Phenol Glycosides in Plant Defense Against Herbivores - American

Chapter 10

Phenol Glycosides in Plant Defense Against Herbivores 1

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Paul B. Reichardt , Thomas P. Clausen , and John P. Bryant 1

Department of Chemistry, University of Alaska, Fairbanks, AK 99775-0520 Institute of Arctic Biology, University of Alaska, Fairbanks, AK 99775-0180

Downloaded by UNIV OF SYDNEY on November 3, 2016 | http://pubs.acs.org Publication Date: November 28, 1988 | doi: 10.1021/bk-1988-0380.ch010

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Phenol glycosides are commonly found as plant metabolites, but little is known about their functional significance in the plants which produce them. We have found that several phenol glycosides are important components of the chemical defenses of two woody plants, Populus balsamifera and P. tremuloides. In these cases the phenol glycosides contribute to plant defense by converting to active defensive chemicals in damaged plant tissues. These findings suggest that phenol glycosides may play similar roles in many plants but that their contributions to defense have gone undetected because of their indirect involvement. Consideration of the phenol glycosides found in agricultural plants as potential precursors of defensive metabolites could lead to a new appreciation of their roles in crop resistance to pests. P h e n o l g l y c o s i d e s a r e among t h e m o s t common a n d w i d e - s p r e a d o f a l l p l a n t m e t a b o l i t e s . From t h e i r f i r s t i s o l a t i o n from p l a n t s b y P i r i a i n 1845 ( 1 ) , t h e y h a v e o c c u p i e d a p r o m i n e n t p l a c e i n phytochemical investigations. Several hundred o f these s u b s t a n c e s a r e now k n o w n ( e . g . 2 ) , r a n g i n g i n s t r u c t u r e f r o m v e r y simple t o v e r y complex ( F i g u r e 1 ) . Even though t h eterm "phenol g l y c o s i d e " h a s been used b y c h e m i s t s f o r o v e r a c e n t u r y , t h e r e r e m a i n s some a m b i g u i t y o v e r i t s e x a c t meaning. Some a u t h o r s h a v e u s e d t h e t e r m t o r e f e r t o any n a t u r a l p r o d u c t h a v i n g a s t r u c t u r e w h i c h i n c l u d e s a p h e n o l i c r e s i d u e bonded t o a carbohydrate w h i l e o t h e r s have e x c l u d e d a n y compounds w h i c h c a n be c l a s s i f i e d a s f l a v o n o i d s . I n t h i s p a p e r we w i l l e m p l o y t h e l a t t e r , m o r e r e s t r i c t i v e , definition.

c

0097-6156/88/0380-0130$06.00/0 1988 American Chemical Society

Cutler; Biologically Active Natural Products ACS Symposium Series; American Chemical Society: Washington, DC, 1988.


REICHARDT ET AL.

Phenol Glycosides in Plant Defense

13 Figure

1.

Examples

of phenol glycosides

produced

°g'u by

plants.


