Biochemistry of Conifer Resistance to Bark Beetles and Their Fungal

Chapter 6

Biochemistry of Conifer Resistance to Bark Beetles and Their Fungal Symbionts Mark A. Johnson and Rodney Croteau

Downloaded by PURDUE UNIV on September 19, 2017 | http://pubs.acs.org Publication Date: December 24, 1987 | doi: 10.1021/bk-1987-0325.ch006

Institute of Biological Chemistry, Washington State University, Pullman, WA 99164-6340

Conifers exhibit resistance to bark beetle attack and fungal invasion because of an induced synthesis and flow of secondary resin which contains high levels of volatile monoterpenes. Monoterpene olefin synthases (geranyl pyrophosphate cyclases) were found in higher levels in fungus-infected lodgepole pine seedlings than in uninfected controls. Cyclases responsible for the formation of (+)-α-pinene and (+)-limonene, monoterpenes which inhibit fungal growth and repel bark beetles, have been studied in herbaceous species. Two distinct types of enzymes synthesize antipodal monoterpene hydrocarbons. The differential expression of the cyclases responsible for co-production of enantiomeric monoterpenes may determine the highly selective resistance response conifers exhibit toward bark beetles and their fungal symbionts. Resin secretion i s part of a resistance mechanism conifers employ against bark beetles and t h e i r associated pathogenic fungi (1-3). Conifer resins are contained i n a system of ducts or c o r t i c a l b l i s t e r s and consist of a mixture of terpenes and benzenoid compounds (A). The terpenoid f r a c t i o n of wood resins of the Pinaceae ( e s p e c i a l l y pines) generally contain 20 to 50% v o l a t i l e mono- (Cio) and sesquiterpenes (C15) and 50 to 80% nonvolatile diterpene acids (Cp.o) (5). Successful resistance to bark beetle attack depends on a complex r e l a t i o n s h i p i n v o l v i n g the quantity and composition of the r e s i n produced, the p h y s i o l o g i c a l condition of the t r e e , the virulence of the fungus and the number of attacks (6-8). A l t e r a t i o n s i n monoterpene content can determine the degree of resistance to bark beetles and, thus, whether or not a tree i s colonized. Resin monoterpenes ( F i g . 1) may be e i t h e r r e p e l l e n t s or attractants to insects or f u n g i s t a t i c or chemotropic stimulants of fungal growth, depending on the precise monoterpene composition and the p a r t i c u l a r herbivore or pathogen (9-13). 0097-6156/87/0325-0076$06.00/0 © 1987 American Chemical Society

Fuller and Nes; Ecology and Metabolism of Plant Lipids ACS Symposium Series; American Chemical Society: Washington, DC, 1987.

6.

JOHNSON AND CROTEAU


a-Pinene

Conifer Resistance to Bark Beetles

0-Pinene

Camphene

11

3-Carene

a

,..OCCH

3

OH c/s-Ocimene

Myrcene

Bornyl Acetate

Terpinen-4-ol

Sabinene

Limonene

£-Phellandrene

y-Terpinene

& & Thujone

Piperitone

Camphor

Fenchone

Figure 1. Major components of the monoterpene f r a c t i o n o f conifer resins.


