Plant Molecular Biology 18: 545-555, 1992. © 1992 Kluwer Academic Publishers. Printed in Belgium.
545
Characterization of cDNAs encoding CuZn-superoxide dismutases in Scots pine Stanislaw Karpinski, Gunnar Wingsle, Olof Olsson and Jan-Erik H~tllgren
Department of Forest Genetics and Plant Physiology, Faculty of Forestry, Swedish University of Agricultural Sciences, S-901 83 Umed, Sweden Received 31 May 1991; accepted in revised form 16 October 1991
Key words: chloroplastic SOD, cytosolic SOD, nucleotide sequences, Pinus sylvestris, sod genes
Abstract
A Scots pine (Pinus sylvestris L.) cDNA library was screened with two heterologous cDNA probes (P31 and T10) encoding cytosolic and chloroplastic superoxide dismutases (SOD) from tomato. Several positive clones for cytosolic and chloroplastic superoxide dismutases were isolated, subcloned, mapped and sequenced. One of the cDNA clones (PS3) had a full-length open reading frame of 465 bp corresponding to 154 amino acid residues and showed approximately 85~o homology with the amino acid sequences of angiosperm cytosolic SOD counterparts. Another cDNA clone (PST13) was incomplete, but encoded a putative protein with 93 ~o homology to pea and tomato chloroplastic superoxide dismutase. The derived amino acid sequence from both cDNA clones matched the corresponding N-terminal amino acid sequence of the purified mature SOD isozymes. Northern blot hybridizations showed that, cytosolic and chloroplastic CuZn-SOD are expressed at different levels in Scots pine organs. Sequence data and Southern blot hybridization confirm that CuZn-SODs in Scots pine belong to a multigene family. The results are discussed in relation to earlier observations of CuZn-SODs in plants.
Introduction
Superoxide dismutases (SODs; EC. 1.15.1.1) are a ubiquitous and diverse class of metalloenzymes found in all plants which catalyse the disproportionation of superoxide radicals into molecular oxygen and H20 2. In addition, these enzymes are
present in all investigated aerobic organisms and are involved in the detoxification of active oxygen species [ 18]. The most abundant SODs in plants are the CuZn-SODs which are found mainly in the cytosol and in the chloroplasts. In the chloroplast stroma, SOD constitutes the first link in an enzymatic scavenging system which protects
The nucleotide sequence data reported will appear in the EMBL, GenBank and DDBJ Nucleotide Sequence Databases under the accession numbers X58578 (clone PS3) and X58579 (clone PST13).
546 the photosynthetic apparatus from noxious oxygen derivatives. The active sites of other plant SODs contain either manganese (Mn-SOD) or iron (Fe-SODs) [8]. Several isozymes ofcytosolic CuZn-SOD have been found in the plants surveyed so far: two in spinach (Spinacia oleracea L.) [12], three in rice (Oryza sativa L.) [ 12], three in maize (Zea mays L.) [4, 5], two in horsetail (Equisetum arvense L.) and in green algae (Spirogyra sp.) [ 12] and at least two in Scots pine (Pinus sylvestris L.) [26]. On the contrary, only one form of CuZn-SOD has been found in chloroplasts in plants [e.g. 14]. The cDNAs encoding cytosolic SODs have been isolated, cloned and sequenced from maize (Zea mays L.) [4, 5] and tomato (Lycopersicon esculentum L.) [17]. The e D N A encoding chloroplastic SOD has been presented from pea (Pisum sativum L.) [ 21 ], tomato [ 17 ] and petunia (Petunia hybrida L.)[24]. CuZn-SODs appear to have been resistant to evolutionary change since the enzymes isolated from a range of organisms differed only slightly in their physical and chemical properties [ 1, 9]. The nucleotide and amino acid sequence showed a great degree of similarity among different plant species [ 1, 4]. We have earlier isolated, purified and shown the subcellular localization of CuZn-SODs from Scots pine needles [26]. Here, for the first time, a description of cytosolic and chloroplastic CuZn-SODs from gymnosperms is presented on the D N A and m R N A level. Derived amino acid sequences of Scots pine SODs and a comparison with other species are discussed.
Materials and methods
Plant material Scots pine (Pinus sylvestris L.) seedlings were grown in a greenhouse in a peat/sand mixture (1:1). The seedlings were fertilized with a nutrient solution containing 4 g N per litre and with other elements in proportion according to Ingestad [ 10]. The photoperiod was 18 h, relative humidity 70 ~o and temperature 20 + 2 ° C day and night.
Extraction of poly(A) ÷ RNA A LiCl-based extraction method was used for RNA preparations [25 ]. R N A was extracted from 6-8-week-old seedlings (roots and cotyledons separated) and from needles of mature trees. The plant material (5 g) was frozen in liquid nitrogen, ground and transferred to 25 ml extraction buffer (0.2 M sodium borate, 30 m M EGTA, 5 m M D T T and 1~o SDS adjusted to pH 9.0 with NaOH), treated with proteinase K, and precipitated with 2.5 M LiC1. Poly(A) + RNA was obtained from the total R N A by two sequential passages through an oligo(dT)-cellulose spun column (Pharmacia, m R N A purification kit). cDNA library construction Extracted and purified poly(A) + RNAs as described above, was sent for library construction to Stratagene Custom Service. Double-stranded, blunt-ended c D N A was synthesized using oligo-dT primer according to the Young and Davis method [27]. After methylation, ligation and digestion of Eco RI linkers, cDNAs were fractionated into three size categories, 500-1000 bp, 1000-2000 bp and >2000 bp cloned into the Eco RI site of the expression vector 2gtl 1. DNA probes The heterologous c D N A probes, cytosolic (P31) and chloroplastic (T10) probes from tomato, were obtained from R. Perl-Treves [17]. Two c D N A Eco RI/Sac ! fragments (for P31 and T10 probes respectively) containing the 5' region of non-coding and coding sequences were used for screening of the e D N A library. Homologous e D N A probes (cytosolic PS3 EcoRI/HincII fragment and chloroplastic PST13 EcoRI/ Hae III fragment) containing 5' non-coding and coding sequences were used for the northern hybridization experiments as well as for Southern hybridization experiments (cytosolic PS3 Eco RI fragment and chloroplastic PST13 Eco RI fragment).
547
Screening of the amplified cDNA library The amplified c D N A library was grown in the Escherichia coli strain Y1090 in LB medium containing 10 mM M g S O 4 and 0.2~o maltose. Duplicate replica filters were prepared and prehybridization and hybridization were performed according to Ausubel et al. [2]. The heterologous probes P31 and T10 were labelled with 32p-dCTP according to the oligolabelling kit protocol of the supplier (Pharmacia). After hybridization the following four washes were done: two times for 20min in 1 × SSC, 0.1~o SDS at room temperature and two times for 10 min in 1 × SSC, 0 . 1 ~ SDS at 55 ° C. After autoradiography (32 h) positive clones were detected and isolated. The same filters were used for screening with different probes. Isolation of the positive clones was done after dilution, plating and rehybridization under the same conditions.
Isolation and subcloning of DNA fragments Preparation of phage D N A was made according to Sambrook et al. [ 19]. The c D N A inserts and the smaller restriction fragments required for sequencing were isolated from agarose gels and ligated with the appropriately cut p U C 18 vector. Competent E. coli DH~5 cells were transformed with the ligated D N A by the CaCI2 method [ 19].
Sequence analys~ The dideoxy method of D N A sequencing was performed according to the description given by Sanger etal. [20] and Chen and Seeburg [6]. Sequence data were analysed on a VAX computer, using software as described [7].
RNA gels and northern blots R N A samples, together with D N A molecular weight markers, were run on 1.4~o agarose gels after glyoxylation in phosphate buffer [ 19]. Blot-
ting to Hybond N membrane was performed according to the protocol of the supplier (Amersham). R N A was fixed on the membrane by baking at 80 °C for 2 h. Filters were prehybridized at 42 °C for 10-12 h in 6 x SSC (1 x SSC is 0.15 M NaC1, 15 mM sodium citrate), 5 x Denhardt's solution, 50 mM sodium phosphate, 0.1 To SDS, 100 #g/ml salmon sperm D N A and 50~o formamide. The filters were hybridized in the same solution plus the radioactive probe (1.2 × 10 6 cpm/ml) at 42 °C for 18 h. After hybridization the following four washes were done: two times for 20 min in 0.1 x SSC, 0.1~o SDS at room temperature and two times for 15 min in 0.1 × SSC, 0.1~o SDS at 58 °C. The filters were exposed to X-ray film for 72 h. Plasmids PS3 and PST13 (100 pg), cut with the restriction enzyme Eco RI, were run in parallel on another 1.4~o agarose gels after glyoxylation under the same conditions as the R N A samples. Following blotting to Hybond N membrane the plasmids D N A s were used as internal hybridization stringency controls in the northern experiment.