BIOLOGICALLY ACTIVE NATURAL PRODUCTS

132


Chemistry Perhaps phenol g l y c o s i d e s have a t t r a c t e d the a t t e n t i o n of chemists p r i m a r i l y because of the c h a l l e n g e s i n v o l v e d i n t h e i r characterization. Many o f t h e m a r e l a b i l e s u b s t a n c e s w h i c h a r e d i f f i c u l t t o i s o l a t e and p u r i f y . F o r many y e a r s i s o l a t i o n o f p h e n o l g l y c o s i d e s was a c c o m p l i s h e d b y t e d i o u s g r a v i t y c o l u m n c h r o m a t o g r a p h y on p o l y a m i d e (3) o r g e l f i l t r a t i o n (4) s u p p o r t s f o l l o w e d by d i f f i c u l t r e c r y s t a l l i z a t i o n s . Often time-consuming p r e t r e a t m e n t o f e x t r a c t s ( c o u n t e r c u r r e n t e x t r a c t i o n and l e a d s u b a c e t a t e t r e a t m e n t s ) were r e q u i r e d p r i o r to chromatography, and t h e s e p r e t r e a t m e n t s commonly r e s u l t e d i n t h e i s o l a t i o n o f a r t i f a c t s ( 5 ) . The r e c e n t d e v e l o p m e n t o f f l a s h c h r o m a t o g r a p h y (6) has d r a m a t i c a l l y s i m p l i f i e d i s o l a t i o n p r o c e d u r e s ( 7 ) , and modern chromatography c o u p l e d w i t h enzymatic t r a n s f o r m a t i o n s o f f e r s promise f o r f u r t h e r s i m p l i f i c a t i o n of the i s o l a t i o n p r o t o c o l s ( C l a u s e n , T.P., U n i v e r s i t y o f A l a s k a F a i r b a n k s , unpublished data). Once p u r i f i e d , p h e n o l g l y c o s i d e s r e m a i n r e l u c t a n t t o reveal their chemical structures. Classically, structure e l u c i d a t i o n has r e s t e d upon c h e m i c a l o r enzymatic h y d r o l y s i s f o l l o w e d by s e p a r a t e s t r u c t u r a l d e t e r m i n a t i o n s o f the aglycone ("genin") and c a r b o h y d r a t e . R e p e t i t i o n of the procedure employing a p r o p e r l y d e r i v a t i z e d phenol g l y c o s i d e i s then u s u a l l y r e q u i r e d to l o c a t e the s i t e at which the g l y c o s i d e i s l i n k e d t o t h e g e n i n (8.). R e c e n t a d v a n c e s i n s p e c t r o s c o p y h a v e , h o w e v e r , p r e s e n t e d more d i r e c t methods f o r s t r u c t u r e elucidation. M o l e c u l a r f o r m u l a s and f r a g m e n t a t i o n p a t t e r n s can now b e o b t a i n e d f r o m h i g h r e s o l u t i o n f a s t a t o m b o m b a r d m e n t a n d f i e l d d e s o r p t i o n mass s p e c t r a l a n a l y s e s (9) o f t h e s e nonv o l a t i l e compounds. A t o m i c c o n n e c t i v i t i e s a n d , i n some c a s e s , c o m p l e t e s t r u c t u r e s c a n be d e t e r m i n e d by a n a l y s i s o f H- a n d C - N M R s p e c t r a ( 7 ) , e s p e c i a l l y w i t h t h e a i d o f 2D NMR techniques (10). S i m i l a r l y , a n a l y s i s o f p h e n o l g l y c o s i d e m i x t u r e s has been d r a m a t i c a l l y f a c i l i t a t e d by modern c h r o m a t o g r a p h i c t e c h n i q u e s . The o l d e r m e t h o d s o f p a p e r o r t h i n l a y e r c h r o m a t o g r a p h y c o u p l e d w i t h s p r a y r e a g e n t s f o r d e t e c t i o n (11) have been r e p l a c e d by gas c h r o m a t o g r a p h i c a n a l y s e s o f d e r i v a t i z e d p h e n o l g l y c o s i d e s (12) o r d i r e c t c h r o m a t o g r a p h i c a n a l y s e s o f p h e n o l g l y c o s i d e s by h i g h p e r f o r m a n c e l i q u i d c h r o m a t o g r a p h y (13.) . The l a t t e r m e t h o d i s p a r t i c u l a r l y e f f e c t i v e when a d i o d e a r r a y d e t e c t o r i s e m p l o y e d b e c a u s e t h e U V / V i s s p e c t r a o b t a i n e d c a n be u s e d t o s u b s t a n t i a t e s t r u c t u r a l assignments f o r peaks otherwise based s o l e l y on r e t e n t i o n times ( 1 4 ) . 13

Biosynthesis I t a p p e a r s t h a t t h e m o s t common b i o s y n t h e t i c r o u t e t o p h e n o l g l y c o s i d e s i n v o l v e s a f i n a l step which couples the preformed g e n i n ( u s u a l l y a s h i k i m a t e ) and c a r b o h y d r a t e . The f i n a l s t e p i s c a t a l y z e d by a t r a n s f e r a s e w h i c h u s u a l l y a l s o has h y d r o l y t i c ("glycosidase") a c t i v i t y (15).


10. REICHARDT ET AL.


B i o l o g i c a l properties o f phenol

133

glycosides

Two g e n e r a l b i o l o g i c a l p r o p e r t i e s seem t o b e a s s o c i a t e d w i t h phenol glycosides: 1) t h e y have a b i t t e r t a s t e , a t l e a s t t o humans ( 1 6 ) a n d 2 ) many p h e n o l g l y c o s i d e s o r t h e i r a g l y c o n e s are t o x i c t o a v a r i e t y o f organisms (15,17). Other s p e c i a l i z e d properties ascribed t o i n d i v i d u a l phenol glycosides include enzymatic i n h i b i t i o n (18), phytoalexic p r o p e r t i e s (19), and r e g u l a t i o n o f p l a n t g r o w t h ( 1 5 ,20, 2JL) .


Agricultural

relevance

o f phenol

glycosides

P h e n o l i c g l y c o s i d e s have been found i n a wide v a r i e t y o f e d i b l e p l a n t s - b o t h w i l d a n d c u l t i v a t e d - a s w e l l a s i n many f o r a g e crops (22,23). Interest i n the a g r i c u l t u r a l l y - r e l e v a n t phenol g l y c o s i d e s has centered on t h e i r g e n e r a l l y b i t t e r t a s t e (16), a p r o p e r t y w h i c h r e n d e r s them u n d e s i r a b l e c o n s t i t u e n t s o f foodstuffs. However, i t i s c l e a r t h a t b i t t e r n e s s i s n o t a uniform t r a i t o f d i e t a r y phenol g l y c o s i d e s , as s t r i k i n g l y exemplified by the f a c i l e conversion o f n a r i n g i n (a b i t t e r p r i n c i p l e o f g r a p e f r u i t ) t o the sweet-tasting isomer, n a r i n g i n chalcone (24). Ecological

