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Changes in the content of unsaturated higher fatty acids in the free lipid fraction of irradiated leave and an investigation of toxic properties of this fraction
Radiation Bofany, 1966, Vol. 6, pp. 351 to 355. Pergamon
Press Ltd. Printed
in Great Britain.
CHANGES IN THE CONTENT OF UNSATURATED HIGHER FATTY ACIDS IN THE FREE LIPID FRACTION OF IRRADIATED LEAVES AND AN INVESTIGATION OF TOXIC PROPERTIES OF THIS FRACTION Bach Institute
E. V, BOUDNITSKAYA of Biochemistry, Academy of Sciences, Moscow, U.S.S.R. (Received 26 August 1965)
Abstract-The action of ionizing radiation upon the content of oleic, linoleic and linolenic acids in the free lipid fraction isolated from bean (“Lutvia”) seedling leaves irradiated with doses of l-50 kR was studied. It was shown that the irradiation of plants with doses of 10, 30 and 50 kR led to a considerable increase (30-50%) in the content of unsaturated higher fatty acids in a free lipid fraction. The irradiation with 1 kR caused a decrease in the quantity of these acids in the fraction tested (20-400/0). The irradiation of bean leaves that had been steamed in order to inactivate enzymic systems showed either a clearly manifested increase or a decrease in the content of the acids investigated (some 7-10%) which proved the enzymic character of the storage of unsaturated higher fatty acids and their peroxides in a free lipid fraction isolated from X-irradiated plants. It was shown that a free lipid fraction isolated from irradiated and unirradiated bean seedlings and containing a large quantity of unsaturated higher fatty acids and their peroxides has a strong toxic effect, inhibiting the growth and development of germinating wheat seeds (“Moscovskaya 2411”). This is in good agreement with the data of other investigators carried out in the same line. R&sum&On a ttudie l’action des radiations ionisantes sur le contenu en acide oltique, linoltioque et linoltnique dans la fraction lipidique libre isolte a Rartir de feuilles de plantules de f&e irradites a des doses de 1 a 50 kR. On a montrt que l’irradiation des plantes par des doses de 10, 30 et 50 kR produit un accroissement considerable (30 ?I 50%) d u contenu en acides gras non satur&s suptrieurs dans la fraction lipidique libre. L’irradiation par 1 kR produit une diminution de la quantitt de ces acides dans la fraction ttudite (20 B 40%). L ‘irradiation de feuilles de f&e prealablement cuites en vue d’inactiver divers sysdmes enzymatiques a montrt soit un accroissement net, soit une diminution du contenu des acides gras ttudib (environ l-10%). Ceci prouve le caractere enzymatique de la conservation des acides gras non sat&s superieurs et de leurs peroxydes dans la fraction lipidique libre provenant de plan& irradites par les rayons X. On a montre que la fraction lipidique libre isolte de plant&s de f&es irradites ou non et contenant une grande quantitt d’acides gras non satur& suptrieurs ainsi que leurs peroxydes possedait un effet toxique accuse se manifestant par I’inhibition de croissance et du dheloppement des graines de blk. Ces faits concordent bien avec les don&es d’autres chercheurs travaillant dans la m&me ligne de recherches. Zusammenfassung-Der Einfluss ionisierender Strahlen auf den Cl-, Lmol- und Linolensluregehalt der Fraktion freier Lipoide, welche sich aus Blattem von Bohnenkeimlingen extrahieren lisst, wurde untersucht. Bestrahlungen mit 10, 30 und 50 kR fuhrten zu einer betr%htlichen Steigerung (30-50%) des Gehaltes dieser Fraktion an ungeslttigten 351
352
E. V. BOUDNITSKAYA Fettsiiuren. Bestrahlung mit 1 kR verursachte eine Abnahme des Gehaltes ungesittigter Bestrahlung von Bohnenbllttern, welche zur Inaktivierung Fettsauren urn 2@-441~0. enzymatischer Systeme eine Hitzebehandlung erhalten hatten, bewirkte entweder eine deutliche Steigerung oder eine Abnahme des Gehaltes der gepriiften Sauren (ca. 7-10%). Dies beweist den enzymatischen Charakter der Speicherung ungesattigter hliherer Fettsauren und ihrer Peroxide in der gepriiften Fraktion. Fraktionen aus bestrahlten und aus unbestrahlten Keimlingen, welche einen hohen Anteil ungesattigter Fettsiuren und ihrer Peroxide enthielten, tibten eine starke Hemmwirkung auf Wachstum und Entwicklung keimender Weizensamen aus. Dies stimmt mit Daten anderer Untersucher gut iiberein.
