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UBC Theses and Dissertations

Some aspects of phenolic metabolism in healthy and rust infected flax cotyledons Lam, Tung Hoi 1971

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SOME ASPECTS OF PHENOLIC METABOLISM IN HEALTHY AND RUST INFECTED FLAX COTYLEDONS by TUNG HOI LAM B.Sc., M.Sc., U n i v e r s i t y of Hong Kong A Thesis Submitted i n P a r t i a l F u l f i l m e n t of the Requirements f o r the Degree of DOCTOR OF PHILOSOPHY i n the Department of Plant Science We accept t h i s t h e s i s as conforming to' the r e q u i r e d standard THE UNIVERSITY OF BRITISH COLUMBIA September, 1971 In p r e s e n t i n g t h i s t h e s i s i n p a r t i a l f u l f i l m e n t of 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 th a t 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 r eference and study. I f u r t h e r agree t h a t permission f o r 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 Depart-ment or by h i s r e p r e s e n t a t i v e s . I t i s understood th a t copy-i n g 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 Plant Science, U n i v e r s i t y of B r i t i s h Columbia, Vancouver 8, B r i t i s h Columbia. ACKNOWLEDGEMENTS I wish to express my s i n c e r e a p p r e c i a t i o n to Dr. Mi c h a e l Shaw, Dean of A g r i c u l t u r a l Sciences, U n i v e r s i t y of B r i t i s h Columbia, under whose s u p e r v i s i o n t h i s t h e s i s was conducted, f o r h i s v a l u a b l e advice and guidance and h i s c r i t i c i s m and help i n the p r e p a r a t i o n of t h i s manuscript. I am deeply indebted to s e v e r a l members of Dean Shaw's h o s t - p a r a s i t e group f o r t h e i r v a l u a b l e advice and encouragement, e s p e c i a l l y Dr. R.K. Ibrahim, Dr. A. K. Chakravorty and Mr. L.A. Scrubb. I am g r a t e f u l to the Commonwealth S c h o l a r s h i p and Fe l l o w s h i p Committee of the A s s o c i a t i o n of U n i v e r s i t i e s and Colleges of Canada f o r f i n a n c i a l support i n the form of a s c h o l a r s h i p , to Mrs. G. Smith f o r t y p i n g the t h e s i s and Mr. I . D e r i c s f o r a s s i s t a n c e i n pre p a r i n g the photographs. Equipment and s u p p l i e s were provided by the N a t i o n a l Research C o u n c i l through a grant to Dean Shaw. I would a l s o l i k e to thank the members of my graduate committee f o r t h e i r i n t e r e s t i n my research and the review-i n g of t h i s t h e s i s . i v ABSTRACT Phytochemical and enzymatic experiments were con-ducted to study the metabolism of phenolic compounds i n the cotyledons of f l a x (Linum u s i t a t i s s i m u m L. TKoto») i n f e c t e d w i t h s t r a i n s #3. and #210 of f l a x r u s t (Melampsora l i n i Pers. Lev.). The primary o b j e c t i v e was to f u r t h e r the understand-i n g of the r o l e of phenolic compounds i n the h o s t - p a r a s i t e r e l a t i o n s h i p w i t h respect to r e s i s t a n c e and s u s c e p t i b i l i t y . The phenolic c o n s t i t u e n t s of f l a x i n c l u d e about 14 est e r s and gl y c o s i d e s of cinnamic a c i d s , v i z . , p-coumaric, c a f f e i c , f e r u l i c and s i n a p i c a c i d s , and 8 g l y c o s i d e s of f l a v o n e s , 4 of which.are of the apigenin-type and 4 of the l u t e o l i n - t y p e . Most of the cinnamic a c i d d e r i v a t i v e s have a f r e e hydroxyl. group and would t h e r e f o r e be good substrates f o r o x i d a t i o n . Except f o r an i n i t i a l drop, the t o t a l s o l u b l e phenolic content i n i n f e c t e d r e s i s t a n t t i s s u e was always higher than i n the healthy c o n t r o l or i n i n f e c t e d s u s c e p t i b l e t i s s u e . This q u a n t i t a t i v e change i n phenolic content a f t e r i n f e c t i o n supports the involvement of phenolics i n r e s i s t a n c e . Tracer s t u d i e s showed that the metabolism of phenyl-a l a n i n e i n f l a x f o l l o w s the order cinnamic ->p-coumaric > c a f f e i c ^ f e r u l i c a c i d s . There was no q u a l i t a t i v e change i n the pathway of phenylalanine metabolism a f t e r i n -f e c t i o n . The. i n c o r p o r a t i o n of phenylalanine-U-^^C i n t o phenolic compounds was higher i n the r e s i s t a n t combination than i n the health y c o n t r o l or the s u s c e p t i b l e combination. • V The r e s i s t a n t r e a c t i n g t i s s u e a l s o showed the highest con-v e r s i o n of monohydric phenols i n t o d i h y d r i c phenols. On the other hand, i n c o r p o r a t i o n of phenylalanine-U-^C i n t o p r o t e i n was highest i n the s u s c e p t i b l e combination. There was a higher accumulation of r a d i o a c t i v i t y from p h e n y l a l a -nine-U-^C i n t o e t h a n o l - i n s o l u b l e , non-proteinaceous m a t e r i a l around the l e s i o n s i n the r e s i s t a n t than i n the s u s c e p t i b l e combination. These f i n d i n g s are i n agreement w i t h the hypothesis t h a t , a f t e r i n f e c t i o n , there i s an enhanced f l o w of aromatic amino a c i d s i n t o p r o t e i n s y n t h e s i s i n the sus-c e p t i b l e t i s s u e whereas i n the r e s i s t a n t r e a c t i n g t i s s u e s there i s a s h i f t i n favour of phenolic metabolism. The enhancement of phenylalanine arnmonia-lyase by as much as 5 - f o l d i n the r e s i s t a n t t i s s u e at 2 days a f t e r inocu-l a t i o n a l s o supports' the above hypothesis. The a c t i v i t i e s of peroxidase, polyphenol oxidase and ^ -glucosidase were a l s o enhanced i n the r e s i s t a n t combination, whereas i n the sus-c e p t i b l e combination polyphenol oxidase and ^ - g l u c o s i d a s e a c t i v i t i e s were lower than i n the h e a l t h y c o n t r o l . There was a s e q u e n t i a l enhancement of phenylalanine ammonia-lyase, t o t a l s o l u b l e phenolic content and polyphenol oxidase i n the r e s i s t a n t r e a c t i n g t i s s u e . These r e s u l t s suggest t h a t oxida-t i o n of phenolic compounds i s important f o r r e s i s t a n c e and tha t the suppression of the o x i d a t i v e enzyme, polyphenol, oxidase', may be e s s e n t i a l f o r the s u r v i v a l of the pathogen i n t h i s biotroph-host combination.. v i The evidence suggests t h a t phenolic metabolism plays an important r o l e i n r e s i s t a n c e and s u s c e p t i b i l i t y i n host-p a r a s i t e r e l a t i o n s . I t i s very l i k e l y t hat phenolic com-pounds and t h e i r o x i d a t i v e products only execute the job of r e s i s t a n c e . The t r i g g e r i n g mechanism f o r the enhance-ment of phenolic metabolism, which remains unknown, and the mechanisms by which phenolic m e t a b o l i t e s act against the pathogen are d i s c u s s e d . v i i TABLE OF CONTENTS Page ACKNOWLEDGEMENTS i i i ABSTRACT i v TABLE OF CONTENTS v i i LIST OF ABBREVIATIONS x LIST OF TABLES x i LIST OF FIGURES x i i INTRODUCTION 1 LITERATURE REVIEW 3 1. Phenolic compounds and pl a n t diseases 3 2. Disease r e s i s t a n t mechanisms of phenolic compounds 16 A. E f f e c t of phenols • 1 6 B. E f f e c t of quinones 1 9 3. Metabolism of ph e n o l i c s i n he a l t h y and diseased t i s s u e s 2 4 A. Metabolism of phen o l i c s i n he a l t h y t i s s u e 2 4 B. E f f e c t of i n f e c t i o n on metabolism of phe n o l i c s 28 4. Some enzymes i n v o l v e d i n phenolic metabolism 3 0 A. Phenylalanine ammonia-lyase 3 0 B. 3 - g l u c o s i d a s e 3 5 C. Polyphenol oxidase 3 7 D. Peroxidase 4 0 5. Phenolic c o n s t i t u e n t s of f l a x and host-p a r a s i t e r e l a t i o n s 43 MATERIALS AND METHODS 4 9 1. P l a n t m a t e r i a l s 4 9 2, Phytochemical s t u d i e s 5 0 A. E x t r a c t i o n of phenolic compounds 5 0 B. H y d r o l y s i s 5 1 C. Chromatography 53 D. U-V ab s o r p t i o n spectrophotometry 55 E. E s t i m a t i o n of phenolic content 57-F. Incubation procedure f o r t r a c e r s t u d i e s 5 7 G. Autoradiography 5 8 H. L i q u i d s c i n t i l l a t i o n counting 5 9 v i i i Page 3 . Enzyme s t u d i e s 60 A. Enzyme e x t r a c t i o n 6 0 B. P r o t e i n e s t i m a t i o n 6 0 C. Enzyme assays 6 1 D. Polyacrylamide g e l e l e c t r o p h o r e s i s 63 E. Isozyme s t u d i e s , 65 F. A n a l y s i s of phenylalanine-U- C l a b e l e d p r o t e i n s " 6 6 RESULTS 6 9 SECTION A: Phenolic compounds of f l a x 6 9 I . The i d e n t i f i c a t i o n of phenolic compounds i n Koto f l a x 6 9 1. Phenolic a c i d d e r i v a t i v e s 6 9 2. Flavonoid d e r i v a t i v e s 7 7 I I . Phenolic compounds and r u s t i n f e c t i o n 7 9 SECTION B: Metabolism of l a b e l e d precursors of phenolic compounds S i I . Metabolism of t y r o s i n e - U - ^ C , D0PA-6 - 1 Z fC, phenylalanine-U-^^C and cinnamate-g --^C S i I I . F u rther s t u d i e s of the metabolism of phenyl-a l a n i n e - U - H c 8 6 1. Accumulation -, , 8 6 2. The metabolism of phenylalanine-U- C i n t o s o l u b l e phenolic compounds 8 9 ( i ) I n c o r p o r a t i o n $ 9 ( i i ) Comparison of the i n c o r p o r a t i o n of l a b e l i n g i n t o mono- and d i - h y d r i c phenolic compounds 9 1 3 . The metabolism of phenylalanine-U- C^ i n t o p r o t e i n s 9 2 ( i ) I n c o r p o r a t i o n 9 2 ( i i ) The l a b e l i n g p a t t e r n of p r o t e i n s 9 4 SECTION C: P r o t e i n s and enzymes 9 8 I . Dowex method 9<3 I I . P r o t e i n and enzymes 1 0 4 1. T o t a l p r o t e i n content 1 0 4 2. Phenylalanine ammonia-lyase 1 0 4 3 . Peroxidase 1 0 7 4. Polyphenol oxidase 110 5. B-glucosidase 110 I I I . E f f e c t of water i n f i l t r a t i o n on enzyme a c t i v i t i e s 1 1 3 i x Page DISCUSSION 1 1 9 SECTION I : Phenolic compounds of f l a x 1 1 9 SECTION I I : Metabolism of phenolic precursors 1 2 3 1. M e t a b o l i c pathway 1 2 3 2. Accumulation 1 2 6 3 . I n c o r p o r a t i o n 1 2 $ (a) Phenolics vs. p r o t e i n 1 2 8 (b) Monohydric phenol vs. d i h y d r i c phenol 1 3 0 SECTION I I I : Enzymes 1 3 2 1. Phenylalanine ammonia-lyase 1 3 2 2. Peroxidase 1 3 5 • 3 . Polyphenol oxidase 1 3 7 4. P-Glucosidase 1 3 9 5. S e q u e n t i a l changes 1 4 0 SECTION IV: General d i s c u s s i o n 1 4 2 SUMMARY AND CONCLUSION 1 4 6 LITERATURE CITED 1 5 1 X LIST OF ABBREVIATIONS Act. D. Actinomycin D n-BAW Normal-butanol:acetic acid:water t-BAW T e r t i a r y - b u t a n o l : a c e t i c acid:water BzAW Benzene:acetic acid:water CMV Cucumber mosaic v i r u s DOPA 3 , 4,-dihydroxyphenylalanine E-4-P Erythrose - 4 -phosphate EMP Embden-Meyerhof pathway HOAc A c e t i c a c i d IAA 3-Indole a c e t i c a c i d NAD Nicotinamide adenine d i n u c l e o t i d e _ NADP Nicotinamide adenine d i n u c l e o t i d e phosphate 0 .D. O p t i c a l d e n s i t y PAL Phenylalanine ammonia-lyase PEP Phosphoenol pyruvic a c i d Phe Phenylalanine PPO Polyphenol oxidase PVP P o l y v i n y l p y r o l i d o n e RNA Ri b o n u c l e i c a c i d TAL Tyrosine ammonia-lyase TEE T o t a l ethanol e x t r a c t TLC Thin l a y e r chromatography (or p l a t e ) TMV Tobacco mosaic v i r u s UV U l t r a - v i o l e t l i g h t H Healthy R I n f e c t e d r e s i s t a n t t i s s u e S I n f e c t e d s u s c e p t i b l e t i s s u e A Apigenin C C a f f e i c a c i d pC p-Coumaric a c i d F F e r u l i c a c i d L L u t e o l i n S i S i n a p i c a c i d LIST OF TABLES x i Table Page I I I I I IV V VI V I I V I I I IX X XI X I I X I I I A l i s t of antipathogenic substances found i n higher p l a n t s . Some r e p o r t s on the changes i n peroxidase i n disease i n f e c t e d p l a n t s . The reagents f o r the p r e p a r a t i o n of poly-acrylamide g e l . Chromatographic p r o p e r t i e s of the phenolic compounds of f l a x . S p e c t r a l p r o p e r t i e s of some blue f l u o r e s c e n t compounds i s o l a t e d from f l a x . S p e c t r a l p r o p e r t i e s of the f l a v o n o i d s i s o -l a t e d from f l a x . The i n c o r p o r a t i o n of phenylalanine-U-^C i n t o phenolic compounds. 1 4 The metabolism of phenylalanine-U- C i n t o c a f f e i c and p-coumaric a c i d s . The i n c o r p o r a t i o n of pheny l a l a n i n e - U - ^ C i n t o p r o t e i n . A comparison of the d i f f e r e n t methods f o r p r o t e i n e x t r a c t i o n . The r e t e n t i o n c a p a b i l i t y of Dowex 1x8 on standard and f l a x phenolic compounds. Peroxidase a c t i v i t i e s i n enzyme e x t r a c t s prepared u s i n g d i f f e r e n t q u a n t i t i e s of Dowex 1x8. The i n c o r p o r a t i o n of phenylalanine-U-^^C i n t o p r o t e i n by i n f i l t r a t i o n method. 4 4 6 4 72 7 6 7 8 9 0 9 2 93 1 0 0 1 0 2 1 0 3 1 1 8 x i i LIST OF FIGURES Figur e Page 1 Host-pathogen r e l a t i o n s w i t h respect to disease r e s i s t a n c e . 4 '2 Reactions between quinone and t h i o l s , amino groups and p r o t e i n . 21 3 The metabolic pathways of phenolic compounds. 25 4 A f l o w chart of e x t r a c t i o n and stepwise h y d r o l y s i s of phenolic compounds. 52 5 Apparatus f o r . s l i c i n g and d r y i n g g e l s from polyacrylamide g e l e l e c t r o p h o r e s i s . 67 6 U V f l u o r e s c e n c e p i c t u r e s of the TEE of h e a l t h y and r u s t e d cotyledons. 70 7 The i d e n t i f i c a t i o n of phenolic m o i e t i e s of f l a x phenolic compounds. 71 8 A t r a c e of a chromatogram of the ether f r a c -t i o n from the a l k a l i n e h y d r o l y s a t e of TEE. 73 9 Photographs of a chromatogram of the ether f r a c t i o n from the a l k a l i n e h y d r o l y s a t e of TEE. 74 10 The t o t a l p h enolic content of h e a l t h y and r u s t - i n f e c t e d f l a x cotyledons. 80 11 Autoradiogram of TLC prepared from some f e e d i n g experiments. $ 3 12 Autoradiogram of TLC prepared from phenyl-alanine-U-l^-C f e e d i n g experiments. 85 13 Autoradiogram of TLC prepared from cinnamate-3 _14Q f e e d i n g experiments. 87 14 Autoradiogram of TLC prepared from the ether f r a c t i o n of h y d r o l y s a t e of TEE from cinnamate-g -14c f e e d i n g experiments, 87 15 Autoradiogram of whole cotyledons from p h e n y l a l a n i n e - U - l ^ c f e e d i n g experiments. 88 Chromoscan pa t t e r n s of the autoradiograms of gels c o n t a i n i n g l a b e l e d p r o t e i n s from 1 day o l d cotyledons. Chromoscan pa t t e r n s of the autoradiograms of g e l s c o n t a i n i n g l a b e l e d p r o t e i n s from 6 day o l d cotyledons. Chromoscan pa t t e r n s of the autoradiograms of g e l s c o n t a i n i n g l a b e l e d p r o t e i n s from 9 day o l d cotyledons. T o t a l p r o t e i n e s t i m a t i o n of health y and ru s t e d f l a x cotyledons. A comparison of PAL a c t i v i t i e s i n he a l t h y and rusted f l a x cotyledons. Peroxidase a c t i v i t y i n h e a l t h y and r u s t e d f l a x cotyledons. The isozyme p a t t e r n s of peroxidase and polyphenol oxidase i n f l a x cotyledons. Polyphenol oxidase a c t i v i t y i n health y and rusted f l a x cotyledons. 3 - G l u c o s i d a s e a c t i v i t y i n health y and rust e d f l a x cotyledons. The e f f e c t of i n f i l t r a t i o n w i t h water and s o l u t i o n s of other compounds on enzyme a c t i v i t i e s . -Temporal changes of enzyme a c t i v i t i e s a f t e r i n f i l t r a t i o n w i t h water, Act. D. or aqueous e x t r a c t of f l a x cotyledons. 1 INTRODUCTION Biochemistry provides a fundamental approach to the study and understanding of h o s t - p a r a s i t e r e l a t i o n s . Amongst the v a r i o u s metabolic systems that f u n c t i o n i n p l a n t s , those concerned w i t h the metabolism of phenolic compounds have pre-dominated i n attempts to e x p l a i n the b a s i s of r e s i s t a n c e or s u s c e p t i b i l i t y . But even so, the r e l a t i o n s h i p between phen-o l i c metabolism and r e s i s t a n c e i s s t i l l f a r from being s e t t l e d and there remain many c o n t r o v e r s i a l and unsolved problems. Do phenolic compounds confer r e s i s t a n c e on a host or do they accumulate as a r e s u l t of r e s i s t a n c e ? Are changes i n phenolic compounds a general or a s p e c i f i c mechanism of r e s i s t a n c e ? What changes occur i n the a c t i v i t y of the en-zymes concerned w i t h p h e n o l i c metabolism i n s u s c e p t i b l e and r e s i s t a n t t i s s u e s a f t e r i n f e c t i o n ? I n order to study changes i n phenolic metabolism i n an i n f e c t e d host, the s i m p l e s t approach i s to employ a s i n g l e host v a r i e t y which responds w i t h s u s c e p t i b l e and r e s i s t a n t r e a c t i o n s to v i r u l e n t and a v i r u l e n t p h y s i o l o g i c a l races of the same species of pathogen. This approach provides a con-sta n t b a s a l host metabolism i n which changes th a t f o l l o w i n -f e c t i o n can be i n t e r p r e t e d i n r e l a t i o n to the s u s c e p t i b l e and r e s i s t a n t responses. For the present work Linum u s i t a -tissimum L. T K o t o f served as the host and was i n o c u l a t e d w i t h race #210 or race #3 of the f l a x r u s t fungus (Melampsora  l i n i Pers. L e v . ) . Race #210 i s v i r u l e n t on Koto and produces a s u s c e p t i b l e r e a c t i o n ; race # 3 i s a v i r u l e n t on Koto, producing a r e s i s t a n t r e a c t i o n . The main o b j e c t i v e s of the i n v e s t i g a t i o n were: 1. To conduct q u a l i t a t i v e and q u a n t i t a t i v e analyses of the phenolic compounds present i n health y and s u s c e p t i b l e and r e s i s t a n t r e a c t i n g t i s s u e s , and to determine whether or not p a r t i c u l a r p h enolic p h y t o a l e x i n s are produced a f t e r i n f e c t i o n . 2. To i n v e s t i g a t e the routes of phenolic metabolism i n i n -f e c t e d and u n i n f e c t e d t i s s u e s by comparing the metabolism of administered p h e n y l a l a n i n e - U - ^ C , t y r o s i n e - U - ^ C , DOPA- B-1Zt-C and cinnamic a c i d - S - 1 4 C . 3. To analyse, q u a n t i t a t i v e l y the metabolism of phenylalanine i n t o phenolic compounds and p r o t e i n s i n the d i f f e r e n t k i nds of t i s s u e s . 4. To conduct a comparative study of the enzymes i n v o l v e d i n phen o l i c b i o s y n t h e s i s and degradation, i n order t o provide a d d i t i o n a l data on which to assess the s i g n i f i c a n c e of any changes observed i n ph e n o l i c l e v e l s or metabolic pathways f o l l o w i n g i n f e c t i o n . The experiments c a r r i e d out do not, of course, pro-v i d e c l e a r cut answers to a l l the problems r e l a t e d to the r o l e of p h e n o l i c s i n disease r e s i s t a n c e . Rather, i t i s hoped t h a t they provide some new ideas and data r e l a t i n g to t h i s d i f f i c u l t and c o n t r o v e r s i a l area of h o s t - p a r a s i t e r e l a -t i o n s h i p s . 3 LITERATURE REVIEW There i s a number of good reviews on phenolic metab-o l i s m i n h o s t - p a r a s i t e systems (Farkas and K i r a l y 1962, Cruickshank and P e r r i n 1964, Goodman et a l . 1967, Wood 1967, Rohringer and Samborski 1967 and Kosuge 1969). Most of these concentrate on the changes i n phenolic compounds that f o l l o w i n f e c t i o n but tend to neglect the enzymatic aspects of phen-o l i c metabolism i n s u s c e p t i b l e and r e s i s t a n t .reactions, as w e l l as the mechanisms by which phenolic compounds exert t h e i r a n t i b i o t i c a c t i v i t i e s . These aspects of what may be c a l l e d the 'phenolic hypothesis' of disease r e s i s t a n c e are t h e r e -f o r e emphasized i n the f o l l o w i n g resume''. 1. PHENOLIC COMPOUNDS AND PLANT DISEASES Disease r e s i s t a n c e and s u s c e p t i b i l i t y are most im-portant aspects of h o s t - p a r a s i t e r e l a t i o n s . Resistance can r e s u l t from three d i f f e r e n t c l a s s e s of mechanism: (1) s t r u c t -u r a l means, such as t h i c k c u t i c l e s or s p e c i a l i z e d stomata; (2) the l a c k of key m e t a b o l i t e s .essential f o r growth of a path-ogen and ( 3 ) substances, produced by the host, and capable of i n h i b i t i n g the growth of the pathogen, or of k i l l i n g i t . These mechanisms are shown s c h e m a t i c a l l y i n Figure 1. As the r e s u l t of years of i n v e s t i g a t i o n phenolic compounds are regarded as the most important agents c o n f e r r i n g r e s i s t a n c e to disease i n higher p l a n t s . The presence of these compounds i n host t i s s u e may prevent a p a r t i c u l a r pathogen from es-t a b l i s h i n g i t s e l f on a p a r t i c u l a r species of host. Very 4 F i g u r e 1. Host-Pathogen R e l a t i o n s w i t h Respect to Disease Resistance (Modified from Shaw 1967). o f t e n , these substances occur i n plant t i s s u e i n a 'bound', non-toxic form. On i n f e c t i o n , the t o x i c moiety i s r e l e a s e d and i n h i b i t s the pathogen, P h y t o a l e x i n s , most of which are phenolic i n nature, are a n t i - f u n g a l substances synthesized de novo i n higher p l a n t s a f t e r i n f e c t i o n . The preformed a n t i f u n g a l ' s u b s t a n c e s present i n p l a n t t i s s u e s can exert t h e i r a c t i v i t y e i t h e r (1) i n the form of exudates moving out of the p l a n t to a f f e c t germination and growth of the pathogen i n the e x t e r n a l environment, (2) i n the p r o t e c t i v e l a y e r s (eg. cork) to stop p e n e t r a t i o n , or (3) w i t h i n host c e l l s or t i s s u e s , a c t i n g a g a i n s t the patho-gens a f t e r they have penetrated epidermal c e l l s or stomatal pores. The best example of the d i f f u s i o n of a n t i f u n g a l sub-stances from host t i s s u e i s seen i n the r e s i s t a n c e of onions to i n f e c t i o n by C o i l e t o t r i c h u m c i r c i n a n s which causes onion smudge (Walker and Link 1935). The fungus i s a s o i l - b o r n e pathogen. F o l l o w i n g a short p e r i o d of saprophytic growth on dead outer s c a l e l e a v e s , the fungus grows i n t o and p a r a s i -t i z e s the i n n e r t h i c k f l e s h y s c a l e l e a v e s . V a r i e t i e s of onion w i t h y e l l o w or red outer s c a l e leaves are r e s i s t a n t . I n s p i t e of the a s s o c i a t i o n between c o l o r and r e s i s t a n c e , the pigments themselves are not the cause of r e s i s t a n c e , as was demonstrated by the f a c t that colored f l e s h y s c a l e s are attacked by the fungus. Resistance i s a c t u a l l y due to the f u n g i t o x i c a c t i o n of protocatechuic a c i d and c a t e c h o l which 6 d i f f u s e out of the dead s c a l e s i n t o the i n f e c t i o n drop and i n h i b i t germination of the c o n i d i a of the pathogen. M a r t i n _et a l . ( 1 9 5 7 ) showed that phenolic a c i d s pre-sent i n the wax l a y e r on the epidermis of apple leaves can i n h i b i t the p e n e t r a t i o n of the i n f e c t i o n hyphae of Podo-sphaera l e u c o t r i c h a . The waxy m a t e r i a l derived from leaves r e s i s t a n t to the mildew, when deposited on the leaves of s u s c e p t i b l e v a r i e t i e s would confer r e s i s t a n c e to the fungus. Kuc' and co-workers ( 1 9 5 6 ) worked on Helminthosporium  carbonum and potato and found t h a t chlorogenic and c a f f e i c a c i d i n the potato peel are f u n g i s t a t i c agents. Kuc' a l s o showed t h a t these compounds could i n h i b i t Cephalothecium  roseum and Myrothecium v e r r u c a r i a which are non-pathogenic to potato whereas two pathogens of potato tubers v i z . S c l e r o t i u m r o l f s i i and Fusarium s o l a n i f . r a d i c o l a are l e s s s e n s i t i v e (Kuc' 1 9 5 7 ) . Lee and Tourneau ( 1 9 5 $ ) showed that i n the case of V e r t i c i l l i u m w i l t disease of potatoes, v a r i e t i e s r e s i s t a n t to i n f e c t i o n c o n t a i n higher amounts of c h l o r o g e n i c a c i d i n the r o o t s than s u s c e p t i b l e ones. P a t i l and co-workers ( 1 9 6 2 ) demonstrated t h a t young potato r o o t s which are p r a c t i c a l l y r e s i s t a n t to i n f e c t i o n by V e r t i c i l l i u m spp. have a r e l a t i v e l y high l e v e l of c h l o r o g e n i c . a c i d u n t i l 5 weeks a f t e r s p r o u t i n g . From the time of sprout-i n g , c h l o r o g e n i c a c i d content decreased c o n t i n u o u s l y i n sus-c e p t i b l e h o s t s . The decrease was c l o s e l y c o r r e l a t e d w i t h an i n c r e a s e i n s u s c e p t i b i l i t y to i n f e c t i o n . They a l s o showed 7 t h a t the higher l e v e l of chlorogenic a c i d i n r e s i s t a n t v a r i e t i e s is.due to t h e i r greater s y n t h e t i c c a p a b i l i t y ( P a t i l et a l . 1 9 6 6 ) . Other common phenolic compounds which are claimed to be f u n g i t o x i c or a n t i b i o t i c and present i n p l a n t s i n c l u d e hydroquinone, j u g l o n e , p h l o r e t i n , isocoumarin, um-b e l l i f e r o n e and s c o p o l e t i n (Table I ) . Sometimes the i n i t i a l c o n c e n t r a t i o n of phenolic com-pounds present i n the t i s s u e i s not high enough t o i n h i b i t f u n g a l growth. A f t e r i n f e c t i o n the c o n c e n t r a t i o n of phenolic compounds i n c r e a s e s and reaches the t h r e s h o l d l e v e l necessary to i n h i b i t the pathogen. Farkas and K i r a l y ( 1 9 6 2 ) working on f u n g a l , b a c t e r i a l and v i r a l diseases found more accumula-t i o n of phen o l i c s i n the incompatible host-pathogen combina-t i o n . They compared the phenolic content of Vernal wheat i n -f e c t e d w i t h two races of P u c c i n i a graminis t r i t i c i v i z . #15B and # 2 1 which are v i r u l e n t and a v i r u l e n t r e s p e c t i v e l y on t h i s v a r i e t y and found t h a t there was an in c r e a s e i n the l e v e l of phenolic compounds at an e a r l i e r stage i n the incompatible combination. Such enhanced s y n t h e s i s of polyphenols and many other s y n t h e t i c processes are made p o s s i b l e by the i n c r e a s e i n r e s p i r a t i o n ( U r i t a n i 1 9 6 3 ) which provides the r e q u i r e d energy. A change i n the r e s p i r a t o r y pathway i n favour of the pentose phosphate pathway (Shaw and Samborski 1 9 5 7 ) can a l s o provide erythrose-4-phosphate f o r phenolic b i o s y n t h e s i s v i a the s h i k i m i c pathway. H y d r o l y s i s i s a very important process i n the r e l e a s e of t o x i c phenolic compounds against the fungus a f t e r i n f e c t i o n . TABLE I . A L i s t of Antipathogenic Substances i n Higher P l a n t s . SUBSTANCES STRUCTURAL FORMULA HOST I . PHENOLIC COMPOUNDS Benzoxazolinones NH C a f f e i c a c i d Catechol Chlorogenic a c i d H O \ / " C H = C H " c O O H H O Dihydroiosocoumarin F i c i n i n OC H = C H - C - O v \ y C O O H H o \ _ y O H HO HO M e O REFERENCES Maize, wheat V i r t a n e n 196$ rye potato C l a r k et a l . 