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UBC Theses and Dissertations

Phenolic metabolism in higher plants : I. Catechol biogenesis in Gaultheria, II. The biogenesis of rosmarinic… Ellis, Brian Edward 1969

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PHENOLIC METABOLISM IN HIGHER PLANTS I . CATECHOL BIOGENESIS IN GAULTHERIA I I . THE BIOGENESIS OF ROSMARINIC ACID IN MENTHA I I I . DEGRADATION OF AROMATIC COMPOUNDS BY STERILE PLANT TISSUES by BRIAN EDWARD ELLIS B . S c , U n i v e r s i t y of New Brunswick, 19&5 A THESIS SUBMITTED IN PARTIAL FULFILMENT OF THE REQUIREMENTS FOR THE DEGREE OF DOCTOR OF PHILOSOPHY i n the Department of Botany We accept t h i s t h e s i s as conforming to the req u i r e d standard THE UNIVERSITY OF BRITISH COLUMBIA November, I 9 6 9 In presenting t h i s t h e s i s in p a r t i a l f u l f i l m e n t o f the requirements f o r an advanced degree at the U n i v e r s i t y of B r i t i s h Columbia, I agree that the L i b r a r y s h a l l make i t f r e e l y a v a i l a b l e f o r reference and study. I f u r t h e r agree tha permission for extensive copying of t h i s t h e s i s f o r s c h o l a r l y purposes may be granted by the Head of my Department or by h i s r e p r e s e n t a t i v e s . It i s understood that copying or p u b l i c a t i o n of t h i s t h e s i s f o r f i n a n c i a l gain s h a l l not be allowed without my w r i t t e n permission. Department of The U n i v e r s i t y of B r i t i s h Columbia Vancouver 8 , Canada Date - i -Abstract I. Previous studies on biogenesis of simple phenols i r plants have been restricted, to hydrocuinone. Ariong the other simple phenols, catechol i s of par-t i c u l a r i n t e r e s t because of i t s p o t e n t i a l role as a ring-cleavage substrate. Tracer studies on the biogenesis of catechol i n Gaultherla l e a f discs showed that i t was formed from s a l i c y l i c acid by oxidative decarboxylation. S a l i c y l a t e decarboxyl-ating a c t i v i t y could be detected i n buffered ex-tra c t s of very young leaves, I I . Among the numerous c a f f e i c acid esters presently known in plants, only 3 - 0-'caffeoylquinic acid (chlor-os;enic acid) has been stud!.ed i n d e t a i l . Rosmarin-ic acid (alpha-O-caffeoyl - 3» ^ -dihydroxyphenyllactlc acid) has been reported to occur i n a number of plants but nothing was known of i t s biosynthesis or metabolic r o l e . Tracer studies demonstrated that ^ n Mentha the c a f f e i c acid moiety was formed from, phenylalanine v i a cihnamic and para-coumarlc acids. In contrast, the .structurally s i m i l a r 3»^-dih.yd.roxy-phenyllactic acid moiety was formed from tyrosine and J>,k-dihydroxyphenylalanine. There was no evid-ence of the p a r t i c i p a t i o n of a para-coumaroyl ester intermediate. Time-course studies and use of - i i -l a b e l l e d rosmarinic a c i d showed that endogenous rosmarinic a c i d was t u r n i n g over s l o w l y . The c a f f e o y l moiety, however, does not appear to be c o n t r i b u t i n g to the formation of i n s o l u b l e polymers, as has been suggested f o r chlorogenic a c i d i n other p l a n t s . I I I . B a c t e r i a and fungi r e a d i l y degrade aromatic com-pounds to carbon d i o x i d e . Despite the l a r g e quan-t i t i e s of aromatic compounds formed i n p l a n t s , l i t t l e a t t e n t i o n has been paid to the a b i l i t y of p l a n t t i s s u e s to degrade aromatic r i n g s . No r e -ported s t u d i e s have used completely s t e r i l e p l a n t s and techniques. This has l e f t open the p o s s i b i l i t y t hat the m i c r o f l o r a a s s o c i a t e d w i t h the p l a n t might be c a r r y i n g out the observed r e a c t i o n s . The a b i l i t y of s t e r i l e p l a n t t i s s u e c u l t u r e s to de-grade aromatic r i n g - l ^ C compounds to carbon d i o x i d e was s t u d i e d . I t was e s t a b l i s h e d that a number of t i s s u e s (Ruta, T r i t i c u m , Phaseolus, M e l i l o t u s ) have the a b i l i t y to cleave the aromatic r i n g of p h e n y l a l -anine. M e l i l o t u s t i s s u e could a l s o degrade cinnamic a c i d - r i n g -1 4 C suggesting that a dihydroxy phenolic a c i d may be the ring-cleavage s u b s t r a t e . Neither Ruta nor M e l i l o t u s t i s s u e s were able to degrade ben-z o i c a c i d or s a l i c y l i c acid-ring--*-^C. Tryptophan-benzene r i n g - C was shown to be degraded to carbon d i o x i d e by both Ruta and M e l i l o t u s . In summary, the a b i l i t y of p l a n t s to cleave the benzene r i n g of aromatic compounds when f r e e of micro-organisms was thus e s t a b l i s h e d . - i v -TABLE OF CONTENTS Page ABSTRACT i TABLE OF CONTENTS i v COMMON AND SYSTEMATIC NAMES OF COMPOUNDS v i i LIST OF TABLES v i i i LIST OF FIGURES i x ACKNOWLEDGEMENT x PREFACE x i I. CATECHOL BIOGENESIS IN GAULTHERIA INTRODUCTION 1 MATERIALS AND METHODS Pla n t m a t e r i a l k Radioactive compounds ^ I s o l a t i o n of cat e c h o l beta-D-glucoside and catechol 5 S a l i c y l a t e hydroxylase assay 6 RESULTS AND DISCUSSION Populus 8 G a u l t h e r i a . 9 LITERATURE CITED -v- Page 16 I I . THE BIOGENESIS OF ROSMARINIC ACID IN MENTHA INTRODUCTION 19 MATERIALS AND METHODS Pl a n t m a t e r i a l 26 L a b e l l e d compounds and t h e i r a d m i n i s t r a t i o n 26 I s o l a t i o n of rosmarinic a c i d and c a f f e i c a c i d 2? S p e c i f i c a c t i v i t y determination 29 RESULTS AND DISCUSSION I s o l a t i o n of rosmarinic a c i d 30 B i o s y n t h e t i c s t u d i e s 31 Time-course s t u d i e s 35 LITERATURE CITED 4-3 I I I . DEGRADATION OF AROMATIC COMPOUNDS BY STERILE PLANT TISSUES INTRODUCTION ^7 MATERIALS AND METHODS Pla n t t i s s u e c u l t u r e s 5? Radioactive compounds 58 Detection of ring-cleavage 59 M i c r o b i a l contamination 6 l - v i -Page RESULTS AND DISCUSSION A s e p t i c a l l y grown pl a n t s 6 2 P l a n t t i s s u e c u l t u r e s 62 Tryptophan 6 3 Phenylalanine 6 3 Tyrosine 68 LITERATURE CITED 75 APPENDIX 80 - v i i -Common and systematic names of compounds Common name Systematic name a n t h r a n i l i c a c i d 2-aminobenzoic a c i d c a f f e i c a c i d 3 » ^ -dihydroxycinnamic a c i d c a t e c h o l 1 , 2-dihydroxybenzene chlorogenic a c i d 3 - 0 - c a f f e o y l q u i n i c a c i d o-coumaric a c i d 2-hydroxycinnamic a c i d p_-coumaric a c i d 4-hydroxycinnamic a c i d DOPA 3 , 4 —dihydroxyphenylalanine dopamine 3 , 4-dihydroxyphenylethylamine DOPL 3 , ^ - d i h y d r o x y p h e n y l l a c t i c a c i d e s c u l e t i n 6 , 7-dihydroxycoumarin f e r u l i c a c i d 3-methoxy-i)--hydroxycinnamic a c i d g e n t i s i c a c i d 2 , 5-dihydroxybenzoic a c i d homogentisic a c i d 2 , 5-dihydroxyphenylacetic a c i d hydroquinone 1 , k -dihydroxybenzene p h l o r o g l u c i n o l 1»3» 5-trihydroxybenzene protocatechuic a c i d 3 , ^ -dihydroxybenzoic a c i d o-pyrocatechuic a c i d 2 , 3-dihydroxybenzoic a c i d rosmarinic a c i d a l p h a - 0 - c a f f eoy 1 - 3,^-di hydroxy-p h e n y l l a c t i c a c i d s a l i c y l i c a c i d 2-hydroxybenzoic a c i d - v i i i -L i s t of t a b l e s Table Page Catechol B i o g e n e s i s i n G a u l t h e r i a I. The I n c o r p o r a t i o n of R a d i o a c t i v e P r e c u r s o r s i n t o C atechol i n Leaf D i s c s of G a u l t h e r i a a d e n o t h r i x a f t e r 24 hr 7777777777 11 I I . D e c a r b o x y l a t i o n of S a l i c y l i c A c i d - 7 - l ^ C by Crude E x t r a c t of G. a d e n o t h r i x Leaves 14 The B i o g e n e s i s of Rosmarinic A c i d i n Mentha I. E s t e r i f i e d Forms of C a f f e i c A c i d i n P l a n t s 22 I I . I n c o r p o r a t i o n of Phenylpropanoid Compounds i n t o Rosmarinic A c i d i n Mentha arvense L 33 I I I . The Turnover of L a b e l l e d Rosmarinic A c i d Readministered to Mentha 41 Degradation of Aromatic Compounds by S t e r i l e P l a n t T i s s u e s I. O x i d a t i o n of DL-Tryptophan-benzene ring_u-l4c to 1^C02 by Ruta and M e l i l o t u s over seven days 64 I I . Metabolism of DL-Phenylalanine-l^C and Cinnamic-l4c by Ruta and M e l i l o t u s over seven days 65 I I I . O x i d a t i o n of Benzoic a c i d and S a l i c y l i c a c i d ring-U-14c by Ruta and M e l i l o t u s over seven days ........ 67 IV. Degradation of T y r o s i n e - 1 * ^ and DOPA-^C by Ruta and M e l i l o t u s over seven days. - i x -L i s t of f i g u r e s F i g u r e Page Catechol Biogenesis i n G a u l t h e r i a 1.. Proposed pathways f o r the formation of catechol i n G-aultheria 12 The Biogenesis of Rosmarinic A c i d i n Mentha 1 . Hydroxycinnamic a c i d s i n p l a n t phenolic metabolism 20 2 . Proposed phenylpropanoid metabolism l e a d i n g to l i g n i n s 23 3 . Rosmarinic a c i d 25 k. Proposed pathway f o r the biogenesis of rosmarinic a c i d i n Mentha 36 5 . Changes i n the s p e c i f i c a c t i v i t y of rosmarinic a c i d i n Mentha a f t e r a two hour exposure to 1^'C02 • • • 38 6 . Changes i n the s p e c i f i c a c t i v i t y of rosmarinic a c i d and the t o t a l a c t i v i t y of i n s o l u b l e e s t e r s i n Mentha a f t e r a pulse-feeding of phenylalanine-^-^C f o r (a) 2 hr and (b) 0 . 5 hr 39 Degradation of Aromatic Compounds by S t e r i l e P l a n t Tissues 1 . Feeding f l a s k . CO2 regeneration system ... 60 -X-Acknowledgement To Dr. G.H.N. Towers, under whose s u p e r v i s i o n t h i s work was c a r r i e d out, goes my deepest a p p r e c i a t i o n f o r h i s guidance and constant encouragement. He has been generous w i t h h i s time as w e l l as h i s research f a c i l i t i e s . The f a c u l t y , s t a f f and students of the Department of Botany have been h e l p f u l i n many ways. I am p a r t i c u l a r l y g r a t e f u l to Dr. B.A. Bohm f o r h e l p f u l d i s c u s s i o n s and ad-v i c e i n the course of t h i s work, and to Dr. C.O. Person f o r the frequent use of h i s s t e r i l e t r a n s f e r f a c i l i t i e s . The f i n a n c i a l a s s i s t a n c e of the Na t i o n a l Research Council of Canada and of the Department of Botany i s g r a t e f u l l y acknowledged. F i n a l l y , I am< very g r a t e f u l f o r the encouragement and as s i s t a n c e of my w i f e , Margaret, both i n the l a b and i n the p r e p a r a t i o n of t h i s manuscript. - x i -Preface Phenolic compounds occur u n i v e r s a l l y i n the p l a n t k i n g -dom. They i n c l u d e a vast v a r i e t y of s t r u c t u r e s ranging from phenol i t s e l f to the h i g h l y polymerized l i g n i n s , and new compounds are being discovered each year as more p l a n t species are examined chemically. More r e c e n t l y the focus of i n t e r e s t has s h i f t e d from the s t r u c t u r a l i d e n t i f i c a t i o n of these compounds to t h e i r biogenesis and metabolic r o l e i n p l a n t t i s s u e s . The l i n k between g l y c o l y t i c metabolism and the aromatic compounds has been demonstrated and the general p a t t e r n of metabolic r e l a t i o n s h i p s has been r e -vealed i n some groups of phenolics such as the f l a v o n o i d s , coumarlns, cinnamic and benzoic a c i d s and the aromatic amino a c i d s . Even the c o m p l e x i t i e s of l i g n i n formation are being g r a d u a l l y u n r a v e l l e d . Other groups of compounds, however, have received l e s s a t t e n t i o n and many questions have remained unanswered i n the metabolism of the simple phenols, the g l y c o s i d e s and the phenolic e s t e r s . L i t t l e s e r i o u s a t t e n t i o n has been paid to the r o l e and extent of aromatic r i n g cleavage i n pl a n t t i s s u e s . As "secondary met a b o l i t e s " , phenolics have g e n e r a l l y been considered to be i s o l a t e d from o x i d a t i v e metabolic pathways and yet considerable carbon and energy i s t i e d up i n these compounds f o r which no r o l e has yet been demonstrated i n many cases. Presumably i t would be an advantage to the p l a n t to be able to o x i d i z e the arom-a t i c compounds as microorganisms do w i t h such ease. Work was begun on some of these questions and ev e n t u a l l y - x i i -three problems proved to be p a r t i c u l a r l y i n t e r e s t i n g and were st u d i e d more or l e s s simultaneously. This l i m i t e d the extent of the area of study i n each t o p i c but the i n -a b i l i t y to c a r r y out experiments on p e r i p h e r a l points of i n t e r e s t has been o f f s e t to some extent by the valuable opportunity to become f a m i l i a r w i t h the s t a t e of knowledge and the techniques i n a number of areas i n phenolic b i o -chemistry. Catechol Biogenesis i n G a u l t h e r i a -1-INTRODUCTION The simple phenols (hydroxy and methoxy-benzene com-pounds) seem r e l a t i v e l y l i m i t e d i n t h e i r d i s t r i b u t i o n i n 1 2 the p l a n t kingdom ' . Hydroquinone, 1,4-dihydroxybenzene, occurs i n a number of Ericaceae"*", and i n t o t a l l y u n r e l a t e d 3 genera such as Pyrus and Bergenia . Catechol, 1,2-dl-hydroxybenzene, has been reported i n A l l i u m ^ , i n Psoro-1 6 7 6 8 9 spermum , i n Populus ' and S a l i x ' and i n G a u l t h e r i a . The other simple phenols are even more l i m i t e d i n t h e i r reported d i s t r i b u t i o n . Catechol i s of p a r t i c u l a r i n t e r e s t i n phenolic meta-bolism because of i t s frequent occurrence as a s h o r t l i v e d 10 intermediate i n ring-cleavage metabolism i n microorganisms In order to o x i d i z e the aromatic r i n g , these organisms u s u a l l y r e q u i r e the presence of orthohydroxyl groups on the compound and t h i s o f t e n r e s u l t s i n higher molecular weight aromatic compounds being hydroxylated and degraded to c a t e c h o l . I t does not normally accumulate, however, "but i s o x i d i z e d to a l i p h a t i c a c i d s and f u r t h e r metabol-l z e d Catechol occurs most prominently i n higher p l a n t s i n 6,7 6,8 G a u l t h e r i a and the S a l i c a c e a e . In Populus and S a l i x i t i s demonstrably present i n small concentrations i n the unhydrolyzed e x t r a c t s of leaves and has been i s o l a t e d from Populus bark"*""1". There are c o n f l i c t i n g r e p o r t s of i t s 8 occurrence i n a bound form i n the p l a n t but to date there i s no r e p o r t of the i s o l a t i o n of a bound form of c a t e c h o l -2-from the Sa l i c a c e a e and ca t e c h o l monoglycosides (the most l i k e l y form of such a compound) are absent from chromato-12 grams of Populus l e a f e x t r a c t s . In c o n t r a s t , c a t echol occurs i n three G a u l t h e r i a spp. i n s u b s t a n t i a l q u a n t i t i e s as the beta-D-glucoside w i t h l i t t l e or no f r e e c a t echol 9 de t e c t a b l e i n the e x t r a c t s . When t h i s study was undertaken the only work on simple phenol biogenesis i n higher p l a n t s had been c a r r i e d out on hydroquinone. I t s beta-D-glucoside, a r b u t i n , occurs i n Pyrus where G r i s d a l e and Towers determined that the hydro-quinone moiety was formed from phenylpropanoid precur-13 sors . This was l a t e r extended by Zenk, who showed that para-hydroxybenzoic a c i d was o x i d a t i v e l y decarboxylated to In-form hydroquinone i n Bergenia The analogous r e a c t i o n could be expected to r e s u l t i n conversion of s a l i c y l i c a c i d to c a t e c h o l , but a number of other i n t e r e s t i n g p o s s i b i l i t i e s e x i s t . These i n c l u d e non-oxidative d e c a r b o x y l a t i o n of 2,3-dihydroxybenzoic 15 , 16 a c i d or 3 .4-dihydroxybenzoic a c i d , and a complex con-v e r s i o n of a n t h r a n i l i c a c i d to catechol e i t h e r v i a 2 , 3 -1? 