WO2000052182A1

WO2000052182A1 - Use of glutathione-s-transferase to increase stress tolerance in plants

Info

Publication number: WO2000052182A1
Application number: PCT/GB2000/000750
Authority: WO
Inventors: Dirk Cooper Drost; Lawrence Lamont Buren; Ian Jepson; Allan Daly
Original assignee: Syngenta Limited
Priority date: 1999-03-03
Filing date: 2000-03-02
Publication date: 2000-09-08
Also published as: AU2924800A

Abstract

The present invention provides a method of preparing a plant which is tolerant to stress comprising incorporation a DNA encoding the GST-II-27 kD subunit into the plant or plant progenitor material such that a glutathione-s-transferase enzyme is produced. Preferred types of plants for use in the method of the invention are maize and rice.

Description

USE OF GLUTATHIONE-S-TRANSFERASE TO INCREASE STRESS TOLERANCE IN PLANTS

FIELD OF THE INVENTION The present invention relates to the use of glutathione-S-transferase (GST) to increase stress tolerance in plants, particularly crop plants. More particularly, the present invention relates to a method of preparing a stress tolerant plant which incorporates the GST-II-27 kD subunit.

BACKGROUND OF THE INVENTION

Glutathione-S-transferases (GSTs) are a heterogeneous family of enzymes that catalyse the conjugation of reduced glutathione (GSH) to electrophilic sites of a variety of target compounds. In plants GSTs are known for their ability to conjugate GSH to a number of pesticides and reduce their toxicity. Expression of GSTs in plants is highly responsive to biotic and abiotic stresses and to a wide variety of stress-associated chemicals including 2,4-D and other synthetic and natural auxins, salicylic acid, methyl jasmonate, abscisic acid and peroxide. This responsiveness has been exploited through the use of chemical safeners that, when applied to plants, induce GST activity and thereby increase herbicide tolerance. GST enzymes have been identified in a range of crop plants including maize, wheat, sorghum and peas. GSTs comprise 1 to 2% of the total soluble protein in etiolated maize seedlings.

Many isoforms of maize GSTs have been identified, examples of which are GST-I, GST-II and GST-III. The major isoform in maize tissue, GST-I, is constitutively expressed and is capable of conjugating glutathione with pre-emergent herbicides such as alachlor. Treatment of maize tissues with chemical safeners (for example, N,N-diallyl-2,2 dichloracetamide) raises the activity of GST-II which participates in the detoxification of the pre-emergent herbicides. Both GST-I and GST-II proteins have a native molecular weight of approximately 50 kD. As in mammals, maize GSTs are dimeric; GST-I has apparently identical polypeptide subunits of 29 kD (GST-I-29), whereas GST-II is a heterodimer of a 29 kD subunit identical to that found in GST-I (GST-I-29) and a 27 kD subunit (GST-II-27). GST-II is detected at a very low basal level in the absence of safener, but its expression is enhanced dramatically by safener treatment. Like GST-I, GST-II confers resistance to certain herbicides. GST-II is known to detoxify chloroactetanilide and thiocarbarbamate herbicides such as alachlor (Mozer et al, 1983, Biochemistry, 22:11068-1072).

A cDNA and a gene corresponding to the 29 kD subunit of GST-I have been cloned previously and sequenced (Wiegand et al. 1986, Plant Mol Biol, 7:235-243). In addition, cDNA corresponding to a 26 kD subunit of a third, minor component of GST activity in maize seedlings (GST-III-26) has been previously cloned and sequenced (Moore et al, 1986, Nucleic Acid Research, 18:7227-7235). GST-III is a homodimer of these 26 kD subunits. Like GST-I and unlike GST-II, GST-III is constitutively expressed. It is known to detoxify herbicides such as atrazine.

U.S. patent 5,589,614 issued December 31, 1996 discloses the GST-II-27KD subunit and DNA encoding the subunit. The patent also discloses transgenic herbicide-resistant plants.