1959 sweet potato U r i t a n i & Akazawa 1955 apple, pear Flood & Kirham I960 c a r r o t LeLaey & V i r t a n e n 195$ onion Walker & Link 1935 same as c a f f e i c a c i d c a r r o t Sondheimer 1961 0 3 -Neorautanenia Brink et a l . 1966 SUBSTANCES STRUCTURAL FORMULA H i r c i n o l 4-hydroxy-3-methoxy-benzoic a c i d ( V a n i l l i c a c i d ) 3-hydroxytyramine (Dopamine) oi -hydrojuglone Hydroquinone I s o p i m p i n e l l i n OMe H ° \ / ~ C O O H MeO HO HO H o Q h O H OMe OMe HOST REFERENCES Orchid sp. Gaumann 1963 apple Fawcett and Spencer 1968 sugar beet Gardner et a l . 1967 walnut Paxton 1964 pear Hildebrand & Schroth 1964 c i t r u s M a r t i n et a l . 1966 SUBSTANCES STRUCTURAL FORMULA Neoedulih N o b i l e t i n O r c h i n o l P h a s e o l l i n P h l o r e t i n P i n o s y l v i n HOST REFERENCES  Neorautanenia Duuren 1 9 6 1 c i t r u s Ben-Aziz 1 9 6 7 Orchid sp. Gaumann 1 9 6 3 French beans Cruickshank 1 9 6 3 apple Avadhani & Towers 1 9 6 1 pines S c h e f f e r & Cowling 1 9 6 6 SUBSTANCES STRUCTURAL FORMULA P i s a t i n MeO Protocatechuic a c i d H O - / V c O O H S c o p o l e t i n T r i c h o c a r p i n B - D - G l u c - O ffVcO-O-CH, HO T r i f o l i r h i z i n B- D - G l u c - O U m b e l l i f e r o n HOST REFERENCES peas Cruickshank 1 9 6 3 onion Walker & Li n k 1 9 3 5 sweet potato Minamikawa et a l . 1 9 6 3 Populus. sp. Leoschke & Franksen 1 9 6 4 red c l o v e r Bredenberg & H i e t a l a 1 9 6 1 sweet potato Minamikawa et a l . 1 9 6 3 H SUBSTANCES STRUCTURAL FORMULA HOST REFERENCES I I . NON-PHENOLIC COMPOUNDS A l l i c i n C H , : C H - C H 2 - S - S - C H 2 - C H : C H . Avenacin C H 2 O H oc-chaconine R O Gossypol Ipomeamarone g a r l i c oats potato R = 2 , 4 - d i - O - L - R h a m - D - G I uc c o t t o n C a v a l l i t o & B a i l e y 1944 Burkhardt et a l . 1964 Kuhn et a l . 1955 H e i n s t e i n et a l . 1962 sweet potato B i r c h et a l . 1954 S U B S T A N C E S S T R U C T U R A L F O R M U L A • I p o m e a n i n e I s o t h i o c y a n a t e s C H 2 = C H - C K 2 - N = C = S ( a l l y l i s o t h i o c y a n a t e ) Q - B - D - G ! u c H O S T R E F E R E N C E S s w e e t ' p o t a t o K u b o t a & I c h i k a w a 1954 C r u c i f e r s E t t l i n g e r & L u n d e n . -1956 b e a n F a w c e t t e t a l . 1965 c i t r u s M u r d o c h & A l l e n 1964 t u l i p S k i n n e r 1955 p o t a t o T o m i y a m a 1968 s m i l a x T s c h e s c h e e t a l . 1967 SUBSTANCES STRUCTURAL FORMULA HOST REFERENCES <* -Solanine Tomatin T u l i p a l i n 15 Hydroquinone, a t o x i c phenol i s u s u a l l y present i n pear as i t s g l u c o s i d e a r b u t i n . A f t e r i n f e c t i o n w i t h E r w i n i a amy-l o v o r a , the enhanced 8-glucosidase a c t i v i t y r e s u l t s i n h y d r o l y s i s of the a r b u t i n to hydroquinone (Hildebrandt and Schroth 1 9 6 3 , 1 9 6 4 ) . I n a s i m i l a r way, the gluc o s i d e p h l o r -i d z i n i s hydrolyzed to give the aglycone p h l o r e t i n , which i s i n h i b i t o r y to V e n t u r i a i n a e q u a l i s , the pathogen of apple scab disease (Holowczak et a l . 1 9 6 2 ) . Very o f t e n the phenolic compounds present i n the he a l t h y p l a n t s are not f u n g i t o x i c but a f t e r i n f e c t i o n they are o x i d i z e d to quinones which are much more t o x i c than the parent phenols. The polyphenol oxidases and peroxidases i n pl a n t t i s s u e s are u s u a l l y a c t i v a t e d by i n f e c t i o n . For exam-p l e , B o t r y t i s c i n e r e a , which causes chocolate spot disease of beans, produces p e c t i c enzymes which l i b e r a t e g a l a c t u r o n i c a c i d d e r i v a t i v e s and p o l y g a l a c t u r o n i c d e r i v a t i v e s from c e l l w a l l s . These compounds unmask the l a t e n t polyphenol oxidase of the host ( D e v e r a l l 1 9 6 1 ) . M u l l e r i n 1 9 5 0 found t h a t when potato tuber t i s s u e was i n o c u l a t e d w i t h an a v i r u l e n t s t r a i n of Phytophthora i n -f e s t a n s i t became r e s i s t a n t to v i r u l e n t s t r a i n s of this^ fungus and a l s o to Fusarium spp. Normally Fusarium spp. can para-s i t i z e l i v i n g t u b e r s . He p o s t u l a t e d the d_e novo sy n t h e s i s of a n t i f u n g a l substances by the host i n response t o i n f e c t i o n . These he c a l l e d p h y t o a l e x i n s . Most phy t o a l e x i n s are phenolic compounds (Table I ) . Cruickshank and P e r r i n (I960) and 1 6 P e r r i n ( 1 9 6 4 ) i s o l a t e d p i s a t i n and p h a s e o l i n from garden pea and French bean pods i n o c u l a t e d w i t h M o n i l i n i a f r u c t i c o l a . There are a l s o other phenolic p h y t o a l e x i n s of simpler molecular s t r u c t u r e s such as isocoumarin, u m b e l l i f e r o n e and s c o p o l e t i n . The c o n c e n t r a t i o n of isocoumarin v a r i e s from 5 -3 4 2 u.g/g c a r r o t t i s s u e depending upon the organism used f o r i n f e c t i o n (Condon et a l . 1 9 6 3 ) . P l a n t s have the p o t e n t i a l to produce p h y t o a l e x i n s but i t i s the pathogen that deter-mines t h e i r r a t e of s y n t h e s i s by the host. 2. DISEASE RESISTANT MECHANISMS OF PHENOLIC COMPOUNDS Knowledge of the mechanisms by which phenolic com-pounds i n h i b i t pathogens i s fragmentary. Many c o n t r a d i c t o r y r e s u l t s have been obtained u s i n g d i f f e r e n t organisms. There-f o r e i t i s d i f f i c u l t at t h i s stage to say e x a c t l y what mech-anisms are i n v o l v e d . Sometimes, phe n o l i c s themselves, act as antipathogenic agents, but quinones, the o x i d a t i v e pro-ducts of phenols, are o f t e n more important i n r e l a t i o n to disease r e s i s t a n c e . A) E f f e c t of Phenols Kuc ( 1 9 6 4 ) pointed out t h a t the r a p i d s y n t h e s i s , accumulation and i n h i b i t o r y a c t i v i t y of the parent phenol or phenol d e r i v a t i v e s at the s i t e of i n f e c t i o n can c o n t r i b u t e to r e s i s t a n c e . Compounds such as p i s a t i n and 3-methyl-6-methoxy-S-hydroxy-3,4-dihydroisocoumarin are not r e a d i l y o x i d i z e d and quinone formation cannot e x p l a i n t h e i r i n h i b i t o r y a c t i v i t y . Flood and Kirkham ( I960) observed that: the q u i n i c a c i d moiety of chlorogenic a c i d has no e f f e c t on growth and s p o r u l a t i o n of V e n t u r i a spp. The c a f f e i c a c i d moiety does have an e f f e c t . Since c a f f e i c a c i d d i f f e r s from p-coumaric which has no i n h i b i t o r y e f f e c t , by only one a d d i t i o n a l OH group i n the meta p o s i t i o n i t seems that such h y d r o x y l a t i o n •causes abnormal growth and s p o r u l a t i o n and t h i s i s found to be t r u e i n many cases. F e r u l i c a c i d has no e f f e c t because there i s methoxylation of the meta hydroxyl group i . e . i t behaves s i m i l a r l y to p-coumaric a c i d . The -CH=CH-COOH group might a l s o be important because, w h i l e protocatechuic a c i d has only a s l i g h t e f f e c t on the s p o r u l a t i o n of V e n t u r i a , i t i s h i g h l y t o x i c to C o l l e t o t r i c h u m c i r c i n a n s . Flood and Kirkham a l s o found t h a t the compounds most t o x i c to Ve n t u r i a were o-coumaric a c i d and cinnamic a c i d i t s e l f . Hulme and Edney ( I 9 6 0 ) , working on the phenolics i n apple p e e l , a l s o found th a t o-coumaric a c i d completely i n h i b i t e d the germination of spores of Gloeosporium perennans whereas p-coumaric a c i d gave only p a r t i a l i n h i b i t i o n . The f a c t that the t o x i c e f f e c t s of some phenols can be g r e a t l y reduced by g l y c o s y l a t i o n of one or more hydroxyl group suggests the importance of the hydroxyl groups i n i n h i b i t i n g f u n g a l growth. This can r e a d i l y be demonstrated .by comparing the germination of broad bean seeds on cotton wool soaked i n equimolar s o l u t i o n s of hydroquinone and a r b u t i n . Both compounds r e a d i l y enter the t i s s u e s but w i t h a r b u t i n germination proceeds normally whereas w i t h hydro-18 quinone the seeds r a p i d l y blacken and d i e (Pridham and S a l t -marsh I 9 6 0 ) . On the other hand, Van Sumere ( i 9 6 0 ) showed tha t p-hydroxybenzoic a c i d and f e r u l i c a c i d were str o n g i n h i b i -t o r s of the germination of wheat stem r u s t uredospores where-as c a f f e i c a c i d , v a n i l l i c a c i d and f e r u l i c acid-B - g l u c o s i d e had l i t t l e e f f e c t . Dabler et a l . ( 1 9 6 9 ) a l s o demonstrated t h a t f e r u l i c a c i d , which has one hy d r o x y l group masked by a methyl group, was the most e f f e c t i v e phenol i n i n h i b i t i n g spore germination of D i p l o d i a zeae at pH 7 . At low pH how-ever, the i n h i b i t o r y e f f e c t was not pronounced. Sondheimer ( 1 9 6 2 ) has reporte d t h a t u n o x i d i z e d c h l o r o -genic a c i d forms complexes w i t h n i t r o g e n c o n t a i n i n g compounds such as c a f f e i n and r i b o f l a v i n . I t a l s o i n h i b i t s a number of enzyme systems such as phosphorylase, another p y r i d o x a l phos-p h a t e - r e q u i r i n g enzyme system, IAA oxidase and a peroxidase-c a t a l y z e d 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 methionine. P o s s i b l y the best s t u d i e d example of a c t i v a t i o n and i n h i b i t i o n of an enzyme system by p h e n o l i c s i s IAA oxidase. Many monohydric phenols act as a c t i v a t o r s of t h i s enzyme system w h i l e d i h y d r i c phenols have i n h i b i t o r y a c t i v i t y . The most e f f e c t i v e a c t i v a -t o r of the pineapple enzyme found by Gortner et a l . ( 1 9 5 8 ) was p-coumaric a c i d and the most potent i n h i b i t o r was c h l o r o -genic a c i d ; c a f f e i c a c i d was somewhat l e s s potent. S e v e r a l p o i n t s emerge from the above account of the e f f e c t of u n o x i d i z e d p h e n o l i c s on f u n g a l growth and enzyme 19 a c t i v i t i e s : 1. The number of OH groups on the benzene r i n g i s important. I n most cases 3 , 4-dihydroxyl phenols are more i n h i b i -t o r y than monohydroxyl ones. 2. A hydroxyl group at the ortho p o s i t i o n to the s i d e chain i s more t o x i c than one at the para p o s i t i o n . 3. The s i d e chain attached to the benzene r i n g a l s o plays some r o l e i n i n h i b i t i o n . 4. Methoxylation and g l y c o s y l a t i o n can reduce t o x i c i t y . 5 Enzyme i n h i b i t i o n may be the most important way i n which phenols exert t h e i r anti-pathogen a c t i v i t i e s . U n f o r t u n a t e l y , these g e n e r a l i z a t i o n s do not always apply. They apply to c e r t a i n organisms under c e r t a i n con-d i t i o n s but not f o r o t h e r s . Thus, f o r example, f e r u l i c a c i d i n h i b i t s spore germination i n D i p l o d i a zeae more than c a f f e i c a c i d does. B) E f f e c t of Quinones Byrde (1963) found t h a t the a c t i v i t y of degradating enzymes of the brown r o t organism, S c l e r o t i n i a f r u c t i c o l a was reduced i n r e s i s t a n t v a r i e t i e s of f r u i t t r e e s owing to pre-c i p i t a t i o n by high molecular weight products, of polyphenol o x i d a t i o n . Increased a c t i v i t y of o x i d a t i v e enzymes may account f o r part of the i n c r e a s e d O2 consumption observed f o l l o w i n g i n f e c t i o n . F o l l o w i n g i n f e c t i o n w i t h Pseudomonas  solanacearum and concomitant w i t h i n c r e a s e s i n O2 uptake and t o t a l phenol content, polyphenol oxidase a c t i v i t y increased 2 0 more i n r e s i s t a n t tomato and tobacco stems than i n s u s c e p t i b l e ones (Maine and Kelman 1 9 6 1 ) . P r i o r to i n f e c t i o n there was no d i f f e r e n c e between the two. Enzyme i n h i b i t o r s such as g l u t a t h i o n e and a s c o r b i c a c i d , fed through the r o o t s , reduced O 2 uptake and polyphenol oxidase a c t i v i t y i n diseased t i s s u e , and reversed r e s i s t a n c e . Chlorogenic a c i d , i n i t i a l l y i n -creased by i n f e c t i o n , was markedly reduced i n the r e s i s t a n t p l a n t s i n the l a t e r stages of disease development, apparently as a r e s u l t of o x i d a t i o n by the enzyme. Resistance to bac-t e r i a l w i l t thus seems to depend on i n f e c t i o n - i n d u c e d produc-t i o n or a c t i v a t i o n of polyphenol oxidase which o x i d i z e s chlorogenic a c i d to t o x i c quinones. Both enzyme and phenol are produced i n greater, q u a n t i t i e s i n r e s i s t a n t s t r a i n s . R esistance t o Pseudomonas p h a s e o l i c o l a i n beans a l s o i n v o l v e s a phenolic o x i d a t i o n system (Hare 1 9 6 6 ) . ' S i m i l a r l y , i n many other cases the t o x i c e f f e c t of p h e n o l i c s i s e x h i b i t e d only a f t e r they have been o x i d i z e d i n -to quinones. I n other words, quinones are the a c t i v e i n h i b i -t o r s of microorganisms. Quinones are very r e a c t i v e and r e a c t r e a d i l y w i t h t h i o l s , amino groups and p r o t e i n s ( F i g . 2 ) . They can thus i n h i b i t enzymes i n the f o l l o w i n g ways: 1 . O x i d a t i o n of f u n c t i o n a l groups of enzymes 2 . Reaction w i t h SH, amino and hydroxyl groups 3. Complexing w i t h metal ions 4 . Reaction w i t h s u b s t r a t e s or c o - f a c t o r s 5. Production of hydrogen peroxide 21 Figure 2. Reactions between quinone and t h i o l s (1), amino group (2) and p r o t e i n (3). (Geiger 1946 and Webb 1966). 2 2 6. N o n s p e c i f i c b i n d i n g through the aromatic r i n g 7 . Competition w i t h q u i n o i d or po l y p h e n o l i c s u b s t r a t e s . In a d d i t i o n to i n h i b i t i n g enzyme a c t i v i t y quinones a f f e c t c e l l u l a r metabolism i n many other ways. Their e f f e c t on e l e c t r o n t r a n s p o r t systems i s q u i t e complicated. They may compete w i t h or d i s p l a c e n a t u r a l or endogenous quinones t h a t are i n v o l v e d i n e l e c t r o n t r a n s p o r t systems. Some q u i -nones can act as a new source of e l e c t r o n donors or acceptors and may thus e s t a b l i s h an a l t e r n a t i v e or bypass pathway f o r e l e c t r o n f l o w , thus u p s e t t i n g normal metabolism. Other metabolic processes t h a t may be a f f e c t e d are o x i d a t i v e phos-p h o r p h o r y l a t i o n , g l y c o l y s i s and l i p i d metabolism. There i s a wide v a r i a t i o n i n the degree of suscept-i b i l i t y of v a r i o u s f u n g i to a p a r t i c u l a r quinone. I t i s evident that some p a r t i c u l a r a c t i v i t y or mechanism must be a s s o c i a t e d w i t h c e r t a i n quinones, inasmuch as they are so much more potent than others f o r a s i n g l e type of fungus. Examples of the r e l a t i o n of growth i n h i b i t i o n to s t r u c t u r e are (Webb 1 9 6 6 ) : 1 . I n 9 out of 1 0 f u n g i , quinone i s more potent than the corresponding hydroquinone; the average r e l a t i v e poten-cy r a t i o i s 5. This value i s greater than t h a t f o r b a c t e r i a . 2 . A d d i t i o n of c h l o r i n e atoms s t r o n g l y i n c r e a s e s potency. 3. Methoxyl groups g e n e r a l l y lower the a c t i v i t y i n the napthoquinone s e r i e s . 23 4. H y d r o x y l a t i o n of 1,4-naphthoquinone i n e i t h e r the 2-or 5 - p o s i t i o n leads to l o s s of a c t i v i t y . Geiger ( 1 9 4 6 ) found t h a t quinones w i t h methyl, hydroxyl or s u l f h y d r y l groups s u b s t i t u t e d i n the benzene r i n g are l e s s i n f l u e n t i a l a g a i n s t b a c t e r i a . I f the 2, 3 , 5 or 6 - p o s i t i o n s of benzoquinone are s u b s t i t u t e d , a n t i m i c r o b i a l a c t i v i t y i s decreased. For example, juglone (2-methyl,5-hydroxy, 1 , 4 -naphthoquinone) i s more a c t i v e a g a i n s t E. c o l i than 2-methyl, 3-hydroxy-l,4-naphthoquinone. This suggests that the a c t i v i t y of quinones agai n s t b a c t e r i a r e q u i r e s the presence of a f r e e p o s i t i o n ortho to a carbonyl group. This f i n d i n g i s f u r t h e r s u b s t a n t i a t e d by the f a c t t h a t the a d d i t i o n of one equivalent of a s u l f h y d r y l c o n t a i n i n g compound almost completely a b o l i s h e s the a n t i m i c r o b i a l a c t i v i t y because i t e l i m i n a t e s the carbonyl group ( F i g . 2 ) . o o o o 1,4-Benzoquinone 1,4-Naphthoquinone 24 3 . METABOLISM OF PHENOLICS IN HEALTHY AND DISEASED TISSUES A) Metabolism of Phenolics i n Healthy Tissue I n p l a n t t i s s u e s a l l phenolic compounds are d e r i v e d from the intermediates of carbohydrate metabolism. There are two main routes f o r s y n t h e s i s of the phenolic nucleus, v i z , the shikimate and the acetate pathways. The carbon-s k e l e t o n f o r the shikimate pathway a r i s e s from phosphoenol-pyruvic a c i d (PEP) and D-erythrose - 4-phosphate (E - 4 - 0 ) which are i n t u r n derived from the metabolism of glucose v i a the Embden-Meyerhof Pathway (EMP) and pentose phosphate pathway ( F i g . 3 ) . PEP and E-4-P condense to give 3-deoxy-D-arabino-heptulosonic acid - 7-phosphate which v i a 5-dehydroquinic and 5-dehydroshikimic a c i d would give r i s e to s h i k i m i c a c i d . On the a d d i t i o n of another PEP, t h i s pathway leads to a whole f a m i l y of C^C^ compounds and t h e i r condensation products. The acetate pathway produces aromatic r i n g s through the head to t a i l condensation of acetyl-CoA and malonyl-CoA b u i l d i n g b l o c k s . Some C^ C-j phenolic a c i d s and acetogenins are formed i n t h i s manner. The c o l l a b o r a t i o n of both pathways produces the f l a v o n o i d s . I n the shikimate pathway, immediately a f t e r the C^C^ u n i t i s formed, there i s a transamination step f o l l o w e d by deamination. This would be a very s u i t a b l e c o n t r o l l i n g point f o r r e g u l a t i n g the f l o w of aromatic r i n g s to n i t r o g e n meta-bol i s m , i n c l u d i n g p r o t e i n s y n t h e s i s , or to the phenolic p o o l . Therefore ever s i n c e Koukol and Conn (1961) discovered 25 Glucose EMP phosphoenol-pyruvic acid (PEP) Pentose phosphate pathway \ Erythrose - 4 - phosphate 1/ i Shikimate pathway Acetyl CoA->Malonyl CoA Shikimate Terpenoids Phenylpyruvic acid p-OH- Phenylpyruvic acid TAL Phenylalanine benzoic acid^ „•„ , » „ , « : , » „ „ : J ^ cinnamic acia coumarin p-OH-benzoic acid<- p-coumaric acid umbelliferone | Chalcone protocatechuic acid <-caffeic acid esculetin phloretin 1 -co I ffe I DO PA flavonoids vanillic acid scopoletin ferulic acid -> coniferyl alcohol 1 syringic acid ^ sinapic acid sinapoyl alcohol Figure 3. The Metabolic Pathways of Some Phenolic Compounds. 2 6 phenylalanine ammonia-lyase (PAL) and Neish ( 1 9 6 1 ) d i s -covered t y r o s i n e ammonia-lyase (TAL) much work has been done on these enzymes both by p l a n t p h y s i o l o g i s t s and biochemists as w e l l as by p h y t o p a t h o l o g i s t s . The a c t i o n of PAL leads to the production of cinnamic a c i d whereas TAL converts t y r o s i n e to p-coumaric a c i d . I n microorganisms and animal t i s s u e s phenylalanine can r e a d i l y be converted i n t o t y r o s i n e but f o r p l a n t m a t e r i a l t h i s conversion has only been reported once by N a i r and V i n i n g ( 1 9 6 5 a ) i n a spinach enzyme p r e p a r a t i o n . They found that the optimum pH was 4.2 and there was an ab-s o l u t e requirement f o r e l e c t r o n donors which was s a t i s f i e d by adding t e t r a h y d r o f o l i c a c i d and a reduced p y r i d i n e nucleo-t i d e . However, McCalla and Neish ( 1 9 5 9 ) and Fuchs et a l . ( 1 9 6 7 ) working w i t h S a l v i a splendens and r u s t - i n f e c t e d wheat leaves r e s p e c t i v e l y found no evidence f o r the i n t e r c o n v e r s i o n of phenylalanine and t y r o s i n e . The h y d r o x y l a t i o n of cinnamic a c i d was demonstrated by Nair and V i n i n g ( 1 9 6 5 b ) w i t h spinach enzyme p r e p a r a t i o n and R u s s e l l and Conn ( 1 9 6 7 ) w i t h pea s e e d l i n g enzyme. The pH optima f o r the two h y d r o x y l a t i n g systems are q u i t e d i f f e r -ent. For the spinach enzyme i t was found to be pH 4.2 where-as a n e u t r a l pH was best f o r the pea enzyme. McCalla and Neish ( 1 9 5 9 ) showed, i n v i v o , the f o l l o w -i n g pathway f o r the metabolism of phenylpropanoid a c i d s : cinnamic a c i d 7 p-coumaric a c i d ^ c a f f e i c a c i d s i n a p i c acid«^ f e r u l i c a c i d < J The i n v i t r o conversion of p-coumaric a c i d to c a f f e i c a c i d was only r e c e n t l y reported by Sato (1969). The enzyme c a t a l y z i n g t h i s r e a c t i o n i s , however, the w i d e l y d i s t r i -buted polyphenol oxidase (PPO). He found that p-coumaric a c i d undergoes a coupled o x i d a t i o n w i t h a s c o r b i c a c i d : p-coumaric a c i d + a s c o r b i c a c i d + Og ? c a f f e i c a c i d + o x i d i z e d a s c o r b i c a c i d . The m e t h y l a t i n g enzymes i n p l a n t s have been s t u d i e d by F i n k l e and Nelson (1963), F i n k l e and Masri (1964), Mann and Mudd (1963) and Mann et a l . (1964). The methyl donor was found to be S-adenosyl-L-methionine. The enzyme su b s t r a t e o r i e n t a t i o n phenomenon i s q u i t e s p e c i f i c f o r p l a n t methyl-t r a n s f e r a s e and l e s s so f o r the animal enzyme (Daly 1967). For example, the catechol-O-methyltransferase from l i v e r t i s s u e c a t a l y z e s the m e t h y l a t i o n of c a f f e i c a c i d to a.mixture of f e r u l i c and i s o f e r u l i c a c i d s whereas the plant methyl-t r a n s f e r a s e gives over 95% m-methylation, i . e . f e r u l i c a c i d . I n p l a n t t i s s u e s m-methylation seems to be predominant and seldom are p-methoxy compounds sy n t h e s i z e d . From these phenylpropionic a c i d s (cinnamic, p-coumaric, c a f f e i c , f e r u l i c and s i n a p i c a c i d s ) many complex phenolic compounds are d e r i v e d . These i n c l u d e v a r i o u s phen-o l i c e s t e r s , g l y c o s i d e s , depsides, coumarins, f l a v o n o i d s , and l i g n i n , ... B) E f f e c t of I n f e c t i o n on Metabolism of Phenolics . -Wheat leaves i n f e c t e d w i t h r u s t showed an increase i n the i n c o r p o r a t i o n of CO2 to shikimate and quinate. This t r e n d was more pronounced i n s u s c e p t i b l e than r e s i s t a n t leaves (Rohringer et a l . 1 9 6 7 ) . -In f e e d i n g experiments u s i n g quinate-U-^C and s h i k i m a t e - U - ^ C , more r a d i o a c t i v i t y was recovered i n the i n s o l u b l e e s t e r s f o r the i n f e c t e d r e s i s t a n t leaves than the h e a l t h y c o n t r o l whereas the i n f e c t e d sus-c e p t i b l e leaves had more r a d i o a c t i v i t y i n the s o l u b l e e s t e r s over the same c o n t r o l . Both Shaw and C o l o t e l o ( 1 9 6 1 ) and Fuchs et a l . ( 1 9 6 7 ) showed that r u s t - i n f e c t e d wheat leaves accumulate t y r o s i n e and phenylalanine. They found t h a t the u t i l i z a t i o n of these amino a c i d s was a l s o i n c r e a s e d . Therefore the production of these amino a c i d s must have incre a s e d tremendously. Together w i t h the q u i n i c and shikimate t r a c e r s t u d i e s i t i s c l e a r t h a t i n f e c t i o n w i t h r u s t enhances the a r o m a t i z a t i o n process l e a d i n g to the s y n t h e s i s of f r e e phenylalanine and t y r o s i n e . I n i n f e c t e d s u s c e p t i b l e leaves a major p o r t i o n of these amino a c i d s i s i n c o r p o r a t e d i n t o p r o t e i n whereas i n r e s i s -t a n t leaves a higher p r o p o r t i o n i s i n c o r p o r a t e d i n t o the non-proteinaceous aromatic compounds. Some examples are c i t e d below to i l l u s t r a t e the syn-t h e s i s of aromatic compounds as a r e s u l t o f • i n f e c t i o n : a) S c o p o l e t i n - Sequeira ( 1 9 6 9 ) , studying the s y n t h e s i s of s c o p o l i n and s c o p o l e t i n i n tobacco p l a n t s i n f e c t e d by 29 Pseudomonas solanacearum, found that i n f e c t e d xylem parenchyma of tobacco p l a n t s accumulated s c o p o l i n and i t s aglycone, s c o p o l e t i n . The r a p i d i n c r e a s e i n scopo-l e t i n i s not due to an i n c r e a s e i n h y d r o l y s i s of sco-p o l i n as the s p e c i f i c a c t i v i t y of S-g l y c o s i d a s e i n these t i s s u e s d i d not i n c r e a s e s i g n i f i c a n t l y over the c o n t r o l . b) P i s a t i n - Hadwiger (1967) suggested t h a t the production of p i s a t i n by pea pod t i s s u e induced by M o n i l i n i a f r u c -t i c o l a or CuClg i s due to the s t i m u l a t i o n of an aux-i l i a r y pathway which u l t i m a t e l y converts phenylalanine to p i s a t i n . I t i s a l s o p o s s i b l e t h a t the inducer i s an agent w i t h the a b i l i t y to block a normal pathway f o r the p r o d u c t i o n of i s o f l a v o n o i d s immediately subsequent to p i s a t i n i n the pathway r e s u l t i n g i n the accumulation of p i s a t i n . c) L i g n i n - Rohringer et a l . (1967) found t h a t i n f e c t e d r e s i s t a n t wheat leaves accumulated more from s h i k i -14 14 mate-U- C and quinate-U- C i n i n s o l u b l e e s t e r s than the h e a l t h y and i n f e c t e d s u s c e p t i b l e l e a v e s . They suggested t h a t at l e a s t some of the components of the i n s o l u b l e f r a c t i o n are intermediates i n l i g n i n s y n t h e s i s . This may r e f l e c t a greater l i g n i f i c a t i o n i n the r e s i s -t ant t i s s u e . From these examples i t can be seen t h a t d e v i a t i o n s i n phenolic metabolism do occur i n diseased t i s s u e when compared w i t h the h e a l t h y . Amongst these v a r i o u s changes, however, 3 0 the most common ones are the enhancement of h y d r o l y s i s and o x i d a t i o n . Very o f t e n g l y c o s i d e s are hydrolyzed t o r e l e a s e t h e i r aglycones which are then o x i d i z e d to t o x i c substances t h a t confer disease r e s i s t a n c e . O x i d a t i o n of p h e n o l i c s leads to the formation of quinones and t h e i r polymers, which may cause the s o - c a l l e d h y p e r s e n s i t i v e r e a c t i o n . The en-zymes r e s p o n s i b l e f o r h y d r o l y s i s and o x i d a t i o n and t h e i r a c t i o n s and changes i n diseased t i s s u e s are reviewed i n the f o l l o w i n g s e c t i o n . 4. SOME ENZYMES INVOLVED IN PHENOLIC METABOLISM A) Phenylalanine Ammonia-Lyase (PAL)(EC 4 . 3 . 1 . 