Conjugation of Allelochemicals by Plants - ACS Symposium Series

Chapter 20

Conjugation of Allelochemicals by Plants Enzymatic Glucosylation of Salicylic Acid by Avena sativa Nelson E. Balke, Michael P. Davis , and Carol C. Lee 1

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Downloaded by UNIV OF SOUTHERN CALIFORNIA on March 25, 2017 | http://pubs.acs.org Publication Date: January 8, 1987 | doi: 10.1021/bk-1987-0330.ch020

Department of Agronomy, University of Wisconsin, Madison, WI 53706

Plants have the ability to conjugate endogenous compounds to most allelochemicals absorbed from the environment. Conjugation reactions are thought to be important for detoxication of secondary products such as allelochemicals because conjugation increases the water solubility and decreases the chemical reactivity of the parent compound. Glucosylation, conjugation with glucose, is one of the most common conjugation reactions in plants. Numerous glucosyltransferase enzymes have been extracted from plants. Uridine diphosphate glucose (UDPG) is the preferred glucose donor for these enzymes. The range of secondary products a particular glucosyltransferase can conjugate has not been determined nor has the ability of allelochemicals to induce different glucosyltransferases in plants. Roots of Avena sativa conjugated glucose to salicylic acid, a phenolic" acid, when the allelochemical was present in solution bathing the tissue. The tissue's capacity to conjugate salicylic acid increased with time suggesting induction of glucosyltransferase activity in the tissue. A glucosyltransferase that transfers glucose from UDPG to the phenolic hydroxyl of salicylic acid was purified about 54-fold.

Organisms produce chemicals to protect themselves from other organisms and to give themselves advantage over other organisms (I) . However, the fact that a chemical is produced and released by a donor does not mean that i t will be effective on the receiver. In addition to loss to the environment during movement from the donor to the receiver, the chemical may become inactive after entering the receiver. This can occur by deposition of active ingredient in insensitive tissues and cellular compartments or by conversion to inactive compounds. Also, the receiver may excrete the compound as either 'Current address: American Malting Barley Association, Milwaukee,WI 53202 Current address: California Institute of Technology, Pasadena, CA 91125

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0097-6156/87/0330-0214$06.00/0 © 1987 American Chemical Society

Waller; Allelochemicals: Role in Agriculture and Forestry ACS Symposium Series; American Chemical Society: Washington, DC, 1987.


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active or inactive compounds. The basic p r i n c i p l e s of what happens to xenobiotic compounds when they enter an organism are the same f o r a l l organisms; only the d e t a i l s d i f f e r . For example, a l l organisms have mechanisms to remove xenobiotics from c e l l u l a r cytoplasm. Higher animals do this by excretion i n feces or urine. Plants have no c i r c u l a t i o n system or s p e c i f i c tissues involved i n excretion; instead the compounds move into vacuoles or are integrated into insoluble residues such as l i g n i n i n the c e l l w a l l . How quickly and to what extent an organism i s able to reduce the concentration of an active chemical at the s i t e of action i s a major determinant of t o x i c i t y . Thus, differences i n the c a p a b i l i t y of receivers to detoxify an active chemical i s of major importance for selective action of allelochemicals. Selective t o x i c i t y w i l l also be important i f allelochemicals are to be put to p r a c t i c a l use. For example, i f allelochemicals are to be used as herbicides, the compounds w i l l have to be toxic to some plants (weeds) but not toxic to others (the crop). Such s e l e c t i v e t o x i c i t y could be accomplished i f the crop, but not the weeds, had the a b i l i t y to convert an administered allelochemical to nontoxic products. The purposes of t h i s paper are to review one of the common mechanisms of detoxication i n plants, namely conjugation, and to present data showing that s a l i c y l i c acid, an a l l e l o p a t h i c phenolic acid, i s enzymatically conjugated by oat roots. Detoxication Reactions i n Plants The various types of reactions that lead to detoxication of xenob i o t i c compounds i n plants have been categorized into three groups 3). Phase I reactions include oxidation, reduction, and hydrolysis. Oxidation introduces an oxygen atom or a hydroxyl group into the molecule and i s catalyzed by peroxidase, mono-oxygenase, or other oxygenase enzymes (2, 4_, 5 ) . Oxidation reactions are common i n plants and can r e s u l t i n e i t h e r t h e detoxication or the a c t i v a t i o n of xenobiotic compounds ( 3 ) . Reduction i s much less prevalent and occurs primarily with n i t r o groups (£). Hydrolysis of esters, amides, and n i t r i l e s i s a common reaction i n plants (2). Carboxylic acid esters are e s p e c i a l l y susceptible to hydrolysis to y i e l d free acids. These free acid forms are thought to be more toxic than the esters (2). Phase I reactions can reduce s i g n i f i c a n t l y the t o x i c i t y of a xenobiotic compound and predispose i t to Phase II reactions. Phase II reactions are conjugation reactions. As applied to detoxication, conjugation can be defined as an i n vivo reaction of a xenobiotic compound, or i t s primary metabolite r e s u l t i n g from a Phase I reaction, with an endogenous substrate to form a new compound of higher molecular weight (6_). To aid i n discussion of conjugates, Dorough (6_) has defined "exocon" as that portion of the conjugate that i s derived from the exogenous compound, and "endocon" as that portion derived from an endogenous compound. Conjugation reactions are catalyzed by many d i f f e r e n t enzymes depending upon the exocon and endocon being conjugated together. Conjugation i s a major determinant of the metabolic a c t i v i t y of a xenobiotic compound ( 7_, 8). One reason i s that conjugation s i g n i f i c a n t l y increases the water s o l u b i l i t y of the exocon. An exocon that could previously d i f f u s e across c e l l u l a r membranes does not d i f f u s e as r e a d i l y once i t i s conjugated. Thus, the conjugate can be compart-



