Regulation of Accumulated Phytoalexin Levels in Ladino Clover Callus

5

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Regulation of Accumulated Phytoalexin Levels in Ladino Clover Callus David L. Gustine U.S. Regional Pasture Research Laboratory, USDA, Agricultural Research Service, University Park, PA 16802

Thiol/disulfide ratios and the interaction of biological disulfides with plasma membrane proteins are postulated as mechanisms by which Ladino clover callus cells regulate biosynthesis and accumulation of the phytoalexin, medicarpin. Evidence is presented to show that organic mercurial sulfhydryl (SH) reagents and N-ethyl maleimide react with free sulfhydryl groups in the cell wall membrane of callus cells. Mersalyl, a non-penetrating SH reagent, was a weak elicitor of medicarpin production in callus, while other sulfhydryl reagents tested in the callus system elicited levels as high or higher than levels reported to be elicited in plants. Biological dithiols, cystine and oxidized glutathione, were also elicitors. Incorporation studies with C-L-phenylalanine and C-acetate showed that medicarpin accumulated in the callus cells because its rate of biosynthesis was increased. Results from these studies support the hypothesis that membrane constituents containing sulfhydryl groups are important factors in regulation of phytoalexin synthesis. 14

14

Plant tissue culture i s an extremely useful tool for investigating biochemical and hormonal aspects of plant metabolism. Such systems have been applied to studies of host-pathogen Interactions and secondary metabolism. My laboratory has been investigating the biochemical regulation of phytoalexin accumulation i n hostpathogen interactions of cultured legumes. Phytoalexins are generally recognized as important components of Induced resistance i n many plants. These metabolites are not found i n healthy plant tissues or are present at very low concentrations. They accumulate as a r e s u l t of interactions between plant c e l l s and e l i c i t o r molecules. Fragments of fungal or plant c e l l walls can act as b i o l o g i c a l e l i c i t o r s , apparently This chapter not subject to U.S. copyright. Published 1986, American Chemical Society

Green and Hedin; Natural Resistance of Plants to Pests ACS Symposium Series; American Chemical Society: Washington, DC, 1986.

56

NATURAL RESISTANCE OF PLANTS TO PESTS

formed by enzymatic hydrolysis of glucan or pectin polysaccharides (1). Phytoalexins are e f f e c t i v e a n t i b i o t i c s because they quickly accumulate i n high concentrations at the s i t e of invasion ( 1). Ample evidence now exists to demonstrate a direct role for phytoalexins i n preventing successful i n f e c t i o n by fungi (2-3-4) and bacteria (5).


Phytoalexins i n A l f a l f a and Clovers At least 10 pterocarpanoid phytoalexins have been i d e n t i f i e d i n a l f a l f a and clover species. These a n t i b i o t i c metabolites have been e l i c i t e d by fungi, b a c t e r i a , and a variety of a b i o t i c substances. Biosynthesis of pterocarpanoid phytoalexins begins with phenylalanine (6) and follows the pathway outlined by Martin and Dewick (2)* depicted i n Figure 1. Medlcarpin, satIvan, isosativan, and v e s t i t o l are produced i n most clovers and a l f a l f a i n response to b i o t i c and a b i o t i c e l i c i t o r s . In clovers, e l i c i t o r s also induce accumulation of maackiain, i s o v e s t i t o l , and arvensin (8). The role of these phytoalexins i n resistance to diseases i n a l f a l f a and clovers has not been f u l l y resolved. Studies of a l f a l f a and red clover showed that medlcarpin or maackiain i s accumulated i n response to pathogens, but data are lacking on the concentration and l o c a l i z a t i o n of these metabolites at the i n f e c t i o n s i t e s (9-10-11-12-13). Limited evidence suggests that rates of synthesis and concentrations of medlcarpin and/or satlvan in a l f a l f a leaf and stem tissues are related to resistance to V e r t i c i l l i u m albo-atrum (14-15-16). Thus i t appears that phytoalexins at least partly r e s t r i c t growth of fungi i n these forage legumes. This i s not unexpected since phytoalexins are components of resistance i n many plant/pathogen interactions involving fungi, b a c t e r i a , nematodes, or viruses (17). Phytoalexins i n Legume Tissue Cultures Tissue Cultures Respond to E l i c i t o r s . Recent investigations i n my laboratory have established that c a l l u s cultures of Canavalia ensiformis (jackbean), Medicago sativa ( a l f a l f a ) , and nine species of T r i f o l i u m (clover) biosyntheeize phytoalexins i n response to e l i c i t o r s i n e s s e n t i a l l y the same way that leaves, hypocotyls, or seedlings do. When c a l l u s tissue cultures were exposed to spores of Pithomyces chartarum (18) or sulfhydryl (SH) reagents (19-20), medlcarpin accumulated at concentrations higher than those reported for whole plants (13) or plant parts treated with other e l i c i t o r s (16-21-22-23-24). The temporal relationships were s t r i k i n g l y s i m i l a r to those observed i n plants. After 6-hour exposure to e l i c i t o r , medlcarpin concentration increased, then reached a maximum by 48 hours, and decreased s l i g h t l y over the next 24 hours (18). A related pterocarpan (maackiain) was also found i n both whole plant and callus tissue. However, some phytoalexins found i n detached, fungus-treated clover l e a f l e t s (Table I) were not found i n e l i c i t e d c a l l u s cultures (Table I I ) .


