窒素・硫黄代謝制御による子実貯蔵タンパク質のサブユニット組成の変化 第一報

NII-Electronic Library Service

J.CropRes.56:17-22

':-

Research

Article

(2011)

Changes

in

the

Subunit

Composition

of

Seed

Storage

Proteins

by

Controlling

Nitrogen

and

Sulfur

Metabolism

L

Rice

plants

in

sandy

soil

and

hydroponic

cultures.

[Ibmoyuki

Katsube-Tanakai),

Hiromichi

Nakashirna2),

Nadar

Khani),

Takeshi

Yamaguchi2)

and

Jyunichi

Nakano2)

i)

Graduate

Schoot

_{ofAgriculture,}

_kyoto

Uhiversity

(Sakyo-ku,

Kyoto

606

e

8502,

Japan)

2)

Eaculty

oLfllgriculture,

7bttori

Uhiversity

(4

"

101

Keyama-minami,

Tbttori

680

-

8553,

Japan)

Summary:

The

major storage

_protein

of rice seeds

is

glutelin,

which comprises various subunits.

Changes

in

the

subunit composition ofglutelin can

greatly

afl7ect

the

nutritional

_quality

ofrice.

Even

though

our

_previous

studies

demonstrated

that

the

subunit composition was changed

by

altered nitrogen and sulfur metabolism using

transgenic

_plants

expressing asulfUr-rich

_protein

and a

detached

ear

eulture

methed,

the

response

ofintact

_plants

was unknown.

In

this

report,

intact

rice

_plants

were analyzed and

found

to

change

their

subimit composition

fo11owing

high

levels

ofnitrogen

fertilization

using conventional sandy soil and

hydroponie

culture systems.

The

excess

nitrogen

treatment

in

the

sandy soil culture reduced

GluA

subunits and

increased

GluB

subunits, which contain rnore

lysine.

And

thus

the

excess nitrogen

treatment

was estimated

to

inerease

the

lysine

content

by

7%

compared

to

the

standard

treatment.

The

efTect of sulfur metabolism on

the

subunit composition was uncertain

in

this

study.

In

consequence,

the

subunit composition modified

by

the

nitrogen rnetabolism centrol was crucial

for

the

nutritional

_quality

improvement

of

the

seeds of

intact

rice

_plants,

Key

words:

Rice,

Storage

_protein,

Subunit

composition,

Nitrogen

/

suifur metabolism

lntroduction

Cereal

grains,

such

as rice and soybean,

have

_proteins

at

10-40%

of

total

dry

weight.

Based

en

this

feature,

these

_grains

are an

important

nutritional

source

for

_people

in

deveLoping

countries.

Storage

_proteins,

howeve4

account

for

a

high

percentage

oftota1 seed

_proteins

and

_generally

require nutritional

improvement

because

the

_proteins

are relatively

defieient

in

essential

amino

acids

compared

to

animal-source

_proteins.

The

nutritional value

of

food

proteins

is

evaluated

using an amino acid score, which

is

calcu]ated

by

their

amino acid composition

(Wbrld

Health

Organization

2007).

Among

the

essential amino acids constituting

food

_proteins,

the

]owest

nutrient adequate or suencient

to

the

_propesed

required

levels

is

called

the

first

limiting

arnino acid

for

the

food

or

the

_proteins.

The

nutrient adequacy of

the

first

limiting

amino acid

is

referred

to

as

the

amino acid score.

In

_general,

the

arnino acid scere of animal-source

_proteins

is

nearly

100

and

hence

the

nutritional

quality

efits

food

is

high,

For

_{grarninaceous}

cereals,

lysine

is

the

first

limiting

amino

acid.

The

arnino acid score

ofrice

is

only

65,

Thus,

increasing

the

lysine

content

is

an

important

_goal

for

the nutritional

improvement

ofrice

_grains.

