Nucleotide sequence of a cDNA clone encoding 1 ...

Plant Physiol. (1993)103: 1019-1020

Plant Gene Register

Nucleotide Sequence of a cDNA Clone Encoding 1-Aminocyclopropane-1 -Carboxylate Synthase in 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 plant hormone, is involved in regulation of various physiological responses during plant growth and development. These include seed germination, abscission, fruit ripening, and plant senescence (Yang and Hoffman, 1984). Plants also produce high levels of ethylene when they are under environmental stresses or pathogen attacks. In ethylene biosynthesis, the precursor Met is converted to SAM via the catalysis of the enzyme SAM synthetase. SAM is further converted to ACC by ACC synthase (EC 4.4.1.14), which is the rate-limiting step of ethylene production. This reaction can be inhibited by exogenous application of aminoethoxyvinylglycine or aminooxyacetic acid, both of which are inhibitors of pyridoxal phosphate, which is required in ACC synthase activity. The last step of ethylene biosynthesis, ACC-ethylene, is catalyzed by ACC oxidase (ethyleneforming enzyme) (Yang and Hoffman, 1984). ACC synthase has been purified to homogeneity from tomato fruits, and the corresponding genes have been isolated (Van Der Straeten et al., 1990), although the enzyme usually is present at low concentrations. To date, genes encoding ACC synthase have been cloned from severa1 plant species including monocots and dicots (Theologis, 1992). In tomato, at least six different genes for ACC synthase have been identified, indicating that the enzyme is encoded by a multigene family (Rottmann et al., 1991). Two of the genes have been shown to be differentially expressed in ripening fruits after wounding. In transgenic experiments, tomato plants expressing antisense ACC synthase RNA displayed a great reduction of ethylene production, concomitant with a delay of the fruit-ripening process (Oeller et al., 1991). We have previously shown the regulatory role of ethylene in shoot morphogenesis of recalcitrant Brassica species in vitro (Chi et al., 1991; Pua, 1993; Pua and Chi, 1993) and have also cloned a cDNA encoding ACC oxidase from mustard (Pua et al., 1992). We report in this study a cDNA clone encoding ACC synthase, designated pMACC, isolated from mustard

Table 1. Characteristics of MACC cDNA from mustard

Organism: Mustard (Brassica juncea [L.] Czern & Coss cv lndia Mustard). Function: Encodes ACC synthase (EC 4.4.1.14). Techniques: The cDNA library was constructed as previously described (Pua et al., 1992).For library screening, a 340-bp homologous probe was amplified from the cDNA library by the polymerase chain reaction using mixed oligonucleotide primers. The upstream primer was CTCGATCCCTCAAA(C/T)CC(C/A/T/ G)(T/C)T(A/G)CCCAC(C/A/T/G)ACand the downstream pri mer was CTCAAGCTTAC(C/A/T/G)A(A/C)(C/A/T/C)CC(A/ G)AA(A/C)CT(C/T)CACAT, as designed by Kim et al. (1992). The clone was identified by Southern blot analysis and linearized, and the insert was restricted with exonuclease III to generate a nested set of deletions using the Erase-a-Base system (Promega Corp., Madison, WI). The nucleotide sequence of t h e insert was determined by the dideoxy chain termination method using Taq DNA polymerase (Promega Corp.). Method of Identification: Sequence comparison showed high homology with the tomato ACC synthase gene LE-ACC2 (Rottmann et al., 1991). Both genes shared the overall identity of 64.1% in nucleotide sequence and 60.5% in deduced amino acid sequence. Feature of t h e cDNA: The clone was 1780 bp in length and possessed an open reading frame of 1560 bp, which encodes a protein of 497 amino acids. The open reading frame began with the first ATG codon at the position of 67 and terminated at the codon TAA at position 1558. The conserved plant polyadenylation signal AATAAT was located downstream of t h e termination codon at the position of 1726. G + C Content: 44.5% for both the coding and entire sequences. Subcellular Localization: N o t determined.

Antibody:

Not available.