GUSTINE

Phytoalexin

Levels in Ladino

Clover

Callus

ο NH I CH -CH-C00H 9


9

3

II C-SCoA I CH , 3C0 2

C

O

O

H

2

HO

SAM OCH,

Formononetin

1

OCH,

Saltan (R»H, R^CHj, R"=CH3) laoaattan (R=CH» R' = H, R'^CH,) Arvanean (R>CH„ W-CHy R'^H)

Vwtttoi (R*H, R^CHg) laovaatHol (R-CK» R H)

F i g . 1. B i o s y n t h e t i c pathway f o r a l f a l f a and phytoalexins. SAM, S-adenosyl m e t h i o n i n e .

,=

clover


58


Table I.

Phytoalexins i n T r i f o l i u m Detached-Leaflet Diffusâtes (48 h o u r s )


Species

Med

T r i f o l i u m fragiferum T. hybridum T. incarnaturn T. miche1lanum T. medium T. pratenee T. repens T. resuplnatum

76 70 Tr Tr 12 42 91 Tr

Maack

C

0 56 0 0 6 45 0 0

a , b

Phytoalexin 4-MM Vest iVest (yg/ml) 0 2 0 0 0 0 0 0

0 0 48 0 0 0 0 0

164 170 115 75 25 0 0 109

Treated with droplets containing Helminthosporlum

Sat

iSat

0 8 Tr 0 45 0 0 0

0 10 71 0 0 0 0 44

carbonum spores.

^Data from Ingham (24) with permission of author and Pergamon Press» Ltd. °Med " medlcarpin, Maack - maackiain, 4-MM - 4-methoyx-maackiain, Vest - v e s i t o l , iVest - i s o v e s t i t o l , Sat - sativan, iSat isosatlvan, Tr » trace.

Table I I .

Phytoalexin Concentrations i n HgCl -stimulated 2

Legume Callus Tissues (48 h o u r s )

Cultured Plant T r i f o l i u m fragiferum L. T. hirtum A l l . T. hybridum L. T. incarnatum L. T. medium L. T. miche 1 lanum Savi T. pratense L. cv. Kenstar T. repens L. cv. Ladino T. resuplnatum L. Canavalia ensiformis L. Medicago satlva L.

&

Medlcarpin Maackiain (yg/g fresh callus) 99 35 6 17 20 100 0 40 34 15 30

0 0 0 0 23 0 8 0 0 0 0

*Data from Gustine and Moyer (20) with permission of authors and Martinus N i j h o f f . ^Other fungitoxic compounds were detected by thin-layer chromatography, but not i n s u f f i c i e n t quantity for i d e n t i f i c a t i o n .



5.