5 ) ( 1 ) General P r o p e r t i e s : PAL was di s c o v e r e d comparatively r e c e n t l y . Koukol and Conn ( 1 9 6 1 ) r e p o r t e d t h a t an enzyme th a t deaminated phenylalanine was present i n b a r l e y . This appeared to be an a s p a r t a s e - l i k e enzyme. I t c a t a l y z e s the e l i m i n a t i o n of one molecule of ammonia from phenylalanine to form an un-sa t u r a t e d a c i d . L-Phenylalanine trans-Cinnamic A c i d PAL can convert a number of r i n g s u b s t i t u t e d phenylalanines to the corresponding cinnamic a c i d s but these phenylalanines must be of the L- or DL-series (Young and Neish 1 9 6 6 , Subba 31 Rao e_t a l . 1967) and the enzyme i s i n a c t i v e towards D-pheny l a l a n i n e s . The optimal pH f o r b a r l e y PAL i s 8.8-9.2 (Koukol and Conn 1961). Other workers have found the same pH optimum f o r the enzyme from other t i s s u e s ; Minamikawa and U r i t a n i (1964) on sweet potato and Subba Rao et a l . (1967) on U s t i l a g o  h o r d e i . Minamikawa and U r i t a n i (1964) reporte d that there are two components f o r PAL. Havir and Hanson (1968) a l s o found two s t a b l e enzyme s p e c i e s , w i t h the minor species having approximately 10% of the t o t a l a c t i v i t y . The p r o v i s i o n a l molecular weight of the major species was found to be 330,000 and the enzyme i s a p p r e c i a b l y a s p h e r i c a l . The minor species of the enzyme may have twice t h i s molecular weight. Apparently no c o f a c t o r s or metal i o n s are r e q u i r e d . The enzyme was s t i m u l a t e d by reduced g l u t a t h i o n e and the i n -h i b i t i o n of the enzyme by s u l f h y d r y l group i n h i b i t o r s suggests t h a t PAL r e q u i r e s the s u l f h y d r y l group f o r a c t i v i t y (Koukol and Conn 1961). The high s u s c e p t i b i l i t y of the enzyme to i n a c t i v a t i o n by heavy metal ions such as Ag+, Hg+ and Cd++ al s o suggests the involvement of s u l f h y d r y l groups i n the r e a c t i o n . Both Koukol and Conn (1961) and Subba Rao et a l . (1967) found t h a t the enzyme was i n h i b i t e d by cyanide, i n d i -c a t i n g that i t could be a m e t a l l o - p r o t e i n . Some aromatic com-pounds such as cinnamic a c i d and p-coumaric a c i d are i n h i b i t o r y to PAL. PAL i s w i d e l y d i s t r i b u t e d i n the pl a n t kingdom as i t i s a key enzyme f o r l i g n i n p r o d u c t i o n . I t occurs i n both 32 d i c o t y l e d o n s as w e l l as monocotyledons, whereas t y r o s i n e ammonia-lyase, an enzyme comparable to PAL but a c t i n g main-l y on t y r o s i n e , i s more or l e s s r e s t r i c t e d to monocotyledons. (2) Enzyme I n d u c t i o n : PAL has been a c t i v e l y s t u d i e d w i t h respect t o enzyme i n d u c t i o n s i n c e i t was found by Zucker i n 1965 t h a t l i g h t induces PAL s y n t h e s i s . By d e f i n i t i o n , i n d u c t i o n i s the de  novo s y n t h e s i s of enzyme molecules as the r e s u l t of a stimu-l a t o r y e f f e c t exerted on the genetic m a t e r i a l ( v i a r e p r e s s o r s ) by the ind u c e r . So f a r , o n l y a few examples of true induc-t i o n have been confirmed i n higher p l a n t s ( F i l n e r et a l . 1 9 6 9 ) . F r e q u e n t l y however data i n d i c a t i n g enhanced enzyme a c t i v i t y which can be i n h i b i t e d by i n h i b i t o r s of the s y n t h e s i s of p r o t e i n s or n u c l e i c a c i d s are considered to i n d i c a t e enzyme i n d u c t i o n . The term i n d u c t i o n i s used i n t h i s t h e s i s to i n c l u d e r e s u l t s of t h i s k i n d . • S e v e r a l f a c t o r s are known to s t i m u l a t e the biosynthe-s i s of PAL. They are exogenous carbohydrates, ethylene, a b s c i s i n I I , temperature, l i g h t , i n j u r y and diseas e . I n t h i s review only the l a s t three e f f e c t s are di s c u s s e d . L i g h t has been found by many workers to enhance PAL a c t i v i t y (Zucker 1965, Ahmed and Swain 1 9 7 0 , Smith and A t t r i d g e 1 9 7 0 ) . At l e a s t two types of l i g h t e f f e c t are be-l i e v e d to r e g u l a t e the PAL l e v e l . One i n v o l v e s the photo-chrome system. Ahmed and Swain (1970) found t h a t PAL a c t i v i t y i n both pea and mung bean s e e d l i n g s i s doubled a f t e r 33 red l i g h t treatment. Smith and A t t r i d g e ( 1 9 7 0 ) a l s o showed th a t short i r r a d i a t i o n a f t e r red l i g h t l e d to marked i n -creases i n PAL l e v e l s . The r a t e constants of phytochrome "decay" under the various, treatments were l i n e a r l y r e l a t e d to the r a t e constant of the e a r l y i n c r e a s e s i n enzyme a c t i v i t y , i n d i c a t i n g t h a t phytochrome "decay" may be an i n t e g r a l part of the mechanism of the a c t i o n of phytochrome. Zucker ( 1 9 6 9 ) found t h a t light-dependent s y n t h e s i s of PAL i s completely i n h i b i t e d by 5 0 \M 3 - ( 4 - c h l o r o p h e n y l ) - 1 , 1 -dimethylurea (CMU) i n d i c a t i n g that photosynthesis i s i n v o l v e d . Creasy ( 1 9 6 8 ) working w i t h strawberry r e p o r t e d a requirement f o r blue l i g h t f o r maximum s t i m u l a t i o n of PAL. This p h o t o s y n t h e t i c requirement i s q u i t e d i s t i n c t from the r e d - f a r - r e d e f f e c t of l i g h t . Zucker ( 1 9 6 8 , 1 9 6 9 ) working on the e f f e c t of l i g h t on PAL i n d u c t i o n proposed a s e q u e n t i a l i n d u c t i o n of PAL and a l y a s e ^ i n a c t i v a t i n g system. By means of cycloheximide and time parameter s t u d i e s , he found t h a t i n s l i c e d potato d i s k s , incubated i n l i g h t , PAL i s the f i r s t enzyme induced. About 1 2 hours a f t e r i n c u b a t i o n , an i n h i b i t o r or degradative agent was a l s o induced and i t s s y n t h e s i s depended on p r o t e i n syn-t h e s i s . This " i n h i b i t o r " may t h e r e f o r e be a protease. Thus the e a r l y phases of i n d u c t i o n i n v o l v e d the s y n t h e s i s of PAL p r o t e i n i n the absence of t u r n o v e r , a f t e r which a l y a s e de-grading or i n a c t i v a t i n g p r o t e i n was s y n t h e s i z e d . He a l s o found t h a t enzymes formed under l i g h t disappeared r a p i d l y 3 4 when d i s k s were placed i n the dark. Thus l i g h t - i n d u c e d syn-t h e s i s coupled w i t h a r a p i d turnover i n the dark can produce a d i u r n a l f l u c t u a t i o n of PAL a c t i v i t y . Induced l y a s e synthe-s i s was a l s o observed i n excised leaves and to a l e s s e r extent i n leaves of whole p l a n t s . The l a r g e changes i n the r a t e of l y a s e s y n t h e s i s i n l e a f d i s k s compared w i t h those of whole p l a n t s suggests that e x c i s i o n i s r e q u i r e d f o r r a p i d i n d u c t i o n of l y a s e s y n t h e s i s . ( 3 ) PAL and Disease: A c t u a l l y the study of PAL i n i n j u r e d or diseased t i s s u e s i s as e a r l y as the studies, of l i g h t i n d u c t i o n on PAL s y n t h e s i s . However, only U r i t a n i ' s group i n Japan and Hadwiger's group i n the United States have been a c t i v e l y working on t h i s problem. Minamikawa and U r i t a n i ( 1 9 6 4 , 1 9 6 5 ) found a marked i n c r e a s e i n PAL a c t i v i t y i n sweet potato s l i c e s a f t e r 6 hours of i n c u b a t i o n or in-sweet potato i n o c u l a t e d w i t h C e r a t o c y s t i s  f i m b r i a t a . The enzyme a c t i v i t y reached a maximum at 2 4 - 2 6 hours and then decreased g r a d u a l l y . There were s i m i l a r p a t t e r n s f o r TAL but the a c t i v i t y was much lower than t h a t of PAL. Concomitant w i t h the i n c r e a s e i n PAL there was a l s o a r i s e i n polyphenol content. Therefore they suggested, that PAL plays an important r o l e i n polyphenol b i o s y n t h e s i s i n wounded or i n f e c t e d t i s s u e s . Hadwiger ( 1 9 6 $ ) found t h a t M o n i l i n i a f r u c t i c o l a spore suspensions and CuClg caused a 1 0 - 1 2 f o l d s t i m u l a t i o n i n PAL 35 a c t i v i t y of pea pods. The f a c t o r s which s t i m u l a t e p i s a t i n f o rmation a l s o caused a r a p i d appearance of PAL a c t i v i t y . Thus he suggested that there was a c l o s e c o r r e l a t i o n between PAL a c t i v i t y and p i s a t i n s y n t h e s i s . Hadwiger et a l . (1970) t r i e d to s u b s t a n t i a t e the c l a i m that p h y t o a l e x i n production i s c o r r e l a t e d w i t h i n c r e a s e s i n PAL a c t i v i t y . They found t h a t PAL increased i n e x c i s e d pea and bean pod t i s s u e s w i t h i n 8 hours a f t e r i n o c u l a t i o n w i t h pathogenic and non-pathogenic organisms. In g e n e r a l , f a c u l t a t i v e p a r a s i t e s are more potent than o b l i g a t e p a r a s i t e s i n s t i m u l a t i n g PAL a c t i v i t y i n these t i s s u e s . They a l s o s t u d i e d the i n d u c t i o n of PAL i n wheat, corn and f l a x s e e d l i n g s by spore suspensions and chemical compounds. The PAL a c t i v i t y i n Bison f l a x ( s u s c e p t i b l e ) was not s i g n i f i c a n t l y a l t e r e d when t i s s u e was incubated f o r 24 hours w i t h spores of M. l i n i , Fusarium s o l a n i f . sp. p i s i or P u c c i n i a s t r i i f o r m i s . PAL was a l s o not s i g n i f i c a n t l y s t i m u l a t e d when Cass-M3, a r e s i s t a n t v a r i e t y of f l a x , was i n o c u l a t e d w i t h race 1 of M. l i n i . The p u b l i s h e d i n f o r m a t i o n on PAL i n diseased t i s s u e i s s t i l l sketchy. More research i n t h i s area i s needed, e s p e c i a l l y s i n c e t h i s enzyme may c o n t r o l p h y t o a l e x i n produc-t i o n . B) B - G l y c o s i d a s e (EC 3.2.1.21). Glycosidases are enzymes c a t a l y z i n g the h y d r o l y s i s of a l k y l and a r y l g l y c o s i d e s : 36 glycosyl-OR + EH- r g l y c o s y l - E + ROH g l y c o s y l - E + HO-RT 7 glycosyl-OR T + EH The enzymatic h y d r o l y s i s of g l y c o s i d e s occurs by f i s s i o n of the bond between C - l of the glycone and the g l y c o s i d e oxygen atom. Those enzymes i n v o l v e d i n t r a n s f e r r e a c t i o n s w i t h n u c l e o t i d e d e r i v a t i v e s of sugars may a l s o be regarded as g l y c o s i d a s e s . H y d r o l y s i s i s r e a l l y a s p e c i a l form of t r a n s -g l y c o s y l a t i o n w i t h water a c t i n g as the acceptor molecule (Pridham 1963) . 3-glucosidase i s one of the gl y c o s i d a s e s and i t a c t s mainly on g l y c o s i d e s w i t h 6 - l i n k a g e s . U s u a l l y the glycone of the su b s t r a t e a f f e c t s the enzyme a c t i v i t y to a much gre a t e r extent than the aglycone. 3- g l y c o p y r a n o s i -dase has a low degree of s p e c i f i c i t y f o r the aglycone a l -though d i f f e r e n c e s i n s t r u c t u r e do a f f e c t the r a t e of reac-t i o n . Glycosidases are u s u a l l y a c i d i c p r o t e i n s and have no p r o s t h e t i c groups or co-enzymes. Maximum h y d r o l y t i c a c t i v i t y normally occurs i n n e u t r a l or a c i d i c s o l u t i o n s . The gl y c o -s i d e undergoes a t t a c k by n u c l e o p h i l i c and e l e c t r o p h i l i c groups on the enzyme surface which r e s u l t s i n e l e c t r o n d i s -placement and rupture of the g l y c o s i d i c bond (Pridham I 9 6 0 ) . One of the b u i l d i n g b l o c k s of l i g n i n , c o n i f e r y l a l c o -h o l , i s g e n e r a l l y present i n the form of c o n i f e r i n i n p l a n t . By the a c t i o n of B-glucopyranosidase, c o n i f e r i n can be hydrolyzed i n t o c o n i f e r y l a l c o h o l which may then be acted on by l a c c a s e or peroxidase to form l i g n i n (Pridham 1 9 6 3 ) . 37 When c e l l s are mechanically i n j u r e d or diseased, contact between g l y c o s i d e s and gl y c o s i d a s e s occurs and sub-sequent h y d r o l y s i s w i t h the l i b e r a t i o n of a n t i m i c r o b i c aglycone could be an important f u n c t i o n .of g l y c o s i d a s e s (see examples on page 15). While t o x i c aglycones may be l i b e r a t e d by 6-gluco-sidase i n the host to act against pathogens, v i r u l e n c e of the l a t t e r may a l s o depend on B-glucosidase a c t i v i t y . Arneson and Durbin (1967) found t h a t S e p t o r i a l y c o p e r s i c i d e t o x i f i e s tomatin both i n v i t r o and i n i n f e c t e d tomato leaves by means of an e x t r a c e l l u l a r enzyme which hydrolyzes one glucose u n i t from the tomatin molecule. Since the enzyme i s e x t r a c e l l u l a r , i t may d i f f u s e ahead of the advancing hyphae w i t h i n the host, d e t o x i f y i n g tomatin and thus a l l o w i n g the fungus to e s t a b l i s h a s u c c e s s f u l p a r a s i t i c r e l a t i o n s h i p . C) Polyphenol Oxidase (PPO)(EC 1.10.3.1) PPO c a t a l y z e s one or both of the f o l l o w i n g r e a c t i o n s : HO ?H HO O 0^—-> o olase or hydroxylase v i t y o catecholase or o-diphenol oxidase a c t i v i t y . + H 20 The most important r o l e of PPO i n the physiology of p l a n t s i s the a b i l i t y of these enzymes to o x i d i z e monophenols to the corresponding o-diphenols. Sato (1969) showed t h a t 3 8 p-coumaric acid, can be converted to c a f f e i c a c i d by PPO i n v i t r o . By means of t h i s r e a c t i o n other monophenols might be converted to complicated polyphenols. F u r t h e r o x i d a t i o n by the o-diphenol oxidase a c t i v i t y of PPO leads to qui n o i d compounds and polymers which are r e s p o n s i b l e f o r the browning r e a c t i o n of p l a n t t i s s u e s . This o x i d a t i o n step i s much more f a m i l i a r than the h y d r o x y l a t i n g a c t i o n of PPO. A c t u a l l y the o x i d a t i o n l e a d i n g to quinone i s commonly encountered i n i n j u r e d t i s s u e or i n i n v i t r o experiments. However i t i s p o s s i b l e that the main f u n c t i o n of PPO i n v i v o i n he a l t h y t i s s u e i s h y d r o x y l a t i o n r a t h e r than complete o x i d a t i o n of phen o l i c compounds. Se v e r a l p o i n t s about PPO i n connection w i t h host-p a r a s i t e r e l a t i o n s h i p are noteworthy: ( 1 ) D i r e c t a c t i o n of PPO on other enzymes: The r o l e of,PPO i n disease r e s i s t a n c e i s t w o - f o l d . The best recognized i s the o x i d a t i o n of phenol to form quinones and polymers which are t o x i c to the pathogen or which form p h y s i c a l b a r r i e r s p r e v e n t i n g f u r t h e r extension of the fungus. The d i r e c t a c t i o n of PPO on other enzymes i s u s u a l l y overlooked. Since PPO can o x i d i z e the t y r o s i n e i n the p r o t e i n molecules of other enzymes ( S i z e r 1 9 5 3 ) , i t i s p o s s i b l e t h a t the con-forma t i o n of these enzymes would be changed and consequently the enzyme a c t i v i t y would be a l t e r e d . Laborzewski (see Rubin's review 1 9 6 4 ) showed th a t PPO from mushroom completely i n a c t i v a t e d c r y s t a l l i n e a l c o h o l dehydrogenase i n 15 min at 3 9 25°C and pH 7 . 5 . The r e s i s t a n c e of potato tuber to Phyto-phthora i n f e s t a n s during the e a r l y stages of i n f e c t i o n i s c h a r a c t e r i z e d by i n c r e a s e d PPO a c t i v i t y and simultaneous i n h i b i t i o n of dehydrogenase a c t i v i t y of the host dur i n g i n v a s i o n , ( 2 ) Latent PPO: Kenten was the f i r s t to study the l a t e n t PPO i n p l a n t m a t e r i a l s . He found t h a t water e x t r a c t s of broad bean leaves contained much l a t e n t PPO a c t i v i t y (Kenten 1 9 5 7 ) . The a c t i v e PPO i s r e l e a s e d by b r i e f exposure of" the e x t r a c t to a c i d (pH 3 - 3 . 5 ) or a l k a l i n e (pOH 2 . 5 - 3 ) c o n d i t i o n s , or by i n c u b a t i o n i n the presence of (NH.)oS0, Hr <~ if at pH 5. F u r t h e r s t u d i e s on the a c t i v a t i n g e f f e c t s of a n i o n i c w e t t i n g agents on PPO (Kenten 1 9 5 $ ) l e d him to suggest that a n i o n i c w e t t i n g agents can combine w i t h the c a t i o n i c group of the p r o t e i n l e a d i n g to the d i s s o c i a t i o n of the PPO-protein i n h i b i t o r complex or c o n f i g u r a t i o n a l changes i n a prophenolase. The a c t i v a t i o n of PPO of the host t i s s u e a f t e r i n -f e c t i o n has been reporte d by a number of workers. D e v e r a l l ( 1 9 6 1 ) found t h a t PPO of bean p l a n t s was a c t i v a t e d a f t e r i n -f e c t i o n w i t h B o t r y t i s c i n e r e a . This a c t i o n i s due to un-masking of the l a t e n t PPO by g a l a c t u r o n i c a c i d d e r i v a t i v e s which could be l i b e r a t e d from the c e l l w a l l as the r e s u l t of the a c t i o n of p e c t i c enzymes secreted by the pathogen. Another example of the a c t i v a t i o n of l a t e n t PPO i n host-p a r a s i t e r e l a t i o n s i s the a c t i v a t i o n of r i c e l e a f PPO by 4 0 o p h i o b o l i n , a t o x i n produced by the pathogen C o c h l i o b o l u s  miyabeanus (Nakamura and Oku I 9 6 0 ) . ( 3 ) I n a c t i v a t i o n of PPO: Rapid i n a c t i v a t i o n of PPO i s always encountered i n the i n v i t r o assay of the enzymes. Van Kammen and Brouwer ( 1 9 6 4 ) used chlorogenic a c i d as a s u b s t r a t e to study PPO a c t i v i t y and found t h a t the decrease i n absorbance as a r e s u l t of PPO a c t i v i t y was not l i n e a r w i t h time. They suggested t h a t t h i s may be due to product i n h i -b i t i o n . The exact mechanism i s s t i l l unknown, but the i n -h i b i t o r y e f f e c t might be a t t r i b u t e d to the r e a c t i v i t y of the quinone which the system generates. This i n v i t r o i n a c t i v a t i o n however could e a s i l y l e a d to the n o t i o n t h a t PPO would not be c a t a l y z i n g such r e a c t i o n s i n the normal h e a l t h y p l a n t c e l l s . This may have been guarded agai n s t by means of compartmentalization (Kosuge 1 9 6 9 ) . D) Peroxidase (EC 1 . 1 1 . 1 . 7 ) This enzyme has been known f o r more than one hundred years and i t s general p r o p e r t i e s are w e l l s t u d i e d (Saunders et a l . 1 9 6 4 ) . I n t h i s review the r e a c t i o n s i n which p e r o x i -dase i s i n v o l v e d are t r e a t e d b r i e f l y and f o l l o w e d by an account of the p o s s i b l e r o l e s of peroxidase i n p l a n t t i s s u e s and t h e i r r o l e i n h o s t - p a r a s i t e r e l a t i o n s . (1) Reactions Hydro x y l a t i o n : Phenylalanine, t y r o s i n e , m-tyrosine, p - c r e s o l , benzoic a c i d and s a l i c y l i c a c i d were reported to be hydroxylated by peroxidase (Saunders et a l . 1 9 6 4 ) . 41 O x i d a t i o n : Many d i f f e r e n t substances i n the p l a n t can act as su b s t r a t e s of peroxidase. Phenolics can be o x i -d i z e d to quinone by peroxidase and hydrogen peroxide: T y p i c a l peroxidase systems are capable of o x i d i z i n g s e v e r a l amino a c i d s and t h e i r d e r i v a t i v e s . The o x i d a t i o n of t y r o s i n e i n v o l v e s a q u i n o n e - l i k e i n t e r m e d i a t e and then leads f i n a l l y to a co l o r e d m e l a n i n - l i k e end product. Peroxidase a l s o c a t a l y z e s the o x i d a t i v e p o l y m e r i z a t i o n of c o n i f e r y l a l c o h o l , and the o x i d a t i o n of NADH, NADPH and IAA. (2) Peroxidase, p l a n t physiology and h o s t - p a r a s i t e r e l a t i o n s Akazawa and Conn (195$) reported that the reduced forms of NAD and NADP are r a p i d l y o x i d i z e d i n the presence of c r y s t a l l i n e h o r s e - r a d i s h peroxidase, c a t a l y t i c amounts of Mn++ and c e r t a i n phenols. One atom of oxygen was con-sumed per molecule of n u c l e o t i d e o x i d i z e d . The phenols which were a c t i v e are e i t h e r monohydric phenols or r e s o r c i n o l . Gamborg et a l . (1961) found that d i a l y z e d e x t r a c t s of pea e p i c o t y l and spruce shoots can a l s o o x i d i z e reduced NAD. They suggested t h a t the f o l l o w i n g r e a c t i o n s occurred: R.OH = phenolic c o f a c t o r . The p o s s i b l e r e l a t i o n s h i p between reducing power and v i r u l e n c e of pathogens i s f r e q u e n t l y mentioned (Kaul and Shaw I 9 6 0 ) . I f there i s a r e l a t i o n s h i p , the c a p a c i t y of peroxidase to destroy reducing power might account f o r the apparent c o r r e l a t i o n between high peroxidase of p l a n t s and r e s i s t a n c e t o c e r t a i n pathogens. IAA can be o x i d i z e d to 3-methylene oxindole by horse-radish peroxidase i n the absence of added H^O^ (Hinman and Lang 1 9 6 5) . Fox and Purves ( 1 9 6 7 ) suggested that the o x i d a t i o n occurred through a f r e e r a d i c a l mechanism. The i n c r e a s e i n peroxidase i n diseased t i s s u e may a f f e c t IAA l e v e l s . I t was a l s o r e p o r t e d t h a t phenolic compounds might r e g u l a t e the growth promoting a c t i v i t y of IAA by v i r t u e of t h e i r e f f e c t s on peroxidase. Chlorogenic a c i d at 5 x 10~^M completely i n h i b i t s the 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 amino ac i d s by peroxidase. The r e l a t i o n s h i p between IAA, p e r o x i -dase and p h e n o l i c compounds and disease i s an i n t e r e s t i n g problem. Yang ( 1 9 6 7 ) demonstrated that ethylene was r a p i d l y formed from a-keto- y -methyl t h i o b u t y r i c a c i d by horse-r a d i s h peroxidase i n the presence of Mn++, SO^-, oxygen and 4 3 a s p e c i f i c phenol. The a c t i v e phenols i n c l u d e some mono-phenols and m-diphenols. Stahmann et a l . ( 1 9 6 6 ) showed that ethylene induced r e s i s t a n c e as w e l l as increased peroxidase a c t i v i t y i n C e r a t o c y s t i s f i m b r i a t a i n f e s t e d sweet potato. I t i s s t i l l unknown whether peroxidase i s r e s p o n s i b l e f o r the p r o d u c t i o n of ethylene i n v i v o , but there seems to be no doubt that there i s some r e l a t i o n s h i p between them. As discussed above peroxidase can hydroxylate as w e l l as o x i d i z e p h e n o l i c s . I n most of the s t u d i e s of p e r o x i -dase i n diseased t i s s u e s (Table I I ) , the i n c r e a s e i n p e r o x i -dase a c t i v i t y i s c o r r e l a t e d w i t h i n c r e a s e d quinone form a t i o n and the t o x i c a c t i o n of the quinone causes n e c r o s i s . I n the n e c r o t i c t i s s u e the pathogens are e i t h e r i n a c t i v a t e d or i n h i b i t e d from spreading through the t i s s u e . Of the s e v e r a l p u t a t i v e r o l e s f o r peroxidase i t i s very d i f f i c u l t to assess the a c t u a l r o l e t h a t t h i s enzyme plays i n v i v o and e s p e c i a l l y i n h o s t - p a r a s i t e r e l a t i o n s . Therefore even though peroxidase i s a w e l l s t u d i e d enzyme there are s t i l l many aspects of i t s r o l e t h a t are worth i n v e s t i g a t i n g i n r e l a t i o n to p l a n t d i s e a s e s . 5 . PHENOLIC CONSTITUENTS OF FLAX AND HOST-PARASITE RELATIONS Cruickshank and Swain ( 1 9 5 6 ) s t u d i e d the phenolic compounds i n e t h a n o l i c e x t r a c t s of f l a x . They reporte d that the phenolic compounds i n f l a x can roughly be d i v i d e d i n t o 3 groups: (1) f l a v o n e - l i k e compounds s i m i l a r to a p i g e n i n ; ( 2 ) chlorogenic a c i d and i t s isomers; and ( 3 ) a miscellaneous group. Except f o r c h l o r o g e n i c a c i d and i t s isomer, they d i d TABLE I I . Some Reports on the Changes of AUTHORS HOST 1. Kanazawa. S c h i c h i & Sweet potato U r i t a n i ( 1 9 6 5 ) r o o t s 2 . Kawashima & U r i t a n i Sweet potato ( 1 9 6 3 ) roots 3 . S t a p l e s & Stahmann Bean leaves ( 1 9 6 4 ) 4. Andreev & Shaw Fl a x var. Bison ( 1 9 6 5 ) Bombay 5. Stahmann, C l a r e & Woodbury ( 1 9 6 6 ) 6. Weber, C l a r e & Stahmann ( 1 9 6 7 ) Sweet potato Sweet potato Peroxidase a f t e r I n f e c t i o n , PATHOGEN CHANGES IN PEROXIDASE AFTER INFECTION C e r a t o c y s t i s  f i m b r i a t a (black r o t ) C e r a t o c y s t i s  f i m b r i a t a (black r o t ) Uromyces p h a s e o l i Melampsora l i n i Increased a c t i v i t y which was i n h i b i t e d by i n h i b i t o r s of p r o t e i n and RNA s y n t h e s i s and o x i d a t i v e p hosphorylation. Increased a c t i v i t y i n both diseased and cut t i s s u e s . There i s q u a l i t a t i v e d i f f e r -ences between the healthy^ diseased and i n j u r e d t i s s u e s . Small i n c r e a s e i n a c t i v i t y . No isozymic changes. By 6 t h day a f t e r i n f e c t i o n new isozyme band formed. The new isozymes formed are s i m i -l a r to those formed i n senescent t i s s u e . C . f i m b r i a t a C. f i m b r i a t a Ethylene induced r e s i s t a n c e and increased peroxidase a c t i v i t y . Increased a c t i v i t y i n r e s i s -t a n t t i s s u e s . AUTHORS HOST 7 . Farkas & Stahmann Bean leaves (1966) 8 . Lovrekovich et a l . Tobacco (1968) 9 . Novacky & Hampton N i c o t i a n a tabacum (1968) Vigna s i e n e n s i s Phaseolus aureus P. v u l g a r i s 1 0 . Novacky & Wheeler Oats (1970) 1 1 . Rautela & Payne Sugar beets (1970) 1 2 . Chant & Bates (1970) N i c o t i a n a  g l u t i n o s a CHANGES IN PEROXIDASE AFTER  INFECTION Increase i n peroxidase and changes i n isozymes. Increased peroxidase a s s o c i a -ted w i t h TMV i n f e c t i o n i n -duced r e s i s t a n c e to tobacco to i n f e c t i o n by P. t a b a c i . Q u a n t i t a t i v e but not q u a l i -t a t i v e changes during i n f e c -t i o n and senescence. Q u a n t i t a t i v e changes i n i s o -zymes. R e s i s t a n t t i s s u e s r e -quired much higher concentra-t i o n of v i c t o r i n to give the same changes. Increase was c o n s i s t e n t l y higher i n r e s i s t a n t than i n s u s c e p t i b l e v a r i e t y . Toward the advanced stages of the disease t h i s p a t t e r n was reversed. Higher a c t i v i t y i n e x t r a c t from leaves showing n e c r o s i s than h e a l t h y or c h l o r o s i s l e a v e s . One more isozyme i n v i r u s i n f e c t e d leaves. AUTHORS HOST 13. Simons & Ross Tobacco (1970) 14. Hussey and Wando.peas Krusberg (1970) PATHOGEN CHANGES IN PEROXIDASE AFTER INFECTION TMV Increase i n peroxidase i n upper leaves i s concomitant w i t h r e s i s t a n c e development induced by v i r u s i n f e c t i o n i n lower l e a v e s . Nematode New isozymes found. Dictylenchus d i p s a c i ON 47 not i d e n t i f y any other compounds. Hegnauer (1965) pointed out t h a t f l a x seed meal contains l i n o c i n n a m a r i n (methyl e s t e r of B-glucosido-p-coumaric a c i d ) , l i n o c a f f e i n (methyl e s t e r of 4- 3 - g l u c o s i d o - c a f f e i c a c i d ) and a l i g n a n g l u c o s i d e a d i g l u c o s i d e of D -2,4-di(3-methoxy -4-hydroxybenzyl) butane-1,4 d i o l (Bakke and Klosterman 1957). Recently Ibrahim (1969) worked on the f l a v o n o i d s of f l a x cotyledons. He claimed t h a t there are 6 f l a v o n e s , two of which are api g e n i n (4 , 5 , 7-trihydroxy-flavone) type and the r e s t are l u t e o l i n - l i k e ( 3 f , 4 T , 5 , 7-tetrahydroxy-flavone). These f l a v o n e s are present i n f l a x cotyledons i n the form of mixed 0- and C-glycoflavones. I n another a r t i c l e , Ibrahim and Shaw (1970) found 9 cinnamic a c i d d e r i v a t i v e s . They were i d e n t i f i e d as p-coumaroyl q u i n i c a c i d , p-coumaroyl g l u -cose, 3 - 0 - c a f f e o y l q u i n i c ( c h l o r o g e n i c ) , glucosido c a f f e i c a c i d , c a f f e o y l glucose, glucosido f e r u l i c a c i d , f e r u l o y l g l u -cose and a g l y c o s i d e and an es t e r of s i n a p i c a c i d whose non-phenolic m o i e t i e s were not i d e n t i f i e d . They d i d not f i n d any f r e e cinnamic a c i d , benzoic a c i d d e r i v a t i v e s or f l a v o n o l g l y c o s i d e s . There was no q u a l i t a t i v e but a s l i g h t q u a n t i t a -t i v e d i f f e r e n c e between cotyledons and young shoots. Cruickshank and Swain (1956) found some r e l a t i o n s h i p between the phenolic contents of s e v e r a l v a r i e t i e s of f l a x and t h e i r r e s i s t a n c e a g a i n s t M. l i n i . They worked on 4 v a r i e t i e s of f l a x , v i z . W i l l i s t o n Golden, Ottawa 770B, Argentine Seln and Bombay, only the f i r s t one being sus-4 8 c e p t i b l e . They found t h a t W i l l i s t o n Golden has a high con-c e n t r a t i o n of chlorogenic a c i d whereas the r u s t r e s i s t a n t group had low c o n c e n t r a t i o n s . Determination of the r a t i o of c h l o r o g e n i c a c i d to t o t a l p h e n o l i c s showed that t h i s r a t i o was highest i n the h i g h l y r e s i s t a n t v a r i e t y Ottawa 770B and l e a s t i n the s u s c e p t i b l e v a r i e t y W i l l i s t o n Golden. A l l a n (1967) s t u d i e d the c o r r e l a t i o n between phen-o l i c content and h i s t o l o g i c a l changes i n s u s c e p t i b l e (Bison) and r e s i s t a n t (Bombay) v a r i e t i e s of f l a x i n f e c t e d w i t h race §3 of M. l i n i . He found t h a t the t o t a l f r e e and f r e e o - d i h y d r o x y l phenolic a c i d s decreased s h a r p l y d u r i n g the perio d of c e l l c o l l a p s e i n the r e s i s t a n t host. A f t e r the r u s t i n f e c t i o n was r e j e c t e d the r e s i s t a n t host underwent a perio d of s t i m u l a t e d phenolic accumulation or s y n t h e s i s which was f o l l o w e d by e a r l y senescence of f l a x cotyledons. T o t a l f r e e and f r e e o-dihydroxy phenolic a c i d s were accumulated d u r i n g the f i r s t 48-60 hours i n the s u s c e p t i b l e host. During t h i s stage the f u n g a l mycelium was spreading throughout the mesophyll t i s s u e . This i n c r e a s e i n phenolic a c i d s was fo l l o w e d by a sharp decrease which corresponded a p p r o x i -mately i n time to the onset of s p o r u l a t i o n by the fungus. 