Received for publication May 4, 1989
and in revised form August 9, 1989
Plant Physiol. (1989) 91, 1586-1593
0032-0889/89/91/1 586/08/$01 .00/0
A Novel Effect in Phycomyces
Phototropism'
Positive Bending and Compensation Spectrum in Far UV
Teodor Popescu, Andreas Roessler, and Leonid Fukshansky*
Institute of Biology I1, University of Freiburg, D-7800 Freiburg i. Br., Federal Republic of Germany
tive to negative tropism has been explained in terms of optical
attenuation of stimulus (1): the gallic acid located in the
vacuole strongly absorbs below X = 305 nm, preventing the
formation of focal bands on the distal side within this spectral
region.
Blue light-induced growth of sporangiophores shows adaptation, which is manifested in different ways depending on
the symmetry of irradiation. Under symmetrical irradiation,
the increase of growth rate due to increase of intensity is only
transient and is followed by complete adaptation (4). In
contrast, the phototropic bending under unilateral irradiation
never adapts (7). This apparent paradox can be resolved only
if a nonlocal signal processing is assumed (9), i.e. the local
growth rate is determined not by the local light intensity
alone, but by the entire light distribution within the sporangiophore. In spite of nonadapting bending, phototropic adaptation and its kinetics can also be demonstrated under
unilateral irradiation. The lag phase (phototropic latency) of
bending under unilateral test irradiation applied after
symmetrical adapting preirradiation has its minimum when
the intensities of test and adapting irradiation are equal (1,
10).
Certain evidence, especially from genetic studies (10, 1),
suggests that the mechanism of adaptation involves at least
two components, one of which is at the level of the photoreceptor, the other at the level of the expression mechanism of
the growth reaction (12). The dichroic photoreceptor (16) is
located at the plasma membrane (22) and is most probably a
flavoprotein (18). In the last few years various results have
accumulated, suggesting that the photoreceptor may be not a
single pigment and that some additional light-induced reactions mediated by their own photoreceptors can interfere with
the transduction chain.
The above inferences have been drawn from comparative
studies of response kinetics in different intensity ranges, studies of photogravitropic equilibrium (in a unilaterally irradiated
sporangiophore the light signal is counteracted by the gravitropic stimulus, resulting in an intensity-dependent equilibrium angle of bending), phototropic balance (vertical growth
under bilateral irradiation with two compensating intensities
of different wavelengths, one of them experimental; the other,
reference wavelength) and dynamics of adaptation, investigated by means of the phototropic latency method, described
above. The most important findings are: (a) action spectra of
photogravitropic equilibrium differ from those of phototropic
balance (1 1); (b) the shape of the phototropic balance action
ABSTRACT
A novel effect-positive phototropic bending under far UV
irradiation (between 260 and 305 nanometers) at low intensitiesis reported. Natural compensation points (intensities which cause
no bending under unilateral irradiation) have been determined for
different wavelengths. The curve connecting these points, the
compensation spectrum, divides the intensity-wavelength plane
into areas of negative and positive tropism. It is further shown
that a highly asymmetrical pattern of light stimulus within the
sporangiophore underlies the symmetrical growth response at
each compensation point. This suggests that some unknown
additional factor is involved in perceiving a UV stimulus at the
level of the photoreceptor. It is also demonstrated here that
positive tropism in the UV range is due to a lens effect. We
conclude that the hypothesis of optical attenuation of the stimulus
(considered until now as the most plausible explanation of negative tropism in the UV spectral range) must be dismissed. The
results presented here represent the first application of our quantitative theoretical consideration of spatial factors in phototropism
heretofore neglected by others.
Phototropism, directional growth under asymmetrical irradiation, has been studied in most detail on giant sporangiophores of the fungus Phycomyces (2). Under unilateral irradiation two strong focal bands occur on the nonirradiated
(distal) side of the cylindrical body of the sporangiophore.
