权利要求

STRESS RESISTANCE GENE

The present invention relates to plant cells, breeding materials, plant parts and plants in which the expression level of the aldose reductase-homologous protein is increased and, therefore, which show higher resistance to stress conditions. Plant cells, breeding materials, plant parts and plants per se are provided and these are advantageously transgenic. The present invention further relates to nucleic acid , eg. DNA and RNA molecules .and vectors suitable for providing expression of aldose reductase-homologous protein in cells and processes for the preparation of plant cells overproducing aldose reductase-homologous protein. The invention has particular use in reducing damage in crops caused by harmful biotic and abiotic stress conditions and for improving harvest prospects thereby.

During their lifetime, plants are exposed to both abiotic (eg. photoinhibition by high light intensity, UV-B irradiation, ozone, heavy metals, high and low temperature, water deficiency, flooding and wounding) and biotic (eg. viral, bacterial and fungal infections, insect action) stress conditions. These stress conditions can cause serious decrease in plant productivity .and economic damage; therefore, increasing plant stress tolerance and producing plants and breeding materials of higher stress resistance are among the major goals of plant breeders.

Aldose reductase (EC. 1.1.1.21) and aldehyde reductase (EC. 1.1.1.2.) enzymes belong to the aldo-keto reductase superfamily. These enzymes are monomeric NADPH-dependent oxidoreductases with broad substrate specificity, ranging from aldose sugars to aromatic aldehydes (Bohren K.M. et al. 1989. J. Biol. Chem. 264: 9547-9551). Until now plant aldose reductase enzymes were isolated only from monocots. The expression of the aldose reductase-homologous gene from b? ley is induced in the late period of embryo development (Bartels, D. et al. 1991. The EMBO J. 10: 1037-1043; Roncarati, R. 1995. The Plant Journal. 7(5): 809-822). Experiments with bromegrass plants suggested a link between the expression of the aldose reductase-homologous gene and the low temperature tolerance of the cells (Lee, S.P. 1993. J. Plant Physiol. 142: 749-753). A series of studies point to an osmoregulatory function of aldose reductases in animal cells, because these enzymes can catalyse the first step of the polyol reaction route: namely the reduction of D-glucose to sorbitol. It is known that the modulation of the intracellular sorbitol level as well as other osmolytes serves to maintain the osmotic balance and the cellular volume (Moriyama, T. et al. 1989. J. Biol. Chem. 264: 16815-16821; Ferraris, J.D. 1996. J. Biol. Chem. 271 : 18318-18321). However, there is still much uncertainty about the physiological function of these enzymes, because the high reactivity of aldose and aldehyde reductases with many aldehydes opens the possibility of a role for these enzymes as detoxificants in animal cells.

The potential mode of a detoxification role of these proteins can be summarised as follows: numerous pathological processes can generate harmful free radicals that can cause cell damage. Most of these free radicals have very short lifetime, their toxic effect being concentrated only to a small area around the site of their generation. However, the reaction of these free radicals with membrane lipids leads to the formation of lipid peroxides and hydroperoxides that subsequently can undergo degradation which results in reactive aldehyde compounds. These aldehydes are much more stable than the free radicals, moreover, they can migrate away from the site of their formation, extending the toxic effect to the whole cell. One of the most frequently formed and most toxic lipid aldehydes is the 4-hydroxy-nonenal (HNE). Because of its reactive α-β unsaturated double bond, HNE can spontaneously form Michael adducts with the -SH groups and lysine or histidine residues of cellular proteins. The aldehyde group of HNE can form Schiff bases with the α or ε amino groups of proteins, thus via crosslinking it can alter their catalytic and structural properties. In nanomolar concentrations HNE can cause DNA damage while in higher, eg. micromolar, concentrations it has a cytotoxic effect. Because HNE has an aldehyde group, a possible way of its detoxification is its reduction to alcohol, catalysed by the members of the aldo-keto reductase superfamily. Reviewing the literature, this detoxification role can be seen to be consistent with experimental results (Van der Jagt, D.L. et al. 1995 Biochim. Biophys. Acta 1249: 117-126; Srivastava, S. et al. 1995. Biochem. Biophys. Res. Commun. 217: 741-746; Spycher S. et al. 1996. Biochem. Biophys. Res. Commun. 226: 512-

516; Spycher S. et al. 1997. FASEB J. 11 : 181-187).

