Jul 11, 1989 - Plant Biology Division, The Samuel Roberts Noble Foundation, P.O. Box 2180, ...... Gustine DL, Sherwood RT, Vance CP (1978) Regulation of.
Plant Physiol. (1990) 92, 440-446
Received for publication July 11, 1989 and in revised form October 3, 1989
Stress Responses in Alfalfa (Medicago sativa L.) 1. Induction of Phenylpropanoid Biosynthesis and Hydrolytic Enzymes in Elicitor-Treated Cell Suspension Cultures Karen Dalkin, Robert Edwards, Brent Edington, and Richard A. Dixon* Plant Biology Division, The Samuel Roberts Noble Foundation, P.O. Box 2180, Ardmore, Oklahoma 73402 ABSTRACT
Although radiolabeling experiments in alfalfa have been employed for the elucidation of several of the steps in Figure 1 (5), no detailed biochemical or molecular genetic analysis of this pathway has been performed in this species. Unlike several other legumes including bean, alfalfa is readily amenable to genetic transformation (27) and is therefore a good candidate for studies on (a) the potential of engineering for increased resistance through modifications to the phytoalexin pathway and (b) the functional analysis of homologous or heterologous defense response gene promoters in transgenic plants or electroporated protoplasts. To provide a basis for such studies, we here describe some of the enzymic changes related to defense metabolism in suspension cultured alfalfa cells exposed to elicitor molecules.
Alfalfa (Medicago sativa L.) cell suspension cultures accumulated high concentrations of the pterocarpan phytoalexin medicarpin, reaching a maximum within 24 hours after exposure to an elicitor preparation from cell walls of the phytopathogenic fungus Colletotrichum lindemuthianum. This was preceded by increases in the extractable activities of the isoflavonoid biosynthetic enzymes L-phenylalanine ammonia-lyase, cinnamic acid 4-hydroxylase, 4-coumarate coenzyme A-ligase, chalcone synthase, chalcone isomerase, and isoflavone 0-methyltransferase. Pectic polysaccharides were weak elicitors of phenylalanine ammonialyase activity but did not induce medicarpin accumulation, whereas reduced glutathione was totally inactive as an elicitor in this system. The fungal cell wall extract was a weak elicitor of the lignin biosynthetic enzymes, caffeic acid 0-methyltransferase and coniferyl alcohol dehydrogenase, but did not induce appreciable increases in the activities of the hydrolytic enzymes chitinase and 1,3-0-D-glucanase. The results are discussed in relation to the activation of isoflavonoid biosynthesis in other legumes and the development of the alfalfa cell culture system as a model for studying the enzymology and molecular biology of plant defense expression.
MATERIALS AND METHODS Buffers
The following buffers were used: (A) 50 mM Tris-HCl (pH 8.5) containing 0.01% (w/v) sodium azide; (B) 50 mm TricineKOH (pH 7.5); (C) 200 mM Tris-HCl (pH 7.5) containing 14 mM 2-mercaptoethanol; (D) 200 mm KH2PO4 (pH 7.5) containing 12 mM DTT; (E) 50 mm KH2PO4 (pH 8) containing 20 mM ascorbic acid; (F) 200 mM Tris-HCl (pH 7.2) containing 14 mm 2-mercaptoethanol; (G) 200 mM Tris-HCl (pH 8.5) containing 14 mM 2-mercaptoethanol, 5 mm EDTA, and 10 mm diethyldithiocarbamate (diethylammonium salt).
Cultivated alfalfa is the host for a wide range of fungal pathogens (1 1). Several reports have suggested that the accumulation of antimicrobial isoflavonoid phytoalexins may be an important factor in resistance to these pathogens (1, 16, 21). In a number of alfalfa cultivars, the pterocarpan medicarpin has been identified as the major phytoalexin. It is synthesized from L-phenylalanine by a pathway (Fig. 1) for which most of the enzymes have been at least partially characterized in other legumes (6, 17, 29). In bean cell suspension cultures, the enzymes of the first stages in phytoalexin biosynthesis (up to chalcone isomerase) have been shown to undergo rapid but transient induction as a result of transcriptional activation of the corresponding genes in response to treatment with fungal cell wall elicitor (6, 9). Resistance to the fungal pathogens Phytophthora megasperma f.sp. medicaginis or Verticillium albo-atrum is retained in callus cultures derived from resistant alfalfa lines (21, 24). This resistance appears to result from a rapid induction of the early enzymes of phytoalexin biosynthesis, with correspondingly early accumulation of medicarpin. In susceptible callus lines, phytoalexin accumulation is slower and may fail to restrict fungal growth (20).