SUMMARY OF THE INVENTION

We have discovered that plants transformed with the nucleic acid encoding the GST- II 27 kD subunit exhibit increased tolerance to environmental stresses. Thus, the present invention provides a method for preparing stress tolerant plants which comprises the incorporation of DNA encoding the GST-II 27 kD subunit into the plant such that a glutathione-S-transferase enzyme is expressed. Another aspect of the invention provides the use of nucleic acid encoding the GST-II 27 kD subunit in the preparation of plants having increased stress tolerance. A preferred nucleic acid sequence encodes the amino acid sequence of SEQ ID NO:2 , more preferably the nucleic acid sequence comprises at least the coding portion of SEQ ID NO: 1, both of which sequences are disclosed in U.S. patent 5,589,614 . The methods of the invention can be used to increase tolerance of plants to environmental stresses. Such stresses include, but are not limited to, heavy metals, ozone, salt stress and temperature extremes. A further aspect of the invention provides a plant transformed with nucleic acid encoding the GST-II 27 kD subunit. These and other aspects of the invention are set out in the appended claims and described in the following detailed description. DET AILED DESCRIPTION OF THE INVENTION

According to the present invention there is provided a method of preparing a plant which is tolerant to stress comprising incorporating a DNA encoding the GST-II-27 kD subunit into the plant or plant progenitor material such that a Glutathione-S-Transferase enzyme is produced. The said DNA may encode the amino acid sequence of SEQ ID NO: 2 or may be a polynucleotide comprising the sequence depicted as SEQ ID No. 1 or a polynucleotide sequence which is the complement of one which hybridises to the sequence depicted as SEQ ID No. 1 at a temperature of about 65°C in a solution containing 6xSSC, 0.01% SDS and 0.25% skimmed milk powder, followed by rinsing at the same temperature in a solution containing 0.2xSSC and 0.1% SDS wherein the said polynucleotide sequence still encodes a Glutathione-S-Transferase enzyme.

The present invention also provides a method as described above wherein the said stress is selected from the group consisting of temperature stress, heavy metal stress or osmotic stress. Preferably, the plant of the present invention is a monocotyledonous plant, more preferably a cereal plant and more preferably a maize or rice plant.

The present invention further provides for the use of DNA encoding the GST-II-27 kD subunit in the preparation of a plant which is tolerant to stress. The said DNA may encode the amino acid sequence of SEQ ID NO: 2 or may be a polynucleotide comprising the sequence depicted as SEQ ID No. 1 or a polynucleotide sequence which is the complement of one which hybridises to the sequence depicted as SEQ ID No. 1 at a temperature of about 65°C in a solution containing 6xSSC, 0.01% SDS and 0.25% skimmed milk powder, followed by rinsing at the same temperature in a solution containing 0.2xSSC and 0.1% SDS wherein the said polynucleotide sequence still encodes a Glutathione-S-Transferase enzyme. Preferably the DNA comprises the sequence depicted as SEQ ID No. 1. Preferably the said DNA for use in the present invention comprises the sequence depicted as SEQ ID No. 1. The present invention also provides for the use as described above wherein the said stress is selected from the group consisting of temperature stress; heavy metal stress or osmotic stress. Preferably, the plant for use in the present invention is a monocotyledonous plant, preferably a cereal plant, preferably a maize or rice plant. Thus, the present invention provides methods of preparing plants having increased stress tolerance comprising the step of incorporating nucleic acid encoding the GST-II 27 kD subunit into the plant such that a glutathione -S-transferase is produced. The present invention also provides the use of nucleic acid encoding the GST-II 27 kD subunit in the preparation of plants having increased stress tolerance.

