Planta (Berl.) 103, 263-266 (1972) 9 by Springer-Verlag 1972
Enzymatic Production of the Plant Growth Inhibitor, Xanthoxin R. D. FIaN and J. FRIEND
Department of Botany, University of I~Iul],England Received November 15, 1971
Summary. Incubation of violaxanthin with lipoxygenase and linoteate gave rise to the plant growth inhibitor, xanthoxin; and the yields were reduced to 1/10 and 1/20 by the omission of lipoxygenase and both lipoxygenase and linoleate respecively. Introduction Xanthoxin, which was identified as an endogenous plant growth inhibitor b y Taylor and Burden (1970a, b), occurs in a number of plant species (Firn et al., 1971) and appears to be similar to abscisic acid in its inhibitory activity (Taylor and Burden, 1970b). Xanthoxin has been characterized as a mixture of the cis-trans and trans-trans isomers, I and I I respectively (Taylor and Burden, 1970b; Burden and Taylor, 1970). and it can be formed in vitro by the photo-oxidation of violaxanthin (Taylor and Burden, 1970b; Taylor and Smith, 1967). In pea seedlings xanthoxin accumulation is induced by red light (Firn, 1971; Burden et al., 1971) land it is likely t h a t in vivo xanthoxin can be formed either b y an enzymatic or a photosensitized oxidation.
~ ~ 0 ~CHO I I t has been shown that the coupled oxidation of fi-earotene by lipoxygenase in the presence of linoleate gives rise to a number of earbonyl compounds (Friend, 1958) including apo-earotenals (Friend, 1959) which can be considered to be analogues of xanthoxin. I t was therefore of interest to determine whether a coupled oxidation of violaxanthin could give rise to xanthoxin. Materials and Methods Violaxanthin was extracted from orange peel and purified chromatographically using the procedure described by Taylor and Burden (1970b). Tween 80 was purified chromatographically to remove small molecular weight material which interfered with the chromatography of the reaction products.
264
R.D. Firn and J. Friend:
Tween 80 dissolved in ethyl acetate/hexane (3/1 v/v) was applied to a silicic acid column which was developed with the same solvent and then with ethyl acetate to elute impurities. The Tween 80 was Muted from the column with methanol. The reaction mixtures were modifications of those used by Grossman et al. (1969) for determination of the rate of carotenoid oxidation by lipoxygenase. A solution of 10 mg violaxanthin and 300 mg Tween 80 in 250 ml 0.2 M Tris-HC1 buffer pH 7.6 was mixed with a solution of 300 mg linoleie acid (Sigma) and 200 rag Tween 80 in 250 ml 0.2 M Tris-HC1 buffer pH 7.6. The mixed solution was divided into two equal parts; to one hMf a solution of 3 rag soy-bean lipoxygenase (Sigma) in 100 ml 0.2 M Tris-HC1 buffer pH 7.6 was added, and to the other 100 ml 0.2 M Tris-HC1 buffer pH 7.6. After 20 rain incubation, 50 g ammonium sulphate was added to each reaction vessel and the solutions were extracted separately with ethyl acetate (2 x 250 ml) until colourless. The ethyl acetate extracts were dried over anhydrous sodium sulphate, filtered, taken to dryness on a rotary evaporator and dissolved in ethyl acetate/hexane (3/1). The solutions were ehromatographed on short columns of silicic acid and developed with ethyl acetate/hexane (3/1); the Twecn 80 was retained on the column and the oxidation products which eluted were purified by the method of Firn et al. (1971), and acetylated. G. L. C. analysis was carried out on a Perkin-Elmer F-11 flame ionization gas chromatograph fitted with a 6' X1/4'' glass column packed with 3% 0V-17 on 80-100 mesh Gasehrom Q using an oven temperature of 190~ and a nitrogen carrier gas flow rate of 60 ml/min. Qualitative analyses were also carried out on columns packed with 2% SE-30 and 3% DC-560 liquid phases.
Results and Discussion There was considerable oxidation of v i o l a x a n t h i n in the presence of the enzyme a n d linoleic acid; the absorbance a t 460 n m fell to approxim a t e l y 25 % of its initial value after 20 rain i n c u b a t i o n . Analysis of the oxidation products (Table 1) revealed the presence of x a n t h o x i n a n d the " b u t e n o n e " which is the c o m p o u n d identified b y Taylor a n d B u r d e n (1970b) as 4 - ( l ' , 2 ' - e p o x y - 4 ' - h y d r o x y - 2 ' , 6 ' , 6 ' - t r i m e t h y l - l ' - c y c l o h e x y l ) trans-3-buten-2-one, I I I .
