Kobe University Repository : Thesis
学位論文題目Tit le
Studies on primary react ion steps of light-induced anthocyaninsynthesis in Sorghum bicolor seedlings(ホウキモロコシ芽生えにおける光誘導アントシアニン生成の初期過程)
氏名Author Shichijo, Chizuko
専攻分野Degree 博士(理学)
学位授与の日付Date of Degree 1993-03-17
資源タイプResource Type Thesis or Dissertat ion / 学位論文
報告番号Report Number 乙1710
権利Rights
JaLCDOI 10.11501/3070675
URL http://www.lib.kobe-u.ac.jp/handle_kernel/D2001710※当コンテンツは神戸大学の学術成果です。無断複製・不正使用等を禁じます。著作権法で認められている範囲内で、適切にご利用ください。
PDF issue: 2020-03-01
Studies on Primary Reaction Steps of Light-induced
Anthocyanin Synthesis in Sorghum bicolor Seedlings
Studies on Primary Reaction Steps of Light-induced
Anthocyanin Synthesis in Sorghum bicolor Seedlings.
Studies on Primary Reaction Steps of Light-induced
Anthocyanin Synthesis in Sorghum bicolor Seedlings
Chizuko Shichijo
January, 1993
CONTENTS
Abbreviations
General Indroduction
Chapter I: Ultraviolet action spectra for the induction
and inhibition of anthocyanin synthesis in
broom sorghum seedlings.
Abstruct
Introduction
Materials and methods
Results
Discussion and conclusion
References
Chapter II: Enhancement of red light-induced anthocyanin
synthesis in sorghum first internodes by
moderate low temperatures given in the
pre-irradiation culture period.
Abstruct
Introduction
Materials and methods
Results
Discussion
References
1
4
5
8
12
22
25
28
29
31
37
52
59
Chapter III: Red light fluence-dependent responses to
pre-irradiation moderate low temperature of
phytochrome-mediated anthocyanin synthesis in
Sorghum bicolor first internodes.
Abstract
Introduction
Materials and methods
Results and discussion
Discussion and conclusion
References
Chapter IV: Storage of red light signal for anthocyanin
synthesis in etiolated Sorghum bicolor
seedlings
Abstract
Introduction
Materials and methods
Results
Discussion
References
General Discussion
63
65
68
73
94
100
105
106
108
110
126
134
138
Acknowledgments 140
i i
FR
HFR
HIR
LFR
LT
MLT
nUV-BL
Pfr
Pfr/Ptot
Pr
PrPfr, PfrPfr
R
a-'
UV
UV-A
UV-B
UV-C
VLFR
Abbreviations
far-red light
high fluence response
high irradiance response
low fluence response
low temperature
moderate low temperature
near-UV-blue light
far-red light-absorbing form of
phytochrome
ratio of Pfr to total phytochrome
red light-absorbing form of
phytochrome
phytochrome dimers
red-light
storage form of red light signal
ultraviolet light
320 - 400 nm ultraviolet light
280 - 320 nm ultraviolet light
ultraviolet light below 280 nm
very low fluence response
iii
General Introduction
Photomorphogenesis in higher plants is one of the
most attracting fields of research in plant physiology.
Plants are the main converter of the solar energy into
organic materials, and develop forms appropriate for this
purpose in their ontogenic processes, sensing the light
environment. This is the photomorphogenesis, which
usually include the synthesis of chlorophyll and
carotenoids as well as anthocyanin and other flavonoids.
To be interesting, photoreceptors for
photomorphogenesis are different from that for
photosynthesis. Thus far, phytochrome, near UV-blue light
photoreceptor,
occurrence has
and UV-B photoreceptor are known or their
been proposed. Besides, the actions of
UV-c which attacks DNA or RNA as a direct target are also
known. Of these photoreceptors some exert coactions, e.g.
phytochrome and near UV-blue photoreceptor, phytochrome
and UV-B photoreceptor in anthocyanin and other flavonoid
synthesis, grown inhibition etc.
The chromophore of phytochrome was almost clarified
for the Pr form as well as Pfr form, and the protein
moiety is bring sequenced. The chemical entity of near
UV-blue photoreceptor has been a subject of debate,
whether carotenoid or ribof lavin, for nearly half
century, and data favouring flavoprotein are now
accumulating, although the possibility of carotenoids has
not completely excluded. Recently pterin compounds have
been also proposed as near UV-blue phtoreceptor
(Ninnemann 1984), and some supportig evidence has been
obtained. However, no mechanism of coaction was even
touched with.
The photomorphogenetic final manifestation of red
light actions was almost fully descr ibed, and a great
deal of work is currently being devoted to the
elucidation of gene expression, at the level of
transcription and translation. Some work was also
directed to the possible change of the permeability of
the plasmalemma (Marme 1977), and the light-induced
increase of Ca 2+ uptake attracted the interest of workers
in the field (Tretyn et al. 1991). Also, as a rapid
reaction, red light-induced pelletability of phytochrome
was studied strenuously (Quail 1983), and it was
interpreted to be associated with pfr-mediated
phosphorylation of phytochrome molecule (Hofmann et. al
1991) However, our knowledge on the processes of red
light signal transmission is very poor.
The present thesis concerns physiological studies on
photomorphogenesis using as the index of light effects
UV-B- and red light-induced anthocyanin synthesis in
etiolated sorghum seeedlings (two cultivars of Sorghum
bicolor Moench). This plant forms a single chemical
species of anthocyanin in the epidermis of the first
internode in response to light, thus minimizing possible
interference by the absorption of light by other cell
layers. Either UV-B or red light given in a single short
pulse is effective, and further, both exert their actions
in a multiplicative way, allowing to assume that the
pathway of anthocyanin synthesis is one and the same for
2
both kinds of light. These properties of the plant makes
it most sui table to analyze the pr imary steps of light
signal transduction.
Chapter 1 will present an UV action spectrum for
anthocyanin synthesis, which confirms the presence of
UV-B photoreceptor distinct from the UV target such as
DNA or RNA. Chapter 2 to 4 will deal with a part of the
process cons ide red to be involved in the primary
reactions of red light signal transmission from
phytochrome to the cell nucleus; chapter 2 will describe
on the presence of a phytochrome-specif ic step which is
amplif ied by moderate low temperature exper ienced during
the growth period before irradiation, chapter 3 will
describe on the presence of very low-f luence as well as
high-fluence responses in red light-induced anthocyanin
synthesis. Finally chapter 4 will describe on the
possible presence of a storage of red light signal other
than Pfr.
References Hofmann, E., Grimm, R., Harter, K., Speth, V., Schafer,
E. (1991) Partial purification of sequestered particles of phytochrome from oat (Avena sativa L.) seedlings. Planta, 183, 265-273.
Marme, D. (1977) Phytochrome: membranes as possible sites of primary action. Ann. Rev. Plant Physiol. 28, 173-198.
Ninnemann, H. (1984) The nitrate reductase system. In: Blue Light Effects in Biological Systems, pp.95-109, Senger, H. ed. Springer-Verlag, Berlin, Heidelberg.
Quail, P.H. (1983) Rapid cation of phytochrome in photomorphogenesis. In: Encyclopedia of Plant physiology, new series 16A Photomorphogenesis, pp.178-212, Shropshire, W. Jr., Mohr, H. eds. Springer-verlag, Berlin.
Tretyn, A., Kendrick, R.E., Wagner, G. (1991) The role(s) of calcium ions in phytochrome action. Photochem. Photobiol. 54, 1135-1155.
3
Chapter I: Ul trav iolet action spectra for the induction and
inhibition of anthocyanin synthesis in broom sorghum
seedlings
Abstract
In the first internode of dark-grown seedlings of broom
sorghum (Sorghum bicolor Moench) UV-B induced anthocyanin
synthesis at low fluences, but inhibited it at high fluences.
Action spectra for the two UV effects were determined so that
the inf luence of one on the other was reduced as much as
possible. The action spectrum for inhibition showed a peak at
or below 260 nm and a shoulder at about 280nm, whereas that
for induction had a peak at about 293 nm, confirming a
previous result. Wavelengths from about 300 to 350 nm were
inductive only and had no inhibitory effect at higher
fluences. Inhibition was observable irrespective of
irradiation sequence, and was photoreactivatable. It was,
thus, concluded that the UV-B photoreceptor proposed for
induction was different from that proposed for inhibition of
anthocyanin synthesis, the latter being presumably the same
as the target of other putatively harmful UV actions.
4
Introduction
The importance of UV-B in the photomorphogenesis of
plants has been demonstrated in addition to the harmful
effects of this radiation (Hashimoto et al. 1984, Hashimoto
and Tajima 1980, Steinmetz and Wellmann 1986, Wellmann 1983).
In the synthesis of anthocyanin and other flavonoids the
involvement of a UV-B photoreceptor with an action maximum at
about 290 nm has been indicated by studies with cut-off
filters, by action spectra, and by coaction with red and blue
light (Arthur 1932, Beggs and Wellmann 1985, Drumm-Herrel and
Mohr 1981, Wellmann 1971, Yatsuhashi et al. 1982). The UV-B
photoreceptors work alone, synergistically with phytochrome
(Beggs and Wellmann 1985, Arakawa 1988, Yatsuhash and
Hashimoto 1985), or enables phytochrome to work (Drumm-Herrel
and Mohr 1981, Wellmann 1971, Beggs et al. 1986) in the
synthesis of anthocyanin or flavonoids.
Synthesis of isoflavonoid derivatives such as resveratrol
(Fritzemeier et ale 1983, Langcake and Pryce 1977) and
coumestrol (Beggs 1985) is induced by UV-B and UV-C. Action
spectra have peaks between 260 and 270 nm and differ from
those for anthocyanin synthesis, but are rather similar to
those for the inhibition of anthocyanin accumulation
(Wellmann 1984). Pisatin, an isoflavonoid derivative, is also
synthesized as a result of exposure to 254 nm light, but not
to 366 nm light (Hadwiger and Jafri 1974, Hadwiger and
Schwochau 1971). The UV actions are reversed by near-UV-blue
light (nUV-BL)(photoreactivation)(Beggs et ale 1985, Hadwiger
and Schwochau 1971). These compounds are all synthesized in
5
plants from pheny lpropanoids, precursors common to those of
anthocyanin and other f lavonoids. Synthesis of the enzymes,
phenylalanine ammonia-lyase and cinnamate 4-hydroxylase,
involved in the phenylpropanoid pathway has been shown to be
induced by UV (Hadwiger and Schwochau 1971, Fritzemeier and
Kindl 1981), as is flavonoid synthesis itself (see, for
example, ref. Beggs et al. 1986).
Ultraviolet light of wavelengths such as 260 and 270 nm
also suppresses anthocyanin synthesis at high fluences.
Wellmann et al. (1984) have obtained a UV action spectrum for
inhibition of red light-induced anthocyanin synthesis in
Sinapis alba. This action spectrum has a peak at or below 260
nm.
These findings indicate that action spectra reported thus
far for the induction of anthocyanin synthesis might be
shifted toward longer wavelengths owing to a complicated
inhibitory effect at shorter wavelengths. Furthermore, it is
suspected that the same photoreceptor might be involved in
both induction and inhibition, acting inductively at low
fluences and inhibitorily at high fluences.
The primary aims of the present study are to test the
above possibility and to determine whether a UV-B
photoreceptor suggested as being involved in
induction is different from the target of UV
action spectrum for induction of anthocyanin
re-examined in a different way to minimize
anthocyanin
damage. The
synthesis is
the possible
inhibitory action of UV. Secondly, an action spectrum for
inhibition of
compared with
anthocyanin synthesis
that for induction. Thus
6
is determined and
far, no study is
available to determine action spectra for both induction and
inhibition of anthocyanin or other flavonoid synthesis with
the same plant material, which is essential for a rigorous
comparison.
7
Materials and methods
Plant material, irradiation and determination of
anthocyanin. Tow cultivars of Broom sorghum (Sorghum
bicolor Moench, cvs. Sekishokuzairai Fukuyama and Acme
Broomcorn) were used without distinction between the two,
since both cultivars responded similarly to light in
anthocyanin synthesis (Yatsuhashi et ale 1982). Seeds
were soaked in running tap water, and germinated on paper
tissues. Seedlings were selected for uniformity (under
room light from white fluorescent tubes), transferred to
plastic boxes, 15 cmX5 cm Xl0 cm (height) with wet
vermiculi te, and grown in total darkness for 3 days at
24.0 + O. 5°C until the seedlings were 7 cm tall, after
which they were irradiated. Irradiation was performed
from the horizontal direction. Twenty four hours later a
part of the first internodes, 20-55 mm below the
coleoptilar node, accumulates anthocyanin. This part was
excised, including the marginal zones, and extracted with
1% hydrochloric acid methanol of (volume 0.2 ml per
segement). The anthocyanin content was determined by the
absorbance difference between 528 and 650 nm. Details of
these procedures are given elsewhere (Yatsuhashi et al.
1982, Yatsuhashi and Hashimoto 1985).
Light sources and determination of fluences rates.
Monochromatic light, 6 nm in bandwidth was provided with
the Large Spectrograph at the National Institute for
Basic Biology, Okazaki, Japan (Watanabe et ale 1982). Red
8
light (R) was obtained from purple fluorescent tubes
(Fishlux, FL20S BRF, Toshiba, Ltd., Tokyo) wrapped with
red polyacrylic resin film (CF109-3, Mitsubishi Rayon,
Ltd., Tokyo) (Yatsuhashi et al. 1982). Near-UV-blue light
(nUV-BL) was obtained from a blue fluorescent tube
(FL20B, Toshiba, Ltd.). This light had an emission
maximum at 410 nm and was half maximal at 350 and 480 nm.
Combined UV and R (induction light A) was provided from
a pane I with two UV31 0 -0 tubes and two red light tubes
(described above) positioned alternately. UV310-0 was a
UV-B source, a UV fluorescent tube (specially
manufactured by Hitachi Ltd.) wrapped with polyvinyl
chloride film, which cut off the wavelengths shorter than
290 nm. The spectral energy distribution of this light
source has been reported as 310-0 previously (Hashimoto
and Tajima 1980).
Photon f luence rates were determined with a photon
density Meter (Model HK-1, Riken, Saitama) (Hashimoto et
al. 1982). The flat-faced sensor of this meter has a
wavelength- dependent sensitivity, and hence the meter
has been designed to correct by factors specific to each
wavelength. When the fluence rates of the combined UV and
R were measured, the correction factor for 660 nm was
used, and, therefore, the fluence rates obtained were
only relative values.
9
Determination of action spectra. In the wavebands where
action spectra are determined, both
inhibition of anthocyanin synthesis are
action spectrum for induction is to
minimizing a possible interference of
induction and
involved. If an
be determined,
the inhibitory
action, two measures were adopted. First, irradiation
with monochromatic UV was followed by an irradiation with
a fixed amount of R. This is based on the finding that
since UV-B and R multiplicatively act in inducing
anthocyanin synthesis (Yatsuhashi and Hashimoto 1985,
Hashimoto and Yatsuhashi 1984), weaker UV, which is less
inhibitory, may suff ice for inducing detectable amounts
of anthocyanin when combined with red light. Secondly, a
fluence rate-response curve for a wavelength where no
inhibitory action of light was considered to be involved,
e.g. 308 nm (Fig. 1-1), was taken as standard curve.
Lines parallel to the standard curve were drawn from
datum points to the line of null action (anthocyanin
levels induced by the respective red light alone), the
fluence rates at the intersections (threshold fluence
rates) were read , multiplied by the irradiation period
and the reciprocals of the products were plotted to
construct an action spectrum. This is justif ied by the
notion that f luence rate-response curves in a log plot
should be parallel, as long as the same photoreceptor is
concerned (see ref. Yatsuhashi and hashimoto 1985).
Threshold fluence rates have previously been used by
Shropshire and Withrow (1958).
For an action spectrum of inhibition, anthocyanin
10
synthesis was
dose) with
induced by a 50
combined UV and
s irradiation
R (induction
(suboptimal
light A),
followed by another 50 s irradiation for inhibition with
monochromatic UV of various wavelengths at various
f luence rates. From the f luence rate-response data
obtained, the threshold fluence rates were read using a
curve for 267 nm as the standard, and processed in the
same way as for the induction spectrum.
11
Results
with
When dark-grown sorghum seedlings were irradiated
pulses of monochromatic UV in the UV-B and UV-C
regions, anthocyanin synthesis was induced and maximal
anthocyanin was found at ca. 100 pmol m-2 (Fig. 1-2). The
curves indicate that the UV action consists of both
induction and inhibition.
Action spectrum for anthocyanin synthesis induction.
Since UV-B
anthocyanin
Hashimoto
and R multiplicatively
synthesis (Yatsuhashi and
and Yatsuhashi 1984),
act in inducing
Hashimoto 1985,
irradiation with
monochromatic UV was followed by irradiation with two
levels of R, and the fluence rates-response curves shown
in Fig. 1-1 were obtained. As seen in Fig. 1-3, 307 nm
light was not inhibiory even at as high a fluence rate as
30 pmol m-2 S-l, nor was it effective in converting
phytochrome (Yatsuhashi et al. 1982, Yatsuhashi and
Hashimoto 1985). This is also true for 303 nm. Hence, the
slope of the curve is postulated to be intrinsic to the
UV-B photoreceptor. The curves for 308 nm in Fig. 1-1
were taken as the standards, threshold fluence rates were
read, and an action spectrum was constructed. At two
different levels of R combined with the monochromatic UV,
virtually identical spectra were obtained (Fig. 1-4).
Action spectrum for the inhibition of anthocyanin
synthesis. Figure 1-3 shows UV fluence rate-response data
12
C)
'" .. <t
... '" '" 't
0.3
o. I
272nm ~
~o 0---- .0 __ ---0
0.03
0.3
o. I
0.03
0.3
0.1
0.03
10
.~. . ..--o-~
278
292 • ___ .-----/ ,//.----~~ ....
o _____ a ..0'
-- 0-- ......
R alone
100 10 100
Pholan Fluence ROle ( n mol m-Z 5-1 )
10 0.000)
100 00,000)
Fig. 1-1. Fluence rate--response relations for the induction
of anthocyanin synthesis in the first internode of broom
sorghum (Sekishokuzairai Fukuyama). Induced by monochromatic
UV followed by R of 1. a (broken line) and 7. a (solid line)
}lmol m 2 irradiation period, 100 s each. Horizontal
short lines on the left in each figure indicate the level by
R alone. Fluence rates in parentheses are for R alone.
13
0.03
I:::) 0.06 ~ ~
~ 0.04 t\i Il)
~
0.02 0
a 5 10 50 100 500
FLUENCE (proal M-2 )
Fig. 1-2. Induction and inhibition of anthocyanin synthesis
by low and high f luence UV in the first internode of broom
sorghum (Sekishokuzairai Fukuyama). UV of 257, 263, 277 and
283 nm, respectively at 0.276, 0.433, 0.616 and 0.749 pmol
m- 2 5-1 was given for 21, 60, 180 and 600 s. UV supplied with
the large spectrograph.
14
1.5
1.0
'-'" 0.5 '" "I:
o
, .. " ,. • r Z~J
.! .J
Z 7'.J I •• .!
2"7', ,,! I
211J ' .•. 1
303 '307
'-"'-' ~, u.' '..wol,------'----'~, J I 7
,,-,-' ...... ' 'u" ...:,_--'----'-~, J I J
'----'---'-_'-' .~.~. ,-.1.1_-11 2 g ,.
. . , '-----'----'--'---'--"-''''''' J....I _-'-~, 29 J
.. '" ,---,--,---,-....L' -'-, -'-, LL' 'J....' _-'-~, 2 9 7'
' . • 1 JOJ
5 10 I 5 10 Fluenc, ror! (Jl mor m- 2 s-r I
Fig. 1-3. Fluence rate-response relations for the inhibition
of anthocyanin synthesis in the first internode of broom
sorghum (Sekishokuzairai Fukuyama). Anthocyanin synthesis was
induced by induction light A I 38 pmol m-2 s -1 (uncorrected) I
then monochromatic UV from the spectrograph was given.
1rradia- tion, both 50 s each. The scale of abscissa is
shifted for each wavelength. Triangle points are values when
the sequence of irradiations was reversed.
15
z o r u
c::C
UJ > r :5 UJ
a::::
10
5
o 270 280
• • o
290 300
HAVELENGTH ( Nt" )
310
Fig. 1-4. Action spectrum for induction of anthocyanin
synthesis in the first internode of broom sorghum
(Sekishokuzairai Fukuyama) in the presence of R of 1.0 (solid
circles) and 7.0 (open circles) }lmol m 2 S-I. The points are
derived from those in Fig. I-i. The values of the solid
circles are normalized to the average of the open circles at
293 nm by a factor of 1.32. The curve represents the means of
both kinds of points; the short bars are standard errors.
16
which were obtained when a pulse irradiation with
induction light A was followed by a pulse irradiation
with monochromatic UV of various wavelengths. Inhibition
of anthocyanin synthesis was found at most wavelengths,
but at 303 nm and longer wavelengths no inhibition was
observed up to as high a f luence rate as 30 jlmol m-2 S-l.
At intermediate fluence rates of some wavelengths
promotion was found. This outcome is assumed to indicate
that the UV component of induction light A was below
saturation.
When the irradiation sequence of induction light A
and the monochromatic light was reversed, almost no
difference was observed (triangles in Fig. 1-3).
Taking the f luence rate-response curve at 267 nm as
the standard, threshold f luence rates were read in Fig.
1-3, and an action spectrum for the inhibition of
anthocyanin synthesis was constructed (Fig. 1-5). This
spectrum was confirmed by three independent experiments.
