Superoxide dismutase expression in plants

Plant species having enhanced superoxide dismutase activity as a result of transformation with a DNA expression cassette comprising an E. coli MnSOD gene are provided. Transportation of the expression product of the gene may be targeted to a specific cell organelle, such as the chloroplast.

INTRODUCTION 
1. Technical Field 
This invention relates to plants, and methods for their preparation, having 
increased tolerance to environmental and chemical stresses. The method 
employs transformation of the plants so as to increase the concentration 
of superoxide dismutase in the plant's cells, especially in the 
chloroplasts. 
2. Background 
In plants, as in other aerobic organisms, superoxide (O.sub.2.sup.-) is a 
commonly encountered intermediate of oxygen reduction. Superoxide is 
extremely toxic to cells because it attacks unsaturated fatty acid 
components of membrane lipids, thus damaging membrane structure. Aerobic 
cells detoxify superoxide by the action of superoxide dismutases, 
metal-containing enzymes that convert the superoxide radical into hydrogen 
peroxide and molecular oxygen. The hydrogen peroxide is subsequently 
converted by catalase into water and molecular oxygen. Superoxide 
dismutases thus provide a defense against the potential cytotoxicity of 
the superoxide radical. 
There are three types of superoxide dismutase (SOD): copper/zinc-containing 
SOD (CuZnSOD), manganese-containing SOD (MnSOD) and iron-containing SOD 
(FeSOD). All three types of SOD have been found in plants. CuZnSODs are 
inhibited by cyanide, while FeSODs and MnSODs are not. In prokaryotic 
organisms containing both Mn and FeSODs, MnSOD is inducible under 
conditions of high oxygen concentration and by O.sub.2.sup.-, while FeSOD 
is constitutively expressed. In plants, CuZnSOD and FeSOD are feedback 
inhibited by H.sub.2 O.sub.2 ; MnSOD is not. MnSODs are usually localized 
within mitochondria. In leaves and fruit of most plants examined, MnSOD 
represents only 3-5% of total SOD activity although it can be as high as 
20% in, for example, peas. FeSOD has been found in only a few families of 
seed plants where it may have arisen by gene transfer from prokaryotes. 
Induction of superoxide dismutase activity in plant cells has been 
correlated with development of increased tolerance to a variety of 
chemical compounds and physical stresses. Plants which show resistance to 
herbicides such as paraquat contain more total SOD activity in a number of 
tissues, including fruit and leaves, than the corresponding 
paraquat-sensitive genotypes. Tolerance to toxic gases (for example, 
ozone, sulfur dioxide), metals (for example, copper, iron, zinc and 
manganese) and damage due to environmental stresses such as photodynamic 
processes and thermal effects such as sun scald have also been correlated 
with increased levels of SOD activity. 
Environmental stresses are considered to decrease crop productivity to 
various extents, depending upon the severity of and the type of stress. 
Enhancing the tolerance of crop plants to adverse effects imposed by 
nonoptimal growing conditions is thus an important objective for 
improvement of crop management. There is therefore substantial interest in 
the ability to increase the concentration of superoxide dismutase in a 
plant cell so as to provide for a plant which has increased tolerance to 
environmental and chemical stresses. 
Relevant Literature 
The gene for manganese superoxide dismutase from Escherichia coli K-12 has 
been cloned. Touati, J. Bacteriol. (1983) 155:1078-1087. The DNA sequence 
of the E. coli K-12 manganese superoxide dismutase gene has been 
determined. Takeda et al., Nucleic Acids Research (1986) 14:4577-4589. The 
amino acid sequence of E. coli B manganese superoxide dismutase is known. 
Steinman, J. Biol. Chem. (1978) 253:8708-8720. The cDNA clones of two 
CuZnSODs from tomato are disclosed in Perl-Treves et al., Plant Molecular 
Biology (1988) 11:609-623. 
The use of a transit peptide of pea small subunit (ssu) 
ribulose-1,5-bisphosphate carboxylase fused to an exogenous gene to 
provide a chimeric gene has been disclosed for targeting a heterologous 
gene product to chloroplasts. See, for example, Van den Broeck et al., 
Nature (1985) 313:358-363; Schreier et al., EMBO J. (1985) 4:25-32. 
A possible protective role for superoxide dismutase against ozone injury in 
snap beans has been reported by Lee et al., Plant Physiol. (1982) 
69:1444-1449. The effect of SOD defense against SO.sub.2 toxicity was 
described by Tanaka et al., Plant and Cell Physiol. (1980) 21:601-611. 
Similarly a protective role for superoxide dismutase against 
photo-oxidative damage in the ripening tomato fruit has been reported. 
Rabinowitch et al., Physiol. Plant. (1982) 54:369-374. Superoxide 
dismutase minus mutants of E. coli are disclosed as having increased 
sensitivity to paraquat and to oxygen. Carlioz and Touati, EMBO J. (1986) 
5:623-630. The role of SOD in drought tolerance of mosses was reported by 
Dhindsa and Matowe, Exp. Botany (1981) 32:79-91. 
Treatment of soybeans with iron and/or manganese is reported to affect 
total SOD activity; MnSOD activity is reported to be modified only by 
manganese concentrations. Leidi et al., Plant and Soil (1987) 99:139-146. 
Palma et al., Plant Physiol. (1987) 85:570-577 disclosed that there is no 
change in mitochondrial SOD activity but that there is increased 
peroxisomal Mn-SOD activity in Cu-tolerant as compared to Cu-sensitive 
plants. Bowler et al., EMBO (1988) 8:31-38 disclosed an increase in SOD 
mRNA following exposure of a plant to ethylene, salicylic acid and 
infection with Pseudomonas syringae. 
Characterization of FeSOD from tomato was described by Kuiakowski et al., 
Eur. Biochem. (1985) 146:459-466. The following review article is related 
to superoxide dismutase in plants: Rabinowitch et al., Photochemistry and 
Photobiology (1983) 37:679-690. 
SUMMARY OF THE INVENTION 
Novel plants, and methods for their preparation, are provided which have 
enhanced superoxide dismutase activity. The plants are regenerated from 
cells transformed using expression cassettes comprising a DNA sequence 
encoding superoxide dismutase attached to a plant promoter. The DNA can 
additionally be attached to chloroplast leader sequences for delivery to 
and expression in the plant chloroplast. The expression cassettes are 
introduced into the plant cell host for integration into the genome so 
that enhanced activity of superoxide dismutase is achieved in the plant 
cell of interest, especially in the chloroplast.

DESCRIPTION OF THE SPECIFIC EMBODIMENTS 
In accordance with the subject invention, methods and compositions are 
provided which allow for modification of superoxide dismutase activity in 
plants. Plant cells are transformed using expression cassettes having 
transcriptional and translational regulatory sequences functional in a 
plant cell and an open reading frame encoding a heterologous or homologous 
superoxide dismutase under the transcriptional and translational control 
of the regulatory regions. The translation of an inserted superoxide 
dismutase DNA sequence will augment levels of superoxide dismutase already 
present in the cell. Thus, the presence of the superoxide dismutase 
resulting from transcription and translation of the added DNA sequence 
will increase the concentration of superoxide dismutase in the plant cell. 
The open reading frame also can include a DNA sequence encoding a transit 
peptide recognized by the plant host and post-processing N-terminal amino 
acids from a mature protein which provides for targeting of the superoxide 
dismutase into a cell organelle. 
The introduction of polypeptides into a cell organelle such as a 
chloroplast or mitochondria is achieved by joining a DNA sequence coding 
for the transit peptide in reading frame with a sequence encoding a 
structural gene for superoxide dismutase. When expressed, the fused 
transit peptide/superoxide dismutase protein will be cleaved by the cell 
organelle for the introduction of the superoxide dismutase enzyme into the 
organelle. In a first embodiment, the transit peptide and the superoxide 
dismutase protein are fused directly to one another. In a second preferred 
embodiment, the transit peptide and the superoxide dismutase protein are 
linked together via a glutamate amino acid. In a third embodiment, the 
transit peptide and the superoxide dismutase are joined together by about 
1-30 post processing amino acids of the N-terminus of a mature, ribulose 
biphosphate carboxylase (RuBPcase) ssu polypeptide. The expression 
cassette will thus include in the 5'-3' direction of transcription, a 
transcriptional and translational initiation region functional in a plant 
cell; a structural gene encoding superoxide dismutase, preferably 
including in reading frame a sequence encoding a transit peptide, wherein 
said transit peptide directs transfer of the superoxide dismutase to a 
plant cell organelle; and a transcriptional and translational termination 
regulatory region. The initiation and termination regulatory regions are 
functional in a plant cell and provide for efficient expression of the 
superoxide dismutase with desirable effects on the viability and 
proliferation of the plant host. 
