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This is to certify that the

thesis entitled
ISOLATION AND CHARACTERIZATION OF
ARAB | DOPS IS THAI. IANA MUTANTS
WITH
ALTERED PHOTOTROPISM

presented by

ZHANGL I NG REN

has been accepted towards fulfillment
of the requirements for

I“ 5~ degree in ice/IQ VLC/Q
Botany 8 Plant Pathology

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Major professor

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ISOLATION AND CHARACTERIZATION OF
ARABIDOPSIS THALIANA MUTANTS
WITH

ALTERED PHOTOTROPISM

By

Zhangling Ren

A THESIS

Submitted to
Michigan State University
in partial fulfillment of the requirements
for the degree of

MASTER OF SCIENCE

_.8
\O
n
.1

ABSTRACT
ISOLATION AND CHARACTERIZATION or
ARABIDOPSIS THALIANA MUTANTS
WITH
ALTERED PHOTOTROPISM
By

Zhangling Ren

A screening procedure has been devised using multiple
pulses of unilateral blue light to identify mutants in

Arabidopsis thaliana with altered phototropism. Thirty five

 

mutants have been isolated with two distinctly different
phototropism phenotypes and with geotropic responses which
are normal or impaired.

A fluence-response curve has been measured for ZR-B,
which has altered phototrOpism and normal geotropism. The
amplitude of the phototropic response to a single light
pulse and multiple pulses by ZR-8 are lower than the
analogous response by the wild-type. The thresholds for
ZR-8 are shifted to higher fluence compared with the
wild-type. The maximum of the first positive response of
ZR-8 and the wild-type are at the same fluence, but the
maximum response of ZR-8 to multiple pulses is at a higher
fluence than that of the wild-type. It is suggested that
such mutants should be useful in gaining an eventual

understanding of phototropism in plants.

ACKNOWLEDGEMENT

I want sincerely to thank my major professor, Dr. Ken
Poff for his guidance and advice in science and also in
English. I also wish to thank Dr. Beni Steinitz for

introducing Arabidopsis to me and guiding me into the world

 

of phototropism. Thanks also to the members of my

committee, Dr Barbara Sears and Dr. Hans Kende.

I am very appreciative of the technical assistance from
Therese Best. I will always remember the good times with
Carol, Brian and Mary. Thanks to all of you who have
patiently explained and helped me to understand American

culture and custom.

Finally, thanks go to my mother, father and

grandmother, whose love is always with me.

ii

TABLE OF CONTENTS

List of Figures .............. ............ ....... ....... iv

IntrOductiOn00000000000.0000000000000000000.00000000000 1

Materials and MethOdS..000000..0.00....000..0.000.00000 6

ReSUItSooooo ...... 000000000.0000000.00000000000000.0000 10

DiSCUSSion ....... 0......00000 00000 00000000000000.000000 36

Literature Cited0.00.00.00.0000.000.0000000000000000000 LI].

Appendix I......... ..... 0.00...00...00.00.000.000000000 “5

iii

Figure

Figure

Figure

Figure

Figure

Figure

LIST OF FIGURES

Frequency distribution for the curvature
of seedlings left in darkness in the

vertical position.......................

Frequency distribution for the curvature
of seedlings exposed to a single pulse

of unilateral light.....................

Frequency distribution for the curvature
of seedlings exposed to nine pulses of

unilateral light at 15 min intervals....

Frequency distribution for the curvature
of mutant Arabidopsis seedlings exposed
to nine pulses of unilateral light at 15

min intervals.0.00.00.000.000.00.000.000

Kinetics for geotropic curvature in

Arabidopsis seedling shoots.............

 

Fluence-response curve for phototropism
of the seedling shoots of Strain ZR-8 in

response to a single pulse of light.....

iv

Page

11

12

13

16

2A

Figure

Figure

Figure

Figure

10.

Page
Fluence-response curve for phototropism of the
seedling shoots of wild-type in response to a

single pulse of light........................3O

Fluence-response curve for phototrOpism of
the seedling shoots of Strain ZR-8 in response to

five pu1SESOflightoooooooooooooooo00000000032

Fluence response curve for phototropism of
the seedling shoots of wild-type in response

to five pUISes Of light.000......00000000000033

Induction of phototropic curvature in seedling
shoots of Strain ZR-8 with sequential pulses

Of light00000000000.000000.00.000.00.0000000.35

INTRODUCTION

The phenomenon of phototropism in plants was observed
and described as early as 1880 by Darwin. He described
phototropic movement and suggested that phototropic
curvature was due to a modification of ordinary
circumnutation. Since that time, the phototropism of stems
has received a great deal of attention. The work on
phototropism has been in three general areas: the nature of
the photoreceptor pigment; the mechanism for detecting light

direction; and the control of the growth response.

The nature of the photoreceptor pigment has been
studied mainly through fluence response curves and action
spectroscopy, and also through the use of inhibitors.
Fluence response curves for plant phototropism are quite
complex with the so-called first positive phototropism at
lower fluence, an indifferent zone or negative phototropism
at intermediate fluence, and the so-called second positive
phototropism at higher fluence. This is further complicated
by the fact that only first positive phototropism follows
the Bunsen-Roscoe law of reciprocity (Dennison, 1979). The
invalidity of reciprocity for second positive phototropism
means that the response is not simply a function of the
number of quanta absorbed, but depends mostly on the time of

irradiation and only slightly on the fluence rate used

1

(Pickard, et al., 1969). It is of interest that essentially
the same complex fluence response curve is found in both
monocotyledonous (Galston, 1959; Briggs, 1963; Zimmerman and
Briggs, 1963a,b; Curry, 1969), and dicotyledonous plants
(Steyer, 1967; Steinitz and Poff, 1985). In addition, the
action spectra are all similar to each other and to other
"blue light responses", with peaks at about 370 nm, 440 nm,
460 nm, and 485 nm (Dennison, 1979; Shropshire, 1980).

Based on the action spectra, carotenoids and flavins have
been suggested as the photoreceptor pigment (Schmidt, 1980).
Although the controversy will likely continue until the
photoreceptor pigment has been isolated and identified, it
is generally thought that the photoreceptor pigment is a
flavoprotein (Dennison, 1979). The flavoprotein hypothesis
is consistent with the data from action spectroscopy and
with work using inhibitors specific to phototropism. Many
such inhibitors have been described but these share one
attribute - - they all in some way combine with flavins or

interfere with flavin physiology or photochemistry.

Although the measurement of light direction could use
either a refraction mechanism or a screening mechanism, the
weight of evidence supports the former in the fungus

Phycomyces blakesleeanus and the latter in flowering plants.

 

Light is thought to be refracted by the Phycomyces

 

sporangiophore growing in air such that the organ acts as a

convergent lens, focusing unilateral light onto the distal

side which is thereby caused to grow faster than the
proximal side (Shropshire, 1962; Bergman, et al., 1969;
Castle, 1955). The shoot of the flowering plant is

considerably more dense than a Phycomyces sporangiophore,

 

and any focusing advantage is overcome by the screening of
light through absorption and scatter of the plant tissue.