Photoreactivation of inhibition of anthocyanin synthesis.
The action spectrum for anthocyanin inhibition (Fig. 1-5)
suggests that DNA damage may be involved. Hence, the
effect of a broad band of nUV-BL was tested to see
whether it causes photoreactivation (Senger 1984) of the
UV inhibition of anthocyanin synthesis.
that nUV-BL partially reversed the
Figure 1-6 shows
inhibition of
anthocyanin synthesis caused by 263 nm light, supporting
the above suggestion.
17
z 0
f-u
c:t:
w > f-
:J LU
0:::
1.5
1.0
0.5
o 250
•
0
• •
2GO 270 280 290 300 310 WAVELENGTH ( NM )
Fig. 1-5. Action spectrum for UV-induced inhibition of antho-
cyan in synthesis in the first internode of broom sorghum
(Sekishokuzairai Fukuyama). Points are derived from those in
Fig. 1-3.
18
TREAH1ENTS 0
NONE A NUV
A-NUV A-L263
A527-A650
0,5 I
+
]-
+
1,0 I
+
+
Fig. 1-6. Inhibition of anthocyanin synthesis by 263 nm light
and its partial reversal by near UV-blue light in -the first
internode of sorghum (Acme Broomcorn). A, induction light A,
35 pmal m 2 c' .> (uncorrected); nUV , near-UV-blue light
(nUV-BL) I 60 pmal m 2 s \; L263 , 263 nm light, 6.1 pmol m- 2
51, obtained from the spectrograph. Irradiations, 50 s each.
Values are the means of quadruplicate experiments with the
syandard errors.
19
Identity of photoreceptors for 305 and 293 nm light
re levant to anthocyanin induction. In Fig. 1-3, 293 nm
light was inhibitory at high fluence rates, whereas light
of 303 nm and of longer wavelengths was not. This finding
indicates that the two wavelengths might be absorbed by
different photoreceptors. To determine whether this is
the case, the effects of 293 nm light at various fluence
rates on anthocyanin synthesis were tested in combination
with phytochrome-saturating R or with
phytochrome-saturating Rand 305 nm light of an intensity
suff icient for anthocyanin synthesis. The results (F ig.
1-7) indicate that the addition of 293 nm light did not
influence the anthocyanin level produced by a combination
of 305 nm light and R. As far as anthocyanin synthesis
induction is concerned, this possibility of different
photoreceptors is excluded. If different photoreceptors
were involved, the addition of 293 nm light should alter
the effect of L305 + R in Fig. 1-7. The action of Lm +
R did not reach the levels of actions of L 305 + R or Lm
+ L~5 + R. This result may be ascribed to an inhibitory
action of Lm at 2 pmol m-2 S-1 and higher f luence
rates.
20
305-H
1.0
R
o
• . ------.--------~:----------~.~------:
L293 + H I I
-----0-
0.05 0.1
.. 0 --I
I
I I
I I
I I
0.5
I
I I
I
?
•
.... 0 ..... .........
1.0
Fig. 1-7. Effects of 293 nm light on anthocyanin synthesis of
Acme Broomcorn induced by a combination of Rand 305 nm
light. The test of 293 nm light combined with R (broken line)
is the control. Two sets of four bars on the left ends of the
curves indicate the level without Lm. Irradiations were
performed in the sequence of 293 nm light (fluence rates
indicated on abscissa), 305 nm light (35 pmol m 2 SI) and R
(24 pmol m-2 S-I), or of 293 nm light and R, 50 s each.
21
Discussion and conclusion
Since UV-B and UV-C inhibit as well as induce
anthocyanin synthesis in sorghum
for induction
seedlings (Fig.
and inhibition
1-2),
were action spectra
determined in
interferences of
a way
the
to minimize the possible
respective counteractions (see
Materials and methods).
Figure 1-4, the action spectrum for induction, has
several scattered points for each wavelength. The
deviations of the points may
addition to the fluctuation of
involve inhibition in
the data. The point
involving no inhibition (probably obtained at low fluence
rates) should come higher than those hav ing inhibition
(probably obtained at high fluence rates). Considering
only the highest points at each wavelength, none of the
points is found to be higher than that for 293 nm, the
action peak. This implies that the true action peak is
not at shorter wavelengths than 293 nm, and Fig. 1-4 is
considered to represent the most plausible action
spectrum. This action spectrum, which was determined in
the presence of red light, is virtually the same as that
previously measured solely with monochromatic UV followed
by far-red irradiation in the same plant (Yatsuhashi et
al. 1982), and also agrees with Beggs and Wellmann's
observation with maize coleoptiles (1985) that
anthocyanin was synthesized most abundantly at 290 nm.
The action spectrum for anthocyanin inhibition (Fig.
1-5) was almost identical with that obtained with the UV
inhibition of red light- induced anthocyanin synthes is in
22
Sinapis alba (Wellmann 1984). It also agreed with the
action spectrum for coiling of the first internode of
sorghum (Hashimoto et al. 1984) and with the generalized
action spectrum for the harmful effects of UV, which
Coohill (1989) compiled from experiments on other
phenomena.
The action spectra determined in the present study
clearly indicate that the induction and inhibition of
UV-induced anthocyanin synthesis are mediated by two
different photoreceptors. The anthocyanin inhibition by
short-wavelength UV did not change whether the
irradiation for inhibition was given before or after the
induction irradiation (triangles in Fig. 1-3). Thus, it
is suggested that the induction and the inhibition are
independent processes at least at early steps of the
phototransduction process.
The results in the present paper verified the view
that a UV-B photoreceptor is involved in anthocyanin
induction (Hashimoto and Tajima 1980, Wellmann 1983,
Drumm-Herrel and Mohr 1981, Wellmann 1971, Beggs et al.
1986, Hashimoto and Yatsuhashi 1984). The absorption band
of the photoreceptor is considered to have a peak at
about 290 nm, which falls slowly toward 270 nm and
sharply toward 310 nm (ref. Yatsuhashi et al. 1982, Fig.
4). Its upper limit is at about 350 nm, as seen from the
waveband where the coaction with phytochrome occurs
(Yatsuhashi and Hashimoto 1985). This excludes the
possibility of the UV-B photoreceptor being a near
UV-blue light photoreceptor (see ref. Senger 1984).
23
The action spectrum for anthocyanin inhibition and
the photoreactivation
I-6), leads us to the
et al. (1984) that
by nUV-BL, though slight (Fig.
same conclusion drawn by Wellmann
the inhibitory action at short
wavelengths of UV in anthocyanin synthesis may be a
result of some DNA damage (Sutherland 1981).
When we consider the effects on the synthesis of
anthocyan ins and flavonoids of the solar UV reaching the
Earth's surface, the difference (roughly 300- 350 nm)
between the longer wavelength end of the action spectrum
for anthocyanin induction and that for inhibition may
have a significance. The solar UV on the Earth's surface
sharply declines to almost zero at 295 nm (Baker et al.
1980), and is considered to act mainly to induce the
synthesis of anthocyan ins or f lavonoids, as long as the
intensity of sunlight is not very strong or the exposure
does not continue very long. In a natural habitat,
seedlings would be exposed to weakened sunlight when they
emerge from the soil, and would synthesize anthocyan ins
or flavonoids that would serve a protective role when
exposed to full sunlight.
In the present paper we have demonstrated that a UV-B
photoreceptor proposed for anthocyanin induction and
other photomorphogenic UV actions exists
DNA, a target of harmful UV. The
in addition to
waveband
approximately 300 to 350 nm is where the
from
UV-B
photoreceptor exerts beneficial actions in response to
UV, keeping out of harmful UV actions such as DNA damage
in this plant.
24
References
Arakawa, o. (1988) Photoregulation of anthocyanin synthesis in
apple fruit under UV-B and red light. Plant Cell Physiol. 29,
1385-1389.
Arthur, J.M. (1932) Red pigment production in apples by means of
artificial light sources. Contr. Boyce Thompson Inst. 4, 1-18.
Baker, K.S., Smith, R.C. and Green, E.S. (1980) Middle ultraviolet
radiation reaching the ocean surface. Photochem. Photobiol. 32,
367-374.
Beggs, C.J., Stolzer-Jehle, A. and Wellmann, E. (1985) Isoflavo
noid formation as an indicator of UV stress in bean (Phaseolus
vulgaris L.) leaves. Plant Physiol. 79, 630-634.
Beggs, C.J. and Wellmann, E. (1985) Analysis of light-controlled
anthocyanin formation in coleoptiles of Zea mays L.: The role of
UV-B, blue, red and far-red light. Photochem. Photobiol. 41 481
-486.
Beggs, C.J., Wellmann, E. and Grisebach, H. (1986) Photocontrol of
flavonoid biosynthesis, In: Photomorphogenesis in Plants,
pp. 467-499, Kendrick, R.E. and Kronenberg, G.H.M. eds. Martinus
Nijhoff, Dordrecht.
Coohill, T.P. (1989) Ultraviolet action spectra (280 to 380 nm)
and solar effectiveness spectra for higher plants. Photochem.
Photobiol. 50, 451-457.
Drumm-Herrel, H. and Mohr, H. (1981) A novel effect of UV-B in a
higher plant (Sorghum vulgare). Photochem. Photobiol. 33, 391-
398.
Fritzemeier, K.-H. and Kindl, H. (1981) Coordinate induction by UV
light of stilbene synthase, phenylalanine ammonia-lyase and
25
cinnamate 4-hydroxylase In leaves of Vitaceae. Planta 151, 48-
52.
Fritzemeier, K.-H., Rolfs, C.-H., Pfau J. and Kindl, H. (1983)
Action of ultraviolet-C on stilbene formation in callus of
Arachis hypogaea. Planta 159, 25-29.
Hadwiger, L.A. and Schwochau, M.E. (1971) Ultraviolet light
induced formation of pisatin and phenylalanine ammonia-lyase.
Plant Physiol. 47, 588-590.
Hadwiger, L.A., Jafri, A., von Broembsen S. and Eddy, R. Jr. (1974)
Mode of pisatin induction. Plant Physiol. 53, 52-63.
Hashimoto, T. and Tajima, M. (1980) Effects of ultraviolet
irradiation on growth and pigmentation in seedlins. Plant Cell
Physiol. 21, 1559-1571.
Hashimoto, T., Yatsuhashi, H. and Kato, H. (1982) Self-corrected,
convertible handy irradiance meter with a photodiode. Abstracts
Ann. Meeting, Jap. Soc. Plant Physiol. p. 38.
Hashimoto, T., Ito S. and Yatsuhashi, H. (1984) Ultraviolet light
induced coiling and curvature of broom sorghum first internodes.
Physiol. Plant. 61, 1-7.
Hashimoto T. and Yatsuhashi, H. (1984) Ultraviolet photoreceptors
and their interaction in broom sorghum - Analysis of action
spectra and fluence-response curves. In: Blue Light Effects in
Biological Systems, pp. 125-136, Senger, H. ed. Springer-Verlag,
Berlin, Heidelberg.
Langcake P. and Pryce, R.J. (1977) The production of resveratrol
and the viniferins by grapevines in response to ultraviolet
irradiation. Phytochemistry 16, 1193-1196.
Senger, H. (1984) Cryptochrome, some terminological thoughts. In:
Blue Light Effects in Biological Systems, p. 72, Senger, H. ed.
26
Springer-Verlag, Berlin, Heidelberg.
Shropshire, W. Jr. and Withrow, R.B. (1958) Action spectrum of
phototropic tip-curvature of Avena. Plant Physiol. 33, 360-365.
Steinmetz, V. and Wellmann, E. (1986) The role of solar UV-B in
growth regulation of cress (Lepidium sativum L.) seedlings.
Photochem. Photobiol. 43, 189-193.
Sutherland, B.M. (1981) Photoreactivation. BioScience, 31, 434-444.
Watanabe, M., Furuya, M., Miyoshi, Y., Inoue, Y., Iwahashi, I. and
Matsumoto, K. (1982) Design and performance of the Okazaki Large
Spectrograph for photobiological research. Photochem. Photobiol.
36, 491-498.
Wellmann, E. (1971) Phytochrome-mediated flavone glycoside synthe
sis in cell suspension cultures of Petroselinum hortense after
preirradiation with ultraviolet light. Planta 101, 283-286.
Wellmann, E. (1983) UV radiation in photomorphogenesis.
In: Encyclopedia of Plant Physiology, NS, Vol 16B, pp. 745-756.
Shropshire, W. Jr. and Mohr, H. ed., Springer-Verlag, Berlin.
Wellmann, E., Schneider-Ziebert, U. and Beggs, C.J. (1984) UV-B
inhibition of phytochrome-mediated anthocyanin formation in
Sinapis alba L. cotyledons. Plant Physiol. 75, 997-1000.
Yatsuhashi, H., Hashimoto, T. and Shimizu, S. (1982) Ultraviolet
action spectrum for anthocyanin formation in broom sorghum first
internodes. Plant Physiol. 70, 735-741.
Yatsuhashi, H. and Hashimoto, T. (1985) Multiplicative action of
a UV-B photoreceptor and phytochrome in anthocyanin synthesis.
Photochem. Photobiol. 41, 673-680.
27
Chapter II: Enhancement of red light-induced anthocyanin
synthesis in sorghum first internodes by moderate low
temperatures given in the pre-irradiation culture period.
Abstract
Anthocyanin synthesis in the broom sorghum, Sorghum
bicolor Moench cvs. Acme Broomcorn and Sekishokuzairai
Fukuyama, is mediated separate ly or synergistically by an
ultraviolet light-B (UV-B) photoreceptor and phytochrome.
When seedlings were exposed to moderate low temperatures
(MLT) ranging from 12 to 20°C before irradiation, only
phytochrome-mediated anthocyanin synthesis was markedly
enhanced compared with the control kept throughout at 24°C,
but UV-B photoreceptor-mediated one was not. The
effectiveness of an exposure to 20°C increased as the
duration of exposure increased up to 24 h and as the time
of exposure was closer to the time of irradiation. When
seedlings were exposed to 20°C after irradiation till
harvest, by contrast, both anthocyanin syntheses induced by
UV-B and red light were equally suppressed, probably due to
the general reduction of metabolism involved in anthocyanin
synthesis that is a consequence of lower temperature. The
results support the view that the signal transduction of
the phytochrome system is different from that of the UV-B
photoreceptor, and suggest that the phytochrome system may
involve a step or steps which are amplified by a previous
exposure to the moderate low temperatures.
28
Introduction
Discovery is often made by unexpected encounters with
seemingly unfavourable results. The possible amplif ication
of phytochrome signals by moderate low temperatures (MLT)
for anthocyanin synthesis in sorghum was discovered by
examining the cause of fluctuation of data. Fluctuation of
data occurred by an non-uniformity of temperature in the
dark room, and the variation was found only in red
light-induced but not UV-induced anthocyanin synthesis.
Promotion by low temperature of anthocyanin synthesis
and other light-induced responses has been reported.
Anthocyanin accumulation in apple skin (Faragher 1983) and
in barley seedlings (Martinez and Favret 1990) increased at
low or moderate low temperature compared with the normal
temperature. The activity of nitrate reductase increased
several fold in white light-grown Sinapis alba seedlings at
10° to 15°C compared with seedlings at 20°C (Moroz et al.
1984). Red light growth inhibition in Sinapis alba also
increased at 15°C (Wall and Johnson 1982). However, these
investigations have not shown whether the low temperatures
to which plants were exposed influence signal transduction
systems of light or physiological reactions themselves
directly involved in the expression of the light effects,
e. g., synthesis of anthocyanin or of related enzymes, and
growth process.
Effects of low temperature on seed germination have
been most extensively studied (VanDerWoude and Toole 1980,
Frankland and Taylorson 1983, Cone and Kendrick 1986).
29
Based on the sensitization effects of low temperature,
VanDerWoude (1985, 1987) has proposed a very elegant,
satisfactory dimeric model on phytochrome action. Even in
the case of germination, however, the possibility that low
temperature might act on the germinabili ty of seeds rather
than on the affinity of phytochrome dimer to its signal
transduction chain has not completely excluded, although it
is very low. Anthocyanin synthesis of Sorghum bicolor
responds to a pulse of red light (R) and UV-B, and these
two parameters and MLT are considered to offer a good tool
to analyze the mechanism of the light actions. The primary
aims of the present paper are 1) to describe the general
characteristic of MLT effects, 2) to show the possible site
of MLT action in the light signal transduction chains
through their expression steps, 3) to show that the signal
transduction chain of phytochrome is distinct from that of
a UV-B phtoreceptor at least at the early steps, 4) to show
that MLT effects are distinct from low temperature (LT)
effects.
30
Materials and methods
Plant materials. Use was made of Broom sorghum (Sorghum
bicolor Moench, cv. Acme Broomcorn), 1987 crop at the
experimental farm, the Aburahi Laboratories, Shionogi
Pharmaceutical Co. Aburahi, Shiga and 1991 crop at the
Experimental Farm of the Faculty of Agriculture, Kobe
University, Kasai, Hyogo and cv. Sekishokuzairai- Fukuyama,
1986 crop at the Aburahi Laboratories.
Since soaking temperature inf luenced results, seeds
were soaked for 24 h at 24 ± lDC throughout, unless
otherwise stated. For this purpose the following three
methods were adopted. Method 1; seeds in a plastic bucket
were supplied with 24 DC-tap water at a ratio of one litre
per 10 g seeds, and were stirred with a magnetic stirrer in
the 24 DC-room. During the soaking period of 24 h, water was
renewed 2 to 3 times. Method 2 • I seeds were placed in a
stainless steel net crate which was shaken in a water bath
with a shaker at a frequency of ca. 60 min- 1• Fresh tap
water was supplied continuously at a rate of one exchange
(16 1) per 17 min. Method 3; seeds were placed in a
open-topped round plastic container having a water inlet at
the corner of the bottom, and the container was submerged
in a water bath. The water outside was pumped into the
container through the water inlet as a jet to stir seeds
and superfluous water was allowed to overflow into the
water bath. The open top of the container was covered with
a plastic net to prevent seeds from flowing out. Fresh tap
water was continuously supplied to the water bath at a rate
of one exchange (8 1) per 7 min.
31
During soaking, some seeds (dehusked seeds) were
germinated. Nongerminated seeds of a uniform size were
selected and sown in wet vermiculite in plastic cups, 7 cm
in diameter and 9 cm in height, or "Seedling Cases", 15 X 5
X 10 (height) cm3• Vermiculite was moistened, placed in
cups or Seedling Cases, and allowed to absorb water from
drain holes of the bottom at 24°C overnight. About 50 seeds
were placed in a cup and 80 seeds in a Seedling Case and
these were then covered with wet vermiculite of 2 cm in
thickness. Application of a suitable pressure to the
covering vermiculite is required for seedlings to grow
upright. Immediately cups or Seedling Cases were placed in
dark boxes kept at 24 ± 1°C and 20 ± PC.
For the purpose of experiments the conditions and
procedures for growing seedlings are complicated, and these
will be stated in "Results", but the general procedure is
as follows. Seedlings were grown to the best he ight for
irradiation, 7 to 10 cm in total length from the seed to
the top of the coleoptile, for 72 to 80 h at 24°C and for
115 to 125 h at 20°C. At the time of irradiation all
seedlings including those grown at 20°C were transfered to
24°C, and then were placed in dark boxes of 24°C and kept
at 24°C for 24 h until harvested for anthocyanin
determination. The ventilation holes of the dark boxes were
taped to keep the humidity high throughout, which assured
reproducible high levels of anthocyanin accumulation. Since
plant height was critical to results (see Results), the
average height of seedlings at the time of irradiation was
determined with 20 to 30 seedlings sampled randomly and
32
indicated in each experiment.
Irradiation. In sorghum, not only R but also UV-B
individually induce anthocyanin synthesis, mediated by
phytochrome and a UV-B photoreceptor (Yatsuhashi et al.
1982, Hashimoto and Yatsuhashi 1984). Since the broad-band
UV-B used in this experiment converted the red
light-absorbing form of phytochrome (Pr) to the far-red
light-absorbing form of phytochrome (Pfr) by the emission
band extending up to around 400 nm (Yatsuhashi et al.
1982), irradiation with the UV-B was always followed by
far-red light (FR) to select only UV-B effects, minimizing
any phytochrome action.
Light intensity was determined with a "Photon density
meter" (HK-l, Riken) (Hashimoto et al. 1982, Yatsuhashi and
Hashimoto 1985) calibrated in photon fluence rate. In some
experiments irradiation was performed from a unilateral
direction, and in others the first unilateral irradiation
was followed by another irradiation of the same period from
the opposite side. These manners of irradiation are
indicated, respectively, as (x s) and ex + x s), where x is
irradiation period in seconda, in figure captions.
Anthocyanin determination. A 5-cm section was excised from
the pigmented part or corresponding non-pigmented part of
the first internode, and 15 to 25 sections per a test were
extracted with 1% hydrochloric acid-methanol of 0.3 ml
segmene 1 overnight in the cold. The amount of anthocyanin
was determined by the difference in absorbances at 528 and
33
650 nm (Yatsuhashi et al. 1982). All data of anthocyanin
content represent the means .±. standard deviations (s.d.)
from 4 to 8 determinations.
