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Received for publication May 3, 1991 Accepted May 14, 1991 Plant Physiol. (1991) 96, 1393-1394 0032-0889/91/96/1 393/02/$01 .00/0 Plant Gene Register Nucleotide Sequence of an Iron Superoxide Dismutase Complementary DNA from Soybean' Dring N. Crowell*2 and Richard M. Amasino Department of Biochemistry, College of Agricultural and Life Sciences, University of Wisconsin-Madison, Madison, Wisconsin 53706 We have reported the isolation of a cDNA (2) for which the corresponding mRNA, called SAM46, accumulates in cultured soybean cells during cytokinin or auxin starvation (see Table I). This mRNA, which decreases rapidly in abundance following treatment of cytokinin-starved cells with 5 mM zeatin or following treatment of auxin-starved cells with 10 mM a-naphthaleneacetic acid, is detectable in expanded primary leaves but not in unexpanded primary leaves of soybean seedlings. The 5' end of the SAM46 cDNA was shown to be 46% identical at the amino acid level to the 5' end of the Escherichia coli iron superoxide dismutase gene (sodB) (1, 2). Furthermore, expression of the SAM46 cDNA in E. coli cells resulted in measurable FeSOD3 activity (2). The nucleotide sequence of the SAM46 cDNA and the deduced amino acid sequence of the SAM46 protein product are shown in Figure 1. The site of translation initiation is presumed to be the ATG at position 24 to 26 of the sequence because: (a) the SAM46 cDNA is a full-length cDNA, or very close to it, suggesting that translation does not begin upstream of position 24 to 26, and (b) the N-terminus of the mature soybean FeSOD is predicted, based on N-terminal amino acid sequences of other known FeSODs, to be the lysine encoded by the AAG codon at position 99 to 101 of the sequence (4), suggesting that translation does not begin downstream of position 24 to 26. The overall sequence identity between the SAM46 product and the FeSOD of E. coli is 41 %, with most of the sequence divergence occurring at the carboxy-terminus (1). In addition, the SAM46 product is 59 and 58% identical to the products of the FeSOD cDNAs from Nicotiana plumbaginifolia and Arabidopsis thaliana, respectively (7). Again, the majority of the sequence divergence is at the carboxyterminus. We are currently studying several aspects of soybean FeSOD gene expression. We are interested, for example, in fully characterizing the induction of the soybean FeSOD gene in response to cytokinin or auxin starvation to determine whether this gene responds generally to stress or specifically Table I. Characteristics of the FeSOD cDNA from Glycine max Organism: Glycine max cv Mandarin (soybean). Location on Chromosomes: Unknown; gene copy number reconstruction experiments suggest between 1 and 5 copies per haploid genome (D. N. Crowell, R. M. Amasino, unpublished observation). Gene, Function, Pathway: Soybean FeSOD gene; FeSOD (EC 1.15.1.1) catalyzes dismutation of superoxide to molecular oxygen and hydrogen peroxide (5). Techniques: cDNA library in Bluescript SKII+ (Stratagene, La Jolla, CA) (2); restriction fragment subcloning; dideoxynucleotide sequencing (6) of plasmid DNA (both strands). Method of Identification: Sequence comparison to GenEMBL database (2, 3); sequence identity to E. coli sodB gene (1, 2); detection of FeSOD activity in E. coli cells expressing SAM46 (2). Expression Characteristics: mRNA of approximately 1 kilobase detected by RNA blot analysis of polyadenylate-enriched RNA and total RNA (2; D. N. Crowell, R. M. Amasino, unpublished observation). Regulation: FeSOD mRNA accumulation induced in cultured soybean cells (G. max cv Mandarin) during cytokinin or auxin starvation; FeSOD mRNA accumulation induced in soybean leaves (G. max cv Elgin) during leaf expansion (2). Codon Usage: Codons not present in FeSOD open reading frame: GCG (A), AGG (R), ACG (T), TGT (C), TCG (S), CGA (R), CGC (R); amino acids with a high bias to one codon: R (AGA), I (ATT), L (CTT). (G + C) Content: 41%. Structural Features of Protein Product: Coding region codes for a polypeptide of 248 amino acids with a predicted Mr of 27,824 D; first 25 amino acids are predicted to be leader sequence for chloroplast import (4, 5); predicted Mr of mature polypeptide, beginning with Lys26, is 25,285 D; plant FeSODs are generally dimers consisting of two equally sized subunits and one to two atoms of iron per dimer (5). Antibodies: Antibodies have not been prepared by this laboratory. Subcellular Location of Protein Product: Chloroplast (5). 