Glutathione -S-transferases are believed to play an important role in determining plant tolerance to a number of environmental stresses. Such stresses include, but are not limited to, heavy metals, ozone, salts, temperature extremes and pathogen attack. GSTs in a number of plants have been found to be induced in response to specific stress conditions, such as heavy metals, potassium and sodium salts, ozone, heat shock and pathogen attack. It has been suggested that these conditions enhance the generation of active oxygen species (AOS) in plant cells and that GSTs act to protect cellular components from oxidation damage. Active oxygen species such as superoxide (O₂ ^"»), hydroxyl radicals (OH^») and hydrogen peroxide (H₂0₂) can interact with cell membranes and DNA to produce highly cytotoxic lipid peroxides (e.g., 4-hydroxyalkenals) and base propenals (e.g., thymidine hydroperoxide), respectively. GSTs can detoxify lipid peroxides by conjugation with glutathione (GSH). Additionally, some GSTs (including GST-27) can function as glutathione peroxidases (GPX) which can detoxify base propenals and catalyze GSH- dependent reduction/inactivation of peroxide. Plant GSTs have also been reported to be induced by ethylene and may function to maintain cell integrity against lipid peroxidation during programmed cell senescence. Roxas et al., Nature Biotechnology, vol. 15, p. 988- 991 , 1997 reported that overexpression of glutathione s-transferase/glutathione peroxidase enhances the growth of transgenic tobacco seedlings during cold and salt stresses. Accordingly, the overexpression of GST-II in plants transformed with GST-II-27 kD subunit should provide such plants with increased tolerance to environmental stresses including heavy metals, ozone, salts, temperature (heat and cold), and pathogen attack. Stress tolerant, increased stress tolerance, tolerance to stress and similar phrases used herein refer to the ability of a plant, or group of plants such as a field planted with a particular crop, that are transformed to contain the GST-II 27 kD subunit to overcome or resist the effects of a stresser to a greater extent than control like plants. Increased stress tolerance may vary from a slight increase in the ability to resist or overcome the effects of a stresser to total tolerance where the plant is unaffected by the stresser. Stress tolerant plants may show physical characteristics that indicate increased tolerance to an environmental stress. For example, plants that are transformed to contain the GST-II 27 kD subunit may be larger than control like plants when grown in the presence of a particular stresser such as hot or cold temperatures. Preferably, plants according to the present invention will be able to tolerate temperatures of at least 1°C higher or lower than the control like plants. More preferably 2°C, more preferably 3°C, more preferably 4°C, more preferably 5°C or higher. Plants according to the invention may also be able to tolerate any such temperature for a longer time period than that of the control like plant. Similarly, transformed plants may emerge sooner than non-transformed plants, or be able to grow in the presence of the stresser when the control like plant is not able to grow under the same conditions. The affected traits of the plant will depend on the type of plant and stresser involved.

Incorporating DNA encoding GST polypeptides into plants will thus confer new or additional tolerance to environmental stresses. Although GSTs are naturally expressed in many plant species, notably maize, production of transgenic plants containing enhanced levels of GSTs may increase the crop's level of stress tolerance. In addition, all the GST isoforms are not normally present in all maize lines. Production of transgenic maize containing one or more additional GST isoforms may confer tolerance to a wider range of environmental stresses. A further advantage comes from placing the GST-II-27 sequence under control of a constitutive promoter, as this will mean that the GST-II enzyme is constitutively expressed within the transgenic maize plant. This avoids the need to apply a chemical safener to the maize seed or plant. Various constitutive promoters are know to the person skilled in the art including CaMN35S, FMN35S, ΝOS, OCS, Patatin and E9 (derived from the small subunit of RUBISCO). Normally, GST-II enzyme activity is inducible. Inducible promoters are also known to the person skilled in the art and include the alcA/alcR gene switch described in published International Patent Application No. WO93/21334; the GST promoter switch described in published International Patent Application Nos. WO90/08826 and WO93/01294 and the RMS switch system described in published International Patent Application No. WO90/08830., A plant expressing GST-I 29 kD polypeptide subunits will show GST-I enzyme activity. A plant co-expressing GST-I 29 kD subunits and GST-II 27 kD subunits will show GST-II enzyme activity, and some GST-I activity. A plant expressing GST-III 26 kD subunits will show GST-III activity. A plant co-expressing GST-1 29 kD, GST-II 27 kD and GST-II 26kD polypeptide will show GST-I, GST-II and GST-III activity.

Plants incorporating the GST-II 27 kD subunit can be prepared, for example, in accordance with U.S. Patent No. 5,589,614 or as described herein. The plants can be transformed with nucleic acid encoding both the GST-I-29 kD subunit and the GST-I-27 kD subunit, or the GST-II-27 kD subunit alone. Amino acid sequencing has shown that the 29 kD subunit of GST-I is identical to the 29 kD subunit of GST-II. Thus a combination of the GST-I-29 subunit and the GST-II-27 subunit will give an active GST-II enzyme. Co- expression of the cDNA encoding GST-I-29 (isolated by Wiegand et al, 1986, Plant Mol

Biol, 7:235-243) and the cDNA encoding GST-II-27 (disclosed in U.S. patent No. 5,589,614) within a transgenic plant will give a plant with GST-II activity. In the case of incorporating only the GST-II-27 kD subunit into the plant, the GST-II- 27 kD subunit produced by the transformed plant will combine with the GST-I- 29 kD subunit normally produced in such plant to form an active GST-II enzyme.