The object of the present invention is to produce novel, advantageously transgenic, plant types that, compared to the starting plants, ie. standard crop or ornamental lines, possess an enhanced resistance against both abiotic and biotic stress conditions and to provide a method for producing these.

The invention is based on the finding that, via overproduction of aldose reductase-homologous enzyme superfamily members having a general detoxificating and osmoregulatory effect, plant cells and plants regenerated from them can be made resistant to a greater extent against number of stress conditions of different origin at the same time. Particularly the effect of the enzyme is correlated to its ability to reduce aldehyde groups of various substrates, eg. 4-hydroxy-nonenal (HNE) and DL- glyceraldehyde.

In reducing the invention to practice, cDNA coding for an alfalfa (Medicago sativa) aldose reductase-homologous enzyme (MsALRh) has been isolated by applying recombinant gene manipulation methods. By attaching this cDNA to a regulatory region, eg. a promoter, ensuring ectopic expression in plants, eg. by constitutive expression, in a functional relationship, eg., in frame, overproduction of the aldose reductase-homologous enzyme has been carried out in cells of transgenic plants, thereby enabling the reduction of damage in said plants exposed to stress conditions of different origin via the detoxificating and/or osmoregulatory function of the aldose reductase-homologous protein.

Thus, in a first aspect the invention there are provided plants, plant cells, plant parts, breeding material and plant product, all of which are advantageously transgenic, which as a consequence of enhanced level of the aldose reductase-homologous enzyme present therein, show an enhanced resistance against the deleterious effects of a number of, and particularly against a wide variety of, stress conditions.

More specifically, the invention concerns plant cells that produce one or more aldose reductase-homologous superfamily enzymes capable of reducing aldehyde groups at an enhanced level and, therefore, which posses an enhanced resistance against damaging effects of stress conditions involving the production of free radicals and/or against drought. Particularly provided is resistance against the damaging effects of free radical generation.

Plant cells, and thus plants, according to the invention advantageously overproduce an aldose reductase-homologous protein comprising the amino acid sequence presented in SEQ ID No 1 , more preferably the alfalfa sequence in SEQ ID No 3 (MsALRh protein) or a functional variant or derivative thereof, said variant being advantageously at least 50% homologous, more advantageously at least 70% homologous and even more advantageously at least 90% homologous to the aldose reductase-homologous protein sequence represented in SEQ ID No 1, and more preferably having such homology to the alfalfa protein of SEQ ID No 3. Still more preferably the cells and plants overproduce such enzymes comprising sequences having at least 50% identity, more preferably at least 70% identity and most preferably at least 90% identity with such sequences, most preferably with SEQ ID No 3.

Plant cells of the invention are advantageously transgenic and are transformed by incorporation therein of a nucleic acid molecule capable of expressing an aldose reductase-homologous protein. Plant cells, plant parts or plants according to the invention, however, can also be produced via traditional non-biological mutation- selection methods wherein, for example, a chemical or UV-light is employed as the mutagenic agent and selection of plants for overproduction of aldose reductase superfamily enzyme of required activity is carried out using assays eg. as described below. The level of aldose reductase expression can be determined by hybridisation (using Northern or Western blot techniques, see Example 2) or via detection of the enzymatic activity, particularly that against HNE and/or DL-glyceraldehyde eg. as described below.

The invention further concerns plants and plant parts comprising plant cells according to the invention. Plants and plant parts according to the invention advantageously posses an enhanced level of resistance against stress conditions involving the production of free radicals, more specifically for example against treatments with herbicides and/or heavy metals and/or producing hydrogen peroxide, and/or with NaCl; and/or infections caused by viruses and/or bacteria and/or fungi; and/or drought.