As a result of this screening, more light is absorbed on the
proximal side than on the distal side of the plant shoot,
permitting the measurement of light direction (Thimann and
Curry, 1961; Seyfried and Fukshansky, 1983; Parsons, et al.,

1984; Piening, 1984)

The most carefully studied aspect of phototropism has
been the mechanism whereby unequal light reception is
translated into unequal growth and thereby into curvature of
the shoot. For example, Blaauw (1915) suggested that the
phototropic response might be caused by a relative
inhibition of cell elongation. However, Went and Thimann
(1937) preposed that the phototropic response is caused by
the redistribution of some plant hormone (Cholodny-Went
theory of tropism). This point continues to be
controversial, but the Blaauw hypothesis seems most favored
recently (Franssen, et al., 1981; Meijer, 1968; Black and
Shuttleworth, 1974; Jose and Vince—Prue, 1977; Gaba and

Black, 1979; Thomas et al., 1980; Cosgrove, 1981).

In most cases, the phototropism of monocotyledonous
plants and fungi has been studied, but not much attention
has been directed to the study of phototropism in
dicotyledonous plants. In 1967, Steyer suggested that the
two types of phototropic responses, first and second
positive phototropism, existed in both monocotyledonous and
dicotyledonous plants. Further studies on phototropic
responses of dicotyledonous plants have been performed by
Bruinsma et a1. (1975), Shuttleworth and Black (1977) and
Hart and MacDonald (1981). Recently, Steinitz and Poff
(1985) characterized the phototropic response in hypocotyls

of a dicotyledenous plant, Arabidopsis thaliana. They

 

showed that the patterns of fluence-response relation (first
and second positive responses) are the same as those of
monocotyledous plants (Axgna), and that sequential multiple
pulses can induce a greater phototropic response than can a

single pulse.

Phototropism and geotropism are the two major sensory
systems responsible for regulating the orientation of aerial
organs of plants (Briggs, 1963; Juniper, 1976; Wilkins,
1977; Dennison, 1979; Hart and MacDonald, 1981). In most
cases, phototropism and geotropism have been considered and
studied separately and independently (Briggs, 1963; Kang and
Burg, 1972, 1974; Mohr and Pichler, 1960; MacArthur and
Briggs, 1979). However, under natural conditions, the

potential for both systems exists within one organ, and the

trOpism of plant organs is determined by the balance of
phototropism and geotropism. Moreover, the two
sensitivities may interact. Hart and MacDonald (1980)
reported that blue light, which induces the phototropic
response, inhibited geotropic bending of Lepidium and
Sinapis hypocotyls. Hart and MacDonald (1981) later
described the degree of interaction or competition between
phototropism and geotropism within a single organ of a

dicotyledous plant, Lepidium sativum L. At best, the

 

interaction of phototropic and geotropic systems is not

clear.

A collection of phototropism mutants would be a
valuable tool both for the study of phototropism itself and
also for the study of the interaction between phototropism
and other sensory responses. Since no such higher plant
phototropism mutants were available, this work was
undertaken to isolate such mutants and to begin their
characterization. A screening technique has been devised
and used for identifying phototropism mutants in Arabidopsis
thaliana. In addition, phototropism and geotropism have

been characterized for some of the mutants isolated.

MATERIALS AND METHODS

Plant Material: The Estland variety of Arabidopsis

 

thaliana (L.) Heynh, a self-fertilizing crucifer, was used
throughout this study. The seed stock was generously

provided by Dr. C. Somerville.

Mutagenesis: The seeds of A. thaliana were mutagenized by
Drs. C. Somerville and B. Steinitz. The seeds were placed
in an erlenmeyer flask and soaked for 12 hr in an aqueous
solution of 0.2% (w/v) ethylmethanesulfonate (EMS; Sigma
Chemical Co., St. Louis, M0.). Following this treatment,
the solution was discarded, and the seeds rinsed with
running distilled water for 8 hr.

m Seed Production: This m1 generation of seed was sown in

2
large flats containing a bottom layer of perlite, overlain
with a pH-balanced peat mixture, sprinkled with a layer of
fine vermiculite, and moistened with distilled water. After
sowing, the flats were covered with clear cellophane to
prevent desiccation, and held at 40 for 2 days to enhance
germination. The seed were then incubated at 21—230 under

16 hrs white light per day from above (104 mW/m2 from

General Electric Delux Cool White fluorescent tubes). The

6

self-pollinating m1 plants were permitted to set seed, and
about 8 weeks after sowing, the second generation mutant
(m2) seeds were harvested by cutting the plants at ground
level and allowing them to dry for about 1 week in paper
bags. The material was then threshed using a wire screen,

and the m seeds collected and pooled from all of the m

2 1

plants.

Growth of Seedlings: To produce material for phototropism,
geotropism or fluence-response relationship tests, 1112 seed
were sown on 0.8% (W/V) agar (Difco Bacto Agar, Difco
Laboratories, Detroit, MI.) containing 1 mM KN03 in 0.3ml
microstrip wells (Lab Systems Inc., Morton Grove, 11) at one
seed per well. Rows of the microstrip wells were set in a
transparent box (3.5 X 17 X 21 cm) which was then wrapped
with black cloth and incubated in darkness at 40 for 4 days
to potentiate germination (Shropshire, et al., 1961). The
black cloth was then removed, and the seeds exposed at 250
for 30 hr to white light (125 mE/mz/sec from General
Electric Delux Cool White fluorescent tubes) also to
potentiate germination. The box of seedlings was moved to a
dark room at 250 and 100% relative humidity for 42 hrs,
after which they were used in phototropism or geotropism

tests.

Phototropic Induction with Multiple Pulses: Seedlings grown

in darkness for 42 hr following the potentiation of

germination, were stimulated unilaterally with nine 2.5 sec
pulses of 450 nm light at 37.6 pE/cmZ/sec for a total
fluence of 94 pE/cm2 for each pulse. The pulses were given
at 15 min intervals. Thus the length of time between the
first and ninth pulse was 120 min. After an additional 30
min in darkness, the experiment was terminated and the

curvature of the stimulated seedlings measured.

Geotropic Induction: Seedlings grown in darkness for 42 hr
following the potentiation of germination were exposed to
red light (500 ergs/cmz/sec from red fluorescent tubes in
combination with a 3-cm thick filter of 5% (w/v) aqueous
CuSO4 (giving a maximum output at 660nm) for one hour. The
microstrips containing the seedlings were then turned within
the plastic box such that the seedlings were in the
horizontal position, and incubated for a period of time in

darkness. The curvature of 100-200 seedlings was measured

2, 8, and 12 hrs after the onset of geotropic stimulus.

