.THESIs

Date

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This is to certify that the

thesis entitled

Mechanisms of Establishing a Light Gradient for
First Positive Phototropism in Z§g_maxs (L.)

presented by

Carol Joyce Piening

has been accepted towards fulfillment
of the requirements for

Master of Science degree in Botany and Plant Pathology

5/ .. “351/ /

Major professor

 

 

August 9, 1984

 

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MECHANISMS OF ESTABLISHING A LIGHT GRADIENT FOR
FIRST POSITIVE PHOTOTROPISM IN ZEA MAYS (L.)

BY

Carol Joyce Piening

A THESIS

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

MASTER OF SCIENCE

Department of Botany and Plant Pathology

1984

ABSTRACT

MECHANISMS OF ESTABLISHING A LIGHT GRADIENT FOR
FIRST POSITIVE PHOTOTROPISM IN ZEA MAYS (L.)

BY

Carol Joyce Piening

Pigment distribution in individual seedlings of
SAN 9789 treated, control, and four "carotenoidless" mutant
lines was mapped in coleoptilar, shoot, mesocotyl, and root
tissue. Total attenuation of light, as well as absorption,
was measured spectrophotometrically. Longitudinal
transmission of three wavelengths (632, 514, and 450 nm) of
light was quantified.

Mechanisms of establishing a light gradient to detect a
dose of light in the first positive range were evaluated.
It is concluded that first and second positive phototropism
share a number of characteristics. Carotenoids play the
role of screening pigments for both responses. Attenuation
by absorption and scatter sets up the gradient of light
necessary for both first and second positive phototropic

responses.

To Ruth Piening and Isabelle Lynch

I could while away the hours

Conferrin' with the flowers

Consultin' with the rain...
E.Y. Harburg

ii

AKNOWLEDGMENTS

There are a number of people whom I wish to thank for
their help, time, advice, and interest during the research
and writing of this thesis. First thanks go to Dr. Ken
Poff, major professor and friend. Thanks also to the
members of my committee, Dr. Norman Good, Dr. Hans Kende,
and Dr. Herb Nakatani for their time and advice.

Technical assistance from Thérése Best, Mary Gregg,
Elliot Light, and the staff of the Abrams Planetarium and
discussions with Brian Parks, Zhangling Ren, and Dr.
Benjamin Steinitz have added to my education and made the
whole process more enjoyable.

Eliot Singer and his computer get a particularly
heartfelt thanks. Without them, this thesis would still be
just so many small dots.

Thanks to Rebecca Grumet for help with statistics and
other perplexities.

Finally, thanks to Mary and Lawrence Piening, who have
been wondering why their daughter spends so much time in the

Idark.

iii

TABLE OF CONTENTS

Page
List of Tables.......................................... vi
List of Figures......................................... ix
Review of Literature
Early experiments.................................. 1
Action spectra..................................... 4
Dose response curves............................... 10
Transduction pathway............................... 15
Introduction............................................ 21
Chapter 1 Distribution of Pigments in Individual Corn
Seedlings
Introduction....................................... 29
Methods............................................ 29
Results and discussion............................. 32
Summary............................................ 44
Chapter 2 The Role of Carotenoids in First Positive
Phototropism of Corn Seedlings
Introduction....................................... 50
Methods............................................ 51
Results and discussion............................. 54

summarYOOOOOOOICO0.0...0..OOOOOOOOOOOOOOOOOOOOOOOOO 71

iv

Chapter 3 Mechanisms of Establishing a Light Gradient:
Lens, Longitudinal Light Transmission, and
Scatter
Introduction : Lens................................ 73
Methods............................................ 74
Results and discussion............................. 74
Introduction : Longitudinal light transmission..... 77
Methods............................................ 79
Results and discussion............................. 83
Introduction : Scatter............................. 93
Methods............................................ 94
Results and discussion............................. 94
Conclusions and Recommendations.........................105
Appendix 1 Seed Multiplication of "Carotenoidless"
Mutants.....................................112
Appendix 2 Geotropism of Corn Seedlings at 60% and 100%
Relative Humidity...........................117

BibliograthOOOOOOOOOOOOOOOOOO0.000.000...00.000.000.000118

Table 2.1.

Table 2.2.

Table 2.3.

LIST OF TABLES

Mean curvature of seedlings irradiated along
long and short transverse axes. Different doses
were evaluated on different days, so comparisons
between different doses should not be made.
Methods given in text. SE: standard error of

the mean. Mean curvature in degrees..........56

Analysis of variance for dose response curves of

4

control and 2 x 10- M SAN 9789 treated

seedlings. Data shown in Figure 2.3..........63

Analysis of variance for dose response curves of

5

control and 2 x 10- M SAN 9789 treated

seedlings. Data shown in Figure 2.4..........63

Table 2.4.

Table 2.5.

Table 2.6.

Table 2.7.

Table 2.8.

a. Geotropism XE SAN 9789 concentration in 100%
relative humidity. Each experiment included
9-11 individuals. Curvature measured in
degrees. SE: standard error of the mean......66
b. Values of Student's t for indicated

comparisonSOOOOOOOOOOO0.0.0.0....00.0.0000000066

Growth rates of control and SAN 9789 treated
seedlings over a three hour interval.

SE: standard error of the mean................66

a. Analysis of variance for dose response curves
of wild type and pale mutant seedlings. Data in
Figure 2.5.0000000000 ..... 0.00.00.00.00000000069

b. Orthogonal contrasts for corn type.........69

Analysis of variance for geotropism of yellow

and pale mutant seedlings.....................70

Growth rates of mutant seedlings. SE: standard

error Of the meanOOOOOIOOOOOO0.0.0.0000000000070

Table 2.9.

Table 3.1.

Table 3.2.

Summary of correlations among carotenoid
concentration, phototropic curvature, and
geotropic curvature. Carotenoid content is
based on absorption at 480 nm of randomly
selected individuals (Figures 1.5, 1.9, and
1.10), phototropic responses on dose response
curves (Figures 2.5, 2.4, and 2.5), and
geotropic responses on geotropic controls used
for dose response curves. For pale mutants,
wild type seedlings served as control, for
SAN 9789 treated seedlings, hybrid seedlings
treated with water were used. Responses are
expressed in terms of percentage of control

responseOOOOOOOOOOOOOOOOOOOOOOOOO0.00.00.00.0070

Analysis of variance of data for longitudinally

transmitted light. Data shown in Figure 3.5..89

Mean Optical density at 450 nm of two randomly
selected corn seedlings. Shoot tip measurements
were taken through both coleoptile and primary
leaf tissue. Coleoptile measurements were made
on excised coleoptiles. Red grown seedlings
received one hour of red light per night. Dark
grown seedlings were completely etiolated. Data
were obtained from measurements made for

Figures 3.8-3.110.000.000000.0.000000000000103

viti

Figure 1.1.

Figure 1.2.

LIST OF FIGURES

Absorption spectra of corn seedlings, taken
through coleoptile and primary leaf, 0.5 cm
from tip. A) Dark grown seedling. Peaks occur
at about 415 nm, 435 nm, 470 nm, and 640 nm.

B) Seedling received one hour of red light per
night. Peaks occur at about 415 nm, 435 nm,
470 nm, and 660 nm. C) Seedling was grown
under fluorescent lights. Peaks occur at about
415 nm, 470 nm, and 660 nm. Curves are offset

along the y axis for clarity in all figures.

0....0.0.0.000...OOOOOOOOOOOCCOO00.0.0000000033

Spectra taken through the short cross-sectional
axis of a red grown seedling, perpendicular to
the vascular bundles. A) extreme tip,
coleoptile tissue alone. B) 1.5 mm from tip.
C) 3 mm from tip. D) 5 mm from tip. E) 7 mm
from tip. F) 9 mm from tip. G) 14 mm from

tip, on node. H) 17 mm from tip, in mesocotyl.

.0...0.00.00.00.00.0COO...00.0.0000000000000034

Figure 1.3.

Figure 1.4.

Figure 1.5.

Figure 1.6.

Six day old excised coleoptile, cross-sectional
spectra. A) Spectrum taken along short axis,
perpendicular to vascular bundles. B) Spectrum
taken along long axis, through vascular

bundleSooo.0.0.0.00000.000000000000000...00.036

Seedlings treated with different concentrations
of SAN 9789. Spectra taken 5 mm from tip of
shoot. A) Control. (B-F) SAN 9789 treated.

B) 4 x 10'7M. C) 4 x 10'6M. D) 4 x 10'5 M.

E) 4 x 10'4M. F) 4 x 10'3M..................37

2 x 10-4

M SAN 9789 treated, red grown seedling.
A) shoot 5 mm from tip. B) mesocotyl.

C) excised coleoptile. D) root..............38

Two control seedlings grown in the same
conditions under red light were selected
randomly to illustrate the variation among
individuals. A) and B) are absorption spectra
taken 5 mm from the tip. C) is the difference

Spectrum (A-B)00.0.0.0...00.00.000.000000000040

Figure 1.7. As with Figure 1.6, but seedlings were treated

with 2 x 10’4

M SAN 9789. A) and B) are
spectra taken 5 mm from the tip of two randomly
selected individuals. C) is the difference

Spectrum (A-B)0.0.0.000...0.0.00.00000000000041

Figure 1.8. Spectra of dry seeds. A) hybrid control.
B) lwl yellow. C) lw2 yellow. D) w7748
yellow. E) clp yellow. F) lwl pale. G) lw2

pale. H) w7748 pale. I) clp pale............42

Figure 1.9. Absorption spectra of different tissues of lwl
seedlings. (A-C) wild type. A) intact shoot
5 mm from tip. B) mesocotyl. C) excised
coleoptile. (D-F) mutant. D) intact shoot
5 mm from tip. E) mesocotyl. F) excised

COleopti-leo.0.0...OOOOOIOOOOIOOOO00.9.000000044

Figure 1.10. Absorption spectra of different tissues of lw2
seedlings. (A-C) wild type. A) intact shoot
5 mm from tip. B) mesocotyl. C) excised
coleoptile. (D-F) mutant. D) intact shoot
5 mm from tip. E) mesocotyl. F) excised

COleOptileoo000.000.000.00000000000000000000045

Figure 1.11.

Figure 1.12.

Figure 2.1.

Absorption spectra of different tissues of
w7748 seedlings. (A-C) wild type. A) intact
shoot 5 mm from tip. B) mesocotyl. C) excised
coleoptile. (D—F) mutant. D) intact shoot

5 mm from tip. E) mesocotyl. F) excised

COleOpti-le.OOOO..0.0.0.0.000...0.0.0.0000000046

Absorption spectra of different tissues of clp
seedlings. (A-C) wild type. A) intact shoot

5 mm from tip. B) mesocotyl. C) excised
coleoptile. (D-F) mutant. D) intact shoot

5 mm from tip. E) mesocotyl. F) excised

COleOptj-le.O..0...OO...0.0.0.000000000000000048

Spectral distribution of the blue light used as
phototropic stimulus and red light used to
promote coleoptile elongation and inhibit

meSOCOtY1 elongation.0.0.0.0.000000000000000053

Figure 2.2.

Figure 2.3.

Figure 2.4.

Time course of bending. Left: 2 x 10-4M

SAN 9789 treated seedlings. Right: control
seedlings. All seedlings were given two
seconds of 12 pW x cm.2 blue light to elicit a
nearly maximal first positive response. After
three hours, SAN 9789 treated seedlings have
reached a maximal response, where control
plants may be capable of some further
curvature....................................58
Dose response curves for control and 2 x 10-4M
SAN 9789 treated seedlings. Experimental
details in Methods. Each point represents the
mean of six experiments, and each experiment
used 9 or 10 seedlings per point. Standard

error for data pooled from six experiments was

+/-1.ZOOOOOOOOCO0......00.0.0000000000000000061

5M

Dose response curves for control and 2 x 10-
SAN 9789 treated seedlings. Methods and
replications as for Figure 2.3. Standard error

for data pooled from six experiments was

+/-1.4o.O...0..OOOOOCOOOOOOOCO0.00.00.00.000062

xiii

Figure 2.5.

Figure 3.1.

Figure 3.2.

Figure 3.3.

Dose response curves for control and SAN 9789
treated hybrid seedlings, yellow and pale
mutant seedlings. Experimental details in
Methods. Each point represents the mean of
three experiments. Each experiment used 7 or 8
seedlings per light point, 5 or 6 seedlings per
dark control. Standard error for data pooled

from three experiments was +/-3.06°..........68

Critical values for absorption coefficient («A

for a Phycomyces sporangiophore (solid line,

 

open circles) and a model corn coleoptile
(dashed line, solid circles). Adapted from

castle (1933b).0...OOOCOOOOOOOOOOOOOOOOO0.00.78

Apparatus used for measuring axial light
transmission through corn coleoptile segments.

For details, see textOOOO00.00.000.000000000081

Light incident on a coleoptile (A) can follow a
number of different paths. Some (B) is
reflected or scattered at the surface, and
never enters the tissue. Of the light which
enters the tissue, some is scattered randomly
(C), some is absorbed (D), and some may be

scattered axially, or "piped" (E)............84

xiv

Figure 3.4.

Figure 3.5.

Figure 3.6.

The apex of a coleoptile segment was
illuminated with red light at the indicated
incident angle (degrees from normal). No
angle dependence was found. Closed circles:
control tissue. Open circles: 2 x 10-4 M

SAN 9789 treated tissue. Error bars: standard

error of the mean............................85

Longitudinal light transmission. Transmission
of three wavelengths of light was measured
through 2.5 cm segments of corn seedlings of
indicated ages. Control seedlings (o—————o),

2 x 10'4

M SAN 9789 treated seedlings (Onvuuuolo
Shaded areas represent the range of dark
current over the several days needed to collect

data. Error bars: standard error of the mean.

OOOO.I.0.00.0I...0.0.0....00.000.000.0000000086

Longitudinal transmission of red light as a
function of tissue length. Control seedlings.
Transmitted light was measured in terms of
current from a photomultiplier tube. Each set
of symbols represents measurements taken from
one individual, with a best fit line through
each set of data. Data separated along x axis

for clarity. Mean slope -0.74...............91

Figure 3.7.

Figure 3.8.

Longitudinal transmission of red light as a

4M SAN 9789

function of tissue length. 2 x 10-
treated seedlings. Transmitted light was
measured in terms of current from a
photomultiplier tube. Each set of symbols
represents measurements taken from one
individual, with a best fit line through each

set of data. Data separated along x axis for

Clarity. Mean SlOpe -0082.oooooooooooooooooogz

Total attenuation of light (absorption plus
scatter) by various tissues of a completely
etiolated control seedling. (0) Tip of
seedling through coleoptile and primary leaf.
(0) Base of seedling through coleoptile and
primary leaf. (0) Mesocotyl. (I) Excised
coleoptile. Measurements of current on the
photomultiplier tube were used to calculate the

ratio of system / sample.....................95

Figure 3.9.

Figure 3.10.

Total attenuation of light (absorption plus
scatter) by various tissues of a control
seedling grown with one hour of red light per
night. (a) Tip of seedling through coleoptile
and primary leaf. (0) Base of seedling through
coleoptile and primary leaf. 1(0) Mesocotyl.

(I) Excised coleOptile. Measurements of‘

current on the photomultiplier tube were used

to calculate the ratio of system / sample....96

Total attenuation of light (absorption plus
scatter) by various tissues of a 2 x 10-4M

SAN 9789 treated, dark grown seedling. (I) Tip
of seedling through coleoptile and primary
leaf. (o) Base of seedling through coleoptile
and primary leaf. (0) Mesocotyl. (I) Excised
coleoptile. Measurements of current on the

photomultiplier tube were used to calculate the

ratio of system / sample.....................97

xvii

Figure 3.11.

Figure 3.12.

Figure 3.13.

