Some Aspects of Phenolic Metabolism in Healthy And

SOME ASPECTS OF PHENOLIC METABOLISM

IN HEALTHY AND RUST INFECTED FLAX COTYLEDONS

by TUNG HOI LAM B.Sc., M.Sc., University of Hong Kong

A Thesis Submitted in Partial Fulfilment

of the Requirements for the Degree of

DOCTOR OF PHILOSOPHY

in the

Department of Plant Science

We accept this thesis as conforming to' the required standard

THE UNIVERSITY OF BRITISH COLUMBIA

September, 1971 In presenting this thesis in partial fulfilment of the

requirements for an advanced degree at the University of

British Columbia, I agree that the Library shall make it

freely available for reference and study. I further agree

that permission for extensive copying of this thesis for

scholarly purposes may be granted by the Head of my Depart• ment or by his representatives. It is understood that copy•

ing or publication of this thesis for financial gain shall not be allowed without my written permission.

Department of Plant Science, University of British Columbia, Vancouver 8, British Columbia. ACKNOWLEDGEMENTS

I wish to express my sincere appreciation to Dr.

Michael Shaw, Dean of Agricultural Sciences, University of

British Columbia, under whose supervision this thesis was conducted, for his valuable advice and guidance and his criticism and help in the preparation of this manuscript.

I am deeply indebted to several members of Dean

Shaw's host-parasite group for their valuable advice and encouragement, especially Dr. R.K. Ibrahim, Dr. A. K.

Chakravorty and Mr. L.A. Scrubb.

I am grateful to the Commonwealth Scholarship and

Fellowship Committee of the Association of Universities and

Colleges of Canada for financial support in the form of a scholarship, to Mrs. G. Smith for typing the thesis and

Mr. I. Derics for assistance in preparing the photographs.

Equipment and supplies were provided by the National Research

Council through a grant to Dean Shaw.

I would also like to thank the members of my graduate committee for their interest in my research and the review• ing of this thesis. iv

ABSTRACT

Phytochemical and enzymatic experiments were con• ducted to study the metabolism of phenolic compounds in the cotyledons of flax (Linum usitatissimum L. TKoto») infected with strains #3. and #210 of flax rust (Melampsora lini Pers. Lev.). The primary objective was to further the understand• ing of the role of phenolic compounds in the host-parasite relationship with respect to resistance and susceptibility.

The phenolic constituents of flax include about 14 esters and glycosides of cinnamic acids, viz., p-coumaric, caffeic, ferulic and sinapic acids, and 8 glycosides of flavones, 4 of which.are of the apigenin-type and 4 of the luteolin-type. Most of the cinnamic acid derivatives have a free hydroxyl. group and would therefore be good substrates for oxidation. Except for an initial drop, the total soluble phenolic content in infected resistant tissue was always higher than in the healthy control or in infected susceptible tissue. This quantitative change in phenolic content after infection supports the involvement of phenolics in resistance.

Tracer studies showed that the metabolism of phenyl• alanine in flax follows the order cinnamic ->p-coumaric

> caffeic ^ferulic acids. There was no qualitative change in the pathway of phenylalanine metabolism after in• fection. The. incorporation of phenylalanine-U-^^C into phenolic compounds was higher in the resistant combination than in the healthy control or the susceptible combination. • V

The resistant reacting tissue also showed the highest con• version of monohydric phenols into dihydric phenols. On the other hand, incorporation of phenylalanine-U-^C into protein was highest in the susceptible combination. There was a higher accumulation of radioactivity from phenylalanine-U-^C into ethanol-insoluble, non-proteinaceous material around the lesions in the resistant than in the susceptible combination. These findings are in agreement with the hypothesis that, after infection, there is an enhanced flow of aromatic amino acids into protein synthesis in the sus• ceptible tissue whereas in the resistant reacting tissues there is a shift in favour of phenolic metabolism.

The enhancement of phenylalanine arnmonia-lyase by as much as 5-fold in the resistant tissue at 2 days after inocu• lation also supports' the above hypothesis. The activities of peroxidase, polyphenol oxidase and ^ -glucosidase were also enhanced in the resistant combination, whereas in the sus• ceptible combination polyphenol oxidase and ^-glucosidase activities were lower than in the healthy control. There was a sequential enhancement of phenylalanine ammonia-lyase, total soluble phenolic content and polyphenol oxidase in the resistant reacting tissue. These results suggest that oxida• tion of phenolic compounds is important for resistance and that the suppression of the oxidative enzyme, polyphenol, oxidase', may be essential for the survival of the pathogen in this biotroph-host combination.. vi

The evidence suggests that phenolic metabolism plays an important role in resistance and susceptibility in host- parasite relations. It is very likely that phenolic com• pounds and their oxidative products only execute the job of resistance. The triggering mechanism for the enhance• ment of phenolic metabolism, which remains unknown, and the mechanisms by which phenolic metabolites act against the pathogen are discussed. vii

TABLE OF CONTENTS Page

ACKNOWLEDGEMENTS iii

ABSTRACT iv

TABLE OF CONTENTS vii

LIST OF ABBREVIATIONS x

LIST OF TABLES xi

LIST OF FIGURES xii

INTRODUCTION 1

LITERATURE REVIEW 3

1. Phenolic compounds and plant diseases 3 2. Disease resistant mechanisms of phenolic compounds 16 A. Effect of phenols • 16 B. Effect of quinones 19 3. Metabolism of phenolics in healthy and diseased tissues 24 A. Metabolism of phenolics in healthy tissue 24 B. Effect of infection on metabolism of phenolics 28 4. Some enzymes involved in phenolic metabolism 30 A. Phenylalanine ammonia-lyase 30 B. 3-glucosidase 35 C. Polyphenol oxidase 37 D. Peroxidase 40 5. Phenolic constituents of flax and host- parasite relations 43 MATERIALS AND METHODS 49 1. Plant materials 49 2, Phytochemical studies 50 A. Extraction of phenolic compounds 50 B. Hydrolysis 51 C. Chromatography 53 D. U-V absorption spectrophotometry 55 E. Estimation of phenolic content 57- F. Incubation procedure for tracer studies 57 G. Autoradiography 58 H. Liquid scintillation counting 59 viii

Page 3. Enzyme studies 60 A. Enzyme extraction 60 B. Protein estimation 60 C. Enzyme assays 61 D. Polyacrylamide gel electrophoresis 63 E. Isozyme studies , 65 F. Analysis of phenylalanine-U- C labeled proteins " 66

RESULTS 69

SECTION A: Phenolic compounds of flax 69

I. The identification of phenolic compounds in Koto flax 69 1. Phenolic acid derivatives 69 2. Flavonoid derivatives 77 II. Phenolic compounds and rust infection 79

SECTION B: Metabolism of labeled precursors of phenolic compounds Si I. Metabolism of tyrosine-U-^C, D0PA-6 -1ZfC, phenylalanine-U-^^C and cinnamate-g --^C Si II. Further studies of the metabolism of phenylalanine-U-Hc 86 1. Accumulation -, , 86 2. The metabolism of phenylalanine-U- C into soluble phenolic compounds 89 (i) Incorporation $9 (ii) Comparison of the incorporation of labeling into mono- and di-hydric phenolic compounds 91 3. The metabolism of phenylalanine-U- ^C into proteins 92 (i) Incorporation 92 (ii) The labeling pattern of proteins 94

SECTION C: Proteins and enzymes 98 I. Dowex method 9<3 II. Protein and enzymes 104 1. Total protein content 104 2. Phenylalanine ammonia-lyase 104 3. Peroxidase 107 4. Polyphenol oxidase 110 5. B-glucosidase 110 III. Effect of water infiltration on enzyme activities 113 ix

Page

DISCUSSION 119

SECTION I: Phenolic compounds of flax 119

SECTION II: Metabolism of phenolic precursors 123

1. Metabolic pathway 123 2. Accumulation 126 3. Incorporation 12$ (a) Phenolics vs. protein 128 (b) Monohydric phenol vs. dihydric phenol 130 SECTION III: Enzymes 132

1. Phenylalanine ammonia-lyase 132 2. Peroxidase 135 •3. Polyphenol oxidase 137 4. P-Glucosidase 139 5. Sequential changes 140

SECTION IV: General discussion 142

SUMMARY AND CONCLUSION 146

LITERATURE CITED 151 X

LIST OF ABBREVIATIONS

Act. D. Actinomycin D n-BAW Normal-butanol:acetic acid:water t-BAW Tertiary-butanol:acetic acid:water BzAW Benzene:acetic acid:water CMV Cucumber mosaic virus DOPA 3,4,-dihydroxyphenylalanine E-4-P Erythrose-4-phosphate EMP Embden-Meyerhof pathway HOAc Acetic acid IAA 3-Indole acetic acid NAD Nicotinamide adenine dinucleotide _ NADP Nicotinamide adenine dinucleotide phosphate 0 .D. Optical density PAL Phenylalanine ammonia-lyase PEP Phosphoenol pyruvic acid Phe Phenylalanine PPO Polyphenol oxidase PVP Polyvinylpyrolidone RNA Ribonucleic acid TAL Tyrosine ammonia-lyase TEE Total ethanol extract TLC Thin layer chromatography (or plate) TMV Tobacco mosaic virus UV Ultra-violet light H Healthy R Infected resistant tissue S Infected susceptible tissue A Apigenin C Caffeic acid pC p-Coumaric acid F Ferulic acid L Luteolin Si Sinapic acid xi

LIST OF TABLES Table Page

A list of antipathogenic substances found in higher plants.

II Some reports on the changes in peroxidase in disease infected plants. 44 III The reagents for the preparation of polyacrylamide gel. 64 IV Chromatographic properties of the phenolic compounds of flax. 72 V Spectral properties of some blue fluorescent compounds isolated from flax. 76

VI Spectral properties of the flavonoids iso• lated from flax. 78 VII The incorporation of phenylalanine-U-^C into phenolic compounds. 90 VIII 14 The metabolism of phenylalanine-U- C into 92 caffeic and p-coumaric acids. IX The incorporation of phenylalanine-U-^C into protein. 93 X A comparison of the different methods for protein extraction. 100 XI The retention capability of Dowex 1x8 on standard and flax phenolic compounds. 102 XII Peroxidase activities in enzyme extracts prepared using different quantities of Dowex 1x8. 103 XIII The incorporation of phenylalanine-U-^^C into protein by infiltration method. 118 xii

LIST OF FIGURES Figure Page

1 Host-pathogen relations with respect to disease resistance. 4 '2 Reactions between quinone and thiols, amino groups and protein. 21 3 The metabolic pathways of phenolic compounds. 25

4 A flow chart of extraction and stepwise hydrolysis of phenolic compounds. 52

5 Apparatus for .slicing and drying gels from polyacrylamide gel electrophoresis. 67

6 UV fluorescence pictures of the TEE of healthy and rusted cotyledons. 70 7 The identification of phenolic moieties of flax phenolic compounds. 71 8 A trace of a chromatogram of the ether frac• tion from the alkaline hydrolysate of TEE. 73 9 Photographs of a chromatogram of the ether fraction from the alkaline hydrolysate of TEE. 74 10 The total phenolic content of healthy and rust-infected flax cotyledons. 80

11 Autoradiogram of TLC prepared from some feeding experiments. $3 12 Autoradiogram of TLC prepared from phenylalanine-U-l^-C feeding experiments. 85

13 Autoradiogram of TLC prepared from cinnamate- 3 _14Q feeding experiments. 87 14 Autoradiogram of TLC prepared from the ether fraction of hydrolysate of TEE from cinnamate- g -14c feeding experiments, 87

15 Autoradiogram of whole cotyledons from phenylalanine-U-l^c feeding experiments. 88 Chromoscan patterns of the autoradiograms of gels containing labeled proteins from 1 day old cotyledons.

Chromoscan patterns of the autoradiograms of gels containing labeled proteins from 6 day old cotyledons.

Chromoscan patterns of the autoradiograms of gels containing labeled proteins from 9 day old cotyledons.

Total protein estimation of healthy and rusted flax cotyledons.

A comparison of PAL activities in healthy and rusted flax cotyledons.

Peroxidase activity in healthy and rusted flax cotyledons.

The isozyme patterns of peroxidase and polyphenol oxidase in flax cotyledons.

Polyphenol oxidase activity in healthy and rusted flax cotyledons.

3-Glucosidase activity in healthy and rusted flax cotyledons.

The effect of infiltration with water and solutions of other compounds on enzyme activities.-

Temporal changes of enzyme activities after infiltration with water, Act. D. or aqueous extract of flax cotyledons. 1

INTRODUCTION

Biochemistry provides a fundamental approach to the study and understanding of host-parasite relations. Amongst the various metabolic systems that function in plants, those concerned with the metabolism of phenolic compounds have pre• dominated in attempts to explain the basis of resistance or susceptibility. But even so, the relationship between phen• olic metabolism and resistance is still far from being settled and there remain many controversial and unsolved problems. Do phenolic compounds confer resistance on a host or do they accumulate as a result of resistance? Are changes in phenolic compounds a general or a specific mechanism of resistance? What changes occur in the activity of the en• zymes concerned with phenolic metabolism in susceptible and resistant tissues after infection?

In order to study changes in phenolic metabolism in an infected host, the simplest approach is to employ a single host variety which responds with susceptible and resistant reactions to virulent and avirulent physiological races of the same species of pathogen. This approach provides a con• stant basal host metabolism in which changes that follow in• fection can be interpreted in relation to the susceptible and resistant responses. For the present work Linum usitatissimum L. TKotof served as the host and was inoculated with race #210 or race #3 of the flax rust fungus (Melampsora lini Pers. Lev.). Race #210 is virulent on Koto and produces a susceptible reaction; race #3 is avirulent on Koto, producing a resistant reaction.

The main objectives of the investigation were:

1. To conduct qualitative and quantitative analyses of the

phenolic compounds present in healthy and susceptible and

resistant reacting tissues, and to determine whether or

not particular phenolic phytoalexins are produced after

infection.

2. To investigate the routes of phenolic metabolism in in•

fected and uninfected tissues by comparing the metabolism

of administered phenylalanine-U-^C, tyro sine-U-^C,

DOPA- B-1Zt-C and cinnamic acid- S -14C.

3. To analyse, quantitatively the metabolism of phenylalanine

into phenolic compounds and proteins in the different

kinds of tissues.

4. To conduct a comparative study of the enzymes involved in

phenolic biosynthesis and degradation, in order to provide

additional data on which to assess the significance of any

changes observed in phenolic levels or metabolic pathways

following infection.

The experiments carried out do not, of course, pro• vide clear cut answers to all the problems related to the role of phenolics in disease resistance. Rather, it is hoped that they provide some new ideas and data relating to this difficult and controversial area of host-parasite rela• tionships . 3

LITERATURE REVIEW

There is a number of good reviews on phenolic metab• olism in host-parasite systems (Farkas and Kiraly 1962,

Cruickshank and Perrin 1964, Goodman et al. 1967, Wood 1967,

Rohringer and Samborski 1967 and Kosuge 1969). Most of these concentrate on the changes in phenolic compounds that follow infection but tend to neglect the enzymatic aspects of phen• olic metabolism in susceptible and resistant .reactions, as well as the mechanisms by which phenolic compounds exert their antibiotic activities. These aspects of what may be called the 'phenolic hypothesis' of disease resistance are there• fore emphasized in the following resume''.

1. PHENOLIC COMPOUNDS AND PLANT DISEASES Disease resistance and susceptibility are most im• portant aspects of host-parasite relations. Resistance can result from three different classes of mechanism: (1) struct• ural means, such as thick cuticles or specialized stomata; (2) the lack of key metabolites .essential for growth of a path• ogen and (3) substances, produced by the host, and capable of inhibiting the growth of the pathogen, or of killing it. These mechanisms are shown schematically in Figure 1. As the result of years of investigation phenolic compounds are regarded as the most important agents conferring resistance to disease in higher plants. The presence of these compounds in host tissue may prevent a particular pathogen from es• tablishing itself on a particular species of host. Very 4

Figure 1. Host-Pathogen Relations with Respect to Disease Resistance (Modified from Shaw 1967). often, these substances occur in plant tissue in a 'bound', non-toxic form. On infection, the toxic moiety is released and inhibits the pathogen, Phytoalexins, most of which are phenolic in nature, are anti-fungal substances synthesized de novo in higher plants after infection.

The preformed antifungal'substances present in plant tissues can exert their activity either (1) in the form of exudates moving out of the plant to affect germination and growth of the pathogen in the external environment, (2) in the protective layers (eg. cork) to stop penetration, or

(3) within host cells or tissues, acting against the patho• gens after they have penetrated epidermal cells or stomatal pores.

The best example of the diffusion of antifungal sub• stances from host tissue is seen in the resistance of onions to infection by Coiletotrichum circinans which causes onion smudge (Walker and Link 1935). The fungus is a soil-borne pathogen. Following a short period of saprophytic growth on dead outer scale leaves, the fungus grows into and parasi• tizes the inner thick fleshy scale leaves. Varieties of onion with yellow or red outer scale leaves are resistant.

In spite of the association between color and resistance, the pigments themselves are not the cause of resistance, as was demonstrated by the fact that colored fleshy scales are attacked by the fungus. Resistance is actually due to the fungitoxic action of protocatechuic acid and catechol which 6 diffuse out of the dead scales into the infection drop and inhibit germination of the conidia of the pathogen.

Martin _et al. (1957) showed that phenolic acids pre• sent in the wax layer on the epidermis of apple leaves can inhibit the penetration of the infection hyphae of Podo- sphaera leucotricha. The waxy material derived from leaves resistant to the mildew, when deposited on the leaves of susceptible varieties would confer resistance to the fungus.

Kuc' and co-workers (1956) worked on Helminthosporium carbonum and potato and found that chlorogenic and caffeic acid in the potato peel are fungistatic agents. Kuc' also showed that these compounds could inhibit Cephalothecium roseum and Myrothecium verrucaria which are non-pathogenic to potato whereas two pathogens of potato tubers viz.

Sclerotium rolfsii and Fusarium solani f. radicola are less sensitive (Kuc' 1957). Lee and Tourneau (195$) showed that in the case of Verticillium wilt disease of potatoes, varieties resistant to infection contain higher amounts of chlorogenic acid in the roots than susceptible ones.

Patil and co-workers (1962) demonstrated that young potato roots which are practically resistant to infection by

Verticillium spp. have a relatively high level of chlorogenic. acid until 5 weeks after sprouting. From the time of sprout• ing, chlorogenic acid content decreased continuously in sus• ceptible hosts. The decrease was closely correlated with an increase in susceptibility to infection. They also showed 7 that the higher level of chlorogenic acid in resistant varieties is.due to their greater synthetic capability (Patil et al. 1966). Other common phenolic compounds which are claimed to be fungitoxic or antibiotic and present in plants include hydroquinone, juglone, phloretin, isocoumarin, umbelliferone and scopoletin (Table I).