H o ~ 9m-
~
Ho~ ~Z
~
There was a barely detectable peak with the R t value of loliolide, c o m p o u n d IV, (Taylor a n d Burden, 1970b); Hedges a n d Porte, 1964), which would indicate t h a t if it were present there was less t h a n 1 ~g produced from 5 mg v i o l a x a n t h i n . The yields of the x a n t h o x i n isomers a n d the " b u t e n o n e " (2 % a n d 8 % respectively) are very similar to those o b t a i n e d b y photo-oxidation of v i o l a x a n t h i n (Taylor a n d B u r d e n , 1970b). The reaction m i x t u r e from which the enzyme was o m i t t e d b u t which contained linoleic acid yielded
Enzymatic Production of Xanthoxin
265
Table 1. Yields of violaxanthin oxidation products obtained from three different reaction mixtures Additions to violaxanthin
Yields of products Cisxanthoxin
Transxanthoxin
"Butenone"
1. Lipoxygenase § linoleic acid 2. Linoleie acid 3. None
20
50
300
2 1
6 3
35 11
In addition to violaxanthin, all reaction mixtures contained Tween 80 and TrisHC1 buffer pH 7.6. Yields expressed as ~zg product/5 mg violaxanthin. For the complete reaction mixture (No. 1), the yields of total x anthoxin and t h e " butenone" are 2 % and 8 % respectively, assuming that one molecule of violaxanthin can give two molecules of xanthoxin or the "butenone".
only 1/10 the a m o u n t of x a n t h o x i n a n d t h e " b u t e n o n e ", a n d since these low yields could h a v e been o b t a i n e d as a result of t h e m a n i p u l a t i v e procedures used, v i o l a x a n t h i n was i n c u b a t e d in buffer w i t h o u t either e n z y m e or f a t t y acid. I n this case t h e yield was o n l y 1/20 of t h a t of t h e c o m p l e t e s y s t e m or 1/2 t h a t w i t h o u t enzyme. I t is a s s u m e d t h a t t h e increased level of o x i d a t i o n p r o d u c t s in t h e presence of linoleic acid w i t h o u t e n z y m e c o m p a r e d with t h a t f o u n d in buffer alone was t h e result of v i o l a x a n t h i n o x i d a t i o n b y the small a m o u n t of linoleate h y d r o p e r o x i d e which was a c o n t a m i n a n t in t h e linoleic a c i d sample. I f this a s s u m p t i o n is correct, it is likely t h a t a n y o x i d a t i o n s y s t e m which will c a t a l y z e t h e f o r m a t i o n of linoleic acid h y d r o p e r o x i d e s will, in the presence of linolieic acd, also oxidize v i o l a x a n t h i n to x a n t h o x i n . V i o l a x a n t h i n o x i d a t i o n coupled to t h e e n z y m a t i c o x i d a t i o n of linoleic acid is therefore as efficient a m e c h a n i s m as p h o t o - o x i d a t i o n for t h e p r o d u c t i o n of x a n t h o x i n a n d m a y well be i n v o l v e d in x a n t h o x i n form a t i o n in vivo. H o w e v e r t h e r e are o t h e r e n z y m a t i c systems p r e s e n t in p l a n t tissues which will oxidize earotenoids in t h e absence of f a t t y acids (Dicks a n d F r i e n d , 1968 ; Dicks, 1970) a n d these are now being investig a t e d to see w h e t h e r t h e y can also oxidize v i o l a x a n t h i n to x a n t h o x i n . We thank the A. R. C. for financial support and Drs. tt. F. Taylor and R. S. Burden for samples of xanthoxin and the "butenone".
Re~erences Burden, l~. S., Firn, I~. D., Hiron, R. W.P., Taylor, H. F., Wright, S. T. C. : Induction of the plant growth inhibitor xanthoxin in seedlings by red light. Nature (Lond.) 234, 95-96 (1971).