18 dihydroxybenzoic a c i d or ortho-amino phenol . The l a t t e r r e a c t i o n has been demonstrated i n v i t r o w i t h an 18, enzyme system from Tecoma stans (Bignoneaceae) but no 19 catechol could be detected i n e x t r a c t s of Tecoma and i t was not shown to be a normal metabolite of the p l a n t . The other r e a c t i o n s have only been demonstrated i n micro-organisms, which have a l s o been found to form catechol by 20 hu d r o x y l a t i o n of phenol and by o x i d a t i v e d e c a r b o x y l a t i o n of s a l i c y l i c a c i d . The l a t t e r r e a c t i o n has been s t u d i e d 21 i n v i t r o w i t h a p a r t i a l l y p u r i f i e d enzyme system In view of the s c a r c i t y of i n f o r m a t i o n regarding sample phenol formation iin p l a n t s , and the p o t e n t i a l r o l e of c a t -echol as a ring-cleavag:e s u b s t r a t e , the biogenesis of c a t -echol i n higher p l a n t t i s s u e s was considered a worthwhile s tudy. MATERIALS AND METHODS Pl a n t m a t e r i a l . Populus leaves were obtained from trees growing on or near the campus of the U n i v e r s i t y of B r i t i s h Columbia, Vancouver. G a u l t h e r i a adenothrlx (Miq.) Mich, was grown i n f l a t s i n the departmental greenhouse under d a y l i g h t supplemented w i t h s i x t e e n hour-day f l u o r e s c e n t l i g h t i n g . Young leaves were used when approximately two-thirds mature s i z e and s t i l l l i g h t green i n c o l o u r . G. o v a t i -f o l i a Gray was c o l l e c t e d near A l t a Lake, B r i t i s h Columbia, i n May. Rad i o a c t i v e compounds. 14 i 4 Benzoic a c i d - r l n g - 1 - C, tryptophan-benzene ring-U- C Inl-and phenylalanine-U- C were purchased from the Radio-3 chemical Centre, Amersham, England, tryptophan-GL- H from 14 ICN, C a l i f o r n i a , s a l i c y l i c a c i d - 7 - C from New England Nuclear, Boston, Massachusetts and s a l i c y l i c a c i d - r i n g -14 U- C from M a l l i n c k r o d t Nuclear Corporation, Orlando, F l o r i d a . T r i t i a t i o n of the r i n g proton p o s i t i o n s of 2,3-dihydroxybenzoic a c i d and 4-hydroxybenzoic a c i d was c a r r i e d out by r e f l u x i n g 1 mM of the compound i n 10 ml of 3 3 CF3COO H (prepared from H2O and t r i f l u o r o a c e t i c anhyd-r i d e ) w i t h 10 mg of Pd on charcoal f o r f o r t y - e i g h t hours. L a b i l e t r i t i u m was removed by successive s o l u t i o n and evaporation and the compound twice c r y s t a l l i z e d from water. The 2,3-d.ihydroxybenzoic a c i d and the 4-hydroxy-benzoic a c i d had s p e c i f i c a c t i v i t i e s of 266 p.C/mM and.752 p-C/mM, r e s p e c t i v e l y . A l l the ac i d s were administered as t h e i r ammonium s a l t s i n aqueous s o l u t i o n . Ten 1.2 cm d i s c s cut from washed leaves were i n f i l t r a t e d w i t h the r a d i o a c t i v e s o l u t i o n (5 ml) usi n g water a s p i r a t o r vacuum. The d i s c s were then f l o a t e d on the r e s i d u a l s o l u t i o n i n small P e t r i dishes under constant i l l u m i n a t i o n (9000 lux) a t 20° f o r twenty-four hours. I s o l a t i o n of cat e c h o l beta-D-glucoside and c a t e c h o l . The r i n s e d d i s c s were ext r a c t e d w i t h hot 95$ ethanol u n t i l c o l o u r l e s s . The solvent was evaporated and the r e s -idue e x t r a c t e d w i t h hot water followed by f i l t r a t i o n through C e l i t e . The f i l t r a t e was concentrated to a few m i l l i l i t e r s and run on to a small column of A v i c e l (micro-c r y s t a l l i n e c e l l u l o s e ) which was then eluted w i t h 2% formic a c i d . The f r a c t i o n s c o n t a i n i n g catechol g l u c o s i d e (ab-sorbing s t r o n g l y a t 278 nm) were taken to dryness and p u r i f i e d by p r e p a r a t i v e t h i n - l a y e r chromatography on 1 mm A v i c e l l a y e r s , using as solvents n-butanol:acetic acid:water (4:1; 2. 2), n-butanol:pyridine:water ( 7 5 ' . 15'. 10) and e t h y l acetate:formic acidswater (75:10:10). The catechol gluco-side was l o c a t e d by spraying a s t r i p of the p l a t e w i t h o d i a z o t i z e d p a r a - n i t r o a n i l i n e reagent and NaOH . (catech o l glucoside-magenta; c a t e c h o l - b l u e ) . The gl u c o s i d e was then - 6 -d i s s o l v e d i n 2 ml water and incubated w i t h emulsin (beta-glucosidase) f o r twenty-four hours. The hydrolysate was banded on A v i c e l p l a t e s and developed i n benzeneJacetic acid:water ( 1 0 : 7 : 3 - o r g a n i c phase). The catechol band was eluted and the c o n c e n t r a t i o n measured spectrophotometri-c a l l y a t 276 nm. The r a d i o a c t i v i t y was determined by l i q u i d s c i n t i l l a t i o n counting. Further chromatography d i d not a l t e r the s p e c i f i c a c t i v i t y . S a l i c y l a t e hydroxylase assay. 2 - 5 gm of young G. adenothrix leaves were washed s u c c e s s i v e l y w i t h d i s t i l l e d water, 0 . 0 5 $ Tween 8 0 , 70% aqueous ethanol and d i s t i l l e d water ( t w i c e ) . They were then b l o t t e d dry and ground w i t h twice t h e i r weight of P o l y c l a r AT ( p o l y v i n y l p y r r o l i d o n e ) and 1 - 2 gm of a c i d -washed sea sand, wetting the mixture w i t h s u f f i c i e n t 0 . 0 5 M phosphate b u f f e r (pH 7 ) to make a t h i c k s l u r r y . The s l u r r y was squeezed through f i v e thicknesses of cheese-c l o t h and c e n t r i f u g e d a t 1 0 , 0 0 0 g f o r ten minutes. The c l e a r supernatant (app. 20 ml) was used as enzyme s o l -u t i o n . The i n c u b a t i o n mixture c o n s i s t e d of 9 ml of the super-natant to which were added 1 5 NADH2» 2 . 5 ^ uM FAD, 5 ^ Ik NADPH2 and 1 . 3 /iM s a l i c y l i c a c i d - 7 - C (3/*C) f o r a f i n a l volume of 10 ml. The i n c u b a t i o n was c a r r i e d out a t room temperature i n a 1 2 5 ml side-arm f l a s k equipped f o r a flow-through of a i r . A slow stream of a i r was passed through the f l a s k during i n c u b a t i o n (2 hours), the r e -a c t i o n mixture a c i d i f i e d w i t h 2 ml cone. HG1 and a r a p i d a i r s t r e a m (app. 100 ml/min.) passed through the f l a s k f o r one-half hour. The C0£ was trapped i n 10 ml 2-phenyl-ethylamine, which was subsequently counted by l i q u i d scin^ t i l l a t i o n counting. The c o n t r o l was t r e a t e d i d e n t i c a l l y except that the supernatant was held a t 100° f o r 20 min-utes p r i o r to being added to the i n c u b a t i o n mixture. -8-RESULTS AND DISCUSSION Populus. The presence of catechol i n unhydrolyzed e x t r a c t s of the leaves of l o c a l species of Populus could be demon-s t r a t e d chromatographically, although the l e v e l s were low. There was no s i g n of c a t e c h o l monoglycosides i n the ex-t r a c t s though, and no g l y c o s i d e f r a c t i o n could be shown to c o n s i s t e n t l y y i e l d c a t e c h o l upon h y d r o l y s i s . Tomaszew-s k i has found a s i m i l a r s i t u a t i o n i n Populus w l l s o n i i , P. 22 tremula and two S a l i x spp . Vazquez et a l found f r e e c a t e c h o l i n S a l i x v i m i n a l i s but they maintain that a bound g form i s a l s o present . P e a r l and D a r l i n g i s o l a t e d cate-11 7 c h o l from e x t r a c t s of P. balsamifera bark and leaves . In the extensive s t u d i e s of the phenolic g l y c o s i d e s of the 23 7 S a l i c a c e a e by Thieme ^ and P e a r l and D a r l i n g , they have never reported a c a t e c h o l g l y c o s i d e or an i n d i c a t i o n of one from h y d r o l y s i s products. I f , however, Populus l e a f d i s c s are exposed to a d i l u t e s o l u t i o n of c a t e c h o l they form a compound chromatograph-12 i c a l l y i d e n t i c a l w i t h catechol-beta-D-glucoside I t would appear, then, that the c a t e c h o l found i n the S a l i c a c e a e i s to a l a r g e extent present i n the f r e e form and must be compartmentalized i n some way w i t h i n the t i s s u e s . E f f o r t s to demonstrate i n c o r p o r a t i o n of l a b e l from aromatic precursors i n t o the c atechol i n P. t r i c h o -carpa leaves of a l l ages were u n s u c c e s s f u l . S i m i l a r d i f f -- 9 -i c u l t i e s have been encountered by Zenk . The reason may be that synthesis i s t a k i n g place elsewhere i n the tree followed by t r a n s l o c a t i o n to the lea v e s , or that some un-suspected b i o s y n t h e t i c pathway i s i n v o l v e d . G a u l t h e r i a . Catechol i s the most prominent phenolic compound i n hydrolyzed e x t r a c t s of G a u l t h e r i a adenothrix (Miq.) Mich. In the o r i g i n a l r e p o r t of the occurrence of catechol-beta-D-glucoside i n G a u l t h e r i a spp. i t was suggested that i t might a r i s e from e i t h e r 2,3-dihydroxybenzoic or 3,4-dihydroxybenzoic a c i d , both of which occur i n G. adenoth-9 r i x . On the b a s i s of the b i o s y n t h e t i c s t u d i e s on hydro-14 quinone , however, the d e c a r b o x y l a t i o n of s a l i c y l i c a c i d seemed a more l i k e l y p o s s i b i l i t y d e s p i t e the apparent ab-o sence of t h i s a c i d from the e x t r a c t s . As can be seen from Table I , s a l i c y l i c a c i d was i n c o r -porated i n t o catechol w i t h l e s s d i l u t i o n of s p e c i f i c ac-t i v i t y than any other compound t r i e d . Both l a b e l l e d ben-z o i c a c i d and phenylalanine served as precursors to cate-chol as w e l l . Phenylalanine i s r e a d i l y converted to cinnamic a c i d i n p l a n t s , and the l a t t e r can undergo beta-o x i d a t i o n to y i e l d benzoic a c i d " ^ . Benzoic a c i d has been shown to be a good precursor to s a l i c y l i c a c i d i n higher 25 p l a n t s . The pathway i n v o l v e d may the r e f o r e be phenyl-alanine—¥ cinnamic a c i d — > benzoic a c i d — > s a l i c y l i c a c i d — > c a t e c h o l . The l a r g e v a r i a t i o n s i n i n c o r p o r a t i o n of -10-benzoic a c i d ( f i v e r e p l i c a t e s ) may be an i n d i c a t i o n that the conversion cinnamic a c i d — > ortho-coumaric a c i d > 26 s a l i c y l i c a c i d plays a dominant r o l e a t times (Figure 1.). The age of the l e a f t i s s u e determined the extent of conversion of s a l i c y l i c a c i d to c a t e c h o l , young leaves possessing a c t i v i t y a t l e a s t ten times that of mature le a v e s . A l l reported work was c a r r i e d out on leaves l e s s than 2 weeks o l d . A s i m i l a r e f f e c t of age has been noted p r e v i o u s l y i n i n v i v o s t u d i e s on h y d r o x y l a t i o n of benzoic 25 13 a c i d s and formation of hydroquinone . In one experiment 14 s a l i c y l i c a c i d - r i n g - U -C was administered to l e a f d i s c s of G. o v a t l f o l i a , one of the other two species of Gaulther-i a reported to c o n t a i n c a t e c h o l . L a b e l l i n g of the cate-chol was again observed (Table I.) the gr e a t e r d i l u t i o n probably being due to the maturity of the leaves obtained. The p o s s i b i l i t y that d e c a r b o x y l a t i o n of 2,3-dihydroxy-benzoic a c i d or 3,4-dihydroxybenzoic a c i d i s i n v o l v e d i n cat e c h o l b i o s y n t h e s i s was examined. The former compound (three r e p l i c a t e s ) and 4-hydroxybenzoic a c i d (three rep-30 l i c a t e s ) , a l i k e l y precursor of the l a t t e r were fed to l e a f d i s c s ; i n n e i t h e r case was any l a b e l detected i n c a t e c h o l . S a l i c y l i c a c i d i s converted i n p a r t to 2,3-dihydroxybenzoic a c i d , however, s i n c e a f t e r feeding 20 ^iG 3 of s a l i c y l i c GL- H (sp. a c t . 238 mC/mM) f o r 24 hours, 9.3 x 10"^ dpm were found i n catechol (sp. a c t . 475 yuC/mM) and 1.34 x 10^ dpm were found i n 2,3-dihydroxybenzoic a c i d . -11-Table I . The I n c o r p o r a t i o n of Ra d i o a c t i v e Precursors i n t o Catechol i n Leaf Discs of G a u l t h e r i a adenothrix a f t e r 24 hr. Compound fed A c t i v i t y fed (jxC) A c t i v i t y taken up (/AC) S p e c i f i c a c t i v i t y of c a techol (/iC/mM) D i l u t i o n i 4 Phenylalanine-U- C, 495 ^C/uM 2 1.53 2.06 2.4x l 0 5 1 4 Benzoic a c i d - r i n g - 1 - C, 2.54 /iC//iM 2 1.80 3.48 730 14 Benzoic a c i d - r i n g - 1 - C, 2.54 ;uC//iM 2 1.94 0.12 2100 S a l i c y l i c a c i d - r i n g -UL- C, 0.95 JuC/uM 2 1.68 5.88 161 S a l i c y l i c a c i d - r i n g -U L - 1 ^ , 0.95 pC/jM 2* 1.26 0.24 3890 S a l i c y l i c a c i d - r i n g -U L - 1 ^ , 0.95 }xQ/jm 2** 1.96 5.90 161 1.83 0.15 6330 2,3-Dihydroxybenzoic acid-3H, 0.27 pC/pM 0.114 0.10 n i l 4-Hydroxybenzoic acid-1 3H , 0.75 p.C/p& 1 0.80 n i l Tryptophan-benz ene-r i n g - U L - ^ C , 52 uC/uM 4 2.99 n i l • Tryptophan-GL-3H, 2.7 C/mM 50 21 0.14 1.93xl0 7 G. o v a t i f o l i a leaves ** Leaves l e s s than 2 weeks o l d *** Leaves more than 6 weeks o l d -12-Pigure 1. Proposed pathways f o r the formation of cate c h o l i n G a u l t h e r i a COOH COOH CINNAMIC ACID PHENYLALANINE 0-COUMARIC ACID COOI-I HO" GENTISIC ACID COOH COOH BENZOIC ACID OH SALICYLIC ACID \ OH COOH OH OH H CATECHOL 0-PYROCATECHUIC ACID -13-Tryptophan degradation by A s p e r g i l l u s n i g e r leads to 17 catechol ' but there was no c l e a r evidence f o r t h i s i n G a u l t h e r i a . The small amount of a c t i v i t y i n catechol a f t e r feeding 50 yuC of tryptophan-GL--% may have been r e -cyc l e d through the shikimate pathway from l o s s of the tryptophan s i d e - c h a i n . The p o s s i b i l i t y of a small amount of degradation to the aromatic nucleus, however, cannot be excluded. U n l i k e c a t e c h o l , s a l i c y l i c a c i d i s q u i t e common i n G a u l t h e r i a , appearing i n some species i n r e l a t i v e l y l a r g e o q u a n t i t i e s as a g l y c o s i d e of methyl s a l i c y l a t e . The sub-group, Amblyandra, however, would seem to have evolved a system f o r converting t h i s s a l i c y l i c a c i d to cat e c h o l which i s then stored as i t s beta-glucoside. I t i s i n t e r -e s t i n g to note that an a c i d which i s apparently absent from e x t r a c t s of the p l a n t i s a c t u a l l y a c t i v e l y i n v o l v e d i n i t s phenolic metabolism and hence does not accumulate i n the t i s s u e s . The formation of a number of methoxyl-27 ated phenols i n higher p l a n t s has r e c e n t l y been reported In each case the corresponding benzoic a c i d was o x i d a t i v e -l y decarboxylated. This r e s u l t confirms the p a t t e r n of simple phenol formation i n higher p l a n t s . An attempt was made to detect s a l i c y l a t e decarboxyl-a t i n g a c t i v i t y i n an i n v i t r o system. By usi n g the super-natant from buffered e x t r a c t s of the very youngest leaves 14 14 and e i t h e r r i n g - C or c a r b o x y l - C s a l i c y l i c a c i d as sub-s t r a t e , the r e a c t i o n could be detected. Using the more -In-s e n s i t i v e CO2 trapping assay, approximately 0 . 2 5 $ of the l a b e l i n the sup p l i e d substrate could be detected i n the CO? trap a f t e r 2 hours (Table I I . ) . Table I I . 