Journal of Chemical Ecology, Vol. 17, No. 1, 1991
COMPARISON OF FURANOCOUMARIN CONCENTRATIONS OF GREENHOUSE-GROWN Ruta chalepensis WITH OUTDOOR PLANTS LATER TRANSFERRED TO A GREENHOUSE
ALICJA
M.
ZOBEL
Department of Chemistry Trent University Peterborough, Ontario, Canada K9J 7B8 (Received February 26, 1990; accepted August 9, 1990) Abstract--Rum chalepensis contained concentrations of furanocoumarins 2550% of those found in R. graveolens both in the whole leaf and on its surface. On the leaf surface of plants grown all year indoors in a greenhouse, they increased steadily between November 1 and December 14 on mature upper and lower leaves. New growth upper leaves on December 14 contained less than mature upper leaves. Plants transferred from outdoors to the greenhouse showed decreased concentrations after the first two weeks, followed by recovery both in the whole leaf and on the leaf surface. Proportions of xanthotoxin and bergapten to psoralen changed during the experiment. On the leaf surfaces and in the whole upper leaves of the indoor plants, the proportions were often similar, but in the transferred plants, in most cases, psoralen was less than bergapten or xanthotoxin in the upper leaves and markedly less in the lower leaves. Implications of these findings for possible effects of environmental changes on secondary plant metabolism are discussed. Key Words--Furanocoumarins, Rum chalepensis, photophytodermatitis, surface deposition, plant defense.
INTRODUCTION
L i n e a r f u r a n o c o u m a r i n s , o r p s o r a l e n s , w h i c h are k n o w n to c a u s e p h o t o p h y t o d e r m a t i t i s , are l o c a l i z e d to s o m e e x t e n t o n t h e l e a f s u r f a c e s o f p l a n t s t h a t e l a b o r a t e t h e m ( Z o b e l a n d B r o w n , 1 9 8 8 b , 1989, 1 9 9 0 a , b ) . A n o u t s t a n d i n g e x a m p l e is R u m graveolens, w h i c h h a s b e e n n o t e d s i n c e a n c i e n t t i m e s f o r i n d u c i n g this d e r m a t i t i s . W e h a v e f o u n d t h a t , in e x t r e m e c a s e s , o v e r 5 5 % o f t h e total fur21 0098-0331/91/0100-0021506.50/0
9 1991 Plenum Publishing Corporation
22
ZOBZL
anocoumarins of the leaf, in micrograms per gram fresh weight, can be extracted from the surface (Zobel and Brown, 1989, 1990a). Studies on Heracleum lanatum (Umbelliferae) have revealed significant changes during the growing season in the concentrations of the three predominant furanocoumarins in this species: psoralen, xanthotoxin, and bergapten, both on the leaf surface and in the whole leaves (Zobel and Brown, 1990b). A hypothesis has been advanced that a function of these surface coumarins is to protect the plant against microbial attack, acting as a defense barrier (Zobel and Brown, 1989). Any such external barrier must be affected by changes in the plant's environment, as well as its developmental changes. A number of workers have hypothesized about the role of these compounds in plants (Camm et al., 1976; Berenbaum, 1978; Berenbaum and Neal, 1985; St/idler and Buser, 1984; Matem et al., 1988), and on their biological activity (Chakraborty et al., 1957; Murray et al., 1982; Towers, 1987; Nitao, 1988), especially under UV irradiation (Pathak and Fitzpatrick, 1959; Grekin and Epstein, 1981; Towers and Yamamoto, 1985; Towers, 1986). Some investigations have dealt with increased concentrations of furanocoumarins as a response to infection (Ashwood-Smith et al., 1985; Surico et al., 1987) and to other stresses (Beier and Oertli, 1983). Recently experiments have demonstrated changes in concentrations of furanocoumarins depending on the plant's developmental stage (Berenbaum and Zangerl, 1986) and on environmental and nutritional conditions (Zangerl and Berenbaum, 1987). In order to ascertain to what extent such changes might occur in a plant in response to changes in its environment, I have examined Rum chalepensis, comparing concentrations in plants grown outdoors that were subsequently transferred to a greenhouse with those of control plants grown in a greenhouse for the whole year.