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mentalized (9^) more r e a d i l y i n the c e l l than can the exocon. Second, the conjugate" i s less reactive because at least one functional group that imparted chemical r e a c t i v i t y to the exocon has been blocked by the endocon. Conjugates formed by Phase II reactions can have additional endocons added to them. The reactions responsible for such secondary conjugations have been c l a s s i f i e d as Phase III reactions (2_, 3_). In addition, primary and secondary conjugates can be incorporated into insoluble bound residues. The reactions responsible for production of these residues are also c l a s s i f i e d as Phase III reactions (2_, 3). Phase III reactions occur primarily i n plants presumably because excretion of Phase II conjugates i s i n s i g n i f i c a n t i n plants. Phase III reactions are mechanisms whereby plants can reduce the e f f e c t i v e concentration of xenobiotic compounds i n the cytoplasm. Thus, conjugation reactions provide mechanisms f o r the elimination of xenobiotic compounds from s i t e s of continuing metabolic a c t i v i t y i n a l l organisms ( 6_). The above c l a s s i f i c a t i o n of detoxication reactions has been developed for the metabolism of synthetic pesticides i n plants. However, the same reactions can occur with natural exocons, such as a l l e l o p a t h i c compounds, that have the same functional groups as synthetic p e s t i c i d e s . Most a l l e l o p a t h i c chemicals contain functional groups that can be conjugated by Phase II reactions. Thus, detoxication of a l l e l o p a t h i c compounds can be expected to proceed by conjugation with the omission of Phase I reactions. The remainder of this review w i l l be concerned with the conjugation of a l l e l o p a t h i c compounds. Conjugation Of Allelochemicals Most allelochemicals i d e n t i f i e d to date can be c l a s s i f i e d as secondary products because they are found only sporadically i n nature and thus do not appear to be involved i n the basic metabolism of organisms (J_0_). That i s not to say that these compounds do not serve a function i n the organism producing them, and their involvement i n both protective functions ( 1 1 ) and i n metabolism (12) i s now recognized. As many as 1 2 , 0 0 0 such compounds may be involved i n ecological interactions between plants, microorganisms, and animals ( 1 3 ) . The types of chemicals active i n these interactions include straight-chain alcohols, aldehydes, ketones, carboxylic acids, quinones, terpenoids, steroids, coumarins, flavonoids, tannins, glucosinolates, and alkaloids ( 1 1 , 1 4 ) . The prominence of a l i p h a t i c hydroxyl, phenolic hydroxyl, and carboxyl groups i n these chemicals i s obvious. These functional groups, as well as amino and mercapto groups, can be involved i n conjugation by plants. In f a c t , conjugates are the c h a r a c t e r i s t i c form i n which phenols, aromatic acids, flavonoids, steroids, and many other secondary products exist i n plants ( 7 ) · The endocons involved i n these conjugations may be derived from monosaccharides (e.g., glucose, galactose, mannose, and apiose), d i saccharides (e.g., gentiobioside and glucosylxylose), oligosaccharides, aromatic acids (e.g., benzoic acid, g a l l i c acid, and c a f f e i c a c i d ) , amines (e.g., putrescine and spermine), a l k y l groups (e.g., methyl, a c e t y l , and d i m e t h y l a l l y l ) , amino acids and peptides (e.g., aspartic a c i d , glycine, and glutathione), a l i p h a t i c acids -



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(e.g., a c e t i c , malic, and malonic acids) c y c l i c hydroxy compounds (e.g., shikimic a c i d ) , and inorganic acids (e.g., s u l f u r i c and phosphoric acids) (7). This great d i v e r s i t y of endocons makes i t d i f f i c u l t to predict what p a r t i c u l a r exocon-endocon conjugate w i l l be present i n a p a r t i c u l a r plant (J_5), and mixtures of conjugates can exist there. Conjugates of secondary products with sugars are e s p e c i a l l y prevalent i n plants (_16_, VJ). S p e c i f i c examples can be found i n almost a l l classes of secondary products including glycosides of simple phenols (18) and phenolic acids (19), cyanogenic glycosides (20), flavonoid gTycosides (22), cardiac~glycosides (23), saponins (~2J\), sesquiterpene lactone glycosides (25), quinone glycosides (26), and glucosinolates ( 27_). In addition, glycosides are often formed when aglycones are fed to plant tissue (17). Such induced formation of glycosides i s important with a l l e l o p a t h i c compounds because the conjugate w i l l most l i k e l y be less toxic to the plant than the aglycone (17). Enzymology of Glucoside