Chemicals and Substrates
All substrates and reference compounds were obtained from Sigma (St. Louis, MO) except for coniferyl alcohol (Aldrich, Milwaukee, WI) and isoflavones (Aapin Chemicals, Oxon, UK). The purity of enzyme substrates was confirmed by TLC prior to use. Isoliquiritigenin had been previously prepared (8), and 4-coumaroyl CoA was synthesized by ester-exchange via the N-hydroxysuccinimide ester of 4CL' (28). Medicarpin was generously donated by Professor W. Barz (Westfalische Wilhelms-Universitat, Munster, FRG). L-[U-'4C]Phenylala' Abbreviations: 4CL, 4-coumarate:CoA ligase (EC 18.104.22.168); CAD, coniferyl alcohol dehydrogenase (EC 1.1. 1.-); CA4H, cinnamic acid 4-hydroxylase (EC 1. 14.13.11); CHI, chalcone isomerase (EC 22.214.171.124); CHS, chalcone synthase; COMT, caffeic acid O-methyltransferase (EC 126.96.36.199); DOMT, daidzein O-methyltransferase; PAL, L-phenylalanine ammonia-lyase (EC 188.8.131.52).
STRESS RESPONSES IN CULTURED ALFALFA CELLS
nine (19 GBq/mmol), [2-'4C]malonyl CoA (2.18 GBq/ mmol), and [3H]acetic anhydride (18.5 GBq/mmol) were obtained from Amersham (Arlington Heights, IL), and adenosyl-L-methionine-S-['4C-methyl] (17 GBq/mmol) from NEN (Boston, MA). [3H]-acetyl]Chitin was prepared to a final specific activity of 5.7 MBq/g by N-acetylation of chitosan with [3H]acetic anhydride as previously described (25). L-[U'4C]Cinnamic acid (2.17 GBq/mmol) was prepared from L[U-'4C]phenylalanine by incubation with a crude (0.4 nkat, 4 mg protein) PAL preparation obtained by ammonium sulfate and Sephacryl S-300 fractionation from an elicited alfalfa cell suspension culture extract (18). Phenylalanine (34 Mm) was incubated with the PAL preparation for 16 h in buffer A at 37°C, the reaction stopped with 0.1 volume of 6 N HCI, and the solution applied to a C18 SEP-PAK (Waters Associates, Milford, MA). After washing with 0.1 N HCI, the bound radioactivity was eluted with ethanol and the product further purified by TLC (in system I below) to a radiochemical purity of 96.5% and final yield of 52.4%.
COOH COOH IILI CIH CH CH
tPAL > t _CA4H PHENYL-
Growth and Elicitation of Plant Cell Cultures
Cell suspension cultures of alfalfa cv Apollo were initiated and maintained as described elsewhere (18). Filter-sterilized elicitor preparations were added to cell cultures on the 5th d after transfer, with an equal volume of sterile water being added to controls. In experiments comparing the effectiveness of different elicitor preparations, a sample of cells treated with elicitor from C. lindemuthianum was always included as an internal control. Cells were harvested by vacuum filtration on Whatman No. 1 filter paper and frozen rapidly in liquid N2. Plant material was stored at -70°C prior to analysis. Enzyme Assays Frozen cells were extracted and assayed directly for PAL (3), CHI (8) and CAD (12) by previously published spectrophotometric assays. Similarly, the assays for glucanase (23) and chitinase (25) have been described; reactions were shown to be linear with respect to the concentration of alfalfa protein extract within the range of 0 to 3 Mg per assay. CA4H was assayed in crude cell-free supernatants following homogenization of cells with a Brinkman Polytron in 1 mL/g fresh weight buffer B containing 14 mM 2-mercaptoethanol. Cell debris was removed by centrifugation and the supernatant
COMT | 12,4,4'TRIHYDROXY
Preparation of Elicitors
Elicitor from Colletotrichum lindemuthianum was prepared by autoclaving partially purified cell walls as described previously (15). The preparation of partially hydrolyzed polygalacturonic acid, partially hydrolyzed polymethylgalacturonic acid (apple pectin), and fractionated pectic oligomers was as described (10). Crude autoclaved alfalfa leaf and stem extracts were prepared by autoclaving plant material for 20 min at 120°C with an equal weight of distilled H20. Extracts were cooled and filtered through Whatman No. 1 paper under vacuum, and the filtrates were centrifuged at l0,OOOg for 10 min. Supernatants were stored at -20°C until use. Glutathione solutions were adjusted to pH 7.0 prior to use.