DNA isolation and Southern blots Total D N A from young seedlings of Scots pine was extracted according to Szmidt et al. [23], digested with the proper restriction enzymes and run on 0.8~o agarose gels. Blotting to Hybond N membranes after depurination in 0.25 M HC1 and denaturation and neutralization was performed according to Sambrook etal. [19]. D N A was fixed on the membrane by baking at 80 ° C for 2 h. The filters were prehybridized at 65 °C for 6 h in 6 × SSC, 5 × Denhardt's solution, 50 mM sodium phosphate, 0.1~o SDS, 100 #g/ml salmon sperm D N A and 50 #g/ml of yeast tRNA. The filters were hybridized in the same solution plus the radioactive probe (2.4 x 10 6 cpm/ml) at 65 °C for 24 h. After hybridization the following five washes were done: three times for 20 min in 0.1 x SSC, 0.1~o SDS at room temperature and two times for 15min in 0.1× SSC, 0.1~o SDS at 56 ° C. The filters were exposed to X-ray film for two weeks.
548 Results
Screening of the amplified cDNA library with heterologous probes The heterologous cytosolic tomato probe P31 was used to screen 200000 transformants from the library with an insert size between 0.5-1 kb and 200000 transformants from the library with an insert size between 1-2 kb. After screening, 19 positive clones hybridizing to the cytosolic probe (P31) were isolated from the c D N A library with an insert size smaller than 1 kb. The other heterologous chloroplastic tomato probe (T10) was used to screen the same library and 13 clones that hybridized to this probe were detected in the c D N A library with an insert size smaller than 1 kb. No hybridization signals were obtained with any of the probes when the library with the insert size 1-2 kb was screened.
Cloning and DNA sequencing The c D N A inserts hybridizing to the cytosolic probe P31 and chloroplastic probe T10 were isolated and cloned into the pUC 18 vector. After restriction and partial sequence analysis of all isolated c D N A inserts, two clones denoted PS3 and PST13 were chosen for further studies and completely sequenced. Other clones contained equal or shorter c D N A inserts (data not shown). The nucleic acid sequence for the cytosolic clone PS3 and the chloroplastic clone PST13 is illustrated together with the sequencing strategy in Fig. 1A and B and in Fig. 2A and B, respectively. From clone PS3, 821 bp of c D N A was sequenced. One of the open reading frames (ORF) was 465 bp corresponding to 154 amino acid residues. For the described ORF, the c D N A contained a 5' end of 112bp and a 3' end of 244 bp. The derived amino acid sequence from c D N A clone PS3 matched the N-terminal amino acid sequence of the purified cytosolic Scots pine CuZn-SOD isozyme with the exception of the two amino acid residues Q and E (position 22 and 23; Fig. 3). Clone PST13 contained a c D N A insert of
676 bp, and the open reading frame corresponded to 424 bp or 141 amino acid residues. The clone contained a 3' end of 252 bp. A small part of the 5' coding region and the whole non-coding region were absent. However, 11 amino acid residues from the N-terminal sequence of the purified CuZn-SOD isozyme (chloroplastic form) fitted perfectly with the derived amino acid sequence of the clone PST13 (Figs. 2 and 3). The derived amino acid sequences of the clones PS3 and PST13 are also shown in Figs. 1 and 2. Both clones encoded polypeptides with a high degree of homology to angiosperm sod gene products (PS3 cytosolic form and PST13 chloroplastic form). Displayed in Fig. 4 is a comparison of amino acid sequence s of CuZn- S O D s from seven plant species, either determined by Edman degradation [ 13, 22] or deduced from nucleotide sequence of cDNAs [5, 17, 21, 24 and this report]. The described c D N A sequences show a high degree of homology to known angiosperm CuZnSOD cDNAs. The homology at the D N A level between corresponding CuZn- SOD in Scots pine, tomato [17], maize [4, 5] and pea [21] was approximately 74~o for the cytosolic and 80~o for the chloroplastic form respectively. On the deduced amino acid level the corresponding values were approximately 8 5 ~ for the cytosolic and 93 ~o for the chloroplastic CuZn-SODs. The homology on the protein level between the cytosolic and chloroplastic forms in Scots pine was higher than that observed in tomato (cf. Fig. 4).
Polyadenylation signal A consensus signal for polyadenylation AATAAA is often present in animal as well as in plant genes. Another conserved sequence, TGTGTTTT, has been found near to the polyadenylation site in plants [11]. In the cytosolic SOD cDNA one AATAAA sequence was found between nucleotide 727 and 732 (Fig. 2). In the chloroplastic SOD cDNA, a T G T G T T T T sequence with one mismatch was observed. The imperfect sequence motif was found starting at position 538 and ending at 545 (Fig. 2).
549 1
61
CAGAAACCAAC TACGATACTGCGATATTGACCACTGTTAGCAAAGGTTTCACTGATCTAG ......... + ......... + ......... + ......... + ......... + ......... C TTGTACAGAGCTCAAGGGG ......... + .........
+
60
TTTCCTGGGATCACATCTTTTATTGATAAAGTATGGGTCT + ......... + ......... + ......... +--- ...... +
M
G
120
L
121
TC TTAAGGCTGTTGTTGTCTTGAATGGTGCTGCTGATGTCAAGGGGGTTGTTCAATTCAC ......... + ......... + ......... + ......... + ......... + ......... + L K A 7 V V L N G A A D V K G V V Q F T
180
181
C CAGGAAGGAGATGGGCCAACAACTGTAACTGGGAAGATCAGTGGTCTGAGCCCTGGTC T ......... + ......... + ......... + ......... + ......... + ......... + Q E G D G P T T 7 T G K I S G L S P G L
Z40
241
CCATGGTTTCCATGTTCATGCACTAGGTGACACAACAAATGGGTGCATGTCAACTGGACC ......... + ......... + ......... + ......... + ......... + .........
H
G
F
H
V
H
A
L
G
D
T
T
N
G
C
M
S
T
G
+
300
P
301
ACATT TTAATC C G TTAGGCAAGGAGCATGG TGCAC C CACAGATGATAAC ......... + ......... + ......... + ......... + ......... H F N P L G K E H G A P T D D N
CGACATGCTGG + ......... + R H A' G
360
361
GGATCTAGGCAATG TAAC TGTAGGAACGGATGGGAC TGTCGAAT TTTCAATCACTGACAG ......... + ......... + ......... + ......... + ......... + ......... + D L G N V T V G T D G T V E F S I T D S
420
421
C CAGATTC C TCTCTC TGGGCCACATTCGATAGTAGGACGAGCTGTAGTTGTC ......... + ......... + ......... + ......... + ......... Q I P L S G P H S ! V G R A V V
CATGC TGA + ......... + V H A D
480
481
TCC TGATGATCTTGGCAAAGGTGGCCATGAAC TGAG TAAGAGCACAGGAAATGCTGG TGG ......... + ......... + ......... + ......... + ......... + ......... + P D D L G K G G H E L S K S T G N A G G
540
541
CAGACTTGCTTGTGGGGTTGTCGGTCTTCAAGGATGAATGTGAGCAGTTGGTTATTTATA ......... + ......... + ......... + ......... + ......... + ......... R L A C G V V G L Q G * M *
+
600
+
660
+
720
TGATG + ......... +
780
601
GGCTGGC TT TTTTCAAGTATGAKTACGTG ......... + ......... + .........
661
TGCGAKAGGGATACAACAGTAKGC CAGATACAAACATTTGGAAGGTGGTTATTAGGTGAA ......... + ......... + ......... + ......... + ......... + .........
721
C C TAACAATAAATTGAGTACGCTGTGCTACATAAGGATTTTGCATGTAATAATTT ......... + ......... + ......... + ......... + .........
781
ATGTTATACAAAC C TATTCAGGC TAAAAAAAAAAAAAAAAA ......... + ......... + ......... + .........
EcoRI
Mbo II Mbo II
Hinc II
strategy
+-
821
Mbo II
II
Restriction map PS3 Seqendng
TATC TATTACTCGTCGC T C TAGTTTGCATTA + ......... + ......... + .........
4
4
D
EcoRI
I
I~
Fig. 1. A (top). Nucleotide sequence and derived amino acid sequence of the cDNA of the clone PS3 encoding cytosolic CuZnSOD. Stop codons are indicated by the asterisks. Underlined are the derived amino acid sequence showing 100~o homology with the N-terminal amino acid sequence of the mature cytosolic SOD protein and the consensus sequence of polyadenylation signal. B (bottom). Sequencing scheme of the PS3 cDNA. The bars denote the coding region. The single line indicates the non-coding region, arrows show the direction and distance of sequencing.