Curvature Measurements: Seedlings were gently affixed to
sticky transparent tape in a way that the curvature angles
were parallel to the tape surface. The tape was mounted in
the slide holder of an enlarger, and an enlarged image of
the seedlings projected onto paper on which two straight
lines were drawn for each seedling, one representing the
shoot, and one the final growth direction of the shoot tip.

The angle of curvature was measured from these lines using a

protractor. In most cases, 100-200 seedlings were measured
for each condition. The experiments were repeated 3-6
times, and the data point is the mean of the pooled data

from these repetitions.

Light Sources: Light for phototropic stimulation was
provided by a Bell and Howell projector equipped with a 300
W tungsten halogen lamp with the light beam filtered through
a 450 nm interference filter (PTR Optics, Waltham, MA.;
half-band width, 8.5 nm) and a 3 cm pathlength of 5% (w/v)
aqueous CuSO4 solution.

All phototropism experiments were performed in complete
darkness, because green light had been previously shown not
to be phototropically "safe" (Appendix I). Some of the
geotropism experiments were conducted in darkness while
others were carried out using a dim green light which had no

apparrent effect on geotropism.

Light Measurements: Light intensities were measured with a
model 68 Kettering Radiometer (Laboratory Data Control,

Riviera Beach, FL.).

RESULTS

To identify mutants with altered phototropism, it is
necessary that the phototropic response of the wild-type
parent be significantly different from the phototropic
response of the mutant. Assuming that the minimum response
would be equivalent to that of the wild—type in darkness
(e.g., zero response), then it is necessary that the
phototrOpic response of the wild-type be significantly
different from the curvature expressed in darkness. For
this reason, the curvature of the wild-type was measured
with and without a phototropic stimulus. The curvature of
seedlings grown in complete darkness was measured, and as
expected, is approximately zero (Fig. 1). In addition, the
curvature of more than 60% of the seedlings was between +50
and ~50. The phototropic curvature of a similar pOpulation
of seedlings treated similarly but exposed to a single pulse
of 846 pE/cm2, was 8.90 +/- 1.10 (Fig. 2). In contrast, the
curvature of a similar population of seedlings, treated
similarly but exposed to nine 2.5 sec pulses of 450 nm blue
light at 94 pE/cm2 per pulse (for a total of 846 pE/cm2) was
800 (Fig. 3). Moreover, only 0.3% of the seedlings showed a
curvature less than 300 and none less than 200. Thus, the

phototropic curvature significantly increased with the

TO

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120 f

80 v

Number of Seedlings

 

 

 

 

 

 

.rl.‘

-20 O 20
Curvature (degrees)

 

Figure 1. Frequency distribution for the curvature of
the shoots of 251 wild-type seedlings prepared for
phototropic stimulation but then left in darkness in the
vertica$ position for 120 min. The megn of the pOpulation
is 0.54 with a standard error of 0.36 .

12

 

 

 

 

 

 

 

1%
100 ‘/
5% IBO:E§%%ZZ
a -%%
2%

Curvature (degrees)

Figure 2. Frequency distribution for the curvature of
the shoots of 236 wild-type seedlings prepared for
phototropic stimulation, exposed £0 a single 22.5 sec pulse
of unilateral light at 37.6 pE/cm /sec, and left in darkness
for 120 min befor the curvatures were measuredb The mean
curvature was 8.9 with a standard error of 1.1 .

l3

 

 

 

 

 

 

 

 

 

 

 

 

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Curvature (degrees)

Figure 3. Frequency distribution for the curvature of
the shoots of 576 wild-type seedlings prepared for
phototropic stimulation, exposgd to nine 2.5 sec pulses of
unilateral light at 37.6 pE/cm /sec given at 15 min
intervals, and left in darkness for 30 min before the
curvatures were measureg 150 min after the first pulsS. The
mean curvature was 79.6 with a standard error of 1.8 .

IA

multiple pulse regime such that there was little overlap
between the curvatures of a population of seedlings in
darkness, and another similar population exposed to the nine

pulses of light.

The (m2) seeds of Arabidopsis thaliana were sown as

 

described above for phototropic induction with multiple
pulses. The seedlings were stimulated with nine 2.5 sec
pulses of 450 nm blue light at 15 min intervals. Thirty min
following the last pulse (150 min after the first pulse),
those seedlings which appeared to have less than 20
curvature degrees were designated as ZR-1, ZR-2, etc. in
sequence as they were found. The plants were allowed to
green under white lights for 2 days and were then
transplanted to clay pots containing moistened peat mixture.
The plants were grown to adults in a growth chamber at 250
under 16 hr daylength. The m seed from each plant were

3
harvested for further testing.

Out of a population of 379,000 m2 seedlings which were
screened, 555 seedlings were identified with a phototropic
curvature less than 20 degrees. Many plants were lost due
to death or inadequate seed set. An adequate amount of seed
were collected from 245 out of the 555 putative mutants.

The m5 seed from each of these 245 strains were sown, the

seedlings exposed to nine pulses of light as described

above, and the curvature of eacn seedling measured. Based

15

on the frequency distributions for these data, 35 strains
were identified with a curvature significantly different
from that of the wild-type parent (Fig. 4). At least two
types of distributions are identifiable: 1). there were 21
mutants for which the peak of the frequency distribution was
close to 00 (Fig. 4a); 2). there were 14 mutants for which
this peak was significantly greater than 00 (Fig. 4b).
Further work was carried out on mutants in the former

category.

Eight of the mutants showing essentially zero
phototropism also were tested for geotropism. The geotropic
responses of ZR-8, ZR-362, ZR-469 and ZR-555 were
essentially the same as the geotropic response of the
wild-type parent (Fig. 5a). In contrast, the geotropic
responses of ZR-19, ZR—162, ZR-196 and ZR-240 were less than

that of the wild-type parent (Fig. 5b).

Assuming that geotropism and phototropism share a
portion of their transduction chains, only those
phototropism mutants which are normal in geotrOpism, are
potential candidates for photoreceptor mutants. Therefore,
a fluence response curve was measured for one of the
photo-minus and geo-plus mutants, ZR-8. Seed (m4) of ZR-8
were sown as before, the seedlings exposed for different
times to a single pulse of light at each of three different

fluence rates, 3.76 pE/cmZ/sec, 37.6 pE/cm2/sec, and 376

16

Figure 4. Frequency distribution for the curvature of
the shoots of Arabidopsis seedlings with a phototropism
phenotype different from that of the wild-type. The
seedlings were prepared for phototropic stimulation, exposed
to ni e 2.5 sec pulses of unilateral light at 37.6
pE/cm /sec given at 15 min intervals, and left in
darkness for 30 min before the curvatures were measured 150
min after the first pulse. Vertical bars represent +/- one
standard error. a. Strains ZR-8, ZR-19, ZR-78, ZR-111,
ZR-161, ZR-162, ZR-190, ZR-196, ZR-207, ZR—214, ZR-225,
ZR-229, ZR-236, ZR-239, ZR-240, ZR-263, ZR-295, ZR-362,
ZR-459; ZR-469, and ZR-555; b. Strains ZR-95. ZR-237,
ZR—288, ZR-293. ZR-301, ZR-328, ZR-337, ZR-338, ZR-422,
ZR-436, ZR-438, ZR-443, ZR-296 and ZR-440.