1 4.7 DIHYDROXY
CHO CH I
/ \ 3-OH
11H CH -
Figure 1. Biosynthesis of medicarpin and lignin precursors. See list of abbreviations in footnote 1 for enzyme names. IFS = isoflavone synthase.
treated with 10% w/v Dowex 1 preequilibrated with buffer B prior to desalting on Sephadex G-25. Conditions for incubation and determination of the ['4C]-4-coumaric acid produced were as previously described (7). 4CL, CHS, COMT, and DOMT were assayed from ammonium sulfate precipitates prepared as follows. Frozen cells (1 g) were allowed to thaw in 4 mL buffer C and homogenized with the Polytron prior to treatment for 10 min with a mixture of Dowex 1 and polyvinylpolypyrrolidone (1:3 w/w) on ice. Following centrifugation (l0,OOOg, 15 min), the supernatant was divided into 1 mL samples that were individually adjusted to 80% saturation with respect to (NH4)2SO4 using saturated (NH4)2SO4 solution (pH 7). Protein precipitates were collected by cen-
DALKIN ET AL.
trifugation (10,OOOg, 20 min) and stored at -70°C prior to assay.
The protein pellet for assay of 4CL was suspended in 0.3 mL buffer D and desalted on a Sephadex G-25 column (10 mL) equilibrated with buffer D. Samples (0.1 mL) were then assayed for production of 4-coumaroyl CoA by measuring the formation of the corresponding hydroxamate from hydroxylamine as described elsewhere (7). Protein precipitates for assay of CHS were taken up in buffer E and desalted through Sephadex G-25. The conditions for the assay of CHS by measurement of incorporation of [2-'4C]malonyl CoA into naringenin have been described elsewhere (7). Reaction products were assayed both by measuring the appearance of radioactivity which could be partitioned into ethyl acetate at pH 8 and by separation of naringenin by TLC in solvent system II. With crude protein extracts from alfalfa cells, the two assays showed a good correlation, with ['4C]naringenin representing 82.6 ± 2.7% of the total radioactivity partitioned into ethyl acetate.
Protein pellets for COMT assays were dissolved and desalted in 0.2 mL buffer F, and 50 ,gL samples were added to 50 nmol of caffeic acid (dissolved in 5 ,uL DMSO) and 150 nmol of S-adenosyl-L-methionine-[14C-methyl] (8.33 MBq/ mmol, dissolved in 5 ytL of distilled H20, pH 3.5). Samples were incubated at 30°C for 30 min, and the reactions were stopped by the addition of 200 nmol of ferulic acid in 40 ,uL
ice-cold ethanol. After centrifugation, 50 gL samples were applied as 3-cm bands to silica TLC plates, and the [14C] ferulic acid was radioassayed following development in solvent III. Samples for DOMT assay were dissolved and desalted in buffer G and assayed with 50 nmol of the isoflavone daidzein under identical conditions to COMT. The reactions were stopped by the addition of 200 nmol of the methylated product formononetin dissolved in ethanol. Products were separated from substrate by TLC in solvent IV and quantified by radioassay. Protein concentrations were determined with the Bio-Rad dye-binding reagent. Analysis of Phenolic Compounds Cells were extracted and analyzed for isoflavones by HPLC (19). Quantification of metabolites was achieved by calibration with authentic standards. In experiments in which different elicitors were compared, medicarpin was separated from ethanolic extracts by TLC in solvent V and estimated semiquantitatively by UV scanning spectrophotometry after elution in ethanol. Phenolic material bound to the cellulosic and hemicellulosic fractions of alfalfa cell walls was extracted and determined as described previously (4). TLC
TLC was performed on aluminum-backed silica gel plates containing fluorescent indicator (Merck, Darmstadt, FRG) using the following solvent systems: I, toluene:ethyl acetate:formic acid (25:75:1, v/v); II, chloroform:ethanol (3:1, v/ v); III, ethyl acetate:methylethylketone:formic acid:water (5:3:1:1, v/v); IV, petroleum ether:ethyl acetate:methanol
Plant Physiol. Vol. 92, 1990