14 Decarboxylation of S a l i c y l i c A c i d - 7 - C by Crude E x t r a c t of G. adenothrix Leaves t o t a l dpm i n trap % of substrate fed supernatant 17,600 0.26 c o n t r o l 580 This i s comparable to the r a t e of deca r b o x y l a t i o n r e -ported f o r the s e m i - p u r i f i e d s a l i c y l a t e hydroxylase i s o -21 l a t e d by K a t a g i r i et a l from a s o i l pseudomonad . Fur-ther s t u d i e s of the a c t u a l c o f a c t o r requirements of the G a u l t h e r i a system have not been p o s s i b l e due to the l a c k of adequate s u p p l i e s of young t i s s u e . The use of Pyrus or Bergenia t i s s u e s as sources of enzyme might be an e a s i e r approach to t h i s enzyme or group of enzymes i n p l a n t s . K a t a g i r i et a l d i d not examine 4-hydroxybenzoic a c i d as a subs t r a t e f o r t h e i r enzyme. They d i d r e p o r t , however, that w h i l e 2,3-dihydroxybenzoic a c i d was a sub-21 s t r a t e , a n i h r a n i l i c and benzoic a c i d s were not Chen st u d i e d d e c a r b o x y l a t i o n of benzoic a c i d s by ace-28 14 tone powder e x t r a c t s of higher p l a n t t i s s u e s . The CO2 was recovered from i n c u b a t i o n mixtures c o n t a i n i n g car--15-14 b o x y l - C-benzoic a c i d s but the expected phenolic prod-u c t s (assuming a non-oxidative decarboxylation) could not be detected. On the b a s i s of the work now published the r e a c t i o n products i n Chen's work could reasonably be ex-pected to be the hydroxylated forms i n s t e a d (e.g. hydroxy-hydroquinone from 3 .4-dihydroxybenzoic a c i d ) . Two simple phenols which may prove to be exceptions to t h i s p a t t e r n of formation are toluhydroquinone (2 , 5 -dihydroxytoluene) a n d ' p h l o r o g l u c i n o l ( l , 3 t 5 - t r i h y d r o x y -29 benzene). The former occurs i n q u a n t i t y i n P y r o l a spp. but has been proposed as a general intermediate i n p l a s t o -30 quinone b i o s y n t h e s i s ^ . The suggested pathway would mean that t y r o s i n e gives r i s e to toluhydroquinone v i a homogen-t i s i c a c i d (2, 5-dihydroxyphenylacetic a c i d ) . This has 24 r e c e n t l y been shown to be the case i n the Ericaceae . I t i s a l s o p o s s i b l e that where p h l o r o g l u c i n o l accumulates i t may be the product of a polyacetate c y c l i z a t i o n , e i t h e r 31 d i r e c t l y or, i n d i r e c t l y , v i a f l a v o n o i d breakdown . - 1 6 -LITERATURE CITED 1. K a r r e r , W. 1958. K o n s t l t u t i o n und Vorkommen der organischen P f l a n z e n s t o f f e . Blrkhauser-Verlag, Basel and S t u t t g a r t . 2. Pridham, J.B. 19&5• L o w molecular weight phenols i n higher p l a n t s , Ann. Rev. P l a n t Physiology 16:13. 3 . F r i e d r i c h , H. 1958. Untersuchungen uber d i e phenolischen I n h a l t s t o f f e von Pyrus communis, Die Pharmazie 13:153. 4 . F r i e d r i c h , H. 1961. Physiology and biochemistry of gl y c o s i d e s y n t h e s i s , P l a n t a med. ( S t u t t g a r t ) 9 : 4 2 5 . 5. L i n k , K.P. and Walker, J.C. 1933. The i s o l a t i o n of catechol from pigmented onion s c a l e s and i t s s i g n i f i c a n c e i n r e l a t i o n to disease r e s i s t a n c e i n onions. J . B i o l . Chem. 1 0 0 ; 3 7 9 . 6. Tomaszewskl, M. i 9 6 0 . Occurrence of p_-hydroxybenzoic a c i d and some other phenols i n v a s c u l a r p l a n t s , B u l l . Acad. Polon. S c i . 8 j 6 l . 7. P e a r l , I.A. and D a r l i n g , S.F. 1968. Studies on the leaves of the f a m i l y S a l i c a c e a e XI. The hot water e x t r a c t i v e s of the leaves of Populus  b a l s a m i f e r a , Phytochem. 7:1845. 8. Vazquez, A., Mendez, J . , Gesto, M.D.V., Seoane, E. and V i e i t e z , E. 1968. Growth substances i s o l a t e d from woody c u t t i n g s of S a l i x v i m i n a l i s L. and Fi c u s c a r i c a L., Phytochem. 7:161. 9. Towers, G.H.N., Tse, Aida and Maass, W.S.G. 1966. Phenolic a c i d s and phenolic g l y c o s i d e s of G a u l t h e r i a s p e c i e s , Phytochem. 5 :677. 10. Towers, G.H.N. 1964. Metabolism of phenolics i n higher p l a n t s and microorganisms, p. 249, Biochem-i s t r y of Phenolic Compounds, ed. J.B. Harborne, Academic Press, London and New York. 11. P e a r l , I.A. and D a r l i n g , S.F. 1968. Studies on the barks of the f a m i l y S a l i c a c e a e XIX. Continued s t u d i e s on the hot water e x t r a c t i v e s of Populus  balsamifera bark, Phytochem. 7:1851. 12. E l l i s , B.E. 1968. Unpublished observations. -17-13. G r i s d a l e , S.K. and Towers, G.H.N. I960. Biosyn-t h e s i s of a r b u t i n from some phenylpropanoid compounds i n Pyrus communism Nature 188:1130. 14. Zenk, M.H. 1964. Einbau von p_-hydroxybenzoesaure i n d i e Hydrochinonkomponente des Ar b u t i n s i n Bergenia c r a s s i f o l l a , Z. Naturforsch, 196s856. 15. Subba Rao, P.V., Moore, K. and Towers, G.H.N. 1967. o-Pyrocatechuic a c i d carboxy-lyase from A s p e r g i l l u s n i g e r , Arch. Biochem. Biophys. 122:466. 16. Cain, R.B., B i l t o n , R.F". and Darrah, J.A. 1968. The metabolism of aromatic compounds by micro-organisms. Metabolic pathways i n the f u n g i , Biochem. J . 1 0 8 : 7 9 7 . 17. Subba Rao, P.V., Moore, K. and Towers, G.H.N. 1967. The conversion of tryptophan to 2 , 3-dihydrox-benzoic a c i d and cat e c h o l by A s p e r g i l l u s n i g e r , Biochem. Biophys. Res. Commun. 28:1008. 18. N a i r , P.M. and Vaidyanathan, C.S. 1966. Conversion of isophenoxazine to catechol i n Tecoma stans, Arch. Biochem. Biophys. 115:515* 19. Subba Rao, P.V. Personal communication. 20. Evans, W.C. 1947. Oxidation of phenol and benzoic a c i d by some s o i l b a c t e r i a , Biochem. J . 41 :373. 21. K a t a g i r i , M., Yamamoto, S., and H a y a i s h i , 0 . 1962. F l a v i n adenine d i n u c l e o t i d e requirement f o r the enzymic h y d r o x y l a t i o n and deca r b o x y l a t i o n of s a l i c y l i c a c i d , J . B i o l . Chem. 237sPC 2413. 22. Tomaszewski, M. Personal communication. 23. Thieme, H. and R i c h t e r , R. I 9 6 6 . I s o l i e r u n g eines neuen Phenolglykosids aus Populus tremula L., Die Pharmazie 21 1 2 5 1 . 24. Zenk, M.H. Personal communication. 25. el-Basyounl, S.Z., Chen, D., Ibrahim, R.K., Neish, A.C. and Towers, G.H.N. 1964. The bi o s y n t h e s i s of hydroxybenzoic a c i d s i n higher p l a n t s , Phyto-chem. 3 : 4 8 5 . 26. Volmer, K . 0 . and Grisebach, H. 1966. Zur Biosynthese der Benzoesauren i n G a u l t h e r i a procumbens I I I , Z. Naturforsch. 216:435. -18-27. B o l k a r t , K.H. and Zenk, M.H. 1968. Zur Biosynthese m e t h o x y l i e r t e r Phenole i n hoheren Pflanzen, Z. f u r P f l a n z e n p h y s i o l o g i e 59'^ 39 28. Chen, D.C.T. 1963. M.Sc. Thesis, Dalhousie Univ., H a l i f a x , N.S. 29. Inouye, H. 1956. Uber d i e B e s t a n d t e i l e der P i r o l -aceae-Pflanzen. V I . Uber d i e B e s t a n d t e i l e von P i r o l a i n c a r n a t a F i s c h . Pharmaceutical B u l l . Japan 4:281. 30. Whistance, G.R. and T h r e l f a l l , D.R. 1968. B i o -synthesis of Phytoquinones. B i o s y n t h e t i c or-i g i n s of the n u c l e i and s a t e l l i t e methyl groups of plastoquinone, tocopherols, and tocopherol-quinones i n maize shoots, bean shoots and i v y leaves, Biochem. J . 109:577. 31. Patschke, V.L., Hess, D. and Grisebach, H. 1964. Uber den Abbau von 4 ,2 ' ,4* ,6'-Tetrahydroxy-chalkon - 2-glucosid und 4 ,2 ' ,4'-Trihydroxychalkon-4-glucosid i n Rotkohlkeimlingen und Petunien, Z. Naturforsch. 19b:1114. The Biogenesis of Rosmarinic A c i d i n Mentha -19-INTRODUCTION The hydroxylated and methoxylated d e r i v a t i v e s of c i n n -amic a c i d form the most important and most studied pool of phenolic intermediates i n p l a n t tissues'''. From the c i n n -amic a c i d s a r i s e the coumarins, f l a v o n o i d s and anthocyanin pigments, the l i g n i n s , the benzoic a c i d s and hence most simple phenols (Figure 1.). P l a n t s form these aromatic compounds almost e x c l u s i v e l y from the amino a c i d s phenyl-2 a l a n i n e and t y r o s i n e , e s p e c i a l l y the former . The a b i l i t y to deaminate phenylalanine to cinnamic a c i d seems to be u n i v e r s a l i n higher p l a n t s but the corresponding a c t i v i t y toward t y r o s i n e to form para-coumaric a c i d (4-hydroxycinn-amic) i s g e n e r a l l y low outside the Gramineae and e f f e c t -3 i v e l y absent i n many groups of p l a n t s . Tyrosine i s hydroxylated i n some p l a n t s to form 3,4-4 dihydroxyphenylalanine (DOPA) . A few p l a n t s have y i e l d e d acetone powders which w i l l deaminate DOPA to c a f f e i c 3»5 a c i d ' (3,4-dihydroxycinnamic) but whether t h i s r e a c t i o n i s of any s i g n i f i c a n c e i n v i v o i s unknown. The reported d i s t r i b u t i o n of DOPA i n the p l a n t kingdom i s l i m i t e d and so f a r i t i s known to be a precursor to some a l k a l o i d s 8 and to the betacyanin pigments . P h e n y l l a c t i c and phenylpyruvic a c i d s are m e t a b o l i c a l -l y a c t i v e i n p l a n t s and have been demonstrated to g i v e 9 r i s e to cinnamic a c i d products such as l i g n i n . There has been no r e p o r t , however, of a d i r e c t dehydration of -20-Fig u r e 1. Hydroxycinnamic a c i d s i n p l a n t phenolic metabolism £22 shikimate pathway simple phenols -21-p h e n y l a c t i c a c i d to cinnamic a c i d w i t h an enzyme system from p l a n t s . With the dis c o v e r y of phenylalanine ammonia-l y a s e i t has g e n e r a l l y been assumed that phenylalanine, p h e n y l l a c t i c and phenylpyruvic a c i d s form a r e a d i l y i n -terchanging pool'*""1' from which phenylalanine provides the main, or s o l e , entrance to the cinnamic a c i d s . The same s i t u a t i o n "occurs w i t h respect to t y r o s i n e , p_-hydroxy-phenylpyruvic and p_-hydroxyphenyllactic a c i d s . In p l a n t s l a c k i n g a p p r e c i a b l e t y r o s i n e ammonia-lyase a c t i v i t y , the d i s t r i b u t i o n of l a b e l from r a d i o a c t i v e phen-y l a l a n i n e and t y r o s i n e i s s t r i k i n g l y d i f f e r e n t w i t h i n the non-nitrogenous m e t a b o l i t e s . Label from phenylalanine i s normally incorporated predominantly i n t o the phenolic compounds w h i l e very l i t t l e l a b e l from t y r o s i n e appears 12 i n these compounds . The l a b e l from t y r o s i n e i s spread throughout the organic a c i d s and carbohydrates, i n d i c -a t i n g extensive degradation of the phenylpropanoid s t r u c -13 -ture, p o s s i b l y i n c l u d i n g aromatic r i n g cleavage . A l -though an enzyme system i s o l a t e d from spinach w i l l hy-14 dr o x y l a t e phenylalanine to form t y r o s i n e , there has been no i n d i c a t i o n from i n v i v o t r a c e r s t u d i e s that t h i s takes place to any extent i n the i n t a c t plant 1''. The f a t e s of these two amino a c i d s are, then, l a r g e l y d i s t i n c t except when t y r o s i n e or DOPA ammonia-lyase a c t i v i t y i s present. The cinnamic a c i d s o r d i n a r i l y do not occur f r e e i n higher p l a n t t i s s u e s i n more than t r a c e q u a n t i t i e s . I n-- 2 2 -16 stead, they are found as g l y c o s i d e s , or e s t e r i f i e d w i t h the hydroxyls of glucose, glucosides and a v a r i e t y of 17 a l i p h a t i c a c i d s (Table ! . ) • Table I . E s t e r i f i e d Forms of C a f f e i c A c i d i n P l a n t s . E s t e r D i s t r i b u t i o n c a f f e o y l - q u i n i c a c i d (mono-and d i c a f f e o y l forms) s h i k i m i c a c i d t a r t a r i c a c i d (mono-and d i c a f f e o y l forms) malic a c i d glucose glucosamine glucose as glu c o s i d e s 3,4-dihydroxyphenyl-l a c t i c a c i d "e t h a n o l - i ns o l u b l e ' compounds widespread 20 prob. widespread 21,22 23 24 V i t i s , Chlcorium Phaseolus 25 widespread 26 17 N i c o t i a n a 27,28 widespread many Labiateae, Boraginaceae and others ' widespread?^ 0 They are a l s o found e s t e r i f i e d w i t h some "ethanol-i n s o l u b l e " component(s) of the c e l l which has not been completely c h a r a c t e r i z e d yet but i s b e l i e v e d to be a poly-peptide or p r o t e i n . -22a-C a f f e i c a c i d i s the most widely accumulated hydroxy-cinnamic a c i d i n the p l a n t kingdom; i n one survey i t was i d e n t i f i e d i n 80$ of the 122 species examined 1^. C a f f e i c a c i d g l y c o s i d e s have been reported much l e s s f r e q u e n t l y than c a f f e o y l e s t e r s , chlorogenic a c i d ( 3 - 0 - c a f f e o l -20 q u i n i c a c i d ) being p a r t i c u l a r l y widespread , The pos-i t i o n a l isomers of mono- and d i c a f f e o y l q u i n i c a c i d are not e a s i l y d i s t i n g u i s h e d chromatographically. They have only r e c e n t l y been unequivocably chemically character-i z e d ^ 1 ' - ^ 2 and i t i s l i k e l y that they w i l l be found to be widespread as w e l l 2 < ^ . C a f f e i c a c i d i s an e s t a b l i s h e d intermediate i n the pathway l e a d i n g from phenylalanine to the c o n i f e r y l and s i n a p y l a l c o h o l s which are the proposed immediate precur-sors to the l i g n i n s 1 . The extent to which the pools of e s t e r i f i e d c a f f e i c a c i d p a r t i c i p a t e l n t h i s pathway, how-ever, i s s t i l l not c l e a r . Tracer stud i e s on phenyl-12 propanoid metabolism i n S a l v i a e s t a b l i s h e d the sequence shown i n F i g u r e 2. The r e s u l t s suggested, however, that .only a small f r a c t i o n of the pool of c a f f e i c a c i d was t u r n i n g over r a p i d l y , p o s s i b l y the f r e e c a f f e i c a c i d as opposed to the bulk of the e s t e r i f i e d compound. Studies i n wheat shoots, on the other hand, have shown that the " e t h a n o l - i n s o l u b l e " cinnamic e s t e r s became l a b e l l e d much more r a p i d l y than the s o l u b l e pools and i t was suggested that these i n s o l u b l e compounds were the 18 30 most l i k e l y precursors to l i g n i n . A study of the -23-F i g u r e 2. Proposed phenylpropanoid metabolism l e a d i n g to l i g n i n s . cinnamic a c i d I p-coumaric a c i d c a f f e i c a c i d i f e r u l i c a c i d > ( c o n i f e r y l a l c o h o l ) ^ J^ ) l i g n i n s s i n a p i c a c i d > ( s i n a p y l a l c o h o l ) response of phenolic metabolism i n wheat to r u s t i n f e c t i o n confirmed the e a r l i e r r e s u l t s i n healthy wheat shoots. S u s c e p t i b l e - r e a c t i n g p l a n t s , however, d i f f e r e d i n showing more a c t i v i t y i n the s o l u b l e esters than the i n s o l u b l e 33 pool when fed l a b e l l e d phenylalanine^ . More r e c e n t l y , work on the b i o s y n t h e s i s of s c o p o l e t i n i n tobacco t i s s u e c u l t u r e s u s i n g an elegant d o u b l e - l a b e l l i n g technique has demonstrated that i n those c u l t u r e s the trace q u a n t i t i e s of f r e e cinnamic aci d s are the metabolic-a l l y most a c t i v e forms, the s o l u b l e esters accumulating l a b e l more slow l y and the i n s o l u b l e esters showing l i t t l e 21 l a b e l l i n g from phenylalanine or cinnamic a c i d . There were very low l e v e l s of i n s o l u b l e e s t e r s , however . The b i o s y n t h e t i c s t u d i e s of the s o l u b l e c a f f e o y l esters have been r e s t r i c t e d to chlorogenic a c i d . Use of t r a c e r s 33 3^ 22 3^ 38 i n a number of p l a n t s ( N i c o t i a n a ' ,Solanum 39»40 Xanthium ' has e s t a b l i s h e d that both d i