In a second aspect of the invention is provided nucleic acid sequences encoding an aldose reductase-homologous protein for use in the invention as well as recombinant nucleic acid molecules comprising such sequences. Particularly this nucleic acid is recombinant, isolated, enriched and/or cell free and encodes for the proteins described above having the aforesaid homology to SEQ ID No 1 or 3. This provides nucleic acid sequences coding for the aldose reductase-homologous protein, particularly for use in the invention and plant cells of the invention for use in producing plants and plant parts according to the invention.

By use of mutagenesis techniques, eg. such as SDM, the nucleic acids of the invention may be designed to encode the functional variants of the reductase proteins of the invention. Oligonucleotides and polynucleotides may also be used as probes and primers to identify further naturally occuring or synthetically produced reductase proteins using eg. southern or northern blotting

Preferably the nucleic acid is DNA or RNA, particularly cDNA or cR A and more preferably is characterised in that where it is a DNA it is a polynucleotide comprising nucleotide sequence having at least 80% identity with SEQ ID No 2 as listed in the sequence listing herein, or a sequence having degenerate substitution of codon nucleotides in that sequence, and where it is an RNA it has a complementary sequence wherein T is replaced by U. Preferably the identity is 90% or more, more preferably 95% or more and most preferably 100%. Preferably non-identical parts of the sequences comprise degenerate substitutions. Most preferred DNA or RNA is that which is capable of hybridizing with one or both strands of the nucleic acid of SEQ ID No 2, and polynucleotide and oligonucleotide fragments thereof of 15 or more contiguous bases, preferably 30 or more and more preferably 100 or more, selected from a characteristic region of the sequence with respect to nucleic acid encoding for other superfamily enzymes, under high stringency conditions, more preferably being capable of such hybridization with two or more of these polynucleotides or oligonucleotides. Most suitable selections of sequences for performing these hybridizations will be selected from coding regions of SEQ ID No 2 coding for parts that are unconserved with respect to the other members of the superfamily.

Thus with reference to Figure 1 below it will be seen that amino acids 41 to 49 and 250 to 256 tend to be conserved within the superfamily and thus these sequences as such are unsuitable for use in selecting hybridizing sequences for selecting the preferred homologs of alfalfa type other than in a screen for superfamily members in general.

In a third aspect of the invention is provided nucleic acid encoding for an aldose reductase homologous protein superfamily member, in the form of a vector or contract, combined in frame with a promoter, activating or otherwise regulating sequence capable of promoting its expression ectopically in plants, either constituitively or locally, eg. in vegetative or root tissues. Conveniently this expression is non-temporal, ie.unlike the Rocarati et al promoter, such that the plant tissue is protected from the damaging effects of free-radicals, eg. generation of HNE, at all times.

It will be realised by those skilled in the art that tissue specific regulatory regions, such as tissue specific promoters, may be used where a specific tissue requires protection from stress related toxins and/or osmoregulation. Examples of tissue specific promoters will be known to those skilled in the art, but may be exemplified by those of WO 97/20057 (incorporated herein by reference) teaching root specific promoters and those of Rocarti et al, being embryo and seed specific. For protection of vegetative tissues, tissue specific promoters may be used but constuitive promoters such as CaMv35S and alfalfa (MsH3gl) (see WO 97/20058 incorporated herein by reference) will have useful application.

In a fourth aspect of the invention there is provided a process for producing plant cells overproducing an aldose reductase-homologous protein, which comprises the transformation of a plant cell with a nucleic acid molecule according to the invention.

In a fifth aspect of the present invention there is provided a transgenic plant or part thereof comprising recombinant nucleic acid, a vector or construct as described above.Preferred plants of the fifth aspect may comprise the nucleic acid of the invention in a construct in functional association with promoter, activating or otherwise regulating sequences. Preferred promoters may be tissue specific such that the resultant expression of protein, and thus its effects, are localised to a desired tisssue. Promoters with a degree of tissue specificity will be known to those skilled in the art of plant molecular biology.