GUSTINE

Phytoalexin

Levels in Ladino

Clover

Callus

Sativan, v e s t i t o l , 4-methoxy-maackiain, i s o v e s t i t o l , and lsosativan were not detected i n callus cultures derived from legumes that normally produce them. These phytoalexins may have been present at very low levels or were not e l i c i t e d by mercuric chloride. The phytoalexin response of callus cultures derived from Ladino clover to various substances i s shown i n Table I I I . Most of the e l i c i t o r s l i s t e d for the callus system have also shown e l i c i t o r a c t i v i t y i n other plant phytoalexin-producing systems. However, most of the a b i o t i c and b i o t i c n o n - e l i c i t o r s for the callus system (Table III) are e l i c i t o r s i n whole plant systems (see j_ and references therein). It i s clear from e a r l i e r reports (18-19-20) and the data i n Tables II and III that production of medlcarpin and maackiain i n response to e l i c i t o r s i s retained i n cultured c e l l s and, most importantly, that biochemical regulation of phytoalexin accumulation i s not l o s t . Mechanisms Regulating Accumulation of Medlcarpin i n Legume Callus Cultures Within 6 hours after exposure of jackbean callus to P. chartarum spores, phenylalanine ammonia lyase (PAL) a c t i v i t y i n crude extracts, measured spectrophotometrically, Increased and reached maximum l e v e l by 24 hours (2-fold Increase). During the next 24 hours, the a c t i v i t y decreased to about 50% of maximum (data not sjjiown). When Sephadex Gy 100 p u r i f i e d fractions were assayed with C-substrates for PAL ( C-L-phenylalanine^ and 0-methyl transferase (OMT) (S-adenoeyl methionine, C-methyl) a c t i v i t i e s , increased levels were also found (18). The radiometric assays showed that PAL and daidzein OMT a c t i v i t i e s increased 2- and 4-fold, respectively, after 36-hour exposure to spores. OMT a c t i v i t i e s also increased 4-fold when i s o l i q u i r i t i g e n i n or genistein were used as substrates. The above enzymes and substrates are considered to be part of the medlcarpin blosynthetic pathway. Caffeic acid, naringenin, and apigenin, although not i n the medlcarpin blosynthetic pathway, were good substrates for OMT. However, they were not methylated at a greater rate i n preparations from spore-treated c a l l u s . Thus, OMT enzymes that are part of the medlcarpin blosynthetic pathway seemed to be s p e c i f i c a l l y stimulated. Stimulation of PAL and other enzyme a c t i v i t i e s associated with biosynthesis of isoflavonoid phytoalexins has been demonstrated i n other plants (25-26). Increased enzyme a c t i v i t y was accompanied by increased synthesis of mRNA's for the s p e c i f i c enzymes (27-28). As shown by elegant experiments conducted i n Lamb's laboratory (29-30), increased de novo protein synthesis, as shown by incorporation of deuterium from D 0 into PAL protein, accounts for the increased enzyme a c t i v i t y during phaseollin accumulation i n French bean. Recent studies u t i l i z i n g genetic engineering technology demonstrated PAL and 4-coumarate CoA ligase mRNAs increased coordinately with enzyme a c t i v i t y i n parsley suspension cultures treated with c e l l wall e l i c i t o r from Phytophthora megasperma (31). S i m i l a r l y , chalcone synthase (CS) 2


59

60


Table I I I .

Response of Trifolium repens L. cv. Ladino Clover


Callus to B i o t i c and Abiotic M a t e r i a l s

0

Elicitors

Non-ellcltors

Biotic

Biotic

Pithomyces chartarum M.B.

Ellis

(Berk. & Curt.)

b

Xanthomonas lespedezae Xanthomonas a l f a l f a e

PseudomonaB corrugata

b

Botrytis cinerea

Helmintho8porium carbonum

Chitin

Cystine

Hexoses^

Oxidized glutathione^ Polygalacturonic acid

b

b

Hexosamlnes fragments

0

Amino acids

Chitosan Abiotic Abiotic

Cycloheximide

Merealyl

b

Actlnomycin D

p-chloromercuribenzoic acid (PCMBA)

Sodium cyanide

p-chloromercuribenzene

Sodium fluoride

sufonic acid

(PCMBS) N-ethylmaleimide

Dimethyl sulfoxide (NEM)

Iodacetamlde (IA) Diamide

b

Mercuric chloride ( H g C l J Oxidized d i t h i o t h r e i t o l Triton X-100 " A l l materials were tested at appropriate concentrations over a 1000-fold range i n a modified Gamborg's medium (19-20). Methyl ethyl ketone (MEK) extracts were examined by s i l i c a gel TLC (MEK-hexanes; 70:30, v/v) or by HPLC (19). b

0 n l y tested i n the c a l l u s system.