METHODS AND MATERIALS Four 2-year-old R. chalepensis subshrubs, each with about 20 long shoots each containing about 40 leaves, growing outdoors in the soil during the summer growing season (outdoor plants), were used. They were transferred to pots on October 1, 1988, before the autumn frosts, and allowed to remain outdoors for the rest of that month to let the plants recover from root damage. Four others grown throughout the year in a greenhouse (indoor plants) were used as controis. The first leaf samples for analysis were collected from both groups on October 1 as a control for how well the potted plants had recovered from transplanting, and the second at the beginning of the experiment on November 1 just before the outdoor plants were transferred to a greenhouse. The next three samples were collected at ca. two-week intervals (November 15 and 30, and
FURANOCOUMARIN CONCENTRATIONS IN
Rbtta
23
D e c e m b e r 14). F r o m each plant, four o f the u p p e r m o s t leaves o f similar size, 2 - 4 c m long, w e r e c o l l e c t e d . T h e s e w e r e not n e w g r o w t h , but small-sized, mature leaves. Separate samples o f such leaves w e r e c o l l e c t e d for analysis f r o m the i n d o o r and o u t d o o r plants, and, in e a c h case, the l o w e s t three u n d a m a g e d g r e e n leaves w e r e c o l l e c t e d f r o m the s a m e plants for c o m p a r i s o n purposes. In contrast to R. graveolens, there w e r e no senescent y e l l o w i s h leaves during the whole experiment. T h e leaves o f each s a m p l e w e r e w e i g h e d and surface c o u m a r i n s extracted by a p r e v i o u s l y d e s c r i b e d m e t h o d ( Z o b e l and B r o w n , 1988a, 1989). The fura n o c o u m a r i n s in the extracts w e r e also purified and d e t e r m i n e d as described earlier ( T h o m p s o n and B r o w n , 1984; Z o b e l and B r o w n , 1988a).
RESULTS Transferring R. chalepensis plants to a g r e e n h o u s e f r o m outdoors resulted in c h a n g e d c o n c e n t r a t i o n s o f f u r a n o c o u m a r i n s both on the l e a f surface and in the w h o l e leaf, c o m p a r e d to the plants g r o w i n g indoors throughout the y e a r (Table 1). T h e i n d o o r plants e x h i b i t e d p h y s i o l o g i c a l changes d e p e n d i n g on the t i m e o f y e a r and the p h o t o p e r i o d , without influence o f other e n v i r o n m e n t a l condi-
TABLE 1. CONCENTRATIONS a OF T O T A L FURANOCOUMARINS IN W H O L E L E A F AND ON L E A F SURFACE OF
Rula
chalepensis
Indoor plants Collection date (1988)
Whole leaf
On surface
Upper
Oct. 1 Nov. 1 Nov. 15 Nov. 30 Dec. 14C
1050 1030 1455 900 630
• 50 • 50 _+ 80 • 80 • 50
390 390 480 660 290
• 20 _+ 30 _+ 30 • 50 _+ 30
Lower, green
Oct. 1 Nov. 1 Nov. 15 Nov. 30 Dec. 14
650 660 700 750 650
_+ 50 • 50 _+ 40 • 50 • 50
320 300 340 400 360
+ • • • •
Leaves
Outdoor plants
ZP + X +B b
/~g/g fresh weight. bp: psoralen, X: xanthotoxin, B: bergapten. "New growth.
EP + X + B
20 30 30 40 30
% on surface
% on surface
Whole leaf
On surface
37 38 33 73 47
1200 1160 850 1130 710
_+ 60 _+ 60 _+ 40 • 50 • 60
480 500 315 610 450
• 40 +_ 40 _+ 30 _+ 40 _+ 30
40 43 37 54 63
49 46 49 54 55
800 760 505 880 525
• 40 ___60 • 40 • 80 • 30
200 170 170 350 260
• • • • •
25 23 35 40 50
15 20 20 30 20
24
ZOBEL
tions such as wind and rain. The outdoor plants (brought into the greenhouse November 1), in contrast, were affected by the transfer indoors, in response to their new environment. All but the December 14 leaves were mature, differing only in size and location on the plant. On December 14, as the upper leaves of both groups of plants were then new growth, the experiment was terminated, because, as shown below, the results obtained suggested a different physiology from that of the mature leaves. Table 1 compares the sum of the concentrations (E values) of psoralen, xanthotoxin, and bergapten (P + X + B). On November 1 the concentrations were similar to those of October 1 in the same group of plants, but the outdoor plants had higher concentrations in the whole leaf than the indoor plants, both in the upper leaves (difference 130 ~g/g) and in the lower leaves (100 t~g/g), and on the surface of the upper leaves (110 ~g/g). On the lower leaves, the indoor group had over 40% more than the outdoor (difference of 130 t~g/g). Upper leaves of both groups contained much more total furanocoumarins than the lower leaves, with a difference of ca. 400 ~g/g, corresponding to more than half of the lower leaves' concentration. The indoor plants showed a steady increase in surface concentrations of the furanocoumarins on the mature leaves. The total concentrations increased until November 15 in the upper leaves, and until November 30 in the lower, followed