Methods of producing vectors and constructs capable of being used in the present invention will occur to those skilled in the art in the light of conventional molecular biology techniques. DNA, RNA and vectors containing or encoding for these may be introduced into target cells in known fashion in the field of plant cell transformation. Particularly preferred is the method of introducing the DNA or RNA into cells, more particularly pollen cells, using techniques such as electroporation or gene gun technology.

It may be preferred to express the DNA or RNA of the invention throughout the plant, but in the event that tissue specific effect is to be exploited then it will be understood by those skilled in the art that tissue specific promoters, enhancers or other activators should be incorporated into the transgenic cells employed in operative relation with the DNA.

Numerous specific examples of methods used to produce transgenic plants and plant cells by the insertion of cDNA in conjunction with suitable regulatory sequences will be known to those skilled in the art. Plant transformation vectors have been described by Denecke et al (1992) EMBO J. 11, 2345-2355 and their further use to produce transgenic plants producing trehalose described in US Patent Application Serial No. 08/290,301. EP 0339009 Bl and US 5250515 describe strategies for inserting heterologous genes into plants (see columns 8 to 26 of US 5250515). Electroporation of pollen to produce both transgenic monocotyledonous and dicotyledonous plants is described in US 5629183, US 7530485 and US 7350356. Further details may be found in reference works such as Recombinant Gene Expression Protocols. (1997) Edit Rocky S. Tuan. Humana Press. ISBN 0-89603-333- 3; 0-89603-480-1 (all these references being incorporated herein by reference). It will be realised that no particular limitation on the type of transgenic plant cell or plant to be provided is envisaged; all classes of plant, monocot or dicot, may be produced in transgenic form incorporating the nucleic acid of the invention such that aldehyde, particularly HNE, reducing activity in the plant is altered, constituitively or ectopically. Further aspects of the invention provide alfalfa homologous protein and

.antibodies raised thereto.

The below-defined terms will be used with the given meaning throughout the whole specification and in the claims even if said given meaning would anyway differ from the meaning of the said term as generally used in the art. An "aldose reductase-homologous protein" is defined in this description as an enzyme being a member of the aldo-keto reductase superfamily and similarly to other members of this family can reduce the aldehyde group of a wide variety of substrates in the presence of NADPH cofactor. The term "aldose reductase-homologous protein" as used herein also includes functional variants and derivatives of the said protein. A "functional variant" or a "functional derivative" of a protein is a protein the amino acid sequence of which can be derived from the amino acid sequence of the original protein by the substitution, deletion and/or addition of one or more amino acid residues in a way that, in spite of the change in the amino acid sequence, the functional variant retains at least a part of at least one of the biological activities of the original protein that is detectable for a person skilled in the art. A functional variant is generally at least 50% homologous (preferably the amino acid sequence is at least 50% identical), advantageously at least 70% homologous and even more advantageously at least 90% homologous to the protein from which it can be derived. A functional variant may also be any functional part of a protein; the function in the present case being particularly but not exclusively aldehyde reduction. Preferably the amino acid sequence differs from SEQ ID No 1 or SEQ ID No

3 mainly or only by conservative substitutions. More preferably the protein comprises an amino acid sequence having 90% or more, still more preferably 95%, sequence identity with SEQ ID No 1 or SEQ ID No 3 and optimally 100% identity with those sequences.

Algorithms and software suitable for use in aligning sequences for comparison and calculation of sequence homology or identity will be known to those skilled in the art. Significant examples of such tools are the Pearson and Lipman search based FAST and BLAST programs. Details of these may be found in Altschul et al (1990), J. Mol. Biol. 215: 403-10; Lipman D J and Pearson W R (1985) Science 227, pl435- 41. Publically available details of BLAST may be found on the internet at 'http://www.ncbi. nlm.nih.gov/BLAST/blast-help.html'. Thus such homology and identity percentages can be ascertained using commercially or publically available software packages incorporating, for example, FASTA and BLASTn software or by computer servers on the internet. An example of the former is the GCG Wisconsin Software package while both Genbank (see http://www.ncbi.nlm.nih.gov/BLAST) .and EMBL: (see http://www.embl-heidelberg.de/Blast2) offer internet services.

