Chapter 47
Chemical Ecology of Quinolizidine Alkaloids Michael Wink
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Institut für Pharmazeutische Biologie der Technischen, Universität Braunschweig, MendelssohnstraBe 1, D-3300 Braunschweig, Federal Republic of Germany
The biochemistry and physiology of quinolizidine alkaloids is reviewed with respect to their role in lupin metabolism. Minor roles of the alkaloids may be nitrogen transport and nitrogen storage, but their main function is that of chemical defense. Alkaloid concentrations in the plant are in the same order or even higher than the inhibitory concentrations against pathogens that have been established experimentally. Alkaloid-free lupins are highly susceptible to herbivore predation, which shows that the alkaloids are obviously important for the survival of a lupin plant. Quinolizidine alkaloids (QA) are thought to be typical natural products of many Leguminosae (1-3) but a few isolated occurrences have been reported also in unrelated families, e.g. Chenopodiaceae (_1 ) , Berberidaceae (JO , Papaveraceae (JO , Scrophulariaceae (£) , Santalaceae (_5) , Solanaceae (2) , and Ranunculaceae (1) . These observations could indicate that the genes for QA biosynthesis are probably not restricted to the Leguminosae but are widely distributed in the plant kingdom; however, they are only rarely expressed in the other families. We could support this belief by recent experiments using plant cell suspension cultures. A short-term and transientQA formation could be detected after induction even in "QAfree" species, such as Daucus, Spinacia, Conium, and Symphytum (6). In order to understand the biological function of QA we have to analyze their physiology and biochemistry f i r s t . Biochemistry and Physiology of Quinolizidine Alkaloids In the first step lysine is decarboxylated to cadaverine. Then three cadaverine units are incorporated into the tetracyclic QA skeleton, such as in lupanine, which serves as a precursor for most of the other QA. Recent tracer experiments have been reviewed (_3, 1) . In y
Current address: Genzentrum der Universitât Munchen, D-8033 Martinsried, Federal Republic of Germany 0097-6156/87/0330-0524$06.00/0 © 1987 American Chemical Society
47.
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Chemical Ecology of Quinolizidine Alkaloids
our l a b o r a t o r y we have c o n c e n t r a t e d on the enzymology o f QA forma t i o n and have been a b l e t o c h a r a c t e r i z e more t h a n f o u r d i s t i n c t new enzymes; a r e v i e w i s a v a i l a b l e ( 8 ) . S i t e o f a l k a l o i d f o r m a t i o n , t r a n s p o r t , and a c c u m u l a t i o n . QA are formed i n the a e r i a l green p a r t s o f legumes, e s p e c i a l l y i n the l e a v e s (9^) . In l u p i n l e a v e s we succeeded i n l o c a l i z i n g the key en zymes o f QA b i o s y n t h e s i s i n the c h l o r o p l a s t (K), _1J_) , where the f o r m a t i o n o f the p r e c u r s o r l y s i n e a l s o t a k e s p l a c e . L i k e most o f the p r o c e s s e s t h a t are l o c a t e d i n the c h l o r o p l a s t , QA b i o s y n t h e s i s i s r e g u l a t e d by l i g h t (8) and QA f o r m a t i o n f o l l o w s a l i g h t - d e p e n d e n t d i u r n a l rhythm (12, 13). The a l k a l o i d s formed i n the l e a v e s are t r a n s l o c a t e d v i a the phloem (_1_3, 14) a l l o v e r a l u p i n p l a n t , so t h a t a l l p l a n t p a r t s c o n t a i n a l k a l o i d s . QA are accumulated and s t o r e d p r e f e r e n t i a l l y i n e p i d e r m a l and s u b e p i d e r m a l t i s s u e s o f stems and l e a v e s (15_, 16). E s p e c i a l l y r i c h i n a l k a l o i d s are the s e e d s , which may c o n t a i n up t o 5% (dry weight) a l k a l o i d ( e q u i v a l e n t t o 200 mmol/ kg) . T u r n o v e r o f q u i n o l i z i d i n e a l k a l o i d s . L i k e many o t h e r n a t u r a l p r o d u c t s , e s p e c i a l l y n i t r o g e n - c o n t a i n i n g compounds, QA are not i n e r t end p r o d u c t s o f m e t a b o l i s m , but compounds w i t h a h i g h degree o f t u r n o v e r . T h i s phenomenon becomes e s p e c i a l l y e v i d e n t d u r i n g g e r m i n a t i o n and s e e d l i n g development. Most o f t h e a l k a l o i d s are me t a b o l i z e d and t h e i r n i t r o g e n i s p r o b a b l y used f o r s e e d l i n g growth (Γ7) . R o l e o f Q u i n o l i z i d i n e A l k a l o i d s i n the M e t a b o l i s m o f
