were averaged, and from this value
the .nean dark control value was subtracted. This corrected value
is defined as the absorbance difference (£A) (Fig. 1).
Sensitivity Changes. Each time a series of response determinations was made, two to three plates were exposed to a standard
energy (24 Joules-m-2) of 455 nm radiation. A correction for
sensitivity variation was calculated by multiplying the AA for
each response by the ratio of the mean standard response for all
experiments (0.40 0.03 SD) to the standard response for that
particular experiment.
In Vivo versus in Vitro Assay. The in vivo assay method was
441
0.8
I
~~~~~~~I0
0.6
z
0.4
m
0
0.2
0.0
400
500
450
550
600
WAVELENGTH (NM)
FIG. 1. Absorption spectra of light induced (1) and dark-grown (2)
Neurospora crassa mycelial pads.
Table I. Reciprocity Determination
Constant exposure (10.5 Joules-m-2) was maintained at a wavelength
of 455 nm by varying irradiance and time.
Irradiance
Time
(w-m-2)
sec
3.5
0.35
0.035
0.0035
1
3
30
300
3000
Carotenoid Level
A'
0.34
0.35
0.31
0.26
Mean values of four separate experiments.
be valid for irradiances of 3.5 mw.m-2 to 3.5 w-m-2 and for
exposure times of 3 to 3000 sec. In constructing the action
spectrum, the maximum exposure time was 1200 sec, and irradiances were less than 3.5 wm-2.
Response Curves as Function of Incident Energy. Two or
more separate determinations were completed for each point
indicated on all dose-response curves. The best first order linear
regression was calculated and plotted for all of the AA values in
the straight line portion of each response versus log,0 dose curve.
Figure 2 typically represents all such curves determined (10).
Table II gives the slope, intercept, and energy value for the
approximately 50% response level (0.3 A) at each wavelength.
Transmission Measurements. The transmission from 250 to
600 nm of a single thickness of a 4-day, dark-grown mycelial pad
was measured against air in a Shimadzu recording spectrophotometer (MPS-50L) (Fig. 3).
Pad Orientation. The orientation of the mycelial pad is important, since there is approximately a 25 % reduction in the amount
of photoinduced carotenoid pigment if the pads are inverted
prior to exposure. Care was taken to maintain top/bottom orientation to reduce variability of photosensitivity.
developed and used mainly to save time and to avoid the difficulty of completely extracting all carotenoid synthesized. Validity was established by comparing several in vitro determinations
of carotenoid pigment removed from the mycelial pads by exhaustive methanol-acetone extraction (11) to the values determined by in vivo spectrophotometry. The ratio of the in vivo
values to in vitro values was nearly constant (+ 5%) for all
RESULTS
incident energies tested.
Reciprocity. Reciprocity was determined by measuring the
Response as Function of Incident Energy Curves. Representaresponse to a constant exposure (455 nm, 10.5 Joules-m-2) by
tive curves plotted from the line equations are given in Figure 2
varying both time and irradiance (I) (Table I). The irradiance
for 455 to 416 nm. Note that the abscissa is shifted for each
values were changed by either changing distance from the source
wavelength to prevent super-position of the response curves.
or by inserting neutral density filters.
to - Published
The absorbance
value observed for near saturation level of
Reciprocity
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Copyright © 1976 American Society of Plant Biologists. All rights reserved.
DE
442
FABO, HARDING,
spectrum for this response (Fig. 4) allows us to rule out a
porphyrin photoreceptor (3), since there is no photosensitivity
for wavelengths greater than 520 nm.
A comparison of this action spectrum with the absorption
spectra of flavins and carotenoids suggests that the photoreceptor is a carotenoid for the following reasons: (a) many carotenoids have low absorption in the near UV (350-380 nm) (34),
while flavins normally have high absorption in this region (ratio
of absorbance of near UV peak/absorbance of main visible peak
is greater than 0.60) (26), and (b) many carotenoids show two or
three absorption peaks in the visible wavelength range (34),
7
> 0.40
-J
o0.30 50ES O E
z
0.200
0100
2.025
4501
2.
3.0
1
2.5
35 40 45
I
3.0
445i
20
I
35
4.0
45
I
2.5
30
435a
Table II. Slope Values and Incident Energies for 50% Level of
Carotenoid Production as Determined by in Vivo Spectrophotometric
assay
I
4403 1
20 25
35
4.0
1
1
3.0 35
1
20
25
,
3.0
430
2.0
45
1
Equations were calculated by first order linear regression, response =
mx + b
1
4.0 45
,
35
4.0
4.5
Wavelength
,
X
l
25
3.0
35
40
1
1
25
3.0
35
40
416
2.0
2.5
3.0
35
2.0
Slope. m (carotenoid
WvltJoulesvcl/'l)
1
426
Plant Physiol. Vol. 57, 1976
AND SHROPSHIRE
45
x = ()
FIG. 2. Response curves for eight representative wavelength determinations. See Table II for slope, intercept, and energy values for approximately 50% maximum response (0.3A) for all wavelengths. All the
response curves showed a similar tendency towards parallelism.
carotenoid production is approximately 0.60. The 50% response
level was chosen as an absorbance of 0.30 and is indicated by a
dashed line. The energy for this response may be determined
from the abscissa for that wavelength, or can be calculated
directly from the line equations. The first order linear regression
equations for all determinations were calculated for each wavelength, and the slope, intercept, and energy values required for
approximately 50% induction are presented in Table II. Note
that the slope values for wavelengths greater than 335 nm are
nearly the same (0.33 + 0.05). For wavelengths shorter than 335
nm the slope values appear to be smaller.
