Journal of Experimental Botany, Vol. 52, No. 357, pp. 681-689,
April 15, 2001
© 2001 Oxford University Press
Original Papers |
Elicitor-induced changes in isoflavonoid metabolism in red clover roots
1 Division of Applied Life Sciences, Graduate School of Agriculture, Kyoto University, Kyoto 606-8502, Japan
2 CREST, Japan Science and Technology Corporation (JST), Japan
Received 14 July 2000; Accepted 21 October 2000
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Abstract |
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When roots of 5-d-old red clover (Trifolium pratense L.) seedlings were treated with chitohexaose and CuCl2, constitutive glucosidic conjugates of formononetin (F) and (-)-maackiain (Ma) promptly disappeared. Free F and Ma, which were not detected in the control tissues, rapidly appeared to reach the maximum levels 24 h after the initiation of treatment and then declined. The pattern of appearance and disappearance was the same between the tissues treated with chitohexaose and CuCl2. The enzyme activities related to isoflavonoid metabolism were investigated using crude extracts from elicitor-treated roots. The conjugate-forming glucosyltransferase and malonyltransferase activities were lost or markedly reduced after elicitor treatment. On the other hand, malonylesterase and glucosidase activities remained unchanged or showed only slight increase. Phenylalanine ammonia-lyase activity disappeared following elicitor treatment. These results indicated that free aglycones were produced from the conjugate pool by hydrolysis under conditions in which the biosynthetic pathway was extinguished. The amount of Ma produced did not explain that of MaGM lost (about 45%). Since Ma, but not its conjugates, served as a substrate for peroxidase from the elicitor-treated roots, Ma was considered to be converted to insoluble materials.
Key words: Phytoalexin, maackiain, Leguminosae, Trifolium pratense, red clover, elicitor.
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Introduction |
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The isoflavones and related pterocarpans constitute a group of secondary metabolites primarily in leguminous plants, and certain pterocarpans are known for their role as phytoalexins (Barz and Welle, 1992
). It has become increasingly clear that they are not only produced in a free form as phytoalexins, but they are also accumulated constitutively in the forms of glycosides and malonylglycosides. Demethylhomopterocarpan and maackiain are produced in the leaves of red clover inoculated with Helminthosporium turcicum (Higgins and Smith, 1972), and maackiain accumulates in the roots when inoculated with Fusarium roseum (McMurchy and Higgins, 1984). In healthy seedlings, these were not at all or only barely detectable, but their glucosidic conjugates were found together with the conjugates of the precursor isoflavone formononetin (Edwards et al., 1997).
Phytoalexins are synthesized by plant tissues in response to various stresses in addition to infection, such as biotic and abiotic elicitors, ultraviolet light, plant hormones, and fungicides (Brooks and Watson, 1985). The metabolic relationship between isoflavonoids produced after elicitation and pre-existing conjugates has been studied in leguminous plants. In chickpea cotyledons, elicitor treatment caused the accumulation of isoflavone and pterocarpan aglycones, but had little effect on the formation of constitutive conjugates (Kessmann and Barz, 1986). When white lupin roots were treated with biotic or abiotic elicitors, the levels of both the prenylated isoflavones and the aglycones and glucosides of precursor isoflavones increased (Gagnon and Ibrahim, 1997; Shibuya et al., 1992). In cell suspension cultures of Pueraria lobata treated with yeast extracts, free isoflavones were formed independently of the rapid decreases in level of constitutive isoflavonoid conjugates (Park et al., 1995).
It has been suggested in some cases that glucosidic conjugates serve as metabolic pools and can be mobilized by hydrolysis to release aglycones during infection (Graham, 1991; Morris et al., 1991; Daniel et al., 1998) and elicitor treatment (Parry et al., 1994; Mackenbrock et al., 1993). In red clover roots, it was found that chito-oligosaccharides and CuCl2 caused rapid and complete loss of formononetin and maackiain conjugates and the accumulation of free aglycones. This prompted the systematic investigation of the biochemical basis of this response.
