Plant Molecular Biology 20: 167-170, 1992. © 1992 Kluwer Academic Publishers. Printed in Belgium.

167

Update section Sequence

Molecular cloning of phenylalanine ammonia-lyase eDNA from Pisum sativum Shinji Kawamata 1, Tetsuji Yamada*, Yoshikazu Tanaka 2, Permpong Sriprasertsak, Hisaharu Kato, Yuki Ichinose, Hidenori Kato, Tomonori Shiraishi and Hachiro Oku

Laboratory of Plant Pathology and Genetic Engineering, College of Agriculture, Okayama University, Tsushirna, Okayama, 700, Japan (* author for correspondence)," Present addresses: 1Takasago Research Institute Inc., 5-31-36 Kamata, Minato, Tokyo, 144, Japan; 2 Toso Inc., Tokuyama, Yamaguchi, Japan Received 23 March 1992; accepted 9 April 1992

Key words: defense response, pea, D N A sequence, elicitor, suppressor, pisatin

Phenylalanine ammonia-lyase (PAL; EC 4.3.1.5) is a key regulatory enzyme in phenylpropanoid pathway including a wide variety of natural products such as isoflavonoid phytoalexins, lignin, flavonoid pigments and UV protectants, furanocoumarin, and wound protectant hydroxycinnamic acid esters [1, 3, 6]. It has been reported that PAL expression is regulated by environmental stimuli such as phytopathogens, elicitors and UV light irradiation [2, 5, 8-10]. Pycnospore germination fluid of Mycosphaerella pinodes (Berk. et Blox.) Stone, a fungus pathogenic on pea, contains both elicitors and suppressors for the accumulation of the pea isoflavonoid phytoalexin, pisatin [ 14]. The elicitors induce pisatin accumulation, accompanying with the gene activation encoding phenylalanine ammonia-lyase (PAL) and chalcone synthase (CHS) [ 16]. Whereas the suppressor have the effects to delay the activation of the defense responses such as a 3-h delay in the accumulation of PAL and CHS m R N A s [ 16]. Very recently, we have shown that the suppressor specifically inhibits pea plasma membrane

(PM)-ATPase but does not inhibit other types of ATPase [ 17]. Furthermore concomitant presence of orthovanadate, a PM-ATPase inhibitor, with fungal elicitor results in delaying the induction of pisatin accumulation [17] and the accumulation of PAL- or C H S - m R N A in a manner very similar to fungal suppressor [ 18]. To analyze the mechanisms of gene activation and suppression by these fungal signal molecules, we first cloned PAL-eDNA and the complete nucleotide sequence was determined. A e D N A library was constructed from poly(A) + R N A extracted from pea epicotyls 4.5 h after elicitor treatment by standard procedures. The resultant independent plaques (5 x 104) were screened by plaque hybridization with 32P-labeled bean PAL-cDNA probe [4], and six positive clones whose c D N A insert sizes were ranging from 2.4 to 0.6 kb were obtained as a result of three rounds of screening. Restriction mapping analysis revealed that they were divided into four groups (data not shown). The lambda clone containing the 2.4 kb insert was designated cPAL-1.

The nucleotide sequence data reported will appear in the EMBL, GenBank and DDBJ Nucleotide Sequence Databases under the accession number D10001.

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Fig. 1. Immunoassay of the expressed protein from PALcDNA in E. coli. Crude lysate from the E. coli 7118 carrying pGEM4Z (lane a), pKT1RV (cDNA insert in the expressible orientation in pGEM4Z) (lane b), and pKT1 (cDNA insert in the orientation opposite to pKT1RV) (lane c) were fractionated on 4-20 % S D S-polyacrylamide gradient gel and blotted onto nitrocellulose membrane filter. The blotted filter was cross-reacted with rabbit antiserum to pea PAL (a gift from Dr L. Hadwiger), washed, and detected according to the supplier's specification (Amersham). The position of putative PAL (ca. 80 kDa) was indicated. The numbers on the left are the molecular mass (in kDa) of the size markers.