Formation

A large number of uridine diphosphate glucose g l u c o s y l transf erases (EC 2.4.1.) (UDPG-GTases) that glucosylate secondary products to produce glucosides or glucose esters have been studied to varying levels of d e t a i l ( 8^. I w i l l summarize these studies, placing emphasis on those enzymes shown to glucosylate phenolic acids such as s a l i c y l i c acid. UDPG-GTases that use phenolic acids as acceptor molecules have been i s o l a t e d from several plant t i s s u e s . These include leaves of sweet clover (28), geranium (29), Cestrum euenthes (30), oak (31, 32), unripe frûTts of tomato T"33, 70", entire seêdiings of radish T~35), and roots of oats ( 36_). Insofar as investigated, a l l the enzymes preferred UDPG as the sugar donor. In many of the studies, UDPG was the only donor used i n the assay (28-30, 33, 34). An extensive study of sugar donors by Gross (32) showed that UDPGrgallic acid-GTase from oak leaves would not use ADP-, CDP-, GDP-, or TDP-glucose, nor UDP-galactose, -galacturonic a c i d , -glucuronic acid, -mannose, -xylose, or -N-acetylglucosamine. A UDPG:sinapic acid-GTase had the same donor s p e c i f i c i t y except that i t used TDPG as well as UDPG (35). Different GTases have d i f f e r e n t acceptor s p e c i f i c i t i e s (8). There are three p o t e n t i a l classes of phenolic acids f o r UDPG:phenolic acid-GTases: true phenolic acids (C/--C-), hydroxyphenylacetic acids (Cg-C ), and hydroxycinnamic acids (δβ-^ο) (J_5) · There are no reports of studies investigating the hydroxyphenylacetic acids as substrates for GTases. Hydroxybenzoic acids and e s p e c i a l l y hydroxycinnamic acids have been studied, often with inclusion of only one of the two classes. Kleinhofs et a l . (37) extracted a UDPG-GTase from sweet clover that would glucosylati~~o-coumaric acid but not o_-coumarinic acid; other phenolic acids were" not tested as substrates. A UDPGGTase from unripe tomato f r u i t s was active with the cinnamic acids p-coumaric, f e r u l i c , c a f f e i c , and sinapic acids (33_, 34). p-Coumaric acid was the best acceptor. Again no other phenolic acids were tested but the enzyme did glucosylate coumarins and flavonols. p-Coumaric acid was the only substrate tested as acceptor f o r the GTase extracted from Cestrum euenthes leaves (30). 2



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For three UDPG-GTases, both hydroxybenzoic and hydroxycinnamic acids have been studied as substrates. UDPG-GTase a c t i v i t y from geranium leaves used both classes of phenolic acids (29), but no quantitation or r e l a t i v e a c t i v i t y with the two were stated. The GTase from radish seedling was active with both hydroxycinnamic acids and hydroxybenzoic acids (38). Compared to sinapic acid a l l other hydroxycinnamic acid derivatives and hydroxybenzoic acid derivatives showed only 30-40% as much a c t i v i t y . The only UDPG-GTase shown p r e f e r e n t i a l l y to use hydroxybenzoic acids was extracted from young leaves of oak trees (_31_, 32). V a n i l l i c acid was the best acceptor of nine benzoic acid derivatives; a l l f i v e cinnamic acid derivatives produced 35% of the a c t i v i t y that v a n i l l i c acid did. The enzyme showed no a c t i v i t y with s a l i c y l i c a c i d . With phenolic acids as substrates for a GTase, the question arises whether the glucoside (phenolic hydroxyl group) or the glucose ester (carboxylic hydroxyl group) i s formed. It appears that both can be produced depending upon the substrate and the enzyme. Only glucose esters were formed from hydroxybenzoic and hydroxycinnamic acids by some GTases (29-32, 38). Both glucose esters and glucosides were formed from hydroxycinnamic acids by other enzymes (33, 34); glucosides were predominant over glucose esters (33), except f o r c a f f e i c acid, from which only the ester was produced (34). o-Coumaric acid produced only the glucoside (28, 37), i n agreement with feeding experiments (39) . In the only i n v i t r o study where s a l i c y l i c acid was tested and found to be glucosylated, the glucoside, rather than the ester, was produced (29). Two general points" need to be made about a l l these UDPG-GTases. The f i r s t i s that they may not be detoxication GTases. A l l these UDPG:phenolic acid-GTases are c o n s t i t u t i v e enzymes; i t i s probable that detoxication GTases are synthesized de novo only when the aglycone i s present. Thus, induction of detoxication GTases i n plants may be s i m i l a r to detoxication UDPglucuronosyltransferases i n animals in that the induced enzyme i s d i f f e r e n t from c o n s t i t u t i v e glucuronosyltransferases (40). The second point i s the p o s s i b i l i t y that more than one GTase was present i n the preparations used to investigate substrate s p e c i f i city. In most of these studies, crude preparations were used and i n no instance was gel electrophoresis used to assess purity. The greatest l e v e l of p u r i f i c a t i o n i n these studies was 45-fold (32). Table I l i s t s several additional GTases, the l e v e l of p u r i f i c a t i o n achieved, and the substrate s p e c i f i c i t y of the enzyme. In general, these more p u r i f i e d GTases displayed high substrate s p e c i f i c i t y ; only substrates with very s i m i l a r structures were glucosylated by the enzyme. The purity of the l a s t two enzymes (Table I) was shown by gel electrophoresis. They both yielded one major protein band; the preparation from Triglochin also yielded one or two minor bands. Because s e l e c t i v e detoxication by a crop plant would be important i f allelochemicals are to be useful as natural herbicides, the f i r s t objective of these studies was to determine i f the cereal oats can metabolize exogenously applied s a l i c y l i c a c i d , a known a l l e l o p a t h i c agent. The second objective was to i d e n t i f y and p a r t i a l l y purify any enzyme responsible f o r such metabolism of s a l i c y l i c acid i n oat roots.


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Table

I.