Light sources. The red light (R) referred to as Rn was
supplied from red filter-coated fluorescent tubes (Coloured
Lamp, FL20S, R-F, Matsushita Electric Co., Osaka), and
another R source, R-IF661, was a slide projector (Master
Lux, Rikagaku Ltd., Tokyo) equipped with a 500 W tangsten
filament lamp (KONDO KP-10, Kondo Silvania Co., Tokyo)
installed with an interference f il ter, 5 cm in diameter,
(BPF ,A max 661 nm, half bandwidth 10 nm, Japan Vacuum Optics
Corporation, Tokyo). Far-red light (FR), FR-CF3024 and
FR-DelaA900, were supplied from far-red fluorescent tubes
(FL20S FR-74, Toshiba Corporation, Tokyo), respectively,
through a sheet of polyacryl resin film (CF3024, Mitsubishi
Rayon Ltd., Tokyo) and through a 2 mm-thick polyacryl resin
sheet (Delaglass A900, Asahikasei Ltd., Tokyo). The
ultraviolet light (UV), UV310-U330-N, was supplied from
ultraviolet fluorescent tubes (FL20S E, Toshiba
Corporation, Tokyo) through a 2-mm thick quarz glass filter
(U330, Hoya Corporation, Akishima, Tokyo) and 0.05-mm thick
polyvinyl resin film (Nangoku, Mitsubishikasei Vinyl, Ltd.
Tokyo). The other UV was 295-nm light from the Large
Spectrograph at National Institute for Basic Biology,
Okazaki (Watanabe et al.1982). Green safe light, G-63-1091
was supplied from green filter-coated fluorescent tubes
(Colored Lamp, FL20S, G-F, Matsushita Electric Co.,
through a sheet of polyacryl resin film (Ryutate
34
Osaka)
No.63
blue, RDS Co. , Tokyo) and double
polyacryllic resin film (No. 109-1
Rayon, Ltd.). The emission spectra
light sources are given in Fig. II-i.
35
layered sheet of
yellow,
of these
Mitsubishi
broad-band
UV310-U330-N G-63-1091 R-IF661
>- ,; I- R .......... 1 u z: ,. w ~
z: , a
l! FR-DelaA900 I-a ::::c 0-
W >-.......... I--c::r -...J W 0::::
300 500 700 900 WAVELENGTH ( nm )
Fig.lI-1. Spectral photon density distribution of the light
sources used for irradiation of plants. ~ max:
UV 31 0 - U 3 3 0 - N , 315 nm ; G - 6 3 - 1 0 9 1 , 5 1 9 nm ; Rn , 6 6 0 nm ;
R-IF661, 663nm; FR-CF3024, 753 nm; FR-DelaA900, 759 nm;
determined with a Spectro-photondensitometer II (Riken,
Wako, Saitama, Japan).
36
Results
Effects of moderate low temperature (MLT) before
irradiation. The capacity for anthocyanin synthesis varied
as seedlings grew, and exposure of seedlings to MLT
necessarily retarded their growth (Fig.II-2). However, it
became clear that the variation of anthocyanin synthesis is
strictly a function of plant height, but is not affected by
the age of plants (time periods in which seedlings grew)
(Fig. 11-3), and further that it is the case with not only
R-induced anthocyanin synthesis, but also with UV-B (Fig.
11-3,11-4). Thus, it is possible to compare unambiguously
the effect of pre- irradiation temperatures on anthocyanin
synthesis.
Figures 11-3 and 11-4 indicate that exposure to 20°C in
the preirradiation culture period significantly increases
R-induced anthocyanin synthesis, compared with that to 24°C
but has no effect on UV-induced anthocyanin synthesis at
all. The same effects were observed wi th cv.
Sekishokuzairai- Fukuyama as well as cv. Acme Broomcorn
(Table II-i). The magnitude of amplification of R-induced
anthocyanin synthesis was greater in the latter than in the
former cultivar. The amplification of anthocyanin synthesis
by MLT was also noticed in the coaction of red light and
UV-B (Table II-i).
In these experiments seedlings grown at 20°C were
transferred to 24°C at the time of irradiation, and hence
some shock effect due to temperature shift was suspected as
a cause of the increased anthocyanin synthesis. But
37
0.4 200
~ 0.3 In ID
c:x: E I 24°C /' E 00 /1 N In
c:x: / f-
0.2 / / 20°C 100 = <..!:)
w z: //1 = z: c:x: f->- z: u
/ :s 0 = / 0-f-Z c:x: 0.1
a <--L--__ --L-___ L-__ --L-___ LJ a 72 96 120 144 168
PLANT AGE (h Fig. 11-2. Red light (R)-induced anthocyanin synthesis
relative to growth period. Seeds were soaked in running tap
water of 24°C for 24 h, and seedlings were grown at 20°C
and 24°C from sowing in the dark. At the indicated times
after sowing, seedlings were irradiated with R (Rn at 20
pmol m-2 S-I) for 30 s from one side, and then for another
30 s from the other (subsequenty shown in such a way as
Rfl , 20 )lmol m-2 S-I, 30 + 30 s) and then kept at 24°C in
the dark for further 24 h until harvested. Open and solid
symbols represent the data with 20°C- and 24°C-seedlings,
respectively. Anthocyanin, o and. plant height as
measured from seed to the top of seedlings at the time of
irradiation, ~ and ~ ; the length of the first internode,
o and _ . A bar on each datum point, s.d .. S. bicolor cv.
Acme Broomcorn, 1987 crop. Exp. 890803.
38
0,4
,..., 0 3 a ' U"J <.0
<t: I !Xl N U"J
<t: '-' 0,2 z z <t: >-u 0 ::c I-z <t: 0,1
o 50 100
PLANT HEIGHT 150 (mm)
200
Fig.II-3. R-induced anthocyanin synthesis relative to plant
height in 20°C- and 24°C-seedlings. Data were derived from
Fig. 1-2. Horizontal and vertical bars on the datum points
are s.d.'s of plant height and of anthocyanin,
respectively. s. bicolor ev. Acme Broomcorn, 1987 crop.
39
0,10
o 20°C
·24°C
,-..
0 ll)
\0 c:t:
I ex> N ll)
c:t: ........ 0,05 2: ....... Z c:t: >-u 0 ::x:: I-z c:t:
a 50 100 150 200
PLANT HE I GHT (mm)
Fig. 11-4. UV-B-induced anthocyanin synthesis relative to
plant height in 20 uC- and 24°C-seedlings. Seedlings were
grmm and irradiated in the same manner as in Fig. I-2
except that irradiation with UV followed by FR. UV:
UV310-U330-N, 2 p.mol m-2 S-I, 45 + 45 s; FR: FR-CF3024, 30
/lmol m-2 S-I, 60 + 60 s. The other explanations are the
same as in Fig. I-2 and 1-3. S. bicolor cv. Acme Broomcorn,
1987 crop. Seeds soaked by method ·1. Exp.900323.
40
Table~-I. Effects of pre-irradiation moderate" low temperature on anthocyanin syntheses induced by
R, UV-B and their combination. Figures in the table are means ± S.D. 's (No. of tests for antho
cyanin or No. of seedlings for plant height). Plant heights are at the time of irradiation.
R: R-IF661, 10~mol m- 2 S-1, 100s; UV: UV310-U330-N, 14umol m- 2 S-1, 60 s; FR: FR-DelaA900,
30~mol 11-2 S-1, 180 s. S. bicolor cv. Sekishokuzairai-Fukuyama, 1986 crop; cv. Acme Broomcorn,
1987 crop. Seeds soaked bymethod 2. Exp.910322.
Light treatments
cv. Sekishokuzairai-Fukuyama
cultured at
cv. Acme Broomcorn
cultured at
Anthocyanin (A"28 - As"o)
R 0.086 ± 0.017 (4) 0.150 ± 0.005 (4) 0.064 :t 0.018 (5) 0.240 i 0.009
UV-FR 0.163 ± 0.004 (4) 0.158 ± 0.027 (4) 0.179 i 0.012 (4) 0.161 ± 0.023
UV-R 0.657 ! 0.015 (4) 0.793 ± 0.033 (4) 0.508 i 0.035 (4) 0.683 ! 0.037
FR 0.008 (2) 0.005 (1) 0.007 (2) 0.012
Dark 0.004 (1) 0.006 (1)
Plant Height (IDI)
(4)
(4)
(4)
(1)
80.8 ± 6.8 (39) 76.0 ± 6.2 (32) 82.0 ± 5.6 (29) 72.7.± 6.6(30)
41
experiments showed that this was not the case; transfer of
seedlings grown at 24° to 28°C being completely ineffective
at all (Fig. 11-5).
Effective time and duration, and optimum temperature range
of the pre- irradiation MLT treatment. First , it was
examined when and how long MLT treatments should be given
during the pre-irradiation period in order to be effective.
Seeds or seedlings were subjected to the temperature
regimes depicted at the left half of Fig. 1-6 and the
culture periods before irradiation were varied in order to
grow seedlings to as equal a height as possible. The
results show: 1) the effect was the greater as the time of
the treatment was closer to the irradiation; 2) exposure
for 18 h was only slightly effective, but exposure for 24
h given immediately before irradiation gave almost a full
effect (compare Treatments 8 and 10). The slightly greater
effect in Treatment 13 than Treatments 9 and 10 is
considered to be due to the greater plant height.
Based on these results seedlings were grown at 24°C,
then exposed to various temperatures for 24 h immediately
before irradiation. Plant heights at the time of
irradiation were shown by the open triangles in Fig. II-7A.
Irradiation with R caused anthocyanin synthesis as shown by
the open circles. In order to know net temperature effects
the effects of plant heights were estimated from the
anthocyanin syntheses observed with seedlings grown without
exposure to MLT (solid circles in Fig. II-7B), and
subtracted from the above-observed anthocyanin synthesis
42
24°C.L.24°C
24°C-L28°C
20° Ci-20° C
20° C.J....24 ° C
Fig.
o
11-5.
+ 1
-I-
I I I I
0,1 0,2 0,3 0,4
ANTHOCYANIN (A528 - A650 )
Ineffectiveness of temperature shift in
promoting R- induced anthocyanin synthes is. Seedlings were
grown at 20°C and 24°C from sowing till irradiation,
irradiated at 24°C (indicated by arrows), and then half of
the seedlings were subjected to higher temperatures by 4°C,
and the rest of the seedlings remained at the same
temperatures as before for 24 h until harvested. Plant
height at the time of irradiation, from the top row, 80.9 ±
8.0, 81.8 ± 6.1, 85.4 ± 11.2, and 87.1 i 9.3 mm. R: Rf}, 40
pmol m-2 S-l, 30 + 30 s. S. bicolor cv. Acme Broomcorn,
1987 crop. Seeds soaked by method 1. Exp.891128.
43
1 2
3
4
5 6
7 8
9
10 11
12
13
TEMPERATURE TREATMENT ( h ANTHOCYANIN (A528 - A650 )
o 24 48 72 96 120 0 0.1 0.2 0.3 0.4 I I
" , ,
t1Jillil" -+- -+-
r-a::a:::JD ,
-t-
~
, '"""""-"-' -t- --+-
.-J ""Ll.lLh...J -+-,
24 h -+- .=I-
1--1 ~'I n ,
+ -+-:'1 n
, .... -t- --t-,
~'I -+-, -t 'I, -t-. -+-,
-t- --, -t- 4-,
""'I- -f-
n ""'I-
o 50 100 PLANT HEIGHT (mm )
Fig. 11-6. Effects of the time and duration of moderate low
temperature treatment on R- induced anthocyanin synthesis.
Seeds were soaked in running tap water at 24°C for 24 h.
Planted seeds were subjected to the temperature regimes
indicated on the left side of the figure until irradiation
(marked by arrows), after which all seedlings were kept at
24~ for 24 h until harvested. Periods at 20°C are shown by
open rectangles with hours, and those at 24°C, by thin
lines. On the right side the bars represent anthocyanin
synthesis; grey parts of the bars, plant heights at the
time of irradiation. The thin short bars, s.d.'s. R: Rfl ,
20 )lmol m-2 S-I, 30+30 s. S. bicolor cv. Acme Broomcorn,
1987 crop. Exp.890826.
44
O.q A B + 200
E E
0.3 t-::r: <.!J
0.2 + 100 ~
0 t-lfl z: '" 20·C :s <I:
a...
'" 0.1 N lfl
<I:
0 0 z: -- 0.2 69 81 93 105 120 120 z: <I: >- PRECULTURE PERIOD ( h ) u 0 ::r: t-z:
0.1 <I:
o
-0.1
q 8 12 16 20 2q 23 TEMPERATURE (·C )
Fig. II-7. Effects of various temperatures given in the pre irradiation culture period on R-induced' anthocyanin synthesis. A. Seedlings were grown at 24°C for 72 h, then exposed to various temperatures for next 24 h immediately before irradiation, and placed again at 24°C for 24 h until harvested. Open circles, anthocyanin synthesis; open triangles, plant heights at the time of irradiation; solid squares, calculated amounts of anthocyanin assumed to be formed in seedlings of corresponding heights which were grown without the temperature treatments (applying the plant heights, open triangles in A to the curve of solid triangles in B, corresponding amount of anthocyanin were read from the curve of solid circles in B). B, Seedlings were grown throughout at 24°C for various periods, hence, to various heights (solid triangles), similarly irradiated and harvested for anthocyanin (solid circles; use the ordinate of A for anthocyanin content). C, Ca lculated net effects of various temperatures on anthocyanin synthesis; obtained by subtracting the curve of solid squares from the curve of open circles in A. The thin short bars, s.d. 'so R: Rfl , 20 rtmol m-2 S-I, 30 + 30 s. S. bicolor cv. Acme
Broomcorn, 1987 crop. Seeds were soaked in running tap water of 23°C for 24 h. Exp.890907.
45
(open circles in A) to obtain Fig. II-7C. It shows that
enhancement was obtained in a range from 12 0 to 20°C with
the optimum of 16 0 to 20°C, and suppression was found below
8°C and at 28°C. Even when exposure to 4°C was shortened to
20 min, no enhancement was obtained, but supprression
increased as the exposure was extended. This was true not
only of R-induced anthocyanin synthesis, but also with
UV-B, suggesting that the inhibitory effects of low
temperature such as 4°C is distinct from the enhancing
effects of MLT (Table 11-2).
Time courses of R- and UV-B-induced anthocyanin synthesis
related to pre-irradiation temperature treatments.
Seedlings grown almost to the same height at 20° and 24°C
were irradiated with a pulse of R or UV- B, and were then
placed at 24°C until harvested at the indicated times after
irradiation (Fig. 11-8). In an early period of ca. 8 h
after R, anthocyanin synthesis in 20°C-seedlings was almost
the same as that in 2 4°C-seedlings, but increased markedly
in a later period after 12 h. In both kinds of seedlings
anthocyanin synthesis reached their respective plateaux
after 30 h. The greater anthocyanin accumulation in
20°C-seedlings was not due to extension of the period for
anthocyanin synthesis. This trend is more clearly seen in
Fig. 8C, in which the time courses of accumutation are
expressed in percent of the plateau leve Is attained. The
UV-induced anthocyanin synthesis in 20°C-seedlings was also
less than that in 24°C-seedlings until ca. 16 h, but
overtook the anthocyanin level of 24°C seedlings ca. 24 h
46
Table U-2. Effects of various periods of 4°C exposure on the R-induced and UV-B-induced antho
cyanin syntheses. Figures in the table are means ± S.O.'s (No. of samples). The 4°C treatments,
were given for the indicated hours immediately before irradiation.Exp.1: R: R-IF661,10umol m- 2 s-',
100s, UV-B: UV310-U330-N, 15 umol m- 2 s-', 50 + 50 s followed by FR, FR-OelaA900, 30 umol m-2 s-',
60 + 60 s. Exps. 2 and 3, R: Rn 40 umol m- 2 S-1 for 30 + 30 s. Plant heights at the time of
irradiation: Exp. 1, 54-58 mm, Exp. 2, 55-70 mm, Exp. 3, 55-65 mm. S. bicolor cv. Acme Broomcorn.
Seeds soaked by method 1 for Exp. 1 and in 24°C running tap water for 24 h for Exps. 2 and 3.
Exp. 1, 910221, Exp. 2, 890809, Exp. 3, 890729.
R-induced UV-B-induced antho-
Periods anthocyanin (A528 - AS50) cyan in (A"28 - AS50~
at 4°C (h) Exp. 1 Exp. 2 Exp. 3 Exp. 1
0 0.118.! 0.011 (5) 0.145 .! 0.014 (4) 0.124 ± 0.012 (7) 0.168 ± 0.006 (5)
1/3 0.132.!0.028 (5) 0.130 ± 0.006 (4) 0.144 i 0.036 (4)
0.121 i 0.008 (5) 0.085 ± 0.004 (4) 0.137 i 0.006 (4)
3 0.081 ± 0.007 (6)
6 0.061 ± 0.004 (5) 0.073 i ,0.004 (6) 0.080 ± 0.010 (4)
24 0.006 i 0.003 (5) 0.020.i 0.003 (4)
47
0,6 !3 8 A ~/B ~
0
AFTER R 8 0
0,4 20°C
0 0 If)
'D « I
l- e ex! .---N 0,2 If)
~ «
= = « 0 >-u 8 a = B 24 ° C .. 0 ,fg,,-- ...... &" I-= « 0,1 AFTER UV-B 7 ·~r ·0 ~:
I ~ · ~ 20°C
0 IN! C ,.t-", ~~ , ,
80 :z: o R 20°C :z: • R 24°C « >- 40 u
A UV-B 20°C a = ~ UV-B 24°C I-= « 0
0 10 20 30 TIME AFTER IRRADIATION h )
Fig. II-8. Time courses of R- and UV-B-induced anthocyanin
synthesis at 24°C in the first internode of 20°C- and 24°C
seedlings. A and B, Actual R- and UV-B-induced anthocyanin
contents expressed as Am - Am; C, anthocyanin contents
expressed as percent of the respective maximum contents in
A and B. Plant heights of 20°C- and 24°C-seedlings at the
time of irradiation were 70.6 ± 7.9 mm (n=118) and 73.6 ± 7.6 (n=117), respectively, for A and 75.6 ± 8.6 (n=125) and
73.1 1. 7.5 mm (n=141), respectively, for B. R: Rfl ,40 }lmol
m-2 s- l, 30 + 30 s ; UV-B: UV310-U330-N, 8 Jlmol m-2
S-I, 60
s followed by FR, FR-DelaA900 70 ,..mol m-2 s- l, 180 s. S.
bicolor cv. Acme Broomcorn, 1987 crop for A and 1991 crop
for B. Seeds soaked by method 1 for A and method 3 for B.
A, Exp.891024; B, Exp.920103.
48
after the irradiation. The slower rises of anthocyanin
content in 20°C-seedling than in 24°C-seedlings are likely
to be due to lower temperature of the vermiculite which had
been kept at 20°C until irradiation. The temperature of
the root is known to influence the shoot (BassiriRad et al.
1991).
Effects of post- irradiation MLT. When seedlings grown at
24°C were subjected to MLT (20 and 18°C for Rand UV,
respectively) after irradiation, both R-induced and
UV-B-induced anthocyanin syntheses were suppressed compared
with the ones at 24°C, but at either temperature
anthocyanin synthesis reached its respective different
plateau at the same per iod after irradiation (F igs. II -9,
11-10). The lower rate of anthocyanin synthesis at the
lower temperature is probably due to the general reduction
of reaction rates at some steps of anthocyanin synthesis
probably including transcription and translation of the
anthocyanin gene. It is note-worthy that the life-time of
the machinery for anthocyanin synthesis seemed not to be
affected by the temperatures applied.
A comparison of Figs 11-9 and 11-10 shows that the
R-induced anthocyanin synthesis rises more slowly and
continues for a longer per iod than the UV- B- induced one,
the half maximam level being attained after 16 h for R at
both temperatures, whereas for UV-B, after 10 h at 24°C and
after 14 h at 18°C. A similar feature of the difference
between R- and UV-B-responses is seen in Fig. II-8C. This
was true of both 20°C- and 24°C-seedlings.
49
o ~ 0.2
c::x:: I CXJ N Ln
c::x::
Z 0.1 Z c::x:: >u o ::c IZ c::x::
o o
Fig. II-9.
AFTER R . , :~
12 24 36
TIME AFTER IRRADIATION ( h )
Effect of post-irradiation moderate low
temperature on the time course of R-induced anthocyanin
synthesis. Seedlings were all grown at 24°C until
irradiation, and, after irradiation, a half of them were
grown at 20 DC until harvested at the indicated times, while
the rest remained at 24°C. Plant height at the time of
irradiation, 73.6 .± 7.6 mm. R: Ru, 40 umol m-2 S-l, 30 +
30 s. s. bicolor cv. Acme Broomcorn, 1987 crop. Seeds
soaked by method 1. Exp.891024.
50
"""0 0,2 • LO AFTER U295 <D
c::::C • • I 24°e ro N • LO
c:r: 0 • 8 • '-" 0 0,1
~o -f3
z 0
z 18°e c::::C >-U 0 ::c t-z c::::C
0 0 12 24 36
TIME AFTER IRRADIATION ( h )
Fig. 11-10. Effect of post-irradiation moderate low
temperature on the time course of UV-B-induced anthocyanin
synthes is. Seeds were soaked in 19°C-running tap water for
22 h, and seedlings were grown to 90-100 mm in height at
24°C for ca 85 h until irradiation, and after irradiation,
a half of them were transferred to 18°C, while the rest
remained at 24°C. UV-B: 295 nm light, 74 pmol m 2 S-I, 20 s
from the Large-Spectrograph. S. bicolor cv. Acme Broomcorn,
1987 crop. Exp.890529.