by a decrease. The lower leaves of the indoor plants had a stable percentage of furanocoumarins on the surface (46-55%), whereas the outdoor plants began with a low percentage (23 % of the total), increasing steadily to 50% at the conclusion. The surface concentrations in the lower leaves of the indoor plants paralleled those of the whole leaf, but the upper leaves did not always follow this pattern, i.e., the 30% increase in the whole leaf concentration on November 15 was unaccompanied by any increase in the surface concentration, which showed a marked increase only after two weeks. Changes were observed after the plants had been transferred into the greenhouse. On November 1 the outdoor plants contained 10% more furanocoumarins in the whole upper leaves and 20% more on the surface than the indoor. The lower leaves contained much smaller concentrations than the upper, both in the whole leaf and on the surface, with the percentage on the surface little more than half that of the upper leaves (23 % vs. 43 %). On November 15, the upper leaves of the outdoor plants showed decreased concentrations in the whole leaves and on the surface. The lower leaves showed decreased concentration in the whole leaves, but it was unchanged on the surface. They recovered in the next two weeks and made up or even exceeded the difference; this recovery was especially visible on the surface. For mature leaves, the highest percentages on the surface were 54 % and 50 %. Ratio of Psoralen to Xanthotoxin and Bergapten. Table 2 shows the pro-
FURANOCOUMARINCONCENTRATIONSIN Ruta
25
TABLE2. RATIOS~ OF CONCENTRATIONSOF FURANOCOUMARINSIN WHOLELEAFAND ON LEAF SURFACEOF Ruta chalepensis PLANTS Indoor plants Collection date (1988)
Xb
Upper
Nov. 1 Nov. 15 Nov. 30 Dec. 14C
0.9 2.1 1.3 0.9
Lower, green
Nov. 1 Nov. 15 Nov. 30 Dec. 14
13.5 25.7 21.6 18.3
Leaves
Whole leaf
Outdoor plants
Leaf surface
Whole leaf
Bb
X
B
X
B
X
B
0.97 2.6 1.0 0.70
0.86 1.1 1.2 0.91
0.93 0.93 0.93 0.76
1.9 1.1 2.6 2.6
1.6 1.6 2.4 1.9
1.6 0.8 2.2 2.6
1.5 1.3 2.4 2.2
5.1 3.1 10.8 13.3
3.1 1.6 5.2 7.9
2.7 4.5 7.5 11.5
3.0 4.6 3.9 7.7
7.0 22.5 8.1 10.6
7.6 28.7 16.5 18.3
7.4 25.0 6.2 10.8
Leaf surface
aBased on psoralen = 1. bX: xanthotoxin,B: bergapten. "New growth.
portions of the three furanocoumarins in the whole leaf and on the leaf surface, with the value for psoralen taken as unity. Four distinct groups of ratios are evident: (1) the upper leaves of the indoor plants, with similar concentrations of the three furanocoumarins (except November 15); (2) their lower leaves, showing a drastic diminution of psoralen (7 to 25 times); (3) upper leaves of the outdoor plants, having bergapten and xanthotoxin concentrations 1.1-2.6 times that of psoralen (except November 15); and (4) lower leaves of the outdoor plants, with bergapten and xanthotoxin concentrations ca. 1.6-13.3 times those of the upper leaves.
DISCUSSION These observations have demonstrated that, after transfer to the greenhouse, the plants responded by increasing furanocoumarin production and steadily increasing extrusion to the surface. As well, new growth after this recovery contained a higher percentage on the surface than new growth of the indoor plants (63 % vs. 47 %); this interesting phenomenon needs more investigation before conclusions can be drawn. The findings of the present experiment, and unpublished data from this laboratory on Ruta graveolens showing similar trends in response, but with concentration values at least twice as high, suggest that there are two parallel
26
ZOBEL
physiological processes influencing furanocoumarin concentrations in the whole leaf and on its surface. The first process, genetically dependent, govems the levels of these compounds and their proportions according to the period of the year and is typical of the species; the second is a physiological and biochemical response to changes in environmental conditions. Further experiments are needed to compare R. chalepensis and R. graveolens to other species with respect to these phenomena, because if, as seems probable, furanocoumarins play a defense role in plants, their levels in the whole leaf and especially on its surface should show changes with varying environmental conditions. Measurements taken over a complete vegetative period on leaves of Heracleum lanatum showed changes in concentrations on the surface and in the whole leaves depending on their developmental stage and on the time within the vegetation period (Zobel and Brown, 1990b). Investigations are now in progress with similar measurements for both Ruta species. Acknowledgments--The studies reported here were financially supported by operating grant A2487 from the Natural Sciences and Engineering Research Council of Canada to Prof. S.A. Brown.