INTRODUCTION earlier that the activity of the lipoxygenase (lipoxidase), increased enzyme, from about 30 to 50 per cent in the leaves of various irradiated plants (wheat, barley, beans, soyabeans and peas). This activation of the enzyme was observed after 24 hr with radiation oflipid doses up to 50 kR. (l sa) The determination peroxides in these leaves showed an increase of about 2 to 6 times more than those found in the unirradiated leaves. @) Since no peroxidation was observed in the preparations from the leaves that had been inactivated by steam prior to radiation,(s) it was established that X-irradiation activates lipoxygenase which in turn brings about the peroxidation of endogenous fatty acid at an accelerated rate. Several workers have observed a sharp increase in the total free lipids of blood and livers of the irradiated animals.(4*6~6) It is not yet known if radiation has a similar effect on the free lipid metabolism in plants. Increased peroxidation by radiation-activated lipoxygenase(s can be brought about if there is a sufficient amount of free lipids already present in the tissue and the total lipid pool is increased following radiation. This study is concerned with changes in the levels of unsaturated fatty acids (oleic, linoleic and linolenic acids) in the irradiated leaves of bean seedlings. IT
WAS
SHOWN
MATERIALS AND METHODS The experimental material consisted of 15-day old bean (“La&“) seedlings grown in a hot house. The first pair of leaves at this time was fully expended. About 24 hr before irradiation, IO-15 seedlings were removed from the soil, washed with water and placed in a beaker containing water. The diameter of the crown of
the leaves did not exceed 8-10 cm. The seedlings were irradiated with a RUP- 1 apparatus (15 mA, 210 kVp without filtration) and then kept in the light. The radiation rate was 550-650 R/min at a target distance of 22 cm. However, when a higher radiation dose was required then the rate was 800 R/min. The leaves were detached, fixed with dry ice and lyophilized 15 or 30 min, 2 or 4, 24 and 48 hr after the irradiation with doses of 1, IO, 30 and 50 kR, respectively. The leaves of unirradiated seedlings served as controls and were treated in the same wa)r as the leaves of the irradiated plants. Lyophilized leaves were ground in a mortar. A sample of the powdered material, not exceeding 200 mg, was extracted with freshly distilled petroleumether (b.p. 50-55°C) in an atmosphere of N, or CO, in a specially designed microextractor. t7e8) Petroleum ether was evaporated in a current of nitrogen and the residue was dissolved in ethanol-ether (2:l) mixture. Fatty acids of this solution were resolved by ascending paper chromatography using acetic acid-formic acid-water (72 : 25 : 2.5 v/v) as a solvent mixture. After 20-24 hr of development, the chromatograms were sprayed with O-1 M KMnO, solution which oxidized the unsaturated fatty acid giving yellow-brown spots. The quantities of the unsaturated fatty acids were determined from these spots with a densitometer (for details of the experimental procedure see Ref. 9). The results are expressed as mg linoleic or linolenic acid/g of dry weight of the leaves. RESULTS AND DISCUSSION The results of the quantitative determination of linoleic and linolenic acids after the irradiation of plants with doses of 1, 10, 30 and 50 kR are given in Table 1. This table shows the average data of 2-5 experimental series for each
CHANGES Table
Dose W
1. Content
Acid
IN CONTENT
of unsaturated
higher
Control mdg dry w. (100%)
OF HIGHER
FATTY
fatQ acids in the free lipid fraction
ACIDS
353
from irradiated
Time after irradiation, 0.5
4
plants*
hr 24
48
(% of control) 1
linoleic linolenic
3.41 13.9
110*3&3*0 100.9 f0.4
10
linoleic linolenic
1.80 12.95
30
linoleic linolenic
3.62 15.22 +
50
linoleic linolenic
3.67 lo.8
* Average
data of 2-5 experimental
70.663.8 86.5 f2.8
60.ak2.a 75.0fl.4
73.3 52.7 96.6 56.2
277.7f 10.0 86.9*2*0
174*7-&-6.0 68+4-&3.6
300-o f9.2 47.451.0
158~0~14~0 89*6&2*0
.io5.a*i5.0 52.2-&10.0
188.6*a.a 109*5*3.0
161.1f15.5 124.3fl.O
209.1 f 16.0 127.0f2.0
74.7 f6-2 95.5 fa-5
124.5*18.0 137.0*8-5
95*5*10*0 119.5f2.0
91*0&2.0 115.7f6.0
series, 3-5 determinations
irradiation dose. Each series includes the determination of the quantity of unsaturated higher fatty acids in the leaves of the control and experimental plants. Both in the control and the experimental plants the content of linolenic acid is much higher than that of linoleic acid. Only traces of oleic acid could be established. As can be seen from Table 1, the quantity of the acids investigated in the free lipid fraction determined in plants grown and assayed under nearly the same conditions, but during different seasons of the year, manifests a certain variation. Thus for the control plants of every experimental series the average data of linoleic acid content varied within the range 1.8-3.67 mg/g of dry weight of leaves; linolenic acid content varied from 10.8 to 15.22 mg/g of dry weight. Those variations can be ascribed to seasonal changes in the fatty acid content in the leaves and to a partial oxidation of acids formed during analysis. However, the percentage of the quantity of unsaturated higher fatty acids in the control and the experimental samples for each irradiation dose in each experimental series show a regular change. Thus, the regularity of the changes in the content of unsaturated higher fatty acids under the effect of irradiation is preserved. Average deviations in the quantity of linoleic and linolenic acids in the free lipid fraction from irradiated plants obtained by
in each series.