51
Discussion
Experimentation. The time courses of anthocyanin
accumulation (either by R or UV) differed with culture
temperatures (Fig. 11-8), but reached their respective
plateaux after 24-30 h. The plateau levels of anthocyanin
accumulation are considered to ref lect the maximum extent
of the light effects; because no destruction of anthocyanin
was observed (data not shown). Accumulation of anthocyanin
varied with the development of seedlings, and the growth of
seedlings were naturally retarded at lower temperatures
(Fig. 11-2), but fortunately the variation in the capacity
for anthocyanin synthesis depended solely on plant height
irrespective of culture temperatures as well as the kind of
light (Figs. 11-3, 11-4). It was thus possible to
unambiguous ly examine the effects of temperature by
comparing anthocyanin content at plateau at the same height
(Figs. 11-3, 11-4).
Effects of MLT distinct from low temperature (LT) effects.
Enhancement of R-induced anthocyanin synthesis was observed
in seedlings grown at 12° to 20°C, with the optimum of 16°
to 20°C, compared with that in seedlings grown at 24°C
(Fig. 11-7). This effective range of temperature is
different from that reported with seed germination of
Lactuca sativa, Betula papyrifera, Arabidopsis thaliana,
and others, which is promoted below 12°C with the optimum
of around 5°C or less (VanDerWoude and Toole 1980,
Bevington and Hoyle 1981, Cone et ale 1985). In fact, such
a LT suppressed anthocyanin synthesis (Fig. 11-7, Table
52
11-2). Another difference from seed germination is that the
MLT effect in anthocyanin synthesis of sorghum is not to
increase
required
the sensitivity to
fluence of R (see
Pfr; i.e. not to
Chapter III), while
lower the
in seed
germination of Lactuca sativa and Arabidopsis thaliana the
LT sens i tizes seeds to respond to very low leve Is of Pfr
(VanDerWoude 1985, Cone et al. 1985). A shift from 24 to
28°C at the time of irradiation was also not promotive.
Thus, the enhancement effect of MLT is also distinct from
so-called heat shock, which occurs with such a short-term
exposure as 15 min to few hours (Brodl 1989).
Enhancement of phytochrome action by MLT. Some papers have
described the promotive effects of LT on anthocyanin
accumulation. Faragher (1983) reported promotion of
anthocyanin accumulation in apple skin by MLT, the optima
being 12°C for unripe fruits ~nd 16 0 to 24°C for ripe ones.
In this work, anthocyanin synthesis was induced by
continuous or intermittent white fluorescent light.
Martinez and Faveret (1990) also observed a great increase
of anthocyanin synthesis in barley (Hordeum vulgare)
seedlings subjected to 10°C for a 16 h-day and SoC for an 8
h-night compared with control seedlings kept continuously
at 18°C for the same regime of day and night. In these
studies, however, no indication has been made whether or
not MLT effects are specific to phytochrome-mediated
anthocyanin induction, nor any distinction made of actions
before and after irradiation. The present paper showed that
pre- irradiation MLT enhanced specif ically phytochrome-
53
mediated anthocyanin synthesis (Figs. 11-3, 11-4, 11-8,
Table II-i).
Effects of MLT on phytochrome actions. In our sorghum
synthesis, phytochrome and a UV-B system of anthocyanin
photoreceptor mediate anthocyanin synthesis not only
individually, but also by a multiplicative coaction
(Yatsuhashi and Hashimoto 1985), and in the three cases one
and the same species of anthocyanin is produced (Yatsuhashi
et al. 1982). The machinery of the anthocyanin synthes is
after transcription of the genes in this plant is,
therefore, assumed to be common to both R- and UV-B-induced
responses. The view is consistent with the findings that
both light responses were equally suppressed by the
post-irradiation MLT treatments (Figs. 11-9, 11-10).
However, the enhancement of anthocyanin synthesis by the
pre-irradiation MLT is limited to R-induced
(Table II -1, Fig. II -8). The time-courses
anthocyanin
of R- induced
anthocyanin
ones (Figs.
to suggest
synthesis differ from those of UV-B-induced
II -8, 11-9 and II -10). These findings lead us
that the signal transduction system from
phytochrome to the anthocyanin genes is different from that
of UV-B photoreceptor, and the former system involves a
step or steps which are enhanced by MLT applied during the
pre-irradiation culture period (Fig. 11-8, Table II-i).
As a consequence of such enhancement of the
phytochrome signal transduction system, enzymes for
anthocyanin synthesis may be produced in larger amounts. In
his work with apple fruit reffered to above, Faragher
54
(1983) observed a several-fold increase of PAL, one of the
key enzymes of anthocyanin synthesis, at MLT ranging from
10° to 20°C compared with at 24° or 28°C, although he
ascribed the increase of PAL activity to less production of
a PAL inactivator rather than to increased production of
PAL at the MLT. Moroz et al. (1984) found with white
light-grown seedlings of Sinapis alba a several-fold
enhancement of nitrate reductase activity, which is known
to be under the control of phytochrome (Whitelam and
Johnson 1981) at 10°C and 15°C compared with at 20°C. These
findings may support the view that in sorghum seedlings MLT
treatments may raise the levels of some enzymes involved in
anthocyanin synthesis, amplifying phytochrome signal
transduction leading to gene expression.
As possible site (s) of MLT action in the
transduction chain of phytochrome signal for anthocyanin
synthesis in sorghum the following possibilities may be
considered: Exposure to MLT 1) increases the total amount
of phytochrome in tissues, 2) promotes the eff iciency of
the conversion of Pr to Pfr, i.e. raises quantum yield of
the conversion, 3) increases the amount of the Pfr
receptor, e. g. X, 4) amplif ies an unknown step or steps
between the formation of pfrX and the transcription of
genes for anthocyanin synthesis enzymes. These
possibilities will be examined in Chapter III.
In summary, the present paper strongly suggests that
exposure of dark-grown seedlings to MLT ranging from 12 to
20°C at least for 24 h immediately before irradiation
55
generates a cellular state to amplify the phytochrome
signal transduction for anthocyanin synthesis induction
which is distinct from that of a UV- B photoreceptor. The
effect of MLT provides a clue to analysis of the former
system.
Moderate low temperature. Since in most laboratory
experiments 25°e and its neighborhood is usually used, we
referred to 12 to 20 0 e as MLT, distinguishing it from LT
below about 10 oe. As pointed out by Moroz et al. (1984)
25°e is too high compared with the usual temperaturs
encountered in the fields in the spring and autumn in
England as well as Japan. Sorghum bicolor may have MLT as
its optimum.
Variation of anthocyanin synthesis ability with the
developmental stage of seedlings. The variation with the
development of seedlings of anthocyanin synthesis induced
by a fixed phytochrome-saturating R (Fig 11-2, 11-3) is
reminiscent of that of phytochrome levels determined by
Kendrick et al. (1969) and in mustard by Schmidt and Mohr
(1981,1982), and also agrees with the variation of
anthocyanin level induced by FR (Small and Pecket 1982),
and hence, seems to be explained in terms of the variation
of phytochrome level. However, it is not so simple, because
Fig. 11-4 indicates that anthocyanin synthesis induced by
UV-B (followed by FR) also showed a similar tendency. Thus,
it is conceivable that the UV-B photoreceptor in addition
56
to phytochrome varies in content with plant height, and/or
that the activity of the machinery for anthocyanin
synthesis which is common to induction by R as well as UV-B
varies with plant height.
Is effect of 20°C in the culture period cancelled by
transfer to 24°C ? The sensitivity of seedlings to a 20°C
treatment seemed to increase towards the end of the
pre irradiation culture period (Fig. 11-6). But comparisons
of treatments No.7, 8 and 9 as well as between No.3 and
4, and No. 9 and 10 in Fig. 6 may suggest that the
enhancement effects of 20°C is cancelled by subsequent
holding at 24°C for 48 h rather than that the sensitivity
of seedlings to the temperature treatment varies with the
developmental stages. The LT effects on the promotion of
transcription of the genes for alcohol dehydrogenase
(Christe et al. 1991) as well as on the suppression of
transcr ipton of the gene for Rubisco subunits were both
cancelled by 24 h shift to the control temperature (Hahn &
Walbot 1989). The LT effect causing very low fluence
response in Lactuca seed germination decayed with a
half-life of about 6 h (VanDerWoude and Toole 1980). These
findings may lead us to assume that in sorghum also, the
MLT effect to promote R-induced anthocyanin synthesis might
be cancelled by holding at the control temperature.
However, our experimental system wi th sorghum first
internodes does not allow such a simple interpretation,
because during the experiment the first internodes grow
producing a new tissue, and the older tissue and the new
57
tissue which come into play for anthocyanin synthesis. If
MLT effects manifest themselves in a tissue which is at a
physiological state sensitive to MLT lit may be that no
conclusive experiment is possible to answer the
above-raised questions as far as intact plants are used.
58
References
BassiriRad, H., Radin, J.W., Matsuda, K. (1991) Temperature
dependent water and ion transport properties of barley and
sorghum roots. Plant Physiol. 97, 426-432
Bevington, J.M., Hoyle, H.C. (1981) Phytochrome action during
prechilling induced germination of Betula papayrifera Harsh.
Plant Physiol. 67, 705-710
Brodl, H.R. (1989) Regulation of the synthesis of normal
cellular proteins during heat shock. Physiol. Plant. 75,
439-443.
Christie, P.J., Hahn, H., Walbot, V. (1991) Low-temperature
accumulation of alchol dehydrogenase-1 mRNA and protein
activity in maize and rice seedlings. Plant Physiol. 95,
699-706.
Cone, J.W., Jaspers, P.A.P.M., Kendrick, R.E. (1985) Biphasic
fluence-response curves for light induced germination of
Arabidopsis thaliana seeds. Plant. Cell and Environment 8,
605-612 .
. Cone, J.W., Kendrick, R.E. (1986) Photocontrol of seed
germination. In: Photomorphogenesis in Plants, pp. 443-465
Kendrick, R.E., Kronenberg, G.H.H. eds. Martinus Nijhoff,
Dordrecht.
59
Faragher, J.D. (1983) Temperature regulation of anthocyanin
accumulation in apple skin. J. Exp. Bot. 34, 1291-1298.
Frankland B., Taylorson R. (1983) Light control of seed
germination. In: Encyclopedia of Plant Physiology, New
Series, Vol.16A,pp.428-456, Shropshire Jr., W., Mohr, H.
eds. Springer-Verlag Berlin Heidelberg
Hahn, M., Walbot, V. (1989) Effects of cold-treatment on
protein synthesis and mRNA levels in rice leaves. Plant
Physiol. 91, 930-938
Hashimoto, T., Yatsuhashi, H., Kato, H. (1982) A convenient
meter to measure the photon and energy fluence rate.
Abstracts of the Annu. Meeting, Japan. Soc. Plant. Physiol.,
p.38. (in Japanese)
Hashimoto, T., Yatsuhashi, H. (1984) Ultraviolet photo
receptors and their interaction in broom sorghum
Analysis of action spectra and fluence-response curves.
In: Blue Light Effects in Biological Systems, pp125-136,
Senger, H. ed. Springer-Verlag Berlin Heidelberg
Kendrick, R.E., Spruit, C.J.P., Frankland, B. (1969) Phyto
chrome in seeds of Amaranthus caudatus. Planta 88, 293-302.
Martinez, A.E., Favret, E.A. (1990) Anthocyanin synthesis and
lengthening in the first leaf of barley isogenic line~.
Plant Science 71, 35-43
60
Moroz, S.M., Alford, E.A., Johnson, C.B. (1984) Effects of
temperature on the development of Sina~ alba L.;
phytochrome-control of nitrate reductase activity at 10°C.
Plant, Cell and Environment 7,45-51
Schmidt, R., Mohr, H. (1981) Time-dependent changes in the
responsiveness to light of phytochrome-mediated anthocyanin
synthesis. Plant Cell Environ. 4, 433-437.
Schmidt, R., Mohr, H. (1983) Time course of signal
transduction in phytochrome-mediated anthocyanin synthesis
in mustard cotyledons. Plant Cell Environ. 6, 235-238
Small, C.J., Pecket, R.C. (1982) Change in sensitivity to far
red irradiation on anthocyanin biosynthesis in red cabbage
seedlings. Plant, Cell Environ. 5, 1-4
VanDerWoude, W.J., Toole, V.K. (1980) Studies of the mechanism
of enhancement of phytochrome-dependent lettuce seed
germination by prechilling. Plant Physiol. 66, 220-224
VanDerWoude, W.J. (1985) A dimeric mechanism for the action of
phytochrome: evidence from photothermal interactions in
lettuce seed germination. Photochem. Photobiol. 42, 655-661
VanDerWoude, W.J. (1987) Application of the dimeric model
of phytochrome action to high irradiance responses. In:
Phytochrome and Photoregulation in plants, pp.249-258,
61
Furuya, M. ed. Academic Press, Tokyo
Wall, J.K., Johnson C.B. (1982) The effect of temperature
on phytochrome controlled hypocotyl extension in Sinapis
Alba L. New Phytol. 91, 405-412
Watanabe, M., Furuya, M., Miyoshi, Y., Inoue, Y. Iwahashi, I.
Matsumoto, K. (1982) Design and performance of the Okazaki
Large Spectrograph for Photobiological research. Photochem.
Photobil. 36, 491-498
Whitelam, G.C., Johnson, C.B. (1981) Temporal separatIon of
two components of phytochrome action. Plant Cell Environ. 4,
53-57
Yatsuhashi, H., Hashimoto, T., Shimizu, S. (1982) UV action
spectrum for anthocyanin formation in broom sorghum first
internodes. Plant Physiol. 70, 735-741
Yatsuhashi, H., Hashimoto, T. (1985) Multiplicative action of
a UV-B photoreceptor and phytochrome in anthocyanin synthes
is of broom sorghum seedlings. Photochem. Photobiol. 41,
673-680.
62
Chapter III: Red light fluence-dependent responses to
pre-irradiation moderate low temperature of phytochrome
mediated anthocyanin synthesis in Sorghum bicolor first
internodes.
Abstract
Red light (R) -specif ic enhancement of anthocyanin
synthesis by a moderate low temperature (MLT) given during
the pre-irradiation culture period (Chapter II) was studied
using seedlings of broom sorghum (Sorghum bicolor Moench,
cvs. Acme Broomcorn and Sekishokuzairai-Fukuyama) grown in
the dark at 20°C and 24°C (control). Spectroscopical
determination of phytochrome revealed that between 20°C
and 24°C-seedlings no appreciable difference was observed
in total phytochrome contents, the quantum eff iciencies of
red light-absorbing form of phytochrome (Pr) to far-red
light-absorbing form of phytochrome (Pfr) conversion, the
rates of destruction of Pfr, nor the rates of escape from
far-red light (FR) reversion. Analysis of fluence-response
curves showed that the rate of enhancement of anthocyanin
synthesis in 20°C- vs. 24°C-seedlings increased as the R
fluence increased starting from the null effect at 20 to 50
pmol m-2• Further below at such very low levels of Pfr as
attained by a R pulse followed by a FR pulse or by a FR
pulse alone, in contrast, anthocyanin synthesis was less in
20°C- than 24°C-seedlings. The findings may be explained
only by assuming that MLT gives distinct responsivities of
plants to PrPfr vs. PfrPfr in the phytochrome dimeric
63
model, supportig that phytochrome dimers are functional.
64
Introduction
Anthocyanin synthes is in the first internodes of
sorghum is induced by an irradiation with either red light
(R) or ultraviolet light-B (UV-B) as well as by a coaction
of the two kinds of light. The Rand UV-B actions are
mediated by phytochrome and a putative UV-B photoreceptor,
respectively (Yatsuhashi et al. 1982, Yatsuhashi and
Hashimoto 1985). Previously it was shown (Chapter II) that
phytochrome action, but not the UV-B action, was enhanced
if seedlings were grown before irradiation at moderate low
temperatures (MLT), 12° to 20°C, suggesting that
phytochrome and/or its signal transduction might be altered
in efficiency by exposure to MLT.
Phytochrome is well established as the R photoreceptor
for plant photomorphogenesis, and the occurrence of two
kinds, unstable phytochrome I and stable phytochrome II,
have been demonstrated, but its action mechanism is still
to be worked out. The presence of low fluence response
(LFR) and very low fluence response (VLFR) as well as high
irradiance response (HIR) have been indicated. VanDerWoude
(1985, 1987) has presented a phytochrome dimeric model
which postulates that PrPfr and PfrPfr are responsible for
VLFR and LFR, respectively, and HIR is resulted by the
product of PrPfr and cycling between PrPr and PrPfr. This
dimeric model has been supported theoretically (Brockmann
et al. 1987) and by experimental results (Shinkle and
Briggs 1984, Cone et al. 1985, De Petter et al. 1985 and
1988). VanDerWoude and Toole (1980) have found that a
pre-irradiation low temperature (LT) sensitizes Lactuca
65
seeds to germinate under a VLF of light, and he (1985) has
explained the LT effect by a possible increase in the
fluidity of the cell membrane, which may ease the access of
PrPfr-X to a reaction partner Y.
In addition, it is known that LT and MLT given during
and/or after irradiation inf luence the destruction of Pfr
(Sch'Afer and Schmidt 1974, Frankland 1972),
photoequilibrium of Pfr and Pr (Jabben et al. 1982), and
escape from FR reversion (Moroz et al. 1984). However,
thus far, no paper is available to examine whether or not
LT or MLT applied during the period of seedling culture
affects phytochrome content and the behaviour of
phytochrome after irradiation, and what step or steps it
affects in the phytochrome signal transduction processes.
Our sorghum system of R-induced anthocyanin synthesis
which is specifically enhanced by a pre-irradiation MLT
seems to be very appropriate to study this problem, because
in this system MLT is very likely to act at the signal
transduction system from phytochrome to the expression of
anthocyanin synthesis genes (Chapter II) and hence, a MLT
treatment may be used as an additional parameter for
investigation of phytochrome action. In the present paper,
with the aim of looking into the primary mechanisms of
phytochrome through locating the action site(s) of the
pre-irradiation MLT,
affects phytochrome
we examine
content and
1 ) whether or not MLT
behaviour, including
destruction of phytochrome after R irradiation and escape
from FR reversal; 2) how MLT modif ies the phytochrome
actions in a wide range of photon fluences, including VLFR
66
and LFR as well as high fluence response (HFR) found over
the phytochrome-saturating f luence. The results
demonstrate that phytochrome I content and behav iour are
not affected, and suggest that the phytochrome action in
the induction of anthocyanin synthesis may consist of VLFR,
LFR and HFR, which are distinguished by the effects of
pre-irradiation MLT.
67
Materials and Methods
Plant materials. Seeds of Broom sorghum, Sorghum bicolor
Moench, cvs. Acme Broomcorn and Sekishokuzairi-Fukuyama
were used. Cv. Acme Broomcorn was grown and harvested at
the experimental farm, the Abrahi Laboratories, Shionogi
Pharmaceutical Co., Aburahi, Shiga in 1987, and at the
Experimental Farm of the Faculty of Agriculture, Kobe
University, Kasai, Hyogo, in 1991; cv. Sekishokuzairi
Fukuyama was also cultivated at Abrahi in 1990.
Seeds were soaked in tap water adjusted to 24DC by one
of the three methods described previously (Chapter II).
From sowing to irradiation, seedlings were grown either at
20° .±. lac or 24° ..± lDC (referred to as 20DC- or
24DC-seedlings, respectively), and from irradiation to
harvest all seedlings were kept at 24°C. The details of the
culture, irradiation and other procedures were described
previously (Chapter II).
Light sources. For irradiation, red light (R) and far-red
light (FR) were mostly used, and details were given
previously (Chapter II). In addition, monochromatic light
of various wavelengths were used which were supplied from a
slide projector (Master Lux, Rikagakuseiki, Ltd., Tokyo)
installed with a 500 W tungsten filament lamp (KONDO KP-l0,
Kondo Sylvania Co., Tokyo) through interference filters,
A max, nm (half bandwidth, nm) 661 (10), 680 (15.5), 691
(18.5),702 (16.5) and 708 (9.5)(the first is of type BPF,
and the others, type W; Japan Vacuum Optics Co., Tokyo).
Photon fluences were determined as previously (Chapter
68
II).
Phytochrome determination. A spectrophotometer (Model 557,
Hitachi, Ltd., Hitachi, Japan) was used with a slit width
of 2 nm. The actinic light was introduced with a 5-mm (~)
glass fibre into the spectrophotometer from a light source
(Technolite KLS-2100R or KHL-150, installed with a 120 or
150 W halogen tungsten lamp, Kenko, Ltd., Osaka). At the
end of the glass fibre was placed a filter revolver which
holds four kinds of f il ters, and the light corning through
was reflected with a concave mirror to project the light on
to the same path as the measuring beam of the spectro
photometer. The details are referred to Hada et ale (1992).
For actinic R an interference filter (IBPF-4 ~ max 641 nm,
half-bandwidth 55 nm, Japan Vacuum Optics, Tokyo) and for
FR a cut-off filter of polyacrylic resin (Delaglass A900,
Asahikasei Ltd., Tokyo) were used. The latter filter gave
light of A max 759 nm, half-bandwidth 68 nm according to
a measurement with a Spectro-photondensitomer II (Riken,
Wako, Saitama, Japan). The other procedures were modified
depending on the purposes, and will be described below on
their appearance.
Phytochrome was determined by ~ ( ~ A730 - 660 ) of
sections excised from first internodes, principally based
on the method of Butler et ale (1959). Unlike other
workers, we placed sections in a cuvette tightly, but
applied no force. For ease of placing sections, a compound
type of cuvette with the front g lass removable was used
throughout.