Total attenuation of light (absorption plus
scatter) by various tissues of a 2 x 10-4M
SAN 9789 treated seedling grown with one hour
of red light per night. (0) Tip of seedling
through coleoptile and primary leaf.

(0) Base of seedling through coleoptile and
primary leaf. (D) Mesocotyl. (I) Excised
coleoptile. Measurements of current on the

photomultiplier tube were used to calculate the

ratio of system / sample ..... ................98

Total attenuation of light (absorption plus
scatter) by shoot tips of control, SAN 9789
treated, yellow and pale mutant seedlings at
selected wavelengths.(l)Control. (I)lw1
yellow. (A)lw2 yellow. (B) 2 x 10-4M

SAN 9789 treated. (o)lw1 pale. (A)lw2 pale.

0.0000.000I.0....O0.0.0.0...0.0.0.0000000000099

Approximating a scatter ramp artificially or
with plant tissue low in pigment. A) control
shoot plus ramp (dashed line). B) control
shoot minus 14 layers of Kimwipes. C) control
shoot minus mesocotyl. D) control shoot minus
excised coleoptile. E) control shoot minus

4

2 x 10' M SAN 9789 treated shoot. F) lw2

yellow shoot minus lw2 pale shoot...........102

indii

REVIEW OF LITERATURE

Early experiments

 

Phototropism is a mystery on any level beyond a
descriptive one. It is well established that a plant
responds to a unilateral dose of light by bending toward or
away from the light source. However, the mechanisms for
detecting light direction, for transducing the stimulus into
differential growth, and the primary photoreceptor all
remain unknown.

Charles Darwin is often credited with the first

methodical observations of phototropism. Power of Movement

 

in Plants, published in 1881, first reported the importance
of the tip to the phototropic response of a coleoptile.
Darwin was also first to observe the transmission of a
physiological effect of light, noting that the response to a
unilateral stimulus appeared at the tip and migrated to the
base of the coleoptile. Darwin was convinced that
phototropism was simply a modified form of circumnutation,
light causing the plant to "modify its movement for a time
in a beneficial manner."

Another early explanation of the mechanism of

phototropism is the ”light growth hypothesis", which, in its

2

simplest form, states that light itself is the stimulus
which works on the plant and that the light gradient across
a unilaterally illuminated shoot could explain its
differential growth (Blaauw, A.H.,1914). Blaauw's work

shows that in the case of Helianthus globosus, an increase

 

in dose results in a decrease in growth on the more brightly
lighted side of the plant. Blaauw's light growth hypothesis
has since been qualified to state that it may be important

only in second positive phototropism (Firn and Digby, 1980).

The Cholodony-Went hypothesis states that tropic
curvatures, whether they are in response to light or to
gravity, are mediated by the establishment of lateral auxin
gradients across the stimulated organ. Went and Thimann
(1937) noted that the discovery and subsequent study of
auxins was a result of work on phototropism.

DuBuy and Nuernbergk (1934) were first to summarize the
body of data which show that plants respond differently to
different doses of light. At low doses of high intensity
and short duration, plants show positive (first positive,
system I, or type a) curvature; at higher doses, a negative
(first negative, system 11, or type b) curvature, and at
still higher doses, (low intensity and long duration)
another positive (second positive, system III, or type c)
response.

The influence of the coleOptile tip on growth, the
asymmetric distribution of auxin in a unilaterally

stimulated coleOptile, the shape of the dose-response curve,

3

and the action spectrum showing maximum phototropic response
to blue light outline the basic studies on the phenomenon of
phototropism. In order to understand phototropism, the
mechanism of detecting a light gradient, the photoreceptor
pigment and the transduction pathway must all be known.

This thesis examines the first step in phototropism, that
is, the mechanism by which a unilateral light stimulus is

detected.

Action spectra

 

Action spectra have been used in many systems to help
identify photoreceptor pigments. An action spectrum is
generated by irradiating a sample with light of different
wavelengths and measuring the relative response to each
wavelength. Assuming that only light which is absorbed can
cause a reaction, the wavelengths of light which are
absorbed most efficiently will elicit the greatest response.
Within certain limits, the action spectrum should parallel
the absorption spectrum of the photoreceptor pigment.

Shropshire (1972) discussed the limitations of action
spectra as a tool for identifying photoreceptor pigments.
An example of a problem in the case of the blue light
photoreceptor is presented by screening pigments. A high
concentration of "foreign" pigment molecules can alter the
response of the plant by absorbing actinic light before it
reaches the photoreceptor. Such screening pigments change
the action spectrum, and make parallels to the absorption
spectrum less obvious.

There are two families of pigments, flavins and
carotenoids, which are the most likely candidates as
photoreceptors for phototropism. Both absorb light
maximally in the 400 - 500 nm range. Carotenoids extracted
in water show a triple peak between 400 - 500 nm like that
seen in action spectra. Riboflavin in water does not show
this fine structure, but when extracted in ethanol and kept

at 770K, the triple peak appears (Sun 23 21., 1972, cited in

5

Dennison, 1979). Both pigment families absorb in the 280 -
300 nm region. Flavins, but not carotenoids, absorb in the
340 - 400 nm region, showing a peak similar to that which

appears in the action spectra of Phycomyces and monocots.

 

However, an identification of the blue light
photoreceptor cannot be made based solely on a correlation
between action and absorption spectra. Action spectra can
be affected by factors other than screening pigments,
including the concentration and distribution of the
photoreceptor and the quantum efficiency with which the
light is absorbed. In addition, the absorption spectrum of
a given pigment can be altered by a number of other factors.
In an extract, solvent and temperature can shift absorption
peaks and change peak heights. Location within a cell or
association with a cell component or with another molecule
contribute to the absorption spectrum.

Action spectra are based on dose response curves and
are valid only over a limited dosage range. To generate
dose-response curves, it is necessary to assume that
reciprocity holds; that response depends only on the total
dose of light given, and is independent of the duration or
intensity of irradiation. For phototropism by plant
seedlings, this is true for only a limited range of doses,
the region known as first positive phototropism. For first
negative and second positive responses, the duration of
stimulus becomes increasingly important (Blaauw and

Blaauw-Jansen, 1970a). Approximate action spectra for

6

systems where reciprocity does not hold have been made, but
must be interpreted cautiously.

Other approaches have been used to distinguish between
flavins and carotenoids as photoreceptors for phototropism.
Presti gt 2l° (1977) found evidence against carotenoids as
the photoreceptor when working with carotenoidless mutants

of Phycomyces. The strain, blocked at each of six steps of

 

biosynthesis between phytoene and B-carotene, had less than

4 x 10--5

of the wild-type carotene concentration, yet had a
normal phototropic response.

Vierstra and Poff (1981b) treated corn seedlings with
SAN 9789, an inhibitor of carotenoid biosynthesis. They
found that seedlings lacking 98% of their carotenoids showed
about 60% of their normal second positive phototropic
response. Vierstra and Poff (1981b) concluded that the
"bulk" carotenoids eliminated by SAN 9789 acted as screening
pigments and increased the light gradient for second
positive phototropism, rather than acting as photoreceptor
pigments. However, they could not rule out a carotenoid
photoreceptor pigment entirely. Similar experiments with
flavinless mutants or tissue treated with inhibitors of
flavin synthesis cannot be performed, since the absence of
flavins would be expected to be lethal.

Several experiments using inhibitors of action rather
than of synthesis of pigments have been performed. Schmidt
gt El: (1977) surveyed several compounds to find a specific

inhibitor of phototropism. They found that chloride (KCl)

7

inhibits neither phototropism nor geotropism, while cyanide
(KCN) inhibits both. Iodide (KI), azide (NaN3), and
phenylacetic acid (PAA) inhibit phototropism specifically.
The authors suggested that iodide, azide, and PAA inhibited
a flavin photoreceptor directly, but did not rule out the
possibility that metabolic processes may have been
inhibited.

Vierstra and Poff (1981a) showed that concentrations of
PAA necessary to cause specific inhibition of phototropism
are lower than those which affect geotropism. Strong auxins
such as IAA inhibited photo- and geo- tropism equally.
PAA's inhibitory effect is probably at the photoreceptor
pigment, rather than as a result of its auxin-like activity.

Vierstra gt 31. (1981) found that xenon specifically
quenched the triplet excited state of flavin molecules, but
did not inhibit phototropism. KI is an effective inhibitor,
but quenches both triplet and singlet excited states of
flavin molecules. Azide and PAA, both specific inhibitors,
quench only the singlet state. Thus, if flavin is the
photoreceptor pigment, the triplet excited state is probably
not the state through which the photochemistry of
phototropism proceeds. Bridges and Wilkins (1971) looked at
another series of compounds including mannitol, carbowax
1540 and KCl. All were effective in inhibiting phototropism
and geotropism, contrary to the results of Schmidt g£_gl.

(1977) for KCl. Glycerol was not an effective inhibitor.

8

The authors concluded that the mechanism for these compounds
is an osmotic inhibition of growth.

Blue-light-induced changes at the chemical level have
been examined in an attempt to identify the photoreceptor
pigment. Song (1980) summarized the molecular changes which
may occur in flavinoids and carotenoids upon irradiation
with blue light. In a more physiological system, Poff and
Butler (1974) noted blue-light-induced absorbance changes in

mycelial mats of Phycomyces, in Dictyostelium discoideum

 

 

slugs and in cell-free extracts derived from D. discoideum.

 

Mufioz and Butler (1975) found similar reversible light

induced absorbance changes in mycelia of Neurospora crassa.

 

The absorbance changes were due to the photoreduction of a
b-type cytochrome and were apparent only in partially
oxidized tissue. Action spectra showed that a flavin
mediated the reduction of the cytochrome. Bleaching the
flavin resulted in an irreversible light-induced absorbance
change and a loss of the reversible cytochrome absorbance
changes. From this latter result, Mufioz and Butler (1975)
proposed that the photoreceptor is a flavin, and that the
first step in the transduction pathway is the flavin-induced
reduction of cytochrome. Brain gt El: (1977) found similar
light induced absorbance changes in membrane fractions of

Neurospora and corn. Other workers (see Briggs and Iino,

 

1984) have further localized the cytochrome to the

peripheral plasma membrane.

9
The identity of the blue light photoreceptor remains
unknown. Spectral evidence points to a flavin or a
carotenoid. Experiments with inhibitors favor a flavin
photoreceptor, but carotenoids have not been decisively

eliminated.

10

Dose-response curves

 

Dose-response curves for most phenomena follow a simple
pattern of increasing response with increasing stimulus, up
to the level of saturation. This is not true of
phototropism to blue light. Instead, for oats and corn,
there are three distinct regions to the dose-response curve.
Response to doses just above threshold, usually given in
short flashes of high intensity, are called first positive
curvature. In this region, the response increases with dose
and is dose dependent. Within limits, the Bunsen-Roscoe law
of reciprocity holds, i;g., the same dose given with varying
duration and intensity elicits the same response. At a dose
in the range of 50 - 100 pE x cm-2 (Zimmerman and Briggs,
1963a, Blaauw and Blaauw—Jansen, 1970a, Iino gt_§l., 1984),
the first positive curvature peaks, and at higher doses the
response decreases with increasing stimulus. In oats,
negative curvature can be induced. This region is known as
first negative, and is also dose-dependent. At still higher
doses, the response is increasingly dependent on exposure
time rather than dose. The curvature in this region is
termed second positive phototropism (Dennison, 1979).

Zimmerman and Briggs (1963a) generated dose response
curves for three different intensities of actinic light.
They found a lack of reciprocity in the second positive
photoresponse. Everett and Thimann (1968) obtained similar
results. Blaauw and Blaauw-Jansen (1970a) mapped the

curvature response for doses from threshold through second

11

positive, at several combinations of intensity and time.

For the second positive response, they also found that
curvature depended mostly on exposure time, although the
steepness of the slope was dependent on intensity. The
failure of reciprocity in the second positive response makes
it impossible to generate true action spectra or the dose-
response curves on which they are based. Relative quantum
efficiency of different wavelengths of light cannot be
evaluated if response is dependent on exposure rather than
dose.

Thimann and Curry (1960) separated Azgtg curvature into
two morphologically different responses. First positive
stimuli trigger a tip response, a curvature which starts
near the apex and migrates basally along the coleoptile.
Second positive doses produce a base response, where
curvature is distributed along the entire length of the
coleOptile. To explain the different responses, they
proposed that two different mechanisms for phototropism
exist, perhaps based on two different photoreceptor
pigments. The distinction between a tip response and a base
response has since been ascribed to uncontrolled red light
effects (Blaauw and Blaauw-Jansen, 1970b), but the
possibility of the involvement of more than one pigment or
mechanism remains.

Recent work has shown that light of wavelengths longer
than 500 nm can also induce a phototropic response.

Iino gt gt. (1984) have described phytochrome-mediated

12
phototropism in response to 667 nm light. Mandoli and
Briggs (1981) have documented growth changes in response to
green "safe" light. Steinitz (personal communication) has

shown that Arabidopsis seedlings respond phototropically to

 

light up to 550 nm.

Even in systems where unilateral blue light is used as
an actinic light, there exists evidence that more than one
pigment is involved. As discussed previously, it appears
that both a flavinoid photoreceptor and a carotenoid
screening pigment are necessary for normal second positive
phototropism in corn. The role of screening pigments in
first positive phototropism will be discussed in the present
work. For many dicots, only tissues which have greened to
some extent are phototropically responsive, raising the
possibility that chlorophyll acts as a screen. Screening
pigments would increase the light gradient across the
tissue, and would also alter the spectral quality of the
light received by the photoreceptor pigment itself.

A second line of evidence supporting the involvement of
more than one pigment in phototropism has been demonstrated
by preirradiating the plants with red light. Asomaning and
Galston (1961) found that preirradiation with red or blue
light increased the carotenoid and flavin content of cats
and barley. Several workers have noticed an effect of red
light on subsequent phototropism (see Briggs 1963b for a
review of early work). Zimmerman and Briggs (1963a) have

shown that oat coleoptiles preirradiated with red light show

13
a less sensitive first positive and a more sensitive second
positive phototropic response. The first negative response
disappears entirely. Blaauw and Blaauw-Jansen (1970a)
summarized the effects of red preirradiation on their
system. Briggs (1963a) found that red light effects
disappeared after about three hours. Chon and Briggs (1966)
showed that the action spectrum for red light preirradiation
resembled that for other phytochrome mediated responses, but
Briggs and Chon (1966) found no simple correlation between
physiological response and the photochemical state of
phytochrome. They proposed that only a small fraction of
the total phytochrome present was active. Kang and Burg
(1974) found that red light enhanced phototropism of
etiolated peas, and prOposed that lateral auxin transport
was affected. Franssen and Bruinsma (1981) proposed that
both phytochrome and a blue-light photoreceptor are involved

in phototrOpic responses of Helianthus, based on a red - far

 

red reversible inhibition of phototropism in preirradiated
plants. Thus, it appears that phytochrome may be involved
in phototrOpism, but whether it has some effect on the
perception of light direction, on auxin distribution, or at
some other point in the transduction pathway remains
unknown. Given the wide variety of experimental techniques,
it is difficult to draw any general rules about the effect
of red light.

Blaauw and Blaauw-Jansen (1970b) proposed that second

positive phototropism has a "tonic" and a ”tropic"

14

component. They showed that a unilateral dose of light can
be replaced by the same dose of light in which a proportion
of the dose is given from above. Depending on the length of
the vertical exposure and the interval between vertical and
unilateral stimuli, the same or greater curvature can be
reached. They proposed that one light dose affects the
tonic condition of the plant, and requires a lag time in
which to work. A separate dose determines the actual
curvature. Thus, first and second positive curvature would
both show reciprocity, except that the second positive
response takes longer to appear because the coleoptile is
desensitized by the amount of light to which it is exposed.