statistical treatment of the data are given in the corresponding columns of Table 1. The data presented in Table 1 show that the radiation action on the plants (the doses of 1 and 30-50 kR) are different. The doses of 30 and 50 kR caused a small decrease in the content of linoleic and linolenic acids in the first 30 min after irradiation followed by a sharp increase in the content of these acids 4 and 24 hr after irradiation. No oleic acid could be detected during this time. The dose of 1 kR leads, as a rule, only to a decrease in. the quantity of unsaturated higher fatty acids without subsequent increase in their content. The increase in the contents of linoleic and linolenic acids in the free lipid fraction from plants irradiated with 10-50 kR observed by the authors fully agree with the data obtained earlier in which the lipid increase could be due to some metabolic disturbance or release of bound fatty acids from the damaged tissue. This seems to be related with considerable damage in the enzymic processes of lipid metabolism in irradiated plants in which an increase in lipid peroxide content and lipoxidase activation in the leaves of irradiated seedlings had been observed. W) Investigation of toxic substances in an isolated free lipid fraction from irradiated leaves was carried out taking into account the data found
E. V. BOUDNITSKAYA
354
in the literature on the presence of water soluble non-lipid substances which, when being isolated from irradiated plants, possess a strong toxic action. (l”-16) The authors studied the toxic action of the free lipid fraction from irradiated leaves upon the growth and development of germinating wheat seeds (“Moscovskaya 2411”). The toxicity of the free lipid fraction from irradiated and non-irradiated bean plants in which the content of oleic, linoleic and linolenic acids had been determined, was investigated. The extract from unirradiated bean leaves contained unsaturated fatty acids in a smaller amount than the irradiated bean seedlings (cf. Table 1). After embedding in a large vessel for 24 hr, germinating wheat seeds were placed in Petri dishes (50 in each dish) and sprayed under CO, pressure, with the extracts of the free lipid fractions from the control (unirradiated) and irradiated plants with the doses from 1 to 50 kR. Wheat seedlings in several Petri dishes were sprayed in the same manner with only an alcohol-ether mixture served as control in which the lipid extracts from u&radiated and irradiated bean leaves were dissolved. A few Petri
Time
after
dishes were not sprayed at all in order to observe their normal development. After 7 days the wheat seedlings were big enough to judge the action of the solvent and the free lipid fraction (alcohol-ether mixture and the extracts from m&radiated and irradiated bean leaves). After being sprayed with plant extracts the wheat seedlings were left in the Petri dishes for 7 days. The plants were then cut off on the level of the seed (the roots not being included), and their length measured in mm. For each dose 3-5 experimental series were carried out with at least 200 seedlings in each experiment. The data after statistical treatment are presented in Fig. 1. Average fluctuations of seedling height (height per plant) do not exceed f5 per cent. The action of the extracts of the free lipid fraction upon unirradiated seedlings served as control. As can be seen from the diagram, the maximum inhibition of the growth of germinating wheat seeds just starting to shoot was brought about by a free lipid fraction isolated from bean seedlings 2-24 hr after their irradiation with doses of 30 and 50 kR. Free lipid fraction extracts from bean leaves irradiated with 1 kR slightly stimulated the growth of the seedlings, a fact that can be explained when comparing
irradiation,
hr
1. The effect of the free lipid fraction isolated from irradiated bean seedlings 0.5, 4, 24 and 48 hr after X-irradiation on the growth and development of germinating wheat seeds. I. Developing normally. II. Action of the solvents (alcohol-ether). III. Action of the extracts of the free lipid fraction upon unirradiated seedlings (control). IV. 1 kR V. 10 lcR Action of the extracts from the seedlings irradiated with a dose of 1, 10, VI. 30 kR 1 30 and 50 kR respectively. VII. 5okRJ FIG.