relevance

o f phenol

glycosides

G l y c o s y l a t i o n a s t h e m o s t common f i n a l s t e p i n t h e b i o s y n t h e s i s o f most p h e n o l g l y c o s i d e s and t h e b i o l o g i c a l a c t i v i t i e s a s c r i b e d t o many p h e n o l g l y c o s i d e s o b v i o u s l y r a i s e t h e q u e s t i o n of t h e i r r a i s o n d'etre ( e s p e c i a l l y v i s a v i s the phenolic genins). C e r t a i n l y t h i s q u e s t i o n has n o t l a c k e d f o r answers. O v e r o n e h u n d r e d y e a r s a g o E r r a r a (2.5) f i r s t a n s w e r e d t h i s question by proposing that phenol glycosides protect plants from "the v o r a c i t y o f animals". However, t h e i n t e r v e n i n g y e a r s h a v e s e e n t h e emergence o f a number o f c o m p e t i n g p r o p o s a l s including: s e c o n d a r y f o o d r e s e r v e s (2£) , w a s t e m e t a b o l i t e s (27), d e t o x i f i e d d e r i v a t i v e s o f phytotoxic aglycones (15), p r o t e c t o r s o f p h e n o l s f r o m o x i d a t i o n ( 2 8 ) , a n d - - i n some c a s e s - p l a n t growth r e g u l a t o r s (20,21)• I n t h i s p a p e r we w i s h t o r e t u r n t o E r r a r a ' s ' s i n i t i a l suggestion and evaluate the current status o f h i s proposal. Compared t o t h e o f t e n a c c e p t e d maxim o f p h e n o l g l y c o s i d e s c o n s t i t u t i n g a common mode o f c h e m i c a l d e f e n s e , h a r d d a t a t o support t h i s contention are sparse. T h e r e i s some e v i d e n c e t o s u g g e s t t h a t p h e n o l g l y c o s i d e s c o n s t i t u t e a p l a n t defense against polyphagous ( g e n e r a l i s t ) and some o l i g o p h a g o u s h e r b i v o r e s , b u t i t i s l a r g e l y c o r r e l a t i v e i n nature. M a r k h a m (16) a n d E d w a r d s ( 2 9 ) d e m o n s t r a t e d t h e a v e r s i o n o f opossums t o P o p u l u s a n d S a l i x s p e c i e s c o n t a i n i n g r e l a t i v e l y h i g h l e v e l s o f s a l i c i n (1) and i t s d e r i v a t i v e s and ascribed the p r o t e c t i o n to the b i t t e r taste o f phenol glycosides. T a h a v a n a i n e n e t a l . (30) l i k e w i s e d i s c o v e r e d a negative r e l a t i o n s h i p between p a l a t a b i l i t i e s o f S a l i x species to t h e mountain hare (Lepus t i m i d u s ) and phenol g l y c o s i d e content. They a d d i t i o n a l l y d e m o n s t r a t e d t h e u n p a l a t a b i l i t i e s