14 These r e s u l t s were supported by t r a c e r s t u d i e s w i t h CO . 49 MATERIALS AND METHODS 1. PLANT MATERIALS F l a x (Linum u s i t a t i s s i m u m L.) v a r i e t y Koto was stu d i e d throughout the present i n v e s t i g a t i o n . Two races'of f l a x r u s t (Melampsora l i n i Pers. Lev.),#3 and#210 were used to give r e s i s t a n t and s u s c e p t i b l e h o s t - p a r a s i t e combinations r e s p e c t i v e l y . In the r e s i s t a n t r e a c t i o n the p a r a s i t e pene-t r a t e s the host t i s s u e and p i n - p o i n t brown f l e c k i n g s occur but there i s no formation of pustules or spores. The sus-c e p t i b l e r e a c t i o n i s c h a r a c t e r i z e d by the s u c c e s s f u l estab-lishment and s p o r u l a t i o n of the p a r a s i t e . The p l a n t s used to produce r u s t i n f e c t e d t i s s u e were grown as f o l l o w s . F l a x seeds were t r e a t e d w i t h 'Arasan 75 T and sown i n 6 " p l a s t i c pots c o n t a i n i n g s o i l and peat moss i n the r a t i o of 4 : 1 . The p l a n t s were grown i n a growth chamber at a l i g h t i n t e n s i t y of $00 -1000 f t . - c . and a photo-p e r i o d of 14 hours w i t h the l i g h t p e r i o d between 6 a.m. and 8 p.m. The temperature of the growth chamber was 22-24°C during the day and l 8 - 2 0°C at n i g h t . P l a n t s used f o r p u r e l y phyto-chemical s t u d i e s and f o r r a i s i n g uredospores were grown i n the greenhouse at a temperature of about 22°C. One week o l d s e e d l i n g s were i n o c u l a t e d by d u s t i n g uredospores onto the cotyledons w i t h a brush. The p l a n t s were sprayed w i t h d i s t i l l e d water before and a f t e r d u s t i n g . A f t e r i n o c u l a t i o n , the pots were covered w i t h moist p l a s t i c bags f o r 18 hours. I n o c u l a t i o n was u s u a l l y c a r r i e d out at the end 50 of the photoperiod so that the p l a n t s could be kept i n dark-ness a f t e r i n o c u l a t i o n f o r 10 hours without i n t e r f e r i n g w i t h the normal photoperiod. The i n f e c t e d s u s c e p t i b l e t i s s u e s showed an average of 21+ pustules and a range of 2 to 62 p u s t u l e s per cotyledon. 2. PHYTOCHEMICAL STUDIES A. E x t r a c t i o n of P h e n o l i c s ( i ) T o t a l ethanol e x t r a c t (TEE) f o r i d e n t i f i c a t i o n (a) About 10 gm of cotyledons were submerged i n 200 ml of b o i l i n g 80% ethanol i n order to stop any enzymatic r e a c t i o n s . The t i s s u e s were allowed to simmer f o r 5 min and then e x t r a c t e d s i m i l a r l y w i t h two more a l i q u o t s (200 ml of 80% e t h a n o l . They were blended i n an o s t e r i z e r d u r i n g the l a s t ethanol e x t r a c t i o n and the residue was removed by suc-t i o n f i l t r a t i o n over a Buchner f u n n e l . (b) The combined e x t r a c t s were d r i e d under vacuum i n a r o t a r y evaporator.. The dry m a t e r i a l was s t i r r e d i n hot water and f i l t e r e d through c e l i t e . (c) The f i l t r a t e from (b) was d e f a t t e d w i t h l i g h t petroleum ether, d r i e d i n vacuo and f i n a l l y taken up i n 2 ml of 80% ethanol or i n 10 ml of d i s t i l l e d water f o r chromato-graphic analyses and h y d r o l y s i s r e s p e c t i v e l y . 5 1 ( i i ) TEE f o r q u a n t i t a t i v e s t u d i e s This e x t r a c t i o n procedure was b a s i c a l l y s i m i l a r t o ( i ) except that about 2 gm of cotyledons were weighed a c c u r a t e l y and e x t r a c t e d three times w i t h 5 0 ml p o r t i o n s of 80% e t h a n o l . The cotyledons were ground d u r i n g the l a s t ex-t r a c t i o n i n a mortar i n s t e a d of homogenizing i n an o s t e r i z e r . The f i n a l volume of the e x t r a c t was brought to 25 ml w i t h 5O70 ethanol i n a v o l u m e t r i c f l a s k . ( i i i ) TEE f o r autoradiography The procedure was s i m i l a r t o that d e s c r i b e d i n ( i ) except t h a t c e n t r i f u g a t i o n was used i n s t e a d of f i l t r a t i o n . The f i n a l d r i e d m a t e r i a l was u s u a l l y taken up i n 1 ml 80% e t h a n o l . B. H y d r o l y s i s Stepwise h y d r o l y s i s and l i q u i d - l i q u i d e x t r a c t i o n (Ibrahim and Towers I960) were c a r r i e d out to i d e n t i f y the phenolic compounds i n f l a x ( F i g . 4 ) . The aqueous phenolic e x t r a c t was a c i d i f i e d t o pH 2.0 and e x t r a c t e d w i t h ether by l i q u i d - l i q u i d e x t r a c t i o n . A l l f r e e phenolic a c i d s p a r t i -t i o n i n the ether phase. The aqueous phase was then hy-d r o l y z e d i n IN NaOH under n i t r o g e n f o r two hours at room temperature. The pH of the h y d r o l y s a t e was adjusted to 2.0 w i t h 5N HCl and again e x t r a c t e d w i t h ether. A l k a l i n e h y d r o l y s i s cleaves the e s t e r l i n k a g e s thereby r e l e a s i n g the phenolic a c i d s . The bulk of the s o l u t i o n was then sub-52 Flax Cotyledons I 80% ethanol extraction Evaporation to dryness I Dissolved in distilled water and filtered i 1 Evaporate to dryness _ i Ether liquid- liquid extraction Total Ethanol Extract (TEE) I f hydrolysis Ether Extract Acid hydrolysis I Ether extraction Ether extraction vf 1 I Ether Extract C Aqueous phase 3L Ether Extract n — Butanol extraction « — L  n - Butanol Extract Aqueous phase A - E : For further analysis Figure l+. A Flow Chart of E x t r a c t i o n and Stepwise H y d r o l y s i s of Phenolic Compounds. 5 3 j e c t e d to a c i d h y d r o l y s i s i n IN HC1 at 100°C f o r 30 min i n order to cleave g l y c o s i d i c l i n k a g e s . This step r e s u l t s i n the fo r m a t i o n of f r e e phenolic a c i d s . The hy d r o l y s a t e was f i r s t e x t r a c t e d w i t h ether and then w i t h n-butanol. The ether f r a c t i o n s c o n t a i n f r e e phenolic a c i d s whereas any phenolic compounds t h a t escape h y d r o l y s i s are e x t r a c t e d i n the n-butanol f r a c t i o n . Each f r a c t i o n was evaporated to dryness i n vacuo and d i s s o l v e d i n 9 5 % ethanol f o r f u r t h e r analyses. C. Chromatography The phenolic compounds were i s o l a t e d by chromatography on TLC p l a t e s ( 5 - 1 0 p l a t e s ) . Bands of the same Rf from d i f f e r e n t p l a t e s were scraped o f f and combined f o r e x t r a c t i o n w i t h 80% eth a n o l . By means of banding and rebanding and ex-p l o i t a t i o n of d i f f e r e n t s olvent systems, the phen o l i c s of f l a x were i s o l a t e d as r e l a t i v e l y pure compounds. One d i s -advantage of t h i s method i s that i t i s very d i f f i c u l t to pre-pare s a t i s f a c t o r i l y l a r g e amounts of each compound. However, the c h a r a c t e r i s t i c s of the compounds on a,chromatogram, i n -c l u d i n g UV f l u o r e s c e n c e , UV fl u o r e s c e n c e under ammonia, Rf values i n d i f f e r e n t s o l v e n t systems and the c o l o r r e a c t i o n s of the compounds w i t h d i f f e r e n t s p r a y i n g reagents provide v a l u a b l e data f o r purposes of i d e n t i f i c a t i o n . Both paper and t h i n l a y e r chromatography were t r i e d . Except f o r f r e e phenolic a c i d s the r e s o l v i n g property of paper (Whatman No. 1 and 3 ) was very poor f o r f l a x p h e n o l i c s . 5 4 For TLC, m a c r o c r y s t a l l i n e c e l l u l o s e " A v i c e l TG-104" was found to be s u p e r i o r to c e l l u l o s e MN 300 G. S i m i l a r r e -s u l t s were reported by M u l l i c k ( 1 9 6 9 ) f o r the s e p a r a t i o n of anthocyanins from c o n i f e r s . Therefore, t h i n l a y e r chro-matography on ' A v i c e l * was adopted as the r o u t i n e procedure. Thin l a y e r p l a t e s were prepared by b l e n d i n g 20 gm ' A v i c e l ' c e l l u l o s e w i t h 80 ml d i s t i l l e d water. For the i d e n t i f i c a t i o n of compounds, a 0 . 2 5 mm l a y e r of ' A v i c e l ' was coated onto the 20 cm x 20 cm g l a s s p l a t e s w i t h a Desaga t h i n l a y e r apparatus. A somewhat t h i c k e r ( 0 . 3 5 mm) l a y e r was used when the e x t r a c t was to be 'banded' on the chromatograms. The s o l v e n t systems u s u a l l y used f o r two dimensional chromatography were: ( i ) t - b u t a n o l : a c e t i c acid:water of r a t i o 3:1:1 (tBAW) and 1 5 % a c e t i c a c i d . This system gave good r e s u l t s w i t h f l a v o n o i d s and was e x c e l l e n t f o r r e s o l v i n g the t o t a l ethanol e x t r a c t . ( i i ) Benzene:acetic acid:water, 2:2:1 or 10 : 7:3 and 2% a c e t i c a c i d . This s o l v e n t gave good r e s u l t s f o r phenolic a c i d s . ( i i i ) n - b u t a n o l : a c e t i c acid:water, 4:2:1 (nBAW) and 2% a c e t i c a c i d . The reagents used f o r c o l o r r e a c t i o n s (Ibrahim and Towers I960) were: ( i ) D i a z o t i z e d p - n i t r o a n i l i n e - 5 ml p - n i t r o a n i l i n e ( 0 . 3 5 % i n 8 % HCl w/v), 1 ml NaN0 2 s o l u t i o n ( 5 % w/v), 1 5 ml 55 sodium acetate (20% w/v), f r e s h l y mixed i n the order des-c r i b e d before. Chromatograms were sprayed w i t h t h i s m i x t u r e , allowed to dry f o r 5 rnin i n the fume hood then over sprayed w i t h 5% Na 2C0^ or NaOH s o l u t i o n . ( i i ) D i a z o t i z e d s u l f a n i l i c a c i d - 2 v o l s , s u l f a n i l i c a c i d ( 9 gm/90 ml cone. HC1 and then d i l u t e d to 1 l i t r e ) , 1 v o l . sodium n i t r i t e s o l u t i o n (5% w/v) and 2 v o l s . NaOH (20% w/v). ( i i i ) F e r r i c c h l o r i d e - 1% s o l u t i o n i n 9 5 % a l c o h o l . D. UV Absorption Spectra Most ph e n o l i c compounds absorb l i g h t i n the UV range. The o p t i c a l p r o p e r t i e s of phe n o l i c compounds and t h e i r ' reac-t i o n s w i t h d i f f e r e n t reagents permit the c h a r a c t e r i z a t i o n of t h e i r f u n c t i o n a l groups. A Unicam SP800 spectrophotometer was used throughout t h i s work. Unless otherwise s p e c i f i e d the s p e c t r a were analyzed i n $0% et h a n o l . The s l i t width of the Unicam was set at 0.02 mm and the machine was set at " f a s t scan". E i t h e r 1 ml or 3 ml quartz cuvettes were used depending on sample s i z e s a v a i l a b l e . Four d i f f e r e n t reagents were used t o study the f u n c t i o n a l groups of the phenolic compounds (Harborne 1 9 6 4 ) . These were: ( i ) Sodium hydroxide - 2 drops of 2N NaOH were added to the sample i n the cuvette and mixed w e l l before the spec-trum was taken. Large bathochromic s h i f t s were noted i n most cases, and. any i n c r e a s e s i n the i n t e n s i t y of the ab-s o r p t i o n bands were a l s o observed. However, the i o n i z a t i o n of aromatic c a r b o x y l i c a c i d by NaOH causes a hypsochromic s h i f t i n the spectrum, so t h a t phenols c o n t a i n i n g f r e e c a r b o x y l i c a c i d groups do not give as l a r g e a bathochromic s h i f t i n a l k a l i n e s o l u t i o n s as the r e l a t e d e s t e r s . This allows the i n v e s t i g a t o r to d i s t i n g u i s h between these two c l a s s e s of compounds. Phenolics such as c a t e c h o l and. p y r o g a l l o l are unstable i n a l k a l i n e s o l u t i o n , ( i i ) Sodium acetate - This was used to d i s t i n g u i s h the f r e e 7 - h y d r o x y l group of f l a v o n o i d s . The l o n g wave-le n g t h band of a l l f l a v o n o i d s i s s h i f t e d bathochromically by t h i s reagent; i t i s the short wave band which undergoes a s h i f t only i f a f r e e 7 - h y d r o x y l group i s present i n the molecule. An excess of s o l i d sodium acetate was added to the cuvette c o n t a i n i n g the sample and thoroughly mixed u n t i l the s o l u t i o n was s a t u r a t e d w i t h sodium ac e t a t e . ( i i i ) Aluminum c h l o r i d e - phenols c o n t a i n i n g a c a t e c h o l (o-dihydroxyl) group-or a hydroxyl group adjacent to a carbonyl group, complex w i t h A l C l ^ i n s o l u t i o n and t h e i r a b s o r p t i o n maxima move toward the v i s i b l e range. Two to 3 drops of 5% A l C l ^ i n 95% ethanol was added to the sample f o r t h i s r e a c t i o n . ( i v ) B o r i c a c i d - A f t e r the absorption s p e c t r a were taken i n the presence of s a t u r a t e d sodium a c e t a t e , excess of b o r i c a c i d was added to the sample i n order to i d e n t i f y the c a t e c h o l groups. The s p e c t r a l s h i f t i s u s u a l l y observed 57 i n the lon g wavelength band and i s of the order of 1 5 - 3 0 mp; the s p e c t r a of a l l other phenols are not appreciably-a f f e c t e d by the presence of t h i s reagent. E. E s t i m a t i o n of Phenolics T o t a l p henolic contents were estimated according to the method of Swain and H i l l i s ( 1 9 5 9 ) . A l i q u o t s of 0.5 ml of the TEE were d i l u t e d w i t h water to 7 ml i n t e s t tubes and 0.5 ml of IN F o l i n reagent was added. The tubes were thoroughly shaken and allowed t o stand f o r e x a c t l y 3 min. One ml of sa t u r a t e d sodium carbonate s o l u t i o n was then added to each tube and the mixture was made up to 10 ml. A f t e r 1 hour, the a b s o r p t i o n was determined at 7 2 5 mp u s i n g a reagent blank. I n s o l u b l e m a t e r i a l s , i f any were c e n t r i f u g e d o f f before the readings were taken. A standard curve was prepared w i t h 0.1, 0.2, 0.4, 0.6, 0.8 and 1.0 ml of chlorogenic a c i d (100 pg/ml). F. I n c u b a t i o n Procedure The r a d i o a c t i v e chemicals used were L-phenylalanine-U- 1 / fC, L- t y r o s i n e - U - 1 / f C , D0PA-g-1/fC (New England Nuclear Corp.) and cinnamic acid-3-^^C ( I n t e r n a t i o n a l Chemical Nuclear Corp.). The r a d i o a c t i v e chemicals were n e u t r a l i z e d w i t h IN NaOH before i n c u b a t i o n . The cotyledons were placed i n a beaker c o n t a i n i n g 10 pC of r a d i o a c t i v e chemical i n 10 ml of d i s t i l l e d water. The uptake of r a d i o a c t i v e pre-• 53 c u r s o r s was f a c i l i t a t e d by vacuum i n f i l t r a t i o n f o r 3-5 m i n . The c o t y l e d o n s and t h e i n c u b a t i o n m i x t u r e was t h e n t r a n s -f e r r e d t o a p e t r i d i s h and i n c u b a t i o n was c o n t i n u e d i n a g r o w t h chamber a t 22°C unde r 800 f t . - c . o f l i g h t f o r 4 -8 h o u r s . I n e x p e r i m e n t s where i n h i b i t i o n o f p r o t e i n s y n -t h e s i s was d e s i r e d , t h e i n c u b a t i o n m i x t u r e a l s o c o n t a i n e d 600 u g / m l o f c y c l o h e x i m i d e . The p r o b l e m o f b a c t e r i a l c o n t a m i n a t i o n i n t h e i n f i l -t r a t i o n - i n c u b a t i o n method was s t u d i e d i n p r e l i m i n a r y e x p e r i -m e n t s . The re was no s i g n i f i c a n t d i f f e r e n c e i n enzyme l e v e l s ( e . g . p e r o x i d a s e ) f o r t r e a t m e n t s w i t h o r w i t h o u t G r a m i c i d i n D i f t h e i n c u b a t i o n p e r i o d was l e s s t h a n 12 h o u r s . F u r t h e r m o r e , t h e i n c o r p o r a t i o n o f p h e n y l a l a n i n e - U - ^ C i n t o p h e n o l i c com-pounds was f o u n d t o be s l i g h t l y h i g h e r i n t i s s u e s i n f i l t r a t e d w i t h G r a m i c i d i n D s o l u t i o n t h a n i n t h e w a t e r i n f i l t r a t e d t i s s u e when t h e i n c u b a t i o n p e r i o d was 8 h o u r s . From t h e s e p r e l i m i n a r y r e s u l t s i t was c o n c l u d e d t h a t b a c t e r i a l c o n t a m i n -a t i o n i s no t l i k e l y t o a f f e c t t h e m e t a b o l i s m o f t h e t r a c e r s i n e x p e r i m e n t s o f l e s s t h a n 8 h o u r s d u r a t i o n . A c c o r d i n g l y no a n t i b i o t i c s were added t o t h e i n c u b a t i o n m e d i a i n t h e e x p e r i m e n t s t o be d e s c r i b e d . Any unknown e f f e c t s o f t h e a n t i b i o t i c on m e t a b o l i s m a r e t h u s a v o i d e d . G. A u t o r a d i o g r a p h y ( i ) A u t o r a d i o g r a p h y o f who le c o t y l e d o n s . A f t e r i n c u -b a t i o n w i t h r a d i o a c t i v e p r e c u r s o r s , t h e c o t y l e d o n s were f i x e d and washed w i t h e t h a n o l (80%) t o remove any r e m a i n i n g p r e c u r s o r . 5 9 The cotyledons were then pressed between l a y e r s of b l o t t i n g paper and g l a s s p l a t e s and d r i e d i n an oven at 60°C. They were t r a n s f e r r e d onto a hard paper and autoradiographed by p l a c i n g a Kodak no-screen medical X-ray f i l m i n contact w i t h the t i s s u e . The exposure p e r i o d was 2 days and the f i l m s were developed w i t h D19 developer and f i x e d w i t h Kodak Rapid F i x . ( i i ) TLC - TLC p l a t e s were a l s o autoradiographed as d e s c r i b e d above. The exposure time v a r i e d from 1 day to 1 week, depending on the r a d i o a c t i v i t y of the compounds under i n v e s t i g a t i o n . H. L i q u i d S c i n t i l l a t i o n Counting R a d i o a c t i v i t y was q u a n t i t a t i v e l y measured i n a Nuclear-Chicago Mark I l i q u i d s c i n t i l l a t i o n counter u s i n g a dioxane based system (Chakravorty 1 9 6 9 ) c o n t a i n i n g dioxane, 800 ml; t o l u e n e , 2 0 0 ml; e t h a n o l , 3 0 ml; 2 , 5 -d i p h e n y l o x a z o l e , 7 gm; 1 , 4 B i s [ 2 - ( 5 - p h e n y l o x a z o l y l )"3 benzene, 1 5 0 mg; naphthalene, 5 0 gm; c a b - o - s i l , 3 6 gm. E i t h e r 0.2 or 0.5 ml samples were taken i n s c i n t i l l a t i o n v i a l s and to each sample 2 drops of IN NaOH and about 15 ml of the s c i n -t i l l a t i o n l i q u i d were added. The v i a l s were cooled to 0 ° and counted f o r 4 or 1 0 min. The counts thus obtained were co r r e c t e d f o r background but not f o r quenching. S i m i l a r channel r a t i o s were obtained i n a l l experiments. 6 0 3 . ENZYME STUDIES A. Enzyme E x t r a c t i o n The e x t r a c t i o n procedure was based on the method reported by Lam and Shaw ( 1 9 7 0 ) . About 1 0 0 cotyledons were ground i n a mortar w i t h a 10%, (w/v) suspension of Dowex 1 - X 8 ( C h l o r i d e form, 2 0 0 - 4 0 0 mesh) i n t r i s - g l y c i n e b u f f e r ( 0 . 0 5 M) at pH 8 . 3 . The r e s i n was washed r e p e a t e d l y w i t h d e i o n i z e d water and e q u i l i b r a t e d w i t h the b u f f e r o v e r n i g h t . The supernatant f r a c t i o n was decanted and f r e s h b u f f e r added to give approximately a 10% (w/v) suspension. The f i n a l pH was about 7 . 6 - 7 . 8 . A f t e r homogenizing, the s l u r r y was c e n t r i f u g e d at 3 0 , 0 0 0 g i n a S o r v a l R C 2 - B r e f r i g e r a t e d c e n t r i f u g e f o r 2 0 min. The supernatant f r a c t i o n represented the crude enzyme p r e p a r a t i o n . B. P r o t e i n E s t i m a t i o n For most of the enzyme work a UV a b s o r p t i o n method (Warburg and C h r i s t i a n 1 9 4 1 ) was used to estimate p r o t e i n c o n c e n t r a t i o n . When a s u b s t a n t i a l contamination by phenolic compounds was suspected, the p r o t e i n s were p r e c i p i t a t e d by TCA and then q u a n t i t a t e d by the method of Lowry et a l . ( 1 9 5 1 ) . The a b s o r p t i o n of the c o l o r complex was measured at 6 0 0 mo.. The p r o t e i n content was c a l c u l a t e d from a standard curve prepared w i t h bovine serum albumin ( F r a c t i o n V., Calbiochem.). 6 1 C. Enzyme Assays ( i ) Phenylalanine ammonia-lyase (PAL) One ml each of enzyme and a s o l u t i o n of 2 0 pMole/ml L-phenylalanine i n 0.1 M borate b u f f e r at pH 8.8, were p i p e t t e d i n t o a t e s t tube and incubated i n a water bath at 40°C. A f t e r 1 hour, the r e a c t i o n was stopped by adding 0.5 ml 5N HC1 and the s o l u t i o n was e x t r a c t e d w i t h 1 5 ml ether by vigorous shaking. The ether l a y e r was q u a n t i t a t i v e l y t r a n s -f e r r e d t o another t e s t tube and a pinch of anhydrous Na^SO, 2 4 was added t o dehydrate the ether e x t r a c t . This was then placed i n a s m a l l beaker and the ether was removed by evaporation i n a fume-hood. The r e s i d u e was r e d i s s o l v e d i n 2 ml of 0.0 1 N NaOH and the a b s o r p t i o n at 2 6 8 mp was measured. The standard curve was prepared from cinnamic a c i d and the a c t i v i t y of PAL- was expressed i n nMole cinnamic a c i d formed per mg p r o t e i n per hour. ( i i ) p -glucosidase 3 - g l u c o s i d a s e a c t i v i t y was estimated by a m o d i f i -c a t i o n of the method of S t e n l i d ( 1 9 5 7 ) . - The reagents used were c i t r a t e phosphate b u f f e r (pH 5.4) at 0.2 M w i t h respect _ 3 t o c i t r i c a c i d and 4 x 10 M p-nitrophenyl-B -glucoside (nipheglu) s o l u t i o n . The assay was done at room temperature (24°C) i n t e s t tubes. Each tube contained 1.0 ml b u f f e r , 0.1 ml nipheglu and 0.2 ml enzyme made up to 2 ml w i t h d i s t i l l e d water. The r e a c t i o n was s t a r t e d by adding nipheglu to the tubes. A f t e r 62 30 min 2 ml of 2.5% NagCO^ was added to each tube, mixed w e l l and the a b s o r p t i o n of the p- n i t r o p h e n o l formed was measured at 400 m|i. The standard curve was p l o t t e d by u s i n g 2, 4, 8, 12 and 15 ng p - n i t r o p h e n o l . ( i i i ) Peroxidase Peroxidase a c t i v i t y was estimated w i t h a f r e s h l y prepared s o l u t i o n of 0 . 1 ml 30% HgOg and 0.1 ml g u a i a c o l (Eastman) made up to 1 0 0 ml w i t h phosphate b u f f e r (pH 6.5) as s u b s t r a t e . For each assay 2.8 ml of the reagent and 0.2 ml of enzyme p r e p a r a t i o n were p i p e t t e d i n t o a 3 ml cuvette and thoroughly mixed. The reference c e l l contained 2.8 ml of reagent and 0.2 ml t r i s - g l y c i n e b u f f e r (pH 8.3). The ab-s o r p t i o n was measured at 470 m\± and the a c t i v i t y was c a l -c u l a t e d as A 0 D 470 mii/min per mg p r o t e i n . • The Unicam S P 8 0 0 spectrophotometer has no tempera-t u r e c o n t r o l but the measurement procedure never took more than 3 minutes and i t was found t h a t the heat generated by the spectrophotometer d i d not a f f e c t the readings appre-c i a b l y . Therefore t h i s study can be considered to have been conducted at room temperature of about 24°C. The same c o n d i t i o n s apply to the assays of PPO. • 63 ( i v ) Polyphenol oxidase (PPO) PPO a c t i v i t y was measured by u s i n g chlorogenic a c i d as s u b s t r a t e (Van Kammen and Brouwer 1964). PPO o x i d i z e s the phenolic h y d r o x y l groups of chlorogenic a c i d and thus causes a decrease i n a b s o r p t i o n i n the r e g i o n of 290-340 m\±. An a l i q u o t of 2.$ ml of 0.005% chlorogenic a c i d i n phosphate b u f f e r (pH 6.5) and 0.5 ml enzyme p r e p a r a t i o n were added to a 3 ml cuvette and the enzyme a c t i v i t y was e s t i -mated by measuring the decrease i n a b s o r p t i o n at 330 m\±. The enzyme a c t i v i t y was l i n e a r f o r the f i r s t l / 2 min only and then reached a p l a t e a u , probably due to product i n h i b i -t i o n . I t was t h e r e f o r e c a l c u l a t e d from the l i n e a r p o r t i o n of the curve. The data were expressed i n AOD/min per mg/protein. D. Polyacrylamide Gel E l e c t r o p h o r e s i s The method used was mo d i f i e d from t h a t of Davis (1964). The reagents and pr o p o r t i o n s of chemicals used to prepare 7.5% polyacrylamide g e l at pH 8.3 are shown i n Table I I I . I n pre p a r i n g both lower and upper gels the reagents were f i r s t mixed w e l l i n p l a s t i c s y r i n g e s (without the needle) and then i n j e c t e d c a r e f u l l y i n t o each of 10 g l a s s tubes (7 cm x 0.5 cm). Fluorescent l i g h t s were used to a l l o w the upper g e l to polymerize r a p i d l y . Sharp and f l a t boundaries between the g e l s and on the top of the upper g e l were achieved by c a r e f u l l y i n t r o d u c i n g a l a y e r of d i s t i l l e d water onto the po l y m e r i z i n g reagents. 