The

predominant

protein

(comprising

60-80%

of

total

Acccepted

:

March

30,

2011

Corresponding

autheT :

Tomoyuki

Katsube-Tattaka

(tanakato@kais.kyoto-u.aejp)

proteins)

in

rice seeds

is

_glutelin,

whese

subunit

consists

of

a

(37-39kl)a,

acidic) and

"

(22-23kDa,

basic)

_polypeptides

that

are

linked

to

each other

by

a

disutfide

bond

in

the

mature

form

(Utsumi

1992).

Like

other seed storage

_proteins,

_gluielin

is

encoded

by

a

multigene

familM

and

six

major

_glutelin

_genes

or

subunits

have

been

identified

in

the

Asian

japonica

subspecies

(Katsube-Tanaka

et al.

2004),

These

_genes

have

been

classified

into

two

subfamilies,

GluA

and

GluB,

according

to

the

degree

of

their

relatedness

in

their

nucleetide sequences

(Takaiwa

et al.

1991),

GluA

and

GluB

subfamilies

consist

of

GluAl,

GluA2,

GluA3,

and

GluB1,

GluB2,

6IuB4,

respectively

(Takaiwa

1990),

The

identities

of amino acid sequences within each subfamily show rnore

than

82%

simi1arity,

but

between

the

subfamilies

this

value

[owers

to

62%

_(Sugimoto

etal.

1986).

Between

the

two

subfamilies,

GluB

is

more nutritious

than

GluA

because

it

contains on average

20%

more

lysine.

Therefore,

ifwe

can

decrease

GluA

_gene

expression and

increase

GluB

_gene

expression, we would

improve

the

nutritienal

_quality

of rice seeds.

In

facg

the

double

knockout

mutant

05TK47e

laeking

GluAl

and

GluA2

showed

9%

more

lysine

than

the

wild

type,

and another mutant

LGCI

_(in

whieh the accumulation

level

of

GluB

subunits were significantly reduced

by

RNAi)

reduced

lysine

by

17%

(Katsube-Tanaka

etal.

2oo7).

These

data

suggest

that

glutelin

subunit compesition

is

very

important

for

the

nutritional value of rice.

Storage

protein

composition of other crops can

be

changed

by

Copyrighturgefttz-ffrebleek

(The

Soc[ety

ofCTop

Science

and

Breeding

in

Kinki,

Japan)

NII-Electronic Library Service

J.

Crop

Res.

56(2011)

plant

nutritien.

In

soybean seeds,

for

example, nitrogefi supply

levels

afTbct

the

accumulation

level

of

the

fi

subunit

of

fi

-conglycinin,

_the

second most

impertant

storage

protein

in

soybean seeds

(Ohtake

2005).

Moreoveg

cultivation

in

a

sulfur-deficient

envi ronrnent results

in

a

decrease

and an

increase

in

the

accumulation of sulfur-rich

and

sulfur-poor

_proteins,

respec-tively

in

_pea,

lupine,

wheat,

soybean,

maize,

cowpea,

and sunflower(FLijiwaraandOtsu2005).

In

rice

plants,

the

detached

ear culture method with medium containing

high

nitrogen and

low

sulfur concentrations was

performed

te

anatyze

the

effects

of nitrogen and

suEfur

metabolisrn

on

the

_glutelin

subunit

composition

in

vitro.

With

the

culture method, we

found

an

increase

in

rnore nutritious

GIuB

subunits

(Katsube-Tanaka

et

al.

2007),

ln

addition,

transgenic

rice

_plants

that

significantly

aecumulated

sulfur-rich

2S

alburnin

and seemed sulfur

deficient

showed a

decrease

in

GluA

and an

increase

GluB

subunits

in

the

seeds

(Nakashima

et

al.

2007).

These

data

led

us

to

the

idea

that

nitrogen and sulfur metabo]ism may affect

glutelin

subunit composition even

in

intact

common rice

_plants.

