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Induction of Anthranilate Synthase Activity by Elicitors in Oats

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Induction of Anthranilate Synthase Activity by Elicitors in Oats Induction of Anthranilate Synthase Activity by Elicitors in Oats Tetsuya Matsukawaa,b,*, Atsushi Ishiharaa,b and Hajime Iwamurab,c a Division of Applied Life Sciences, Graduate School of Agriculture, Kyoto University, Kyoto 606-8502, Japan. Fax: +81(75) 7536408. E-mail: [email protected] b CREST, Japan Science and Technology Corporation (JST) c Department of Bio-Technology, School of Biology-Oriented Science and Technology, Kinki University, Uchita-cho, Naga-gun, Wakayama 649-6493, Japan * Author for correspondence and reprint requests Z. Naturforsch. 57c, 121Ð128 (2002); received September 10/October 12, 2001 Anthranilate Synthase, Avenanthramide, Oats Oat phytoalexins, avenanthramides, are a series of substituted hydroxycinnamic acid am- ides with anthranilate. The anthranilate in avenanthramides is biosynthesized by anthranilate synthase (AS, EC 4.1.3.27). Induction of anthranilate synthase activity was investigated in oat leaves treated with oligo-N-acetylchitooligosaccharide elicitors. AS activity increased transiently, peaking 6 h after the elicitation. The induction of activity was dependent on the concentration and the degree of polymerization of the oligo-N-acetylchitooligosaccharide elicitor. These findings indicate that the induction is part of a concerted biochemical change required for avenanthramide production. The elicitor-inducible AS activity was strongly in- hibited by l-tryptophan and its analogues including 5-methyl-dl-tryptophan, and 5- and 6- fluoro-dl-tryptophan, while the activity was not affected by d-tryptophan. The accumulation of avenanthramide A was also inhibited by treatment of elicited leaves with these AS inhibi- tors, indicating that a feedback-sensitive AS is responsible for the avenanthramide pro- duction. In elicited leaves, the content of free anthranilate remained at a steady, low level during avenanthramide production. Moreover, administration of anthranilate to elicited oat leaves resulted in an enhanced avenanthramide accumulation. AS may play a role as a rate- limiting enzyme in the biosynthesis of avenanthramides. Introduction (HHT, EC 2.3.1.-), which catalyzes the final con- densation reaction to form the amide bond, was Avenanthramides, a series of hydroxycinnamic identified in elicitor-treated oat leaves (Ishihara et acid amides with hydroxyanthranilates, have been al., 1997; 1998). These findings indicate that aven- well characterized as phytoalexins in oats (May- anthramides are de novo synthesized from ama et al., 1981; 1982). Avenanthramides accumu- anthranilate and l-phenylalanine supplied by the late in oat leaves in incompatible interactions with shikimate pathway that branches at chorismate. crown rust fungus (Puccinia coronata f. sp. Thus, the enzymes catalyzing the first steps from avenae), and are considered an important factor in the branch point may have important regulatory the defense against pathogens. An accumulation functions for avenanthramide biosynthesis. of avenanthramides is also evoked by treatment The first step leading to the biosynthesis of hy- of oat leaves with various elicitors including N- droxyanthranilate is catalyzed by anthranilate syn- acetylchitooligosaccharides (Bordin et al., 1991). thase (AS, EC 4.1.3.27). AS is involved in the bio- The hydroxyanthranilate in avenanthramides synthesis of many secondary metabolites in plants, has been demonstrated to be derived from and is indicated to play a regulatory role in the anthranilate by feeding experiments with labeled biosynthesis of tryptophan (Delmer and Mills, precursors (Ishihara et al., 1999a). The hydroxycin- 1968; Widholm, 1974). Plant AS has been demon- namoyl moieties have been shown to be biosyn- strated to be composed of two subunits, ASα and thesized via the phenylpropanoid pathway. The AS, by enzyme purification (Poulsen et al., 1993; enzymes of this pathway are activated concertedly Bohlmann et al., 1995) and by cDNA cloning (Niy- following elicitor treatment (Ishihara et al., ogi and Fink, 1992; Niyogi et al., 1993). ASα cata- 1999b). In addition, hydroxycinnamoyl-CoA:hy- lyzes the conversion of chorismate to anthranilate droxyanthanilate N-hydroxycinnamoyltransferase with ammonia as amino donor, while AS 0939Ð5075/2002/0100Ð0121 $ 06.00 ” 2002 Verlag der Zeitschrift für Naturforschung, Tübingen · www.znaturforsch.com · N 122 T. Matsukawa et al. · Anthranilate Synthase Activity Induced by Elicitors in Oats transfers the amino group from glutamine to ASα. Enzyme extraction Genes encoding the α-subunits of AS isozymes All procedures were carried out at 4 ∞C. Oat have been cloned from Arabidopsis (Niyogi and leaves (ca. 0.25 gFW) were frozen in liquid nitro- Fink, 1992) and Ruta graveolens (Bohlmann et al., gen and ground until well powdered. The powder 1995). The expression of a specific isozyme is in- was extracted with 5 volumes of buffer A, and duced by environmental stimuli, suggesting the in- centrifuged