C

01igomer mixture from Incubation of Rhizopus s t o l i n i f e r endopolygalacteronaee with polygalacturonic acid (from Dr. Charles A. West). Tested with Canavalia ensiformis (Jackbean) c a l l u s .



5.

GUSTINE

Phytoalexin

Levels in Ladino Clover

61

Callus

a c t i v i t y and mRNA levels were coordlnately Increased i n soybean seedlings inoculated with Ρ. megasperma (32). Increased CS mRNA was demonstrated i n Phaseolus vulgaris suspension cultures i n p a r a l l e l with Increased a c t i v i t y of the enzyme after treatment with e l i c i t o r from Colletotrichum lindemuthianum (33). Increased mRNAs encoding for PAL, CS, and chalcone isomerase were recently reported for the same system (34). These findings provide firm evidence to support the proposed sequence, mRNA synthesis, protein synthesis, and phytoalexin synthesis, i n parsley and bean tissues following stimulation with e l i c i t o r s . This chain of events has yet to be demonstrated i n a l f a l f a or clover tissue cultures. Cline and Albershelm (33) suggested that the primary event i n activating the biochemical processes leading to increased concentrations of phytoalexins i n plants i s the binding of e l i c i t o r s to receptors i n the plasma membrane. I n i t i a l evidence for this was reported by Yoshikawa et a l . ^ 3 6 ) when they found that soybean membrane preparations bound C-laminaran (a β-1,3glucan e l i c i t o r from Phytophthora spp.). The receptor, while not yet characterized, appears to be a protein or glycoprotein. Small, b i o l o g i c a l l y active 6-1,3-glucan oligosaccharide e l i c i t o r s can also be released from larger plant or fungal polysaccharides by β-l,3-endoglucanases found i n the plant c e l l wall (37). Thus i t i s l i k e l y that e l i c i t o r s i n contact with i n cultured plant c e l l s act by mechanisms involving membrane receptors, β-1,3endoglucanases, or both. In addition, other membrane components may be c r u c i a l to e l l c i t o r - s t i m u l a t e d responses. Sulfhydryl Reagents and Thiols E l i c i t Medlcarpin i n Ladino Clover Callus and Soybean Hypocotyls

Accumulation

Sulfhydryl (SH) reagents are excellent e l i c i t o r s for stimulating medlcarpin accumulation In c a l l u s cultures (Tables II and I I I ; 19). The SH reagents l i s t e d i n Table I I I (abiotic e l i c i t o r s ) vary i n their a b i l i t y to penetrate the plasma membrane and are shown i n the order of non-penetrating (mersalyl) to rapidly penetrating (HgCl^) reagents. When Ladino callus was treated with mersalyl and tested for medlcarpin, the l e v e l of phytoalexin was increased to only one-fourth of that f o r 6.3 mM HgCl (Table IV). The 12-fold increase observed i n the same experiment with HgCl^ was t y p i c a l of the c a l l u s response to t h i o l reagents. A l l SH reagents tested so f a r , with the exception of mersalyl, have Induced a 12- to 15-fold increase i n medlcarpin concentration i n c a l l u s . These data suggest that the i n t e r i o r , but not the outer surface, of the plasma membrane contains components that are sensitive to SH reagents. The data further suggest that a l t e r a t i o n of these membrane components by SH reagents activates the biosynthesis of medlcarpin. S i m i l a r l y , Stoeesel (38) has demonstrated g l y c e o l l i n accumulation i n soybean hypocotyls treated with PCMBS, PCMBA, and NEM. He also concluded that the reactive SH groups are associated with the plasma membrane, and they are on the outer surface rather than i n the i n t e r i o r membrane. Further evidence that SH groups can regulate phytoalexin accumulation was obtained by Gustine (19) and Stoeseel (38) who found that SH groups i n the plasma membrane could be protected from SH reagents by DTT pretreatment. 2


62


Table IV. Effect of Penetrating (HgCl^ and Non-penetrating (Mersalyl Acid) Sulfhydryl Reagents on Medlcarpin Accumulation HgCl (mM)"

Medlcarpin (ug/g f r . wt.)