Sometimes the initial concentration of phenolic com• pounds present in the tissue is not high enough to inhibit fungal growth. After infection the concentration of phenolic compounds increases and reaches the threshold level necessary to inhibit the pathogen. Farkas and Kiraly (1962) working on fungal, bacterial and viral diseases found more accumula• tion of phenolics in the incompatible host-pathogen combina• tion. They compared the phenolic content of Vernal wheat in• fected with two races of Puccinia graminis tritici viz. #15B and #21 which are virulent and avirulent respectively on this variety and found that there was an increase in the level of phenolic compounds at an earlier stage in the incompatible combination. Such enhanced synthesis of polyphenols and many other synthetic processes are made possible by the increase in respiration (Uritani 1963) which provides the required energy. A change in the respiratory pathway in favour of the pentose phosphate pathway (Shaw and Samborski 1957) can also provide erythrose-4-phosphate for phenolic biosynthesis via the shikimic pathway.

Hydrolysis is a very important process in the release of toxic phenolic compounds against the fungus after infection. TABLE I. A List of Antipathogenic Substances in Higher Plants.

SUBSTANCES STRUCTURAL FORMULA HOST REFERENCES I. PHENOLIC COMPOUNDS

Benzoxazolinones Maize, wheat Virtanen 196$ rye

Caffeic acid potato Clark et al. 1959 sweet potato Uritani & Akazawa 1955 apple, pear Flood & Kirham I960 HO \ /"CH=CH"cOOH carrot LeLaey & Virtanen 195$

Catechol HO onion Walker & Link 1935

Chlorogenic acid same as caffeic acid

HO CH=CH-C-O \v yCOO H HOO Ho\_yOH Dihydroiosocoumarin carrot Sondheimer 1961 MeO

03- Ficinin Neorautanenia Brink et al. 1966 SUBSTANCES STRUCTURAL FORMULA HOST REFERENCES

Hircinol Orchid sp. Gaumann 1963

4-hydroxy-3-methoxy- OMe apple Fawcett and benzoic acid Spencer 1968 (Vanillic acid) H° \ /~COOH

3-hydroxytyramine MeO sugar beet Gardner et al. 1967 (Dopamine)

HO HO oi -hydrojuglone walnut Paxton 1964

Hydroquinone pear Hildebrand & Schroth 1964 HoQhOH

Isopimpinellin OMe citrus Martin et al. 1966

OMe SUBSTANCES STRUCTURAL FORMULA HOST REFERENCES

Neoedulih Neorautanenia Duuren 1961

Nobiletin citrus Ben-Aziz 1967

Orchinol Orchid sp. Gaumann 1963

Phaseollin French beans Cruickshank 1963

Phloretin apple Avadhani & Towers 1961

Pinosylvin pines Scheffer & Cowling 1966 SUBSTANCES STRUCTURAL FORMULA HOST REFERENCES

Pisatin MeO peas Cruickshank 1963

Protocatechuic acid onion Walker & Link 1935 HO-/ VcOOH

Scopoletin sweet potato Minamikawa et al. 1963

Trichocarpin B-D-Gluc-O Populus. sp. Leoschke & Franksen 1964 ffVcO-O-CH,

HO Trifolirhizin red clover Bredenberg & B- D-Gluc-O Hietala 1961

Umbelliferon sweet potato Minamikawa et al. 1963

H SUBSTANCES STRUCTURAL FORMULA HOST REFERENCES II. NON-PHENOLIC COMPOUNDS

Allicin garlic Cavallito & Bailey 1944 CH,: CH-CH2-S-S -CH2 -CH:CH.

Avenacin oats Burkhardt et al. 1964

CH2OH

oc-chaconine potato Kuhn et al. 1955

R = 2,4-di-O-L-Rham-D-GI uc

RO Gossypol cotton Heinstein et al. 1962

Ipomeamarone sweet potato Birch et al. 1954 SUBSTANCES STRUCTURAL FORMULA HOST REFERENCES

•Ipomeanine sweet' potato Kubota & Ichikawa 1954

Crucifers Ettlinger & Lunden . Isothiocyanates CH2= CH-CK2-N= C=S (allylisothiocyanate) -1956

bean Fawcett et al. 1965

citrus Murdoch & Allen 1964

tulip Skinner 1955

potato Tomiyama 1968

smilax Tschesche et al. 1967

Q-B-D-G!uc SUBSTANCES STRUCTURAL FORMULA HOST REFERENCES <* -Solanine

Tomatin

Tulipalin 15

Hydroquinone, a toxic phenol is usually present in pear as its glucoside arbutin. After infection with Erwinia amylovora, the enhanced 8-glucosidase activity results in hydrolysis of the arbutin to hydroquinone (Hildebrandt and

Schroth 1963, 1964). In a similar way, the glucoside phloridzin is hydrolyzed to give the aglycone phloretin, which is inhibitory to Venturia inaequalis, the pathogen of apple scab disease (Holowczak et al. 1962).

Very often the phenolic compounds present in the healthy plants are not fungitoxic but after infection they are oxidized to quinones which are much more toxic than the parent phenols. The polyphenol oxidases and peroxidases in plant tissues are usually activated by infection. For exam• ple, Botrytis cinerea, which causes chocolate spot disease of beans, produces pectic enzymes which liberate galacturonic acid derivatives and polygalacturonic derivatives from cell walls. These compounds unmask the latent polyphenol oxidase of the host (Deverall 1961).

Muller in 1950 found that when potato tuber tissue was inoculated with an avirulent strain of Phytophthora infestans it became resistant to virulent strains of this^ fungus and also to Fusarium spp. Normally Fusarium spp. can para• sitize living tubers. He postulated the d_e novo synthesis of antifungal substances by the host in response to infection.

These he called phytoalexins. Most phytoalexins are phenolic compounds (Table I). Cruickshank and Perrin (I960) and 16

Perrin (1964) isolated pisatin and phaseolin from garden pea and French bean pods inoculated with Monilinia fructicola.

There are also other phenolic phytoalexins of simpler molecular structures such as isocoumarin, umbelliferone and scopoletin. The concentration of isocoumarin varies from 5 -

342 u.g/g carrot tissue depending upon the organism used for infection (Condon et al. 1963). Plants have the potential to produce phytoalexins but it is the pathogen that deter• mines their rate of synthesis by the host.

2. DISEASE RESISTANT MECHANISMS OF PHENOLIC COMPOUNDS Knowledge of the mechanisms by which phenolic com• pounds inhibit pathogens is fragmentary. Many contradictory results have been obtained using different organisms. There• fore it is difficult at this stage to say exactly what mech• anisms are involved. Sometimes, phenolics themselves, act as antipathogenic agents, but quinones, the oxidative pro• ducts of phenols, are often more important in relation to disease resistance.

A) Effect of Phenols Kuc (1964) pointed out that the rapid synthesis, accumulation and inhibitory activity of the parent phenol or phenol derivatives at the site of infection can contribute to resistance. Compounds such as pisatin and 3-methyl-6- methoxy-S-hydroxy-3,4-dihydroisocoumarin are not readily oxidized and quinone formation cannot explain their inhibitory activity. Flood and Kirkham (I960) observed that: the quinic

acid moiety of chlorogenic acid has no effect on growth and

sporulation of Venturia spp. The caffeic acid moiety does

have an effect. Since caffeic acid differs from p-coumaric

which has no inhibitory effect, by only one additional OH

group in the meta position it seems that such hydroxylation

•causes abnormal growth and sporulation and this is found to

be true in many cases. Ferulic acid has no effect because there

is methoxylation of the meta hydroxyl group i.e. it behaves

similarly to p-coumaric acid. The -CH=CH-COOH group might

also be important because, while protocatechuic acid has only

a slight effect on the sporulation of Venturia, it is highly

toxic to Colletotrichum circinans. Flood and Kirkham also

found that the compounds most toxic to Venturia were

o-coumaric acid and cinnamic acid itself. Hulme and Edney

(I960), working on the phenolics in apple peel, also found

that o-coumaric acid completely inhibited the germination of

spores of Gloeosporium perennans whereas p-coumaric acid

gave only partial inhibition.

The fact that the toxic effects of some phenols can

be greatly reduced by glycosylation of one or more hydroxyl

group suggests the importance of the hydroxyl groups in

inhibiting fungal growth. This can readily be demonstrated

.by comparing the germination of broad bean seeds on cotton

wool soaked in equimolar solutions of hydroquinone and

arbutin. Both compounds readily enter the tissues but with

arbutin germination proceeds normally whereas with hydro- 18 quinone the seeds rapidly blacken and die (Pridham and Salt- marsh I960).

On the other hand, Van Sumere (i960) showed that p-hydroxybenzoic acid and ferulic acid were strong inhibi• tors of the germination of wheat stem rust uredospores where• as caffeic acid, vanillic acid and ferulic acid-B -glucoside had little effect. Dabler et al. (1969) also demonstrated that ferulic acid, which has one hydroxyl group masked by a methyl group, was the most effective phenol in inhibiting spore germination of Diplodia zeae at pH 7. At low pH how• ever, the inhibitory effect was not pronounced.

Sondheimer (1962) has reported that unoxidized chloro• genic acid forms complexes with nitrogen containing compounds such as caffein and riboflavin. It also inhibits a number of enzyme systems such as phosphorylase, another pyridoxal phos• phate-requiring enzyme system, IAA oxidase and a peroxidase- catalyzed oxidative decarboxylation of methionine. Possibly the best studied example of activation and inhibition of an enzyme system by phenolics is IAA oxidase. Many monohydric phenols act as activators of this enzyme system while dihydric phenols have inhibitory activity. The most effective activa• tor of the pineapple enzyme found by Gortner et al. (1958) was p-coumaric acid and the most potent inhibitor was chloro• genic acid; caffeic acid was somewhat less potent.

Several points emerge from the above account of the effect of unoxidized phenolics on fungal growth and enzyme 19 activities:

1. The number of OH groups on the benzene ring is important.

In most cases 3,4-dihydroxyl phenols are more inhibi•

tory than monohydroxyl ones.

2. A hydroxyl group at the ortho position to the side chain

is more toxic than one at the para position.

3. The side chain attached to the benzene ring also plays

some role in inhibition.

4. Methoxylation and glycosylation can reduce toxicity.

5 Enzyme inhibition may be the most important way in which

phenols exert their anti-pathogen activities.

Unfortunately, these generalizations do not always apply. They apply to certain organisms under certain con• ditions but not for others. Thus, for example, ferulic acid inhibits spore germination in Diplodia zeae more than caffeic acid does.

B) Effect of Quinones

Byrde (1963) found that the activity of degradating enzymes of the brown rot organism, Sclerotinia fructicola was reduced in resistant varieties of fruit trees owing to pre• cipitation by high molecular weight products, of polyphenol oxidation. Increased activity of oxidative enzymes may account for part of the increased O2 consumption observed following infection. Following infection with Pseudomonas solanacearum and concomitant with increases in O2 uptake and total phenol content, polyphenol oxidase activity increased 20 more in resistant tomato and tobacco stems than in susceptible ones (Maine and Kelman 1961). Prior to infection there was no difference between the two. Enzyme inhibitors such as glutathione and ascorbic acid, fed through the roots, reduced

O2 uptake and polyphenol oxidase activity in diseased tissue, and reversed resistance. Chlorogenic acid, initially in• creased by infection, was markedly reduced in the resistant plants in the later stages of disease development, apparently as a result of oxidation by the enzyme. Resistance to bac• terial wilt thus seems to depend on infection-induced produc• tion or activation of polyphenol oxidase which oxidizes chlorogenic acid to toxic quinones. Both enzyme and phenol are produced in greater, quantities in resistant strains.

Resistance to Pseudomonas phaseolicola in beans also involves a phenolic oxidation system (Hare 1966). '

Similarly, in many other cases the toxic effect of phenolics is exhibited only after they have been oxidized in• to quinones. In other words, quinones are the active inhibi• tors of microorganisms. Quinones are very reactive and react readily with thiols, amino groups and proteins (Fig. 2).

They can thus inhibit enzymes in the following ways:

1. Oxidation of functional groups of enzymes

2. Reaction with SH, amino and hydroxyl groups

3. Complexing with metal ions

4. Reaction with substrates or co-factors

5. Production of hydrogen peroxide 21

Figure 2. Reactions between quinone and thiols (1), amino group (2) and protein (3). (Geiger 1946 and Webb 1966). 22

6. Nonspecific binding through the aromatic ring

7. Competition with quinoid or polyphenolic substrates.

In addition to inhibiting enzyme activity quinones affect cellular metabolism in many other ways. Their effect on electron transport systems is quite complicated. They may compete with or displace natural or endogenous quinones that are involved in electron transport systems. Some qui• nones can act as a new source of electron donors or acceptors and may thus establish an alternative or bypass pathway for electron flow, thus upsetting normal metabolism. Other metabolic processes that may be affected are oxidative phos- phorphorylation, glycolysis and lipid metabolism.

There is a wide variation in the degree of suscept• ibility of various fungi to a particular quinone. It is evident that some particular activity or mechanism must be associated with certain quinones, inasmuch as they are so much more potent than others for a single type of fungus.

Examples of the relation of growth inhibition to structure are

(Webb 1966):

1. In 9 out of 10 fungi, quinone is more potent than the

corresponding hydroquinone; the average relative poten•

cy ratio is 5. This value is greater than that for

bacteria.

2. Addition of chlorine atoms strongly increases potency.

3. Methoxyl groups generally lower the activity in the

napthoquinone series. 23

4. Hydroxylation of 1,4-naphthoquinone in either the 2-

or 5-position leads to loss of activity.

Geiger (1946) found that quinones with methyl, hydroxyl or sulfhydryl groups substituted in the benzene ring are less influential against bacteria. If the 2, 3, 5 or 6-positions of benzoquinone are substituted, antimicrobial activity is decreased. For example, juglone (2-methyl,5-hydroxy, 1,4- naphthoquinone) is more active against E. coli than 2-methyl,

3-hydroxy-l,4-naphthoquinone. This suggests that the activity of quinones against bacteria requires the presence of a free position ortho to a carbonyl group. This finding is further substantiated by the fact that the addition of one equivalent of a sulfhydryl containing compound almost completely abolishes the antimicrobial activity because it eliminates the carbonyl group (Fig. 2).

o o

1,4-Benzoquinone 1,4-Naphthoquinone 24

3. METABOLISM OF PHENOLICS IN HEALTHY AND DISEASED TISSUES

A) Metabolism of Phenolics in Healthy Tissue

In plant tissues all phenolic compounds are derived from the intermediates of carbohydrate metabolism. There

are two main routes for synthesis of the phenolic nucleus, viz, the shikimate and the acetate pathways. The carbon-

skeleton for the shikimate pathway arises from phosphoenol-

pyruvic acid (PEP) and D-erythrose-4-phosphate (E-4-0) which

are in turn derived from the metabolism of glucose via the

Embden-Meyerhof Pathway (EMP) and pentose phosphate pathway

(Fig. 3). PEP and E-4-P condense to give 3-deoxy-D-arabino-

heptulosonic acid-7-phosphate which via 5-dehydroquinic and

5-dehydroshikimic acid would give rise to shikimic acid. On

the addition of another PEP, this pathway leads to a whole

family of C^C^ compounds and their condensation products.

The acetate pathway produces aromatic rings through the head

to tail condensation of acetyl-CoA and malonyl-CoA building

blocks. Some C^C-j phenolic acids and acetogenins are formed

in this manner. The collaboration of both pathways produces

the flavonoids.

In the shikimate pathway, immediately after the C^C^

unit is formed, there is a transamination step followed by

deamination. This would be a very suitable controlling point

for regulating the flow of aromatic rings to nitrogen meta•

bolism, including protein synthesis, or to the phenolic pool.

Therefore ever since Koukol and Conn (1961) discovered 25

Glucose

EMP Pentose phosphate pathway

\ phosphoenol- Erythrose - 4- phosphate pyruvic acid (PEP) 1/ Shikimate pathway i Acetyl CoA->Malonyl CoA Shikimate

Phenylpyruvic acid p-OH- Phenylpyruvic acid TAL Terpenoids Phenylalanine

benzoic acid^ „•„,»„,«:,» „„:J ^ cinnamic acia DO PA coumarin 1 p-OH-benzoic acid<- p-coumaric acid -co umbelliferone |

protocatechuic acid <-caffeiI c acid esculetin ffe Chalcone I vanillic acid ferulic acid -> coniferyl alcohol phloretin scopoletin

flavonoids 1 syringic acid ^ sinapic acid sinapoyl alcohol

Figure 3. The Metabolic Pathways of Some Phenolic Compounds. 26 phenylalanine ammonia-lyase (PAL) and Neish (1961) dis• covered tyrosine ammonia-lyase (TAL) much work has been done on these enzymes both by plant physiologists and biochemists as well as by phytopathologists. The action of PAL leads to the production of cinnamic acid whereas TAL converts tyrosine to p-coumaric acid. In microorganisms and animal tissues phenylalanine can readily be converted into tyrosine but for plant material this conversion has only been reported once by Nair and Vining (1965a) in a spinach enzyme preparation.

They found that the optimum pH was 4.2 and there was an ab• solute requirement for electron donors which was satisfied by adding tetrahydrofolic acid and a reduced pyridine nucleo• tide. However, McCalla and Neish (1959) and Fuchs et al.

(1967) working with Salvia splendens and rust-infected wheat leaves respectively found no evidence for the interconversion of phenylalanine and tyrosine.

The hydroxylation of cinnamic acid was demonstrated by Nair and Vining (1965b) with spinach enzyme preparation and Russell and Conn (1967) with pea seedling enzyme. The pH optima for the two hydroxylating systems are quite differ• ent. For the spinach enzyme it was found to be pH 4.2 where• as a neutral pH was best for the pea enzyme.

McCalla and Neish (1959) showed, in vivo, the follow• ing pathway for the metabolism of phenylpropanoid acids:

cinnamic acid 7 p-coumaric acid ^caffeic acid

sinapic acid«^ ferulic acid < J The in vitro conversion of p-coumaric acid to caffeic acid was only recently reported by Sato (1969). The enzyme catalyzing this reaction is, however, the widely distri• buted polyphenol oxidase (PPO). He found that p-coumaric acid undergoes a coupled oxidation with ascorbic acid:

p-coumaric acid + ascorbic acid + Og ? caffeic

acid + oxidized ascorbic acid.

The methylating enzymes in plants have been studied by Finkle and Nelson (1963), Finkle and Masri (1964), Mann and Mudd (1963) and Mann et al. (1964). The methyl donor was found to be S-adenosyl-L-methionine. The enzyme substrate orientation phenomenon is quite specific for plant methyltransferase and less so for the animal enzyme (Daly 1967).

For example, the catechol-O-methyltransferase from liver tissue catalyzes the methylation of caffeic acid to a.mixture of ferulic and isoferulic acids whereas the plant methyltransferase gives over 95% m-methylation, i.e. ferulic acid.

In plant tissues m-methylation seems to be predominant and seldom are p-methoxy compounds synthesized.

From these phenylpropionic acids (cinnamic, p-coumaric, caffeic, ferulic and sinapic acids) many complex phenolic compounds are derived. These include various phen• olic esters, glycosides, depsides, coumarins, flavonoids, and lignin, ... B) Effect of Infection on Metabolism of Phenolics .

-Wheat leaves infected with rust showed an increase in the incorporation of CO2 to shikimate and quinate. This trend was more pronounced in susceptible than resistant leaves (Rohringer et al. 1967). -In feeding experiments using quinate-U-^C and shikimate-U-^C, more radioactivity was recovered in the insoluble esters for the infected resistant leaves than the healthy control whereas the infected sus• ceptible leaves had more radioactivity in the soluble esters over the same control.