Action Spectrum. The action spectrum was obtained by plotting the reciprocal of the number of incident quanta required to
produce a 50% response versus wavelength. The reciprocal
value is designated as the relative quantum effectiveness (14).
Figure 4 is the action spectrum with the ordinate value for the
most effective wavelength set at 100.
The highlights of this action spectrum are: (a) the major peaks
in the blue at 450 and 481 nm, (b) the minimum at 463 nm, (c)
the major peaks in the UV at 280 and 370 nm, and (d) the
generally very low response across the UV region.
520
540
6002
7002
8002
Slope mean
Slope SD
I
Dose for 50%
Response
mJoules -cm-
nm
260
265
280
290
305
325
330
335
345
350
355
360
365
370
380
385
390
405
411
416
426
430
435
440
445
450
455
463
467
472
481
485
490
496
500
LOG DOSE (p&j.cm-2)
DISCUSSION
Advantages of in Vivo Carotenoid Measurements. Because of
its time saving aspect, the in vivo method allowed determinations
to be made rapidly and eliminated the problems of exhaustive
extraction of carotenoids in in vitro solutions. Elimination of
both of these factors resulted in a more detailed and precise
action spectrum.
Interpretation of Action Spectrum. Action spectra for photoinduction of carotenoid biosynthesis in nonphotosynthetic organisms suggest either porphyrin-like or flavin versus carotenoid-like photoreceptors (3). Photoinduction of carotenoid biosynthesis in Neurospora is a blue light response. The action
Intercept, b
0.21
0.23
0.25
0.30
0.00
0.00
-
0.00
0.00
-
0.32
0.28
0.30
0.33
0.32
0.34
0.34
0.35
0.31
0.32
0.35
0.32
0.34
0.34
0.33
0.38
0.35
0.34
0.33
0.32
0.34
0.34
0.37
0.33
0.32
0.34
0.33
0.00
0.00
0.00
0.00
0.00
0.32
0.03
-
-
-
-
0.66
0.81
0.76
0.97
0.00
0.00
0.00
0.00
1.01
0.87
0.86
0.92
0.86
0.93
0.97
1.02
0.82
0.86
0.91
0.85
0.88
0.85
0.78
0.90
0.76
0.74
0.71
0.69
0.75
0.73
0.79
0.72
0.74
0.92
0.91
0.00
0.00
0.00
0.00
0.00
44.9
81.7
16.3
19.4
001
001
11.4
15.5
8.29
5.02
4.22
4.03
5.13
5.81
4.24
4.97
3.11
3.48
2.70
2.33
1.85
1.42
1.10
1.05
1.17
1.24
1.17
1.02
0.92
1.23
1.69
3.48
4.35
001
ool
001
001
001
No detectable response
Cutoff filters
(for example, the 600 nm filter transmits all wavelengths greater than 600 nm and excludes wavelengths less than 600
nm).
2
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Plant Physiol. Vol. 57, 1976
443
CAROTENOID BIOSYNTHESIS IN NEUROSPORA
z
0
u)
(n
z
4c
I--
WAVELENGTH (nm)
FIG. 3. Transmission curve measured through one 4-day, dark-grown
mycelial pad of Neurospora crassa.
H
100
90
80
C
9
70
v-60
E
C
50
a
0
* 40
& 30
20
10
0
I
W
I
Il_
I
I
I
I
I
I
I
I
260 280 300 320 340 360 380 400 420440 460 480 500 520 540 600 700 800
Wavelength (nm)
FIG. 4. Action spectrum of photoinduction of carotenoid biosynthesis in Neurospora crassa.
whereas flavins generally have a major peak, with or without
shoulders (26). ,8-carotene, a carotenoid synthesized by Neurospora (31), has an absorption spectrum (34) which is very similar
to the action spectrum for photoinduction of carotenoid biosynthesis in this organism (Fig. 4) (5).
In a previously published action spectrum on light-induced
carotenoid biosynthesis in Neurospora (32), only radiation in the
400 to 500 nm range was used, and no statistically significant
peaks were seen. Zalokar (32) reported, "There was a plateau
between 449 and 488 mg in which analysis of variance did not
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Copyright © 1976 American Society of Plant Biologists. All rights reserved.
4444 DE FABO, HARDING, AND SHROPSHIREPPlant Physiol. Vol. 57, 1976
reveal any particular peak, although the mean values were highest at 480 m,u." Within this region our action spectrum (Fig. 4)
clearly shows two definite peaks, one at 450 nm and the other at
481 nm.