CHANGES
IN CONTENT
the data on the toxicity of this fraction with those on the change in the content of linoleic and linolenic acids in the same fraction isolated from irradiated plants. The higher the content of linoleic and linolenic acids in the fraction the more inhibited were the growth and development of germinating wheat seeds and vice versa. Previously, the author asserted that higher radiation leads to the higher amount of peroxide production.(r*sl That means that toxicity is due to the amount of peroxide present in the extracts with higher concentrations of unsaturated fatty acids. The toxicity is a function of the amounts of peroxide present in the extracts of free lipid fractions. Untreated wheat seeds grow and develop better than those treated with an extract from unirradiated bean leaves. The irradiation of leaves in which enzymic systems were inactivated by steam heating prior to irradiation did not result in a change in the content of unsaturated higher fatty acids. The data obtained confirm the hypothesis put forth earlier that changes in the quantity of unsaturated higher fatty acids in the free lipid fraction from irradiated plants are due to considerable damage to the enzymic processes in lipid metabolism, which, however, is not related to the enzyme lipoxygenase, but could be due to some other metabolic disturbance.
REFERENCES 1. BOUDNI-IXAYA E. V. and BORNOVA I. G. (1960) Radiation effect on the lipid metabolism of plants. pp. 203-205. In G. POPJAK (Ed.), Biochemistry of Lipids, Biochem. Probl. Lipids. PTOC. Intern. Conf., 5th, Vienna, 1958. Pergamon Press, London. 2. BOUDNITUUYA E. V. and BORI~~OVA I. G. (1960) Formation of peroxide-s and activation of enzyme oxidation of lipids after irradiation of plants. pp. 85-91. In SBORNIK, Rol perekisei i kisloroda v nachalnihk stadiakh radiobiologischeskogo effect (The role of peroxides and oxygen in the initial stages of radiations effect). Soviet Press, Izd. AN USSR, Moscow.
OF HIGHER
FATTY
ACIDS
355
E. V. (1961) Effect of ionizing 3. BOUDNIT~KAYA radiation on oxidation ‘processes in plants. pp. 367-370. In B. C. CHRISTENSEN and B. BUCHMANN (Eds.), Progress in Photobiology. PTOC. Intern. Congr. Photobiol., 3rd, Copenhagen, 1960. 4. SMIRNOV K. V. and SHATERNIKIV A. A. (1960) Effect of external ionizing irradiation on the participation of liver and intestine in lipid metabolism. Vop. Med. Khim. 6, 464-470. 5. TRETIAKOVA K. A. and GRODZENSKY D. E. (1959) Effect of ionizing irradiation on the biosynthesis of cholesterol and of fatty acids. Vopr. Med. Khim. 5, 363-369. 6. ME~~ERD R. B. JR., WEBSTER W. W. and NYMAN M. A. (1958) Lipid metabolism in X-irradiated mice and rats. Radiation Res. 8,461-465. A. and POLESOP~KY W. 7. ZIRM K. L., PONGRATZ (1955) Beitrag zur konstitution pflanzlicher lipoide, im besonderen der lipoidkomponente des chloroplastins. Biochem. <. 326, 405-412. A. N. and PROSKURYAKOV N. I. 8. BELOZER~KY ( 195 1) Prakticheskoe rukovodstvo po biochimii rastenii. Soviet Press, Izd. Sov. Nauka, Moscow. E. V. (1963) 9. BORISO~A I. G. and BOUDNIT~KAYA A method for quantitative paper chromatography of unsaturated higher fatty acids. Biokhimiya, USSR 28, 497-500. English translation by Cons&ants Bureau Enterprises, New York (1964), pp. 405-407. 10. KRWKOVA L. M. (1961) Production of antimitotic substances in different plant species after irradiation. Radiobiologea, USSR 1, 139-144. 11. KOPYLOV V. A. (1961) Influence of antimitotic substances from irradiated plants on the growth of mice. Radiobiologiya, USSR 1, 358-363. 12. KRWKOVA L. M., LOMAKIN M. S. and KUSIN A. M. (1961) Effect of extracts from irradiated and non-irradiated plants (Vicia Phaba) on growth of tumor and homologous normal tissue. Radiobiologja,
13. SIEGEL S. toxicity in 15,21-26. 14. BOWEN H. from seed
USSR
1, 365-372.
M. (1962) Observations on peroxide seed germination. Physiol. Planturum.
J. M. and THICK J. (1960) Factors extracts that modify radiosensitivity. Radiation Res. 13, 234-241. 15. Effects of seed extracts on radiosensitivity. In Effects of ionizing radiation on see& (1961) Proceedings of the Symposium on the effect of ionizing radiation on seeds. IAEA, Vienna, Austria, pp. 75-82. 1961.
