Substances of Legume SeedsCaused Absorbance ... - J-Stage

Japanese Society for Cryobiology and Cryotechnology

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zamegktzI7ftSv(Cryobiology and Cryotechnology] ,Vbl. 61, No. 1,59--64,2015 JSCC - Since19S9

Specific Changes in Dynamic States ofWater and Digestion of Storage

Substances of Legume Seeds Caused by Water Absorbance

Koichiro [IANAMACHI,i" Misaki HISAMP(TSU,2" Takashi YUASA,3

YUshi ISHIBASHIi･2t and Mari IWAYA-INOUEi･2

iGraduate School ofBioresouree andBioenvironmentat SZriences, 10]ushu Uhivensi4J4 Fukuoka 812-858J, Jbpan

2SZ]hool

ofAgriculture, 1<),ushu CinivensiijL Fleikuoka 812-8581. Jixpan

3School

ofAgriculture, Miyùaki Clhivensi(z Miyazaki 889-2192, lapan

("Equal contributors, tCorresponding

author, e-mail: [email protected])

The seeds of the soybean Glycine max (L.) Merr. show high sensitivity to excessive water in the

germination stage, and this susceptibility reduces germination rate, plant growth and yield.

Therefbre, it is important to determine the relationship between germination and water conditions.

In this study, we investigated whether nuclear magnetic resonance (NMR) relaxation time (71?)could be used to characterize germination in two soybean cultivars,

`Fukuyutaka' (yellow seed

coat) and `Wase-kurodaizu'

(black seed coat), and one kidney bean cultivar, Àmerica-saito',

The

germination index (op of `Fukuyutaka'

was the highest, fbllowed by that of `Wase-kurodaizu',

and

Àmerica-saito,'

The water contents and 71? values of long fraction in the two soybean cultivars

increased faster than those in the kidney bean, and T> values more clearly explained the difference

than water content. We also observed the digestion ofseed storage substances, Starch granules were

digested 72 hours after imbibition in the cotyledons of `Fukuyutaka,'

while still observed in those

of `Wase-kurodaizu'

and Àmerica-saito.'

Additionally, most oil bodies were digested in the

cotyledons of `Fukuyutaka,'

while abundant oil bodies were still observed in those of

CWase-kurodaizu'

72 hours after imbibition, This suggests that the difference in Gl can be explained

by water states indicated by NMR 7I? and the degradation of the storage substances of seeds after

imbibition,

(Received Aug. 6, 2014; Accepted Feb. 10, 2015)

INTRODUCTION

Most mature seeds have extremely lower water

content than the various plant organs. This low

moisture condition enables seeds to be stored fbr a

long period of time and still remain viable.

asFg:"Rfs,

[Key words: Germination, GJ,

Water contentl

NMR, Soybean,

However, seeds need to absorb a considerable

amount of water in order to germinate.i) Previous

reports have shown that nuclear magnetic resonance

(NMR) could be used to detect the state ofwater in

seeds,2,3) The dynamic state of water has been

shown to be an irnportant characteristic fbr

pre-harvest sprouting and germination in wheat.4) In

the study by Tanaka et al.,4) the water content of the

seeds of the two wheat cultivars,

`Shirogane-Komugi' (sprouting susceptible) and

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CNorin61'

(sprouting resistant), were not

significantly difTerent during their ripening stages.

On the other hand, the relaxation time (71?) values of

long fraction in `Shirogane-Komugi' were about

130 ms while that of `Norin61'

was about 60 ms at

28 days after pollination (DAP), Among jmportant

crops in the world, soybean plants are susceptible

to excessive water, especially in the germinationstage,S> and this sensitivity causes a lower

germination rate and a reductien in crop growth and

yield.5,6) A previous report suggests that

imbibitional damage in germination may bring

about the mechanical destruction of cotyledons by

the swelling of stored materials associated with the

rapid sorption of water into dry soybean seeds.i) It

has also been reported that there is a direct

relationship between the amount of soluble

carbohydrates exuded by a germinating seed and

seed rot caused by bacteria.S} However, underlying

physiological responses are poorly understood.

Furthermore, it has been reported that some

varieties of soybean with black seed coats exhibit a

high tolerance of flooding stress, while all varieties

with a yellow seed coat failed completely to

gerrninate,5i9) Therefbre, in the present study, we

investigated the relationship between germination

and the dynamic state of water (712 value) in two

soybean cultivars with diffbrent colors of seed coat,

compared to a kidney bean cultivar that can

germinate even by overnight soaking. iO)

MAI-ERIALS AND METHODS

Plant materials

TWo cultivars of the soybean [Glycine niax (L.)Merr.], `Fulcuyutaka'

with a yellow seed coat, and`Wase-kurodaizu'

with a black seed coat, and one

kidney bean cultivar (Phaseolus vzaLgaris L,)`America-saito'

were used in the present study.