64 TABLE I I I . The Reagents f o r the P r e p a r a t i o n of Polyacrylamide G e l . P r e p a r a t i o n of Gel Pr e p a r a t i o n of Reagents Pa r t s Reagents Chemicals Quantity 1 A pH(8.9) 1 N HCl T r i s Temed r Water 24 ml 15.$5 gm 0.32 ml to 100 ml Lower Gel 1 • 2 C Acrylamide BIS Water 30 gm 0.8 gm to 100 ml 1 Deionized water -1 G Ammonia p e r s u l -f a t e ; water 0.14 gm to 100 ml 1 B(pH 6.7) IN HCl T r i s TEMED Water 48 ml 5.9$ gm 0.32 ml to 100 ml Upper Gel 2 D Acrylamide GIS Water 10.5 gm 2.5 gm to 100 ml 2 E R i b o f l a v i n Water 4 mg to 100 ml 3 F Sucrose Water 40 gm to 100 ml 65 The r e s e r v o i r s were each f i l l e d w i t h 250 ml of pre-c h i l l e d t r i s - g l y c i n e b u f f e r ( 0 . 5 M, pH 8.3) and the whole set up was kept i n a r e f r i g e r a t o r at 4°C du r i n g e l e c t r o -p h o r e s i s . The enzyme i n 10% sucrose was c a r e f u l l y l a y e r e d on the specimen g e l . Two to three drops of 1% bromophenol blue were used as a t r a c k i n g dye. The power supply was maintained at 2.5 m i l l i Amp. per tube and at a vol t a g e l e s s than 600 V. E. Isozyme Studies A f t e r e l e c t r o p h o r e s i s the g e l s were removed from the tubes and s t a i n e d f o r p a r t i c u l a r enzymes. There i s no known method f o r the d e t e c t i o n of the isozymes of PAL and 3 - g l u c o s i d a s e on polyacrylamide g e l s . I n the present work the author u n s u c c e s s f u l l y t r i e d to s t a i n PAL isozymes by i n c u b a t i n g the g e l s w i t h phenylalanine and then s t a i n i n g w i t h n i n h y d r i n reagent or d i a z o t i z e d p - n i t r o a n i l i n e . Isozymes of peroxidase were detected by the method of S i e g e l and Galston (1966) u s i n g g u a i a c o l reagent i n 3.5% a c e t i c a c i d . Benzidine reagent was a l s o t r i e d but was not as good as g u a i a c o l . This may be due to the f a c t t h a t t h i s reagent was o r i g i n a l l y developed f o r haemoglobin or myco-g l o b i n peroxidases.' PPO isozymes were s t u d i e d by the method of Hyodo and U r i t a n i (1965) u s i n g a s o l u t i o n c o n t a i n i n g equal volumes of 0.9% c a f f e i c a c i d and 0.1% p-phenylene-diamine. The c a f f e i c a c i d i s very s p a r i n g l y s o l u b l e i n water but i t can 6 6 be s o l u b i l i z e d by n e u t r a l i z i n g i t w i t h IN NaOH to sodium c a f f e a t e (pH 6.0). By u s i n g Hyodo and U r i t a n i ' s method the c o l o r e d r e a c t i o n product formed was found to d i s s o l v e i n the s o l u t i o n e a s i l y . Therefore the reagent was made up to 3 . 5 % w i t h respect to a c e t i c a c i d to give a b e t t e r f i x a -t i o n of the c o l o r e d product i n the g e l . F. A n a l y s i s of Phenylalanine-U-^C-Labeled P r o t e i n s The e l e c t r o p h o r e s i s p a t t e r n of l a b e l e d p r o t e i n s was s t u d i e d a c c o r d i n g t o the method by Fairbanks et a l , ( 1 9 6 5 ) . The gels were s t a i n e d w i t h Amido black to l o c a t e the bands and then s l i c e d w i t h an apparatus devised by Fairbanks and co-workers. S l i c e s l / l 6 i n . t h i c k w i t h two f l a t surfaces were s e l e c t e d f o r study. The method of d r y i n g the g e l was m o d i f i e d by the present author ( F i g . 5 ) . The g e l s l i c e s were d r i e d over a "Nalgene" f i l t e r i n g f u n n e l w i t h the rim of the f u n n e l cut down t o about 1 cm h i g h . The s l i c e s were arranged on f i l t e r paper on the f u n n e l and covered w i t h ' Saran wrap'. A rubber band was used to f a s t e n the Saran wrap to the f u n n e l to keep i t a i r - t i g h t . The f u n n e l was f i t t e d i n t o a s u c t i o n f l a s k which was connected t o a water pump. This apparatus was placed 1 f o o t below a 60 watt lamp which provided heat f o r f a s t e r d r y i n g . I t u s u a l l y took 1 2 - 1 $ hours to dry the g e l s l i c e s completely. The d r i e d g e l s were arranged on b l o t t i n g paper and autoradiographed. The autoradiograms were then cut to the 67 F i g u r e $. Apparatus f o r s l i c i n g (A) and d r y i n g (B) g e l s i n poly-acrylamide g e l e l e c t r o p h o r e s i s . 6 8 s i z e of a microscope s l i d e and scanned w i t h a densitometer (Joyce-Loebel Chromoscan) u s i n g 10 mm x 0.5'mm s l i t width w i t h a 1:3 gear r a t i o of g e l to c h a r t . For the determination of phenolic and p r o t e i n con-t e n t s of f l a x , the data presented i n t h i s t h e s i s represent the averages of t r i p l i c a t e experiments. There were however wide v a r i a t i o n s i n the l e v e l of metabolic a c t i v i t y between experiments. Therefore i n t h i s t h e s i s the data presented f o r enzyme a c t i v i t y and phenylalanine i n c o r p o r a t i o n were from s i n g l e r e p r e s e n t a t i v e experiments. 69 v. ' RESULTS S e c t i o n A: Phenolics of F l a x and F l a x I n f e c t e d w i t h Rust I . The I d e n t i f i c a t i o n of Phenolics i n Koto F l a x I n studying the phenolic compounds of f l a x , i t was found t h a t they migrated only s l i g h t l y on the TLC p l a t e s developed w i t h BzAW. In t-BAW, the phe n o l i c s i n the t o t a l ethanol e x t r a c t (TEE) were separated i n t o two main groups ( F i g . 6 ) . One group c o n s i s t s of 24 spots which showed blue f l u o r e s c e n c e under UV l i g h t . The second group c o n s i s t e d of 8 spots which absorbed UV l i g h t and are t h e r e f o r e brown i n c o l o r . By means of chromatographic c h a r a c t e r i s t i c s (Table IV) and UV a b s o r p t i o n s p e c t r a (Table V) the blue f l u o r e s c e n t compounds were i d e n t i f i e d as e s t e r s and ethers of phenolic a c i d s and those of the other group as d e r i v a t i v e s of flav o n e s (Table IV and V I ) . 1. .Phenolic a c i d d e r i v a t i v e s ( i ) The b a s i c phenolic a c i d s of f l a x No f r e e phenolic a c i d s were detected on the chromato-gram of TEE, even a f t e r c o n c e n t r a t i o n by ether e x t r a c t i o n . During stepwise h y d r o l y s i s , however, phenolic a c i d s were r e -lea s e d . They were i d e n t i f i e d as C^-C^ phenolic a c i d s : p-coumaric, c a f f e i c , f e r u l i c and s i n a p i c a c i d s ( F i g s . 8 & 9 ) . A l l of them showed c i s - t r a n s isomerism i n 2% a c e t i c a c i d but not i n organic s o l v e n t s such as BzAW or n- or t-BAW. By v i s u a l judgement of the fl u o r e s c e n c e under UV l i g h t and c o l o r Figure 6. The UV f l u o r e s c e n t p i c t u r e s of the TEE of (A) h e a l t h y and (B) r u s t i n f e c t e d ( r e s i s t a n t ) cotyledons. o 71 15% HOAc—> F i g u r e 7. The I d e n t i f i c a t i o n of the Phenolic M o i e t i e s of F l a x Phenolic Compounds. 1-24: d e r i v a t i v e s of phenolic a c i d s , p-coumaric a c i d ( p c ) , c a f f e i c a c i d I c ) , f e r u l i c a c i d (F) and s i n a p i c a c i d ( S i ) . F1-F8: d e r i v a t i v e s of f l a v o n e s , apigenin (A) and l u t e o l i n ( L ) . TABLE IV. Chromatographic P r o p e r t i e s of the Phenolic Compounds of F l a x . Rf Fluorescence Color Reaction tBAW 15%H0Ac UV UV w i t h NH^ p - n i t r o a n i l i n e (PNA) PNA + Na 2C0^ Flavonoids F l 9 19 brown l i g h t y e llow y e l l o w F2 11 27.5 brown l i g h t y e l l o w y e l l o w -F3 19 7 brown l i g h t y e l l o w y e l l o w y e l l o w F4 21 35 brown ye l l o w brown - y e l l o w F5 24 47 brown ye l l o w brown - y e l l o w F6 33.5 13 brown ye l l o w brown - yellow F7 37 22.5 brown y e l l o w brown yel l o w orange FS 45 35 brown b r i g h t yellow yellow y e l l o w c 6 - c 3 Phenolics 1 2 3 4 5 6 7 $ 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 oi 32.5 45 46 45 53 55.5 56 56 57.5 59 62 62 65 65 69 69 75 75 $2.5 83 82.5 82.5 87.5 87.5 77 70 77.5 $2.5 5$ 75 77.5 82 86 65 72.5 $2.5 90 67.5 $0 $7.5 32.5 47.5 65 77.5 $5 90 90 97 blue blue blue blue blue blue blue blue blue blue blue blue blue blue blue blue blue blue blue blue green green green green green green green green green green green green green green green green green y e l l o w green y e l l o w green y e l l o w green y e l l o w green blue y e l l o w green y e l l o w y e l l o w y e l l o w y e l l o w y e l l o w purple y e l l o w y e l l o w y e l l o w blue blue y e l l o w y e l l o w y e l l o w yellow purple purple green green green green green green green green green green green green orange orange orange orange pink orange orange orange orange orange orange orange orange orange y e l l o w y e l l o w yellow y e l l o w grey grey y e l l o w y e l l o w y e l l o w y e l l o w y e l l o w y e l l o w yellow yellow yellow purple purple Figure 8 . A Trace of a Chromatogram of A l k a l i n e Hydrolysate (ether s o l u b l e f r a c t i o n ) . Figure 9. Photographs of a chromatogram of a l k a l i n e h y d r o l y s a t e (ether s o l u b l e f r a c t i o n ) of f l a x phenolic a c i d s as (A) viewed under UV, (B) sprayed w i t h p - n i t r o a n i l i n e and (C) sprayed w i t h p - n i t r o a n i l i n e and then 5% Na 2 C 0 o . (See F i g . 8 f o r the i d e n t i f i c a t i o n of these compounds'). 75 r e a c t i o n s the r e l a t i v e c o n c e n t r a t i o n s of these f o u r phen-o l i c a c i d s were found to be c a f f e i c > p-coumaric > f e r u l i c s s i n a p i c . No C^ -C-^  phenolic a c i d s were detected. ( i i ) The phenolic a c i d d e r i v a t i v e s Since many of the compounds were present i n small amounts and ran very c l o s e together on TLC, only some of them could be i s o l a t e d as pure compounds. Nearly a l l these major compounds were e s t e r s . This was shown by the l a r g e bathochromic s h i f t of the A. max. of the longer a b s o r p t i o n band of most of these compounds when NaOH was added (Table V). Further evidence came from stepwise h y d r o l y s i s . Most of the f r e e phenolic a c i d s were r e l e a s e d by a l k a l i n e hy-d r o l y s i s , whereas a c i d h y d r o l y s i s r e l e a s e d only a moderate amount of c a f f e i c a c i d and a t r a c e amount of f e r u l i c a c i d . No t r a c e s of p-coumaric or s i n a p i c a c i d were detected. The most e a s i l y i d e n t i f i e d compounds on the TLC were chlorogenic acid.and i t s isomer. I d e n t i f i c a t i o n was based on Rf values and f l u o r e s c e n c e under UV l i g h t and UV w i t h ammonia. The other prominent compounds were d e r i v a t i v e s of p-coumaric a c i d . These could not be detected under UV l i g h t but showed blue v i o l e t f l u o r e s c e n c e under UV when exposed to ammonia. The major compounds were i d e n t i f i e d by i s o l a t i o n and study of UV a b s o r p t i o n s p e c t r a and h y d r o l y t i c products. I n order to i d e n t i f y the minor compounds and those running very c l o s e together on TLC, a method was devised TABLE V . S p e c t r a l P r o p e r t i e s o f Some B l u e F l u o r e s c e n t Compounds I s o l a t e d f rom F l a x . UV a b s o r p t i o n maxima (mp,) Compounds EtOH NaOH B o r i c A c i d 1 (290) 327 280 410 - (290)34$ 2 (295) 328 275 400 255(300)345 3 (295) 325 273 3$7 255(305)347 4 (288) 325 275 394 347 11 (295) 327 - 381 256(300)348 12 (295) 328 273 382 257(303)347 13 (290) 312 - 370 315 15 (295) 327 275 394 256(303)350 17 (295) 328 ' 273 389 255(305)350 19 (295)*327 273 390 255(305)350 21 (293) 320 - 375 320 24 290-315 b r o a d p l a t e a u 370 same as E tOH *Numbers w i t h i n p a r e n t h e s i s i n d i c a t i n g " s h o u l d e r s " . 7 7 by the present author. TEE vras banded on TLC p l a t e s and developed i n t-BAW as w e l l as i n 1 5 % HOAc s e p a r a t e l y . The compounds i n the bands were i s o l a t e d and e x t r a c t e d and hy-d r o l y z e d . The ph e n o l i c a c i d s r e l e a s e d were i d e n t i f i e d . The r e s u l t s were then t a b u l a t e d i n a checker board f a s h i o n . With the help of the knowledge of the p o s i t i o n s of the major com-pounds which had been al r e a d y i d e n t i f i e d , the i n d i v i d u a l spots of the chromatogram of the TEE were p i n - p o i n t e d w i t h respect to the phenolic a c i d moiety ( F i g . 7 ) . However, even w i t h t h i s method some spots could not be i d e n t i f i e d e.g. # 5 , 8 and 1 0 . 2. F l a v o n o i d d e r i v a t i v e s The f l a v o n o i d compounds of f l a x were found t o c o n s i s t of o n l y two kinds of f l a v o n e n u c l e i , namely a p i g e n i n and l u t e o l i n . Compounds F l , F 2, F3 and F& are l u t e o l i n d e r i v a -t i v e s and the r e s t are ap i g e n i n d e r i v a t i v e s . They were i d e n -t i f i e d by t h e i r c h a r a c t e r i s t i c s p e c t r a and chromatographic p r o p e r t i e s . A l l the ap i g e n i n d e r i v a t i v e s of f l a x have f r e e 7-h y d r o x y l groups as i n d i c a t e d by the bathochromic s h i f t s of the A. max. of the short UV a b s o r p t i o n band when sodium acetate was added to the s o l u t i o n s of the compounds (Table V I ) . I n a l l apigenin-type compounds the 5-hydroxyl group was found t o be bonded because there was no prominent bathochromic s h i f t of the A. max of the long UV a b s o r p t i o n band w i t h A l C l ^ . As expected, these apigenin-type compounds TABLE V I . S p e c t r a l P r o p e r t i e s o f t h e F l a v o n o i d s o f F l a x . UV a b s o r p t i o n maxima(mp) Compounds EtOH NaOH NaOAC B o r i c A c i d A l C l ^ F l 273 350 275 414 270 407 270 371 278(300)348 F2 272 350 275 414 271 407 268 370 278 345 F3 270 352 . 272 412 276 400 263 380 275 390 F4 274 336 283(336)408 283(305)386 283(305)335 281(305)345 F5 274 335 283(336)407 283(305)390 282 340 281(305)343 F6 271 335 280(332)402 280(303)272 271(305)337 279(304)342 F7 274 336 283 408 283(305)392 282(305)342 278(300)345 F8 269 350 273 406 270 412 264 376 279(298)386 ''^Numbers w i t h i n p a r e n t h e s i s i n d i c a t e " s h o u l d e r s " . 7 9 are not responsive t o b o r i c a c i d because there i s no c a t e c h o l group ( o r t h o - d i h y d r o x y l ) present. On the other hand none of the l u t e o l i n d e r i v a t i v e s of f l a x has a f r e e 7-hydroxyl group and compounds F l and F2 have a l s o t h e i r ^-hydroxyl groups bonded. The s p e c t r a of a l l these compounds showed a bathochromic s h i f t i n the l o n g wavelength band w i t h b o r i c a c i d . This i n d i c a t e s t h a t t h e r e 3 , ' 4 f - h y d r o x y l groups of these compounds are f r e e (Table V ) . I I . Phenolic Compounds and Rust I n f e c t i o n of F l a x The 80% ethanol s o l u b l e phenolic compounds of h e a l t h y f l a x and r e s i s t a n t and .susceptible combinations of rusted f l a x at 1, 2, 4 and 6 days a f t e r i n o c u l a t i o n were s t u d i e d by chromatography. The r e s u l t s presented i n F i g u r e 10 r e -v e a l no s i g n i f i c a n t d i f f e r e n c e s were detected i n the number of spots or t h e i r running p r o p e r t i e s on TLC. However, some q u a n t i t a t i v e changes i n the t o t a l s o l u b l e phenolic content were found. For h e a l t h y cotyledons there was an i n c r e a s e i n p h enolic content as the cotyledons aged from 7 days on-ward ( F i g . 10). By the 13th day a f t e r seed germination there was a drop of 12% when compared w i t h the 11 day o l d c o t y l e -dons. As the cotyledons aged f u r t h e r and senesced the phenolic content i n c r e a s e d again. For the i n f e c t e d t i s s u e both the r e s i s t a n t and s u s c e p t i b l e combinations showed an i n i t i a l drop i n phenolic content i n the f i r s t day a f t e r i n -80 A J , 1 1 1 1 1 1 1 I 2 3 4 5 6 7 8 Days after inoculation F i g u r e 1 0 . The T o t a l P h e n o l i c s of Healthy and I n f e c t e d F l a x Cotyledons. • © Healthy (H) 0 . . . . 0 R e s i s t a n t combination (R) x x S u s c e p t i b l e combination (S) Same symbols f o r f o l l o w i n g graphs unless s p e c i f i e d . 81 o c u l a t i o n . I n the case of the r e s i s t a n t , the phenolic con-t e n t rose to the l e v e l of the h e a l t h y c o n t r o l i n the second day and t h e r e a f t e r remained above the c o n t r o l l e v e l . The s u s c e p t i b l e remained lower than the c o n t r o l i n the second day a f t e r i n o c u l a t i o n and d i d not r i s e to the c o n t r o l l e v e l u n t i l the 4 t h day. Although the p a t t e r n of the curves f o r a l l three types of t i s s u e i s very s i m i l a r , the l e v e l of phen-o l i c content i n the s u s c e p t i b l e combination was always lower than t h a t i n the r e s i s t a n t ( F i g . 1 0 ) . S e c t i o n B: Metabolism of Labeled Precursors of Phenolics I . Metabolism of Tyrosine-U-^C , DOPA-g - 1 / fC , Phenylalanine- U-^C and Cinnamate- g-^C In the s h i k i m i c a c i d pathway the key step c o n t r o l l i n g the f l o w of the benzene r i n g i n t o phenolic compounds or pro-t e i n i s guarded by ammonia-lyases. In most dicotyledonous p l a n t s phenylalanine ammonia-lyase i s the main enzyme r e s -p o n s i b l e f o r t h i s c o n t r o l l i n g p o i n t . However the deamina-t i o n of t y r o s i n e and DOPA could not be overlooked. Thus i n the present i n v e s t i g a t i o n of the metabolism of phenolic com-pounds the th r e e aromatic amino a c i d s were administered to the h e a l t h y and r u s t e d cotyledons and the metabolic products were s t u d i e d by chromatography and autoradiography. 82 1. Tyrosine: Tyrosine was found to be metabolized i n t o a number of compounds ( F i g . 1 1 ) . The amino f r a c t i o n c o l l e c -ted from a c a t i o n i c exchange r e s i n Dowex 50W-X4 column con-t a i n e d unmetabolized r a d i o a c t i v e t y r o s i n e as w e l l as 6 other compounds. A l l were n i n h y d r i n p o s i t i v e . On h y d r o l y s i s of the TEE w i t h both a c i d and a l k a l i , no phenolic a c i d s were detected. In one dimensional autoradiographs of the 'non-amino' compounds there were two major and s e v e r a l weakly l a b e l e d spots. Some of these spots were a l s o n i n h y d r i n p o s i -t i v e . Chromatography i n the second dimension w i t h 1 5 % a c e t i c a c i d showed th a t a l l these compounds moved very q u i c k l y , forming patches near the f r o n t . Again, no l a b e l e d phenolic a c i d s were r e l e a s e d by h y d r o l y s i s . 2. DO PA: 1 / fC from DOPA- B -ll*C was not found i n phenolic compounds. I t was however metabolized i n t o a number of n i n -h y d r i n p o s i t i v e and other compounds which n e i t h e r reacted w i t h n i n h y d r i n nor f l u o r e s c e d . The n i n h y d r i n negative pro-p e r t y of some l a b e l e d spots may be due to low concentrations even though the s p e c i f i c a c t i v i t i e s were high enough to be detected by autoradiography ( F i g . 1 1 ) . H y d r o l y s i s of the TEE showed no phenolic a c i d s . 3 . Phenylalanine: The amino f r a c t i o n from phenylalanine-1 4 U- C f e e d i n g showed the presence of only unmetabolized p h e n y l a l a n i n e . This i s d i f f e r e n t from t y r o s i n e and DOPA metabolism, i n which there were a number of amino-bearing 83 9 f t-BAW B Figure 11. The autoradiogram of some f e e d i n g experiments -A - 'phenolic f r a c t i o n ' of DOPA f e e d i n g B - 'amino a c i d f r a c t i o n ' of DOPA f e e d i n g C - 'amino a c i d f r a c t i o n ' of t y r o s i n e f e e d i n g D - 'phenolic f r a c t i o n ' of t y r o s i n e f e e d i n g E - 'amino a c i d f r a c t i o n ' of phenylalanine f e e d i n g . 84 compounds formed. This may be because there i s no h y d r o x y l group on the benzene r i n g which i s t h e r e f o r e not very r e a c t i v e . Phenylalanine on the other hand was metabolized i n t o a number of phenolic d e r i v a t i v e s ( F i g . 12A). The l a b e l i n g p a t t e r n of the autoradiogram of the TLC i s s i m i l a r to the p a t t e r n of blue f l u o r e s c e n t spots. The d i s t r i b u t i o n of l a b e l between the d i f f e r e n t compounds was very uneven. Thus the exposure p e r i o d necessary to r e v e a l some of the weakly l a b e l e d spots r e s u l t e d i n the over-development of the others on the autoradiogram. Except f o r one l u t e o l i n - t y p e compound, the other f l a v o n e s were not l a b e l e d . The ether e x t r a c t of TEE showed only cinnamic a c i d and t r a c e amounts of p-coumaric a c i d as l a b e l e d f r e e p h e n o l i c a c i d s . A f t e r h y d r o l y s i s p-coumaric a c i d was found to be more h e a v i l y l a b e l e d than c a f f e i c and f e r u l i c a c i d s which were approximately e q u a l l y l a b e l e d . No r a d i o a c t i v i t y was detected i n s i n a p i c a c i d ( F i g . 12B). There were no q u a l i t a t i v e d i f f e r e n c e s between the h e a l t h y , r e s i s t a n t and s u s c e p t i b l e cotyledons as f a r as the metabolism of phenylalanines i s concerned. 4. Cinnamic a c i d : p-Coumaric a c i d and i t s d e r i v a t i v e s were the major l a b e l e d compounds when the cotyledons were fed w i t h cinnamate-B-"^C ( F i g . 13). The spots observed on the autoradiogram d i d not correspond to the f l u o r e s c e n t compounds on the chromatogram of the TEE. The f l a v o n o i d s were not l a b e l l e d . Therefore i t seems tha t under the A 1$ Figure 12. Autoradiograms of TLC prepared from TEE of phenylalanine-U- C fed cotyledons (A) and the ether e x t r a c t of the a l k a l i n e h y d r o l y s a t e of the same TEE (B). 00-VJ-> 86 c o n d i t i o n s o f c i n n a m i c a c i d f e e d i n g t h e m e t a b o l i s m vras n o t n o r m a l . T h i s may be due t o t h e h i g h a c c u m u l a t i o n o f f r e e p - c o u m a r i c a c i d and i t s d e r i v a t i v e s , w h i c h a r e n o t n o r m a l l y p r e s e n t i n t h e f l a x c o t y l e d o n s . The p a t t e r n o f r a d i o a c t i v e s p o t s f o r t h e s u s c e p t i b l e c o m b i n a t i o n i s s i m i l a r t o t h a t o f t h e h e a l t h y c o n t r o l . I n t h e r e s i s t a n t c o m b i n a t i o n t h e r e i s one s p o t ( F i g . 1 3 C ) w h i c h i s more r a d i o a c t i v e t h a n t h e c o r r e s -p o n d i n g s p o t i n t h e h e a l t h y and s u s c e p t i b l e . C h r o m a t o g r a p h y o f t h e e t h e r e x t r a c t o f t h e h y d r o l y -s a t e o f TEE showed t h a t by c o m p a r i s o n w i t h p - c o u m a r i c a c i d w h i c h was h e a v i l y l a b e l e d , c a f f e i c and f e r u l i c a c i d s were o n l y s l i g h t l y l a b e l e d ( F i g . 1 4 ) . I I . F u r t h e r S t u d i e s on t h e M e t a b o l i s m o f P h e n y l a l a n i n e - U - " ^ C 1 . A c c u m u l a t i o n The a c c u m u l a t i o n o f m e t a b o l i t e s a r o u n d l e s i o n s a f t e r i n f e c t i o n i s a v e r y common phenomenon. I n t h e p r e s e n t i n -v e s t i g a t i o n r a d i o a c t i v i t y f rom t h e p h e n y l a l a n i n e - U - ^ C f e d t o t h e f l a x c o t y l e d o n s was f o u n d t o a c c u m u l a t e a r o u n d t h e l e s i o n s b o t h i n r e s i s t a n t and s u s c e p t i b l e c o m b i n a t i o n s ( F i g . 15A). S i n c e p h e n y l a l a n i n e i s a p r e c u r s o r f o r b o t h p r o t e i n and p h e n o l i c compounds i t w o u l d be more m e a n i n g f u l I f t h e n a t u r e o f t h e compounds a c c u m u l a t e d were known . I n v i e w o f t h i s p o i n t a p r o t e i n i n h i b i t o r , c y c l o h e x i m i d e , was added 1 LL t o t h e p h e n y l a l a n i n e - U - C s o l u t i o n f o r t h e f e e d i n g e x p e r i -men t . A f t e r f e e d i n g t h e c o t y l e d o n s were f i x e d w i t h b o i l i n g Figure 13. Autoradiograms of TLC prepared from TEE of cinnamate - 2 - H C f e d cotyledons. A - Healthy, B - S u s c e p t i b l e and C - R e s i s t a n t . Figure 14. Autoradiogram of TLC prepared from ether exr t r a c t of h y d r o l y s a t e of TEE of cinnarnate - 2 - ^C f e d cotyledons. 8 8 Figure 15. Autoradiogram of whole cotyledons f e d w i t h phenylalanine-U- (A) and p h e n y l a l a n i n e -U-^C plus cycloheximide (B). H - h e a l t h y , R - r e s i s t a n t and S - s u s c e p t i b l e . 89 95% ethanol to remove unmetabolized phenylalanine and the s o l u b l e phenolic compounds, l e a v i n g the p r o t e i n s and i n -s o l u b l e p henolic compounds i n the t i s s u e . The au t o r a d i o -grams i n Figu r e 1$B showed th a t the accumulation of r a d i o -a c t i v i t y at i n f e c t i o n s i t e s i n the s u s c e p t i b l e combination was i n h i b i t e d by cycloheximide. Thus the accumulation shown i n F i g . 1$A i s probably mainly due t o i n c o r p o r a t i o n of phenylalanine i n t o p r o t e i n . The r e s i s t a n t combination, how-ever, showed high accumulation at i n f e c t i o n s i t e s even i n the presence of cycloheximide. This suggests that there was a much higher degree of i n c o r p o r a t i o n of phenylalanine i n t o i n s o l u b l e p henolic m a t e r i a l s than i n the s u s c e p t i b l e combin-a t i o n . 