ECOLOGY AND METABOLISM OF PLANT LIPIDS


78

Monoterpenes are derived from the c l a s s i c a l mevalonate pathway, and c o n t r o l of monoterpene composition i s presumed to occur, at l e a s t i n part, at the l a t t e r stages of the pathway, during which the trans Cxo prenyl pyrophosphate (geranyl pyrophosphate (GPP)) i s converted to a c y c l i c and c y c l i c products (14). The conversion of geranyl pyrophosphate to parent compounds of the various c y c l i c types i s catalyzed by enzymes c o l l e c t i v e l y referred to as monoterpene cyclases. The cyclases of conifers have received l i t t l e experimental attention thus f a r , but these enzymes are probably s i m i l a r to the numerous monoterpene cyclases which have been i s o l a t e d from herbaceous species (15,16). In general, monoterpene cyclases are soluble enzymes with molecular weights i n ^ t h e raiy*e of 50,000-100,000. They require a divalent cation (Mg or Mn ) for a c t i v i t y , have pH optima between 6 and 7, and are capable of c y c l i z i n g geranyl pyrophosphate as w e l l as the cis-isomer, n e r y l pyrophosphate (NPP), and the t e r t i a r y a l l y l i c isomer, l i n a l y l pyrophosphate (LPP) (17). Studies with p a r t i a l l y p u r i f i e d cyclases have focused on the mechanism and stereochemistry of the c y c l i z a t i o n process, and o f f e r promising i n s i g h t s i n t o the manner by which the monoterpene composition i n conifer wood resins may be controlled. Detailed reviews of the l i t e r a t u r e on terpene biochemistry can be found i n two volumes edited by Porter and Spurgeon (18,19). A volume on plant terpenes has been edited by Nes and associates (20), and s i m i l a r reviews by Loomis and Croteau (21) and Banthorpe and Charlwood (22) are a v a i l a b l e . Terpenes occurring i n trichomes and i n wood extractives have been described by Croteau and Johnson (15,23). S p e c i f i c accounts of monoterpene biochemistry can be found i n reviews by Croteau (16,17,24), C o r i (25), Charlwood and Banthorpe (26) and Bernard-Dagan and co-workers (27). Bark Beetle Attack The r o l e of conifer resins i n resistance to i n f e s t a t i o n by bark beetles and t h e i r fungal symbionts has been discussed extensively by Shrimpton (1), Raffa and Berryman (6), Cates and Alexander (8) and others (28,29), and a b r i e f summary of the i n t e r a c t i o n s between c o n i f e r s , beetles and fungi i s presented here. T r e e - k i l l i n g bark beetles, which attack conifers by boring into the phloem, belong p r i m a r i l y to the genera Dendroctonus, Ips and Scolytus of the Scolytidae (30). Death follows successful c o l o n i z a t i o n of the tree which i s brought about by fungal invasion of xylem t i s s u e . Tree m o r t a l i t y i s usually recessary i f the beetles and t h e i r progeny are to survive, since excavation of egg g a l l e r i e s and subsequent l a r v a l development can only take place a f t e r host defensive reactions have stopped. Pioneer beetles which i n i t i a t e an attack may encounter c o n s t i t u t i v e (preformed) r e s i n i n c o r t i c a l b l i s t e r s or when r e s i n ducts are severed. Contact with the r e s i n leads to emission of v o l a t i l e aggregation pheromones, which i n many cases are derived from host monoterpenes, and t h i s i n turn leads to a mass attack by f l y i n g beetles. Mass attack may deplete the flow of



6. JOHNSON AND CROTEAU


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c o n s t i t u t i v e r e s i n i n 1-2 days, and i n the absence of further r e s i n o s i s , the beetles w i l l successfully colonize the tree. Healthy trees react to wounding and fungal invasion by a series of well-documented responses. F i r s t , the wound s i t e develops a layer of autolysed c e l l s (necrotic lesion) which serves to deprive the pathogen of nutrients and compartmentalize the damage (this i s the hypersensitive response which i s common i n higher plants (3,31)). Second, adjacent wood parenchyma d i f f e r e n t i a t e into traumatic r e s i n ducts and secrete a secondary r e s i n i n t o the wound s i t e and sapwood, creating a reaction zone around the wound (3,6). Bark beetles respond to r e s i n o s i s e i t h e r by attempting to clear r e s i n from the wound or by abandoning the wound s i t e . In the absence of r e s i n o s i s , beetles e a s i l y bore through the necrotic l e s i o n , allowing the fungus to spread unhindered. Secondary r e s i n o s i s and the hypersensitive response are also important factors i n fungal development. I f the flow of r e s i n i s s u f f i c i e n t l y i n t e n s i v e , fungal growth can be prevented (1,32,33). However, since secondary r e s i n i s derived from xylem carbohydrate reserves, a pervasive fungal attack, mediated by numerous beetle penetrations, w i l l deplete t h i s carbon source, leading to reduced secondary flow and increased tree v u l n e r a b i l i t y (6). Resin Composition and Host Resistance While other r e s i n components, such as diterpene acids, show little i n t r a s p e c i f i c v a r i a t i o n , monoterpene content varies greatly from tree to tree even w i t h i n small stands. This v a r i a b i l i t y has been a t t r i b u t e d to s e l e c t i o n f o r resistance to bark beetle i n f e s t a t i o n . Monoterpenes of conifer oleoresins may have opposing functional e f f e c t s as both insect r e p e l l e n t s and as pheromone precursors and synergists. For example, Sturgeon and Mitton (34) have d e t a i l e d the complex balance between the repellent r o l e of limonene i n ponderosa pine o i l and the attractant r o l e of a-pinene as the aggregation pheromone precursor of the western pine beetle (Dendroctonus brevicomis). A high concentration of limonene alone i s i n s u f f i c i e n t to deter attack by the pine beetle; however, trees bearing both high limonene and low cx-pinene content avoid c o l o n i z a t i o n . Bark beetles may encounter oleoresin having d i f f e r e n t monoterpene compositions at three d i f f e r e n t stages of c o l o n i z a t i o n . Constitutive r e s i n stored i n c o r t i c a l b l i s t e r s and r e s i n ducts may serve to deter pioneer beetles, and c o r t i c a l monoterpene content has often been used as an i n d i c a t o r of insect resistance (13,35). Beetle attack initiates a hypersensitive wound response i n which parenchyma around the wound s i t e d i f f e r e n t i a t e s and secretes new r e s i n . Subsequent fungal invasion elicits a strong resinous infusion ( f u n g a l - e l i c i t e d resin) from the sapwood at a considerable distance from the wound s i t e , and t h i s process continues u n t i l either the fungus or the tree dies (6). Thus, a c a r e f u l measure of resistance must account f o r r e s i n quantity and composition at each i n f e c t i o n stage. Studies on the i n t e r a c t i o n s between grand f i r (Abies grandis), the f i r engraver beetle (Scolytus v e n t r a l i s ) and the