Northern blot hybridizations Northern hybridizations were performed at high stringency, and under these conditions no crosshybridizations were observed. Two northern blot
experiments were performed to analyse size (Fig. 5) and abundance of m R N A species in different tissues of Scots pine (Fig. 6). The external controls (Eco RI-cut plasmids PS3 and PST13) indicated that the two probes did not cross-
550
1
61
121
181
241
301
361
&21
ACAGGTCGAGGGTGTTGTCACTCTCTCGCAGG~GAC~CGGTCCCAC~CAGTG~GGT ......... + ......... + ......... + ......... + ......... Q V E G V V T L S Q E D N G P T T V CCGTTTGACAGGATTGACTCCTGGG~GCATGGCTTTCATCTACATGAGTTTGGTGACAC ......... + ......... + ......... + ......... + ......... R L T G L , T P G K H G F H L H E F G
K
+ ......... V
+ ..... D T
+
A---+
~CCAATGGCTGCATGTcAACAGGATCACATTTT~TCCAAAAAAATTGACACATGGTGC ......... + ......... + ......... + ......... + ......... T N G C M S T G S H F N P K K L
+ ......... T H G
A
TCCTGAGGATGATGTACGCCATGCGGGTGACCT~GAAACATAGTTGCGGGTTCTGATGG ......... + ......... + ......... + ......... + ......... P E D D V K H A G D L G N I V A
+ ......... G S D,
G
60
120
+
180
+
2k0
AGTTGCAGAGGCAAC~TTGTTGAT~TCAGATTC~TTGAGTGGACCTGATTCAGTTAT ......... + ......... + ......... + ......... + ......... V A E A T I V D N Q I P L S G P D S
+ ......... I
+
300
V
TGGTAGGGCACTTGTTGTCCATGAGTTAGAGGATGACCTGGGGAAAGGTGGGCATGAACT ......... + ......... + ......... + ......... + ......... G K A L V V H E L E D D L G K G G H
+ ......... E ~
+
360
TAGTCTGAC~CTGGC~TGCTGGGGGCAGGTTGGCTTGTGGTGTGGTTGGACTCACTCC ......... + ......... + ......... + ......... + ......... $ L T T G N A G G K L A C G V V G L
+ ......... P
+
420
T
+ .........
+
~80
CATTT~GGCCCAGTCAAATATGATGATCTTCAAAGGTCATGGACATCGTATGATACCAG ......... + ......... + ......... + ......... + ......... I *
481
TGACTGCAATAATAATTCAAATATATAGTTCTCTATCCTCGC~GATTGTTAGC~TTGT ......... + ......... + ........ .+ ......... + .........
+ .........
+
540
541
GATTTGTTTTTGGCATTAGCGAGTTGCACTTTGGGACATT~TGTTTGGGCGCAGAT~G ......... + ......... + ......... + ......... + .........
+ .........
+
500
+ .........
+
560
ATTGAGAKACATTTGTAATTCATG~TCTCT~TAATAGTCTCCTTTTTGAAGGGAAAAA
601
.........
+ .........
661
.........
+ ......
+ .........
+ .........
+ .........
676
EcoRI Re;tp;;°n
I
Seqencing strategy
JI
Hae III
EcoRI
I IL
IL
~,
Fig. 2. A (top). Nucleotide sequence and derived amino acid sequence of the eDNA of the clone PST13 encoding incomplete chloroplastic CuZn-SOD. The asterisk indicates the stop codon. The derived amino acid sequence (underlined), matched the N-terminal amino acid sequence of the mature chloroplastic SOD protein. B (bottom). Sequencing scheme of the PST13 eDNA. The bars denote the coding region. The single line indicates the non-coding region, arrows show the direction and distance of sequencing.
AA number Chloroplast N-terminal PST13 AA-sequence
! 5 l0 AAKKAVAVLKGDS
Cytosolic N-terminal
GLLKAVVVLNGAAVKGVVQFTDG GL L KAV VV LNGAAVKGVVQ
PS3 AA-sequence
15 20 25 QVEGVVT L S Q E Q v E G v v T L S Q E
F T QE GDG
Fig. 3. Comparison of N-terminal amino acid sequences of mature SOD chloroplastic and cytosolic proteins, purified from Scots pine [26] with the derived amino acids sequence (AA) from the e D N A of the chloroplastic and the cytosolic clones PST13 and PS3, respectively.
551 Chloroplast CuZn SOD Pine Pea Spinach Tomato Petunia Cytosolic CuZn SOD Pine Tomato Cabbage Maize2 Chloroplast CuZn SOD Pine Pea Spinach Tomato Petunia Cytosolic CuZn SOD Pine Tomato Cabbage Maize2 Chloroplast CuZn SOD Pine Pea Spinach Tomato Petunia Cytosolic CuZn SOD Pine Tomato Cabbage Maize2 Chloroplast CuZn SOD Pine Pea Spinach Tomato Petunia Cytosolic CuZn SOD Pine Tomato Cabbage Maize2
u PI T T v K TIV T LIIIQIEIDIEIG PIT T V S V L K G I T ISt N v m ;? vlv T LITIQIEIDI D G PI T T v N VlV T LlSlQIDIDIDIG PI T T v N V~.KGNISINVE vlv T '.ITIQIDIOIDIG PIT T V K V~.KGTISlNV v,.
TltqKAv TKt_~KAV GLLIKAV VIKAV AIKAV VIKAV
VLN VLN SSA VLA
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GLHGFHLHEY GKHGFHLHEF ~G L H G P H L H E Y GLHGFHLH F
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L
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DTTNGC DNTNGC DTTNGCi
A ~.IG T D IG A DIG A SIG
VlR H
I S T GIP H F N PIN K A I H M S T GIP H F N PID K K T H M S T G I A H F N PIN K L T H H M S T G TP H F I N PIN G K
G G G G
A A A A
P P P P
E E E G
D D D D
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NIR VIR fIR VIR
H H H H
i~'s T G,P . P N Pl,.~--~. H ~,s T G,P H ~ N PIHIG KI~. H MIS T GIP H F N P DIG KIT H M,S T GIP H F iN P V[G KIE H
G G G G
A A E E
P P P P
T E E E
D D D D
D E A E
NIR V|R NIR DIR
H H H H
i
N!Q I P
i
mlVlTILIVlD
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D s vIIIG R AILIV v H N s V]V]G R AILIV V H N s VlVlG R AlZlV v a N s vIVIG a AILI v V . N s v ~ v J G a AI_LIV V .
LI S G P
V A ~.IAITII I v F N Q I P LI T S P v A mlAITIX IVlD N,Q I P L' T G P V A
P K IIGLIS NISGLIKP TVSGLIRP S I G LIK P
T N T T
M S T GIS H F N PIK K L T H G A P E P D
A G D L G N I V A]G S D ~ A ~ I ] V l D
A G D L G N I V AIN A G D L G N r V A N AGD L GN I V A N A G D L G N I VA N
G LiT ~G LIT P G L!A P G LIA P GLAPI
E I I I
NIQ I P LJ T G P
V A EIA[T[L VV~D NIQ I P L] S G P
i A G D L G N I T VIGIEIDIG T A S F T A G D L G N I I VIG[D[DIG T A T F T AG D L G N V T AIGIEIDIG V V N V N
~ ~
SIQ I P L S G P K Q I P L, T G P Q SQ I P L S GP SQ
H S I I G R AIVIV V H N S I V G R A|IIV V H
I P L AGP
H S
EID D L GIKIG G H E L SIL TIT G N A G G R L A C G I V
VG
QIDD EID D EID D EID D
RILACGV L G1LIGG~ELSILSITGNAGG L G I K I G G H E L SIP TIT G N A GG RILACGV L GIKIG G H E L SIL TIT G N A G G R L A C G ~ V G H E g Sr~T G N A e G RLACGV L G~G
VG VG VG VG
P DD
L
IA DIP D o L G iA o iP o D ~. G IAD[ADD
L G
RLACGV
GGHELSK GGHELSL GGHELSK
J
TGNAGG TGNAGG IGNAGG
RIACGI RVACGI RVACGI
I G R AIVIV V H
LTPI LTPV LTPV LTPI LTPI
VGLQG IGLQG IGLQG IGLQG
Fig. 4. Comparison of the amino acid sequences of the CuZn-SODs from pine [this report and 26], pea [21], spinach [13], tomato [17], petunia [24], cabbage [22] and maize [5]. Boxed regions indicate 100% conservation. Asterisks indicate amino acid residues coordinating copper and zinc.
hybridize. Each probe recognized an abundant m R N A species from the poly(A) + fraction. These experiments showed that m R N A species, for both isozyme forms, were expressed in seedlings and mature Scots pine needles. The sizes of the mRNAs in Scots pine tissues were about 850 bp and 1000 bp for clones PS3 and PST13, respec-
tively (Fig. 5). In the case of clone PS3 the size was similar to the cloned insert (Fig. 5A). The observed size discrepancy between the detected m R N A species and the cloned insert for PST13 could be due to the lack of part of the coding and full non-coding 5' region in the c D N A insert (Fig. 5B).