D)

17

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

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Figure 5. Kinetics for geotropic curvature in
Arabidopsis seedling shoots. Curvature was measured 2, 8,
and 12 hours after the onset of the geotropic stimulus.
Vertical bars represent +/- one standard error. a. Strains
ZR-8, ZR-362, ZR-469, and Zr-555; b. Strains ZR-19, ZR-162,
ZR-196, and ZR-240.

 

 

 

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D 0 I O 0 //..I.M..~,.l. 1 +2
.LA/
0 0 .1 1 .r O
m m m m m 0

Time (hours)

Curvature (degrees)

26

 

0 Wild-type

A ZR-19

o ZR-162

O ZR- 1 96
so . A ZR-24O
4o ‘-

 

 

 

 

2 I: is
Time (hours)

27

pE/cmZ/sec, and the resultant curvature measured. These
data are plotted as fluence response curves (Fig. 6) which
show that the amplitude of the phototropic response was
decreased, but that the maximum first positive responso is
at the same fluence as in the wild-type (c.f., Fig. 6 and
Fig. 7). In addition, the threshold of the first positive
response may be higher in ZR-8 (about 5 pE/cm2) than in the
wild-type parent (about 0.3 pE/cm2). It is also evident
that the amplitude of the second positive response is

decreased in ZR-8 relative to the wild-type parent.

A fluence response curve was also measured for ZR-8
with multiple pulses. Seed (m4) of ZR-8 were sown as
before, the seedlings exposed for different times to five
pulses of light at 37.6 pE/cmZ/sec, the resultant curvature
measured, and the data plotted in the form of a fluence
response curve. The results (Fig. 8) show that the amplitude
of the response was increased and that fluence required for
the maximum response was shifted toward higher fluence
compared with the results obtained using a single pulse
(c.f., Fig. 6 and Fig. 8). The maximum response obtained
using five pulses of light is less for ZR-8 than for the
wild-type under comparable conditions, and the fluence
required for the maximum response using five pulses is
shifted for ZR-8 to higher fluence compared with the
wild-type under comparable conditions (c.f., Fig. 8 and Fig.

9).

28

Figure 6. Fluence response curve for phototropism of
the seedling shoots of Strain ZR-8 in rgsponse to a single
pulse of light at 3.76, 37.6, 376 pE/cm /sec. Curvatures
were measured 120 min after the onset of stimulation.
Vertical bars represent +/- one standard error.

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30

Figure 7. Fluence response curve for phototropism of
the seedling shoots of wild-type in responge to a single
pulse of light at 3.76. 37.6 and 376 pE/cm /sec. Curvatures
were measured 120 min after the onset of stimulation.
Vertical bars represent +/- one standard error (From
Steinitz and Poff, 1985).

31

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Fluence of 450 nm Light (pE-cmz)

Figure 8. Fluence response curve for phototropism of
the seedling shoots of StrainZZR—8 in response to five
pulses of light at 37.6 pE/cm /sec. Curvatures were
measured 120 min after the onset of stimulation. Vertical
bars represent +/- one standard error.

 

 

 

 

 

80<~ I
L I
o 3.76
70» , I
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0 I 376
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Fluence 01 450 nm Light (DE-cm )

Figure 9. Fluence response curve for phototropism of
the seedling shoots of wild-type in Eesponse to five pulses
of light at 3.76, 37.6 and 376 pE/cm /sec. Curvature were
measured 120 min after the onset of stimulation. Vertical
bars represent +/- one standard error (From Steinitz and
Poff, 1985).

34

The additivity of phototropic curvature with multiple
pulses was measured for ZR-8. Seed (m4) of ZR-8 were sown
as before, the seedlings exposed to different numbers of
pulses of light at 37.6 pE/cmZ/sec, and the resultant
curvature measured. The results (Fig. 10) show that
curvature increased about 4.50 for each additional pulse for
ZR—8 in contrast with an approximately 100 increase per

pulse reported for the wild-type (Steinitz & Poff, 1985).

35

 

ZR-8

30 ..

 

 

 

13
33
I: +
"o 20‘» 1 ,
e +
+
a 10‘-
C)
O

 

 

 

 

 

 

 

 

 

 

 

0 7 1 f '2 3 4 5 6 7
Number of Pulses

Figure 10. Induction of phototropic curvature in
seedling shoots of Strain ZR- with sequential 2.5 sec
pulses of light at 37.6 pE/cm /sec. The interval between
sequential pulses was 15 min with curvature measured 120 min
after the onset of stimulation. Vertical bars represent +/-
one standard error.

DISCUSSION

It would be difficult at best to attempt any screening
of phototropism mutants based on the differences measured
here between the maximum phototropic curvature obtained in
first positive phototropism (Fig. 2), and the curvature
which might be expected by plants not exposed to any
unilateral light (Fig. 1). Clearly, greater differences
must be found to provide the basis for a screening
procedure. Although a far greater amplitude of phototropic
curvature can be obtained in second positive phototropism,
it must be remembered that reciprocity is not valid for this
response for which the amplitude of the response is
primarily time dependent and only slightly dependent on the
fluence rate. Thus it is doubtful whether or not one could
find, using such a screen, mutants with a decreased amount
of photoreceptor pigment. To locate such mutants, one must
work under conditions in which the organism is a quantum

counter - - conditions in which reciprocity is valid.

A second possible approach to the problem of how to
screen for phototropism mutants was presented by the report
of Steinitz and Poff (1985) that large angles of curvature
could be induced under conditions where reciprocity is

valid, if the organism is exposed to multiple pulses of

36

37

light at 15-20 min intervals. The value of this approach
was supported by the frequency distribution obtained for
seedlings exposed to nine pulses of light at 15 min
intervals (Fig. 3). Since there is little overlap between
this distribution and one for a similar population held in
darkness, it appeared feasible to proceed with a screen.
The validity of this decision was demonstrated by the

mutants obtained.

The usefulness of mutants in gaining an understanding
of phototropism requires ideally that they be stable, that
every step in the transduction pathway be represented by a
mutant, and that the mutants be characterized both with
respect to genetics and with respect to biophysics. The
firsst point has been partially accomplished by the fact that
eack) of the 35 strains described have been carried at least
II>‘the m4 generation, and some to the m6 generation. It is
difiiicult to respond to the second point without
consxiderably more information. Once it is known from the
genetic characterization how many genes are involved in the
existing collection of mutants, it may be possible to guess
whether or not all of the steps are represented. If only a
few 1cmfl.are found again and again, it is likely that most
0f the possibilities have been located. 0n the other hand,
if few loci are represented more than once, it is likely
that; the pathway has not been saturated. In addition, this

screening procedure has been designed for identifying

38

non-lethal mutants. If some specific step is critical for
the plant, then the loss of that step may be lethal, and
that step would not be represented in this collection of
phototrOpism mutants. The ultimate way around this
limitation would be the isolation of temperature-sensitive
conditional mutants. Since the genetic characterization of
these mutants will require a considerable amount of time,
the remainder of this work was devoted to beginning the

biophysical characterization.