Plants can be transformed with constructs containing sequences which encode GST subunits according to a variety of known methods, such as Agrobacterium Ti plasmids, electroporation, microinjection, microprojectile gun, and whisker technology (Frame et al. The Plant Journal 6(6): 941-948, 1994). The transformed cells may then in suitable cases be regenerated into whole plants in which the new nuclear material is stably incorporated into the genome. Both transformed monocot and dicot plants may be obtained in this way. Examples of genetically modified plants which may be produced include field crops such as canola, sunflower, tobacco, sugarbeet, cotton and cereals such as wheat, barley, rice, sorghum and also maize. A preferred type of plant for use in the present invention is maize. DNA encoding the GST-I 29 kD subunit or the GST-II 27 kD subunit can be incorporated into a vector under the control of a suitable promoter such as the 35S CaMN promoter. Plants may be transformed with vectors containing either the GST-I-29 or the GST-II-27 expression cassette. Transformants expressing the respective GST-II subunits (29 kD or 27 kD) may be crossed to produce progeny expressing both GST-I-29 and GST-II-27, resulting in a stress tolerant phenotype. In a modification of this method, each plant may be co-transformed with vectors containing the GST-I-29 expression cassette and with vectors containing the GST-II-27 expression cassette. Alternatively, DNA encoding the GST-I 29 kD subunit and the GST-II 27 kD subunit may be included in a single plant transformation vector under the control of a single promoter. Transformants expressing both GST-II subunits (29 kD and 27 kD) will show a stress tolerant phenotype; transformants expressing only one of the respective GST-II subunits (29 kD or 27 kD) may be crossed to produce progeny expressing both subunits.

Alternatively, in species that naturally contain GST enzymes, GST-II can be expressed by incorporating DNA encoding the GST-II 27 kD subunit into the plant without also incorporating DNA encoding the GST-I 29 kD subunit or later crossing with plants transformed to express the 29 kD subunit.

The above methods can be adapted to produce herbicide resistant plants expressing the GST-I enzyme (transformation with DNA encoding GST-(-29), the GSTII enzyme (transformation with DNA encoding GST-III-26, as isolated by Moore et al, 1986, Nucleic Acid Research, 18:7227-7235), or some selection of GST-I/GST-II/GST-III activity. Nucleic acid, preferably DNA, encoding the GST subunit(s) is incorporated into a plant under the control of a constituitive or inducible promoter. Preferably, DNA encoding the GST subunits is introduced into the plant under control of a constitutive promoter, such as the 35S CaMN promoter, the maize polyubiquitin promoter (Christensen, AH et al, Plant. Mol. Biol., 18: 675-689, 1992) or the rice actin promoter (McElroy et al., Plant Cell 2: 163- 171, 1990). This avoids any need for external induction of GST expression. DΝA encoding the GST subunits may also be included in a plant transformation vector under the control of an inducible promoter, to give inducible herbicide resistance in the transgenic plants. Such a promoter includes the chemically-inducible GST-II-27 promoter as disclosed in U.S. patent No. 5,589,614. Resistance may be switched on by application of a suitable inducer (such as a chemical safener). In certain circumstances, the ability to increase stress tolerance only when required may be advantageous and more metabolically efficient as the plant would be producing GST only when required.

The transformation vector may also contain a suitable selection marker such as the phosphinothricin acetyl transferase (PAT) gene (U.S. Patent 5,561,236), or neomycin phosphotransferase II (npll), acetolactate synthase, EPSPS (which confers resistance to glyphosate) genes or the manA gene which encodes phosphomannose isomerase which provides the plant with the ability to convert mannose-6-phosphate into fructose-6- phosphate. The selection marker gene can be deleted from the transformation vector or plants if it is not needed to select plants transformed with the GST-II 27 kD subunit.