69
To determine the distribution of phytochrome along the
internode, 10-mm sections were excised from the first
internode so as to represent various parts, and sections
from the same part were placed horizontally in a compound
cuvette, 10 X 45 (height) X 5 (thickness) mm3, with the
removable front. To reduce the number of sections needed,
unnecessay room in the cuvette proper was f illed with two
pieces of silicone rubber, leaving a room for plant
material, 2 mm to 18.5 mm above bottom and 10 mm wide. In
this room 100 sections were tightly placed, the front glass
was placed back and fastened. In order to prevent stray
light scattered at the cuvette from entering the
photomultiplier, the photomultiplier side of the cuvette
was covered with a black paper frame which had the same
size as the cuvette and the opening, 8 mm in width and 13
mm in height. When the cunette, thus-prepared, was set
upright in the spectrophotometer, the measuring beam, 1 mm
in width and 9 mm in height in cross-section, intersected
the sections at a l-mm part in the middle to determine
phytochrome content there. Immediately behind the black
frame for the cuvette was always placed a diffuser of thin
translucent plastic sheet. These handlings were done under
dim green safe-light. In this phytochrome determination the
double beam mode was used, and the intensity of the
reference beam was reduced with a neutral filter to balance
it with the measuring beam.
The cuvette thickness of 5 mm comprised average 5.3
and 5. a sections for 20 °C- and 24°C-seedlings,
respectively, and the measured values of.6( 4 A730 - 660 ) were
70
divided by either relevant section number to obtain
phytochrome content / section.
For determining Pfr/Ptot established by various
irradiations the top about 30-mm part of the first
internode was excised from irradiated seedlings immediately
after irradiation, and 20 sections were tightly placed
ups ide down in a compound cuvette, lOX· 45 (he ight) X 2
(thickness) mm3 (see above). Thus-prepared, the cuvette was
placed upright in the spectrophotometer, and a sheet of
dry , white f il ter-paper was placed on the photomultiplier
side of the cuvette as diffuser for the measuring beam.
This handling was done under a dim green safe-light. The
measuring beam, 1 mm in width and 9 mm in height, targetted
the 5 to 14 mm (from top) part of the internode sections.
Using the dual wavelength mode, differences in absorbance
between 730 and 660 nm were recorded three times, first
before additonal actinic
finally after R. The
phytochrome-saturating.
irradiation, then after FR, and
Rand FR were confirmed to be
Actinic irradiation and absorption
measurement were made at room temperature. Pfr /Ptot was
calculated with the following equation:
Pfr/Ptot = 0.876 {lst D (.6 Ano-660 ) - 2nd.A (A A730-66o )} /
{ 3rd A ( .6. Ano-66o) - 2 nd A ~ A730-66o ) }
where 0.876 is the Pfr/Ptot in photoequilibrium at 660 nm
observed with a highly purified Avena sativa 124-kd
phytochrome (Kelly and Lagarias 1985).
Anthocyanin determination. The procedures were described
previously (Chapter II), but slight modifications were made
71
depending on the purpose, which will be described in
relevant places in "Results". Such small amounts of
anthocyanin as induced by FR and R + FR as well as values
of dark controls were determined by reading the peak height
rising at 528 nm above the background absorption in each
scanned spectrum.
72
Results and Discussion
Distribution of phytochrome and anthocyanin synthesis.
Since enhancement by a pre-irradiation exposure of
seeedlings to 20°C is restricted to R-induced (Chapter II),
it was first suspected that the exposure to 20°C might
affect the total content and / or behaviour of phytochrome.
As the first step, the distribution of phytochrome was
examined in a comparison between 20°C- and 24°C-seedlings
which grew to the size to be used for irradiation
experiments. Sequential 10-mm sections were excised from
first internodes as indicated in Table 111-1, and 100
sections were placed tightly and horizontally in a cuvette,
which was then subjected to spectroscopy. The measuring
beam of 9 mm height and 1 mm width· intersected the
internode sections in the center, and so thus-obtained
absorbance change represented phytochrome content in the
middle of the segments. The thickness of the first
internode was greater in 20°C-seedlings than in
24°C-seedlings, and the absorbance changes were corrected
for the unit fresh weight, i.e. converted into relative
concentrations and also into the values per section (Table
111-1). The results show no significant difference of
phytochrome content between 20°C- and 24°C-seedlings.
Figure 111-1 (left side) depicts the content and
distribution of phytochrome at the time of irradiation. It
was highest near the top of the internode (coleoptilar
node), and sharply decreased downward along the internode
to stay at a constant low level. No significant difference
was found between 20°C- and 24°C-seedlings.
73
PHYTOCHROME t.(t.A73o-66o ) X 10-3
10 5 eO· ..
SECTION -1
0 0
- ,.20
-40
I I
I I
I
I
I
I I
I I
I I
I I
I
I
I
I
I
40 ~ ~ il
j)
20 ~ ANTHOCYANIN ~. ( AS28 - A6s8 ) 1,,:0. 0,2 0, 4 , 6
Fig. III-i. The local distributions of phytochrome before R
and of R-induced anthocyanin synthesis along the first
internode of Sorghum bicolor (cv. Acme Broomcorn).
Seedlings were grown at 24°C for 71 h and at 20nc foi 118 h
before a R pulse, and after the R pulse both kinds of
seedlings were kept at 24°C for 33 h till anthocyanin
determination. The ordinate represents the distance from
the point where the coleoptilar node has been at the time
of irradiation and phytochrome determination (see methods).
The drawings in the center represent the sizes of seedlings
at the times of irradiation and anthocyanin determination;
grey portions, the coleoptiles; white portions, the first
internodes. Phytochrome content .6(.6/\730-660) section-1;
the values of anthocyanin, Am - Am obtained when a 5-mm
section were excised from the indicated zone of the first
internode, and 100 sections were extracted with 4 ml of 1%
HCI-MeOH. R: Rfl , 50 pm m 2 Sl, 30 + 30 s; seeds, 1991
crop, soaked by method 3.
74
Table Dr-l. Comparison of phytochrome contents in the first internode of sorghum seedlings grown
at 20°C and 24°C in the dark. Figures in the Table represent the means ± S.D.·s (No. of tests).
~. bicolor cv. Acme Broomcorn. 1991 crop. Plant heights, 78 - 98 mm 20°C-seedlings; 78 - 100 mm
24°C-seedlings. Seeds were soaked by method 3.
Phytochrome content
at the zones of first internode indicated
Zones 0 - -10 mm -10 - -20 mm -15 - -25
20°C-seedlings
Fresh weight (mg/100 sections) 597 i 23 (3) 608 ± 22 (3) 608 1. 13
l:; ( l:; A730- .... 0 ) X 10-3 .) 50.9 ± 8.9 (3) 15.0 f- LO (3) 14.9 ± 1.7
l:; ( l:; Ano- .... o) X 10-3/ g Fit 85.1 ± 15.9 24.9 i 1.8 24.6 ± 2.5
l:; ( l:; A730-~60) X 10-3/ section b, 9.6 ±. 1.7 2.8 ± 0.2 2.8 ± 0.3
24°C-seedlings
Fresh weight (mg/100 sections) 547 ± 6 (3) 532 ± 6 (3) 538 1. 10
l:; ( l:; A730- SS0) X 10-3 ., 50.1 i 1.1 (3) 13.0 i 8.3 (3) 12.4 ±. 1.4
l:; (l:; Ano-6so) X 10-3/ g Fit 91.5 1. 1.0 24.3 ± 1.8' 23.0 ± 2.3
l:; ( l:; A730-6S0) X 10-3 / section b, 10.0 .± 0.2 2.6 ± 0.2 2.5 1. 0.3
·'Values at a cuvette thickness of 5 mm.
b'The cllvette thickness comprises average 5.3 sections for 20°C-sedlings and 5.0 for
24°C-seedlings. which were used as denominators.
75
mm
(7)
(7)
(7)
(7)
Next, the distribution of anthocyanin synthesis was
determined. Between harvest and irradiation at 24°C,
20°C-seedlings grew slightly less than for 24°C-seedlings
as indicated by the illustrations. Starting at the
coleoptilar node, 5-mm sections were excised consecutively
from an internode, and 100 sections from a corresponding
zone were extracted with 4 ml
acid-methanol for quantification
111-1, right side).
of
of
1% hydrochloric
anthocyanin (Fig.
For an precise comparison of the anthocyanin profile
with the phytochrome distribution the following measure was
taken. The growth of the first internode is confined to
the top 5 -mm part (Hashimoto et al. 1984), and the base
point was marked with india ink in lanolin before growth,
and the length from the node to the mark was determined
with 20 to 30 seedlings each for 20°C- and 2 4°C-seedlings.
The average lengths obtained were, respectively, applied to
20°C- and 24°C-seedlings at the time of harves to locate
the original 5-mm base points.
Most of the anthocyanin synthesis occurred at the zone
from +5 to -35 mm, which had ceased to grow at the time of
irradiation, peaking at 10 to 15 mm below the previous
node. The peak of anthocyanin synthesis corresponded with
the border of the sharp decline and constant level of
phytochrome (Fig. 111-1).
Figure 111-1 also shows that the 20°C-treatment
amplif ied anthocyanin synthes is without broadened the zone
of anthocyanin synthesis downward without changing the peak
position, confirming the previous results (Chter II).
76
The characteristic phytochrome distribution along the
first internode is similar to that in Avena sativa first
internode. The concentrations and contents per indiv idual
are also of the same order as those previously reported
(Briggs and Siegelman 1965, Kondo et al. 1973). It is to
be noted that most anthocyanin synthesis occurred below the
top part of the internode which was richest in phytochrome.
Behaviour of phytochrome. The photon efficiencies of Pr to
Pfr photoconversion with various fluences of R are shown in
Fig. III-2. No difference was observed between 20°C- and
24°C-seedlings. Next, a pulse of 10 s or 100 s was given to
20°C- and 24°C-seedlings with varied wavelengths of
monochromatic light in the red region, and the resulting
Pfr/Ptot ratios were determined. Although wavelength
dependent convers ions of Pr were observed, there were no
appreciable differences in Pfr/Ptot ratios between 20°C
seedlings and 24°C- seedlings (Fig. III-3).
After an irradiation with saturating R the destruction
of Pfr was followed, using 10-mm sections which span from
15 to 25 mm below the node, and thus gave the data at 20 mm
below the node (Fig. III-4). The reason why this part of
the internode was chosen is that this part still actively
synthesized anthocyanin,
uniform distribution of
though not the maximum, and had a
phytochrome, which minimized the
fluctuation of data due to an aberration of the position
for phytochrome determination. Pfr decreased with time in a
sigmoidal manner with an initial delay, the half lives
77
I,D o 20°C • 24°C 8 0 .- ~
"/~ +J 0 +J 8 a... o/?J ........... D,S H ~ 4-l 8/" a...
~/~ ~
0 20 50 100 200 500 1000
R FLUENCE ( ,umol m-2 )
Fig.III-2. Photon eff iciency of Pr to Pfr conversion with
various f luences of R in seedlings grown in the dark at
R (R-IF661) of 10 pmol m-2 S-l was given
for the indicated periods of time, then the top part of ca.
30 mm was excised from the first internode, and placed
vertically upside down in a cuvette. Plant heights of 20°C-
and 24°C-seedlings: 62 to 86 mm and 62 to 88 mm,
respectively. s. bicolor cv. Acme Broomcorn, 1987 crop;
seed soaked by method 1.
78
1.0 10 S IRRADIATION 100 S IRRADIATION • •
C=:J 20°C ~i;~ifJ;l 24° C
+J 0 +J
D-
"- 0.5 r-~
r-
!-I I" ~ ~ .~ D-
r~ ~~ •
~ . \", • \.:
,.1. ". ~r ~\ • t.~ ~;'
I ~ .~ f.~
I r-~ 0 :~ :~ ~~ e.,~-
r-
.~ • ~ ~ :-\~~ :t~ • • ",~" ,;, .7i ~~ 'i".;~ f!i ;;~J -if ...... "" I ~\~ ~~~
~'.* ~~
~1i ~~
~ .'" },,~ ~~,
~~ • ~' • )j. ~~ ttP ~l{ rea
~ ~ • • ~ ,- 4 .~
IT~ ~ I ~,~ ;.~~
~'{
j (iii ,~. ~i .~ ;~ ~~ ~)
m ~ ~J ~ ~ ~r' ~ ,;..~ ~}~ ~~!
661 680 691 702 661 680 691 702 708
WAVELENGTH ( nm )
Fig. III-3. Pfr/Ptot established by various wavelengths of
monochromatic light in 20°C- (open bars) ~nd 24~-seedlings
(grey bars). Seedlings were irradiated with 661, 680, 691,
702 and 708 nm light (10 pmol m-2 SI) for 10 s (A) and 100
s (B), and then immediately top part of ca. 30 mm of the
first internode was excised, and placed verticcally upside
down in a cuvette. Each phytochrome determination
comprised 3 to 4 replications. Plant heights of 20°C- and
24°C-seedlings both: 61 to 91 mm. S. bicolor cv. Acme
Broomcorn, 1987 crop; seeds soaked by method 1.
79
100 -'0 '0
" " \ • \ \ 20°C
• • 0\ 0 \
\
......, ...J « ..... f-
z
u.. 0 50
IN!
'-"
H 4-l
a..
'- - -0 •
O~--~--~----J_--~----~--~--~ o 60 120 180
TI ME AFTER R (min)
Fig.III-4. Destruction of Pfr after a R pulse in the dark
at in and 24°C-seedlings. 20°C- and
24°C-seedlings were irradiated with R pulse at time 0, then
kept at 24°C in the dark until harvest. A 10-mm section
which spans 15 mm to 25 mm below the node was excised from
the first internode, and 100 sections were placed
horizontally in a cuvette for spectroscopy. R: Rfl , 50 pmol
30 + 30 s. Plant heights of and
24°C-seedlings: 89 to 98 mm and 78 to 92 mm, respectively.
Solid and open circles, from Exp. 920618; triangles, Exp.
920619; squares, Exp. 920625. S. bicolor cv. Acme
Broomcorn, 1991 crop; seeds soaked by method 3.
80
be ing 120 min for both 20DC- and 2 4DC-seedlings. In
20DC-seedlings the destruction was found to be slower in
the initial 90 min than in 24DC-selings. This may be
ascribed to a possible delay in the temperature rise of the
sUbstratum for seedlings after transfer from 20DC to 24DC,
because the rate of the destruction of Pfr is greatly
retarded at low temperatures (Schl:ifer and Schmidt 1974).
The initial lag of Pfr destruction is already found in
monocoty ledonous seedlings (Sch~fer et al. 1975) and this
is not a unique case, although many other cases showed a
sharp decline according to the monomolecular reaction (cf.
Sch~fer and Schmidt 1974). The half-lives observed here in
both kinds of seedlings are much longer than 30 to 40 min
thus-far reported with Cucurbita ~ (Sch~fer and Schmidt
1974), Amaranthus caudatus (Heim et al. 1981, Brockmann and
Schafer 1982), and Cucumis sativus (Peters et al. 1991).
The reason for the discrepancies is not known. However, the
present results show that no significant difference of the
Pfr destruction was observed between and
2 4DC-seedlings.
Figure III-SA shows time courses of the escapes of Pfr
from FR reversion in 20DC- and 24°C-seedlings. The percent
escapes (Fig. 1II-5B) shows more clearly that there was no
difference between the two kinds of seedlings.
R fluence-dependent amplification by an 20°C-exposure
Fig. 111-6 shows fluence-response curves for R-induced
anthocyanin synthesis in 20DC- and 24°C-seedlings. In the
range of R fluence tested the reciprocity law held (data
81
0 A y{r-t---? LO ID 0.1 cx:: I 2/,? co N LO
cx:: ? z: 0.2 /' .-.----.-. .. z: /'----'--;0 c cx:: >-u b-e/ 0 = t-z: cx:: a
100 .,JI----~ • B
/~ N o •
w 50 . ?o a...
/" cx:: u en w
a a 6 12 18 24 30
PERIOD FROM R TO FR h )
Fig. 111-5. Escape of Pfr from FR reversion in anthocyanin
synthesis in 20°C- and 24°C-seedlings. A, actual amounts of
anthocyanin synthesis; B, percent of the respective
anthocyanin levels without FR. All open marks represent
20°C-Seedlings; solid marks, 24°C-seedlings; open circles,
from Exp. 900109; solid circles, EXp. 891219; triangles,
EXp. 890815; squares, Exp.890705 performed at Okazaki.
20°C- and 24°C-seedlings were irradiated with R at time 0,
then kept at 24°C tn the dark for 30 h until harvest,
during which FR was once given at the indicated times
except for 30 h. R: Rn , 40 pmol m- 2 S-I, 30 s + 30 s; FR:
FR-CF3024, 30 pmol m-2 S', 60 s + 60 s. Plant heights of
20°C- and 24"C-seedlings: 62.0 + 6.8 mm and 72.0 ± 8.5 mm,
respectively. S. bicolor cv. Acme Broomcorn,' 1987 crop;
seeds soaked. by method 1.
82
not shown). The enhancement by an exposure to 20°C
compared with an exposure to 24°C increased with increase
in R fluence, starting at a fluence of 20 to 50 pmol m-2•
Either curves in Fig.III-6 diverged from a straight line in
a log-linear plot (cf. Drumm and Mohr 1974), while in a
double log plot both curves gave straight lines, as
reported previously (Yatsuhashi et al. 1982).
Anthocyanin synthesis and PfrjPtot
Seedlings grown at 20°C and 24°C were irradiated with a
R pulse of various fluences, and the resulting PfrjPtot
values were determined, against which anthocyanin levels
obtained 24 h after the irradiations were plotted (Fig.
111-7). Likewise, anthocyanin levels resulting from
irradiations at various wavelengths were plotted against
PfrjPtot obtained with the same irradiations (Fig. 111-8).
The two curves thus-obtained were identical in general
shape. Figures 111-7 and 111-8 indicate that the
amplification
20°C-seedlings
PfrjPtot with
0.28. The
of anthocyanin synthesis occurs in
compared with 2 4°C-seedlings, depending on
null amplification at PfrjPtot of 0.13 to
curves for either 20°C- or 24°C-seedlings
consisted of two components having different slopes, gentle
at lower PfrjPtot and steep at higher PfrjPtot. The
gentle-sloped component corresponds with LFR, and the
steep-sloped one appears to arise from some other action
mechanism of phytochrome, which may be preferably called a
high fluence response (HFR), but not the so-called HIR.
83
o LO \0
c:x::
0.3
I 0.2 co N LO
c:x::
z: z: c:x:: >-~ 0.1 ::c Iz: c:x::
o 10
o
o
20 50 100 200 500 1000 FLUENCE ( JJ mo I / m2 )
Fig. 111-6. Effects of R photon fluences on anthocyanin
synthesis in 20°C- and 24°C-seedlings. R pulse was given
for the indicated periods of time. Anthocyanin was
determined 24 h after R. R: R-IF661, 10 pmol m-2 S-I. Plant
heights of 20°C- and 24°C-seedlings: 82.2 .±. 11.7 mm and
73.9 ~ 8.6 mm, respectively. S. bicolor cv. Acme
Broomcorn, 1987 crop; seeds soaked by method 1.
84
0.3 ,........
0 If)
~
c:::r.:
00 0.2 N If)
c:::c ...........
z ....... z: 0.1 c:::r.: >-u 0 :r: I-z: <t: ~-~
0 0 0.2
r -- --~ --(-~ , --, ," , L ...... ___ ___ ....
--r----~:;~ Lu_.".. __ u_,
'"
0.4 0.6
Pfr / Ptot
~
, ,
F6 .... ··-l·--· oJ
I , :----t----; 0L-! :.. .. !- .. - ----: ,
:~------; , , , ,
1---.... _ .. -...,._. , , i --.: ~ ____ L ___ .:
0.8
Fig. I II -7 . Anthocyanin synthes is re lative to Pf:r/Ptot
estalished with a R pulse of various photon fluences in
20°C- and 24°C-seedlings. Anthocyanin was determined 24 h
after R. A rectangle at each datum point represent
standard deviations of Pfr/Ptot and of anthocyanin
synthesis. Other explanations are the same as in Fig. 11-6.
85
0,4
".....
0 Lfl
0,3 \0 c:x:
co N Lfl
c:x: '-' 0,2 z: ....... z: c:x: >-u 0 ::c 0,1 I-z: c:x:
o o
Fig. 1II-8.
Pfr/Ptot
0,2 0,4
Pfr / Ptot
;78"·'· .-.-~ : I ; I I : 1./.. __ • __ .....
I I "--··_·1· ... .{-..
: I :
:--Q-r---J ; J I : '- ..... -- -i .......... J
I I
I
20°C / /
~~-~ .-... ;.:-.:2 ~~~ ~ .. ~ . .-... ~;
0,5 0,8
Anthocyanin synthesis relative to varied
established with a light pulse of various
wavelengths in 20°C- and 24°C-seedlings. Anthocyanin was
determined 24 h after R. A rectangle at each datum point
represent standard deviations of Pfr/Ptot and of
anthocyanin synthesis. Grey rectangles show the results
with a 10 s pulse. Other explanations are the same as in
Fig. II-3.
86
Effects of the pre-irradiation 20 D C treatment on action of
very low levels of Pfr
In order to examine the action of very low levels of Pfr in
anthocyanin synthesis of 20 D C- and 24 DC-seedlings, a
phytochrome-saturating f luence of R (50 pmol m-2 S-l for 10
min) was given followed by varied periods of FR (70 pmol
m-2 S-l). Effects of irradiations with FR alone for varied
periods of time were also tested. These irradiations are
based on the methods of Small et al. (1979) and VanDerWoude
(1980). In seedlings thus irradiated, the levels of Pfr are
too low to detect spectroscopically. A 10-min R pulse
alone gave anthocyanin of A528- 660 0.461 ..± 0.036 and 0.216
.±. 0.033 for 20 D C- and 24 DC-seedlings, respectively. When
the R was followed by varied periods of FR, anthocyanin
synthesis was minimum at 13 to 17 mmol m- 2 of FR, but never
reached null although non-irradiated control had null
anthocyanin, and increased slightly with extension of FR
pulse (Fig. 111-9). Corresponding f luences of FR pulses
alone also formed anthocyanin of very low levels, which
slightly increased with increased fluences FR. A test with
cv. Sekishokuzairai-Fukuyama also gave a similar result
(Fig. 111-10). In both cases anthocyanin synthesis was
definitly less in 20 DC than 24DC-seedlings.