It is well known that both phototropism and geotropism
control the aerial orientation of plant organs. Given the
fact that the expression of both responses requires a
modification of the growth of the two sides of the shoot, it
is likely that the two transduction pathways share common
elements. Although this has not been proven, it has been
sufficiently accepted to have been used as the basis for
tests of the specificity of various potential inhibitors of
phototropism. Given a collection of phototropism mutants,
it was therefore desirable to test each strain for
geotropism. Two types of geotropic responses were detected,
one with the wild-type or "normal" response (Fig. 5a), and a
second with impaired geotropism (Fig. 5b). This identifies
at least two phenotypes, the phototropism minus, geotropism
plus; and phototropism minus, geotropism minus. In
addition, if the latter phenotype can be shown to result

from single gene mutation, this supports the contention that

39

there are common elements in the phototropism and geotropism

transduction pathways.

A single strain, ZR-8 mutant, which is phototropism
minus and geotropism plus, was further characterized through
the measurement of a fluence-response curve. For a simple
photochemical sequence, where the response is regulated by a
single photoreceptor pigment and proportional to the number
of quanta absorbed by that pigment, it is clear that
decreasing the "capture cross section" of the pigment should
shift the fluence-response curve toward higher fluence. In
contrast, for that simple case, altering the efficiency at
some point "downstream" of the photoreceptor pigment should
cause a decrease in the amplitude of the fluence response
curve, but no shift along the fluence axis. The results
obtained for ZR-8 do not easily fit into either category.

It is clear that the amplitude of the response is decreased
and that the response maximum for first positive
phototropism is not shifted from that of the wild-type
parent, and this is consistent with a modification
"downstream" of the photoreceptor pigment. However, it
appears from the fluence response measured using a single
pulse that the threshold fluence for first positive
phototropism is shifted toward higher fluence in comparison
with that of the wild-type. It is possible that this
apparent shift is actually statistical variation resulting

from the extremely low curvatures measured in ZR-8. The

40

fluence-response curve measured using multiple pulses shows
a decreased amplitude of response, a shift in the maximum
response toward higher fluence, and an increase in the

threshold fluence.

It is evident that these results are not easily
reconciled with those expected for the simple hypothetical
photoresponse system discussed above. One possible
explanation for the threshold shift is the loss of one
photoreceptor pigment out of a system containing two or more
pigments. It is not clear what effect this should have on
the fluence-response curve measured using multiple pulses,
and this points to a need for much more work in this area.
Any model which is developed to explain phototropism in
response to multiple pulses of light must also include
provision for the various changes in fluence response curve

seen in ZR-8.

10.

11.

LITERATURE CITED

Bergman K P, V Burke, E Cerda-Olmedo, C N David, M
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Blaauw A H 1915 Licht Und Wachstum II. Z. Bot. 7:
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Black M, J Shuttleworth 1974 The role of the
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in Cucumis sativus L. Planta 117: 57-66

 

Briggs W R 1963 The phototropic responses of higher
plants. Ann. Rev. Plant Physiol. 14: 311-352

Bruinsma J, C M Karssen, M E Benschop, J B Van Dort
1975 Hormonal regulation of phototropism in the
light-grown sunflower seedling, Helianthus annuus L.:
immobility of endogenous indoleacetic acid and
inhibition of hypocotyl growth by illuminated
cotyledons. J. Exp. Bot. 26: 411-418

 

Castle E S 1933 The physical basis of the positive
phototropism of Phycomyces. J. Gen. Physiol. 17:
49-62

 

Cosgrove D J 1981 Rapid suppression of growth by blue
light. Occurrence, time course and general
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Curry G M 1969 Phototropism. In M B Wilkins ed.
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McGraw-Hill, London, pp. 243-273

Darwin C 1880 The Power of Movements in Plants. John
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Dennison D S 1979 Phototropism. IQ W Haupt, M E
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504-566

Franssen J M, S A Cooke, J Digby, R D Firn 1981
Measurements of differential growth causing phototrOpic
curvature of coleoptiles and hypocotyls . Z.
Pflanzenphysiol. 103: 207-216

41

120

13.

14.

15.

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A2

Gaba V, M Black 1979 Two separate photoreceptors
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Galston A W 1959 Phototropism of stems, roots and
coleoptiles. In W Ruhland ed. Encyclopedia of plant
physiology, vol 17-1, Springer, Berlin Heidelberg New
York. PP. 492-529

Hart J W, I R MacDonald 1980 Photoregulation of
hypoocotyl growth: geotropic evidence for the
Operation of two photosystems. Plant Cell
Environ. 3: 189-193

Hart J W, I R MacDonald 1981 Phototropism and
geotropism in hypocotyls of cress (Lepidium sativum
L.). Plant Cell Environ. 4: 197-201

 

Jose A M, D Vince-Prue 1977 Action spectra for the
inhibition of growth in radish hypocotls. Planta 136:
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Juniper B E 1976 Geotropism. Ann. Rev. Plant
Physiol. 27: 385-406

Kang B G, S P Burg 1972 Relation of
phytochrome-enhanced geotropic sensitivity to ethylene
production. Plant Physiol. 50: 132-135

Kang B G, S P Burg 1974 Red light enhancement of the
phototropic response of etiolated pea stems. Plant
Physiol. 53: 445-448

MacAuthur J A, W R Briggs 1979 Effect of red light
on geotropism in pea epicotyls. Plant Physiol. 63:
218-220

Meijer G 1968 Rapid growth inhibition of gherkin
hypocotyls in blue light. Acta Bot. Neerl. 17: 9-14

Mohr H, Z Pichler 1960 Der Einfluss hellroter und
dunkelroter Strahlung auf die Geotropische Reaktion der
Keimlinge Von Sinapis alba L. Planta 55: 57-66

 

Parsons A, K Macleod, R D Firn, J Digby 1984 Light
gradients in shoots subjected to unilateral
illumination - implications for phototr0pism.

Plant Cell Environ. 7: 325-332

Pickard B G, K Dutson, V Harrison, E Donegan 1969
Second positive phototropic response patterns of the
oat cole0ptile. Planta 88: 1-33

25.

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U3

Piening C 1984 Mechanisms of establishing a light
gradient for first positive phototropism in Zea mays L.
M. S. Thesis, East Lansing, Michigan 132 p.

