Chemical Manipulation of Soybean (Glycine max L. Merr.)

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Chemical Manipulation of Soybean (Glycine max L. Merr.) Oil Quality JUDITH B. ST. JOHN, MERYL N. CHRISTIANSEN, and DANIEL E. TERLIZZI Beltsville Agricultural Research Center, U.S. Department of Agriculture, Beltsville, MD 20705

Action of the enzyme lipoxygenase (E.C. 1.13.11.12) on l i n o l e n i c acid causes soybean oil to develop rancidity and reduces oil q u a l i t y . The formation of l i n o l e n i c acid and the enzyme lipoxygenase were i n h i b i t e d . This chapter reports the potential for chemical enhancement of soybean oil quality by treatment of growing plants with pyridazinones. The substituted pyridazinones are a class of chemicals with multiple actions i n higher plants. These actions include: Inhibition of the Hill reaction and CO f i x a t i o n ; i n h i b i t i o n of the formation of chloroplast pigments, ribosomes, and membrane l i p i d s ; and a l t e r ations i n chloroplast u l t r a - s t r u c t u r e (1). These actions may occur singly or i n combination, dependent upon the structure of the p a r t i c u l a r pyridazinone (2, 3). BASF 13 338 l4-chloro-5(dimethylamino)3(2H)-pyridazinone = Sandoz 9785 = BASF 105 00W] i s repre sentative of the group of pyridazinones that have the strongest action on membrane l i p i d s . These pyridazinones s p e c i f i c a l l y block the conversion of l i n o l e i c (18:2) acid to l i n o l e n i c (18:3) acid. Sandoz 6706 [4-chloro-5-(dimethylamino)-2-(α,α α-trifluoro-m-tolyl)3(2H)-pyridazinone] i s representative of the pyridazinones referred to as bleaching agents because of their potent i n h i b i t i o n of chloro plast pigment formation. These pyridazinones also block l i n o l e n i c acid formation, but are not as e f f e c t i v e as the 4-chloro-5-dimethylamino-pyridazin-3-ones. The molecular structure of pyrazon [5-amino-4-chloro- 2-phenyl-3(2H)-pyridazinone] accounts for i n h i b i t i o n of the Hill reaction and photosynthetic CO f i x a t i o n by the pyridazinones. Polyunsaturated fatty acids i n soybean (Glycine max L. Merr.) seeds contain cis,cis-l,4-pentadiene systems that serve as sub strates for the formation of hydroperoxides by the enzyme lipoxygen ase (E.C. 1.13.11.12) and by a i r (autoxidation). Hydroperoxides lead to the development of rancid oil, greatly reducing oil q u a l i t y . Our interest was i n the potential to improve oil quality by using the pyridazinones to prevent the formation of l i n o l e n i c acid and/or lipoxygenase a c t i v i t y . The pyridazinones have thus far only been 2

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This chapter not subject to U.S. copyright Published 1984, American Chemical Society

Ory and Rittig; Bioregulators ACS Symposium Series; American Chemical Society: Washington, DC, 1984.

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used to prevent l i n o l e n i c acid formation i n vegetative tissues of a number of crop species (4_, _5, 6).


Pyridazinone Action on Lipids of Soybean Cotyledons

In. V i t r o

Experiments to determine the effects of pyridazinones on l i n o l e n i c acid production i n soybean seeds were carried out by Drs. John Thompson and James Madison at the USDA laboratory i n Ithaca, New York, using their previously published procedures (_7) · B r i e f l y , 0.01 M stock solutions of the test compounds were prepared. An aliquot of this stock solution (80 y l ) was then combined with nutrient culture medium (8 ml) to give a f i n a l pyridazinone concent r a t i o n of 0.1 mM. The bottles were capped and s t e r i l i z e d by autoclaving. Pods were harvested from the plants and s t e r i l i z e d with 10-fold diluted Chlorox. The embryos were then removed from the pods a s e p t i c a l l y and cut into two r e l a t i v e l y equivalent halves (cotyledons). One-half was placed i n a bottle containing only the nutrient solution. The other h a l f was placed i n a b o t t l e containing the nutrient solution plus the test compound. These bottles were incubated for 6 days at 27°C with slow reciprocal shaking. At the end of the incubation period, the cotyledons were removed from the b o t t l e s , washed with water, blotted and weighed. The cotyledons were then lyophilized and l i p i d s were extracted and analyzed by gas chromatography (4)· This split-cotyledon test system eliminates genetic v a r i a b i l i t y between control and treated tissues. In this i n v i t r o system BASF 13 338 and Sandoz 6706 reduced the r e l a t i v e proportion of l i n o l e n i c acid i n soybean l i p i d s (Table I ) . No reduction in cotyledon weight occurred as a result of pyridazinone treatment. The formation of l i n o l e n i c acid i s generally accepted to occur by elongation of palmitic acid (16:0) to stearic acid (18:0) and subsequent sequential desaturation to o l e i c acid (18:1), l i n o l e i c acid (18:2), and l i n o l e n i c acid (18:3). The data i n Table I show that the r e l a t i v e proportion of l i n o l e i c acid increases when l i n o l e n i c acid decreases, without a change i n the overall r a t i o of saturated to unsaturated fatty acids. Thus the r a t i o of 18:2/18:3 i s a r e f l e c t i o n of the i n h i b i t i o n of l i n o l e i c acid desaturation to l i n o lenic acid. Conversion of l i n o l e i c acid to l i n o l e n i c acid i n soybean cotyledons (seeds) i s inhibited by pyridazinones when the cotyledons are i n d i r e c t contact with the pyridazinones i n nutrient cultures·