This lens effect somehow promotes the growth rate on the
distal side to a level higher than that of the proximal side,
which causes bending toward the light source (positive tropism). The decisive role of the lens effect has been proved by
immersion of sporangiophores in media with different refractive indices (26). With increased external refractive index, the
lens effect as well as phototropic bending decrease until, at
some critical value (slightly below the refractive index of
sporangiophore), the residual lens effect is counterbalanced
by attenuation within the sporangiophore, and the phototropic bending is resumed (compensation point). Any further
increase in refractive index results in a negative bending
response. As discovered by Curry and Gruen (3) in the range
below X = 305 nm, even sporangiophores placed in air show
negative phototropism. At X = 305 nm no bending occurs
(the natural compensation point). This switching from posi' Supported by the Deutsche Forschungsgemeinschaft (SFB 206
Projekt D5).
1586
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A NOVEL EFFECT IN PHYCOMYCES: POSITIVE UV-PHOTOTROPISM
spectrum depends on the reference wavelength and intensity
range (13); (c) phototropic dark adaptation kinetics depend
on wavelength (14); (d) action spectra of different characteristics of response kinetics are different in high and low intensity ranges (15); (e) antagonistic interaction is seen between
subsequently applied short pulses of low intensity in mediating the photogravitropic reaction (17).
Unfortunately, a straightfoward interpretation of these findings in terms of multireceptor systems appears premature
until important spatial factors are accounted for. Neither the
light perception nor the signal processing in phototropism is
concentrated in a point or spread homogeneously in space.
The phototropic signal processing appears to be a sequence
of four inhomogeneous spatial distributions emerging one
from another as follows. (a) First, the light distribution within
the sporangiophore results from contributions of a lens effect
and other effects accompanying light propagation. The fact
that under unilateral irradiation the distal side, which receives
less light due to attenuation within sporangiophore, grows
faster (light promotes growth in Phycomyces) implies a decisive role for the shape of the light distribution. Indeed, immersion in a medium with a higher refractive index, which
changes only the shape of the light distribution, can invert
the relationship between the growth rates of the distal and
proximal sides. (b) On the basis of the light distribution, the
distribution of excitation (energy absorbed per second)
emerges at the location of the photoreceptor. Since the receptor is located at the plasma membrane (22), one can speak of
one-dimensional light and excitation profiles at the circular
boundary of the cross-section of a cylindrical sporangiophore.
A dichroic photoreceptor is characterized not only by an
absorption coefficient but also by two angles, polar and azimuthal, specifying the direction of the transition dipole, which
is wavelength dependent. Since the light energy absorbed
depends on the orientation of the transition dipole, one can
easily conclude that neither the shape of an excitation profile
must coincide with that of the light profile, nor are the shapes
of the excitation profiles at different wavelengths identical. (c)
On the basis of the excitation profile, the adaptation profile
emerges. (d) The interaction of excitation and adaptation
profiles brings about, in the course offurther signal processing,
the profile of potential growth rates. The measured phototropic bending is the result of superposition of the profile of
potential growth rates by two constraints, one of which arises
from the topological continuity of a cylinder, the other from
the elasticity of the cell wall.
The picture of spatial transformations is further complicated by nonlocal signal processing (see above). This means
that some steps of the phototropic mechanism are spatiotemporal processes: specifically, they involve communication
between different azimuthal points.
This qualitative consideration of spatial factors leads directly to two questions: what errors are introduced when the
spatial factors are neglected, and what can be gained when
they are treated quantitatively? We were able to give some
answers to these questions after developing the optical theory
of light profiles (21). Profiles were then calculated on the basis
of the measured optical parameters of sporangiophores (9,
23). We believe now that the phototropic action spectra
1 587
cannot be interpreted in a classical way; furthermore, our
data indicate that even for a single oriented photoreceptor the
adaptation cannot be expected to have the same kinetics
under symmetrical and asymmetrical irradiation, as well as
under irradiation with different wavelengths. It has been
shown that a single oriented photoreceptor molecule can also
mimic other features which are considered as evidence for a
multipigment receptor: the action spectra of phototropic balance can be wavelength and intensity-dependent and can
strongly deviate from the action spectrum of photogravitropic
equilibrium (9). At the same time, a special procedure has
been developed for extracting information about an oriented
photoreceptor from special balance experiments and profile
calculations (9). Furthermore, profile calculations applied to
old immersion experiments by Zankel et al. (26) delivered
the proof that the photoreceptor is located at the plasma
membrane (22) by demonstrating that a photoreceptor associated with the tonoplast (the alternative hypothesis) would
be unable to sense changes in external refractive index, which
in fact do cause switching from positive to negative bending.