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associated fungus Trichosporium symbioticum have shown that monoterpenes which are p a r t i c u l a r l y r e p e l l e n t to the beetle and t o x i c to the fungus occur p r i n c i p a l l y i n the secondary (induced) r e s i n rather than i n c o n s t i t u t i v e r e s i n (9,36). Components which are found to s p e c i f i c a l l y increase i n the induced r e s i n include myrcene, limonene, a-pinene and 3-carene. (The t o t a l monoterpene content increases 25-fold over p r e - i n f e s t a t i o n r e s i n l e v e l s . ) In a r e l a t e d study, r e s i s t a n t lodgepole pine ( P . contorta) was shown to produce p a r t i c u l a r l y high l e v e l s of a-pinene and limonene i n the secondary r e s i n . In t h i s case, however, the major f a c t o r i n resistance to the mountain pine beetle (Dendroctonus ponderosae) and associated fungus (Europhium clavigerum) appeared to be a rapid and vigorous secondary r e s i n o s i s with increased production of a l l c o n s t i t u t i v e monoterpenes (6,32,37). This finding correlates w e l l with f i e l d studies i n which only stressed, weakened or old trees, which are u n l i k e l y to mount a vigorous defensive response, were observed to be s u c c e s s f u l l y colonized by endemic bark beetle populations. Survival of healthy trees during epidemics, however, depends on a highly v a r i a b l e i n t r a s p e c i f i c r e s i n composition which increases the p r o b a b i l i t y that some trees w i l l have the r i g h t combination of r e s i n components to be e f f e c t i v e against the attacking beetles (34). Thus, genetic e f f e c t s which determine subtle differences i n r e s i n composition are probably the c r i t i c a l factors i n conifer s u r v i v a l during epidemics, since both p h y s i o l o g i c a l l y weak trees and healthy trees which produce the precursors of insect a t t r a c t a n t s are l i k e l y to be eliminated. The o p t i c a l p u r i t y of r e s i n terpenoids can have a significant e f f e c t on the success or f a i l u r e of beetle c o l o n i z a t i o n since generally only one enantiomer i s e f f e c t i v e as a pheromone precursor or synergist. Ips typographis bark beetles produce the aggregation pheromone (-)-cis-verbenol from host r e s i n (-)-a-pinene. This insect also produces the b i o l o g i c a l l y i n a c t i v e (+)-trans-verbenol from host r e s i n (+)-a-pinene. Since both enantiomers of a-pinene often co-occur i n wood r e s i n s , the proportion of (-)-a-pinene would be expected to be a f a c t o r i n tree v u l n e r a b i l i t y to mass attack. This expectation was confirmed by Klimetzek and Francke (38) i n a study of a l l the verbenol stereoisomers produced by beetles exposed to natural oleoresins containing varying proportions of (+)and (-)-a-pinene. The proportion of (-)-a-pinene i n the r e s i n was shown to be d i r e c t l y r e l a t e d to the amount of (-)-cis-verbenol produced, providing a chemical basis f o r beetle-host s p e c i f i c i t y . The isomeric compound (-)-trans-verbenol i s produced from host-derived (-)-a-pinene by Dendroctonus brevicomis and functions as a r e p e l l e n t to female beetles, presumably to reduce i n t r a s p e c i f i c competition. The antipodal (+)-trans-verbenol produced from (+)-a-pinene, i s without b i o l o g i c a l e f f e c t (39). The e f f e c t s of enantiomeric monoterpenes on bark beetle a c t i v i t y have been reviewed by Wood (30), Francke and V i t e (40) and S i l v e r s t e i n (41). In some cases the presence of as l i t t l e as 2-5% of the enantiomeric product completely i n h i b i t s the e f f e c t of the active antipodal form (30).