Variation of VLFR with the development of seedlings
The capacity of anthocyanin synthesis varies with the
development of seedlings, peaking at a plant height of 70
to 90 mm (Chapter II). It was examined whether or not
anthocyanin synthesis induced at very low levels of
87
".... 30 o z :::> o 0::: l!)
~ 20 u c:(
1=0
I co N Lfl
ex: M I C> 10 r-I
><
2:
2: ex: >-g 5 = f-2: ex:
Sorghum bicoZor cv. Acme Broomcorn
l..t' All
" A A A
r-'~~ 24°C R-FR A A : . .....~ ..... -- .. A ', ..... - ____ A _------..P-A' .... , ___ --
-• --
0
-<I>
0
o
o
5
20°C R-FR
24°C FR
20°C FR
10
• o o
o
o o o
o
o
o
20
FR PHOTON FLUENCE ( mmol m-2 )
o
30
Fig. III-9. Anthocyanin synthesis induced by very low
levels of Pfr in 20°C- and 24°C-seedlings. Very low levels
of Pfr were produced with 10 min R (Rn, 50 pmol m-2 S-l)
followed by 1, 3, 4 and 6 min FR (FR-DelaA900, 70 pmol m-2
S-l) (open triangles for 20°C-seedlings, solid triangles
for 24°C-seedlings), and by the same FR alone (open circles
for 20°C-seedlings, solid circles for 24°C-seedlings) .
Anthocyanin was determined 24 h after R. The anthocyanin
levels induced by R alone were 0.461 ± 0.036 for
20°C-seedlings and 0.216 ± 0.033 for 24°C-seedlings, and
non-irradiated seedlings grown at 20°C or 24°C had no
anthocyanin at all. Plant heights of and
24°C-seedlings: 78.5 ± 8.2 mm and 77.5 ±11.1 mm,
respectinely. s. bicolor cv. Acme Broomcorn, 1991 crop;
seeds soaked by method 3.
88
Sorghum bicolor A cv. Sekishokuzairai-50 A Fukuyama
... ... .t 0 i ,
Z A ... ... :::l ""24°C R-FR 0 , A 0:: , (!)
, :><: Il ,
A U \ A A
« I A , ~- - -"\ A
~ I A Il' • t,
I A .' 10 Il \ ,
<Xl \ ,
N \ L() \
c::( I
'<I' \
I C>
I rl 20°C I >< ---, R-FR \
'-'
Z
Z c::(
>-u C> ::c I
l- I I Z
, c::(
, ,
5 10 20 FR PHOTON FLUENCE ( mmol m-2 )
Fig. III-l0. Anthocyanin synthesis induced by very low
levels of Pfr in S. bicolor cv. Sekishokuzairai-Fukuyama
seedlings grown at 20°C and 24°C in the dark. Very low
levels of Pfr were produced with 1 min R (Rn, 50 }lmol m-2
S-I) followed by 1, 2, 3, 4 and 5 min FR (FR-DelaA900, 46
}lmol m-2 S-I) (open tr iang les for 20°C-seedlings, solid
triangles for 24°C-seedlings), and by the same FR alone
(open circles for 20°C-seedlings, solid circles for
24°C-seedlings) • Anthocyanin was determined 24 h after R.
Anthocyanin levels induced by R alone were 0.197 + 0.044
for 2 DOC-seedlings and 0.051 + 0.010 for 24°C-seedlings
and non-irradiated seedlings grown at 20°C or 24°C had no
anthocyanin at all. Plant heights of and
24°C-seedlings; 123.1 + 7.2 mm and 103.1 + 8.0 mm,
respectively. Seeds, 1990 crop, soaked by method 2.
89
Pfr/Ptot varies in parallel with anthocyanin synthesis in
LFR. Seedlings of cv. Acme Broomcorn (Fig. 111-11) and of
cv. Sekishokuzairai-Fukuyama (Fig. 111-12) were grown at
20°C or 24°C to various plant heights, and were irradiated
with FR alone. In both cultivars 20°C- as well as 24°C
seedlings showed plant height-dependent var iations of the
responses similar to those for LFR (Chapter II), except
that 20°C-seedlings responded to the very low level of
Pfr/Ptot with less anthocyanin synthesis at all stages
(plant heights) tested.
The responsivity in VLFR of Sekishokuzairai-Fukuyama
was noted to be poorer than in cv. Acme Broomcorn, although
both cultivars formed almost the same amount of anthocyanin
in LFR (See also Figs. 111-9, 111-10). In particular, the
response of 20°C-seedlings of the former cuI tivar was very
poor, making greater the difference in response between
20°C- and 24°C-seedlings. Thus, these results show that
VLFR of anthocyanin synthesis is distinguished by the
opposite effects of the preirradiation MLT, and that it is
not a phenomenon restricted to a particular developmental
stage, although its extent of expression may vary among
cuI tivars. This is the first report in the literature to
describe the occurrence of a VLFR distinct from LFR in
phytochrome- mediated anthocyanin synthesisis, although
data are available to show anthocyanin synthesis at very
low levels of Pfr/Ptot or by FR (Drumm and Mohr 1974; Small
and Pecket 1982).
Since the data in Fig. 111-9 show that the photo
equilibrium established by FR seems to be the same as that
90
,,-...
~ z ::> 0 a::: (!J
~ u ~
a::l
00 N lJ)
c:::r:: M I C> ~
>< '-'
z ....... z c:::r:: >-u C> ::c r-z c:::r::
10
8
6
4
2
Sorghum bicolor -L ev. Acme Broomcorn
20°C
o 40 60 80
PLANT HE I GHT mrn
Fig. III-110 Variation of FR-induced anthocyanin synthesis
with the development of S. bicolor cv. Acme Broomcorn
seedlings grown at 20°C and 24°C in the dark. FR:
FR-DelaA900, 65 }lmol m-2 S-l, 180 s. Seeds, 1991 crop,
soaked by method 2.
91
........
0 Z ::J 0 oc: l!) ~ u <t co
00 N lJ)
c:::c M I 0 ...-i
>< '-"
z -z c:::c >-u 0 :::c I-z c:::c
4
2
0
Sorghum bicoZor cv. SekishokuzairaiFukuyama
~ 20°C
_~-Y,,\ I -i--i-
40 60 80 100 120 140
PLANT HEIGHT ( mm )
Fig. III-12. Variation of FR-induced anthocyanin synthesis
with the development of S. bicolor cv.
Sekishokuzairai-Fukuyama seedlings grown at 20°C and 24°C
in the dark. Seeds, 1990 crop, soaked by method 2.
Irradiation conditions and other explanations are the same
as in Fig. 11-11.
92
established by R + 17 mmol m- 2 FR, the increased FR
irradiation must maintain the same photoequilibria in R +
FR as well as FR alone, and the slight increases in
anthocyanin levels by the increased FR are the subject of a
future paper. When the curves for R + FR with 20°C and
24°C-seedlings cross over (F ig. III -9) or co- incided (F ig.
111-10), respectively at about 6 and 3 mmol m~ of FR after
R, it is probable that amplification of LFR and suppression
of VLFR in 20°C-seedlings were compensatory. It is very
likely that the PfrjPtot ratio at this point may be 0.13 to
0.28, where the curves for LFR in 20°C- and 24°C-seedlings
also crossed (Fig. 111-5, 1II-7, 111-8), and this point may
be the transition point between LFR and VLFR.
93
Discussion and conclusions
In the literature few papers describe on the photon
f luence- or f luence rate-response at LT or MLT. Wall and
Johnson (1982) reported that in the growth inhibition of
Sinapis alba seedlings under continuous R the f luence rate
dependency disappeared at MLT, the effectiveness of the
light being raised at lower fluence rates. In Lactuca
sativa and Arabidopsis thaliana seeds cold inbibition
raised the sensitivity of seeds to VLF's of R, giving a
biphasic fluence-response curve (VanDerWoude 1985, Cone et
al. 1985).
Similar sensitization to R was also observed following
treatments by other means. Lactuca sativa seeds made
dormant by a high temperature treatment (Small et al.
1979), Kalanchoe blossfeldiana seeds treated by gibberellin
or KN0 3 (De Petter et al. 1985) and Dryopter is f ilix-mas
spores treated with nitrates (Haas and Scheuerlein 1990)
germinated in response to R of 4 to 5 orders of magnitude
lower fluences than the fluences effective without such
sensitization treatments. Red light-induced growth
promotion of Avena sativa coleoptiles was also caused by 4
orders of magnitude less intense light, if the seedlings
were treated with indoleacetic acid (Schinkle and Briggs
1984) • All these sensitizations are such that the
fluence-response curves plotted on semi-log scale axes are
shifted toward lower fluence.
The enhancement of R-induced anthocyanin synthesis in
sorghum by the pre-irradiation MLT is completely different
94
from the above-cited sensitizations. Anthocyanin synthesis
was amplified at the same range of photon fluence without a
downward shift of the effective range of photon fluence
(F ig. III -6), and thus, the enhancement of R effect was
observed at the photon fluence range of LFR, but not at
that of VLFR (Figs. 111-11, 111-12). In VLFR an exposure
to 20°C resulted in the opposite effect to that in LFR,
i.e. the anthocyanin synthesis was signficantly less in
20°C- than 24°C-seedlings.
VanDerWoude (1985) has proposed that phytochrome
endogenously acts as dimers of identical subunits, PrPfr
and PfrPfr for VLFR and LFR, respectively. At such low
f luences of R as 10-3 to 1 pmol m-2 or at the photo
equilibrium established by FR, a phytochrome dimer exists
as heterodimer PrPfr, and at higher f luences than 10 pmol
m-2 (LFR) it assumes a form of homodimer PfrPfr (Small et
al. 1979, Cone et al. 1985, De Petter et al. 1985). Each
dimer binds a putative receptor X before it can act
(VanDerWoude 1985). Although the endogenous amount of X is
assumed to be limited, the active phytochrome complex can
exert full action if the seeds of Lactuca sativa,
Arabidopsis thaliana and Kalanchoe blossfeldiana are fully
sensitized by prechilling, KN03 , or gibberellin treatments.
However, the maximum responses vary depending on the extent
of sensitization. In Avena coleoptile growth promotion,
also, the extent of the action of PrPfr-X likewise depends
on auxin concentrations (Shinkle and Briggs 1984). Even in
partial sensitizations the effective photon f luence range
does not vary, thus giving biphasic photon fluence response
95
curves for VLFR and LFR.
The present results with R-induced anthocyanin
synthesis in sorghum are that the pre-irradiation MLT
treatment only enhances it at the photon f luence range of
LFR, but does not "sensitize" it, and even suppresses VLFR.
Since in both VLFR and LFR one and the
anthocyanin 1S formed, it is very likely
same species of
that a single
biosynthetic sistem is functional, and the presence of two
distinct biosynthetic systems for VLFR and LFR is unlikely.
It is, therefore, tempting to speculate that PrPfr and
PfrPfr are responsible for VLFR and LFR, respectively, and
these two active forms of phytochrome dimer bind the
receptor X before these two X-conjugates transmit their
signals to the next step in the signal transduction chain.
The pre- irradiation 20°C treatment suppresses the aff ini ty
of the signal transduction chain to PrPfr-X, and enhances
that to PfrPfr-X compared with 24°C treatment. The
f luence-response curves for 20°C- and 24°C-seedlings cross
at a R photon f luence or R + FR to satisfy the following
equation:
PrPfr-X/PfrPfr-X = (A2o - A2.)/(a24 - a20)
where A20 and A24 represent the PfrPfr-X aff ini ties of
20°C- and 24°C-seedlings; and an and au,
affinities for 20°C- and 24 cC-seedlings.
point was found at 20 to 50 pmol m-2 (Fig.
the PrPfr-X
The crossing
III -6), where
the Pfr/Ptot determined was 0.13 to 0.28 (Fig. 111-7,
III -8), and correspcnding points are also seen in Figs.
111-9, 111-10. The aff ini ties may be assumed to be such
entities as the amounts of two different transmitters Y and
96
y for PfrPfr-X and PrPfr-X, respectively, adopted according
to VanDerWoude (1985) although he assumes a single
transmi tter Y. It may then be possible to state that Y is
larger and y is smaller in 20°C- than 24°C-seedlings (F ig.
111-13).
Fluence-dependent magnification of LFR
In LFR, anthocyanin synthesis was increased by
additional R irradiation after the maximum Pfr/Ptot was
established. This is the case not only with 20°C- but also
24°C-seedlings with greater increases in the former than
the latter (Figs. 111-7, 111-8). According to Drumm and
Mohr (1974) and Hecht and Mohr (1990) anthocyanin synthesis
and enzyme synthes is are linear against Pfr /Ptot, but the
curves for anthocyanin synthesis in 20°C- and
24°C-seedlings are diverged upward even before Pfr/Ptot
reaches the maximum (Figs. 111-7, 111-8). Similar upward
divergence of photoaction in LFR has been described with
the hypocotyl extension of Sinapis alba (Wall and Johnson
1982). We refer to this phenomena as a high fluence
response (HFR). In order to explain this we assume that the
cycling of phytochrome between PfrPfr and PrPfr may exert
an additional action to enhance the phytochrome signal
transduction, and the cycling occurs not only after the
Pfr/Ptot is maximum, but also even before, though to much
lesser extent. This effect of cycling is also assumed to be
expressed more in 20°C- than in 24°C-seedlings.
VanDerWoude (1987) assumes cycling between PrPfr-X and
PrPr-X for so-called high irradiance response, but Hecht
97
PrPr-X -.... ---II~- PrPfr-X -... -----~- PfrPfr-X
ANTHOCYANIN SYNTHESIS
Fig. III-13. A scheme to explain VLFR and LFR in
phytochrome mediated anthocyanin synthesis of ~Q_rghum
bicolor.
98
and Mohr (1990) cast a doubt on this concept which assumes
qnly one kind of receptor X. More detailed evidence for and
discussions on our view on HFR will be presented in the
next paper.
In conclusion, the present investigation indicated that a
pre-irradiation MLT treatment of sorghum seedlings did not
affect the content and behaviour of phytochrome itself, but
it affected phytochrome-mediated anthocyanin synthesis in
different ways depending on the R fluences. Analyses of the
effects on phytochrome actions over a wide range of
f luence-response curves indicated the presence of distinct
VLFR, LFR and HFR in light-pulse-induced anthocyanin
synthesis. HFR does not refer to so-called HIR, but to
additional phytochrome action surpassing the action that
would be given by PfrjPtot. VLFR and LFR are well
explained by the dimeric model of phytochrome, but require
an involvement of multiple signal transmitters in the
primary step of the signal transduction chain even if only
anthocyanin synthesis' is taken into account as phytochrome
action. HFR may be explicable by cycling of phytochrome
between PrPfr and PfrPfr.
99
References
Briggs, W.R., Siegelman, H.I'/. (1965) Distribution of
phytochrome in etiolated seedlings. Plant Physiol., 40,
934-941
Brockmann, J., Schafer, E. (1982) Analysis of Pfr
destruction in Amaranthus caudatus L.- Evidence for two
pools of phytochrome. Phtochem. Photobiol. 35, 555-558
Brockmann, J., Riehle, S., Kazarinova-Fukshansky, N.,
Seyfried, H., Schafer, E. (1987) Phytochrome behaves as
a dimer in vivo. Plant, Cell and Environment 10,
105-111
Butler, W.L., Norris, K.H., Siegelman, H.W., Hendricks, S.B.
(1959) Detection, assay, and preliminary purification of
the pigment controlling photoresponsive development of
plants. Proc. Natl. Acad. Sci. USA, 45,1703-1708
Cone, J.W., Jaspers, P.A.P.H., Kendrick, R.E. (1985)
Biphasic fluence-response curves for light induced
germination of Arabidopsis thaliana seeds. Plant. Cell
and Environment 8, 605-612
De Petter, E., Van Wiemeersch, L., Rethy, R., Dedonder, A.,
Fredericq, H., De Greef, J., Steyaert, H., Stevens, H.
(1985) Probit analysis of low and very-low fluence
responses of phytochrome-controlled Kalanchoe
blossfeldiana seed germination. Phytochem. Phytobiol.
42, 697-703
100
De Petter, E., Van Wiemeersch, L., Rethy, R., Dedonder, A.,
Fredericq, H., De Greef, J. (1988) Fluence-response
curves and action spectra for the very low fluence and
the low fluence response for the induction of Kalanchoe
seed germination. Plant Physiol. 88, 276-283
Drumm, H., Mohr, H. (1974) The dose response curve in
phytochrome-mediated anthocyanin synthesis in the
mustard seedling. Photochem Photobiol. 20, 151-157
Frankland, B. (1972) Biosynthesis and dark transformations
of phytochrome. In: Phytochrome, pp.195-225, Mitrakos,
K., W. Shropshire, Jr., eds. Academic press London
Haas, C.J., Scheuerlein, R. (1990) Phase-specific effect of
nitrate on phytochrome-mediated germination in spores of
Dryopteris filix-mas L. Photochem Photobiol. 52, 67-72
Hada, M., Tada, M., Hashimoto, T. (1992) UV-B light-induced
absobance changes in the yeast Rhodotorula minuta.
J. Photochem. Photobiol. B: BioI. (in press)
Hashimoto, T., Ito, S., Yatsuhashi, H. (1984) Ultraviolet
light-induced coiling and curvature of broom sorghum
first internodes. Physiol. Plant. 61, 1-7.
101
Hecht, U., Mohr, H. (1990) Relationship between phytochrome
photoconversion and response. Photochem. Photobiol. 51,
369-373.
He i m . B., Jab ben, M., S c hlA fer, E . (1981) Ph y t 0 c h rom e
destruction in dark- and light-grown Amaranthus
caudatus seedlings. Photochem. Photobiol. 34, 89-93.
Jabben, M., Beggs, C., Sch~fer, E. (1982) Dependence of
Pfr/Ptot-ratios on light quality and light quantity.
Photochem. Photobiol. 35, 709-712
Kelly, J.M., Lagarias, J.C. (1985) Photochemistry of
124-kilodalton Avena phytochrome under constant
illumination in vitro. Biochemistry 24, 6003-6010
Kondo, N., Inoue, Y., Shibata, K. (1973) Phytochrome
distribution in Avena seedlings measured by scanning a
single seedling. Plant Science Letters, 1, 165-168
Moroz, S.M., Alford, E.A., Johnson, C.B. (1984) Effects of
temperature on the development of Sinapis alba L.;
phytochrome-control of nitrate reductase activity at
10 g C. Plant, Cell and Environment 7, 45-51
Peters, J.L., Kendrick, R.E., Mohr, H. (1991) Phytochrome
content and hypocotyl growth of long-hypocotyl mutant
and wild-type cucumber seedlings during de-etiolation.
J. Plant Physiol. 137, 291-296
102
Sch~fer, E., Schmidt, 'rI. (1974) Temperature dependence of
phytochrome dark reactions. Planta 116, 257-266
Sch~fer, E., Lassig, T.-U., Schopfer, P. (1975) Phtocontrol
of phytochrome destruction in grass seedlings. The
influence of wavelength and irradiance. Photochem.
Photobiol. 22, 193-202
Sch~fer, E., Apel, K., Batschauer, A., Mosinger, E. (1986)
The molecular biology of action. In: Photomorphogenesis
in Plants, pp.83-98, Kendrick, R.E., Kronenberg, G.H.M.
eds. Martinus Nijhoff Publishers, Dordrecht
Shinkle, J .R., Briggs, W.R. (1984) Indole-3-acetic acid
sensitization of phytochrome-controlleed growth of
coleoptile sections. Proc. Natl. Acad. Sci. USA 81,
3742-3746
Small, C.J., Pecket, R.C. (1982) Change in sensitivity to
far-red irradiation on anthocyanin biosynthesis in red
cabbage seedlings. Plant, Cell Environ. 5, 1-4
Small, J.G.C., Spruit, C.J.P., Blaauw-Jansen, G., Blaauw,
D.H. (1979) Action spectra for light-induced germination
in dormant lettuce seeds. I. Red Region. Planta 144,
125-131
VanDerWoude, 'rI.J., Toole, V.K. (1980) Studies of the
mechanism of enhancement of phytochrome-dependent
lettuce seed germination by prechilling. - Plant
Physiol. 66, 220-224 103
VanDerWoude, W.J. (1985) A dimeric mechanism for the action
of phytochrome: Evidence from photothermal interactions
In lettuce seed germination. Photochem. and Photobiol.
42, 655-661
VanDerWoude, W.J. (1987) Application of the dimeric model
of phytochrome action to high irradiance responses. In:
Phytochrome and Photoregulation in plants, pp.249-258,
Furuya, M. ed. Academic Press, Tokyo
Wall, J.K., Johnson C.B. (1982) The effect of temperature
on phytochrome controlled hypocotyl extension in Sinapis
Alba L. New Phytol. 91, 405-412
Yatsuhashi, H., Hashimoto, T., Shimizu, S. (1982) UV action
spectrum for anthocyanin formation in broom sorghum
first internodes. Plant Physiol. 70, 735-741
Yatsuhashi, H., Hashimoto, T. (1985) Multiplicative action
of a UV-B photoreceptor and phytochrome in anthocyanin
synthesis of broom sorghum seedlings. Photochem.