for 10 min at 12,000¥g. The superna- volvement of the isozyme in the activated pro- tant was passed over a PD-10 column (Pharmacia) duction of secondary metabolites in these plants. equilibrated with buffer A to remove low molecu- To elucidate the function of AS in the biosyn- lar weight compounds. The fraction containing thesis of avenanthramides, the induction of AS by proteins was used for the enzyme assay as the elicitor treatment was investigated in oat leaves. crude extract. Protein concentration was deter- In addition, the elicitor-inducible AS was purified mined by the method of Bradford (1976) using bo- and characterized. vine serum albumin (BSA) as the protein stan- dard. Materials and Methods Enzyme assay Plant material Anthranilate synthase assay was carried out ac- Oat (Avena sativa L. cv. Shokan 1) caryopses cording to the method of Bücker et al. (1995) with were soaked in distilled water for 24 h. They were a slight modification. A reaction mixture contain- then sown in wet vermiculite and maintained at ing 0.5 ml of 4 mm chorismate and 0.5 ml of the Ð 20 ∞C for 7 d under continuous light (15 W m 2) crude extract was incubated at 30 ∞C for 30 min. in growth chambers. At this growth stage, the The reaction was stopped by boiling water. Preci- average height of seedlings was around 7 cm. Af- pitated proteins were removed by centrifugation ter the lower epidermis had been peeled off, pri- (12,000¥g, 30 min), and the resulting supernatant mary leaves were floated on a solution of 1 mm was subjected to reversed-phase HPLC (column: oligo-N-acetylchitooligosaccharides as described Polymer C18, 150¥4.6 mm, YMC, Kyoto). previously (Ishihara et al., 1998). Anthranilate was eluted with 45% methanol in water containing 0.1% phosphoric acid at a flow Chemicals rate of 0.8 ml minÐ1, and was detected with a fluo- Chorismic acid (barium salt) and -phenylpyru- rescence detector (Shimadzu RF-10AXL; Ex = vic acid were obtained from Sigma. Oligo-N-ace- 340 nm, Em = 400 nm). In this system, anthranilate tylchitooligosaccharides were purchased from Sei- was eluted with a retention time of 15.2 min. kagaku Kogyo, Tokyo. All other chemicals were from Wako Pure Chemical Industries, Osaka. Partial purification of AS All operations were carried out at 4 ∞C. A solu- Buffers tion of 50% (w/v) polyethylene glycol 6000 was The following buffers were used for enzyme added to the crude extract to give a final concen- preparation and purification: Buffer A, 100 mm tration of 5%. After stirring for 30 min, the precip- tris(hydroxymethyl)aminomethane (Tris)-HCl itate was collected by centrifugation (10,000¥g,30 buffer (pH 7.5) containing 1 mm EDTA, 2 mm min). The supernatant was adjusted to a final con- mercaptoethanol, 20 mm glutamine, 4 mm MgCl2 centration of 18% polyethylene glycol 6000, and and 10% glycerol; Buffer B, 50 mm Tris-HCl buffer the precipitate was collected by centrifugation (pH 7.5) containing 1 mm EDTA, 2 mm mercapto- (10,000¥g, 30 min). The resulting pellet was dis- ethanol, 20 mm glutamine, 4 mm MgCl2 and 10% solved in buffer B. This solution was subjected to glycerol; Buffer C, 20 mm Tris-HCl buffer (pH 7.5) preparative electrophoresis using a Mini Prep Cell containing 1 mm EDTA, 2 mm mercaptoethanol, (Bio Rad) instrument (elution buffer: buffer B, 20 mm glutamine, 4 mm MgCl2 and 10% glycerol. polyacrylamide gel: 5%, 5 cm, pH 7.5, constant current: 7.5 mA). The active fractions were col- T. Matsukawa et al. · Anthranilate Synthase Activity Induced by Elicitors in Oats 123 lected and applied to a Mono Q HR 5/5 column (Pharmacia) equilibrated with buffer C. AS was eluted with a 40 ml linear gradient of 0 to 0.3 m NaCl at a flow rate of 0.5 ml/min. Fractions con- taining AS activity were concentrated by ultra- filtration (Centricon-10, Amicon), and finally loaded onto a Superdex 200 HR 10/30 column (Pharmacia) equilibrated with buffer B. Proteins were eluted with buffer B at a flow rate of 0.2 ml/min. For determination of the native molecular weight of the purified enzyme, transfer in (MW = 81 kDa), BSA (MW = 66 kDa), lactoglobin (MW = 35 kDa), and myoglobin (MW = 17.6 kDa) were used as molecular weight markers. Analysis ofanthranilic acid and avenanthramides in elicitor-treated oat leaves For analysis of free anthranilic acid, elicitor- treated oat leaves frozen in liquid nitrogen were ground until well powdered. After the addition of p-aminobenzoic acid as internal standard, the powder was extracted twice with 10 volumes of methanol. The solution was centrifuged for 10 min at 12,000¥g. The combined supernatant fraction was evaporated to dryness in vacuo. The residue was dissolved in 5 ml of distilled water, and ex- tracted twice with 5 ml of ethyl acetate. The com- bined ethyl acetate phase was evaporated, and the residue was dissolved in 1 ml of methanol. The so- lution was analyzed by reversed-phase HPLC un- der the same conditions described in the enzyme assay section. The amounts of avenanthramides were deter- mined by HPLC analysis of the elicitor solution 24 h after elicitation as described previously (Ishi- hara et al., 1998).
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