FEBS Letters 358 (1995) 62-66
FEBS 15009
Structure and differential response to abscisic acid of two promoters for
the cytosolic copper/zinc-superoxide dismutase genes, zyxwvutsrqponmlkjihgfedcbaZYXW
SodCcl and
SodCc2, in rice protoplasts
Atsushi
Sakamoto*,
Takekazu
Okumura,
Hironori
Kaminaka,
Kazuhiko
Sumi, Kunisuke
Tanaka
Depurtment of‘ Biochemistry, College of Agriculture, Kyoto Prejkctural University. Shimogamo. Kyoto 606, Japan
Received 14 November 1994; revised version received 5 December 1994
mizing oxidative damage to plant cells. Three types of SOD are
Abstract
We determined the 5’-flanking sequences of two nudistinguished by their associating metals: copper/zinc- (CulZn),
clear genes (SodCcl and SodCc2) encoding cytosolic copper/
manganese(Mn-), and iron- (Fe-) isozymes [2]. CulZn- and
zinc-superoxide dismutase zyxwvutsrqponmlkjihgfedcbaZYXWVUTSRQPONMLKJIHGFEDCBA
in rice (Oryza sativa L.). Utilizing
Mn-metalloenzymes
are ubiquitously distributed among plants.
transient P-glucuronidase (GUS) reporter assays, functional proCulZn-SOD
is the most abundant,
occurring as two distinct
moter-GUS analysis was performed in rice protoplasts exposed
isoforms
that
are
targeted
to
the
cytosol
and the chloroplast
to the phytohormone abscisic acid (ABA) or the antioxidant
stroma. Mn-SOD typically appears in the mitochondrial
masulfhydryl reagent, dithiothreitol (DTT). Transcriptional activitrix, whereas Fe-SOD is found within the plastids of limited
ties from both &Kc-GUS
fusions were stimulated by DTT,
plant species. SOD activities in plants are not only controlled
which induces the promoter activity of the tobacco SodCc gene
[Proc. Natl. Acad. Sci. USA 90 (1993) 310131121. ABA had no
in a developmental
manner, but they are also differentially
effect on SodCcl-GUS
expression but specifically induced the
induced by a number of environmental
cues which can cause
gene expression of the SodCcZ-GUS fusion. The simultaneous
oxidative stress in living cells (for a review, see [I]). Each cDNA
application of ABA and gibberellin A,, however, abolished the
for three plant SOD genes (Sod) has been cloned and sequenced
enhancing effect of ABA. These results indicated that two rice
from a variety of plant species. Because of its potential imporSodCc promoters differentially respond to externally supplied
tance, cloned cDNAs have been used in transgenic studies to
ABA and that one of the regulatory factors for plant SodCc
achieve the enhancement
of stress tolerance in plants (for a
expression is ABA in addition to cellular redox-modulating antireview, see [3]). Gel blot analyses of RNA using isolated
oxidants.
cDNAs as probes have shown that the differential and developmental regulation of Sod expression primarily operates at the
Key words: Abscisic acid; Differential gene regulation;
Inducible promoter; Superoxide dismutase; Cellular redox;
transcript
level [4,5]. While studies have extensively focused
Rice
upon the isolation of cDNA clones, genetic engineering
for
antioxidative
protection and analyses of the steady state-transcript levels, characterization
of the Sod genes [6-91 and functional promoter studies [9-l l] have just begun. Recently, the
1. Introduction
transcriptional
regulation of two plant Sod promoters has been
investigated
in transgenic tobacco. HCrouart et al. [lo] have
Plants are constantly challenged by various types of stress
reported that reduced forms of sulfhydryl antioxidants,
such as
resulting from climatic and other environmental
fluctuations,
glutathione
and dithiothreitol
(DTT), can stimulate the tranand generate specific gene products that confer resistance and
scriptional activity of the tobacco SodCc promoter, suggesting
adaptation under severe conditions. One protein intimately asthe modulation of the SodCc expression by cellular redox levels.
sociated with stress tolerance is superoxide dismutase (SOD;
also directs developmentally
regulated
The SodCc promoter
superoxide:superoxide
oxidoreductase,
EC 1.15.1.1) [ 11. SOD
[l 11.Using a short promoter segment, Kardish
gene expression zyxwvutsrqponmlkjihgfedcbaZYXWVUTSRQPO
is a metalloenzyme
that promotes the disproportionation
of the
et al. [9] have shown the spatial and light-responsive
regulation
superoxide anion radical into molecular oxygen and hydrogen
of the tomato chloroplastic
CulZn-SOD
gene.
peroxide. Cooperating
with other enzymes involved in hydroTo gain a better understanding
of the regulation of plant Sod
gen peroxide-detoxification
systems, SOD contributes to miniexpression, we set out to characterize
individual members of
Sod families in rice (Oryza sativa L.) plants. We isolated cDNA
*Corresponding author. Present address.. Department of Regulation
clones coding for Cu/Zn- and Mn-isoproteins
from developing
Biology, National Institute for Basic Biology, Myodaiji,
two rice genes (SodCcl and
seeds [ 12,131. We also characterized
Okazaki, 444 Japan. Fax: (81) (564) 54 4866.