Photobiol. 41, 673-680
104
Chapter IV: Storage of red light signal for anthocyanin
synthesis in etiolated Sorghum bicolor seedlings
Abstract
Red light (R) pulse-induced anthocyanin synthesis in
etiolated Sorghum bicolor seedlings did not plateau, but
increased at a reduced s lope with increase in the photon
f luence of R even after phytochrome reached the maximum
Pfr/Ptot ratio. It also increased with increasing numbers
of alternations of R and far-red light (FR) pulses despite
complete FR reversal, if the irradiation was terminated by
a R pulse. These phytochrome paradoxes led to assume the
storage of R signal, 0- , which is generated by Pr to Pfr
photoconversion and manifests itsslf by a multiplicative
coaction with Pfr or PfrPfr. Exper imental data are
presented to indicate that 0- is generated depending on the
R fluence in a wide range, and the action of a fixed amount
of 0-' is expressed depending on the amount of Pfr or
Pfr/Ptot ratio. The assumption of 0-- may explain the
fluence rate dependency of R action not only in the low and
high f luence responses but also in the action at the red
waveband of the so-called high irradiance response.
105
Introduction
In red light-induced photomorphogenesis in plants
extensive work on the role of phytochrome has been done and
its s ignif icance established. Dur ing the course of these
studies several paradoxes have been pointed out on the
functions of phytochrome (Hillman 1972), some of the
paradoxes are settled and others not. A paradox is an
indication of the incompleteness of the current theory, and
settling a paradox may lead to improve our current
understanding.
In Chapter III, we have described another paradox, i.e.
in a pulse irradiation R-induced anthocyanin synthesis of
etiolated Sorghum bicolor seedlings increased with increase
in the fluence of R even after the maximum Pfr/Ptot ratio
is established;
the number of
effect of a
also, anthocyanin synthesis increased with
alternations of Rand FR pulses with the
R pulse being maximally nullified by a
subsequent FR pulse in each alternation. Analysis of the
paradoxical phenomena led us to assume that each time when
phytochrome Pr (or PfrPr) is converted to Pfr (or PfrPfr),
it stores R signal in a storage form, proposed to refer to
as ~, which is insusceptible to FR, and hence the storage
of the signal continues to increase through cycling of
phytochrome even after the maximum Pfr/Ptot ratio is
established; the 0-' manifests its signal by a coaction of
Pfr (or PfrPfr). This chapter describes experimental data
to support the assumption, and discuss its significance in
measuring photon fluence and fluence rate of light in the
106
low fluence response as well as in the high irradiance
response.
In most experiments in this chapter, both seedlings
grown at 20°C and 24°C are used. It is because in
20°C-seedlings the R response is amplif ied compared with
24°C-seedlings (chapter II), and the use of 20°C-seedlings
first directed our attention to the phenomena dealt with
here, although experiments have disclosed that the storage
of R signal occured equally irrespective of the
pre-irradiation culture temperature.
107
Materials and Methods
Plant mater ials. Seeds of broom sorghum, Sorghum bicolor
Moench, cvs. Acme Broomcorn and Sekishokuzairai-Fukuyama
were used. Acme Broomcorn was grown and seeds were
harvested at the experimental farm, the Aburahi
Laboratories, Shionogi Pharmaceutical Co., Aburahi, Shiga
in 1987, and at the Experimental Farm of the Faculty of
Agriculture, Kobe University, Kasai, Hyogo in 1991;
Sekishokuzairai-Fukuyama was grown and seeds were harvested
at Aburahi in 1990.
Seeds were soaked in tap water adjusted to 24°C by
Method 3 except that Method 2 was used in Figs. IV-2 and
IV-4. The soaking methods were described prev ious ly
(Chapter II). From sowing to irradiation, seedlings were
grown in the dark either at 20 0 ± l°C for 115 to 125 h or
24 0 .±. PC for 72 to 80 h (referred to as 20°C- or
24°C-seedlings, respectively) for both to become 70 to 95
mm tall (length from seed to the coleoptile tip), and from
irradiation to harvest all seedlings were kept in the dark
at 24°C. The details of soaking, culture, irradiation and
other procedures were described previously (Chapter II).
Light sources and irradiation. For irradiation, red light
(R: Rfl and R-IF661), far-red light (FR: FR-CF3024 and
FR-DelaA900) and UV-B light (UV310-U330-N) were mostly used
(Chapter II). In some exper iments, 660 nm light from the
Large Spectrograph of the National
Biology at Okazaki (Watanabe et al.
Institute for
1982) was used,
Basic
being
referred to as so. Various fluence rates were obtained with
108
neutral density filters having 0.39, 0.85, 2.7, 11.2, 25.7,
29.9 and 45.0 % transmittances (Yatsuhashi and Hashimoto
1985). Irradiation with the broad band UV-B was always
followed by an adequate f luence of FR to se lect only UV- B
effects, negating
Yatsuhashi 1984,
phytochrome
Yatsuhashi
action (Hashimoto and
and Hashimoto 1985) .
Irradiation was performed from horizontal direction.
Anthocyanin determination. Anthocyanin was extracted with
1% hydrochloric acid-methanol 24 h after irradiation, and
was determined by absorbance at 528 nm unless otherwise
stated. The details of the procedure were described
previously (Chapter II and III).
Phytochrome determination. Sections of 10 mm long were
excised from the first internode, and A ( A A730 - 66o ) was
determined at the middle part of the sections. The details
of the procedure were described previously (Chapter III).
109
Results
Possible storage of R signal after FR. Figure IV-l shows
f luence-responce curves for a R pulse- induced anthocyanin
synthesis of the first internode of etiolated sorghum
seedlings grown at 20°C and 24°C. Anthocyanin synthesis
increased with increase in R fluence even over 500 to 1000
)lmol m-2 which gave the maximum Pfr /Ptot ratio (Chapter
III). It is true of both 20°C- and 24°C-seedlings.
Greater amounts of anthocyanin were formed in 20°C- than
24°C-seedlings, conf irming our prev ious results. The curves
appear biphasic with inflection points at ca. 700 and 2500
pmol m-2•
The action observable at R f luences higher than the
phytochrome-saturating fluence is defined high fluence
response (HFR) (also, cf. Chapter III), and it can be
assumed that the R signal is stored in a form distinct from
Pfr, tentatively named 0- mediated by Pr to Pfr
photoconversion and 0- is not active by itself, but be
expressed in the presence of Pfr (probably PfrPfr, see
Discussions).
In order to make the R signal storage more substantial,
various fluences of R were given, reverted with a
saturating FR, and then a R pulse of 1 mmol a
saturating minimum fluence of R, was given at the end of
the irradiation. Such irradiation regimes are subsequentlly
indicated as Rivar -FR-R2 • Resulting anthocyanin synthesis
increased with increase in the f luences of the Ri pulses
despite R2 being the same (Fig. IV-2). The reciprocity law
held. It is clear that the effects of Rim were not due to
110
0.4 r-------------------------------------------~o~
o l!)
d' 0.3
'-' 0.2
z z c::r:: >u §E 0.1 Iz c::r::
o
100 1000 R FLUENCE ( )Jmol m-2 )
Fig. IV -1. Effects of R f luences on anthocyanin synthes is
in etiolated broom sorghum seedlings grown at 20 DC and
24DC. R: 660 nm light, at 1.0, 3.2, 13.4, and 35.9 pmol m-2
S-1 X 20 s each and at .120 pmol m-2 S-1 X 20, 60 and 200 s
for 20DC-seedlings; at 1.1, 3.5, 14.6 and 38.9 pmol m- 2 S-1
X 20 s each and at 130 }lmol m-2 S-1 X 20, 60 and 200 s.
Plant height at the time of irradiation of 20DC- and
24DC-seedlings: 91.3 ± 6.9 mm and 76.4 ± 3.5 mm,
respectively. s. bicolor cv. Acme Broomcorn, 1987 crop.
111
~ 0.30 1.0
c:::c I OJ N LI'l
c:::c 0.10
z
~ 0.05 >u
~ 0.03 z c:::c
1--
I'
I
1000
Rl FLUENCE
I
10.1000 ( ,umol m-2 )
Fig. IV -2. Effects of various fluences of the first R
pulse (Rim) on 0- generation in the multiple irradiation
The amount of 0-
was manifested as anthocyanin synthesis by R2 of a definite
fluence (----). The responses to only Rlvar are shown by
solid lines, and those to FR-R2, by the bars on the left
side of the figure (upper and lower bars, respectively, for
20 DC- and 24DC-seedlings). Anthocyanin levels given by
Rlvar-FR were less than 0.01 in absorbance unit. Open and
solid symbols represent the data with and
24DC-seedlings, respectively. R1var, R (Rn), 14 pmol m-2
S-1 X 20, 180 and 600 s o and. for R1nr-FR-R2 and
6. and .. for Rim) and 50 jlmol m-2 S-1 X 20, 180 and 600 s
({ and. o and. for R1var );
R2 , R(Rn) 50 ,umol m--2 S-1 X 20 s; and FR, FR (FR-CF3024),
30 pmol m-2 S-1 X 180 s. Plant height at the time of
irradiation, 88-93 mm and 85-108 mm for 20 DC- and 24DC-
seedlings, respectively. S. bicolor cv. Sekishokuzairai-
Fukuyama, 1990 crop. Seeds soaked by method 2.
112
incomplete reversion by the FR, since the FR was saturating
(see Fig. IV-4). In a double log plot, the curves for
R1nr-FR-R2 is linear against the Rl fluences, and the
slopes of the curves (for 20°C- and 24°C-seedlings)
corresponded with those of the curve parts at higher Rl
f luences than 2500 !mOl where phytochrome was to be
saturated. The curves for 20°C- and 24°C-seedlings had an
identical slope, although the response of 20°C-seedlings
was greater than that of 24°C-seedlings.
In order to exclude the possible involvement of other
light than R, which might be contained as a trace
contaminant in the R source, similar experiments were
repeated with 660 nm light supplied from the Okazaki Large
Spectrograph, and gave virtually an identical results (Fig.
IV-3), excluding the above possibility.
Storage of R signals in repeated alternations of Rand FR
pulses. Various numbers of red light pulses of a
satuarating f luence, 3 mmol m-2 each, were applied with a
saturating FR pulse interposed. The results show that the
signal of each R pulse was accumulated although the FR
reversion was maximum each time (Fig. IV-4A). Seedlings
grown at 20°C responded with greater anthocyanin synthesis
than 24°C-seedlings. When we closely examined the very low
anthocyanin levels observable if the irradiation was
terminated with FR (Fig. IV-4B), accumulations of very low
fluence responses (VLFR) were also noted. It is clear,
however, that the accumulation of VLFR does not account for
the above-stated accumulation of R signal, because the
113
1,OOr-----------------------------------------------------~
(Xl
N U1
ex::
'-' 0.10
z z ex:: >u o = I-z ex::
R t Rlvar-FR-R2 --
2 A_ --.,.----- A- ------ ------.-FR-R2
R2
FR-R2t- ----t------
Rl FLUENCE ( )Jmol rrr2 )
Fig. IV-3. Effects of various fluences of the first R pulse
(R1m ) on 0- generation in the multiple irradiation
R1var-FR-R2. The amount of 0- was manifested as anthocyanin
synthesis by R2 of a constant fluence (-----); the
responses to R1m alone are shown by solid curves; those
to FR-R2 and to R2 are shown by the horizontal bars on the
left side of the figure (upper and lower bars,
respectively, for 20°C- and 24°C-seedlings). R1var : 660 nm
light, 0.67, 1.4, 3.5, 12.7, 31.2, -35.8, 58.3 and 133.0
pmol m-2 Sl X 60 s, R2 : 660 nm light, 1040 pmol m 2 and
FR: FR-DelaA900, 65 umol m-2 S-l X 180 s. Plant height at
the time of irradiation, 91 ±.. 6.8 mm and 71.2 .±.. 6.1 mm for
20°C- and 24°C-seedlings, respectively. Datum points are
means ± S.D. 's (n=4). S. bicolor cv. Sekishokuzairai
Fukuyama, 1990 crop ..
114
24°C-SEEDLINGS R R.:.FR R-FR-R R-FR-R-FR R-FR-R-FR-R R-FR-R-FR-R-FR
20° C- SEEDLI NGS R R-FR
~
~
~
+ A
-+-
--=i-
-+-
R-FR-R R-FR-R-FR R-FR-R-FR-R R-FR-R-FR-R-FR
-t-
-=::t-
I I I
0 0.1 0.2 0.3 0.4
24"C-SEEDLINGS ANTHOCYANIN ( AS28 - A6s0 )
FR B R-FR
R-FR-R-FR R-FR- R- FR- R-FR
, 20°C-SEEDLINGS FR R-FR R-FR-R-FR R-FR-R-FR-R-FR
0 0.01 0.02 ANTHOCYANIN ( AS28 - BACKGROUND )
Fig. IV -4. Effects of repeated alternations of Rand FR
on anthocyanin synthesis in 20°C- and 24°C-seedlings (A),
and 10 times magnification of the bottom part of A (B).
R: Rn, 50 )lmol m-2 S-1 X 60 s; FR: FR-DelaA900, 42 }lmol
m- 2 S-1 X 180 s. Plant height at the time of irradiation,
85.0 + 7.1 mm and 87.8 ± 7.8 mm, for 20°C- and
24°C-seedlings, respectively. A short bar on each thick
bar, .± S.E. (n=5 or 6). S. bicolor cv. Sekishokuzairai-
Fukuyama, 1990 crop. Seeds soaked by method 2.
115
anthocyanin levels due to the VLFR were extremely low, and
furthermore, they were lower in 20°C- than 24°C-seedlings
in contrast to the cases of the R signal accumulation.
The storage of R signal was observed with a very short
period (10 s) of 660 nm light (133 Jlmol m- 2 S-1) from the
Large Spectrograph, indicating that the reaction of R
signal storage is very fast (Fig. IV-5).
Capacity of Sigma generation along with the developmental
stages of seedlings. Applying a regime R1-FR-R2 to --~~----------------=-
seedlings at various developmental stages, the capacity of
S generation was followed (F ig. IV-6). The capacity of
anthocyanin synthesis varied along with the development of
seedlings as represented by plant height, as shown by the
curves for R1 alone or R2 alone, but the ratios of
anthocyanin induced by R1-FR-R2 and that induced by R2
alone were almost constant (almost parallel curves in a
double-log plot), implying that the capacity of~generation
stayed almost constant throughout the developmental stages
tested. This was true of both 20°C- and 24°C-seedlings.
The phytochrome content of the lower part of the
internode involved in anthocyanin synthesis (Chapter III)
was also constant during the developmental stages of
seedlings tested, although the phytochrome content of the
upper part of the internode which was shown to be less
involved in anthocyanin synthesis was varied (Fig. IV-7).
The correspondence of the constant 0- generation with the
constant phytochrome content along with the development of
seedlings may have some significance.
116
24°C-SEEDLINGS R
FR-R
R-FR-R
20°C-sEEDLINGS R
FR-R
R-FR-R
o· b
~ .
o I I
0,1
ANTHOCYANIN
• I •
• J • • I-
1 I I
0,2
( AS28 - A6s0 )
Fig. IV-5. Effects on ~ generation of a short (10 s) pulse
of R followed by FR within ca. 10 s. The generation of
cr- is represented by the difference between R-FR-R and
FR-R. R: 660 nm light, 133 pmol m- 2 S-I X 10 s; FR:
FR-DelaA900, 65 }lmol m-2 S-I X 180 s. Plant height at the
time of irradiation, 91.1 ± 6.8 mm and 71.2 ± 6.1 mm, for
20°C- and 24°C-seedlings, respectively. s. bicolor cv.
Sekishokuzairai-Fukuyama, 1990 crop.
117
I 0,3
RI-FR-R2.1 20"( 0.4 8~
......... Rl.l 20"( " ~~ 0 0.3
R2, 20·C ~ It')
\0 <I:
I 0-------0 ',~ co Rb 24" ___ ~ N It') 0.2 <I: ..... --~ ... -",
'-" -- , RI-FR-R2.1 24"( ............ z .. ........ .-----<, 24·C z <I: >-W 0 ::c .-z 0.1 <I:
60 70 80 90 100 110
PLANT HEIGHT (mm )
Fig. IV-6 . Ubiquitous r;--- production irrespective of the
developmental stages of the seedlings. Seedlings were grown
at 20°C and 24°C in the dark for various periods, hence to
various heights, and was generated by high f luence R\,
and manifested as anthocyanin synthesis by low f luence R2
in the regime R\-FR-R2 (------). R\: Rn, 45 umol m~2 S~1
X 600 s; FR: FR-DelaA900, 65 pmol m~2 s~\ X 180 s; R2 :
Rn, 45 pmol m 2 S~1 X 30 s. S. bicolor cv. Acme Broomcorn,
1991 crop.
118
r-l I
Z 0
8 ....... I-u UJ (/)
M I o~ C> r-I 0
>< " ~ 4 0
/. .. 1.0 e 1.0 ee I e-e-- e_e
0 e e e M e I'
c::(
-5 7 <J a
50 100 150 PLANT HEIGHT ( mm )
Fig. IV -7. Variation of phytochrome content with the
development of etiolated seedlings. Total phytochrome
contents at 5 mm (0) and 15 mm (.) below the coleoptilar
node were followed as the seedlings grew at 24°C.
Consecutive 2 parts 'of 10-mm sections were excised from the
top part of the first internode and were subjected to
spectroscopy. ~. bicolor cv. Acme Broomcorn, 1991 crop.
119
Decay of Ov Seedlings grown at 20°C and 24°C were
subjected to an irradiation regime of Rl-FR-(x h)-R2, where
Rl was a pulse of an excessive f luence (31 to 32 mmol m-2)
and FR, a pulse of saturating fluence (70 umol m-2 SI X
180 s). After Rl followed by FR was given, the plants were
kept in the dark at 24°C for various periods before R2
slightly over the saturating fluence (1.4 to 1.5 mmol m-2)
was given (Fig. IV-8). Resulting anthocyanin was
determined 24 h after R2 • In control regime, FR-(x h)-R2 ,
FR was given at 0 time and a same R2 pulse was given in
parallel during the experimental period. The remaining
(1tw may be estimated as percent of the amount at time 0 by
the following equation:
[Rl-FR-(x h)-R2 J _ 1 [FR - (x h) - R2 J
Xloo [Rl-FR- (0 h) -R2 J
- 1 [FR-(O h)-R2 J
where [ J means the anthocyanin synthesis caused by the
indicated treatment. In 24°C-seedlings 0- was kept without
loss at least for ca. 1 h, then decayed with a half-life of
ca. 3 h to disappear after 6 h. In 20°C-seedlings 0- was
more stable than in 24°C-seedlings, survived till after ca.
3 h, and some remained after 6 h.
Multiplicative coaction of ~ with Pfr. Seedlings grown at
24°C were subjected to an irradiation regime Rl-FR-R2var,
and effects of a fixed amount of 0..... to be generated by Rl
on the actions caused by various amounts of Pfr or PfrjPtot
ratios were examined. For control without Rl , FR-R2var was
120
Rl alone • 20°C-SEEDLI NGS • 013 •
~!----! l-FR- (x h) -R2 • • •
0
........ 0.2 0
0 0 ________ 8 lfl ----8 0 \D c::t:
0
I
~ 0.1 0
Ifl c::t: 0
0 '-'
z 0
Rl-FR z c::t: • 24°C-SEEDLINGS >- • 1..J 0
~~ :I: r z
Rl alCOne c::t:
0.1 I Rl-FR- (x h) -R2
5~ RI-FR F~
8
0 0 3 6
TIME AFTER Rl-FR ( h )
Fig. IV-8. Life time of (j' : the effects of the interval
between RI and Rz on anthocyanin synthesis in 20°C- and
24°C-seedlings.
R1-FR-(x h)-Rz
Seedlings
and, as
were
a
irradiated in the regimes,
control, of FR-(x
Anthocyanin was determined 24 h after the last pulse in any
irradiation regime. Anthocyanin levels of RI and R1-FR were
shown by the bars on the left s ide of the figures. R1: Rfl ,
50 ,umol m~2 S~I X 600 s· , the same X 30 s, and FR:
FR-DelaA900, 70 ,umol m-2 S-I X 180 s. The plant heights of
20°C- and 24°C-seedlings at the first irradiation, 84 mm
and 70 mm, respectively, and at the last irradiation, 92 mm
and 83 mm, respectively. s. bicolor cv. Acme Broomcorn,
1987 crop.
121
given. Rl was a pulse of an excessive f luence (30 mmol
m-2) and R2 was varied in f luence. Figure IV-9 shows that
in a double-log plot the curve for R1-FR-R2vBr is parallel
to that for FR-RhBr , indicating that the actions of
various amounts of Pfr or Pfr/Ptot ratios were multiplied
by a certain factor, i.e. by the amount of ~-.
To see the effects of S on UV-B-induced anthocyanin
synthesis, a similar experiment was performed with a regime
R1-FR-UVvor-FR. A pulse of UV-B was varied in fluence, and
the last FR is to reverse the Pfr which is generated by
UV-A contained in the UV-B source. As controls regimes
UV-FR and FR-UV-FR were administered (Fig. IV-lO).
Compared with Fig. IV-9, the extent of amplification of the
UV-B actions by the \}---.. was only slight.
Time courses of anthocyanin synthesis induced by the
coaction of Pfr and G'-. As shown in Fig. IV-ll, the time
course of anthocyanin synthesis induced by R1-FR-R2 was not
different from that by R2 alone. This was true of both
20 a C- and 2 4a C-seedlings. The findings suggest that the
action site of ~ __ and Pfr are close in the R signal
transduction chain.