In this paper we report new findings made feasible by
quantitative treatment of light and excitation profiles: positive
tropism in the far UV region, and some evidence in favor of
a separate UV receptor.
MATERIALS AND METHODS
Strain and Culturing
Wild-type Phycomyces (strain NRRL 1555) were grown on
a minimal medium: potato dextrose agar with 1.5 Ag thiamine/mL medium added at 60°C after sterilization (1.2 atmosphere, 20 min at 120°C). The stock spore solution was
diluted to 50 spores per mL, heat shocked for 15 min at 48°C,
and inoculated into vials 4 cm high and 1 cm in diameter (50
AL per vial).
After inoculation, the vials were kept in darkness and then
placed in a constant temperature growing room (T = 22 ±
1°C, humidity 60%) under broadband blue light (3 x 20 W
Osram L 25 fluorescent tubes with PVC filters Xmax = 500
nm, AX = 100 nm, intensity 0.17 W m-2) for 5 to 7 d. Only
IV/B stage sporangiophores (1), preselected with respect to
their growth rate (more than 20 Am/min-'), were used.
Phototropic Measurements and Data Processing
Experiments were carried out at a constant temperature 23
± 2°C, in a setup especially designed for kinetic testing of
Phycomyces phototropism (for detailed description see [19]).
This construction permits continuous automatic monitoring
of bending angle as a function of time (real-time processing).
For measurements of light induced growth and phototropic
responses, a vial containing a single sporangiophore is
mounted on a turntable with arrangement providing rotation
(4 rpm) and vertical passage adjusted by the computer to the
growth rate of the sporangiophore.
A computer-controlled CCD video camera transfers images
of the sporangiophore to the monitor and digitizer, where
bending is calculated according to a specially designed program. The system provides automatic control and observation
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1 588
POPESCU ET AL.
of experiments over a long time and produces a large amount
of data which can be quickly computerized, assuring good
statistics. In all experiments, the irradiation light beam was
horizontal, i.e. the beam angle with respect to the long axis
of sporangiophore is maintained at 90°C.
Growth rates were measured by means of a horizontal
microscope equipped with an ocular scale. In the 'immersion
type' experiments, the sporangiophore was placed upside
down in a special quartz cuvette 2 x 2 x 4 cm filled with a
liquid which permits the normal growth ofthe sporangiophore
(for many hours) and has a refractive index close to the
refractive index of cytoplasm.
Actinic Light Sources
The values of actinic light intensity will be indicated where
used. For UV irradiation a deuterium lamp (H3 ODS, Zeiss)
with a stabilized adjustable power supply and appropriate
interference filters has been applied. However, in experiments
with weak monochromatic UV fluxes, like those reported in
this paper, one cannot rely on a conventional combination of
a lamp and an interference filter. A standard transmission
curve of an interference filter shows some background energy
and secondary peaks (up to 10-2 of the main peak) located
outside the band nominally selected by the filter. The spectral
distribution of irradiation produced by the combination of
this filter with the standard deuterium lamp is not suitable
for spectrally specified phototropic irradiation in UV area. A
photoreceptor as little as 1O' times more sensitive outside the
selected band can cause a measurable effect due to this
background radiation. To suppress more strongly the background radiation we used interference filters in combination
with a special liquid absorption filter consisting of a cm
high cylindrical quartz tube filled with an aqueous solution
of different chemicals. The chemical and its concentration,
C, are chosen according to the required wavelength range and
intensity (in all experiments reported in this paper, 4-aminoantipyrine has been applied). The spectral transmission, T,
(X), of the liquid filter is calculated according to the formula
T, (X)
exp[-t (X) *c/c0]
where M(X) is the absorption experimentally determined at the
concentration c0, and c is the concentration used. Usually c0
< c, since measurements performed with the standard equipment, a Uvikon 930, are much more exact at higher intensities
(lower concentrations). Because the liquid filter is insufficiently stable over longer time intervals, we carried through a
spectroscopic check before each application and also used
each filter no longer than 2 d. This was necessary because
measurable deviations in absorption could sometimes be registered 4 to 5 d after preparation of the solvents. The spectral
energy density ofthe actinic light source, dw, is then calculated
as a product of spectral characteristics of its components:
dw
=
=
K.E(X, I).- Tl(X)- Tf(X)-d
(1)
where E(X, I) is the spectral energy distribution of the lamp
(depending also through the discharge temperature on the
electric current), Tf(X) is the spectral transmission of the
interference filter, and k is a factor depending on the irradia-
Plant Physiol. Vol. 91, 1989
tion geometry. This factor was elucidated as usually by means
of more precise measurements in high intensity range. The
data are provided by a Tektronix J 16 digital photometer
fitted for the UV range with a photodiode (S 1337 BQ
Hamamatsu) calibrated with an Optronic 742 photometer.