Thus,

this

study was conducted

to

analyze

the

effect of nitrogen and suLfur control

ln

jntact

_pLants,

utilizing sandy soil and

hydroponic

cultures, which would

provide

plant

production

systems more comparahle

to

a

_paddy

field,

Materials

and

Methods

Plant

materiats

and

cultlvation

i)

Sandy

soil

culture

Rice

seeds

(var.

Koshihikari)

were sterilized and

pre-incubated

in

the

dark

at

30

℃

for

_{gertnination.}

The

seeds were

sown

into

disinfected

granular

soil using

_paper

_pots

and

_grewn

to

seedlings

for

four

weeks.

The

seedlings

were

transpianted

into

1

/5ooOa

Wagner

_pot

fi11ed

with sand, whose

particle

size was

less

than

1

rnm.

IXventy

_plants

were

_grown

in

each

_pot

with no

tillers

until maturity.

Conventional

chemical

fertilizer

(N:P:K=14:14:14)

at

the

rate of3

_g

_per

_pot

or

the

sulfUr-1imiting

chemicals consisting of urea

(O.9

g

_per

pot)

/

sodium

dihydrogen

_phosphate

(O.69

g

per

pot)

/potassium

chloride

(O.92

g

per

pot)

were applied as a

basa]

fenilizer.

In

addition, urea was either not applied er applied at arate ofO.9

g

per

pot

as a

panicle

fertilizer

twe

weeks

before

heacling.

The

above-mentioned

procedures

were combined

to

construct

the

foLLowing

four

types

of

treatments:

₍₁₎

standard

treatment

_(STD,

conventional

basal

ehemical

fertilizer

and no

_panicle

fertilizer

used),

(2)

excess

nitrogen

treatment

(+N,

conventional

basal

chemical

fertMzer

and urea of

_panicle

fertMzer

used),

(3)

sulfUr

limiting

treatment

(-S,

sulfur-limiting

basal

chemicals and no

panicte

fertilizer

used), and

(4)

excess nitrogen and sulfur

Limiting

treatment

(+N-S,

sulfur-lirniting

basal

chemicals and urea of

_paniele

fert11izer

used).

Panicles

were sampled at maturity and used

for

further

analysis

after

drying

in

acirculation

drier

at

80

℃

.

ii)

Hydroponic

culture

Rice

seedlings were

_prepared

similar

to

sandy soil

cu!ture.

The

seedlings

were

fixed

on

Styrofoam

boards

for

hydroponic

culture

with sponges at

40

_plants

_per

30-liter

container.

The

hydroponic

culture medium was

_prepared

by

fo11owing

Kasugai's

A-type

mediurn

(typical

one

for

rice

_plants

hydroponic

culture) with

some

modifications.

The

following

four

types

of

treatments

were constructed;

(1)

standard

treatment

_(STD,

N:

40

mg

/

1,

S:

46

mg

/

1),

(2)

excess nitrogen

treatment

_(+N,

N:

160

mgr'1,

S:

46

mg/1),

(3)

sulftur

1imiting

treatment

_(-S,

N:

40

rn[Yl,

S:

O.46

mg/1),

and

(4)

excess

nitrogen

and

sulfur

limiting

treatment

(+N-S,

N:

160

mg/1,

S:

O,46

rng/1).

The

culture mediurn was

_prepared

fresh

every week with

tap

water

during

the

vegetative stage and with

distilled

water

during

tlie

reprQductive stage.

T-lers

were removed on a

time!y

basis.

Panicles

were sampled

at

maturity and used

for

further

analysis

after

drying

in

a circulation

drier

at

80

℃

.

Analysis

ot

_glutelin

subunit

composition

Tota1

proteins

were extracted

from

the

superior spikelets at

the

uppermost

_portien

of

the

_panicles

with

700

_flI

of

SDS-urea

buffer

against

20

mg of seeds

(Khan

et al,

2008a),

Five

_ttEof

the

extract was

subjected

to

SDS-PAGE

_to

compare

the

_glutelin

subunit

compositien,

The

sarnple

_preparation

fbr

2D-PAGE

was

conducted according

to

the

_protocol

of

Khan

et al.