In order to verify cPAL-1 as PAL-specific, the immunoassay was carried out. That is, the crude protein extracts from the lysate of Escherichia coli carrying the cDNA insert subcloned into pGEM4Z in both directions (pKT1 and pKT1RV) were fractionated by electrophoresis on a 4-2070 SDS-polyacrylamide gradient gel and blotted onto nitrocellulose filter. Immunoassay was carried out with the Amersham SuperScreen immunoscreening system using antiserum to Pisurn sativurn PAL (a gift from Dr L.A. Hadwiger) (Fig. 1). Approximately 80 kDa of polypeptide bound to anti-PAL was detected in the lysate of E. coli carrying pKT1RV, but not pKT1 or pGEM4Z. According to the molecular mass of the purified pea PAL (81 kDa) deter-

mined by Loschke and Hadwiger [11 ], this clone was presumed to contain closely full-length cDNA. The nucleotide sequence of the cDNA shows that it contains 2367 bp encoding 724 amino acids with a deduced molecular weight of 78827 (Fig. 2). The deduced amino acid sequence was compared with that from cDNA of Phaseolus vulgaris [4], Solanurn tuberosum [ 151, Petroselinurn crispum [10], Arabidopsis thaliana [13] and Oryza sativa [ 12]. When gaps that permit maximum homology were introduced, amino acid homology of 88.1, 77.1, 85.1, 77.0, and 65 70, respectively, were scored. We have not observed poly(A) tail at the 3' end, although no obvious polyadenylation signal [7] was detected. The disappearance of poly(A) tail might be due to the incomplete second-strand synthesis during cDNA construction. To analyze the organization of pea pal genes, high-molecular-mass genomic DNAs digested with Barn HI, Bgl II, Eco RI, Eco RV and Hind III were separated by 0.770 agarose gel electrophoresis, and transferred to Hybond N + nylon membrane filter (Amersham). Hybridization was carried out using 32p-labeled Barn HI fragment (349bp) from cPAL-1. At least three to five bands, one to two of which were strong bands, were observed in restriction endonuclease-digested total genomic DNA under relatively lowstringency hybridization conditions (Fig. 3). These results and a grouping of PAL-cDNAs suggest that PAL is encoded by a small gene family with at least four closely related pal genes in pea.

Acknowledgements The authors deeply thank Dr Chris J. Lamb of the Salk Institute, UCSD, USA, and Dr Lee A. Hadwiger, Dept. of Plant Pathology, Wash-

Fig. 2. The nucleotide sequence and the deduced amino acid sequence of cPAL-1. A. A restriction map. Specific Barn HI fragment (349 bp) used in Southern blot hybridization analysis is indicated as closed box below. Restriction enzymes: B, Barn HI; C, Hinc II; H, Hind III; G, Bgl II; R, Eco RV; P, Pst I. B. The nucleotide sequence and the deduced amino acid sequence of cPAL-1.

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Fig. 3. DNA gel blot analysis of pea genomic DNA probed with a fragment from PAL-cDNA. Per lane 15/~g of DNA was digested with Barn HI (lane 1), BgllI (lane2), Eco RI (lane 3), Eco RV (lane 4), and Hind III (lane 5), separated by electrophoresis on a 0.7 % agarose gel, blotted, and hybridized with a labeled 349 bp Barn HI fragment as indicated in Fig. 2A. The blot was washed in 0.1 x SSC, 0.1% SDS at 65 °C and autoradiographed for 3 days with intensifying screen.

ington State University, for the gift of the bean PAL-cDNA probe and anti-pea PAL, respectively. This work was supported in part by Grants-in-Aid for Scientific Research from the Ministry of Education, Science and Culture of Japan and by Takasago Research Institute. References 1. Cramer CL, Edwards K, Dron M, Liang X, Dildine SL, Bolwell GP, Dixon RA, Lamb CJ, Schuch W: Phenylalanine ammonia-lyase gene organization and structure. Plant Mol Biol 12:367-383 (1989). 2. Dangle JL, Hauffe KD, Lipphardt S, Hahlbrock K, Scheel D: Parsley protoplasts retain differential responsiveness to u.v. light and fungal elicitor. EMBO J 6: 2551-2556 (1987). 3. Dixon RA, Dey PM, Lamb CJ: Phytoalexins; enzymology and molecular biology. Adv Ezymol Relat Areas Mol Biol 55:1-135 (1983). 4. Edwards K, Cramer CL, Bolwell GP, Dixon RA, Schuch W, Lamb CJ: Rapid transient induction of phenylalanine ammonia-lyase mRNA in elicitor-treated bean cells. Proc Natl Acad Sci USA 82:6731-6735 (1985).