Table I. Effects of BASF 13 338 and Sandoz 6706 on Relative Proportions of Linolenic Acid i n Total Lipids of Soybean Cotyledons Ratio of Treatment Linolenic Acid (18:3) L i n o l e i c Acid (18:2) to (0.1 mM) Linolenic Acid (18:3) S/U % by wt 0.16 Control 14.8 3.41 0.15 BASF 13 338 5.04 10.8 0.15 Jandoz 6706 4.14 11.8 S/U = palmitic (16:0) + stearic (18:0) acids / o l e i c (18:1) + l i n o l e i c (18:2) + l i n o l e n i c (18:3) acids. 1


6. ST. J O H N E T A L .

Manipulation of Soybean Oil Quality

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Pyridazinone Action on Lipids of Soybean Seeds From Plants Grown and Treated i n a Greenhouse Soybeans were grown in J i f f y Mix i n 7 inch pots i n the greenhouses at USDA, B e l t s v i l l e , Maryland. Treatments, replicated six times, are indicated in Table I I . Sandoz 6706 applied as a 5 ppm s o i l drench 3 days after flower induction was the only pyridazinone/treatment combination that r e sulted i n reduced l i n o l e n i c acid in o i l from seeds of greenhousegrown soybeans. This treatment with BASF 13 338 increased l i n o l e n i c acid l e v e l s . Experiments replicated i n time v e r i f i e d the results for the 5 ppm s o i l drench 3 days after induction of flowering. The percentage of l i n o l e n i c acid i n the o i l was measured at 5.7 for the control and 6.3 and 4.8 for BASF 13 338 and Sandoz 6706, respect i v e l y . These data are indicative of the complications that can arise i n attempts to move from predictable results i n an i n v i t r o system (Table I) to the more complex set of variables present i n a greenhouse s i t u a t i o n (Table I I ) .

Table I I . Effects of Pyridazinones on Relative Proportions of Linolenic Acid i n Soybean O i l of Seeds from Greenhouse Plants Linolenic acid % by wt. of t o t a l l i p i d Treatment 5.0 Control BASF 13 338 5.0 S o i l incorporated, 5ppm Spray, 10 ppm 3 days after 6.0 flower induction S o i l drench, 5 ppm 3 days after 6.0 flower induction Sandoz 6706 4.9 S o i l incorporated, 5 ppm Spray, 10 ppm 3 days after 4.9 flower induction S o i l drench, 5 ppm 3 days after 4.4 flower induction

Effects of BASF 13 338 on In Vivo Lipoxygenase A c t i v i t y of Soybean Seeds from Field-Grown Plants A l l of our experiments to this point had involved attempts to prevent the formation of l i n o l e n i c acid i n the o i l of soybean seeds. Dr. Robert Ory, USDA, New Orleans, LA, personally communicated to us his preliminary observation on pyridazinone i n h i b i t i o n of l i p oxygenase in peanuts. This communication led us into studies of pyridazinone action on soybean lipoxygenase. We f i r s t determined the lipoxygenase a c t i v i t y i n soybean seeds from plants grown and treated at the BASF experimental farms i n Greenville, M i s s i s s i p p i . In vivo lipoxygenase a c t i v i t y was assayed using soybean homogenates prepared according to Vick and Zimmerman (8) with an 18:2 substrate solution described by Surry ( 9_) · Lipoxygenase a c t i v i t y was measured with a YSI oxygraph at 30°C with a Clark 02~electrode. The data in Table III show that a BASF 13 338 treatment of 2.24 kg ai/ha


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applied as a f o l i a r spray at flowering reduced lipoxygenase a c t i v i t y to 78 percent of the control l e v e l . This 22 percent reduction in lipoxygenase a c t i v i t y was s i g n i f i c a n t l y different from the control at the 5% confidence l e v e l .