The superoxide dismutase DNA sequence may be native to the plant host or 
heterologous, and may be derived from prokaryotic or eukaryotic sources. 
Prokaryotic sources are preferred. The structural gene for the superoxide 
dismutase may be obtained in a variety of ways. The gene may be 
synthesized in whole or in part, particularly where it is desirable to 
provide plant preferred codons. Thus, all or a portion of the open reading 
frame may be synthesized using codons preferred by the plant host. Plant 
preferred codons may be determined from the codons of highest frequency in 
the proteins expressed in the largest amount in the particular plant 
species of interest. 
Methods for synthesizing sequences and bringing the sequences together are 
well established in the literature. Where a portion of the open reading 
frame is synthesized, and a portion is derived from natural sources, the 
synthesized portion may serve as a bridge between two naturally occurring 
portions, or may provide a 3'-terminus or a 5'-terminus. Particularly 
where the transit peptide and the open reading frame encoding the 
superoxide dismutase are derived from different genes, synthetic adapters 
commonly will be employed. In other instances, linkers may be employed, 
where the various fragments may be inserted at different restriction sites 
or substituted for a sequence in the linker. 
For the most part, some or all of the super-oxide dismutase structural gene 
will be from a natural source and is preferentially E. coli MnSOD, since 
this enzyme is not feedback inhibited by hydrogen peroxide. Methods for 
identifying sequences of interest have found extensive exemplification in 
the literature, although in individual situations, different degrees of 
difficulty may be encountered. Various techniques include the use of 
probes where genomic or cDNA libraries may be searched for complementary 
sequences. 
When the structural gene to be inserted is derived from, for example, 
bacterial MnSOD, it may be desirable to eliminate a large portion of the 
3' non-coding region of the bacterial gene. Thus, in some instances, a 
truncated gene may be employed, where up to 0.8 kb of the bacterial 3' 
untranslated region of MnSOD may be removed. Elimination of this 
untranslated region may have a positive effect on the transcription, 
stability, and/or translation of the mRNA in the plant cells. 
The transcriptional and translational initiation regulatory regions may be 
homologous or heterologous to the plant host. Of particular interest are 
transcriptional initiation regions from genes which are present in the 
plant host or other plant species, for example, the tobacco ribulose 
biphosphate carboxylase ssu transcriptional initiation region; those 
present in viruses such as the cauliflower mosaic virus (CaMV) 35S 
transcriptional initiation region, including a "double" 35S CaMV promoter 
("MAC") described in co-pending U.S. application Ser. No. 339,755 filed 
Apr. 18, 1988; and those associated with T-DNA, such as the opine synthase 
transcriptional initiation region, e.g., octopine, mannopine, agropine, 
and the like. 
Any one of a number of regulatory sequences may be preferred in a 
particular situation, depending upon whether constitutive or induced 
transcription is desired, the particular efficiency of the promoter in 
conjunction with the heterologous superoxide dismutase, the ability to 
join a strong promoter with a control region from a different promoter 
which allows for inducible transcription, ease of construction and the 
like. These regulatory regions find ample precedence in the literature. 
Transport of the heterologous superoxide dismutase into other cellular 
compartments may be accomplished by the use of a transit peptide to target 
a desired cellular compartment such as a chloroplast. The transit peptide 
may be from the same gene as the transcriptional initiation regulatory 
region and/or the host organism or from a gene foreign to both the 
transcriptional initiation region and the host organism. 
Of particular interest is the use of a transit peptide which provides for 
transport of the superoxide dismutase to a plant cell chloroplast or 
mitochondria. Otherwise, the superoxide dismutase expressed in the 
transgenic plant will be found in the cytoplasm. Transit peptides which 
may be employed can be obtained from genes encoding chloroplast proteins 
produced in the cytoplasm, then translocated to the chloroplast. Such 
proteins include tobacco and soybean ribulose biphosphate carboxylase ssu, 
acyl carrier protein (ACP), chlorophyll A/B binding protein and other 
components of fatty acid synthesis. 
To enhance the efficiency of transport, a DNA sequence encoding the transit 
peptide can be fused in reading frame with a DNA sequence encoding a 
peptide comprising post-processing N-terminal amino acids of a 
nuclear-encoded chloroplast protein such as the mature ribulose 
biphosphate carboxylase ssu polypeptide from pea. The optimum number of 
post-processing amino acids will generally be 10 to 30, preferably 10 to 
20 amino acids. The DNA sequence is generally inserted at the border 
between the 3' end of the transit peptide sequence and the 5' end of the 
superoxide dismutase gene. It may be inserted directly if a convenient 
restriction site is available, or a synthetic restriction site can be 
created at the 3'-5' border. One or more amino acids can be added to the 
5' end of the SOD coding region. In the most preferred mode, a glutamine 
is added to the SOD. In other, less preferred embodiments, the additional 
glutamine codon may be removed by techniques known in the art, and 
replaced with a codon(s) for other amino acid(s). Methods for preparing 
chimeric genes comprising transit peptides and post-processing amino acids 
include those described in copending U.S. application Ser. No. 912,408 
filed Sep. 26, 1986, which disclosure is hereby incorporated by reference. 
The termination region may be derived from the 3'-region of the gene from 
which the initiation region was obtained or from a different gene. 
Preferably the termination region will be derived from a plant gene, 
particularly the tobacco ribulose biphosphate carboxylase ssu termination 
region; a gene associated with the Ti-plasmid such as the octopine 
synthase termination region; or the tml termination region. 
In developing the expression cassette, the various fragments comprising the 
regulatory regions and open reading frame may be subjected to different 
processing conditions, such as ligation, restriction, resection, in vitro 
mutagenesis, primer repair, use of linkers and adapters, and the like. 
Thus, nucleotide transitions, transversions, insertions, deletions, or the 
like, may be performed on the DNA which is employed in the regulatory 
regions and/or open reading frame. 
During the construction of the expression cassette, the various fragments 
of the DNA will usually be cloned in an appropriate cloning vector, which 
allows for amplification of the DNA, modification of the DNA or 
manipulation by joining or removing of the sequences, linkers, or the 
like. Normally, the vectors will be capable of replication in at least a 
relatively high copy number in E. coli. A number of vectors are readily 
available for cloning, including such vectors as pBR322, pUC series, M13 
series, etc. The cloning vector will have one or more markers which 
provide for selection of transformants. The markers will normally provide 
for resistance to cytotoxic agents such as antibiotics, heavy metals, 
toxins, or the like. By appropriate restriction of the vector and 
cassette, and as appropriate, modification of the ends, by chewing back or 
filling in overhangs, to provide for blunt ends, by addition of linkers, 
by tailing, complementary ends can be provided for ligation and joining of 
the vector to the expression cassette or component thereof. 
After each manipulation of the DNA in the development of the cassette, the 
plasmid will be cloned and isolated and, as required, the particular 
cassette component analyzed as to its sequence to ensure that the proper 
sequence has been obtained. Depending upon the nature of the manipulation, 
the desired sequence may be excised from the plasmid and introduced into a 
different vector or the plasmid may be restricted and the expression 
cassette component manipulated, as appropriate. 
The manner of transformation of E. coli with the various DNA constructs 
(plasmids and viruses) for cloning is not critical to this invention. 
Conjugation, transduction, transfection or transformation, for example, 
calcium phosphate mediated transformation, may be employed. 
In addition to the expression cassette, depending upon the manner of 
introduction of the expression cassette into the plant cell, other DNA 
sequences may be required. For example when using the Ti- or Ri-plasmid 
for transformation of plant cells, as described below, at least the right 
border and frequently both the right and left borders of the T-DNA of the 
Ti- or Ri-plasmids will be joined as flanking regions to the expression 
cassette. The use of T-DNA for transformation of plant cells has received 
extensive study and is amply described in Genetic Engineering, Principles 
and Methods (1984) Vol 6 (Eds. Setlow and Hollaender) pp. 253-278 [Plenum, 
N.Y.]; A. Hoekema, in: The Binary Plant Vector System (1985) 
Offsetdrukkerij Kanters, B. V. Alblasserdam. 