(10:10:1, v/v); V, petroleum ether:ethyl acetate:methanol (60:40:1, v/v). Samples were over-spotted on reference standards and, following development, products were visualized under UV light. Quantification was achieved by eluting the excised zone of interest in methanol (0.5 mL) followed by liquid scintillation counting. RESULTS AND DISCUSSION Relative Effects of Elicitors on Alfalfa Cell Suspension Cultures
The relative activities of a range of elicitor preparations, of both fungal and host origin, are shown in Table I. A dose response curve was constructed for each elicitor preparation, and the data presented were measured at the optimal concentration as assessed by determination of the extent of elicitation of extractable PAL activity. An elicitor preparation obtained from autoclaved cell walls of the phytopathogenic fungus C. lindemuthianum induced the largest increase in extractable PAL activity when compared to H20-treated control alfalfa cells (see Fig. 3 for absolute values). Molecules of host plant origin, termed endogenous or constitutive elicitors, have been shown to induce defense responses in a number of plant species (reviewed in ref. 6); such elicitors include defined oligogalacturonides of chain length 11 to 13 residues. Crude preparations of material released during autoclaving of alfalfa stems or leaves exhibited elicitor activity; in the case of leaf extracts, this was maximally just over 30% of that obtained with the fungal elicitor. Mixtures of pectic fragments produced by acid hydrolysis of polygalacturonic acid or polymethylgalacturonic acid appeared to be less potent elicitors, on the basis of PAL induction, than the autoclaved leaf extract components. Analysis of polygalacturonic acid hydrolysates, fractionated into discrete oligomers, indicated that the optimal size for elicitor activity was approximately 9 to 11 galacturonosyl units. Smaller oligogalacTable 1. Relative Effects of Elicitors on Alfalfa Cell Suspension Cultures Concen- PAL! MediElicitor tration
Untreated 5 Water-treated control 9 C. lindemuthianum cell wall elicitor 50c 100 ++ Crude autoclaved alfalfa stem extract 1 OOC 15 Crude autoclaved alfalfa leaf extract 1 0OC 32 + Polygalacturonic acid fragments (partial 1 50d 23 hydrolysate) Methyl polygalacturonic acid fragments 1 50d 19 (partial hydrolysate) Pectic oligomers (9-11 galacturonosyl 1 00d 21 units) Glutathione [1 mM] 8 a Activity induced in cultured alfalfa cells relative to that induced by elicitor from C. lindemuthianum. Determined 8 h after exposure to elicitor. b Determined 24 h after exposure to elicitor (semiquantitative analysis by TLC). +, presence; -, not detected. c Glucose d Uronic acid equivalents. equivalents.
STRESS RESPONSES IN CULTURED ALFALFA CELLS
turonides were inactive in the alfalfa cultures. Previous studies support the finding that oligogalacturonides are less potent elicitors than the glucans or galactoglucomannans prepared from fungal cell walls (6). PAL induction was followed by accumulation of medicarpin in response to the elicitor from C. lindemuthianum or autoclaved alfalfa leaf components but not in response to pure pectic fragments. In contrast, in bean cell suspension cultures, a mixture of oligomers obtained by hydrolysis of polymethylgalacturonic acid at a concentration of 150 ,ug mL-' elicited very high levels of the phytoalexin phaseollin (10). Treatment of bean cell suspension cultures with reduced glutathione causes a rapid and selective induction of defense gene transcripts, including those encoding phenylpropanoid biosynthetic enzymes, closely resembling the response to fungal elicitor (30). Alfalfa suspension cultured cells exposed to glutathione at concentrations in the range 0.01 to 10.0 mM showed no change in extractable PAL activity compared with control cells and no detectable accumulation of medicarpin. Studies with labelled glutathione or its methyl ester suggest that this may, at least in part, be due to the inability of alfalfa cells to take up the tripeptide (R Edwards, J Blount, RA Dixon, manuscript in preparation).