Seeds of these cultivars were stored at 40C fbr one

month befbre use in the fbliowing experiments.

Germination assay

Seeds of each cultivar were placed on a wet

Kim-towel in a petri dish and were incubated at

30bC under well-watered conditions. The nurnber of

grains that germinated was counted daily fbr 7 days,

The germination index (GD was calculated using

the fo11owing equation:ii)

7 × ni +6 × n2 +...+I × n7 GI Total days x Total seeds (OsGIsl)

Here, ni, n2, ..., n7 are the number of seeds that

germinated on the first, second, and subsequent

days through 7 days. Sixty seeds frorn each cultivar

were used for the analysis.

Water content of seeds and iH-NMR

7"> values

The fresh weights ofthe seeds of each cultivar

were measured, and the seeds were dried fbr 24

hours at 90eC and used fbr dry weight measurement.

Water content was expressed as the ratio of the

weight of water to the weight of fresh tissue,

Spin-spin (7I?) relaxation times of the samples were

measured by a iH-NMR

spectrometer with a

magnet operating at 25 MHz fbr iH (Mp 25A;

JEOL, Ltd., Tokyo, Japan). The 71? values of the

samples were measured based on the procedure

described.7) Samples were prepared and put into an

NMR tube (10 mm in diameter) and the tube was

then put into the NMR spectrometer. The probe

temperature was maintained at 300C by thermostat,

71? was measured by the solid eco method until 10

hours after imbibition in the two soybean cultivars

and until 12 hours after imbibition in the kidney

bean cultivar. The Carr-Purcell-Meiboom-Gill

(CPMG) method was used after 12 hours after

imbibition in the two soybean cultivars and 24

hours after imbibition in the kidney bean. For

detailed analysis, a two-component analysis was

carried out and used as the long fraction of 71?.

There were five replications fbr this treatment.

Histochemical analysis

Seeds of `Fukuyutaka,'

`Wase-kurodaizu'

and

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JapaneseSociety foi Ciyobiology and Ciyotechnology

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CAmerica-saito'

that had imbibed for O, 8, 24, and

72 hours were fixed in fbrmalin acetic alcohol

(FAA, 80% ethanol: 100% acetic acid:

fbrmalin=90:5:5, vfv). Tissues were cut into 16-pm

sections and placed in a cryostat (HM-505E;Microm Co., Ltd,, Japan, Nagano). The sections

were stained with 1.5% iodine-potassium iodide

solution and O.5% Sudan III ethanol solvate, The

morphological features of the seeds of each cultivar

were observed under a microscope (ECLISE 80i;Nikon Co., Ltd,, Japan, Tokyo). Three or fbur seeds

from each treatment were used,

RESULTS AND DISCUSSION

Germination rate and germination index

Flooding during the sowing phase often inhibits

the gerrnination and emergence of soybeans.i2)

However, in the present study, all seeds ofthe three

cultivars reached 100% germination within 6 days

(Fig. IA). The (l4 which reflects gemination speed,

was larger in soybean seeds than in kidney bean

seeds (Fig. IB), SpecificallM `Fukuyutaka'

seeds,

which have a yellow seed coat, showed a higher Gl

value than `Wase-kurodaizu'

seeds, which have a

black seed coat. In a previous study, some varieties

of soybean with a black seed coat germinated

properly even after 10 days of soaking, while all

yellow seeds failed to germinate completely after 8

days of soaking.6) This suggests that there may be a

relationship between germination speed and color

ef seed coat.

Water content and dynamic states of water in

seeds

The water contents of the twe soybean cultivars

started to increase 30 minutes after imbibition, and

reached approximately two-fold of the dry seeds

after one hour of imbibition (Fig. 2A). There was

no significant difference between the two soybean

cultivars, On the other hand, the water content of

the kidney bean cultivar did not significantly

increase until 6 hours after imbibition. This

suggests that there is a specific difference in water

A t"eiA

1oo*x

ooNg

oog.E

4eEG'

2o

o

B

Ol134SS7

Time after imbibition (deys)

Germination lndex O O.2 OA O.S O.S

Fukuyutaka

Wase-kurodaizu

America-saito

{deys)

1

Fig. 1. Germination assay of two soybean cultivars and one

kidney bean cultivar. (A) Germination rate after

imbibition (days). (B) Germination index,

absorption in seeds between soybeans and kidney

beans. In a previous studM it was suggested that the

spin-lattice (n) and spin-spin (L) relaxation timesof NMR could be used to characterize pre-haryestsprouting in wheat (71-iticum aestivum L,)

cultivars,7) Another study clarified the water

distribution in soybean seeds during the first 24

hours after imbibition.i) [Ib analyze the dynamic

state of water in seeds among the present three

cultivars, we also measured NMR relaxation time.