Both Shaw and Colotelo (1961) and Fuchs et al. (1967) showed that rust-infected wheat leaves accumulate tyrosine and phenylalanine. They found that the utilization of these amino acids was also increased. Therefore the production of these amino acids must have increased tremendously. Together with the quinic and shikimate tracer studies it is clear that infection with rust enhances the aromatization process leading to the synthesis of free phenylalanine and tyrosine.

In infected susceptible leaves a major portion of these amino acids is incorporated into protein whereas in resis• tant leaves a higher proportion is incorporated into the non- proteinaceous aromatic compounds.

Some examples are cited below to illustrate the syn• thesis of aromatic compounds as a result of•infection:

a) Scopoletin - Sequeira (1969), studying the synthesis of

scopolin and scopoletin in tobacco plants infected by 29

Pseudomonas solanacearum, found that infected xylem

parenchyma of tobacco plants accumulated scopolin and

its aglycone, scopoletin. The rapid increase in scopo-

letin is not due to an increase in hydrolysis of sco•

polin as the specific activity of S-glycosidase in

these tissues did not increase significantly over the

control.

b) Pisatin - Hadwiger (1967) suggested that the production of pisatin by pea pod tissue induced by Monilinia fructicola or CuClg is due to the stimulation of an aux• iliary pathway which ultimately converts phenylalanine to pisatin. It is also possible that the inducer is an agent with the ability to block a normal pathway for the production of isoflavonoids immediately subsequent to pisatin in the pathway resulting in the accumulation of pisatin.

c) Lignin - Rohringer et al. (1967) found that infected resistant wheat leaves accumulated more from shiki- 14 14 mate-U- C and quinate-U- C in insoluble esters than

the healthy and infected susceptible leaves. They

suggested that at least some of the components of the

insoluble fraction are intermediates in lignin synthesis.

This may reflect a greater lignification in the resis•

tant tissue.

From these examples it can be seen that deviations in

phenolic metabolism do occur in diseased tissue when compared with the healthy. Amongst these various changes, however, 30 the most common ones are the enhancement of hydrolysis and oxidation. Very often glycosides are hydrolyzed to release their aglycones which are then oxidized to toxic substances that confer disease resistance. Oxidation of phenolics leads to the formation of quinones and their polymers, which may cause the so-called hypersensitive reaction. The en• zymes responsible for hydrolysis and oxidation and their actions and changes in diseased tissues are reviewed in the following section.

4. SOME ENZYMES INVOLVED IN PHENOLIC METABOLISM A) Phenylalanine Ammonia-Lyase (PAL)(EC 4.3.1.5) (1) General Properties:

PAL was discovered comparatively recently. Koukol and Conn (1961) reported that an enzyme that deaminated phenylalanine was present in barley. This appeared to be an aspartase-like enzyme. It catalyzes the elimination of one molecule of ammonia from phenylalanine to form an un• saturated acid.

L-Phenylalanine trans-Cinnamic Acid

PAL can convert a number of ring substituted phenylalanines to the corresponding cinnamic acids but these phenylalanines must be of the L- or DL-series (Young and Neish 1966, Subba 31

Rao e_t al. 1967) and the enzyme is inactive towards D-

phenylalanines.

The optimal pH for barley PAL is 8.8-9.2 (Koukol and

Conn 1961). Other workers have found the same pH optimum

for the enzyme from other tissues; Minamikawa and Uritani

(1964) on sweet potato and Subba Rao et al. (1967) on Ustilago

hordei. Minamikawa and Uritani (1964) reported that there are

two components for PAL. Havir and Hanson (1968) also found

two stable enzyme species, with the minor species having

approximately 10% of the total activity. The provisional molecular weight of the major species was found to be 330,000

and the enzyme is appreciably aspherical. The minor species

of the enzyme may have twice this molecular weight.

Apparently no cofactors or metal ions are required.

The enzyme was stimulated by reduced glutathione and the in•

hibition of the enzyme by sulfhydryl group inhibitors suggests

that PAL requires the sulfhydryl group for activity (Koukol

and Conn 1961). The high susceptibility of the enzyme to

inactivation by heavy metal ions such as Ag+, Hg+ and Cd++

also suggests the involvement of sulfhydryl groups in the

reaction. Both Koukol and Conn (1961) and Subba Rao et al.

(1967) found that the enzyme was inhibited by cyanide, indi•

cating that it could be a metallo-protein. Some aromatic com•

pounds such as cinnamic acid and p-coumaric acid are inhibitory

to PAL.

PAL is widely distributed in the plant kingdom as it

is a key enzyme for lignin production. It occurs in both 32 dicotyledons as well as monocotyledons, whereas tyrosine ammonia-lyase, an enzyme comparable to PAL but acting main• ly on tyrosine, is more or less restricted to monocotyledons.

(2) Enzyme Induction:

PAL has been actively studied with respect to enzyme induction since it was found by Zucker in 1965 that light induces PAL synthesis. By definition, induction is the de novo synthesis of enzyme molecules as the result of a stimu• latory effect exerted on the genetic material (via repressors) by the inducer. So far, only a few examples of true induc• tion have been confirmed in higher plants (Filner et al.

1969). Frequently however data indicating enhanced enzyme activity which can be inhibited by inhibitors of the synthesis of proteins or nucleic acids are considered to indicate enzyme induction. The term induction is used in this thesis to include results of this kind.

• Several factors are known to stimulate the biosynthe• sis of PAL. They are exogenous carbohydrates, ethylene, abscisin II, temperature, light, injury and disease. In this review only the last three effects are discussed.

Light has been found by many workers to enhance PAL activity (Zucker 1965, Ahmed and Swain 1970, Smith and

Attridge 1970). At least two types of light effect are be• lieved to regulate the PAL level. One involves the photo• chrome system. Ahmed and Swain (1970) found that PAL activity in both pea and mung bean seedlings is doubled after 33 red light treatment. Smith and Attridge (1970) also showed that short irradiation after red light led to marked in• creases in PAL levels. The rate constants of phytochrome "decay" under the various, treatments were linearly related to the rate constant of the early increases in enzyme activity, indicating that phytochrome "decay" may be an integral part of the mechanism of the action of phytochrome. Zucker (1969) found that light-dependent synthesis of PAL is completely inhibited by 50 \M 3-(4-chlorophenyl)-1,1- dimethylurea (CMU) indicating that photosynthesis is involved. Creasy (1968) working with strawberry reported a requirement for blue light for maximum stimulation of PAL. This photosynthetic requirement is quite distinct from the red-far-red effect of light.

Zucker (1968, 1969) working on the effect of light on PAL induction proposed a sequential induction of PAL and a lyase^inactivating system. By means of cycloheximide and time parameter studies, he found that in sliced potato disks, incubated in light, PAL is the first enzyme induced. About 12 hours after incubation, an inhibitor or degradative agent was also induced and its synthesis depended on protein syn• thesis. This "inhibitor" may therefore be a protease. Thus the early phases of induction involved the synthesis of PAL protein in the absence of turnover, after which a lyase de• grading or inactivating protein was synthesized. He also found that enzymes formed under light disappeared rapidly 34 when disks were placed in the dark. Thus light-induced syn• thesis coupled with a rapid turnover in the dark can produce a diurnal fluctuation of PAL activity. Induced lyase synthe• sis was also observed in excised leaves and to a lesser extent in leaves of whole plants. The large changes in the rate of lyase synthesis in leaf disks compared with those of whole plants suggests that excision is required for rapid induction of lyase synthesis.

(3) PAL and Disease:

Actually the study of PAL in injured or diseased tissues is as early as the studies, of light induction on PAL synthesis. However, only Uritani's group in Japan and

Hadwiger's group in the United States have been actively working on this problem.

Minamikawa and Uritani (1964, 1965) found a marked increase in PAL activity in sweet potato slices after 6 hours of incubation or in-sweet potato inoculated with Ceratocystis fimbriata. The enzyme activity reached a maximum at 24-26 hours and then decreased gradually. There were similar patterns for TAL but the activity was much lower than that of

PAL. Concomitant with the increase in PAL there was also a rise in polyphenol content. Therefore they suggested, that

PAL plays an important role in polyphenol biosynthesis in wounded or infected tissues.

Hadwiger (196$) found that Monilinia fructicola spore suspensions and CuClg caused a 10-12 fold stimulation in PAL 35 activity of pea pods. The factors which stimulate pisatin formation also caused a rapid appearance of PAL activity.

Thus he suggested that there was a close correlation between

PAL activity and pisatin synthesis. Hadwiger et al. (1970) tried to substantiate the claim that phytoalexin production is correlated with increases in PAL activity. They found that PAL increased in excised pea and bean pod tissues within

8 hours after inoculation with pathogenic and non-pathogenic organisms. In general, facultative parasites are more potent than obligate parasites in stimulating PAL activity in these tissues. They also studied the induction of PAL in wheat, corn and flax seedlings by spore suspensions and chemical compounds. The PAL activity in Bison flax (susceptible) was not significantly altered when tissue was incubated for 24 hours with spores of M. lini, Fusarium solani f. sp. pisi or

Puccinia striiformis. PAL was also not significantly stimulated when Cass-M3, a resistant variety of flax, was inoculated with race 1 of M. lini.

The published information on PAL in diseased tissue is still sketchy. More research in this area is needed, especially since this enzyme may control phytoalexin produc• tion.

B) B-Glycosidase (EC 3.2.1.21).

Glycosidases are enzymes catalyzing the hydrolysis of alkyl and aryl glycosides: 36

glycosyl-OR + EH- r glycosyl-E + ROH

glycosyl-E + HO-RT 7 glycosyl-ORT + EH

The enzymatic hydrolysis of glycosides occurs by fission of the bond between C-l of the glycone and the glycoside oxygen atom. Those enzymes involved in transfer reactions with nucleotide derivatives of sugars may also be regarded as glycosidases. Hydrolysis is really a special form of trans- glycosylation with water acting as the acceptor molecule

(Pridham 1963) . 3-glucosidase is one of the glycosidases and it acts mainly on glycosides with 6-linkages. Usually the glycone of the substrate affects the enzyme activity to a much greater extent than the aglycone. 3-glycopyranosi- dase has a low degree of specificity for the aglycone al• though differences in structure do affect the rate of reac• tion. Glycosidases are usually acidic proteins and have no prosthetic groups or co-enzymes. Maximum hydrolytic activity normally occurs in neutral or acidic solutions. The glyco• side undergoes attack by nucleophilic and electrophilic groups on the enzyme surface which results in electron dis• placement and rupture of the glycosidic bond (Pridham I960).

One of the building blocks of lignin, coniferyl alco• hol, is generally present in the form of coniferin in plant.

By the action of B-glucopyranosidase, coniferin can be hydrolyzed into coniferyl alcohol which may then be acted on by laccase or peroxidase to form lignin (Pridham 1963). 37

When cells are mechanically injured or diseased, contact between glycosides and glycosidases occurs and sub• sequent hydrolysis with the liberation of antimicrobic aglycone could be an important function .of glycosidases (see examples on page 15).

While toxic aglycones may be liberated by 6-glucosidase in the host to act against pathogens, virulence of the latter may also depend on B-glucosidase activity. Arneson and Durbin (1967) found that Septoria lycopersici detoxifies tomatin both in vitro and in infected tomato leaves by means of an extracellular enzyme which hydrolyzes one glucose unit from the tomatin molecule. Since the enzyme is extracellular, it may diffuse ahead of the advancing hyphae within the host, detoxifying tomatin and thus allowing the fungus to establish a successful parasitic relationship.

C) Polyphenol Oxidase (PPO)(EC 1.10.3.1) PPO catalyzes one or both of the following reactions: HO ?H

olase or hydroxylase vity

HO O o catecholase or o-diphenol 0^—-> oxidase activity. + H o 20 The most important role of PPO in the physiology of plants is the ability of these enzymes to oxidize monophenols to the corresponding o-diphenols. Sato (1969) showed that 38

p-coumaric acid, can be converted to caffeic acid by PPO in

vitro. By means of this reaction other monophenols might

be converted to complicated polyphenols. Further oxidation

by the o-diphenol oxidase activity of PPO leads to quinoid

compounds and polymers which are responsible for the browning

reaction of plant tissues. This oxidation step is much more familiar than the hydroxylating action of PPO. Actually

the oxidation leading to quinone is commonly encountered in

injured tissue or in in vitro experiments. However it is

possible that the main function of PPO in vivo in healthy

tissue is hydroxylation rather than complete oxidation of

phenolic compounds.

Several points about PPO in connection with host-

parasite relationship are noteworthy:

(1) Direct action of PPO on other enzymes: The role

of,PPO in disease resistance is two-fold. The best recognized

is the oxidation of phenol to form quinones and polymers

which are toxic to the pathogen or which form physical

barriers preventing further extension of the fungus. The

direct action of PPO on other enzymes is usually overlooked.

Since PPO can oxidize the tyrosine in the protein molecules

of other enzymes (Sizer 1953), it is possible that the con•

formation of these enzymes would be changed and consequently

the enzyme activity would be altered. Laborzewski (see

Rubin's review 1964) showed that PPO from mushroom completely

inactivated crystalline alcohol dehydrogenase in 15 min at 39

25°C and pH 7.5. The resistance of potato tuber to Phyto- phthora infestans during the early stages of infection is characterized by increased PPO activity and simultaneous inhibition of dehydrogenase activity of the host during invasion,

(2) Latent PPO: Kenten was the first to study the latent PPO in plant materials. He found that water extracts of broad bean leaves contained much latent PPO activity

(Kenten 1957). The active PPO is released by brief exposure of" the extract to acid (pH 3-3.5) or alkaline (pOH 2.5-3)

conditions, or by incubation in the presence of (NH.)oS0, Hr <~ if at pH 5. Further studies on the activating effects of anionic wetting agents on PPO (Kenten 195$) led him to suggest that anionic wetting agents can combine with the cationic group of the protein leading to the dissociation of the PPO-protein inhibitor complex or configurational changes in a prophenolase. The activation of PPO of the host tissue after in• fection has been reported by a number of workers. Deverall (1961) found that PPO of bean plants was activated after in• fection with Botrytis cinerea. This action is due to un• masking of the latent PPO by galacturonic acid derivatives which could be liberated from the cell wall as the result of the action of pectic enzymes secreted by the pathogen. Another example of the activation of latent PPO in host- parasite relations is the activation of rice leaf PPO by 40

ophiobolin, a toxin produced by the pathogen Cochliobolus

miyabeanus (Nakamura and Oku I960).

(3) Inactivation of PPO: Rapid inactivation of PPO

is always encountered in the in vitro assay of the enzymes.

Van Kammen and Brouwer (1964) used chlorogenic acid as a

substrate to study PPO activity and found that the decrease

in absorbance as a result of PPO activity was not linear with

time. They suggested that this may be due to product inhi•

bition. The exact mechanism is still unknown, but the in•

hibitory effect might be attributed to the reactivity of

the quinone which the system generates. This in vitro

inactivation however could easily lead to the notion that

PPO would not be catalyzing such reactions in the normal

healthy plant cells. This may have been guarded against by means of compartmentalization (Kosuge 1969).

D) Peroxidase (EC 1.11.1.7) This enzyme has been known for more than one hundred years and its general properties are well studied (Saunders et al. 1964). In this review the reactions in which peroxi• dase is involved are treated briefly and followed by an account of the possible roles of peroxidase in plant tissues and their role in host-parasite relations.

(1) Reactions

Hydroxylation: Phenylalanine, tyrosine, m-tyrosine,

p-cresol, benzoic acid and salicylic acid were reported to

be hydroxylated by peroxidase (Saunders et al. 1964). 41

Oxidation: Many different substances in the plant

can act as substrates of peroxidase. Phenolics can be oxi• dized to quinone by peroxidase and hydrogen peroxide:

Typical peroxidase systems are capable of oxidizing

several amino acids and their derivatives. The oxidation of tyrosine involves a quinone-like intermediate and then leads finally to a colored melanin-like end product. Peroxidase

also catalyzes the oxidative polymerization of coniferyl

alcohol, and the oxidation of NADH, NADPH and IAA.

(2) Peroxidase, plant physiology and host-parasite

relations

Akazawa and Conn (195$) reported that the reduced forms of NAD and NADP are rapidly oxidized in the presence of crystalline horse-radish peroxidase, catalytic amounts of Mn++ and certain phenols. One atom of oxygen was con• sumed per molecule of nucleotide oxidized. The phenols which were active are either monohydric phenols or resorcinol. Gamborg et al. (1961) found that dialyzed extracts of pea epicotyl and spruce shoots can also oxidize reduced NAD. They suggested that the following reactions occurred: R.OH = phenolic cofactor.

The possible relationship between reducing power and virulence of pathogens is frequently mentioned (Kaul and

Shaw I960). If there is a relationship, the capacity of peroxidase to destroy reducing power might account for the apparent correlation between high peroxidase of plants and resistance to certain pathogens.

IAA can be oxidized to 3-methylene oxindole by horse-radish peroxidase in the absence of added H^O^ (Hinman and Lang 1965) . Fox and Purves (1967) suggested that the oxidation occurred through a free radical mechanism. The increase in peroxidase in diseased tissue may affect IAA levels. It was also reported that phenolic compounds might regulate the growth promoting activity of IAA by virtue of their effects on peroxidase. Chlorogenic acid at 5 x 10~^M completely inhibits the oxidative decarboxylation of amino acids by peroxidase. The relationship between IAA, peroxi• dase and phenolic compounds and disease is an interesting problem.

Yang (1967) demonstrated that ethylene was rapidly formed from a-keto- y -methyl thiobutyric acid by horse• radish peroxidase in the presence of Mn++, SO^-, oxygen and 43 a specific phenol. The active phenols include some monophenols and m-diphenols. Stahmann et al. (1966) showed that ethylene induced resistance as well as increased peroxidase activity in Ceratocystis fimbriata infested sweet potato.

It is still unknown whether peroxidase is responsible for the production of ethylene in vivo, but there seems to be no doubt that there is some relationship between them.

As discussed above peroxidase can hydroxylate as well as oxidize phenolics. In most of the studies of peroxi• dase in diseased tissues (Table II), the increase in peroxi• dase activity is correlated with increased quinone formation and the toxic action of the quinone causes necrosis. In the necrotic tissue the pathogens are either inactivated or inhibited from spreading through the tissue.

Of the several putative roles for peroxidase it is very difficult to assess the actual role that this enzyme plays in vivo and especially in host-parasite relations.

Therefore even though peroxidase is a well studied enzyme there are still many aspects of its role that are worth investigating in relation to plant diseases.

5. PHENOLIC CONSTITUENTS OF FLAX AND HOST-PARASITE RELATIONS

Cruickshank and Swain (1956) studied the phenolic compounds in ethanolic extracts of flax. They reported that the phenolic compounds in flax can roughly be divided into 3 groups: (1) flavone-like compounds similar to apigenin;

(2) chlorogenic acid and its isomers; and (3) a miscellaneous group. Except for chlorogenic acid and its isomer, they did TABLE II. Some Reports on the Changes of Peroxidase after Infection,

AUTHORS HOST PATHOGEN CHANGES IN PEROXIDASE AFTER INFECTION

1. Kanazawa. Schichi & Sweet potato Ceratocystis Increased activity which was Uritani (1965) roots fimbriata (black inhibited by inhibitors of rot) protein and RNA synthesis and oxidative phosphorylation.