r e c t e s t e r i f -i c a t i o n of c a f f e i c a c i d w i t h q u i n i c a c i d , or the form--24-a t i o n of 3-0-p_-coumaroyl q u i n i c a c i d w i t h subsequent hy-d r o x y l a t i o n of t h i s e s t e r can be i n v o l v e d , Recent s t u d i e s on chlorogenic a c i d metabolism i n 39 40 Xanthiunr *^ have shown that i n t h i s p l a n t the c a f f e o y l e s t e r s are not metabolic end-products but are t u r n i n g over, w i t h a h a l f - l i f e of about fourteen hours f o r 3-0-c a f f eoyl q u i n i c a c i d . This e s t e r appeared to be s e r v i n g as a precursor to 3 t 5 - d i - 0 - c a f f e o y l q u i n i c a c i d , and i t was suggested that the l a t t e r , and perhaps both, compounds were s e r v i n g as substrates f o r p o l y m e r i z a t i o n r e a c t i o n s which would produce l i g n i n s . The " a l c o h o l - i n s o l u b l e " es-t e r s contained comparatively l i t t l e l a b e l from phenyl-propanoid precursors but the use of aqueous methanol i n the e x t r a c t i o n procedure-^ makes i t d i f f i c u l t to compare t h i s r e s u l t w i t h the e a r l i e r work on wheat where only 80% 30 ethanol was used f o r e x t r a c t i o n of the " s o l u b l e " esters-' . I f c a f f e i c a c i d esters are r e a l l y p l a y i n g an a c t i v e metabolic r o l e i n p l a n t s i t becomes of i n t e r e s t to examine the other l e s s well-known e s t e r s . Rosmarinic a c i d i s the c a f f e o y l e s t e r of 3 , 4 - d i h y d r o x y p h e n y l l a c t i c a c i d (DOPL) e s t e r i f i e d through the alpha hydroxyl, and i s p a r t i c u l a r l y i n t e r e s t i n g because nothing i s known concerning the source of the DOPL moiety (Figure 3 . ) . This e s t e r was o r i g i n a l l y i s o l a t e d from Rosmarinus 4l o f f i c i n a l i s . I t occurs together w i t h chlorogenic a c i d i n S a n i c u l a spp. where the d i s t r i b u t i o n of the two esters has been studied q u a n t i t a t i v e l y w i t h i n the p l a n t and - 2 5 -42 w i t h i n the genus . I t s d i s t r i b u t i o n i n the p l a n t k i n g -dom has been examined chromatographically i n higher 29 43 p l a n t s 'and i n ferns . These repo r t s show rosmarinic a c i d to be widespread w i t h i n a l i m i t e d number of p l a n t f a m i l i e s , notably the Labiateae and Boraginaceae, and a t present i t i s the second most common c a f f e o y l e s t e r r e -ported i n p l a n t s . This study was undertaken to determine the biogenesis of t h i s c a f f e i c a c i d e s t e r and, i f p o s s i b l e , to see i f i t plays a r o l e s i m i l a r to that of chlorogenic a c i d i n Xanthlum. Figure 3. Rosmarinic a c i d . OH -26-MATERIALS AND METHODS Pl a n t m a t e r i a l . Rosmarinus o f f i c i n a l i s was grown on the campus of the U n i v e r s i t y of B r i t i s h Columbia. Mentha arvense was normally grown i n f l a t s i n the de-partmental greenhouses under d a y l i g h t supplemented by sixteen-hour-day f l u o r e s c e n t l i g h t i n g . M. p i p e r i t a and M. arvense used i n feeding experiments w i t h l a b e l l e d r o s -m a r i n i c a c i d were growing outdoors i n June and J u l y . Shoots were chosen which c a r r i e d 5-7 p a i r s of f u l l y ex-panded leaves but which had no flower heads. The f r e s h l y cut shoots had t h e i r ends recut under water before use. L a b e l l e d compounds and t h e i r a d m i n i s t r a t i o n . 14 14 L-Phenylalanine-U- C, DL-phenylalanine-2- C, 14 3 L - t y r o s i n e - 3 - C, L-phenylalanine-G- H and L - t y r o s i n e -3 G- H were purchased from the Radiochemical Centre, Amer-14 sham, England. Cinnamic a c i d 2- C was purchased from 14 14 ICN, C a l i f o r n i a . DL-DOPA-2- C, malonic a c i d - 2 - C and 14 barium carbonate- C were purchased from New England 14 Nuclear, Boston. The radiochemical p u r i t y of the C-amino a c i d s was checked by paper chromatography and s t r i p -14 14 scanning. p_-coumaric a c i d - 2 - C and c a f f e i c a c i d - 2 - C were prepared by the condensation of the appropriate benzalde-14 hyde w i t h malonic a c i d - 2 - C i n p y r i d i n e w i t h a t r a c e of 44 p i p e r i d i n e . The products were p u r i f i e d by c r y s t a l l i z --27-a t i o n from water, and i n the case of c a f f e i c a c i d , by TLC and l e a d acetate p r e c i p i t a t i o n as w e l l . The a c i d s were administered as t h e i r ammonium s a l t s i n 1 aqueous s o l u t i o n (0.5-1.0 ml) to 5-10 gm of mint shoots under constant i l l u m i n a t i o n (9000 l u x ) a t 22°. The s o l -u t i o n was normally completely taken up i n one-half to two hours and was fol l o w e d w i t h d i s t i l l e d water. I s o l a t i o n of rosmarinic a c i d and c a f f e i c a c i d . Rosmarinic a c i d was i s o l a t e d on a l a r g e s c a l e u s i n g the c l a s s i c a l l e a d acetate p r e c i p i t a t i o n technique employed i n 41 i t s o r i g i n a l i s o l a t i o n The e s t e r could be i s o l a t e d from 5-10 gm of mint shoots by use of the above technique on a much reduced s c a l e . A f t e r the appropriate i n c u b a t i o n period w i t h a l a b e l l e d precursor, the shoots were homogenized i n b o i l i n g 95% eth-anol and ext r a c t e d u n t i l c o l o u r l e s s . The ethanol e x t r a c t was taken to dryness, taken up i n a small volume of hot water (15 ml) and f i l t e r e d through C e l i t e . The f i l t e r cake was washed w i t h hot water and the combined f i l t r a t e s were t r e a t e d w i t h 20% (w/v) aqueous n e u t r a l lead acetate u n t i l no f u r t h e r p r e c i p i t a t e appeared. The p r e c i p i t a t e was separated by c e n t r i f u g a t i o n and washed twice w i t h d i s -t i l l e d water by resuspension and c e n t r i f u g a t i o n . The r e -suspended p r e c i p i t a t e was then decomposed by bubbling H 2S through the suspension. Decomposition was completed by heating on the steam bath and the p r e c i p i t a t e of l e a d -28-sulphide was removed by f i l t r a t i o n w h i l e s t i l l hot. The r e s u l t i n g s o l u t i o n of "catechols" or polyphenols was ex-tr a c t e d three times w i t h a small volume of ether. The ether e x t r a c t was taken to dryness, taken up i n a minimal volume of hot water and f i l t e r e d through d e c o l o u r i z i n g charcoal on a small s i n t e r e d g l a s s f u n n e l . The f i l t r a t e was stored a t 4 ° and c r y s t a l l i z a t i o n of the rosmarinic a c i d i n i t i a t e d by seeding w i t h a minute p a r t i c l e of the pure compound. The es t e r was r e c r y s t a l l i z e d from water and was s u f f i c i e n t l y pure a f t e r one r e c r y s t a l l i z a t i o n . The y i e l d of p u r i f i e d rosmarinic a c i d from one batch of shoots v a r i e d from 2 to 1 0 mg. The d i s t r i b u t i o n of l a b e l between the two moieties of the e s t e r was determined by h y d r o l y s i s and chromatographic i s o l a t i o n of the c a f f e i c a c i d by TLC on A v i c e l p l a t e s . Most hydrolyses were c a r r i e d out i n 2N NaOH under N 2 f o r 2 2 2 hr. L a t e r , i t was found that pectinase hydrolyzed the e s t e r overnight w i t h b e t t e r y i e l d s of products. The h y d r o l y s i s products were ext r a c t e d i n t o ether a f t e r a c i d -i f i c a t i o n and the e x t r a c t chromatographed on A v i c e l p l a t e s u s i n g as sol v e n t chloroform shaken w i t h 0 . 2 volume of water methanol:formic a c i d ( 7 1 : 1 2 5 : 4 ) . M u l t i p l e dev-elopment separated c a f f e i c a c i d from r e s i d u a l rosmarinic a c i d , DOPL and e s c u l e t i n (the coumarin formed by l i g h t -4 5 a c t i v a t e d c y c l i z a t i o n of c l s - c a f f e i c a c i d ). The c a f f e i c a c i d band, e a s i l y detected by i t s b r i g h t blue fluorescence under 3 6 6 nm UV l i g h t , was el u t e d w i t h 9 5 $ ethanol. - 2 9 S p e c i f i c a c t i v i t y determination. The c o n c e n t r a t i o n of rosmarinic a c i d or c a f f e i c a c i d i n 95$ ethanol s o l u t i o n was determined spectrophotometri-c a l l y a t 331 nm and 325 nm r e s p e c t i v e l y . An a l i q u o t of the same s o l u t i o n was used f o r l i q u i d s c i n t i l l a t i o n counting. Blanks f o r compounds i s o l a t e d by TLC were pre-pared by e l u t i o n of bands a t the same R^ on blank p l a t e s developed i n the app r o p r i a t e s o l v e n t . - 3 0 -RESULTS AND DISCUSSION I s o l a t i o n of rosmarinic a c i d . Attempts to o b t a i n a sample of pure rosmarinic a c i d from Mentha by p r e p a r a t i v e paper chromatography were un-s u c c e s s f u l because of the i n e v i t a b l e o x i d a t i o n of the o r t h o - d i hydroxyl s t r u c t u r e s during manipulation. The e s t e r appeared to be immobile on polyamide and charcoal columns us i n g the usual s o l v e n t s . By u s i n g the l e a d acetate p r e c i p i t a t i o n technique a p p l i e d i n the o r i g i n a l i s o l a t i o n of rosmarinic a c i d from R. o f f i c i n a l i s a white c r y s t a l l i n e s o l i d was obtained i n 0.1$ y i e l d ( f r e s h wt.) from R. o f f i c i n a l i s (reported 0.01-41 0.02$) and i n 0.2$ y i e l d from Mentha arvense. The i s o -l a t e d products were compared w i t h samples of a u t h e n t i c rosmarinic a c i d (courtesy of Dr. M.L. S c a r p a t i and Dr. K. H i l l e r ) and were shown to have the same melt i n g p o i n t , u l t r a v i o l e t and i n f r a r e d spectra and chromatographic be-h a v i o r . The m e l t i n g p o i n t of 204° reported by S c a r p a t i 41 and Oriente must be i n c o r r e c t s i n c e the sample s u p p l i e d by S c a r p a t i , a sample s u p p l i e d by H i l l e r and both of the present products a l l melted a t 172-174°. Hy d r o l y s i s of the i s o l a t e d e s t e r by NaOH or p e c t i n -ase y i e l d e d c a f f e i c a c i d ( i d e n t i f i e d by m.p., U.V. spect-rum and chromatographic behavior) and an u n c r y s t a l l i z a b l e compound w i t h ^X m ax 2 8 ^ ^ (EtOH) and colour r e a c t i o n s of an unconjugated c a t e c h o l nucleus. This compound, 3,4-- 3 1 -d i h y d r o x y p h e n y l l a c t i c a c i d (DOPL), was very water s o l u b l e and unstable to high pH. A reference sample f o r chroma-tographic purposes was i s o l a t e d from hydrolyzed rosmarinic a c i d by column chromatography ( A v i c e l , 2% formic a c i d ) of the et h e r - e x t r a c t e d hydrolysate. B i o s y n t h e t i c s t u d i e s . While on the b a s i s of cu r r e n t knowledge of cinnamic a c i d biochemistry, the c a f f e i c a c i d moiety was expected to a r i s e from phenylalanine v i a cinnamic a c i d , the poss-i b l e routes to form DOPL were more numerous. From phenyl-a l a n i n e a route could be envisioned passing through phen-y l l a c t i c a c i d to 3 , 4 - d i h y d r o x y p h e n y l l a c t i c a c i d , or the corresponding phenylpyruvic a c i d d e r i v a t i v e s . An u n l i k e l y but i n t r i g u i n g p o s s i b i l i t y was the hyd r a t i o n of the double bond of c a f f e i c a c i d or some other cinnamic a c i d . More l i k e l y were the p o s s i b i l i t i e s a r i s i n g from t y r o s i n e . The hy d r o x y l a t i o n of 4 - h y d r o x y p h e n y l l a c t i c or 4-hydroxyphenyl-pyruvic a c i d s , f o l l o w e d by r e d u c t i o n i n the l a t t e r case, would y i e l d DOPL. F i n a l l y , the conversion of t y r o s i n e to DOPA fol l o w e d by transamination and re d u c t i o n of the keto group would form the simples t and most e a s i l y demonstrated pathway. Any t r a c e r s t u d i e s i n v o l v i n g p h e n y l l a c t i c or phenylpyruvic a c i d s , or the hydroxy analogues, would be made d i f f i c u l t by t h e i r i n t e r c o n v e r s i o n s w i t h phenylalan-, 1 5 ine and t y r o s i n e , r e s p e c t i v e l y In a p r e l i m i n a r y experiment, both l a b e l l e d phenylalan-ine and l a b e l l e d t y r o s i n e fed to Mentha arvense f o r 24 hr. were incorporated i n t o rosmarinic a c i d . Radioautography of the chromatographed h y d r o l y s i s products i n d i c a t e d that phenylalanine was l a b e l l i n g the c a f f e o y l moiety while t y r o s i n e was l a b e l l i n g the DOPL. This was confirmed i n subsequent feedings f o r 8 hr. (Table I I . ) . The problems inherent i n a c c u r a t e l y measuring c a f f e i c a c i d concentra-t i o n s s p e c t r o p h o t o m e t r i c a l l y (the r e a d i l y i n t e r c o n v e r t i b l e 46 x c i s and trans isomers have d i f f e r e n t £ values ) make i t d i f f i c u l t to decide i f the s l i g h t i n t e r c o n v e r s i o n of phen-y l a l a n i n e and t y r o s i n e i m p l i e d by the l a b e l d i s t r i b u t i o n i s r e a l . 14 14 Administered C-cinnamic a c i d and C-p_-coumaric a c i d were incorporated more e f f i c i e n t l y than phenylalanine and l a b e l l e d only the c a f f e o y l moiety, as expected. On the 14 other hand, C-DOPA proved to be as good a precursor to rosmarinic a c i d as t y r o s i n e (Table I I . ) , i n d i c a t i n g that the pathway d i d not d i r e c t l y i n v o l v e 4-hydroxyphenyllactic or '4-hydroxyphenylpyruvic a c i d s . D i r e c t e s t e r i f i c a t i o n of c a f f e i c a c i d w i t h a l i p h a t i c 17 a l c o h o l s i s one known mode of c a f f e i c e s t e r formation Studies on chlorogenic a c i d b i o s y n t h e s i s have suggested that an a l t e r n a t e route to these esters i s v i a the _-22 coumaryl q u i n i c e s t e r , and t h i s may be the major route 3 5 - 3 7 i n some cases . With DOPA as an intermediate i n the formation of DOPL, the involvement of p_-coumaroyl-DOPL i s a p o s s i b i l i t y . E a r l i e r e s t e r s are u n l i k e l y s i n c e the - 3 3 -Table I I Inco r p o r a t i o n of Phenylpropanoid Compounds i n t o Rosmarinic A c i d i n Mentha arvense L. Precursor fed Rosmarinic A c i d A c t i v i t y s p e c i f i c $ l a b e l i n fed a c t i v i t y c a f f e i c (jiC) (;AC/mM) D i l u t i o n moiety 14 P h e n y l a l a n i n e - 2 - C 2 2 . 5 mC/mM 1 . 8 8 1 2 0 0 9 6 . 8 $ 14 T y r o s i n e - 3 - C ' 6 . 7 mC/mM 3 . 6 7 1 8 5 0 3 . 7 $ 14 Cinnamic a c i d - 2 - C 2 1 . 1 5 mC/mM 3 . 0 0 407 1 0 2 $ 4-OH Cinnamic a c i d - 2 - ^ C 6 5 . 8 ;aC/mM 0 . 6 2 126 9 9 $ C a f f e i c acid - 2- l i ('C 1 121 /iC/mM 0 . 5 1 236 9 8 $ DOPA-2-^C 3 . 3 2 1 2 8 5 4 . 