REFERENCES
ASHWOOD-SMITH,M.J., CESKA,O., and CHAUDHARY,S.K. 1985. Mechanism of photosensitivity reactions to diseased celery. Br. Med. J. 290:1249. BEIER, R.C., and OERTLI, E.H. 1983. Psoralen and other linear furanocoumarins as phytoalexins in celery (Apium graveolens). Phytochemistry 22:2595-2597. BERENBAUM,M. 1978. Toxicity ofa furanocoumarin to armyworms: A case of biosynthetic escape from insect herbivores. Science 201:532-534. BERENBAUM,M., and NEAL, J.J. 1985. Synergism between myristicin and xanthotoxin, a naturally occurring plant toxicant. J. Chem. Ecol. 11:1349-1358. BERENBAUM, M.R., and ZANGERL, A.R. 1986. Variation in seed furanocoumarin content within the wild parsnip (Pastinaca sativa). Phytochemistry 25:659-661. CAMM, E.L., WAT, C.-K., and TOWERS, G.H.N. 1976. An assessment of the roles of furanocoumatins in Heracleum lanatum. Can. J. Bot. 54:2562-2566. CHAKRABORTY, D.P., DAS GUPTA, A., and BosE, P.H. 1957. On the antifungal action of some natural coumarins. Ann. Biochem. Exp. Med. 17:59-62. GREKIN, D.A., and EPSTEIN, J.H. 1981. Psoralens and UV-A (PUV-A) and photocarcinogenesis. Photochem. Photobiol. 36:857-860. MATERN,U., STRASSER,H., WENDORFF,H., and HAMERSKI,D. 1988. Coumarins and furanocoumatins, pp. 3-21, in I. Vasil and F. Constabel (eds.). Cell Culture and Somatic Cell Genetics of Plants, Vol. 5. Academic Press, New York. MURRAY, R.D.H., MI~NDEZ, J., and BROWN, S.A. 1982. The Natural Coumarins: Occurrence, Chemistry and Biochemistry. Wiley, Chichester. NITAO, J.K. 1988. Artificial defloration and furanocoumarin induction in Pastinaca sativa (Umbelliferae). J. Chem. Ecol. 14:1515-1522. PATHAK, M.A., and FITZPATRICK, T.B. 1959. Bioassay of natural and synthetic furocoumarins (psoralens). J. Invest. Dermatol. 32:509-518.
FURANOCOUMARINCONCENTRATIONS IN Ruta
27
ST.~DLER,E., and BUSER,H.-R. 1984. Defense chemicals in the leaf surface wax synergistically stimulate oviposition by a phytophagous insect. Experientia 40:1157-1159. SURICO, G., VARVANO,L., and SOLFRIZZO, M. 1987. Linear furanocoumarins accumulation in celery plants infected with Erwinia carotovera pathovar carotovera. J. Agric. Food Chem. 35:406-409. THOMPSON,H J . , and BROWN,S.A- 1984. Separations of some coumarins of higher plants by liquid chromatography. J. Chromatogr. 314:323-336. TOWERS, G.H.N. 1986. Induction of cross-links in viral DNA by naturally occurring photosensitizers. Photochem. Photobiol. 44:187-192. TOWERS, G.H.N. 1987. Fungicidal activity of naturally occurring photosensitizers. Am. Chem. Soc. Syrup. Ser. 339:231-240. TOWERS, G.H.N., and YAMAMOTO,E. 1985. Interactions of cinnamic acid and its derivatives with light. Ann. Proc. Phytochem. Soc. Eur. 25:271-288. ZAN~ERL, A.R., and BERENBAUM,M. 1987. Furanocoumarins in wild parsnip. Effects of photosynthetically active radiation, ultraviolet light and nutrients. Ecology 68:516-520. ZOBEL, A.M., and BROWN, S.A. 1988a. Determination of furanocoumarins on the leaf surface of Ruta graveolens with an improved extraction technique. J. Nat. Prod. 51:941-946. ZOBEL, A.M., and BROWN, S.A. 1988b. Furanocoumarins on plant surfaces. Bulletin de Liaison no. 14 du Groupe Polyphenols. Compte-rendu des Journdes lnternationales d'Etude et de l 'AssembIde G~ndrale, pp. 65-68. ZOBEL, A.M., and BROWN, S.A. 1989. Histological localization of furanocoumarins in Rum graveolens. Can. J. Bot. 67:915-921. ZOBEL, A.M., and BROWN, S.A. 1990a. Dermatitis-inducing furanocoumarins on the leaf surfaces of rutaceous and umbelliferous plants. J. Chem. Ecol. 16:693-700. ZOBEL, A.M., and BROWN, S.A. 1990b. Seasonal changes of furanocoumarin concentrations in leaves of Heracleum lanatum. J. Chem. Ecol. 16:1623-1634.