2. Kawashima & Uritani Sweet potato Ceratocystis Increased activity in both (1963) roots fimbriata (black diseased and cut tissues. rot) There is qualitative differ• ences between the healthy^ diseased and injured tissues.

3. Staples & Stahmann Bean leaves Uromyces phaseoli Small increase in activity. (1964) No isozymic changes. 4. Andreev & Shaw Flax var. Bison Melampsora lini By 6th day after infection (1965) Bombay new isozyme band formed. The new isozymes formed are simi• lar to those formed in senescent tissue.

5. Stahmann, Clare & Sweet potato C . fimbriata Ethylene induced resistance Woodbury (1966) and increased peroxidase activity.

6. Weber, Clare & Sweet potato C. fimbriata Increased activity in resis• Stahmann (1967) tant tissues. AUTHORS HOST CHANGES IN PEROXIDASE AFTER INFECTION

7. Farkas & Stahmann Bean leaves Increase in peroxidase and (1966) changes in isozymes.

8. Lovrekovich et al. Tobacco Increased peroxidase associa• (1968) ted with TMV infection in• duced resistance to tobacco to infection by P. tabaci.

9. Novacky & Hampton Nicotiana tabacum Quantitative but not quali• (1968) Vigna sienensis tative changes during infec• Phaseolus aureus tion and senescence. P. vulgaris 10. Novacky & Wheeler Oats Quantitative changes in iso• (1970) zymes. Resistant tissues re• quired much higher concentra• tion of victorin to give the same changes.

11. Rautela & Payne Sugar beets Increase was consistently (1970) higher in resistant than in susceptible variety. Toward the advanced stages of the disease this pattern was reversed.

12. Chant & Bates Nicotiana Higher activity in extract (1970) glutinosa from leaves showing necrosis than healthy or chlorosis leaves. One more isozyme in virus infected leaves. AUTHORS HOST PATHOGEN CHANGES IN PEROXIDASE AFTER INFECTION

13. Simons & Ross Tobacco TMV Increase in peroxidase in (1970) upper leaves is concomitant with resistance development induced by virus infection in lower leaves.

14. Hussey and Wando.peas Nematode New isozymes found. Krusberg (1970) Dictylenchus dipsaci

ON 47 not identify any other compounds. Hegnauer (1965) pointed out that flax seed meal contains linocinnamarin (methyl ester of B-glucosido-p-coumaric acid), linocaffein (methyl ester of 4- 3-glucosido-caffeic acid) and a lignan glucoside a diglucoside of D-2,4-di(3-methoxy-4-hydroxybenzyl) butane-

1,4 diol (Bakke and Klosterman 1957).

Recently Ibrahim (1969) worked on the flavonoids of flax cotyledons. He claimed that there are 6 flavones, two of which are apigenin (4,5,7-trihydroxy-flavone) type and the rest are luteolin-like (3f,4T,5,7-tetrahydroxy-flavone).

These flavones are present in flax cotyledons in the form of mixed 0- and C-glycoflavones. In another article, Ibrahim and Shaw (1970) found 9 cinnamic acid derivatives. They were identified as p-coumaroyl quinic acid, p-coumaroyl glu• cose, 3-0-caffeoyl quinic (chlorogenic), glucosido caffeic acid, caffeoyl glucose, glucosido ferulic acid, feruloyl glu• cose and a glycoside and an ester of sinapic acid whose non- phenolic moieties were not identified. They did not find any free cinnamic acid, benzoic acid derivatives or flavonol glycosides. There was no qualitative but a slight quantita• tive difference between cotyledons and young shoots.

Cruickshank and Swain (1956) found some relationship between the phenolic contents of several varieties of flax and their resistance against M. lini. They worked on 4 varieties of flax, viz. Williston Golden, Ottawa 770B,

Argentine Seln and Bombay, only the first one being sus- 48

ceptible. They found that Williston Golden has a high con•

centration of chlorogenic acid whereas the rust resistant

group had low concentrations. Determination of the ratio of chlorogenic acid to total phenolics showed that this ratio was highest in the highly resistant variety Ottawa

770B and least in the susceptible variety Williston Golden.

Allan (1967) studied the correlation between phen•

olic content and histological changes in susceptible (Bison)

and resistant (Bombay) varieties of flax infected with race

§3 of M. lini. He found that the total free and free

o-dihydroxyl phenolic acids decreased sharply during the

period of cell collapse in the resistant host. After the

rust infection was rejected the resistant host underwent a

period of stimulated phenolic accumulation or synthesis which was followed by early senescence of flax cotyledons. Total

free and free o-dihydroxy phenolic acids were accumulated

during the first 48-60 hours in the susceptible host. During

this stage the fungal mycelium was spreading throughout the mesophyll tissue. This increase in phenolic acids was

followed by a sharp decrease which corresponded approxi• mately in time to the onset of sporulation by the fungus. 14 These results were supported by tracer studies with CO . 49

MATERIALS AND METHODS

1. PLANT MATERIALS

Flax (Linum usitatissimum L.) variety Koto was studied throughout the present investigation. Two races'of flax rust (Melampsora lini Pers. Lev.),#3 and#210 were used to give resistant and susceptible host-parasite combinations respectively. In the resistant reaction the parasite pene• trates the host tissue and pin-point brown fleckings occur but there is no formation of pustules or spores. The sus• ceptible reaction is characterized by the successful estab• lishment and sporulation of the parasite.

The plants used to produce rust infected tissue were grown as follows. Flax seeds were treated with 'Arasan 75 T and sown in 6" plastic pots containing soil and peat moss in the ratio of 4:1. The plants were grown in a growth chamber at a light intensity of $00-1000 ft.-c. and a photo• period of 14 hours with the light period between 6 a.m. and

8 p.m. The temperature of the growth chamber was 22-24°C during the day and l8-20°C at night. Plants used for purely phytochemical studies and for raising uredospores were grown in the greenhouse at a temperature of about 22°C.

One week old seedlings were inoculated by dusting uredospores onto the cotyledons with a brush. The plants were sprayed with distilled water before and after dusting. After inoculation, the pots were covered with moist plastic bags for 18 hours. Inoculation was usually carried out at the end 50

of the photoperiod so that the plants could be kept in dark• ness after inoculation for 10 hours without interfering with

the normal photoperiod. The infected susceptible tissues

showed an average of 21+ pustules and a range of 2 to 62

pustules per cotyledon.

2. PHYTOCHEMICAL STUDIES A. Extraction of Phenolics

(i) Total ethanol extract (TEE) for identification

(a) About 10 gm of cotyledons were submerged in 200 ml of boiling 80% ethanol in order to stop any enzymatic reactions. The tissues were allowed to simmer for 5 min and then extracted similarly with two more aliquots (200 ml of

80% ethanol. They were blended in an osterizer during the

last ethanol extraction and the residue was removed by suc• tion filtration over a Buchner funnel.

(b) The combined extracts were dried under vacuum

in a rotary evaporator.. The dry material was stirred in hot water and filtered through celite.

of 80% ethanol or in 10 ml of distilled water for chromato•

graphic analyses and hydrolysis respectively. 51

(ii) TEE for quantitative studies

This extraction procedure was basically similar to

(i) except that about 2 gm of cotyledons were weighed accurately and extracted three times with 50 ml portions of

80% ethanol. The cotyledons were ground during the last ex• traction in a mortar instead of homogenizing in an osterizer.

The final volume of the extract was brought to 25 ml with

5O70 ethanol in a volumetric flask.

(iii) TEE for autoradiography

The procedure was similar to that described in (i) except that centrifugation was used instead of filtration.

The final dried material was usually taken up in 1 ml 80% ethanol.

B. Hydrolysis

Stepwise hydrolysis and liquid-liquid extraction (Ibrahim and Towers I960) were carried out to identify the phenolic compounds in flax (Fig. 4). The aqueous phenolic extract was acidified to pH 2.0 and extracted with ether by liquid-liquid extraction. All free phenolic acids parti• tion in the ether phase. The aqueous phase was then hydrolyzed in IN NaOH under nitrogen for two hours at room temperature. The pH of the hydrolysate was adjusted to 2.0 with 5N HCl and again extracted with ether. Alkaline hydrolysis cleaves the ester linkages thereby releasing the phenolic acids. The bulk of the solution was then sub- 52

Flax Cotyledons I

80% ethanol extraction

Evaporation to dryness I Dissolved in distilled water and filtere1 d Evaporate to i Ether liquid- liquid extraction dryness

_i f Total Ethanol Ihydrolysi s Ether Extract Extract (TEE)

Ether extraction

vf 1 I Acid hydrolysis Ether Extract C I Ether extraction 3L Aqueous phase Ether Extract

n — Butanol extraction «—L n - Butanol Extract Aqueous phase A - E : For further analysis

Figure l+. A Flow Chart of Extraction and Stepwise Hydrolysis of Phenolic Compounds. 53 jected to acid hydrolysis in IN HC1 at 100°C for 30 min in order to cleave glycosidic linkages. This step results in the formation of free phenolic acids. The hydrolysate was first extracted with ether and then with n-butanol. The ether fractions contain free phenolic acids whereas any phenolic compounds that escape hydrolysis are extracted in the n-butanol fraction.

Each fraction was evaporated to dryness in vacuo and dissolved in 95% ethanol for further analyses.

C. Chromatography

The phenolic compounds were isolated by chromatography on TLC plates (5-10 plates). Bands of the same Rf from different plates were scraped off and combined for extraction with 80% ethanol. By means of banding and rebanding and ex• ploitation of different solvent systems, the phenolics of flax were isolated as relatively pure compounds. One dis• advantage of this method is that it is very difficult to pre• pare satisfactorily large amounts of each compound. However, the characteristics of the compounds on a,chromatogram, in• cluding UV fluorescence, UV fluorescence under ammonia, Rf values in different solvent systems and the color reactions of the compounds with different spraying reagents provide valuable data for purposes of identification.

Both paper and thin layer chromatography were tried.

Except for free phenolic acids the resolving property of paper (Whatman No. 1 and 3) was very poor for flax phenolics. 54

For TLC, macrocrystalline cellulose "Avicel TG-104" was

found to be superior to cellulose MN 300 G. Similar re•

sults were reported by Mullick (1969) for the separation

of anthocyanins from conifers. Therefore, thin layer chro• matography on 'Avicel* was adopted as the routine procedure.

Thin layer plates were prepared by blending 20 gm

'Avicel' cellulose with 80 ml distilled water. For the

identification of compounds, a 0.25 mm layer of 'Avicel' was coated onto the 20 cm x 20 cm glass plates with a

Desaga thin layer apparatus. A somewhat thicker (0.35 mm)

layer was used when the extract was to be 'banded' on the

chromatograms.

The solvent systems usually used for two dimensional

chromatography were:

(i) t-butanol:acetic acid:water of ratio 3:1:1

(tBAW) and 15% acetic acid. This system gave good results with flavonoids and was excellent for resolving the total

ethanol extract.

(ii) Benzene:acetic acid:water, 2:2:1 or 10:7:3 and

2% acetic acid. This solvent gave good results for phenolic acids.

(iii) n-butanol:acetic acid:water, 4:2:1 (nBAW) and

2% acetic acid.

The reagents used for color reactions (Ibrahim and

Towers I960) were:

(i) Diazotized p-nitroaniline - 5 ml p-nitroaniline

(0.35% in 8% HCl w/v), 1 ml NaN02 solution (5% w/v), 15 ml 55 sodium acetate (20% w/v), freshly mixed in the order des• cribed before. Chromatograms were sprayed with this mixture, allowed to dry for 5 rnin in the fume hood then over sprayed

with 5% Na2C0^ or NaOH solution.

(ii) Diazotized sulfanilic acid - 2 vols, sulfanilic acid (9 gm/90 ml cone. HC1 and then diluted to 1 litre), 1 vol. sodium nitrite solution (5% w/v) and 2 vols. NaOH

(20% w/v).

(iii) Ferric chloride - 1% solution in 95% alcohol.

D. UV Absorption Spectra

Most phenolic compounds absorb light in the UV range.

The optical properties of phenolic compounds and their' reac• tions with different reagents permit the characterization of their functional groups. A Unicam SP800 spectrophotometer was used throughout this work. Unless otherwise specified the spectra were analyzed in $0% ethanol. The slit width of the Unicam was set at 0.02 mm and the machine was set at

"fast scan". Either 1 ml or 3 ml quartz cuvettes were used depending on sample sizes available.

Four different reagents were used to study the functional groups of the phenolic compounds (Harborne 1964).

These were:

(i) Sodium hydroxide - 2 drops of 2N NaOH were added to the sample in the cuvette and mixed well before the spec• trum was taken. Large bathochromic shifts were noted in most cases, and. any increases in the intensity of the ab• sorption bands were also observed. However, the ionization of aromatic carboxylic acid by NaOH causes a hypsochromic shift in the spectrum, so that phenols containing free carboxylic acid groups do not give as large a bathochromic shift in alkaline solutions as the related esters. This allows the investigator to distinguish between these two classes of compounds. Phenolics such as catechol and. pyrogallol are unstable in alkaline solution,

(ii) Sodium acetate - This was used to distinguish the free 7-hydroxyl group of flavonoids. The long wave• length band of all flavonoids is shifted bathochromically by this reagent; it is the short wave band which undergoes a shift only if a free 7-hydroxyl group is present in the molecule. An excess of solid sodium acetate was added to the cuvette containing the sample and thoroughly mixed until the solution was saturated with sodium acetate.

(iii) Aluminum chloride - phenols containing a catechol (o-dihydroxyl) group-or a hydroxyl group adjacent to a carbonyl group, complex with AlCl^ in solution and their absorption maxima move toward the visible range. Two to 3 drops of 5% AlCl^ in 95% ethanol was added to the sample for this reaction.

(iv) Boric acid - After the absorption spectra were taken in the presence of saturated sodium acetate, excess of boric acid was added to the sample in order to identify the catechol groups. The spectral shift is usually observed 57 in the long wavelength band and is of the order of 15-30 mp; the spectra of all other phenols are not appreciably- affected by the presence of this reagent.

E. Estimation of Phenolics

Total phenolic contents were estimated according to the method of Swain and Hillis (1959). Aliquots of 0.5 ml of the TEE were diluted with water to 7 ml in test tubes and 0.5 ml of IN Folin reagent was added. The tubes were thoroughly shaken and allowed to stand for exactly 3 min.

One ml of saturated sodium carbonate solution was then added to each tube and the mixture was made up to 10 ml. After 1 hour, the absorption was determined at 725 mp using a reagent blank. Insoluble materials, if any were centrifuged off before the readings were taken.

A standard curve was prepared with 0.1, 0.2, 0.4,

0.6, 0.8 and 1.0 ml of chlorogenic acid (100 pg/ml).

F. Incubation Procedure

The radioactive chemicals used were L-phenylalanine-

U-1/fC, L-tyrosine-U-1/fC, D0PA-g-1/fC (New England Nuclear

Corp.) and cinnamic acid-3-^^C (International Chemical

Nuclear Corp.). The radioactive chemicals were neutralized with IN NaOH before incubation. The cotyledons were placed in a beaker containing 10 pC of radioactive chemical in 10 ml of distilled water. The uptake of radioactive pre- • 53

cursors was facilitated by vacuum infiltration for 3-5 min.

The cotyledons and the incubation mixture was then trans• ferred to a petri dish and incubation was continued in a

growth chamber at 22°C under 800 ft.-c. of light for 4-

8 hours. In experiments where inhibition of protein syn• thesis was desired, the incubation mixture also contained

600 ug/ml of cycloheximide.

The problem of bacterial contamination in the infil•

tration-incubation method was studied in preliminary experi• ments. There was no significant difference in enzyme levels

(e.g. peroxidase) for treatments with or without Gramicidin D

if the incubation period was less than 12 hours. Furthermore, the incorporation of phenylalanine-U-^C into phenolic com•

pounds was found to be slightly higher in tissues infiltrated with Gramicidin D solution than in the water infiltrated

tissue when the incubation period was 8 hours. From these

preliminary results it was concluded that bacterial contamin•

ation is not likely to affect the metabolism of the tracers

in experiments of less than 8 hours duration. Accordingly no antibiotics were added to the incubation media in the

experiments to be described. Any unknown effects of the

antibiotic on metabolism are thus avoided.

G. Autoradiography

(i) Autoradiography of whole cotyledons. After incu• bation with radioactive precursors, the cotyledons were fixed

and washed with ethanol (80%) to remove any remaining precursor. 59

The cotyledons were then pressed between layers of blotting

paper and glass plates and dried in an oven at 60°C. They were transferred onto a hard paper and autoradiographed by

placing a Kodak no-screen medical X-ray film in contact with

the tissue. The exposure period was 2 days and the films were developed with D19 developer and fixed with Kodak Rapid

Fix.

(ii) TLC - TLC plates were also autoradiographed as

described above. The exposure time varied from 1 day to 1 week, depending on the radioactivity of the compounds under investigation.

H. Liquid Scintillation Counting

Radioactivity was quantitatively measured in a Nuclear-Chicago Mark I liquid scintillation counter using a dioxane based system (Chakravorty 1969) containing dioxane, 800 ml; toluene, 200 ml; ethanol, 30 ml; 2,5- diphenyloxazole, 7 gm; 1,4Bis[2-(5-phenyloxazolyl )"3 benzene, 150 mg; naphthalene, 50 gm; cab-o-sil, 36 gm. Either 0.2 or 0.5 ml samples were taken in scintillation vials and to each sample 2 drops of IN NaOH and about 15 ml of the scin• tillation liquid were added. The vials were cooled to 0° and counted for 4 or 10 min. The counts thus obtained were corrected for background but not for quenching. Similar channel ratios were obtained in all experiments. 60

3. ENZYME STUDIES A. Enzyme Extraction

The extraction procedure was based on the method reported by Lam and Shaw (1970). About 100 cotyledons were ground in a mortar with a 10%, (w/v) suspension of Dowex

1-X8 (Chloride form, 200 - 400 mesh) in tris-glycine buffer

(0.05 M) at pH 8.3. The resin was washed repeatedly with deionized water and equilibrated with the buffer overnight.

The supernatant fraction was decanted and fresh buffer added to give approximately a 10% (w/v) suspension. The final pH was about 7.6-7.8. After homogenizing, the slurry was centrifuged at 30,000 g in a Sorval RC2-B refrigerated centrifuge for 20 min. The supernatant fraction represented the crude enzyme preparation.

B. Protein Estimation

For most of the enzyme work a UV absorption method (Warburg and Christian 1941) was used to estimate protein concentration. When a substantial contamination by phenolic compounds was suspected, the proteins were precipitated by TCA and then quantitated by the method of Lowry et al. (1951). The absorption of the color complex was measured at 600 mo.. The protein content was calculated from a standard curve prepared with bovine serum albumin (Fraction V., Calbiochem.). 61

C. Enzyme Assays

(i) Phenylalanine ammonia-lyase (PAL)

One ml each of enzyme and a solution of 20 pMole/ml

L-phenylalanine in 0.1 M borate buffer at pH 8.8, were pipetted into a test tube and incubated in a water bath at

40°C. After 1 hour, the reaction was stopped by adding 0.5 ml 5N HC1 and the solution was extracted with 15 ml ether by vigorous shaking. The ether layer was quantitatively trans• ferred to another test tube and a pinch of anhydrous Na^SO, 2 4 was added to dehydrate the ether extract. This was then placed in a small beaker and the ether was removed by evaporation in a fume-hood. The residue was redissolved in

2 ml of 0.01 N NaOH and the absorption at 268 mp was measured.