2 . The Metabolism of Pheny l a l a n i n e - U - ^ C i n t o S o l u b l e  P h e n o l i c s ( i ) I n c o r p o r a t i o n : The i n c o r p o r a t i o n of pheny l a l a n i n e -U-^C i n t o s o l u b l e p h e n o l i c compounds i s shown i n Table V I I . The i n c o r p o r a t i o n i n the r e s i s t a n t t i s s u e was always higher than i n h e a l t h y t i s s u e except at a very l a t e stage a f t e r i n f e c t i o n . At t h i s time the cotyledons have s t a r t e d to senesce. R e s i s t a n t t i s s u e senesced e a r l i e r and f a s t e r than the h e a l t h y . This may account f o r the lower i n c o r p o r a t i o n f o r r e s i s t a n t t i s s u e at the 9th day a f t e r i n o c u l a t i o n . Furthermore the highest l e v e l of phenolic compounds i n the r e s i s t a n t a l s o a f f e c t e d the c a l c u l a t i o n of the s p e c i f i c a c t i v i t y . The peak of i n c o r p o r a t i o n f o r the r e s i s t a n t TABLE V I I . The I n c o r p o r a t i o n of Phenylalanine-U- C i n t o Phenolic Compounds. Days a f t e r Healthy R e s i s t a n t S u s c e p t i b l e I n o c u l a - Sp. Act. Sp. Act. % of Healthy Sp. Act. % of Healthy t i o n cpm/|j,g phenolics 1 2377 2906 122.2 2791 117.4 2 2509 3340 133.1 225$ 90.0 4 2325 2663 114.6 2358 101.2 6 2655 2657 100.0 2167 81.6 9 2428 2139 $8.0 1141 46.9 MO o 91 t i s s u e was 2 days a f t e r i n o c u l a t i o n . I n s u s c e p t i b l e t i s s u e , i n c o r p o r a t i o n was a l s o higher than i n he a l t h y at the e a r l y stage of i n f e c t i o n , but dropped below the he a l t h y c o n t r o l by the 2nd day a f t e r i n o c u l a t i o n . A value of y $0% lower than the he a l t h y c o n t r o l at the 9th day co i n c i d e d w i t h a c t i v e s p o r u l a t i o n of the r u s t and maximum expansion of the host cotyledons. ( i i ) A comparison of the i n c o r p o r a t i o n of ^ ^C i n t o  mono- and di-hydroxy p h e n o l i c s : As dihydroxy-phenolic compounds are more r e a c t i v e c h e m i c a l l y than mono-hydroxy p h e n o l i c s the r a t i o of i n c o r p o r a t i o n of the phenyl-a l a n i n e - U - ^ C i n t o mono- and di-hydroxy phenols i n he a l t h y and i n f e c t e d t i s s u e s was s t u d i e d . A l k a l i n e h y d r o l y s i s of the TEE was f o l l o w e d by e x t r a c t i o n of the phenolic a c i d s w i t h ether. The p-coumaric a c i d and c a f f e i c a c i d , as repre-s e n t a t i v e s of mono- and di-hydroxy phenols r e s p e c t i v e l y , were separated by TLC and the spots were marked under UV l i g h t . The spots were scraped and t r a n s f e r r e d q u a n t i t a t i v e l y i n t o v i a l s and r a d i o a c t i v i t y was measured by l i q u i d s c i n t i l -l a t i o n counting. Since only the r a t i o of cpm i n c a f f e i c a c i d : cpm i n p-coumaric a c i d was under i n v e s t i g a t i o n , the steps from e x t r a c t i o n u n t i l s p o t t i n g were not q u a n t i f i e d . The observed counts are t h e r e f o r e of no s i g n i f i c a n c e . The r e s u l t s are shown i n Table V I I I . The r e s i s t a n t t i s s u e always had a higher r a t i o than the s u s c e p t i b l e e s p e c i a l l y by the 6th day a f t e r i n o c u l a t i o n . This i n d i c a t e s that more mono-hydroxy 9 2 phenols were converted i n t o di-hydroxy phenols i n r e s i s t a n t than i n s u s c e p t i b l e t i s s u e s . The r e s i s t a n t t i s s u e had the highest r a t i o at the 2 n d day a f t e r i n o c u l a t i o n and was a l s o always higher than the he a l t h y c o n t r o l . TABLE V I I I . The Metabolism of Pheny l a l a n i n e - U - ^ C i n t o C a f f e i c and p-Coumaric A c i d s . Tissues C a f f e i c a c i d p-Coumaric Ra t i o c a f f e i c a c i d (cpm) a c i d p-coumaric a c i d (cpm)  H - -2 R 519 1 $ 8 0 0 . 3 2 8 days S 4 2 3 1 5 1 3 0 . 2 7 9 H 4 4 9 1 7 4 1 O.258 4 R 3 7 4 1 2 8 6 0 . 2 9 1 days S 7 2 5 2 6 2 6 0 . 2 7 6 H 3 1 4 119.9 0 . 2 6 2 6 R 4 3 1 2 1 4 0 0 . 2 9 3 days S 7 6 8 4 3 2 0 0 . 1 7 7 3 . The Metabolism of Phenylalanine-U- C i n t o P r o t e i n ( i ) I n c o r p o r a t i o n : I n the he a l t h y t i s s u e (Table I X ) , the r a t e of phenylalanine i n c o r p o r a t i o n dropped from the 8 t h day a f t e r seeding, when the cotyledons were mature. The rea d i n g on the 8 t h day probably represented the peak of i n c o r p o r a t i o n f o r the cotyledons and t h e n c e f o r t h the i n -c o r p o r a t i o n slowed down as the cotyledons aged and senesced. TABLE I X . The I n c o r p o r a t i o n o f P h e n y l a l a n i n e - U - C i n t o P r o t e i n . Age o f C o t y l e d o n s (days ) Days a f t e r I n o c u l a t i o n H e a l t h y s p . a c t . (cpm/mg p r o t e i n ) R e s i s t a n t s p . a c t . % o f h e a l t h y S u s c e p t i b l e s p . a c t . % o f h e a l t h y 8 1 4 0 2 5 5 4 9 0 8 0 ' 1 2 1 . 9 5 3 0 3 0 1 3 1 . 7 9 2 2 2 9 9 0 2 3 8 0 0 1 0 3 . 5 4 3 0 0 0 1 8 7 . 0 1 1 4 2 5 1 7 0 2 8 3 3 2 1 1 2 . 2 3 1 2 0 5 1 2 3 . 9 13 6 30398 2 6 4 7 0 8 7 . 0 2 7 7 5 7 9 1 . 3 1 6 9 2 2 3 9 0 1 6 3 1 9 7 2 . 8 1 8 8 6 0 8 4 . 2 9 4 I n f e c t e d t i s s u e s of both r e s i s t a n t and s u s c e p t i b l e types showed a higher i n c o r p o r a t i o n than the h e a l t h y c o n t r o l during the f i r s t 4 days a f t e r i n o c u l a t i o n . This was more remarkable i n the s u s c e p t i b l e combination. On the second day a f t e r i n -o c u l a t i o n the i n c o r p o r a t i o n In the s u s c e p t i b l e was as much as 8 7 % higher than i n the h e a l t h y c o n t r o l . The trend to a decrease i n i n c o r p o r a t i o n w i t h i n c r e a s e i n age observed i n h e a l t h y t i s s u e a l s o occurred i n the i n f e c t e d t i s s u e s . I n the s u s c e p t i b l e combination t h i s d e c l i n e was more gradual duri n g the f i r s t f o u r days a f t e r i n o c u l a t i o n . 1 4 Contrary to the i n c o r p o r a t i o n of phenylalanine-U- C i n t o phenolic compounds, the s u s c e p t i b l e combination always showed a higher i n c o r p o r a t i o n of t h i s amino a c i d i n t o pro-t e i n than the r e s i s t a n t r e a c t i n g t i s s u e (Table I X ) . ( i i ) The l a b e l i n g p a t t e r n of p r o t e i n s : In h e a l t h y t i s s u e s the l a b e l i n g p a t t e r n of p r o t e i n s changed as the cotyledons aged. I n 8 day o l d cotyledons there was more l a b e l i n g i n those p r o t e i n s which migrated only s l o w l y i n polyacrylamide g e l e l e c t r o p h o r e s i s . As the cotyledons aged, more of the f a s t moving p r o t e i n became l a b e l e d and by the 1 6 t h day, these p r o t e i n s were the most h e a v i l y l a b e l e d ones ( F i g s . 1 6 - 1 8 ) . The l a b e l i n g p a t t e r n s were the same i n h e a l t h y , r e s i s t a n t and s u s c e p t i b l e t i s s u e s f o r the f i r s t 6 days a f t e r i n o c u l a t i o n . The only d i f f e r e n c e observed between them was the r e l a t i v e degree of l a b e l i n g i n the v a r i o u s p r o t e i n bands. Figure 16. Chromoscan Patterns of the Autoradiograms of Gels c o n t a i n i n g Labeled P r o t e i n from 1 Day Old Cotyledons. vO Figure 17. Chromoscan Patterns of the Autoradiograms of Gels Containing Labeled P r o t e i n from 6 Day Old Cotyledons. F i g u r e 18. Chromoscan Patterns of the Autoradiograms of Gels con-t a i n i n g Labeled P r o t e i n f o r 9 Day Old Cotyledons. 98 An example of t h i s i s shown i n F i g u r e 1 6 . For healthy-t i s s u e the r a t i o of the r a d i o a c t i v i t y of peaks a and b i s 1 . 0 . For the s u s c e p t i b l e combination t h i s r a t i o i s l e s s than 1 .0 whereas f o r the r e s i s t a n t combination i t i s greater than 1 . 0 . By the 9 t h day a f t e r i n o c u l a t i o n the p a t t e r n s f o r the h e a l t h y and r e s i s t a n t combination were s t r i k i n g l y s i m i l a r , but the p a t t e r n f o r the s u s c e p t i b l e combination was n o t i c e a b l y d i f f e r e n t ( F i g . 1 8 ) . S e c t i o n C: P r o t e i n s and Enzymes I . Dowex Method I t was found t h a t the phenolic compounds i n f l a x i n t e r f e r e d w i t h the e x t r a c t i o n and determination of p r o t e i n and enzymes. Even w i t h p r e c i p i t a t i o n by t r i c h l o r o - a c e t i c a c i d the phenolic compounds could not be removed completely and the p r e c i p i t a t e d p r o t e i n s t i l l had a y e l l o w t i n g e and r e - d i s s o l v e d i n b u f f e r to give a y e l l o w i s h s o l u t i o n . This phenolic contamination a f f e c t s p r o t e i n determination by e i t h e r Warburg's (Warburg and C h r i s t i a n 1941) or Lowry's (Lowry et_ a l . 195-1) method. The y e l l o w pigment i s probably formed by the breaking down of c e l l compartmentation. During e x t r a c t i o n the phenolic compounds are t h e r e f o r e exposed to the a c t i o n of oxidases w i t h the formation of quinones and polymers which complex w i t h p r o t e i n . Once the complexes were formed the phenolic compounds could not be removed 99 even w i t h repeated p r e c i p i t a t i o n . I n s o l u b l e p o l y v i n y l p y r o l i d o n e (PVP) has been em-ployed as an i n s o l u b l e adsorbant f o r c l e a r i n g p r o t e i n ex-t r a c t s by Loomis and B a t t a i l e (1966) and i s now w i d e l y used f o r t h i s purpose. I t has, however, c e r t a i n disadvantages. I t probably binds only those phenolic compounds of molecular weight g r e a t e r than chlor o g e n i c a c i d (MW 354). Moreover, i t i n h i b i t s some enzymes (Harel et_ a l . 1964) and i t s c a p a c i t y f o r adsorbing p h e n o l i c s i s very low, n e c e s s i t a -t i n g i t s use i n l a r g e amounts so t h a t i t i s sometimes d i f f i -c u l t to g r i n d the t i s s u e i n a mortar. Dowex 1 - X 8 anion exchange r e s i n was used to remove the phenolic compounds d u r i n g e x t r a c t i o n before formation of the y e l l o w complex. Peroxidase, polyphenol oxidase, t o t a l p r o t e i n and t o t a l phenolic content were determined u s i n g both PVP and Dowex r e s i n e x t r a c t i o n methods. The r e s u l t s i n Table X show t h a t the Dowex r e s i n i s an e f f i c i e n t adsorbant f o r phenolic compounds. P r o t e i n content was apparently the lowest i n ( c ) , but i t must be emphasized t h a t the higher readings i n (a) and (b) were at l e a s t p a r t l y due to i n t e r f e r e n c e caused by phenolic compounds.bound to the p r o t e i n . This view i s supported by the observation t h a t the p r o t e i n prepared from (b) and e s p e c i a l l y t h a t from (a) had a y e l l o w i s h c o l o r . I t cannot t h e r e f o r e be concluded t h a t (c) had the lowest p r o t e i n content and i t i s c l e a r t h a t removal of phenolic compounds was l a r g e l y r e s p o n s i b l e TABLE X. A Comparison of the D i f f e r e n t Methods f o r P r o t e i n E x t r a c t i o n . PEROXIDASE A 0 D 4 7 0 / A0D470/mg.. E x t r a c t i o n . 2 ml Extract/min* protein/min. (a) B u f f e r 0 . 6 0 0 1 . 8 7 5 (b) B u f f e r PVP + 0 . 3 9 0 1 . 8 1 4 (c) B u f f e r Dowex + 0 . 7 5 0 3 . 8 4 6 E x t r a c t i o n POLYPHENOL A O D 3 3 0 / 0 . 2 ml Extract/min* OXIDASE AOD 330/mg protein/min • • PROTEIN CONTENT (mg/ml) PHENOLIC CONTENT (ug/ml) (a) B u f f e r 0 . 0 7 5 0 . 2 3 4 1 . 7 0 0 5 0 0 (b) B u f f e r PVP + 0 . 0 6 5 0 . 3 0 2 1 . 0 7 5 1 6 4 (c) B u f f e r Dowex + 0 . 1 0 3 0 . 5 2 8 0 . 9 7 5 . 0 -I'Crude e x t r a c t s were prepared from 2 gm f r e s h wt. of f l a x cotyledons and made up to 1 2 ml w i t h b u f f e r . A c t i v i t y per mg p r o t e i n depends on p r o t e i n estimations which are a f f e c t e d by the degree t o which phenolics are complexed and r e t a i n e d w i t h the p r o t e i n i n (a) and (b). 1 0 1 f o r i n c r e a s i n g the apparent a c t i v i t y per mg. p r o t e i n f o r both enzymes. I n a d d i t i o n , the a c t i v i t i e s of both p e r o x i -dase and polyphenol oxidase expressed per u n i t volume of e x t r a c t were d i s t i n c t l y higher i n (c) than i n (a) or ( b ) . This may r e f l e c t the more complete removal of i n h i b i t o r y phenolic compounds In (c) and p o s s i b l y a l s o an i n h i b i t o r y e f f e c t of PVP. The a b i l i t y of Dowex r e s i n to remove phenolic com-pounds was a l s o s t u d i e d by passing standard ph e n o l i c s through a column of Dowex 1-X 8. Compounds such as simple phenolic a c i d s fp-coumaric, c a f f e i c , f e r u l i c , s i n a p i c and c h l o r o -genic) and f l a v o n o i d s ( r u t i n , q u e r c e t i n and f l a v o n e ) were s t u d i e d . A l i q u o t s ( 2 ml i n $ 0 % ethanol) of (a) a known mixture of p h e n o l i c s , (b) a f r e s h phenolic e x t r a c t of f l a x and (c) an 'aged' e x t r a c t of f l a x l e f t on the bench f o r 2-3 weeks were each passed through a separate column of 6 x 1 cm Dowex which was e q u i l i b r a t e d i n 5 0 % ethanol. Twenty ml e f f l u e n t was c o l l e c t e d and concentrated to 2 ml. The phenolic content of the o r i g i n a l s o l u t i o n s and the e f f l u e n t s were estimated u s i n g c h l o r o g e n i c a c i d as a standard. The r e s u l t s i n Table XI show the r e t e n t i o n power of the Dowex r e s i n . I t i s not as e f f i c i e n t f o r the p l a n t e x t r a c t es-p e c i a l l y the 'aged' e x t r a c t . . This i s probably due to some of the phenolic compounds i n the p l a n t e x t r a c t having been denatured, polymerized or complexed w i t h other plant chemicals d u r i n g e x t r a c t i o n . Exposure of the e x t r a c t to 102 a i r would l e a d to more o x i d a t i o n and complex formation. TABLE X I . The Retention Capacity of Dowex-l-Xg f o r Standard and Pla n t Phenolic Compounds. Phenolic Mixture Quercetin Old E x t r a c t s New E x t r a c t P h e n o l i c s added 424.5 203 .0 57.5 42.5 P h e n o l i c s i n e f f l u e n t 7.0 5.5 12.5 6.5 % recovery 1.65 2.70 21.73 15.29 % absorbed 98.35 97.30 78.27 84.71 The amount of Dowex r e s i n r e q u i r e d f o r s a t i s f a c t o r y e x t r a c t i o n of enzymes was s t u d i e d by u s i n g 5 ml, 3 ml and 1 ml of 10% Dowex suspension plus the r e q u i r e d amount of p l a i n t r i s - g l y c i n e b u f f e r to make a b u f f e r : t i s s u e r a t i o of about 5 ml to 1 gm f r e s h weight. The r e s u l t s shown i n Table X I I suggest t h a t 5 ml -of 10% Dowex per gm t i s s u e i s the best f o r the e x t r a c t i o n of peroxidase. Concentrations higher than 5 ml per gm t i s s u e were not st u d i e d because i n other e x p e r i -ments t h i s 5:1 r a t i o was found to remove p r a c t i c a l l y a l l the i n t e r f e r i n g phenolic compounds and the enzyme prepara-t i o n showed UV a b s o r p t i o n spectrum c l o s e l y s i m i l a r to that of bovine serum albumen. TABLE X I I . Peroxidase A c t i v i t i e s i n Enzyme E x t r a c t s prepared by u s i n g D i f f e r e n t Q u a n t i t i e s of Dowex 1X8. T r i s - g l y c i n e T r i s - g l y c i n e T r i s - g l y c i n e T r i s - g l y c i n e B u f f e r pH 8.3 B u f f e r pH 7.6 B u f f e r pH 7.6 B u f f e r pH 7.6 + 5 ml Dowex + 5 ml Dowex + 3 ml Dowex + 1 ml Dowex AOD/min/ 0.2 ml enzyme 0.413 0.333 0.393 0.413 Protein / 0 . 2 ml enzyme 0.476 0.372 0.492 0.736 OD/min/mg p r o t e i n 0.868 0.895 0.799 O.56I 1 0 4 I I . P r o t e i n and Enzymes 1. T o t a l p r o t e i n content The curves f o r t o t a l p r o t e i n content vs. days a f t e r i n o c u l a t i o n were s i m i l a r f o r h e a l t h y and r e s i s t a n t and sus-c e p t i b l e r e a c t i n g t i s s u e s ( F i g . 1 9 A ) . There was a peak of p r o t e i n l e v e l at 2 days a f t e r i n o c u l a t i o n (equivalent to 9 day o l d c o t y l e d o n s ) . From the 4 t h day onward, the p r o t e i n content of the h e a l t h y t i s s u e l e v e l l e d out, whereas t h a t of the s u s c e p t i b l e i n c r e a s e d s l i g h t l y and the r e s i s t a n t de-creased f u r t h e r . Repeated v i s u a l observations i n d i c a t e d t h a t the cotyledons were f u l l y extended by the 9 t h day a f t e r seeding. By the 1 3 t h day, the t o t a l p r o t e i n l e v e l of the cotyledons was lower than on the 8 t h day, i n d i c a t i n g t h a t the metabolism of the cotyledons had slowed down and that senescence would soon f o l l o w . When compared w i t h the h e a l t h y c o n t r o l ( F i g . 1 9 B ) r e s i s t a n t cotyledons always had a lower p r o t e i n content a f t e r i n o c u l a t i o n but f o r s u s c e p t i b l e j u s t the opposite was t r u e . The d i f f e r e n c e between the three kinds of t i s s u e i n c r e a s e d as the i n f e c t i o n s aged. 2. Phenylalanine ammonia-lyase (PAL) The PAL a c t i v i t y was found to be very low i n h e a l t h y cotyledons o l d e r than 8 days. The a c t i v i t y was h a r d l y de-t e c t a b l e by the 1 3 t h day. In the s u s c e p t i b l e combination there was an e a r l y d e r e p r e s s i o n i n PAL a c t i v i t y which r e -turned to normal by the 2 n d day ( F i g . 2 0 ) . The a c t i v i t y was then maintained s l i g h t l y above the h e a l t h y c o n t r o l but F i g u r e 1 9 . T o t a l P r o t e i n Content and I n f e c t e d F l a x Cotyledons. Symbols are the•same as i n Figu r e 1 0 . 106 Days a f te r inoculat ion F i g u r e 20. A Comparison of PAL A c t i v i t i e s i n Healthy and Rusted F l a x Cotyledons. Symbols are the same as i n Figure 10. 107 was s t i l l very low. In the r e s i s t a n t combination, there was an i n c r e a s e o l more than 5 - f o l d when compared w i t h the hea l t h y c o n t r o l on the second day a f t e r i n o c u l a t i o n . The d e c l i n e i n a c t i v i t y on the 4 t h day was as abrupt as i t s r a p i d b u i l d up e a r l i e r . By the 6 t h day a f t e r i n o c u l a t i o n , the a c t i v i t y i n the r e s i s t a n t was not much higher than i n the s u s c e p t i b l e and by the 8 t h day the a c t i v i t y i n the r e s i s t a n t combination was h a r d l y d e t e c t a b l e . 3. Peroxidase There was a s i m i l a r p a t t e r n f o r the s p e c i f i c ac-t i v i t y of peroxidase i n h e a l t h y , s u s c e p t i b l e and r e s i s t a n t t i s s u e s when p l o t t e d a g a i n s t time a f t e r i n o c u l a t i o n ( F i g . 21). U n l i k e PAL, there was low a c t i v i t i e s f o r a l l three kinds of t i s s u e s f o r the f i r s t two days. There was a grad-u a l i n c r e a s e i n a c t i v i t y i n the he a l t h y cotyledons as they aged. Both r e s i s t a n t and s u s c e p t i b l e always had higher ac-t i v i t y than the h e a l t h y c o n t r o l w i t h the r e s i s t a n t being d i s t i n c t l y higher than the s u s c e p t i b l e a f t e r the 4 t h day. Three isozyme bands of peroxidase were detected ( F i g . 22A). There was no d i f f e r e n c e among the three k i n d s of t i s s u e s . There was a g u a i a c o l p o s i t i v e f r a c t i o n of the enzyme p r e p a r a t i o n l o c a t e d on top of the spacer g e l which d i d not move i n t o the g e l . This f r a c t i o n was b a r e l y de-t e c t a b l e i n the e a r l y stages a f t e r i n o c u l a t i o n but by the 8 t h day i t was q u i t e i n t e n s e l y s t a i n e d as judged by eye. I t was a l s o more i n t e n s e l y s t a i n e d i n the i n f e c t e d t i s s u e than i n h e a l t h y t i s s u e . 108 Figure 21. Comparative Graphs of Peroxidase i n Healthy and Rusted F l a x Cotyledons. Symbols are the same as i n Figure 10. 109 F i g u r e 22 . The Isozyme Bands of A - Peroxidase and B - PPO, i n F l a x Cotyledons. 1 1 0 4. Polyphenol oxidase. Polyphenol oxidase i n h e a l t h y cotyledons i n c r e a s e d as the cotyledons aged ( F i g . 2 3 ) . A f t e r i n o c u l a t i o n , the s u s c e p t i b l e had a lower l e v e l of t h i s enzyme than the h e a l t h y and i n s t e a d of i n c r e a s i n g f u r t h e r a f t e r the 4 t h day, l i k e the h e a l t h y c o n t r o l , i t d e c l i n e d . By the 6 t h day i t was 3 0 % below the healthy, c o n t r o l . On the other hand the en-zyme l e v e l i n r e s i s t a n t t i s s u e i s higher than i n the h e a l t h y c o n t r o l and there was a peak of enhancement on the 4 t h day when the a c t i v i t y was 2 7 % higher than the h e a l t h y . The e a r l y senescence observed i n the r e s i s t a n t cotyledons c o i n -cided w i t h a very high value of PPO a c t i v i t y on the 8th day. There were 7 isozyme bands detected f o r the f l a x PPO ( F i g . 2 2 B ) . Most of them moved very s l o w l y down the g e l . There were no d e t e c t a b l e d i f f e r e n c e s between the h e a l t h y , r e s i s t a n t and s u s c e p t i b l e . 5. S-Glucosidase The a c t i v i t y of t h i s enzyme a l s o i n c r e a s e d w i t h the age of the cotyledons u n t i l the 8 t h day a f t e r i n o c u l a t i o n , when the h e a l t h y and s u s c e p t i b l e d e c l i n e d s l i g h t l y . As w i t h PPO, the r e s i s t a n t t i s s u e always had a higher l e v e l of g-glucosidase than the h e a l t h y c o n t r o l . On the other hand the a c t i v i t y i n the s u s c e p t i b l e combination was always lower than i n h e a l t h y t i s s u e . F i g u r e 2 3 . Comparative Graphs of Polyphenol Oxidase i n Healthy and Rusted F l a x Cotyledons. S y m b o l s 60 J i 1 5 1 1 1 r r — I 2 3 4 5 6 7 8 Days after inoculation F i g u r e 24. Comparative Graphs of ft-Glucosidase i n Healthy and Rusted F l a x Cotyledons. Symbols are the same as i n Figure 10. 1 1 3 I I I . E f f e c t of Water I n f i l t r a t i o n on Enzyme A c t i v i t i e s Since an i n f i l t r a t i o n method was used to study the metabolism of r a d i o a c t i v e precursors i t i s appropriate to examine the e f f e c t of water i n f i l t r a t i o n on the enzyme a c t i v i t i e s . Furthermore, i t i s p o s s i b l e t h a t i n f i l t r a t i o n w i t h water under reduced pressure causes some mechanical i n j u r y so t h a t a study of the e f f e c t of water i n f i l t r a t i o n may provide some i n s i g h t i n t o host response to i n j u r y as compared w i t h r u s t i n f e c t i o n . Since the present i n v e s t i g a t i o n centres on phenolic compounds the e f f e c t s of a standard phenolic s o l u t i o n , c h l o r o -genic a c i d and a hot aqueous e x t r a c t of f l a x cotyledons were a l s o s t u d i e d . Another set of experiments was conducted u s i n g Actinomycin D, Actinomycin D plus chlorogenic a c i d and Actinomycin D plus f l a x p l a n t e x t r a c t as i n f i l t r a t i n g s o l u t i o n s . The c o n c e n t r a t i o n of chlorogenic a c i d was 2 x 10"^M and the f l a x e x t r a c t had an e q u i v a l e n t amount of phenolic m a t e r i a l . The c o n c e n t r a t i o n of Actinomycin D was about 5 0 [ig per ml. The i n f i l t r a t e d cotyledons were t r a n s -f e r r e d to a p e t r i d i s h and incubated i n a growth chamber under $ 0 0 f t . - c . at 22°C f o r 1 2 hr. Ex c i s e d cotyledons i n -cubated on moist f i l t e r paper i n a p e t r i d i s h were used as c o n t r o l s . The r e s u l t s are shown i n Fig u r e 25.. Water i n f i l t r a -t i o n caused an i n c r e a s e of more than 1 5 0 % i n the a c t i v i t y of both PAL and peroxidase as compared to the c o n t r o l . The Control H2O Chlorogenic Flax acid Extract Act. D Act. D Act. D + + Chlorogenic Flax acid Extract F i g u r e 2 5 . The E f f e c t of I n f i l t r a t i o n w i t h Water and S o l u t i o n s of other Compounds on Enzyme A c t i v i t i e s : A, PAL and B, Peroxidase. 1 1 5 a d d i t i o n of c h l o r o g enic a c i d had no f u r t h e r e f f e c t . On the other hand f l a x e x t r a c t s caused a f u r t h e r i n c r e a s e of 1 5 0 % i n PAL and of about 60% i n peroxidase i n a d d i t i o n to the e f f e c t of p l a i n water. Actinomycin D i n h i b i t e d the i n c r e a s e i n PAL and peroxidase a c t i v i t i e s caused by water i n f i l t r a -t i o n . I t s enzyme i n h i b i t o r y e f f e c t i s more pronounced i n the case of f l a x e x t r a c t i n f i l t r a t i o n than the p l a i n water i n f i l t r a t i o n . There was 6 7 % i n h i b i t i o n of PAL and 80% i n -h i b i t i o n of peroxidase i n the former but o n l y 2 6 % i n h i b i t i o n of PAL and 60% i n h i b i t i o n of peroxidase i n the l a t t e r . A study of the e f f e c t of water, f l a x e x t r a c t and Actinomycin D s o l u t i o n s vs. time of i n c u b a t i o n a f t e r i n f i l -t r a t i o n showed th a t there was a l a g phase of 2 hours f o r a l l three treatments ( F i g . 2 6 ) . For PAL an i n c r e a s e f o l l o w e d t h i s l a g phase i n a l l treatments. A f t e r the 4 t h hour, the i n c r e a s e i n PAL on the treatment w i t h Actinomycin D d e c l i n e d . The a c t i v i t y i n the water t r e a t e d cotyledons d e c l i n e d a f t e r the 6 t h hour and t h a t i n the f l a x e x t r a c t t r e a t e d cotyledons remained very h i g h f o r 1 2 hours. These temporal s t u d i e s a l s o i n d i c a t e t h a t b a c t e r i a l contamination i s not r e s p o n s i -b l e f o r the observed i n c r e a s e s i n enzyme a c t i v i t y . I f i n f i l t r a t i o n induced b a c t e r i a l growth i n c r e a s e s i n enzyme-a c t i v i t i e s would be expected to be continuous throughout the p e r i o d of i n c u b a t i o n . There was a l s o a l a g p e r i o d of 2 hours f o r the per-oxidase a c t i v i t i e s ( F i g . 2 6 ) , which then i n c r e a s e d r a p i d l y , 1 1 6 Figure 2 6 . Temporal Changes of Enzyme A c t i v i t i e s (A, PAL and B, Peroxidase) a f t e r I n f i l t r a -ted w i t h o © water, o o f l a x e x t r a c t and x -x Actinomycin D. 1 1 7 the r a t e s of i n c r e a s e being i n i t i a l l y comparable f o r both water and f l a x phenolic e x t r a c t treatments. By the 8 t h hour a higher l e v e l of a c t i v i t y was c l e a r l y e s t a b l i s h e d i n the phenolic treatment. The Actinomycin D t r e a t e d t i s s u e showed a low peroxidase a c t i v i t y at a l l times. P r e l i m i n a r y evidence that the i n c r e a s e i n enzyme a c t i v i t i e s i s due to enzyme s y n t h e s i s was provided by a f e e d i n g experiment. Phenylalanine-U-^C was f e d by i n f i l -t r a t i o n to two set s of cotyledons i n the presence and ab-sence of Actinomycin D. A 3 r d set of cotyledons was water i n f i l t r a t e d and incubated i n l i g h t i n a growth chamber f o r 4 hours before being i n f i l t r a t e d w i t h phenylalanine-U-~^C s o l u t i o n . A l l 3 s e t s of cotyledons were incubated i n the r a d i o a c t i v e s o l u t i o n f o r 4 hours. Table X I I I shows t h a t ther e was 2 7 % l e s s i n c o r p o r a t i o n of phenylalanine-U-^^C i n t o p r o t e i n i f Actinomycin D was added to the i n f i l t r a -t i o n s o l u t i o n . I n c o r p o r a t i o n i n t o p r e - i n f i l t r a t e d t i s s u e was 3 1 % higher than i n t i s s u e which was not p r e - i n f i l t r a t e d . In the second p r e c i p i t a t i o n of the p r o t e i n , by which more non-protein r a d i o a c t i v e contaminants were removed, the d i f f e r e n c e s between the treatments were even more prominent (Table X I I I ) . 118 14 TABLE X I I I . The I n c o r p o r a t i o n o f P h e n y l a l a n i n e - U - C i n t o P r o t e i n by I n f i l t r a t i o n M e t h o d . P r e - i n f i I t r a t e d 1 4 c + A c t . D P h e - U - ^ C P h e - U - ^ C P h e - U - i y f o r 4 h r F i r s t P r e c i p i t a t i o n Second P r e c i p i t a t i o n S p . A c t . cpm/mg 1 5 4 5 2 2 1 2 2 2 27880 p r o t e i n % 7 2 . 8 1 0 0 1 3 1 . 3 S p . A c t . cpm/mg 5 3 5 1 8 5 9 6 I 4 5 2 O . p r o t e i n % 6 2 . 2 1 0 0 1 6 9 . 