SodCc2)’
encoding cytosolic Cu/Zn-SOD,
which brought the
Internet: atsushi@nibb.ac.jp
first information
on the Sod exon/intron
organization
from
plant sources [6,7]. As one approach to define the molecular
The nucleotide sequence data reported in this paper have been deposited in the GenBank/EMBL/DDBJ
Nucleotide
Sequence Databases
mechanisms responsible for plant Sod regulation and to idenunder the accession
numbers
L19434 (SodCc2) and i19435
(SodCcl).
Abbreviations: ABA, abscisic acid: ABRE, abscisic acid-responsive
element; Cu/Zn-SOD,
copper/zinc-superoxide
dismutase; GA,, gibberellin
A,; GUS, /?-glucuronidase;
SOD, superoxide dismutase; Sod, superoxide dismutase
gene; SodCc2, cytosolic
copper/zinc-superoxide
dismutase gene.
0014-5793/95/$9.50
0 1995 Federation
SSDl 0014-5793(94)01396-9
of European
Biochemical
Societies.
’ Two rice cytosolic Cu/Zn-SOD genes, SodCcI and SodCc2, were previously called sodA and sods, respectively, but have been renamed in
accordance
with the recommended
nomenclature
of plant genes
(Zilinskas, B.A., Asada, K., Galun, E.. In& D. and Tanaka, K. (1994)
Plant Mol. Biol. Rep. 12, S73-S74).
All rights
reserved
4. Sakamoto
et cd. I FEBS Letters
63
358 11995) 62-66
tion (PCR). A 3 kb SacI-Ncol
fragment which corresponds
to the
tify the actual inducers of the gene expression, we constructed
5’ region of the SodCcZ gene was recloned into Bluescript from the
two chimeric /3-glucuronidase
(GUS) reporter genes under the
genomic /%clone gSOD27 (the Sac1 site is shown in Fig. I A and the Ncol
SodCcl and SodCc2
transcriptional
control of the 5’ regions of zyxwvutsrqponmlkjihgfedcbaZYXWVUTSRQPONMLKJIHGFEDCBA
site is located in the middle of the third exon of the gene). PCR was
genes. Here, we investigated
the structures of the S-flanking
performed using the plasmid subclone as a template, the Ml3 primer
sequences of rice SodCc genes and examined the effects of the
M4 (Takara Shuzo, Otsu, Japan), and the mutated antisense oligonucleotide primer (5’~CTCACTGCTAGCAAGCACA-3’;
the Nhrl site is
phytohormone,
abscisic acid (ABA), on the transcriptional
acunderlined) to introduce an Noel
the second exon of the SodCcI
tivities of the SodC‘c promoters
in rice protoplasts.
The study
gene. The NheI site was placed at the position identical to that found
provided evidence that the two SodCc promoters differentially
in the SodCc2 gene. An amplified 2.3 kb fragment was first cloned into
respond to ABA, and that the 5’ region of the SodCc2 gene
the pGEM-T vector (Promega, Madison, WI). Following PsrI (in the
polylinker) and NheI digestion, the excised SodCcI 5’ fragment was
conferred
enhanced
reporter gene expression
in a transient
inserted between the PslI and XhaI sites in pBI221 (NheI and XhrrI
GUS assay. This is the first demonstration
of the involvement
produce compatible 5’ overhangs).
Plasmid pSodCc2-GUS
I was conof ABA in the regulation of plant Sod expression by a funcstructed as follows. A 7 kb-DNA fragment encompassing
the entire
tional promoter analysis.
SodCc2 gene and its flanking regions was obtained by BamHl digestion
2. Materials and methods
2.1. Nucleotide sequence determination oj’rice SodCc S-flanking regions
The rice SodO genes have been isolated and characterized
[6,7].
Successively deleted plasmids were generated by means of exonuclease
digestion
after each 5’-flanking
sequence was subcloned
into the
Bluescript vector (Stratagene,
La Jolla, CA). Double-stranded
DNA
templates were sequenced in both directions using the ABI373A sequenator (Applied Biosystems, Foster City, CA).
2.2.
of the SodCcpGUSjiision
genes
fusions between SodCc and GUS genes (pSodCclGUSI and pSodCc2-GUSI)
were constructed
in the transient GUS
expression vector pBI221 [l4]. To construct
pSodCcl-GUSI,
site-directed mutagenesis was performed by means of polymerase chain reac-
of gSOD7 1DNA and integrated
into Bluescript at the appropriate
restriction site. Following digestion at the Hind111 site in the polylinker
and at the NhrI site located in the second exon, the released 2.2 kb
fragment corresponding
to the SodCc2 5’ region was fused translationally to the coding sequence of the GUS gene by ligation with the
HindIIIlXhaI-cut
pBI22 I. Both fusion constructs contain the 5’-flanking sequence, the 5’ non-coding
exonhntron,
and the coding sequence
for up to 9 NH,-terminal
amino acids of the corresponding
SOD polypeptide instead of the cauliflower mosaic virus (CaMV) 35s RNA
promoter in the original construct (Fig. 2). Translational
fusion junctions in these chimeric constructa were verified by supercoil sequencing.
Consrrucrion
Two translational
2.3. Introduction oj ,/usion genes into rice pro~oplusrs
Fusion plasmids were introduced
into rice protoplasts
according to
Li et al. [I51 with slight modifications.
