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