Received for publication July 2, 1990
Accepted September 1, 1990
Plant Physiol. (1990) 94, 1605-1608
0032-0889/90/94/1 605/04/$01 .00/0
A Common Fluence Threshold for First Positive and Second
Positive Phototropism in Arabidopsis thaliana'
Abdul Janoudi and Kenneth L. Poff*
Michigan State University, Department of Energy Plant Research Laboratory, Michigan State University,
East Lansing, Michigan 48824
second positive curvature (Fig. 1). We do not understand the
basis for the descending arm of first positive curvature, or the
basis for second positive curvature, although several models
have been proposed to account for these complexities (6, 7,
12, 13, 19).
In the ascending arm of the fluence-response for first positive phototropism, curvature is proportional to the number
of quanta . That is, for a given curvature, there is a reciprocal
relationship between the fluence rate of the light and the time
of irradiation (fluence rate x time = a constant). Therefore,
curvature in the ascending arm obeys the Bunsen-Roscoe law
of reciprocity (3). In contrast, the apparent fluence threshold
for second positive curvature varies, depending upon the
fluence rate of the light. Thus, in second positive phototropism, the response is primarily a function of the time of
irradiation. This time threshold, which can be calculated for
second positive curvature, varies depending on the report but
is in the range of 4 to 10 min (2, 5, 14, 18).
We have reasoned for second positive phototropism that
the plant is measuring the amount of time over which it is
irradiated, and that there must be a time threshold. However,
we also reason that the plant cannot measure the duration of
irradiation without measuring the light itself (i.e. fluence of
blue light). Based on this reasoning, we have measured the
fluence-response relationship for Arabidopsis thaliana under
conditions which satisfy the apparent time threshold requirement for second positive phototropism. Under these conditions, we find that first positive and second positive phototropism have the same quantum threshold. Based on these
data, we suggest that the fluence threshold for both first
positive and second positive phototropism is set by a single
photoreceptor pigment system.
ABSTRACT
The relationship between the amount of light and the amount
of response for any photobiological process can be based on the
number of incident quanta per unit time (fluence rate-response)
or on the number of incident quanta during a given period of
irradiation (fluence-response). Fluence-response and fluence
rate-response relationships have been measured for second positive phototropism by seedlings of Arabidopsis thaliana. The fluence-response relationships exhibit a single limiting threshold at
about 0.01 micromole per square meter when measured at fluence rates from 2.4 x 10-5 to 6.5 x 10-3 micromoles per square
meter per second. The threshold values in the fluence rateresponse curves decrease with increasing time of irradiation, but
show a common fluence threshold at about 0.01 micromole per
square meter. These thresholds are the same as the threshold of
about 0.01 micromole per square meter measured for first positive
phototropism. Based on these data, it is suggested that second
positive curvature has a threshold in time of about 10 minutes.
Moreover, if the times of irradiation exceed the time threshold,
there is a single limiting fluence threshold at about 0.01 micromole
per square meter. Thus, the limiting fluence threshold for second
positive phototropism is the same as the fluence threshold for
first positive phototropism. Based on these data, we suggest that
this common fluence threshold for first positive and second
positive phototropism is set by a single photoreceptor pigment
system.
Although phototropism has been intensively studied since
its description by Darwin (4), this physiological process is
poorly understood. A fluence-response relationship is one of
the most basic measurements that can be made on a photobiological system, showing the relationship between the response of the organism and the number of quanta incident
upon or absorbed by the organism. However, for phototropism, the fluence-response relationship is extremely complex
(Fig. 1). The curvature that is produced in response to low
fluence and short irradiation times is referred to as first
positive curvature. Curvature increases with increasing fluence of blue light for approximately 1.5 orders of fluence
above a threshold of about 0.01 tmol m-2, and then decreases
with increasing fluence for an additional 1.5 orders of fluence
to an indifferent zone ( 14). Curvature increases again at higher
fluence and longer irradiation times, and this is referred to as
MATERIALS AND METHODS
Plant Growth and Phototropic Stimulation
Seeds of Arabidopsis thaliana (L.) Heynh. strain 'Estland'
were sown in strips of micro-assay wells containing 0.7% (w/
v) agar as previously described (16). The micro-assay strips
were placed in clear plastic boxes, lined with moistened paper
to maintain a high RH. Seed germination was potentiated by
chilling at 5 + 1°0C in darkness for 3 d, and then exposing to
white light for 20 h at 25 ± 1°C at a RH greater than 90%.
At the end of the white light irradiation, the boxes containing
the micro-assay strips were transferred into darkness at 25 ±
1°C for 42 h, at the end of which time the seedlings were
exposed to the phototropic stimulus. Since green light is
' Supported by the U.S. Department of Energy under contract No.
DE-ACO2-76ERO- 1 338.