Press Ltd. Printed
in Great Britain.
CHANGES IN THE CONTENT OF UNSATURATED HIGHER FATTY ACIDS IN THE FREE LIPID FRACTION OF IRRADIATED LEAVES AND AN INVESTIGATION OF TOXIC PROPERTIES OF THIS FRACTION Bach Institute
E. V, BOUDNITSKAYA of Biochemistry, Academy of Sciences, Moscow, U.S.S.R. (Received 26 August 1965)
Abstract-The action of ionizing radiation upon the content of oleic, linoleic and linolenic acids in the free lipid fraction isolated from bean (“Lutvia”) seedling leaves irradiated with doses of l-50 kR was studied. It was shown that the irradiation of plants with doses of 10, 30 and 50 kR led to a considerable increase (30-50%) in the content of unsaturated higher fatty acids in a free lipid fraction. The irradiation with 1 kR caused a decrease in the quantity of these acids in the fraction tested (20-400/0). The irradiation of bean leaves that had been steamed in order to inactivate enzymic systems showed either a clearly manifested increase or a decrease in the content of the acids investigated (some 7-10%) which proved the enzymic character of the storage of unsaturated higher fatty acids and their peroxides in a free lipid fraction isolated from X-irradiated plants. It was shown that a free lipid fraction isolated from irradiated and unirradiated bean seedlings and containing a large quantity of unsaturated higher fatty acids and their peroxides has a strong toxic effect, inhibiting the growth and development of germinating wheat seeds (“Moscovskaya 2411”). This is in good agreement with the data of other investigators carried out in the same line. R&sum&On a ttudie l’action des radiations ionisantes sur le contenu en acide oltique, linoltioque et linoltnique dans la fraction lipidique libre isolte a Rartir de feuilles de plantules de f&e irradites a des doses de 1 a 50 kR. On a montrt que l’irradiation des plantes par des doses de 10, 30 et 50 kR produit un accroissement considerable (30 ?I 50%) d u contenu en acides gras non satur&s suptrieurs dans la fraction lipidique libre. L’irradiation par 1 kR produit une diminution de la quantitt de ces acides dans la fraction ttudite (20 B 40%). L ‘irradiation de feuilles de f&e prealablement cuites en vue d’inactiver divers sysdmes enzymatiques a montrt soit un accroissement net, soit une diminution du contenu des acides gras ttudib (environ l-10%). Ceci prouve le caractere enzymatique de la conservation des acides gras non sat&s superieurs et de leurs peroxydes dans la fraction lipidique libre provenant de plan& irradites par les rayons X. On a montre que la fraction lipidique libre isolte de plant&s de f&es irradites ou non et contenant une grande quantitt d’acides gras non satur& suptrieurs ainsi que leurs peroxydes possedait un effet toxique accuse se manifestant par I’inhibition de croissance et du dheloppement des graines de blk. Ces faits concordent bien avec les don&es d’autres chercheurs travaillant dans la m&me ligne de recherches. Zusammenfassung-Der Einfluss ionisierender Strahlen auf den Cl-, Lmol- und Linolensluregehalt der Fraktion freier Lipoide, welche sich aus Blattem von Bohnenkeimlingen extrahieren lisst, wurde untersucht. Bestrahlungen mit 10, 30 und 50 kR fuhrten zu einer betr%htlichen Steigerung (30-50%) des Gehaltes dieser Fraktion an ungeslttigten 351
352
E. V. BOUDNITSKAYA Fettsiiuren. Bestrahlung mit 1 kR verursachte eine Abnahme des Gehaltes ungesittigter Bestrahlung von Bohnenbllttern, welche zur Inaktivierung Fettsauren urn 2@-441~0. enzymatischer Systeme eine Hitzebehandlung erhalten hatten, bewirkte entweder eine deutliche Steigerung oder eine Abnahme des Gehaltes der gepriiften Sauren (ca. 7-10%). Dies beweist den enzymatischen Charakter der Speicherung ungesattigter hliherer Fettsauren und ihrer Peroxide in der gepriiften Fraktion. Fraktionen aus bestrahlten und aus unbestrahlten Keimlingen, welche einen hohen Anteil ungesattigter Fettsiuren und ihrer Peroxide enthielten, tibten eine starke Hemmwirkung auf Wachstum und Entwicklung keimender Weizensamen aus. Dies stimmt mit Daten anderer Untersucher gut iiberein.