COMPARISON OF FURANOCOUMARIN CONCENTRATIONS OF GREENHOUSE-GROWN Ruta chalepensis WITH OUTDOOR PLANTS LATER TRANSFERRED TO A GREENHOUSE
ALICJA
M.
ZOBEL
Department of Chemistry Trent University Peterborough, Ontario, Canada K9J 7B8 (Received February 26, 1990; accepted August 9, 1990) Abstract--Rum chalepensis contained concentrations of furanocoumarins 2550% of those found in R. graveolens both in the whole leaf and on its surface. On the leaf surface of plants grown all year indoors in a greenhouse, they increased steadily between November 1 and December 14 on mature upper and lower leaves. New growth upper leaves on December 14 contained less than mature upper leaves. Plants transferred from outdoors to the greenhouse showed decreased concentrations after the first two weeks, followed by recovery both in the whole leaf and on the leaf surface. Proportions of xanthotoxin and bergapten to psoralen changed during the experiment. On the leaf surfaces and in the whole upper leaves of the indoor plants, the proportions were often similar, but in the transferred plants, in most cases, psoralen was less than bergapten or xanthotoxin in the upper leaves and markedly less in the lower leaves. Implications of these findings for possible effects of environmental changes on secondary plant metabolism are discussed. Key Words--Furanocoumarins, Rum chalepensis, photophytodermatitis, surface deposition, plant defense.
INTRODUCTION
L i n e a r f u r a n o c o u m a r i n s , o r p s o r a l e n s , w h i c h are k n o w n to c a u s e p h o t o p h y t o d e r m a t i t i s , are l o c a l i z e d to s o m e e x t e n t o n t h e l e a f s u r f a c e s o f p l a n t s t h a t e l a b o r a t e t h e m ( Z o b e l a n d B r o w n , 1 9 8 8 b , 1989, 1 9 9 0 a , b ) . A n o u t s t a n d i n g e x a m p l e is R u m graveolens, w h i c h h a s b e e n n o t e d s i n c e a n c i e n t t i m e s f o r i n d u c i n g this d e r m a t i t i s . W e h a v e f o u n d t h a t , in e x t r e m e c a s e s , o v e r 5 5 % o f t h e total fur21 0098-0331/91/0100-0021506.50/0
9 1991 Plenum Publishing Corporation
22
ZOBZL
anocoumarins of the leaf, in micrograms per gram fresh weight, can be extracted from the surface (Zobel and Brown, 1989, 1990a). Studies on Heracleum lanatum (Umbelliferae) have revealed significant changes during the growing season in the concentrations of the three predominant furanocoumarins in this species: psoralen, xanthotoxin, and bergapten, both on the leaf surface and in the whole leaves (Zobel and Brown, 1990b). A hypothesis has been advanced that a function of these surface coumarins is to protect the plant against microbial attack, acting as a defense barrier (Zobel and Brown, 1989). Any such external barrier must be affected by changes in the plant's environment, as well as its developmental changes. A number of workers have hypothesized about the role of these compounds in plants (Camm et al., 1976; Berenbaum, 1978; Berenbaum and Neal, 1985; St/idler and Buser, 1984; Matem et al., 1988), and on their biological activity (Chakraborty et al., 1957; Murray et al., 1982; Towers, 1987; Nitao, 1988), especially under UV irradiation (Pathak and Fitzpatrick, 1959; Grekin and Epstein, 1981; Towers and Yamamoto, 1985; Towers, 1986). Some investigations have dealt with increased concentrations of furanocoumarins as a response to infection (Ashwood-Smith et al., 1985; Surico et al., 1987) and to other stresses (Beier and Oertli, 1983). Recently experiments have demonstrated changes in concentrations of furanocoumarins depending on the plant's developmental stage (Berenbaum and Zangerl, 1986) and on environmental and nutritional conditions (Zangerl and Berenbaum, 1987). In order to ascertain to what extent such changes might occur in a plant in response to changes in its environment, I have examined Rum chalepensis, comparing concentrations in plants grown outdoors that were subsequently transferred to a greenhouse with those of control plants grown in a greenhouse for the whole year.