Protoplasts
were released from
suspension cultured cells derived from embryogenic
calli (Ori~rr .wtiw
B
A
zyxwvutsrqponmlkjihgfedcbaZYXWVUTSRQPONMLKJ
TICAGCAAGT
T-XGT-TAGGA TTKTGATGA
TGAAAACAGG ATTAATGATA zyxwvutsrqpo
-2069
zyxwvutsrqponmlkjihgfedcbaZYXWVUTSRQPONMLKJIHGFEDCBA
zyxwvutsrqponmlkjihgfedcbaZYXWVUTSRQPONMLKJIHGFEDCBA
TCTATCATAG
ATGAGTTCIT -2196
ACTT&&mGTAITGTA
TTGTCCTCCT TGAGCTTATT CATXGAGAT
ACCGGTARAT -2009
ATITAGT'ITA ACTCGTGTCA -2136
GATGGATTGG TCTGGCGCAA -2076
PStI zyxwvutsrqponmlkjihgfedcbaZYXWVUTSRQPONM
sac1
.@mCATCA
GAGATG’lTTA
TGTGTCGGTCCTTATATAAA
ACATGTGGCT
GAGACAAATT
TGLLTTGTGT
AGTCATTTCC
ITTTKGTCC
TGCITGATCT
TTGAGCTTCA
ATCGTAGATA
GGATCTA?TG
TTTAATTCZT
CAGCAGCATI
CGGGTG'IGGT CGGTGAGTAT
AGTGTCAACA AAACGTGGTT
==zz=zz
GZ,&W&CCG
GTCCTTKAG
AATCGGTGTA
GTAATAACGT AAGAGGGTTA GATGGCAGTA
-C
GATQWSAXLATTATACGAG
CTATCGBBAA mT-%CCGT
GCTTCTAAAA
TAAAACACIT TATKTACCA
TAZTATTTAT
TGTCCAAAAA CATCAAAAAA GTcTmCAT
ATCGTI'AAGA ATACACAACA CCGTCGTTTC
T'lTACAAACG AAhAATAATI TATAAATAAA
A'FXAAAGAT GAAAAAAAAA CTTCGATGAA
mm
CAAAAT-ITGG TIGTGATAAA
TTACAACGGG
GACCGTCATA
GTCAATP.AA
ECORV
GCCA@TATF
GGAAATCCGT
CACACAAGAC
TTACGGCCAT
TAGTA-LTTGA
AT-m
AGTACTACTC
CAGAGGTTAT
TCTTATTCGG
ACmTATAT
AAAAATCTTA
CATGAGCGAA
Bana
,~~~CTGAC
ATGAAGTGCT
CCCAGCTGAG
-2016
CCTCATATTA
CT-T~~~!YA
CCTTKACAC
CAAAAAAAAA
ACACTGCRG
AACGTGGTTG
zY==zzz
GCTCGGGAGA
GCATARATTG
TCAAGCTCAT
AAAATATTCT
AAACATTAGA
TAACAT-ITAA
AAACAACATA
AGCGATCTAA
TAAZTTAAGA
CCCAAAI-RI
-1956
-1896
ATXCAACAT
TCT-KGTACG
GGGACGTGTC
TATCATCTGC ATTACAATGT
GAAGAAT-ITC ATGATCCAGG
TTCRAGTCAG GCAAATTAAT
AAGCAAACTT
RAT-AAA
GGAAATAATT
GACTGCAAAG
TTGRAGTCTT
TGTTGCCTTA
GATGGTACTC
ATKAGTAGA
AGCCTAATCT
TTCCCCGAAA
TCCCTCCGAC
TTI-KAGTAT
GAAAAAAGCG
ATGTGITm
AAATAAACTA
AAGATGAGAC
-1836
-1776
-1716
-1656
-1596
-1536
-1476
-1416
-1356
-1296
npar
TmAATE+A
CAACTGCAAG
-CAGAAG
A'ITATTATTA
CTGCCAGTTT
CTCCAGATTC
.QCIl
TCGTCGTC?Z_c.E@SGGGTC
HldlII
cacccaagca.?~tqcttctagg
gggtctccct ccctccctgc
gctgcttcgc gwgatttgg
gatgttgatt gatcgatcgg
acggctcgtc tcqtgtgttt
tgcgtggacc tgctgcttcc
ggagggatac ggatttgtta
ggctctggct gctcatgatg
gttttttctg caagtgtqga
gggtgqatgt agagaattgt
TAATAAATAA
GACTACAART
CAAXCAGCA
ACGAGGGcK
ACCACCGCAC
GGAGTCGCCT
CCAATGTTGG
ACCGCTGGAG
CGCCTCCTCC
CTGGTGGCTG
GGAGGGGAAC
T-KATCCTCC
-1236
-1176
-1116
GCCTGAGqta
gqaatcccaa
gttttcgtga
gtgtggatct
gTctt'=TTTc
tgttgttggt
gaatccatat
tttttttttt
gttgccgatt
cgatgggctt
gttgattgca
agatttggga
cttgtttcct
cagatggatc
TggctgWW
tcggggcgat
gcaagttcaa
ttccttttga
actagtaata
tgtgatatgt
gagtgtcctt
gttattggac
caacccccc~
cccccccccc
-1056
ccggtttcgt
ggtgtttcct
t'3WW'gggT
tggttggttg
gtgatgcgag
gcgatgattc
atcattggaa
tcatgattag
ccctgatatg
tagccaacag
gggaggcggt
tgttggtctt
TattTttTtg
gtggggagga
agtgttttga
gtgctgctgc
ttgtgtactg
atttagacat
tcgggggatt
acagtttcct
-996
-936
-676
-816
-756
-696
-636
-576
-516
-456
EC&I
aggatgaaat
atcccttcat gcagaaatga
atggagggat
T~?l9tT~g~~
atcccaaacc
ctaagattga aacttaccct ttagatgggt gggaaatcat
gaatggatag