Lupins
P r i m a r y m e t a b o l i s m . Having b r i e f l y r e v i e w e d the p h y s i o l o g y and b i o c h e m i s t r y o f QA i n the s e c t i o n s above, we can c o n s i d e r t h e q u e s t i o n as t o why do l u p i n s produce a l k a l o i d s . F i r s t o f a l l we can ask whether the QA may p l a y a r o l e i n the p r i m a r y m e t a b o l i s m o f a l u p i n plant. A l k a l o i d s a r e t r a n s l o c a t e d i n t h e phloem sap l i k e o t h e r p h o t o s y n t h a t e s , and QA c o n t r i b u t e about 8 % t o the o v e r a l l n i t r o g e n . S i n c e QA are r e a d i l y m e t a b o l i z e d by c e l l s , t h e a l k a l o i d s c o u l d t h u s p l a y a r o l e as a minor means o f n i t r o g e n t r a n s p o r t . S p e c i e s t h a t produce r a t h e r few and heavy seeds s t o r e up t o 5 % a l k a l o i d s i n a d d i t i o n t o c a . 30 % s t o r a g e p r o t e i n . We have e s t i m a t e d t h a t QA c o n t r i b u t e c a . 10 % o f t h e t o t a l n i t r o g e n s t o r e d i n l u p i n s e e d s . T h e r e f o r e a n o t h e r minor r o l e o f QA c o u l d be n i t r o g e n s t o r a g e (_Γ7 , _18) . We c o u l d not f i n d o t h e r a r e a s o f p r i m a r y m e t a b o l i s m i n which QA might p l a y a p a r t . S i n c e we can assume t h a t a l l m e t a b o l i t e s found i n organisms have a d e f i n i t e f u n c t i o n (19) , we s u g g e s t t h a t t h e n i t r o g e n s t o r a g e f u n c t i o n o f QA i s o f o n l y minor importance and p r o b a b l y not s u f f i c i e n t t o e x p l a i n t h e complex p h y s i o l o g y o f QA. C h e m i c a l e c o l o g y . I t has been g e n e r a l l y a c c e p t e d t h a t many o f the s o - c a l l e d "secondary m e t a b o l i t e s " p l a y a r o l e i n the i n t e r r e l a t i o n s h i p o f p l a n t - p l a n t s , p l a n t - m i c r o b e s , and p l a n t - h e r b i v o r e s (20-23). In a s e r i e s o f e x p e r i m e n t s we have sought t o d e t e r m i n e whether l u p i n a l k a l o i d s are i m p o r t a n t i n an e c o l o g i c a l c o n t e x t .
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ALLELOCHEMICALS: ROLE IN AGRICULTURE AND FORESTRY
What s h o u l d be t h e r e q u i r e m e n t s 1. 2. 3. 4.
o f a p l a n t defense
chemical?
I t s h o u l d d i s p l a y a s i g n i f i c a n t a c t i v i t y under e x p e r i m e n t a l conditions. I t s c o n c e n t r a t i o n i n t h e i n t a c t p l a n t s h o u l d e q u a l o r exceed the e x p e r i m e n t a l l y d e f i n e d i n h i b i t o r y c o n c e n t r a t i o n s . I t s h o u l d be p r e s e n t i n t h e p l a n t a t t h e r i g h t time and t h e right place. I t s h o u l d be e c o l o g i c a l l y r e l e v a n t .
We have i s o l a t e d pure a l k a l o i d s and s t u d i e d t h e i r e f f e c t on t h e m u l t i p l i c a t i o n o f p o t a t o - X - v i r u s ( F i g u r e 1 ) , on t h e growth o f gramp o s i t i v e and gram-negative b a c t e r i a ( 2 4 , T a b l e I) and o f f u n g i ( 2 4 , 2 5 , T a b l e I ) , t h e g e r m i n a t i o n o f l e t t u c e seeds (18_, 26), and t h e f e e d i n g o f i n s e c t s and m o l l u s c s (2_7, 2 8 , T a b l e I ) . F u r t h e r m o r e , QA are t o x i c f o r mammals, as has been r e v i e w e d ( 2 ^ 2j5, 27) . I t i s r e markable t h a t QA a r e a c t i v e , n o t i n one, b u t i n a l l t h e i n t e r a c t i o n s s t u d i e d . The s t r u c t u r e o f t h e i n d i v i d u a l a l k a l o i d s d e t e r m i n e d t h e e f f e c t o n l y t o some d e g r e e . F o r example, t h e e s t e r s o f 13-hydroxyl u p a n i n e , such as 1 3 - t i g l o y l o x y l u p a n i n e , seemed t o be more t o x i c and r e p e l l e n t t h a n l u p a n i n e (_27, _28) . V e r t e b r a t e t o x i c i t y seemed t o be h i g h e s t i n t h e o