552
Fig. 6. Northern blot hybridization of Scots pine RNA (5/~g) with homologous cDNA (used as a radioactive probe) clone PS3 (A) and PST13 (B). Slot designations: 1, poly(A) + RNA isolated from cotyledons of 8-week-old seedlings; 2, poly(A) + RNA isolated from roots of 8-week-old seedlings.
Fig. 5. Northern blot hybridization of Scots pine RNA with homologous cDNA (used as a radioactive probe) clone PS3 (A) and PST13 (B). A and B: 1, total RNA (15 ~g) from Scots pine seedlings; 2, poly(A) + RNA (4 #g) isolated from needles from mature trees; 3, poly (A) ÷ RNA (4/zg) isolated from Scots pine seedlings. Southern blot controls of hybridization stringency C and D: 100 pg of recombinant plasmid PS3 (line 1) and PST13 (line 2) digested by Eco RI.
Northern hybridization experiments showed that RNA species, for the cytosolic form, were detectable in appreciable amounts in both needles and roots of eight-week-old seedlings and that the abundance was higher in the roots (Fig. 6A). On the contrary, when using chloroplastic SOD c D N A as a probe (clone PST13) we were able to detect a strong signal in m R N A isolated from needles and approximately a 10 times weaker signal in m R N A isolated from roots (Fig. 6B).
Southern blot hybridization
In order to get some impression of the complexity of the CuZn-SOD gene organisation, two
Southern blot experiments were performed. Figure 7 shows sizes of DNA fragments hybridizing to cytosolic CuZn-SOD c D N A (Fig. 7A) and chloroplastic CuZn-SOD c D N A (Fig. 7B) in Scots pine. Each probe recognized specific restriction fragments of the DNA, hence no cross hybridization was observed. The hybridization showed that the genes, for both isozyme forms, were present on different restriction fragments. The sizes of the D N A fragments hybridizing to the cytosolic CuZn-SOD c D N A in Scots pine tissues were about 7.0 kb and 2.6 kb for Eco RI fragments; 5.6 kb, 2.1 kb and 1.9 kb for Hind III fragments; 3.5 kb and 2.9 kb for Hinc II fragments. In the case of chloroplastic CuZn-SOD the sizes were approximately 10 kb, 3.0 kb and 1.7 kb for Eco RI fragments; 4.0 kb, 3.2 kb and 1.2 kb for Hind III fragments; 1.7 kb and 1.2 kb for Hinc II fragments.
Discussion
Earlier we purified and localized CuZn-SODs from Scots pine needles [26]. This report presents a complete cDNA sequence data of CuZn cytosolic SOD from a coniferous tree species (Fig. 1A). We also show the sequence of an in-
553
Fig. 7. Southernblot hybridizations of Scots pine DNA to homologouscDNA (Eco RI fragment used as radioactive probes) clone PS3 (A) and PST13 (B). Slot designations: lines 1, 2 and 3, 25 #g of Scots pine DNA restricted with Eco RI (1), Hind III (2) and Hin cII (3); lines 4 and 5, 30 pg of recombinant plasmid PS3 and PST13 restricted with Eco RI; line 6 contains the molecularweight
markers.
complete c D N A clone of the CuZn chloroplastic SOD (Fig. 2A). The match between the amino acid sequences of the mature SOD proteins isolated from cytosol and chloroplasts, respectively, of Scots pine needles and the deduced amino acid sequence from the c D N A clones (PS3 and PST 13) support this statement. In the case of the deduced amino acid sequence derived from the clone PS3, two amino acids Q and E (position 22 and 23) differ compared with the N-terminal sequence of the mature cytosolic SOD (Fig. 3). This result supports the previous observation of the existence of several isoforms of cytosolic SOD in Scots pine and might indicate that we isolated c D N A for the cytosolic CuZn-SOD-2 as defined in Wingsle e t a l . [26]. Even though the partial chloroplastic clone lack 13 most N-terminal amino acids the other 11 amino acids match per-
fectly the N-terminal amino acid sequence of the mature chloroplastic protein (Figs. 2 and 3). Both c D N A clones showed high sequence homology to their angiosperm counterparts. These results thus confirm earlier indications of the conserved nature of CuZn-SODs in plants. Generally, CuZn-SODs appear to be quite resistant to evolutionary change since recent nucleotide and amino acid sequence comparisons show a great degree of similarity [5, 9]. It has earlier been pointed out that the chloroplastic SOD is much more conserved than the cytosolic form [e.g. 12, 14, 17]. The results presented here (Fig. 4) support these observations. The differences between the approximately 250 million-year-old conifer species and the much younger tomato [ 17], spinach [ 13], petunia [24] and pea [21] are small when the chloroplastic
554 forms are compared (homology approximately 90-94% at the amino acid level) (Fig. 4). The comparison presented in Fig. 4 indicates that the rate of mutation for CuZn-SOD is higher for the cytosolic than for the chloroplastic form. Several different hypotheses were proposed to explain this phenomenon. Kanematsu and Asada [ 12] suggested that a high rate of production of superoxide radicals in the chloroplast would have a lethal effect if mutations in CuZn-SOD bring about a decrease in or loss of their activity to scavenge superoxide. Another possible explanation for the different rates of mutation could be the different hypothetical origins of these two genes. At an early stage of evolution, chloroplastic CuZn-SOD could have been encoded by the chloroplast genome which evolve at a significantly lower rate than most nuclear genes [15, 16]. The strongest sequence differences between the SODs are located in the N-terminal region of the protein. Hence, the C-terminal end is much more conserved (Fig. 4). The reason for this is not known, but it has been suggested that the contact area of the two subunits and the local guide of O f to the active site for CuZn-SODs, involve several residues from the C-terminal part of the protein [1 ]. The data in Fig. 4 also clearly indicate that the metal-binding sites are highly conserved. Northern experiments (Figs. 5, 6] showed that both genes are expressed in Scots pine. A study of needles and roots of Scots pine seedlings indicate distinct differences in expression level of m R N A encoding cytosolic and chloroplastic CuZn-SODs. In the case of chloroplastic SOD, we were able to detect a signal in m R N A isolated from needles and a much weaker (ca. 10-fold) signal in roots (Fig. 6B). This result confirms the expression of chloroplastic CuZn-SOD gene and existence of protein earlier detected in roots of Scots pine [26]. The CuZn-SODs are encoded by a multi-gene family in Scots pine, containing at least three members [26]. Furthermore, we have isolated c D N A clones that only differ a few bp to the D N A sequence of the c D N A clone PS3 presented in this paper (data not shown). Presently, we do
not know whether they originate from different non-allelic genes, or from different alleles on the same gene, since our c D N A library was constructed from a mixture of seedlings. In addition, the isozymes of SOD in maize have been shown to be encoded by distinct, unlinked nuclear genes [3]. For the chloroplastic SOD, corresponding data are lacking. The Southern hybridization experiments show that the cDNAs were encoded by separate genes since they were found on different DNA restriction fragments and they did not cross-hybridize. As we were able to detect only one form of chloroplastic CuZn-SOD [26], the complex hybridization pattern for this gene could be due to the presence of introns. However, hybridization to PS3 and PST13 c D N A confirm our previous observation that CuZn-SODs belong to a multigene family. On the basis of these data it is impossible to predict the exact number of genes present in the Scots pine CuZn-SODs family.
Acknowledgements We are indebted to Dr Rafael Perl-Treves, Department of Plant Genetics, The Weizmann Institute of Science, Rehovot, Israel, for kindly providing us with the plasmids T10 and P31. We are thankful to Stefan Jansson, Department of Plant Physiology, Ume~ University, for comments on the manuscript. The work was supported by a grant from the Swedish Council for Forestry and Agricultural Research.
References 1. Asada K: Superoxide dismutase. In: Otsuka S, Yamanaka T (eds) Metalloproteins, pp. 331-341. Elsevier, Amsterdam (1988). 2. Ausubel FM, Brent R, Kingston RE, Moore DD, Seidman JG, Smith JA, Struhl K: Screening of recombinant DNA libraries. In: Current Protocols of Molecular Biology. Green Publishing Associates/Wiley-Interscience, New York (1989). 3. Baum JA, Scandalios JG: Multiple genes controlling superoxide dismutase expression in maize. J Hered 73: 95100 (1982).