Conjugation

of Allelochemicals

Qr leaves

45-X

V C

C

110-X


219

Acceptor S p e c i f i c i t y of Several P a r t i a l l y P u r i f i e d G l u c o s y l t r a n s f e r a s e s from P l a n t s

a/ Source— P u r i f i c a t i o n C l a s s

C

cells

120-X

Ca roots

C

200-X Tm seedling

1

C

6" 3 6- 3 C

C

6- 1 C

isoflavone

C

Pa sap

by Plants

6- 1 6" 3 C

C

cyanohydrin C

1700-X

6- 1 C

6- 3 V 3 C

C

C

6~ 3 flavonoid C

C

Acceptor S p e c i f i c i t y Example

Relative Activity

Ref.

v a n i l l i c acid s a l i c y l i c acid caffeic acid ferulic acid

32

s a l i c y l alcohol p-OH-benzyl a l c o h o l hydroquinone

41

biochanin A benzoic acids cinnamic a c i d s

42

4-0H-mandelonitrile p-OH-benzyl a l c o h o l

43

coniferyl alcohol coniferaldehyde p-OH-benzoic a c i d cinnamic a c i d naringenin

++ +

44

a/ — A b b r e v i a t i o n s o f p l a n t s p e c i e s : Q r , Quercus r u b r a ; G j , Gardenia^ j a s m i n o i d e s ; C a , C i c e r a r i e t i n u m ; Tm, T r i g l o c h i n m a r i t i m a ; " Pa, P i c e a a b i e s . Materials

and Methods

P l a n t M a t e r i a l s . Oat seeds (Avena s a t i v a L . c v . G o o d f i e l d ) were germ i n a t e d and grown a e r o p o n i c a l l y on m o i s t e n e d c h e e s e c l o t h s t r e t c h e d a c r o s s the top o f a 4 - L beaker c o n t a i n i n g 3 L 1 mM CaSOn. P l a n t s were grown 5 days i n the dark a t room t e m p e r a t u r e ( c a . 21 C) w i t h c o n t i n u o u s a e r a t i o n o f the s o l u t i o n . The a p i c a l 5 cm o f the r o o t s were used t o measure the a b s o r p t i o n o f s a l i c y l i c a c i d . The e n t i r e , e x c i s e d r o o t was used f o r measurements o f m e t a b o l i s m o f s a l i c y l i c a c i d and f o r p r o t e i n e x t r a c t i o n s . S a l i c y l i c Acid Absorption. The a p i c a l 5 cm o f t h e p r i m a r y and two s e m i n a l r o o t s from e a c h o f t h r e e p l a n t s were c u t i n t o 1-cm segments t o form an e x p e r i m e n t a l u n i t ( c a . 0.08 g ) . Incubation s o l u t i o n c o n t a i n e d 0 . 5 mM K C 1 , 0.25 mM CaSO^, 0 . 5 mM s a l i c y l i c a c i d , 10 nCi/mL [ C ] - s a l i c y l i c a c i d , w i t h 25 mM T r i s and 25 mM Mes b u f f e r s mixed t o o b t a i n pH 6 . 5 . Because t h e s a l i c y l i c a c i d was d i s s o l v e d i n a b s o l u t e e t h a n o l , the f i n a l c o n c e n t r a t i o n o f e t h a n o l i n t h e i n c u b a t i o n s o l u t i o n was 1ί ( v / v ) . Root segments were t r a n s f e r r e d t o t e s t tubes c o n t a i n i n g 10 mL c o n t i n u o u s l y a e r a t e d i n c u b a t i o n s o l u t i o n . A f t e r the p r e d e t e r m i n e d a b s o r p t i o n t i m e , segments were c o l l e c t e d from t h e i n c u b a t i o n s o l u t i o n by r a p i d f i l t r a t i o n on Whatman No. 2 f i l t e r p a p e r ,


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washed f o r 2 min i n unaerated, i c e - c o l d , incubation solution lacking s a l i c y l i c acid, collected again, and weighed. Radioactive content of the segments was determined by l i q u i d s c i n t i l l a t i o n spectroscopy using a dioxane/methylcellosolve s c i n t i l l a t i o n f l u i d (45). In Vivo Metabolism of S a l i c y l i c Acid. Entire oat roots were cut into segments 2-3 cm long and poolea ~to"give 5 g f o r each experimental u n i t . Segments were intubated i n 100 mL incubation solution containing 16 nCi/mL [ C ] - s a l i c y l i c acid with aeration. At predetermined times, the segments were collected by f i l t r a t i o n and washed as f o r the absorption experiments. Segments were frozen with l i q u i d nitrogen and ground to a fine powder with a mortar and pestle. The powder was extracted with 10 mL 80% (v/v) methanol with gentle s t i r r i n g f o r 30 min. The homogenate was centrifuged at 20x10 xg f o r 20 min and the p e l l e t was extracted twice more with 80% methanol. The methanol extracts were combined, concentrated with a f l a s h evaporator, and evaporated to 1 to 1.5 mL under a stream^of nitrogen gas. Aliquots of the concentrate containing at least 10 dpm were spotted on 250-^i cellulose-coated thin layer (250 μ) chromatography plates and developed with either 6% (v/v) acetic acid or 6:1:2 1-butanol, acetic acid, water (BAW). Radioactive spots were located with a radiochromatogram scanner and R^ values were calculated. Radioactive content of the spots was quantified by l i q u i d s c i n t i l l a t i o n spectrometry a f t e r each spot was scraped o f f the plate.