METHODS AND MATERIALS Four 2-year-old R. chalepensis subshrubs, each with about 20 long shoots each containing about 40 leaves, growing outdoors in the soil during the summer growing season (outdoor plants), were used. They were transferred to pots on October 1, 1988, before the autumn frosts, and allowed to remain outdoors for the rest of that month to let the plants recover from root damage. Four others grown throughout the year in a greenhouse (indoor plants) were used as controis. The first leaf samples for analysis were collected from both groups on October 1 as a control for how well the potted plants had recovered from transplanting, and the second at the beginning of the experiment on November 1 just before the outdoor plants were transferred to a greenhouse. The next three samples were collected at ca. two-week intervals (November 15 and 30, and
FURANOCOUMARIN CONCENTRATIONS IN
Rbtta
23
D e c e m b e r 14). F r o m each plant, four o f the u p p e r m o s t leaves o f similar size, 2 - 4 c m long, w e r e c o l l e c t e d . T h e s e w e r e not n e w g r o w t h , but small-sized, mature leaves. Separate samples o f such leaves w e r e c o l l e c t e d for analysis f r o m the i n d o o r and o u t d o o r plants, and, in e a c h case, the l o w e s t three u n d a m a g e d g r e e n leaves w e r e c o l l e c t e d f r o m the s a m e plants for c o m p a r i s o n purposes. In contrast to R. graveolens, there w e r e no senescent y e l l o w i s h leaves during the whole experiment. T h e leaves o f each s a m p l e w e r e w e i g h e d and surface c o u m a r i n s extracted by a p r e v i o u s l y d e s c r i b e d m e t h o d ( Z o b e l and B r o w n , 1988a, 1989). The fura n o c o u m a r i n s in the extracts w e r e also purified and d e t e r m i n e d as described earlier ( T h o m p s o n and B r o w n , 1984; Z o b e l and B r o w n , 1988a).
RESULTS Transferring R. chalepensis plants to a g r e e n h o u s e f r o m outdoors resulted in c h a n g e d c o n c e n t r a t i o n s o f f u r a n o c o u m a r i n s both on the l e a f surface and in the w h o l e leaf, c o m p a r e d to the plants g r o w i n g indoors throughout the y e a r (Table 1). T h e i n d o o r plants e x h i b i t e d p h y s i o l o g i c a l changes d e p e n d i n g on the t i m e o f y e a r and the p h o t o p e r i o d , without influence o f other e n v i r o n m e n t a l condi-
TABLE 1. CONCENTRATIONS a OF T O T A L FURANOCOUMARINS IN W H O L E L E A F AND ON L E A F SURFACE OF
Rula
chalepensis
Indoor plants Collection date (1988)
Whole leaf
On surface
Upper
Oct. 1 Nov. 1 Nov. 15 Nov. 30 Dec. 14C
1050 1030 1455 900 630
• 50 • 50 _+ 80 • 80 • 50
390 390 480 660 290
• 20 _+ 30 _+ 30 • 50 _+ 30
Lower, green
Oct. 1 Nov. 1 Nov. 15 Nov. 30 Dec. 14
650 660 700 750 650
_+ 50 • 50 _+ 40 • 50 • 50
320 300 340 400 360
+ • • • •
Leaves
Outdoor plants
ZP + X +B b
/~g/g fresh weight. bp: psoralen, X: xanthotoxin, B: bergapten. "New growth.
EP + X + B
20 30 30 40 30
% on surface
% on surface
Whole leaf
On surface
37 38 33 73 47
1200 1160 850 1130 710
_+ 60 _+ 60 _+ 40 • 50 • 60
480 500 315 610 450
• 40 +_ 40 _+ 30 _+ 40 _+ 30
40 43 37 54 63
49 46 49 54 55
800 760 505 880 525
• 40 ___60 • 40 • 80 • 30
200 170 170 350 260
• • • • •
25 23 35 40 50
15 20 20 30 20
24
ZOBEL
tions such as wind and rain. The outdoor plants (brought into the greenhouse November 1), in contrast, were affected by the transfer indoors, in response to their new environment. All but the December 14 leaves were mature, differing only in size and location on the plant. On December 14, as the upper leaves of both groups of plants were then new growth, the experiment was terminated, because, as shown below, the results obtained suggested a different physiology from that of the mature leaves. Table 1 compares the sum of the concentrations (E values) of psoralen, xanthotoxin, and bergapten (P + X + B). On November 1 the concentrations were similar to those of October 1 in the same group of plants, but the outdoor plants had higher concentrations in the whole leaf than the indoor plants, both in the upper leaves (difference 130 ~g/g) and in the lower leaves (100 t~g/g), and on the surface of the upper leaves (110 ~g/g). On the lower leaves, the indoor group had over 40% more than the outdoor (difference of 130 t~g/g). Upper leaves of both groups contained much more total furanocoumarins than the lower leaves, with a difference of ca. 400 ~g/g, corresponding to more than half of the lower leaves' concentration. The indoor plants showed a steady increase in surface concentrations of the furanocoumarins on the mature leaves. The total concentrations increased until November 15 in the upper leaves, and until November 30 in the lower, followed