1605
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1 606
JANOUDI AND POFF
Plant Physiol. Vol. 94, 1990
second positive
phototropism
0
0
2
-
0
10
la
0
0
first positive phototropism
0
.*
4..
so
>,/
/
4.
a
.
_s
/
, ,/
\\
vo
/~/
SW
/
/
co
/
"__/__~~~~~~//
v
fluence
threshold
0.01
indifferent
0.1
1.0
10
Fluence (,umol. m-2)
zone
Fluence (,umol. mi2)
Figure 1. Diagrammatic representation of the fluence-response relationship for phototropism of A. thaliana. Based on data presented
by Steinitz and Poff (16) and Khurana and Poff (8).
known not to be phototropically 'safe' (17), all manipulations
performed in complete darkness.
were
Figure 3. Fluence-response relationship for first positive phototropism of A. thaliana. Etiolated seedlings were irradiated with 450-nm
light at 0.065 Amol mr2 s-' for times from 0.15 to 200 s. Curvature
was measured 60 min after the irradiation. Each data point represents
the mean curvature of 90 to 1 10 seedlings ± 1 SE.
nation with a LI- 1000 Datalogger. The duration of irradiation
was controlled with a Uniblitz (Vincent Associates, Rochester,
NY) shutter.
Light Sources
White light at 50 Amol m-2 s-', used to potentiate seed
germination, was provided by two General Electric (Cleveland, OH) Delux Cool-White fluorescent tubes. A slide projector equipped with a Sylvania 900 W BVA tungsten-halogen
lamp, in combination with a 450-nm interference filter (10nm half-bandwidth) was used as the light source in the phototropism experiments. The fluence rate was varied with
neutral density filters. Fluence rates were measured using a
Li-Cor (Lincoln, NE) LI-190SA quantum sensor in combi-
Measurement of Curvature
If the time of irradiation is varied, it is not possible to keep
the time from the beginning of the irradiation to the measurement of curvature a constant along with the time from the
end of the irradiation to the measurement of curvature. The
results from a preliminary experiment indicated that phototropic curvature reaches a maximum 60 min after the end of
the unilateral irradiation (Fig. 2). Therefore, subsequent ex-
30
310 _
7
0
07.1
10-3
24 x 10-4/
6~~.51x10-3
x
07
0
02.4 x10-5
io5
A4.8x105
0D20
120
2
0
1-
I
I0
C.)
(iIII
30
60
90
I
120
Time after irradiation (min.)
Figure 2. Kinetics for the development of phototropic curvature in
seedlings of A. thaliana. Curvature was measured at the indicated
times following the end of the irradiation. Seedlings were irradiated
with unilateral 450-nm light for 1.5 s (0.5 ,umol m2) for first positive
phototropism (0), or 40 min (0.3 umol m-2) for second positive
phototropism (0). Each data point represents the mean curvature of
120 to 320 seedlings ± 1 SE.
0.1
0.01
1.0
10
Fluence (,Lmol. m2)
Figure 4. Fluence-response relationship for second positive phototropism of A. thaliana. Etiolated seedlings were irradiated with 450nm light at the indicated fluences in MAmol m-2 si1, and times of 2 min
or longer. Curvature was measured 60 min after the irradiation. Each
data point represents the mean curvature of 90 to 110 seedlings +
1 SE.
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COMMON THRESHOLD FOR FIRST AND SECOND POSITIVE PHOTOTROPISM
Fluence (prmol. m2)
102
AX
100
1i D-1
101
103
102
:60 min
D
1 607
periments were terminated 60 min after the end of the light
stimulus, although this resulted in a variable time after the
beginning of the light stimulus. The seedlings were then gently
mounted on transparent adhesive tape with the direction of
bending in the plane of the tape surface. The tape was inserted
into a photographic enlarger and the hypocotyl curvature
traced, and later measured using a protractor. Only seedlings
that emerged upright from the agar were used.
RESULTS
:1,
io
I
io-3
4io2
40
20:
40
100
0m
102
i~~~±
io0i
10-3
10-5
,
l
1
:240minC
0
0
0
0
W-S.
10-2
0
C.)
o
1-
100
102
0
2mi
20 _-
40
.
,
,
.
20
DISCUSSION
Based on these data (Fig. 6), second positive curvature by
Arabidopsis thaliana seedlings has a minimum time requirement (threshold) of about 10 min. This is consistent with the
time thresholds which can be calculated from previously
reported fluence-response relationships for A. thaliana (8, 9,
16), Avena (1), Zea (2), and Pilobolus ( 11).
Under conditions in which the time of irradiation exceeds
the time threshold, it can be demonstrated that second posi-
20
10-3
io5
10-2
10-1
102
100
I, 10 minI+I
20
Fluence-response curves were measured at variable times
and fluence rates for first positive and second positive phototropism by Arabidopsis thaliana seedlings to 450-nm light.