INTRODUCTION earlier that the activity of the lipoxygenase (lipoxidase), increased enzyme, from about 30 to 50 per cent in the leaves of various irradiated plants (wheat, barley, beans, soyabeans and peas). This activation of the enzyme was observed after 24 hr with radiation oflipid doses up to 50 kR. (l sa) The determination peroxides in these leaves showed an increase of about 2 to 6 times more than those found in the unirradiated leaves. @) Since no peroxidation was observed in the preparations from the leaves that had been inactivated by steam prior to radiation,(s) it was established that X-irradiation activates lipoxygenase which in turn brings about the peroxidation of endogenous fatty acid at an accelerated rate. Several workers have observed a sharp increase in the total free lipids of blood and livers of the irradiated animals.(4*6~6) It is not yet known if radiation has a similar effect on the free lipid metabolism in plants. Increased peroxidation by radiation-activated lipoxygenase(s can be brought about if there is a sufficient amount of free lipids already present in the tissue and the total lipid pool is increased following radiation. This study is concerned with changes in the levels of unsaturated fatty acids (oleic, linoleic and linolenic acids) in the irradiated leaves of bean seedlings. IT
WAS
SHOWN
MATERIALS AND METHODS The experimental material consisted of 15-day old bean (“La&“) seedlings grown in a hot house. The first pair of leaves at this time was fully expended. About 24 hr before irradiation, IO-15 seedlings were removed from the soil, washed with water and placed in a beaker containing water. The diameter of the crown of
the leaves did not exceed 8-10 cm. The seedlings were irradiated with a RUP- 1 apparatus (15 mA, 210 kVp without filtration) and then kept in the light. The radiation rate was 550-650 R/min at a target distance of 22 cm. However, when a higher radiation dose was required then the rate was 800 R/min. The leaves were detached, fixed with dry ice and lyophilized 15 or 30 min, 2 or 4, 24 and 48 hr after the irradiation with doses of 1, IO, 30 and 50 kR, respectively. The leaves of unirradiated seedlings served as controls and were treated in the same wa)r as the leaves of the irradiated plants. Lyophilized leaves were ground in a mortar. A sample of the powdered material, not exceeding 200 mg, was extracted with freshly distilled petroleumether (b.p. 50-55°C) in an atmosphere of N, or CO, in a specially designed microextractor. t7e8) Petroleum ether was evaporated in a current of nitrogen and the residue was dissolved in ethanol-ether (2:l) mixture. Fatty acids of this solution were resolved by ascending paper chromatography using acetic acid-formic acid-water (72 : 25 : 2.5 v/v) as a solvent mixture. After 20-24 hr of development, the chromatograms were sprayed with O-1 M KMnO, solution which oxidized the unsaturated fatty acid giving yellow-brown spots. The quantities of the unsaturated fatty acids were determined from these spots with a densitometer (for details of the experimental procedure see Ref. 9). The results are expressed as mg linoleic or linolenic acid/g of dry weight of the leaves. RESULTS AND DISCUSSION The results of the quantitative determination of linoleic and linolenic acids after the irradiation of plants with doses of 1, 10, 30 and 50 kR are given in Table 1. This table shows the average data of 2-5 experimental series for each
CHANGES Table
Dose W
1. Content
Acid
IN CONTENT
of unsaturated
higher
Control mdg dry w. (100%)
OF HIGHER
FATTY
fatQ acids in the free lipid fraction
ACIDS
353
from irradiated
Time after irradiation, 0.5
4
plants*
hr 24
48
(% of control) 1
linoleic linolenic
3.41 13.9
110*3&3*0 100.9 f0.4
10
linoleic linolenic
1.80 12.95
30
linoleic linolenic
3.62 15.22 +
50
linoleic linolenic
3.67 lo.8
* Average
data of 2-5 experimental
70.663.8 86.5 f2.8
60.ak2.a 75.0fl.4
73.3 52.7 96.6 56.2
277.7f 10.0 86.9*2*0
174*7-&-6.0 68+4-&3.6
300-o f9.2 47.451.0
158~0~14~0 89*6&2*0
.io5.a*i5.0 52.2-&10.0
188.6*a.a 109*5*3.0
161.1f15.5 124.3fl.O
209.1 f 16.0 127.0f2.0
74.7 f6-2 95.5 fa-5
124.5*18.0 137.0*8-5
95*5*10*0 119.5f2.0
91*0&2.0 115.7f6.0
series, 3-5 determinations
irradiation dose. Each series includes the determination of the quantity of unsaturated higher fatty acids in the leaves of the control and experimental plants. Both in the control and the experimental plants the content of linolenic acid is much higher than that of linoleic acid. Only traces of oleic acid could be established. As can be seen from Table 1, the quantity of the acids investigated in the free lipid fraction determined in plants grown and assayed under nearly the same conditions, but during different seasons of the year, manifests a certain variation. Thus for the control plants of every experimental series the average data of linoleic acid content varied within the range 1.8-3.67 mg/g of dry weight of leaves; linolenic acid content varied from 10.8 to 15.22 mg/g of dry weight. Those variations can be ascribed to seasonal changes in the fatty acid content in the leaves and to a partial oxidation of acids formed during analysis. However, the percentage of the quantity of unsaturated higher fatty acids in the control and the experimental samples for each irradiation dose in each experimental series show a regular change. Thus, the regularity of the changes in the content of unsaturated higher fatty acids under the effect of irradiation is preserved. Average deviations in the quantity of linoleic and linolenic acids in the free lipid fraction from irradiated plants obtained by
in each series.