JOHNSON AND CROTEAU


Optical p u r i t y has also been shown to influence the f u n g i s t a t i c properties of oleoresin monoterpenes. The growth of b l u e - s t a i n fungi (Ceratocytis spp.) and Fomes annosus i s i n h i b i t e d by c h i r a l monoterpenes from ponderosa pine r e s i n (e.g., limonene, 3-pinene and 3-phellandrene and a-pinene). The greatest enantiomeric e f f e c t i s observed with (+) -a-pinene which i s three times more i n h i b i t o r y against Ceratocystis than the (-)-antipode (42,43). In another study using Pinus radiata monoterpenes, (+)-limonene was shown to be a potent i n h i b i t o r of Diplodea pinea spore germination while (-)-limonene was i n a c t i v e (44). The role of monoterpene enantiomers in imparting d i f f e r e n t i a l conifer resistance to bark beetle attack has received l i t t l e attention because of the d i f f i c u l t i e s i n p u r i f y i n g the i n d i v i d u a l monoterpenes and i n quantitating the o p t i c a l antipodes. Techniques which have been used to determine o p t i c a l p u r i t y include preparative g a s - l i q u i d chromatography (GLC) followed by polarimetry or NMR analysis using c h i r a l s h i f t reagents, GLC analysis of diastereomeric d e r i v a t i v e s , and, more recently, d i r e c t separation of enantiomers by c h i r a l phase c a p i l l a r y GLC (30,40,45,46). The l a t t e r technique i s both convenient and very s e n s i t i v e , and with t h i s development, new information concerning the r o l e s of antipodal monoterpenes should be forthcoming. Fungal E l i c i t o r s Wounding alone, caused by insects or mechanical damage, w i l l induce a resinous wound response that includes formation of traumatic r e s i n ducts i n the sapwood (1,47). However, an early study by Hepting showed that conifers respond more vigorously to fungal i n f e c t i o n than to simple wounding (48). Live canker fungus (Fusarium sp.) induced 30 times the r e s i n flow from wounds on Pinus v i r g i n i a n a than wounds where dead fungus was applied. Later studies, using a r t i f i c i a l inoculations of bark beetle fungal symbionts (Ceratocystis, Europhium and Trichosporium) have c l e a r l y supported the r o l e of the fungus i n induction of secondary r e s i n production i n conifers (1,6,7,32,37). Further support for fungal e l i c i t a t i o n has been obtained recently from tissue culture studies. Laszlo and Heinstein (49) have shown that heat inactivated Fusarium sp. induce a 4- to 10-fold increase i n diterpene r e s i n acid production (especially dehydroabietate) by pine c e l l suspension cultures. Further consideration of conifer resistance mechanisms must take into account the nature of the chemical signals involved (fungal e l i c i t o r s ) , t h e i r o r i g i n and t h e i r e f f e c t s on plant metabolism and c e l l d i f f e r e n t i a t i o n . D a r v i l l and Albersheim (50), Dixon and associates (51), Hadwiger and co-workers (52,53) and West (54) have reviewed the extensive literature on elicitors responsible for the biosynthesis of phytoalexins (newly-synthesized compounds which impart resistance). I t i s now clear that e l i c i t o r s of p h y s i o l o g i c a l o r i g i n are soluble carbohydrates derived by enzymatic hydrolysis of either plant cell walls