Alternatively, to enhance integration into the plant genome, terminal 
repeats of transposons may be used as borders in conjunction with a 
transposase. In this situation, expression of the transposase should be 
inducible, so that once the expression cassette is integrated into the 
genome, it should be relatively stably integrated and avoid hopping. 
The expression cassette will normally be joined to a marker for selection 
in plant cells. Conveniently, the marker may be resistance to a biocide, 
particularly an antibiotic, such as Kanamycin, G418, Bleomycin, 
Hygromycin, Chloramphenicol, or the like. The particular marker employed 
will be one which will allow for selection of transformed plant cells as 
compared to plant cells lacking the DNA which has been introduced. 
A variety of techniques are available for the introduction of DNA into a 
plant cell host. These techniques include transformation with Ti-DNA 
employing A. tumefaciens or A. rhizogenes as the transforming agent, 
protoplast fusion, injection, electroporation, DNA particle bombardment, 
and the like. For transformation with agrobacterium, plasmids can be 
prepared in E. coli which plasmids contain DNA homologous with the 
Ti-plasmid, particularly T-DNA. The plasmid may be capable of replication 
in agrobacterium, by inclusion of a broad spectrum prokaryotic replication 
system, for example RK290, if it is desired to retain the expression 
cassette on an independent plasmid rather than having it integrated into 
the Ti-plasmid. By means of a helper plasmid, the expression cassette may 
be transferred to the A. tumefaciens and the resulting transformed 
organism used for transforming plant cells. The plant cells may be any 
photosynthetic plant cells, or other cells in which elevated 
concentrations of the superoxide radical are produced. 
Conveniently, explants may be cultivated with the A. tumefaciens or A. 
rhizogenes to allow for transfer of the expression cassette to the plant 
cells, the plant cells dispersed in an appropriate selection medium. The 
agrobacterium host will contain a plasmid having the vir genes necessary 
for transfer. 
After transformation, the cell tissue (for example protoplasts, explants or 
cotyledons) is transferred to a regeneration medium, such as 
Murashige-Skoog (MS) medium for plant tissue and cell culture, for 
formation of a callus. Cells which have been transformed may be grown into 
plants in accordance with conventional ways. See, for example, McCormick 
et al., Plant Cell Reports (1986) 5:81-84. The transformed plants may then 
be analyzed to determine whether the desired gene product is still being 
produced in all or a portion of the plant cells. After expression of the 
desired product has been demonstrated in the plant, the plant can be 
grown, and either pollinated with the same transformed strain or different 
strains and the resulting hybrid having the desired phenotypic 
characteristic identified. Two or more generations may be grown to ensure 
that the subject phenotypic characteristic is stably maintained and 
inherited. 
Various techniques exist for determining whether the desired DNA sequences 
are present in the plant cell and are being transcribed. Techniques such 
as the Northern blot can be employed for detecting messenger RNA which 
codes for MnSOD. In addition, the presence of expression can be detected 
in a variety of ways. The expression of MnSOD in transformed plants may be 
detected by several means including solution enzyme assay, Western 
analysis and native electrophoresis with activity staining. Furthermore, 
antibodies specific for mature MnSOD may be employed. 
The response of the transformed plants to various stresses such as high or 
low (chilling) temperature, water deficit, high metal content of the 
rhizosphere, exposure to herbicides such as paraquat dichloride 
(1,1-dimethyl-4,4-bipyridilium dichloride), salinity or conditions 
favoring photooxidative damage or photoinhibition can be measured in a 
variety of ways. The stress one of the above factors alone, or in 
combination with at least one other factor; or with high light intensity. 
The transgenic plants can be evaluated directly, for example, transgenic 
plants can be evaluated for tolerance/resistance particularly to chemical 
stresses such as herbicides and metals, by the ability of the plant to 
grow in the presence of higher concentrations of the toxic compound as 
compared to non-transgenic plants, or plants transformed with other than 
an expression cassette providing for increased SOD activity. 
Exposure of plants to stress conditions (see relevant review from S. Powles 
(1984) Annual Review of Plant Physiology, 35; 15-44) results in inhibition 
of photosynthesis. Thus the effects of oxygen toxicity and photoinhibition 
are strongly directed towards photosynthesis. Under normal, physiological 
conditions, photosynthesis is defined as the vectorial photosynthetic 
electron transport (P.E.T.) and the subsequent CO.sub.2 reduction. The 
relatively high susceptibility of photosystem II is frequently related to 
a loss of chlorophyll variable fluorescence. Since P.E.T. is affected by 
various stresses it can be used for "in situ" evaluation of the stress 
resistance of the transgenic plants. 
Plants engineered to produce increased levels of SOD activity in response 
to environmental stresses find use in being able to grow under conditions 
which inhibit growth of the parental strain, in particular under 
conditions which increase plant superoxide (O.sub.2.sup.-) to growth 
inhibitory levels. Examples of such conditions include increased salinity, 
drought and elevated metal concentration of the rhizosphere. These plants 
may also find use where it is desirable to use herbicides such as the 
salts of bipyridylium quaternary ammonium compounds, for example paraquat, 
for weed control. 
Evaluation of the amount of SOD activity in the transgenic plants may also 
be used as an indicator of nutritional deficiency of the soil, an increase 
reflecting a stress-inducing deficiency. Thus, the plants themselves may 
find use as indicator crops for presence of undesirable components in the 
soil or absence of desirable nutrients where the desirable or undesirable 
components affect SOD activity. The relatively large response to 
environmental stress due to increased copy number of the SOD gene and the 
use of an inducible enzyme (e.g. MnSOD), provide for a substantially more 
reliable indication than can be obtained using standard plants. 
The following examples are offered by way of illustration and not by way of 
limitation. 
EXPERIMENTAL 
Standard laboratory techniques of restriction, ligation, transformation, 
and analysis (Maniatis et al., (1982) Molecular Cloning: A Laboratory 
Manual, Cold Spring Harbor Laboratory, New York) were used. 
Single-stranded DNA template was prepared and DNA sequence was determined 
using the Sanger dideoxy technique (Sanger et al., Proc. Nat'l. Acad. Sci. 
USA (1977) 74: 5463-5467). Double-stranded DNA was sequenced using the 
technique of Maxam and Gilbert (1980) Methods Enzymol. 65:499-560). 
Sequence analysis was performed using a software package from 
IntelliGenetics, Inc. 
Cloning vectors used include pUC vectors, pUC8 and pUC9 (Vieira and 
Messing, Gene (1982) 19:259-268), pUC18 and pUC19 (Norrander et al., Gene 
(1983) 26:101-106; Yanisch-Perron et al., Gene (1985) 33:103-119), and 
analogous vectors exchanging chloramphenicol resistance (CAM) as a marker 
for the ampicillin resistance of the pUC plasmids described above (pUC-CAM 
[pUC12-Cm, pUC13-Cm] Buckley, K., Ph.D. Thesis, U.C.S.D., CA 1985). The 
multiple cloning sites of pUC18 and pUC19 vectors were exchanged with 
those of pUC-CAM to create pCGN565 and pCGN566 which are CAM resistant. 
Phage vectors used included M13mp8 and M13mp9 (Messing and Vieira, Gene 
(1982) 19, 269-276 and M13mp18 and M13mp19 (Norrander et al., supra; 
Yanish-Perron et al., supra). 
EXAMPLE 1 
Superoxide Dismutase Purification and Antibody Production 
Manganese superoxide dismutase (MnSOD) was purified from E. coli B which 
had been grown under conditions designed to induce production of the 
enzyme (100 .mu.M FeSO.sub.4.7H2O, 100 .mu.M MnSO.sub.4, 100 .mu.M 
8-hydroxyquinoline, 10 .mu.M methylviologen). The purification scheme was 
based on that of Keele et al., J. Biol. Chem. (1970) 245:6176-6181. Enzyme 
activity was monitored based on the assay described by Misra et al., Arch. 
Biochem. Biophys. (1977) 181:308-312. The purified enzyme was lyophilized 
and used for antibody production. Since there may be cross-reactivity 
between the antibody prepared against the bacterial MnSOD and that present 
in chloroplasts, the plant and bacterial MnSOD for Western blotting are 
separated on an isoelectric focusing gel or using a normal SDS-gel system. 
Briefly, intact chloroplasts were isolated from tobacco leaves using an 
isolation protocol as described by Jensen et al., Proc. Nat'l. Acad. Sci. 