Schmidt W 1980 Physiological blue light reception.
In P Hemmerich, ed. Molecular structure and sensory
physiology. Structure and bonding V 41, Springer-
Verlag, Berlin, pp. 1-41

Seyfried M, L Fukshansky 1983 Light gradients in
plant tissue. Appl. Optics. 22: 1402-1408.

Shropshire W, Jr 1962 The lens effect and
phototropism of Phycomyces. J. Gen. Physiol. 45:
949-958

 

Shropshire W, Jr 1980 Carotenoids as primary
photoreceptors in blue light responses. In H Senger
ed. The blue light syndrome. Springer-Verlag, Berlin,
pp. 172-186

Shropshire W, Jr, W H Kleir, V B Elstad 1961 Action
spectra of photomorphogenic induction and photoinaction
of germination in Arabidopsis thaliana. Plant Cell
Physiol. 2: 63-69

 

Shuttleworth J Z, M Black 1977 The role of cotyledons
in phototropism of de-etiolated seedlings. Planta
135: 51-55

Steinitz B, K L Poff 1985 Phototropic bending by
Arabidopsis seedlings to pulsed light - a new approach

 

to higher plant phototropism. Planta in Press

Steyer B 1967 Die Dosis-wirkungsrelation bei
geotrOper und phototroper Reizung: Vergleich von
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Thimann K V, G M Curry 1961 Phototropism and
Phototaxis. In W McElroy, 8 Glass eds. Light and Life.
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647-670

Thomas B, S E Tull, T J Warmer 1980 Light dependent
gibberellin responses in hypocotyls of Lactuca sativa
L. Plant Sci. Letters, 19: 355-362

 

Went F W, K V Thimann 1937 Phytohormones. MacMillan,
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Wilkins M B 1977 Light and gravity-sensing guidance
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513-524

38.

39.

A4

Zimmerman B K, W R Briggs 1963a Phototropic
dosage-response curves for oat coleoptiles. Plant
Physiol. 38: 248-253

Zimmerman B K, W R Briggs 1963b A kinetic model for
phototropic response of oat coleoptiles. Plant
Physiol. 38: 253-261

APPENDIX I

Plant Physiol. (1985) 77, 248-251
0032-0889/85/77/0248/04/301.00/0

flgrt qumgnigtigg

Blue and Green Light-Induced Phototropism in Arabidopsis
thaliana and Lactuca sativa L. Seedlings‘

Received for publication September IO. 1984 and in revised form October 18. 1984

BENJAMIN Srrsmrrz’, ZHANGLING REN, AND KENNETH L. Porr"
Michigan State University-Department of Energy Plant Research Laboratory. Michigan State Uni versit y.

East Lansing. Michigan 48824

ABSTRACT

Exposure time-response curves for blue and green light-induced pho-
totropic bending In hypocotyls of Arabidopsis thaliana (I...) Heynh. and
Lactuca sativa L. seedlings are presented. These seedlings show signifi-
cant phototropic sensitivity up to 540 to 550 nanometers. Since wave-
lengths longer than 560 nanometers do not induce phototropic bending.
it is suggested that the response to 510 to 550 nanometers light Is
mediated by the specific blue light photoreceptor of phototropism. We
advise care in tbe use of green 'safeligbts’ for studies of phototropism.

 

There are several action spectra for the phototropic response
in Avena coleoptiles. and all show that the activity of visible light
is limited to the 400 to 500 nm region, with the major peak of
activity being near 450 nm. Some of the action spectra end at
about 500 to 520 nm (1, 7, 17, 19) making it difficult to assess
whether these are indeed the longest wavelengths for phototropic
activity. However, Everett and Thimann (7) and Elliot and Shen-
Miller (6) state that there is no activity above 510 nm. Action
spectra for phototropism of other higher plants are not availble.
Recently lino er al. (11) showed that phytochrome mediates red
light-induced phototropism in corn, indicating that wavelengths
longer than 510 nm induce phototropic bending.

While initiating a detailed study on phototropism in dicotyle-
donous plants, we encountered a high level of irregularity in our
results, and tested the possibility that a green safelight might
induce phototropic response. We report here results showing that
green light induces phototropism in two dicotyledonous plants,
- and advise care in the use of green ‘safelights’ for studies of
phototropism.

MATERIAIS AND METHODS

Plant Growth. Seeds of Arabidopsis thaliana (L.) Heynh. were
sown on 0.8% (w/v) mr (Difco Bacto-Agar) supplemented with
1.0 mM KNO, in rows of 0.3-ml wells prepared from Falcon
3911 microtest III flexible assay plates. Sowing was done at a
density of one seed per well making it possible to expose individ-
ual rows of seedlings and to isolate single seedlings for measure-
ment of curvature. The plant assay rows were placed in trans-

' Supported by the United States Department of Energy under contract
no. DE-AC02-76ER01338.

3 Permanent address: Agricultural Research Organization. The Vol-
cani Center. Bet Dagan, Israel.

115

parent plastic boxes (21 x 16 x 3 cm) lined with moistened
paper to maintain high humidity. These incubation boxes were
kept in the dark for 4 d at 3' :1: PC to increase germination (16)
and then transferred to a Sherer Gro Lab (Sherer-Gilbert, Mar-
shall, M1) at 25°C and continuous white light for 30 h. This
preirradiation was required to induce phytochrome-mediated
germination (16). By the end of the preirradiation, only the
radicle emerged from the seed coat. At this stage, seedlings were
moved and kept in the dark at 25’ :1: 0.5' C, until the end of the
experiment.

Lactuca sativa seeds (6058 Grand Rapids—Burme’s Green-
hard brand; W. Atlee Burpee Co., Warminster, PA) were sown
on Whatman 1 filter paper moistened with distilled water and
incubated for 48 h in transparent plastic boxes at 25°C in dark.
The germinated seedlings, while remaining in the boxes, were
then transferred to 25'C and continuous white light for 8 h. This
preinadiation was shown in preliminary experiments to increase
the response to the subsequent phototropic stimulus (cf 9). At
the end of the preinadiation period, seedlings were selected for
uniformity, transplanted into vials containing moistened vermi-
culite (one plant per vial), replaced in boxes, and kept in dark-
ness.

The onset of the stimulus was at 72 h after transfer of the
imbibed Arabidopsis seeds from 3' to 25'C, or at 96 h after
sowing of lettuce seeds. For stimulation, Arabidopsis seedling
rows or lettuce seedlings in vials were taken out from their
incubation box, and the phototropic stimulus applied in a room
with a controlled RH of more than 90%. Following stimulation,
seedlings were again placed in the box and incubated in darkness
for development of curvature. Curvature was measured 120 min
alter onset of the stimulus (Figs. 1 and 2).