The standard curve was prepared from cinnamic acid and the activity of PAL- was expressed in nMole cinnamic acid formed per mg protein per hour.

(ii) p-glucosidase

3-glucosidase activity was estimated by a modifi• cation of the method of Stenlid (1957).- The reagents used were citrate phosphate buffer (pH 5.4) at 0.2 M with respect _3 to citric acid and 4 x 10 M p-nitrophenyl-B -glucoside

(nipheglu) solution.

The assay was done at room temperature (24°C) in test tubes. Each tube contained 1.0 ml buffer, 0.1 ml nipheglu and 0.2 ml enzyme made up to 2 ml with distilled water. The reaction was started by adding nipheglu to the tubes. After 62

30 min 2 ml of 2.5% NagCO^ was added to each tube, mixed well and the absorption of the p-nitrophenol formed was measured at 400 m|i.

The standard curve was plotted by using 2, 4, 8, 12 and 15 ng p-nitrophenol.

(iii) Peroxidase

Peroxidase activity was estimated with a freshly prepared solution of 0.1 ml 30% HgOg and 0.1 ml guaiacol (Eastman) made up to 100 ml with phosphate buffer (pH 6.5) as substrate.

For each assay 2.8 ml of the reagent and 0.2 ml of enzyme preparation were pipetted into a 3 ml cuvette and thoroughly mixed. The reference cell contained 2.8 ml of reagent and 0.2 ml tris-glycine buffer (pH 8.3). The ab• sorption was measured at 470 m\± and the activity was cal• culated as A 0D 470 mii/min per mg protein.

• The Unicam SP800 spectrophotometer has no tempera• ture control but the measurement procedure never took more than 3 minutes and it was found that the heat generated by the spectrophotometer did not affect the readings appre• ciably. Therefore this study can be considered to have been conducted at room temperature of about 24°C. The same conditions apply to the assays of PPO. • 63

(iv) Polyphenol oxidase (PPO)

PPO activity was measured by using chlorogenic acid as substrate (Van Kammen and Brouwer 1964). PPO oxidizes the phenolic hydroxyl groups of chlorogenic acid and thus causes a decrease in absorption in the region of 290-340 m\±.

An aliquot of 2.$ ml of 0.005% chlorogenic acid in phosphate buffer (pH 6.5) and 0.5 ml enzyme preparation were added to a 3 ml cuvette and the enzyme activity was esti• mated by measuring the decrease in absorption at 330 m\±.

The enzyme activity was linear for the first l/2 min only and then reached a plateau, probably due to product inhibi• tion. It was therefore calculated from the linear portion of the curve. The data were expressed in AOD/min per mg/protein.

D. Polyacrylamide Gel Electrophoresis

The method used was modified from that of Davis (1964).

The reagents and proportions of chemicals used to prepare

7.5% polyacrylamide gel at pH 8.3 are shown in Table III. In preparing both lower and upper gels the reagents were first mixed well in plastic syringes (without the needle) and then injected carefully into each of 10 glass tubes (7 cm x 0.5 cm). Fluorescent lights were used to allow the upper gel to polymerize rapidly. Sharp and flat boundaries between the gels and on the top of the upper gel were achieved by carefully introducing a layer of distilled water onto the polymerizing reagents. 64

TABLE III. The Reagents for the Preparation of Polyacrylamide Gel.

Preparation of Gel Preparation of Reagents

Parts Reagents Chemicals Quantity 1 N HCl 24 ml 1 A pH(8.9) Tris 15.$5 gm Temed 0.32 ml

r Water to 100 ml

Lower 1 Acrylamide 30 gm Gel • 2 C BIS 0.8 gm Water to 100 ml

1 Deionized water -

Ammonia persul- 0.14 gm 1 G fate; water to 100 ml IN HCl 48 ml B(pH 6.7) Tris 5.9$ gm 1 TEMED 0.32 ml Water to 100 ml

Upper Acrylamide 10.5 gm Gel 2 D GIS 2.5 gm Water to 100 ml

2 E Riboflavin 4 mg Water to 100 ml

3 F Sucrose 40 gm Water to 100 ml 65

The reservoirs were each filled with 250 ml of pre- chilled tris-glycine buffer(0.5 M, pH 8.3) and the whole set up was kept in a refrigerator at 4°C during electro• phoresis. The enzyme in 10% sucrose was carefully layered on the specimen gel. Two to three drops of 1% bromophenol blue were used as a tracking dye. The power supply was maintained at 2.5 milli Amp. per tube and at a voltage less than 600 V.

E. Isozyme Studies

After electrophoresis the gels were removed from the tubes and stained for particular enzymes. There is no known method for the detection of the isozymes of PAL and 3 -glucosidase on polyacrylamide gels. In the present work the author unsuccessfully tried to stain PAL isozymes by incubating the gels with phenylalanine and then staining with ninhydrin reagent or diazotized p-nitroaniline. Isozymes of peroxidase were detected by the method of Siegel and Galston (1966) using guaiacol reagent in 3.5% acetic acid. Benzidine reagent was also tried but was not as good as guaiacol. This may be due to the fact that this reagent was originally developed for haemoglobin or myco- globin peroxidases.'

PPO isozymes were studied by the method of Hyodo and Uritani (1965) using a solution containing equal volumes of 0.9% caffeic acid and 0.1% p-phenylene-diamine. The caffeic acid is very sparingly soluble in water but it can 66 be solubilized by neutralizing it with IN NaOH to sodium caffeate (pH 6.0). By using Hyodo and Uritani's method the colored reaction product formed was found to dissolve in the solution easily. Therefore the reagent was made up to 3.5% with respect to acetic acid to give a better fixa• tion of the colored product in the gel.

F. Analysis of Phenylalanine-U-^C-Labeled Proteins

The electrophoresis pattern of labeled proteins was studied according to the method by Fairbanks et al, (1965).

The gels were stained with Amido black to locate the bands and then sliced with an apparatus devised by Fairbanks and co-workers. Slices l/l6 in. thick with two flat surfaces were selected for study.

The method of drying the gel was modified by the present author (Fig. 5). The gel slices were dried over a

"Nalgene" filtering funnel with the rim of the funnel cut down to about 1 cm high. The slices were arranged on filter paper on the funnel and covered with ' Saran wrap'. A rubber band was used to fasten the Saran wrap to the funnel to keep it air-tight. The funnel was fitted into a suction flask which was connected to a water pump. This apparatus was placed 1 foot below a 60 watt lamp which provided heat for faster drying. It usually took 12-1$ hours to dry the gel slices completely.

The dried gels were arranged on blotting paper and autoradiographed. The autoradiograms were then cut to the 67

Figure $. Apparatus for slicing (A) and drying (B) gels in poly• acrylamide gel electrophoresis. 68

size of a microscope slide and scanned with a densitometer

(Joyce-Loebel Chromoscan) using 10 mm x 0.5'mm slit width with a 1:3 gear ratio of gel to chart.

For the determination of phenolic and protein con• tents of flax, the data presented in this thesis represent

the averages of triplicate experiments. There were however wide variations in the level of metabolic activity between

experiments. Therefore in this thesis the data presented for enzyme activity and phenylalanine incorporation were

from single representative experiments. 69 v.

' RESULTS

Section A: Phenolics of Flax and Flax Infected with Rust

I. The Identification of Phenolics in Koto Flax

In studying the phenolic compounds of flax, it was found that they migrated only slightly on the TLC plates developed with BzAW. In t-BAW, the phenolics in the total ethanol extract (TEE) were separated into two main groups

(Fig. 6). One group consists of 24 spots which showed blue fluorescence under UV light. The second group consisted of

8 spots which absorbed UV light and are therefore brown in color. By means of chromatographic characteristics (Table

IV) and UV absorption spectra (Table V) the blue fluorescent compounds were identified as esters and ethers of phenolic acids and those of the other group as derivatives of flavones

(Table IV and VI).

1. .Phenolic acid derivatives

(i) The basic phenolic acids of flax

No free phenolic acids were detected on the chromato• gram of TEE, even after concentration by ether extraction.

During stepwise hydrolysis, however, phenolic acids were re• leased. They were identified as C^-C^ phenolic acids: p- coumaric, caffeic, ferulic and sinapic acids (Figs. 8 & 9).

All of them showed cis-trans isomerism in 2% acetic acid but not in organic solvents such as BzAW or n- or t-BAW. By visual judgement of the fluorescence under UV light and color Figure 6. The UV fluorescent pictures of the TEE of (A) healthy and (B) rust infected (resistant) cotyledons. o 71

15% HOAc—>

Figure 7. The Identification of the Phenolic Moieties of Flax Phenolic Compounds. 1-24: derivatives of phenolic acids, p-coumaric acid (pc), caffeic acid Ic), ferulic acid (F) and sinapic acid (Si). F1-F8: derivatives of flavones, apigenin (A) and luteolin (L). TABLE IV. Chromatographic Properties of the Phenolic Compounds of Flax.

Rf Fluorescence Color Reaction tBAW 15%H0Ac UV UV with NH^ p-nitroaniline PNA +

(PNA) Na2C0^

Flavonoids Fl 9 19 brown light yellow yellow F2 11 27.5 brown light yellow yellow - F3 19 7 brown light yellow yellow yellow F4 21 35 brown yellow brown - yellow F5 24 47 brown yellow brown - yellow F6 33.5 13 brown yellow brown - yellow F7 37 22.5 brown yellow brown yellow orange FS 45 35 brown bright yellow yellow yellow

c6-c3 1 32.5 77 blue green yellow green 2 45 70 blue green yellow green orange yellow Phenolics 3 46 77.5 blue green yellow green orange yellow 4 45 $2.5 blue green yellow green orange yellow 5 53 5$ blue blue 6 55.5 75 blue green yellow green orange yellow 7 56 77.5 grey $ 56 82 blue green yellow green 9 57.5 86 blue green yellow green pink grey 10 59 65 blue green yellow green orange yellow 11 62 72.5 blue green yellow green orange yellow 12 62 $2.5 blue green yellow green 13 65 90 purple 14 65 67.5 blue green yellow green orange yellow 15 69 blue green yellow green orange yellow 16 $0 69 $7.5 blue green yellow green orange yellow 17 75 blue blue 18 32.5 75 47.5 blue blue 19 .5 blue green yellow green orange yellow 20 $2 65 83 77.5 blue green yellow green orange yellow 21 82.5 $5 blue green yellow green orange yellow 22 82.5 90 blue green yellow green orange yellow 23 87.5 90 purple purple oi 87.5 97 purple purple Figure 8. A Trace of a Chromatogram of Alkaline Hydrolysate (ether soluble fraction). Figure 9. Photographs of a chromatogram of alkaline hydrolysate (ether soluble fraction) of flax phenolic acids as (A) viewed under UV, (B) sprayed with p-nitroaniline

and (C) sprayed with p-nitroaniline and then 5% Na2C0o . (See Fig. 8 for the identification of these compounds'). 75 reactions the relative concentrations of these four phen• olic acids were found to be caffeic > p-coumaric > ferulic

s sinapic. No C^-C-^ phenolic acids were detected.

(ii) The phenolic acid derivatives

Since many of the compounds were present in small amounts and ran very close together on TLC, only some of them could be isolated as pure compounds. Nearly all these major compounds were esters. This was shown by the large bathochromic shift of the A. max. of the longer absorption band of most of these compounds when NaOH was added (Table

V). Further evidence came from stepwise hydrolysis. Most of the free phenolic acids were released by alkaline hy• drolysis, whereas acid hydrolysis released only a moderate amount of caffeic acid and a trace amount of ferulic acid.

No traces of p-coumaric or sinapic acid were detected.

The most easily identified compounds on the TLC were chlorogenic acid.and its isomer. Identification was based on Rf values and fluorescence under UV light and UV with ammonia. The other prominent compounds were derivatives of p-coumaric acid. These could not be detected under UV light but showed blue violet fluorescence under UV when exposed to ammonia. The major compounds were identified by isolation and study of UV absorption spectra and hydrolytic products. In order to identify the minor compounds and those running very close together on TLC, a method was devised TABLE V. Spectral Properties of Some Blue Fluorescent Compounds Isolated from Flax.

UV absorption maxima (mp,) Compounds EtOH NaOH Boric Acid

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

24 290-315 370 same as EtOH broad plateau

*Numbers within parenthesis indicating "shoulders". 77 by the present author. TEE vras banded on TLC plates and developed in t-BAW as well as in 15% HOAc separately. The compounds in the bands were isolated and extracted and hydrolyzed. The phenolic acids released were identified. The results were then tabulated in a checker board fashion. With the help of the knowledge of the positions of the major com• pounds which had been already identified, the individual spots of the chromatogram of the TEE were pin-pointed with respect to the phenolic acid moiety (Fig. 7). However, even with this method some spots could not be identified e.g. #5,

8 and 10.

2. Flavonoid derivatives

The flavonoid compounds of flax were found to consist of only two kinds of flavone nuclei, namely apigenin and luteolin. Compounds Fl, F2, F3 and F& are luteolin deriva• tives and the rest are apigenin derivatives. They were iden• tified by their characteristic spectra and chromatographic properties.

All the apigenin derivatives of flax have free 7- hydroxyl groups as indicated by the bathochromic shifts of the A. max. of the short UV absorption band when sodium acetate was added to the solutions of the compounds (Table

VI). In all apigenin-type compounds the 5-hydroxyl group was found to be bonded because there was no prominent bathochromic shift of the A. max of the long UV absorption band with AlCl^. As expected, these apigenin-type compounds TABLE VI. Spectral Properties of the Flavonoids of Flax.

UV absorption maxima(mp) Compounds EtOH NaOH NaOAC Boric Acid AlCl^

Fl 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 within parenthesis indicate "shoulders". 79 are not responsive to boric acid because there is no catechol

group (ortho-dihydroxyl) present.

On the other hand none of the luteolin derivatives of flax has a free 7-hydroxyl group and compounds Fl and

F2 have also their ^-hydroxyl groups bonded. The spectra of all these compounds showed a bathochromic shift in the long wavelength band with boric acid. This indicates that there 3,'4f-hydroxyl groups of these compounds are free

(Table V).

II. Phenolic Compounds and Rust Infection of Flax

The 80% ethanol soluble phenolic compounds of healthy flax and resistant and .susceptible combinations of rusted flax at 1, 2, 4 and 6 days after inoculation were studied by chromatography. The results presented in Figure 10 re• veal no significant differences were detected in the number of spots or their running properties on TLC. However, some quantitative changes in the total soluble phenolic content were found. For healthy cotyledons there was an increase in phenolic content as the cotyledons aged from 7 days on• ward (Fig. 10). By the 13th day after seed germination there was a drop of 12% when compared with the 11 day old cotyle• dons. As the cotyledons aged further and senesced the phenolic content increased again. For the infected tissue both the resistant and susceptible combinations showed an initial drop in phenolic content in the first day after in- 80

J , 1 1 1 1 1 1 1 I 2 3 4 5 6 7 8

Days after inoculation

Figure 10. The Total Phenolics of Healthy and Infected Flax Cotyledons. • © Healthy (H) 0....0 Resistant combination (R) x x Susceptible combination (S) Same symbols for following graphs unless specified. 81 oculation. In the case of the resistant, the phenolic con• tent rose to the level of the healthy control in the second day and thereafter remained above the control level. The susceptible remained lower than the control in the second day after inoculation and did not rise to the control level until the 4th day. Although the pattern of the curves for all three types of tissue is very similar, the level of phen• olic content in the susceptible combination was always lower than that in the resistant (Fig. 10).

Section 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 shikimic acid pathway the key step controlling the flow of the benzene ring into phenolic compounds or pro• tein is guarded by ammonia-lyases. In most dicotyledonous plants phenylalanine ammonia-lyase is the main enzyme responsible for this controlling point. However the deamination of tyrosine and DOPA could not be overlooked. Thus in the present investigation of the metabolism of phenolic com• pounds the three aromatic amino acids were administered to the healthy and rusted cotyledons and the metabolic products were studied by chromatography and autoradiography. 82

1. Tyrosine: Tyrosine was found to be metabolized into a number of compounds (Fig. 11). The amino fraction collec• ted from a cationic exchange resin Dowex 50W-X4 column con• tained unmetabolized radioactive tyrosine as well as 6 other compounds. All were ninhydrin positive. On hydrolysis of the TEE with both acid and alkali, no phenolic acids were detected. In one dimensional autoradiographs of the 'non- amino' compounds there were two major and several weakly labeled spots. Some of these spots were also ninhydrin posi• tive. Chromatography in the second dimension with 15% acetic acid showed that all these compounds moved very quickly, forming patches near the front. Again, no labeled phenolic acids were released by hydrolysis.

2. DO PA: 1/fC from DOPA- B -ll*C was not found in phenolic compounds. It was however metabolized into a number of nin• hydrin positive and other compounds which neither reacted with ninhydrin nor fluoresced. The ninhydrin negative pro• perty of some labeled spots may be due to low concentrations even though the specific activities were high enough to be detected by autoradiography (Fig. 11). Hydrolysis of the TEE showed no phenolic acids.

3. Phenylalanine: The amino fraction from phenylalanine- 14 U- C feeding showed the presence of only unmetabolized phenylalanine. This is different from tyrosine and DOPA metabolism, in which there were a number of amino-bearing 83

t-BAW B

Figure 11. The autoradiogram of some feeding experiments - A - 'phenolic fraction' of DOPA feeding B - 'amino acid fraction' of DOPA feeding C - 'amino acid fraction' of tyrosine feeding D - 'phenolic fraction' of tyrosine feeding E - 'amino acid fraction' of phenylalanine feeding. 84 compounds formed. This may be because there is no hydroxyl group on the benzene ring which is therefore not very reactive.

Phenylalanine on the other hand was metabolized into a number of phenolic derivatives (Fig. 12A). The labeling pattern of the autoradiogram of the TLC is similar to the pattern of blue fluorescent spots. The distribution of label between the different compounds was very uneven. Thus the exposure period necessary to reveal some of the weakly labeled spots resulted in the over-development of the others on the autoradiogram. Except for one luteolin-type compound, the other flavones were not labeled. The ether extract of

TEE showed only cinnamic acid and trace amounts of p-coumaric acid as labeled free phenolic acids. After hydrolysis p-coumaric acid was found to be more heavily labeled than caffeic and ferulic acids which were approximately equally labeled. No radioactivity was detected in sinapic acid

(Fig. 12B). There were no qualitative differences between the healthy, resistant and susceptible cotyledons as far as the metabolism of phenylalanines is concerned.

4. Cinnamic acid: p-Coumaric acid and its derivatives were the major labeled compounds when the cotyledons were fed with cinnamate-B-"^C (Fig. 13). The spots observed on the autoradiogram did not correspond to the fluorescent compounds on the chromatogram of the TEE. The flavonoids were not labelled. Therefore it seems that under the A 1$

Figure 12. Autoradiograms of TLC prepared from TEE of phenylalanine-U- C 00- fed cotyledons (A) and the ether extract of the alkaline VJ-> hydrolysate of the same TEE (B). 86 conditions of cinnamic acid feeding the metabolism vras not normal. This may be due to the high accumulation of free p-coumaric acid and its derivatives, which are not normally present in the flax cotyledons. The pattern of radioactive spots for the susceptible combination is similar to that of the healthy control. In the resistant combination there is one spot (Fig. 13C) which is more radioactive than the corres• ponding spot in the healthy and susceptible.