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Materials and methods |
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Plant materials
Seeds of red clover (Trifolium pratense L. cv. Kenland, Yukijirushi Shubyo, Sapporo, Japan) were sown on moistened filter paper and maintained at 25 °C with a 12 h period of illumination from fluorescent lamps (15 W m-2, photosynthetic irradiance) in growth chambers.
Chemicals
Chito-oligosaccharides were purchased from Seikagaku Kogyo (Tokyo, Japan). Formononetin (F) and formononetin-7-O-glucoside (FG) were obtained from Extrasynthése. The phenylalanine ammonia-lyase inhibitor S-84702 [1-amino-1-hydroxycarbonylmethyliden-(3-chloro-2-methyl)aniline] was a gift from Sumitomo Chemical Co., Ltd.
Formononetine-7-O-glucosyl-6''-malonate (FGM), formononetin-7-O-glucosyl-6''-methyl malonate (FGMM) and (-)-maackiain-3-O-glucosyl-6''-malonate (MaGM) were obtained from roots of red clover. The roots (60 g) were extracted seven times with 150 ml of methanol containing 2% (v/v) acetic acid by ultrasonication for 10 min at 50 °C, and the combined extract was fractionated on an ODS column (Cosmosil 75C18-OPN, 100 mm length, 40 mm i.d., Nacalai Tesque, Kyoto, Japan) by stepwise elution. FGM and MaGM were eluted in the 50% (v/v) methanol–water fraction, and FGMM was eluted in the 60% methanol fraction. Each compound was further purified by preparative reversed phase HPLC [column, Wakosil II 5C18HG, 250 mm length, 20 mm i.d. (Wako, Osaka, Japan); solvent, 25% (v/v) acetonitrile in water containing 3% (v/v) acetic acid at 10 ml min-1; detection, 280 nm] to give FGM (21.2 mg), FGMM (5.1 mg) and MaGM (22.3 mg).
(-)-Maackiain-3-O-glucoside (MaG) was prepared by alkaline hydrolysis of MaGM. A solution of MaGM (14 mg) in methanol (1 ml) was added to 0.1 M NaHCO3 (19 ml), and the reaction mixture was stirred at 60 °C for 12 h. After evaporation, the concentrate was subjected to preparative reversed phase HPLC to give MaG (9.7 mg, 82% yield). The HPLC conditions were the same as those described above.
(-)-Maackiain (Ma) was prepared by enzymatic hydrolysis of MaG using sweet almond glucosidase (Nacalai, Kyoto, Japan). The glucosidase (100 µg) was added to the solution of MaG (10 mg) in McIlvain buffer (pH 5.5, 20 ml). After incubation at 30 °C for 18 h, the reaction was stopped by adding 5 ml of 1 N HCl. The mixture was applied to an ODS column (20 mm long, 10 mm i.d.). The column was first washed with 10 ml of water and then eluted with 10 ml of methanol. The eluate was concentrated and further purified by preparative reversed phase HPLC [column, Wakosil II 5C18HG, 250 mm long, 20 mm i.d.; solvent, 40% (v/v) acetonitrile in water containing 3% (v/v) acetic acid at 10 ml min-1; detection, 280 nm] to give Ma (5.3 mg, 83% yield).
The identity of the compounds was confirmed by 1H NMR, UV, and ion-spray mass spectroscopy (FGM, Dakora et al., 1993; FGMM, Beck and Knox, 1971; MaGM, Yamamoto et al., 1991; MaG and Ma, Yagi et al., 1992). The optical rotations of MaG, and Ma were identical with the reported values (Bredneberg and Hirtala, 1961).
Elicitor treatment
Seedlings (5-d-old) were wounded on their roots with carborundum and immediately placed on 24-well tissue culture plates (10x16 mm) containing solutions of chito-oligosaccharides or CuCl2 (0.5 ml well-1). After incubation at 25 °C with a 12 h photoperiod for 24 h, the seedlings were separated into roots and shoots and extracted for analyses of metabolites and enzyme activities.