266
1~. D. Firn and J. Friend: Enzymatic Production of Xanthoxin
Burden, R. S., Taylor, H . F . : The structure and chemical transformations of xanthoxin. Tetrahech'on Letters 47, 40714074 (1970). Dicks, J. W. : The participation of peroxidase and oxalic acid in crocin destruction by a particulate system from sugar beet leaves. Phytochemistry 9, 1433-1441 (1970). Dicks, J. W., Friend, J. : The oxidation of carotenoids by mitochondria from sugar beet leaves. III. Crocin oxidation by a peroxidase system. Phytochemistry 7, 1933-1947 (1968). Firn, 1%.D.: Studies on some natural and synthetic plant growth inhibitors. Ph. D. Thesis, University of London (1971). Firn, R.D., Burden, R. S., Taylor, H . F . : The detection and estimation of the growth inhibitor xanthoxin in plants. Planta (Berl.) 102, 115-126 (1972). Friend, J. : The coupled oxidation of fl-carotene by a linoleate-lipoxidasesystem and by autoxidizing linoleate. Chemy. Ind. 597-598 (1958). Friend, J. : Studies on the biological oxidation of fl-carotene. Ph. D Thesis, University of Cambridge (1959). Grossmann, S., Ben Aziz, A., Budowski, P., Ascarelli, I., Gertler, A., Birk, Y., Bondi, A. : Enzymic oxidation of carotene and linoleate by alfalfa: Extraction and separation of active fractions. Phytochemistry 8, 2287-2293 (1969). Hodges, R., Porte, A. L. : The structure of Loliolide: A terpene from Lolium perenne. Tetrahedron Letters 20, 1463-1467 (1964). Taylor, H. F., Burden, R. S. : Xanthoxin, a new naturally occurring plant growth inhibitor. Nature (Lond.) 227, 302-304 (1970a). Taylor, H. F., Burden, R. S. : Identification of plant growth inhibitors produced by photolysis of violaxanthin. Phytochemistry 9, 2217-2223 (1970b). Taylor, H. F., Smith, T. A. : Production of plant growth inhibitors from xanthophylls. Nature (Lond.) ~15, 1513-1514 (1967). Dr. J. Friend Department of Botany University of Hull, Hull HU6 7 RX England
Enzymatic Production of the Plant Growth Inhibitor, Xanthoxin R. D. FIaN and J. FRIEND
Department of Botany, University of I~Iul],England Received November 15, 1971
Summary. Incubation of violaxanthin with lipoxygenase and linoteate gave rise to the plant growth inhibitor, xanthoxin; and the yields were reduced to 1/10 and 1/20 by the omission of lipoxygenase and both lipoxygenase and linoleate respecively. Introduction Xanthoxin, which was identified as an endogenous plant growth inhibitor b y Taylor and Burden (1970a, b), occurs in a number of plant species (Firn et al., 1971) and appears to be similar to abscisic acid in its inhibitory activity (Taylor and Burden, 1970b). Xanthoxin has been characterized as a mixture of the cis-trans and trans-trans isomers, I and I I respectively (Taylor and Burden, 1970b; Burden and Taylor, 1970). and it can be formed in vitro by the photo-oxidation of violaxanthin (Taylor and Burden, 1970b; Taylor and Smith, 1967). In pea seedlings xanthoxin accumulation is induced by red light (Firn, 1971; Burden et al., 1971) land it is likely t h a t in vivo xanthoxin can be formed either b y an enzymatic or a photosensitized oxidation.
~ ~ 0 ~CHO I I t has been shown that the coupled oxidation of fi-earotene by lipoxygenase in the presence of linoleate gives rise to a number of earbonyl compounds (Friend, 1958) including apo-earotenals (Friend, 1959) which can be considered to be analogues of xanthoxin. I t was therefore of interest to determine whether a coupled oxidation of violaxanthin could give rise to xanthoxin. Materials and Methods Violaxanthin was extracted from orange peel and purified chromatographically using the procedure described by Taylor and Burden (1970b). Tween 80 was purified chromatographically to remove small molecular weight material which interfered with the chromatography of the reaction products.