gattttctag ggataattcc tctatactag acgtgtgttt tgtttqtatc tagtatgttt
gcagt9T3ca gtggcatttg ttctatgatg cctttggttt acatgctttq gaaaaaacaa
tagatcattgggccatattgttatcttttt ctgactagta gtaaatgatg cgtctgtgtt
gcttctgctt taacatcttt tcaatcttca ctaggataat ttcatagttc aatatgttct
CgtS?Pt.%9
ttgggaactt
tqttttgtgg
GTAGCAGTGA
ggttgggttt
aatttgtfta
CaZTGTTAAG
xba1
tasgacctc~..?g~ggctact tctgccactt gctaagtaac
cagATCACAT TAACAATGGT GAAGGCTGT-I GTTGKXTTG
GGCACTATCC ACT-ITGTCCA AGAGGGAGAT Ggtatgccat
T-IGTTGTACA
CCTTCCAAAC AAACAACAAA
TTGCCATGTC TGCTTACCAA
C'ITAGGCAAG AmGGGAAA
CGGAAATGAA ACAACTGlTT
AACTTATGGT CTCCACITGT
GCT'I-ITCCX GCAAAAGAAA
CAATATGAAC AAGAGCAATT
npar
ACCTAGCCAA AgmJCGTC
TAAATGAGTG CA'EACATAT
TACGAAGTGT GGAAAGATTG
GCTTCTGCGG
AACTET
GTAATCCTTI
CCTGATTGAT
GATGCCCTGT
AGAAGTGTGG
TTGTGTATAA
ACTGATTAAC
GTCATGCTCC
TITGATGGGA ATITCG'I-IGG TATGGTAGGA
'ITCTGAAATT ATTGAGCTAT TITGATGGAG
CCTGTGTTTT CAACTTTTCT
ATGTCTTGTT
TGTGTCTTGT
TGTAGGGATG TCTCTGCTGA
GCCCAAGATA CGTTGGCATT ATCCTCACTI
TTAATATGAA AAGTACTAGA TGAGACCCCT
TA?TAAAATT TGTAAAT-XA TTATGGTAAA
AAAGGAACTT GATTGCTATC ATACAAAAGA
CTAAGACATT 'ITGAATGGGC CTAGCCATCA
GGAGTCTTAT
CCTA-
CGGAGAGGGA
TKTXAAGA
GTAGGTAACC
AAAGAAAAGC
CCTCCTCCCT
CAGATCGCCT
cccccaactt
gctccgattc
cgattcggcc
atctcaggct
catgtggtag
TCTGGAGTCT
TCTCGTCGCG
tctagggttc
cgccggagta
gcgcttcggt
cgacatqtgg
ggqggctttt
TCCTCATCAG
CTCGCGCCGC
taatcgcctc
gctggstctg
ttgttctcgc
acggccacaa
caggatcgga
-AGGGATACAA
TATATGTATA
TCAACTTTTC
CTGTAGGGAC
GT-I-KCGTTG
TATTATCTGT
CGGACAAACC
-1949
-1889
-1829
-1769
AGAATCACTA
GGCmAAT-I
TAATATAACA
AGGTACAGTE
TTCITATA~
CTACCAAACA
-1709
-1649
-1589
TAATAAATTG
GTRCCTAGG
GCAAAGTGTG
ATGGCCATAC
ATTAAAAGAA
AAACGCTCCT
*par
TTGGA@GCC
-1529
-1469
-1409
TGCTATIGTC AGCATCAAAG
GCA?Q+TTAAG TGTAAGAAGG
GGCCACAGGT GGGATATTGA
GTI-I-ITGTGCTAGGCTGGGA
-1349
AGGAGACTCC
GTATTTACCA
AAGGGTATGT
ATTTAGTTTC
CTTCTATGTI
CTICAAATTT
AGAAACRACA
CGCAAGTCAG
GCGGGAAATC
AAATAT-EAA
GAACGAAAGA
TAACAGACAG
GA_@TQCTT
GTGTCCACGT
GATCATGGAC
CTGCAAGCCA
GCGTGTTGCA
CATATI'AGTA
CTGACWTC
GATITTGATC
EC&"
CTGATGAGAA
GGACTGGACT
-1289
-1229
-1169
-1109
-1049
-989
-929
-869
-803
TAAGCAAATC
CTQJ,A?TIT
-749
-689
AAATCAGAAG
AGGGGTCGCC
tgctcgctcg
tgtgccccgt
gcgtgattgc
acataaaaaa
ggtttaggtg
AGGAGAGGGT GGGCAACTCG
TGAGqtatqc aqcttcacct
gattatqcgt ggtgggtatq
gcta?.tttalJgtttqgcttt
ttcgttcggc catagggctc
tccttgttaa atttacggtt
atttggagta gcaaaacgat
-629
-569
-509
-449
-389
-329
-269
-396
-336
-276
-216
-156
-96
-36
25
85
-209
-149
-89
-29
ttcqgcttct
qtgcagAACA
CATAGACAAT
GGTGAAGGCT
GTTGCTGTGC
m_GCC&GCAG
32
Fig. I. Nucleotide sequences of the 5’ regions of rice SodCc genes. (A) The SodCcI gene. (B) The .SodCc,Z gene. Nucleotide residues are numbered
relative to translation
start site as +I. Bold-face indicates presumptive
CAAT boxes, the 5’ end of the longest cDNA and the translation
startmethionine codon. The 5’ non-coding
intron is shown in lower letter case. Motifs similar to the heat shock elements (GAANNTTC)
are underlined.