Chromatography of the ether extract of the hydroly• sate of TEE showed that by comparison with p-coumaric acid which was heavily labeled, caffeic and ferulic acids were only slightly labeled (Fig. 14).

II. Further Studies on the Metabolism of Phenylalanine-U-"^ C

1. Accumulation

The accumulation of metabolites around lesions after infection is a very common phenomenon. In the present in• vestigation radioactivity from the phenylalanine-U-^C fed to the flax cotyledons was found to accumulate around the lesions both in resistant and susceptible combinations (Fig.

15A). Since phenylalanine is a precursor for both protein and phenolic compounds it would be more meaningful If the nature of the compounds accumulated were known. In view of this point a protein inhibitor, cycloheximide, was added

1 LL to the phenylalanine-U- C solution for the feeding experi• ment. After feeding the cotyledons were fixed with boiling Figure 13. Autoradiograms of TLC prepared from TEE of

cinnamate-2 -HC fed cotyledons. A - Healthy, B - Susceptible and C - Resistant.

Figure 14. Autoradiogram of TLC prepared from ether exr tract of hydrolysate of TEE of cinnarnate-2- ^C fed cotyledons. 88

Figure 15. Autoradiogram of whole cotyledons fed with phenylalanine-U- (A) and phenylalanine- U-^C plus cycloheximide (B). H - healthy, R - resistant and S - susceptible. 89

95% ethanol to remove unmetabolized phenylalanine and the soluble phenolic compounds, leaving the proteins and in• soluble phenolic compounds in the tissue. The autoradiograms in Figure 1$B showed that the accumulation of radio• activity at infection sites in the susceptible combination was inhibited by cycloheximide. Thus the accumulation shown in Fig. 1$A is probably mainly due to incorporation of phenylalanine into protein. The resistant combination, how• ever, showed high accumulation at infection sites even in the presence of cycloheximide. This suggests that there was a much higher degree of incorporation of phenylalanine into insoluble phenolic materials than in the susceptible combin• ation .

2. The Metabolism of Phenylalanine-U-^C into Soluble Phenolics

(i) Incorporation: The incorporation of phenylalanine-

U-^C into soluble phenolic compounds is shown in Table VII.

The incorporation in the resistant tissue was always higher than in healthy tissue except at a very late stage after infection. At this time the cotyledons have started to senesce. Resistant tissue senesced earlier and faster than the healthy. This may account for the lower incorporation for resistant tissue at the 9th day after inoculation.

Furthermore the highest level of phenolic compounds in the resistant also affected the calculation of the specific activity. The peak of incorporation for the resistant TABLE VII. The Incorporation of Phenylalanine-U- C into Phenolic Compounds.

Days after Healthy Resistant Susceptible Inocula- Sp. Act. Sp. Act. % of Healthy Sp. Act. % of Healthy tion 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 tissue was 2 days after inoculation. In susceptible tissue, incorporation was also higher than in healthy at the early stage of infection, but dropped below the healthy control by the 2nd day after inoculation. A value of y $0% lower than the healthy control at the 9th day coincided with active sporulation of the rust and maximum expansion of the host cotyledons.

(ii) A comparison of the incorporation of ^^C into

mono- and di-hydroxy phenolics: As dihydroxy- phenolic compounds are more reactive chemically than mono- hydroxy phenolics the ratio of incorporation of the phenylalanine-U-^C into mono- and di-hydroxy phenols in healthy and infected tissues was studied. Alkaline hydrolysis of the TEE was followed by extraction of the phenolic acids with ether. The p-coumaric acid and caffeic acid, as repre• sentatives of mono- and di-hydroxy phenols respectively, were separated by TLC and the spots were marked under UV light. The spots were scraped and transferred quantitatively into vials and radioactivity was measured by liquid scintil• lation counting. Since only the ratio of cpm in caffeic acid: cpm in p-coumaric acid was under investigation, the steps from extraction until spotting were not quantified.

The observed counts are therefore of no significance. The results are shown in Table VIII. The resistant tissue always had a higher ratio than the susceptible especially by the 6th day after inoculation. This indicates that more mono-hydroxy 92 phenols were converted into di-hydroxy phenols in resistant than in susceptible tissues. The resistant tissue had the highest ratio at the 2nd day after inoculation and was also always higher than the healthy control.

TABLE VIII. The Metabolism of Phenylalanine-U-^C into Caffeic and p-Coumaric Acids.

Tissues Caffeic acid p-Coumaric Ratio caffeic acid (cpm) acid p-coumaric acid (cpm)

H - - 2 R 519 1$80 0.328 days S 423 1513 0.279

H 449 1741 O.258 4 R 374 1286 0.291 days S 725 2626 0.276

H 314 119.9 0.262 6 R 431 2140 0.293 days S 768 4320 0.177

3. The Metabolism of Phenylalanine-U- C into Protein

(i) Incorporation: In the healthy tissue (Table IX), the rate of phenylalanine incorporation dropped from the 8th day after seeding, when the cotyledons were mature. The reading on the 8th day probably represented the peak of incorporation for the cotyledons and thenceforth the in• corporation slowed down as the cotyledons aged and senesced. TABLE IX. The Incorporation of Phenylalanine-U- C into Protein.

Age of Days Healthy Resistant Susceptible Cotyledons after sp. act. sp. act. % of healthy sp. act. % of healthy (days) Inoculation (cpm/mg protein)

8 1 40255 49080 ' 121.9 53030 131.7 9 2 22990 23800 103.5 43000 187.0 11 4 25170 28332 112.2 31205 123.9 13 6 30398 26470 87.0 27757 91.3 16 9 22390 16319 72.8 18860 84.2 94

Infected tissues of both resistant and susceptible types showed a higher incorporation than the healthy control during the first 4 days after inoculation. This was more remarkable in the susceptible combination. On the second day after in• oculation the incorporation In the susceptible was as much as 87% higher than in the healthy control. The trend to a decrease in incorporation with increase in age observed in healthy tissue also occurred in the infected tissues. In the susceptible combination this decline was more gradual during the first four days after inoculation. 14 Contrary to the incorporation of phenylalanine-U- C into phenolic compounds, the susceptible combination always showed a higher incorporation of this amino acid into pro• tein than the resistant reacting tissue (Table IX).

(ii) The labeling pattern of proteins: In healthy tissues the labeling pattern of proteins changed as the cotyledons aged. In 8 day old cotyledons there was more labeling in those proteins which migrated only slowly in polyacrylamide gel electrophoresis. As the cotyledons aged, more of the fast moving protein became labeled and by the

16th day, these proteins were the most heavily labeled ones

(Figs. 16-18).

The labeling patterns were the same in healthy, resistant and susceptible tissues for the first 6 days after inoculation. The only difference observed between them was the relative degree of labeling in the various protein bands. Figure 16. Chromoscan Patterns of the Autoradiograms of Gels containing Labeled Protein from 1 Day Old Cotyledons.

vO Figure 17. Chromoscan Patterns of the Autoradiograms of Gels Containing Labeled Protein from 6 Day Old Cotyledons. Figure 18. Chromoscan Patterns of the Autoradiograms of Gels con• taining Labeled Protein for 9 Day Old Cotyledons. 98

An example of this is shown in Figure 16. For healthy- tissue the ratio of the radioactivity of peaks a and b is

1.0. For the susceptible combination this ratio is less than 1.0 whereas for the resistant combination it is greater than 1.0. By the 9th day after inoculation the patterns for the healthy and resistant combination were strikingly similar, but the pattern for the susceptible combination was noticeably different (Fig. 18).

Section C: Proteins and Enzymes I. Dowex Method

It was found that the phenolic compounds in flax interfered with the extraction and determination of protein and enzymes. Even with precipitation by trichloro-acetic acid the phenolic compounds could not be removed completely and the precipitated protein still had a yellow tinge and re-dissolved in buffer to give a yellowish solution. This phenolic contamination affects protein determination by either Warburg's (Warburg and Christian 1941) or Lowry's (Lowry et_ al. 195-1) method. The yellow pigment is probably formed by the breaking down of cell compartmentation. During extraction the phenolic compounds are therefore exposed to the action of oxidases with the formation of quinones and polymers which complex with protein. Once the complexes were formed the phenolic compounds could not be removed 99

even with repeated precipitation.

Insoluble polyvinylpyrolidone (PVP) has been em•

ployed as an insoluble adsorbant for clearing protein ex•

tracts by Loomis and Battaile (1966) and is now widely used for this purpose. It has, however, certain disadvantages.

It probably binds only those phenolic compounds of molecular weight greater than chlorogenic acid (MW 354). Moreover,

it inhibits some enzymes (Harel et_ al. 1964) and its

capacity for adsorbing phenolics is very low, necessita• ting its use in large amounts so that it is sometimes diffi•

cult to grind the tissue in a mortar.

Dowex 1-X8 anion exchange resin was used to remove

the phenolic compounds during extraction before formation

of the yellow complex. Peroxidase, polyphenol oxidase,

total protein and total phenolic content were determined using both PVP and Dowex resin extraction methods. The results in Table X show that the Dowex resin is an efficient adsorbant for phenolic compounds. Protein content was apparently the lowest in (c), but it must be emphasized that

the higher readings in (a) and (b) were at least partly due to interference caused by phenolic compounds.bound to the

protein. This view is supported by the observation that

the protein prepared from (b) and especially that from (a) had a yellowish color. It cannot therefore be concluded that (c) had the lowest protein content and it is clear

that removal of phenolic compounds was largely responsible TABLE X. A Comparison of the Different Methods for Protein Extraction.

PEROXIDASE A0D470/ A0D470/mg.. Extraction .2 ml Extract/min* protein/min.

(a) Buffer 0.600 1 .875 (b) Buffer + PVP 0.390 1 .814

POLYPHENOL OXIDASE PROTEIN PHENOLIC AOD330/0.2 AOD 330/mg • CONTENT CONTENT Extraction ml Extract/min* protein/min • (mg/ml) (ug/ml)

(a) Buffer 0.075 0.234 1.700 500

(b) Buffer + PVP 0.065 0.302 1.075 164 (c) Buffer + Dowex 0.103 0.528 0.975 . 0 -I'Crude extracts were prepared from 2 gm fresh wt. of flax cotyledons and made up to 12 ml with buffer. Activity per mg protein depends on protein estimations which are affected by the degree to which phenolics are complexed and retained with the protein in (a) and (b). 101

for increasing the apparent activity per mg. protein for

both enzymes. In addition, the activities of both peroxi•

dase and polyphenol oxidase expressed per unit volume of

extract were distinctly higher in (c) than in (a) or (b).

This may reflect the more complete removal of inhibitory

phenolic compounds In (c) and possibly also an inhibitory

effect of PVP.

The ability of Dowex resin to remove phenolic com• pounds was also studied by passing standard phenolics through a column of Dowex 1-X 8. Compounds such as simple phenolic acids fp-coumaric, caffeic, ferulic, sinapic and chloro• genic) and flavonoids (rutin, quercetin and flavone) were studied. Aliquots (2 ml in $0% ethanol) of (a) a known mixture of phenolics, (b) a fresh phenolic extract of flax and (c) an 'aged' extract of flax left on the bench for 2-3 weeks were each passed through a separate column of 6 x 1 cm Dowex which was equilibrated in 50% ethanol. Twenty ml effluent was collected and concentrated to 2 ml. The phenolic content of the original solutions and the effluents were estimated using chlorogenic acid as a standard. The results in Table XI show the retention power of the Dowex resin. It is not as efficient for the plant extract es• pecially the 'aged' extract. . This is probably due to some of the phenolic compounds in the plant extract having been denatured, polymerized or complexed with other plant chemicals during extraction. Exposure of the extract to 102 air would lead to more oxidation and complex formation.

TABLE XI. The Retention Capacity of Dowex-l-Xg for Standard and Plant Phenolic Compounds.

Phenolic Mixture Quercetin Old Extracts New Extract

Phenolics added 424.5 203 .0 57.5 42.5 Phenolics in effluent 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 resin required for satisfactory extraction of enzymes was studied by using 5 ml, 3 ml and 1 ml of 10% Dowex suspension plus the required amount of plain tris-glycine buffer to make a buffer: tissue ratio of about 5 ml to 1 gm fresh weight. The results shown in Table XII suggest that 5 ml -of 10% Dowex per gm tissue is the best for the extraction of peroxidase. Concentrations higher than 5 ml per gm tissue were not studied because in other experi• ments this 5:1 ratio was found to remove practically all the interfering phenolic compounds and the enzyme prepara• tion showed UV absorption spectrum closely similar to that of bovine serum albumen. TABLE XII. Peroxidase Activities in Enzyme Extracts prepared by using Different Quantities of Dowex 1X8.

Tris-glycine Tris-glycine Tris-glycine Tris-glycine Buffer pH 8.3 Buffer pH 7.6 Buffer pH 7.6 Buffer 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 protein 0.868 0.895 0.799 O.56I 104

II. Protein and Enzymes

1. Total protein content

The curves for total protein content vs. days after inoculation were similar for healthy and resistant and sus• ceptible reacting tissues (Fig. 19A). There was a peak of protein level at 2 days after inoculation (equivalent to 9 day old cotyledons). From the 4th day onward, the protein content of the healthy tissue levelled out, whereas that of the susceptible increased slightly and the resistant de• creased further. Repeated visual observations indicated that the cotyledons were fully extended by the 9th day after seeding. By the 13th day, the total protein level of the cotyledons was lower than on the 8th day, indicating that the metabolism of the cotyledons had slowed down and that senescence would soon follow. When compared with the healthy control (Fig. 19B) resistant cotyledons always had a lower protein content after inoculation but for susceptible just the opposite was true. The difference between the three kinds of tissue increased as the infections aged.

2. Phenylalanine ammonia-lyase (PAL)

The PAL activity was found to be very low in healthy cotyledons older than 8 days. The activity was hardly de• tectable by the 13th day. In the susceptible combination there was an early derepression in PAL activity which re• turned to normal by the 2nd day (Fig. 20). The activity was then maintained slightly above the healthy control but Figure 19. Total Protein Content and Infected Flax Cotyledons. Symbols are the•same as in Figure 10. 106

Days after inoculation

Figure 20. A Comparison of PAL Activities in Healthy and Rusted Flax Cotyledons. Symbols are the same as in Figure 10. 107

was still very low. In the resistant combination, there

was an increase ol more than 5-fold when compared with the

healthy control on the second day after inoculation. The

decline in activity on the 4th day was as abrupt as its

rapid build up earlier. By the 6th day after inoculation,

the activity in the resistant was not much higher than in

the susceptible and by the 8th day the activity in the

resistant combination was hardly detectable.

3. Peroxidase

There was a similar pattern for the specific ac•

tivity of peroxidase in healthy, susceptible and resistant

tissues when plotted against time after inoculation (Fig.

21). Unlike PAL, there was low activities for all three

kinds of tissues for the first two days. There was a grad•

ual increase in activity in the healthy cotyledons as they

aged. Both resistant and susceptible always had higher ac•

tivity than the healthy control with the resistant being

distinctly higher than the susceptible after the 4th day.

Three isozyme bands of peroxidase were detected

(Fig. 22A). There was no difference among the three kinds

of tissues. There was a guaiacol positive fraction of the

enzyme preparation located on top of the spacer gel which

did not move into the gel. This fraction was barely de•

tectable in the early stages after inoculation but by the

8th day it was quite intensely stained as judged by eye. It was also more intensely stained in the infected tissue than

in healthy tissue. 108

Figure 21. Comparative Graphs of Peroxidase in Healthy and Rusted Flax Cotyledons. Symbols are the same as in Figure 10. 109

Figure 22. The Isozyme Bands of A - Peroxidase and B - PPO, in Flax Cotyledons. 110

4. Polyphenol oxidase. Polyphenol oxidase in healthy cotyledons increased as the cotyledons aged (Fig. 23). After inoculation, the susceptible had a lower level of this enzyme than the healthy and instead of increasing further after the 4th day, like the healthy control, it declined. By the 6th day it was

30% below the healthy, control. On the other hand the en• zyme level in resistant tissue is higher than in the healthy control and there was a peak of enhancement on the 4th day when the activity was 27% higher than the healthy. The early senescence observed in the resistant cotyledons coin• cided with a very high value of PPO activity on the 8th day.

There were 7 isozyme bands detected for the flax

PPO (Fig. 22B). Most of them moved very slowly down the gel. There were no detectable differences between the healthy, resistant and susceptible.

5. S-Glucosidase

The activity of this enzyme also increased with the age of the cotyledons until the 8th day after inoculation, when the healthy and susceptible declined slightly. As with

PPO, the resistant tissue always had a higher level of

g-glucosidase than the healthy control. On the other hand the activity in the susceptible combination was always lower than in healthy tissue. Figure 23. Comparative Graphs of Polyphenol Oxidase in Healthy and Rusted Flax Cotyledons. Symbols J 60 i 1 5 1 1 1 r r— I 2 3 4 5 6 7 8 Days after inoculation Figure 24. Comparative Graphs of ft-Glucosidase in Healthy and Rusted Flax Cotyledons. Symbols are the same as in Figure 10. 113

III. Effect of Water Infiltration on Enzyme Activities Since an infiltration method was used to study the metabolism of radioactive precursors it is appropriate to examine the effect of water infiltration on the enzyme activities. Furthermore, it is possible that infiltration with water under reduced pressure causes some mechanical injury so that a study of the effect of water infiltration may provide some insight into host response to injury as compared with rust infection.

Since the present investigation centres on phenolic compounds the effects of a standard phenolic solution, chloro• genic acid and a hot aqueous extract of flax cotyledons were also studied. Another set of experiments was conducted using Actinomycin D, Actinomycin D plus chlorogenic acid and Actinomycin D plus flax plant extract as infiltrating solutions. The concentration of chlorogenic acid was 2 x 10"^M and the flax extract had an equivalent amount of phenolic material. The concentration of Actinomycin D was about 50 [ig per ml. The infiltrated cotyledons were trans• ferred to a petri dish and incubated in a growth chamber under $00 ft.-c. at 22°C for 12 hr. Excised cotyledons in• cubated on moist filter paper in a petri dish were used as controls. The results are shown in Figure 25.. Water infiltra• tion caused an increase of more than 150% in the activity of both PAL and peroxidase as compared to the control. The Control H2O Chlorogenic Flax Act. D Act. D Act. D acid Extract + + Chlorogenic Flax acid Extract

Figure 25. The Effect of Infiltration with Water and Solutions of other Compounds on Enzyme Activities: A, PAL and B, Peroxidase. 115 addition of chlorogenic acid had no further effect. On the other hand flax extracts caused a further increase of 150% in PAL and of about 60% in peroxidase in addition to the effect of plain water. Actinomycin D inhibited the increase in PAL and peroxidase activities caused by water infiltra• tion. Its enzyme inhibitory effect is more pronounced in the case of flax extract infiltration than the plain water infiltration. There was 67% inhibition of PAL and 80% in• hibition of peroxidase in the former but only 26% inhibition of PAL and 60% inhibition of peroxidase in the latter.