264
R.D. Firn and J. Friend:
Tween 80 dissolved in ethyl acetate/hexane (3/1 v/v) was applied to a silicic acid column which was developed with the same solvent and then with ethyl acetate to elute impurities. The Tween 80 was Muted from the column with methanol. The reaction mixtures were modifications of those used by Grossman et al. (1969) for determination of the rate of carotenoid oxidation by lipoxygenase. A solution of 10 mg violaxanthin and 300 mg Tween 80 in 250 ml 0.2 M Tris-HC1 buffer pH 7.6 was mixed with a solution of 300 mg linoleie acid (Sigma) and 200 rag Tween 80 in 250 ml 0.2 M Tris-HC1 buffer pH 7.6. The mixed solution was divided into two equal parts; to one hMf a solution of 3 rag soy-bean lipoxygenase (Sigma) in 100 ml 0.2 M Tris-HC1 buffer pH 7.6 was added, and to the other 100 ml 0.2 M Tris-HC1 buffer pH 7.6. After 20 rain incubation, 50 g ammonium sulphate was added to each reaction vessel and the solutions were extracted separately with ethyl acetate (2 x 250 ml) until colourless. The ethyl acetate extracts were dried over anhydrous sodium sulphate, filtered, taken to dryness on a rotary evaporator and dissolved in ethyl acetate/hexane (3/1). The solutions were ehromatographed on short columns of silicic acid and developed with ethyl acetate/hexane (3/1); the Twecn 80 was retained on the column and the oxidation products which eluted were purified by the method of Firn et al. (1971), and acetylated. G. L. C. analysis was carried out on a Perkin-Elmer F-11 flame ionization gas chromatograph fitted with a 6' X1/4'' glass column packed with 3% 0V-17 on 80-100 mesh Gasehrom Q using an oven temperature of 190~ and a nitrogen carrier gas flow rate of 60 ml/min. Qualitative analyses were also carried out on columns packed with 2% SE-30 and 3% DC-560 liquid phases.
Results and Discussion There was considerable oxidation of v i o l a x a n t h i n in the presence of the enzyme a n d linoleic acid; the absorbance a t 460 n m fell to approxim a t e l y 25 % of its initial value after 20 rain i n c u b a t i o n . Analysis of the oxidation products (Table 1) revealed the presence of x a n t h o x i n a n d the " b u t e n o n e " which is the c o m p o u n d identified b y Taylor a n d B u r d e n (1970b) as 4 - ( l ' , 2 ' - e p o x y - 4 ' - h y d r o x y - 2 ' , 6 ' , 6 ' - t r i m e t h y l - l ' - c y c l o h e x y l ) trans-3-buten-2-one, I I I .
H o ~ 9m-
~
Ho~ ~Z
~
There was a barely detectable peak with the R t value of loliolide, c o m p o u n d IV, (Taylor a n d Burden, 1970b); Hedges a n d Porte, 1964), which would indicate t h a t if it were present there was less t h a n 1 ~g produced from 5 mg v i o l a x a n t h i n . The yields of the x a n t h o x i n isomers a n d the " b u t e n o n e " (2 % a n d 8 % respectively) are very similar to those o b t a i n e d b y photo-oxidation of v i o l a x a n t h i n (Taylor a n d B u r d e n , 1970b). The reaction m i x t u r e from which the enzyme was o m i t t e d b u t which contained linoleic acid yielded
Enzymatic Production of Xanthoxin
265
Table 1. Yields of violaxanthin oxidation products obtained from three different reaction mixtures Additions to violaxanthin
Yields of products Cisxanthoxin
Transxanthoxin
"Butenone"
1. Lipoxygenase § linoleic acid 2. Linoleie acid 3. None
20
50
300
2 1
6 3
35 11
In addition to violaxanthin, all reaction mixtures contained Tween 80 and TrisHC1 buffer pH 7.6. Yields expressed as ~zg product/5 mg violaxanthin. For the complete reaction mixture (No. 1), the yields of total x anthoxin and t h e " butenone" are 2 % and 8 % respectively, assuming that one molecule of violaxanthin can give two molecules of xanthoxin or the "butenone".