Table I I I . Effects of BASF 13 338 on In Vivo Lipoxygenase A c t i v i t y of Soybean Seeds From Plants Grown and Treated Under F i e l d Conditions Lipoxygenase A c t i v i t y Treatment % of Control Pre-emergence, s o i l incorporated 4.48 kg ai/ha 100 Post-emergence, broadcast spray 2.24 kg ai/ha 100 Flowering, f o l i a r spray 2.24 kg ai/ha 78

We have since obtained data from greenhouse experiments conducted at B e l t s v i l l e , Maryland, v e r i f y i n g pyridazinone reduction of in vivo lipoxygenase a c t i v i t y of soybean seeds (Table IV).

Table IV. Effects of Pyridazinones on In Vivo Lipoxygenase A c t i v i t y of Soybean Seeds From Plants Grown and Treated i n Greenhouses Lipoxygenase A c t i v i t y % of Control Treatment F o l i a r spray, 50 ppm with 1% ethanol and 0.01% Tween 20 1 BASF 13 338 70 Sandoz 6706 76 Norflurazon S i g n i f i c a n t l y d i f f e r e n t from the control at the 5% confidence .evel. Norflurazon = 4-chloro-5-(methylamino)-2-(a ,a ,a trifluoro-mtolyl)-3(2H)-pyridazinone. A

2

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A l l three of the pyridazinones tested s i g n i f i c a n t l y reduced l i p oxygenase a c t i v i t y i n the seeds from soybean plants treated with a f o l i a r spray at flowering. The most promising potential for enhancement of soybean o i l quality with the pyridazinones appears to be by reduction of i n vivo lipoxygenase a c t i v i t y i n the seed. Ory et a l . i n a preliminary report (10) and i n the detailed report (11) in this volume have reached similar conclusions based on their work with peanuts. Effects of Pyridazinones on JaV-ii-ro Lipoxygenase

Activity

The Jji v i t r o assays were conducted to obtain information on the nature of pyridazinone i n h i b i t i o n of lipoxygenase a c t i v i t y . The i n v i t r o assays were carried out as described for the _in vivo assays except that " c r y s t a l l i n e " soybean lipoxygenase was obtained from



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ST. J O H N E T A L .


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Sigma Chemical Company, St, Louis, Missouri. Approximately 30 pyridazinones, including those i n Table IV, were evaluated for i n h i b i t i o n i n the i n v i t r o system. Pyrazon and BASF 37827 [2-(4-fluorophenyl)-4-bromo-5-amino-3(22U-pyridazinone] reduced i n v i t r o lipoxygenase a c t i v i t y to 56 and 73 percent, respectively, of the control a c t i v i t y at an i n h i b i t o r concentration of 0.1 mM and a substrate concentration of 0.5 mM. The double-reciprocal plot ( F i g . 1) of pyrazon action on the " c r y s t a l l i n e " soybean lipoxygenase suggests that pyrazon i s a competitive i n h i b i t o r i n this system. The calculated Michaelis-Menton constants for the control were: V max = 0.373 moles/min. ^ = 1.108 mM. The constants for 0.1 mM Pyrazon were: V max = 0.392 moles/min.; ^ = 1.817 mM; = 0.16 mM. Pyrazon and BASF 37827 were not included i n our i n vivo studies. This, combined with the lack of i n v i t r o i n h i b i t i o n of " c r y s t a l l i n e " soybean lipoxygenase by BASF 13 338, Sandoz 6706 and Norflurazon, suggests caution i n extrapolating between the Jji vivo and in_ v i t r o systems. Thus pyridazinones may competitively i n h i b i t soybean l i p oxygenase or, conversely, the action may involve a reduced concent r a t i o n of the enzyme i n vivo. Ory et a l . (11) have concluded that BASF 105 00W reduced the total lipoxygenase a c t i v i t y i n peanuts by reducing the concentration of the enzyme. The combined data indicate that pyridazinones may enhance soybean o i l quality by two mechanisms. Pyridazinones may i n h i b i t the formation of l i n o l e n i c acid (Tables I and II) and/or decrease the a c t i v i t y of lipoxygenase (Tables I I I , IV, and Figure 1). Methods and timing of application of the appropriate pyridazinones must be developed so that the enhancement of soybean o i l quality i s not at the expense of reduced y i e l d s . Other Uses for Pyridazinones as Bioregulators Sunflowers. Substituted pyridazinones may also have potential as bioregulators to bring about desirable changes i n the composition of the fatty acids i n sunflower (Helianthus annuus L.) seed o i l (Table V). The sunflowers were grown and treated by Dr. Marvin Heilman, USDA, Weslaco, TX. BASF 13 338 was sprayed d i r e c t l y on the sunflower seed heads at the f i r s t bud stage. Fatty acid analyses were performed i n B e l t s v i l l e , Maryland.