Administration of PAL inhibitor S-84702
Seedlings (4.5-d-old) were wounded on their roots and immersed in 0.5 ml of 1 mM S-84702 solution. After incubation at 25 °C for 12 h, 5 µl of 100 mM chitohexaose [(GlcN)6] or CuCl2 was added to the solution. The seedlings were incubated under the same conditions for an additional 24 h and extracted for analysis of secondary metabolites.
Analysis of isoflavonoids and pterocarpans
Plant materials (40–60 mg) were extracted with 0.5 ml of methanol containing 2% (v/v) acetic acid by ultrasonication at 50 °C for 10 min. The extracts were subjected to reversed phase HPLC analyses (Köster et al., 1984). Isoflavonoids and pterocarpans released into the elicitor solutions were also analysed. The following HPLC conditions were used: column, Wakosil II 5C18HG, 150 mm long, 4.6 mm i.d.; elution, linear gradient from 20–60% (v/v) B/A for 35 min [solvent A: 3% (v/v) acetic acid in water, solvent B: acetonitrile]; flow rate, 0.8 ml min-1; detection, 280 and 310 nm.
Enzyme extraction and assays
Extraction of enzyme activities was carried out using the method described previously (Daniel et al., 1988) with slight modifications. Roots (40–60 mg) were homogenized with sea sand and 5 vols of 100 mM TRIS-HCl buffer (pH 7.5) containing 10 mM mercapteothanol and 5% (w/v) polycral SB 100. The homogenate was centrifuged twice (25 000 g, 10 min and 25 000 g, 20 min at 4 °C), and the supernatant was used in assays for enzyme activities. Three different buffer systems were used for the enzyme reactions: 100 mM K-Pi buffer (pH 7.5) containing 10 mM mercaptoethanol (buffer A), 100 mM TRIS-HCl buffer (pH 8.0) containing 10 mM mercaptoethanol (buffer B), and 100 mM TRIS-HCl buffer (pH 9.0) containing 10 mM mercaptoethanol (buffer C). The compositions of the reaction mixtures are described below.
(i) FG glucosidase (FG-GLC): 5 µl of 10 mM FG, 20 µl of enzyme preparation, and 275 µl of buffer A (Burmeister and Hösel, 1980). For measurement of MaG glucosidase (MaG-GLC) activity, a modified reaction mixture was used (5 µl of 10 mM MaG, 40 µl of enzyme preparation, and 305 µl of buffer A). (ii) FGM and MaGM malonylesterase (FGM-, MaGM-ME): 5 µl of 10 mM FGM or MaGM, 10 µl of enzyme preparation, and 285 µl of buffer B (Hinderer et al., 1986). (iii) F and Ma glucosyltransferase (F-, Ma-OGT): 5 µl of 10 mM F or Ma, 5 µl of 20 mM UDP-glucose, 20 µl of enzyme preparation, and 270 µl of buffer B (Daniel et al., 1988). (iv) FG and MaG malonyltransferase (FG-, MaG-MT): 5 µl of 10 mM FG or MaG, 5 µl of 20 mM malonyl-CoA, 20 µl of enzyme preparation, and 270 µl of buffer B (Köster et al., 1984). (v) Phenylalanine ammonia-lyase (PAL): 5 µl of 100 mM L-phenylalanine, 20 µl of enzyme preparation, and 275 µl of buffer C (Southerton and Deverall, 1990; Mackenbrock and Barz, 1991).
All reactions were carried out at 37 °C for 30 min and stopped by adding 50 µl of 2 N HCl to the mixture. The enzyme activities were determined by quantification of the products by reversed phase HPLC. The following HPLC conditions were used: column, Wakosil II 5C18HG, 150 mm length, 4.6 mm i.d.; elution, linear gradient from 40% to 75% (v/v) B/A for 12 min [solvent A: 3% (v/v) acetic acid in water, solvent B: acetonitrile]; flow rate, 0.8 ml min-1; detection, 280 and 310 nm.