The fluence threshold at about 0.01 smol m-2 for first positive
phototropism (Fig. 3) coincides with the single limiting threshold fluence at about 0.01,Itmol m-2 for second positive
phototropism (Fig. 4).
In addition, second positive phototropism by A. thaliana
seedlings to 450-nm light was measured as a function of
fluence rate for different exposure times (10, 20, 40, and 60
min). The fluence rate-response curves (Fig. 5) at each exposure time show increasing curvature above some fluence rate
threshold to a maximum, and then decreasing curvature with
still higher fluence rates. The threshold fluence rate decreases
with increasing exposure time. However, within the limits to
which a threshold can be determined, the threshold fluence
rates multiplied by the exposure times show a constant single
fluence threshold at about 0.01 zImol m-2 (Fig. 5). This fluence
threshold is the same as the fluence threshold for first positive
curvature (Fig. 3), and the same as the limiting fluence
threshold for second positive phototropism from the fluence
response curves (Fig. 4).
Finally, curvature of A. thaliana seedlings to 450-nm light
at a constant fluence of 0.5 ,umol m2 has been measured as
function of the time of irradiation from 3 to 3600 s. The
results (Fig. 6) show a constant curvature of about 9° for
irradiation times from 3 to 600 s, and a rapid increase in
amount of curvature for longer irradiation times.
I0-
I
I0
1I
02
10-5 10-4 10-3 10-2 10-1
Fluence rate (,lmol. m-2 s-1)
100
Figure 5. Fluence rate-response relationships for second positive
phototropism of A. thaliana. Etiolated seedlings were irradiated with
450-nm light at the indicated fluence rates for A, 10 min; B, 20 min;
C, 40 min; D, 60 min. Curvature was measured 60 min after the
irradiation. Each data point represents the mean curvature of 90 to
110 seedlings ± 1 SE. Fluence rate is indicated on the lower abscissa
of each panel. The corresponding fluence is indicated on the upper
abscissa of each panel.
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JANOUDI AND POFF
1 608
IV 40
0
I
Um
.U 20 F
I
10
the only system in which a failure of reciprocity has raised
major questions. Under conditions of short irradiation times,
phytochrome-regulated photomorphogenesis obeys the law of
reciprocity. However, under longer irradiation times, reciprocity fails and this failure has generated problems concerning the nature of the photoreceptor pigment(s) involved (15).
In summary, second positive phototropism exhibits a time
threshold of about 10 min. In addition, if the irradiation times
exceed the time threshold, second positive phototropism exhibits a limiting fluence threshold of about 0.01 gmol m-2.
Thus, the limiting fluence threshold for second positive phototropism is insufficiently different from that for first positive
phototropism to warrant postulating regulation by different
photoreceptor pigment systems.
1000
100
Time (seconds)
1.
Figure 6. Phototropic curvature of A. thaliana seedlings as a function
of time of exposure to 450-nm light at 0.5 Mmol m-2. Curvature was
measured 60 min after the irradiation. Each data point represents the
mean curvature of 90 to 110 seedlings ± 1 SE.
tive phototropism shows a single threshold fluence of about
0.01 ,umol m-2 (Figs. 4 and 5). This fluence threshold coincides with the fluence threshold for first positive phototropism
(8, 9, 16; Fig. 2). The Bunsen-Roscoe law of reciprocity (3) is
valid for the threshold of second positive phototropism. However, based on the fluence-response curves measured for different fluence rates (Fig. 4), reciprocity ceases to be valid at
fluences slightly above the threshold of 0.01 ,umol m-2. Thus,
at the threshold for second positive phototropism, when the
time exceeds the time threshold, the plant is indeed measuring
the number of quanta.
These results lead to a model for second positive phototropism with two thresholds, one in fluence and one in the
time of irradiation. Although first positive phototropism appears to use more than one pigment at different wavelengths
and fluence rates (10), the common threshold for first positive
and second positive phototropism can be interpreted to mean
that the element that limits first positive phototropism and
sets its fluence threshold, is the same element setting the
fluence threshold for second positive phototropism. Because
the quantum threshold for second positive phototropism is
the same as that for first positive phototropism, and because
reciprocity is valid for both thresholds, it seems unlikely that
the fluence threshold for first and second positive phototropism is set by different pigment systems.
Since Bunsen and Roscoe (3) defined the law of reciprocity,
an emphasis has been placed on defining the set of conditions
under which that law is valid for a particular biological
response. Based on the results presented here, there clearly is
much to be gained from a consideration of the possible
mechanisms for the failure of reciprocity. Phototropism is not
Plant Physiol. Vol. 94, 1990
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3.
4.
5.
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7.
8.
9.
10.
11.
12.
13.
14.
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18.
19.
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