0 7 mC/mM -34-r i n g h y d r o x y l a t i o n e v i d e n t l y precedes the transamination and r e d u c t i o n to y i e l d an e s t e r i f i a b l e alpha-hydroxyl. C a r e f u l examination of the phenolics e x t r a c t e d from Mentha f a i l e d to show any s i g n of such an e s t e r , which could be expected to show t y p i c a l p_-coumaric a c i d f l u o r -escence (blue when viewed under 366 nm U.V. l i g h t w i t h ammonia) and a strong c o l o u r r e a c t i o n between the DOPL 47 14 hydroxyls and d i a z o t i z e d p _ - n i t r o a n i l i n e . When C s h i k i m i c a c i d , phenylalanine and t y r o s i n e were fed to Mentha f o r three hours and the compounds w i t h catechol h y d r o x y l a t i o n were i s o l a t e d and chromatographed f o r auto-radiography, the r e s u l t s showed that a l l three precursors l a b e l l e d rosmarinic a c i d w e l l but no other phenolics con-tained a p p r e c i a b l e l a b e l . The feeding time may have been s u f f i c i e n t l y long, however, to allow the l a b e l to pass through a small pool of p_-coumaroyl e s t e r . One of the main c r i t e r i a , used i n st u d i e s on c h l o r o -genic a c i d formation, f o r the involvement of a p_-coumaroyl precursor has been the r e l a t i v e i n e f f i c i e n c y of adminis-tered c a f f e i c a c i d as a precursor to the e s t e r . The r e s u l t s have not been c o n s i s t e n t , however, and have v a r i e d from c a f f e i c a c i d being incorporated w i t h l e s s d i l u t i o n than ^ c o u m a r i c a c i d to i t s being incorporated w i t h much gre a t e r d i l u t i o n and p o s s i b l y even degradation and 35 r e s y n t h e s i s . The p i c t u r e i s complicated by the f r e -quent d e s t r u c t i o n of part of the administered c a f f e i c a c i d by polyphenol oxidases as i t passes through c e l l -35-membranes or comes i n contact w i t h i n j u r e d t i s s u e such as l e a f d i s c s , tuber d i s c s or cut shoots. 14 C - c a f f e i c a c i d administered to Mentha was i n c o r p o r -ated s p e c i f i c a l l y i n t o the c a f f e o y l moiety of rosmarinic a c i d w i t h an average d i l u t i o n of about 240 x (four r e p l i -c a t e s ) , about twice that of p_-coumaric a c i d . However, co n s i d e r i n g the l o s s e s encountered i n feeding such a l a b i l e compound, the observed i n c o r p o r a t i o n suggests that d i r e c t e s t e r i f i c a t i o n of c a f f e i c a c i d w i t h DOPL i s the main route of b i o s y n t h e s i s of rosmarinic a c i d i n Mentha (Figure 4.). Time-course s t u d i e s . Recent r e s u l t s showing that chlorogenic a c i d i n Xanthium leaves and SPlanum tubers i s a c t i v e l y t u r n i n g 39.40 o v e r J / ' prompted an examination of the turnover of r o s -marinic a c i d i n Mentha. In a closed i l l u m i n a t e d system, 50 shoots of M. arvense were exposed f o r two hours to 14 continuously c i r c u l a t i n g a i r c o n t a i n i n g 150 )xC of CO2 14 (released from Ba CO3 by a d d i t i o n of d i l u t e a c i d ) . The s p e c i f i c a c t i v i t y of the e s t e r and of i t s c a f f e o y l moiety were checked over 40 hours (Figure 5 . ) . In experiment 1, the l a b e l reached the rosmarinic a c i d more slowly than i n experiment 2, which may r e f l e c t d i f f e r e n c e s i n age or p h y s i o l o g i c a l c o n d i t i o n of the Mentha a t that time. The short d e c l i n e i n s p e c i f i c a c t i v i t y l e v e l l e d o f f and l i t t l e change was seen over 40 hours. The i n i t i a l peak may have -36-F i g u r e 4. Proposed pathway f o r the biogenesis of rosmarinic a c i d i n Mentha. H O SHIKIMIC ACID PREPHENIC ACID N H 2 H O -H O H O Rosmarinic a c i d V N H 2 C O O H C O O H C O O H C O O H DOPL O H O H -37-been produced by the pulse of l a b e l passing through the pools of f r e e precursors such as monosaccharides and a r -omatic amino a c i d s . The l a b e l i n i t i a l l y i ncorporated i n t o other compounds would reach these precursors more slowly through metabolic turnover and help to maintain the l e v e l of a c t i v i t y i n the e s t e r . On the other hand the uptake 14 of COg among the 40-50 shoots i n the chamber was un-doubtedly not completely uniform and some s p e c i f i c a c t i v -i t y v a r i a t i o n from t h i s i s unavoidable. The general p i c t -ure, then, i s one of r a p i d l a b e l l i n g from C0 2 followed by r e l a t i v e s t a b i l i t y of the amount of l a b e l i n t h i s p o o l . The d i s t r i b u t i o n of l a b e l between c a f f e i c a c i d and DOPL v a r i e d from about 70% i n the c a f f e o y l moiety i n the e a r l y p a r t of the experiments, to 52-60$ i n the c a f f e o y l moiety a f t e r 40 hr., i n d i c a t i n g that the cinnamoyl com-pounds become l a b e l l e d somewhat f a s t e r than the DOPL-DOPA pool. The r e l a t i v e l y l a r g e d i l u t i o n of DOPA as a l a b e l l e d precursor (Table I I ) suggests that the l i m i t i n g step may be transamination and/or r e d u c t i o n . 14 Using phenylalanine-U- C as a precursor, there i s a d e f i n i t e i n d i c a t i o n of turnover i n the rosmarinic a c i d pool (Figure 6>.), but i t i s s t i l l not very r a p i d . The i n c o r p o r a t i o n of l a b e l from phenylalanine i n t o the " e t h a n o l - i n s o l u b l e " e s t e r pool i n Mentha was demonstrated (Figure 6b.) w i t h a t l e a s t as much l a b e l i n t h i s pool as i n the estimated t o t a l pool of rosmarinic a c i d over 2 hr. The r e l a t i o n s h i p between these pools i s of great i n t e r e s t -38-Experiment 1 0 4 8 12 16 20 24 28 32 36 40 44 48 52 Hours Experiment'2 F i g u r e 5. Changes i n the s p e c i f i c a c t i v i t y of rosmarinic a c i d i n Mentha a f t e r a two hour exposure to 14 CO-s p e c i f i c a c t i v i t y rosmarinic a c i d •-s p e c i f i c a c t i v i t y c a f f e o y l moiety A -Hours Hours Figure 6 . Changes i n the s p e c i f i c a c t i v i t y of rosmarinic a c i d and the t o t a l a c t i v i t y of i n s o l u b l e esters i n Mentha a f t e r a pulse-feeding of p h e n y l a l a n i n e - l 1 ^ f o r (a) 2 hr and (b) 0.5 hr. S p e c i f i c a c t i v i t y rosmarinic a c i d T o t a l a c t i v i t y i n s o l u b l e e s t e r s -40-but was not pursued i n t h i s study. F i n a l l y , i n an attempt to see i f rosmarinic a c i d was being used as a substrate f o r the formation of i n s o l u b l e polymers such as l i g n i n , l a b e l l e d rosmarinic a c i d was pre-pared b i o s y n t h e t i c a l l y . The comparatively low s p e c i f i c 14 a c t i v i t i e s obtained from C-precursors made i t necessary 3 to r e s o r t to H - l a b e l l i n g . 3 3 Phenylalanine-G- H (250 ;uC: 0 . 2 mg) and tyrosine-G- H (250 J A C : 0 . 2 mg) were administered to separate l o t s of M. p i p e r i t a f o r 2 hr. and the rosmarinic a c i d i s o l a t e d and p u r i f i e d . In each case the rosmarinic a c i d was s p e c i f -i c a l l y l a b e l l e d i n one moiety or another. Each sample was readministered to Mentha f o r two feeding periods, ( i ) the len g t h of time r e q u i r e d to take up the s o l u t i o n of rosmar-i n i c a c i d , and ( i i ) 24 hours l a t e r (Table I I I ) . 3 The l a b e l from rosmarinic a c i d ( c a f f e i c - H) remained e n t i r e l y i n the s o l u b l e compounds over 24 hours. The s p e c i f i c a c t i v i t y of the i s o l a t e d rosmarinic a c i d showed a small decrease, suggesting some turnover i n the c a f f e o y l moiety. The l a b e l from rosmarinic a c i d (DOPL- H) i n the s o l u b l e compounds, however, showed a marked decrease over 24 hours, as d i d the s p e c i f i c a c t i v i t y of the i s o l a t e d e s t e r . An attempt was made to l o c a t e the i n s o l u b l e l a b e l w i t h l i t t l e success. An increase i n the l a b e l l i n g of the 30 " i n s o l u b l e " e s t e r p o o l ^ was evident but was apparently too small to account f o r the decrease noted above. No 48 trace of l a b e l could be detected i n the Klason l i g n i n - 4 1 -Table I I I . The Turnover of L a b e l l e d Rosmarinic A c i d Readministered to Mentha Rosmarinic % fed a c i d amount a c t i v i t y time a c t i v i t y s p e c i f i c Compound fed fed fed ethanol a c t i v i t y fed (mg) (dpm) (hr) s o l u b l e (dpm/mM) RA ~ ( c a f f e i c - ^ H ) * 1 . 5 ( 0 . 3 3 V 1 . 5 ( 0 . 2 9 ) 2 . 5 ( 0 . 4 5 ) 2 . 5 ( 0 . 5 2 ) 6.8xlo| 6.8x105 1.1x10° 1.1x10° 1 24 2 24 88 83 85 82 3.28x10^ 2.86x10° RA o (D0PL- JH)** 1 .5( 0 . 36 ) 1 .5( 0 . 33 ) 2 .5 ( 0 . 42 ) 2 . 5 ( 0 . 4 5 9.5x10-5 9.5x105 1 . 6 x 1 0 ° 1 . 6 x 1 0 ° 1 24 2 24 82 53 75 55 1.28x10? 8 . 3 0 x 1 0 ° Rosmarinic a c i d ( c a f f e i c moiety w i t h greater than 96$ of the 3 H ) , sp. a c t . 73.5yiC/mK •* Rosmarinic a c i d (D0PL moiety w i t h greater than 98$ of the 3 H ) , sp. a c t . 102 ^ uC/mM t f i g u r e s i n parentheses (mg fed/gm f r e s h wt. mint) -42-(or s o l u b l e hydrolysate) prepared from the 24 hour rosmar-3 i n i c a c i d (DOPL- H) sample. Since the shoots used were young a c t i v e l y growing m a t e r i a l c o n t a i n i n g l i g n i n (app-roximately \% f r e s h wt.) i t i s reasonable to assume that l i g n i n was a c t u a l l y being synthesized during the feeding p e r i o d . I t i s p o s s i b l e that the l a b i l i t y of DOPL i t s e l f , or of i t s t r i t i a t e d protons, a t high pH produces l a r g e l o s s e s i n procedures such as " i n s o l u b l e e s t e r " h y d r o l y s i s and thus y i e l d s low r e s u l t s . 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Metabolism of cinnamic a c i d i n p l a n t s : chlorogenic a c i d formation, Phytochem. 7 : 1 7 1 1 . 3 7 . K o j i n a , M., Minamikawa, T., Hyodo, H. and U r i t a n l , I . 1 9 6 9 . I n c o r p o r a t i o n of some p o s s i b l e r a d i o a c t i v e intermediates i n t o chlorogenic a c i d i n s l i c e d sweet potato t i s s u e , P l a n t and C e l l P h y s i o l . 1 0 : 4 7 1 . -46-3 8 . Gamborg, O.L. 1 9 6 7 . Aromatic metabolism i n p l a n t s . V. The b i o s y n t h e s i s of chlorogenic a c i d and l i g n i n i n potato c e l l c u l t u r e s , Can. J . Biochem. 4 5 : 1 4 5 1 . 3 9 . Taylor, A.O. and Zucker, M. 1 9 6 6 . Turnover and met-abolism of chlorogenic a c i d i n Xanthium leaves and potato tubers, P l a n t P h y s i o l ^ 41 : 1 3 5 Q » 40. Taylor, A.O. I 9 6 8 . The d i s t r i b u t i o n and turnover r a t e of s o l u b l e and i n s o l u b l e c a f f e o y l esters i n Xanthium, Phytochem. 7 s 6 3 . 41. S c a r p a t i , M.L. and Oriente, G. 1 9 5 8 . Isolament e c o n s t i t u z i o n e d e l l ' acido rosmarinico (del r o s -marinus o f f . ) , La R i c e r c a S c i e n t i f i c a 2 8 : 2 3 2 9 . 42. H i l l e r , K. and Kothe, N. I 9 6 7 . Chlorogen- und Rosmarinsaure-Vorkommen und q u a n t i t a t i v e V e r t e i l u n g i n Pflanzen der Saniculoideae, Phar-mazie 2 2 : 2 2 0 . 4 3 . Bohm, B.A. 1 9 6 8 . Phenolic compounds i n f e r n s . I I I . An examination of some ferns f o r c a f f e i c a c i d d e r i v a t i v e s , Phytochem. 7 : 1 8 2 5 . 4 4 . A u s t i n , D.J. and Meyers, M.B. 1 9 6 5 . The formation of 7-oxygenated coumarins i n hydrangea and l a v -ender, Phytochem. 4 : 2 4 5 . 45. Kagan, J . I 9 6 6 . The photochemical conversion of c a f f e i c a c i d to e s c u l e t i n . A model f o r the synthesis of coumarins i n v i v o , J . Am. Chem. Soc. 8 8 : 2 6 1 7 . 46. Kahnt, G. 1 9 6 6 . Uber das Gleichgewicht zwischen den Stereoisomeren e i n i g e r Zimtsaurederivate i n Abhangigkeit von der molaren Konzentration und i h r e q u a n t i t a t i v e spektrophotometrische Messung bei Pflanzenanalysen, B i o l o g i s c h e s Z e n t r a l b l a t t . 8 5 : 5 4 5 . 4 7 . Bray, H.G., Thorpe, W.V. and White, K. 1 9 5 0 . The f a t e of c e r t a i n organic a c i d s and amides i n the r a b b i t . 1 0 . The a p p l i c a t i o n of paperchromatography to metabolic s t u d i e s of hydroxyacids and amides, B i o -chem. J . 46 : 2 7 1 . ^ 8 . Modern methods of P l a n t A n a l y s i s . I I . p. 2 0 5 , 1 9 5 5 , Springer V e r l a g . 4 9 . Wang, C.H. and W i l l i s , D.L. I 9 6 5 . Radiotracer meth-odology i n b i o l o g i c a l science. P r e n t i c e - H a l l Inc., New Jersey. Degradation of Aromatic Compounds by S t e r i l e P l a n t T i s s u -47-INTRODUCTION Aromatic compounds are ubiquitous i n higher p l a n t s , p r i n c i p a l l y as p h e n o l i c s . Aside from the usual accumu-l a t i o n of s o l u b l e phenolic compounds, most p l a n t s convert an a p p r e c i a b l e p a r t of t h e i r carbon pool i n t o the l i g n i n polymers which provide a matrix f o r secondary c e l l walls'*'. A second pool of aromatic compounds i s the f r e e and protein-bound aromatic amino a c i d s , phenylalanine, t r y o -sine and tryptophan. Some p l a n t s a l s o accumulate complex nitrogenous aromatics, notably the a l k a l o i d s formed from 2 the aromatic amino a c i d s . A great deal of work has been devoted to r e v e a l i n g the pathways of formation of the aromatic amino a c i d s and the 3 4 many c l a s s e s of phenolics i n c l u d i n g the a l k a l o i d s . A l l can be s a i d to be formed from the pools of g l y c o l y t i c m e t a b o l i t e s , notably the monosaccharides, pyruvate and acetate, through such well-known routes as the shikimate pathway, acetate ( p o l y k e t i d e ) condensation and the 3 mevalonate-isoprenoid pathway . Many of the accumulated products have s t r u c t u r e s which d i f f e r g r e a t l y from those of the f i r s t aromatic Intermediates, so i t i s not easy to draw a d i s t i n c t i o n between pathways of metabolism and cat-abolism i n t h i s group of compounds. In pl a n t s these f i r s t intermediates are phenylpyruvic a c i d , para-hydroxy-phenylpyruvic a c i d and (enroute to tryptophan) a n t h r a n i l i c a c i d . Aside from i t s r o l e as an intermediate i n tryptophan -48-b i o s y n t h e s i s , the only i n f o r m a t i o n on a n t h r a n i l i c a c i d metabolism i n p l a n t s i s the presence of an enzyme i n Tec-oma stans which can convert t h i s compound to c a t e c h o l , i n 5 v i t r o . In microorganisms the degradation of tryptophan can g i v e r i s e to a n t h r a n i l i c a c i d and hence catechol , to 7 8 3 - h y d r o x y a n t h r a n i l i c a c i d or to kynurenate d e r i v a t i v e s . In a l l three cases, the f i n a l steps i n v o l v e aromatic r i n g f i s s i o n and complete o x i d a t i o n of the f i s s i o n products to CC>2. In p l a n t s , however, i n t e r e s t i n the metabolic f a t e of tryptophan has been l a r g e l y r e s t r i c t e d to the formation 9 of i n d o l e a c e t i c a c i d and the numerous a l k a l o i d s which are 4 g e n e r a l l y regarded as i n e r t metabolic end-products . There i s considerable evidence f o r a wide-spread a b i l i t y to o x i d i z e I A A 1 0 , a r e a c t i o n which has been suggested as one means of c o n t r o l l i n g the profound p h y s i o l o g i c a l e f f -ects of that auxin. The products of t h i s o x i d a t i o n have not been w e l l c h a r a c t e r i z e d , however, and there i s l i t t l e reason to b e l i e v e that the r e a c t i o n or r e a c t i o n s i n v o l v e aromatic r i n g f i s s i o n . The f u r t h e r metabolism of phenylpyruvate and 4-hydroxy-phenylpyruvate i n p l a n t s i s probably dominated by t h e i r transamination to form phenylalanine and t y r o s i n e respec-t i v e l y 1 1 . Both of these compounds undergo extensive mod-i f i c a t i o n s i n t h e i r f u r t h e r metabolism, i n c l u d i n g r i n g - and s i d e - c h a i n h y d r o x y l a t i o n s , s i d e - c h a i n c y c l i z a t i o n , deamin-3 a t i o n and b e t a - o x i d a t i o n . Microorganisms r e a d i l y degrade both phenylalanine and -49-t y r o s i n e , u s u a l l y v i a the corresponding keto compound or 12 cinnamate . The s i d e - c h a i n may be degraded to any ex-tent from l o s s of one carbon to l o s s of a l l three, and most microorganisms have the a b i l i t y to hydroxylate the r i n g . A f t e r i n t r o d u c t i o n of a hydroxyl, e i t h e r ortho to another hydroxyl (or para to another hydroxyl and ortho to a s i d e - c h a i n ) , microorganisms can r e a d i l y o x i d i z e the r i n g w i t h atmospheric oxygen to produce a l i p h a t i c 12 13 ac i d s ' . Few microorganisms seem to r e q u i r e polyphen-o l s compounds f o r any s t r u c t u r a l or p h y s i o l o g i c a l r o l e . These r e a c t i o n s , t h e r e f o r e , must be assumed to be d i r e c t e d toward scavenging the carbon and energy t i e d up i n the considerable q u a n t i t i e s of aromatic compounds i n the en-vironment, notably i n dead p l a n t m a t e r i a l . In p l a n t s , phenylalanine and t y r o s i n e form the aromatic 4 n u c l e i of extensive s e r i e s of a l k a l o i d s . Of more i n t e r -est w i t h respect to degradative metabolism, however, are the conversions of phenylalanine, and to some extent tyro-. 