134

BIOLOGICALLY ACTIVE NATURAL PRODUCTS o f p h e n o l g l y c o s i d e - c o n t a i n i n g e x t r a c t s o f S a l i x t o L. t i m i d u s . L i n d r o t h et a l . have demonstrated the n e g a t i v e e f f e c t s of p h e n o l g l y c o s i d e s f r o m P o p u l u s t r e m u l o i d e s on t h e p e r f o r m a n c e o f P a p i l l o g l a u c u s l a r v a e (31) and h a v e a s c r i b e d t h e e f f e c t t o t h e l e v e l s o f s a l i c o r t i n (2) and t r e m u l a c i n (3) i n t h e l e a v e s ( 3 2 ) . Z u c k e r (.33) h a s t e n u o u s l y s u g g e s t e d t h a t p h e n o l g l y c o s i d e s of Populus a n g u s t i f o l i a leaves i n h i b i t the s u c c e s s f u l c o l o n i z a t i o n of t h i s p l a n t by a g a l l - f o r m i n g aphid (Pemphigus b e t a e ) . Some o l i g o p h a g o u s a n d m o n o p h a g o u s ( s p e c i a l i s t ) i n s e c t h e r b i v o r e s , h o w e v e r , a p p a r e n t l y u t i l i z e p h e n o l g l y c o s i d e s as p o s i t i v e cues f o r f e e d i n g . T a h a v a n a i n e n e t a l . (34) have reported that four l e a f beetle species select t h e i r favored h o s t p l a n t s ( S a l i x sp.) b a s e d upon the p l a n t ' s s u i t e o f p h e n o l glycosides. When t h e f a v o r e d h o s t p l a n t was r e m o v e d i n f e e d i n g t r i a l s , i n s e c t s s h i f t e d to the S a l i x species having a phenol g l y c o s i d e c o n t e n t most l i k e the p r e f e r r e d h o s t . A very r e v e a l i n g study of e x p l o i t a t i o n of phenol g l y c o s i d e s by h e r b i v o r e s has been r e p o r t e d i n a s e r i e s o f p a p e r s by R o w e l l - R a h i e r a n d P a s t e e l s (3j>-41) . T h e y d i s c o v e r e d several oligophagous chrysomelid beetles which prefer S a l i x h o s t s r i c h i n s a l i c i n (37.f4Q) a n d one b e e t l e w h i c h u t i l i z e s a s a l i c o r t i n - c o n t a i n i n g S a l i x species (35). I n these cases the p h e n o l g l y c o s i d e does n o t s e r v e as a f e e d i n g cue; b u t i t i s m e t a b o l i z e d by the b e e t l e t o produce s a l i c a l d e h y d e ( 4 ) , a d e f e n s i v e c h e m i c a l u t i l i z e d by the i n s e c t , and g l u c o s e , w h i c h s e r v e s as a s i g n i f i c a n t e n e r g y s o u r c e ( 4 1 ) . S m i l e y e t a l . (42) have extended t h i s work to a N o r t h American ecosystem. They found t h a t the l a r v a e of the C a l i f o r n i a n S i e r r a Nevada b e e t l e (Chrvsomela a e n i c o l l i s ) p r e f e r S a l i x leaves which have h i g h s a l i c i n l e v e l s and t h a t l a r v a e p l a c e d on S a l i x l e a v e s w i t h h i g h s a l i c i n content have a h i g h e r s u r v i v a l r a t e than l a r v a e p l a c e d on l e a v e s low i n s a l i c i n . I n summary, we may s a y t h a t t h e f e w r e p o r t s o n p h e n o l g l y c o s i d e s as m e d i a t o r s o f p l a n t / h e r b i v o r e i n t e r a c t i o n s s u g g e s t t h a t E r r a r a ' s ' s p r o p o s a l h a s some m e r i t b u t t h a t i t s g e n e r a l i t y r e m a i n s t o be d e t e r m i n e d . Furthermore, beyond the work of R o w e l l - R a h i e r and P a s t e e l s t h e r e i s a g e n e r a l l a c k o f i n f o r m a t i o n on t h e c h e m i s t r y b e h i n d t h e o b s e r v e d r e s p o n s e s o f herbivores to phenol g l y c o s i d e s . Phenol glycosides

and

defense of Alaskan

woody

plants

O v e r t h e p a s t s e v e r a l y e a r s we h a v e i n v e s t i g a t e d t h e r o l e s o f p h e n o l g l y c o s i d e s i n two w o o d y p l a n t / h e r b i v o r e i n t e r a c t i o n s which have s i g n i f i c a n t i m p l i c a t i o n s i n h i g h - l a t i t u d e ecosystems of North America. I n e a c h c a s e we w e r e d r a w n t o s t u d y p h e n o l g l y c o s i d e s by r e s u l t s o b t a i n e d f r o m b i o a s s a y e x p e r i m e n t s , and we h a v e b e e n a b l e t o d e f i n e t h e e c o l o g i c a l r o l e s o f t h e s e compounds o n l y by p a y i n g a t t e n t i o n t o t h e i r d e t a i l e d s t r u c t u r e s and the dynamic n a t u r e o f t h e i r e x i s t e n c e i n p l a n t s . Our i n i t i a l e n c o u n t e r came a b o u t d u r i n g a t t e m p t s t o c h e m i c a l l y d e f i n e the r e a s o n s f o r the s e l e c t i v e use o f balsam p o p l a r ( P o p u l u s b a l s a m i f e r a ) p a r t s and g r o w t h s t a g e s by the



10.

REICHARDT ET AL.