The interference filters type UV-M-IL (Schott, Mainz,
FRG) used had the following characteristics (according to the
manufacturer):
The 260 nm wavelength filter had a half-bandwidth of 15
nm and 20% transmission; 277.2 nm wavelength, 11 nm halfbandwidth and 22% transmission; 291 nm wavelength, 17
nm half-bandwidth and 18.5% transmission; and 298 nm
wavelength, 13 nm half-bandwidth and 18% transmission.
The spectral distributions of actinic light calculated according to formula (1) for these interference filters and a special
adjusted liquid filter are shown in Figure 1. The maxima of
these distributions are slightly shifted as compared to those of
the interference filters used. From formula (1) we can estimate
what fraction of energy is contained in any given interval
around the maximum (see legend to Fig. 1). Even more
important, the background radiation of these combined light
sources in the blue is supressed to a level much below the
phototropic threshold; thus, any artefact due to the blue
background radiation is completely excluded.
Optical Characteristics and Profile Calculations
Refractive indices and attenuation coefficients of vacuole
and cytoplasm were measured as described previously (23).
Refractive indices in the UV are obtained by extrapolation
from the visible region. Attenuation coefficients are in good
agreement with the global absorption of sporangiophores as
measured by Jesaitis (16) and by Wolken (25) (transmission
of the order of 10-15% at the absorption maximum at X =
280 nm) and in disagreement with global transmission of only
1% estimated earlier by Delbruck and Shropshire (5).
The refractive index of fluorocarbon (FC-43 totally fluorinated tributyl amine) applied in immersion experiments was
measured with an Abbe-Refractometer equipped with a special prism designed to measure refractive index in the interval
1.17 to 1.56. The refractive index was measured in monochromatic light (monochromator GM 252) in the visible and
extrapolated into the UV according to Hartmann's relation:
n = no + c-(X -o)a
where no is the refractive index measured at the wavelength
Xo. This relation is valid outside the domain of absorption
maxima-normal dispersion domain.
Profile calculations were performed on the basis of theory
developed in Steinhardt and Fukshansky (21) with the help
of programs implemented on the Sperry-Univac computer in
the Freiburg University Computer Center.
RESULTS AND DISCUSSION
Natural Compensation Point Is the Only Case of
Symmetrical Vertical Growth under Strongly
Asymmetrical Spatial Pattern of Light Stimulus on the
Plasma Membrane
For any irradiation geometry the light distribution within
sporangiophores can be calculated on the basis of the meas-
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A NOVEL EFFECT IN PHYCOMYCES: POSITIVE UV-PHOTOTROPISM
1589
E
U)
0-
240
280
320
240
280
320
240
280
320
240
280
320
Wavelength [nm]
Figure 1. Spectral energy distributions of four UV-sources constructed as a combination of deuterium lamp with interference and liquid filters.