The similarity in action spectra for a wide variety of
organisms has led to the hypothesis that there is one
primary blue light photoreceptor pigment (see Dennison,
1979). Briggs and Iino (1983) raised the possibility that
there is more than one blue light photoreceptor pigment,
though the difference may be as slight as a flavin in
different environments. Their arguments for more than one
photoreceptor pigment are based mostly on differences in
action spectra for a number of blue light triggered
responses in a variety of organisms.

Thus, it appears that more than one pigment is involved
in phototropism, whether the second pigment be a screen,
receptor pigment for a different phenomenon, or an auxillary
pigment which primes the response of the photoreceptor

pigment.

15

Transductiongpathway

 

Early observers of phototropism noticed that
light-induced curvature appears to start at the tip of a
unilaterally irradiated coleoptile and progresses, with
time, down the axis (Darwin, 1881). This observation led to
the postulate that some diffusible substance manufactured at
the tip of a plant was necessary for phototropism to occur.
Experiments with coleoptiles which had their tips removed, a
block of gelatin inserted, and tips replaced confirmed that
a diffusible substance was necessary for phototropism
(Boysen Jensen, cited in Went and Thimann, 1937). Went
later found evidence that an asymmetric distribution of the
growth substance was responsible for the curvature of the
shoot. The Cholodony-Went hypothesis states that an auxin
gradient through a shoot or a root is responsible for the
differential growth which leads to curvature (Went and
Thimann, 1937). Went and Thimann (1937) list four possible
ways of distributing auxin to lead to differential growth:
increased transmission of a growth promoter on the dark side
of a unilaterally irradiated plant, decreased transmission
of a promoter on the light side, increased transmission of
an inhibitor on the light side, or decreased transmission of
an inhibitor on the dark side. Cholodony and Went (Went and
Thimann, 1937), working separately, established that the
first two possibilities above occur.

Galston (1959) pointed out that, if auxin were

responsible for phototropic curvature, there must be a

l6
4000-fold amplification step somewhere in the transduction
pathway. He based this figure on an estimate of the number
of quanta incident in the near side of a unilaterally
illuminated coleoptile and the approximate concentration of
auxin, extrapolated from experiments using the gyggg
curvature test. He discussed three possible mechanisms by
which this amplification could occur: auxin destruction,
redistribution based on electrophoretic migration or
light-induced alteration of auxin synthesis.

A series of experiments using split coleoptiles gave
rise to the idea that perhaps auxin transport, rather than
synthesis or destruction, is necessary for a gradient to be
established. Briggs (1963a) found that corn coleoptiles
exposed to light and those kept in darkness gave the same
amount of auxin as measured by the gtgtg curvature test.
Coleoptiles were slit longitudinally except for the top
0.5 mm, and exposed to unilateral light. Briggs found more
auxin in receiver blocks from the shaded side of the
partially split coleoptile tip than in the blocks from the
lighted side. Receiver blocks from the bases of totally
split tips showed no difference in auxin content, whether
from the lighted or the shaded side.

Radiolabelled auxin enabled workers to measure the
amount of auxin transported more accurately than the gyggg
curvature test allowed. Experiments using radiolabelled IAA
in agar blocks also showed that lateral auxin transport

occurred in response to gravity (reviewed in Wilkins, 1977).

17

Further studies using asymmetrically applied auxin were
made by Gardner gt gt. (1974). In this work, a micropipette
was used to apply high specific activity auxin to one side
of the tip of intact or excised corn or oat shoots. In
many, but not all, experiments performed, a correlation
between auxin redistribution and curvature was found.
Unilateral illumination with a first positive dose of blue
light caused significant lateral movement of auxin away
from the lighted side of both corn and oat coleoptiles.
Second positive doses, however, caused lateral movement of
auxin in corn but not in oats. In intact gtggg seedlings,
inhibition of basipetal auxin transport by second positive
dosages was also found. Even though lateral gradients of
auxin were found to be induced by unilateral light, under
certain conditions curvature toward a first positive dose of
blue light occurred, but no auxin gradient was seen.

Elliott and Shen-Miller (1976) found a correlation
between phototropism and photoinhibition of growth, based on
dose response curves and action spectra. They proposed that
the growth inhibition is a result of photoinhibition of
basipetal auxin transport. Blaauw (1915) had previously set
forth the light growth hypothesis, predicting that light was
responsible for phototropic curvature by causing inhibition
of growth.

Studies of the growth rate of seedlings have been
employed in an attempt to determine the role of auxin in

phototropism, but results and conclusions of different

18

workers are contradictory. Franssen gt_gt. (1981), using
marker beads and sucessive photographs of a phototropically
responding £2222! Lepidium, or Cucumis shoot, found that
growth stopped on the illuminated side, and that no
statistically significant increase in growth occurred on the
shaded side. The authors argued against auxin mediation of
the observed patterns of growth. They proposed that if a
high concentration of auxin were present in the responding
tissue, a large drop in concentration would be necessary on
the lighted side in order for growth to stop. If
concentrations were low, a small increase should lead to
increased growth on the shaded side. An intermediate auxin
concentration should lead to a coordinated change in the
growth rates of both sides. As an alternative theory to
explain their data, they accepted Blaauw's light growth
hypothesis, that is, that light brings about a direct
inhibition of growth in the lighted side of the seedling.

IFranssen gt_gt. (1982) continued to examine the
applicability of the Cholodony-Went hypothesis by studying
the curvature of intact, black-capped, and decapitated gyggg
coleoptiles. They found that the apex is not essential to
the perception of a unilateral light stimulus, but that
subapical regions responded independently to unilateral
light. Initial growth responses are not different in intact
and black-capped coleoptiles, and even decapitated

coleoptiles show curvature after a lag. Franssen gt gt.

19

(1982) concluded that the role of the apex has been
overemphasized in explanations of phototropism.

Firn and Digby (1980) provided a review of mechanisms
of phototropism and geotropism with an emphasis on the
"behavior" of the plant during curvature. They criticized
the Cholodony-Went hypothesis on four counts: 1) the role of
the apex is overemphasized, 2) phototropism has been known
to occur in the absence of auxin gradients, 3) the magnitude
of auxin redistribution, when it does occur, has not been
shown to be great enough to account for the changes in
growth rate observed, and 4) the lag time before
phototropism begins is often shorter than would be necessary
for the establishment of an auxin gradient. They advocated
consideration of Blaauw's light growth hypothesis for an
explanation of second positive phototropism.

Iino and Briggs (1984) examined relative growth rates
on shaded and lighted sides of phototropically stimulated
corn coleoptiles. They performed their experiments under
red light in order to stabilize any contribution by
phytochrome mediated processes. Under these conditions,
they found that first positive curvature was the result of
increased growth on the shaded side and decreased growth on
the illuminated side of the coleOptile. They correlated
this lateral redistribution of growth with lateral
redistribution of auxin.

Working on the assumption that auxin indeed is

responsible for phototropic curvature, Galston and Hand

20
(1949) found that riboflavin could sensitize the
photooxidation of IAA, as well as the oxidation of other
compounds. This, along with the presence of riboflavin in
coleoptiles and the similarity of the riboflavin absorbtion
spectrum with the action spectrum of coleOptiles, first led
to consideration of flavin, rather than carotenoid, as the
photoreceptor pigment for phototropism.

Evidence about the nature of the transduction pathway
for phototropism remains largely circumstantial. Further
work, including the mechanism for Galston's (1959)
hypothesized "amplification step" is necessary before the

immediate cause of phototropic curvature is known.

I NTRODUCT I ON

The first step in responding to a directional stimulus
is to detect the direction from which it comes. For plants
responding to light, this entails establishing a gradient of
light across the organ doing the sensing. Once the gradient
has been created, the organism can measure it either
spatially (at one moment in time) or temporally (by
comparing the intensity of light on a photoreceptor at two
different moments) (Feinleib, 1980).

Three potential mechanisms for establishing a spatial
gradient of light are refraction, absorption, and scatter.
This thesis will examine the relative contribution of these
mechanisms to the detection of a light gradient for first
positive phototropism in corn (Zea mays L.).

Refraction was proposed as a mechanism for establishing

a gradient of light in Phycomyces by Blaauw in 1915. A

 

cylindrical sporangiophore in air has a refractive index of
1.38, independent of cell diameter (Castle, 1933a). The
sporangiophore thus refracts light, bringing it to a focus
within the cell on the side distal to the light source, thus

acting as a lens. Phycomyces shows a transient increase in

 

21

22
growth rate when given a brighter light than that to which
it is adapted, and the sporangiophore twists as it grows.

These two characteristics of Phycomyces' growth combine so

 

that a unilaterally irradiated sporangiophore grows toward

the light source, but away from the most brightly

illuminated area within the sporangiophore (Dennison, 1979).
Several experiments have been performed which

manipulate the lens and alter Phycomyces' phototropism.

 

They are reviewed in Bergman gt gt. (1969). Briefly, Buder

immersed Phycomyces sporangiophores in mineral oil with a

 

refractive index much higher than that of the cell, and
found negative phototropism. Shropshire (reviewed in
Bergman gt gt., 1969) modified this approach by using media
of intermediate refractive indices, and found a medium which
rendered the sporangiophores phototropically neutral. He
placed a cylindrical lens in front of a sporangiophore at
the proper distance to turn the sporangiophore into a
diverging lens, and found negative phototropism. However,
he found that sporangiophores also avoided the cylinder in
the dark, though to a lesser degree. This "avoidance
response" complicates the question of the existence of a
lens as a focusing mechanism, as do objections raised that
sporangiophores immersed in liquids of high refractive index
are subjected to changes in oxygen levels.

Dictyostelium discoideum may use a lens as a mechanism

 

for establishing a gradient. When a lens is at work, the

organism which shows positive phototropism or phototaxis

23
turns away from the most brightly lit portion of itself.
Francis (1964) illuminated only one side of migrating

Dictyostelium pseudoplasmodia, and found that they would

 

indeed turn away from the most brightly illuminated region.
Bonner and Whitfield (1965) found that slime molds immersed
in a liquid of high refractive index showed a negative photo
response. Further support for the lens effect in

Dictyostelium comes from an experiment in which an

 

artificial screening pigment, neutral red, was incorporated

into Dictyostelium pseudoplasmodia (Hader and Burkhart,

 

1983). The artificial screening pigment increased the
amount of unilateral light absorbed or scattered by the
slug, shifting the balance of light intensities in the
proximal and distal halves such that the proximal half was
more brightly illuminated. The slug, in turning away from
the more brightly lit side, showed negative phototaxis.

ColeOptiles are nearly cylindrical, light sensitive
organs. The primary leaf inside the coleoptile is dense
enough to eliminate the possibility of a lens effect along
most of its length. If a focusing advantage exists in
coleoptilar tissue, it would be at the extreme apex, above
the primary leaf. Lange (1928) showed that the greatest
phototropic sensitivity resides in the most apical 500
microns of an gtggg shoot. Some sensitivity is present in
lower portions as well (Franssen gt gt., 1982).

Converging and diverging beams of light have been

directed at Avena coleoptiles, which then show positive and

24
negative phototropism, respectively (Shropshire, cited in
Dennison 1979). gtggg has also been the subject of immersion
experiments, and has shown negative phototropism when
immersed in a liquid of high refractive index. Ziegler

(1950) observed inversion of phototropism in Phycomyces

 

sporangiophores, liverwort rhizoids, dicot seedlings and
gtggg coleOptiles under paraffin oil. Others have observed
negative phototropism in gtggg coleoptiles immersed in
liquids of a high refractive index. There is some question
whether this reversal is due to a focusing effect at the
organ or cellular level, or to changes in the cell wall
structure caused by penetration of oil (Dennison, 1979).

Below the extreme apex of a coleoptile, where both
coleOptilar and primary leaf tissue are present, the most
likely mechanisms for establishing a gradient of light are
absorption and scatter. Attenuation through absorption can
take two forms. For self screening, the photoreceptor
pigment is present in high enough concentration that it can
establish a gradient of light itself. For foreign
screening, the photoreceptor pigment is less concentrated; a
second pigment acts as a screen, absorbing light before it
reaches the photoreceptor, thus altering the amount and
possibly the spectral quality of the light received by the
photoreceptor.

BUnning gt _t. (1953) found a decrease in phototropism

when the primary leaf was removed from a coleoptile.

Replacing the primary leaf with a dye solution restored

25
phototropic capability. Meyer (1969) attempted to increase
the absorption gradient across an gtggg coleoptile by
infiltrating the coleoptiles with neutral red, but did not
see a significant change in first positive phototropism.
She took advantage of the natural asymmetry in pigmentation
and pathlength which arises from the elliptical
cross-section of a coleoptile, and found a slight increase
in phototropic response when the plants were illuminated
along the long axis. She saw a greater response when the
apical 500 microns of the coleoptile were illuminated than
when the next lower increment of 500 microns received the
unilateral dose, which agrees with earlier observations that
the greatest phototropic sensitivity resides in the tip of a
coleOptile.

Vierstra and Poff (1981) have examined the role of
carotenoids in second positive phototropism of corn
seedlings. Since seedlings treated with SAN 9789 retained
about 60% of a normal second positive phototropic response,
but had only 2% of their normal carotenoid concentration,
Vierstra and Poff (1981) concluded that carotenoids acted as
screening pigments.

Attenuation of light by scattering must also play a
role in establishing a gradient across an organ. Seyfried
and Fukshansky (1983) have developed a model for calculating
light gradients in scattering, layered tissue. Based on the
morphology, reflective and absorptive properties of

Cucurbita pepo cotyledons, they performed a series of

 

26
mathematical manipulations which allowed them to approximate
the scatter attenuation of different wavelengths of light
through the tissue.
Seyfried and Schafer (1983) measured reflectance and
transmittance of green, etiolated, and SAN 9789 treated

Cucurbita cotyledons (two to seven days old). They

 

calculated absorption and scattering coefficients for the
tissues at the different ages and treatments. Developmental
changes which occurred between the second and seventh days
were reflected in changes in the Optical properties of the
plants. Seyfried and Schafer (1983) discussed the effect
the changes in optics might have on light absorption by
phytochrome.

Parsons gt gt. (1984) attributed most of a plant's
phototropic capability to scatter. They have measured the
percent transmission of red and blue light through different
thicknesses of tissues. Seyfried and Fukshansky (1983)
pointed out that this method does not account for changes in
light intensity due to backscatter or specular reflection,
but Parsons _t _t. (1984) assumed that these changes would
probably enhance the gradient. Their data showed that light
is attenuated quickly with increasing tissue thickness.

They found that the light intensity at the shaded side is
only 2% of that at the lighted side. The attenuation of
light with tissue thickness is about the same for both
etiolated and de-etiolated hypocotyls of ytgtg and

Helianthus hypocotyls. Treatment with SAN 6706 to inhibit

 

27

carotenoid synthesis in Avena and Helianthus did not alter

 

the light gradient appreciably. Parsons gt gt. (1984)
altered the scattering properties of plant tissue by
infiltration with cedarwood oil, which makes the tissue more
optically homogeneous and much more transparent. They
illustrated how the path of a normal and an oblique ray of
light diverges through five cell layers. From these data,
Parsons gt gt. (1984) concluded that absorption is
relatively unimportant to light attenuation across a
coleoptile.

Poff (1983) pointed out that neither absorption nor
scatter alone would allow an organism to detect a light
stimulus. By definition, some light must be absorbed for a
light—dependent response to occur. Unless all wavelengths
of incident light are absorbed by the photoreceptor, some
light will be scattered by the molecule itself. In
addition, organisms are inherently light-scattering objects.
Subcellular components, changes in tissue composition, and
changes in index of refraction as a light ray travels
through cytoplasm, cell wall, and vacuole could all
contribute to attenuation by scatter. Thus, in any
biological system, scatter and absorption will both
contribute to the gradient of light.