Assay for glucosidase activities in elicitor solutions
The glucosidase activities released into the elicitor solution were determined after a 24 h incubation of the seedlings with the elicitor. The reaction mixture contained 5 µl of 10 mM FG or MaG and 295 µl of filtrated elicitor solution. The mixture was incubated at 37 °C for 3 h, and the reaction was terminated by adding 50 µl of 2 N HCl. The products were quantified by reversed phase HPLC analysis.
Assay for peroxidase activity
Preparation and measurement of peroxidase activity were conducted by a method described earlier (Kristensen et al., 1999). Roots (40–60 mg) were homogenized with sea sand, 10 vols of 200 mM K-Pi buffer (pH 6.0) and 5% (w/v) polycral SB 100. The homogenate was centrifuged twice (25 000 g, 10 min, and 25 000 g, 20 min at 4 °C), and the supernatant was used for the reaction. The reaction mixture consisted of 10 µl of 5 mM substrate, 10 µl of 10 mM H2O2, 2–20 µl of enzyme preparation, and 200 mM K-Pi buffer (pH 6.0) in a total volume of 500 µl. The mixture was incubated for 10 min at 25 °C, and the reaction was stopped by adding 100 µl of 2 N HCl and 400 µl of methanol. The enzyme activity was determined by quantification of the amounts of reduced substrates by reverse phase HPLC.
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Identification of isoflavonoids in roots
The extracts from roots of 5-d-old seedlings were analysed by reversed phase HPLC. The most abundant isoflavonoid was FGM (about 4 µmol g-1 FW), and next one was MaGM (3–4 µmol g-1 FW). FGMM was found in a significant amount (about 1.5 µmol g-1 FW), but FG and F were barely detectable. Only trace amounts of Ma and MaG were detected.
Effects of elicitor treatments
The changes in the contents of F and Ma conjugates during the experimental period were essentially the same between the wounded and intact roots, with F, Ma and MaG being barely detectable (Figs 1A, 2A).
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), FG (
), FGM (•), and FGMM (
) in wounded roots treated with H2O (A), 1 mM (GlcN)6 (B), and 1 mM CuCl2 (C). Each data point indicates the mean of four experiments±SD.
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Treatment with 1 mM (GlcN)6 caused a marked decrease and disappearance of FGM, FGMM and MaGM by 24 h (Figs 1B
, 2B). FG, initial level of which was very low, also disappeared 24 h after the start of treatment. On the other hand, F appeared to increase steeply to reach a maximum level of about 2 µmol g-1 FW 24 h after the start of treatment, and then decreased. Ma appeared and increased to a maximum level (0.5 µmol g-1 FW) 12–24 h after the start of treatment and then gradually declined. The level of Ma was about one-fifth that of F at maximum. The situation was essentially the same after treatment with 1 mM CuCl2 (Figs 1C, 2C).
When F conjugates disappeared in the roots treated with 1 mM (GlcN)6 and CuCl2, the amount of F was only 55–75% that of the conjugates at time 0 (Fig. 1B, C). A similar calculation showed that the missing Ma amounted to about 80%. The missing amount of F conjugates was found in the elicitor solutions in which the roots were immersed mostly as aglycone and only partly as glucosidic conjugates (see Fig. 3 for dose–response experiments). Ma and its conjugates were also found in the elicitor solutions (Fig. 4). However, the total amounts of Ma in the tissues and elicitor solutions did not explain the amount of MaGM lost (about 45%).
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As shown in Figs 3
and 4, both (GlcN)6 and CuCl2 showed some effects at 0.1 mM on both the disappearance of the conjugates and accumulation of aglycones. Amounts of isoflavonoids were determined 24 h after the start of treatment. The results were essentially the same at concentrations higher than 0.5 mM, and between (GlcN)6 and CuCl2 treatments.
In the solutions used for treatment with (GlcN)6 at concentrations higher than 0.1 mM, F and Ma were found, together with very small or barely detectable amounts of conjugates (Figs 3B, 4B). In the CuCl2 solutions, F conjugates (FG and FGM) were found at slightly higher levels than those in the (GlcN)6 solutions, and Ma conjugates (MaG and MaGM) were present in considerably larger amounts (Figs 3D, 4D). Only aglycones were markedly accumulated in the (GlcN)6 solutions, and their amounts did not change significantly with increasing concentrations of the elicitor. In CuCl2 solution, Ma and MaGM decreased and MaG increased with increasing concentration. Thus treatment with CuCl2 was in this respect different to that with (GlcN)6.