a, A = 263nm (interference filter at A = 260 nm). Total transmitted energy per second (with 200 mA current in the lamp power supply) (5.3 ±
0.5). 1 -6 J * m-1. Fractions of transmitted energy per second in different intervals: between 240 and 290 nm, (5.0 ± 0.5). 10-6 J * m2; between
305 and 350 nm, (5.7 ± 0.5).10-9 J. m-2 (below the threshold for blue-light phototropism for light grown sporangiophors). b, A = 281 nm
(interference filter at A = 277 nm). Total transmitted energy per second with: 80 mA current in the lamp supply, (2.4 ± 0.2)- 1 0-6 J * m2; 100 mA
current in the lamp power supply, (2.7 ± 0.3).10-6 J.m-2; 200 mA current in the lamp power supply, (4.6 + 0.5). 10-6 J-. m2. Fractions of
transmitted energy in different intervals for 100 mA current: between 250 and 300 nm, 98%; between 305 and 350 nm, 0.6%, i.e. (1.7 ± 0.2).
1 0-8 J-. m2. c, A = 301 nm (interference filter at A = 291 nm). Total transmitted energy per second with: 100 mA current in the lamp power
supply, (5.6 ± 0.6). 1 0-5 J - m2; 260 mA current in the lamp power supply, (1.2 ± 0.1). 1 0-4 J m2; 440 mA current in the lamp power supply:
(1.9 ± 0.2). 1 0-4 J * m. Fractions of transmitted energy in different intervals for 260 mA current: between 280 and 320 nm, 99%; between 305
and 350 nm, 33%, i.e. (2.0 ± 0.2). 1 0-5 J * m2. d, A = 304 nm (interference filter at X = 298 nm). Total transmitted energy per second with: 100
mA current in the lamp power supply, (5.1 ± 0.5). 10-4 J. m-2; 250 mA current in the lamp power supply, (1.0 ± 0.1). 10- J. m-2; 400 mA current
in the lamp power supply, (1.6 ± 0.2). 1 0-3 J. m2. Fractions of transmitted energy in different intervals for 250 mA current: between 290 and
330 nm, 99%; between 305 and 350 nm, 53%, i.e. (5.7 ± 0.6). 10-3 J. m-2.
ured refractive indices and attenuation coefficients of vacuole
and cytoplasm (21, 23), as shown in Figures 2 and 3. This
calculation takes into account light focusing, rays crossing,
external reflection, multiple internal reflections, interference
and, if necessary, polarization effects. Figure 2 gives a graphic
representation of the propagation of the incoming parallel
rays within the horizontal cross-section of a vertical sporangiophore and indicates the positions of the irradiated, or
proximal, side (00 < (P < 180°) and nonirradiated, or distal
side ( 1800 < P < 3600) on the azimuthal scale used throughout
the following discussion. In Figure 3a the light profile on the
periphery of the cylinder (at the plasma membrane) is plotted
as intensity versus azimuth (P. The azimuthal scale here is
from 0 to 180°; this corresponds to the proximal side of the
sporangiophore. The dashed line presents the proximal part
of the light profile. The continuous line presents the distal
part of the light profile mapped onto the azimuthal interval
of the proximal side so that the ordinate of the continuous
line in a point (P shows the intensity at the corresponding
point on the opposite side of the sporangiophore, i.e. at point
2ir - 'P. Figure 3b shows, in the same way, the excitation
profile obtained from the light profile shown in Figure 3a if a
transversely tangential orientation of the transition dipole is
assumed. When distal and proximal parts of a profile are
-IT
*900
no
2700
Figure 2. Picture of ray propagation within sporangiophore. The
proximal side corresponds to the azimuthal interval (0-1800), distal
side-to the interval (180-3600). The unilateral irradiation comes
under azimuthal angle 900 (arrow).
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Plant Physiol. Vol. 91,1989
POPESCU ET AL.
1 590
0
rO VI
b l+,.
to
b
0
c
0
co
L
L
C.)
0
w
L
L:
L
..
0n
X
ED
I-
CD
n
CD
a:
Ez
L
tY
0
'-C
z
E_
z
0.