USA (1966) 56:1095-1101. The chloroplasts were osmotically lysed by 
resuspending in extraction buffer containing 100 mM sodium citrate, pH 
5.5, 10 mM EDTA, 150 mM NaCl, 0.05% (v/v) Nonident P-40, 25 mg/ml BSA, 1 
mM DTT, 10 mM thiourea, 10 .mu.M leupeptin. The samples were then 
subjected to immunoprecipitation of cross-reacting proteins using the 
antiserum to the purified bacterial SOD as follows. To 1 ml of the 
supernatant liquid was added 25 .mu.l of antiserum and 125 .mu.l S. aureus 
(freeze dried cells). The samples were incubated at room temperature for 
45 minutes, centrifuged at 3000.times.g for 5 minutes and the pellet 
washed twice with 20 mM Tris pH 7.5, 1 mM EDTA, 50 mM NaCl and 0.05% (v/v) 
Nonident P-40. The precipitated protein was resuspended and denatured in 
electrophoresis sample buffer, (Anderson and Anderson, Proc. Nat'l. Acad. 
Sci. USA (1977) 74:5421-5425) containing 2% (w/v) SDS, 2% (v/v) Triton 
X-100 , 5% (v/v) mercaptoethanol, 20% (v/v) glycerol and 2% (w/v) LKB 
Ampholines (pH 3.5-10). To one sample was added 0.5 .mu.g of purified 
MnSOD from E. coli. In the isoelectric focusing gel method, samples were 
then subjected to isoelectric focusing in 9 M urea at 500 v in a 1.5 mm 
thick 4.25% polyacrylamide gel (5.7% cross-linked, pH 3.5-10) at 0.degree. 
C. for 18 hours, otherwise the samples were separated using typical 
SDS-PAGE system for Westerns as discussed in Example 8. 
The resolved proteins were transferred to nitrocellulose (BA 85 Schleicher 
and Schuell) as described by Burnette (Anal. Biochem. (1981) 112:195-203) 
at 100 v for three hours in a Hoefer TE42 transfer unit. The 
nitrocellulose filter was then incubated in Blotto for 1 hour at room 
temperature followed by an overnight incubation at 4.degree. C. in 50:1 
Blotto:antiserum. The filter was washed for 10 minutes in 20 mM Tris, pH 
7.5, 150 mM NaCl, for 20 minutes in the same buffer containing 0.05% 
Tween-20 and for another 10 minutes in buffer without detergent. Blotto 
containing 10.sup.6 cpm/ml of .sup.125 I-labeled protein A (9 .mu.Ci/mg; 
NEN) was then added to the filter and incubated at room temperature for 
two hours. The filter was then washed overnight in 50 mM Tris, pH 7.5, 1M 
NaCl and 0.4% (w/v) Sarkosyl. After rinsing and drying, filters were 
exposed for 24 hours to Kodak XAR x-ray film at -70.degree. C. using a 
DuPont Cronex intensifying screen. 
EXAMPLE 2 
Subcloning and Sequencing of the MnSOD Gene 
A MnSOD gene (sodA) from E. coli K12 was obtained from Danielle Touati on 
plasmid pDT1-5 (Touati, J. Bacteriol. (1983) 155: 1078-1087). The plasmid 
contained a 4.8 kb EcoRI, BamHI fragment of E. coli genomic DNA. The SOD 
gene nucleotide sequence has been published (Takeda and Avila, Nucl. Acids 
Res. (1986) 14:4577-4589). A nucleotide sequence independently derived at 
Calgene from pDT1-5 is identical to that reported except for a C residue 
in place of the published T at the third nucleotide of codon #58. This 
creates an additional BClI restriction site. A 3.5 kb HincII/BamHI 
fragment of pDT1-5 was subcloned to M13mp8 cut with BamHI and SmaI to 
create pCGN1300a. 
EXAMPLE 3 
Creation of an SphI Site for Insertion of a Chloroplast Leader Sequence 
Construction of pCGN1303 
Single-stranded DNA from clone pCGN1300a was subjected to in vitro 
mutagenesis (Adelman et al., DNA (1983) 2:183-193) to insert a SphI site 
at the initiation codon of the MnSOD gene. The synthesized 35 base 
oligonucleotide 5'-GCAGGGTATAGCTCTGCATGCATTGTCGGGCGCCA-3'. After in vitro 
mutagenesis, one clone pCGN1303 was selected and the sequence of the 
mutated region was confirmed by both Maxam and Gilbert sequencing from the 
NcoI site 68 nucleotides from the SOD start codon and by subcloning the 
SphI-BamHI fragment of pCGN1303 into M13 and subsequent Sanger dideoxy 
sequencing. The in vitro mutagenesis to add the SphI site to the 5'-end of 
SOD added one amino acid (glutamine) to the coding region. 
Expression of Altered MnSOD in an SOD.sup.- Mutant 
To investigate the effect of the extra amino acid on SOD activity, the 
EcoRI-HindIII fragment of pCGN1303 containing the SOD gene and its own 
promoter was transferred from its M13mp8 background to pUC19 that had been 
digested with EcoRI and HindIII to give pCGN1309. pCGN1309 was then used 
to transform an SOD.sup.- E. coli strain similar to the double mutant 
(MnSOD.sup.- and FeSOD.sup.-) described by Carlioz and Touati, EMBO J. 
(1986) 5:623-630. SOD activity was detected in the pCGN1309-transformed 
SOD.sup.- cells by electrophoresis of extracts prepared as described by 
Touati, J. Bacteriol. (1983) 155:1078-1087 on non-denaturing 
polyacrylamide gels and staining for activity (Beauchamp and Fridovich, 
Anal. Biochem. (1971) 44:276-287). No detectable activity was seen in 
SOD.sup.- cells alone or when transformed with pUC19. SOD.sup.- cells 
transformed with pCGN1309 showed approximately the same amount of 
activity, 1 unit/30 .mu.g of total protein, as E. coli 7118 cells (Messing 
et al., Proc. Nat'l. Acad. Sci. USA (1977) 74:3642-3646). 
EXAMPLE 4 
MnSOD Expression Constructs for Chloroplast Targeting 
Both plant gene expression signals (promoter and 3'-sequences) and a leader 
sequence must be added to the bacterial MnSOD gene to have the gene 
product expressed and transported to plant chloroplasts. A binary vector, 
named pCGN1332, containing a tobacco ssu ribulose bisphosphate carboxylase 
(RuBPcase) promoter and leader sequences, the MnSOD gene and tobacco ssu 
3'-regulatory sequences with a plant selectable kanamycin resistance 
marker, was constructed as follows: A 3.4 kb EcoRI fragment of lambda 
clone TSSU3-8 (O'Neal et al., Nucl. Acids Res. (1987) 15:8661-8677 
containing a ssu RuBPCase gene from tobacco was subcloned into M13mp18 
digested with EcoRI to give plasmid 2018. The promoter and leader of the 
tobacco ssu sequences were removed from plasmid 2018 by digestion with 
HindIII and SphI and ligated to HindIII/SphI digested pPMG72 (described in 
copending U.S. application Ser. No. 097,498, filed Sep. 16, 1987, which 
application is hereby incorporated by reference) to make pCGN650. pPMG72 
contains the leader sequences of the soybean carboxylase attached to a 
modified aroA gene. In pCGN650 the soybean ssu leader is replaced by the 
tobacco ssu promoter and leader. 
The aroA gene was replaced in pCGN650 by SphI-BamHI digestion and ligation 
with SphI-BamHI digested pCGN1303. The resulting plasmid, pCGN1304 now 
contains the tobacco ssu TSSU3-8 promoter and leader sequence and MnSOD 
coding region. A polyadenylation signal was added to EcoRI digested 
pCGN1304 by isolation and ligation of an EcoRI partial digest fragment 
from pCGN632. pCGN632 contains a 1.6 kb HaeIII fragment of TSSU3-2 (O'Neal 
et al, (1987) supra) adapted using Sall linkers into SalI digested pUC18. 
The entire HaeIII fragment includes 123 nucleotides of exon 2, intron 2, 
exon 3, the polyadenylation site and 1 kb#3' to the polyadenylation site 
of a tobacco ssu gene. The expression construct containing the tobacco ssu 
promoter and leader, the mutant MnSOD gene and tobacco ssu 3'-sequences is 
designated pCGN1305. 