Direct repeat sequences are shown in italic letters. A complementary
sequence to the mutated oligonucleotide
primer used for pSodCcl-GIJSI
construction
is also indicated in italics (Fig. IA). The double underline refers to the putative ABA-responsive
element (ACGTG) or the as-l motil
(TGACG).
The dotted line indicates the restriction site with its name above the sequence.
64
A. Sakamoto et ul. IFEBS
Letters 358 (1995)
6246
was found between repeated motifs. ABREs in the SodCcl 5’
region appeared within the direct repeats. Although the structural organization was quite similar between the two genes [6,7],
the 5’ upstream regions had only limited sequence homology,
except for a stretch of 38 bp between positions -1360 and
- 1323 bp in the SodCcl gene and between -98 1 and -946 bp
in the SodCc2 gene (Fig. 2A).
L. cv. Nipponbare) during a 3-hour incubation at 28°C without shaking
in enzyme solution containing 4% Cellulase RS, 1% Macerozyme R-10
(both from Yak&, Tokyo, Japan) and 0.4 M mannitol. The protoplast
suspension was filtered through a 30pm sieve to remove undigested cell
clumps and debris, then mixed with a two volumes of KMC solution
[15]. The filtrates were centrifuged at 130 x g for 5 min and protoplast
pellets were washed twice with KMC. Protoplasts were resuspended
(4x 10” protoplasts/ml) in MaMg solution [15]. Aliquots (1 ml) of
protoplast suspension were mixed with plasmid DNA and carrier DNA
(each 40 pug/ml). After incubating protoplast/DNA mixtures for 15 min
at room temperature, 1.85 ml of 40% polyethyleneglycol (PEG) 6,000
in MaMg was gently applied. After an incubation for 30 min at room
temperature, protoplasts were suspended in 40 ml KMC and centrifuged at 130 x g for 5 min. Protoplast pellets were resuspended in
General medium [15] supplement with sucrose (106.9 g per liter) and
cultured at 28°C for 48 h in the dark.
3.2. Transcriptional activities
of rice the
SodCc promoters
in
protoplasts
To analyze SodCc promoter functions, the expression of
chimeric genes containing 5’-flanking regions of rice SodCcl
and SodCc2 genes linked to the coding sequence of GUS was
examined in protoplasts prepared from suspension cultured
2.4. zyxwvutsrqponmlkjihgfedcbaZYXWVUTSRQPONMLKJIHGFEDCBA
Assay for transient GUS uctivity
cells after PEG-mediated DNA uptake. Fusion constructs beTo suppress endogenous GUS activity, a modified protocol [16] was
tween rice SodCc 5’ regions and the GUS gene are shown in Fig.
employed for the fluorimetric determination of 4-methylumbelliferone
3.
Translational fusion allowed the inclusion of the first intron,
(4-MU) produced from the cognate glucuronide (4-methylumbelliferylwhich occurred in the 5’ untranslated region of each SodCc
B-D-glucuronide) in protoplast extracts. The protein concentration was
measured by the method of Bradford [17]. GUS activity was normalized
according to the expression derived from pB1221 in control experiments
(average 204 pmol of 4-MU produced per minute per pg of protein
obtained from four independent experiments = 1).
gene, in the chimeric constructs.
Fig. 4 shows the results of
transient GUS assays for rice protoplasts transformed with
pB1221 and two SodCc-GUS fusions. Since the CaMV 35s
promoter confers constitutive expression to a downstream reporter gene [22], pB1221 was used as the positive control plas3. Results
mid of PEG-mediated transformation and also for the negative
standard of inducible or responsive gene expression. As shown
3.1. Characterization
of S- flanking
regions of rice SodCcgenes
in Fig. 4A, the application of ABA and DTT to transformed
We determined
the nucleotide
sequences of more than lprotoplasts did not substantially alter the pattern of GUS activSodCc genes from rice
kilobase at the S-flanking ends of two zyxwvutsrqponmlkjihgfedcbaZYXWVUTSRQPONMLKJIHGFEDCBA
ities from this constitutive expression cassette. Basal levels of
(Fig. 1). The 5’ upstream regions of these genes contained
GUS expression from pSodCcl-GUS1
and pSodCc2-GUS1
sequences with homology to known regulatory elements identiwere 1.6- and 0.6-fold, respectively, compared to that of
fied in other plant genes. Neither upstream region displayed
pB1221. Expression of both SodCc-GUS constructs was stimuconsensus TATA signals, but both had a CAAT box. Two and
lated by DTT (1 mM), a known inducer of tobacco SodCc
three putative ABA-responsive elements (ABREs) [ 18,191were
transcription [lo]. Since both rice SodCc-promoter sequences
found in the SodCcl and SodCc2 5’ regions, respectively. Ancontained ABRE motifs, we added ABA (100 PM) for the
other plant c&acting factor, as-l [20], was found in the SodCc2
transient assay. The GUS activity derived from pSodCclpromoter, but not in the SodCcl region. Several putative heatGUS1 remained at the same level as those of controls, and
shock elements [21] were found in each promoter region
therefore the SodCcl promoter did not respond to ABA. How(SodCcl, 7; SodCc2, 4). Complete direct repeats, consisting of
ever, exogenous ABA enhanced the reporter gene expression
11 and 24 bp-units, were identified in SodCcl and SodCR
from pSodCQ-GUS1 with the average increase in enzyme ac5’ upstream sequences, respectively. No nucleotide similarity
tivity being about 7-fold. This was in marked contrast to the
A
??? ? ? ? ? ? ? ? ? ? ??? ? ?