(Brassica juncea). The cDNA contained one open reading frame encoding a

' This research was supported by grants RP920358 (to E.-C.P.and C.-J.G.) and RP910401 (to M.W.) of the National University of Singapore. * Corresponding author; fax 65-779-5671.

Abbreviation: SAM, S-adenosylmethionine 1019

Wen et al.

1020

protein of 497 amino acids, with an estimated molecular mass of 55.5 kD (Table I). Both Leu (9.05%) and Ala (7.85%), as in ACC oxidase (Pua et al., 1992), were the most abundant amino acids in the MACC protein. Comparison of a highly conserved region (amino acid positions 211-311) of the MACC cDNA with ACC synthase genes of other plant species showed the highest homology, 69%, with AT-ACCI of Arabidopsis thaliana (Van Der Straeten et al., 1992), 65% with LE-ACC2 of tomato (Rottmann et al., 1991), and 52% with OS-ACCI of rice (Theologis, 1992). The degree of homology may be related to the phylogenetic relationship between mustard and these plant species, among which A. thaliana is closer to mustard. Nevertheless, MACC contained domains highly conserved in ACC synthase genes, including the active site of pyridoxal phosphate attachment, of both monocots and dicots (Theologis, 1992), indicating the common evolutionary origin of the genes. Received May 25, 1993; accepted June 7, 1993. Copyright Clearance Center: 0032-0889/93/103/1019/02. The GenBank/EMBL accession number for the sequence reported in this article is X72676 BJMACC.

LITERATURE ClTED

Chi G-L, Pua E-C, Goh C-J (1991) Role of ethylene on de novo shoot regeneration frorn cotyledonary explants of Brassica campestris ssp. pekinensis (Lour) Olsson in vitro. Plant Physiol96: 178-183

Plant Physiol. Vol. 103, 1993

Kim WT, Silverstone A, Yip WK, Dong JG, Yang SF (1992) Induction of 1-aminocyclopropane-1-carboxylatesynthase mRNA by auxin in mung bean hypocotyls and cultured apple shoots. Plant Physiol98: 465-471 Oeller PW, Lu M-W, Taylor LP, Pike DA, Theologis A (1991) Reversible inhibition of tomato fruit senescence by antisense RNA. Science 254 437-439 Pua E-C (1993) Cellular and molecular aspects of ethylene on plant morphogenesis of recalcitrant Brassica species in vitro. Bot Bull Acad Sin 3 4 191-209 Pua E-C, Chi G-L (1993) De novo shoot morphogenesis and plant growth of mustard (Brassica juncea) in vitro in relation to ethylene. Physiol Plant 88: 467-474 Pua E-C, Sim G-E, Chye M-L (1992) Isolation and sequence analysis of a cDNA clone encoding ethylene-forming enzyme in Brassica juncea (L.) Czern & Coss. Plant Mo1 Biol 19: 541-544 Rottmann WH, Peter GF, Oeller PW, Keller JA, Shen NF, Nagy BP, Taylor LP, Campbell AD, Theologis A (1991) 1-Aminocyclopropane-1 -carboxylate synthase in tomato is encoded by a multigene family whose transcription is induced during fruit and floral senescence. J Mo1 Biol 222: 937-961 Theologis A (1992) One rotten apple spoils the whole bushtrl: the role of ethylene in fruit ripening. Cell 70: 181-184 Van Der Straeten D, Rodrigues-Pousada RA, Villarroel R, Hanley S, Goodman HM, Van Montague M (1992) Cloning, genetic mapping, and expression analysis of an Arabidopsis gene that synthase. Proc Natl encodes 1-aminocyclopropane-1-carboxylate Acad Sci USA 8 9 9969-9973 Van Der Straeten D, Van Wiemeersch L, Goodman HM,, Van Montague M (1990) Cloning and sequence of two different c'DNAs encoding 1-aminocyclopropane-l -carboxylate synthase in tomato. Proc Natl Acad Sci USA 87: 4859-4863 Yang SF, Hoffman NE (1984) Ethylene biosynthesis and its regulation in higher plants. Annu Rev Plant Physiol 3 5 155-189