0

0 2.9 6.3 12.6

7.6 93.9 101.2 82.1

i n Ladino Clover C a l l u s

0

Mersalyl Acid (mM)

Medlcarpin (ug/g f r . wt.)

0 1 2 4 8 12 16 20

2.8 8.9 12.1 12.5 20.1 19.8 23.2 24.9

g Callus tissue was mixed, divided into 12 equal portions, and placed i n incubation v i a l s containing Gamborg's modified medium (19-20) and indicated concentrations of a b i o t i c e l i c i t o r . V i a l s were incubated f o r 24 hours. Callus was separated from medium, extracted with MEK, and medlcarpin concentration determined by HPLC analysis of extracts (19). Gustine (19) found that p r i o r incubation of c a l l u s for one hour i n DTT prevented stimulation of medlcarpin accumulation by NEM. To further explore t h i s observation, c a l l u s was incubated for one hour with 50 mM DTT and washed with DTT-free medium to remove the reagent and any oxidized DTT. The c a l l u s was then Incubated for 24 hours with 0 to 80 mM NEM. The amount of medlcarpin accumulated was determined and compared with that found i n c a l l u s e l i c i t e d with NEM but not preincubated with DTT (Figure 2). These r e s u l t s show that c a l l u s preincubated with DTT and washed, required about 36 mM NEM to produce half-maximal response, whereas callus untreated with DTT gave the same response at 10 mM NEM. These data suggest that during pretreatment DTT converted a l l accessible protein and cytoplasmic d i s u l f i d e s to free SH groups, thus greatly increasing the number of reactive s i t e s for NEM. This means that e l i c i t o r a c t i v i t y of SH reagents i s associated with s p e c i f i c free SH groups i n the cytoplasmic membrane rather than with non-specific c e l l t o x i c i t y . Thus, for DTT-treated c a l l u s , the lack of response to NEM at concentrations less than 25 mM was due to t i t r a t i o n of SH groups, which were not associated with the e l i c i t o r - s t i m u l a t e d response. Stoessel (38) found that 10 mM DTT reduced the amount of e l i c i t e d g l y c e o l l i n i n soybean hypocotyls pretreated with 10 mM PCMBS or PCMBA. In contrast to the c a l l u s system, pretreatment with DTT had no e f f e c t i n hypocotyls exposed to 10 mM NEM. Since the r e a c t i v i t y of membrane protein SH groups with t h i o l reagents i s variable, i t i s possible that some or a l l regulatory SH groups are i n the cytoplasm. A l l the SH reagents tested w i l l react with membrane SH groups before they enter the cytoplasm (39). Thus, IA and HgCl may be e f f e c t i v e e l i c i t o r s at concentrations less than 9



GUSTINE

Phytoalexin

Levels *'n Ladino

Clover

Callus

N-Ethylmaleimide, mM

Fig. 2. Accumulation of medlcarpin i n Ladino clover callus e l i c i t e d with N-ethyl maleimide. Data are from three separate experiments. 1) Callus was prepared as described (19) and divided into v i a l s containing the indicated concentrations of NEM (one v i a l for each NEM concentration). After 24-hour incubation period, callus was extracted with methyl ethyl ketone, and medlcarpin concentrations determined i n the extracts by HPLC (19); 2) Same as experiment 1, except the callus was incubated for 1 hour with 50 mM DTT, and washed with DTT-free medium p r i o r to addition of NEM; 0-0. 3) Same conditions as experiment 2; ·-·.



64


5 mM (19) because they react better with cytoplasmic than membraneous SH groups. The p o s s i b i l i t y s t i l l remains that cytoplasmic SH groups may be important i n regulating medlcarpin biosynthesis. When callus was incubated with 50 mM diamide, a reagent that reacts p r e f e r e n t i a l l y with glutathione (GSH) (40) and i s an e l i c i t o r (Table I I I ) , the medlcarpin l e v e l after 24-hour exposure was 40% of that found for 6.3 mM HgCl (Gustine, unpublished). I further found that oxidized GSH (GSSGj and cystine, both b i o l o g i c a l d i t h i o l s , and 2,3-dihydroxy-l,4-dithiolbutane (oxidized DTT) demonstrated e l i c i t o r a c t i v i t y i n Ladino c a l l u s . GSSG and cystine induced medlcarpin levels to about 60% of that obtained with HgCl., while oxidized DTT produced a l e v e l of about 33% of that found i n HgCL^-treated tissue. These r e s u l t s may indicate SH groups associated with the phytoalexin response are regulated by cellular dithiols.

