Cloning and Nucleotide Sequence of a cDNA ...

Plant Physiol.

(1995) 107: 1021-1022

Plant Gene Register

Cloning and Nucleotide Sequence of a cDNA Encoding S-Adenosyl-L-Methionine Synthetase from Mustard (Brassica juncea [L.] Czern & COSS)' Chao-Ming Wen, Mian Wu, Chong-Jin Goh, and Eng-Chong Pua*

Department of Botany, Faculty of Science, National University of Singapore, Lower Kent Ridge Road, Singapore 051 1, Republic of Singapore Ethylene, a gaseous hormone in plants, is involved in the regulation of a wide range of plant physiological processes, including leaf abscission, fruit ripening, and flower senescence (Yang and Hoffman, 1984). SAM or Adomet synthetase (ATP:L-methionine S-adenosyltransferase; EC 2.5.1.6) catalyzes the conversion of Met to SAM in the presence of ATP and Mg2+, which is the first step in ethylene biosynthesis. Changes in the leve1 of endogenous SAM can greatly affect ethylene synthesis. In tomato (Lycopersicon esculentum), transgenic plants that overproduced bacteriophage T3 SAM hydrolase, the enzyme that degrades SAM to a-ketobutyrate and ammonia, showed a 60 to 80% decrease in ethylene production (Kellogg et al., 1994). Apart from being a precursor in ethylene biosynthesis, SAM also serves as a propylamine group donor in polyamine synthesis and as a methyl group donor in a variety of reactions including transmethylation of proteins, nucleic acids, lipids, and polysaccharides (Tabor and Tabor, 1984). SAM synthetase is ubiquitous in both prokaryotes and eukaryotes. Genes encoding this enzyme have been cloned from Escherichia coli, yeast, and rat (see Van Doorsselaere et al., 1993) and severa1 plant species including Avabidopsis thaliana (Peleman et al., 1989), parsley (Kawalleck et al., 1992), poplar (Van Doorsselaere et al., 1993), carnation (Larsen et al., 1991), and tomato (Espartero et al., 1994). In plants, SAM synthetase is encoded by a small gene family (Peleman et al., 1989; Van Doorsselaere et al., 1993). Two SAM synthetase homologs, sam-1 and sam-3, isolated from A. thaliana exhibited a similar expression pattern. Further study revealed that the sam-2 gene was preferentially expressed in vascular tissues, stem sclerenchyma, and root cortex in transgenic plants (Peleman et al., 1989). In addition, environmental stresses and hormones may have a regulatory role in sam expression. This is suggested by the accumulation of sam-1 and sam-3 transcripts in tomato roots in response to NaC1, mannitol, or ABA treatment (Espartero et al., 1994).

We are interested in the molecular mechanism of ethylene biosynthesis in plants. Genes encoding ACC synthase (Wen et al., 1993) and ACC oxidase (Pua et al., 1992), which are the key enzymes of ethylene biosynthesis (Yang and Hoffman, 1984), have previously been cloned from mustard in our laboratory. In this study, we report the isolation of a cDNA encoding SAM synthetase, designated as msams, from mustard. Severa1 cDNA clones with similar insert size (1.5 kb) were isolated from an amplified library. Restriction and sequence analysis revealed that a11 clones carried an identical insert, which contained an open reading frame encoding a protein of 393 amino acids, with an estimated molecular mass of 43 kD (Table I). msams was highly homologous to the sam-1 gene of A. thaliana, with 82.8% homology in nucleotide sequence and 96.2% in deduced amino acid sequence. The homology in amino acid sequence between msams and those of other organisms is 91.8% with tomato, 89.0% with poplar, 88.5% with parsley, 87.5% with carnation, 62.9% with rat, 59.0% with yeast, and 58.3% with E. coli. Hydrophobic analysis of the amino acid sequence showed the lack of a transit peptide region at the N terminus of the msams protein, indicating that SAM synthetase is a cytosolic enzyme. ACKNOWLEDCMENTS

We are grateful to Professor Marc Van Montagu for providing us with the sam-l gene and to Dixon Yeong Ching Yuen for assistance in the use of the computer program for sequence comparison. Received July 25, 1994; accepted September 26, 1994. Copyright Clearance Center: 0032-0889/95/lO7/l021/02. The GenBank/EMBL accession number for the sequence reported in this article is X80362. LITERATURE ClTED