'This work was supported by grant DCB-8957036 from the National Science Foundation and by the College of Agricultural and Life Sciences, University of Wisconsin-Madison. R.M.A. is a James D. and Dorothy Shaw scholar. 2Current Address: Department of Biology, Indiana UniversityPurdue University at Indianapolis, Indianapolis, IN 46205. 'Abbreviation: FeSOD, iron superoxide dismutase. EMBL Accession No.: M64267 1393 Downloaded from on June 16, 2017 - Published by www.plantphysiol.org Copyright © 1991 American Society of Plant Biologists. All rights reserved. Plant Physiol. Vol. 96, 1991 CROWELL AND AMASINO 1394 TCCAGCCACACGCTTCAGAAGACATGGCCTCATTGGGTGGGTTACAAAATGTGAGCGGCATCAATTTTCTTATCAAAGAGGGTCCAAAAGTCAATGCAAA 100 S A M L G G L N Q S V I G N L F K I K G P 20 E V A N K GTTCGAGCTGAAGCCGCCACCATATCCACTGAATGGTTTGGAGCCGGTGATGA(,CCAGCA(,ACACT'FGAG1_-TTTCACTGGGGGAAGCACCACAAGACTTAT 200 F E K L P P Y P N L P L G E P S M V Q Q T E L H F W K G H H T K Y 40 GTGGAAAATCTGAAAAAACAAGTTGTTGGGACAGAGCTTGATGGGAAGTCACTAGAAGAGATTATTGTCACATCATACAATAAGGGTGACATTCTTCCAG 300 E V K L N K Q V G V E T L D K G L S E V I I 80 E 60 S T Y K N D G L I P CTTTCAACAATGCAGCACAGGTATGGAACCATGACTTCTTCTGGGAGTGCATGAAACCAGGTGGAGGTGGAAAGCCATCCGGGGAGCTTCTAGAACTGAT 400 F A N N A V Q A N W D H F F E W C K M P G G G G P K S L E G L L E I 120 100 TGAAAGAGACTTTGGTTCATTTGTAAAATTCCTTGATGAGTTCAAGGCTGCTGCTGCAACACAATTTGGTTCAGGGTGGGCTTGGCTAGCATATAGAGCA 500 E R D F S G F K V F L D E F K 140 A A A T A F Q G S A W G W A L R Y A AGAAAATTTGATGGGGAAAATGTAGCAAATCCTCCTTCACCCGATGAGGACAACAAGCTAGTGGTGCTCAAGAGTCCCAATGCTGTGAACCCCCTTGTTT 600 R K D F G E A V N N P P P S D D E K N L V K L V S P N A N V L P V 180 160 GGGGAGGTTACTACCCACTTCTTACCATTGATGTTTGGGAGCATGCTTACTACCTTGATTTTCAGAACCGGCGTCCTGATTATATATCAGTGTTCATGGA 700 W Y G G P Y L I T L V D E W H Y A Y F D L N Q R R P 200 D Y I 220 S V F M D TAAGCTTGTTTCCTGGGATGCAGTGAGCTCTAGACTTGAACAAGCTAAGGCTTTAATTACCAGTGCATGATGCTGAATTAAATGCAGAATAAGTGATTTA K L V S W D A V S S R L E Q A 240 K A L I T S A 800 * TCCTGATAGTGATGATGAATTGGATGGCGTCATGCGGAATAGTGAATTATTTTTATCTAGAAAGTGTAAGCAGGCACATCTTTTGTACTTTAAATAGGTG 900 TTGTGGTATCAGGGCTAAATCTCAGATTATTATGTTCTATGGTTAGAAATCTTAGTTATGGTTCTCTTTGTGTGACAATGTGAACAATAAGATTGCTTAT 1000 GCATGAATAATCAACAAAATAATACTATGGTGTAGAGCCTTTTCATATAAAAAAAAAAAAAAAAAAA 1067 Figure 1. Nucleotide sequence of the SAM46 cDNA and deduced amino acid sequence of the SAM46 protein product The amino acid assumed to be the N-terminus of the mature soybean FeSOD (Lys26) is in bold face. This assumption is based on N-terminal amino acid sequences of other known FeSODs (4). in cultured soybean cells during cytokinin or auxin starvation. to phytohormones. We are also interested in studying FeSOD Plant Physiol 95: 711-715 would we In leaf induction expansion. particular, during gene 3.Devereux J, Haeberli P, Smithies 0 (1984) A comprehensive set liketo dtermne wethe a cusalrelaionsip coincicausal relationship or a cinci whether of sequence analysis programs for the VAX. Nucleic Acids Res like to determine 12: 387-395 dental relationship exists between leaf expansion and FeSOD 4. Harris JI, Auffret AD, Northrop FD, Walker JE (1980) Strucgene induction. Finally, we are beginning studies of the import tural comparisons of superoxide dismutases. Eur J Biochem of the SAM46 protein product into chloroplasts and its assem- 297-303 bly into aa functional functional enzyme. with iron iron into bly with 5. Salin ML (1988) Toxic oxygen species and protective systems of the chloroplast. Physiol Plant 72: 681-689 a o a enzyme.106: LITERATURE CITED 1. Carlioz A, Ludwig ML, Stallings WC, Fee JA, Steinman HM, Touati D (1988) Iron superoxide dismutase: nucleotide sequence of the gene from Escherichia coli K 12 and correlations with crystal structures. J Biol Chem 263: 1555-1562 2. Crowell DN, Amasino RM (I1991) Induction of specific mRNAs 6. Sanger F, Nicklen S, Coulson AR (1977) DNA sequencing with chain-terminating inhibitors. Proc Natl Acad Sci USA 74: 5463-5467 7. Van Camp W, Bowler C, Villarroel R, Tsang EWT, Van Montagu M, Inze D (1990) Characterization of iron superoxide dismutase cDNAs from plants obtained by genetic complementation in Escherichia coli. Proc Natl Acad Sci USA 87: 9903-9907 Downloaded from on June 16, 2017 - Published by www.plantphysiol.org Copyright © 1991 American Society of Plant Biologists. All rights reserved.