The relative contributions of a lens effect, scatter,
and absorption differ among organisms. It is also possible
that within an organism, different mechanisms could

predominate in first positive, first negative, and second

28

positive phototropism. The contributions of a lens effect,
scatter, and absorption by screening pigments to the
gradient of light for first positive phototropism will be
considered in this thesis. Chapter 1 contains a
spectrophotometric analysis of pigment distribution in
individual control, mutant, and SAN 9789 treated seedlings.
Chapter 2 examines the contribution of bulk carotenoids to
first positive phototropism. Chapter 3 considers the
contribution of scatter to the light gradient of intact
seedlings, both axially and longitudinally. Results are

summarized and recommendations for future work given.

CHAPTER 1

Distribution of Pigments in Individual Corn Seedlings

Introduction

 

Most tg tttg absorption spectra have been obtained
using tissue pooled from many plants (see Vierstra and Poff
1981b for example). Pooled measurements can be used to
estimate an average spectrum for a population, but there are
difficulties in localizing pigments or in gauging the
variation among seedlings. All spectra in this thesis were
obtained from individual seedlings. In this chapter,
spectra of different tissues from individual SAN 9789
treated, mutant and control seedlings are presented in order
to illustrate relative pigment distributions. In later
chapters, the relationships among pigmentation, other
optical qualities, and phototropism will be examined.
Methods

The instrument used to generate these absorption
spectra is a modified Cary 14 single beam spectrophotometer
with double monochromator. The spectrophotometer was on
line with a Hewlett-Packard 21MX minicomputer, a system
similar to that described by Davis gt gt. (1973). Light

from the monochromator fell on a seedling which was taped in

29

30

place over a square hole about 0.5 mm x 0.5 mm. Except
where noted, the corn seedling was oriented so that the
measuring beam passed through the short cross-sectional axis
of the coleoptile, between the vascular bundles. A large
portion of the light which passed through the sample was
collected by an EMI 9659 QA photomultiplier tube with a 5 cm
diameter cathode 3 cm from the sample. The current from the
photomultiplier was converted to a voltage of -log i,
digitized, and stored by the computer. The resulting
spectra consisted of absorbance measurements as a function
of wavelength at 0.4 nm intervals. The spectra could be
manipulated by the computer and output directly for figures,
using an x-y plotter (Hewlett Packard 7047A).

Hybrid corn seeds (Egg ggyg hybrid MS WFg x Bear 38,
Custom Farm Seed Research, Decatur, IL.) were soaked
overnight in distilled water or in a solution containing a
known amount of SAN 9789. SAN 9789, trade name Norflurazon,
is an inhibitor of carotenoid biosynthesis and is available
as an 80% wettable powder from Sandoz, Inc., Crop Protection
(San Diego, CA). In preliminary experiments, it was found
that SAN 9789 treated seedlings which were sown in soil
produced a substantial amount of pigment, but those sown on
Kimpak germinating paper or in vermiculite did not. For
subsequent experiments, seeds were sown, embryos up, on
Kimpak or in coarse vermiculite which had been soaked with
the same solution in which the seeds had been imbibed.

Boxes containing seeds sown on Kimpak were covered with

31
plastic wrap and placed in dark chambers where they received
no light before use (dark grown), or in chambers where they
received one hour of red light per night (red grown). Red
light was supplied by a General Electric red fluorescent

2 at plant height, as

bulb. Intensity was 22 pW x cm-
measured with a Kettering radiometer (model number 7720).
The spectral distribution of this red light is shown in
Figure 2.1. Plants grown in red light were straighter and
shorter than those grown in darkness. All plants were grown
at 240 +/- 20 C, and the humidity was maintained at 80 -
100%. The seeds were started to imbibe on day zero, planted
on day one, and used on days 4-9.

Mutant seeds for initial testing and for seed
multiplication were the gift of Dr. D.S. Robertson, Iowa
State University, Ames, Iowa. The methods for and results
of seed multiplication are detailed in Appendix 1. The
mutants were from four class I "carotenoidless" lines, with
single mutations at either the lwl, lw2, w7748, or clp
allele. The phenotype of class I mutants is a seed with a
white or pale yellow endosperm and a seedling lacking
carotenoids. Wild type seeds are yellow, and give normally
pigmented seedlings. Seeds from the four mutant lines were
imbibed overnight in distilled water and planted, embryos
up, in 8-dram vials filled with coarse vermiculite soaked
with distilled water.

The contribution of scatter to light attenuation was

approximated by drawing a slanted baseline with the angle of

32

the slant determined by using the red region of the spectrum
where little light is absorbed. Attenuation above this line
is assumed to be due to absorption. The height of peaks
above the slanted baseline is used as a basis for comparing
absorption by different tissues.

Results and discussion

 

Spectra of seedlings grown in darkness, with one hour
of red light per night, or in white fluorescent light all
have absorption maxima at about 415 nm and 470 nm (see
Figure 1.1). Red- and dark-grown seedlings show an
additional peak at about 435 nm. Blue-light absorbing
pigments known to be present in corn include B-carotene,
zeaxanthin, and lutein (Fong _t _t., 1982). Seedlings from
all three light treatments have a smaller peak in the red.
In dark grown corn, the peak is around 640 nm, and at 660 nm
in red and light grown seedlings. These peaks are probably
due to protochlorophyll and chlorophyll, respectively
(Shibata, 1957). The 660 nm peak is most pronounced in
white light grown tissue.

Figure 1.2 shows a series of absorption spectra taken
through the seedling at the points indicated. Using the
470 - 480 nm peak as a basis for comparison, and assuming
that other peaks do not contribute to absorption at 470 nm,
the excised coleoptile (curve A) shows an absorbance of
0.05 A. Five millimeters from the tip (curve D), the

absorbance is 0.3 A, and in the mesocotyl, 0.17 A.

Coleoptile and mesocotyl tissue are less heavily pigmented

33

 

 

A Absorbance

 

 

 

j l l l l l J

400 450 500 550 600 650 700 750
Wavelength (nm)

 

Figure 1.1. Absorption spectra of corn seedlings, taken

through coleoptile and primary leaf, 0.5 cm from tip. A)
Dark grown seedling. Peaks occur at about 415 nm, 435 nm,
470 nm, and 640 nm. B) Seedling received one hour of red
light per night. Peaks occur at about 415 nm, 435 nm,

470 nm, and 660 nm. C) Seedling was grown under fluorescent
lights. Peaks occur at about 415 nm, 470 nm, and 660 nm.

Curves are offset along the y axis for clarity in all

figures.

34

 

 

750

l
700

l
O

Wavelength (nm)

500

450

 

O
-m
o
O
D
O
-m
ID
I

<

9

O

P———————-—-4
(:1: 00m LLG I

eouaqnocqvv

 

400

 

Figure 1.2. Spectra taken through the short cross-sectional
axis of a red grown seedling, perpendicular to the vascular
bundles. A) extreme tip, coleoptile tissue alone. B) 1.5
mm from tip. C) 3 mm from tip. D) 5 mm from tip. E) 7 mm
from tip. F) 9 mm from tip. G) 14 mm from tip, on node.

H) 17 mm from tip, in mesocotyl.

35

than the primary leaf. In intact shoots, the orientation of
the plant makes little difference in the absorption
spectrum, since the primary leaf is so heavily pigmented
compared to the coleoptile. However, in an excised
coleoptile, a slight increase in absorption is seen (Figure
1.3) when the spectrum is taken along the long
cross-sectional axis, through the vascular bundles. This
may be a result of the higher concentration of carotenoids
around the vascular bundles (Galston, 1959). Scatter
intensification over the longer pathlength may also add to
the difference.

Absorption spectra of seedlings grown with different
concentrations of SAN 9789 were measured (Figure 1.4).
Vierstra and Poff (1981b) reported that a solution of

4

l x 10- M SAN 9789 inhibited carotenoid production 98 -

99%. For experiments presented in this thesis, a

4 M SAN 9789 was the standard

concentration of 2 x 10-
concentration for inhibiting carotenoid synthesis. In
higher concentrations of SAN 9789 the plants were crooked
and grew slowly and at lower concentrations more carotenoids

4 M SAN 9789, many of the

were synthesized. At 2 x 10-
plants had short primary leaves, so that the coleOptile tips
were hollow. Absorption spectra for the intact coleoptile,
mesocotyl, excised coleoptile, and root of a seedling grown

4

with 2 x 10- M SAN 9789 were generated (Figure 1.5). Based

on a comparison with absorption by a control shoot (Figure

 

A (15 A

 

 

AAbsorbanco
u:

 

 

 

 

l l l I 1

400 450 500 550 600 650 700 750
Wavelength (nm)

 

Figure 1.3. Six day old excised coleoptile, cross—sectional

spectra. A) Spectrum taken along short axis, perpendicular

to vascular bundles. B) Spectrum taken along long axis,

through vascular bundles.

37

 

 

AAbsorbance

 

 

 

1 l _l l 1

 

400 450 500 550 600 650 700
Wavelength (nm)

Figure 1.4. Seedlings treated with different concentrations

of SAN 9789. Spectra taken 5 mm from tip of shoot. A)

Control. (B-F) SAN 9789 treated. B) 4 x 10-7M. C)

3

4 x 10'6M. o) 4 x 10'5 M. E) 4 x 10’4M. F) 4 x 10‘ M.

38

 

*7

-4
2x10 M SAN treated
q

A
(L6 A

 

m
g
I

 

AAbsorbance

 

 

1 1
1 n
l I
1

400 450 500 550 600 650 700 750
Wavelength (nm)

 

Figure 1.5. 2 x 10-4M SAN 9789 treated, red grown seedling.

A) shoot 5 mm from tip. B) mesocotyl. C) excised

coleoptile. D) root.

39

1.6), it appears that carotenoid synthesis in the shoot is
inhibited by about 80%.

There is some variation among individual corn seedlings
grown under the same conditions. Figures 1.6 and 1.7
illustrate the extent of the variation. For the control
seedlings, the difference in absorption at 480 nm is about

0.06 A, for 2 x 10-4

M SAN 9789 treated, 0.02 A. In each
case the variation is about 10%. Based on observations made
in the course of other experiments, the pigmentation of

SAN 9789 treated seedlings may be more variable than
indicated in Figure 1.7. It was noted that SAN 9789 treated
seedlings appeared to accumulate pigment as they aged, so
for subsequent experiments, seedlings were used when they
were as young as possible.

The four carotenoidless mutant lines used are
characterized by seeds which have pale endosperm and which
give rise to albino seedlings. The endosperm of pale seeds
was usually distinctly lighter than that of yellow seeds
within the same genotype, but occasionally the distinction
between pale and yellow endosperm was difficult to make. In
these cases, spectra of individual dry seeds were used to
differentiate between the two. Spectra were generated using
a method similar to that used for seedlings, but with a
"pinhole" slightly smaller than the seed. The seed was held
in place with modeling clay. Although the broad,

flat-topped curves observed for yellow seeds (Figure 1.8)

are characteristic when the measuring system is saturated,

 

Control

A

 

AAbsorbance

 

 

 

 

l l L l l l l l

400 450 500 550 600 650 700 750
Wavelength (nm)

 

Figure 1.6. Two control seedlings grown in the same
conditions under red light were selected randomly to
illustrate the variation among individuals. A) and B) are
absorption spectra taken 5 mm from the tip. C) is the

difference spectrum (A-B).

41

 

 

 

 

 

 

 

 

 

 

SAN 9789
A I
(15 A
3 I
C B ‘
Q
‘3.
o /\
O ‘—
.0
:3.
\ fi-
0
l J l 1 l L l 1

 

400 450 500 550 600 650 700 750
Wavelength (nm)

Figure 1.7. As with Figure 1.6, but seedlings were treated

4

with 2 x 10- M SAN 9789. A) and B) are spectra taken 5 mm

from the tip of two randomly selected individuals. C) is the

difference spectrum (A-B).

Figure 1.8.

42

 

 

AAbsorbance

 

 

 

450 500
Wavelength (nm)

Spectra of dry seeds.

A) hybrid control.

B)

lwl yellow.

E) clp yellow.

I) clp pale.

C)

F)

lw2 yellow.

lwl pale.

G)

D) w7748 yellow.

lw2 pale. H) w7748 pale.

43

such saturation did not occur. In a highly scattering
sample, wavelengths of light which would normally not be
absorbed will be attenuated, making absorption peaks less
distinct. This may explain the shape of the absorption
spectra of seeds.

Spectra from intact coleoptiles, excised coleoptiles
and mesocotyls were measured for pale and yellow seedlings
of all four mutant lines. Results are shown in Figures
1.9 - 1.12. In each of these figures, curves A, B, and C
represent the absorption spectrum of intact coleoptile,
mesocotyl, and excised coleOptile of a yellow seedling,
respectively. Curves D, E, and F are spectra for similar
tissues of a pale seedling. Comparisons of absorption at
the 470 - 480 nm peak based on these spectra show that for
mutant line lwl, the carotenoid content in the pale seedling
is reduced about 72% from that of the wild type. For lw2,
the reduction is 67%, for w7748, 61% , and for clp, 53%.
Variation among individual seedlings is also about 10% for
the mutant lines. Absorption spectra for hybrid corn and
for wild type seedlings of the four mutant lines are
qualitatively similar. Wild type seedlings may be more
heavily pigmented than hybrid control seedlings. Pale
mutant seedlings show a more distinct set of peaks in the
blue region of the spectrum than do seedlings treated with

4

2 x 10- M SAN 9789. The mutants' pigment content relative

to their respective wild type is not reduced as much as

4

2 x 10- M SAN 9789 treated seedlings relative to controls.

 

lw1

 

AAbsorbance'

 

 

 

1 1 1 1 l 1 1 1

400 450 500 550 600 650 700 750
Wavelength (nm)

 

Figure 1.9. Absorption spectra of different tissues of lwl
seedlings. (A-C) wild type. A) intact shoot 5 mm from tip.
B) mesocotyl. C) excised coleoptile. (D-F) mutant.

D) intact shoot 5 mm from tip. E) mesocotyl. F) excised

coleoptile.

45

 

 

 

 

 

 

Q)
U
C
(U
E
O
(D
D
<
" o

E

F

400 450 500 550 600 650 700 750

Wavelength (nm)

Figure 1.10. Absorption spectra of different tissues of lw2
seedlings. (A-C) wild type. A) intact shoot 5 mm from tip.
B) mesocotyl. C) excised coleoptile. (D-F) mutant.

D) intact shoot 5 mm from tip. E) mesocotyl. F) excised

coleoptile.

 

  

015A

:§

1 l I I l J l I

400 450 500 550 600 650 700 750
Wavelength (nm)

 

 

AAbsorbance

 

 

 

 

 

Figure 1.11. Absorption spectra of different tissues of

w7748 seedlings. (A-C) wild type. A) intact shoot 5 mm from

tip. B) mesocotyl. C) excised coleoptile. (D-F) mutant.

D) intact shoot 5 mm from tip. E) mesocotyl. F) excised

coleoptile.

47

Figure 1.12. Absorption spectra of different tissues of clp
seedlings. (A-C) wild type. A) intact shoot 5 mm from tip.
B) mesocotyl. C) excised coleoptile. (D-F) mutant.

D) intact shoot 5 mm from tip. E) mesocotyl. F) excised

coleoptile.

AAbsorbance

 

 

 

 

 

 

I l L I I

_____~__~

I I l

 

 

400 450 500 550 600

Wavelength (nm)

Figure 1.12

650 700 750

49
Summary

Bulk pigments have been mapped in a number of seedlings
from different genetic lines and growth regimes. Two sets
of seedlings with reduced pigments, one generated by
treatment with SAN 9789, the other a group of mutants with
similar phenotypes, have been characterized. These two
groups of seedlings with reduced pigments will be used to
assess the role of carotenoids in first positive
phototropism.