Espartero J, Pintor-Toro JA, Pardo JM (1994) Differential accumulation of S-adenosylmethionine synthetase transcripts in response to salt stress. Plant Mo1 Biol 2 5 217-227 Kawalleck P, Plesch G , Hahlbrock K, Somssich IE (1992) Induction by fungus elicitor of 5-adenosyl-L-methionine synthetase and 5-adenosyl-L-homocysteine hydrolase mRNAs in cultured cells and leaves of Detroselinum crispum. Proc Natl Acad Sci USA 89: 4713-4717

' This research was supported by grant No. RP930305 of the National University of Singapore. * Corresponding author; e-mail botpuaec8nus3090; fax 65-7795671. Abbreviation: SAM, 5-adenosyl-L-methionine. 1021

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Plant Physiol. Vol. 107, 1995

Table 1. Characteristics of msams cDNA from mustard Organism: Mustard (Brassica juncea [L.] Czern & Coss cv lndia Mustard). Function: Encodes SAM synthetase (EC 2.5.1.6). Techniques: A cDNA library was constructed as previously described (Pua et al., 1992). For library screening, a 1.4-kb cDNA clone sam-7 encoding SAM synthetase of A. thaliana (Peleman et al., 1989) was used as a heterologous hybridization probe. Severa1 positive clones were obtained and nucleotide sequence was determined as previously described (Wen et al., 1993). Methods of Identification: Sequence comparison showed extensive homology with sam-7. Tht? two genes, msams and sam-7, shared the overall identity of 82.8% in nucleotide sequence and 96.2% in deduced amino acid sequence. Features of the cDNA: The clone was 1474 bp in length and possessed an open reading frame of 1 1 78 bp. msams encodes a protein of 393 amino acids with an estimated molecular mass of 43 kD. Gly (9.7%), Asn (8.9%), and Val (8.7%) were the most abundant amino acidr; in the msams protein. The coding region was flanked by 99 bp of noncoding sequence at the 5' end and 197 bp at the 3' end. An in-frame stop codon TGA was located 4 to 6 bp upstream of the coding sequence. The conserved polyadenylation signal AATAAT was located 87 bp downstream of the termination codon. Expression : In northern analysis, a 1.4-kb transcript was detected in leaf, stem, and root of mustard using msams as a homologous hybridization probe. G + C Content: 51.5% for the coding sequence. SubcelI ular Local ization : Not determined. Anti body: Not available.

Kellogg J, Good X, Cohen C, Langhoff D, Matsumura W, Bestwick RK (1994) Regulated expression of S-adenosylmethionine hydrolase using the tomato E4 promoter reduces ethylene synthesis in ripening tomatoes. In Abstracts 4th International Congress of Plant Molecular Biology, 19-24 June 1994, Amsterdam, abstract number 1977 Larsen PB, Woodson WR (1991) Cloning and nucleotide sequence of a S-adenosylmethionine synthetase cDNA from carnation. Plant Physiol96 997-999 Peleman J, Boerjan W, Engler G, Seurinck J, Botterman J, Alliotte T, Van Montagu M, Inze I (1989) Strong cellular preference in the expression of a housekeeping gene of Arubidopsis thaliunu encoding S-adenosylmethionine synthetase. Plant Cell 1: 81-93 Pua E-C, Sim G-E, Chye M-L (1992) Isolation and sequence anal-

ysis of a cDNA clone encoding ethylene-forming enzyme in Brassica juncen tL.1 Czern & Coss. Plant Mo1 Biol 19: 541-544 Tabor CW, Tabor H (1984) Methionine adenosyltransferase (Sadenosylmethionine synthetase) and S-adenosylmethionine decarboxylase. Adv Enzymol 56: 251-282 Van Doorsselaere J, Gielen J, Van Montagu M, Inz,e D (1993) A cDNA encoding S-adenosyl-L-methionine synthetase from poplar. Plant Physiol 102: 1365-1366 Wen C-M, Wu M, Goh C-J, Pua E-C (1993) Nucleotide sequence of a cDNA clone encoding 1-aminocyclopropane-1-carboxylate synthase in mustard (Brussicu junceu [L.] Czern &: Coss). Plant Physiol 103: 1019-1020 Yang SF, Hoffman NE (1984) Ethylene biosynthesis and its regulation in higher plants. Annu Rev Plant Physiol 35: 155-189