The effects of degree of polymerization of chito-oligosaccharides were examined 24 h after the start of treatment with a fixed concentration (1 mM) of the compounds (Fig. 5). The effects were observed from trisaccharide, with free F in the tissues and elicitor solution composing of about 40% of the total of F and its conjugates, and free Ma about 30% of the total of Ma and its conjugates. Oligomers larger than tetrasaccharide gave essentially the same results, with the species found being mostly aglycones. Hexasaccharide was used as an elicitor throughout the experiments.
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Effects of PAL inhibitor on the induced changes in contents of isoflavonoids
The induced changes in the isoflavonoid pattern were investigated in roots incubated with the PAL inhibitor S-87402 (Tanigaki et al., 1993) for 12 h before treatment with elicitors. The inhibitor showed near complete inhibition of PAL activity in the extracts from the roots of 6-d-old seedlings at 0.1 and 1 mM (6% and 5% of the control, respectively). When the inhibitor was applied at 1 mM to wounded roots of 4.5-d-old seedlings, the sum total of F conjugates was reduced to about half of that of the control after 36 h, and that of Ma conjugates to about two-thirds (Fig. 6). When roots preincubated with the inhibitor were treated with 1 mM (GlcN)6 and CuCl2, the remaining conjugates disappeared, and the accumulated amounts of free F and Ma were the same as those observed in the absence of the inhibitor.
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Changes in enzyme activities in elicitor-treated roots
To investigate the mechanisms of accumulation of the aglycones and disappearance of the conjugates, the enzyme activities pertinent to the conjugation and hydrolysis of the series of isoflavonoids in the tissues treated with the elicitors for 24 h were measured. The results are summarized in Table 1.
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FG-GLC activity was relatively low in the control roots, and did not change markedly following treatment with 1 mM (GlcN)6 or CuCl2. MaG-GLC activity increased slightly (about 0.025 nkat g-1 FW) in roots treated with (GlcN)6, but the activity did not changed in roots treated with CuCl2. FGM- and MaGM-ME activities remained at a high level after elicitor treatment. F- and Ma-OGT activities, which were well detected in the control (0.2–0.3 nkat g-1 FW), were lost in the treated roots. FG- and MaG-MT activities were reduced to about 20–30% in the (GlcN)5-treated roots and to 2% or less in the CuCl2-treated roots. PAL activity, which was detected at a high level in the control roots, was not found after treatment with elicitors for 24 h.
The changes in activities of F-OGT, FG-GLC and MaGM-ME during the CuCl2 treatment (1 mM) were also investigated. The decrease in activity of F-OGT (0.04 nkat g-1 FW) had already started at 12 h after the initiation of treatment, whereas the activities of FG-GLC and MaGM-ME remained at high levels (0.3 and 0.88 nkat g-1 FW, respectively). The enzyme activities did not change significantly from 24 h after the start of treatment until 48 h.
Both (GlcN)6 and CuCl2 had little effect on the glucosidase and esterase activities in the crude extracts from roots of 6-d-old plants. Both were somewhat inhibitory for Ma-OGT. F-OGT was inhibited by CuCl2, to a level about 30% that of the control, but not by (GlcN)6. CuCl2, but not (GlcN)6, enhanced FG-MT activity by about 2-fold. Ma-MT and PAL were not affected by either compound. Thus, the enzymes that were significantly affected in vitro were F-OGT and FG-MT, but only by CuCl2.
Glucosidase activity in elicitor solutions
As hydrolases may have been released into the elicitor solutions, the FG- and MaG-GLC activities were measured in 1 mM elicitor solutions in which the roots were immersed for 24 h (Table 2). The glucosidase activities were very weak in the control solution, 1–2 pkat g-1 FW. In the (GlcN)6 solution, the FG- and MaG-GLC activities were increased about 20- and 10-fold, respectively, whereas, in the CuCl2 solution, the levels were 2- to 3-fold those of the control.