The technique presented in this chapter could be used
to separate individual seedlings with pigmentation
differences too subtle to be culled visually. Changes in
pigmentation with time or light regime could be monitored in
an individual seedling, since sample seedlings are left
intact. Pigments present in specific areas of the plant can
be mapped more accurately than is possible with pooled
material. For these reasons, the ability to generate
absorption spectra from individual seedlings will be useful

in further studies of phototropism.

CHAPTER 2

The Role of Carotenoids in First Positive Phototropism of
Corn Seedlings

Introduction

 

The complex way in which monocots respond to increasing
doses of unilateral blue light (light impinging on the plant
from one direction, normal to the longitudinal axis) has led
to several hypotheses about the mechanism used to sense the
light. The possibility that more than one pigment or
response system is involved in phototropism has been
discussed. Poff (1983) suggested that, at an even more
fundamental level, there may be more than one mechanism for
establishing the light gradient necessary for detecting a
unilateral light stimulus. He proposed a lens and a
mechanism of screening by absorption and scatter as two
alternate possibilities. Vierstra and Poff (1981b) showed
that absorption by screening pigments is important for
second positive phototropism. Removal of screening pigments
by treatment with an inhibitor of carotenoid synthesis
decreased phototropic response 40% without affecting
geotropism. Bandurski and Galston (1951) reported that
mutant albino corn seedlings with no detectable carotenoids
showed 50% of the wild type's first positive phototropic

response.
50

51

In this chapter, the role of carotenoids in first
positive phototropism will be examined. The phototropic
response of inhibitor treated or mutant seedlings with
reduced carotenoid content will be assessed. Geotropism and
growth rates are used to verify that reduced carotenoid
content affects only the phototropic response.
Methods

Hybrid corn seeds (tgg_ggyg hybrid MS WFg x Bear 38,
Custom Farm Seed Research, Decatur, IL) were soaked

overnight in distilled water or in a solution of 2 x 10-4M

or 2 x 10-5

M SAN 9789. Seeds were planted, embryos up,
about 5 cm deep in 10 cm x 20 cm x 8 cm boxes filled with
vermiculite which had been saturated with water or a
solution of SAN 9789. Seeds were planted in diagonal rows,
so that seedlings would not shade each other. Twenty seeds
were planted per box. Before exposure to actinic light, ten
straight seedlings with coleoptiles still intact were
selected for use. The rest of the seedlings were snapped
off at the soil line. For experiments with mutant
seedlings, seeds were planted individually, 5 cm deep in 8
dram vials containing wet vermiculite. Planting in vials
allowed a maximum number of a limited supply of seedlings to
be used.

Of the several mutants known (see Robertson, 1975, for
review), two lines were used to assess further the
contribution of carotenoids to first positive phototropism.

Mutants with carotenoidless lwl and lw2 alleles (pale

52

seedlings) and the counterpart wild types (yellow seedlings)
were used to generate dose response curves and were compared
to control and SAN 9789 treated hybrid seedlings. A limited
number of mutant seeds was available (see Appendix 1), so
only three light doses in addition to a dark control were
evaluated. The three doses chosen correspond to the peak of
first positive response for hybrid corn, a point in first
negative, and a point in second positive. Three experiments
were run.

All plants were grown for five days in 90 - 100%
relative humidity at 240 +/- 20 C. They received one hour
of red light per night (intensity 22 pW x cm-Z, spectral
distribution as shown in Figure 2.1). A flashlight covered
with a piece of green plexiglas and an interference filter
(560 nm peak, 10 nm half-band) was used as a "safe"
worklight.

To measure phototropic and geotropic responses,
seedlings were photographed before any stimulus was given,
and again three hours after commencement of the stimulus.
Kodak high speed infrared 2481 film was used to take the
photographs. Light for photography was provided by a flash
covered with a Kodak 87C Wratten filter with a cutoff at
800 nm. Plants did not respond in any discernable manner to
the infrared light used for photography. During the
interval between photographs, when the response to a
stimulus was developing, plants were kept in the dark, humid

growth chamber. Curvatures were measured with a protractor

53

 

Red

Intensity (pWecm'zenm'1)

 

 

 

 

I

4 1

400 450 500 550 600 650 700
Wavelength (nm)

 

 

Figure 2.1. Spectral distribution of the blue light used as

phototropic stimulus and red light used to promote

coleoptile elongation and inhibit mesocotyl elongation.

54

from the projected image of the negative. Mean curvature
before stimulus was subtracted from mean curvature after
stimulus and a three hour development time to give net
curvature.

The actinic light used to generate the dose reSponse
curves was provided by a Beseler Slide King II projector.
Light passed through a DT Blaauw broad band blue filter and
3.5 cm of 5% (w/v) copper sulfate, giving a peak at 470 nm
(spectral distribution is shown in Figure 2.1). For all
exposure times shorter than three hours, light intensity was
12 pW x cm-Z. Dose was varied by changing exposure time
from 0.1 to 180 seconds, using a shutter and timing unit
(Uniblitz). When a stimulus for second positive curvature
was needed, the light intensity was cut to 0.9 pW x cm-2
with neutral density filters, and exposure time was
increased to three hours.

For all dose response curves measured, seedlings were
oriented randomly with respect to their transverse axes.
The straightest seedlings were selected, or seedlings were
oriented so the actinic light was normal to the straightest
longitudinal axis.

Results and discussion

 

 

Meyer (1969) reported a slight increase in the
curvature of gtggg coleoptiles when they were oriented so
that an actinic beam of light passed along the long
transverse axis. Recently, Iino and Briggs (1984) have

taken care to orient plants so the long transverse axis was

55

parallel to the direction of irradiation. In the present
experiments, a comparison of mean curvature for plants
illuminated along the long and the short transverse axes was
made. Means were compared by a two-tailed Student's t test
(see Table 2.1). No significant difference in curvature was
found at any dosage tested. For subsequent experiments, no
attempt was made to orient seedlings with respect to their
transverse axes.

In order to choose an appropriate time interval after
stimulus at which curvature could be evaluated, it was
necessary to know the rate at which curvature occurred. A
dose of light to elicit maximal first positive response was

4M SAN 9789 treated seedlings.

given to control and 2 x 10-
The seedlings were photographed at 30 minute intervals for
three hours, and curvature of individual seedlings followed
(see Figure 2.2). For both control and SAN 9789 treated
seedlings, curvature began about 60 minutes after the
stimulus was given. SAN 9789 treated plants reached a peak
response by three hours, but control plants had not.
Geotropic straightening does not begin until after five
hours or more (data not shown).

In order to assess the role of carotenoids in first
positive phototropism, two sets of experiments were
performed using plants with reduced carotenoid content. In
the first set of experiments, dose response curves for

seedlings treated with SAN 9789 were compared to those for

control (water treated) seedlings. SAN 9789 has a number of

56

Table 2.1. Mean curvature of seedlings irradiated along
long and short transverse axes. Different doses were
evaluated on different days, so comparisons between
different doses should not be made. Methods given in text.
SE: standard error of the mean. Mean curvature in degrees.

mean curvature (SE) t

log(dg§e) df
short axis long axis

(chm x t)

 

-5.3 24 9.3 (2.1) 9.5 (1.5) 0.08
-5.0 26 5.8 (1.8) 6.6 (2.6) 0.24
-4.5 26 8.3 (1.5) 9.7 (2.4) 0.49
-4.5 21 15.3 (2.5) 12.5 (2.7) 0.76
-4.5 14 11.4 (3.3) 6.4 (3.5) 1.03
-3.3 18 5.6 (2.2) 1.9 (2.3) 1.20
-.75 10 25.0 (4.8) 22.3 (2.6) 0.49
-.69 17 2.3 (1.4) 1.1 (1.1) 0.65

 

57

Figure 2.2. Time course of bending. Left: 2 x 10-4M

SAN 9789 treated seedlings. Right: control seedlings. All
seedlings were given two seconds of 12 pw x cm"2 blue light
to elicit a nearly maximal first positive response. After
three hours, SAN 9789 treated seedlings have reached a
maximal response, where control plants may be capable of

some further curvature.

58

 

 

 

SAN 9789

 

 

 

(0901509) emu/(mo

Figure 2.2

120 150 180

90

80

120 150 180 0

30 80 90

0

Time after tlaeh (min)

59
effects aside from inhibiting carotenoid synthesis. In
plant tissue with no carotenoids, photobleaching occurs, and
neither chlorophyll nor chloroplast proteins accumulate
(Quarrie and Lister, 1984). Ribosomal RNA (Bartels and
Watson, 1978, Quarrie and Lister, 1984) and abscisic acid
(Quarrie and Lister, 1984, Henson, 1984) also do not
accumulate in light grown SAN 9789 treated tissue. SAN 9789
and related compounds alter the fatty acid composition of
galactolipids (St. John, 1976). How these alterations in
plant metabolism affect phototropism is not known, but these
non-specific effects of SAN 9789 make it necessary to run
controls to Show that the effect on phototropism is due to
reduced carotenoid concentration, and not some more
generalized physiological factor.

If SAN 9789 treatment affects phototropism only by
reducing carotenoid content, then one would expect a
correlation between carotenoid content and response.
Unfortunately, the range of effective SAN 9789
concentrations is limited. At concentrations of 10-3M or
greater, seedlings are generally crooked and unhealthy. At

concentrations of 10-6

M or less, carotenoid content and
response to a peak first positive stimulus are
indistinguishable from untreated seedlings. Within the
concentration range where the major effect of SAN 9789
appeared to be on carotenoid content, two dose response

curves were generated. The first, comparing control

seedling curvature to that of seedlings treated with

60

2 x 10-4

M SAN 9789 (Figure 2.3) showed that an approximately
90% reduction in carotenoid content (Figure 1.4)
corresponded to a 39% reduction in the peak of the first
positive response (Table 2.9). Treatment with 2 x 10-5M
SAN 9789 resulted in a 31% reduction in peak first positive
response (Figure 2.4). The results for reduction in
carotenoid content agree qualitatively with those of
Vierstra and Poff (1981b), but a lower concentration of
SAN 9789 appears to reduce curvature more effectively in the
present work. The shape of the dose response curves at both
concentrations is similar, with a peak response occurring at
a log (dose) of -5.0. This dose corresponds to an intensity
of 12 uW x cm.2 and time of 0.7 seconds. Analyses of
variance for the two sets of experiments show that at both
concentrations of SAN 9789, differences in response for
control and SAN 9789—treated seedlings are highly
significant (0.01 level) (Tables 2.2 and 2.3). Both the
dose of light to which a seedling had been exposed and the
herbicide treatment affected its response. In addition, at
the higher SAN 9789 concentration, there was an interactive
effect between light dose and herbicide treatment
significant at the 0.05 level.

Both control and SAN 9789 treated seedlings which had
been kept in darkness (except for infrared flash and
safelight) were used as a measure of undirected movement by

the seedlings. Since they received no directional stimulus,

only the absolute value of their net curvature is

61

 

  

 

  

   

 

 

 

 

5 I
2
G e: I d ‘1‘
- 2 a
g a 3 I
22:“
a a g l
e<>m
I - ‘7’
I
I a?
I
I - : g
l 5
S
I a
o
3
I 8
.J
| ‘ '1’
I
I
I l 3°
I It
Jzfl I f
l ' L 8
J 1
9 3 m o ?
(eeemep) emuuno to"
4

Figure 2.

SAN 9789
Methods.
and each
Standard

+/-10200

3. Dose response curves for control and 2 x 10- M
treated seedlings. Experimental details in
Each point represents the mean of six experiments,
experiment used 9 or 10 seedlings per point.

error for data pooled from six experiments was

62

 

 

 

 

 

 

I I
In ‘ “‘
a S’.’ I I
a... I
-5 Q
so» a
5% ° ~——£—" I
b
0032
I c m '—
l
I ‘ ‘P
l
l
l
's
.4 l ‘ 1‘ g
l g
v
I 3
8
I '0
V
O
O
l .l
b a: '0
l I I
, l
l
l
" ' l
o
I I .
\\
l I;
,—4 .
p:?** a
1:
. l
o J ,1 I
N 9 S ' m o
(seetbep) etntauno ten
5

Figure 2.4. Dose response curves for control and 2 x 10- M

SAN 9789 treated seedlings. Methods and replications as for

Figure 2.3. Standard error for data pooled from six

experiments was +/-1.4°.

63

Table 2.2. Analyst? of variance for dose response curves of
control and 2 x 10 M SAN 9789 treated seedlings. Data
shown in Figure 2.3.

 

 

source df F
**

treatments 15 21.25**

doses _ 7 34.60**

SAN 9789 1 58.90*

dose x SAN 9789 7 2.53

* *1: . . _
, Significant at 0.05 and 0.01 levels.

Table 2.3. Analystg of variance for dose response curves of
control and 2 x 10 M SAN 9789 treated seedlings. Data
shown in Figure 2.4.

 

source df F
**
treatment 17 15.80**
doses 8 3l.l9**
SAN 9789 1 7.48
dose x SAN 9789 8 1.45

 

* significant at the 0.01 level.

64
meaningful. Up to 30 of curvature (Figure 2.3) was
attributed to random movement by the plant.

Geotropism serves as a second control for the
specificity of SAN 9789. Since phototropism and geotropism
both involve a unilateral redistribution of growth across
the plant, it is likely that they share some common steps in
the transduction pathway. Detecting the two different
stimuli involves different mechanisms, but the changes in
growth rate necessary to bring about curvature should be the
same for both responses. If phototropism is affected by an
inhibitor to a greater degree than is geotropism, then the
inhibitor must work at a point not held in common between
the two responses. In preliminary experiments, it was found
that SAN 9789 treated seedlings required high humidity for
expression of a full geotropic response. Appendix 2
presents data showing the geotropic responses of control and
SAN 9789 treated seedlings at 60% and 100% humidity.
Thereafter, all geotropic experiments, like phototropic
experiments, were carried out in 100% relative humidity.

Once the humidity requirement was met, SAN 9789 treated
seedlings responded as strongly to a geotropic stimulus as
control seedlings (Table 2.4). Using a Student's t test, no
significant differences in geotropic curvature for seedlings
given different SAN 9789 treatments were found.

A final estimate of non-specific effects of SAN 9789 was
made by measuring the growth rate for control and SAN 9789

treated seedlings. It was observed that seedlings treated

65

4

with 2 x 10- M SAN 9789 grew more slowly than control plants

over the course of the five days before they were used.

However, when the growth increments of control, 2 x 10-5M,

and 2 x 10'4

M SAN 9789 treated seedlings which had received
no actinic irradiation were measured over a three hour
period, no differences in growth rate were observed (Table
2.5). If phototropism and geotropism are the result of a
lateral redistribution of growth (Iino and Briggs, 1984) and
if this distribution of growth remains constant despite
changes in growth rate, then a plant which shows a reduced
growth rate could still be capable of normal tropic
curvature.

From these data, it appears that SAN 9789 inhibits first
positive phototropism without affecting geotropism, as long
as high humidity is maintained. An 80% reduction in
carotenoid content reduced the phototropic response only
39%, leading to the conclusion that carotenoids contribute
as screening pigments to the gradient of light for first
positive phototropism.

Another approach to reducing the carotenoids in a corn
seedling which avoids the problems inherent in using an
inhibitor is to introduce a mutant allele which gives a
"carotenoidless" plant. For mutant seedlings, as with
control and SAN 9789 treated seedlings, a first positive
response is separated from a second positive response by an

indifferent or negative response (Figure 2.5).

66

Table 2.4a. Geotropism 3g SAN 9789 concentration in 100%
relative humidity. Each experiment included 9-11
individuals. Curvature measured in degrees. SE: standard

error of the mean.