(2008b).

Briefiy,

_glutelin

fractions

were extractecl and

dissolved

in

100

_u1

lysis

buffer

contaming

9.5

M

urea,

2%

Triten

X-100,

5%

2-rnercaptoethanol

and

5%

BIo-Lyte

_(Bio-Rad)

_pH

ranges

from

3

te

10.Nitrogen

and

sulfur

analy$es

Nitrogen

and sulfur contents were aEso analyned

for

the

same

spikeiets

to

evaluate

the

effects

ef

culture medium on nitrogen and

sulfur

metabolism

of

rice

_plants.

The

Kieldahl

method and

indophenol

reaction were

performed

to

determine

nitrogen

contenL

Nitric

acid

decomposition

and

inductively-coupled

plasma

emission spectrochemical ana]ysis

(RIGAKU

C[ROS

CCD,

Japan)

were used

to

deterrnine

sulfur content.

2D-RAGE

and

quantification

ef

subunit

content

2D-PAGE

was

perfbrmed

to

analyze

the

detailed

difllerence

in

the

glutefin

subunit composltion

about

the

samples

_prepared

in

the

sandy soil culture.

Gels

fbr

NEPHGE

(non-equilibrium

_pH

gradient

gel

electrophoresis> were

_prepared

in

_glass

tubes

(75

x

1

mm)

according

to

the

method of

Khan

et al.

(2oo8b).

Electro-phoresis

in

first

dimension

_(NEPHGE)

was carried out at

200

V

for

15

minutes,

300

V

for

15

minutes,

400

V

for

15

minutes,

500

V

for

15

minutes and

750

V

for

5

hrs.

The

second electropheresis was conducted at constant voltage of

200

M

Afier

staining with

CBB,

the

_gel

was

_photographed

and each spot on

the

_gel

was

18

NII-Electronic Library Service

Cbanges

in

the

SubunitCornpositionofSeedStorageProteinsbyControllingNitrogenandSu1fur

Metabelism

I.

ruce

_plants

in

sandy soiland

hydroponic

cultures.

quantified

by

ImageJ

1.38x

computer software

developed

by

Wayne

Rasband

at

the

Research

Services

Branch,

National

Institute

ofMenta]

Health,

Bethesda,

Maryland,

USA

Results

Glutelin

subunit

composition

analyzed

by

SDS-IzaGE

SDS-PAGE

analysis of

rice

seeds

_prepared

by

sandy soil culture

demonstrated

the

subunit

composition

was changed

in

the

excess nitrogen

treatment

and

the

excess nitrogen and sulfur

limiting

treatment

_(Fig.

IA).

The

intensity

of

the

band

al

(GluB4)

was

very

low

in

the

standard

and

the

sulfur-limiting

treatments,

while

that

was

alrnost

equal

to

the

intensity

of

the

band

a

2

(GLuA2)

in

the

excess

nitrogen and

the

excess

nitrogen

and sulfur

limiting

treatments.

The

intensity

of

the

Eowest-size

bands

a5

(GluB2)

and a6

(GliiBl)

was also

increased

in

the

excess nitrogen and

the

excess nitrogen and sulfur

limiting

treatments.

Although

a

detached

ear culture method

has

shown

that

a sulfur-limiting

treatment

decreased

GIuA

and

GluB4

subunits

(Katsube-TleLnaka

et al,

2007),

the

sulfur

limiting

treatment

in

sandy soil culture

did

not affect

the

_glutelin

subunit composition.

In

the

meantime, rice seeds

_prepared

by

hydroponic

culture showed ne apparent

differences

among

the

four

treatments.

When

compared

to

the

_pattern

ofthe standard

treatment

in

the

sandy soil culture, a

decrease

in

GluA

and an

increase

in

GluB

subunits

were

observed

not

only

in

the

excess

nitrogen

treatment,

the

sulfur-limiting

treatment,

and

the

excess nitrogen and

su]fur-lirniting

treatment,

but

also

in

the

standard

treatment

_(Fig.