3 s i n e , to numerous p l a n t phenolics . The p r i n c i p a l route i s through the formation of cinnamic and 4-hydroxycinnamic a c i d which are both e x t e n s i v e l y modified by r i n g h y d r o x y l -14 15 16 a t i o n * and methoxylation , b e t a - o x i d a t i o n of the 17 si d e - c h a i n and d e c a r b o x y l a t i o n of the r e s u l t i n g benzoic acids'*" 0 ,'"^ . The l a t t e r r e a c t i o n s i n p a r t i c u l a r could be considered degradative, as three of the nine carbon atoms i n the o r i g i n a l precursor have been made a v a i l a b l e f o r f u r t h e r metabolism. The products of phenylpyruvate-- 5 0 -phenylalanine metabolism i n c l u d e 2 , 3 - and 3 ,4-dihydroxy-20 21 benzoic a c i d , 3,4-dihydroxycinnamic a c i d , 2,3-dihydroxy-22 19 phenylacetic a c i d and catechol . These compounds bear ortho d i h y d r o x y l s and are r e a d i l y r i n g - c l e a v e d by micro-organisms. Some or a l l of them could p o s s i b l y serve as ring-cleavage substrates i n p l a n t c e l l s . Other p o t e n t i a l substrates are 2,5-di-hydroxybenzoic and 3 , ^ , 5 - t r i h y d r o x y -benzoic a c i d s . P o s s i b l e ring-cleavage substrates known to be formed from t y r o s i n e i n c l u d e 3,4-dihydroxyphenylalanine 23 24 (DOPA) , 3 , 4 - d i h y d r o x y p h e n y l l a c t i c a c i d (DOPL) , dopa-4 22 mine and 3,b- and 2,5-d.ihydroxyphenylacetic a c i d More complex s t r u c t u r e s such as f l a v o n o i d s and a l k a -l o i d s can a l s o bear ortho-dihydroxyls and t h e i r degrad-a t i v e metabolism could p o t e n t i a l l y i n c l u d e r i n g - f i s s i o n . While degradation and r e u t i l i z a t i o n of phenolics i n p l a n t s i s o f t e n assumed to occur to some extent, r e l a t i v e -l y l i t t l e experimental work has been c a r r i e d out on e i t h e r general degradation of phenolics or on aromatic r i n g cleavage i n p l a n t s . No work has been published on r i n g -f i s s i o n by p l a n t t i s s u e s growing i n the absence of micro-organisms. This c o n d i t i o n i s e s s e n t i a l because of the f a c i l i t y w ith which b a c t e r i a and fungi hydroxylate and degrade aromatic s t r u c t u r e s , and the g e n e r a l l y low l e v e l s of such a c t i v i t y one may expect to f i n d i n p l a n t t i s s u e s . E s s e n t i a l l y a l l published s t u d i e s on phenolic degradation have used the technique of a d m i n i s t e r i n g the l a b e l l e d com-pound i n aqueous s o l u t i o n to l e a f d i s c s , cut shoots or - 5 1 -25 26 l e a f c u t t i n g s ' . Some st u d i e s have apparently ignored any p o s s i b i l i t y of m i c r o b i a l metabolism e i t h e r w i t h i n the 27 p l a n t t i s s u e s or on the surface of the submerged t i s s u e , s i n c e no precautions are described to minimize such a 26,28 p o s s i b i l i t y The metabolism of t y r o s i n e i n p l a n t s has a t t r a c t e d the most a t t e n t i o n . In c o n t r a s t to phenylalanine, which pre-dominantly forms p h e n o l i c s , t y r o s i n e i s apparently de-graded q u i t e r e a d i l y . Label from uni f o r m l y l a b e l l e d t y -r o s i n e q u i c k l y appears i n monosaccharides, malate and com-pounds d e r i v e d from these precursors. This phenomenon has 25 29 been examined i n a number of st u d i e s ( N i c o t i a n a , Pyrus , 28 30 Eucalyptus ) and commented on i n others . In only one case was any evidence of r i n g - f i s s i o n presented, when i n two of three cases more than 33$ of the l a b e l from t y r o s i n e -14 U- C was recovered i n non-aromatic compounds a f t e r 7-23 29 hours of metabolism i n the l i g h t . This i s an e x t r a -ordinary demonstration of aromatic degradation c o n s i d e r i n g the apparent s t a b i l i t y of other aromatic compounds ( i n -c l u d i n g 4-hydroxyphenylpropanoids) and the f a c t that t y r o -s i n e i s a p r o t e i n amino a c i d . In other s t u d i e s , the l a b e l appearing i n non-aromatics could e a s i l y have a r i s e n by s i d e - c h a i n degradation without invoking ring-cleavage. In no case could the p a r t i c i p a t i o n of m i c r o b i a l metabol-ism be excluded, although the magnitude and s p e c i f i c i t y of the degradation observed makes i t l i k e l y that i t was, a t l e a s t i n p a r t , a p l a n t t i s s u e phenomenon. Unfortunately, - 5 2 -there have been no reported s t u d i e s on the metabolic route or routes i n v o l v e d . Recent work on the bi o s y n t h e s i s of the b e t a n i d i n nu-cleus of the betacyanin pigments i n the Centrospermae has provided evidence of the a b i l i t y of p l a n t s to o x i d i z e the aromatic r i n g of DOPA, as p r e v i o u s l y p o s t u l a t e d on the 30 bas i s of the s t r u c t u r e of betanin . I t remains to be seen i f the non-aromatic r i n g - f i s s i o n product i s u t i l i z e d i n r e a c t i o n s other than b e t a n i d i n formation. There has been no r e p o r t of s t u d i e s on r i n g - f i s s i o n of DOPL or dopa-mine . I t has been suggested that 2,3- and 3,4-dihydroxy-phe n y l a c e t i c a c i d s undergo r i n g - f i s s i o n i n A s t i l b e and 22 S i n a p i s . Using u n i f o r m l y - l a b e l l e d compounds i n a f i v e day feeding experiment, about 5% of the administered l a b e l appeared i n sucrose. Considering the apparent l a c k of c o n t r o l s over m i c r o b i a l contamination during t h i s extended p e r i o d , t h i s r e s u l t i s suggestive, but h a r d l y convincing evidence of r i n g - f i s s i o n . The l o s s of a l l or part of the l a b e l l e d s i d e - c h a i n could produce the same r e s u l t . Sim-14 i l a r l y , the r e s u l t s of feeding alpha- C-hydroxycinnamic 14 ac i d s and phenylalanine-U- C to tobacco l e a f d i s c s over f o r t y hours show that the side-chains of these compounds 25 are r e a d i l y degraded and i t i s reasonable to suspect b e t a - o x i d a t i o n of the cinnamates, a well-known r e a c t i o n i n 17 plants The case of 2,5-dihydroxyphenylacetic a c i d (homo-- 5 3 -g e n t i s i c a c i d ) may be d i f f e r e n t . I t i s a well-documented subs t r a t e f o r ring-cleavage i n the degradation of phenyl-a l a n i n e and t y r o s i n e i n animals and microorganisms , In pl a n t s i t has r e c e n t l y been demonstrated to be an i n t e r -mediate i n the formation of plastoquinone from t y r o s i n e ^ . There i s l i t t l e reason to suspect that plastoquinone i t -s e l f i s a subs t r a t e f o r a p p r e c i a b l e r i n g - f i s s i o n but i t has been suggested that the homogentisic a c i d intermediate may be an immediate substrate f o r r i n g - c l e a v a g e 2 ^ . Such a r e a c t i o n could e x p l a i n the r a p i d degradation of t y r o s i n e i n p l a n t s but t h i s i n t e r e s t i n g p o s s i b i l i t y has yet to be examined. Pr e p a r a t i o n of r i n g - l a b e l l e d t y r o s i n e and homo-g e n t i s a t e and the use of s t e r i l e p l a n t s w i l l be necessary to o b t a i n an unequivocal answer. Studies on the degradation of f l a v o n o i d s i n p l a n t s have been r e s t r i c t e d to the catechins and chalcones. C-14 catechins prepared b i o s y n t h e t i c a l l y from COg were read-14 m i n i s t e r e d to tea shoots. The observed r e l e a s e of COg amounted to ca. 10$ of the fed l a b e l over the f i r s t 25-40 26 hours and a remarkable 80$ a f t e r 50-75 hours . I f t h i s i s any i n d i c a t i o n of the r a t e of turnover of the l a r g e pool of catechins i n tea shoots, they are m e t a b o l i c a l l y 14 extremely a c t i v e . Even i f only the e a r l y phase of C 0 2 r e l e a s e i s a t t r i b u t a b l e to p l a n t metabolism, the Cg-Cj-Cg s t r u c t u r e s are obviously being degraded very r a p i d l y to at l e a s t and Cg-C-^ components. Catechins have been 28 reported to be m e t a b o l i c a l l y a c t i v e i n other work as w e l l -54-With no r e p o r t of the d i s t r i b u t i o n of -^C w i t h i n the c a t -echins, and the very long f e e d i n g p e r i o d , no c o n c l u s i v e case can be made f o r r i n g - f i s s i o n i n t h i s experiment. An i n t e r e s t i n g r e p o r t t h a t a s p e c i f i c a l l y l a b e l l e d chalcone, 4,2',4',6'-tetrahydroxychalcone-2'-glucoside f was being s p l i t by Petunia to form p h l o r o g l u c i n o l and 32 para-coumaric a c i d ^ has never been pursued, A - r i n g -l a b e l l e d 5,7,4'-trihydroxyflavanone was found to g i v e r i s e 14 to CO2, presumably a f t e r f o r m a t i o n of p h l o r o g l u c i n o l J - > . Both aspects of t h i s work need to be confirmed u s i n g d i f f -erent f l a v o n o i d s and s t e r i l e t i s s u e s . A number of micro-organisms can break f l a v o n o i d s down to p h l o r o g l u c i n o l and a corresponding phenylpropionate d e r i v a t i v e - ^ , or p h l o r o -g l u c i n o l c a r b o x y l i c a c i d , carbon monoxide and a CgC-^ 35 a c i d . The aromatic products can then be f u r t h e r met-a b o l i z e d , although the mode of cleavage of p h l o r o g l u c i n o l has not been c l e a r l y demonstrated i n any organism. 14 U n i f o r m l y - l a b e l l e d p h l o r o g l u c i n o l - C incubated asept-i c a l l y w i t h nine s p e c i e s of marine algae was p a r t l y de-graded to "^CC^ i n e a c h c a s e ^ . The e f f i c i e n c y of t h i s pathway was i n c r e a s e d by p r i o r i n c u b a t i o n of the alga e w i t h c o l d p h l o r o g l u c i n o l . An attempt to d e t e c t r i n g - c l e a -14 14 vage of C-catechol and p h e n y l a l a n i n e - r i n g - C by asept-37 i c a l l y c u l t u r e d tomato s e e d l i n g s was u n s u c c e s s f u l . T h i s suggests t h a t c a t e c h o l i s not a widespread s u b s t r a t e f o r r i n g cleavage i n p l a n t s , whereas i t i s i n microorgan-isms . -55-14 The production of CCv, from toluene and benzene ri n g -14 38 C by avocado-" cannot be d e f i n i t e l y attributed to plant metabolism. Exposure of i n t a c t , u n s t e r i l i z e d , tree-grown f r u i t to vapours of the aromatic substrate (100 JJLC) gave 14 r i s e to only minute amounts of COg over four hours. Much larger amounts of l a b e l were found i n compounds other than benzene and toluene, but they were not i d e n t i f i e d . 14 14 Benzene- C has been shown to give r i s e to COg (2-3$ over 72 hours) when fed through the s t e r i l i z e d roots 39 of tea plants, or d i r e c t l y into the stem y /. Considerable quantities of the l a b e l appeared i n non-aromatic compounds throughout the plant. S i m i l a r l y , leaf homogenates (Thea, 40 V i t i s ) converted benzene to phenol and muconic acid . The pathway known for the conversion of benzene to carbon d i -oxide i n microorganisms involves phenol, catechol and c i s , sen .40 41 cis-muconic acid ; the same general route has been proposed for the observed degradation of benzene i n plants Since benzene i s not a normal metabolite of these plants the enzymes involved may normally act on other substrates but possess low substrate s p e c i f i c i t y . On the other hand, the s t e r i l i z a t i o n procedures (0.01$ mercuric bichloride or bromine water) may have been inadequate, leaving a micro-b i a l f l o r a quite capable of carrying out such degradations. While reference i s made to the use of sterilely-grown 39 plants i n other cases, no d e t a i l s are given^ . One of the best controlled studies to date involved the 14 administration of phenylalanine-ring-UL- C to cut shoots -56-of a number of p l a n t s and measurement of evolved " ^ C O g ^ . A n t i b i o t i c s were used i n the feeding s o l u t i o n and attempts were made to reduce the l i k e l i h o o d of m i c r o b i a l metabolism being i n v o l v e d . The r e s u l t s showed that T r i t i c u m , Hordeum and Picea could r e s p i r e as much as 7.3$ of fed -^C. No attempt was made to determine the pathway being followed to 14 r i n g - f i s s i o n . The r e l e a s e of CO^ seemed to vary w i t h the s t a t e of development of the shoots i n the one species ex-amined i n d e t a i l (Hordeum). While gross contamination was r u l e d out, and the l i k e l i -hood of the p l a n t t i s s u e s themselves c a r r y i n g out the r i n g -f i s s i o n was high, the v a l i d i t y of the r e s u l t s of any ex-periments such as these u s i n g non-aseptic t i s s u e s and tech-niques w i l l remain i n doubt. Two approaches which could e l i m i n a t e t h i s doubt are the use of a s e p t i c a l l y grown p l a n t s or t i s s u e c u l t u r e s . With the recent a v a i l a b i l i t y of a number of aromatic r i n g -l a b e l l e d compounds, a p r e l i m i n a r y study of t h e i r p o s s i b l e ring-cleavage and o x i d a t i o n to COg was undertaken us i n g both approaches. - 5 7 -MATERIALS AND METHODS Pl a n t t i s s u e c u l t u r e s . The two t i s s u e s used i n the present work were deri v e d from Ruta graveolens and M e l i l o t u s a l b a . The c u l t u r e s were i n i t i a t e d by Dr. O.L. Gamborg, P r a i r i e Regional Laboratory, NRC, Saskatoon and i n o c u l a were sent to the U n i v e r s i t y of B r i t i s h Columbia. They were maintained on the standard m e d i a ^ (Appendix A), B5 (Ruta) and B 5 C 2 ( M e l i l o t u s ) , both as c a l l u s c u l t u r e s and as l i q u i d suspension c u l t u r e s . C a l l u s c u l t u r e s were grown on 30 ml of the appropriate medium plus 0 . 6 $ agar i n 50 ml cotton-stoppered Erlenmeyer f l a s k s . Grown a t 2 3 ° under continuous f l u o r e s c e n t l i g h t -i n g ( 9 0 0 0 lux) they r e q u i r e d s u b c u l t u r i n g every 2 - 4 weeks. L i q u i d suspension c u l t u r e s were grown i n 2 5 0 ml cotton-stoppered Erlenmeyer f l a s k s w i t h 50 ml of medium. They were e s t a b l i s h e d by i n o c u l a t i o n w i t h one or two small pieces of c a l l u s t i s s u e . Stocks of l i q u i d suspension c u l -tures were maintained on r e c i p r o c a t i n g and gyrotary shakers but a l l experiments were run on the l a t t e r , which produced b e t t e r growth. S u b c u l t u r i n g was necessary a t 1 - 3 week i n -t e r v a l s . The two t i s s u e s are very d i f f e r e n t i n morphology. Ruta t i s s u e i s uni f o r m l y green and i n c a l l u s o f t e n forms a mass of minute shoots. In suspension, the lumps of i n o c u l a i n -crease i n s i z e , producing extensions of t i s s u e i n a l l d i r -e c t i o n s . These u s u a l l y do not spontaneously break up i n t o -58-smaller p i e c e s . Experiments w i t h Ruta t y p i c a l l y ended when the mass of t i s s u e clogged the r o t a r y flow i n the feeding f l a s k . The M e l i l o t u s , i n c o n t r a s t , was not a green t i s s u e and grew i n l i q u i d suspension as s i n g l e c e l l s and small clumps of c e l l s . R a d i o a c t i v e compounds. DL-Phenylalanine-ring-U--*-^C, DL-phenylalanine-ring-1-14 1 4 I LL T LL C, D-glucose-U- C, DL-DOPA-2- C and DL-tyrosine-2- X 4 ,C were purchased from New England Nuclear, Boston, Mass. 14 Cinnamic a c i d - 2 - C was purchased from ICN, C a l i f o r n i a . 