SodCc Z
(rice)
SodCcl
(rice)
SodCc
(tobacco)
? ? ? ? ??
??
???
???
zyxwvutsrqponmlkjihgfedcbaZYXWVUTSRQPONMLK
-946
-981 TAATATT-AAAATTTGTAAATT-GATTATGGTAAACAT
IIIII
I II II
IIIIIII
IIIIIII
III III zyxwvutsrqponmlkjihgfedcbaZYXWVUTSRQPONMLKJIHGFEDCBA
- 1360
TAAGATTGAAAATTTCAAAATTTGGTTGTGATAAACAT
- 1323
II I I I I IIlIIlIIII
I I
I I
II
-386
-423 CAAWATAATATTTTCAAAATTAAGGTAAGGTGCACGC
B
SodCcl
Trxh
- 1491
- 455
AAGCGAAACAACATATTTACAAACG~T~TTTATAIlATAAAACTTTTATATA-TGTGTTTTTAGCGATCT~T
-1414
III I I I I I I I I I
IIIIIlIlIIIIl zyxwvutsrqponmlkjihgfedcbaZYXWVUTSRQPONMLKJ
III III
III1
ll/IIlIIlI
IIIIlIIII
IIIIIIIIII
III
-383
AAGCGAAACGACATATTTACGAACACAAAATAATTTGTAACTTTTATATACTGTG-----AGCGATCTAAAT
Fig. 2. Homologous sequences in the S-flanking regions between rice SodCc and other genes, The vertical bars represent nucleotide identity and the
minus signs show gaps. Numbers refer to nucleotide positions relative to start of translation (SodCcl and SodCc2) or transcription (tobacco SodCc
and rice thioredoxin h) as +l. (A) Homology among rice SodCcl, SodCc2 and tobacco SodCc genes. Conserved residues are marked with asterisks
above the alignment. (B) Homology in the 5’ upstream regions between SodCcl and thioredoxin h (Trxh) genes from rice plants. The genomic sequence
for rice thioredoxin h was cited from the GenBank database (D26547 deposited by Y. Ishiwatari and M. Chino).
A. Sakamolo
CI al. I FEBS Lrttrrs
v
s
E
A CM
65
358 (1995) 62456
X
sion of the SodCc2-GUS
chimeric gene is specific to the
5’ region of the SodCc2 gene. We therefore concluded that the
2.2 kb fragment from the SodCc2 5’ region at least partly
comprised the &-acting
element(s) necessary for ABA-mediated hormonal regulation.
BM
pSodCcl-GUSl
BP
A
A
V
E
0M
4. Discussion
pSodCcZGUS1
To identify the regulatory factors of plant Sod families, we
determined the nucleotide sequences of the 5’-flanking regions
YVKAVAVLNRGSPGGQSLM zyxwvutsrqponmlkjihgfedcbaZYXWVUTSRQPONMLKJIHGFEDCBA
of two Sod0
genes from rice and investigated the transient
OSkbp
transcriptional
activities of the promoters
in a homologous
gene expression system. Both SodCc promoters were activated
Fig.
3. Two SodC’c~GUS
chimeric constructs
(pSodCcl-GUS1
and
by exogenous DTT, but the responsiveness
of the promoters to
pSodCcZ-GUSl)
used for transient transformation
of rice protoplasts.
Hatched and open boxes refer to exons and introns, respectively, while
ABA was markedly different. The SodCc2 promoter was stimshaded boxes indicate the 5’-flanking regions. The GUS coding sepromoter was not.
ulated in response to ABA, but the Sod01
quence shown by a closed box is not represented in scale. The amino
This
differential
expression
of
two
similar
genes
indicates that
acid sequence of a translational
fusion between SodCc and GUS genes
they play somewhat different roles in the stress response and
is indicated in one letter code below each construct. Boldface residues
denote SodQ and GUS translation
initiation methionines and fusion
that different mechanisms
occur in the control of the SodC’c,
junctions
are indicated
by slashes. Restriction
sites are: A, A@;
expression in rice under some environmental
conditions. Also,
B, BumHI; C, SacII; E, EcoRI; H, HindIII; M, SmaI; P, &I; S, SacI;
our results confirmed the activation of plant SodCc promoters
V, EcoRV; X, Xhal.
by antioxidant sulfhydryl molecules, suggesting that the cellular
redox-mediated
regulation is a common regulatory aspect of
in which there was
results from pBI221 and pSodCcIGUS1,
the SodCc expression in plants.
no apparent increase in GUS activity in the presence of ABA.