Light Sources. White light during preirradiation, at 125 mE-
m’2.s", was provided by General Electric Delux Cool White
fluorescent tubes (F48Tl2/CWX/l-IO, 800 ma) and measured
with a Li-Cor 1.1-185A radiometer. Phototropism was induced
by a unilateral light stimulus. The source consisted of a projector
equipped with a General Electric 300 w ELH multi-mirror
quartzline bulb. The light was passed through 3 cm of 5% (w/v)
cupric sulfate solution and through an interference filter. The
wavelength maxima of the interference filters used were at 450,
500, 510, 521, 53l, 540, 550, and 560 um, (PTR Optics, Wal-
tham, MA) with a half band width of 8.5. 8.2, 11.6, 9.6, 10.0.
12.0, 10.2, and 9.6 nm, respectively. To increase the ability to
distinguish between 450 nm light and all other wavelengths used,
and to avoid blue light contamination due to stray light, all the
interference filters used for wavelengths of 500 nm and above
were used in combination with a Corning 3-70 sharp cut filter
(Corning Glass Works), which has greater than 30% transmission
from 500 to 700 nm and less than 0.1% transmission at 480 nm.

ll 6
SEEDLING PHOTOTROPISM IN BLUE AND GREEN LIGHT

 

 

 

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Tlrne (min) Time (min)

FIG. 3. a, Time course for bending by Lactuca seedling hypocotyls

‘°‘ ’ ’ ”a ‘ exposed to unilateral light of either 0.1 w-m" 450 nm light (0) or 1.0

631 . w-m" 531 our light (0). Onset of exposure was at time zero. SE ranged

m/nsoo / .. from 0.3 to 10% of the value. b, Time course for growth increment below

r7 5 5 3 "1’ 5 3 3 r3 5 5 3 ’f 5 5 3 the tip of Lactuca hypocotyls. Onset ofexperiment in the dark at time

Emosue Time (“0103) -60 min. Arrow at time zero indicates onset of vertical irradiation.

Seedlingswere irradiated vn‘th either 0.1 w-m'2 450 our light (0) or with

FIG. 1. Exposure time-response curves for phototropism by A. rhal- 10 w-m" 531 um light (0). (O, 0), Growth in the dark. sr: ranged from
iana seedling hypocotyls. Seedlings were exposed to unilateral light with 0.1 to 7.6% of the value.

a fluence rate of 0.001 w-m‘z (A), 0.01 w-m’2 (B). 0.1 w-m"2 (C). and

1.0 w.m" (D). Curvature was measured 120 min afieronset of exposure. To m phototropic sensitivity above 560 nm, seedlings were
senngedfrom 2to 9% of the value. Numbers adjacent to curves indicate exposed to 58m mm through two Corning 2-62 sharp cm
theactrnrc wavelength "‘ nm. filters, which have greater than 50% transmission from about
600 nm above and less than 0.2% transmission from 400 to 580
nm. The fluence rate was varied by changing the distance be-
tween the plants and the light source and by using neutral density
filters. Measurements of the monochromatic light were made
using a model 68 Kettering radiometer (Laboratory Data Con-
trol, Riviera Beach, FL).

Measurement of Curvature and Growth Increment. At 120 min
after onset of stimulus, Arabidopsis and Lactuca seedlings were
gently adhered to a sticky transparent tape, with the direction of
bending parallel to the tape surface. The tape with Arabidopsis
seedlings was inserted into an enlarger and the curvature traced
and measured from the enlarged image of the seedling. The tape
with lettuce seedlings was photocopied and the curvature traced
and measured from the copy. in preliminary experiments we
found that the elongation of the lettuce hypocotyl between 56 h
(end of the white light preirradiation) and 100 h after sowing was
confined to a zone which, at 56 h after sowing, is within the first
mm below the seedling’s apical shoot meristem. At the end of
the preirradiation period, a black Indian lnk mark was placed 1
mm below the tip. The time course of the growth increment of
m this region was followed by time lapse photography (Fig. 3b).
Time lapse photography was also used to follow the development
of curvature (Fig. 3a). Photographs were taken using a Minolta
X-700 camera with Kodak IR film. 1R light was provided by a
”i l Minolta auto 280 PX flash lamp equipped with an IR 87 C

Kodak Wratten filter. Curvature and growth measurements were

made on the enlarged negative images.
3 ,/. f . {,1. A. . ,7. . . Twenty four Arabidopsis seedlings, sown in two rows, were
° ‘2 2‘ ° ‘2 2‘ '2 ‘2 2‘ used for each exposure. The rows were positioned to prevent
Exposure Time (sec '10 ) shading and curvature was measured for seedlings with hyocotyls

FIG. 2. Exposure time-response curves for phototropism in L. sativa emerging vertically from the agar. A mean curvature value was
seedling hypocotyls. Seedlings were exposed to unilateral light with a calculated from measurements made on 15 to 20 seedlings at
fluence rate of 0.01 w-m‘2 (A). 0.1 w-m’ (B). and 1.0 w-m" (C). each exposure. Experiments were repeated four to six times.
Curvature was measured 120 min after onset of exposure. SE ranged from Thus, for each exposure, at least four mean values were obtained.
I to 7% or the value. Numbers adjacent to curves indicate the actinic The data in the figures represented a final mean value of these
wavelength in nm. means. In experiments with lettuce, only two seedlings could be

 

 

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0

 

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70"

450
460

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LI 7
Shawn ET AL.

used at a time because of the use of a macro lens for the time
lapse photography of elongation (Fig 3b). All experiments with
lettuce seedlings were done in triplicate.

RESULTS AND DISCUSSION

The only appropriate way to evaluate the relative effectiveness
of different wavelengths of light in the induction of a response is
to compare fluence response curves for each wavelength. How-
ever, this can be done only when one can establish reciprocity
for that response. For phototropism, reciprocity holds only over
the range of fluences to which the plant exhibits what is tradi-
tionally referred to as the ‘first positive‘ response (5). Unfortu-
nately, the peak of the first positive response is only 9 to 10°
curvature for Arabidopsis seedlings which does not make the
desired quantitative approach feasible. A larger curvature can be
induced with relatively longer unilateral exposures to the stimu-
lating light (a condition in which reciprocity is not valid) and the
magnitude of the resulting response depends on the fluence-rate
used, as is known to occur in the ‘second positive’ response (5).
Moreover, the dependency on the fluence rate is complex (see
Fig. 5 in Ref. 7). Given these factors, comparison of fluence rate-
response curves would be misleading We have therefore chosen
to present results in the form of exposure time-response curves.
(Figs I and 2).