E l i c i t o r s Stimulate Biosynthesis of Medlcarpin i n Ladino Clover Callus Another aspect of regulation of medlcarpin l e v e l s i n Ladino c a l l u s i s control of biosynthesis versus catabolism. As previously mentioned, recent evidence indicates that the primary c e l l response to e l i c i t o r s i s the t r a n s c r i p t i o n of new mRNAs and t r a n s l a t i o n of new protein required f o r phytoalexin biosynthesis. This has been demonstrated i n soybean e l i c i t e d with P. megasperma glucan or HgCl (41-42), i n French bean e l i c i t e d with heatreleased e l i c i t o r from C. lindemuthianum (43), and i n parsley suspension cultures e l i c i t e d with P. megasperma glucan (31). Results from callus experiments arc,consistent with those reports. Callus was allowed to incorporate C-L-phenylalanine or C-acetate at d i f f e r e n t times following treatment with 6.3 mM HgCl , f o r one hour pulse^periods. The concentrations of medlcarpin and amount of C-medicarpin were determined and are compared i n Figure 3. The rates of incorporation of r a d i o a c t i v i t y into medlcarpin from either precursor Increased between 10 and 15 hours, and decreased thereafter. The maximum rate of incorporation occurred between 14 and 18 hours, and preceded the point at which maximum medlcarpin concentration occurred by 10 to 12 hours. Because medlcarpin remained at the maximum l e v e l from 20 to 50 hours, while i t s rate of synthesis decreased, I concluded that i t was not further metabolized. Furthermore, the s p e c i f i c r a d i o a c t i v i t i e s of medlcarpin synthesized from either precursor declined over time (Table V). I f medlcarpin was further metabolized, the s p e c i f i c a c t i v i t i e s would have attained a steady-state l e v e l . Thus, increased medlcarpin concentration i n HgCl^-treated Ladino clover c a l l u s appears to be the result of increased biosynthesis, a finding observed f o r to other phytoalexin-accumulating systems. 2

2



5.

GUSTINE

Phytoalexin

Levek in Ladino

Clover

Callus

65

Time After Addition of Elicitor, hr.

££g. 3. Rate of incorporation of radiolaljgl from C-L-phenylalanine and C-acetate into C-medicarpin. S u f f i c i e n t c a l l u s was mixed i n medium to provide about 1 g of callus per time point. At zero time, callus was added to medium containing 6.3 mM HgCl . At 8, 13, 24, 36, and 50 fyuirs, 5 uCi of C-L-phenylalanine (500 yCi/umole) or 5 uCi of C-acetate (50 yCl/umole) was added to c a l l u s . After 1 hour, the callus was extracted and medlcarpin determined (see Figure 2). Medlcarpin peaks were collected during HPLC analyses and r a d i o a c t i v i t y determined by s c i n t i l l a t i o n counting. 2


66


Table V.

Specific Radioactivity of Medlcarpin After One-hour 14 14 Incorporation of C-L-phenylalanine or C-acetate


Post-Elicitor Time (Hours) 8-9 13-24 24-25 36-37 50-51

S p e c i f i c Radioactivity (yCi/ymole medlcarpin) 14 C-L-phenylalanine 16.0 15.0 11.0 5.0 4.0