snowshoe h a r e (Lepus a m e r i c a n u s ) . F i e l d observations (Bryant, J.P., U n i v e r s i t y of A l a s k a Fairbanks, unpublished data) i n d i c a t e d t h a t i n t e r n o d e s (stems between buds) and b a r k a r e the o n l y p a r t s of winter-dormant balsam p o p l a r eaten by hares. Furthermore, o n l y o l d e r internodes from mature s a p l i n g s are u t i l i z e d ; a l l i n t e r n o d e s f r o m j u v e n i l e p l a n t s and c u r r e n t - y e a r growth i n t e r n o d e s from mature p l a n t s are i g n o r e d by the hares. Guided by p a l a t a b i l i t y b i o a s s a y r e s u l t s from e x t r a c t s o f p o p l a r , we t e n t a t i v e l y c o n c l u d e d t h a t e t h e r s o l u b l e m e t a b o l i t e s i n the i n t e r n o d e s (stems between buds) were r e s p o n s i b l e f o r h a r e s ' s e l e c t i v e use o f t h i s p o t e n t i a l f o o d s o u r c e . S u b s e q u e n t l y we f o u n d t r i c h o c a r p i g e n i n ( b e n z y l g e n t i s a t e , !)) t o be a m a j o r component o f t h i s e x t r a c t , as h a d b e e n p r e v i o u s l y r e p o r t e d by P e a r l and D a r l i n g ( 4 3 ) . We w e r e e n c o u r a g e d w h e n we f o u n d t h i s s u b s t a n c e , w h i c h has a p e p p e r y f l a v o r and c a u s e s n u m b n e s s t o t h e m o u t h a n d gums w h e n t a s t e d , t o b e a d e t e r r e n t to hare feeding. H o w e v e r , a p r o b l e m d e v e l o p e d w h e n we d i s c o v e r e d t h a t h a r e s r e a d i l y consume p o p l a r i n t e r n o d e s w h i c h our a n a l y s e s showed c o n t a i n e d c o n c e n t r a t i o n s o f 5 w e l l above those which caused complete a v e r s i o n i n the b i o a s s a y s (Mattes, B.R., U n i v e r s i t y of Alaska Fairbanks, unpublished r e s u l t s ) . More c a r e f u l i n v e s t i g a t i o n r e v e a l e d t h a t l i t t l e , i f any, f r e e 5 occurs i n the p l a n t but t h a t i t i s produced from t r i c h c o c a r p i n (6) d u r i n g e x t r a c t i o n o f f r e s h p l a n t m a t e r i a l w i t h d i e t h y l ether. Further experiments revealed that t h i s h y d r o l y s i s d u r i n g e x t r a c t i o n i s a p p a r e n t l y c a t a l y z e d by a p l a n t - c o n t a i n e d enzyme w h i c h i s p r e s u m a b l y s e g r e g a t e d f r o m t h e a p p a r e n t l y p a l a t a b l e s u b s t r a t e (6) b u t i s r e l e a s e d d u r i n g t h e d i s r u p t i v e events a s s o c i a t e d w i t h the e x t r a c t i o n process (44). We concluded, however, t h a t t h i s p o t e n t i a l l a t e n t defense o f p o p l a r t o h a r e s i s i n e f f e c t i v e due t o t h e r e l a t i v e l y s l o w r a t e of r e a c t i o n experienced i n plant material during herbivory. F u r t h e r i n v e s t i g a t i o n o f the l i p i d s and p h e n o l g l y c o s i d e s o f P. b a l s a m i f e r a p r o v i d e d a n e v e n m o r e i n t e r e s t i n g a n d e c o l o g i c a l l y relevant aspect of phenol glycoside biochemistry o f t h i s p l a n t . Among t h e e t h e r - s o l u b l e m e t a b o l i t e s o f b a l s a m p o p l a r i n t e r n o d e s , we d i s c o v e r e d 6 - h y d r o x y c y c l o h e x e n o n e ( 7 ) . A s e c o n d m a j o r p h e n o l g l y c o s i d e p r o v e d t o be s a l i c o r t i n (2) ( 4 4 ) . Based upon s t r u c t u r a l c o n s i d e r a t i o n s and the r e p o r t by P e a r l and D a r l i n g (45) t h a t 2 p r o d u c e s c a t e c h o l (8) upon b a s i c h y d r o l y s i s , we i n v e s t i g a t e d t h e b i o c h e m i c a l r e l a t i o n s h i p b e t w e e n 2 a n d 7. We w e r e g r a t i f i e d t o f i n d t h a t a n e n z y m e p r e p a r a t i o n from p o p l a r i n t e r n o d e s r e a d i l y c o n v e r t s 2 t o 7 and 1. I n t h i s c a s e , we f o u n d t h a t t h e u n p a l a t a b l e p r o d u c t ( 7 ) i s n o t an a r t i f a c t . T h u s t h e p h e n o l g l y c o s i d e (2) i s a b i o s y n t h e t i c p r e c u r s o r o f a d e f e n s i v e c h e m i c a l as w e l l as a l a t e n t a d d i t i o n a l source of the d e t e r r e n t which i s produced from 2 at some u n k n o w n r a t e d u r i n g h e r b i v o r y . Furthermore, the other p r o d u c t f r o m t h i s r e a c t i o n (1) c o u l d w e l l s e r v e as t h e b i o s y n t h e t i c p r e c u r s o r o f a n o t h e r d e f e n s i v e m e t a b o l i t e (4) o f P. b a l s a m i f e r a ( C l a u s e n , T.P., U n i v e r s i t y o f A l a s k a F a i r b a n k s , unpublished results). A t t h i s p o i n t o u r w o r k o n t h e d e f e n s i v e c h e m i s t r y o f P. b a l s a m i f e r a p a i d d i v i d e n d s i n our p a r a l l e l study o f the l a r g e