A
.--, 1 I; AlI
\8\
(
>
E\ zZ
z
I
AZ I MUTH
( V)
45
90
135
190
AZ I MUTH ( P)
AZIMUTH (|Q)
Figure 3. Light and excitation profiles in a sporangiophore. a, Light
profile. The continuous curve shows the distal side; the dashed curve,
the proximal side. This profile calculated for X = 305 nm (the natural
compensation point) has a similar shape to profiles for unilateral
irradiation at different wavelengths in the blue-light interval. b, Excitation profile emerging from the light profile shown in Figure 3a,
assuming the transversely tangential orientation of the transition
dipole.
mirror images of one another, the dashed and continuous
curves coincide completely. We say in this case that the profile
shows absolute distal-proximal symmetry. Obviously, the degree of distal-proximal symmetry is closely connected to the
phototropic response. More specifically, in all cases when no
bending occurs, one should expect a high degree of such
symmetry. This has also been observed in profiles for bilateral
irradiations in the blue spectral range providing phototropic
balance (23). Another case of a profile with a high degree of
distal-proximal symmetry underlying symmetrical growth is
shown in Figure 4. This profile, calculated for the conditions
of the compensation point under unilateral irradiation with X
= 510 nm of a sporangiophore immersed in an external
medium with a critical refractive index, has almost coinciding
central (the most important) parts of the distal and proximal
curves. The only case where symmetrical growth is accompanied by an extremely asymmetrical light profile is the
natural compensation point in the UV at X = 305 nm (20).
This is the profile shown in Figure 3. One glance at Figure 2
reveals why the profile in Figure 3 has two strong focal bands
on the distal side, in obvious contradiction to the optical
attenuation hypothesis: the most irradiation contained in
focal bands passes by the strongly absorbing vacuole. One can
argue that the attenuation coefficient and diameter of the
vacuole are not exactly measured and that a small increase of
these parameters will remove the focal bands. This argument
does not work, as Figure 5 shows that even for negative
bending (at X = 280 nm) there are still large focal bands, in
spite of assuming an increased vacuole diameter and an
attenuation coefficient that is 100 times stronger than measured. Another argument might be that the excitation profile,
which depends on the (unknown) orientation of the transition
dipole, possesses at X = 305 nm the required level of symmetry
AZ I MUTH
Figure 4. Light (a) and excitation (b) profiles calculated for a sporangiophore immersed in fluorocarbon with refractive index n = 1.312
and irradiated unilaterally at A = 510 nm. Under these conditions no
bending occurs (compensation point). Both profiles show a high
degree of distal-proximal symmetry. (The excitation profile is calculated here for the transversely tangential orientation of the transition
dipole).
absent in the light profile. However, calculations of excitation
profiles based on the light profile from Figure 3 and three
extremely different assumed orientations of the transition
dipole (longitudinal, transversely radial, transversely tangential) exclude this possibility (one of these profiles is shown in
Figure 3b). Thus, the theory challenges the optical attenuation
hypothesis and calls for an alternative explanation of the
natural compensation point.
Positive Tropism in the UV Range and the Compensation
Spectrum
At this point the evident question is whether the natural
compensation point may result from counteraction of two
photoreceptors: the 'regular' blue-light receptor providing positive tropism and a novel UV receptor which mediates negative bending. This working hypothesis appears reasonable,
because first, the absorption of riboflavin continues down to
240 nm, and second, a UV receptor not located at the plasma
membrane escapes the influence of the lens effect and can
promote negative bending due to light attenuation. In this
context one should also remember that the response kinetics
of positive and negative bending differ considerably. As our
measurements show, in accordance with results of other
workers (5, 6), negative bending is 4 to 6 times faster
and its lag phase lasts only about 2 to 3 min, compared to
about 6 min for that of positive bending (for light-adapted
sporangiophores).
The rather artificial explanation of these differences in
terms of the optical attenuation hypothesis-gallic acid absorbs UV light before it can reach the distal side, so that the
asymmetry between proximal and distal irradiation is more
pronounced-can hold no longer than the hypothesis itself.
Further discrimination between positive and negative tropism
could be given in terms of adaptation, since the adaptation is
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A NOVEL EFFECT IN PHYCOMYCES: POSITIVE UV-PHOTOTROPISM
1591
b
a
I
(A
Figure 5. Dependence of the light profile at X =
280 nm on the radius (r) and attenuation (e) of
the vacuole. a, r8 = 21 Am, e = 0.02 Am-', as
measured in Steinhardt and Fukshansky (21); b,
rb = 24 Mm, (b = E.100; c, r, = 27 Mm, E, = 100.