^C-acetate 7.2 5.1 2.1 0.8 0.6

B i o l o g i c a l D i s u l f i d e s as Regulators of Medlcarpin Accumulation Coordinated regulation of metabolic processes i s required by plants to adapt to perceived r i s k of i n f e c t i o n . The f i r s t step i n exercising control of the metabolic processes i s recognition of the perceived r i s k , presumably a direct consequence of an e l i c i t o r binding to a receptor. Once recognition occurs, the processes leading to phytoalexin accumulation would be stimulated. These events could be activated by a l l o s t e r l c modification of the receptor, a l t e r a t i o n of the polymerization state of protein subunits of an enzyme, or covalent attachment of an effector molecule to the receptor or an enzyme. Besides modulation of protein function by phosphorylation/dephosphorylatlon, methylation, carboxylation, and r l b o s y l a t l o n , changes In the t h i o l / d i s u l f i d e r a t i o may also a l t e r enzyme a c t i v i t y . Gilbert (44) recently proposed that b i o l o g i c a l d i s u l f i d e s may act as a third messenger. That i s , the second messenger, cAMP, induces metabolic changes i n the t h i o l / d i s u l f i d e r a t i o , which acts as a third messenger to modulate enzyme a c t i v i t y (44). Protein SH groups are often important factors i n enzyme a c t i v i t y — c a t a l y t i c function can be inhibited by SH reagents or can be maintained by reducing reagents such as DTT or mercaptoethanol. It i s clear from t h i s and e a r l i e r evidence (19-38), that oxidation l e v e l of SH groups i n plant c e l l s , most l i k e l y i n the plasma membrane, i s important i n modulating phytoalexin biosynthesis. The proposal that b i o l o g i c a l d i t h i o l s act as a t h i r d messenger to stimulate accumulation of medlcarpin i s highly speculative. Current knowledge suggests several areas to investigate to test this hypothesis. Do plant e l i c i t o r receptors contain SH groups, and must they be oxidized or reduced for binding to occur? Do SH reagents prevent e l i c i t o r binding to receptors and, i f so, does DTT reverse the inhibition? Another area of interest i s whether p e c t i n o l y t i c or c e l l u l o l y t i c enzymes associated with plant membranes or c e l l walls are affected by SH reagents or DTT. Yoshikawa et a l . (36) found that 45 mM NEM, mM iodoacetamide, and 14 mM PCMBA did not prevent binding of C-mycolaminarin to soybean membrane f r a c t i o n s , while 44 mM HgCl reduced binding to 8% of the control. They also reported that


2


5. GUSTINE

Phytoalexin

Levels in Ladino

Clover

Callus

soybean endoglucanase was inhibited more than 90% by 5 mM PCMBA or PCMBS. Data from Gustine (19) and from the present report indicated that a l l of these SH reagents would have e l i c i t e d medlcarpin accumulation In Ladino c a l l u s , as would PCMBS, PCMBA, and NEM In soybean hypocotyls (38). I t i s further shown i n this report that b i o l o g i c a l d i t h i o l s , oxidized GSH, and cystine were e f f e c t i v e e l i c i t o r s of medlcarpin synthesis and accumulation (Table I I I and t e x t ) . Thus, i t i s a t t r a c t i v e to suggest that SH reagents act as e l i c i t o r s by any one or combination of the following three mechanisms: 1) they a l t e r the t h i o l / d i s u l f i d e r a t i o and thus activate the " t h i r d messenger" pathway, 2) they react with free SH groups on a receptor protein to activate the phytoalexin response, or 3) they react with free SH groups i n endoglucanases or endopolygalacturonases, stimulating the enzymatic formation of oligosacharide "endogenous" e l i c i t o r s . Since preliminary data indicates SH reagents have no e f f e c t or i n h i b i t endoglucanase from soybean (36), the l a s t p o s s i b i l i t y appears u n l i k e l y . The data summarized here provide preliminary evidence f o r regulation of phytoalexin accumulation by t h i o l and/or d i s u l f i d e groups i n the i n t e r i o r of the plasma membrane. A hypothesis i s proposed: oxidation state of the membrane SH groups regulating the phytoalexin response i s maintained by b i o l o g i c a l t h i o l / d i s u l f i d e r a t i o s i n the cytoplasm and by a c c e s s i b i l i t y of these compounds to the membrane SH moieties. Acknowledgments I thank Christopher S. Halliday and Leigh Jacoby for technical assistance.

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5. GUSTINE

Phytoalexin

Levels in Ladino

Clover

Callus

42. Moesta, P.; Grisebach, H. Arch. Biochem. Biophys. 1981, 212, 462-7. 43. Lawton, M.A.; Dixon, R.A.; Hahlbrock, K.; Lamb, C. Eur. J. Biochem. 1983, 129, 593-601. 44. Gilbert, H.F. J. Biol. Chem. 1982, 257, 12089-91. 1985


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Regulation of Accumulated Phytoalexin Levels in Ladino Clover Callus

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