135


136


OH

OC OR

6

R = glu



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aspen t o r t r i x (Choristoneura c o n f l i c t a n a (Walker))/quaking aspen (P. t r e m u l o i d e s ) i n t e r a c t i o n . I n t h i s s t u d y we h a d s e t out t o examine the reasons b e h i n d the p o p u l a t i o n c y c l e s o f t h i s moth s p e c i a l i s t . Our h y p o t h e s i s was t h a t h e r b i v o r y - i n d u c e d changes i n h o s t p l a n t c h e m i s t r y causes a drop i n d i e t a r y q u a l i t y o f the l e a v e s , l e a d i n g t o a p r e c i p i t o u s d e c l i n e i n moth p o p u l a t i o n s i n years f o l l o w i n g severe d e f o l i a t i o n of the plants. Our d i s c o v e r i e s t h a t t h e m a j o r p h e n o l g l y c o s i d e s o f quaking aspen f o l i a g e were s a l i c i n ( 1 ) , s a l i c o r t i n ( 2 ) , t r e m u l a c i n ( 3 ) , a n d t r e m u l o i d e n ( 9 ) a n d t h a t t h e same l e a v e s c o n t a i n e d 7 l e d us t o e v a l u a t e the i m p o r t a n c e o f t h e i r b i o c h e m i c a l r e l a t i o n s h i p s to the s u i t a b i l i t y of quaking aspen f o l i a g e as f o o d f o r t h e l a r g e a s p e n t o r t r i x l a r v a e . Initially we d e m o n s t r a t e d t h a t p h e n o l g l y c o s i d e - c o n t a i n i n g f r a c t i o n s f r o m a s p e n l e a v e s r e d u c e d t h e p e r f o r m a n c e o f C. c o n f l i c t a n a l a r v a e (46). S u b s e q u e n t l y we d i s c o v e r e d t h a t a l l p h e n o l g l y c o s i d e s e x c e p t 9 a d v e r s e l y a f f e c t l a r v a l p e r f o r m a n c e b u t t h a t 2 and 3 are p a r t i c u l a r l y d e t r i m e n t a l i n t h i s r e g a r d (Bryant, J.P., U n i v e r s i t y of Alaska Fairbanks, unpublished r e s u l t s ) . Furthermore, d i s r u p t i o n of aspen l e a f t i s s u e leads to the p r o d u c t i o n o f 7 f r o m b o t h 2 a n d 3. The p r o d u c t o f t h e s e t r a n s f o r m a t i o n s apparently reduces l e a f q u a l i t y to the l a r v a e b y d i s r u p t i n g t h e i n s e c t s ' m e t a b o l i s m due t o i t s r e a c t i v i t y a s a n e l e c t r o p h i ] •» o r a f t e r i t s f a c i l e c o n v e r s i o n t o p h e n o l o r catechol (Figure 2). Experiments w i t h i n t a c t aspen s a p l i n g s r e v e a l e d another f a c e t of the r o l e phenol g l y c o s i d e s p l a y i n the p l a n t ' s r e s p o n s e t o l e a f damage. P e r f o r a t i o n o r c l i p p i n g the edges o f Baves, as m i g h t o c c u r d u r i n g i n s e c t a t t a c k , c a n e l i c i t t h e w r a n s l o c a t i o n o f 2 and 3 f r o m i n t e r n o d e s t o l e a v e s . Thus the p l a n t ' s r e s p o n s e t o l e a f damage i s a t l e a s t t w o - f o l d : 1) c o n v e r s i o n o f two p h e n o l g l y c o s i d e s t o a d e f e n s i v e c h e m i c a l and 2) r e p l a c e m e n t a n d e n h a n c e m e n t o f p h e n o l g l y c o s i d e r e s e r v e s i n d a m a g e d l e a v e s b y t r a n s l o c a t i o n ( C l a u s e n , T.P., U n i v e r s i t y of Alaska Fairbanks, unpublished r e s u l t s ) . I t i s i n t e r e s t i n g to consider these f i n d i n g s i n l i g h t of t h e r e s u l t s o f two o t h e r s t u d i e s . Among t h e i n s e c t - m e d i a t e d t r a n s f o r m a t i o n s d e s c r i b e d by R o w e l l - R a h i e r and P a s t e e l s i s the c o n v e r s i o n o f 2 t o 4 a n d g l u c o s e b y P. v i t e l l i n a e ( 3 6 ) . Based u p o n t h i s i n f o r m a t i o n , a n d o u r f i n d i n g s , one c a n s u r m i s e t h a t 1_ i s a l s o produced during these transformations. What w o u l d be t h e e f f e c t o f i t s p r o d u c t i o n o n t h e i n s e c t ? The two o p t i o n s s e e m t o b e t h a t 1) i t s a d v e r s e e f f e c t s a r e t h e p r i c e t h e i n s e c t p a y s f o r m e t a b o l i c e n e r g y a n d d e f e n s i v e c h e m i c a l ( 4 ) o r t h a t 2) the i n s e c t i n c o r p o r a t e s 7 i n t o i t s d e f e n s i v e s e c r e t i o n , thus m a k i n g t h r e e - f o l d u s e o f d i e t a r y 2. The r e l e v a n c e o f o u r f i n d i n g s t o t h e P. glauca/P. t r e m u l o i d e s s y s t e m s t u d i e d b y L i n d r o t h e t a l . (3JL) s e e m s m o r e clearcut. They r e p o r t t h a t o f the f o u r p h e n o l g l y c o s i d e s ( 1 , 2, 3, a n d 9) f o u n d i n P. t r e m u l o i d e s f o l i a g e , o n l y two (2 a n d 3) c a u s e d e c r e a s e s i n p e r f o r m a n c e o f P. g l a u c a l a r v a e (32.) . I t i s h i g h l y l i k e l y t h a t the b i o c h e m i c a l t r a n s f o r m a t i o n s o f 2 and 3 to 7 described here e x p l a i n t h i s observation. -