3c
m
E
>.
0;
,. s3cn
z
u
o
45
90
19o
135
45
0
AZIMUTH ( f )
90
AZIMUTH (
5 10-3
I
I * 10-
-
5.10-'
_
1.10-'
-
5*10-'
_
1*10-'
-
I
135
1s6
)
I
I
I
I7I
I
I
I
I
I
I
I
290
I
00
*
E
IN
Q)
e
-u
5*1004 _
1*10-'
l
250
I60
270
280
290
310
Wavelength [nm]
Figure 6. Compensation spectrum. The curve connects the four compensation points, separating regions of negative (above) and positive
(below) tropism. Open circles above and below each compensation point estimate the boundaries of compensation intervals. The higher/lower
boundaries (in W m-2) at different wavelengths are located as follows: 263 nm, (7.5 ± 0.8)10-6/(3.2 ± 0.3)1 0-6; 281 nm, (4.6 ± 0.5)10-6/(2.4 ±
0.2)10-6; 301 nm, (1.9 ± 0.2)10-4/(5.6 ± 0.6)10-5; 304 nm, (1.6 ± 0.2)10-3 /(5.1 ± 0.5)10-4.
at least partially bound to the photoreceptor. Our preliminary
measurements (T Popescu, T Richter, A Roessler, L Fukshan-
sky, unpublished data) suggest that UV causes much less (if
any) adaptation, rather that a symmetrical preirradiation with
UV sensitizes the phototropic mechanism. Since changes of
state under UV preirradiation seem to be nonsaturable over
hours of irradiation and also since even under UV irradiation
both assumed receptors would contribute to the response,
quantifying and clarifying the origin of the adaptation under
UV irradiation will require a major effort. Therefore, we
carried out an additional direct test of the two-pigment hypothesis based on the following idea. If the compensation
point is caused by counteraction of two receptors, this counteraction should not be restricted to one wavelength. In the
neighborhood of X = 305 nm, one should expect other compensation points, which must appear for each wavelength at
a specific intensity value. Furthermore, given that a compensation point can be found, an increase of intensity should
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Copyright © 1989 American Society of Plant Biologists. All rights reserved.
POPESCU ET AL.
1592
Plant Physiol. Vol. 91, 1989
a
90 -
I ,l
a
4.)
70-
c
0
co
a)
<1)
50-
-o
!I
a)
L
C.)
In
~
~
~~
~
~
~
~
~
~
~
~~~~~~~~~~~~~~~~~~~~~~~~~"I
*1- -~
_I
0
C.,
.)~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~.
S
30-
0,
z
Cc
C9
z
10-
z
EL)
-100
I
I
I
I
I
10 20 30 40 50 60 70 80 90
I
I
I
0
10 20 30 40 50 60 70 80 90
Time [min]
Figure 7. Kinetics of positive tropism mediated by blue light at X =
450 nm, 1 -2 W. m-2 (b) and UV irradiation at X = 281 nm (filter 277
nm) (1.6 ± 0.2).10-6 W. m-2 (a). Each curve is the average of five
experiments. The phototropic latency under blue light is estimated as
23 ± 2 min; under UV, as 8 ± 3 min. Vertical bars show the mean
square deviations.
3~ ~~~
~bt 1
~a
Q~~~~~~~~~~~~
Figure 9. Light (a) and excitation (b) profile for a sporangiophore
irradiated at X = 281 nm (in fluorocarbon).
estimate the limits of these intervals up to the open circles
above and below each compensation point. Higher open
circles represent the lowest intensity providing negative bending; lower open circles, the highest intensity at which positive
bending was observed. Thus, the open circles around a compensation point mark an 'uncertainty' area. The actual compensation intervals can be equal to or smaller than the distances between higher and lower open circles.