0 119 DISCUSSION S e c t i o n I: P h e n o l i c Compounds o f F l a x O x i d a t i o n and l i g n i f i c a t i o n a r e g e n e r a l l y c o n s i d e r e d t o be t h e m a j o r r o u t e s f o r c o n v e r s i o n o f p h e n o l i c s i n t o d i s e a s e r e s i s t a n t p r i n c i p l e s i n p l a n t s . Q u i n o n e s fo rmed by o x i d a t i o n o f p h e n o l i c compounds can i n a c t i v a t e enzymes p r o d u c e d by p l a n t p a t h o g e n s (By rde 1 9 6 3 , P a t i l and Dimond 1 9 6 7 ) . I n o r d e r t o make o x i d a t i o n p o s s i b l e , t h e p h e n o l i c m o i e t i e s o f t h e compounds must have c e r t a i n f u n c t i o n a l g r o u p s s u c h as f r e e h y d r o x y l and c a t e c h o l g r o u p i n g s f o r t h e o x i d a t i v e enzymes t o a c t o n . I n t h e p r e s e n t s t u d i e s , t h e 24 s p o t s o f p h e n y l p r o -p a n o i d d e r i v a t i v e s d e t e c t e d on TLC ( F i g . 6) r e p r e s e n t no more t h a n 14 compounds . Many o f t h e s p o t s on t B A W / a c e t i c a c i d two d i m e n s i o n a l TLC were i n p a i r s as c i s - and t r a n s -i s o m e r s o f t h e same compounds. O n l y one o f t h e 12 m a j o r s p o t s o f t h e s e C^-C-^ d e r i v a t i v e s was f o u n d t o have a f r e e c a r b o x y l g roup ( T a b l e V ) . On t h e o t h e r hand 10 o f t h e s e 12 s p o t s showed o - d i h y d r o x y l g r o u p s . F o r t h o s e compounds s h o w i n g o n l y one s p o t t h e d o u b l e bonds be tween t h e B -and Y -carbon atoms must have been b r o k e n and a d d i t i o n o r c o n d e n s a t i o n p r o d u c t s were f o r m e d . Bakke and K l o s t e r m a n (1957) showed t h a t i n f l a x seed m e a l t h e r e i s a l i g n a n d i -g l u c o s i d e (two f e r u l i c g l u c o s i d e m o l e c u l e s l i n k e d a t t h e 6 - c a r b o n ) . 1 2 0 Thus i t was found t h a t most of the C ^ - Q ^ phenolic compounds i n f l a x are c a f f e o y l and p-coumaroyl i n nature. These compounds are present mainly as est e r s r a t h e r than g l y c o s i d e s . Their hydroxyl groups are t h e r e f o r e f r e e and they provide good s u b s t r a t e s f o r o x i d a t i o n . The f l a v o n o i d s of f l a x have the same p a t t e r n of hydro x y l groupings as the major C^-C^ d e r i v a t i v e s . How-ever whether t h e i r h ydroxyl groups could be o x i d i z e d or condense w i t h other compounds i s not known. ' Some f u n g i and b a c t e r i a can degrade f l a v o n o i d s such as r u t i n to simpler phenolics and carbon monoxide. These phenolic fragments, i f leached i n t o the s o i l , would become bound as humic a c i d (Harborne 196$). The problem of o x i d a t i o n and condensa-t i o n of f l a v o n o i d s . i n the n e c r o t i c l e s i o n s i s worth st u d y i n g . Clark et a l . (1959), working w i t h potato p e e l , found that a d e r i v a t i v e of C^-G^ phenolic a c i d , chlorogenic a c i d , was r e a d i l y combined w i t h amino acid s such as phenyl-a l a n i n e , t y r o s i n e , tryptophane, methionine, v a l i n e and i s o -l e u c i n e to form a d d i t i o n a l products. They suggested that the a d d i t i o n of amino a c i d s to the r e a c t i v e centres on the ben-zene r i n g a f t e r o x i d a t i o n of the ortho phenol to a quinone could prevent p o l y m e r i z a t i o n of the quinone. They showed tha t the amino-chlorogenic a c i d a d d i t i o n product i s h i g h l y t o x i c to the growth of Helminthosporium carbonum. The t o x i c compound i s th e r e f o r e produced by o x i d a t i o n of a phenolic compound and subsequently complexed w i t h amino 1 2 1 a c i d s . They speculated t h a t i n the r e s i s t a n t combination t h i s process i s very l i k e l y to occur. A l l the b u i l d i n g blocks of l i g n i n such as p-coumaroyl, f e r u l o y l and s i n a p o y l d e r i v a t i v e s are present i n f l a x . Many of them have double bonds between the g- and y - carbon i n t a c t (as shown by c i s - and t r a n s - i s o m e r i z a t i o n ) . Rohringer et a l . ( 1 9 6 7 ) and Fuchs and DeVries ( 1 9 6 9 ) working on rusted wheat and Fusarium i n f e c t e d tomato p l a n t s r e s p e c t i v e l y found t h a t there was i n c r e a s e d i n c o r p o r a t i o n of "*"^C from shikimate-and quinate-U-^C i n t o i n s o l u b l e , non-proteinaceous m a t e r i a l s and they suggested t h a t there was enhanced l i g n i f i c a t i o n . I n the present s t u d i e s most of the c o n d i t i o n s were favourable f o r l i g n i f i c a t i o n i n the r e s i s t a n t combination a f t e r i n o c u l a -t i o n , i n c l u d i n g the dehydropolymerization step (as oxida-t i o n i s u s u a l l y enhanced at the i n f e c t i o n s i t e ) except t h a t the conversion of cinnamic a c i d d e r i v a t i v e s to t h e i r cinnamoyl a l c o h o l s - r e q u i r e s enzymatic r e d u c t i o n (Freudenberg and Neish 1968) which i s not p r e v a l e n t i n the n e c r o t i c t i s s u e s . Therefore the present author p r e f e r s the theory t h a t o x i d a t i o n and o x i d a t i v e p o l y m e r i z a t i o n of polyphenols predominate i n the formation of the i n s o l u b l e bound phen-o l i c compounds at the i n f e c t i o n s i t e r a t h e r than the theory that l i g n i f i c a t i o n i s enhanced. The t o t a l s o l u b l e phenolic content of the r e s i s t a n t combination decreased immediately a f t e r i n o c u l a t i o n but by the 2 n d day i t was a l r e a d y higher than the h e a l t h y c o n t r o l 1 2 2 ( F i g . 1 0 ) . These r e s u l t s are s i m i l a r to those found by A l l a n ( 1 9 6 7 ) with Bombay f l a x i n f e c t e d w i t h race #3 of M. l i n i .• The po i n t at which the phenolic l e v e l i n i n f e c t e d t i s s u e overtakes the he a l t h y c o n t r o l s was a l s o on the 2 n d day a f t e r i n o c u l a t i o n i n h i s s t u d i e s . The present f i n d i n g s f o r the s u s c e p t i b l e combination do, however, d i f f e r from A l l a n ' s i n tha t th e r e was al s o an i n i t i a l drop i n phenolic content r a t h e r than the i n i t i a l i n c r e a s e t h a t he re p o r t e d . Such d i f f e r e n c e s may be due to d i f f e r e n c e s i n the degree of s u s c e p t i b i l i t y of Koto/#210 and Bison/#3. Shaw ( 1 9 6 7 ) showed th a t even i n a s u s c e p t i b l e f l a x / r u s t combination there are aborted l e s i o n s . A b o r t i o n of the pathogen occurred mainly between 21+ and 1+8 hours a f t e r i n o c u l a t i o n and the number of l i v i n g i n f e c t i o n s was as low as 6 3 % of the t o t a l number of i n f e c t i o n centres per cotyledon. I t i s p o s s i b l e t h a t aborted l e s i o n s may have a biochemistry l i k e that of r e s i s t a n t l e s i o n s . An i n t e r p r e t a t i o n of the changes i n t o t a l s o l u b l e phenolic content a f t e r i n o c u l a t i o n i s tha t the p h e n o l i c s are o x i d i z e d and polymerized to form the i n s o l u b l e m a t e r i a l s bound i n the c o l l a p s e d c e l l s e i t h e r to the c e l l w a l l or complexed w i t h the p r o t o p l a s t s . Therefore there was an e a r l y drop i n the phenolic content. Farkas et a l . ( 1 9 6 2 ) have a l s o shown tha t the o - d i h y d r i c phenols decreased d u r i n g l e s i o n f o r m a t i o n i n v i r u s i n f e c t e d p l a n t s showing l o c a l l e s i o n s . At about the same time, phenolic b i o s y n t h e s i s 123 was turned on and by the 2nd day the. phenolic content was higher than the h e a l t h y c o n t r o l ( F i g . 10), This i n d i c a t e s t h a t at t h i s stage the s y n t h e t i c r a t e i s higher than the r a t e of removal from the s o l u b l e pool i n t o bound phenolic compounds. The f a c t that the phenolic l e v e l i n the sus-c e p t i b l e combination did. not reach the l e v e l i n h e a l t h y c o n t r o l s u n t i l the 4th day a f t e r i n o c u l a t i o n ( F i g . 10) i n d i c a t e s a slower s y n t h e s i s or accumulation i n the sus-c e p t i b l e as compared to the r e s i s t a n t combination. To conclude t h i s s e c t i o n , the f o l l o w i n g p o i n t s are noteworthy i n r e l a t i n g p h e n o l i c s and r e s i s t a n c e : (1) The phenolic c o n s t i t u e n t s of f l a x are favour-able s u b s t r a t e s f o r o x i d a t i o n and l i g n i f i c a t i o n . (2) The q u a n t i t a t i v e changes i n phenolic content a f t e r i n f e c t i o n provide evidence to support the involvement of p h e n o l i c s i n r e s i s t a n c e . S e c t i o n I I : Metabolism of Phenolic Precursors 1. M e t a b o l i c Pathway Tyrosine can be metabolized i n t o p h e n o l i c s under the a c t i o n of t y r o s i n e ammonia-lyase which converts i t to p-coumaric a c i d . The l a t t e r i s then metabolized to other phenolic compounds. However, by means of t r a c e r s t u d i e s , no conversion of t y r o s i n e t o p-coumaric a c i d was detected i n the present work and t h e r e f o r e TAL i s probably absent 1 2 4 1 4 i n f l a x cotyledons. The metabolism, of t y r o s i n e - U - C was q u i t e s i m i l a r to that of DOPA. P o s s i b l y some of the t y r o -s i n e was converted to DOPA and then metabolized i n t o a group of amino-bearing compounds. Young and Neish ( 1 9 6 6 ) were able to show deamination of DOPA by acetone powders of wheat shoot but not by Pte r i d i u m . Even though they c l a i m t h a t there was deamina-t i o n (based on the r a d i o a c t i v e counts of the ether e x t r a c -t a b l e m a t e r i a l from the enzyme assay s o l u t i o n ) , they were unable to i d e n t i f y the product. I f the enzyme simply cleaves NH^ from the phenolic moiety the product should be c a f f e i c a c i d . I n the present i n v e s t i g a t i o n no r a d i o -a c t i v e c a f f e i c a c i d was detected. There i s t h e r e f o r e no evidence f o r a DOPA-ammonia l y a s e i n f l a x cotyledons. Phenylalanine i s the precursor of the C^-C^ u n i t s of the phenolic compounds of f l a x . This i s shown by the s i m i l a r i t y of the autoradiogram of the phenolic e x t r a c t 1 4 a f t e r phenylalanine-U- C f e e d i n g ( F i g . 12A) and the f l u o r e s c e n t spots on the TLC ( F i g . 6 ) . Furthermore, on h y d r o l y s i s , the cinnamic a c i d s commonly found as t h e i r d e r i v a t i v e s i n f l a x such as p-coumaric, c a f f e i c and f e r u l i c a c i d s were r a d i o a c t i v e . The metabolic pathway, t h e r e f o r e , i s probably c o n s i s t e n t w i t h t h a t found by McCal l a and Neish ( 1 9 5 9 ) : P henylalanine > cinnamic a c i d > p-coumaric a c i d s i n a p i c a c i d ^ f e r u l i c a c i d 4 c a f f e i c a c i d 4--1 2 5 1 4 The f e e d i n g of cinnamate- B- C, the second member i n t h i s pathway should t h e r e f o r e give a l a b e l i n g p a t t e r n s i m i l a r to t h a t obtained by f e e d i n g p h e n y l a l a n i n e . However, t h i s i s not the case when cinnamate-B -~^C was f e d t o the cotyledons ( F i g . 1 3 ) . There was an accumulation of f r e e p-coumaric a c i d and of some p-coumaryl d e r i v a t i v e s . No f r e e p-coumaric a c i d was formed when phenylalanine was f e d . In a d d i t i o n , the l a b e l i n g i n c a f f e o y l and f e r u l o y l d e r i v a -1 4 t i v e s i s very low when cinnamate-B - C was f e d . Apparent-l y the metabolism of cinnamate- B -~^C d i d not f o l l o w the u s u a l metabolic pathway i n f l a x cotyledons. This may be accounted f o r by the h y p o t h e t i c a l scheme proposed by Zucker et a l . ( 1 9 6 7 ) . I n t h e i r scheme they suggested t h a t cinnamic a c i d i n h i b i t s the conversion of p-coumaric a c i d i n t o c a f f e i c a c i d . This was i n d i c a t e d by the f a c t t h a t a d d i t i o n of cinnamic a c i d to potato tuber d i s c s would i n -h i b i t .the s y n t h e s i s of chlorogenic a c i d , w i t h a concomitant accumulation of 3-O-p-coumaroyl q u i n i c a c i d and other p-coumaroyl conjugates as w e l l as f r e e p-coumaric a c i d . The present f i n d i n g s seem to f i t i n t o t h i s scheme very w e l l . The conversion of o - d i p h e n o l i c compounds such as c a f f e i c a c i d to O-methyiated d e r i v a t i v e s i s c a t a l y z e d by O-methyltransferase. I n the present experiments only f e r u l i c a c i d was found to be l a b e l e d when both phenylalanine-U-^^C and cinnamate- B. -"^C were used as precursors whereas 126 s i n a p i c a c i d was not. E i t h e r the h y d r o x y l a t i o n and methy-l a t i o n of the 5 - p o s i t i o n of the benzene r i n g d i d not occur under the present experimental c o n d i t i o n s or the product, s i n a p i c a c i d , was formed i n very low amounts and i t com-plexed w i t h the i n s o l u b l e f r a c t i o n as soon as i t was pro-duced. 2. Accumulation Accumulation of me t a b o l i t e s and other compounds i n . diseased areas of leaves was a f a v o u r i t e subject of i n -v e s t i g a t i o n by plant p a t h o l o g i s t s d u r i n g the 1950's. Amongst the v a r i o u s m e t a b o l i t e s , carbohydrates were f r e -quently observed to accumulate i n the host t i s s u e surround-i n g the p a r a s i t i c colonies. (Yarwood. and Jacobson 1955, Shaw and Samborski 1956 and Tanaka and Akai I960). The accumu-l a t i o n of phenolic m a t e r i a l around disease l e s i o n s has, however, r e c e i v e d l i t t l e a t t e n t i o n . I n t h i s t h e s i s , w i t h 14 the help of phenylalanine-U- C t r a c e r , accumulation of r a d i o a c t i v i t y was found i n both s u s c e p t i b l e and r e s i s t a n t . combinations. By means of a p r o t e i n i n h i b i t o r , c y c l o -heximide, i t was shown that the m a t e r i a l that accumulated around the r e s i s t a n t l e s i o n s i s . d i f f e r e n t -in nature from that around s u s c e p t i b l e l e s i o n s ( F i g . 15). The r e s i s t a n t l e s i o n s accumulated l a b e l i n g i n t r e a t -ments w i t h or without cycloheximide. This i n d i c a t e s t h a t some of the accumulated compounds were not p r o t e i n s . They., could be: -127 (1) l i g n i n (2) polymers of o x i d i z e d phenolics ( 3 ) o x i d i z e d simple p h e n o l i c s complexed w i t h p r o t e i n or c e l l w a l l m a t e r i a l and t h e r e f o r e not removed by hot e t h a n o l . The accumulation of i n s o l u b l e phenolic compounds around the l e s i o n s i n r e s i s t a n t t i s s u e i s su p p o r t i n g evidence f o r one of the f o l l o w i n g t h e o r i e s of r e s i s t a n c e : (1) the r e s i s t a n t r e a c t i o n i s due to the formation of t o x i c o x i d i z e d p h e n o l i c s ( i . e . quinones) which p r e c i p i -t a t e and i n a c t i v e exogenous enzymes of the pathogen by complexing w i t h them as w e l l as k i l l i n g the host c e l l and hence d e p r i v i n g the pathogen of n u t r i e n t . (2) d e p o s i t i o n of phenolic polymers or l i g n i f i c a t i o n may r e s u l t i n the formation of p h y s i c a l b a r r i e r s t h a t c o n t a i n the pathogen. ( 3 ) a combination of the two. Rohringer et a l . ( 1 9 6 7 ) , i n studying r u s t - i n f e c t e d primary leaves of wheat, found t h a t r e s i s t a n t leaves i n -14 corporated more r a d i o a c t i v i t y from shikimate-U- C and quinate-U-^C i n t o the non-hydrolyzable, i n s o l u b l e residue than the s u s c e p t i b l e l e a v e s . They suggested t h a t some of the i n s o l u b l e e s t e r f r a c t i o n s are intermediates i n the syn-t h e s i s of wheat l e a f l i g n i n . F u r t h e r work from t h i s group on the metabolism of phenylalanine-U-^^C (Fuchs et a l . 1967) a l s o showed t h a t the p r o p o r t i o n i n i n s o l u b l e e s t e r s 1 2 8 i n c r e a s e d more markedly i n r e s i s t a n t leaves than i n sus-c e p t i b l e l e a v e s . For the s u s c e p t i b l e combination, i n c u b a t i o n w i t h phenylalanine without cycloheximide r e s u l t i n the accumula-t i o n of r a d i o a c t i v i t y at the l e s i o n s . I n the presence of cycloheximide o n l y a few i s o l a t e d l e s i o n s accumulated small q u a n t i t i e s of l a b e l i n g ( F i g . 1 5 ) . This suggests t h a t , i n the s u s c e p t i b l e , the major m a t e r i a l accumulated i s p r o t e i n -aceous. I n the cycloheximide t r e a t e d cotyledons most of the l e s i o n s showed a halo w i t h even lower r a d i o a c t i v i t y than the surrounding t i s s u e s . This may be because the area occupied by the fungus has an even lower a b i l i t y to convert s o l u b l e p h e n o l i c s i n t o i n s o l u b l e ones than the surrounding h e a l t h y t i s s u e . The few l e s i o n s showing accumulation may represent aborted i n f e c t i o n s i t e s . 3. I n c o r p o r a t i o n fa) Phenolics" vs. p r o t e i n The r a t e of i n c o r p o r a t i o n of phenylalanine-U-^C i n -to p h e n o l i c s i n d i c a t e s that there was an enhanced s y n t h e s i s of phenolic compounds immediately a f t e r i n f e c t i o n i n both the r e s i s t a n t and s u s c e p t i b l e combinations (Table V I I ) . The low t o t a l s o l u b l e p h e n o l i c s at t h i s e a r l y stage a f t e r i n -o c u l a t i o n t h e r e f o r e means t h a t i n i t i a l l y the r a t e of b i n d i n g of phenolic compounds i s f a s t e r than the r a t e of s y n t h e s i s , l e a v i n g a d e f i c i t i n the t o t a l s o l u b l e phenolic content. This probably s i g n i f i e s a stage of n o n s p e c i f i c response of 129 the host to the presence of a f o r e i g n organism i n the p l a n t t i s s u e . On the 2nd day a f t e r i n o c u l a t i o n the i n c o r p o r a t i o n i n the r e s i s t a n t reached i t s peak. At t h i s stage the r a t e of s y n t h e s i s was a l r e a d y higher than the r a t e of consump-t i o n (binding) and the t o t a l s o l u b l e phenolic content was t h e r e f o r e higher than i n the c o n t r o l . This i s l i k e l y to be a c o n t i n u a t i o n of the process of r e j e c t i n g a f o r e i g n organism. The s u s c e p t i b l e , however, showed a drop i n the i n c o r p o r a t i o n r a t e on the 2nd day. At t h i s stage the host-p a r a s i t e r e l a t i o n s h i p would have entered a compatible Tphase T and the metabolism of the host was no longer h o s t i l e to the pathogen.' The e f f e c t of i n f e c t i o n on the i n c o r p o r a t i o n of 14 phenylalanine-U- C i n t o p r o t e i n ( F i g . IX) i s the reverse of that on i t s i n c o r p o r a t i o n i n t o phenolic compounds. The p r o t e i n content of the t i s s u e s under study f o l l o w s the order: i n f e c t e d s u s c e p t i b l e ^ h e a l t h y ^ i n f e c t e d r e s i s t a n t . I t i s l i k e l y t h a t i n the i n f e c t e d s u s c e p t i b l e cotyledons a major p o r t i o n of the phenylalanine i s i n c o r p o r a t e d i n t o p r o t e i n . In i n f e c t e d r e s i s t a n t cotyledons a higher propor-t i o n of the phenylalanine i s d i v e r t e d to the production of p h e n o l i c s . These f i n d i n g s are i n agreement w i t h those of Fuchs et a l . (1967) . The change of the l a b e l i n g p a t t e r n of the p r o t e i n bands i n g e l e l e c t r o p h o r e s i s as the cotyledons aged ( F i g . 16 and 17) suggests a change of enzyme c o n s t i t u e n t s i n favour 130 of degradative enzymes as the cotyledons senesce. At the l a t e stage of i n f e c t i o n the p r o t e i n bands of the s u s c e p t i b l e t i s s u e are d i s t i n c t l y d i f f e r e n t from those i n the health y and r e s i s t a n t t i s s u e ( F i g . 1 8 ) . This i s i n part due to the presence of a high p r o p o r t i o n of fu n g a l p r o t e i n s which have d i f f e r e n t m i g r a t o r y p r o p e r t i e s i n g e l e l e c t r o p h o r e s i s . (b) Monohydric vs. d i h y d r i c phenols Cruickshank and Swain (1956) s t u d i e d the chlorogenic a c i d content of a number of f l a x v a r i e t i e s and found t h a t the r e s i s t a n t ones had a higher r a t i o of chl o r o g e n i c a c i d to t o t a l phenolic content. Simons and Ross (1970) found that o r t h o - d i h y d r i c phenols decreased more r a p i d l y d u r i n g l e s i o n f o r m a t i o n i n tobacco leaves r e s i s t a n t to TMV than i n sus-c e p t i b l e ones. I n the present s t u d i e s on the metabolism of p h e n y l a l a n i n e - U - ^ C , there was a higher conversion of mono-h y d r i c phenol i n t o d i h y d r i c phenol i n the r e s i s t a n t coty-ledons- than i n t h e - h e a l t h y and s u s c e p t i b l e t i s s u e s (Table V I I I ) . The r e s i s t a n t t i s s u e would t h e r e f o r e have a l a r g e r supply of s u b s t r a t e f o r o x i d a t i o n and f o r complexing w i t h p r o t e i n . On the b a s i s of data showing an i n i t i a l low l e v e l of d i h y d r i c phenols i n the r e s i s t a n t combination and a high t o t a l s o l u b l e phenolic content between 2-1+ days a f t e r i n -o c u l a t i o n , A l l a n (1967) suggested t h a t the low d i h y d r i c phenol content would favour high a c t i v i t y of IAA oxidase and a low f u n c t i o n a l auxin content. Low auxin l e v e l s i n 131 t u r n would favour the d e s t r u c t i o n of RNA and induce early-senescence i n the r e s i s t a n t v a r i e t y . But he a l s o showed that senescence occurred between 7 and 8 days a f t e r i n o c u -l a t i o n and t h a t the d i h y d r i c phenol content b u i l t up r a p i d -l y between the 4th and 6th days a f t e r i n o c u l a t i o n . These two l a t t e r f i n d i n g s do not f i t very w e l l i n t o the theory th a t d i h y d r i c phenols are r e s p o n s i b l e f o r e a r l y senescence v i a an e f f e c t on auxin l e v e l . I n a d d i t i o n the e a r l y low d i h y d r i c phenol l e v e l t h a t A l l a n observed a f t e r i n o c u l a t i o n i n h i s r e s i s t a n t combination was only i n the e x t r a c t a b l e s o l u b l e phenolic f r a c t i o n . The low l e v e l of d i h y d r i c phenols may have been due to the f a c t t h a t d i h y d r i c phenols are the major compounds that are o x i d i z e d and bound to the i n s o l u b l e residue as shown i n the present r e s u l t s ( F i g . 1$). The e a r l y senescence of r e s i s t a n t cotyledons may not have any r e l a t i o n w i t h the d i h y d r i c phenol l e v e l but may be due t o other mechanisms which l e a d to the i n c r e a s e d production of degradative enzymes such as peroxidase, polyphenol o x i -dase, B-glucosidase and IAA oxidase. Se v e r a l p o i n t s discussed above are r e l e v a n t to our understanding of h o s t - p a r a s i t e r e l a t i o n s : (1) In the r e s i s t a n t combination there was an accumu-l a t i o n of i n s o l u b l e phenolic compounds around the l e s i o n s whereas the s u s c e p t i b l e accumulated p r o t e i n . (2) I n c o r p o r a t i o n experiments a l s o showed that more phen y l a l a n i n e - U - ^ C was metabolized i n t o phenolic compounds 132 i n the r e s i s t a n t whereas i n the s u s c e p t i b l e there was more phenylalanine i n c o r p o r a t e d i n t o p r o t e i n . (3) The r e s i s t a n t showed a higher degree of i n c o r -p o r a t i o n of ph e n y l a l a n i n e - U - ^ C i n t o d i h y d r i c phenols than the s u s c e p t i b l e . S e c t i o n I I I : Enzymes 1. Phenylalanine ammonia-lyase I n order to understand the change i n PAL a f t e r i n -f e c t i o n and i t s s i g n i f i c a n c e i n the metabolism of r e s i s t a n t and s u s c e p t i b l e t i s s u e s we should a l s o examine the changes i n p henylalanine l e v e l , s i n c e phenylalanine i s the sub s t r a t e f o r t h i s enzyme. Although the author d i d not measure the l e v e l or s y n t h e s i s of phenylalanine i n f l a x , some r e s u l t s put f o r t h by other workers are worth d i s c u s s i n g i n r e l a t i o n to the present r e s u l t s . Pegg and Sequeira (1968), working on tobacco t i s s u e i n f e c t e d by Pseudomonas solanacearum, found pronounced i n c r e a s e s i n the amount of phenylalanine and tryptophan w i t h i n 24 hours of i n o c u l a t i o n . Since concen-t r a t i o n s of most of the other amino acids decreased d u r i n g t h i s p e r i o d , i t appears t h a t an in c r e a s e i n the concentra-t i o n of the aromatic amino a c i d s was not the r e s u l t of p r o t e i n breakdown but was due to s p e c i f i c s y n t h e s i s of these com-pounds. They also found that there was a marked i n c r e a s e i n PAL and enhanced s y n t h e s i s of s c o p o l e t i n . E a r l i e r work by Shaw and C o l o t e l o (1961) showed th a t aromatic amino a c i d s 133 were increased i n both r e s i s t a n t and s u s c e p t i b l e combina-t i o n s of r u s t - i n f e c t e d wheat. In the p r o t e i n hydrolysate there was a higher phenylalanine l e v e l f o r the s u s c e p t i b l e t i s s u e when compared w i t h the h e a l t h y c o n t r o l . On the other hand the l e v e l f o r the r e s i s t a n t i s lower and the r e s i s t a n t / h e a l t h y r a t i o i s about 0.55 at 2 days a f t e r i n -o c u l a t i o n . Furthermore Rohringer et_ a l . (1967) demonstra-ted that i n r u s t - i n f e c t e d wheat leaves there was an i n - • creased carbon f l o w from CO to shikimate and quinate. This phenomenon was more pronounced i n the s u s c e p t i b l e i n -t e r a c t i o n than the r e s i s t a n t one. These f i n d i n g s demonstrate c o n c l u s i v e l y t h a t there i s enhancement i n the production of phenylalanine a f t e r i n f e c t i o n , e s p e c i a l l y i n the s u s c e p t i b l e t i s s u e . These r e s u l t s together w i t h the present f i n d i n g s (Tables VII and IX) a l s o suggest that most of the phenyl-a l a n i n e i s i n c o r p o r a t e d i n t o p r o t e i n i n the s u s c e p t i b l e whereas there i s a s t r o n g f l o w of aromatic amino acid s to phenolic compounds i n the r e s i s t a n t combinations. This l a t t e r process might have a f f e c t e d the p r o t e i n s y n t h e s i s i n favour of those having low7 aromatic amino a c i d c o n s t i t u e n t s . This would be an i n t e r e s t i n g problem f o r f u r t h e r research. Since s u s c e p t i b l e t i s s u e has an even higher f r e e phenylalanine pool than the r e s i s t a n t phenylalanine i s pro-bably not a f a c t o r i n i n d u c i n g the PAL enhancement i n the r e s i s t a n t t i s s u e s . The i n i t i a l low l e v e l of phenolics 134 would not be a f a c t o r e i t h e r because t h i s occurs i n both r e s i s t a n t and s u s c e p t i b l e i n t e r a c t i o n s . However, the t r i g g e r i n g mechanism f o r enhancement of PAL a c t i v i t y has yet to be dis c o v e r e d . Hadwiger at a l . (1969) reported that the a c t i v i t y of PAL i n Bison ( s u s c e p t i b l e ) and Cass M-3 ( r e s i s t a n t ) was not s i g n i f i c a n t l y a l t e r e d a f t e r i n o c u l a t i o n w i t h race #1 of M. l i n i . However t h e i r experiment was done on 14-day-old s e e d l i n g s t r e a t e d w i t h a spore suspension and incubated f o r 24 hours. The d i f f e r e n c e between these r e s u l t s and the pre-sent f i n d i n g s ( F i g . 