555 4. Cannon RE, White JA, Scandalios JG: Cloning ofcDNA for maize superoxide dismutase 2 (SOD2). Proc Natl Acad Sci USA 84:179-183 (1987). 5. Cannon RE, Scandalios JG: Two cDNAs encode two nearly identical Cu/Zn superoxide dismutase proteins in maize. Mol Gen Genet 219:1-8 (1989). 6. Chen EY, Seeburg PH: Supercoil Sequencing: A fast and simple method for sequencing plasmid DNA. DNA 4: 165-170 (1985). 7. Devereux J, Haeberli P, Smithies O: A comprehensive set of sequence analysis programs for the VAX. Nucl Acids Res 12:387-395 (1984). 8. Fridrovich I: Superoxide dismutases. Adv Enzymol 58: 62-97 (1986). 9. Hassan HM, Scandalios JG: Superoxide dismutases in aerobic organisms. In: Alscher RG, Cumming JR (eds) Stress Responses in Plants: Adaptation and Acclimation Mechanisms, pp. 175-199. Wiley-Liss, New York (1990). 10. Ingestad T: Mineral nutrient requirements for Pinus sylvestris and Picea abies seedlings, Physiol Plant 45: 373380 (1979). 11. Joshi CP: Putative polyadenylation signals in nuclear genes of higher plants: A compilation and anlysis. Nucl Acids Res 15:9627-9641 (1987). 12. Kanematso S, Asada K: Characteristic amino acid sequences of chloroplast and cytosol isozymes of CuZnsuperoxide dismutase in spinach, rice and horsetail. Plant Cell Physiol 31:99-112 (1990). 13. Kitagava Y, Tsunasawa S, Tanaka N, Katsube Y, Sakiyarna F, Asada K: Amino acid sequence of copper, zincsuperoxide dismutase from spinach leaves. J Biochem 99: 1289-1298 (1986). 14. Kwiatowski JA, Kaniuga Z: Isolation and characterization of cytosolic and chloroplast isoenzymes of CuZnsuperoxide dismutase from tomato leaves and their relationship to other CuZn-superoxide dismutase. Biochim Biophys Acta 874:99-115 (1986). 15. Oseki H, Umesono K, Inokuchi H: The chloroplast genome of plants: a unique origin. Genome 31:169-174 (1989). 16. Palmer JG: Evolution of chloroplast and mitochondrial DNA in plants and algae. In: Maclntyre RJ (ed), Molecular Evolutionary Genetics, pp. 131-240. Plenum Press, New York/London (1985).
17. Perl-Treves R, Nacimas B, Aviv D, Zeelon EP, Galun E: Isolation of two cDNA clones from tomato containing two different superoxide dismutase sequences. Plant Mol Biol 11:609-624 (1988). 18. Rabinowich HD, Fridrovich I: Superoxide radicals, superoxide dismutase and oxygen toxicity in plants. Rev Photochem Photobiol 37:679-690 (1983). 19. Sambrook J, Fritsch T, Maniatis F: Molecular Cloning: A laboratory Manual, 2nd ed. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY (1989). 20. Sanger F, Nicklen S and Coulson AR: DNA sequencing with chainterminating inhibitors. Proc Natl Acad Sci USA 74:5463-5467 (1977). 21. Scioli JR, Zilinskas BA: Cloning and characterization of cDNA encoding the chloroplastic copper/zinc-superoxide dismutase from pea. Proc Natl Acad Sci USA 85: 76617665 (1988). 22. Steffens GJ, Michelson AM, (~tting F, Puget K, Strassburger W, Floh6 L: Primary structure of Cu-Zn superoxide dismutase of Brassica oleracea proves homology with corresponding enzymes of animals, fungi and procaryotes. Biol Chem Hoppe-Seyler 367:1007-1016 (1986). 23. Szmidt AE, Lidholm J, Hallgren J-E: DNA extraction and preliminary characterisation of chloroplast DNA from Pinus sylvestris and Pinus contorta. In: Lindgren D (ed) Provenances and Forest Tree Breeding for High Latitudes, pp. 269-280. Proceedings of the Frans Kempe symposium, Ume~t, 10-1l June (1986). 24. Tepperman J, Katayama C, Dunsmuir P: Cloning and nucleotide sequence of a petunia gene encoding a chloroplast-localized superoxide dismutase. Plant Mol Biol 11:871-872 (1988). 25. Whitmore FW, Kriebel HB: Expression ofa gene in Pinus strobus ovules associated with fertilization and early embryo development. Can J Forest Res 17:408-412 (1987). 26. Wingsle G, Gardestr~m P, H~llgren J-E, Karpinski S: Isolation, purification and subcellular localization of isozymes of superoxide dismutase from Scots pine (Pinus sylvesOqs L.) needles. Plant Physiol 95:21-28 (1991). 27. Young RG, Davis RW: Efficient isolation of genes by using antibody probes. Proc Natl Acad Sci USA 80: 1194-1198 (1983).
545
Characterization of cDNAs encoding CuZn-superoxide dismutases in Scots pine Stanislaw Karpinski, Gunnar Wingsle, Olof Olsson and Jan-Erik H~tllgren
Department of Forest Genetics and Plant Physiology, Faculty of Forestry, Swedish University of Agricultural Sciences, S-901 83 Umed, Sweden Received 31 May 1991; accepted in revised form 16 October 1991
Key words: chloroplastic SOD, cytosolic SOD, nucleotide sequences, Pinus sylvestris, sod genes
Abstract
A Scots pine (Pinus sylvestris L.) cDNA library was screened with two heterologous cDNA probes (P31 and T10) encoding cytosolic and chloroplastic superoxide dismutases (SOD) from tomato. Several positive clones for cytosolic and chloroplastic superoxide dismutases were isolated, subcloned, mapped and sequenced. One of the cDNA clones (PS3) had a full-length open reading frame of 465 bp corresponding to 154 amino acid residues and showed approximately 85~o homology with the amino acid sequences of angiosperm cytosolic SOD counterparts. Another cDNA clone (PST13) was incomplete, but encoded a putative protein with 93 ~o homology to pea and tomato chloroplastic superoxide dismutase. The derived amino acid sequence from both cDNA clones matched the corresponding N-terminal amino acid sequence of the purified mature SOD isozymes. Northern blot hybridizations showed that, cytosolic and chloroplastic CuZn-SOD are expressed at different levels in Scots pine organs. Sequence data and Southern blot hybridization confirm that CuZn-SODs in Scots pine belong to a multigene family. The results are discussed in relation to earlier observations of CuZn-SODs in plants.
Introduction
Superoxide dismutases (SODs; EC. 1.15.1.1) are a ubiquitous and diverse class of metalloenzymes found in all plants which catalyse the disproportionation of superoxide radicals into molecular oxygen and H20 2. In addition, these enzymes are
present in all investigated aerobic organisms and are involved in the detoxification of active oxygen species [ 18]. The most abundant SODs in plants are the CuZn-SODs which are found mainly in the cytosol and in the chloroplasts. In the chloroplast stroma, SOD constitutes the first link in an enzymatic scavenging system which protects
The nucleotide sequence data reported will appear in the EMBL, GenBank and DDBJ Nucleotide Sequence Databases under the accession numbers X58578 (clone PS3) and X58579 (clone PST13).
546 the photosynthetic apparatus from noxious oxygen derivatives. The active sites of other plant SODs contain either manganese (Mn-SOD) or iron (Fe-SODs) [8]. Several isozymes ofcytosolic CuZn-SOD have been found in the plants surveyed so far: two in spinach (Spinacia oleracea L.) [12], three in rice (Oryza sativa L.) [ 12], three in maize (Zea mays L.) [4, 5], two in horsetail (Equisetum arvense L.) and in green algae (Spirogyra sp.) [ 12] and at least two in Scots pine (Pinus sylvestris L.) [26]. On the contrary, only one form of CuZn-SOD has been found in chloroplasts in plants [e.g. 14]. The cDNAs encoding cytosolic SODs have been isolated, cloned and sequenced from maize (Zea mays L.) [4, 5] and tomato (Lycopersicon esculentum L.) [17]. The e D N A encoding chloroplastic SOD has been presented from pea (Pisum sativum L.) [ 21 ], tomato [ 17 ] and petunia (Petunia hybrida L.)[24]. CuZn-SODs appear to have been resistant to evolutionary change since the enzymes isolated from a range of organisms differed only slightly in their physical and chemical properties [ 1, 9]. The nucleotide and amino acid sequence showed a great degree of similarity among different plant species [ 1, 4]. We have earlier isolated, purified and shown the subcellular localization of CuZn-SODs from Scots pine needles [26]. Here, for the first time, a description of cytosolic and chloroplastic CuZn-SODs from gymnosperms is presented on the D N A and m R N A level. Derived amino acid sequences of Scots pine SODs and a comparison with other species are discussed.
Materials and methods
Plant material Scots pine (Pinus sylvestris L.) seedlings were grown in a greenhouse in a peat/sand mixture (1:1). The seedlings were fertilized with a nutrient solution containing 4 g N per litre and with other elements in proportion according to Ingestad [ 10]. The photoperiod was 18 h, relative humidity 70 ~o and temperature 20 + 2 ° C day and night.