The MnSOD gene contained in the initial construct, pCGN1303, and the 
expression construct, pCGN1305, contain the entire MnSOD gene from the 
initial ATG codon (plus one extra amino acid) through the stop codon of 
the gene and 0.8 kb of bacterial 3' untranslated region. To eliminate a 
large part of the 3'-noncoding bacterial sequences in vitro mutagenesis 
was performed on pCGN1303 to insert a linker containing Xbal, BamHI and 
EcoRI restriction sites 18 nucleotides downstream of the stop codon. The 
42 base oligonucleotide 5' ATACGCCTCATGAATTCGGATCCTCTAGAGCAGCAGGCGGC-3' 
was used to perform the mutagenesis and the resulting clone is pCGN1329. 
The long MnSOD sequence contained in the expression construct pCGN1305 was 
substituted with the shorter MnSOD sequence from the SphI site (at the 
ATG) to the new BamHI site downstream of the stop codon by SphI, BamHI 
digestion of pCGN1329 and ligation to pCGN1305 digested with SphI and 
BamHI to create pCGN1331. pCGN1331 was digested with HindIII to linearize 
the plasmid at the unique HindIII site at the 5' end of the tobacco ssu 
promoter and the entire plasmid was ligated to the HindIII digested binary 
vector pCGN783 to create the construct pCGN1332. (For the construction of 
pCGN783 see copending U.S. application Ser. No. 078,538, filed Jul. 28, 
1987, which application is incorporated herein by reference). The binary 
vector includes the T-DNA borders (left border, LB and right border, RB) 
which delineate the DNA to be transferred into the plant, a bacterial 
marker, (gentamicin resistance), and a plant selectable marker that is 
co-transferred with the gene of interest, consisting of a kanamycin 
resistance gene expressed from the constitutive cauliflower mosaic virus 
35S promoter and 3'-polyadenylation signal from transcript 7 of 
agrobacterium T-DNA. pCGN1331 can be cloned in either of two orientations 
at the HindIII site of pCGN783. pCGN1332 is in the orientation with the 
MnSOD gene and the kanamycin resistance gene being transcribed in opposite 
directions. 
A second MnSOD expression construct in a binary vector was constructed. In 
this construct, pCGN1328, the MnSOD gene is expressed from the 
constitutive CaMV35s promoter and utilizes the soybean ssu leader for 
chloroplast targeting. pCGN1328 was constructed as follows: The 
mutagenized MnSOD gene in construct pCGN1303 was combined with the soybean 
ssu leader sequence by digestion with SphI and BamHI and ligation to 
pCGN350 digested with SphI and BamHI to make pCGN1306. Plasmid pCGN350 
contains the leader sequence of the ssu of ribulose bisphosphate 
carboxylase from soybean and was constructed as follows: Plasmid pPMG70 
(described in copending U.S. application Ser. No. 097,498, filed Sep. 16, 
1987) was restricted with DdeI, blunted with Klenow fragment of DNA 
polymerase, digested with BamHI, and the ssu promoter fragment isolated. 
The fragment was cloned into pUC8 cut with SmaI-BamHI. 
The soybean leader/MnSOD gene from pCGN1306 was cloned into the expression 
cassette pCGN986 (described below) by digestion with EcoRI, filling in the 
ends with DNA polymerase I and restricting with BamHI. The fragment was 
ligated to pCGN986 which had been digested with XbaI, filled in with DNA 
polymerase I and BamHI digested. Clones were isolated that contained both 
the regenerated XbaI and EcoRI sites from the ligation (pCGN1308a) and the 
clones which contained neither site (pCGN1308b). 
pCGN986 contains a cauliflower mosaic virus 35S (CaMV35) promoter and a 
T-DNA tml 3'-region with multiple restriction sites between them. pCGN986 
was derived from another cassette, pCGN206, containing a CaMV35S promoter 
and a different 3'-region, the CaMV region VI 3'-end. The CaMV 35S 
promoter was cloned as an AluI fragment (bp 7144-7734) (Gardner et al., 
Nucl. Acids Res. (1981) 9:2871-2888) into the HincII site of M13mp7 
(Messing et al., Nucl. Acids Res. (1981) 9:309-321) to create C614. An 
EcoRI digest of C614 produced the EcoRI fragment from C614 containing the 
35S promoter which was cloned into the EcoRI site of pUC8 (Vieira and 
Messing, Gene (1982) 19:259) to produce pCGN147. 
pCGN148a containing a promoter region, selectable marker (KAN with 2 ATG's) 
and 3'-region, was prepared by digesting pCGN528 with BglII and inserting 
the BamHI-BglII promoter fragment from pCGN147. This fragment was cloned 
into the BglII site of pCGN528 so that the BglII site was proximal to the 
kanamycin gene of pCGN528. 
The shuttle vector used for this construct pCGN528, was made as follows: 
pCGN525 was made by digesting a plasmid containing Tn5 which harbors a 
kanamycin gene (Jorgenson et al., Mol. Gen. Genet. (1979) 177:65) with 
HindIII-BamHI and inserting the HindIII-BamHI fragment containing the 
kanamycin gene into the HindIII-BamHI sites in the tetracycline gene of 
pACYC184 (Chang and Cohen, J. Bacteriol. (1978) 134: 1141-1156). pCGN526 
was made by inserting the BamHI fragment 19 of pTiA6 (Thomashow et al., 
Cell (1980) 19:729-739), modified with XhoI linkers inserted into the SmaI 
site, into the BamHI site of pCGN525. pCGN528 was obtained by deleting the 
small XhoI fragment from pCGN526 by digesting with XhoI and religating. 
pCGN149a was made by cloning the BamHI-kanamycin gene fragment from 
pMB9KanXXI into the BamHI site of pCGN148a. pMB9KanXXI is a pUC4K variant 
(Vieira and Messing, Gene (1982) 19:259-268) which has the XhoI site 
missing, but contains a functional kanamycin gene from Tn903 to allow for 
efficient selection in agrobacterium. 
pCGN149a was digested with HindIII and BamHI and ligated to pUC8 digested 
with HindIII and BamHI to produce pCGN169. This removed the Tn903 
kanamycin marker. pCGN565 and pCGN169 were both digested with HindIII and 
Pstl and ligated to form pCGN203, a plasmid containing the CaMV 35S 
promoter and part of the 5'-end of the TN5 kanamycin gene (up to the Pstl 
site, Jorgenson et al., (1979), supra). A 3'-regulatory region was added 
to pCGN203 from pCGN204 (an EcoRI fragment of CaMV (bp 408-6105) 
containing the region VI 3' cloned into pUC18 (Gardner et al., (1981), 
supra) by digestion with HindIII and Pstl and ligation. The resulting 
cassette, pCGN206, was the basis for the construction of pCGN986. 
The pTiA6 T-DNA tml 3'-sequences were subcloned from the Bam19 T-DNA 
fragment (Thomashow et al., (1980), supra) as a BamHI-EcoRI fragment 
(nucleotides 9062 to 12,823, numbering as in Barker et al., Plant Mol. 
Biol. (1982) 2:335-350) and combined with the pACYC184 (Chang and Cohen 
(1978), supra) origin of replication as an EcoRI-HindIII fragment and a 
gentamycin resistance marker (from plasmid pLB41), obtained from D. 
Figurski) as a BamHI-HindIII fragment to produce pCGN417. 
The unique SmaI site of pCGN417 (nucleotide 11,207 of the Bam19 fragment) 
was changed to a SacI site using linkers and the BamHI-SacI fragment was 
subcloned into pCGN565 to give pCGN971. The BamHI site of pCGN971 was 
changed to an EcoRI site using linkers. The resulting EcoRI-SacI fragment 
containing the tml 3'regulatory sequences was joined to pCGN206 by 
digestion with EcoRI and SacI to give pCGN975. The small part of the Tn5 
kanamycin resistance gene was deleted from the 3'-end of the CaMV 35S 
promoter by digestion with SalI and BglII, blunting the ends and ligation 
with SalI linkers. The final expression cassette pCGN986 contains the CaMV 
35S promoter followed by two SalI sites, an XbaI site, BamHI, SmaI Kpnl 
and the tml 3'-region (nucleotides 11207-9023 of the T-DNA). 
The CaMV 35s expression construct with the soybean leader/MnSOD gene, 
pCGN1308b, was transferred into a chloramphenicol resistant (Cam.sup.r) 
background by insertion into the chloramphenicol resistant vector, 
pCGN566, at the EcoRI and HindIII sites to create pCGN1312. 
Similar to pCGN1305, the tobacco ssu/MnSOD expression construct, pCGN1312, 
contains the entire MnSOD gene and a long bacterial 3'-untranslated 
region. To remove much of the 3'-untranslated region, pCGN1312 was 
partially digested with BclI, completely digested with BamHI and 
religated. 