The 71r and 7b relaxation times indicate the water

status in tissues since they reflect the motien of

water molecules.9) In a previous report, the 71?

values of long fraction in a sprouting-susceptible

wheat cultivar were higher than those in a

sprouting-resistant cultivar at 28 days after

pollination, indicating that the difference in 7> value

could explain the characteristics of sprouting

resistance among cultivars,7) In the present studM

befbre imbibition, the 7> values of the dry seeds of

the `Fukuyutaka' and

`Wase-kurodaizu' cultivars

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JapaneseSociety for Cryobiology and Ciyotechnology

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r

1･S}

1,6

s- II:vltO 1,O.O.

o.s=UO

O.6v

. O.49)

e',2

10oo

g.?-,.k.- re 10re )s-tne

E. ,{-rez

O.1

O O.5 1 1 ] 4 5 S 10 11 14 4S 72 th) Tjme after imbi bition {hours)

O O.S 1 13 4 6 8 10 12 24 de 72 {h) Time after imbibition (hours)

Fig. 2. Water content ofseeds and iH-NMR

re[axation time

(T>) (A) Water content (W. C.) in the seeds of two

soybean cu[tivars and one kidney bean cultiyars after

imbibition (hours) (n=5). (B) 71] of long ftaction

(n=5), Different letters indicate significant difftirence

among three p]ants at the 5% level (Tukey's test).

were approximately 1.5 ms and 2.5 ms, respectively,

and the vaiue of `America-saito'

dry seeds was

about O.5 ms (Fig, 2B). The T> value of soybean

seeds was prolonged to about 140 ms at 10 hours

after imbibition, while that of kidney bean seeds

was prolonged to 29 ms at 24 hours after imbibition,

suggesting that the free water of soybeans increases

more than that ofkidney beans. However, we fbund

no significant difference in the dynamic states of

water between `Fukuyutaka'

and `Wase-kurodaizu,'

A previous report suggests that imbibitional damage

during seed germination may bring about the

mechanical destruction of cotyledons associated

with the rapid absorption ofwater into the dry seeds

ofsoybeans.`) We predict that under excessive water

conditions, the rapidly increasing water content and

dynamic state of water may cause the physical

breakdown ofthe soybean cotyledon,

Digestion of seed storage substances among

the three cultivars

To determine whether there were any

differences in the starch and lipid spatial patterns in

the seed cotyledons among the three examined

cultivars, their cotyledons were examined by both

iodine-potassium iodide solution and Sudan 11I

stainings (Fig. 3). A large number of starch granules

in the cotyledons of `America-saito'

stained by

iodine-potassium iodide solution at 72 hours after

imbibition indicated that the seeds had not been

digested significantly at that time (Fig. 3A). On the

other hand, starch granules were fu11y digested 72

hours after imbibition in the `Fukuyutaka'

cotyledons, but not in the `Wase-kurodaizu'

cotyledons (Fig. 3A). Thus, starch digestion fbr

A Oh Sh 24h 72h

cege3,gg9iditsl.-e-$.8inE<

:Eg::.g:9isi;.e-$.8thE(

sxi .. ).,,

.ttVdi..l

,,e.tX

B Oh

'r

:.1ee, beS'/ '-..

'

L- t./v

di8h

.1 ..di..fft

{ lF,'tif

' ' itlj-if

SbX,:"''ll' ., vt., ,.t-1 . e.

r- ."

24h

tsls..}ec g

.rk･

'I",.','.ewvn･'F:,g 't-.Ii ..- x

t.. :,r,tht"-

.L '-'

k . ･+'-

i'" "

72h

' ''i

t../..

}

,,,..w..//'de;s'giiFipsl/,.

Fig. 3. Morphologieal features of soybean cotytedon tissues of

dry seeds (O hours), and at 8, 24 and 72 hours after

imbibition. Tissues were fixed in FAA. Scale bar/ SO

Fm. (A) Tissues were stained by iodine-potassiurn iodide

solution. (B) Tissues were stained by Sudan III.