statistical treatment of the data are given in the corresponding columns of Table 1. The data presented in Table 1 show that the radiation action on the plants (the doses of 1 and 30-50 kR) are different. The doses of 30 and 50 kR caused a small decrease in the content of linoleic and linolenic acids in the first 30 min after irradiation followed by a sharp increase in the content of these acids 4 and 24 hr after irradiation. No oleic acid could be detected during this time. The dose of 1 kR leads, as a rule, only to a decrease in. the quantity of unsaturated higher fatty acids without subsequent increase in their content. The increase in the contents of linoleic and linolenic acids in the free lipid fraction from plants irradiated with 10-50 kR observed by the authors fully agree with the data obtained earlier in which the lipid increase could be due to some metabolic disturbance or release of bound fatty acids from the damaged tissue. This seems to be related with considerable damage in the enzymic processes of lipid metabolism in irradiated plants in which an increase in lipid peroxide content and lipoxidase activation in the leaves of irradiated seedlings had been observed. W) Investigation of toxic substances in an isolated free lipid fraction from irradiated leaves was carried out taking into account the data found
E. V. BOUDNITSKAYA
354
in the literature on the presence of water soluble non-lipid substances which, when being isolated from irradiated plants, possess a strong toxic action. (l”-16) The authors studied the toxic action of the free lipid fraction from irradiated leaves upon the growth and development of germinating wheat seeds (“Moscovskaya 2411”). The toxicity of the free lipid fraction from irradiated and non-irradiated bean plants in which the content of oleic, linoleic and linolenic acids had been determined, was investigated. The extract from unirradiated bean leaves contained unsaturated fatty acids in a smaller amount than the irradiated bean seedlings (cf. Table 1). After embedding in a large vessel for 24 hr, germinating wheat seeds were placed in Petri dishes (50 in each dish) and sprayed under CO, pressure, with the extracts of the free lipid fractions from the control (unirradiated) and irradiated plants with the doses from 1 to 50 kR. Wheat seedlings in several Petri dishes were sprayed in the same manner with only an alcohol-ether mixture served as control in which the lipid extracts from u&radiated and irradiated bean leaves were dissolved. A few Petri
Time
after
dishes were not sprayed at all in order to observe their normal development. After 7 days the wheat seedlings were big enough to judge the action of the solvent and the free lipid fraction (alcohol-ether mixture and the extracts from m&radiated and irradiated bean leaves). After being sprayed with plant extracts the wheat seedlings were left in the Petri dishes for 7 days. The plants were then cut off on the level of the seed (the roots not being included), and their length measured in mm. For each dose 3-5 experimental series were carried out with at least 200 seedlings in each experiment. The data after statistical treatment are presented in Fig. 1. Average fluctuations of seedling height (height per plant) do not exceed f5 per cent. The action of the extracts of the free lipid fraction upon unirradiated seedlings served as control. As can be seen from the diagram, the maximum inhibition of the growth of germinating wheat seeds just starting to shoot was brought about by a free lipid fraction isolated from bean seedlings 2-24 hr after their irradiation with doses of 30 and 50 kR. Free lipid fraction extracts from bean leaves irradiated with 1 kR slightly stimulated the growth of the seedlings, a fact that can be explained when comparing
irradiation,
hr
1. The effect of the free lipid fraction isolated from irradiated bean seedlings 0.5, 4, 24 and 48 hr after X-irradiation on the growth and development of germinating wheat seeds. I. Developing normally. II. Action of the solvents (alcohol-ether). III. Action of the extracts of the free lipid fraction upon unirradiated seedlings (control). IV. 1 kR V. 10 lcR Action of the extracts from the seedlings irradiated with a dose of 1, 10, VI. 30 kR 1 30 and 50 kR respectively. VII. 5okRJ FIG.