138


A proposal in plants

f o r the

defensive

s i g n i f i c a n c e of phenol

glycosides

I f t h e r e i s any g e n e r a l i t y t o a d e f e n s i v e r o l e o f p h e n o l g l y c o s i d e s i n p l a n t s , i t a p p e a r s t o be i n t h e i r a b i l i t y t o f u n c t i o n as m o b i l i z a b l e d e f e n s e s . G l y c o s y l a t i o n of phenolic m e t a b o l i t e s o f f e r s two a d v a n t a g e s t o b i o l o g i c a l s y s t e m s : 1) g e n e r a l l y , although not always, i t increases the water s o l u b i l i t y o f t h e m e t a b o l i t e ( 1 5 ) a n d 2) i t a t t e n u a t e s the t o x i c (15., 17) a n d d e s t r u c t i v e p r o p e r t i e s (15.) o f t h e f r e e phenol. Thus t h e p h e n o l g l y c o s i d e s c a n r e a d i l y be t r a n s l o c a t e d by p l a n t s and e n z y m a t i c a l l y c o n v e r t e d t o d e f e n s i v e s u b s t a n c e s at the s i t e of a t t a c k . W h i l e t h i s p r o p o s a l i s b a s e d upon d a t a f r o m o n l y t h r e e p h e n o l g l y c o s i d e s ( 2 , 3, a n d 6 ) , one can e n v i s i o n a t l e a s t one o t h e r m e c h a n i s t i c s c e n a r i o , e x e m p l i f i e d by s a l i c i n (1) - p e r h a p s t h e most commonly f o u n d p h e n o l glycoside i n plants. B i o c h e m i c a l c o n v e r s i o n o f 1 t o 10 ( w i t h R = PO3H" , f o r e x a m p l e ) c o u l d e a s i l y l e a d t o 11 a s d e p i c t e d i n F i g u r e 3. T h i s p r o d u c t w o u l d u n d o u b t e d l y be an e x c e l l e n t e l e c t r o p h i l e ( F i g u r e 3; 4 7 - 4 9 ) a n d t h u s i n t e r f e r e w i t h h e r b i v o r e m e t a b o l i s m i n a manner s i m i l a r t o t h a t a s c r i b e d t o s e s q u i t e r p e n e l a c t o n e s (50) and o t h e r M i c h a e l a c c e p t o r s (49). The p o t e n t i a l i m p o r t a n c e o f t h e p r o c e s s d e p i c t e d i n F i g u r e 3 g o e s w e l l b e y o n d i t s a p p l i c a t i o n t o s a l i c i n i n t h a t many o f t h e k n o w n p h e n o l g l y c o s i d e s c o n t a i n p a r t s t r u c t u r e s ( e . g . 12 a n d 13) w h i c h c o u l d s e r v e a s p r e c u r s o r s t o a n a l o g u e s o f 11 (quinonemethides; 48). An a p p r e c i a t i o n o f t h e p r a c t i c a l c o n s e q u e n c e s o f the b i o c h e m i c a l l a b i l i t y o f p h e n o l g l y c o s i d e s a l l o w s one t o c o n s i d e r t h e i r r o l e s i n p l a n t d e f e n s e f r o m a new perspective. We c a n move f r o m t h e c l a s s i c a l a p p r o a c h o f c o r r e l a t i n g h e r b i v o r e b e h a v i o r w i t h c h e m i c a l c o n s t i t u e n t s o f p l a n t s t o one which i s based, at l e a s t i n p a r t , upon a fundamental understanding of the chemistry b e h i n d the i n t e r a c t i o n s . The p o t e n t i a l p a y o f f o f t h i s a p p r o a c h i s t h a t i t has p r e d i c t i v e as w e l l as r e t r o s p e c t i v e p r o p e r t i e s . T h u s we b e l i e v e t h a t E r r a r a ' s ' s (25) o l d p r o p o s a l t h a t p h e n o l g l y c o s i d e s p r o v i d e p l a n t s w i t h d e f e n s e f r o m h e r b i v o r e s c a n o n l y be f u l l y evaluated by s t u d i e s w h i c h a r e b a s e d upon d e t a i l e d c o n s i d e r a t i o n s o f p h e n o l g l y c o s i d e s t r u c t u r e and e l u c i d a t i o n o f b i o c h e m i c a l transformations. The a p p l i c a t i o n o f s u c h a n a p p r o a c h t o a g r i c u l t u r a l p l a n t s c o u l d have a dramatic e f f e c t upon the p r e s e n t v i e w t h a t phenol g l y c o s i d e s are g e n e r a l l y undesirable c o n s t i t u e n t s of crops. Consider, f o r example, the p o t e n t i a l e c o l o g i c a l r o l e s o f two p h e n o l g l y c o s i d e s , v i c i n e a n d c o n v i c i n e , i n f a v a b e a n s (Vicia faba). T h e s e two s u b s t a n c e s r a p i d l y u n d e r g o b o t h e n z y m a t i c and c h e m i c a l h y d r o l y s i s t o /3-D-glucose and a g l y c o n e s - d i v i c i n e a n d i s o v a m i l , r e s p e c t i v e l y . The l a t t e r two s u b s t a n c e s a r e p o t e n t r e d u c i n g a g e n t s (51,52) and c a u s e d e c l i n e s i n g l u t a t h i o n e a n d ATP l e v e l s i n human b l o o d c e l l s (53). T h e i r r o l e s i n t h e a c u t e human h e m o l y t i c a n e m i a k n o w n a s f a v i s m h a v e b e e n e x t e n s i v e l y i n v e s t i g a t e d (52,54,.55), and t h e y a r e known t o i n h i b i t the g r o w t h o f c e r t a i n p l a n t p a t h o g e n s