r

Preparation of Protein Extracts. Excised intact roots (175 g) were incubated i n 5.25 L incubation solution lacking radiolabelled s a l i c y l i c acid f o r 20 hr with aeration. Incubation solution was decanted and the roots were rinsed i n 5.25 L cold d i s t i l l e d water f o r 10 min. Water was decanted and the roots were frozen with l i q u i d nitrogen and pulverized with a mortar and pestle. Extraction buffer (1.5 mL/g root) containing 10 mM 2-mercaptoethanol, and 25 mM Tris and 25 mM Mes mixed to give pH 7.0 was added and the homogenate was allowed to thaw at room temperature with gentle s t i r r i n g . The homogenate was f i l t e r e d immediately through four layers of cheesecloth and the supernatant was centrifuged at 20x10 xg f o r 20 min. The r e s u l t i n g supernatant was brought to 50% (w/v) ammonium s u l f a t e , s t i r r e d , and centrifuged again. Supernatant was brought to 65% (w/v) ammonium sulfate and centrifuged once more. The p e l l e t was dissolved i n 4 mL extraction buffer with the addition of NaCl to 0.8 mM and g l y c e r o l to 10% (v/v) (elution b u f f e r ) . The protein solution was eluted through a G-100 Sephadex column (1.9 x 65 cm) with elution buffer at 8 mL/hr. Fractions containing GTase a c t i v i t y were pooled, concentrated to 10 mL by d i a l y s i s i n polyethylene g l y c o l 8000 MW, and eluted through G-75 Sephadex under the same conditions. Fractions containing GTase a c t i v i t y were pooled and applied to a DEAE-Sephacel column (1.22 χ 14 cm), which was then washed with 80 ml elution buffer. Proteins were eluted from the column by l i n e a r l y increasing the NaCl concentration in the elution buffer to 0.5 M over 10 h at 30 ml/h. Fractions containing GTase a c t i v i t y were pooled. Protein content of various fractions was determined with Coomassie Blue by the method of Bradford (46) as refined by Spector (47). ~~



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In Vitro S a l i c y l i c Acid Metabolism. Protein fractions were assayed for their a b i l i t y to produce the same metabolite of s a l i c y l i c acid as the root tissue produced. Protein fractions (50-185 pL) were incubated i n an assay mixture containing 0.4 mM s a l i c y l i c acid, 25 pCi [ C ] - s a l i c y l i c acid, 1 mM UDPG, 25 mM Tris-Mes buffer to adjust to pH 7.0 (total volume 200 μΐ), f o r 1 h at 30 °C. The reaction was stopped by adding 200 /iL absolute methanol. S a l i c y l i c acid and i t s metabolite were separated by two methods. The f i r s t was thin layer chromatography on c e l l u l o s e with BAW solvent as for the i n vivo metabolism studies. A quicker separation was achieved with a polyamide column. The entire 400 μΐ, from an individual assay was placed on top of a 0.8 χ 2.0 cm column packed with Polyamide-6 (Accurate Chemical and S c i e n t i f i c Corp.). The s a l i c y l i c acid metabolite was eluted with 6 mL water but s a l i c y l i c acid was retained. 3a70B s c i n t i l l a t i o n f l u i d (Research Product International Corp.) was used to determine the radioactive content of the entire 6 mL of eluant. Separation of s a l i c y l i c acid and i t s metabolite by polyamide column chromatography was v e r i f i e d by thin layer chromatography. The a b i l i t y of an esterase or a J3-glucosidase to hydrolyze the in v i t r o generated metabolite was tested. An assay mixture that had been incubated f o r 22 h (ca. 50% conversion of s a l i c y l i c acid) was incubated with either 10 units of hog-liver esterase (E.C. 3.1.1.1, Sigma Chemical Co.) at pH 8.0 or 20 units of ^-glucosidase (E.C. 3.2.1.21, Sigma) at pH 5.0 f o r 1 h at 37 °C. S a l i c y l i c acid and the metabolite were separated by thin layer chromatography with BAW and quantified by l i q u i d s c i n t i l l a t i o n chromatography. Results Absorption of S a l i c y l i c Acid. Excised oat roots absorbed s a l i c y l i c acid i n two distinct" phases (Figure 1). Upon exposure to s a l i c y l i c acid the root segments rapidly absorbed the compound to a t t a i n a concentration of about 0.5 /imole/g of t i s s u e . On the assumption that 1 g of tissue equals 1 mL of tissue, t h i s translates to 0.5 mM s a l i c y l i c acid inside the tissue, the same concentration as the external solution. This concentration of s a l i c y l i c acid was present in the tissue a f t e r 1 h and was maintained f o r over 3 h. By 4 h a second phase of absorption was evident (Figure 1). During the second phase, s a l i c y l i c acid was absorbed at a greater rate that lasted f o r at least 24 h. At that time, enough s a l i c y l i c acid had been absorbed that the concentration i n the tissue was 8.0 mM. Thus, the tissue accumulated s a l i c y l i c acid to concentrations greater than that i n the external solution. An additional experiment (not shown) showed that the tissue would continue to absorb s a l i c y l i c acid u n t i l the compound was depleted from the external solution. In Vivo Metabolism of S a l i c y l i c Acid. A l l the s a l i c y l i c acid absorbed by the tissue T F i g u r e IT did not remain as that a c i d . I n i t i a l l y (< 2 h), most of i t remained as such (Table I I ) . However, gradually more parent compound was converted to a metabolite, so that between 4 and 20 h most of the absorbed s a l i c y l i c acid was thus converted. At 20 h, 6.79 jmmole/g of metabolite was present i n the tissue. Thus, the accumulation of " s a l i c y l i c a c i d " observed i n the absorption


222


Table

II.