Extraction of poly(A) ÷ RNA A LiCl-based extraction method was used for RNA preparations [25 ]. R N A was extracted from 6-8-week-old seedlings (roots and cotyledons separated) and from needles of mature trees. The plant material (5 g) was frozen in liquid nitrogen, ground and transferred to 25 ml extraction buffer (0.2 M sodium borate, 30 m M EGTA, 5 m M D T T and 1~o SDS adjusted to pH 9.0 with NaOH), treated with proteinase K, and precipitated with 2.5 M LiC1. Poly(A) + RNA was obtained from the total R N A by two sequential passages through an oligo(dT)-cellulose spun column (Pharmacia, m R N A purification kit). cDNA library construction Extracted and purified poly(A) + RNAs as described above, was sent for library construction to Stratagene Custom Service. Double-stranded, blunt-ended c D N A was synthesized using oligo-dT primer according to the Young and Davis method [27]. After methylation, ligation and digestion of Eco RI linkers, cDNAs were fractionated into three size categories, 500-1000 bp, 1000-2000 bp and >2000 bp cloned into the Eco RI site of the expression vector 2gtl 1. DNA probes The heterologous c D N A probes, cytosolic (P31) and chloroplastic (T10) probes from tomato, were obtained from R. Perl-Treves [17]. Two c D N A Eco RI/Sac ! fragments (for P31 and T10 probes respectively) containing the 5' region of non-coding and coding sequences were used for screening of the e D N A library. Homologous e D N A probes (cytosolic PS3 EcoRI/HincII fragment and chloroplastic PST13 EcoRI/ Hae III fragment) containing 5' non-coding and coding sequences were used for the northern hybridization experiments as well as for Southern hybridization experiments (cytosolic PS3 Eco RI fragment and chloroplastic PST13 Eco RI fragment).
547
Screening of the amplified cDNA library The amplified c D N A library was grown in the Escherichia coli strain Y1090 in LB medium containing 10 mM M g S O 4 and 0.2~o maltose. Duplicate replica filters were prepared and prehybridization and hybridization were performed according to Ausubel et al. [2]. The heterologous probes P31 and T10 were labelled with 32p-dCTP according to the oligolabelling kit protocol of the supplier (Pharmacia). After hybridization the following four washes were done: two times for 20min in 1 × SSC, 0.1~o SDS at room temperature and two times for 10 min in 1 × SSC, 0 . 1 ~ SDS at 55 ° C. After autoradiography (32 h) positive clones were detected and isolated. The same filters were used for screening with different probes. Isolation of the positive clones was done after dilution, plating and rehybridization under the same conditions.
Isolation and subcloning of DNA fragments Preparation of phage D N A was made according to Sambrook et al. [ 19]. The c D N A inserts and the smaller restriction fragments required for sequencing were isolated from agarose gels and ligated with the appropriately cut p U C 18 vector. Competent E. coli DH~5 cells were transformed with the ligated D N A by the CaCI2 method [ 19].
Sequence analys~ The dideoxy method of D N A sequencing was performed according to the description given by Sanger etal. [20] and Chen and Seeburg [6]. Sequence data were analysed on a VAX computer, using software as described [7].
RNA gels and northern blots R N A samples, together with D N A molecular weight markers, were run on 1.4~o agarose gels after glyoxylation in phosphate buffer [ 19]. Blot-
ting to Hybond N membrane was performed according to the protocol of the supplier (Amersham). R N A was fixed on the membrane by baking at 80 °C for 2 h. Filters were prehybridized at 42 °C for 10-12 h in 6 x SSC (1 x SSC is 0.15 M NaC1, 15 mM sodium citrate), 5 x Denhardt's solution, 50 mM sodium phosphate, 0.1 To SDS, 100 #g/ml salmon sperm D N A and 50~o formamide. The filters were hybridized in the same solution plus the radioactive probe (1.2 × 10 6 cpm/ml) at 42 °C for 18 h. After hybridization the following four washes were done: two times for 20 min in 0.1 x SSC, 0.1~o SDS at room temperature and two times for 15 min in 0.1 × SSC, 0.1~o SDS at 58 °C. The filters were exposed to X-ray film for 72 h. Plasmids PS3 and PST13 (100 pg), cut with the restriction enzyme Eco RI, were run in parallel on another 1.4~o agarose gels after glyoxylation under the same conditions as the R N A samples. Following blotting to Hybond N membrane the plasmids D N A s were used as internal hybridization stringency controls in the northern experiment.
DNA isolation and Southern blots Total D N A from young seedlings of Scots pine was extracted according to Szmidt et al. [23], digested with the proper restriction enzymes and run on 0.8~o agarose gels. Blotting to Hybond N membranes after depurination in 0.25 M HC1 and denaturation and neutralization was performed according to Sambrook etal. [19]. D N A was fixed on the membrane by baking at 80 ° C for 2 h. The filters were prehybridized at 65 °C for 6 h in 6 × SSC, 5 × Denhardt's solution, 50 mM sodium phosphate, 0.1~o SDS, 100 #g/ml salmon sperm D N A and 50 #g/ml of yeast tRNA. The filters were hybridized in the same solution plus the radioactive probe (2.4 x 10 6 cpm/ml) at 65 °C for 24 h. After hybridization the following five washes were done: three times for 20 min in 0.1 x SSC, 0.1~o SDS at room temperature and two times for 15min in 0.1× SSC, 0.1~o SDS at 56 ° C. The filters were exposed to X-ray film for two weeks.
548 Results
Screening of the amplified cDNA library with heterologous probes The heterologous cytosolic tomato probe P31 was used to screen 200000 transformants from the library with an insert size between 0.5-1 kb and 200000 transformants from the library with an insert size between 1-2 kb. After screening, 19 positive clones hybridizing to the cytosolic probe (P31) were isolated from the c D N A library with an insert size smaller than 1 kb. The other heterologous chloroplastic tomato probe (T10) was used to screen the same library and 13 clones that hybridized to this probe were detected in the c D N A library with an insert size smaller than 1 kb. No hybridization signals were obtained with any of the probes when the library with the insert size 1-2 kb was screened.
Cloning and DNA sequencing The c D N A inserts hybridizing to the cytosolic probe P31 and chloroplastic probe T10 were isolated and cloned into the pUC 18 vector. After restriction and partial sequence analysis of all isolated c D N A inserts, two clones denoted PS3 and PST13 were chosen for further studies and completely sequenced. Other clones contained equal or shorter c D N A inserts (data not shown). The nucleic acid sequence for the cytosolic clone PS3 and the chloroplastic clone PST13 is illustrated together with the sequencing strategy in Fig. 1A and B and in Fig. 2A and B, respectively. From clone PS3, 821 bp of c D N A was sequenced. One of the open reading frames (ORF) was 465 bp corresponding to 154 amino acid residues. For the described ORF, the c D N A contained a 5' end of 112bp and a 3' end of 244 bp. The derived amino acid sequence from c D N A clone PS3 matched the N-terminal amino acid sequence of the purified cytosolic Scots pine CuZn-SOD isozyme with the exception of the two amino acid residues Q and E (position 22 and 23; Fig. 3). Clone PST13 contained a c D N A insert of
676 bp, and the open reading frame corresponded to 424 bp or 141 amino acid residues. The clone contained a 3' end of 252 bp. A small part of the 5' coding region and the whole non-coding region were absent. However, 11 amino acid residues from the N-terminal sequence of the purified CuZn-SOD isozyme (chloroplastic form) fitted perfectly with the derived amino acid sequence of the clone PST13 (Figs. 2 and 3). The derived amino acid sequences of the clones PS3 and PST13 are also shown in Figs. 1 and 2. Both clones encoded polypeptides with a high degree of homology to angiosperm sod gene products (PS3 cytosolic form and PST13 chloroplastic form). Displayed in Fig. 4 is a comparison of amino acid sequence s of CuZn- S O D s from seven plant species, either determined by Edman degradation [ 13, 22] or deduced from nucleotide sequence of cDNAs [5, 17, 21, 24 and this report]. The described c D N A sequences show a high degree of homology to known angiosperm CuZnSOD cDNAs. The homology at the D N A level between corresponding CuZn- SOD in Scots pine, tomato [17], maize [4, 5] and pea [21] was approximately 74~o for the cytosolic and 80~o for the chloroplastic form respectively. On the deduced amino acid level the corresponding values were approximately 8 5 ~ for the cytosolic and 93 ~o for the chloroplastic CuZn-SODs. The homology on the protein level between the cytosolic and chloroplastic forms in Scots pine was higher than that observed in tomato (cf. Fig. 4).
