Download Nucleotide Sequence of an Iron Superoxide Dismutase

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
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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
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Copyright © 1991 American Society of Plant Biologists. All rights reserved.