Distribution of C between S a l i c y l i c A c i d and M e t a b o l i t e i n Oat Roots F o l l o w i n g Exposure f o r V a r i o u s Times — Salicylic Metabolite acid (jimole/g) 0.06 0.34


Exposure Time (h) 1 2 4 6 20

0.23 0.04 0.00

0.27 0.66 1.60

0.51

6.79

a/ — The r a d i o l a b e l e d compounds were s e p a r a t e d by t h i n l a y e r chromatography on c e l l u l o s e u s i n g 6% a c e t i c a c i d . R^ o f s a l i c y l i c a c i d was 0 . 6 3 and o f t h e m e t a b o l i t e was 0 . 7 6 . e x p e r i m e n t ( F i g u r e 1) r e s u l t e d from m e t a b o l i s m o f t h e p a r e n t compound as i t c o n t i n u e d t o d i f f u s e i n t o t h e t i s s u e . The e x t e n t o f m e t a b o l i s m o f absorbed s a l i c y l i c a c i d i n c r e a s e d from 0 t o 6 h , and e s s e n t i a l l y a l l t h e absorbed s a l i c y l i c a c i d was c o n v e r t e d t o t h e m e t a b o l i t e a f t e r 6 h (Table I I ) . In V i t r o M e t a b o l i s m o f S a l i c y l i c A c i d . The p r o t e i n p r e c i p i t a t i n g between 50 and 65% (w/v) ammonium sûTfate p o s s e s s e d t h e c a p a c i t y t o produce t h e same m e t a b o l i t e a s t h e o a t r o o t t i s s u e ( T a b l e I I I ) . When the p r o t e i n f r a c t i o n was h e a t e d a t 100 °C f o r 5 min i t no l o n g e r c o n v e r t e d s a l i c y l i c a c i d t o t h e m e t a b o l i t e . The enzyme t h a t c a t a l y z e d

Table

III.

S a l i c y l i c A c i d M e t a b o l i t e P r o d u c t i o n by a 50-65% Ammonium S u l f a t e F r a c t i o n E x t r a c t e d from Oat Roots Exposed t o S a l i c y l i c A c i d f o r 20 h -

Assay C o n d i t i o n

Metabolite

+ [

, H

C]-Salicylic

a c i d , - UDPG

+ [

1 4

C]-Salicylic

a c i d , + UDPG

+ Unlabelled + [

1 4

salicylic acid,

C ] - S a l i c y l ^ acid, + esterase— ^ + ^-glucosidase— b

+ UDP([

Production

+ 14

C]-G)

+

+ UDPG +

— Enzymatic a c t i v i t y was measured i n t h e p r e s e n c e o f v a r i o u s r a d i o labelled substrates. S a l i c y l i c a c i d and m e t a b o l i t e were s e p a r a t e d on c e l l u l o s e w i t h BAW. R ^ ' s f o r s a l i c y l i c a c i d , UDPG, and m e t a b o l i t e w^re 0 . 9 2 , 0 . 0 0 , and 0 . 6 8 , r e s p e c t i v e l y . — E s t e r a s e o r ^ - g l u c o s i d a s e were added a f t e r t h e m e t a b o l i t e had formed.


20.

Conjugation

BALKE ET AL.

of AUelochemicab

223

by Plants


the metabolite production required UDPG (Table I I I ) . The same m e t a b o l i c was produced when the protein f r a c t i o n was incubated with e i ^ e r [ C ] - s a l i c y l i c acid and unlabelled UDPG or with UDP([ C]-glucose) and unlabelled s a l i c y l i c a c i d . Addition of the p-glucosidase following production of the metabolite resulted i n conversion of the metabolite back to s a l i c y l i c a c i d . However, the esterase did not hydrolyze the metabolite. Thus, the metabolite appears to be p - g l u c o s y l s a l i c y l i c acid, which i s produced by UDPG:salicylic acid glucosyltransferase (SA-GTase). P a r t i a l P u r i f i c a t i o n of SA-GTase. Proteins extracted from oat roots incubated f o r 20 h i n s a l i c y l i c acid were separated by s a l t p r e c i p i tation and gel exclusion and anion exchange chromatography (Table IV). A 54-fold p u r i f i c a t i o n of the SA-GTase was achieved with this

Table IV. P a r t i a l P u r i f i c a t i o n of the Glucosyltransferase —

Fraction

Crude homogenate 50-65% (NHn) S0 Sephadex G-100 Sephadex G-75 DEAE-Sephacel 2

u

Total Protein (mg)

Total (mU)

142.4 56.1 9.2 2.3 0.4

259.0 159.2 114.2 64.1 36.2

Glucosyltransferase A c t i v i t y Purification Yield Specific (fold) (mU/mg) (%) 1.82 2.83 12.37 27.45 97.90

100 62 44 25 14

1 1.6 6.8 15.1 53.8

— The metabolite and s a l i c y l i c acid were separated on a polyamide column following incubation of the enzyme with r a d i o l a b e l e d s a l i c y l i c acid and UDPG.

separation scheme. However, the SA-GTase was not homogeneous as shown by the presence of several protein bands on polyacrylamide g e l electrophoresis. Total protein recovered from the anion exchange column was 0.4 mg and represented 14% of the SA-GTase a c t i v i t y present i n the crude homogenate that contained 142 mg protein. Thus, less than 2.86 mg SA-GTase was present i n the crude homogenate. Or, less than 2% of the protein extracted from the tissue was SA-GTase. Hence, the enzyme i s a minor protein i n the tissue. Discussion These experiments demonstrate that oat roots can metabolize s a l i c y l i c a c i d . Metabolism r e s u l t s i n more s a l i c y l i c acid being absorbed by the tissue and i t s accelerated metabolism with time of exposure to the parent compound (Figure 1, Table I I ) . The appearance of only one metabolite (Table II) and the hydrolysis of the metabolite by JB-glucosidase but not esterase (Table III) suggests that the glucoside and not the glucose ester of s a l i c y l i c acid i s produced by oat roots. Both metabolites of s a l i c y l i c acid have been found i n other plants (16). Some species produce only the glucose ester (48), others produceToth the glycoside and the ester (49), and others