IB),

Nitrogen

ana{ysis

of

rice

seeds

Nitrogen

content measurernents of rice seeds were conducted

to

see

whether

or

not

fertilizer

management

could

control

nitrogen

metabolism

in

sandy

soil

culture.

The

nitrogen

concentration

of

both

the

standard

treatment

and

the

sulfur-Aachain

Bchain

Bq

chain

Pchain

STD

+N

-S

+N-S

Subunit

ai

GluB4

a

2

GluA2

a3

GluAf

a

4

GluA3

a5

GluB2

a6

GluBt

limiting

treatment

was

O,7%.

Meanwhile,

the

nitrogen

concen-tration

was

1.0%

in

the

excess nitrogen

treatment

and

1.1%

in

the

excess nitrogen and sulfur-limlting

treatment.

Thus

nitrogen concentration

increased

by

43%

in

the

excess nitfogen

treatment

and

by

57%

in

the

excess

nitrogen

and

sulfur-1imiting

treatment,

Namely,

only

high

rates of nitrogen

fertilization

raised

the

nitrogen concentration

in

seeds

(Fig,

2A).

In

the

hydroponic

culture, allof

the

four

treatments

showecl

high

nitrogen

cencentrations

in

seeds

with

values

of

1.3%

_,

1,4%,

1.5%,

and

1.1%

for

the

standard,

the

excess nitrogen,

the

sulfur

lirniting,

and

the

excess nitrogen and sulftu;

limiting

treatments,

respectively

(Fig.

2B).

Sulfur

analysis

ot rice seeds

SulfUr

content

of

the

rice

seeds

was

measured

to

deterrnine

whether or not

fenilizer

management could control

the

sulfur rnetabolism

in

sandy soil culture.

The

sulfur concentration of

both

the

standard

treatment

and

the

sulfur-limiting

treatment

was

O.28%,

while

the

excess nitrogen

treatment

and

the

excess

nhrogen and sulfur-limiting

treatment

were

O,40%

and

O.39%,

respectively.

These

values

increased

by

43%

and

39%,

respectively.

Regardless

of sulfur-limiting

treatment,

a

hjgh

rate ofnitrogen

fertilization

raised

the

sulfur concerrtration

in

seeds

(Fig.2C).

In

the

hydroponic

culture,

the

sulfhr concentratien

in

seeds was relatively

low

and was

O.21%,

O.28%,

O.20%,

and

O.19%

for

the

standard,

the

excess nitrogen,

the

sulfur-limiting, and

the

excess

nitrogen

and

sulfur-limiting

treatments,

respectively

(Fig.

2D).2D-RAGE

and

estimat{on

of

lysine

content

2D-PAGE

overall

_patterns

ef

_glutelin

fractions

_prepared

by

the

standard and

the

excess

nitrogen

treatments

in

the

sandy soil

culture

were

simi]ar

to

that

reported

_by

_Khan

et al.

(2008b).

However,

significant

differences

were

detected

between

the

twe

treatments

in

this

study

(Fig.

3),

The

intensities

of

the

spots

B4,

mole%

Lys

Mceyt,'

3.15

2,52

2.53

2.53

2.53

2.11

2.97

2,54

3.60

1,48

3,37

t,89

Fig.

1

_SDS-PAGE

_patterns

of

rice

seed

proteins

prepared

by

sandy

soil

culture

(A)

and

hydroponic

culture

(B).

Lysine

content

and

methionine and cysteine content of six major

subunits of

_glutelin

are

shown

with

_the

_position

of a

_polypeptides

at

the

right

_part

of

the

tlgure.

STD,

+N,

-S,

and

+N-S

are

abbreviatbns

of

the

treatments

(see

texO.

NII-Electronic Library Service

J.

Crop

Res.

56(2011)

Fig.