 

treatment # of experiments mean curvature SE
controt5(water) 9 53.44 1.6
2 x 10_4M SAN 9789 5 55.1 6.2
2 x 10 M SAN 9789 10 59.4 2.0

 

Table 2.4b. Values of Student's t for indicated comparisons

comparison t

 

5

control Ki 2 x 10:4M SAN 9789 0.55
controt 3g 2 x 10 MSSAN 9789 0.85
2 x 10 M.X§ 2 x 10 M SAN 9789 1.79

 

Table 2.5. Growth rates of control and SAN 9789 treated
seedlings over a three hour interval. SE: standard error of
the mean.

 

treatment growth increment SE
controt5 0.15 0.02
2 x 10_4M SAN 9789 0.14 0.02
2 x 10 M SAN 9789 0.19 0.02

 

67
A statistical analysis (Table 2.6) shows that the

difference in curvature between normally pigmented seedlings
and pale or SAN 9789 treated seedlings is significant. By
analysis of variance (Table 2.6a), most of the difference
between the dose response curves shown in Figure 2.5 could
be attributed to an effect of dose. However, the "corn

4M SAN 9789 treated hybrid,

type" (whether control or 2 x 10-
yellow or pale mutant) made a small contribution,
significant at the 0.1 level. Since it was the effect of
"corn type" that was considered most interesting, a further
analysis of the differences between dose response curves due
to corn type was made using orthogonal contrasts (Table
2.6b). This showed that the difference in phototropic
responsiveness (significant at the 0.5 level) is between
fully pigmented seedlings and seedlings with reduced
carotenoid content. At the first positive dose tested, the
response of pale mutant seedlings was reduced 62% from that
of the wild type (Table 2.9).

Geotropism and growth rate were used as controls for
mutant seedlings as well. Because of the geometry of the
holders for individual vials used in these experiments,
geotropic and phototropic stimuli were given in the same
plane. An analysis of variance (Table 2.7) shows that the
previous light dose has an effect on the subsequent
geotropic curvature. This is probably because seedlings
which responded strongly to a phototropic stimulus started

responding to a geotropic stimulus from a negative

68

 

20

0 Control

I Yellow

0 SAN 9789

? Pale

Error barsztt SEM
15 '-

O
I

 

Net curvature (degrees),
0|
T

O
I

 

 

 

I
0|
I
»y——-——u—-1

II 1 1 1 1 1

 

 

//
dark —6 -5 -4 _2 -3 -2
Log(dose) (w-cm )

Figure 2.5. Dose response curves for control and SAN 9789
treated hybrid seedlings, yellow and pale mutant seedlings.
Experimental details in Methods. Each point represents the
mean of three experiments. Each experiment used 7 or 8
seedlings per light point, 5 or 6 seedlings per dark
control. Standard error for data pooled from three

experiments was +/-3.06°.

69

Table 2.6a. Analysis of variance for dose response curves
of wild type and pale mutant seedlings. Data in Figure 2.5.

 

source df . F
**
treatments 15 4.20**
doses 3 14.93+
corn type 3 2.57
dose x corn type 9 1.17

 

**
+, significant at 0.1, 0.01 levels.

Table 2.6b. Orthogonal contrasts for corn type.

 

source df F
control and yellow 1 7.06*
XE SAN and pale

control 3g yellow 1 0.36
SAN 3g pale 1 0.28

 

* significant at 0.05 level

70

Table 2.7. Analysis of variance for geotropism of yellow
and pale mutant seedlings.

 

source df F
**
treatments 7 4.80**
doses 3 8.42
pigmentation 1 1.60
dose x pigmentation 3 2.24

 

significant at 0.01 level.

Table 2.8. Growth rates of mutant seedlings. SE: standard
error of the mean.

 

phenotype growth increment SE
lwl pale 0.1 0
lw2 pale 0.19 0.03
lw2 yellow 0.06 0.02

 

Table 2.9. Summary of correlations among carotenoid
concentration, phototropic curvature, and geotropic
curvature. Carotenoid content is based on absorption at
480 nm of randomly selected individuals (Figures 1.5, 1.9,
and 1.10), phototropic responses on dose response curves
(Figures 2.5, 2.4, and 2.5), and geotropic responses on
geotropic controls used for dose response curves. For pale
mutants, wild type seedlings served as control, for SAN 9789
treated seedlings, hybrid seedlings treated with water were
used. Responses are expressed in terms of percentage of
control response.

 

corn carotenoid phototropic geotropic
type content response response
lwl angslwz 3o 1 38 107
2 x 10_4M SAN 9789 ND 69 103
2 x 10 M SAN 9789 13 61 111

 

1 mean of two individuals. ND: no data.

71
curvature. More importantly, the analysis of variance also
shows that pale and yellow seedlings did not differ in their
geotropic responses.

Growth rates from one experiment (Table 2.8) indicate
that pale mutants may elongate more quickly than wild type
seedlings. Thus, carotenoidless mutants appear to be
capable of normal growth and geotropic curvature, but like
SAN 9789 treated seedlings show a reduced response to doses
of light which stimulate first and second positive
phototropism.

Summary

Nonspecific effects in plant tissue treated with
SAN 9789 which may alter phototropic capability. It is also
possible that pleiotropic effects in the mutants could
affect phototropism. One method to estimate the effects of
SAN 9789 in systems other than carotenoid synthesis would be
to treat pale mutant seedlings with SAN 9789. Since pale
seedlings are low in carotenoids and carotenoid-derived
metabolic products, further reduction in phototropic
response could be attributed to nonspecific SAN effects.

Further comparisons between pale mutants and SAN 9789
treated seedlings might help to define effects not
specifically related to carotenoid concentration which alter
phototropism. The mutant's response to gravity in 60%
humidity and the kinetics of bending for mutant seedlings
are data which would allow further comparison of the

metabolism of SAN 9789 treated and pale seedlings.

72
Carotenoids appear to act as screening pigments in first
positive phototropism, a role suggested by Vierstra and Poff
(1981b) in second positive phototropism. A decrease in
carotenoid content correlates with a decrease in phototropic

response, but does not affect geotropism (Table 2.9).

 

CHAPTER 3

Mechanisms of Establishing a Light Gradient: Lens,
Longitudinal Light Transmission, and Scatter

Introduction : Lens

 

If a cylinder is sufficiently transparent, and has an
index of refraction sufficiently different from the
surrounding medium, it can act as a lens. The refractive
index relative to the surrounding medium will determine
whether the cylinder will cause unilateral light to converge
or diverge. However, the amount of absorption and scatter
will determine how quickly light is attenuated. In a

Phycomyces sporangiophore in air, light is focused on the

 

side of the sporangiophore away from the light source. The
attenuation across the sporangiophore is sufficiently small
that the absolute intensity in the distal half is larger
than in the proximal half. Thus, there is a "focusing
advantage" within the sporangiophore, and a "lens effect"
exists (Dennison, 1979). This "lens effect" is the

mechanism which Phycomyces sporangiophores use to determine

 

the direction of a unilateral light stimulus (see

Introduction). Poff (1983) and others have suggested that a

73

74

focusing advantage may also exist in the nearly-cylindrical
shoot tips of monocots.

The shoot of a corn plant consists of mesocotyl, node,
and tightly rolled primary leaves encased in the coleoptile.
The primary leaves are dense enough to eliminate the
possibility of a lens effect along much of the length of the
shoot. In the apical 0.5 to 1 mm of the shoot, only
coleoptile tissue is present. If a lens contributes to
detection of light direction in a corn seedling, it would be
here, where the tissue is most transparent.

The tip of a coleoptile is a cone with an elliptical
base and an off-center apex. The two vascular bundles
converge at the tip, and approximately intersect the long
axis of the ellipse. Because of the different path length
and tissue composition along the long and short transverse
axes of the shoot, measurements of light intensity, like
spectra generated in previous chapters, were made along the
short axis of the ellipse, perpendicular to the vascular
bundles.

Methods

Using a modified Cary 14 spectrophotometer with a
picoammeter attached to the photomultiplier tube, currents
from the phototube representing initial intensity (IO) and
final intensity (12) were measured. I1 is the intensity at
the border between the front half and the back half of the
cylindrical "coleOptile". The difference between I and I

0 2
is due to both absorption and scatter.

75
Results and discussion
A number of simplifying assumptions make it possible to
evaluate the possibility of a lens effect in corn
coleoptiles in a manner similar to Castle's treatment of

Phycomyces sporangiophores (Castle, 1933b). Castle (1933a)

 

measured a refractive index of 1.38 for Phycomyces.

 

Assuming a circular cross-section, he found that the total
path length of focused light in the rear half of a
sporangiophore was 1.26 times greater than the path length
in the front half. These values, and a set of similar
assumptions, are used here to evaluate corn seedlings.

1. Rather than an ellipse, the cross-section of a model
"coleoptile" is assumed to be a circle. Ten coleoptiles
measured just above the primary leaves averaged 1.01 mm
along the long axis, 0.78 mm along the short axis, so the
diameter of the model coleoptile is taken to be 0.9 mm.

2. Effects of cell boundaries are ignored: it is
assumed that the coleoptile refracts light as a single cell.

3. Index of refraction is taken to be 1.38, as for

Phycomyces.

 

4. The ratio of path length in the back half to the

front half is taken to be 1.26, as for Phycomyces.

 

5. All light incident on the coleoptile is assumed to
enter the shoot. Reflection and scatter at the surface are
ignored.

These assumptions favor the existence of a lens effect.

The following equations were used to calculate the

76

intensity midway through the coleoptile (11) using measured

values of IO and 12.

_ -¢11
Il - Ioe (a)

- -ah_
I2 - Ile (b)

where<x= absorption coefficient, 11 = path length in front

half of coleoptile, 12 = path length in back half of

coleoptile = 1.2611

Substituting into equation (b),

-1.26a11)

I = I e'°‘11)(e

2 0‘
1.75 x 10-6 amps = 1.13 x 10-5 amps (e

-2.264g_)

0.55 = e

0.825 = «11

Substituting into equation (a),

e-0.825
1 0

= -0.825

1n I - ln (1.13 x 10'5 amps) = -0.825
1n I = -12e2
1 = 5.03 x 10'6 amps

Then, absorption in front half = IO - I1
5 6 6.27 x 10.6 amps

= 1.13 x 10’ - 5.03 x 10'

Absorption in back half = Il - I2
6

- 1.75 x 10’6

6

= 5.03 x 10’ 3.28 x 10' amps

absorption in back half = 0 52
absorption in front half '

 

77
The absorption coefficient can be estimated from the

incident and final light intensities: I
log -0 = OD
I2

—5
log 1.13 x 10 amps

 

1.75 x 10'5 amps = 0.8 OD over 0.9 mm or

(X: 8.9 for a standard pathlength of 1 cm.

Castle (1933b) generated a curve which shows the
dependence of relative absorption in the front and back
halves of a cell on CX, and stated that similar curves can
be generated for cells of different diameters, larger cells
giving curves with steeper slopes. Castle's (1933b) figure
is adapted in Figure 3.1. The curve from his data, using

Phycomyces sporangiophores with a diameter of 0.08 mm, is

 

shown by the solid line. The corresponding curve for a
model coleoptile 0.9 mm in diameter is shown by the dashed
line. The critical value oft: for a pathlength of 0.9 mm is
2.6, or an optical density of 0.23 for a standard pathlength
of 1 cm. Therefore, for a cylinder 0.9 mm in diameter, cx
must be less than 2.6, and the ratio of absorption in the
back half to absorption in the front half must be greater
than 1.0 for a focusing advantage to exist. With values of
Cr: 8.9 and the ratio of absorption in the back half to
absorption in the front half = 0.52, there can be no lens
effect.

Introduction : Longitudinal light transmission

 

 

Light gradients exist longitudinally, as well as

transversely, along a unilaterally irradiated coleoptile.

78

 

  
       

 

 

 

"7;

= 1.2

E

2

c 1.0 ------ x ------- - -----

.9: §\ :

9 . \ I

o ' '

30.8 1 \\ g

S i \t

.. : :\\

" I

20.6» g g \

'3 I : \

3 i i \\

c 0.4’ l I \

.2 g g \\

e i E \

30.2b : . \\

.D 1 : \

( : I \

. I

1_2 1 1: 1 1 J I 4
2 4 6 8 1o 12 14 16

Absorption coefficient (I

Figure 3.1. Critical values for absorption coefficient (a)

for a Phycomyces sporangiophore (solid line, open circles)

 

and a model corn coleoptile (dashed line, solid circles).

Adapted from Castle (1933b).

79
Mandoli and Briggs (1982b) have shown that plant tissues are
capable of axial light transmission over 20-25 mm, and
attribute this transmission to internal reflection, or
"piping", of the light. Scatter must also have an effect on
the longitudinal distribution of light. Light rays will
scatter off particles and/or when moving through cell
components of different refractive indices. Both scattered
and piped light are attenuated log-linearly with distance.
Unlike randomly scattered light, piped light is transmitted
coherently through the tissue (Mandoli and Briggs, 1982b).
Mandoli and Briggs proposed that light transmitted axially
could trigger photomorphogenesis (1982a) or phototropism
(1982b) by exciting pigments at a location distant from the
area directly illuminated.

The axial transmission of light is wavelength
dependent. Scatter and absorption would tend to attenuate
short wavelengths of light more quickly than longer
wavelengths. In addition, Mandoli and Briggs (1984)
attributed some attenuation to less effective "piping" of
blue light. Their results are qualitative, and trace
relative transmission of light from 400 - 750 nm through
several plant tissues. The experiments presented here are
an attempt to quantify the amount of light transmitted at
selected wavelengths.

Methods
Corn seeds were grown on Kimpak as described in

Chapter 1. Plants from six to ten days old were used.

80

Seedlings at least 2.5 cm long, with primary leaves
extending the entire length of the coleoptile, were chosen
for use. A segment of a coleoptile plus primary leaf 2.5 cm
long was placed in a holder, with 1 cm of tissue extending
above the holder. The coleoptile was held in place with
black plasticine, which also prevented light leaks between
the plant tissue and the holder (see Figure 3.2). The light
sources used were a helium - neon laser with an output at
632 nm, or the 514 nm and 458 nm lines of an argon laser.
The different bands of light from the argon laser were
separated by a prism. Each wavelength of light was passed
through a slit and an interference filter of appropriate
wavelength (Baird atomic, 630, 510, or 460 nm peak, 10 nm
half-band) to eliminate stray light of other wavelengths.
The intensities of the green and red bands were adjusted
with neutral density filters (Inconel) so that the incident
intensity of the three wavelengths used was nearly equal.
The beam of light was then passed through a lens and brought
to a focus on the tip of the coleoptile, and normal to its
longitudinal axis. The bottom of the coleoptile was abutted
against an optical fiber, which led to a photomultiplier
tube. Current from the photomultiplier was measured with a
picoammeter and subsequently converted to units of
intensity. Incident light intensity was measured with a
Kettering radiometer (model 7720).

When light was incident on the tip of the coleoptile,

the top 1 - 1.5 cm "glowed" from light scattered from the

81

   

 

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:6 «209::

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Ecmmv

Ecwnm

Ento

   

 

 

coma. ozIeI

 

 

 

come. 2

 

 

Apparatus used for measuring axial light

Figure 3.2.

For details,

transmission through corn coleoptile segments.

see text .

82

sides of the tissue. To prevent this scattered light as
well as any stray light from striking the photomultiplier
tube, the bottom of the coleoptile was inserted into a hole
in a cap on top of the optical fiber. Each coleOptile was
exposed to 632 nm, 510 nm, and 460 nm beams of light.
Measurements were taken from ten control and ten SAN 9789
treated plants for each of the five ages used.

The photomultiplier tube was run at 600 V. At this

voltage, dark current on the phototube was 9 x lo-llA to

-10A. Dark current was subtracted from measurements

1.2 x 10
of transmitted light before converting the measurements of
current (A) to units of intensity (pW). The system could
measure intensities over eight orders of magnitude before
dark current became limiting.