A preferred DNA sequence for use in the present invention encodes GST-II-27 shown in U.S. Patent 5,589,614 which has the amino acid sequence shown in SEQ ID NO: 2. More preferably the sequence is SEQ ID NO: 1 which is also disclosed in U.S. Patent 5,589,614. cDNA encoding GST-I-29 is disclosed in Wiegand et al, 1986, Plant Mol Biol, 7:235-243. Alternatively the sequence used in the present invention may be a polynucleotide sequence which is the complement of one which hybridises to the sequence depicted as SEQ ID No. 1 at a temperature of about 65°C in a solution containing 6xSSC, 0.01 % SDS and 0.25% skimmed milk powder, followed by rinsing at the same temperature in a solution containing 0.2xSSC and 0.1% SDS wherein the said polynucleotide sequence still encodes a GST enzyme.

Other sequences which may be used in the present invention include those sequences which encode functional variants of the basic sequence described as SEQ ID No. 2. The expression "functional variants" refers to any peptide which has some amino acids in common with the basic sequence and still provides for a functional GST enzyme which is capable of conferring stress tolerance to plants. Preferably at least 60% of the amino acids will be similar to the sequence depicted as SEQ ID No. 2, more preferably at least 70%, more preferably at least 80%), more preferably at least 90% and most preferably at least 95%.

Amino acids which differ from those present in the basic sequence may be substituted with conservative or non-conservative amino acids. A conservative substitution is to be understood to mean that the amino acid is replaced with an amino acid with broadly similar chemical properties. In particular conservative substitutions may be made between amino acids within the following groups:

(i) Alanine, Serine, Glycine and Threonine; (ii) Glutamic acid and Aspartic acid; (iii) Arginine and Lysine; (iv) Asparagine and Glutamine; (v) Isoleucine, Leucine, Naline and Methionine;

(vi) Phenylalanine, Tyrosine and Tryptophan. In general, more conservative than non-conservative substitutions will be possible without destroying the ability of the protein to confer stress tolerance in plants. The present invention will now be described by way of the following non limiting examples with reference to the figures and sequence listing of which: Figure 1 is a monocotyledonous transformation vector containing GST-27 and phosphinothricin referred to as pCATl 1.

SEQ ID No. 1 - Polynucleotide encoding a GSTII-27.

SEQ ID No. 2 - Amino acid sequence encoded by SEQ ID No. 1.

SEQ ID Nos. 3 and 4 - Linker sequences. SEQ ID nos. 5 and 6 - PCR primer sequences.

EXAMPLES

Example 1 - Preparation of plants transformed with GST-27 A.) A monocotyledonous transformation vector containing GST-27 and phosphinothricin referred to as pCATll (Figure 1) is generated as follows: pUBl, a pUC-based vector containing the maize ubiquitin promoter and the first intron (Bruce et al., Proc. Natl. Acad. Sci. 86: 9692, 1989) was digested with Hind III and a Hind Ill-Age I - Hind III linker (5'AGCTTGTACCGGTCTACA 3', SEQ ID NO: 3) inserted. GST-II 27 cDNA obtained as described in U.S. patent 5,589,614 and provided with a 3' Kpn I site and a 5' Ban HI site was inserted into the BamHI-Kpn I site. A Kpnl -Pac I -Kpnl linker (5' CGGACAATTAAT-TAATTGTCCGGTAC 3', SEQ ID NO: 4) is self annealed and inserted into the Kpnl site. The Agrobacterium nos terminator provided with Sma I ends and blunt end cloned into the Eco RN site. Orientation of the nos terminator was confirmed by restriction digestion with Eco RI and Bam HI. Confirmation of all junctions was determined by sequence analysis.

The ubiquitin - GST-27- nos cassette was removed by digestion with Agcl and Pad, and inserted into the ampicillin minus vector pIGPAT which contains the phosphinothricin acetyl transferase (PAT) gene (U.S. Patent 5,561,236) under the control of the 35S CaMN promoter. pIGPAT is a pUC18 derivative with the ampicillim resistance gene replaced by the yeast imidazole glycerol phosphate dehydratase (IGPD) gene. Recombinants are detected by colony hybridization with the GST-27 cDNA insert from pIJ21-3A. Recombinants are oriented with Ncol restriction digestion to form pCATlO.