The initial sluggish rise of anthocyanin synthesis in
2 DoC-seedlings compared with 24a C-seedlings. (F ig. IV-llB)
was previously discussed to be most probably due a slow
rise in temperature of the substratum (Chapter III).
122
,-...
~0,20 f- __ Rl alone , ~~.-t------.
I 0 ,10 r-
00 N If)
c:I:
Rl-FR-R2var .. ~ .. ~ ~ . ,Q,---<&~ ~ .-----~ ",f/···· !~ . ,
.,' ~
z:: z:: c:I: >U o ::c
f;;:' . \-. --. - ~ -- -. - ~ . ~
t----·· •. --------- FR-R2var ~ ___ o
I-z:: c:I: 0,01~~~'~--~~~~~~~'----~~~~~~~'
10 100 1000 R2 FLUENCE ( }Jmol m-2 )
Fig. IV-9. Manifestation of a def ini te amount of 0-- by
varied levels of Pfr in R-induced anthocyanin synthesis in
24°C- seedlings. Sedlings were irradiated in the regimes,
R1 - FR-Rzvar ( • and FR-Rzvar o ). The anthocyanon
level caused by R1 alone is indicated on the upper left of
the figure. Means ±. S. E. ' s of anthocyanin leve ls by FR
alone and R1-FR were O. 0085 + o. 013 (n=6) and O. 0212 ±.
0.0030 (n=4), respectively. R1 : Rf 1 , 50 )lmo 1 m-z s -t X
600 s; Rzvar : R-IF661, 1.2 )lmol m-z S-l X 5, and 10 sand
10 pmol m-z S-l X 5, 10, 20, 50 and 100 s. Plants heights
at the time of irradiation, 89-98 mm. S. bicolor cv. Acme
Broomcorn, 1991 crop.
123
......... 0,50 0 If)
\0 f-c:::r:
Rl (Xl
N If)
c:::r: '-" 0,10 f-
z .......... z c:::r: 0,05 >-u C> :r: I-z c:::r:
I I I
100 1000 UV FLUENCE ( )Jmol m-2 )
Fig. IV-l0. Effects of a def ini te amount of 0- on
UV-B-induced anathocyanin synthesis in 24°C-seedlings.
Seedlings were irradiated in regimes, R1-FR-UV-FR (- -.- -),
FR-UV-FR (-0--) and UV-FR (-----6------). Anthocyanin level
produced by Rl alone is indicated by tp.e bar at the left
s ide of the figure, and those by FR a lone and R1- FR were
o • 0073 + 0.0007 and 0.0159 + 0.0015 { Means + SE (n= 6) } ,
respectively. Rt : Rfl , 50 pmol m-2 S-1 X 600s; FR:
FR-DelaA900, 60 }lmol m-2 S-l X 180 s; UV: UV310-U330-N,
5 Jlmol m-2 S-l X 10, 30, 90, 240 and 600 s. Plant height at
the time of irradiation, 72-82 mm. S. bicolor cv. Acme
Broomcorn, 1991 crop.
124
~ 0.4-"" c::c I co N L()
c::c
:z: .......... z c::c >u o ::c Iz c::c
0.2
A IJ
6 /i"'" t RI-FR-R2.1 200( !"", 6 .~
,-,,'if. 6
O~~~--L---~--~--~--~--~~
z .......... z c::c >u o ::c Iz: c::c
100 B
50
10 20 30 TIME AFTER IRRADIATION ( h )
Fig. IV-ii. Time courses of anthocyanin synthesis induced
by R1 -FR-R2 (--/::;.--and-A- , for 20°C- and 24°C-seedlings,
respectively) relative to those by R2 alone (--o--and-___ ,
for 20°C- and 24°C-seedlings, respectively). (A), actual
anthocyanin leve Is; (B), percent of the anthocyanin leve Is at
35 h after irradiation. Rl and R2 : Rfl , 50 pmol m-2 SI X
600 sand 30 s, respectively; FR: FR-DelaA900, 70 pmol m-2
S-1 X 180 s. Plant height of at the time of irradiation,
72-80 mm and 67-79 mm for 20°C- and 24°C-seedlings,
respectively. s. bicolor cv. Acme Broomcorn, 1991 crop.
125
Discussion
The present paper described experimental data to
suggest that the photomorphogenic signal of R might be
stored in a form which escapes FR reversion and be
expressed by a multiplicative coaction with Pfr. Phenomena
of promotion of a R effect by a previously given R were
first pointed out in Sinapis alba by Whitelam and Johnson
(1981) and Schmidt and Mohr (1981), but the effects have
been dealt with in a different context from the present
paper (Schmidt and Mohr 1982). Yatsuhashi et al. ( 1982 )
also described similar effects, but no further analysis has
been made. Prior to these papers some data had been
reported to show the accumulation of R effect, though
slight and unnoticed, after repeated alternations of Rand
FR pulses terminated by R in Lactuca sativa seed
germination (Table 7 of Borthwick et ale 1954) and in
hypocotyl elongation inhibition and primary leaf expansion
of Phaseolus vulgaris (Table II of Downs 1955). Thus, such
R signal storage seems to occur ubiquitously. The present
paper claims the possible presence of R signal storage even
in a LFR caused by a pulse irradiation, and propose to
refer to this storage form of R signal as rr-, meaning
summation.
Generation of q--..-. Since (J'- is generated by an irradiation ~~~~~=-~~----
with R very quickly (Fig.IV-5) escaping from the reversion
of Pfr by a FR pulse given after 10 s, it may be argued
that some photoreceptor other than phytochrome may be
responsible for generating [7-. However, the action
126
spectrum for anthocyanin synthesis of this plant is
considered to involve the generation and action of V--,
and yet agrees with the absorption spectrum of Pr. If
other photoreceptor (s) having an absorption peak at other
wavelengths than 660 nm is involved, the action spectrum of
the anthocyanin synthesis should differ. But it is not the
case (Yatsuhash et al. 1982). Also, the constant ability
for r;--- generation paralleled with the constant content of
phytochrome, al though anthocyanin synthesis and other
activities usually vary along with the developmental stages
of seedlings (F igs. IV-6, IV-7). These findings are bases
on which to assume that 0-- generation is mediated by
phytochrome photoconversion.
On irradiation with a R pulse, Pfr/Ptot ratio increases
linearly against the log fluence of the pulse (Steinitz et
al. 1979, Chapter III) before reaching the maximum. The
generation of cr- is, in contrast, linear against log R
f luences not only be low the phytochrome-saturating f luence
but also above it (Figs. IV-2, IV-3). The reciprocity law
hold (Fig. IV-2). These findings imply that V- generation
does not depend on Pfr/Ptot ratio nor Pfr content, but may
probably depend on the rate of phytochrome photoconversion.
After reaching the maximum Pfr/Ptot ratio the
photoconversion continues by cycling.
Previously we (Chapter III) described the presence of
both very low and low f lUence responses (VLFR and LFR) in
anthocyanin synthesis of sorghum, which were most suitably
explained by VanDerWoude's dimeric model of phytochrome
(1985). Further we proposed "high f luence response"
127
distinct from low fluence in a pulse irradiation as well as
so-called high irradiance response. The present paper
deals only with low and high f luence responses, and the
cycling refers to the one between PrPfr-X and PfrPfr-X in
the dimeric model. Whether or not r.;.- is generated also by
the photoconversion from PrPr to PrPfr, i.e.in the very low
fluence response, is subject to future studies.
Property of (/'-- Sigma is nonsusceptible to FR, fairly
stable in tissues, and cumulative (Fig. IV-4). It survived
without loss at least for 1 h with a half-life of ca. 3 h
to disappear after 6 h in 24°C-seedlings (F ig. IV-8). In
20°C-seedlings it was more stable. At present no further
information on its property is available . Although quite
speculative, some transmembrane localization, uptake or
release of Ca 2+ as well as the generation of inositol di
phosphate and inositole triphosphate or the activation of a
GTP-binding protein might be possible candidates for o-(cf.
Tretyn et al. 1991). Further studies will be awaited.
Action of r:;-- Sigma exerts its action wi th a
multiplicative coaction with PfrPfr (Fig. IV-9), but no
action in the absence of PfrPfr, i. e. after FR reversion
(Fig. IV-4).
The multiplicative nature of the coaction of 0'- with
PfrPfr is indicated in Fig.
IJ'---. ampl if ied the act ions
nearly by a fixed factor.
IV-9, where a fixed amount of
of var ious amounts of PfrPfr
Al though the idea of such a
128
multiplicative action was
and Johnson (1981), the
evidence it experimentally.
proposed previously by Whitelam
present paper is the first to
If observed in magnification, the action of D'- is not
absolutely null in the absence of PfrPfr. When a R pulse
was followed by FR, extremely small amounts of anthocyanin
were synthesized, thus ~ might coact with PrPfr-X in a
different way, because anthocyanin synthesis was
s ignif icantly less in 20°C- than 24°C-seedlings (F ig.
IV-4B, Fig.III-9 of Chapter III). However, VLFR is beyond
the scope of the present paper
Sigma is likely to interact with UV-B also, but the
magnitude of its amplification is only slight (compare Fig.
IV-l0 with Fig. IV-9). The anthocyanin synthesis induced
by the coaction of U- and PfrPfr followed the same time
course as that induced by a single pulse of R (Fig. IV-ll).
This finding may suggest that the action sites of 0-- and
PfrPfr-X are close in the R signal transduction chain, but
not a general amplif ication at the process of anthocyanin
synthesis (Fig. IV-12).
Relation of :7-- to the amplification effect of MLT given
prior to irradiation. Moderate low temperature (MLT) given
in the pre-irradiation culture period amplified R-induced.
anthocyanin synthesis (Figs. IV-l, IV-2, IV-3, IV-4, IV-5,
IV-6, IV-8, IV-9), and it was first expected that a
possible greater generation of ~ might be an attribute of
MLT-grown seedlings. However , it was not the case (F igs.
IV-2, IV-3). The slower decay of rr- in 20°C- than 24°C-
129
FR or R MLT
PrPfr-X PfrPfr-X t .. Y
R
Fig. IV -12. Proposed scheme for the generation and action
of cr. based on the dimeric model for low fluence response
of phytochrome. Phytochrome molecules are shown as
conjugate forms with receptor X. PrPfr-X is converted to
PfrPfr-X by R, generating (j" from its precursor <ro, and
PfrPfr-X is reverted by FR or R. PfrPfr-X and 0- interact
with a reaction partner Y, whose affinity or level is
modulated by moderate low temperature.
130
seedlings is to be noted (Fig. IV-8), but seems not
adequate to ascribe the MLT effect to.
We propose the following scheme (Fig. IV-12):
phytochrome photoconversion reacton from PrPfr-X to
PfrPfr-X generates ('--. The r.7'-- generation occurs through
phytochrome cycling even when the maximum Pfr/Ptot ratio is
established. Sigma manifests itself by multiplicative
coaction with PfrPfr-X, and thus the R action in LFR and
HIR depends on the amounts of V'- and PfrPfr-X, ~ being
inactive by itself. Thus, phytochrome can measure fluence
rate in an inductive pulse irradiation, even if phytochrome
is saturated. Since 0-- is kept without loss at least for
an hour, phytochrome can also sum up the light fluences of
irradiations intermittently given, as far as each pause is
within the period in which o--is stable.
Relation with HIR
The high irradiance responses (HIR) requiring prolonged
irradiation had the action peaks in the blue and FR
wavebands (Mohr 1957, Siege Iman and Hendr icks 1957), and
were fluence rate-dependent. Detailed studies showed that
the action spectra for the growth inhibition of etiolated
Sinapis alba seedlings (Beggs et ale 1980, Holmes and
Sch~fer 1981) showed the main peak at 653-655 nm in
addition to peaks at 712-716 nm and in the blue-near UV
waveband in a 24 h continuous irradiation. In order to
explain the fluence rate dependency of HIR, SchMfer (1975)
has proposed the open cycling model of phytochrome, in
which Pfr-X' is slowly formed from Pfr-X, and Pfr-X'
131
represents the f luence rate dependency. Sigma does not
correspond with Pfr-X I, because the phytochrome conjugate
is reversed by FR. Johnson and Tasker (1979) and Wall and
Johnson (1983) stressed the importance of phytochrome
cycling in explaining the fluence rate dependency of HIR,
and postulated an entity, XO, which is generated by
phytochrome cycling, and acts multiplicatively with Pfr.
Their postulated XO seems to be identical in notion with
our cr- despite of the lack of enough data on XO to
compare with r:r-. Obviously the v generation by a R
pulse is dependent on fluence rate or fluence (Figs. IV-2,
IV-3) (the reciprocity law held in pulse irradiation).
However, it seems that 0-- coacts only with PfrPfr, but not
with PrPfr or dose in a distinct way (Figs. 111-9, 111-10
of Chapter III). According to VanDerWoude (1987) PrPfr-X is
assumed to act in HIR, and it may be premature to apply the
possible coaction of S or XO with PrPfr-X to HIR.
The action at the 653 nm peak is also fluence
rate-dependent (Holmes and Sch'Afer 1981), although in the
24 h irradiation, phytochrome is considered to be
saturated. VanDerWoude (1987) has proposed that the peak in
this red waveband is due to PfrPfr. The fluence rate
dependency of this waveband in HIR may be explained by the
coaction of ~- and PfrPfr proposed in the present paper.
In conclusion, R-induced anthocyanin synthesis of etiolated
Sorghum bicolor seedlings does not plateau even after
phytochrome reached the maximum Pfr/Ptot ratio in a pulse
irradiation, and R signal is accumulated in repeated
132
alternations of Rand FR pulses. These effects and other
findings presented in this paper led to assume f1'-- as a
quantitative pool of R effect, which may manifest itself by
a multiplicative coaction with PfrPfr. The assumption of
the 0'-- generation may provide an explanation for that
plants respond through the mediation of phytochrome
depending on a wide range of f luence rates of R in an
inductive action as well as in a prolonged irradiation of R.
133
References
Beggs, C.J., Holmes, M.G., Jabben, M., Schafer, E. (1980)
Action spectra for the inhibition of hypocotyl growth by
continuous irradiation in light and datk-grown Sinapis
alba L. seedlings. Plant Physiol. 66,615-618.
Borthwick, H.A., Hendricks, S.B., Toole, E.H., Toole, V.K.
(1954) Action of light on lettuce-seed germination. Bot.
Gaz.115, 205-225.
Downs, R.J. (1955) Photoreversibility of leaf and hypocotyl
elongation of dark grown red kidney bean seedlings. Plant
Physiol. 30, 468-473
Hillman, W.S. (1972) On the physiological significance of in
vivo phytochrome assays. In: Phytochrome. pp573-584,
Mitrakos, K., W. Shropshire, Jr., eds. Academic press
London
Holmes, M.G., Schafer, E. (1981) Action spectra for changes
in the "high irradiance reaction" in hypocotyls of
Sinapis alba L. Planta 153, 267-272.
Johnson, C.B., Tasker, R. (1979) A scheme to account
quantitatively for the action of phytochrome in etiolated
and light-grown plants. Plant Cell Environ. 2, 259-265
Mohr, H. (1957) Der Einfluss Monochromatischer Strahlung auf
134
das Langenwachstum des Hypokotyls und auf die Anthocyan
bildung bei Keimlingen von Sinapis alba L. Planta 49, 389
-405.
Schafer, E. (1975) A new approach to explain the "High Irrad
iance Responses" of photomorphogenesis on the basis of phyto
chrome. J. Math. BioI. 2, 41-56.
Schmidt, R., Mohr, H. (1981) Time-dependet changes in the
responsiveness to light of phytochrome-mediated
anthocyanin synthesis. Plant Cell Environ. 4, 433-437
Schmidt, R., Mohr, H. (1982) Evidence that a mustard
seedling responds to the amount of Pfr and not the
Pfr/Ptot ratio. Plant Cell Environ. 5, 495-499
Schmidt, R., Mohr, H. (1983) Time course of signal
transduction in phytochrome-mediated anthocyanin
synthesis in mustard cotyledons. Plant Cell Environ. 6,
235-238
Siegelman, H.W., Hendricks, S.B. (1957) Photocontrol of
anthocyanin formation in turnip and red cabbage seedlings.
Plant Physiol. 32, 393-398.
Steinitz, B., Schafer, E., Drumm, H., Mohr, H. (1979)
Correlation between far-red absorbing phytochrome and
response in phytochrome-mediated anthocyanin sinthesis.
Plant Cell Environ. 2, 159-163.
135
Tretyn, A., Kendrick, R.E., Wagner, G. (1991) The role(s) of
calcium ions in phytochrome action. Phorochem. Phorobiol.
54, 1135-1155.
VanDerWoude, W.J. (1985) A dimeric mechanism for the action
of phytochrome: evidence from photothermal interactions
in lettuce seed germination. Photochem. and Photobiol.
42, 655-661
VanDerWoude, W.J. (1987) Application of the dimeric model
of phytochrome action to high irradiance responses. In:
Phytochrome and Photoregulation in plants, pp.249-258,
Furuya, M. ed. Academic Press, Tokyo
Wall, J.K., Johnson C.B. (1981) Phytochrome action in light
-grown plants: the influence of light quality and fluence
rate on extension growth in Sinapis alba L. planta 153,
101-108
Watanabe, M., Furuya, M., Miyoshi, Y., Inoue"Y. Iwahashi,I.
Matsumoto, K. (1982) Design and performance of the
Okazaki Large Spectrograph for Photobiological research.
Photochem. Photobil. 36, 491-498
~lhitelam, G.C., Johnson, C.B. (1981) Temporal separation of
two components of phytochrome action. Plant Cell Environ.
4, 53-57
136
Yatsuhashi, H., Hashimoto, T., Shimizu, S. (1982) UV action
spectrum for anthocyanin formation in broom sorghum
first internodes. Plant Physiol. 70, 735-741
Yatsuhashi, H., Hashimoto, T. (1985) Multiplicative action
of a UV-B photoreceptor and phytochrome in anthocyanin
synthesis of broom sorghum seedlings. Photochem.
Photobiol. 41, 673-680
137
General Discussion
The present thesis has described several new
findings and conceptions concerning the primary reaction
steps in the R signal transduction chain leading from
phytochrome to the gene coding enzymes involved in
light-induced anthocyanin synthesis in etiolated sorghum
seedlings: the presence of a step enhanced or suppressed
by pre- irradiation MLT (chapter 2 and 3), a different
type of very low fluence response (VLFR) and high fluence
response (HFR) distinct from the so-called HIR (chapter
3). The presence of HFR led to assume a storge of R
signal, sigma, other than Pfr (chapter 4). The opposite
effects of pre-irradiation MLT between LFR anf VLFR, i.e.
enhancement and suppression, respectively, led to the
suggestion of the presence of respective distinct
reaction partners, Y and y. The presence of the
MLT-enhanced step
point in LFR (at
chain the two
multiplicatively)
suggests that the possible merging
some step of the signal transduction
signal should merge to work in
of the signals from phytochrome and
UV-B photoreceptor may be located after the MLT-sensitive
step, because the UV-B action was not affected by MLT at
all.
All evidence for these conceptions are
data by physiological experiments. To
conceptions we should substanciate them
biochemical entities of sigma as well
MLT-sensi tive step. These will be subject
studies.
138
based on the
prove these
by finding
as of the
to my future
The physiological findings presented in the thesis
certainly offer the means by which to find and identify
the biochemical entities as such; i.e. choice of the
induction light, i. e. out of UV- B, R for LFR or FR for
VLFR and application of pre-irradiation MLT and
combination of them would give several situations,
through which correlation or noncorrelation could be
found. For example, the opposite effect of MLT on LFR vs.
VLFR will be very helpful to identifying Y and y; the
combination of various fluence of R and 24°C and 20DC,
for identifying sigma.
Elucidation of molecular mechanism of phytochrome
signal transduction is to be done hereafter, and I hope
that this thesis will supply keys to break through the
fascinating, but difficult problem.
139
Acknowledgements
I wish to express my sincere thanks to Professors Tohru
Hashimoto (Chief referee), Yoshikiyo Ohji, and Teruo Iwasaki,
Graduate School of Science and Technology, Kobe university
for their kind judgements, to which the present thesis is to
be submitted. My special thanks are directed to Professor
Hashimoto, under whose auspices and guidance the research
work of the thesis was performed and the thesis was prepared.
Dr. Christopher B. Johnson, Department of Botany, University
of Reading, UK, enlightened my knowledge of phytochrome and
performed some experiments (Chapters 2 and 3) with me during
his stay in kobe University as invited Professor from
September to December 1989. Mr. Tohru Hamada contributed a
part of Chapter 2, which formed his master thesis.
In the course of the research work I obtained many
assistances from the following persons and thank all of them:
Dr. Seiji Tsurumi, our laboratory with general encouragement
and understanding; Dr. M. Watanabe and Mr. M. Kubota,
National Institute for Basic Biology, Okazaki, with the Larg
Spectrograph exper iments; Professor Guruprasad K. N., Indore
University, India and Mr. Y. Tsujino, our laboratory, with
spectrograph experiments; Mr. M_ Hiraoka, with a UV-B
exper iment, and Ms. C. Shibata, our laboratory , with a part
of phytochrome determination; Drs. Y. Takeuchi, Shionogi
Pharmaceutical Company, Aburahi, Shiga and K. Hosaka, the
Experimental Farm, Kobe University, Kasai with sorghum seeds;
and Professor K. Manabe, Yokohama City University, Yokohama
with the technique of phytochrome determination.
My thanks is also due to my parents for their affection
and understanding.
140