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Detection of peroxidase activity
To examine whether a portion of Ma or its conjugates was lost by oxidation, peroxidase activity was measured. When the extract from control roots was used as crude enzyme in the presence of H2O2, Ma decreased rapidly (about 96.3 µkat g-1 FW) (Table 3). The activity for F was 5.5 µkat -1 g FW, and those for the conjugates of Ma and F were only marginal. The activity for Ma in the extracts from the roots treated with (GlcN)6 and CuCl2 were about 80% and 40% of that in the control, respectively.
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In the present study, it was found that (GlcN)6 and CuCl2 caused a marked reduction in levels of constitutive F and Ma conjugates and an accompanying increase of their aglycones in red clover roots within 24 h after the start of treatment. In roots treated with the elicitors for 24 h, PAL activity disappeared, and the activities of conjugate-forming glucosyl- and malonyltransferases were markedly diminished or abolished. On the other hand, the activities of malonylesterases and glucosidases that catalyse degradation of conjugates were unchanged or slightly increased. Constitutive FGM and MaGM that remained unaffected by application of PAL inhibitor alone disappeared when the root tissues were treated with the elicitors, and the aglycones were formed. These findings indicated that, while the transferases and PAL activities disappeared resulting in blocking carbon flows to conjugates, esterases hydrolysed the malonylglucosides, and the glucosides formed were converted to aglycones by glucosidases. Neither elicitor had significant inhibitory effects on the in vitro activities of the enzymes except for F-OGT, which was inhibited to 30% of the control by 1 mM CuCl2, but not by (GlcN)6. Since the changes in isoflavonoid metabolism were substantially the same between the tissues elicited by CuCl2 and (GlcN)6, the inhibitory property of CuCl2 on the transferase activity was considered to have little effect on isoflavonoid metabolism in the elicited tissues.
Copper ion (Kokubun and Harborne, 1994; Rouxel et al., 1991; Dewick, 1975; Parry et al., 1994) and chitosan (Ebel and Mithöfer, 1998) have been shown to induce defence reactions including phytoalexin accumulation in many plant species. On the other hand, these compounds have toxic effects against plant cells, involving disintegration of membranes (Luna et al., 1994; Doncheva, 1998; Young et al., 1982). The stresses that cause the disintegration of membranes are not limited to treatment with CuCl2 and chitosan. Toxins produced by pathogens frequently affect membrane functions (Walton, 1996). In addition, membrane perturbation would occur in cells carrying out hypersensitive reactions. The disintegration of membranes probably results in the contact of conjugates with hydrolytic enzymes leading to the release of free aglycones. Detection of isoflavonoids and glucosidase activity in the elicitor solutions after 24 h incubation indicates membrane damage. The pattern of enzyme activities after 24 h treatment with CuCl2 or (GlcN)6 may also be related to membrane perturbation since mixing components stored in different compartments causes inactivation of enzymes (Cooke et al., 1980). It is conceivable that red clover has a mechanism leading to the release of toxic aglycones by default under stress conditions, and that hydrolytic enzymes that are relatively resistant to inactivation function in this mechanism, although the possibility of involvement of transcriptional regulation cannot be excluded. Accumulation of toxic aglycones in damaged tissues is probably beneficial to the plant for preventing the invasion of pathogens from the damaged site.
The effects of treatment with CuCl2 and (GlcN)6 on isoflavonoid metabolism were similar. However, the compositions of compounds released into the elicitor solutions were different. In accordance with this, glucosidase activities released into the (GlcN)6 solution were higher than those in CuCl2 solution. The effects of elicitors on the plasma membrane are probably dependent on the nature of elicitors in terms of release of hydrolytic enzymes into the solution.
