Vol.. 25 No,. 1 (1973)
A GERMINATION INHIBITOR FRACTION FROM SOYBEANS A PRELIMINARY REPORT
Yoshiko KAKUTANI* and Sin'itirB KAWAMURA
There are some germination inhibitors in plants.(l) According to EVENARI(~) such inhibitors were detected from about 100 species of plants, of which 22 species (including Pirum and Vzcza)
contained them in seeds They were relatively low in molecular weight. He listed u p un- saturated lactones, organic acids, aldehydes, essential acids, and alkaloids, besides hydrogen cyanide, ethylene, and ammonia.
Concerning leguminous seeds, inbitition in water prior to germination has various effects.@) This treatment is generally beneficial for germination of various seeds However, some adverse eff'ects have been noted in case of leguminous seeds They might have some connection with the presence of germination inhibitors.
Nearly all species of leguminous seeds germinate after a n induction period of some days. For this phenomenon, GAL ST ON(^) speculated that some substance in leguminous seeds inhibited germination, and it was removed by dissolution into water or was metabolized during soaking of seeds Germination inhibitors might be also adsorbed on soil particles (5)
According to G A L I T Z ( ~ > ~ ) immature soybean seeds contained water-soluble germination inhib- itor, which is probably non-volatile, heat-stable compound However, mature soybean seeds did not contain similar compounds. Phareolur vulgarzr contained low-molecular growth inhib- itors such as caffeic, protocatechuic, fl-cumaric, salicylic, ferulic, and 6-hydroxybenzoic acids, and coumarin (8) Pea plants contained two germination inhibitors, whose chemical natures were not decided.@)
We observed that the germination of soybeans was inhibited by a fraction of some substance(s) which was extracted from soybeans, and amino acids were contained in the hydrolyzate of this fraction.
This preliminary report describes the methods of extraction and partial purification of the fraction which inhibits the germination of soybeans.
Experimental 1 Extraction and Fractionation Procedures
One kg of soybeans immersed in 4 L of distilled water for 24 hr was hulled and ground with a chopper. The oil in the ground meal was extracted with 2 5 L of acetone The acetone solu- tion containing oil was separated from the mixture by squeezing through 4 sheets of thin cotton cloth and was clarified by centrifugation at 3500 rpm for 30 min.
The precipitate was collected and dried to remove acetone for 48 hr at rooin temperature The dried matter thus obtained was suspended in distilled water and the pII of the suspension was adjusted to 4 5 with dilute hydrochloric acid The resulting precipitate was removed by centrifugation at 3500 rpm for 30 min Solid ammonium sulfate was slowly added by mixing with the supernatant fraction to 50% saturation After allowing to stand overnight a t 5O, the
*
Present address: Kobe Women's University, Suma-ku, Kobe The request for reprints and other communicationsshould be addressed to this author
114 Tech Bull Fac Agr Kagawa Univ
precipitate was centrifuged off (3500 rpm for 30 min), and the supernatant fluid was dialyzed against distilled water overnight
The dialyzate was concentrated by passing through a column of DEAE-cellulose (7 x 7 cm) which was equilibrated with 0 01 M potassium phosphate buffer (pH 9 0) The following buffers were mainly used as eluting solvents: buffer A, 0 01 M potassium phosphate buffer ( p H 9.0), and buffer B, M potassium phosphate buffer (pH 5 1) containing M sodium chloride. The eluate with buffer B was dialyzed against distilled water overnight, and then it was placed on a top of a CM-cellulose column (3 x 20 cm) which was equilibrated with 0 01 M acetate buffer a t p H 4 0 The buffers used were 0 01 M acetate buffer (pH 4 0) and 0 01 M, 0 1 M, 0
2
M, and 0 5 M acetate buffers (pH 5 2), successively.2 Protein content
The protein content of each fraction was determined by absorbance at 280 nm 3. Germination Test
After washing under running water for 5 hr, soybean seeds were immersed in the protein solu- tion at p H 7 0 for 24 hr The degree of inhibition of germination was measured by observing the decrease in the number of seeds sprouted on the cotton cloth which was immersed in the solution Water was used instead of the solution as the control (pH 7.0) for observing germina- tion.
Results and Discussion
The precipitate and supernatant fluid obtained by adding ammonium sulfate to 50% satura- tion were separated by centrifugation at 3500 rpm for 30 min They were tested for the inhib- itory effect on germination of soybeans after dialysis. The result is shown in Table 1.
Table 1 Measurement of ratio of germination for the fractions separated with ammonium sulfate The precipitate and supernatant fluid which were separated with ammonium sulfate (50% saturation) were examined for the effect of inhibition on germination The test was carried out by counting the number of sprouted soybeans on the cotton cloth wetted by the three media Concentration of each fraction was presumed by measure- ment of the absorbance a t 280 nm
- - -- --
-Concentration Germination rate (%)
Medium of solution
0 D 280 nm/ml 16 hr 24 hr 40 hr
Water - 20 44 72
Precipitate* 1 0 5 12 36 60
Supernatant solution 1 02 0 12 20
*
The precipitate suspended in distilled waterFrom this result, we recognized that the fraction which inhibited germination of soybeans was contained in the supernatant fluid The supernatant fluid was concentrated by DEAE- cellulose column chromatography, and the concentrated fraction was examined for the effect of inhibition for sprouting of soybeans after dialysis.