The partial digestion was necessary because pCGN1312 contains 3 BclI sites, 
a BclI site in the coding region of the MnSOD clone, one in the 
3'-untranslated region 95bp from the stop codon and one in the 3'-tml 
polyA fragment. A clone in which only the 3'-noncoding bacterial MnSOD 
BclI/BamHI fragment was identified by size and restriction pattern and 
named pCGN1322. pCGN1322 contains the entire MnSOD gene and only 95 
nucleotides of 3'-noncoding sequence. pCGN1322 was cloned into the binary 
vector pCGN783 by digestion at the unique HindIII site and ligation to 
HindIII digested pCGN783. pCGN1328, the final binary vector, contains the 
MnSOD gene being transcribed in the same direction as the kanamycin 
resistant selectable marker. 
The two binary vectors pCGN1328 and pCGN1332 were introduced into 
Agrobacterium tumefaciens strains LBA4404 (Ooms et al., Plasmid (1982) 
7:15-29) and K61 (described below) respectively by transformation. A 10 ml 
culture of agrobacterium was grown overnight in MG/L broth (Garfinkel and 
Nester, J. Bacteriol. (1980) 144:732-743) at 30.degree. C. and then 
diluted to 100 ml with MG/L broth and grown in a shaking incubator for 5 
hours at 30.degree. C. Cells were pelleted by centrifugation at 4,000 rpm 
for 10 minutes and the pellet resuspended in 1 ml MG/L and either placed 
on ice or frozen (-70.degree. C.) in 200 .mu.l aliquots. 
Approximately l.mu.g of plasmid DNA (pCGN1328 or pCGN1332) in water or MG/L 
broth (about 100 .mu.l) and 200 .mu.l of competent agrobacterium cells 
were placed in a tube and immediately frozen in a dry ice-ethanol bath for 
5 minutes. The tube was thawed in a 37.degree. C. water bath for 5 minutes 
and 2 ml of MG/L broth was added to the cells. The culture was placed in a 
30.degree. C. shaking incubator for 3 hours. The cells were plated on MG/L 
containing 100 mg/l gentamicin. Plasmid DNA was isolated from individual 
gentamicin resistant colonies, transformed into E. coli and characterized 
by restriction enzyme analysis to verify that the gentamicin resistant 
EHA101 contained intact copies of pCGN1328 or pCGN1332. 
EXAMPLE 5 
MnSOD Construct For Cytoplasmic Targeting 
The construction of a plasmid to express MnSOD in the cytoplasm was as 
follows: The MnSOD gene found in pCGN1329 (described above) was inserted 
into an ampicillin resistant background by digestion with HindIII and SphI 
and ligation to pUC18 digested with HindIII and SphI. The resulting 
plasmid, pCGN1363, was then digested with SalI and BamHI and ligated to 
pCGN1308a (described above) digested with SalI and BamHI to give pCGN1366. 
This replaced the soybean leader/MnSOD gene found in pCGN1308a with the 
shortened version of the MnSOD gene (with only 18 nucleotides of 
3'-noncoding bacterial sequence). The MnSOD gene in pCGN1366 is flanked by 
the CaMV 35S promoter and the polyadenylation sequences of the tml gene of 
the T-DNA to make an expression cassette. The CaMV 35S promoter/MnSOD/tml 
3'-sequences were moved to a chloramphenicol resistant background by 
digestion of pCGN1366 with HindIII and EcoRI and ligation to pCGN566 
digest with EcoRI and HindIII to give pCGN1367. pCGN1367 was cloned into 
the binary vector pCGN783 at the unique HindIII site by digestion with 
HindIII and ligation to HindIII-digested pCGN783. The resulting binary 
vector, pCGN1371, contains the SOD transcription unit in the same 
orientation as the kanamycin transcription unit. pCGN1371 was transformed 
into A. tumefaciens strain K61 (as described below) to be cocultivated 
with tobacco leaf discs. 
EXAMPLE 6 
Construction of Disarmed Agrobacterium Strain K61 
Construction of pCGN567 
The left and right border regions of Ti plasmid pTiA6, Garfinkel, J. Bact. 
(1980) 144:732-743, were ligated together into pUC8. The resulting 
plasmid, pCGN503, has the left border region from bases 625-2212 and the 
right border from bases 13367-15298 (using the numbering system of Barker, 
et al.) fused in the proper orientation and flanked by HindIII sites. The 
HindIII fragment of pCGN503 was transferred to the broad host range cosmid 
pVCK102 Knauf, Plasmid (1982) 8:40-54, inactivating the kanamycin 
resistance locus of that plasmid and creating pCGN506. pCGN567 was made by 
ligating the kanamycin resistance locus from pUC4K Messing, Gene (1982) 
19:269-272, into the unique BamHI site between the two border regions. 
Final Construction of K61 
pCGN567 was introduced into agrobacterium strain A722 (which contains 
pTiA6, Garfinkel, J. Bact. (1980) 144:732-743, by transformation, Holsters 
et al., Mol. Gen. Genet. (1978) and selection for kanamycin resistance. 
Plasmid pPH1J1, Hirsch, Plasmid (1984) 12:139-141 was introduced by 
conjugation, Ditta et al., Proc. Nat'l. Acad. Sci. (1980) 77:7347-7351, 
and agrobacteria were selected on gentamicin and kanamycin in minimal 
medium. Since pCGN567 and pPH1J1 are incompatible plasmids, selection for 
both markers results in very slow-growing colonies as the two plasmids 
tend to segregate to separate daughter cells. A double recombination 
event, which occurs at a low frequency between the homologous border 
regions of pCGN567 and pTiA6, results in the kanamycin resistance locus 
replacing the oncogenes of pTiA6. Since pTiA6 and pPH1J1 are compatible 
replicons, the pCGN567 recombinant containing pPH1J1 and the disarmed Ti 
plasmid grow relatively quickly under kanamycin and gentamicin selection. 
The construction of K61 is shown in FIG. 1. 
Strain K61 may have been the result of a three step process as outlined in 
FIG. 1. Since the kanamycin resistance gene of pUC4K is homologous to the 
inactivated kanamycin resistance locus of PVCK102, an internal 
recombination in pCGN567 (D) would delete the right border region (E). 
Upon introduction of pPH1J1, the deleted plasmid could recombine with the 
left border region of pTiA6 (F) thus stabilizing the kanamycin resistance 
locus although the presence of the pCGN567 replicon in the recombinant 
pTiA6 would still destabilize pPH1J1 replication, slowing growth until a 
spontaneous deletion removed the pCGN567 replicon (G). In the case of 
strain K61, the spontaneous deletion apparently removed not only the 
incompatibility-coding portions of the cointegrate but also the oncogenes 
and adjacent regions. A deposit of strain K61 was made at the American 
Type Culture Collection on May 20, 1987 and assigned accession number ATCC 
53621. 
EXAMPLE 7 
Transformation of Tobacco Plants 
Transformation of tobacco plants was performed using agrobacterium 
containing constructs pCCN1328 and pCGN1332 as described in Comai et al., 
Nature (1985) 317:741-744, which publication is incorporated herein by 
reference. 
EXAMPLE 8 
Analysis of Transgenic Tobacco Plants for MnSOD Activity In the Chloroplast 
When total leaf extracts were analyzed by Western Blot, plants transformed 
from either construct pCGN1328 or pCGN1332 showed a band corresponding to 
the size of properly processed MnSOD. Enzymes for processing the ssu 
transit peptide are found only in the chloroplast, as the presence of a 
properly processed MnSOD protein indicates that plants from construct 
pCGN1332 or 1328 have the enzyme in their chloroplast. Additional work was 
done, as discussed below, which further verified this finding. 
Protocol for Isolation of Protoplasts 
Leaves from plants transformed with pCGN1328, and leaves from untransformed 
plants for control, about two centimeters long and one centimeter across, 
were selected from 3-4 week old in vitro grown tobacco plants (var. 
xanthi). The midrib was dissected out and discarded. The remaining tissue 
was then placed in an enzyme solution (0.04% y23 pectolyase; 0.42% 
cellulase RS; 0.5% potassium dextran sulphate; 6% sorbitol; pH 5.5), 
approximately 15-20 leaves per 30 ml of enzyme solution. The tissue was 
infiltrated with the enzyme solution using vacuum to 300 millitorr. This 
was done by placing leaves and enzyme solution in a beaker, placing a 
screen on top of the leaves to prevent them from bubbling up while under 
vacuum, then placing the beaker in a desiccator and applying vacuum. The 
vacuum was then slowly released to atmospheric pressure. 