The 35S-PAT- nos cassette was removed from pCATIO by digestion with Ascl and the vector religated. pCATIO was then digested with Ascl and the Ascl maize ubiquitin -PAT- nos Asc I fragment from pUN #14 inserted in pCATIO to form pCATl 1 (Figure 1).

B. Transformation of cells and generation of plants pCATl 1 was transformed into maize cell suspension cells using the whisker technology as described in Frame et al. The Plant Journal 6(6): 941-948, 1994. The cells were then transferred to culture medium containing bialophos in order to select for calli expressing the PAT gene. Calli which grew on bialophos containing media were then checked for the presence of the GST-27 gene by PCR using oligos GST-II/7 (5' cca aca agg tgg cgc agt tea 3'; SEQ ID NO: 5) and NOS3' (5' cat cgc aag ace ggc aac ag 3'; SEQ ID NO: 6). The calli which contained the GST-27 expression cassette were transferred to plant regeneration media and maize plants recovered.

The transformed maize plants were confirmed to have constitutive levels of the GST-27 protein present by extracting total leaf protein and using this to perform western blot analysis. The plants identified to contain high constitutive levels of GST-27 protein were cross pollinated with an elite maize inbred line and seed recovered.

Example 2 Stress Tolerance Testing

Two GST-transformed corn (Zea mays L.) lines, COP50 (Line I) and CUI50 (Line II), were used in these experiments. Lines I and II were test crosses with one copy of the GST gene segregating 1:1 for the GST gene. Within each line, the plants which were expressing the GST gene were compared to the plants not expressing the GST gene to determine the effects the GST gene expression on the plant growth. The PAT gene included in the construct conveys tolerance to glufosinate and is used as a marker to identify plants expressing the GST gene. In addition to the transformed lines, the parent line (BP32) used in the test crosses and a commercial glufosinate tolerant hybrid (8692LL obtained from Garst Seed Company) were planted. The soil used was a sandy clay loam (pH=6.5 and OM=l .4%). Forty seeds from each transformed line and the BP32 inbred and 8 seeds of 8692LL were seeded into seven different environmental treatments (Table 1). Four of the treatment regimes followed a sequence of warm-cold- warm, two of cold- warm and one of warm-hot. The cold (50°F) and hot (100790° F, day/night) environments were in growth chambers and the warm (80768° F) environment was in a glasshouse. Light levels in the growth chambers are very low (200 microEinsteins/m2) compared to the glasshouse, but plants grow well in both the chambers and glasshouse. Day length in the glasshouse and chambers was 14 hours. The experimental design was completely randomised and the data were subjected to analysis of variance and then to a significance test.

Table 1. Environmental treatment regimes.

(Example: A: After seeding, flats were placed in the warm environment for 3 days then moved to the cold environment for 3 days then to the warm environment for 22 days.)

Plant emergence was recorded daily from 3 to 18 days after planting and plant height was recorded each time the plants were moved to a new environment and at the end of the test, before spraying with a 1% glufosinate solution. After 28 days the plants were all placed in the glasshouse and sprayed with 1% solution of glufosinate. One week later the plants were rated for glufosinate tolerance. Data was separated into groups of populations expressing the GST gene (glufosinate tolerant) and populations without the GST gene (glufosinate susceptible).

The experimental design was completely randomised. Plant heights were subjected to analyses of variance. Significance tests were conducted to compare populations expressing the GST gene to those not expressing the gene within each line. For emergence data, it was necessary to use non-parametric methods to compare the time to emergence of tolerant and non-tolerant plants. Ordered chi-squared tests were therefore conducted to compare the time to emergence for non-tolerant and tolerant plants for each of the two lines within each environment separately.

The differences among corn lines within an environment were small. At least 90% of the plants had emerged in environments B, C, D and G within 4 days, in environment A within

6 days, and in environment F within 9 days. In the environment with the longest cold period, E, 14 days were required to reach 90% emergence. Emergence patterns were similar for GST expressing and non-GST expressing plant populations.

Plant heights at the end of the 28 day treatment regimes were averaged for GST expressing and non-GST expressing plants (Table 2).