8


82

ECOLOGY AND METABOLISM OF PLANT LIPIDS (polygalacturonates) or fungal walls (chitosans) and that these signals a r i s e from pathogen attack or plant counterattack. E l i c i t o r s from mycelial walls of a pathogenic fungus induce the rapid appearance of chalcone synthetase mRNA i n bean c e l l s , thus showing that phytoalexin synthesis i s c o n t r o l l e d at the t r a n s c r i p t i o n a l l e v e l (55). The postulated mechanism of e l i c i t o r action involves binding to a s p e c i f i c c e l l membrane receptor which t r i g g e r ^ the release of an i n t r a c e l l u l a r second message (possibly Ca ) and i n i t i a t e s new mRNA synthesis f o r the enzyme(s) of phytoalexin biosynthesis (51). In a preliminary study, the presumptive induction of monoterpene cyclases, and the r e s u l t i n g changes i n monoterpene content, were examined i n lodgepole pine saplings i n response to fungal i n f e c t i o n (56). Two year-old lodgepole pine were inoculated with Europhium clavigerum, a symbiont of the mountain pine beetle, and t o t a l monoterpere content was monitored over a 12-day period. Infected stems accumulated three times the monoterpene l e v e l of untreated controls ( F i g . 2, Table I ) . Aseptic wounds led to a lesser increase (1.5-fold) i n monoterpene l e v e l which i s a c h a r a c t e r i s t i c wound response i n c o n i f e r s . S p e c i f i c increases i n the l e v e l s of the monoterpene o l e f i n s a-pinene, 3-pinene, 3-carene and 3-phellandrene, which are t o x i c to t h i s fungus, were found i n infected tissues compared to controls. Two weeks a f t e r i n f e c t i o n , monoterpene cyclase l e v e l s were measured and compared to untreated controls. (Crude stem homogenates were assayed with [ H]geranyl pyrophosphate as substrate under standard conditions f o r monoterpene cyclases (57).) Infected tissue exhibited a corresponding 3-fold increase i n monoterpene hydrocarbon cyclase a c t i v i t y . Thus, both monoterpene cyclase a c t i v i t i e s and monoterpene l e v e l s increased i n lodgepole pine i n response to fungal i n f e c t i o n , i n a manner reminiscent of the t y p i c a l phytoalexin response. E l i c i t o r - s t i m u l a t e d r e s i n production i s u l t i m a t e l y l i m i t e d by the a v a i l a b i l i t y of carbohydrate reserves, which serve as r e s i n precursors (58). The death of sapwood parenchyma due to fungal toxins or other p h y s i o l o g i c a l malfunction (59,60) w i l l obviously also l i m i t r e s i n production. The r e l a t i o n s h i p between the extent of fungal attack and the quantity of monoterpenes synthesized has been examined by Raffa and Berryman (32). Lodgepole pine exhibited a 6-fold increase i n monoterpene content over c o n s t i t u t i v e l e v e l s at the wound s i t e when subjected to up to 10 inoculations of Europhium per 0.3 m , and a 30-40% decrease i n t h i s maximum when subjected to 20-35 inoculations per 0.3 m . Given these inherent l i m i t a t i o n s of tree physiology (due to age, vigor and s i z e ) , an understanding of the o r i g i n and mechanism of action of fungal e l i c i t o r s i s e s s e n t i a l to the development of protective strategies against bark beetle attack and subsequent fungal invasion. 3

2

2

Biosynthesis of Monoterpenes i n Conifers The v a r i a t i o n i n monoterpene s k e l e t a l types found i n conifer oleoresins i s determined to a large degree by d i f f e r e n t i a l expression of the corresponding monoterpene cyclases. These




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Days

Figure 2. Effect of time on the accumulation of monoterpenes i n i n fected or wounded lodgepole pine stems. Two year-old lodgepole pine (P. contorta) were infected with the vegetative form of Europhium olavigerum or wounded a s p e t i c a l l y . Monoterpenes were quantified following GLC analysis of steam d i s t i l l a t e s of stem t i s s u e .