As shown in Table 2, the 4 fractions were separated from supernatant fluid by DEAE-cellulose column chromatography The inhibition of germination was found in the last fraction which was eluted with M phosphate buffer (pH 5 1) containing M sodium chloride. Further purifica- tion by CM-cellulose column chromatography was carried out, and the results are shown in
Vol. 25, No.. 1 (1973) 115
Table 2 DEAE-cellulose column chromatography
DEAE-cellulose was packed in the column ( 7 x 7 cm) after equilibrating with 0 01 M phosphate buffer at pH 9 0. The supernatant fluid which was separated by ammonium sulfate (50% saturation) was applied to that column. The eluting buffer was M phosphate buffer containing M sodium chloride (pH 5 1) Each fraction was examined for the effcct of the inhibition on germination after dialysis The measurement of concentration of solution and germination test are as shown in Table 1
- -
Concentration of Germination rate
Fraction fraction Conc of fraction 16 hr 24 hr
0 D */ml 0 I) */total 0 D */ml % % 1 1.54 394 0 53 24 60 2 5 23 1830 0 53 28 72 3 4 21 1310 0 53 44 80 4 0 54 140 0 52 24 36 water - - - 68 96
*
Optical density of fraction was measured at 280 nmAcetate ( M ) +O. 0 1 4 0 . 0 1 4 + 0 . . l & O . 2-#+0..5&-
buffer 0 5 +O 5 M NaCl
PH b4..0--52-
Fig 1 CM-cellulose column chromatography
The fraction 4 (Table 2) eluted from the column of DEAE-cellulose was rechromatographed by CM-cellulose column Eluting busers used were 0 01 A4 (pH 4 0) and 0 01 M, 0 1 M, 0 2 M, 0 5 M acetate buffers (pH 5.2) and 0,5 M acetate buffer containing 0 5 M sodium chloride (pH 5 2) Each fraction (1 50 ml) was examined for the effect of repression (i e inhibition) on germination after dialysis The measurement of concentration of the fraction and the germination test are as shown in Table 1
Optical density at 280 nm % of repression compared to that of the control
Tech Bull Fac. Agr Kagawa Univ.
Fig. 1.
The three fractions (Nos. 3, 5, and 6) which were eluted with 0 01 M and 0 1 M acetate buffers a t p H 5.2 were found to inhibit germination of soybeans The fraction No. 3 was called "A", and the fractions No 5 plus 6 were called "B" I t was observed that "B" had the inhibitory effect about 100 times as high as the supernatant fluid separated with ammonium sulfate
After concentrating, "B" was hydrolyzed with 6 M hydrochloric acid in sealed tubes at 110' for 24 hr, and the hydrolyzate was chromatographed on filter paper No. 50 of Toyo Roshi Co. with ascending method by use of the mixed solvent of acetic acid, butanol, and distilled water (4: 4: 1). Spraying with the ninhydrin reagent showed the presence of amino acids in "B".
The proteins in soybeans have been studied by many investigators Among them, trypsin inhibitors, hemagglutinins, and intraperitoneal toxicity protein have been found in soybeans.(l0- 12) I t is possible that the fraction which inhibits germination of soybeans might be the same protein as one of them
However, we cannot exclude the possibility that this fraction may be some peptide or high- molecular prosthetic group of some conjugated protein.
An outline of this paper was presented before the second Annual Meeting of Chugoku-Shikoku Branch of Japanese Society of Food and Nutrition at Okayama on December 3, 1969 (13)
We wish to express thanks to Prof Daizo Yonezawa of Osaka Prefectural University for carry- ing out a portion of the experiment.
References NAKAYAMA, K : Hatuga Seirigaku (Physiology of Seed Germination), 239-58, Tokyo, Uchida Rokakuho (1960)
EVENARI, M : Bat Reo., 15 (1949) ; through (1). NAKAYAMA, K : NBrin Syusi no Hatuga (Seed Germination of Useful Plants), 15- 2 1, Tokyo, Uchida Rokakuho (1966)
GALSTON, A W : The Life of the Green Plant (ed GALSTON, A M.' ), 70, Englewood Cliffs, N J , U S A , Prentice Hall (1961)
NAKAYAMA, K : (3), 46
GALIIZ, D S : Plant Phyrzol, 33, Suppl xxxi (1958)
G ~ r r r z , D S
,
HOWELL, R W : Plant Physzol,34, Suppl x (1959)
PANNIER, R. F. de: Acta Czent Venerolana, 15,
16 (1964); throughChem Abrtr ,62, 55736 (1965) ( 9 ) LIBBERT, E
,
IJIEBENOW, H : Plants, 60, 582 (1964) ; through Chem Abrtr,
61, 1186J (1964) (10) RACKIS, J J , SESAME, H A., ANDERSON, R L,
SMITH, A K : J Amer. Chem Sac
,
81, 6265 (1959).(1 1) STEAD, R H
,
MTULENAERE, H J,
QUICKE, G. V : Arch Bzochem Bzoptyr,
113, 703 (1966). (12) RACKIS, J J : Biologically active components, Soybeans, Chemistry and Technology, Vol 1, Proteins (ed SMITH, A. K., CIRCLE, S. J.), 158- 202, Westport, Conn,
U S A Avi Pub1 Co,
(1972)(13) KAKUTANI, Y , MIYAIAKE, H : lliyo To Shokuryo,
23, 225 (1970)
~ B . ~ c $ ; ~ ~ & ~ ~ ' T ~ B . o % & Q P H % ~ & @ ~ Q H Y : L ,
D E A E - ~ I ~ D - x ~ 4 ~ l . Z . l 0, 7 9 b . T CM-..tt)k D - X ? J 4 ~ t z d : ?I 'I D ? b 'I"? 7 7 , 3%#4L7:. t%%L7:iX%kLhfl7k%%,?& 2 ' 7<
1 @%&.L7:9T,4 Y.*'IRT&&+, 47°F l z ' T & & x 3 ~ , ;ff:kL%?ZBb ~ . * 3 ~ 9 % % 3 ~ % % % . 3 % ' C & & 7 f 5 , 3 k , E h l z & . (Received M a y 31,1973)