Elicitor-induced Changes in Phenolic Compounds Acetone/acetonitrile extracts of cells harvested at various times up to 48 h after exposure of cultures to elicitor from C. lindemuthianum were analyzed by HPLC (Fig. 2). Untreated cell samples had a relatively simple profile of phenolic metabolites, which was effectively unaltered in H20-treated control cells. In contrast, fungal elicitation produced a complex pattern of phenolic compounds, the changes beginning as early
as 2 h after treatment. The accumulation of medicarpin reached a maximum value of nearly 400 nmol/g fresh weight by 24 h. The kinetics of medicarpin accumulation in the culture medium was very similar to that observed in the cell extracts (H Kessman, AD Choudhary, RA Dixon, manuscript in preparation). The related isoflavan phytoalexins sativan and vestitol were not detected in the alfalfa cells. In bean cell suspension cultures treated with elicitor from C. lindemuthianum, both 5-hydroxylated (kievitone) and 5deoxy (phaseollin) isoflavonoid-derived phytoalexins are formed. Kievitone accumulation occurs rapidly to a maximum level of approximately 40 nmol/g fresh wt at between 8 and 12 h postelicitation. Phaseollin, which is biosynthetically related to medicarpin in terms of its A-ring hydroxylation pattern and pterocarpan structure, is present in very low levels up to 24 h postelicitation, but then it accumulates to levels in excess of 500 nmol/g fresh weight at around 48 h postelicitation (26). Thus, marked differences exist in the kinetics of accumulation of closely related phytoalexins in bean and alfalfa cell cultures growing under the same conditions and exposed to the same elicitor. Cochromatography with authentic standards failed to provide evidence for the accumulation of significant levels of any of the precursors of medicarpin in the elicited alfalfa cell cultures. The isolation, purification, and structural elucidation of the elicitor-induced phenolics from the alfalfa cultures is currently under way in this laboratory. Visible browning of the alfalfa cells was observed at 12 to 16 h postelicitation, and changes in cell wall phenolic material were therefore monitored. A twofold increase, compared to control cells, of phenolics associated with the hemicellulosic fraction occurred 16 to 24 h after exposure to fungal elicitor (data not shown). No significant increase was detected in the levels of phenolics associated with the cellulosic fraction. In contrast, treatment of bean cell suspension cultures with elicitor from C. lindemuthianum has been shown to lead to rapid and extensive increases in phenolic material bound to both cellulosic and hemicellulosic fractions of the cell wall
(4). z n
Figure 2. HPLC profiles of solvent extracts of suspension cultured alfalfa cells. A. Untreated; B, 24 h after addition of H20; C, 24 h after addition of elicitor from C. lindemuthianum (50 Ag glucose equivalents/mL culture); D, levels of medicarpin in elicitor-treated cells (0) and in H20-treated control cells (0). M = medicarpin (retention time approximately 36 min). The data points here and in Figures 3 through 5 are the average values of duplicate determinations made on extracts from two separately treated cell culture batches. The bars are the actual values for the replicate cultures.
Induction of Phenylpropanoid Biosynthetic Enzymes Exposure of cell cultures to fungal elicitor resulted in large increases in the extractable activities of enzymes involved in the biosynthesis of medicarpin (Fig. 3). PAL, CA4H, and 4CL are common to the biosynthesis of all phenylpropanoid compounds, whereas CHS and CHI are involved specifically in the formation of flavonoid and isoflavonoid precursors (Fig. 1). DOMT is specific for the isoflavonoid branch pathway leading to medicarpin. With the exception of 4CL, basal activities of all enzymes were low relative to the maximum induced activities. The absolute basal activities of the six enzymes varied from less than 0.5 ,ukat/kg protein (CHS, DOMT) to 1.25 mkat/kg protein (CHI), a difference of more than 2000-fold. Differences were also observed in the timing of attainment of maximum activity and the rate of decline in activity after the maximum; induction of PAL, and particularly CA4H, was transient, whereas CHI and DOMT activities remained elevated up to 48 h postelicitation. With the possible exception of 4CL, the kinetics of enzyme induction were
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DALKIN ET AL.