by a decrease. The lower leaves of the indoor plants had a stable percentage of furanocoumarins on the surface (46-55%), whereas the outdoor plants began with a low percentage (23 % of the total), increasing steadily to 50% at the conclusion. The surface concentrations in the lower leaves of the indoor plants paralleled those of the whole leaf, but the upper leaves did not always follow this pattern, i.e., the 30% increase in the whole leaf concentration on November 15 was unaccompanied by any increase in the surface concentration, which showed a marked increase only after two weeks. Changes were observed after the plants had been transferred into the greenhouse. On November 1 the outdoor plants contained 10% more furanocoumarins in the whole upper leaves and 20% more on the surface than the indoor. The lower leaves contained much smaller concentrations than the upper, both in the whole leaf and on the surface, with the percentage on the surface little more than half that of the upper leaves (23 % vs. 43 %). On November 15, the upper leaves of the outdoor plants showed decreased concentrations in the whole leaves and on the surface. The lower leaves showed decreased concentration in the whole leaves, but it was unchanged on the surface. They recovered in the next two weeks and made up or even exceeded the difference; this recovery was especially visible on the surface. For mature leaves, the highest percentages on the surface were 54 % and 50 %. Ratio of Psoralen to Xanthotoxin and Bergapten. Table 2 shows the pro-
FURANOCOUMARINCONCENTRATIONSIN Ruta
25
TABLE2. RATIOS~ OF CONCENTRATIONSOF FURANOCOUMARINSIN WHOLELEAFAND ON LEAF SURFACEOF Ruta chalepensis PLANTS Indoor plants Collection date (1988)
Xb
Upper
Nov. 1 Nov. 15 Nov. 30 Dec. 14C
0.9 2.1 1.3 0.9
Lower, green
Nov. 1 Nov. 15 Nov. 30 Dec. 14
13.5 25.7 21.6 18.3
Leaves
Whole leaf
Outdoor plants
Leaf surface
Whole leaf
Bb
X
B
X
B
X
B
0.97 2.6 1.0 0.70
0.86 1.1 1.2 0.91
0.93 0.93 0.93 0.76
1.9 1.1 2.6 2.6
1.6 1.6 2.4 1.9
1.6 0.8 2.2 2.6
1.5 1.3 2.4 2.2
5.1 3.1 10.8 13.3
3.1 1.6 5.2 7.9
2.7 4.5 7.5 11.5
3.0 4.6 3.9 7.7
7.0 22.5 8.1 10.6
7.6 28.7 16.5 18.3
7.4 25.0 6.2 10.8
Leaf surface
aBased on psoralen = 1. bX: xanthotoxin,B: bergapten. "New growth.
portions of the three furanocoumarins in the whole leaf and on the leaf surface, with the value for psoralen taken as unity. Four distinct groups of ratios are evident: (1) the upper leaves of the indoor plants, with similar concentrations of the three furanocoumarins (except November 15); (2) their lower leaves, showing a drastic diminution of psoralen (7 to 25 times); (3) upper leaves of the outdoor plants, having bergapten and xanthotoxin concentrations 1.1-2.6 times that of psoralen (except November 15); and (4) lower leaves of the outdoor plants, with bergapten and xanthotoxin concentrations ca. 1.6-13.3 times those of the upper leaves.
DISCUSSION These observations have demonstrated that, after transfer to the greenhouse, the plants responded by increasing furanocoumarin production and steadily increasing extrusion to the surface. As well, new growth after this recovery contained a higher percentage on the surface than new growth of the indoor plants (63 % vs. 47 %); this interesting phenomenon needs more investigation before conclusions can be drawn. The findings of the present experiment, and unpublished data from this laboratory on Ruta graveolens showing similar trends in response, but with concentration values at least twice as high, suggest that there are two parallel
26
ZOBEL
physiological processes influencing furanocoumarin concentrations in the whole leaf and on its surface. The first process, genetically dependent, govems the levels of these compounds and their proportions according to the period of the year and is typical of the species; the second is a physiological and biochemical response to changes in environmental conditions. Further experiments are needed to compare R. chalepensis and R. graveolens to other species with respect to these phenomena, because if, as seems probable, furanocoumarins play a defense role in plants, their levels in the whole leaf and especially on its surface should show changes with varying environmental conditions. Measurements taken over a complete vegetative period on leaves of Heracleum lanatum showed changes in concentrations on the surface and in the whole leaves depending on their developmental stage and on the time within the vegetation period (Zobel and Brown, 1990b). Investigations are now in progress with similar measurements for both Ruta species. Acknowledgments--The studies reported here were financially supported by operating grant A2487 from the Natural Sciences and Engineering Research Council of Canada to Prof. S.A. Brown.