Comparison of Positive Tropism Mediated by UV and
Blue Light
LLS
0n
z
0-z
CD
C.,
45
0
AfRZI MUTH
M
135
P)
180
15
AZIMUTH ( Y )
Figure 8. Light (a) and excitation (b) profile for a sporangiophore
irradiated at X = 281 nm (in air).
cause negative, and a decrease of intensity, positive bending,
since the receptor providing negative bending has smaller (if
any) adaptation compared to that of the receptor providing
positive bending. Direct measurements with light-grown, 1 h
dark-adapted, sporangiophores at four wavelengths, as shown
in Figure 6, have confirmed all these predictions.
The line in Figure 6-we call this curve the compensation
spectrum-connects the points representing the intensity values at the four different wavelengths at which unilateral
irradiation causes symmetrical vertical growth. Each of these
compensation points has been determined in at least five
independent experiments. Above the compensation spectrum
is the area of negative tropism; below it, the domain of positive
tropism. By stepwise varying of the intensity around each of
the compensation points it was revealed that they are, in
reality, not points but small intervals of intensity. We could
Like UV-mediated negative tropism, the positive tropism
in the UV range has a much shorter phototropic latency and
faster kinetics of bending as compared to those characteristics
of the blue light-mediated tropism. Kinetics of bending measured on light-grown, 1 h dark-adapted, sporangiophores are
shown in Figure 7b (irradiation at X = 450 nm) and Figure
7a (irradiation at X = 281 nm). Each curve represents the
average of five sporangiophores; the vertical bars correspond
to the mean square deviation of individual points.
Immersion experiments with irradiation at compensating
(in air) intensities have determined that the positive UVmediated bending is promoted by a lens effect as is the bluelight induced tropism. Sporangiophores immersed in fluorocarbon (refractive indices at actinic wavelengths: n(X = 263
nm) = 1.3091, n(X = 281 nm) = 1.3070, n(X = 301 nm) =
1.3061, and n(X = 304 nm) = 1.3059) were irradiated with
intensities equal to those providing natural compensation in
sporangiophores placed in air. Since the fluorocarbon itself
absorbs UV radiation (transmissions of the fluorocarbon layer
at actinic wavelengths: T(X = 263 nm) = 45%; T(X = 281
nm) = 68%; T(X = 301 nm) = 70%; T(X = 304 nm) = 79%);
the actinic intensities in all these experiments were considerably below the compensation points. These intensities cause
positive tropism in sporangiophores placed in air. In all immersion experiments, on the contrary, we observed strong
negative bending.
Changes in the light and excitation profiles due to immer-
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Copyright © 1989 American Society of Plant Biologists. All rights reserved.
A NOVEL EFFECT IN PHYCOMYCES: POSITIVE UV-PHOTOTROPISM
sion shown in Figures 8 and 9 for X = 281 nm are also
representative of those at other wavelengths. Figure 8 presents
light and excitation (for a transversely tangential oriented
transition dipole) profiles in air; Figure 9, those profiles in
fluorocarbon. A comparison of these pictures brings out unmistakably the discrepancy between the symmetries of stimulus and response envisaged in Figure 3. Not only a totally
asymmetrical stimulus (Fig. 8) causes symmetrical response,
but also a strong increase in the symmetry of stimulus (Fig.
9) breaks the symmetry of response. This implies that some
unknown spatially asymmetrical factor counterbalances the
asymmetrical excitation profile shown in Figure 8b (a differently postulated orientation of transition dipole would not
affect this argument). Furthermore, this factor, unlike the lens
effect, is not removed by immersion in fluorocarbon; it causes
the asymmetry of response under these conditions. The nature
of this factor remains to be clarified. Another consequence of
our findings does not require any further justification: since
the lens effect under UV irradiation has been derived theoretically (focal bands in calculated profiles) and demonstrated
experimentally (inversion of the direction of bending upon
immersion in fluorocarbon), the optical attenuation hypothesis should be abandoned.
ACKNOWLEDGMENTS
We are deeply indebted to Rainer Hertel (Freiburg) for fruitful
discussions. We are thankful to Walter Shropshire (Maryland) for
supplying us with fluorocarbon, to Paul Galland (Marburg) for spores
of Phycomyces, and to Randall Cassada (Freiburg) for critical reading
of the text.
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Copyright © 1989 American Society of Plant Biologists. All rights reserved.