Polyadenylation signal A consensus signal for polyadenylation AATAAA is often present in animal as well as in plant genes. Another conserved sequence, TGTGTTTT, has been found near to the polyadenylation site in plants [11]. In the cytosolic SOD cDNA one AATAAA sequence was found between nucleotide 727 and 732 (Fig. 2). In the chloroplastic SOD cDNA, a T G T G T T T T sequence with one mismatch was observed. The imperfect sequence motif was found starting at position 538 and ending at 545 (Fig. 2).
549 1
61
CAGAAACCAAC TACGATACTGCGATATTGACCACTGTTAGCAAAGGTTTCACTGATCTAG ......... + ......... + ......... + ......... + ......... + ......... C TTGTACAGAGCTCAAGGGG ......... + .........
+
60
TTTCCTGGGATCACATCTTTTATTGATAAAGTATGGGTCT + ......... + ......... + ......... +--- ...... +
M
G
120
L
121
TC TTAAGGCTGTTGTTGTCTTGAATGGTGCTGCTGATGTCAAGGGGGTTGTTCAATTCAC ......... + ......... + ......... + ......... + ......... + ......... + L K A 7 V V L N G A A D V K G V V Q F T
180
181
C CAGGAAGGAGATGGGCCAACAACTGTAACTGGGAAGATCAGTGGTCTGAGCCCTGGTC T ......... + ......... + ......... + ......... + ......... + ......... + Q E G D G P T T 7 T G K I S G L S P G L
Z40
241
CCATGGTTTCCATGTTCATGCACTAGGTGACACAACAAATGGGTGCATGTCAACTGGACC ......... + ......... + ......... + ......... + ......... + .........
H
G
F
H
V
H
A
L
G
D
T
T
N
G
C
M
S
T
G
+
300
P
301
ACATT TTAATC C G TTAGGCAAGGAGCATGG TGCAC C CACAGATGATAAC ......... + ......... + ......... + ......... + ......... H F N P L G K E H G A P T D D N
CGACATGCTGG + ......... + R H A' G
360
361
GGATCTAGGCAATG TAAC TGTAGGAACGGATGGGAC TGTCGAAT TTTCAATCACTGACAG ......... + ......... + ......... + ......... + ......... + ......... + D L G N V T V G T D G T V E F S I T D S
420
421
C CAGATTC C TCTCTC TGGGCCACATTCGATAGTAGGACGAGCTGTAGTTGTC ......... + ......... + ......... + ......... + ......... Q I P L S G P H S ! V G R A V V
CATGC TGA + ......... + V H A D
480
481
TCC TGATGATCTTGGCAAAGGTGGCCATGAAC TGAG TAAGAGCACAGGAAATGCTGG TGG ......... + ......... + ......... + ......... + ......... + ......... + P D D L G K G G H E L S K S T G N A G G
540
541
CAGACTTGCTTGTGGGGTTGTCGGTCTTCAAGGATGAATGTGAGCAGTTGGTTATTTATA ......... + ......... + ......... + ......... + ......... + ......... R L A C G V V G L Q G * M *
+
600
+
660
+
720
TGATG + ......... +
780
601
GGCTGGC TT TTTTCAAGTATGAKTACGTG ......... + ......... + .........
661
TGCGAKAGGGATACAACAGTAKGC CAGATACAAACATTTGGAAGGTGGTTATTAGGTGAA ......... + ......... + ......... + ......... + ......... + .........
721
C C TAACAATAAATTGAGTACGCTGTGCTACATAAGGATTTTGCATGTAATAATTT ......... + ......... + ......... + ......... + .........
781
ATGTTATACAAAC C TATTCAGGC TAAAAAAAAAAAAAAAAA ......... + ......... + ......... + .........
EcoRI
Mbo II Mbo II
Hinc II
strategy
+-
821
Mbo II
II
Restriction map PS3 Seqendng
TATC TATTACTCGTCGC T C TAGTTTGCATTA + ......... + ......... + .........
4
4
D
EcoRI
I
I~
Fig. 1. A (top). Nucleotide sequence and derived amino acid sequence of the cDNA of the clone PS3 encoding cytosolic CuZnSOD. Stop codons are indicated by the asterisks. Underlined are the derived amino acid sequence showing 100~o homology with the N-terminal amino acid sequence of the mature cytosolic SOD protein and the consensus sequence of polyadenylation signal. B (bottom). Sequencing scheme of the PS3 cDNA. The bars denote the coding region. The single line indicates the non-coding region, arrows show the direction and distance of sequencing.
Northern blot hybridizations Northern hybridizations were performed at high stringency, and under these conditions no crosshybridizations were observed. Two northern blot
experiments were performed to analyse size (Fig. 5) and abundance of m R N A species in different tissues of Scots pine (Fig. 6). The external controls (Eco RI-cut plasmids PS3 and PST13) indicated that the two probes did not cross-
550
1
61
121
181
241
301
361
&21
ACAGGTCGAGGGTGTTGTCACTCTCTCGCAGG~GAC~CGGTCCCAC~CAGTG~GGT ......... + ......... + ......... + ......... + ......... Q V E G V V T L S Q E D N G P T T V CCGTTTGACAGGATTGACTCCTGGG~GCATGGCTTTCATCTACATGAGTTTGGTGACAC ......... + ......... + ......... + ......... + ......... R L T G L , T P G K H G F H L H E F G
K
+ ......... V
+ ..... D T
+
A---+
~CCAATGGCTGCATGTcAACAGGATCACATTTT~TCCAAAAAAATTGACACATGGTGC ......... + ......... + ......... + ......... + ......... T N G C M S T G S H F N P K K L
+ ......... T H G
A
TCCTGAGGATGATGTACGCCATGCGGGTGACCT~GAAACATAGTTGCGGGTTCTGATGG ......... + ......... + ......... + ......... + ......... P E D D V K H A G D L G N I V A
+ ......... G S D,
G
60
120
+
180
+
2k0
AGTTGCAGAGGCAAC~TTGTTGAT~TCAGATTC~TTGAGTGGACCTGATTCAGTTAT ......... + ......... + ......... + ......... + ......... V A E A T I V D N Q I P L S G P D S
+ ......... I
+
300
V
TGGTAGGGCACTTGTTGTCCATGAGTTAGAGGATGACCTGGGGAAAGGTGGGCATGAACT ......... + ......... + ......... + ......... + ......... G K A L V V H E L E D D L G K G G H
+ ......... E ~
+
360
TAGTCTGAC~CTGGC~TGCTGGGGGCAGGTTGGCTTGTGGTGTGGTTGGACTCACTCC ......... + ......... + ......... + ......... + ......... $ L T T G N A G G K L A C G V V G L
+ ......... P
+
420
T
+ .........
+
~80
CATTT~GGCCCAGTCAAATATGATGATCTTCAAAGGTCATGGACATCGTATGATACCAG ......... + ......... + ......... + ......... + ......... I *
481
TGACTGCAATAATAATTCAAATATATAGTTCTCTATCCTCGC~GATTGTTAGC~TTGT ......... + ......... + ........ .+ ......... + .........
+ .........
+
540
541
GATTTGTTTTTGGCATTAGCGAGTTGCACTTTGGGACATT~TGTTTGGGCGCAGAT~G ......... + ......... + ......... + ......... + .........
+ .........
+
500
+ .........
+
560
ATTGAGAKACATTTGTAATTCATG~TCTCT~TAATAGTCTCCTTTTTGAAGGGAAAAA
601
.........
+ .........
661
.........
+ ......
+ .........
+ .........
+ .........
676
EcoRI Re;tp;;°n
I
Seqencing strategy
JI
Hae III
EcoRI
I IL
IL
~,
Fig. 2. A (top). Nucleotide sequence and derived amino acid sequence of the eDNA of the clone PST13 encoding incomplete chloroplastic CuZn-SOD. The asterisk indicates the stop codon. The derived amino acid sequence (underlined), matched the N-terminal amino acid sequence of the mature chloroplastic SOD protein. B (bottom). Sequencing scheme of the PST13 eDNA. The bars denote the coding region. The single line indicates the non-coding region, arrows show the direction and distance of sequencing.
AA number Chloroplast N-terminal PST13 AA-sequence
! 5 l0 AAKKAVAVLKGDS
Cytosolic N-terminal
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Fig. 3. Comparison of N-terminal amino acid sequences of mature SOD chloroplastic and cytosolic proteins, purified from Scots pine [26] with the derived amino acids sequence (AA) from the e D N A of the chloroplastic and the cytosolic clones PST13 and PS3, respectively.