224

convert s a l i c y l i c acid to g e n t i s i c acid and produce the glucoside of g e n t i s i c acid (j_9, ^0) . Positive i d e n t i f i c a t i o n of the metabolite from oats w i l l require p u r i f i c a t i o n of s u f f i c i e n t amounts of the metabolite f o r structure elucidation. The metabolite i s enzymatically produced i n the root tissue (Table I I I ) . In one other study (29), as already mentioned a protein extract from plants was shown to glucosylate s a l i c y l i c a c i d ; the glucoside rather than the glucose ester was produced. The protein extracts from oat roots also produced the glucoside and not the ester of s a l i c y l i c acid (Table I I I ) . The enzyme uses UDPG as the glucose donor, but we do not yet know i f UDPG i s the preferred sugar donor. If so, the enzyme w i l l be UDPG:salicylic acid glucosyltransferase. By analogy with s i m i l a r enzymes (8), the enzyme probably catalyzes formation of a ^-linkage between glucose and s a l i c y l i c acid. Thus, the reaction can be v i s u a l i z e d as shown i n Figure 2. The enzyme has not yet been p u r i f i e d to homogeneity (Table IV). Because the enzyme represents < 2% of the extractable protein, further p u r i f i c a t i o n w i l l require more s t a r t i n g tissue as well as additional techniques. Conclusion Because of the presence of reactive hydroxyl groups on most a l l e l o chemicals i d e n t i f i e d to date, the l i k e l i h o o d i s great that these compounds can be glycosylated by plants. Such glycosylation may result i n detoxication of allelochemicals because of blockage of the reactive group and increased water s o l u b i l i t y of the conjugate. Glycosyltransferases that catalyze glycosylation of secondary products i n plants have been i d e n t i f i e d and the a b i l i t y of such enzymes to glycosylate certain a l l e l o p a t h i c compounds as they are absorbed by plants has been v e r i f i e d . The substrate range of glycosyltransferases has not been extensively studied. Thus, i t i s not known i f a family of d i f f e r e n t glycosyltransferases are necessary to glycosylate the various allelochemicals plants might encounter. P r a c t i c a l use of glycosyltransferases such as the glucosyltransferase that glucosylates s a l i c y l i c acid i n oat roots may be made to provide s e l e c t i v e phytotoxicity f o r allelochemicals. Differences i n the constitutive l e v e l of glycosyltransferases among crops and weeds might provide that s e l e c t i v i t y . Selection of crop v a r i e t i e s with higher levels of glycosyltransferases could lead to crops more r e s i s tant to allelochemicals. More rapid induction of glycosyltransferases in desirable plants might be exploitable. And f i n a l l y , transfer of genes encoding f o r glycosyltransferases with high a c t i v i t y toward a p a r t i c u l a r phytotoxic allelochemical might be possible i n the future. Thus, modification of an allelochemical with a glycosyltransferase to detoxify the allelochemical may be a useful approach f o r making p r a c t i c a l use of allelochemicals i n a g r i c u l t u r e . Further c h a r a c t e r i zation of the types and a c t i v i t i e s of glycosyltransferases i n plants must be done before such uses of these enzymes can be pursued. Acknowledgments Research supported by the College of A g r i c u l t u r a l and L i f e Sciences, University of Wisconsin, Madison and SEA/USDA Grant 85-CRCR-1-1572 from the Competitive Research Grants Program.



20.

Conjugation

BALKE ET AL.

sI Ο

1

1 4

1

of Allelochemicals

1 8

ι

1 12

by Plants

ι

I 16

ι

225

I 20

ι

Ι 24

Time ( hr )

Figure 1. Absorption of s a l i c y l i c acid f o r 1 to 24 hr by excised oat roots.

o

UDP-glucose Salicylic Acid

UDP /3-Glucosylsalicylic Acid

Figure 2. Reaction scheme f o r UDPG:salicylic acid glucosyltransferase extracted from oat roots incubated f o r 20 hr i n solution containing s a l i c y l i c a c i d .