only 1/10 the a m o u n t of x a n t h o x i n a n d t h e " b u t e n o n e ", a n d since these low yields could h a v e been o b t a i n e d as a result of t h e m a n i p u l a t i v e procedures used, v i o l a x a n t h i n was i n c u b a t e d in buffer w i t h o u t either e n z y m e or f a t t y acid. I n this case t h e yield was o n l y 1/20 of t h a t of t h e c o m p l e t e s y s t e m or 1/2 t h a t w i t h o u t enzyme. I t is a s s u m e d t h a t t h e increased level of o x i d a t i o n p r o d u c t s in t h e presence of linoleic acid w i t h o u t e n z y m e c o m p a r e d with t h a t f o u n d in buffer alone was t h e result of v i o l a x a n t h i n o x i d a t i o n b y the small a m o u n t of linoleate h y d r o p e r o x i d e which was a c o n t a m i n a n t in t h e linoleic a c i d sample. I f this a s s u m p t i o n is correct, it is likely t h a t a n y o x i d a t i o n s y s t e m which will c a t a l y z e t h e f o r m a t i o n of linoleic acid h y d r o p e r o x i d e s will, in the presence of linolieic acd, also oxidize v i o l a x a n t h i n to x a n t h o x i n . V i o l a x a n t h i n o x i d a t i o n coupled to t h e e n z y m a t i c o x i d a t i o n of linoleic acid is therefore as efficient a m e c h a n i s m as p h o t o - o x i d a t i o n for t h e p r o d u c t i o n of x a n t h o x i n a n d m a y well be i n v o l v e d in x a n t h o x i n form a t i o n in vivo. H o w e v e r t h e r e are o t h e r e n z y m a t i c systems p r e s e n t in p l a n t tissues which will oxidize earotenoids in t h e absence of f a t t y acids (Dicks a n d F r i e n d , 1968 ; Dicks, 1970) a n d these are now being investig a t e d to see w h e t h e r t h e y can also oxidize v i o l a x a n t h i n to x a n t h o x i n . We thank the A. R. C. for financial support and Drs. tt. F. Taylor and R. S. Burden for samples of xanthoxin and the "butenone".
Re~erences Burden, l~. S., Firn, I~. D., Hiron, R. W.P., Taylor, H. F., Wright, S. T. C. : Induction of the plant growth inhibitor xanthoxin in seedlings by red light. Nature (Lond.) 234, 95-96 (1971).
266
1~. D. Firn and J. Friend: Enzymatic Production of Xanthoxin
Burden, R. S., Taylor, H . F . : The structure and chemical transformations of xanthoxin. Tetrahech'on Letters 47, 40714074 (1970). Dicks, J. W. : The participation of peroxidase and oxalic acid in crocin destruction by a particulate system from sugar beet leaves. Phytochemistry 9, 1433-1441 (1970). Dicks, J. W., Friend, J. : The oxidation of carotenoids by mitochondria from sugar beet leaves. III. Crocin oxidation by a peroxidase system. Phytochemistry 7, 1933-1947 (1968). Firn, 1%.D.: Studies on some natural and synthetic plant growth inhibitors. Ph. D. Thesis, University of London (1971). Firn, R.D., Burden, R. S., Taylor, H . F . : The detection and estimation of the growth inhibitor xanthoxin in plants. Planta (Berl.) 102, 115-126 (1972). Friend, J. : The coupled oxidation of fl-carotene by a linoleate-lipoxidasesystem and by autoxidizing linoleate. Chemy. Ind. 597-598 (1958). Friend, J. : Studies on the biological oxidation of fl-carotene. Ph. D Thesis, University of Cambridge (1959). Grossmann, S., Ben Aziz, A., Budowski, P., Ascarelli, I., Gertler, A., Birk, Y., Bondi, A. : Enzymic oxidation of carotene and linoleate by alfalfa: Extraction and separation of active fractions. Phytochemistry 8, 2287-2293 (1969). Hodges, R., Porte, A. L. : The structure of Loliolide: A terpene from Lolium perenne. Tetrahedron Letters 20, 1463-1467 (1964). Taylor, H. F., Burden, R. S. : Xanthoxin, a new naturally occurring plant growth inhibitor. Nature (Lond.) 227, 302-304 (1970a). Taylor, H. F., Burden, R. S. : Identification of plant growth inhibitors produced by photolysis of violaxanthin. Phytochemistry 9, 2217-2223 (1970b). Taylor, H. F., Smith, T. A. : Production of plant growth inhibitors from xanthophylls. Nature (Lond.) ~15, 1513-1514 (1967). Dr. J. Friend Department of Botany University of Hull, Hull HU6 7 RX England