Table 2. Comparison of average plant heights for GST expressing and non-GST expressing plants. NT = Non-GST expressing plants; T = GST expressing plants

Mean plant height (mm)

Line I Line II

Code Environment NT¹ T p-value NT T p-value

A W3-C3-W22 475 476 96% 427 441 57%

B W9-C3-W16 610 618 56% 592 628 23%

C W3-C9-W16 552 551 94% 480 484 89%

D W9-C9-W10 586 587 90% 542 598 7%

E C14-W14 379 415 0.01% 356 369 61%

F C7-W21 561 586 6.10% 550 569 46%

G W10-H18 307 408 1.10% 395 472 23% all na na na 477 509 0.01% environments p- value for 0.06% 72% interactions ' NT = Non-GST expressing plants; T = GST expressing plants

There is strong evidence of an interaction between environment and GST expression (p=0.06%) for Line 1. This means that the relationship between GST expressing and non- GST expressing plant height is dependent upon the environment for Line 1. Comparisons of the heights of non-tolerant and tolerant plants must be made for each environment separately and the results of the factorial analysis are not presented. Analysis shows strong evidence that Line I GST expressing plants were larger than non-GST expressing plants in environments E and F where cold stress started the treatment regimes. There is also evidence to support that Line I GST expressing plants were larger than non-GST expressing plants in environment G where plants were subjected to heat stress.

In Line II, there was no interaction between environment and GST expression which allows us to average across all of the various temperature stress environments. Averaged across the various stress environments, Line II plants expressing the GST gene were larger than plants not expressing the GST gene.

Example 3 - Stress Tolerance Testing

A selfed line of the COP50 event (Line Is) segregating 3:1 for the GST gene was used to evaluate the effect of GST expression under conditions of continuous cold stress. The experiment was conducted in a growth chamber set at 10° C for 29 days. After 29 days, the temperature was raised to 24°C for 2 days. The parent line, BD68, was also included in the experiment. Plants which were expressing the GST gene were compared to the plants not expressing the GST gene to determine the effects of the GST gene expression on plant growth. The PAT gene included in the construct used conveys tolerance to glufosinate and was used as a marker to identify plants expressing the GST gene. The height of GST- expressing plants and non GST-expressing plants was measured at the end of the experiment (Table 3).

Table 3. Effect of cold stress on growth of Line Is

Mean Plant Height (mm)

Environment Assessment Date Non GST- GST-Expressing P-Value for

(days after Expressing Comparison planting)

Warm 6 105 104 88% 13 151 151 99% 20 382 384 93%

Cold 31 120 144 0.6%

Statistical analysis supports that on average GST-expressing plants were larger than non GST-expressing plants in the cold conditions of the experiment. Emerged plants were counted every two days and percent emergence was determined using the final emergence count taken after 2 days in the warm environment. By 17 days after planting 100%) of the GST-expressing Line Is plants had emerged. Germination of non- GST-expressing plants appeared to peak at 88% 19 days after planting. At 29 days after planting, the flats containing the seeds were moved to a warm environment in which non GST-expressing and BD68 seed continued to emerge. The transformed plants had a greater rate of emergence and greater mean plant height (37%) compared to the non-transformed plants. All of the non GST-expressing plants did not emerge until the flats were put into a warm environment.

Claims

1. A method of preparing a plant which is tolerant to stress comprising incorporating a DNA encoding the GST-II-27 kD subunit into the plant or plant progenitor material such that a Glutathione-S-Transferase enzyme is produced.

2. A method according to claim 1 wherein the DNA encodes the amino acid sequence of SEQ ID NO: 2.

3. A method according to claim 1 or claim 2 wherein the DNA comprises a polynucleotide having the sequence depicted as SEQ ID No 1 or a polynucleotide sequence which is the complement of one which hybridises to the sequence depicted as SEQ ID No. 1 at a temperature of about 65°C in a solution containing 6xSSC, 0.01%) SDS and 0.25% skimmed milk powder, followed by rinsing at the same temperature in a solution containing 0.2xSSC and 0.1 % SDS wherein the said polynucleotide sequence still encodes a Glutathione-S-Transferase enzyme.

4. A method according to any one of claims 1 to 3 wherein the said stress is selected from the group consisting of temperature stress, heavy metal stress or osmotic stress.