The tissue with the enzyme solution was then transferred to petri dishes 
and placed in an orbital shaker at 50 rpm for 2 to 4 hours. Under 
magnification, the appearance of cellular rounding within the tissue and 
the releasing of protoplasts from the tissue was an indication that 
digestion was nearing completion. Using a 10 ml Japanese pipette, the 
tissue was slowly pipetted and agitated to further aid digestion. Tissue 
debris and undigested cells were then removed by passage through a 52 
micron filter. The enzyme solution was removed by centrifugation at 
150.times.g. The supernatant was discarded and the protoplast pellet 
rinsed with wash solution (0.5% potassium dextran sulfate; 6% sorbitol; pH 
5.5) then centrifuged and the supernatant discarded. The washing step was 
repeated twice. The protoplasts were then counted using a hemocytometer. 
Test indicated that a minimum of 19 ml protoplast were needed to proceed 
with the analysis. 
Chloroplast Isolation from Protoplasts 
The protoplast suspension was passed through a 30 micron mesh screen 
attached to a 5 cc syringe until the protoplast were lysed. The suspension 
was then loaded onto a Percoll gradient, 15 ml 40% PBF-Percoll (3% 
polyethyleneglycol (PEG) 4000; 1% bovine serum albumin (BSA); 1% ficoll; 
in Percoll) over 6 ml 80% PBF-Percoll in a 30 ml Corex tube. The gradient 
was centrifuged in a swinging bucket rotor at 6000.times. g for 15 
minutes. Chloroplast form into bands, one of broken chloroplasts near the 
top of the gradient, and a second of intact chloroplasts lower in the 
gradient. The broken band was removed and discarded. The intact band was 
transferred to a 50 ml culture tube. The intact chloroplasts were 
resuspended in 5-10 volumes of GR buffer (50 mM HEPES-KOH, pH 7.5; 0.33M 
sorbitol; 2.0 mM EDTA; 1.0 mM MgCl.sub.2 ; 1.0 mM MnCl.sub.2 ; 5.0 mM 
sodium ascorbate. The resuspended chloroplasts were then centrifuged at 
1800 rpm in an IEC centrifuge for 80 seconds, starting timing at 1600 rpm. 
The supernatant was removed and the chloroplasts resuspended in 5-10 
volumes of GR buffer. The centrifugation was then repeated and the 
chloroplasts resuspended in 2 ml sorbitol-HEPES (0.33M sorbitol; 50 mM 
HEPES-KOH, pH 7.5). 
One half of each chloroplast preparation (1 ml) was subjected to digestion 
with protease (Trypsin, TPCK treated at 25 .mu.g/ml for 45 minutes at 
4.degree. C.) to destroy proteins exterior to the chloroplast membrane. As 
a control, chloroplasts prepared from untransformed N. Tabacum `xanthi` 
were "spiked" with purified MnSOD prior to protease treatment to verify 
the effectiveness of the treatment. The protease was diluted by addition 
of 30 volumes of sorbitol-HEPES. The digested chloroplasts were then 
washed twice with sorbitol-HEPES buffer. 
Analysis of Chloroplasts for MnSOD by Western Blot 
The chloroplasts were resuspended in 1 ml Western Extraction Buffer (WEB 
(100 mM sodium citrate, pH#5.6; 10 mM EDTA; 150 mM NaCl; 25 mg/ml (BSA); 
0.05% Tween 20(polyoxyethylene sorbitan monolaurate; 2 mM phenylmethyl 
sulfonylfluoride (PMSF) dissolved in 100 ml ethanol before addition; 10 mM 
leupeptin; 10 mM; 10 mM dithiothreitol (DTT); 10 mM thiourea)) and 
homogenized using a Polytron, two bursts for five seconds at moderate 
speed to disrupt the chloroplasts. The samples were immunoprecipitated 
with 100 .mu.l anti-MnSOD polyclonal antibody (1 mg/ml) and 250 .mu.l 
Staphylococcus A cells overnight at 4.degree. with shaking. The samples 
were then washed three times with TBST (10 mM Tris, pH 8.0; 150 mM NaCl; 
0.05% Tween 20) and resuspended with 30 .mu.l SDS-PAGE sample buffer 
(0.125M, Tris pH 6.8; 10% SDS; 20% Glycerol; 10% 2-.beta.-mercaptoethanol 
(BME); Bromphenol Blue for color), boiled for three minutes and stored at 
4.degree. C. for 1-6 hours. 
The samples were then loaded onto an 11% SDS-polyacrylamide gel with 5% 
stacking gel and electrophoresed at 50 volts (constant voltage) overnight 
(approximately 18 hours). The gel was electroblotted for three hours at 
100 volts in Electroblot Buffer (For 6 Liters, 18.2 g Tris base; 86.5 g 
Glycine; 1200 mls methanol; to 6 L with distilled water). After blotting, 
the nitrocellulose was washed in distilled water and incubated in Blotto 
(20 mM Tris, pH 7.5; 5% dehydrated skim milk; 0.5M NaCl; 0.1% antifoam A; 
10 mM sodium azide) for one hour at room temperature with gentle shaking. 
The nitrocellulose was then incubated in antibody/Blotto (anti-MnSOD 
polyclonal serum in Blotto 1:50, v/v) overnight at 4.degree. C. with 
gentle shaking. The nitrocellulose was then washed for twenty minutes in 
TS buffer (20 mM Tris, pH 7.5; 150 mM NaCl), again for 20 minutes in T5 
buffer plus 0.05% between 20, then again for 20 minutes in TS buffer. The 
nitrocellulose was then incubated for 2 hours at room temperature in 
Blotto +.sup.125 I-protein A (NEN Research Products, 0.5 .mu.Ci/ml Blotto) 
100 ml, with gentle shaking. The nitrocellulose was then rinsed in 
approximately 100 ml of distilled water briefly, then incubated for four 
hours in Wash Buffer #1 (50 mM Tris, pH 7.5; 1M NaCl; 0.4% lauryl 
sarcosine) at room temperature with gentle shaking. The nitrocellulose was 
rinsed quickly in distilled water then dried under a heat lamp. 
Autoradiography was then performed for an appropriate time period. This 
analysis demonstrated the presence of properly processed E. coli MnSOD in 
the chloroplasts of transgenic plants transformed using construct 1328. 
EXAMPLE 9 
Analysis of SOD Activity in Transgenic Plants 
Total leaf extracts from transgenic plants obtained from pCGN1328 were 
analyzed for SOD activity in a system of native gel electrophoresis 
followed by an SOD activity stain assay, capable of resolving total SOD 
activity into multiple component bands. MnSOD bands were then detected by 
binding to anti-MnSOD polyclonal antibody. 
The activity stain assays were done as follows: Leaf samples were harvested 
and quick frozen using liquid nitrogen, then stored at -70.degree. C. 
until use. 0.5 g of the frozen leaf tissue was then ground to a fine 
powder in a mortar and pestle pre-cooled with liquid nitrogen. 
Polyvinylpyrollidone (Polyclar AT) was then added to the ground samples 
(0.15 g/0.5g leaf tissue). Sodium citrate, 0.1M (pH 5.6) was then mixed in 
(750 .mu.l/0.5 g leaf tissue). The samples were thawed and the tissue 
mixture transferred immediately to Corex centrifuge tubes and incubated on 
ice for 10 minutes. The samples were then centrifuged at 20,000.times.g 
for 15 minutes at 4.degree. C. 120 .mu.l of the supernatant was then 
removed from each sample and 20 .mu.l of sucrose dye were added (25 g 
sucrose; 5 ml 1% Bromphenol Blue brought to 50 ml with distilled water; 
prepared fresh) and the samples electrophoresed on a native gel. 
The solutions for the SOD activity stain for the native gel are as follows. 
Solution A: 30% acrylamide (acrylamide 58.4 g; bis acrylamide 1.6 g; 
brought to 200 ml); Solution B: 8.times. Laemmli resolving gel buffer 
(LRB) 3M Tris-HCl, pH 8.8; Solution C: 4.times. Laemmli stacking gel 
buffer (LSB), 0.5M Tris-HCl, pH 6.8); Solution D: 10.times. chamber buffer 
(0.25M Tris-HCl, pH 8.5; 1.92M glycine). 