.f3 cb IC -J
Time (h) 2(
24 Time (h)
Figure 3. Changes in the extractable activities of PAL, CA4H, 4CL, CHS, CHI, and DOMT in cultures treated with elicitor from C. lindemuthianum (50 ,g glucose equivalents/mL culture) (0) and in control H20-treated alfalfa cell cultures (0).
consistent with a role for the enzyme increases in medicarpin synthesis, with the increased CHS and DOMT activities of particular significance in view of their extremely low basal levels. The changes in enzyme activity were not closely coordinated, as observed within the enzymes of general phenylpropanoid metabolism or specific flavonoid metabolism in parsley cell cultures ( 14). Rather, the relative induction kinetics of PAL, CA4H, CHS, and CHI were very similar to those observed in elicitor-treated bean suspension cultures (26). Essentially similar enzyme induction kinetics have been observed in elicited suspension cells of the alfalfa cultivar "Calwest" (H Kessmann, AD Choudhary, RA Dixon, manuscript in preparation). In these cells, increased extractable activities of isoflavone synthase (Fig. 1) and isoflavone 2'hydroxylase were also measured. These enzymes, like CA4H, are membrane-associated Cyt P450s (9, 17). Although the small quantities of substrates available precluded a detailed time course study, the activities of isoflavone reductase and pterocarpan synthase (the final enzyme in the biosynthesis of medicarpan) were also measured in the alfalfa cultures used in this study; both enzymes were significantly induced, from low basal activities, by fungal elicitor (R Edwards, unpublished results). It would appear from our results, and recent work with chickpea cell cultures (2), that induction of pterocarpan biosynthesis in cultured legume cells involves the induction of all the enzymes in the pathway from L-phenylalanine. Induction of isoflavone 0-methyltransferase activity has previously been observed in relation to medicarpin accumu-
lation in fungus-infected jack bean callus (13). The induced activity catalyzed the O-methylation of the isoflavones daidzein (4',7-dihydroxyisoflavone, Fig. 1) and genistein (4',5,7trihydroxyisoflavone), although the activity was not further purified. However, an isoflavone O-methyltransferase has been purified from chickpea cell cultures (29) and shown to be active specifically toward daidzein and genistein. Crude extracts from alfalfa cells likewise contained both daidzein and genistein O-methyltransferase activity, and the activity against genistein was induced in a similar manner to the DOMT activity (data not shown). Preliminary results suggest that the alfalfa DOMT and genistein O-methyltransferase activities are functions of the same enzyme, whereas COMT, an enzyme involved in the biosynthesis of hydroxycinnamic acids and lignin precursors, exhibits different induction kinetics (see below). Although it is assumed that the DOMT activity catalyzes the methylation of the isoflavone 4'-hydroxyl group, this remains to be demonstrated; TLC analysis of methylation products from incubations with partially purified enzyme preparations indicated the presence of more than one methylated product. COMT and CAD are involved in the biosynthesis of wallbound phenolics and lignin. In jack bean callus, fungal infection led to medicarpin accumulation but no increase in COMT activity (13). In the alfalfa cells, the extractable activity of COMT underwent an initial rapid increase upon elicitation, followed by an even more rapid decline and then a second, more gradual, increase (Fig. 4). In contrast, the activity in control cells slowly declined after 12 h following transfer to
STRESS RESPONSES IN CULTURED ALFALFA CELLS
fresh culture flasks. This pattern ofexpression is very different from that observed for DOMT activity in these cells. In the case of CAD activity, a small elicitor-induced increase was observed from around 15 h postelicitation (Figure 4). This is in strong contrast to the situation in elicitor-treated bean cells, where CAD induction is exceedingly rapid, preceding that of PAL and CHS (12). It is possible that this might be related to the formation of biologically active signal molecules rather than the synthesis of lignin precursors per se (dehydrodiconiferyl glucosides have recently been shown to exhibit plant cell division promoting activity ). In the alfalfa cultures, the late appearance of COMT and CAD activities is more likely to be associated with the appearance of wall-bound phenolic material. Induction of Glucanase and Chitinase
Elicitor-mediated induction of the hydrolytic enzymes, 1,3-
f3-D-glucanase and chitinase, has been reported in several cell
suspension systems (reviewed in ref. 9). Especially in combination (23), these enzymes may exhibit antifungal activity by hydrolyzing the polymers of hyphal walls. In bean cell suspension cultures, elicitor from C. lindemuthianum induced a steady, coordinated, three-fold increase in chitinase and glucanase extractable activities over 48 h, resulting from 10-fold increases in transcript levels by 8 h postelicitation (B Edington, RA Dixon, unpublished results). In the alfalfa cell cultures growing under identical conditions, chitinase and glucanase activities were induced weakly, if at all, from relatively high basal levels (Fig. 5). The data in Figure 5 do not take into account the possibility of release of enzyme into the culture medium; in alfalfa seedlings infected with Colletotrichum trifolii, a large percentage of the induced chitinase activity is found in the intercellular spaces (E Maher, RA Dixon, unpublished results). CONCLUDING REMARKS Elicited alfalfa cell suspension cultures are characterized by a rapid accumulation, to relatively high levels, of the phyto-
40 4( 06 0%
30 .5 0~ ._
o0Q C6 0 ,l, -.r Time(h) Figure 4. Changes in the extractable activities of COMT and CAD in fungal elicitor-treated (0) and control (0) alfalfa cell cultures.