The ratio of (light in / light out) when converted to
log terms gives the optical density (A) of the sample. In
this experiment, transmission losses include losses at the
surface, so are an overestimate of the actual density of the
tissue.

To measure the attenuation of transmitted light as a
function of distance, coleOptiles were placed in the same
holder apparatus and irradiated at the tip with 632 nm
light. Transmitted light was measured, a segment of known
length was cut from the coleoptile, and transmitted light

was measured again. Three to six sequential measurements

were made from each plant.

Results and discussion

Light incident on a coleoptile can be reflected,
absorbed, or scattered. Some of the scattered component
will be transmitted axially, as well as in other directions
through the tissue (see Figure 3.3). The proportion of the
light which is transmitted axially has been reported to
depend strongly on the incident angle of light, with
different tissues and plants showing different acceptance
angles (Mandoli and Briggs, 1982b). Preliminary
experiments with the present system did not show this
angular dependence (Figure 3.4), so the incident angle used
in subsequent experiments was always 0°, normal to the
longitudinal axis, as with actinic light for phototropic
experiments.

Dark current varied over the several days necessary to
collect data, so it is presented as a shaded area, 7.6 to
8.0 orders of magnitude less than incident intensity (Figure

3.5). A representative dark current of 1.2 x 10-10A can be

expressed using conversion constants as 0.17 pW x cm-2

(9 x 10-5pE x cm.2 x sec—1) for 632 nm light, 0.14 pW x cm-2

5pE x cm”2 x sec-1) for 514 nm light, or 0.22

(6 x 10-
-2 -5 -2 -1 .

pW x cm (8.3 x 10 pE x cm x sec ) for 460 nm light.

Thus, the lower limit of sensitivity of the system is on the

4 2 x sec-1, two

order of 0.5 pW x cm"2 or 1 x 10- pE x cm-
orders of magnitude more sensitive than the light levels
reported by Mandoli and Briggs (1981) to be necessary to

evoke a threshold "very low irradiance response" in Avena.

 

 

 

 

 

 

 

 

 

 

Figure 3.3. Light incident on a coleoptile (A) can follow a

number of different paths. Some (B) is reflected or

scattered at the surface, and never enters the tissue. Of

the light which enters the tissue, some is scattered

randomly (C), some is absorbed (D), and some may be

scattered axially, or "piped" (E).

85

 

 

 

 

 

3 ’6'

E

a)

t-7-

:

3

U)

2 _8I.
1 1 1 L
0 30 60 90

Incident angle (degrees)

Figure 3.4. The apex of a coleoptile segment was
illuminated with red light at the indicated incident angle
(degrees from normal). No angle dependence was found.
Closed circles: control tissue. Open circles: 2 x 10-4M

SAN 9789 treated tissue. Error bars: standard error of the

mean.

 

10

6 7 8 9
458 nm

 

10

8
514 nm

 

10

632 nm

 

 

 

l 1 1

N (D

 

-5
6

I l
(MN) (100 IIIBII / U! lll5!l)50l

Age(days)6 7 8 9

Wavelength

Figure 3.5. Longitudinal light transmission. Transmission
of three wavelengths of light was measured through 2.5 cm
segments of corn seedlings of indicated ages. Control

seedlings (e————e), 2 x 10"4

M SAN 9789 treated seedlings
(Ouou-o). Shaded areas represent the range of dark current
over the several days needed to collect data. Error bars:

standard error of the mean.

87

There is some age-dependence to the longitudinal
transmission of light, though the dependence is not direct.
Differences attributed to age may be due to mean cell
length, the degree to which tissues were hydrated, or some
other factor not directly measured.

More interesting is the relative transmission of red,
green, and blue light through 2.5 cm of tissue. Acceptance
losses should be equal for all three wavelengths of light,
so a comparison based on wavelength shows the relative
efficiency with which each wavelength is "piped". Red light
is transmitted most efficiently, but over 2.5 cm is
attenuated 5.3 to 6.1 orders of magnitude (Figure 3.5).

Over the same length of tissue, green light is attenuated
5.8 to 6.6 orders of magnitude, and blue light between 6.5
and 7.9 orders of magnitude. The final blue light intensity
is very low, approaching the limit of measurement. It seems
unlikely that such extremely low intensities of transmitted
blue light would affect a plant's phototropic response when
a much stronger stimulus is present elsewhere along the
shoot. Red light, on the other hand, may be transmitted
strongly enough to have some physiological effect; Parks and
Poff (personal communication) find that 0.4 W x cm-2 red
light incident at one point in a corn shoot can cause
conversion of phytochrome at a location at least 1.4 cm
away.

Absorption and/or scatter are potential mechanisms to

explain the additional attenuation of blue light over red.

88
To test the possibility that absorption by carotenoids
affected the longitudinal transmission of blue light,

4M SAN 9789

transmission by seedlings treated with 2 x 10-
was also measured. No significant difference between

SAN 9789 treated and control tissue was found (Figure 3.5,
Table 3.1). Measurements of the transmission of green light
were included as a second method to try to separate
attenuation by absorption from scatter. Green light should
be absorbed very little, so if absorption were the source of
the attenuation, transmission of green light should resemble
that of red. On the other hand, green light should be
scattered similarly to blue light. A statistical analysis
using orthogonal contrasts showed that the transmission of
red, blue, and green light are all different from one
another. Transmission of green light is intermediate to
that of red and blue, so that it is not particularly useful
in distinguishing between scatter and absorption as
mechanisms of attenuation. The similarity of light
transmission in control and SAN 9789-treated tissue
eliminates absorption as a source of the additional
attenuation of blue light, leaving scatter as an
alternative.

An analysis of variance (Table 3.1) shows that the
wavelength of light and the age of the tissue both have a
highly significant effect on the longitudinal transmission
of light. Interactive effects of light x age, also highly

significant, may be due to the tendency of SAN treated

89

Table 3.1. Analysis of variance of data for longitudinally
transmitted light. Data shown in Figure 3.5.

 

source df F
**
treatments 29 18.07**
light 2 208.65**
age 4 13.34
SAN 9789 1 1.05
light x age 8 0.827
light x SAN 9789 2 1.285**
age x SAN 9789 4 5.782
light x age x SAN 9789 8 2.49

 

**
*, significant at 0.05 and 0.01 levels, respectively.

90
seedlings to accumulate pigments as they age. Third order
interactions (light x age x SAN 9789) are statistically
significant, but difficult to explain.

In order to estimate transmission losses with distance,
sequential measurements were made from a number of
individual seedlings. The tip of a seedling of known length
was irradiated with 632 nm light, and transmission was
measured. A segment was sliced from the bottom of the
seedling, and transmission was measured again. Since
acceptance losses are the same for all measurements, the
slope of the line plotted for attenuation with distance
gives an accurate measure of the density of the tissue. The
mean slope for control seedlings (Figure 3.6) is -0.74 A,

for 2 x 10'-4

M SAN 9789 treated seedlings, -0.82 A (Figure
3.7).

Mandoli and Briggs (1982b) have measured light
transmission from the proximal and distal sides of a
unilaterally irradiated coleoptile, and find a higher
intensity on the distal side, when irradiating along the
short axis. They suggested that, due to the shape of the
coleOptile tip, unilateral light was closer to the
acceptance angle of the cells on the distal side, such that
they intercept and transmit more light. However, Vogelman
and Haupt (in press), using a fiber optic to probe the
distribution of blue light across a corn coleoptile found

that light intensity was greatest on the proximal side of

the plant for all angles measured.

91

 

 

 

 

 

 

-4, __
-5. .
§ -6I-
0
O.
E
3
a -7-
-8" _ A RA
-9:- v-
I I A I 1 I
1 2 3 1 2 3

Length (cm)

Figure 3.6. Longitudinal transmission of red light as a
function of tissue length. Control seedlings. Transmitted
light was measured in terms of current from a
photomultiplier tube. Each set of symbols represents
measurements taken from one individual, with a best fit line
through each set of data. Data separated along x axis for

clarity. Mean slope -0.74.

92

 

 

 

 

 

 

’5 I" I’
‘9
\ I\\‘l\
’3 '6" B '
9 \
A \
(U
.. A «A ........
-8 p r-
..9 i- ,_ A ‘ ..
A3”;
L 1 1 I 1 1 4
1 2 3 1 2 3 4

Length (cm)

Figure 3.7. Longitudinal transmission of red light as a

4M SAN 9789 treated

function of tissue length. 2 x 10-
seedlings. Transmitted light was measured in terms of

current from a photomultiplier tube. Each set of symbols
represents measurements taken from one individual, with a

best fit line through each set of data. Data separated

along x axis for clarity. Mean slope -0.82.

93

If "piping"-contributes to the light gradient for
phototropism, it seems likely that it will be because of an
effect on the way light is intercepted by the plant, rather
than because of transmission over a distance. Blue light is
attenuated quickly, so probably does not contribute much to
a gradient of light distant from the point of incidence.

Introduction : Scatter

 

Manipulation of the scattering properties of a plant
affect the plant in other physiologically important ways.
Parsons _t gt. (1984) have estimated the contribution of
scattered light to perception of direction by measuring
attenuation through different thicknesses of tissue. They
have reduced scatter, and thus the gradient of light across
a shoot, by infiltrating tissue with oil of high refractive
index.

Seyfried and Fukshansky (1983) have developed a method
for calculating light gradients in layered tissue. They
point out that measuring light inside an object, although
necessary, interferes with the optical properties being
measured. Their model allows estimates of light gradients
to be made unobtrusively. Using a similar method, Seyfried

and Schaefer (1983) monitored changes in reflectance and

transmittance of Cucurbita cotyledons giving special

 

attention to scattering properties of the tissue.
In a corn coleoptile, attenuation due to scatter comes
not only from light scattered off molecules and other

cellular components, but also from refractive changes as

94

each ray of light moves from cell to cell. A model of the
attenuation of light due to scatter by refraction is
presented by Parsons gt gt. (1984). They argued that most
of the light gradient in a coleoptile was due to refraction
and that absorption played a relatively minor role.
Methods

Measurements of light attenuation due to scatter and
absorption in different tissues of corn seedlings were made
with a modified Cary 14 spectrophotometer. Spectra were
generated as in Chapter 1. In addition, measurements of
current were taken every 10 nm from a picoammeter connected
to the photomultiplier tube. Similar measurements were made
without any sample in place for the system response. The
ratio of system / sample gives a measure of total
attenuation by the sample.

Results and Discussion

 

Total attenuation (absorption plus scatter) can be
measured directly for various tissues of a seedling. When
plotted as a ratio of system response to sample 3g
wavelength, data corresponding to absorption spectra are
generated (Figure 3.8 - 3.12). The location along the
y-axis is a function of absolute attenuation. Although
absorption of shoot and mesocotyl tissue differs, total
attenuation by these two tissues is quite similar in both
dark and red-grown, control and SAN 9789-treated tissue.
Variations in absorption in seedlings grown under different

conditions are apparent, but there also appears to be a

 

 

 

 

95
darkcontrol
)-
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O
I
O
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100.0)— 0
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Doaooogogon
I
.llll.
10.0b ..'.
. I
I
. 'III
> l..IIII.
I
1 1 1 I 1 P 1 1
400 450 500 550 600 650 700 750

Wavelength (nm)

Figure 3.8. Total attenuation of light (absorption plus
scatter) by various tissues of a completely etiolated
control seedling. (0) Tip of seedling through coleoptile
and primary leaf. (0) Base of seedling through coleoptile
and primary leaf. (0) Mesocotyl. (I) Excised coleoptile.
Measurements of current on the photomultiplier tube were

used to calculate the ratio of system / sample.

96

 

Red control

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O ODD 099
3; oocBoooo
m

10.0—

5.0- a.

.I.-
_ -I'I.I-I'.- I
I.. ......

 

 

I I I I I I I I

400 450 500 550 600 650 700 750
Wavelength (nm)

 

Figure 3.9. Total attenuation of light (absorption plus
scatter) by various tissues of a control seedling grown with
one hour of red light per night. (0) Tip of seedling
through coleoptile and primary leaf. (0) Base of seedling
through coleoptile and primary leaf. (0) Mesocotyl.

(I) Excised coleoptile. Measurements of current on the
photomultiplier tube were used to calculate the ratio of

system / sample.

97

 

 

 

 

dark SAN
o
1000 0
" 0898
)-
Pa o
o
- 0
DD
50.0L ° ° 0 '.
o
A 0 O
2 ’ o ."oo
o o.
g o 0 0.0.0000
. .000 o
3 00000 on...
E 00000
2’. 00000
a °°°880
m b Dmgmmmmoo
v o
g . 008039838038
a
m III
I
II
III
I—I— .-
1OD_ .....--. .
.I .I. -
b - ....II.III
5D~
1 1 1 1 l l 1
400 450 500 550 600 650 700 750

Wave|ength (nm)

Figure 3.10. Total attenuation of light (absorption plus
scatter) by various tissues of a 2 x lO-4M SAN 9789 treated,
dark grown seedling. (0) Tip of seedling through coleoptile
and primary leaf. (0) Base of seedling through coleoptile
and primary leaf. (0) Mesocotyl. (I) Excised coleoptile.

Measurements of current on the photomultiplier tube were

used to calculate the ratio of system / sample.

98

 

 

 

 

Red SAN
100.0”0
_o
.0
~ 9880
o
50.0)- .
... O
r- .00
Do
A .0 000
g . 00 noon
2. .0 DD
0
g 0800 DuooooDDDDD o
\ .0 0000000
5 L aggoggogooo IOII
; o 000. o 0......
z 000 00
3 00000
2
E
E
10°F
L
“I
5_o.— ..II-
.II-
III
'I'IIII. .
' ..I-I I..-
I I I I I l L J
400 450 500 550 600 650 700 750

Wavelength (nm)

Figure 3.11. Total attenuation of light (absorption plus

4M SAN 9789 treated

scatter) by various tissues of a 2 x 10-
seedling grown with one hour of red light per night.

(I) Tip of seedling through coleoptile and primary leaf.

(0) Base of seedling through coleoptile and primary leaf.
(D) Mesocotyl. (I) Excised coleoptile. Measurements of

current on the photomultiplier tube were used to calculate

the ratio of system / sample.

 

 

 

99
O
100.0—I ‘3
:. ‘m.
E D
a ..
s 501) ..
a) - A
E
b D
+3
A
E - '.
DA
:2 I At)
a: ..
10.0: T
51)-
I I l .

 

400 500 600 700
Wavelength (nm)

Figure 3.12. Total attenuation of light (absorption plus

scatter) by shoot tips of control, SAN 9789 treated, yellow

and pale mutant seedlings at selected wavelengths.

(0)1w1 yellow. (A)lw2 yellow. (D) 2 x 10-4M

(I)Control.

SAN 9789 treated. (0)1w1 pale. (A)lw2 pale.

100
constant contribution to light attenuation by scatter,
independent of growth conditions.

Estimates of the portion of attenuation of light by a
corn seedling which is due to absorption can be made from
computer-generated difference spectra. By subtraction of a
baseline "scatter spectrum" from a tissue spectrum such an
estimate can be given. There are three possible ways to
generate a baseline (Figure 3.13).

1. A line congruent with the part of the spectrum which
shows no peaks could be drawn and extended through
wavelengths where absorption occurs (Figure 3.13A).
Attenuation above this baseline is attributed to absorption,
below, to scatter. This method overestimates the amount of
absorption in the blue, since the baseline has a constant
slope. In reality, shorter wavelengths of light scatter
more than longer wavelengths, giving a curved baseline
rather than a straight line. Using this method, the
seedling represented in Figure 3.13A has an apparent
absorption at 470 nm of 0.52 A.