551 Chloroplast CuZn SOD Pine Pea Spinach Tomato Petunia Cytosolic CuZn SOD Pine Tomato Cabbage Maize2 Chloroplast CuZn SOD Pine Pea Spinach Tomato Petunia Cytosolic CuZn SOD Pine Tomato Cabbage Maize2 Chloroplast CuZn SOD Pine Pea Spinach Tomato Petunia Cytosolic CuZn SOD Pine Tomato Cabbage Maize2 Chloroplast CuZn SOD Pine Pea Spinach Tomato Petunia Cytosolic CuZn SOD Pine Tomato Cabbage Maize2
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Fig. 4. Comparison of the amino acid sequences of the CuZn-SODs from pine [this report and 26], pea [21], spinach [13], tomato [17], petunia [24], cabbage [22] and maize [5]. Boxed regions indicate 100% conservation. Asterisks indicate amino acid residues coordinating copper and zinc.
hybridize. Each probe recognized an abundant m R N A species from the poly(A) + fraction. These experiments showed that m R N A species, for both isozyme forms, were expressed in seedlings and mature Scots pine needles. The sizes of the mRNAs in Scots pine tissues were about 850 bp and 1000 bp for clones PS3 and PST13, respec-
tively (Fig. 5). In the case of clone PS3 the size was similar to the cloned insert (Fig. 5A). The observed size discrepancy between the detected m R N A species and the cloned insert for PST13 could be due to the lack of part of the coding and full non-coding 5' region in the c D N A insert (Fig. 5B).
552
Fig. 6. Northern blot hybridization of Scots pine RNA (5/~g) with homologous cDNA (used as a radioactive probe) clone PS3 (A) and PST13 (B). Slot designations: 1, poly(A) + RNA isolated from cotyledons of 8-week-old seedlings; 2, poly(A) + RNA isolated from roots of 8-week-old seedlings.
Fig. 5. Northern blot hybridization of Scots pine RNA with homologous cDNA (used as a radioactive probe) clone PS3 (A) and PST13 (B). A and B: 1, total RNA (15 ~g) from Scots pine seedlings; 2, poly(A) + RNA (4 #g) isolated from needles from mature trees; 3, poly (A) ÷ RNA (4/zg) isolated from Scots pine seedlings. Southern blot controls of hybridization stringency C and D: 100 pg of recombinant plasmid PS3 (line 1) and PST13 (line 2) digested by Eco RI.
Northern hybridization experiments showed that RNA species, for the cytosolic form, were detectable in appreciable amounts in both needles and roots of eight-week-old seedlings and that the abundance was higher in the roots (Fig. 6A). On the contrary, when using chloroplastic SOD c D N A as a probe (clone PST13) we were able to detect a strong signal in m R N A isolated from needles and approximately a 10 times weaker signal in m R N A isolated from roots (Fig. 6B).
Southern blot hybridization
In order to get some impression of the complexity of the CuZn-SOD gene organisation, two
Southern blot experiments were performed. Figure 7 shows sizes of DNA fragments hybridizing to cytosolic CuZn-SOD c D N A (Fig. 7A) and chloroplastic CuZn-SOD c D N A (Fig. 7B) in Scots pine. Each probe recognized specific restriction fragments of the DNA, hence no cross hybridization was observed. The hybridization showed that the genes, for both isozyme forms, were present on different restriction fragments. The sizes of the D N A fragments hybridizing to the cytosolic CuZn-SOD c D N A in Scots pine tissues were about 7.0 kb and 2.6 kb for Eco RI fragments; 5.6 kb, 2.1 kb and 1.9 kb for Hind III fragments; 3.5 kb and 2.9 kb for Hinc II fragments. In the case of chloroplastic CuZn-SOD the sizes were approximately 10 kb, 3.0 kb and 1.7 kb for Eco RI fragments; 4.0 kb, 3.2 kb and 1.2 kb for Hind III fragments; 1.7 kb and 1.2 kb for Hinc II fragments.
Discussion
Earlier we purified and localized CuZn-SODs from Scots pine needles [26]. This report presents a complete cDNA sequence data of CuZn cytosolic SOD from a coniferous tree species (Fig. 1A). We also show the sequence of an in-
553
Fig. 7. Southernblot hybridizations of Scots pine DNA to homologouscDNA (Eco RI fragment used as radioactive probes) clone PS3 (A) and PST13 (B). Slot designations: lines 1, 2 and 3, 25 #g of Scots pine DNA restricted with Eco RI (1), Hind III (2) and Hin cII (3); lines 4 and 5, 30 pg of recombinant plasmid PS3 and PST13 restricted with Eco RI; line 6 contains the molecularweight
markers.
complete c D N A clone of the CuZn chloroplastic SOD (Fig. 2A). The match between the amino acid sequences of the mature SOD proteins isolated from cytosol and chloroplasts, respectively, of Scots pine needles and the deduced amino acid sequence from the c D N A clones (PS3 and PST 13) support this statement. In the case of the deduced amino acid sequence derived from the clone PS3, two amino acids Q and E (position 22 and 23) differ compared with the N-terminal sequence of the mature cytosolic SOD (Fig. 3). This result supports the previous observation of the existence of several isoforms of cytosolic SOD in Scots pine and might indicate that we isolated c D N A for the cytosolic CuZn-SOD-2 as defined in Wingsle e t a l . [26]. Even though the partial chloroplastic clone lack 13 most N-terminal amino acids the other 11 amino acids match per-
fectly the N-terminal amino acid sequence of the mature chloroplastic protein (Figs. 2 and 3). Both c D N A clones showed high sequence homology to their angiosperm counterparts. These results thus confirm earlier indications of the conserved nature of CuZn-SODs in plants. Generally, CuZn-SODs appear to be quite resistant to evolutionary change since recent nucleotide and amino acid sequence comparisons show a great degree of similarity [5, 9]. It has earlier been pointed out that the chloroplastic SOD is much more conserved than the cytosolic form [e.g. 12, 14, 17]. The results presented here (Fig. 4) support these observations. The differences between the approximately 250 million-year-old conifer species and the much younger tomato [ 17], spinach [ 13], petunia [24] and pea [21] are small when the chloroplastic
554 forms are compared (homology approximately 90-94% at the amino acid level) (Fig. 4). The comparison presented in Fig. 4 indicates that the rate of mutation for CuZn-SOD is higher for the cytosolic than for the chloroplastic form. Several different hypotheses were proposed to explain this phenomenon. Kanematsu and Asada [ 12] suggested that a high rate of production of superoxide radicals in the chloroplast would have a lethal effect if mutations in CuZn-SOD bring about a decrease in or loss of their activity to scavenge superoxide. Another possible explanation for the different rates of mutation could be the different hypothetical origins of these two genes. At an early stage of evolution, chloroplastic CuZn-SOD could have been encoded by the chloroplast genome which evolve at a significantly lower rate than most nuclear genes [15, 16]. The strongest sequence differences between the SODs are located in the N-terminal region of the protein. Hence, the C-terminal end is much more conserved (Fig. 4). The reason for this is not known, but it has been suggested that the contact area of the two subunits and the local guide of O f to the active site for CuZn-SODs, involve several residues from the C-terminal part of the protein [1 ]. The data in Fig. 4 also clearly indicate that the metal-binding sites are highly conserved. Northern experiments (Figs. 5, 6] showed that both genes are expressed in Scots pine. A study of needles and roots of Scots pine seedlings indicate distinct differences in expression level of m R N A encoding cytosolic and chloroplastic CuZn-SODs. In the case of chloroplastic SOD, we were able to detect a signal in m R N A isolated from needles and a much weaker (ca. 10-fold) signal in roots (Fig. 6B). This result confirms the expression of chloroplastic CuZn-SOD gene and existence of protein earlier detected in roots of Scots pine [26]. The CuZn-SODs are encoded by a multi-gene family in Scots pine, containing at least three members [26]. Furthermore, we have isolated c D N A clones that only differ a few bp to the D N A sequence of the c D N A clone PS3 presented in this paper (data not shown). Presently, we do
not know whether they originate from different non-allelic genes, or from different alleles on the same gene, since our c D N A library was constructed from a mixture of seedlings. In addition, the isozymes of SOD in maize have been shown to be encoded by distinct, unlinked nuclear genes [3]. For the chloroplastic SOD, corresponding data are lacking. The Southern hybridization experiments show that the cDNAs were encoded by separate genes since they were found on different DNA restriction fragments and they did not cross-hybridize. As we were able to detect only one form of chloroplastic CuZn-SOD [26], the complex hybridization pattern for this gene could be due to the presence of introns. However, hybridization to PS3 and PST13 c D N A confirm our previous observation that CuZn-SODs belong to a multigene family. On the basis of these data it is impossible to predict the exact number of genes present in the Scots pine CuZn-SODs family.
Acknowledgements We are indebted to Dr Rafael Perl-Treves, Department of Plant Genetics, The Weizmann Institute of Science, Rehovot, Israel, for kindly providing us with the plasmids T10 and P31. We are thankful to Stefan Jansson, Department of Plant Physiology, Ume~ University, for comments on the manuscript. The work was supported by a grant from the Swedish Council for Forestry and Agricultural Research.
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