Table V. Effect of BASF 13 338 on Fatty Acid Composition of Sunflower Seed Lipids Fatty acid composition (% by wt.) 1

Treatment 16-.0 18:0 18:1 18:2 18:1/18:2 Control 5.62 4.78 41.05 48.58 0.85 BASF 13 338 2 2 2 50 ppm spray, 1st buds 5.83 4.55 36.50 53.10 0.69 116:0 = palmitic acid. 18:0 = stearic acid. 18:1 = o l e i c a c i d . 18:2 = l i n o l e i c acid. Significantly

different

from the c o n t r o l at the 5% confidence

level.



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Figure 1·

Double-reciprocal plot of lipoxygenase pyrazon.

i n h i b i t i o n by



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ST. J O H N E T A L .

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Sunflower o i l from seeds produced i n cool northern climates normally contain 70% or more l i n o l e i c acid, while o i l from seeds produced i n warmer southern climates may contain as l i t t l e as 30% l i n o l e i c acid. The data i n Table V suggest that pyridazinones might be useful i n producing sunflower o i l s with, higher levels of l i n o l e i c acid. This could be p a r t i c u l a r l y useful i n warmer climates and suggests an alternative approach to selective breeding for highl i n o l e i c acid sunflower l i n e s . It i s interesting to note that BASF 13 338 increased levels of l i n o l e i c acid i n a seed that does not contain l i n o l e n i c a c i d . B o l l Weevils. The a c t i v i t y of pyridazinones i n b i o l o g i c a l systems other than plants further j u s t i f i e s the term bioregulator as the descripter for their action. The p o s s i b i l i t y of using a single bioregulator to enhance the quality of a plant product and bioregulate the l i f e cycle of a pest insect i s i n t r i g u i n g . B i o l o g i c a l a c t i v i t y of the pyridazinones against b o l l weevils (Anthonomus grandis) i s documented i n Table VI. These experiments were conducted at the USDA Boll Weevil Research Laboratory, M i s s i s s i p p i State, M i s s i s s i p p i .

Table VI. Effects of Feeding Two Substituted Pyridazinones to B o l l Weevils (Anthonomus grandis) BASF 13 338 (% wt/wt added to d i e t ) 0.075 0.03 0.01 Parameter Measured Control 50.0a 59.8a 64.2a Y i e l d , adults/dish 67.8a& 72.8b 82.3ab 93.4a 100a Y i e l d , % of control 10.47c 12.01b 12.80a 13.23a Wt. of adults (mg) 3.52b 5.07ab 7.19a 8.18a Eggs/fyday 43.1c 66.0b 91.3a 100a Eggs, % of control 69.6b 78.3ab 81.3ab 86.4a Hatch, % 2 6b 4.0ab 5.9ab 7.2a Viable eggs/day Sandoz 6706 (% wt/wt added to diet) 0.075 0.03 0.01 Control 44.6b 44.7b 53.3b 76.5a Y i e l d , adults/dish ^ 59.2b 59.2b 70.6b 100a Y i e l d , % of control 10.74b 11.66b 12.71a 13.07a Wt. of adults (mg) 2.79b 4.77ab 6.62a 6.60a Eggs/$/day 41.3b 72.3b 100.3a 100a Eggs/% of control^ 3.04a 59.6a 65.9a 74.2a Hatch, % 1.94a 3.04a 4.46a 4.95a Viable eggs/day Xarval + adult d i e t s . Used to eliminate v a r i a t i o n i n number of eggs and hatch from rep to rep i n time. 12 consecutive days (50 pair/rep). ^Eliminated v a r i a t i o n i n time. 'Five samples; 300 eggs/sample/replicate. 'Means followed by the same l e t t e r are not s i g n i f i c a n t l y different. 1