A study of the effect of water, flax extract and

Actinomycin D solutions vs. time of incubation after infil• tration showed that there was a lag phase of 2 hours for all three treatments (Fig. 26). For PAL an increase followed this lag phase in all treatments. After the 4th hour, the increase in PAL on the treatment with Actinomycin D declined.

The activity in the water treated cotyledons declined after the 6th hour and that in the flax extract treated cotyledons remained very high for 12 hours. These temporal studies also indicate that bacterial contamination is not responsi• ble for the observed increases in enzyme activity. If infiltration induced bacterial growth increases in enzyme- activities would be expected to be continuous throughout the period of incubation.

There was also a lag period of 2 hours for the per• oxidase activities (Fig. 26), which then increased rapidly, 116

Figure 26. Temporal Changes of Enzyme Activities (A, PAL and B, Peroxidase) after Infiltra• ted with o © water, o o flax extract and x -x Actinomycin D. 117 the rates of increase being initially comparable for both water and flax phenolic extract treatments. By the 8th hour a higher level of activity was clearly established in the phenolic treatment. The Actinomycin D treated tissue showed a low peroxidase activity at all times.

Preliminary evidence that the increase in enzyme activities is due to enzyme synthesis was provided by a feeding experiment. Phenylalanine-U-^C was fed by infil• tration to two sets of cotyledons in the presence and ab• sence of Actinomycin D. A 3rd set of cotyledons was water infiltrated and incubated in light in a growth chamber for

4 hours before being infiltrated with phenylalanine-U-~^C solution. All 3 sets of cotyledons were incubated in the radioactive solution for 4 hours. Table XIII shows that there was 27% less incorporation of phenylalanine-U-^^C into protein if Actinomycin D was added to the infiltra• tion solution. Incorporation into pre-infiltrated tissue was 31% higher than in tissue which was not pre-infiltrated.

In the second precipitation of the protein, by which more non-protein radioactive contaminants were removed, the differences between the treatments were even more prominent

(Table XIII) . 118

14 TABLE XIII. The Incorporation of Phenylalanine-U- C into Protein by Infiltration Method.

Pre-infiItrated 14c Phe-U- iy for 4 hr + Act. D Phe-U-^C Phe-U-^C

Sp. Act. cpm/mg 15452 21222 27880 protein First Precipitation % 72.8 100 131.3

Sp. Act.

cpm/mg 5351 8596 I452O . protein Second Precipitation % 62.2 100 169.0 119

DISCUSSION

Section I: Phenolic Compounds of Flax

Oxidation and lignification are generally considered

to be the major routes for conversion of phenolics into

disease resistant principles in plants. Quinones formed

by oxidation of phenolic compounds can inactivate enzymes

produced by plant pathogens (Byrde 1963, Patil and Dimond

1967). In order to make oxidation possible, the phenolic moieties of the compounds must have certain functional

groups such as free hydroxyl and catechol groupings for

the oxidative enzymes to act on.

In the present studies, the 24 spots of phenylpro-

panoid derivatives detected on TLC (Fig. 6) represent no

more than 14 compounds. Many of the spots on tBAW/acetic

acid two dimensional TLC were in pairs as cis- and trans-

isomers of the same compounds. Only one of the 12 major

spots of these C^-C-^ derivatives was found to have a free

carboxyl group (Table V). On the other hand 10 of these

12 spots showed o-dihydroxyl groups. For those compounds

showing only one spot the double bonds between the B-

and Y-carbon atoms must have been broken and addition

or condensation products were formed. Bakke and Klosterman

(1957) showed that in flax seed meal there is a lignandi-

glucoside (two ferulic glucoside molecules linked at the

6 -carbon). 120

Thus it was found that most of the C^-Q^ phenolic compounds in flax are caffeoyl and p-coumaroyl in nature.

These compounds are present mainly as esters rather than glycosides. Their hydroxyl groups are therefore free and they provide good substrates for oxidation.

The flavonoids of flax have the same pattern of hydroxyl groupings as the major C^-C^ derivatives. How• ever whether their hydroxyl groups could be oxidized or condense with other compounds is not known. ' Some fungi and bacteria can degrade flavonoids such as rutin to simpler phenolics and carbon monoxide. These phenolic fragments, if leached into the soil, would become bound as humic acid

(Harborne 196$). The problem of oxidation and condensa• tion of flavonoids.in the necrotic lesions is worth studying.

Clark et al. (1959), working with potato peel, found that a derivative of C^-G^ phenolic acid, chlorogenic acid, was readily combined with amino acids such as phenyl• alanine, tyrosine, tryptophane, methionine, valine and iso- leucine to form additional products. They suggested that the addition of amino acids to the reactive centres on the ben• zene ring after oxidation of the ortho phenol to a quinone could prevent polymerization of the quinone. They showed that the amino-chlorogenic acid addition product is highly toxic to the growth of Helminthosporium carbonum. The toxic compound is therefore produced by oxidation of a phenolic compound and subsequently complexed with amino 121

acids. They speculated that in the resistant combination

this process is very likely to occur.

All the building blocks of lignin such as p-coumaroyl, feruloyl and sinapoyl derivatives are present in flax. Many

of them have double bonds between the g- and y - carbon

intact (as shown by cis- and trans-isomerization). Rohringer

et al. (1967) and Fuchs and DeVries (1969) working on rusted wheat and Fusarium infected tomato plants respectively found that there was increased incorporation of "*"^C from shikimate- and quinate-U-^C into insoluble, non-proteinaceous materials and they suggested that there was enhanced lignification.

In the present studies most of the conditions were favourable for lignification in the resistant combination after inocula• tion, including the dehydropolymerization step (as oxida• tion is usually enhanced at the infection site) except that the conversion of cinnamic acid derivatives to their cinnamoyl alcohols-requires enzymatic reduction (Freudenberg and Neish 1968) which is not prevalent in the necrotic tissues. Therefore the present author prefers the theory that oxidation and oxidative polymerization of polyphenols predominate in the formation of the insoluble bound phen• olic compounds at the infection site rather than the theory that lignification is enhanced.

The total soluble phenolic content of the resistant combination decreased immediately after inoculation but by the 2nd day it was already higher than the healthy control 122

(Fig. 10). These results are similar to those found by

Allan (1967) with Bombay flax infected with race #3 of

M. lini .• The point at which the phenolic level in infected tissue overtakes the healthy controls was also on the 2nd day after inoculation in his studies. The present findings for the susceptible combination do, however, differ from

Allan's in that there was also an initial drop in phenolic content rather than the initial increase that he reported.

Such differences may be due to differences in the degree of susceptibility of Koto/#210 and Bison/#3. Shaw (1967) showed that even in a susceptible flax/rust combination there are aborted lesions. Abortion of the pathogen occurred mainly between 21+ and 1+8 hours after inoculation and the number of living infections was as low as 63% of the total number of infection centres per cotyledon. It is possible that aborted lesions may have a biochemistry like that of resistant lesions.

An interpretation of the changes in total soluble phenolic content after inoculation is that the phenolics are oxidized and polymerized to form the insoluble materials bound in the collapsed cells either to the cell wall or complexed with the protoplasts. Therefore there was an early drop in the phenolic content. Farkas et al. (1962) have also shown that the o-dihydric phenols decreased during lesion formation in virus infected plants showing local lesions. At about the same time, phenolic biosynthesis 123 was turned on and by the 2nd day the. phenolic content was higher than the healthy control (Fig. 10), This indicates that at this stage the synthetic rate is higher than the rate of removal from the soluble pool into bound phenolic compounds. The fact that the phenolic level in the sus• ceptible combination did. not reach the level in healthy controls until the 4th day after inoculation (Fig. 10) indicates a slower synthesis or accumulation in the sus• ceptible as compared to the resistant combination.

To conclude this section, the following points are noteworthy in relating phenolics and resistance:

(1) The phenolic constituents of flax are favour• able substrates for oxidation and lignification.

(2) The quantitative changes in phenolic content after infection provide evidence to support the involvement of phenolics in resistance.

Section II: Metabolism of Phenolic Precursors

1. Metabolic Pathway

Tyrosine can be metabolized into phenolics under the action of tyrosine ammonia-lyase which converts it to

p-coumaric acid. The latter is then metabolized to other

phenolic compounds. However, by means of tracer studies,

no conversion of tyrosine to p-coumaric acid was detected

in the present work and therefore TAL is probably absent 124

14 in flax cotyledons. The metabolism, of tyrosine-U- C was quite similar to that of DOPA. Possibly some of the tyro• sine was converted to DOPA and then metabolized into a group of amino-bearing compounds.

Young and Neish (1966) were able to show deamination of DOPA by acetone powders of wheat shoot but not by

Pteridium. Even though they claim that there was deamina• tion (based on the radioactive counts of the ether extractable material from the enzyme assay solution), they were unable to identify the product. If the enzyme simply cleaves NH^ from the phenolic moiety the product should be caffeic acid. In the present investigation no radio• active caffeic acid was detected. There is therefore no evidence for a DOPA-ammonia lyase in flax cotyledons.

Phenylalanine is the precursor of the C^-C^ units of the phenolic compounds of flax. This is shown by the similarity of the autoradiogram of the phenolic extract 14 after phenylalanine-U- C feeding (Fig. 12A) and the fluorescent spots on the TLC (Fig. 6). Furthermore, on hydrolysis, the cinnamic acids commonly found as their derivatives in flax such as p-coumaric, caffeic and ferulic acids were radioactive. The metabolic pathway, therefore, is probably consistent with that found by McCalla and

Neish (1959):

Phenylalanine > cinnamic acid > p-coumaric acid

sinapic acid ^ ferulic acid 4 caffeic acid 4-- 125

14 The feeding of cinnamate- B- C, the second member in this pathway should therefore give a labeling pattern similar to that obtained by feeding phenylalanine. However, this is not the case when cinnamate-B -~^C was fed to the cotyledons (Fig. 13). There was an accumulation of free p-coumaric acid and of some p-coumaryl derivatives. No free p-coumaric acid was formed when phenylalanine was fed.

In addition, the labeling in caffeoyl and feruloyl deriva- 14 tives is very low when cinnamate-B - C was fed. Apparent• ly the metabolism of cinnamate- B -~^C did not follow the usual metabolic pathway in flax cotyledons. This may be accounted for by the hypothetical scheme proposed by

Zucker et al. (1967). In their scheme they suggested that

cinnamic acid inhibits the conversion of p-coumaric acid into caffeic acid. This was indicated by the fact that addition of cinnamic acid to potato tuber discs would in• hibit .the synthesis of chlorogenic acid, with a concomitant accumulation of 3-O-p-coumaroyl quinic acid and other p-coumaroyl conjugates as well as free p-coumaric acid.

The present findings seem to fit into this scheme very well.

The conversion of o-diphenolic compounds such as

caffeic acid to O-methyiated derivatives is catalyzed by

O-methyltransferase. In the present experiments only ferulic acid was found to be labeled when both phenylalanine-

U-^^C and cinnamate- B. -"^C were used as precursors whereas 126 sinapic acid was not. Either the hydroxylation and methy• lation of the 5-position of the benzene ring did not occur under the present experimental conditions or the product, sinapic acid, was formed in very low amounts and it complexed with the insoluble fraction as soon as it was pro• duced.

2. Accumulation

Accumulation of metabolites and other compounds in. diseased areas of leaves was a favourite subject of in• vestigation by plant pathologists during the 1950's.

Amongst the various metabolites, carbohydrates were fre• quently observed to accumulate in the host tissue surround• ing the parasitic colonies. (Yarwood. and Jacobson 1955, Shaw and Samborski 1956 and Tanaka and Akai I960). The accumu• lation of phenolic material around disease lesions has, however, received little attention. In this thesis, with 14 the help of phenylalanine-U- C tracer, accumulation of radioactivity was found in both susceptible and resistant . combinations. By means of a protein inhibitor, cycloheximide, it was shown that the material that accumulated around the resistant lesions is. different -in nature from that around susceptible lesions (Fig. 15).

The resistant lesions accumulated labeling in treat• ments with or without cycloheximide. This indicates that some of the accumulated compounds were not proteins. They., could be: - 127

(1) lignin

(2) polymers of oxidized phenolics

(3) oxidized simple phenolics complexed with

protein or cell wall material and therefore

not removed by hot ethanol.

The accumulation of insoluble phenolic compounds around the lesions in resistant tissue is supporting evidence for one of the following theories of resistance:

(1) the resistant reaction is due to the formation of toxic oxidized phenolics (i.e. quinones) which precipi• tate and inactive exogenous enzymes of the pathogen by complexing with them as well as killing the host cell and hence depriving the pathogen of nutrient.

(2) deposition of phenolic polymers or lignification may result in the formation of physical barriers that contain the pathogen.

(3) a combination of the two.

Rohringer et al. (1967), in studying rust-infected primary leaves of wheat, found that resistant leaves in- 14 corporated more radioactivity from shikimate-U- C and quinate-U-^C into the non-hydrolyzable, insoluble residue than the susceptible leaves. They suggested that some of the insoluble ester fractions are intermediates in the syn• thesis of wheat leaf lignin. Further work from this group on the metabolism of phenylalanine-U-^^C (Fuchs et al.

1967) also showed that the proportion in insoluble esters 128 increased more markedly in resistant leaves than in sus• ceptible leaves.

For the susceptible combination, incubation with phenylalanine without cycloheximide result in the accumula• tion of radioactivity at the lesions. In the presence of cycloheximide only a few isolated lesions accumulated small quantities of labeling (Fig. 15). This suggests that, in the susceptible, the major material accumulated is proteinaceous. In the cycloheximide treated cotyledons most of the lesions showed a halo with even lower radioactivity than the surrounding tissues. This may be because the area occupied by the fungus has an even lower ability to convert soluble phenolics into insoluble ones than the surrounding healthy tissue. The few lesions showing accumulation may represent aborted infection sites.

3. Incorporation

fa) Phenolics" vs. protein

The rate of incorporation of phenylalanine-U-^C in• to phenolics indicates that there was an enhanced synthesis of phenolic compounds immediately after infection in both the resistant and susceptible combinations (Table VII). The low total soluble phenolics at this early stage after in• oculation therefore means that initially the rate of binding of phenolic compounds is faster than the rate of synthesis, leaving a deficit in the total soluble phenolic content.

This probably signifies a stage of nonspecific response of 129 the host to the presence of a foreign organism in the plant tissue. On the 2nd day after inoculation the incorporation in the resistant reached its peak. At this stage the rate of synthesis was already higher than the rate of consump• tion (binding) and the total soluble phenolic content was therefore higher than in the control. This is likely to be a continuation of the process of rejecting a foreign organism. The susceptible, however, showed a drop in the incorporation rate on the 2nd day. At this stage the host- parasite relationship would have entered a compatible TphaseT and the metabolism of the host was no longer hostile to the pathogen.'

The effect of infection on the incorporation of 14 phenylalanine-U- C into protein (Fig. IX) is the reverse of that on its incorporation into phenolic compounds. The protein content of the tissues under study follows the order: infected susceptible ^ healthy ^ infected resistant.

It is likely that in the infected susceptible cotyledons a major portion of the phenylalanine is incorporated into protein. In infected resistant cotyledons a higher propor• tion of the phenylalanine is diverted to the production of phenolics. These findings are in agreement with those of

Fuchs et al. (1967) .

The change of the labeling pattern of the protein bands in gel electrophoresis as the cotyledons aged (Fig. 16 and 17) suggests a change of enzyme constituents in favour 130 of degradative enzymes as the cotyledons senesce. At the late stage of infection the protein bands of the susceptible tissue are distinctly different from those in the healthy and resistant tissue (Fig. 18). This is in part due to the presence of a high proportion of fungal proteins which have different migratory properties in gel electrophoresis.

(b) Monohydric vs. dihydric phenols

Cruickshank and Swain (1956) studied the chlorogenic acid content of a number of flax varieties and found that the resistant ones had a higher ratio of chlorogenic acid to total phenolic content. Simons and Ross (1970) found that ortho-dihydric phenols decreased more rapidly during lesion formation in tobacco leaves resistant to TMV than in sus• ceptible ones. In the present studies on the metabolism of phenylalanine-U-^C, there was a higher conversion of mono• hydric phenol into dihydric phenol in the resistant coty• ledons- than in the-healthy and susceptible tissues (Table

VIII). The resistant tissue would therefore have a larger supply of substrate for oxidation and for complexing with protein.

On the basis of data showing an initial low level of dihydric phenols in the resistant combination and a high total soluble phenolic content between 2-1+ days after in• oculation, Allan (1967) suggested that the low dihydric phenol content would favour high activity of IAA oxidase and a low functional auxin content. Low auxin levels in 131 turn would favour the destruction of RNA and induce early- senescence in the resistant variety. But he also showed that senescence occurred between 7 and 8 days after inocu• lation and that the dihydric phenol content built up rapid• ly between the 4th and 6th days after inoculation. These two latter findings do not fit very well into the theory that dihydric phenols are responsible for early senescence via an effect on auxin level. In addition the early low dihydric phenol level that Allan observed after inoculation in his resistant combination was only in the extractable

soluble phenolic fraction. The low level of dihydric phenols may have been due to the fact that dihydric phenols are the major compounds that are oxidized and bound to the insoluble residue as shown in the present results (Fig. 1$).

The early senescence of resistant cotyledons may not have any relation with the dihydric phenol level but may be due to other mechanisms which lead to the increased production of degradative enzymes such as peroxidase, polyphenol oxi• dase, B-glucosidase and IAA oxidase.

Several points discussed above are relevant to our understanding of host-parasite relations:

(1) In the resistant combination there was an accumu•

lation of insoluble phenolic compounds around the lesions whereas the susceptible accumulated protein.

(2) Incorporation experiments also showed that more

phenylalanine-U-^C was metabolized into phenolic compounds 132 in the resistant whereas in the susceptible there was more phenylalanine incorporated into protein.

(3) The resistant showed a higher degree of incor• poration of phenylalanine-U-^C into dihydric phenols than the susceptible.

Section III: Enzymes

1. Phenylalanine ammonia-lyase

In order to understand the change in PAL after in• fection and its significance in the metabolism of resistant and susceptible tissues we should also examine the changes in phenylalanine level, since phenylalanine is the substrate for this enzyme. Although the author did not measure the level or synthesis of phenylalanine in flax, some results put forth by other workers are worth discussing in relation to the present results. Pegg and Sequeira (1968), working on tobacco tissue infected by Pseudomonas solanacearum, found pronounced increases in the amount of phenylalanine and tryptophan within 24 hours of inoculation. Since concen• trations of most of the other amino acids decreased during this period, it appears that an increase in the concentra• tion of the aromatic amino acids was not the result of protein breakdown but was due to specific synthesis of these com• pounds. They also found that there was a marked increase in

PAL and enhanced synthesis of scopoletin. Earlier work by

Shaw and Colotelo (1961) showed that aromatic amino acids 133 were increased in both resistant and susceptible combina• tions of rust-infected wheat. In the protein hydrolysate there was a higher phenylalanine level for the susceptible tissue when compared with the healthy control. On the other hand the level for the resistant is lower and the resistant/healthy ratio is about 0.55 at 2 days after in• oculation. Furthermore Rohringer et_ al. (1967) demonstra• ted that in rust-infected wheat leaves there was an in- • creased carbon flow from CO to shikimate and quinate.