2

Fig

3

A

1.4A

1.2l;

to8

o.s-S

:16,8m

O.2e.

o.e2'

B

_t.6

t.4ge

t.2:

1.eE

o.s-g

o.62

o.48

O,2g

o,o2'

STD

+N

.S

+N-S

STD

+N

-S

+N-S

c

O.40A*

O.30==-O

O.20=8

o.le.6'

o.oo

DA*x=

¢ "=eoL= ± =co

O.30

O.20

O.10

o.oo

STD

+N

-S

+N.S

STD

+N

-S

+N-S

Nitrogen

and sulfur content of rice seeds

_prepared

by

sandy soil culture

{A,

C)

and

hydroponic

eu[ture

(B,

D),

Values

are

indicated

as

mean

±

SD

(n==4,

2,

2,

1

for

panels

A,

B,

C,

and

D,

respectively),

_STD,

+N,

-S,

and

+N-S

are

abbreviations

of

the

treatments

(see

text).

A

B

acidic basic

2D-PAGE

of rice

glutelin.

The

glutelin

fractions

extracted

from

seeds

prepared

under

standard

(A}

and excess nitrogen

(B)

conditiens

by

asandy soilculture were electrophoresed

and

detected

by

_CBB.

_The

a

polypeptides

of

six

maior

subunits

glutelin

were

ind[cated

by

abbreviations

_(Al,

A2.

A3,

Bl,

B2,

and

B4}.

G[obulin

polypeptide

was

also compared as

internal

control.

B2,

and

Bl

were

increased

in

the

excess

nitrogen

treatment

when

compared

to

that

of

the

spots

Al,

A2,

and

A3.

The

relative accurnulatien

Levels

ofthe spots

Al,

A2,

A3,

B4,

B2,

and

Bl

were

quantified

to

be

O.32,

O.32,

O.08,

O.10,

O.07.

and

O.10

for

the

staridard

treatment

and

O,24,

O.19,

O,10,

O.23,

O.l2,

and

O.12

for

the

excess nitrogen

treatment

_(data

were

from

singie

quantification).

Thus,

the

ratio of

GluB

to

GluA

subunits was

increased

frorn

37.5%

of

the

standard

to

nltrogentreatment.

Discussion

zz.7%ef

theexcess

The

subunit cornpesition ofglutelin, which

is

the

rnajor storage

protein

accoLmting

for

60-80%

of

the

total

_protein

of rice seeds,

NII-Electronic Library Service

Changes

in

the

Subuni,t

Composition

ofSeed

Storage

Proteins

by

Contrelling

Nitrogen

and

Sulfur

Metabolism

I.

kice

_plants

in

sandy soiland

hydroponic

cultures.

was

altered

by

the

_plant's

nutrition.

Namely,

high

expression

levels

Qf

sunflower

2S

albumin,

which

{s

rich

in

sulfur-centalning

amino

acids,

induced

a

decrease

in

GluA

subunits which contain a

relatively

higher

arnount of sulfur-containing arnino acids

(Nakashima

et aL

2007).

Also,

the

detached

ear culture method, whiclt

_preduces

_partially

matured seeds

in

a

test

tube

fi11ed

with a

liquid

culture mediurn,

increased

GluBl

and

GluB2

subunits which centain

fewer

sulfur-containing amino acids when

the

nitrogen concentration ofthe medium

inereased

by

four-foId

and

/or

the

sulfur concentration

decreased

by

O.Ol-fold

cornpared

to

a

typical

culture medium

like

Murashige

and

Skoog

medium

(Katsube-Tanaka

et

al.

2007>.

These

results

imply

that

nitrogen and

sulfUr

metabolism would

affbct

the

glutelin

subunit

composition.

That

is,

rice

_plants

try

to

assimilate

as

much

nitrogen

as

_possible

by

decreasing

the

sulfur-requiring subunit

in

a relatively su}fUr-deficient environment.

Hewever,

the

above-mentioned

studies

were

achieved

with special experimental

systems

and

the

response under normal cultivation with

intact

plants

remains unknown.