5. A method according to any one of claims 1 to 4 wherein said plant is a monocotyledonous plant.

6. A method according to claim 5 wherein said plant is a cereal.

7. A method of claim 6 wherein said plant is a maize or rice plant.

8. The use of DNA encoding the GST-II-27 kD subunit in the preparation of a plant which is tolerant to stress.

9. The use of claim 8 wherein said DNA encodes the amino acid sequence of SEQ ID NO: 2.

10. The use according to claim 8 or claim 9 wherein the DNA comprises a polynucleotide having the sequence depicted as SEQ ID No. 1 or a polynucleotide sequence which is the complement of one which hybridises to the sequence depicted as SEQ ID No. 1 at a temperature of about 65°C in a solution containing 6xSSC, 0.01% SDS and 0.25% skimmed milk powder, followed by rinsing at the same temperature in a solution containing 0.2xSSC and 0.1 %> SDS wherein the said polynucleotide sequence still encodes a Glutathione-S-Transferase enzyme.

11. The use according to any one of claims 8 to 10 wherein the said stress is selected from the group consisting of temperature stress; heavy metal stress or osmotic stress.

12. The use according to any one of claims 8 to 11 wherein said plant is a monocotyledonous plant.

13. The use according to claim 12 wherein said plant is a cereal.

14. The use according to claim 13 wherein said plant is maize or rice plant.

AMENDED CLAIMS

[received by the International Bureau on 21 August 2000 (21.08.00); original claims 4 and 11 cancelled; original claims 1 and 8 amended; new claims 7 and 14 added; original claims 5-7 and 12-14 renumbered as claims 4-6 and 11-13; other claims unchanged (2 pages)]

1. A method of preparing a plant which is tolerant to stress comprising incorporating a DNA encoding the GST-II-27 kD subunit into the plant or plant progenitor material such that a Glutathione-S-Transferase enzyme is produced wherein said stress is induced by an agent selected from the group consisting of: Ozone; Potassium and Sodium salts; Temperature and Pathogens.

2. A method according to claim I wherein the DNA encodes the amino acid sequence of SEQ ID NO: 2.

3. A method according to claim 1 or claim 2 wherein the DNA comprises a polynucleotide having the sequence depicted as SEQ ID No 1 or a polynucleotide sequence which is the complement of one which hybridises to the sequence depicted as SEQ ID No. 1 at a temperature of about 65°C in a solution containing 6xSSC, 0.01% SDS and 0.25% skimmed milk powder, followed by rinsing at the same temperature in a solution containing 0.2xSSC and 0.1% SDS wherein the said polynucleotide sequence still encodes a Glutathione-S-Transferase enzyme.

4. A method according to any one of claims 1 to 3 wherein said plant is a monocotyledonous plant.

5. A method according to claim 5 wherein said plant is a cereal.

6. A method of claim 6 wherein said plant is a maize or rice plant.

7. A method according to any one of claims 1 to 3 wherein said plant is a canola, sunflower, tobacco, sugarbeet or cotton plant.

8. The use of DN encoding the GST-II-27 kD subunit in the preparation of a plant which is tolerant to stress wherein said stress is induced by an agent selected from the group consisting of: Ozone; Potassium and Sodium salts; Temperature and Pathogens.

9. The use of claim 8 wherein said DNA encodes the amino acid sequence of SEQ ID NO; 2.

10. The use according to claim 8 or claim 9 wherein the DNA comprises a polynucleotide having the sequence depicted as SEQ ID No. 1 or a polynucleotide sequence which is the complement of one which hybridises to the sequence depicted as SEQ ID No. 1 at a temperature of about 65°C in a solution containing 6xSSC, 0.01% SDS and 0.25% skimmed milk powder, followed by rinsing at the same temperature in a solution containing 0.2xSSC and 0.1% SDS wherein the said polynucleotide sequence still encodes a Glutathione-S-Transferase enzyme.

11. The use according to any one of claims 8 to 10 wherein said plant is a monocotyledonous plant.

12. The use according to claim 11 wherein said plant is a cereal.

13. The use according to claim 12 wherein said plant is maize or rice plant .

14. The use according to any one of claims 8 to 10 wherein said plant is a canola, sunflower, tobacco, sugarbeet or cotton plant.