This phenomenon was more pronounced in the susceptible in• teraction than the resistant one. These findings demonstrate conclusively that there is enhancement in the production of phenylalanine after infection, especially in the susceptible tissue. These results together with the present findings

(Tables VII and IX) also suggest that most of the phenyl• alanine is incorporated into protein in the susceptible whereas there is a strong flow of aromatic amino acids to phenolic compounds in the resistant combinations. This latter process might have affected the protein synthesis in favour of those having low7 aromatic amino acid constituents.

This would be an interesting problem for further research.

Since susceptible tissue has an even higher free phenylalanine pool than the resistant phenylalanine is pro• bably not a factor in inducing the PAL enhancement in the resistant tissues. The initial low level of phenolics 134 would not be a factor either because this occurs in both resistant and susceptible interactions. However, the triggering mechanism for enhancement of PAL activity has yet to be discovered.

Hadwiger at al. (1969) reported that the activity of

PAL in Bison (susceptible) and Cass M-3 (resistant) was not significantly altered after inoculation with race #1 of M. lini. However their experiment was done on 14-day-old seedlings treated with a spore suspension and incubated for

24 hours. The difference between these results and the pre• sent findings (Fig. 20) may be due to the difference in ex• perimental design. They only studied the PAL activities

24 hours after inoculation and the present findings indi• cate that the rapid build up of activity occurred between 24 and 4$ hours after inoculation. It was shown by Chakravorty and Shaw (1971) that a significant increase in the rate of

RNA synthesis occurred between 24 to 4$ hours after inocula• tion of flax with M. lini. If PAL synthesis per se is res• ponsible for the enhanced activity, it should not occur before the production of RNA.

Towers and co-workers have found that many wood destroying basidiomycetes produce PAL. These fungi are

Collybia velutipes, Lentinus, Lepideus, Polyporus, Trametes and Ganoderma lucidum (Power at al. 1965) and Schizophyllum commune (Moore and Towers 1967). However, Jackson et al.

(1970) could not detect PAL in wheat rust uredospores. They 135 showed that radioactivity was not present in free phenolic acids when uredospores were fed with phenylalanine-U-^C.

In the present work the low PAL activity in the susceptible tissue, especially at a late stage of infection when there would be a significant amount of fungal material in the host tissue, also supports the idea that the rust fungus does not produce PAL.

2. Peroxidase

Novacky and Wheeler (1970) found that victorin from

Helminthosporium victoriae induced quantitative changes in peroxidase in susceptible oat leaves and a higher concentra• tion of victorin was required for similar alterations in resistant leaves. Since they found that the enhancement of peroxidase in the susceptible is more sensitive to victorin than in the resistant, they suggested that peroxidase acti• vity is not related to resistance. However the host-parasite combination they studied is a necrotrophic type and the virulent pathogen has to produce degradative enzymes to turn the susceptible host tissue into a necrotic lesion on which the pathogen can feed. Therefore the production of a high level of peroxidase in such interaction is to be expected and bears no significance in disease resistance.

On the other hand the biotrophs which feed on living cells generally cause a larger enhancement of peroxidase in resistant hosts than the susceptible ones. In this case only the resistant interactions show necrotic lesions. - 136

Lovrekovich et al. (1968) showed that injection of heat- killed cells of Pseudomonas tabaci or cell free extracts of bacteria into tobacco leaves increased peroxidase activity.

They also showed that injecting a solution of commercial peroxidase into leaves resulted in increased resistance to the disease. Macko et al. (196$) showed a marked increase in peroxidase activity in rust-infected resistant wheat at about the time of sporulation, followed by a decline. Sus• ceptible wheat showed only a small increase in activity at

24 hours after inoculation. Other observations on increased peroxidase activity in resistant interactions include those of Rudolph and Stahmann (1964) on bean leaves infected with

Pseudomonas phaseolicola and Simmons and Ross (1970) on local lesions on tobacco caused by TMV. The latter authors found that a marked increase of peroxidase was detectable only in the necrogenic phase of the disease. They suggested a direct role for peroxidase in the localization of TMV".

The increase of peroxidase in the resistant flax- flax rust interaction (Fig. 21) is consistent with the above data for biotrophic pathogens. In other words, infection with avirulent biotroph is accompanied with an enhancement1 of peroxidase in the host tissue thus resulting in a necrotic lesion and abortion of the parasite.

As already discussed in the literature review, peroxidase can catalyze the oxidation and hydroxylation of phenolics as well as the oxidation of protein and amino 137 acids. These reactions would lead to products Inactivating enzymes and precipitating proteins. The result is generally a necrotic lesion. For the necrotrophs this would happen for both the susceptible and resistant interaction whereas this occurs only in the resistant reaction for the biotrophs. In the susceptible host-biotroph combination, there would be either no enhancement of peroxidase or the enhancement is required for functions other than oxidation and necrosis is not an initial feature of the reaction.

3. Polyphenol Oxidase

Deverall (1961) found that Botrytis cinerea, the causal fungus for the chocolate spot disease of bean plants, produced pectic enzymes which liberate galacturonic acid and polygalacturonic derivatives from the cell walls. These compounds unmasked the latent polyphenol oxidase. Some• times it is the fungal metabolite that activates the en• zyme e.g., ophiobolin, a toxin produced by Cochliobolus was reported to be able to activate polyphenol oxidase of rice leaves (Nakamura and Oku i960). Zucker et al. (196$) suggested that cinnamic, p-coumaric and ferulic acid would inhibit the oxidase site of polyphenol oxidase. The higher conversion of monohydric phenol to dihydric phenol would therefore reduce the inhibitory effect. In the flax- rust combination the enhancement of PPO (Fig. 23) in the resistant interaction is probably due to this mechanism. The conversion of monohydric to dihydric phenols is highest 13$

in the resistant combination and would enhance PPO which

then oxidizes the o-dihydric phenols. The result would be

enhanced PPO activity, but not a high level of dihydric

phenol. After the 4th day the pathogen is already in•

activated or contained and some host cells are also dead.

Consequently the PPO in the lesion may be inactivated

gradually and an accumulation of o-diphenols as observed

by Allan (1967) would be expected. These results support

the suggestion of Hyodo and Uritani (1966) who worked with

sliced sweet potato tissue and proposed the idea that in•

creased polyphenol content might be involved in the enhance• ment of PPO activity.

The lower activity of PPO found in the susceptible

combination than in healthy tissue may be. of significance

in preventing the oxidation of phenols thus preventing the

formation of necrotic lesions. Therefore the inhibition of

this enzyme may be essential for susceptibility.

As with peroxidase, those virus-host systems showing

necrotic local lesions also exhibit enhanced PPO activity

(Farkas, Kiraly and Solymosy I960). Van Kammen and Brouwer

(1964) have demonstrated that in the local lesions of Nico-

tiana tabacum caused by TMV there is an increase in poly•

phenol oxidase. The increase was not restricted to the

inoculated parts of the leaves where virus multiplication

occurred and local lesions developed, but was also found

in the uninoculated parts. 139

4. 3-Glucosidase

The findings presented in this dissertation (Fig.

24) are quite similar to those reported by Allan (1967).

He found that in rusted Bison cotyledons (susceptible) the

3-glucosidase activity was below the healthy control throughout the entire process of rust infection. For the resistant Bombay race #3 combination, the enzyme activity always remained above the control level. However in Sec• tion A (Table V) it can be seen that most of the soluble phenolics of flax are esters and their hydroxyl groups are free. Therefore the significance of the enhancement of

3 -glucosidase is not very clear. There are two ways in which this enhancement may be of advantage to the host:

(a) one of the few phenolic glycosides or a non-phenolic glycoside may be the main resistance factors and therefore enhancement of 3-glucosidase is essential for resistance, b) the enhancement is part of the general increase of the degradative and oxidative enzymes resulting from the primary

(unknown)- resistant reaction. In the susceptible combina• tion this enzyme is suppressed and this is a safeguard against any possibility of the production of any resistant principles such as the aglycone, 2,4-dihydroxy-7-methoxy

1,4 benzoxazin-3-one, released in wheat leaves infected with wheat rust (El Naghy and Shaw 1966). 5. Sequential Changes

The temporal changes in the phenolics and enzymes after infection suggest a sequential synthesis or activation of PAL, total soluble phenolics and the oxidative and degradative enzymes. Initially after infection, there is a lag phase of one to two days for all enzymes studied. PAL was enhanced first on the 2nd day after inoculation. The build up in phenolic content was very fast between the 2nd and 4th day. The oxidative enzymes showed two days of lag and high activities were detectable- by the 4th day after inoculation.

Hyodo and Uritani (1966) found that polyphenol oxi• dase was enhanced by the. 40th hour after the sweet potato tissue was sliced and the peak of enzyme activity occurred between 80 and 100 hours. On the other hand a 4-fold in• crease of polyphenols was found by the 20th hour and the peak at .the 40th hour. Minamikawa and Uritani (1965) showed that PAL was increased by the 12th hour and reached a plateau of high activity between 12-36 hours. These data also implied the same sequential changes as those found by me in flax. The infiltration injury of the cotyledons also increased PAL more than peroxidase at an early stage after infiltration. Peroxidase always took a longer time to show the same percentage of enhancement when compared, with PAL.

These results suggest that the oxidative enzymes 141 may be enhanced by the phenolics produced as a result of the high PAL activity. However, this is speculation at this stage of our knowledge.

To conclude this section, the following points are noteworthy:

(1) PAL was enhanced in the resistant combination but not appreciably in the susceptible one. This is evi• dence for the enhanced synthesis of phenolic compounds in the resistant reacting tissue.

(2) PPO and peroxidase were also enhanced in the resistant combination. It is possible that oxidation of phenolic compounds was also enhanced as these enzymes are generally considered to be responsible for the oxidation of phenolic compounds.

(3) The suppression of PPO and g-glucosidase in the susceptible combination may play an important role in allow• ing the pathogen to proliferate in the host tissue.

(4) There is probably a sequential induction of PAL, phenolic content and PPO in the resistant reacting tissue. 142

Section IV: General Discussion

With the evidence obtained from three different types of experiments, namely, phytochemical study of phen• olic constituents, tracer studies and enzyme assays

(Sections I-III), it can be concluded that the resistance and susceptibility of flax to flax rust are related to the metabolism of phenolic compounds. Even though no phenolic phytoalexin was detected after infection, the characters of the phenolics in flax provide a very good basis for resistance to be built on this group of compounds. With the enhanced activities of peroxidase and polyphenol oxi• dase and possibly the breakdown of cellular compartmentation, these phenolics can easily be converted into resistant principles such as enzyme inhibiting quinones and polymers of phenolics and quinones.

Tracer studies provide good evidence to show that in the resistant there was high conversion of soluble phen• olics into 80% ethanol-insoluble ones which would either be polymers or simple phenolics bound to the cell wall or protoplast. The incorporation experiments also support the idea that phenolics are involved in resistance.

The triggering mechanism for the first step in the series of enhancements of PAL, total phenolic content, and degradative enzymes remains unknown. From the water infil• tration experiments it is obvious that injury could lead to the enhancement of PAL. The higher enhancement effect for 143 the infiltration with phenolic extract over the water infil• tration indicates that some compounds in the extract would cause enhancement. It is possible that mechanical injury or fungal infection may cause a breakdown of cellular compartmentation and that 'leakage' may bring phenolic compounds or other substances into contact with the protein synthe•

sizing machinery of the host cells. A part of the effect may also be at the level of the cell nucleus, because actin• omycin D, as inhibitor of RNA polymerase prevents the en• hancement effect of infiltration with water or flax phenolic

extract.

Susceptibility seems to be mainly due to the suppres• sion of the production- of phenolics and especially of oxida• tive enzymes. The channeling of a large quantity of aromatic amino acid into protein synthesis therefore serves two pur• poses: a) it reduces the amount of substrate for PAL and b) it allows more protein to be used for the benefit of the pathogen. The mechanism for the suppression of the oxi• dative enzymes is unknown but probably the compatibility between the haustoria and the membrane systems of the host cells plays an important role.

Based on the above discussion I would like to pro• pose a model for the resistant and susceptible reactions:

After infection, the avirulent rust produces the normal infection structures and attacks some cells. It is probably the haustoria of the avirulent rust that trigger changes in 144 the host cells such as membrane permeability. These would lead to the mixing of the phenolics and the oxidative enzymes and the formation of quinones and phenolic polymers. Con• sequently the haustoria would be inactivated. The fungus would then produce more haustoria and affect other cells but more of them would be inactivated until finally the pathogen would be starved of substrate for further growth.

Finally due to the deposition of more phenolic polymers the pathogen would be fixed and killed.

The number of cells collapsed and the area being affected would probably depend on the rate and degree to which the haustoria are inactivated and this would in turn be an exhibition of the degree of incompatibility. Thus for Bombay flax and race #3 of M. lini the interaction is quite drastic leading to a hypersensitive reaction with microscopic flecks consisting of only a few collapsed cells

(Allan 1967). For Koto and race #3 I found that the brown flecks were larger. This is probably because the reaction is not as violent so that the haustoria can function at least partially, and the fungus therefore affects more

cells before it becomes exhausted. Nevertheless, in any resistant combination, the point at which the pathogen be•

comes exhausted and fixed must occur before sporulation.

In susceptible tissue the inactivation of haustoria is probably a slow process. Thus the fungus can ramify ex• tensively and take control of the metabolism of the host 145 cells. The slowing down of the oxidation of the phenolic compounds is probably essential for the fungus to obtain nutrition from the host cells.

To conclude, this thesis deals only with the phyto• chemical and biochemical changes of phenolic metabolism in healthy and rust-infected flax with respect to resistance and susceptibility. I prefer the idea that phenolic com• pounds are agents executing the actual process of resis• tance. The results presented suggest that if the phenolic compounds can be maintained in their normal 'states', as in the healthy tissue, the pathogen would be able to survive.

It is still not known what triggers the biochemical change in phenolic metabolism in the resistant reacting tissue or how these changes are suppressed in the susceptible combin• ation. Answers to these problems are most likely to be obtained from studies on the membrane biochemistry of the host cell and the haustoria, and changes in nucleic acid metabolism. 146

SUMMARY AND CONCLUSION

1. Eight flavonoids and 14 esters and glycosides of phen•

olic acids were found in Koto flax cotyledons. The

flavonoids are of two major types, namely, apigenin

and luteolin and they are present as glycosides. The

phenolic esters and glycosides are the derivatives of

p-coumaric, caffeic, ferulic and sinapic acids. Chloro•

genic acids and esters of p-coumaric and caffeic acids

are the major compounds. No benzoic acids or their

derivatives or anthocyanins were detected in the coty•

ledons.

2. There were no new phenolic compounds i.e., phenolic

phytoalexins, detected in either the infected sus•

ceptible or infected resistant flax cotyledons as a

result of rust infection.

3. Total phenolic contents of healthy cotyledons increased

gradually with age between one to two weeks after seeding.

Infection with rust caused an initial decline in total

phenolic content but later rose above the healthy control.

This occurred on the 2nd day for the resistant reacting

tissue and the 4th day for the susceptible. The resis•

tant combination maintained the highest phenolic content

from the 2nd day onward.

4. Phenolic metabolism in flax cotyledons probably follows

the following route which has been suggested by McCalla

and Neish (1959): 147

phenylalanine ^cinnamic acid ^-p-coumaric acid

ferulic acidf caffeic acid£ ^

This was shown by feeding experiments. Tyrosine-U-^C

and DOPA- $-~^C were metabolized only into some ninhydrin

positive compounds. Cinnamate-3 -"^C was found to inhi•

bit the metabolic steps beyond the formation of p-coumaric

acid.

5. When flax cotyledons were fed with phenylalanine-U-^C,

the labeling was found to accumulate in lesions of both

the resistant and susceptible combinations. If the feed•

ing was accompanied by treatment with cycloheximide the

accumulation was found to be higher in lesions of the

resistant combination than the susceptible one.

6. In phenolic metabolism, phenylalanine-U-^C was mainly

metabolized into a number of derivatives of p-coumaric,

caffeic and ferulic acids. Very little labeling was found

in the flavonoids. There were no qualitative differences

in phenylalanine-U-^C metabolism between H, R and S

Tissues.

7. Quantitative studies on the metabolism of phenylalanine- 14 U- C showed that incorporation into phenolic compounds

is highest in the resistant reacting tissue. On the

other hand, the incorporation into protein showed a re•

verse trend and the susceptible combination showed the

highest incorporation. 8. In the resistant reacting tissue there was a higher

conversion of monohydric to dihydric phenol than in the

healthy tissue or the susceptible combination.

9. Gel electrophoresis of phenylalanine-U-"^ C labeled pro•

tein showed that there was a change in labeling patterns

as the flax cotyledons aged. The R and S showed slight

deviation from H at an early stage after inoculation.

By the 9th day after inoculation the patterns of the H

and R were very similar whereas S was distinctly

different.

10. The anion exchange resin, Dowex 1X8, was found to be

capable of binding the phenolic compounds from flax as

well as standard phenolic compounds. This finding has

been applied to remove phenolic materials during enzyme

preparation.

11. PAL activity was found to be very low in full-grown

healthy flax cotyledons. However, 2 days after inocula•

tion, the resistant combination showed an increase as

much as 5-fold over that of the healthy control. By the

6th day after inoculation, the enzyme activity dropped

back to a low level. There was no remarkable enhance•

ment of PAL in the susceptible combination.

12. There was a gradual increase in peroxidase activity as

the cotyledons aged. For the first 2 days after inocula•

tion, the peroxidase activities of both resistant and

susceptible combinations were comparable to the activity 149 in healthy control. After the 4th day, there was a clear trend of R ) S ) H. This difference in activi• ties increased with time and, by the 8th day, R was nearly 2-fold of H and S was 50% higher than the same healthy control. Gel electrophoresis showed 3 'iso• zyme' bands for the acidic protein fraction of the peroxidase. The overall patterns were the same for

H, R and S.

The activity of PPO in susceptible cotyledons was always lower than that of the healthy tissue. The enzyme ac• tivity of resistant reacting tissues was similar to that of the healthy control for the first two days but in• creased rapidly by the 4th day after inoculation to about 30% above the control. Seven PPO 'isozyme' bands were detected. No differences were found in the isozymes patterns of H, R and S.

The 3-glucosidase level of R was always higher than that of H. The level in the susceptible combination did not change for the first two days after inoculation but then declined as the fungus established itself and sporulated.

Peroxidase and PAL activities of the cotyledons were found to be enhanced by water infiltration. Chlorogenic acid did not cause further enhancement whereas an aqueous extract of flax cotyledons produced a pronounced enhancement in addition to the effect of water infiltra• tion. The effect of infiltration with both water and 150 plant extract could be abolished by adding actinomycin

D to the infiltration solution.

Based on the above findings it is suggested that phenolic compounds play an important role in disease resistance and susceptibility in Koto flax infected with flax rust races #3 and #210. In the resistant combination both the synthesis and oxidation of phenolic compounds were probably enhanced. On the other hand, in the susceptible reacting tissue phenolic precursors such as aromatic amino acids are directed to the synthesis of protein and the phenol oxidizing system is probably suppressed.

Thus the enhanced synthesis and oxidation of phenolic compounds may be essential for resistance. 151

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