REFERENCES
ASHWOOD-SMITH,M.J., CESKA,O., and CHAUDHARY,S.K. 1985. Mechanism of photosensitivity reactions to diseased celery. Br. Med. J. 290:1249. BEIER, R.C., and OERTLI, E.H. 1983. Psoralen and other linear furanocoumarins as phytoalexins in celery (Apium graveolens). Phytochemistry 22:2595-2597. BERENBAUM,M. 1978. Toxicity ofa furanocoumarin to armyworms: A case of biosynthetic escape from insect herbivores. Science 201:532-534. BERENBAUM,M., and NEAL, J.J. 1985. Synergism between myristicin and xanthotoxin, a naturally occurring plant toxicant. J. Chem. Ecol. 11:1349-1358. BERENBAUM, M.R., and ZANGERL, A.R. 1986. Variation in seed furanocoumarin content within the wild parsnip (Pastinaca sativa). Phytochemistry 25:659-661. CAMM, E.L., WAT, C.-K., and TOWERS, G.H.N. 1976. An assessment of the roles of furanocoumatins in Heracleum lanatum. Can. J. Bot. 54:2562-2566. CHAKRABORTY, D.P., DAS GUPTA, A., and BosE, P.H. 1957. On the antifungal action of some natural coumarins. Ann. Biochem. Exp. Med. 17:59-62. GREKIN, D.A., and EPSTEIN, J.H. 1981. Psoralens and UV-A (PUV-A) and photocarcinogenesis. Photochem. Photobiol. 36:857-860. MATERN,U., STRASSER,H., WENDORFF,H., and HAMERSKI,D. 1988. Coumarins and furanocoumatins, pp. 3-21, in I. Vasil and F. Constabel (eds.). Cell Culture and Somatic Cell Genetics of Plants, Vol. 5. Academic Press, New York. MURRAY, R.D.H., MI~NDEZ, J., and BROWN, S.A. 1982. The Natural Coumarins: Occurrence, Chemistry and Biochemistry. Wiley, Chichester. NITAO, J.K. 1988. Artificial defloration and furanocoumarin induction in Pastinaca sativa (Umbelliferae). J. Chem. Ecol. 14:1515-1522. PATHAK, M.A., and FITZPATRICK, T.B. 1959. Bioassay of natural and synthetic furocoumarins (psoralens). J. Invest. Dermatol. 32:509-518.
FURANOCOUMARINCONCENTRATIONS IN Ruta
27
ST.~DLER,E., and BUSER,H.-R. 1984. Defense chemicals in the leaf surface wax synergistically stimulate oviposition by a phytophagous insect. Experientia 40:1157-1159. SURICO, G., VARVANO,L., and SOLFRIZZO, M. 1987. Linear furanocoumarins accumulation in celery plants infected with Erwinia carotovera pathovar carotovera. J. Agric. Food Chem. 35:406-409. THOMPSON,H J . , and BROWN,S.A- 1984. Separations of some coumarins of higher plants by liquid chromatography. J. Chromatogr. 314:323-336. TOWERS, G.H.N. 1986. Induction of cross-links in viral DNA by naturally occurring photosensitizers. Photochem. Photobiol. 44:187-192. TOWERS, G.H.N. 1987. Fungicidal activity of naturally occurring photosensitizers. Am. Chem. Soc. Syrup. Ser. 339:231-240. TOWERS, G.H.N., and YAMAMOTO,E. 1985. Interactions of cinnamic acid and its derivatives with light. Ann. Proc. Phytochem. Soc. Eur. 25:271-288. ZAN~ERL, A.R., and BERENBAUM,M. 1987. Furanocoumarins in wild parsnip. Effects of photosynthetically active radiation, ultraviolet light and nutrients. Ecology 68:516-520. ZOBEL, A.M., and BROWN, S.A. 1988a. Determination of furanocoumarins on the leaf surface of Ruta graveolens with an improved extraction technique. J. Nat. Prod. 51:941-946. ZOBEL, A.M., and BROWN, S.A. 1988b. Furanocoumarins on plant surfaces. Bulletin de Liaison no. 14 du Groupe Polyphenols. Compte-rendu des Journdes lnternationales d'Etude et de l 'AssembIde G~ndrale, pp. 65-68. ZOBEL, A.M., and BROWN, S.A. 1989. Histological localization of furanocoumarins in Rum graveolens. Can. J. Bot. 67:915-921. ZOBEL, A.M., and BROWN, S.A. 1990a. Dermatitis-inducing furanocoumarins on the leaf surfaces of rutaceous and umbelliferous plants. J. Chem. Ecol. 16:693-700. ZOBEL, A.M., and BROWN, S.A. 1990b. Seasonal changes of furanocoumarin concentrations in leaves of Heracleum lanatum. J. Chem. Ecol. 16:1623-1634.