Thus,

this

study was conducted with

sandy soil and

hydroponic

culture systems similar

to

field

conditions

in

an attempt

to

centrol nitrogen and sulfur metabolism.

In

the

sandy

seil

culture

of

this

studM

higher

rates

ofnitrogen

fertilization

(the

excess

nitrogen

treatrnent

and

the

excess

nitrogen

and suifur-llmiting

treatment)

tended

to

increase

GluB

subunits,

but

the

sulfur-limiting

treatment

had

no apparent effect on

the

subunit composition.

The

nitrogen concentration of

the

seeds was

high

with

higher

rates of nitregen

fertilization

treatments.

'

Howeveg

changes

in

the

sutfur

concentration

of

the

seeds

were

'similar

to

the

nitrogen

concentration,

regardless of

the

sulfur abundance or scarcity.

Based

on

these

results, we anticipated

that

the

higher

raie of nitrogen

fertilization

treatments

would

increase

root

biomass

and/or

_{physiological}

activity as well as

the

nitrogen concentration of

the

roots, which would resutt

in

higher

amounts of sulfur abserption.

Because

Japan

is

_general]y

volcanic and

is

speculated

to

be

naturally

_provided

with sulfur

by

rainfa11 and

irrigated

water,

the

sulfur-limiting

treatment

in

this

study might

have

no effect on

the

sulfur content.

However,

TsLiji

(20(K))

reported sulfur-deficient symptoms

in

rice

_plants

in

a

_ponion

of

paddy

fields

in

Shiga

prefecture,

where sulfur-less

fertilizer

had

been

applied over a

long

_period

of

time.

Therefore,

sulfur

metabolism

control might

be

possible

only

if

_paddy

fields

were maintained with

the

use ofurea or ammonium

_phosphorous

under

Long-term

cultivation management.

Giutelin

ftactions

prepared

from

the

sandy soil culture were applied

to

2D-PAGE

and

then

image

analysis was

_performed

to

estimate

the

accumulation

Eeyels

ofeach

_glutelin

subunit.

Because

the

lysine

content ofeach subunit was already

known

(Fig.

1),

the

total

lysine

content of

_the

_glutelin

_fraction

could

be

estimated

by

integrating

the

accumulation

level

weighted with

the

lysine

content,

The

result

of

_preliminary

estimation showed

that

the

excess nitrogen

treatment

increased

the

lysine

content

by

7%

compared

to

the

standard

treatment.

Therefore,

the

subunit composition modified

by

the

nitrogen control was crucial

for

the

nutritionaE

_quality

improvement

of rice seeds.

In

the

future,

growth

and

_yieLd

_perfbrmance

scores

as

welt as

_the

subunit

compesitien should

be

assessed

to

alIow

for

the

optimization of

nitrogen and sulfur conclitions

to

enhance

the

Iysine

centent under

paddy

field

condjtions.

In

the

hydroponic

culture of

this

study,

all

the

treatments

demonstrated

a

decrease

in

GluA

and

an

increase

in

GluB

subunits

when compared

to

the

standard

treatrnent

in

the

sandy seil

cu]ture,

suggesting

that

the

nitrogen

concelltration of

culture

mediurn

was relatively

high

compared

to

the

biomass

accumulation.

Note

that

the

removal of

tillers

_(as

described

in

Materials

and

Methods)

might repress

the

biomass

increase

and

cause

imbalance

between

the

demand

and

the

supply ofnutrients

in

this

study.

The

nitrogen concentration ofthe seeds was actuaLLy more

tha'n

1.1%

in

al1

the

treatments,

which

is

comparable

to

that

ofthe excess nitrogen and

sulfu-1imiting

treament

in

the

sandy soil

culture

(1.1%).

The

su]fur concentratlon of

the

seeds was,

however,

low

in

all

the

treatments

with

the

highest

value of

O.28%

in

the

excess

nitrogen

treatment.

This

value was

the

same as

the

standarcl and

sulfur-Iimiting

treatments

in

the

sandy soil culture

(O.28%).