Figure 5. Changes in the extractable activities of chitinase and 1,3,B-D-glucanase in fungal elicitor-treated (0) and control (0) alfalfa cell cultures.
DALKIN ET AL.
alexin medicarpin, preceded by large increases, from low basal levels, of a number of important biosynthetic enzymes. The system is therefore ideal for studying the enzymology of isoflavonoid biosynthesis in legumes, both in terms of the early enzymes common to other areas of phenylpropanoid metabolism and, perhaps more importantly, the later enzymes specific for isoflavonoid synthesis. We have recently purified PAL from this source and described some properties of its multiple forms (18). Work is now in progress on the purification and/or cloning of isoflavone O-methyltransferase and the Cyt P450 enzymes involved in the conversion of 4',7dihydroxyflavanone to medicarpin. Elicitation leads to a rapid and massive switch in the pattern of mRNA species in the cultures, including the appearance of species encoding distinct subunit isoforms of PAL and CHS (K Dalkin, J Jorrin, RA Dixon, unpublished results); this should facilitate the cloning of stress response cDNAs. We have recently developed a transient assay system in which a chimaeric gene containing the bean chalcone synthase promoter is expressed in an elicitor-dependent manner in electroporated alfalfa protoplasts (AD Choudhary, H Kessmann, CJ Lamb, RA Dixon, manuscript in preparation). This, in conjunction with the ability of alfalfa to undergo stable Agrobacterium-mediated transformation and regeneration through somatic embryogenesis, will make this plant a valuable model system for studying stress response gene promoters, in addition to its being an important target crop for improvement through gene transfer technology. ACKNOWLEDGMENTS We thank Scotty McGill for preparation of the manuscript, and Carla Thomas for providing fractionated oligogalacturonides. LITERATURE CITED 1. Baker CJ, O'Neill NR, Bauchan GR (1988) Production of phytoalexins in race-clone interactions of alfalfa and Colletotri-
chum trifolii. Phytopathology 78: 1590 2. Barz W, Daniel S, Hinderer W, Jacques U, Kessmann H, Koster J, Otto C, Tiemann K (1988) Elicitation and metabolism of phytoalexins in plant cell cultures. Ciba Found Symp 137: 178-198 3. Bolwell GP, Bell JN, Cramer CL, Schuch W, Lamb CJ, Dixon RA (1985) L-Phenylalanine ammonia-lyase from Phaseolus vulgaris. Characterization and differential induction of multiple forms from elicitor-treated cell suspension cultures. Eur J Biochem 149: 411-419 4. Bolwell GP, Robbins MP, Dixon RA (1985) Metabolic changes in elicitor-treated bean cells: enzymic responses associated with rapid changes in cell wall components. Eur J Biochem 148: 571-578 5. Dewick PM, Martin M (1979) Biosynthesis of pterocarpan, isoflavone and coumestan metabolites of Medicago sativa: chalcone, isoflavone and isoflavanone precursors. Phytochemistry 18: 597-602 6. Dixon RA (1986) The phytoalexin response: Elicitation, signalling and the control of host gene expression. Biol Rev 61: 239291 7. Dixon RA, Bendall DS (1978) Changes in the levels of enzymes of phenylpropanoid and flavonoid synthesis during phaseollin production in cell suspension cultures of Phaseolus vulgaris. Physiol Plant Pathol 13: 295-306