TABLE I . Effect of Wounding and Infection on the Monoterpene Composition of Lodgepole Pine Stems 5

Monoterpene*

Control

Aseptic Wound

Infected Wound

a-Pinene 8-Pinene 3-Carene 3-Phellandrene a-Phellandrene Limonene Camphene

0.44 1.55 0.27 1.22 0.14 0.85 tr

0.69 2.79 0.34 2.14 0.14 0.70 tr

1.68 5.97 1.33 3.14 0.08 0.22 0.09

Total Monoterpenes

4.47

6.79

12.51

Two year-old Lodgepole pine saplings were infected with Europhium clavigerum or wounded a s e p t i c a l l y . Monoterpene content of pine stems was determined 10 days a f t e r treatment following steam d i s t i l l a t i o n and quantitation of peak areas obtained from g a s - l i q u i d chromatography. ^Composition i s given i n mg/g dry weight.



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enzymes catalyze the crucial ring-forming reactions in monoterpene biosynthesis by e x p l o i t i n g a v a r i e t y of e l e c t r o p h i l i c c y c l i z a t i o n s of geranyl pyrophosphate which involve carbonium ion attack on a double bond as the primary C-C bond-forming event (61). Geranyl pyrophosphate (Fig. 3) i s derived by l i g a t i o n of two 5-carbon isoprenoid u n i t s , isopentenyl pyrophosphate (IPP) and d i m e t h y l a l l y l pyrophosphate (DMAPP), under the influence of the enzyme prenyl transferase ( s p e c i f i c a l l y GPP synthetase) (15,62). Monoterpene biosynthesis has been studied i n conifers using labeled precursors such as carbon dioxide, acetate and mevalonate (63,64). S p e c i f i c a l l y labeled precursors have been employed to deduce mechanistic features of a-pinene (65,66) and 3-carene (67,68) biosynthesis i n pine species. Gleizes and co-workers (69) have argued, by way of time-course studies, that the i n i t i a l formation of a c y c l i c hydrocarbons (ocimene, myrcene) from C02 i n Pinus pinaster needles indicated that these o l e f i n s serve as precursors to c y c l i c monoterpenes (a-pinene, 3-pinene) by a reversible protonation mechanism. These suggestions, however, are without precedent, and run counter to d i r e c t evidence demonstrating that the c y c l i z a t i o n of geranyl pyrophosphate occurs without the involvement of free intermediates (17). Studies on monoterpene biosynthesis using c e l l - f r e e extracts from conifers have been few i n number, due i n large part to the l i m i t a t i o n s i n propagating conifers ( i . e . , slow growth) and to the d i f f i c u l t i e s i n extracting enzymes from woody t i s s u e . C y c l i c terpenes (Cio, C15) are reportedly synthesized from IPP or NPP i n p a r t i c u l a t e f r a c t i o n s of Pinus pinaster and Douglas f i r needles (70,71); yet Cori (72) has demonstrated that soluble enzymes from Pinus radiata seedlings convert GPP and NPP to c y c l i c monoterpene o l e f i n s (a-pinene, 3-pinene and limonene). In none of these studies have the products or the responsible enzymes been adequately characterized. Recently developed procedures for i s o l a t i n g terpene cyclases (57) , and methods for obtaining active enzymes from sapwood extracts (73), should f a c i l i t a t e studies on the enzymology of monoterpene biosynthesis i n c o n i f e r s . 14

Mechanism of Monoterpene C y c l i z a t i o n Monoterpene c y c l i z a t i o n processes have been studied p r i m a r i l y i n herbaceous species, and cyclase preparations have been obtained which catalyze the c y c l i z a t i o n of geranyl pyrophosphate to e s s e n t i a l l y a l l major s t r u c t u r a l classes. M u l t i p l e cyclases, each producing a d i f f e r e n t s k e l e t a l arrangement from the same a c y c l i c precursor co-occur i n higher plants, while s i n g l e cyclases which synthesize a v a r i e t y of stereochemically d i s t i n c t s k e l e t a l types are also known (24,57). I n d i v i d u a l cyclases, each generating a simple d e r i v a t i v e or p o s i t i o n a l isomer of the same s k e l e t a l type, have been described, as have d i s t i n c t cyclases catalyzing the synthesis of enantiomeric products (74). The mechanistic outlines of the c y c l i z a t i o n of geranyl pyrophosphate have been the subject of some controversy (17) because GPP i s precluded from d i r e c t c y c l i z a t i o n (to cyclohexanoid products) by the C2-trans double bond. Various isomerization schemes have



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Figure 3. Unified stereochemical mechanism of monoterpene c y c l i z a t i o n from geranyl pyrophosphate.