5. Hahlbrock K, Scheel D: Physiology and molecular biology of phenylpropanoid metabolism. Annu Rev Plant Physiol Plant Mol Biol 40:347-369 (1989). 6. Jones DH: Phenylalanine ammoniaqyase: Regulation of its induction, and its role in plant development. Phytochemistry 23:1349-1359 (1984). 7. Joshi CP: Putative polyadenylation signals in nuclear genes of higher plants: A compilation and analysis. Nucl Acids Res 15:9627-9640 (1987). 8. Lamb CJ, Lawton MA, Dron M, Dixon RA: Signals and transduction mechanisms for activation of plant defenses against microbial attack. Cell 56:215-224 (1989). 9. Liang X, Dron M, Cramer CL, Dixon RA, Lamb CJ: Differential regulation of phenylalanine ammonia-lyase genes during plant development and environmental cues. J Biol Chem 264:14486-14492 (1989). 10. Lois R, Dietrich A, Hahlbrock K, Schulz W: A phenylalanine anlmonia-lyase gene from parsley: Structure, regulation and identification of elicitor and light responsive cis-acting elements. EMBO J 8:1641-1648 (1989). 11. Loschke DC, Hadwiger LA: Effects of light and of Fusarium solani on synthesis and activity of phenylalanine ammonia-lyase in peas. Plant Physiol 68: 680685 (1981). 12. Minami E, Ozeki Y, Matsuoka M, Koizuka N, Tanaka Y: Structure and some characterization of the gene for phenylalanine ammonia-lyase from rice plants. Eur J Biochem 185:19-25 (1989). 13. Ohl S, Hedrick SA, Chory J, Lamb CJ: Functional properties of a phenylalanine ammonia-lyase promoter from Arabidopsis. Plant Cell 2:837-848 (1990). 14. Shiraishi T, Yamada T, Oku H, Yoshioka H: Suppressor production as a key factor for fungal pathogenesis. In: Patil SS, Ouchi S, Mills D, Vance C (eds) Molecular Strategies of Pathogens and Host Plants. Springer-Verlag, New York (1991). 15. Tanaka Y, Matsuoka M, Yamamoto N, Ohashi Y, KanoMurakami Y, Ozeki Y: Structure and characterization of a cDNA clone for potato. Plant Physiol 90:1403-1407 (1989). 16. Yamada T, Hashimoto H, Shiraishi T, Oku H: Suppression ofpisatin, phenylalanine ammonia-lyase mRNA, and chalcone synthase mRNA accumulation by a putative pathogenicity factor from the fungus Mycosphaerella pinodes. Mol Plant-Microbe Inter 2:256-261 (1989). 17. Yoshioka H, Shiraishi T, Yamada T, Ichinose Y, Oku H: Suppression ofpisatin production and ATPase activity in pea plasma membranes by orthovanadate, verapamil and suppressor from Mycosphaerella pinodes. Plant Cell PhysioI 31:1139-1146 (1990). 18. Yoshioka H, Shiraishi T, Kawamata S, Nasu K, Yamada T, Ichinose Y, Oku H: Orthovanadate suppresses accumulation of phenylalanine ammonia-lyase mRNA and chalcone synthase mRNA in pea epicotyls induced by elicitor from Mycosphaerella pinodes. Plant Cell Physiol, in press.

Molecular cloning of phenylalanine ammonia-lyase cDNA from Pisum sativum.

Plant Molecular Biology 20: 167-170, 1992. © 1992 Kluwer Academic Publishers. Printed in Belgium. 167 Update section Sequence Molecular cloning of...
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