3

5

T

3

5

Animal Systems. Lipoxygenase i s found i n animals as well as plants. Arachidonic (20:4) acid serves as a substrate for lipoxygenase i n animal systems. Products of lipoxygenase action on arachidonic acid have been implicated i n a l l e r g i c and inflammatory reactions i n



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mammals (12). Pyrazon was a more e f f e c t i v e i n h i b i t o r of soybean lipoxygenase a c t i v i t y when arachidonic acid was used as the substrate (Table VII). Soybean lipoxygenase i s the only commercially available form of the enzyme. It has been used as a model system for studying i n h i b i tors of i n vivo mammalian lipoxygenase (13, 14). Wallach and Brown (14) concluded that the response to i n h i b i t o r s of soybean lipoxygenase and human p l a t e l e t lipoxygenase were q u a l i t a t i v e l y s i m i l a r . Our results therefore suggest a potential pharmacological role for the pyridazinones as lipoxygenase i n h i b i t o r s .

Table VII. Effect of Pyrazon on In V i t r o Lipoxygenase of " C r y s t a l l i n e " Commercial Soybean Enzyme Treatment Substrate (0.1 mM) % of C o n t r o l (0.5 mMO) Pyrazon 62 L i n o l e i c acid Pyrazon 36 Arachidonic acid Control = 0.125 moles 02/min. with l i n o l e i c acid as substrate and 0.177 moles 02/min. with arachidonic acid as substrate.

1

Summary. The substituted pyridazinones are bioregulators with the potential for numerous applications i n agriculture. The quality of soybean products may be improved through the reduction of undesirable compounds ( l i n o l e n i c acid) and reduction of undesirable enzymic a c t i v i t y (lipoxygenase). The pyridazinones o f f e r the potent i a l for chemically enhancing l i n o l e i c acid levels i n sunflowers. A d d i t i o n a l l y , the possible pharmacological role i n animal systems for the pyridazinones suggests the d e s i r a b i l i t y of developing chemi c a l s with broad applications. Acknowledgment s This work was supported in part by competitive grant #80433 from the American Soybean Association to Judith B. St. John and Meryl N. Christiansen. Literature Cited 1. 2.

3.

4. 5. 6.

Eder, F.A. Z. Naturforsch, 1979, 34C, 1052-54. St. John, J.B. i n "Biochemical Responses Induced by Herbicides;" Moreland, D.E., St. John, J.B., and Hess, F.D., Ed.; ACS SYMPOSIUM SERIES NO. 2, American Chemical Society: Atlanta, Georgia, 1981; pp. 97-110. St. John, J.B.; Schirmer, U.; R i t t i g , F.R.; and Bleiholder, H. i n "Rational Approaches to Pesticide Synthesis;" Kohn, G.K., and Menn, J.J., Ed.; ACS SYMPOSIUM SERIES, American Chemical Society: Washington, D.C., 1983; In Press. St. John, J.B.; Christiansen, M.N. Plant Physiol., 1976, 57, 257-59. St. John, J.B.; Christiansen, M.N.; Ashworth, E.N.; Gentner, W.A. Crop Sci., 1979, 19, 65-9. Willemot, C.; Slack, C.R.; Browse, J.; and Roughan, P.G. Plant Physiol., 1982, 70, 78-81.


6. ST. JOHN ET AL. Manipulation of Soybean Oil Quality

7. 8. 9. 10.


11.

12. 13. 14.

Thompson, J.F.; Madison, J.T.; Muenster, A.E. Ann. Bot., 1977, 41, 29-39. Vick, Β.Α.; Zimmerman, D.C. Plant Physiol., 1976,57,780788. Surry, K. Plant Physiol., 1964, 39, 65-70. Ory, R.L.; Conkerton, E.J.; St. Angelo, A.J.; Vincent, C.H.; R i t t i g , F.R.; Schroeder, M. Proc., Amer. Peanut Res. Educ. Soc., 1981, 13, 86. Ory,R.L.;St.Angelo, A.J.; Conkerton, E.J.; Chapital, D.C.; and R i t t i g , F.R. i n "Bioregulators: Chemistry and Uses;" Ory, R.L., R i t t i g , F.R., Ed.; ACS SYMPOSIUM SERIES, American Chemi c a l Society, Washington, D.C., 1983; this book. Marx, J.L. Science, 1982, 215, 1380-3. Blackwell, G.J.; Fowler, R.J. Prostaglandins, 1978, 16, 41725. Wallach, D.P.; Brown, V.R. Biochim. Biophys. Acta, 1981, 663.

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Chemical Manipulation of Soybean (Glycine max L. Merr.)

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