226



Literature Cited 1. Whittaker, R.H.; Feeny, P.P. Science 1971, 171, 757-69. 2. Shimabukuro, R.H.; Lamoureux, G.L.; Frear, D.S. In "Biodegradation of Pesticides"; Matsumura, F.; Murti, C.R.K., Eds.; Plenum: New York, 1982; pp. 21-66. 3. Shimabukuro, R.H. In "Weed Physiology"; Duke, S.O.,Ed.; CRC: Boca Raton, FL, 1985; Vol. II, pp. 215-40. 4. Lamoureux, G.L.; Frear, D.S. In "Xenobiotic Metabolism, In Vitro Methods"; Paulson, G.D.; Frear, D.S.; Marks, E.P., Eds.; ACS SYMPOSIUM SERIES No. 97, American Chemical Society: Washington, D. C., 1979; Chap. 3. 5. Sanderman, H.; Diesperger, H.; Scheel, D. In "Plant Tissue Culture and Its Biotechnological Applications"; Barz, W.; Reinhard, E.; Zenk, M.H., Eds.; Springer-Verlag: New York, 1977; pp. 178-210. 6. Dorough, H.W. In "Bound and Conjugated Pesticide Residues"; Kaufman, D.D.; S t i l l , G.G.; Paulson, G.D., Eds.; ACS SYMPOSIUM SERIES No. 29, American Chemical Society: Washington, D.C., 1976; Chap. 2. 7. Barz, W.; Koster, J. In "The Biochemistry of Plants, A Comprehensive Treatise"; Stumpf, P.K.; Conn, E.E., Eds.; Academic: New York, 1981; Vol. 7, Chap. 3. 8. Hösel, W. In "Biochemistry of Plants, A Comprehensive Treatise", Stumpf, P.K.; Conn, E.E., Eds.; Academic: New York, 1981; Vol. 7, Chap. 23. 9. Conn, E.E. In "Cellular and Molecular Biology of Plant Stress"; Key, J.L.; Kosuge, T., Liss: New York, 1985; . 10. Rice, E.L. "Allelopathy"; Academic: New York, 1984; p. 266. 11. Bell, E.A. In "The Biochemistry of Plants, A Comprehensive Treatise"; Stumpf, P.K.; Conn, E.E., Eds.; Academic: New York, 1981; Vol. 7, Chap. 1. 12. Robinson, T. Science 1974, 184, 430-5. 13. Harborne, J.B. "Introduction to Ecological Biochemistry"; Academic: New York, 1982; p.3. 14. Putnam, A.R. In "Weed Physiology"; Duke, S.O., Ed.; CRC: Boca Raton, FL, 1985; Vol. I, pp. 131-55. 15. Harborne, J.B. In "Encyclopedia of Plant Physiology"; Bell, E. A.; Charlwood, B.V., Eds.; Springer-Verlag: New York, 1980; Vol. 8, pp. 329-402. 16. Towers, G.H.N. In "Biochemistry of Phenolic Compounds"; Harborne, J.B., Ed.; Academic: New York, 1964; Chap. 7. 17. Miller, L.P. In "Phytochemistry", Miller, L.P., Ed.; Reinhold: New York, 1973; Chap. 11. 18. Pridham, J.B. Ann. Rev. Plant Physiol. 1965, 16, 13-36. 19. Cooper-Driver, G.; Corner-Zamodits, J.J.; Swain, T. Z. Naturforsch. 1972, 27b, 943-6. 20. Cutler, A.J.; Conn, E.E. Recent Adv. Phytochem. 1982, 16, 249-271. 22. Cosio, E.G.; McClure, J.W. Plant Physiol. 1984, 74, 877-81. 23. Kopp, B.; Loffehardt, W.; Kubelka, W. Z. Naturforsch. 1978, 33c, 646-50. 24. Aoki, T.; Suga, T. Phytochemistry 1978, 17, 771-3. 25. Kiesel, W. Phytochemistry 1984, 23, 1955-8. 26. Muller, W-U; Leistner, E. Phytochemistry 1978, 17, 1739-42.



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Conjugation

of Allelochemicals

by Plants

227

27. Matsuo, M.; Underhill, E.W. Phytochemistry 1971, 10, 2279-86. 28. Poulton, J.E.; McRee, D.E.; Conn, E.E. Plant Physiol. 1980, 65, 171-5. 29. Corner, J.J.; Swain, T. Nature 1965, 207, 634-5. 30. Nagels, N.; Molderez, M.; Parmentier, F. Phytochemistry 1981, 20, 965-7. 31. Gross, G.G. FEBS Lett. 1982, 148, 67-70. 32. Gross, G.G. Phytochemistry 1983, 22, 2179-82. 33. Fleuriet, A.; Macheix, J.J. Phytochemistry 1980, 19, 1955-8. 34. Fleuriet, Α.; Macheix, J.J.; Suen, R.; Ibrahim, K. Z. Naturforsch. 1980, 35c, 967-72. 35. Nurmann, G.; Strack, D. Z. Pflanzenphysiol. 1981, 102s, 11-17. 36. Balke, N.E.; Lee, C.C.; Davis, M.P. Plant Physiol. 1983, 72s, 153. 37. Kleinhofs, A.; Haskins, F.Α.; Gorz, J.H. Phytochemistry 1967, 6, 1313-18. 38. Strack, D. Z. Naturforsch. 1980, 35c, 204-8. 39. Kosuge, T; Conn, E.E. J. Biol. Chem. 1959, 234, 2133-7. 40. Burchell, B. Rev. Biochem. Toxicol. 1981, 3, 1-32. 41. Mizukami, H.; Terao, T.; Ohashi, H. Planta Med. 1985, pp.104-7. 42. Koster, J.; Barz, W. Arch. Biochem. Biophys. 1981, 212, 98-104. 43. Hosel, W.; Schiel, O. Arch. Biochem. Biophys. 1984, 229, 177-86. 44. Schmid, G.; Grisebach, H. Eur. J. Biochem. 1982, 123, 363-70. 45. Bruno, G.A.; Christian, J.E. Anal. Chem. 1961, 33, 1216-18. 46. Bradford, M.M. Anal. Biochem. 1976, 72, 248-54. 47. Spector, T. Anal. Biochem. 1978, 86, 142-6. 48. Barz, W.; Schlepphorst, R.; Wilhelm, P.; Kratl, K.; Tengles, E. Z. Naturforsch. 1978, 33c, 363-7. 49. Tabata, M.; Ikeda, F.; Hiraoka, N.; Konoshima, M. Phytochemistry 1976, 15, 1225-9. 50. Zenk, M.H. Phytochemistry 1967, 6, 245-52. RECEIVED January 21, 1986


Conjugation of Allelochemicals by Plants - ACS Symposium Series

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