Phytohormones
act positively upon plant Sod expression at
Since ABA and gibberellin A,(GA,) often function as antagosteady state mRNA levels. Ethylene and salicylic acid stimulate
nistic regulators, we examined whether the effect of ABA in the
Mn-SOD gene expression in tobacco and rubber trees [8,23].
SodCcZLGUS expression is hampered by GA,. incubation
in
Ethylene also induces the accumulation
of tomato SodCc tranthe presence of both phytohormones,
indeed, resulted in no
scripts [4]. Our results showed that ABA promotes the reporter
enhancement
of the activity, thereby suggesting that the trangene expression driven by the 5’ region of an rice SodCc gene,
scriptional activity is regulated by both phytohormones.
The
providing direct evidence that this phytohormone
is involved
ABA concentrations
(0. 1, 5, 10 and 100 PM) were titrated to
in the transcriptional
activation of the plant Sod promoter.
induce the full activity of the SodCc2 promoter. Fig. 4B shows
Kanematsu and Asada commented [24] that rice cytosolic Cu/
that 10pM ABA is sufficient to maximally induce the SodCc2Zn-SODS III and IV, either of which is the putative SodCc2
GUS expression. The expression of GUS activities was proporgene product [12], are dominant in the seed embryos but their
tional to ABA concentration
from 1 to 10 PM. These results
activities decrease following seed germination.
The pattern
clearly demonstrated
that hormonal regulation of the expresof transient SodCc.2-GUS expression correlated well with this
B zyxwvutsrqponmlkjihgfedcbaZYXWVUTSRQPONMLKJIHGFED
A
T
pBl221
pSodCcl-GUS1
1
T
pSodCcP-GUS1
0
1
10
5
ABA
100
WI
Fig. 4. Transient GUS expression in rice protoplasts
transformed
with chimeric constructs.
(A) The effects of ABA and DTT on transient GUS
expression from pB1221. pSodCcl-GUSI
and PSodCcZ-GUSI.
Transformed
rice orotoolasts
were incubated at 28°C for 48 h without or with rhr
foilowing at the indicated’concentrations:
ABA, IOOflM; GA,, 100pM: DTT, I m&M.Thereafter, GUS activities (pmol 4-MU produced per minute
per microgram
protein) were fluorimetrically
determined and normalized by comparison
with control experiments of pB122 I. Mean values from four
Independent experiments are shown. Vertical lines refer to standard errors. (B) The effect of various concentrations
of ABA on GUS activity from
pSodCQ-GUS
1. Protoplasts were incubated with the indicated concentrations
of ABA for 48 h immediately after transformation
with pSodCc2-GUS.
The relative GUS activity is shown from two independent
experiments. The results are independent
from those shown zyxwvutsrqponmlkjihgfedc
in Fig. 4A, so the degree of
induction by DTT at 100 PM is somewhat altered.
A. Sakamoto et ul. IFEBS
zyxwvutsrqponmlkjihgfedcbaZ
Letters 358 (1995) 62-66
66
activity profile since the transcriptional
activity of the SodCc2
regulatory sequences involved in sulfhydryl reagent- and ABA5’ region was elevated by ABA , but the effect was abolished
responses and characterization
of trans-acting
factors associby the germination-promotive
GA,. ABA differentially
actiated with each promoter region should provide more insight
into the mechanisms of plant Sod expression.
vates the expression of other antioxidant
defense multigenes
encoding catalase in maize endosperms
[25]. These observaAcknowledgements: This work was supported in part by a Grant-in-Aid
tions imply that specific member(s) of antioxidant defense mulfor Scientific Research on Priority Areas (No. 04273102) from the
tigene families such as those of Sod and Cat are organized in
Ministry of Education,
Science and Culture, Japan. AS. is grateful to
an ABA-regulated
manner in the plant genome and that their
the Japan Society for the Promotion
of Science for Japanese Junior
expression is coordinately
conducted in response to increased
Scientists for a postdoctoral
fellowship.
ABA levels within plant cells during seed maturation
or under
environmental
stresses such as desiccation, high osmotic presReferences
sure and low temperature.
We found several putative cis-acting elements which are sup[l] Bowler, C., Van Montagu,
M. and Inz&, D. (1992) Annu. Rev.
posed to be involved in the regulation of rice SodCc genes. Each
Plant Physiol. Plant Mol. Biol. 43, 83-I 16.
[2] Asada, K., Kanematsu,
S., Okada, S. and Hayakawa,
T. (1980) in:
promoter contained several heat shock-resemble
elements. ToChemical and Biochemical Aspects of Superoxide and Superoxide
bacco SodCc expression is induced by heat shock and there are
Dismutase (Bannister, J. V. and Hill, H.A.O. Eds.) pp. 136153,
several heat shock elements in its promoter region [5,1 I]. Motifs
Elsevier, Amsterdam.
like ABREs have been identified in both rice SodCc promoters,
[3] Htrouart,
D., Bowler, C., Willekens, H., Van Camp, W., Slooten,
L., Van Montagu
M. and Inze, D. (1993) Phil. Trans. R. Sot.
but the elements in the SodCcl sequence seemed not to function
Lond. B 342, 235-240.
since this promoter
was not responsive to ABA. One of the
[4] Perl-Treves, R. and Galun, E. (1991) Plant Mol. Biol. 17,745%760.