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8. Dixon RA, Dey PM, Whitehead IM (1982) Purification and properties of chalcone isomerase from cell suspension cultures of Phaseolus vulgaris. Biochim Biophys Acta 715: 25-33 9. Dixon RA, Harrison MJ (1990) Activation, structure and organization of genes involved in microbial defense in plants. Adv Genet (in press) 10. Dixon RA, Jennings AC, Davies LA, Gerrish C, Murphy DL (1989) Elicitor-active components from French bean hypocotyls. Physiol Mol Plant Pathol 34: 99-115 11. Graham JH, Frosheiser FI, Stuteville DL, Erwin DC (1979) A Compendium of Alfalfa Diseases. APS Press, St. Paul, MN 12. Grand C, Sarni F, Lamb CJ (1987) Rapid induction by fungal elicitor of the synthesis of cinnamyl alcohol dehydrogenase, a specific enzyme of lignin synthesis. Eur J Biochem 169: 73-77 13. Gustine DL, Sherwood RT, Vance CP (1978) Regulation of phytoalexin synthesis in jack bean callus cultures. Stimulation of phenylalanine ammonia-lyase and O-methyltransferase. Plant Physiol 61: 226-230 14. Hahlbrock K, Knobloch K-H, Kreuzaler F, Potts JRM, Wellmann E (1976) Co-ordinated induction and subsequent activity changes of two groups of metabolically interrelated enzymes. Light-induced synthesis of flavonoid glycosides in cell suspension cultures of Petroselinum hortense. Eur J Biochem 61: 199-206 15. Hamdan MAMS, Dixon RA (1986) Differential biochemical effects of elicitor preparations from Colletotrichum lindemuthianum. Physiol Mol Plant Pathol 28: 329-344 16. Higgins VJ (1972) Role of the phytoalexin medicarpin in three leaf spot diseases of alfalfa. Physiol Plant Pathol 2: 289-300 17. Hinderer W, Flentje U, Barz W (1987) Microsomal isoflavone 2' and 3'-hydroxylases from chickpea (Cicer arietinum L.) cell suspension cultures induced for pterocarpan phytoalexin formation. FEBS Lett 214: 101-106 18. Jorrin J, Dixon RA (1989) Stress responses in alfalfa (Medicago sativa L.). II. Purification, characterization, and induction of phenylalanine ammonia-lyase isoforms from elicitor-treated cell suspenion cultures. Plant Physiol 92: 447-455 19. Koster JA, Zuzok A, Barz W (1983) High performance liquid chromatography of isoflavones and phytoalexins from Cicer arietinum. J Chromatogr 270: 392-395 20. Latunde-Dada AO, Dixon RA, Lucas JA (1987) Induction of phytoalexin biosynthetic enzymes in resistant and susceptible lucerne callus lines infected with Verticillium albo-atrum. Physiol Mol Plant Pathol 31: 15-23 21. Latunde-Dada AO, Lucas JA (1985) Involvement of the phytoalexin medicarpin in the differential response of callus lines of lucerne (Medicago sativa) to infection by Verticillium alboatrum. Physiol Plant Pathol 26: 31-42 22. Lynn DG, Chen RH, Manning KS, Wood HN (1987) The structural characterization of endogenous factors from Vinca rosea crown gall tumors that promote cell division of tobacco cells. Proc Natl Acad Sci USA 84: 615-619 23. Mauch F, Mauch-Mani B, Boller T (1988) Antifungal hydrolases in pea tissue. II. Inhibition of fungal growth by combinations of chitinase and ,3-l,3-glucanase. Plant Physiol 88: 936-942 24. Miller SA (1985) Tissue culture methods in phytopathology. II. Fungi. In RA Dixon, ed, Plant Cell Culture-A Practical Approach. IRL Press, Oxford, pp 215-229 25. Molano J, Duran A, Cabib E (1977) A rapid and sensitive assay for chitinase using tritiated chitin. Anal Biochem 83: 648-656 26. Robbins MP, Bolwell GP, Dixon RA (1985) Metabolic changes in elicitor-treated bean cells. Selectivity of enzyme induction in relation to phytoalexin accumulation. Eur J Biochem 148: 563-569 27. Shahin EA, Spielmann A, Sukhapinda JK, Simpson RB, Yashar M (1986) Transformation of cultivated alfalfa using disarmed Agrobacterium tumefaciens. Crop Sci 26: 1235-1239 28. Stockigt J, Zenk MH (1975) Chemical syntheses and properties of hydroxycinnamoyl-coenzyme A derivatives. Z Naturforsch 30C: 352-358 29. Wengenmayer H, Ebel J, Grisebach H (1974) Purification and properties of a S-adenosylmethionine: isoflavone 4'-O-methyltransferase from cell suspension cultures of Cicer arietinum L. EurJ Biochem 50: 135-143 30. Wingate VPM, Lawton MA, Lamb CJ (1988) Glutathione causes a massive and selective induction of plant defense genes. Plant Physiol 86: 206-210