Up until now, no data have been published for the photoinduction of carotenoid pigment in Neurospora by light in the UV
range. Our action spectrum shows 17 experimentally determined
points between 260 and 390 nm. The generally low response
shown in this region is characteristic of the absorption spectra of
carotenoids and not flavins (26, 34).
In the present study, an attempt has been made to correct
specifically for problems involved in interpreting action spectra
(4, 7, 14, 15, 25). The effect of temperature following irradiation
on carotenoid production (11), reciprocity (Table I), and parallelism of dose-response curves (Fig. 2) have been measured. In
order to estimate how much screening of the photoreceptor
could occur in Neurospora, a transmission measurement through
a single dark-grown mycelial pad was determined (Fig. 3). For
wavelengths longer than 300 nm, transmission is greater than
10%. This is significant because if the photoreceptor is within
the upper 10% of the mycelial pad, and the screen is homogeneously distributed, then it can be calculated that 80% of the
incident energy at 300 nm reaches the photorecepter. The distribution of the photoreceptor in the dark-grown mycelial pads is
unknown. Two observations have been made which suggest that
the photoreceptor may be localized on the upper surface. (a) If
part of the lower level of mycelial pad is irradiated, little or no
carotenoid production occurs in the lower portion. (b) If the
dark-grown mycelial pad is inverted prior to exposure to light,
about 25% less carotenoid pigment is formed. Morphological
and biochemical differences between the surface layer and the
deeper layer of mycelial pads have been described (33).
Nature of Photoreceptor. In arguing for a flavin (flavoprotein)
photoreceptor, studies with albino mutants are often cited, since
blue light responses occur even though carotenoid pigments are
present at low levels (18, 24). It has been shown that these
results alone cannot be used to rule out a carotenoid photoreceptor (18, 24).
In recent studies, an in vivo blue light-induced absorbance
change in Neurospora mycelium has been demonstrated (20).
This absorbance change is due to the photoreduction of a b-type
cytochrome. It has not been shown that this reduction is coupled
directly with such physiological responses as carotenoid induction or other blue light responses. So far, the strongest circumstantial evidence consists of action spectrum data. Furthermore,
a flavin has not been demonstrated to be directly involved in the
initial light reaction which leads to photoreduction of the Cyt b.
It could be argued that light, mediated by some compound other
than a flavin, might be activating an enzyme (such as a dehydrogenase) which could subsequently lead to reduction of a Cyt by
means of a flavin.
Another argument cited often in favor of a flavin photoreceptor is the high photoreactivity of flavins versus that of carotenoids. Flavins have been demonstrated in vitro to photosensitize
the oxidation of a wide variety of biological compounds, whereas
carotenoids do not carry out such intermolecular reactions (26).
These intermolecular reactions need not necessarily be the only
way photoresponses are regulated. An alternative suggestion is
that the photoresponse is triggered by a different mechanism,
namely direct photoisomerization of a carotenoid. As some investigators have previously stated, "Surprisingly, practically no
attention has been paid to the possible implications of the intramolecular photodecomposition of riboflavin and the photoisomerization of carotene in the growth response of plants in general and phototropism in particular" (26). Carotenoids are
known to undergo isomerization when exposed to light (34), and
activation of a biological system by photoisomerization is known
to occur in vertebrate vision (29).
One way that a carotenoid could act as a photoreceptor for the
biosynthesis of carotenoids in Neurospora is as follows. Assume
that a small amount of an isomer of ,8-carotene, synthesized in
the dark, feedback inhibits an early enzyme in the carotenoid
pathway. As a result, carotenoid accumulation stops. When this
isomer of 1-carotene absorbs light, formation of a different 18carotene isomer occurs and feedback inhibition is removed. This
new carotenoid isomer, or a metabolic product of it, acts as an
inducer leading to de novo synthesis of enzymes in the carotenoid pathway which are absent (or at low levels) in the darkgrown cultures. These enzymes would continue to synthesize
carotenoids for some time even in the dark. Thus, 18-carotene
would have a dual role: (a) to act as a photoreceptor and (b) to
induce de novo synthesis of enzymes in the carotenoid pathway.
Since inhibitor studies have shown that protein synthesis is
required in Neurospora and other organisms in order for carotenoid production to take place, it was proposed that light
induces de novo synthesis of an enzyme(s) required for carotenoid production (3, 12, 13, 19, 22, 23). It was postulated that the
initial light reaction involves either the production of an inducer
or the direct inactivation of a repressor. 18-carotene has been
shown to be involved in the induction of carotenoid synthesis in
Blakeslea trispora by acting as a precursor of trisporic acids (2)
which are inducers of carotenogenesis in that organism (27), and
recently, in Phycomyces, 18-carotene has been shown to directly
regulate its own synthesis (8). The model proposed in the present investigation emphasizes the regulation by a carotenoid photoreceptor and could be tested by extensive in vitro studies of
carotenoid biosynthesis in Neurospora similar to those carried
out using tomato plastids (21). In support of this model, a recent
report by Gressel et al. (10) is of considerable interest in that it
shows oxygen is not needed for blue light induction of sporulation in Trichoderma.
Acknowledgments - Appreciation is expressed to the shop personnel of the Radiation Biology
Laboratories and to J. Azzara for his technical assistance.
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