The

above

data

indicated

that

al1

the

treatrnents

in

this

hydroponic

study

were under excess nitrogen and sulfur-1imiting conditions, which

resulted

in

an

increase

in

Glul3

subunits.

In

consequence,

the

hydroponic

culture of rice

_plants

requires more stricteptimization

of

treatment

to

control

the

nitrogen and sulfur metabolisrn.

'

Acknowledgements

This

work was supported

in

_part

by

_grants

to

T.K.-T.

from

the

Ministry

ofEducation,

Culture,

Sports,

Scienee,

and

Tlechnology,

Japan

for

Scientific

Research

_(C).

20580013,

2008-2010

and

fe11owship

to

N.

K.

from

Japan

Society

for

the

Promotion

of

Science.

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284

．

窒素

・

硫 黄代謝制御

に よ る子

実貯蔵

タ

ン パ

ク

質

の

サ

ブ

_{ユ ニ}

ッ

ト

組

成

の

変

化

第

一

報 　砂耕 栽培 と水耕 栽培

に お け るイ

ネ

の

応 答

田

中朋

之

D

・

中 嶋 祐

道

2）

・Nadar

　

KhanD ・

_{山 口}

_{武 視}

2〕

・

_{中 野 淳}

一

2） 1 ）

京 都 大 学 大 学 院 農 学 研 究 科

（

〒

606

−

8502

　

京 都 市 左 京 区 北 白川 追 分 町

）

2）

鳥 取 大 学 農 学 部 （

〒

680

−8553　

鳥取 市 湖 山 町 南

4

丁 目

101

）

要 旨：_{イ ネ 子 実}におい て 主要 な

財

蔵タン パク

質

である グル テ リン のサ ブユ ニ ッ

F

組 成は

，

その栄 養 性に大 き く影 響 する

．

こ れ ま で に

，

そ の サ ブユ ニ ッ ト

組

成は窒

素

・

硫 黄 代 謝

に よ

り

変

化す

る こ とを

，

含硫

ア ミ ノ

酸

を

多 く含

む

外 来

タ ンパ ク

質

遺伝

子によ る 形

質転 換

や穂

培 養

とい っ た

特殊

な 実 験

系

におい て

明

らかに し た が

，

インタク トな 植 物

体

による応

答

は

不 明

であっ た

．

そこ で

，

圃 場 栽 培に より近い砂

耕

栽 培

・

水

耕 栽

培

での応

答

を解 析 し た と こ ろ

．

イン タ ク トな

普

通 品 種

植

物 体 に おい て

も高 窒 素施 肥

に よ

りサ

ブユ ニ ット

組 成

が

変化 す

る ことが

明

ら か と

な

っ た

，

砂 耕 栽培

の

高 窒素 施肥

に よ

り

，

GluA

サ ブユ ニ ッ トが

減

少 し

，

制

限ア ミノ酸であ る リ ジンを

多

く含 む

GluB

サ ブユ ニ _{ッ トが 増 加 し た}

．

_{これによ り} t リジ ン

含

有

率 は

7

％ 増 加 す る と 予 測 さ れ た

．

硫 黄

制

限 処 理の

効

果 は 不 明 で あっ た

．

以 上のこ とか ら

，

インタ ク トな イ ネ

植 物体

に おい て

も

，

高 窒 素 施 肥

に より グル テリン サ ブユ ニ ッ ト

組 成

を

改 変 出 来

る こと

，

そし て

栄 養性

を

改 善

で

き

る ことが

明

らか と なっ た

．

キ

ー

ワ

ー

ド ：イ

ネ

，

貯 蔵

タン パ ク

質

，

サ ブユ ニ ッ ト

組

成

，

窒

素

・

硫 黄 代謝

　 　 　 作

物 研 究

　

56

：

17

−

22

_（

2011

_）

連 絡責 任者 ：田 中朋 之 （

tanakato

_＠

kais

、

kyoto

−

u

．

ac

．

jp

）

22