It has been reported that labelled phenylalanine was incorporated into F and Ma in red clover seedlings treated with 3 mM CuCl2 for 12 h prior to application (Dewick, 1975), indicating the production of Ma by de novo synthesis. In previous experiments, whole seedlings were subjected to extraction with ethanol after treatment with CuCl2 on roots. However, the response of the plant to CuCl2 treatment on roots was different between roots and shoots. In shoots, the amounts of MaGM increased together with Ma, indicating the involvement of de novo synthesis (S-I Tebayashi, unpublished data). Thus, the activation of de novo synthesis in shoots probably contributed to the incorporation of phenylalanine into Ma and F in the previous experiment.
It was not possible to estimate the contribution of the pathway from F to Ma in accumulation of Ma after treatment with CuCl2. Formononetin 3'-hydroxylase activity was measured according to the method of Hinderer et al. (Hinderer et al., 1987). However, the enzyme activity was not detected in the microsomal preparations from either CuCl2-treated or untreated roots, although trans-cinnamic acid 4-hydrolylase (C4H) activity was detected in untreated roots (52 pkat g-1 FW). C4H activity was not detected in CuCl2-treated roots. Feeding of CuCl2-treated roots with [methyl-13C] formononetine was also carried out, but significant incorporation of 13C was not observed in an ion-spray mass spectrum of Ma (data not shown). This implies that the contribution of the pathway from F to Ma may be small, but before forming conclusions, more sensitive methods including feeding with radiolabelled formononetin need to be applied.
In chickpea cell suspension culture, a fungal elicitor induced accumulation of medicarpin and maackiain glucosides at low concentrations, whereas the elicitor induced accumulation of their aglycones at high concentrations (Mackenbrock et al., 1993). The activities of enzymes involved in pterocarpan conjugation and hydrolysis were correlated with the changing ratio of accumulating aglycones and conjugates; glucosyltransferase and malonyltransferase activities decreased, while glucosidase and malonylesterase activities increased along with increases in the elicitor concentration. The changes in the enzyme activities after treatment with high concentration of elicitor were similar to those in red clover roots treated with CuCl2 or (GlcN)6 in terms of decreases in biosynthetic enzyme activities. However, preferential accumulation of conjugates after treatments with low concentrations of CuCl2 or (GlcN)6 was not observed in red clover roots. Moreover, PAL activity, which linearly increased with elicitor concentration in chickpea cells, disappeared, indicating that de novo synthesis is not responsible for accumulation of F and Ma. These findings suggest that the mode of action of CuCl2 and (GlcN)6 is different from that of the fungal elicitor in chickpea.
The increase in amount of free F after 24 h treatment and the accompanying decrease in the amount of its conjugates were similar, but about 45% of MaGM was missing. Park et al. reported that about 90% of the isoflavonoids produced in elicitor-treated Pueraria lobata cell suspension cultures were converted to insoluble materials in the lignocellulosic fraction (Park et al., 1995). In elicitor-treated soybean cotyledons, increases in anionic peroxidases have been reported with the accumulation of phenolic polymers (Graham and Graham, 1991). In this study, Ma but little F served as substrates for peroxidase activity extracted from roots, and this suggested the conversion of Ma to insoluble product(s) as a likely fate of the missing Ma. Although peroxidase showed a decrease in activity after treatment with CuCl2 and (GlcN)6, disintegration of membranes presumably results in the contact of Ma and pre-existing peroxidase in other compartments. The accumulated insoluble material in damaged tissue is considered to function as a phenolic barrier against pathogens together with the accumulated free aglycones.
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3 To whom correspondence should be addressed. Fax: +81757536408. aishiha{at}kais.kyoto\|[hyphen]\|u.ac.jp
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Abbreviations |
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F, formononetin; FG, formononetin-7-O-glucoside; FGM, formononetin-7-O-glucosyl-6''-malonate; FGMM, formononetin-7-O-glucosyl-6''-methyl malonate; GLC, glucosidase; (GlcN)6, chitohexaose; Ma, (-)-maackiain; MaG, (-)-maackiain-3-O-glucoside; MaGM, (-)-maackiain-3-O-glucosyl-6''-malonate; ME, malonyl esterase; MT, malonyltransferase; OGT, O-glucosyltransferase; PAL, phenylalanine ammonia-lyase..
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References |
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