2. The scatter ramp can be approximated by generating a
baseline spectrum using a highly-scattering, non-absorbing
substance such as layered Kimwipes (Figure 3.133). This
gives a curve of the appropriate shape, but it is difficult
to choose the proper thickness of Kimwipes which will most
accurately approximate the amount of scatter seen in plant

tissue. Apparent absorption at 470 nm is 0.39 A.

101

3. Plant tissue can be used to approximate scatter, if it
does not absorb blue light. Mesocotyl tissue, excised
coleoptiles, carotenoidless mutants, or SAN 9789 treated
tissue, of a thickness similar to the pigmented sample, can
be used (Figure 3.13 C, D, and E, respectively). This
method would tend to underestimate the contribution of
absorption, since such tissue is not completely pigmentless.
Residual pigments (carotenoids, flavins, or cytochromes)
which are present in the tissue used for the baseline are
subtracted from the sample. The tissue chosen for the
baseline changes the shape of the absorption spectrum. At
470 nm, apparent absorption using mesocotyl tissue as a
baseline gives an an absorption of 0.32 A, coleoptile tissue
0.45 A, and SAN 9789 treated tissue 0.41 A. A wild type
(yellow) lw2 shoot, using a mutant (pale) shoot as a
baseline, has an apparent absorption of 0.29 A (Figure
3.13F).

The actual contribution of absorption to total light
attenuation probably falls between the approximations
provided by method one and method three above, that is,
between 0.3 A and 0.5 A at 470 nm.

With a measurement of total light attenuation and an
approximation of the attenuation due to absorption, it is
possible to estimate the contribution of scatter to the
light gradient. At 450 nm, scatter accounts for about 1.5 A
of the attenuation across the shoot (Table 3.2) in both

control and SAN 9789 treated tissue. Treatment with

102

 

 

AAbsorbance

 

 

 

 

I I 1 I I I I

 

 

400 450 500 550 600 650 700 750
Wavelength (nm)

Figure 3.13. Approximating a scatter ramp artificially or
with plant tissue low in pigment. A) control shoot plus
ramp (dashed line). B) control shoot minus 14 layers of
Kimwipes. C) control shoot minus mesocotyl. D) control
shoot minus excised coleoptile. E) control shoot minus

4

2 x 10- M SAN 9789 treated shoot. F) lw2 yellow shoot minus

lw2 pale shoot.

103

Table 3.2. Mean optical density at 450 nm of two randomly
selected corn seedlings. Shoot tip measurements were taken
through both coleoptile and primary leaf tissue. Coleoptile
measurements were made on excised coleoptiles. Red grown
seedlings received one hour of red light per night. Dark
grown seedlings were completely etiolated. Data were
obtained from measurements made for Figures 3.8 - 3.11.

herbicide treatment

 

tissue light treatment control 2 x 10 M SAN 9789
shoot tip red grown 2.1 1.5

shoot tip dark grown 2.15 1.6

coleoptile red grown 0.8 0.75

coleoptile dark grown 0.95 0.95

 

104
SAN 9789 does not affect the light scattering properties of
the plant. By subtraction of the optical density of an
excised coleoptile from an intact shoot, an estimate of the
attenuation of the primary leaf can be obtained. In control
plants, the primary leaf is 1.3 A, in SAN 9789 treated
plants, 0.75 A. Scattering properties of lwl and lw2
seedlings are comparable to control and SAN 9789 treated
hybrid seedlings (Figure 3.12).

Thus, it appears that scatter and absorption in corn
seedlings establishes a gradient of light for first positive
phototropism. The shoot tip as a whole is not sufficiently
transparent to allow a "lens effect". As in second positive
phototropism, light incident on a seedling will be
attenuated by absorption and scatter, giving a gradient with

the highest intensity on the proximal side of the plant.

 

CONCLUSIONS AND RECOMMENDATIONS

A method has been developed to monitor the light
attenuation and pigment distribution in individual
seedlings. Using this method, the distribution of bulk
pigments in corn seedlings from different genetic lines and
from different growth regimes has been mapped. Total light
attenuation by various tissues of the seedlings has also
been measured. The contributions of absorption and scatter
to the gradient of light for first positive phototropism
have been assessed.

The possibility that different photoreceptor pigments
or different mechanisms for establishing a gradient of light
are used in first positive and in second positive
phototropism has been raised (Poff, 1983). Evidence
presented in this thesis supports the hypothesis that there
is no such fundamental difference between first and second
positive phototropism (see Chapter 3). Instead, it appears
that carotenoids play a similar role in first positive to
that in second positive, and that the gradient for detecting
a unilateral light stimulus is established in the same way

for both responses.

105

106

Carotenoids act as screening pigments, rather than
photoreceptor pigments, in first positive phototropism.

SAN 9789 treated seedlings with an 80 - 90% reduction in
carotenoid content show only a 40% reduction in response.
The response of carotenoidless mutants is more variable, but
data presented here show that albino seedlings with a 70%
reduction in carotenoids show a 60% reduction in response.
Thus, it seems likely that the photoreceptor pigment remains
unaffected by manipulations of the carotenoid content. The
reduced response is due to a reduced gradient of light
across the coleoptile.

Attenuation is the mechanism used to establish the
gradient of light for first positive phototropism. Even the
most transparent part of a corn seedling, the apical 0.5 mm
of the shoot tip, is too dense to allow refraction to
produce a "focusing advantage". Instead, light incident on
a seedling is absorbed and scattered as it passes through
the tissue. "Piping" effects may modify this distribution.

Absorption by screening pigments contributes 20 - 25%
of the total gradient of light across the seedling
(approximately 0.5 A out of a total 2.0 - 2.5 A). The
remaining 75% is due to scatter. However, based on
experiments with SAN 9789 treated seedlings, it appears that
the 25% of the gradient established by carotenoid absorption
mediates about 40% of the response. This discrepancy raises
the possibility that absorption and scatter do not

contribute additively to the gradient of light. It is

107

possible that a threshold level of attenuation, established
by scatter, exists. If this is so, it should be possible to
find a very low intensity of light which evokes no
phototropic response, regardless of the length of time for
which it is presented.

Within a plant, the amount of light scattered is
intrinsic to the tissue, and is largely a function of
changes in refraction as light moves through cytoplasm,
vacuoles, and cell walls (Parsons gt 31., 1984). Changes in
pigment content, on the other hand, are a response to light
and are controlled by the metabolism of the plant. It is
possible that a seedling could use absorption as a method of
"fine tuning" to control its response or level of adaptation
to light.

Recent work on "light piping" (Mandoli and Briggs 1982a
and b, 1984) and on the distribution of light within plant
tissue (Seyfried and Fukshansky, 1983, Seyfried and Schafer,
1983, Parsons 33 31., 1984, Vogelman and Haupt, in press)
has led to consideration of light gradients along the long
axis of the shoot, as well as transverse light gradients.
Data presented here show that light is attenuated more
quickly when traveling transversely through shoot tissue
than when traveling longitudinally along the seedling.

Shoot tissue attenuates red light at a rate of 0.7 - 0.8 A
per cm in the longitudinal direction. Blue light is
attenuated more quickly. Because blue light is attenuated

so quickly, no measurement of longitudinal optical density

108
in the blue was made. Axially, shoot tissue is
approximately 6.5 - 7.5 A per cm in the red, 10 A per cm in
the blue. These values are an overestimate since they
include light losses at the surface of the tissue. Still,
shoot tissue attenuates light up to 10 times more per cm in
the transverse than in the longitudinal direction.

The contribution of absorption to total attenuation has
been measured in the transverse direction. Longitudinally,
it appears that absorption does not contribute to the
gradient of light; however, given the variability of the
data, a small difference due to absorption could have been
missed. Most absorption occurs in the primary leaves, which
have been shown to transmit little light longitudinally
(Mandoli and Briggs, 1982a). This could account for the
apparent lack of contribution of absorption in altering the
longitudinal transmission of light. It is also possible
that absorption contributes proportionately less to the
longitudinal than to the transverse gradient of light.

A normally pigmented control shoot is about 2.0 to
2.5 A in the blue. The range of the first positive
response, from threshold to indifferent or first negative,
is about two orders of magnitude. If there is a correlation
between light attenuation and the breadth of first positive
response, then plants which attenuate light differently
should show correspondingly different widths of doses for
first positive phototropism. Such a correlation could be

based on excitement of the photoreceptor pigment or another

109

component of the transduction chain. As dose increases, the
ratio of excited component in the front to that in the back
would increase until saturation was reached in the front
half. This would correspond to the peak of first positive
response, and could be expected to depend in some way on the
rate at which incident light is attenuated. As light dose
increases beyond the peak, the excited component in the
front half would remain saturated, but an increasing portion
of the component would be excited in the back half,
decreasing the gradient and the response. When all of the
component was excited, no phototropic response would be
seen. A first step in testing this model would be to look
at correlations between the width of the dosage range for
first positive phototropism and the total attenuation of
light by a variety of plants with different total
attenuations. If such a correlation were important, one
might expect SAN 9789 treated seedlings to show a narrower
peak of first positive response than control seedlings.
From data presented here, it appears that SAN 9789 treated
and control tissue show the same breadth of response.
SAN 9789 treatment alters only the height of the peak. An
experiment with phototropically responsive plants which
attenuate much more (or much less) light than control or
SAN 9789 treated corn is appropriate.

Further work with "carotenoidless" mutant tissue is
desirable. A better defined dose response curve and more

replications to minimize the effect of variable responses

 

110

are needed. Further comparisons between carotenoidless
seedlings generated from mutant stock and from SAN 9789
treatment would help to insure that the reduced response of
pale seedlings is a result of carotenoid concentration and
not some pleiotropic effect.

The difference between transverse and longitudinal
transmission of light could be carried out further. A first
step would be to measure acceptance losses from the surface
of a transversely illuminated seedling, or to eliminate such
losses from calculations of attenuation by measuring the
density of different thicknesses of tissue (similar to
Parsons E£.El° 1984).

To see whether cell elongation has an effect on
longitudinal transmission of light, transmission through
dwarf (compact cells), gibberellin treated (elongated cells)
and normal tissue could be compared. Such measurements
would also help define how much of the difference in
longitudinal and transverse light transmission is due to
cell geometry.

Changes in pigmentation and possible correlations to
phototropic capability could be monitored. Does the
pigmentation of a seedling change after exposure to a first
or second positive dose of light? Would such changes affect
its subsequent phototropic capability? A normal hybrid
population of corn seedlings varies both in pigmentation and
in phototrOpic response. How closely linked are these

variations? On a simpler level, it has been observed that

.111

completely etiolated dicots do not respond to phototropic
stimuli. Monitoring the synthesis of pigment and the
appearance of phototrOpic capability might help to establish
how large a gradient of light is necessary for phototropism
to occur.

Attenuation due to scatter and absorption is the source
of the gradient of light necessary for a plant to detect
unilateral light stimuli. This is true for both first and
second positive phototropism. Further studies of light
gradients, along with other components of the transduction
pathway, are necessary for a complete understanding of

phototropism.

APPENDICES

APPENDIX 1

Seed Multiplication of "Carotenoidless" Mutants

"Carotenoidless" mutant corn lines lwl, lw2, clp, and
w7748 show simple dominance. None of the four lines are
allelic (Robertson, personal communication).
Phenotypically, double recessive seeds have a white or pale
yellow endosperm, and germinate to give white seedlings
lacking much of the normal complement of carotenoids.
Two-thirds of the yellow seeds from these lines should be
heterozygous for the "carotenoidless" allele.

Approximately forty yellow seeds from each of the four
lines (generous gift of Dr. D.S. Robertson, Iowa State
University, Ames, Iowa) were planted on August 31 -
September 1, 1983. Each seed was planted in an 8-inch
diameter plastic pot in Sunshine Peat Complete mix
(micronutrients added to the peat), and placed in a
greenhouse. Daylight was supplemented by 1000 W metal
halide or 400 W high pressure sodium lamps. Light intensity
in the greenhouse in full sun, but away from any lamps, was

0.06 W x cm"2 x sec-1. Intensity about 30 cm below a metal

halide lamp was 0.07 W x cm.2 x sec-l, and

0.08 W x cm.2 x sec.1 below a sodium lamp. Intensities were

112

113

measured using a Kettering radiometer (model 7720, Yellow
Springs, Ohio). Daylength was 16 hours (6 am to 10 pm).
Temperature was maintained at or above 800 F (270 C). Plants
were watered daily with half-strength modified Hoagland's
solution from a dripline (see Table A1.1). Plants were
sprayed periodically with Pirimore to control aphids, and
with a mix of Paramex and Pentac to kill spider mites.

The first pollinations were performed November 3, 1983.
Tassels which were shedding pollen were covered with a paper
bag held in place with a paper clip. The husks surrounding
an ear were clipped just above the ear once silks had
appeared, and the ear covered with another paper bag.

Pollen shaken into tassel bags was poured directly on silks.
Pollinations were performed between 0800 and 0930, in order
to use the pollen shortly after it had been shed, since
pollen remains viable for only 10 - 30 minutes after
shedding (Neuffer, 1982). A given ear was pollinated on
several successive days, until silks began to dry. Crosses
were made over a period of three weeks. Plants were then
allowed to dry, and ears were harvested two to three weeks
after the end of pollination. Yields are summarized in
Table A1.2.

Whenever possible, plants were self-pollinated.
However, self-pollination was not always possible, either
because a plant was male-sterile, or because the tassel had
matured before silks appeared on the ear. If a plant had

receptive silks but no pollen, pollen from another plant of

the same line was

four lines, lwl,

114

used for fertilization. For three of the

lw2, and w7748, production of silk and

pollen were synchronous enough for several successive

fertilizations to
were very slow to
of pollen, excess
and high humidity
line was set from

impossible to say

be made. Plants of the fourth line, clp,

produce ears. To extend the availability

pollen was stored in sealed vials at 4° C

or in liquid nitrogen. No seed of the clp
either fresh or stored pollen, so it is

whether storage would have been useful.

 

Table Al.l. Modified half-strength Hoagland's solution

115

Light, personal communication).

Nutrient
Solution A (pH 4.5)

' 4H O

Ca(N03)2 2

NaFe, 13% or EDTA

Solution B (pH 3.5)

KHZPO4

KNO3

MgSO4 7H20

ZnSO4 7H20

MnSO4 H20

CuSO4 SHZO

(E.

grams per liter stock solution

295.0

38.44

34.25
126.65
126.65

0.056
0.391
0.021
0.725

0.005

 

Both stock solutions A and B were used at 1:500,

and the pH

of final solution adjusted to 6.0 - 6.4 using about 0.40 ml

1N KOH.

Table A1.2. Yield of greenhouse-grown "carotenoidless" corn

 

line total heterozygous number of
ears ears pale seeds
clp 0 0 0
w7748 4 2 27
lwl 8 3 45
lw2 11 8 134

 

Only seeds from lwl and lw2 were used in subsequent

experiments.

 

116

APPENDIX 2

Geotropism of Corn Seedlings at 60% and 100% Relative
Humidity

Humidity is important to a plant's response to
phototrOpic stimuli (Nathansohn and Pringsheim, 1908).
SAN 9789 treated plants, which wilt easily, seemed
especially sensitive to humidity. Table A2.1 presents the
results of a number of experiments which contrasted the

4M SAN 9789

response of control, 2 x 10-5M, and 2 x 10-
treated plants to 60% and 100% relative humidity. Based on
these results, subsequent experiments were performed in 100%

relative humidity (RH).

Table A-1 Humidity effects on control and SAN 9789 treated
corn seedlings. Means compared using Student's t test.

 

 

treatment mean curvature (degrees) df t
60% RH 100% RH
**
control 39.0 53.6 23 4.74
2 x 10'5M **
SAN 9789 35.7 55.1 5 3.65
2 x 10-4M **
SAN 9789 22.7 59.4 20 11.10
* **

, significant at 0.05 and 0.01 levels.

 

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