14 14 14 DL-tyrosine-1- C, DL-tyrosine-3- C, L-tyrosine-U- C, 14 DL-tryptophan-benzene ring-U- C and benzoic a c i d - r i n g - U -14 C were purchased from the Radiochemical Centre, Amersham, 14 England. S a l i c y l i c a c i d - r i n g - U - C was purchased from M a l l i n c k r o d t Nuclear, Orlando, F l a . 14 Cinnamic a c i d - r i n g - 1 - C was prepared from DL-phenyl-14 a l a n i n e - r i n g - 1 - C by use of phenylalanine ammonia-lyase. The enzyme was prepared from U s t i l a g o hordel E^ and p u r i f -i e d to the end of the f i r s t ammonium s u l f a t e f r a c t i o n -a t i o n ^ . By u s i n g no c o l d phenylalanine, a l a r g e excess of enzyme and overnight i n c u b a t i o n a t 35°, more than 90$ of the L-isomer was converted to cinnamic a c i d . The prod-uc t was p u r i f i e d by ether e x t r a c t i o n from the i n c u b a t i o n mixture and TLC of the ether e x t r a c t . A l l compounds were made up i n aqueous s o l u t i o n and - 5 9 -s t e r i l i z e d by M i l l i p o r e f i l t r a t i o n . D e tection of ring-cleavage. A feeding f l a s k c o n s i s t e d of a cotton-stoppered 250 ml Erlenmeyer f l a s k w i t h a center w e l l (Figure 1 . ) . To begin an experiment, a heavy inoculum of t i s s u e and an a l i q u o t of a r a d i o a c t i v e compound were added to the 50 ml of med-ium. A f t e r 24 hours' i n c u b a t i o n on the gyrotary shaker (100 rpm, 2 2 - 2 3 °, continuous low l i g h t ) , the center w e l l was charged w i t h 2 ml 5N KOH and a s t e r i l e f i l t e r paper wick (4x5 cm, Whatman #50). In e a r l y experiments the C0 2 trapping was c a r r i e d on continuously, changing the KOH and wick a t 24 hr. i n t e r v a l s . This was found to a l l o w app-r e c i a b l e l o s s e s of COg through the stopper, however. L a t e r experiments (most of those reported) were run by trapping COg f o r 24 hr, s e a l i n g the f l a s k w i t h a t i g h t f o i l cap over the stopper. This was f o l l o w e d by 24 hr i n -cubation without t r a p p i n g . There was no i n d i c a t i o n that the 24 hr f l a s k s e a l i n g depressed growth of the c u l t u r e s . In the course of an experiment (7-14 days) the Ruta t i s s u e increased i n mass about Jx, w h i l e the mass of M e l i l o t u s increased from 5 to lOx. The KOH and wick a s e p t i c a l l y removed from a feeding f l a s k were placed i n a 125 ml side-arm f l a s k which was then connected to the apparatus shown i n Figure 1. The COg was regenerated from the carbonate by a d d i t i o n of 2 ml of 50$ l a c t i c a c i d . The system was then f l u s h e d w i t h a i r ( c a . / F i g u r e 1. - 6 0 -Feeding f l a s k CO2 Regeneration system - 6 1 -60 ml/min) f o r 20 min and the C O 2 trapped i n 10 ml of 46 2-phenylethylamine . A f t e r 20 min, 2 ml of water were added to the 2-phenylethylamine to a i d i n d i s s o l v i n g c a r -bonate deposits and 2 ml of the f i n a l 1 2 ml volume were counted i n d u p l i c a t e by l i q u i d s c i n t i l l a t i o n counting (to 14 a minimum of 1 0 0 0 cpm.). Tests w i t h Na 2 C O 3 i n the medium of the feeding f l a s k and 16 hr trapping a f t e r a c i d i f -i c a t i o n showed that the recovery of a c t i v i t y i n the com-p l e t e procedure was 7 0 - 8 0 $ . At the end of a feeding experiment, the r e s i d u a l r a d i o -a c t i v i t y i n the medium was determined, a f t e r removal of the t i s s u e by f i l t r a t i o n . M i c r o b i a l contamination. Contamination of feeding experiments, stock c u l t u r e s or radiochemical s o l u t i o n s was checked by p l a t i n g the t i s s u e or s o l u t i o n on two media-yeast e x t r a c t - g l u c o s e and malt . o e x t r a c t (Appendix A ) . The p l a t e s were incubated a t 3 3 o f o r two days and then 23 f o r three days. -62-RESULTS AND DISCUSSION A s e p t i c a l l y grown p l a n t s . A number of attempts were made to grow sunflower seed-l i n g s a s e p t i c a l l y and examine t h e i r a b i l i t y to o x i d i z e the r i n g of aromatic compounds. Using various growth systems and feeding p l a n t s phenylalanine-ring-U- C e i t h e r through the roots or through cut stems, r a d i o a c t i v e COg was u s u a l l y detected. Close examination of the system a t the end of each run f r e q u e n t l y showed some m i c r o b i a l contamination, l e a v i n g the e n t i r e s e r i e s of r e s u l t s i n doubt. This approach was, t h e r e f o r e , abandoned i n favour of p l a n t t i s s -ue c u l t u r e s . P l a n t t i s s u e c u l t u r e s . P l a n t t i s s u e s grown a s e p t i c a l l y i n appropriate media can be induced to grow without d i f f e r e n t i a t i o n . This i s f r e q u e n t l y accompanied by an a l t e r e d metabolism, as demon-s t r a t e d by t h e i r requirement f o r more or l e s s complex nut-r i e n t media ' and growth f a c t o r s . A two week study w i t h Dr. O.L. Gamborg a t the P r a i r i e Regional Laboratory (NRC), Saskatoon made i t p o s s i b l e to 48 l e a r n h i s t i s s u e c u l t u r e techniques and to attempt some p r e l i m i n a r y s t u d i e s on suspension c u l t u r e s which he pro-vided. In two experiments, c u l t u r e s of T r i t i c u m , soybean and mung bean could be shown to y i e l d small amounts of 14 14 COg from phenylalanine-ring-U- C. The procedures used - 6 3 -i n t h i s work were improved upon and the studi e s were con-tinued a t UBC using Ruta graveolens and M e l i l o t u s a l b a . Tryptophan. DL-Tryptophan-benzene r i n g - U - ^ C administered to both 14 species was metabolized to a small extent to CO2 (Table I . ) . The uptake of t h i s amino a c i d was c o n s i s t e n t l y poorer than that of other compounds used, but t h i s does not app-ear to be the reason f o r the one low value f o r Ruta. The c u l t u r e s i n use a t that time were growing more slowly than usual and there may have been d i f f e r e n t demands on the met-a b o l i c pool of tryptophan, r e s u l t i n g i n l e s s degradation of the compound. 14 Because of the low l e v e l s of released CGv,, no attempt was made to tr a c e the route of degradation. Phenylalanine. D L - P h e n y l a l a n i n e - r i n g - l - x ^ C was degraded to ^CCv, by both t i s s u e s (Table I I . ) . This confirms the a b i l i t y of p l a n t s to o x i d i z e the r i n g of phenylalanine as suggested 4 3 by e a r l i e r work i n a n o n - s t e r i l e system. J I f i t i s assumed that only the L-isomer i s a c t i v e l y metabolized and the D-isomer i s stored as an N-acetyl or N-malonyl d e r i v a t i v e ^ 9 , the percent recovery of a c t i v i t y i n CO^ can be doubled. There are a number of routes through which phenylalanine could be degraded. Deamination to cinnamic a c i d could l e a d 21 to ortho-dihydroxy compounds such as c a f f e i c a c i d . Fur--64-Table I . 14 14 Oxidation of DL-Tryptophan-benzene ring-U- C to CO by Ruta and M e l i l o t u s over seven days. a c t i v i t y fed % a c t i v i t y % a c t i v i t y Tissue (JULC) taken up recovered i n C0 2* Ruta 1 67 0 . 2 2 1 70 0 . 2 6 4 8 9 0 . 0 3 M e l i l o t u s 2 5 1 . 5 0 . 2 3 * as percent of a c t i v i t y taken up. -65-Table I I . Metabolism of D L - P h e n y l a l a n i n e - 1 ^ and C i n n a m i c - 1 ^ by Ruta and M e l i l o t u s over seven days t Compound fed Tissue a c t i v i t y fed (/AC) % a c t i v i t y taken up % a c t i v i t y recovered i n C0 2* DL-phenylalanine-r i n g - l - X M C 3.44 mC/mM M e l i l o t u s 2.8 7.5 86 79 0.14 0.14 cinnamic a c i d -Ruta 2.5 4.2 5.0 94.9 95.9 95.3 0.31 0.17 0.12 14 r i n g - 1 - C 3.44 mC/mM M e l i l o t u s 1.2 3.5 84 71.1 0.04 0.39 Ruta 2.1 89.9 N.D.** DL-phenylalanine-14 2 - C 2.5 mC/mM Ruta 2 88 1.1 cinnamic a c i d -14 2 - C 50 mC/mM M e l i l o t u s 1 73.5 3.8*** Ruta 2 88.2 0.71 * as percent of a c t i v i t y taken up. ** not de t e c t a b l e , l e s s than 0.02$. *** i n c u b a t i o n f o r four days. -66-ther degradation of the cinnamyl s i d e - c h a i n by beta-17 o x i d a t i o n would y i e l d acetate and benzoic or hydroxy-20 benzoic a c i d s . On the other hand, o x i d a t i v e deamin-a t i o n or transamination of phenylalanine would y i e l d phenylpyruvic acid'*"''". The l a t t e r could g i v e r i s e to 2-hydroxy- and 2,3-dihydroxyphenylacetic a c i d s as has 22 been observed i n A s t i l b e . There i s a l s o a p o s s i b i l i t y of conversion of 2-hydroxyphenylacetic a c i d to homogen-t i s i c a c i d (2,5-dihydroxyphenylacetic a c i d ) . Although t h i s l a s t r e a c t i o n has not been observed i n p l a n t s , i n an a n a l -gous r e a c t i o n s a l i c y l i c a c i d i s hydroxylated to y i e l d g e n t i s i c a c i d ^ . Cinnamic a c i d - r i n g - 1 - C was degraded to C0 2 by M e l i l o t u s q u i t e w e l l i n one experiment and only i n traces i n a second experiment. The reason f o r t h i s v a r i a t i o n i s not known. This t i s s u e d e f i n i t e l y has the a b i l i t y to de-grade cinnamoyl compounds, both r i n g - and s i d e - c h a i n l a b -14 e l l e d . The g r e a t l y increased y i e l d of C0 2 when using 14 cinnamic a c i d - 2 - C i s probably i n d i c a t i v e of beta-oxid-a t i o n of the s i d e - c h a i n . The same t i s s u e , however, i s apparently unable to degrade the benzoic or s a l i c y l i c a c i d s (Table I I I . ) . This suggests that the r i n g - f i s s i o n s u b s t r a t e i s a C^C^ compound, such as c a f f e i c a c i d , but more work i s r e q u i r e d to confirm t h i s . The one attempt to detect r i n g cleavage of cinnamic a c i d by Ruta was u n s u c c e s s f u l . While t h i s r e q u i r e s con-f i r m a t i o n , i t may mean that phenylalanine i s being de-- 6 7 -Table I I I . Oxidation of Benzoic a c i d and S a l i c y l i c a c i d r i n g - U - ^ C by Ruta and M e l i l o t u s over seven days. a c t i v i t y % a c t i v i t y Compound fed % a c t i v i t y recovered fed Tissue {jxC) taken up i n C0 2* Benzoic a c i d -14 ring-U- C 4 5 mC/mM Ruta Ruta M e l i l o t u s 5 5 3 9 6 . 7 96.. 5 6 5 . 9 N.D.* * N.D. N.D. S a l i c y l i c a c i d -14 ring-U- C 0 . 9 5 mC/mM Ruta M e l i l o t u s 5 3 82.1 88.2 N.D. N.D. * as percent of a c t i v i t y taken up. »* not d e t e c t a b l e , l e s s than 0 . 0 2 $ . -68-graded through the phenylpyruvic acid-hydroxyphenylacetic a c i d s route i n Ruta. In support of t h i s , the r e l e a s e of 14 14 COg from cinnamic a c i d - 2 - C was s t r i k i n g l y lower i n Ruta than i n M e l i l o t u s . No degradation of the r i n g of benzoic or s a l i c y l i c a c i d s could be detected (Table I I I . ) . The c u l t u r e s of Ruta i n use have an a c t i v e cinnamate metabolism s i n c e they accumulate coumarins and r u t i n ^ x . There i s no s i g n of an accumulation of cinnamic or benzoic a c i d s , however, when hydrolyzed e x t r a c t s of the t i s s u e are chromatographed. The same i s true of the M e l i l o t u s c u l -t u r e s , which a l s o l a c k the coumarins and r u t i n . I t would be i n t e r e s t i n g to extend t h i s work to t i s s u e s which accum-u l a t e the pools of hydroxycinnamic and hydroxybenzoic a c i d s more t y p i c a l of higher p l a n t s . Tyrosine. Because of the great i n t e r e s t i n t y r o s i n e degradation i n p l a n t s , an attempt was made to measure ring-cleavage of 14 t h i s amino a c i d . R i n g - l a b e l l e d t y r o s i n e - C i s u n a v a i l a b l e 14 at present, but comparison of the extent of COg r e l e a s e 14 from c u l t u r e s fed tyrosine-1,2,3-(side-chain) C and c u l -14 tures fed tyrosine-U- C (same t o t a l and s p e c i f i c a c t i v i t y ) might be expected to demonstrate ring-cleavage i n d i r e c t l y . The i n e v i t a b l e experimental v a r i a t i o n makes traces of ring-cleavage undetectable, but e a r l i e r work i n n o n - s t e r i l e systems suggested that ring-cleavage of t y r o s i n e i n p l a n t s 29 may be q u i t e extensive . -69-Using Ruta, a 1:3 r a t i o of a c t i v i t y from tyrosine-U-14 14 C and tyrosine-1,2,3- C was not exceeded, d e s p i t e the remarkably high r a t e of degradation of the s i d e - c h a i n (Table I V . ) . S i m i l a r l y , the s l i g h t increase i n t h i s r a t i o u s i n g M e l i l o t u s may not be s i g n i f i c a n t . The r a t e of s i d e -chain degradation w i t h M e l i l o t u s was lower but s t i l l con-s i d e r a b l e . Ring-cleavage of t y r o s i n e does not appear to be o c c u r r i n g to any great extent i n these t i s s u e s . Phenyl-a l a n i n e i s not being converted to t y r o s i n e very r e a d i l y , 14 as i s shown by the comparatively low r a t e of CO^ r e l -14 ease from phenylalanine-2- C (Table I I . ) . To o b t a i n some i n d i c a t i o n of the extent of degradation 14 of the s i d e - c h a i n of t y r o s i n e , t y r o s i n e - 2 - C and t y r o -14 s i n e - 3 - C were compared as s u b s t r a t e s . As can be seen from Table IV., the degradation of the s i d e - c h a i n extends to a l l three carbons. The second carbon i s l o s t most r e a d i l y , w h i l e carbon three i s l o s t to a smaller extent (approx. 60% of carbon two). In experiments run beyond 14 seven days the r a t i o of C0£ recovery from t y r o s i n e - 3 -14 14 C vs. t y r o s i n e - 2 - C s t e a d i l y i n c r e a s e s . This suggests 14 that the r e l e a s e of C from both p o s i t i o n s might occur i n the i n i t i a l r e a c t i o n , but the subsequent metabolic f a t e of the two carbons d i f f e r s . On the other hand, the two carbons may be l o s t i n a stepwise f a s h i o n i n d i s t i n c t r e a c t i o n s . Tyrosine i s known to be a precursor to the p l a s t o -32 quinones . This route i n v o l v e s 2,5-dihydroxyphenyl-- 7 0 -Table IV. 14 14 Degradation of Tyrosine- C and DOPA- C by Ruta and M e l i l o t u s over seven days. a c t i v i t y % a c t i v i t y Compound fed % a c t i v i t y recovered fed T i s s u e (yuC) taken up i n COg* DL-tyrosine-14 1,2.3- C 5.14 mC/mM Ruta 2.16 n.m.#* 19.69 L - t y r o s i n e -14 U- C 5.14 mC/mM Ruta 2.0 n.m. 6.65 DL-tyrosine-14 1 , 2 , 3 - C 5.14 mC/mM Ruta 1 9 3 . 9 19.20 L - t y r o s i n e -U-^C 5.14 mC/mM Ruta 1 9 2 . 3 4 .35 DL-tyrosine-l ^ ^ - 1 ^ M e l i l o t u s 1 81 7.07 L- t y r o s i n e -U-^C M e l i l o t u s 1 83 2.58 -71-Table IV. (cont'd.) Compound fed a c t i v i t y % a c t i v i t y fed % a c t i v i t y recovered Tissue (p.c) taken up i n C0 2* DL-tyrosine-14 2 - C 5 0 mC/mM Ruta 2 n.m. 1 9 . 5 0 DL-DOPA-2 - ^ c 4.1 mC/mM Ruta 2 n.m. 0 . 5 0 DL-tyrosine-2-^C 5 0 mC/mM Ruta 1 n.m. 21.00 DL-tyrosine-3 - ^ c 6 . 8 5 mC/mM Ruta 1 n.m. 10.3 0 DL-tyrosine-2-^C 0.90 mC/mM Ruta 1 9 3 . 9 1 7 . 3 5 DL-tyrosine-3 - l 4 C 0.90 mC/mM Ruta 1 9 4 . 8 1 2 . 