REICHARDT ET AL.

F i g u r e 2. biochemistry

E c o l o g i c a l l y relevant aspects of 6-hydroxycyclohexenone.

glu

of the chemistry

and

glu

+ glu

F i g u r e 3. of s a l i c i n

139


• OR"

H y p o t h e t i c a l pathway f o r b i o c h e m i c a l f o r defensive purposes.

mobilization


140


(.56). However, the generality of the roles of these four compounds in the defense of Vicia faba is unknown. As agricultural practice moves away from use of "hard" pesticides, the efficacies of intrinsic defensive systems of crops will become more important. If phenol glycosides prove to be significant components of the chemical defenses of cultivated plants (as they appear to be in at least some wild plants), no longer will they be viewed as totally undesirable constituents. In fact, they could become important ingredients of integrated pest management systems.


Acknowledgments The experimental work described here has been supported by the National Science Foundation (BSR8416461 and BSR8500160). Literature Cited 1. 2. 3. 4. 5. 6. 7. 8. 9.

10. 11. 12. 13. 14. 15. 16. 17.

Armstrong, E. F.; Armstrong, K. F. The Glycosides: Longmans: London, 1931; p 12. Devon, T. K.; Scott, A. I. Handbook of Naturally Occurring Compounds: Academic Press: New York, 1975; Vol. 1. Estes, T. K.; Pearl, I. A. Tappai 1967, 50, 318-323. Repas, A.; Nikolin, B.; Dursun, K. J. Chromatog. 1969, 44, 184-187. Steele, J. W.; Bolan, M.; Audette, R. C. S. Chromatog. 1969, 40, 370-376. Still, W. C.; Kahn, W.; Mitra, A. J. Org. Chem. 1978, 43, 2923-2925. Lindroth, R. L.; Hsia, M. T. S.; Scriber, J. M. Biochem. Syst. Ecol. 1987, 15, 677-680. Pridham, J. B. In Adv. Carbohydrate Chem.:. Wolfram, M. L.; Tipson, R. S., Eds.; Academic Press: NY, 1965; Vol. 20, pp 371-408. Howe, I.; Jordan, M.; In Fortshritte der Chemie organischer Naturstoffe: Herz, W.; Grisebach, H.; Kirby, G. W.; Tamm. Ch., Eds.; Springer-Verlag: NY, 1985; Vol. 47, pp 107-152. Benn, R.; Gunther, H. Agnew. Chem. Int. Ed. 1983, 22, 350-380. Audette, R. C. S.; Blunden, G.; Steele, J. W.; Wong, C. S. C. J. Chromatog. 1966, 25, 367-372. Julkunen-Tiitto, R. J. Agric. Food Chem. 1985, 33, 213217. Meier, B.; Sticher, O.; Bettschart, A. Deutsche Apotheker Zeitung 1985, 125, 341-347. Meier, B.; Lehmann, D.; Sticher, O.; Bettschart. A. Pharm. Acta Helv. 1985, 60, 269-275. Hopkinson, S. M. Quart. Rev. (London) 1969, 23, 98-124. Markham, K. R. N. Z. J. Sci. 1971, 14, 179-186. Vickery, M. L.; Vickery, B. Secondary Plant Metabolism: Macmillan: Baltimore, MD, 1981.


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Phenol Glycosides in Plant Defense Against Herbivores - American

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