Arabidopsis seedlings were exposed to durations of 10 to 40
min of unilateral light of different wavelengths and subsequently
returned to darkness. Curvature was measured 120 min after
onset of the stimulus. A low fluence rate of l mw-m“2 at 450
nm was sufficient to induce a very strong bending response (Fig
1). Using the same fluence rate at 500 nm induced a minute
response, yet the strong curvature was readily induced at 500,
510, and 521 nm given the fluence rate of 10 mw- m”. Similarly,
this latter fluence rate at 531 nm induced a significantly smaller
bending response, but an increase of the fluence rate to 100 mw-
rn‘2 led to a response comparable to that induced by 1 mw. rn‘2
at 450 nm. The degree of curvature was consistently lower for
S40 and 550 nm light although a considerable response could be
induced with l w rn'2 at 550 nm. At wavelengths of 560 nm
and above, no bending was observed at any fluence rate from
10'" to 10’ mw- rn'2

The response of lettuce seedlings was significantly less than
that of Arabidopsis seedlings in the green light region (Fig. 2). At
all wavelengths above 500 nm, a fluence rate 1 order of magni-
tude higher was required in order to detect the phototropic
response. When the yellow-orange Corning cut—off filters were
used (see “Material and Methods”), no curvature was observed
in either Arabidopsis and Lactuca seedlings. Thus, 560 nm
probably represents the upper limit of phototropic sensitivity in
the visible region for both species.

Given the report of Elliot and Shea-Miller (6) that the fluence-
response curves and action spectra are similar for growth inhi-
bition and phototropism in Avena coleoptiles, and given the
report of Hartmann (10) that 531 nm light induced no growth
inhibition in lettuce hypocotyls, we were surprised to find green
light-induced phototropism in lettuce (Fig. 2). To investigate
further, we followed the time course for the blue light- and green
light-induced growth inhibition and curvature development in
the hypocotyls of lettuce seedlings. The first measurable blue
light-induced growth inhibition and curvature development ap-
peared about 10 min after onset of light. Green light-induced
curvature development and inhibition of elongation could be
detected 30 to 40 min after onset of light (Fig. 3, a and b). Hence,
as in A vcna (6). Lactuca spectral sensitivity for phototropism
and photomorphogenesis is similar in the blue-green region.

This reported responsiveness of lettuce to 531 nm does not
necessarily stand in disagreement with Hartmann's observations
(10). The fact that he could not find an inhibitory growth

Plant Physiol. Vol. 77, 1985

response to 531 nm light might have resulted from a difference
in experimental approach. It has been previously observed that
the response by lettuce hypocotyls to red light is qualitatively
different in different regions of the hypocotyl. This results in a
zero net effect when a red light-induced growth response is
measured on the level of the entire organ (8). Our elongation
measurements were confined to that region of the hypocotyl in
which the curvature response to unilateral light developed. In
contrast, in the work where no inhibition of elongation by 531
nm light was reported, growth of the entire hypocotyl was
measured (10).

Phytochrome is thought to be the photoreceptor mediating the
photomorphogenic influence of green light (3, 4, 15). In some
cases, a specific reversible green-orange-red (13) or far-red-green
light photoreceptor has been suggested (18). In phototropism, we
see no reason to postulate a photoreceptor pigment for the green
region different from the blue light photoreceptor of phototro-
pism. The sharp drop in the 480 to 510 our region as seen in the
action spectra of A vena coleoptile phototropism (19) may simply
be red-shifted into the 520 to 550 nm region in Arabidopsis and
Lactuca. This significant extension of phototropic activity into
the green region resembles a similar extension of phototropic
activity into the green region in Celosia cristata (2). These results
definitely corroborate the variation in spectral phototropic sen-
sitivity among different higher plant species (reported by Atkins
[2]). A precise comparison of the spectral sensitivites of Arabi-
dopsis, Lactuca, and Avena must await the development of a
reasonable method for measuring an action spectrum in Arabi-
dopsis and Lactuca, overcoming the limited first positive pho-
totropic response.

The present work demonstrates that 500 to 550 nm light can
serve as a vectorial signal for inducing phototropic responses.
Green light has also been shown to have a scalar effect on
subsequent gravitropic response (13, 14), and to have a potential
photomorphogenic effect via reception by photochrome. There-
fore, we suggest caution in the use of green light as safelight not
only in studies on photomorphogenesis (12) but also in work on
phototropism. The ‘safety’ of any light should be established for

any given species.
Wedgmam—We thank T. R. Best for excellent teehnial asistance.

LITERATURE GTE!)

l. AsoatANrNG EJA, AW Cm 1961 Comparative study of phototropic
responseandpigrnentcontentinoatand coleoptilesPlantPhysiol
36: 453-464

2. ATKINS GA 19361'he effect of pigment on phototropic response: A comparative
study of relations to monochromatic light. Ann Bot 50: 197-218

3. Buauw Jansen G 1973 Dose-response curves for phytochrome-mediated
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Need 23: 513-519

4. BLAAUW-JANSEN G l983 Thoughts on the possible role of phytochrome
destnrction in phytochrome-controlled responses Plant Cell Environ 6: 173-
179 ‘

5. DENNISON DS 1979 Phototropism. In W Haupt, ME Feinleib, eds, Encyclo-
pedia of Plant Physiology, Vol 7. Physiology of Movements. Springer-Veda;
Berlin, pp 506-566

6. ELuor'r WM. J SHEN-MILLER 1976 Similarity in dose-response. action spectra
and red light responses between phototropism and photoinhibition of growth.
Photochem Photobiol 23: 195-199

7. Evertm M. KV THIHANN 1968 Second positive phototropism in the Avena
coleoptiles Plant Physiol 43: 1786-1792

8. HACKER M, KH HARTIIANN. H Moan 1964 Zellteilung und Zellwachstum im
Hypoltotyl von Lactuca sativa L unter dem Einflus des Lichtec Plants 63:
253-268

9. Han JW, IR MACDONALD 1981 Phototropic responses of hypocotyls of
etiolated and green seedling. Plant Sci Lett 21:151—158

10. HARTMANN KM 1966 A general hypothesis to interpret ‘high energy phenom-
ena‘ of photomorphogenesis on the basis of phytochrome. Photochem Pho-
tobiol 5: 349—366

11. IINo H. WR Bums. E, Scrum I984 Phytochromemediated phototropism
in maize seedlingsshoots Planta 160 41-51

12. IINO M. DJ Can 1981 Safel'ght for photomorphogenetic studies; Infrared

L18

SEEDLING PHOTOTROPISM IN BLUE AND GREEN LIGHT

radiation and infrared-scope. Plant Sci let! 23: 263—268 W5C ‘3’:me I:hlttlplhtzittzmac‘t;vatiorl of germination in Arabidopsis
K. an RM eversi m m and orange- radia ' plant mac. n ysio : 3-6
'3' cell «“9131: $1”, 1:5,“ 63-‘Ifl4-l 16 M ”on on 17. Snaorsmas W In, RB WITHIOW l958 Actijr‘isr; spectrum of phototropic tip
' . ' . . - curvature of Avena. Plant Phya'ol 33: 360-
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Act- Bot New 17:437-440 . , closure ofAlbizzia leaflets Plant Physiol 70: 901-904
15. MANDou DP. WR Bum I981 Phytochrome control of two low-madam r9. rummn xv. GM Cum I960 Phototropism and phototaxis. In HS Mason,
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