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ECOLOGY AND METABOLISM OF PLANT LIPIDS been proposed f o r the preliminary conversion of geranyl pyrophosphate to either NPP or LPP (or for the independent o r i g i n of these compounds), both of which can c y c l i z e d i r e c t l y . However, since i t i s now clear that GPP i s e f f i c i e n t l y c y c l i z e d without formation of free intermediates, i t follows that monoterpene cyclases must be capable of c a t a l y z i n g both the required isomerization to a bound intermediate competent to c y c l i z e and the c y c l i z a t i o n reaction i t s e l f . The mechanism of c y c l i z a t i o n of geranyl pyrophosphate i s now considered to involve the i n i t i a l i o n i z a t i o n of the pyrophosphate moiety, i n which a divalent metal ion i s presumed to a s s i s t , followed by stereospecif i c s_yn-isomerization to a l i n a l y l intermediate ( i . e . , l i n a l y l pyrophosphate, the corresponding i o n - p a i r , or other bound equivalent), with r o t a t i o n about the C2-C3 single bond and subsequent c y c l i z a t i o n of the c i s o i d rotamer i n the anti-endo conformation. The c h i r a l a - t e r p i n y l cation so produced, may then undergo e l e c t r o p h i l i c addition to the remaining double bond, as w e l l as hydride s h i f t s and rearrangements, to provide the various other c a t i o n i c c y c l i c parents. The i n i t i a l l y formed c y c l i z a t i o n products, c o n s i s t i n g of one or more r i n g s , are generated as o l e f i n s ( v i a deprotonation) or simple alcohol or ether d e r i v a t i v e s (via capture by a nucleophile). Subsequent transformations, frequently o x i d a t i o n , u l t i m a t e l y give r i s e to the vast number of monoterpene metabolites. This u n i f i e d i s o m e r i z a t i o n - c y c l i z a t i o n scheme i s completely consistent with the r e s u l t s of numerous model studies of terpenoid c y c l i z a t i o n s , r e a d i l y allows for the observed d i r e c t c y c l i z a t i o n of both LPP and NPP as alternate substrates, and, most importantly, accounts for the formation of e s s e n t i a l l y a l l c y c l i c types as w e l l as the regio- and enanticselectivities inherent i n cyclase catalysis (75). I m p l i c i t i n t h i s stereochemical model i s the postulated f o l d i n g of GPP by the enzyme on binding, which determines the absolute configuration of the intermediate LPP, which i n turn d i c t a t e s the stereochemical outcome of the subsequent cyclization. Accordingly, antipodal monoterpenes would be derived from d i f f e r e n t enzymes which generate opposite enantiomers of LPP i n the isomerization step of the coupled sequence (74,75). Direct evidence f o r the above proposal was obtained with the demonstration that separable cyclases derived from sage (Salvia officinalis) synthesize monoterpene olefins of opposite stereochemistry (74,76). Thus, cyclase I , of MW 96,000, converted GPP to (+)-a-pinene, (+)-camphene and (+)-limonene of related configuration, whereas cyclase I I , of MW 55,000, transformed the same a c h i r a l precursor to (-)-3-pinene i n addition to (-)-a-pinene, (-)-camphene and (-)-limonene. Extensive purification of each enzyme and differential i n a c t i v a t i o n studies ensured that each set of stereochemically related products was synthesized by a s i n g l e , d i s t i n c t enzyme (74). Since (±)-LPP had been shown to serve as a precursor for both enzymes (76) i t was possible to d i r e c t l y assess the absolute configuration of the t e r t i a r y intermediate c y c l i z e d , by the preparation and separate t e s t i n g of each enantiomer. As predicted by the general model ( F i g . 3), 3R-LPP p r e f e r e n t i a l l y



6.