ABRE (positions -716 to -705) in the SodCc2 promoter is of
[5] Tsang, E.W.T., Bowler, C., Herouart,
D., Van Camp,
W.,
particular interest. The motif and surrounding nucleotides form
Villarroel, R., Genetello, C., Van Montagu, M. and Inz&, D. (1991)
a perfect 12-bp palindromic structure (5’-GTCCACGTGGACPlant Cell 3, 783-792.
[6] Sakamoto,
A., Okumura,
T., Ohsuga, H. and Tanaka, K. (1992)
3’) with the G-box core, CACGTG, in its center. The hexameric
FEBS Lett. 301, 185-189.
sequence is classified into the A type G-box according to the
[7] Sakamoto,
A., Okumura,
K., Kaminaka,
H. and Tanaka,
K.
sequence analogy of the flanking regions [26]. HCrouart et al.
(1995) Plant Physiol., in press.
[1 1]
zyxwvutsrqponmlkjihgfedcbaZYXWVUTSRQPONMLKJIHGFEDCBA
pointed out the presence of two homologous boxes between
[S] Miao, Z. and Gaynor, J.J. (1993) Plant Mol. Biol. 23, 267-277.
[9] Kardish, N., Magal, N., Aviv, D. and Gahm, E. (1994) Plant Mol.
the promoters
for the tobacco SodCc and the bean chalcone
Biol. 25, 887-897.
synthase genes. Apparent homology to these sequences, how[IO] Htrouart,
D., Van Montagu,
M. and Inzk, D. (1993) Proc. Nat].
ever, was not detected in the rice promoters.
Instead, the toAcad. Sci. USA 90, 3108-3112.
bacco SodCc sequence showed moderate homology to the con[l l] Htrouart,
D., Van Montagu, M. and Inzk, D. (1994) Plant Physiol.
served region between two rice promoters (Fig. 2A). Whether
104, 873-880.
[12] Sakamoto, A., Ohsuga, H. and Tanaka, K. (1992) Plant Mol. Biol.
or not the region plays a role in plant SodCc regulation remains
19, 323-327.
to be proven, but it might be responsible for coordinated induc[13] Sakamoto,
A., Nosaka, Y. and Tanaka, K. (1993) Plant Physiol.
tion by thiol molecules. A search for sequence similarities of
103, 1147-l 148.
rice SodCc S-flanking sequences in the GenBank database re[14] Jefferson, R.A., Kavanagh,
T.A. and Bevan, M.W. (1987) EMBO
J. 6, 3901-3907.
vealed the striking homology of over 70-nucleotide residues in
[15] Li, Z., Burow, M.D. and Murai, N. (1991) Plant Mol. Biol. Rep.
h
the promoter sequences of the SodCcl and rice thioredoxin
8, 276-291.
genes (GenBank Accession Number D26547) (Fig. 2B). This
[16] Kosugi, S., Ohashi, Y., Nakajima,
K. and Arai, Y. (1990) Plant
long stretch of nucleotide
similarity was not found in the
Sci. 70, 133-140.
SodCc2 promoter.
Thioredoxin
h is a cytosolic form of the
[17] Bradford, M.M. (1976) Anal. Biochem. 72, 248-254.
[I81 Marcotte Jr., Russell, W.R. and Quatrano,
R.S. (1989) Plant Cell
protein in higher plant cells and it participates in a number of
I, 969-976.
cellular redox reactions where disulfide linkages formed as a
[19] Yamaguchi-Shinozaki,
K., Mundy, J. and Chua, N.-H. (1990)
consequence of oxidative damage may be reversibly reduced to
Plant Mol. Biol. 14, 29-39.
a dithiol. Although the significance of the conserved sequence
[20] Lam, E.. Benfey, P.N., Gilmartin,
P.M., Fang, R.X. and Chua,
N.-H. (19891 Proc. Natl. Acad. Sci. USA 86. 7890-7894.
in the regulation of the SodCcl gene is yet to be tested, the
[21] Gurley, W.B. and Key, J.L. (1991) Biochemistry
30, l-12.
expression patterns of the SodCcl and the thioredoxin h genes
[22] Skriver, K., Olsen, F.L., Rogers, J.C. and Mundy, J. (1991) Proc.
in plants should be examined since the state of cellular redox
Natl. Acad. Sci. USA 88, 7266-7270.
appears to involve the regulation of plant SodCc genes.
[23] Bowler. C.. Alliotte. T.. De Loose, M., Van Montagu,
M. and zyxwvutsr
In conclusion, we examined the structure and function of two
InzC, d. (1489) EMBO j. 8, 31-38.
S. and Asada, K. (1989) Plant Cell Physiol. 30, 381~241Kanematsu,
promoters of the rice SodCc genes in rice protoplasts
by tran391.
sient assays for GUS activity after transformation
with proJ.G. (1992) Proc. Natl. Acad.
~251Williamson, J.D. and Scandalios,
moter-reporter
gene fusions. Both promoter activities were coSci. USA 89, 8842-8846.
ordinately
induced by an antioxidant
sulfhydryl reagent but
WI Williams, M.E., Foster, R. and Chua, N.-H. (1992) Plant Cell 4,
485496.
their responses to ABA markedly differed. Dissecting cis-acting
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