3 0 * as percent of a c t i v i t y taken up. ** n.m. not measured: a c t i v i t y i n C0 2 i s % a c t i v i t y f e d . -72-a c e t i c a c i d (homogentisic a c i d ) and i s probably common to a l l photosynthetic higher p l a n t s . Tyrosine a l s o gives r i s e to 3 » k - d i h y d r o x y p h e n y l a c e t i c a c i d i n A s t i l b e but the extent of t h i s r e a c t i o n i n the p l a n t kingdom i s not known. In a number of p l a n t s , t y r o s i n e i s hydroxylated 23 to form 3>4-dihydroxyphenylalanine (DOPA) . iii 14 Ruta was incubated w i t h D0PA-2- C and the C0 2 14 re l e a s e compared w i t h that from t y r o s i n e - 2 - C (Table IV.) Although some of the DOPA may be l o s t by o x i d a t i v e poly-m e r i z a t i o n w i t h i n the c e l l s , i t seems u n l i k e l y that t y -r o s i n e i s being degraded v i a i t s hydroxy d e r i v a t i v e i n t h i s case. The p o s s i b i l i t y of 2,5-dihydroxyphenylacetic a c i d (DPA) involvement i n the t y r o s i n e s i d e - c h a i n degradation was checked i n two ways. ( i ) 2 5 yuM of DPA was added to one of two c u l t u r e s of 14 M e l i l o t u s m e t a b o l i z i n g t y r o s i n e - 2 - C. Over the subse-14 quent 24 hr i n c u b a t i o n , a 30% decrease i n the C0 2 out-put by the tr e a t e d c u l t u r e ( r e l a t i v e to the c o n t r o l ) was observed. The same amount of DPA added to one of two 14 c u l t u r e s m e t a b o l i z i n g glucose-U- C had no e f f e c t on 14 C0 2 r e l e a s e . ( i i ) A heavy inoculum of M e l i l o t u s t i s s u e was incubated 14 f o r 24 hr w i t h 2 ^ uC DL-tyrosine-2- C and 25 jm DPA. The t i s s u e was then e x t r a c t e d w i t h 95$ ethanol (10 dpm eth-anol s o l u b l e ) . This e x t r a c t was taken up i n hot water, f i l t e r e d , hydrolyzed w i t h a c i d and extracted w i t h ether -73-f o r 48 hours. The ether e x t r a c t was chromatographed on A v i c e l TLC p l a t e s i n the f o l l o w i n g solvent systems: (1) benzene:acetic acid:water (10:7:3 org. phase) 2x dev. (2) methyl e t h y l ketone:acetone:formic acid:water (80:4:2:12) (3) 2% formic a c i d The DPA band a f t e r t h i s p u r i f i c a t i o n procedure s t i l l con-tained a t l e a s t 20,000 dpm, d e s p i t e considerable l o s s e s of the compound by o x i d a t i o n . These experiments i n d i c a t e that t y r o s i n e i s forming 2,5-dihydroxyphenylacetic a c i d i n a p l a n t t i s s u e that i s p r e s e n t l y non-photosynthetic and presumably has no r e q u i r e -ment f o r plastoquinone s y n t h e s i s . I t i s tempting to sugg-est that the formation of DPA normally serves as a route to the plastoquinones as w e l l as f o r the degradation of t y r o s i n e . The c o n t r o l over the extent of t y r o s i n e con-v e r s i o n to DPA may be c h l o r o p l a s t - and light-dependent, as 25 29 e a r l i e r s t u d i e s on t y r o s i n e degradation have suggested The Ruta used i n the present s t u d i e s i s a green t i s s u e w i t h s t r u c t u r a l l y normal c h l o r o p l a s t s ^ 2 , and i t shows a notably greater tendency to degrade t y r o s i n e than does M e l i l o t u s (Table I V . ) . While these experiments are p r e l i m i n a r y i n nature, the a b i l i t y of p l a n t t i s s u e s to c a r r y out aromatic r i n g -f i s s i o n seems e s t a b l i s h e d . Since both phenylalanine-r i n g - l - ^ C and tryptophan-benzene ring-U-^^C are prepared by s p e c i f i c chemical syntheses, there i s no reason to sus--74-pect s u b s t a n t i a l l a b e l l i n g of these compounds i n non-aromatic p o s i t i o n s . The p o s s i b i l i t y e x i s t s that a v o l a -t i l e aromatic a c i d i s being synthesized from phenylalan-ine and tryptophan i n these t i s s u e c u l t u r e s . The low vapor pressures of the known aromatic a c i d s , however, and the two step CO2 i s o l a t i o n procedure make i t very u n l i k e l y that the a c t i v i t y recovered i n the 2-phenylethylamine i s due to compounds other than CO2. In a t l e a s t one case, the aromatic r i n g of cinnamic a c i d can a l s o be degraded, an important p o i n t i n p l a n t phenolic metabolism. Whether or not the low percentage of ring-cleavage observed i n the s t e r i l e c u l t u r e s i s t y p i c a l of the true l e v e l i n i n t a c t p l a n t s can only be a s c e r t a i n e d by use of s t e r i l e l y c u l t u r e d p l a n t s and s t e r i l e procedures. I t i s p o s s i b l e that a r e -duced emphasis on "secondary metabolism" i n the c u l t u r e s reduces the observed r i n g - f i s s i o n . D e f i n i t i v e s t u d i e s on t y r o s i n e degradation await the a v a i l a b i l i t y of r i n g - ^ C -t y r o s i n e . - 7 5 -LITERATURE CITED 1. Brown, S.A. 1 9 6 4 . L i g n i n and tannin b i o s y n t h e s i s , Biochemistry of Phenolic Compounds, p. 3 6 1 , J.B. Harborne, ed., Academic Press, London and New York. 2. The A l k a l o i d s , Chemistry and Physiology. 1 9 6 4 . ' Manske, R.H.F., ed., Academic Press, London and New York. 3. Neish, A.C. 1 9 6 4 . Major pathways of bi o s y n t h e s i s of phenols, Biochemistry of Phenolic Compounds, p. 2 9 5 , J.B. Harborne, ed., Academic Press, London and New York. 4 . Leete, E. 1 9 6 7 . A l k a l o i d biogenesis, Biogenesis of Natur a l Compounds (2nd Ed.). P. B e r n f e l d , ed., P« 9 5 3 , Pergamon Press, Oxford. 5. N a i r , P.M. and Vaidyanathan, C.S. 1 9 6 6 . Conversion of isophenoxazine to catechol i n Tecoma stans, Arch. Biochem. Biophys. 1 1 5 ; 5 1 5 « 6. Subba Rao, P.V., Moore, K. and Towers, G.H.N. I 9 6 7 . The conversion of tryptophan to 2,3-dihydroxy-benzoic a c i d and catechol by A s p e r g i l l u s n i g e r , Biochem. Biophys. Res. Comm. 28:1008~ 7. Jakoby, W.B. and Bonner, D.M. 1 9 5 3 . Kynureninase from Neurospora: p u r i f i c a t i o n and p r o p e r t i e s , J . B i o l . Chem. 2 0 5 : 6 9 9 . 8. Taniuchi, H. and Ha y a i s h i , 0. 1 9 6 3 . Studies on the metabolism of kynurenic a c i d . I I I . Enzymatic formation of 7 ,8-dihydroxykynurenic a c i d from kynurenic a c i d . J . B i o l . Chem. 2 3 8 : 2 8 3 . 9 . Moore, T.C. and Shaner, C.A. I 9 6 7 . B i o s y n t h e s i s of i n d o l e a c e t i c a c i d from tryptophan - l 4 c i n c e l l -f r e e e x t r a c t s of pea shoot t i p s , P l a n t P h y s i o l . 42 : 1 7 8 7 . 1 0 . Galston, A.W., Bonner, J . and Baker, R.S. 1 9 5 3 . F l a v o p r o t e i n and peroxidase as components of the i n d o l e a c e t i c a c i d oxidase system of peas, Arch. Biochem. Biophys. 42 : 4 5 6 . 11. Gamborg, O.L. and Wetter, L.R. 19^3. An aromatic amino a c i d transaminase from mung bean, Can. J . Biochem. P h y s i o l . 4 l : 1 7 3 3 . -76-12. Towers, G.H.N, and Subba Rao, P.V. 1969. Degrad-a t i v e metabolism of phenylalanine, t y r o s i n e and DOPA, i n press 9th Annual Symposium of Phyto-chemical S o c i e t y of North America, Banff, A l t a . 13. Cain, R.B. B i l t o n , R.F. and Darrah, J.A. I 9 6 8 . The metabolism of aromatic compounds by microorgan-isms. Metabolic pathways i n the f u n g i , Biochem. J . 108:797. 14. N a i r , P.M. and V i n i n g , L.C. 1 9 6 5 . Phenylalanine hydroxylase from spinach leaves, Phytochem. 4:401. 1 5 . Vaughan, P.F.T. and Butt, V.S. 1 9 6 9 . The hydroxyl-a t i o n of p_-coumaric a c i d by an enzyme from leaves of spinach beet (Beta v u l g a r i s L . ) , Biochem. J . 113 : 1 0 9 . 16. Byerrum, R.U., F l o k s t r a , H.H., Dewey, L . J . and B a l l , C D. 195^. In c o r p o r a t i o n of formate and the methyl group of methionine i n t o methoxyl groups of l i g n i n , J . B i o l . Chem. 210:633. 17. Vollmer, K.O., Reisener, H.J. and Grisebach, H. 1965. The formation of a c e t i c a c i d from p_-hydroxycinnamic a c i d during i t s degradation to 2-hydroxybenzoic a c i d i n wheat shoots, Biochem. Biophys. Res. Comm. 21:221. 18. Zenk, M.H. 1964. Einbau von p_-hydroxybenzoesaure i n d i e Hydrochinonkomponente des Ar b u t i n s i n Bergenia c r a s s i f o l i a , Z. Naturforsch. 1 9 6 : 8 5 6 . 19. E l l i s , B.E. and Towers, G.H.N. 1969. The biogenesis of catechol i n G a u l t h e r i a , Phytochem. 8 : l 4 l 5 . 20. Vollmer, K.0. and Grisebach, H. 1966. Zur biosyn-these der Benzoesauren i n G a u l t h e r i a procumbens. I I I . Z. Naturforsch. 2 1 6 : 4 3 5 . 21. McCalla, D.R. and Neish, A.C. 1 9 5 9 . Metabolism of phenylpropanoid compounds i n S a l v i a . I I . B i o -synthesis of phenolic cinnamic a c i d s , Can. J . Biochem. P h y s i o l . 37:537. 22. K i n d l , H. 1 9 6 9 . B i o s y n t h e s i s and metabolism of hy-droxyphenylacetic a c i d s i n higher p l a n t s , European J . Biochem. 7:340. -77-23. Kovacs, P. and J i n d r a , A. 1964. B i o s y n t h e s i s of a l k a l o i d s . On the transformation of t y r o s i n e to 3,'4-dihydroxyphenylalanine i n Pa paver somnif-erum L. p l a n t s , E x p e r i e n t i a 21:18. 24. E l l i s , B.E. and Towers, G.H.N. 1969. The biogenesis of rosmarinic a c i d i n Mentha, i n prep. 25. Runeckles, V.C. I 9 6 3 . Formation of sugars from phenylpropanoid compounds i n tobacco l e a f d i s c s , Can. J . Botany 41:823. 26. Zaprometov, M.N. 1959. On the a b i l i t y of higher p l a n t s to cleave the benzene r i n g . Deep o x i d -a t i o n of l^C-catechins i n tea, Doklady Academii Nauk SSSR 125:1359. 27. Henry, E.W., Valdovinos, J.G. and Jensen, T.E. 1968. Invasion of p l a n t t i s s u e by b a c t e r i a under i n v i t r o c o n d i t i o n s , P l a n t . P h y s i o l . 43:1730. 28. H i l l i s , W.E. and I s o i , K. 1965. The b i o s y n t h e s i s of polyphenols i n Eucalyptus species, Phytochem. 4 : 9 0 5 . 29. Ibrahim, R.K., Lawson, S.G. and Towers, G.H.N. 1961. Formation of l a b e l l e d sugars from L - t y r o s i n e -l^C i n some higher p l a n t s , Can. J . Biochem. P h y s i o l . 3 9 : 8 7 3 . 30. Dougall, D.K. and Shimbayashi, K. i 9 6 0 . Factors a f f e c t i n g growth of tobacco c a l l u s t i s s u e and i t s i n c o r p o r a t i o n of t y r o s i n e , P l a n t P h y s i o l . 25:396. 31. M i l l e r , H.E., R o s i e r , H., Wohlpart, A., Wyler, H., Wilcox, M.E., Frohofer, H., Mabry, T.J. and D r e i d i n g , A.S. 1968. Biogenese der B e t a l a i n e . B i o t r a n s f o r m a t i o n von DOPA und Tyrosin i n den B e t a l a m i n s a u r e t e i l des Betanins, Helv. Chim. Acta ^51:1470. 32. Whistance, G.R. and T h r e l f a l l , D.R. 1968. Biosyn-t h e s i s of phytoquinones. B i o s y n t h e t i c o r i g i n s of the n u c l e i and s a t e l l i t e methyl groups of plastoquinone, tocopherols, and tocopherol-quinones i n maize shoots, bean shoots, and i v y leaves, Biochem. J . 109:577. -78-33. Patschke, V.L., Hess, D. and Grisebach, H. 1964. Uber den A'b'bau von 4 , 2 ' , 4 * , 6'-Tetrahydroxy-chalkon - 2-glucosid und 4 , 2 ' , 4'-Trihydroxy-cha l k o n - 4 - g l u c o s i d i n Rotkohlkeimlingen und Petunien, Z. Naturforsch. 19° :1114. 34. Patschke, U.L., Barz, W. and Grisebach, H. 1964. Uber den Einbau von 5 . 7 . 4'-Trihydroxyflavanon-2,6 ,8 ,10- l4c i n Cyanidin und d i e Isoflavone Biochanin-A und Formononetin, Z. Naturforsch. 19bs1110. 35. Jayasankar, N.P., Bandoni, R.J. and Towers, G.H.N. 1969. Fungal degradation of p h l o r i d z i n , Phyto-chem. 8;379. 36. Westlake, D.W.S. and Spencer, J.F.T. 1966. The u t i l i z a t i o n of f l a v o n o i d compounds by yeasts and y e a s t - l i k e f u n g i , Can. J . M i c r o b i o l . 12;165• 37. C r a i g i e , J.S., McLachlan, J . and Towers, G.H.N. 1965. A note on the f i s s i o n of an aromatic r i n g by algae, Can. J , Botany 43:1589. 38. Garay, A.S. and Towers, G.H.N. 1965. On the a b i l i t y of tomato seedlings to cleave the benzene r i n g , unpublished r e s u l t s . 39. Jansen, E.F. and Olson, A.C. 1969.Metabolism of car-bon - l 4-labeled benzene and toluene i n avocado f r u i t , P l a n t P h y s i o l . 44:766. 40. Durmishidze, S.V. and Ugrekhelidze, D. Sh. 1969. S p l i t t i n g of phenol by the tea p l a n t , Doklady Akademii Nauk SSSR 184:228. 4 1 . Durmishidze, S.V., Ugrekhelidze, D. Sh., D z h i k i a , A.N. and Ts e v e l i d z e , D. Sh. 1969. Intermediate products of the fermentative o x i d a t i o n of benzene and phenol, Doklady Akademii Nauk SSSR 184:466. 42. Evans, W.C., Smith, B.S.W., Li n s t e a d , R.P. and El v i d g e , J.A. 1951- Chemistry of the o x i d a t i v e metabolism of c e r t a i n aromatic compounds by micro-organisms, Nature 168:772. 43. Rosa, N. 1966. Ph.D. t h e s i s , Dalhousie U n i v e r s i t y , H a l i f a x , N.S. 44. Gamborg, O.L., M i l l e r , R.A. and Ojima, K. 1968. N u t r i e n t requirements of suspension c u l t u r e s of soybean root c e l l s , Exp. C e l l Research 50:151. - 7 9 -4 5 . Towers, G.H.N, and Subba Rao, P.V. I 9 6 9 . Phenyl-a l a n i n e ammonia-lyase ( U s t i l a g o hordei) i n press Methods i n Enzymology. 46. Wang, C.H. and W i l l i s , D.L. 1 9 6 5 . R a d i o t r a c e r methodology i n b i o l o g i c a l science, p. 168, P r e n t i c e - H a l l , Inc^., New Jersey. 4 7 . Staba, E.J. 1 9 6 9 . P l a n t t i s s u e c u l t u r e as a tech-nique f o r the phytochemist. Recent Advances i n Phytochemistry v o l . I I . S e i k e l , M.K. and Runeckles, V.C. ed, Appleton-Century-Crofts, New York. 48 . Gamborg, O.L. and Ev e l e i g h , D.E. 1 9 6 8 . C u l t u r e methods and d e t e c t i o n of glucanases i n suspen-s i o n c u l t u r e s of wheat and b a r l e y , Can. J . Biochem. 4 6 : 4 1 7 . 4 9 . Rosa, N. and Neish, A.C, 1 9 6 8 . Formation and occurrence of N-malonylphenylalanine and r e l -ated compounds i n p l a n t s , Can, J . Biochem. 46:797 . 50. el-Basyouni, S.Z., Chen, D., Ibrahim, R.K., Neish, A.C. and Towers, G.H.N. 1 9 6 4 . The b i o s y n t h e s i s of hydroxybenzoic a c i d s i n higher p l a n t s , Phytochem. 3 : 4 8 5 . 51. Gamborg, O.L. Personal communication. 52. McBride, D.L. I 9 6 9 . Unpublished observations. -80-Appendix "A" ( i ) Tissue c u l t u r e media B5 medium* p e r l i t e r per l i t e r g l a s s - d i s t i l l e d g l a s s - d i s t i l l e d Compound water Compound water NaH 2P0k.H 20 150 mg Sucrose 20 gm KNO^ 2500 mg 2,4-D*** 1 mg (NHj^gSO^ 1 3 k mg Vitamin s o l u t i o n 10 ml MgS0 k.?H 20 250 mg M i c r o n u t r i e n t s CaCl 2.2H 20 150 mg s o l u t i o n 1 ml Fe(EDTA)** 28 mg PH 5 . 5 KI 0 . 7 5 mg Add 10$ excess water to compensate f o r lo s s e s i n auto-c l a v i n g and i n c u b a t i o n . * B5C2 medium c o n s i s t s of B5 medium plus 2 g m / l i t e r N-Z. Amine Type A ca s e i n hydrolysate, S h e f f i e l d Chem-i c a l , Norwich, N.Y. (B5C2 medium thus contains 102 mg phenylalanine /.L, 62 mg t y r o s i n e /L and 28 mg t r y -ptophan /.L. ) Sequestrene 330 Fe, G e i g i A g r i c u l t u r a l Chemicals, A r d s l e y , N.Y. *** 2 , 4-dichlorophenoxyacetic a c i d d i s s o l v e d i n 95$ ethanol (1 mg/ml). £ Stock s o l u t i o n . D i s s o l v e d i n 100 ml d i s t i l l e d water: 1000 mg MnS04-H 2 0, 300 mg H3BO3, 300 mg ZiiSOk. 7H2O, 25 mg Na 2 M o 0 k . 2 H 2 0 , 25 mg CuSOk, 25 mg CQC1 2 .6H20 stored f r o z e n . t Stock s o l u t i o n . D i s s o l v e d i n 100 ml d i s t i l l e d water: 10 mg n i c o t i n i c a c i d , 100 mg thiamine HC1, 10 mg pyr-i d o x i n e HC1 and 1000 mg m y o - i n o s i t o l stored f r o z e n i n p l a s t i c b o t t l e . -81-( i i ) P l a t i n g media f o r contamination monitoring. (a) D i f c o malt e x t r a c t agar 4 . 5 $ (b) D i f c o yeast e x t r a c t 1$ Glucose 1$ agar 1.5$ 

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