JOHNSON AND

CROTEAU


gave r i s e to the (+)-olefins generated by cyclase I , whereas (3S)-LPP p r e f e r e n t i a l l y gave r i s e to the ( - ) - o l e f i n series generated by cyclase I I . The c y c l i z a t i o n scheme posits the formation of a- and 3-pinene by alternate deprotonations of a p i n y l cation (76). Banthorpe and associates have suggested, on the basis of i n vivo studies (65,66), that (-)-a-pinene could be formed indirectly via the thermodynamically favorable isomerization of (-)-3-pinene. No support for t h i s proposal was obtained with the c e l l - f r e e systems described above (74,76), nor could an alternate proposal by Gleizes and associates (69) for the r e v e r s i b l e protonation of ocimene and myrcene to a- and 3-pinene, respectively, be substantiated in cell-free preparations. Support f o r the c r u c i a l r o l e of the isomerization step of the reaction sequence was also obtained from studies on geranyl pyrophosphate:(-)-endo-fenchol cyclase from fennel (Foeniculum vulgare) (77-79). Direct t e s t i n g of ( 3 R ) - l i n a l y l pyrophosphate afforded a K value lower than that obtained with geranyl pyrophosphate,—and a r e l a t i v e v e l o c i t y nearly three-times higher. These r e s u l t s are clearly consistent with the proposed stereochemical model and further suggest that the isomerization step i s rate l i m i t i n g i n the coupled i s o m e r i z a t i o n - c y c l i z a t i o n of geranyl pyrophosphate to (-)-endo-fenchol (Fig. 3). ( 3 S ) - L i n a l y l pyrophosphate was not an e f f e c t i v e substrate for (-)-endo-fenchol biosynthesis but d i d , by an anomalous c y c l i z a t i o n , give r i s e to low l e v e l s of the enantiomeric (+)-(1R)-endo-fenchol. I t was therefore concluded that enzymatic recognition of the t e r t i a r y intermediate i n an approach from s o l u t i o n was not s u f f i c i e n t to completely discriminate between the s i m i l a r o v e r a l l p r o f i l e s presented by the l i n a l y l pyrophosphate enantiomers. While the formation of (+)-endo-fenchol from ( 3 S ) - l i n a l y l pyrophosphate i s c l e a r l y an aberrant reaction sequence, the stereochemical outcome i s s t i l l e n t i r e l y consistent with the c y c l i z a t i o n model. More importantly, the complete absence of the (+)-isomer with the a c h i r a l geranyl pyrophosphate as substrate serves to confirm the absolute regio- and stereochemical c o n t r o l over the i n i t i a l isomerization step as w e l l as the subsequent c y c l i z a t i o n of the normal, coupled reaction sequence. Thorough stereochemical analysis of the c y c l i z a t i o n s of geranyl pyrophosphate to (+)- and (-)-bornyl pyrophosphate ( F i g . 3) (by enzymes from S a l v i a o f f i c i n a l i s and Tanacetum vulgare, respectively) have also been c a r r i e d out (80,81). These reactions, which generate the c y c l i c parents of (+)- and (-)-camphor (82-84), are unique among monoterpene c y c l i z a t i o n s i n that the pyrophosphate moiety of the a c y c l i c precursor i s retained i n the c y c l i c product. In t h i s instance i t was demonstrated that geranyl pyrophosphate ( c h i r a l l y labeled) was cyclized to bornyl pyrophosphate with net retention of configuration at CI of the a c y c l i c precursor, whereas s i m i l a r l y labeled n e r y l pyrophosphate was c y c l i z e d to product with inversion of configuration at CI (85). The observed stereochemistry i s a consequence of the reaction mechanism whereby geranyl pyrophosphate i s f i r s t stereospecifically isomerized to l i n a l y l pyrophosphate which, following r o t a t i o n


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about C2-C3 to the c i s o i d conformer, c y c l i z e s from the anti-endo configuration. Neryl pyrophosphate c y c l i z e s either d i r e c t l y or v i a the l i n a l y l intermediate without the attendant r o t a t i o n . The r o l e of i o n p a i r formation i n monoterpene c y c l i z a t i o n s was revealed by a d d i t i o n a l studies with the (+)- and (-)-bornyl pyrophosphate cyclases i n which the migrations of the pyrophosphate moiety were examined (86,87). Separate incubation of [1- H ;

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