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JapaneseSociety folCiyobiology and Ciyotechnology

germination was faster in `Fukuyutaka'

cotyledons

than in `Wase-kurodaizu'

cotyledons. Sudan III

staining also demonstrated that there was no

staining in the cotyledons of `America-saito'

dry

seeds (Fig. 3B), The lipid component in matured

kidney seeds is less than 89'6,i3) indicating that there

are few lipid components as seed storage nutrition,

In contrast, most oil bodies were digested in`Fukuyutaka'

cotyledons, while abundant oil bodies

were observed in `Wase-kurodaizu' cotyledons 72

hours after imbibition. Thus, the speed of digestion

of oil bodies in `Fukuyutaka' cotyledons in the

germination stage was faster than that of

CWase-kurodaizu'cotyledons.

In conclusion, the dynamic state of water and

the metabolism of storage substances such as starch

and lipids contribute to gerrnination characteristics.

When water treatment is appropriate, the difference

in germination speed between soybeans and kidney

beans can be explained by water content and NMR

relaxation time during germination. Furthermore,

the difierence in the GI between the two tested

soybean cultivars can be explained by differences in

the digestion by cotyledons of seed storage

oil-bodies and starch after imbibitien. We suggest

that the combination of NMR analysis and a

metabolic approach to seed storage can be used to

predict GI.

REFERENCES

1) Pietrzak LN, Fregeau-Reid J, Chatson B, Blackwell

B: Observation on water distribution in soybean

seed during hydration processes using nuclear

magnetic resonance imaging, Can J PIant Sci, 82,

513-519(2002)

2) Iwaya-Inoue M, Nonami H: Effects of trehalose on

fiower senescence from the viewpoint of physical

states of water, Environ Control Biol, 41, 3-15

(2003)3) Iwaya-Inoue M, Matsui R, Sultana N, Saitou K,

Fula]yama M: iH-NMR

method enables early

identification of degeneration in the quality of sweet

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potato tubeTs, J Agron Crop Sci, 190, 65-72 (2004)4) Tanaka M, Ishibashi Y; Yuasa T, Iwaya-Inoue M:

Analysis of pre-harvest sprouting during seed

maturation using iH-NMR,

Cryobiol Cryotechnol,

58, 87-91 (2012)

5) Sayama T, Nakazaki T, Ishikawa G, Yagasaki K,

Yamada N, Hirota N, Hirata K, Ybshikawa T, Saito

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soybean (Glysine mex [L,] Merr.), Plant Sci, 176,

514-521 (2009)6) Oosterhuis DM, Scott HD, Hampton RE,

Wullschleger SD: Physiolegical responses of two

soybean [Glycine mczx (L,) Merr1 cultivars to

short-term flooding, Environ and Experimental

Botany, 30, 85-92 (1990)

7) Ishida N, Kano H, Kobayashi T, Hamaguchi H,

Ybshida T: The relationship between imbibitional

damage and initial water content of soybeans, Agric

Biol Chem, 52, 2771-2775 (1988)8) Keeling BL: Soybean seed rot and the relation of

seed exudates to host susceptibilitM PhytopathoL 64,

1445-1447 (1 974)

9) Hou FF, Thseng FS: Studies on the fiooding

toleranee of soybean seed: varietal difTerences,

Euphytica, 57, 169-173 (1991)10) Pusxtai A, Duncan I: Changes in proteolytic enzyme

aetivities and transforrnation of nitrogenous

cornpounds in the germinating seeds of kidney bean

(Phaseo lus vuigaris), Planta, 96, 317-325 (1971)11) Walker-Simmons M, Sesing J: Temperature effects

on embryonic abscisic acid levels during

development of wheat grain dormancM J PIant

Growth Regul, 9, 51-56 (1990)12) Tian XH, Nakamura T, Kokubun M: The role of

seed structure and oxygen responsiveness in

pre-germination fiooding tolerance of soybean

cultivars, Plant Prod Sci, 8, 157-165 (2005)13) Megat Rusydi MR, Nraliza CWI Azrina A, Zulkhairi

A: Nutritional changes in germinated legumes and

rice varieties, International Food Res J, 18, 705-713

(2011)

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マメ科種子の吸水過程における水分動態と貯蔵物質の特異的変化：棚町弘一郎1，久松美咲

2，湯

浅高志3

，石橋勇志’・2

，井上眞理1，2 （

⊥

九州大学大学院生物資源環境科学府，2九州大学農学部，

3

宮崎大学農学部）匚キー

ワー

ド：発芽，発芽勢， NMR ，ダイズ，含水率］

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Substances of Legume SeedsCaused Absorbance ... - J-Stage

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