The gel was a 20.times.16.times.0.15.times.cm vertical slab. The resolving 
gel was a 10% acrylamide gel (11.7 mls acrylamide, 30:0.8); 4.38ml 
8.times.LRB (pH 8.8); 18.8 ml water; 150 .mu.l 10% APS, fresh (ammonium 
persulfate in water) 35 .mu.l TEMED; brought to 35 ml. The gel was 
polymerized under n-butanol. 
The stacking gel was a 6% acrylamide gel (4 ml acrylamide 30:08); 5 ml 
4.times. LSB (pH 6.8); 10.7 ml water; 0.3ml APS (10%); 20.mu.l TEMED. 
The gel was run slowly (at about 5-6 mA) overnight at 4.degree. C. The gel 
was then stained by immersion in activity stain (50 mM Tris, pH 8; 0.003% 
riboflavin; 2.times.10.sup.-4 M EDTA; 0.01% of nitro blue tetrazolium 
(NBT) and incubation for 30 min. in the dark with gentle shaking. The 
liquid was then poured off and the gel immediately illuminated with a 
bright light. When the spots were most intense, the gel was rinsed quickly 
with water and photographed. 
The native gels for activity assay were then complexed with anti-MnSOD 
antibody as follows. The gels were soaked in 25 mM Tris-HCl pH 8.5, 0.192M 
glycine, 0.1% (w/v) SDS for 1.5 hours with gentle shaking. The gels were 
then electroblotted and hybridized as described above for the Western 
procedure. 
An additional band of SOD activity, not present in wild-type N. Tabacum 
`xanthi` plants, was detected in transformants by binding of the 
anti-MnSOD antibody after blotting. These data indicate that the E. coli 
MnSOD protein expressed in the transgenic tobacco plants is active. 
EXAMPLE 10 
Increased Drought Tolerance of Transgenic Tobacco Plants 
Description of Genetic Material 
Tobacco plant Xanthi-nc 1328-10-C was regenerated from cocultivation of 
Xanthi-nc leaf tissue with pCGN1328 using the leaf disk method and after 
selection on medium containing Kanamycin. Southern and Northern blot tests 
and a specific ELISA antibody test revealed that the novel MnSOD genetic 
sequences were integrated into the genome of regenerated T1 plants and 
that the corresponding mRNA and MnSOD protein were produced. 
After selfing the T1 plant, T2 generation seeds were obtained and displayed 
a Mendelian segregation pattern when germinated on a medium containing 
Kanamycin, as shown in the Table I, suggesting that the new gene was 
integrated at a single locus. 
TABLE 1 
______________________________________ 
Transmission To Progeny.sup.1 
Of Kanamycin Resistance 
Segregation 
Pollination 
Seeds Non Sen- Ratio 
Condition 
Total Germinated 
sitive 
Resistant 
S:R 
______________________________________ 
1328-10-C 
200 14 40 146 1:3.sup.2 
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.sup.1 Seeds were surface sterilized and germinated under aseptic 
conditions on solid medium containing 100 mg/l kanamycin and incubated fo 
12 days under light. 
.sup.2 Chisquare analysis indicates observed values are reasonable for a 
1:3 segregation ratio (sensitive:resistant) indicating a genotype of 
1:2:1. 
The T2 generation is a population of segregative plants composed of three 
groups of genotypes: 
Group A: Genotype +,+; Homozygous resistant transformed Xanthi-nc (2 novel 
MnSOD gene doses) 
Group B: Genotype +, -; Heterozygous resistant transformed Xanthi-nc (1 
novel MnSOD gene doses ) 
Group C: Genotype -,-; Homozygous recessive transformed Xanthi-nc (No novel 
MnSOD gene) 
If one assumes that no somaclonal variations have occurred during the 
regeneration process, all the above mentioned plants are considered as 
isolines: they display the same genetic background except the novel MnSOD 
gene. 
Comparison of the different phenotypes may reveal the effect of the novel 
gene on the agronomic performances of the plants when cultivated under an 
environmental stress such as drought stress (hydric stress combined or not 
with heat stress). Overexpression of the Mn superoxide-dismutase enzyme in 
the transformed plants may contribute to a better protection of the 
photosynthetic apparatus from the negative action of the superoxide 
O.sub.2.sup.- generated when plants are so stressed. 
The resistance of the T2 plants to stress was tested as follows. T2 
1328-10-C segregating plants were grown in the field under different 
drought stress treatments. Xanthi-nc wild type plants were also cultivated 
under the same conditions (group D, Xanthinc not transformed); treatments 
were applied at the same location. Three "environmental conditions" were 
used as follows: Environmental condition 1: "severe stress": plants were 
grown in an open field below a plastic field greenhouse; severe hydric and 
heat stresses were expected. Environmental condition 2: "natural stress": 
plants were grown in an open field. Local agro-climatic conditions 
indicated a high risk of drought stress from flowering to maturity stages. 
Environmental condition 3: "well watered": plants were grown in the open 
field, irrigation was scheduled in order to maintain the soil moisture 
profile at the field capacity. 
Individual plants will be evaluated based upon the following 
characteristics: Total leaf number; Dates of beginning and completion of 
flowering stages; Date of senescence of specific organs; Height at 
maturity; Total biomass and fertility at harvest time. 
ELISA tests performed on a subsample of T2 plants chosen at random will 
give an estimation of the content of the MnSOD protein in the plants. 
Progeny study on T3 seeds harvested on all the T2 plants will determine 
the corresponding genotypes. 
EXAMPLE 11 
Increased Metal Tolerance of Transgenic Tobacco Plants 
Transgenic N. Tabacum Xanthi-nc transformed with pCGN1328 was obtained as 
described in Example 7 to provide transgenic plants 1328-4. Plants were 
grown in standard growth medium (Murashige and Skoog salts with 1 mg/l IAA 
and 1.5 mg/l kinetin). The standard growth medium contains 0.025 mg/l 
(CuSO.sub.4.5H.sub.2 O and 8.6 mg/l ZnSO.sub.4.7H.sub.2 O. Prior to 
transfer to experimental medium, shoot tips were taken from the 
sterile-grown plants (3-4 weeks old), including 3-4 nodes. Leaves were 
removed, except from the meristems. 
To evaluate the response of the transgenic plants to increasing 
concentrations of copper and zinc, the concentration of copper and zinc in 
standard growth medium was increased 10 fold, 20 fold, 40 fold, 60 fold or 
100 fold the normal concentration. Each type of medium was adjusted to 
0.7% Phytoagar. One hundred ml of medium were added to a 1 quart Mason jar 
which was covered with plastic food wrap and sterilized by autoclaving for 
20 minutes. After the medium had cooled, the plantlets were added to the 
jars, 2 plants/jar in duplicate. As controls, non-transformed Xanthi-nc 
plants and plants transformed with a glyphosate resistance gene (aroA) 
were used. 
The plants were grown at 25.degree. C. with a 12 hour photoperiod. Light 
intensity during the photoperiod was 60-80 microeinsteins/m.sup.2 /sec. 
Plants were evaluated 24 days after planting. At 40 fold the normal 
Cu.vertline.Zn concentration, the transgenic 1328 plants clearly outgrew 
the Xanthi-nc and transgenic aroA plants, as determined by visual 
observation of the height of the plants. The results are summarized in 
Table 2 below. 
TABLE 2 
______________________________________ 
Relative Growth of Plants 
Exposed to Increased Cu.vertline.Zn 
Normal Cu.vertline.Zn Concentration 
Plant Medium 10x 20x 40x 60x 100x 
______________________________________ 
SOD+ ++++ ++++ ++++ +++ + + 
AroA ++++ ++++ ++ + + + 
Xanthi-nc 
++++ ++++ +++ ++ + + 
______________________________________ 
The above results demonstrate that plant species can be transformed 
efficiently with constructs which provide for expression of superoxide 
dismutase in a cell organelle such as a chloroplast. As evidenced by the 
above disclosure, plants are provided which have increased tolerance to 
environmental and chemical stresses such as drought and metal toxicity. 
All publications and patent applications mentioned in this specification 
are indicative of the level of skill of those skilled in the art to which 
this invention pertains. All referenced publications and patent 
applications are herein incorporated by reference to the same extent as if 
each individual publication or patent application was specifically and 
individually indicated to be incorporated by reference. 
The invention now being fully described, it will be apparent to one of 
ordinary skill in the art that many changes and modifications can be made 
thereto without departing from the spirit or scope of the appended claims.