20) may be due to the d i f f e r e n c e i n ex-perimental design. They only s t u d i e d the PAL a c t i v i t i e s 24 hours a f t e r i n o c u l a t i o n and the present f i n d i n g s i n d i -cate t h a t the r a p i d b u i l d up of a c t i v i t y occurred between 24 and 4$ hours a f t e r i n o c u l a t i o n . I t was shown by Chakravorty and Shaw (1971) t h a t a s i g n i f i c a n t i n c r e a s e i n the r a t e of RNA s y n t h e s i s occurred between 24 to 4$ hours a f t e r i n o c u l a -t i o n of f l a x w i t h M. l i n i . I f PAL syn t h e s i s per se i s r e s -p o n s i b l e f o r the enhanced a c t i v i t y , i t should not occur before the p r o d u c t i o n of RNA. Towers and co-workers have found that many wood d e s t r o y i n g basidiomycetes produce PAL. These f u n g i are C o l l y b i a v e l u t i p e s , L e n t i n u s , Lepideus, Polyporus, Trametes and Ganoderma lucidum (Power at a l . 1965) and Schizophyllum  commune (Moore and Towers 1967). However, Jackson et a l . (1970) could not detect PAL i n wheat r u s t uredospores. They 135 showed that r a d i o a c t i v i t y was not present i n f r e e phenolic a c i d s when uredospores were fed w i t h p h e n y l a l a n i n e - U - ^ C . I n the present work the low PAL a c t i v i t y i n the s u s c e p t i b l e t i s s u e , e s p e c i a l l y at a l a t e stage of i n f e c t i o n when there would be a s i g n i f i c a n t amount of f u n g a l m a t e r i a l i n the host t i s s u e , a l s o supports the i d e a that the r u s t fungus does not produce PAL. 2. Peroxidase Novacky and Wheeler (1970) found t h a t v i c t o r i n from Helminthosporium v i c t o r i a e induced q u a n t i t a t i v e changes i n peroxidase i n s u s c e p t i b l e oat leaves and a higher concentra-t i o n of v i c t o r i n was r e q u i r e d f o r s i m i l a r a l t e r a t i o n s i n r e s i s t a n t l e a v e s . Since they found that the enhancement of peroxidase i n the s u s c e p t i b l e i s more s e n s i t i v e to v i c t o r i n than i n the r e s i s t a n t , they suggested t h a t peroxidase a c t i -v i t y i s not r e l a t e d t o r e s i s t a n c e . However the h o s t - p a r a s i t e combination they s t u d i e d i s a n e c r o t r o p h i c type and the v i r u l e n t pathogen has to produce degradative enzymes to t u r n the s u s c e p t i b l e host t i s s u e i n t o a n e c r o t i c l e s i o n on which the pathogen can feed. Therefore the production of a high l e v e l of peroxidase i n such i n t e r a c t i o n i s to be expected and bears no s i g n i f i c a n c e i n disease r e s i s t a n c e . On the other hand the biotrophs which feed on l i v i n g c e l l s g e n e r a l l y cause a l a r g e r enhancement of peroxidase i n r e s i s t a n t hosts than the s u s c e p t i b l e ones. In t h i s case only the r e s i s t a n t i n t e r a c t i o n s show n e c r o t i c l e s i o n s . - 136 Lovrekovich et a l . (1968) showed th a t i n j e c t i o n of heat-k i l l e d c e l l s of Pseudomonas t a b a c i or c e l l f r e e e x t r a c t s of b a c t e r i a i n t o tobacco leaves i n c r e a s e d peroxidase a c t i v i t y . They a l s o showed that i n j e c t i n g a s o l u t i o n of commercial peroxidase i n t o leaves r e s u l t e d i n increased r e s i s t a n c e to the dise a s e . Macko et a l . (196$) showed a marked increase i n peroxidase a c t i v i t y i n r u s t - i n f e c t e d r e s i s t a n t wheat at about the time of s p o r u l a t i o n , f o l l o w e d by a d e c l i n e . Sus-c e p t i b l e wheat showed only a sm a l l i n c r e a s e i n a c t i v i t y at 24 hours a f t e r i n o c u l a t i o n . Other observations on increa s e d peroxidase a c t i v i t y i n r e s i s t a n t i n t e r a c t i o n s i n c l u d e those of Rudolph and Stahmann (1964) on bean leaves i n f e c t e d w i t h Pseudomonas p h a s e o l i c o l a and Simmons and Ross (1970) on l o c a l l e s i o n s on tobacco caused by TMV. The l a t t e r authors found th a t a marked increase of peroxidase was detectable only i n the necrogenic phase of the disease. They suggested a d i r e c t r o l e f o r peroxidase i n the l o c a l i z a t i o n of TMV". The inc r e a s e of peroxidase i n the r e s i s t a n t f l a x -f l a x r u s t i n t e r a c t i o n ( F i g . 21) i s c o n s i s t e n t w i t h the above data f o r b i o t r o p h i c pathogens. In other words, i n f e c t i o n w i t h a v i r u l e n t b i o t r o p h i s accompanied w i t h an enhancement 1 of peroxidase i n the host t i s s u e thus r e s u l t i n g i n a n e c r o t i c l e s i o n and a b o r t i o n of the p a r a s i t e . As al r e a d y discussed i n the l i t e r a t u r e review, peroxidase can c a t a l y z e the o x i d a t i o n and h y d r o x y l a t i o n of phenolics as w e l l as the o x i d a t i o n of p r o t e i n and amino 1 3 7 a c i d s . These r e a c t i o n s would lead to products I n a c t i v a t i n g enzymes and p r e c i p i t a t i n g p r o t e i n s . The r e s u l t i s g e n e r a l l y a n e c r o t i c l e s i o n . For the necrotrophs t h i s would happen f o r both the s u s c e p t i b l e and r e s i s t a n t i n t e r a c t i o n whereas t h i s occurs only i n the r e s i s t a n t r e a c t i o n f o r the b i o -trophs. I n the s u s c e p t i b l e h o s t - b i o t r o p h combination, there would be e i t h e r no enhancement of peroxidase or the enhancement i s r e q u i r e d f o r f u n c t i o n s other than o x i d a t i o n and n e c r o s i s i s not an i n i t i a l f e a t u r e of the r e a c t i o n . 3 . Polyphenol Oxidase D e v e r a l l ( 1 9 6 1 ) found t h a t B o t r y t i s c i n e r e a , the ca u s a l fungus f o r the chocolate spot disease of bean p l a n t s , produced p e c t i c enzymes which l i b e r a t e g a l a c t u r o n i c a c i d and p o l y g a l a c t u r o n i c d e r i v a t i v e s from the c e l l w a l l s . These compounds unmasked the l a t e n t polyphenol oxidase. Some-times i t i s the f u n g a l m e t a b o l i t e that a c t i v a t e s the en-zyme e.g., o p h i o b o l i n , a t o x i n produced by Co c h l i o b o l u s was reporte d to be able to a c t i v a t e polyphenol oxidase of r i c e leaves (Nakamura and Oku i 9 6 0 ) . Zucker et a l . ( 1 9 6 $ ) suggested t h a t cinnamic, p-coumaric and f e r u l i c a c i d would i n h i b i t the oxidase s i t e of polyphenol oxidase. The higher conversion of monohydric phenol to d i h y d r i c phenol would t h e r e f o r e reduce the i n h i b i t o r y e f f e c t . In the f l a x -r u s t combination the enhancement of PPO ( F i g . 2 3 ) i n the r e s i s t a n t i n t e r a c t i o n i s probably due to t h i s mechanism. The conversion of monohydric to d i h y d r i c phenols i s highest 1 3 $ i n the r e s i s t a n t combination and would enhance PPO which then o x i d i z e s the o - d i h y d r i c phenols. The r e s u l t would be enhanced PPO a c t i v i t y , but not a high l e v e l of d i h y d r i c phenol. A f t e r the 4 t h day the pathogen i s a l r e a d y i n -a c t i v a t e d or contained and some host c e l l s are a l s o dead. Consequently the PPO i n the l e s i o n may be i n a c t i v a t e d g r a d u a l l y and an accumulation of o-diphenols as observed by A l l a n ( 1 9 6 7 ) would be expected. These r e s u l t s support the suggestion of Hyodo and U r i t a n i ( 1 9 6 6 ) who worked w i t h s l i c e d sweet potato t i s s u e and proposed the idea t h a t i n -creased polyphenol content might be i n v o l v e d i n the enhance-ment of PPO a c t i v i t y . The lower a c t i v i t y of PPO found i n the s u s c e p t i b l e combination than i n h e a l t h y t i s s u e may be. of s i g n i f i c a n c e i n p r e v e n t i n g the o x i d a t i o n of phenols thus p r e v e n t i n g the formation of n e c r o t i c l e s i o n s . Therefore the i n h i b i t i o n of t h i s enzyme may be e s s e n t i a l f o r s u s c e p t i b i l i t y . As w i t h peroxidase, those v i r u s - h o s t systems showing n e c r o t i c l o c a l l e s i o n s a l s o e x h i b i t enhanced PPO a c t i v i t y (Farkas, K i r a l y and Solymosy I960). Van Kammen and Brouwer (1964) have demonstrated that i n the l o c a l l e s i o n s of Nico-t i a n a tabacum caused by TMV there i s an i n c r e a s e i n poly-phenol oxidase. The i n c r e a s e was not r e s t r i c t e d to the i n o c u l a t e d p a r t s of the leaves where v i r u s m u l t i p l i c a t i o n occurred and l o c a l l e s i o n s developed, but was a l s o found i n the u n i n o c u l a t e d p a r t s . 139 4. 3-Glucosidase The f i n d i n g s presented i n t h i s d i s s e r t a t i o n ( F i g . 24) are q u i t e s i m i l a r to those reported by A l l a n (1967). He found t h a t i n rusted Bison cotyledons ( s u s c e p t i b l e ) the 3-glucosidase a c t i v i t y was below the h e a l t h y c o n t r o l throughout the e n t i r e process of r u s t i n f e c t i o n . For the r e s i s t a n t Bombay race #3 combination, the enzyme a c t i v i t y always remained above the c o n t r o l l e v e l . However i n Sec-t i o n A (Table V) i t can be seen th a t most of the s o l u b l e p h e n o l i c s of f l a x are e s t e r s and t h e i r h y d r o x y l groups are f r e e . Therefore the s i g n i f i c a n c e of the enhancement of 3 -gluco s i d a s e i s not very c l e a r . There are two ways i n which t h i s enhancement may be of advantage to the host: (a) one of the few phenolic g l y c o s i d e s or a non-phenolic g l y c o s i d e may be the main r e s i s t a n c e f a c t o r s and t h e r e f o r e enhancement of 3-glucosidase i s e s s e n t i a l f o r r e s i s t a n c e , b) the enhancement i s part of the general i n c r e a s e of the degradative and o x i d a t i v e enzymes r e s u l t i n g from the primary (unknown)- r e s i s t a n t r e a c t i o n . I n the s u s c e p t i b l e combina-t i o n t h i s enzyme i s suppressed and t h i s i s a safeguard against any p o s s i b i l i t y of the production of any r e s i s t a n t p r i n c i p l e s such as the aglycone, 2,4-dihydroxy-7-methoxy 1,4 benzoxazin - 3-one, r e l e a s e d i n wheat leaves i n f e c t e d w i t h wheat r u s t ( E l Naghy and Shaw 1966). 5 . S e q u e n t i a l Changes The temporal changes i n the phenolics and enzymes a f t e r i n f e c t i o n suggest a s e q u e n t i a l s y n t h e s i s or a c t i v a t i o n of PAL, t o t a l s o l u b l e p henolics and the o x i d a t i v e and de-gra d a t i v e enzymes. I n i t i a l l y a f t e r i n f e c t i o n , there i s a l a g phase of one to two days f o r a l l enzymes s t u d i e d . PAL was enhanced f i r s t on the 2nd day a f t e r i n o c u l a t i o n . The b u i l d up i n phenolic content was very f a s t between the 2nd and 4th day. The o x i d a t i v e enzymes showed two days of l a g and high a c t i v i t i e s were detectable- by the 4th day a f t e r i n o c u l a t i o n . Hyodo and U r i t a n i ( 1 9 6 6 ) found t h a t polyphenol o x i -dase was enhanced by the. 40th hour a f t e r the sweet potato t i s s u e was s l i c e d and the peak of enzyme a c t i v i t y occurred between 80 and 100 hours. On the other hand a 4 - f o l d i n -crease of polyphenols was found by the 20th hour and the peak at .the 40th hour. Minamikawa and U r i t a n i ( 1 9 6 5 ) showed that PAL was increased by the 12th hour and reached a plat e a u of high a c t i v i t y between 1 2 - 3 6 hours. These data a l s o i m p l i e d the same s e q u e n t i a l changes as those found by me i n f l a x . The i n f i l t r a t i o n i n j u r y of the cotyledons a l s o i n c r e a s e d PAL more than peroxidase at an e a r l y stage a f t e r i n f i l t r a t i o n . Peroxidase always took a longer time to show the same percentage of enhancement when compared, w i t h PAL. These r e s u l t s suggest that the o x i d a t i v e enzymes 141 may be enhanced by the p h e n o l i c s produced as a r e s u l t of the h i g h PAL a c t i v i t y . However, t h i s i s s p e c u l a t i o n at t h i s stage of our knowledge. To conclude t h i s s e c t i o n , the f o l l o w i n g p o i n t s are noteworthy: (1) PAL was enhanced i n the r e s i s t a n t combination but not a p p r e c i a b l y i n the s u s c e p t i b l e one. This i s e v i -dence f o r the enhanced s y n t h e s i s of phenolic compounds i n the r e s i s t a n t r e a c t i n g t i s s u e . ( 2 ) PPO and peroxidase were a l s o enhanced i n the r e s i s t a n t combination. I t i s p o s s i b l e t h a t o x i d a t i o n of phenolic compounds was a l s o enhanced as these enzymes are g e n e r a l l y considered to be r e s p o n s i b l e f o r the o x i d a t i o n of phenolic compounds. (3) The suppression of PPO and g-g l u c o s i d a s e i n the s u s c e p t i b l e combination may p l a y an important r o l e i n a l l o w -i n g the pathogen to p r o l i f e r a t e i n the host t i s s u e . (4) There i s probably a s e q u e n t i a l i n d u c t i o n of PAL, phenolic content and PPO i n the r e s i s t a n t r e a c t i n g t i s s u e . 142 S e c t i o n IV: General D i s c u s s i o n With the evidence obtained from three d i f f e r e n t types of experiments, namely, phytochemical study of phen-o l i c c o n s t i t u e n t s , t r a c e r s t u d i e s and enzyme assays (Sections I - I I I ) , i t can be concluded t h a t the r e s i s t a n c e and s u s c e p t i b i l i t y of f l a x t o f l a x r u s t are r e l a t e d to the metabolism of phenolic compounds. Even though no phenolic p h y t o a l e x i n was detected a f t e r i n f e c t i o n , the c haracters of the p h e n o l i c s i n f l a x provide a very good b a s i s f o r r e s i s t a n c e to be b u i l t on t h i s group of compounds. With the enhanced a c t i v i t i e s of peroxidase and polyphenol o x i -dase and p o s s i b l y the breakdown of c e l l u l a r compartmenta-t i o n , these p h e n o l i c s can e a s i l y be converted i n t o r e s i s t a n t p r i n c i p l e s such as enzyme i n h i b i t i n g quinones and polymers of p h e n o l i c s and quinones. Tracer s t u d i e s provide good evidence to show t h a t i n the r e s i s t a n t there was h i g h conversion of s o l u b l e phen-o l i c s i n t o 80% e t h a n o l - i n s o l u b l e ones which would e i t h e r be polymers or simple p h e n o l i c s bound to the c e l l w a l l or p r o t o p l a s t . The i n c o r p o r a t i o n experiments a l s o support the i d e a t h a t p h e n o l i c s are i n v o l v e d i n r e s i s t a n c e . The t r i g g e r i n g mechanism f o r the f i r s t step i n the s e r i e s of enhancements of PAL, t o t a l phenolic content, and degradative enzymes remains unknown. From the water i n f i l -t r a t i o n experiments i t i s obvious t h a t i n j u r y could l e a d to the enhancement of PAL. The higher enhancement e f f e c t f o r 1 4 3 the i n f i l t r a t i o n w i t h phenolic e x t r a c t over the water i n f i l -t r a t i o n i n d i c a t e s t h a t some compounds i n the e x t r a c t would cause enhancement. I t i s p o s s i b l e t h a t mechanical i n j u r y or f u n g a l i n f e c t i o n may cause a breakdown of c e l l u l a r compart-mentation and t h a t 'leakage' may b r i n g phenolic compounds or other substances i n t o contact w i t h the p r o t e i n synthe-s i z i n g machinery of the host c e l l s . A part of the e f f e c t may a l s o be at the l e v e l of the c e l l nucleus, because a c t i n -omycin D, as i n h i b i t o r of RNA polymerase prevents the en-hancement e f f e c t of i n f i l t r a t i o n w i t h water or f l a x phenolic e x t r a c t . S u s c e p t i b i l i t y seems to be mainly due to the suppres-s i o n of the production- of p h e n o l i c s and e s p e c i a l l y of o x i d a -t i v e enzymes. The channeling of a l a r g e q u a n t i t y of aromatic amino a c i d i n t o p r o t e i n s y n t h e s i s t h e r e f o r e serves two pur-poses: a) i t reduces the amount of s u b s t r a t e f o r PAL and b) i t a l l o w s more p r o t e i n to be used f o r the b e n e f i t of the pathogen. The mechanism f o r the suppression of the o x i -d a t i v e enzymes i s unknown but probably the c o m p a t i b i l i t y between the h a u s t o r i a and the membrane systems of the host c e l l s p l a y s an important r o l e . Based on the above d i s c u s s i o n I would l i k e to pro-pose a model f o r the r e s i s t a n t and s u s c e p t i b l e r e a c t i o n s : A f t e r i n f e c t i o n , the a v i r u l e n t r u s t produces the normal i n f e c t i o n s t r u c t u r e s and a t t a c k s some c e l l s . I t i s probably the h a u s t o r i a of the a v i r u l e n t r u s t that t r i g g e r changes i n 144 the host c e l l s such as membrane p e r m e a b i l i t y . These would l e a d to the m i x i n g of the p h e n o l i c s and the o x i d a t i v e enzymes and the formation of quinones and phenolic polymers. Con-sequently the h a u s t o r i a would be i n a c t i v a t e d . The fungus would then produce more h a u s t o r i a and a f f e c t other c e l l s but more of them would be i n a c t i v a t e d u n t i l f i n a l l y the pathogen would be starved of s u b s t r a t e f o r f u r t h e r growth. F i n a l l y due to the d e p o s i t i o n of more phe n o l i c polymers the pathogen would be f i x e d and k i l l e d . The number of c e l l s c o l l a p s e d and the area being a f f e c t e d would probably depend on the r a t e and degree to which the h a u s t o r i a are i n a c t i v a t e d and t h i s would i n t u r n be an e x h i b i t i o n of the degree of i n c o m p a t i b i l i t y . Thus f o r Bombay f l a x and race #3 of M. l i n i the i n t e r a c t i o n i s q u i t e d r a s t i c l e a d i n g t o a h y p e r s e n s i t i v e r e a c t i o n w i t h microscopic f l e c k s c o n s i s t i n g of only a few c o l l a p s e d c e l l s ( A l l a n 1967). For Koto and race #3 I found t h a t the brown f l e c k s were l a r g e r . This i s probably because the r e a c t i o n i s not as v i o l e n t so that the h a u s t o r i a can f u n c t i o n at l e a s t p a r t i a l l y , and the fungus t h e r e f o r e a f f e c t s more c e l l s before i t becomes exhausted. Ne v e r t h e l e s s , i n any r e s i s t a n t combination, the p o i n t at which the pathogen be-comes exhausted and f i x e d must occur before s p o r u l a t i o n . I n s u s c e p t i b l e t i s s u e the i n a c t i v a t i o n of h a u s t o r i a i s probably a slow process. Thus the fungus can ramify ex-t e n s i v e l y and take c o n t r o l of the metabolism of the host 145 c e l l s . The slowing down of the o x i d a t i o n of the phenolic compounds i s probably e s s e n t i a l f o r the fungus to o b t a i n n u t r i t i o n from the host c e l l s . To conclude, t h i s t h e s i s deals o n l y w i t h the phyto-chemical and biochemical changes of phenolic metabolism i n h e a l t h y and r u s t - i n f e c t e d f l a x w i t h respect to r e s i s t a n c e and s u s c e p t i b i l i t y . I p r e f e r the idea that phenolic com-pounds are agents executing the a c t u a l process of r e s i s -tance. The r e s u l t s presented suggest that i f the phenolic compounds can be maintained i n t h e i r normal ' s t a t e s ' , as i n the h e a l t h y t i s s u e , the pathogen would be able to s u r v i v e . I t i s s t i l l not known what t r i g g e r s the biochemical change i n phenolic metabolism i n the r e s i s t a n t r e a c t i n g t i s s u e or how these changes are suppressed i n the s u s c e p t i b l e combin-a t i o n . Answers to these problems are most l i k e l y to be obtained from s t u d i e s on the membrane bio c h e m i s t r y of the host c e l l and the h a u s t o r i a , and changes i n n u c l e i c a c i d metabolism. 146 SUMMARY AND CONCLUSION 1. Eight f l a v o n o i d s and 14 e s t e r s and g l y c o s i d e s of phen-o l i c a c i d s were found i n Koto f l a x cotyledons. The f l a v o n o i d s are of two major types, namely, a p i g e n i n and l u t e o l i n and they are present as g l y c o s i d e s . The phenolic e s t e r s and g l y c o s i d e s are the d e r i v a t i v e s of p-coumaric, c a f f e i c , f e r u l i c and s i n a p i c a c i d s . Chloro-genic a c i d s and e s t e r s of p-coumaric and c a f f e i c a c i d s are the major compounds. No benzoic a c i d s or t h e i r d e r i v a t i v e s or anthocyanins were detected i n the coty-ledons. 2. There were no new p h e n o l i c compounds i . e . , phenolic p h y t o a l e x i n s , detected i n e i t h e r the i n f e c t e d sus-c e p t i b l e or i n f e c t e d r e s i s t a n t f l a x cotyledons as a r e s u l t of r u s t i n f e c t i o n . 3. T o t a l phenolic contents of h e a l t h y cotyledons i n c r e a s e d g r a d u a l l y w i t h age between one t o two weeks a f t e r seeding. I n f e c t i o n w i t h r u s t caused an i n i t i a l d e c l i n e i n t o t a l p h e n o l i c content but l a t e r rose above the h e a l t h y c o n t r o l . This occurred on the 2nd day f o r the r e s i s t a n t r e a c t i n g t i s s u e and the 4th day f o r the s u s c e p t i b l e . The r e s i s -t a n t combination maintained the highest phenolic content from the 2nd day onward. 4. Phenolic metabolism i n f l a x cotyledons probably f o l l o w s the f o l l o w i n g route which has been suggested by McCalla and Neish (1959): 147 phenylalanine ^cinnamic a c i d ^-p-coumaric a c i d f e r u l i c a c i d f c a f f e i c acid£ ^ This was shown by f e e d i n g experiments. Tyrosine-U-^C and DOPA- $-~^C were metabolized only i n t o some n i n h y d r i n p o s i t i v e compounds. Cinnamate-3 -"^C was found to i n h i -b i t the metabolic steps beyond the formation of p-coumaric a c i d . 5. When f l a x cotyledons were fed w i t h p h e n y l a l a n i n e - U - ^ C , the l a b e l i n g was found to accumulate i n l e s i o n s of both the r e s i s t a n t and s u s c e p t i b l e combinations. I f the feed-i n g was accompanied by treatment w i t h cycloheximide the accumulation was found to be higher i n l e s i o n s of the r e s i s t a n t combination than the s u s c e p t i b l e one. 6. I n phenolic metabolism, phenylalanine-U-^C was mainly metabolized i n t o a number of d e r i v a t i v e s of p-coumaric, c a f f e i c and f e r u l i c a c i d s . Very l i t t l e l a b e l i n g was found i n the f l a v o n o i d s . There were no q u a l i t a t i v e d i f f e r e n c e s i n p h e n y l a l a n i n e - U - ^ C metabolism between H, R and S Tiss u e s . 7 . Q u a n t i t a t i v e s t u d i e s on the metabolism of phenylalanine-14 U- C showed th a t i n c o r p o r a t i o n i n t o phenolic compounds i s highest i n the r e s i s t a n t r e a c t i n g t i s s u e . On the other hand, the i n c o r p o r a t i o n i n t o p r o t e i n showed a r e -verse trend and the s u s c e p t i b l e combination showed the highest i n c o r p o r a t i o n . 8. I n the r e s i s t a n t r e a c t i n g t i s s u e there was a higher conversion of monohydric to d i h y d r i c phenol than i n the h e a l t h y t i s s u e or the s u s c e p t i b l e combination. 9. Gel e l e c t r o p h o r e s i s of phenylalanine-U-"^ C l a b e l e d pro-t e i n showed th a t there was a change i n l a b e l i n g p a t t e r n s as the f l a x cotyledons aged. The R and S showed s l i g h t d e v i a t i o n from H at an e a r l y stage a f t e r i n o c u l a t i o n . By the 9 t h day a f t e r i n o c u l a t i o n the patterns of the H and R were very s i m i l a r whereas S was d i s t i n c t l y d i f f e r e n t . 10. The anion exchange r e s i n , Dowex 1X8, was found to be capable of b i n d i n g the phenolic compounds from f l a x as w e l l as standard phenolic compounds. This f i n d i n g has been a p p l i e d to remove phenolic m a t e r i a l s d u r i n g enzyme p r e p a r a t i o n . 11. PAL a c t i v i t y was found to be very low i n f u l l - g r o w n h e a l t h y f l a x cotyledons. However, 2 days a f t e r i n o c u l a -t i o n , the r e s i s t a n t combination showed an i n c r e a s e as much as 5 - f o l d over t h a t of the h e a l t h y c o n t r o l . By the 6 t h day a f t e r i n o c u l a t i o n , the enzyme a c t i v i t y dropped back to a low l e v e l . There was no remarkable enhance-ment of PAL i n the s u s c e p t i b l e combination. 12. There was a gradual i n c r e a s e i n peroxidase a c t i v i t y as the cotyledons aged. For the f i r s t 2 days a f t e r i n o c u l a -t i o n , the peroxidase a c t i v i t i e s of both r e s i s t a n t and s u s c e p t i b l e combinations were comparable to the a c t i v i t y 1 4 9 i n h e a l t h y c o n t r o l . A f t e r the 4 t h day, there was a c l e a r trend of R ) S ) H. This d i f f e r e n c e i n a c t i v i -t i e s i n c r e a s e d w i t h time and, by the 8 t h day, R was n e a r l y 2 - f o l d of H and S was 5 0 % higher than the same he a l t h y c o n t r o l . Gel e l e c t r o p h o r e s i s showed 3 ' i s o -zyme' bands f o r the a c i d i c p r o t e i n f r a c t i o n of the peroxidase. The o v e r a l l patterns were the same f o r H, R and S. The a c t i v i t y of PPO i n s u s c e p t i b l e cotyledons was always lower than that of the h e a l t h y t i s s u e . The enzyme ac-t i v i t y of r e s i s t a n t r e a c t i n g t i s s u e s was s i m i l a r to t h a t of the h e a l t h y c o n t r o l f o r the f i r s t two days but i n -creased r a p i d l y by the 4 t h day a f t e r i n o c u l a t i o n to about 3 0 % above the c o n t r o l . Seven PPO 'isozyme' bands were detected. No d i f f e r e n c e s were found i n the isozymes p a t t e r n s of H, R and S. The 3-glucosidase l e v e l of R was always higher than that of H. The l e v e l i n the s u s c e p t i b l e combination d i d not change f o r the f i r s t two days a f t e r i n o c u l a t i o n but then d e c l i n e d as the fungus e s t a b l i s h e d i t s e l f and s p o r u l a t e d . Peroxidase and PAL a c t i v i t i e s of the cotyledons were found to be enhanced by water i n f i l t r a t i o n . Chlorogenic a c i d d i d not cause f u r t h e r enhancement whereas an aqueous e x t r a c t of f l a x cotyledons produced a pronounced enhancement i n a d d i t i o n to the e f f e c t of water i n f i l t r a -t i o n . The e f f e c t of i n f i l t r a t i o n w i t h both water and 1 5 0 p l a n t e x t r a c t could be a b o l i s h e d by adding actinomycin D t o the i n f i l t r a t i o n s o l u t i o n . 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