CHANGES
IN CONTENT
the data on the toxicity of this fraction with those on the change in the content of linoleic and linolenic acids in the same fraction isolated from irradiated plants. The higher the content of linoleic and linolenic acids in the fraction the more inhibited were the growth and development of germinating wheat seeds and vice versa. Previously, the author asserted that higher radiation leads to the higher amount of peroxide production.(r*sl That means that toxicity is due to the amount of peroxide present in the extracts with higher concentrations of unsaturated fatty acids. The toxicity is a function of the amounts of peroxide present in the extracts of free lipid fractions. Untreated wheat seeds grow and develop better than those treated with an extract from unirradiated bean leaves. The irradiation of leaves in which enzymic systems were inactivated by steam heating prior to irradiation did not result in a change in the content of unsaturated higher fatty acids. The data obtained confirm the hypothesis put forth earlier that changes in the quantity of unsaturated higher fatty acids in the free lipid fraction from irradiated plants are due to considerable damage to the enzymic processes in lipid metabolism, which, however, is not related to the enzyme lipoxygenase, but could be due to some other metabolic disturbance.
REFERENCES 1. BOUDNI-IXAYA E. V. and BORNOVA I. G. (1960) Radiation effect on the lipid metabolism of plants. pp. 203-205. In G. POPJAK (Ed.), Biochemistry of Lipids, Biochem. Probl. Lipids. PTOC. Intern. Conf., 5th, Vienna, 1958. Pergamon Press, London. 2. BOUDNITUUYA E. V. and BORI~~OVA I. G. (1960) Formation of peroxide-s and activation of enzyme oxidation of lipids after irradiation of plants. pp. 85-91. In SBORNIK, Rol perekisei i kisloroda v nachalnihk stadiakh radiobiologischeskogo effect (The role of peroxides and oxygen in the initial stages of radiations effect). Soviet Press, Izd. AN USSR, Moscow.
OF HIGHER
FATTY
ACIDS
355
E. V. (1961) Effect of ionizing 3. BOUDNIT~KAYA radiation on oxidation ‘processes in plants. pp. 367-370. In B. C. CHRISTENSEN and B. BUCHMANN (Eds.), Progress in Photobiology. PTOC. Intern. Congr. Photobiol., 3rd, Copenhagen, 1960. 4. SMIRNOV K. V. and SHATERNIKIV A. A. (1960) Effect of external ionizing irradiation on the participation of liver and intestine in lipid metabolism. Vop. Med. Khim. 6, 464-470. 5. TRETIAKOVA K. A. and GRODZENSKY D. E. (1959) Effect of ionizing irradiation on the biosynthesis of cholesterol and of fatty acids. Vopr. Med. Khim. 5, 363-369. 6. ME~~ERD R. B. JR., WEBSTER W. W. and NYMAN M. A. (1958) Lipid metabolism in X-irradiated mice and rats. Radiation Res. 8,461-465. A. and POLESOP~KY W. 7. ZIRM K. L., PONGRATZ (1955) Beitrag zur konstitution pflanzlicher lipoide, im besonderen der lipoidkomponente des chloroplastins. Biochem. <. 326, 405-412. A. N. and PROSKURYAKOV N. I. 8. BELOZER~KY ( 195 1) Prakticheskoe rukovodstvo po biochimii rastenii. Soviet Press, Izd. Sov. Nauka, Moscow. E. V. (1963) 9. BORISO~A I. G. and BOUDNIT~KAYA A method for quantitative paper chromatography of unsaturated higher fatty acids. Biokhimiya, USSR 28, 497-500. English translation by Cons&ants Bureau Enterprises, New York (1964), pp. 405-407. 10. KRWKOVA L. M. (1961) Production of antimitotic substances in different plant species after irradiation. Radiobiologea, USSR 1, 139-144. 11. KOPYLOV V. A. (1961) Influence of antimitotic substances from irradiated plants on the growth of mice. Radiobiologiya, USSR 1, 358-363. 12. KRWKOVA L. M., LOMAKIN M. S. and KUSIN A. M. (1961) Effect of extracts from irradiated and non-irradiated plants (Vicia Phaba) on growth of tumor and homologous normal tissue. Radiobiologja,
13. SIEGEL S. toxicity in 15,21-26. 14. BOWEN H. from seed
USSR
1, 365-372.
M. (1962) Observations on peroxide seed germination. Physiol. Planturum.
J. M. and THICK J. (1960) Factors extracts that modify radiosensitivity. Radiation Res. 13, 234-241. 15. Effects of seed extracts on radiosensitivity. In Effects of ionizing radiation on see& (1961) Proceedings of the Symposium on the effect of ionizing radiation on seeds. IAEA, Vienna, Austria, pp. 75-82. 1961.