By the term identity is meant that the stated percentage of the claimed amino acid sequence or base sequence is to be found in the reference sequence in the same relative positions when the sequences are optimally aligned, notwithstanding the fact that the sequences may have deletions or additions in certain positions requiring introduction of gaps to allow alignment of the highest percentage of amino acids or bases. Preferably the sequence are aligned by using 10 or less gaps, ie. the total number of gaps introduced into the two sequences when added together is 10 or less. The length of such gaps is not of particular importance as long as the aldehyde reducing activity is retained but generally will be no more than 10, and preferably no more than 5 amino acids, or 30 and preferably no more than 15 bases.

Preferred parameters for BLAST searches are the default values, ie. for EMBL Advanced Blast2: Blastp Matrix BLOSUMS, Filter default, Echofilter X, Expect 10, Cutoff default, Strand both, Descriptions 50, Alignments 50. For BLASTn defaults are again preferably used. GCG Wisconsin Package defaults are Gap Weight 12, Length weight 4. FASTDB parameters used for a further preferred method of homology calculation are mismatch penalty = 1.00, gap penalty =1.00, gap size penalty = 0.33 and j oining penalty =30.0.

The expression 'high stringency conditions' will be understood by those skilled in the art, but are conveniently exemplified as set out in US 5202257, Col 9- Col 10, which is incorporated herein by reference.

The expression 'degenerative substitution' refers to substitutions of nucleotides by those which result in codons encoding for the same amino acid; such degenerative substitutions being advantageous where the cell or vector expressing the protein is of such different type to the DNA source organism cell that it has different codon preferences for transcription/translation to that of the cDNA source cell. Such degenerative substitutions will thus be host specific. The expression 'conservative substitutions' as used with respect to amino acids relates to the substitution of a given amino acid by an amino acid having physicochemical characteristics in the same class. Thus where an .amino acid in the SEQ ID No 1 or SEQ ID No 3 has a hydrophobic characterising group, a conservative substitution replaces it by another amino acid also having a hydrophobic characterising group; other such classes are those where the characterising group is hydrophilic, cationic, anionic or contains a thiol or thioether. Such substitutions are well known to those of ordinary skill in the art, i.e. see US 5380712 which is incorporated herein by reference, and are only contemplated where the resultant protein has activity as an aldehyde reducing enzyme, particularly acting upon HNE, eg. in the presence of NADPH.

A protein or RNA is said to be "produced at enhanced level" or "overproduced" if the concentration of the said protein in the cell where it is produced is at least 20% higher than that in the original cell, ie. a cell of the original plant line which has now been transformed. Similarly, a plant, a plant tissue or a plant cell is defined as having an "enhanced level of resistance" to a harmful condition if it can withstand a 20% enhanced effect of the same harmful condition, without detectable damage, than the original plant, plant tissue or plant cell. Overproduction of a molecule in a cell can be achieved via both traditional mutation and selection techniques and genetic manipulation methods.

The term "ectopic expression" is used herein to designate a special realisation of overproduction in the sense that, for example, an ectopically expressed protein is produced at a spatial point of a plant where it is naturally not at all (or not detectably) expressed, that is, said protein is overproduced at said point. Particular types of ectopic expression will comprise eg. expression of the aldo-keto reductase protein in vegetative tissues or roots of a plant, whereas normally it may be found only temporally expressed through transcripts in embryo and/or pollen (see Roncarati et al).

The present invention will now be further described by way of illustration only by reference to the following non-limiting Examples, Figures and Sequence listing. Further Examples falling within the scope of the invention will occur to those skilled in the art in the light of these.

SEQUENCE LISTING: The sequences of the sequence listing attached hereto are those as follows:

SEQ ID No 1 : Amino acid sequence shared by preferred aldose reductase- homologous enzymes to which homology of the enzymes for use in the present invention is related. SEQ ID No 2: DNA including cDNA encoding for an alfalfa (Medicago satvia) aldose reductase-homologous enzyme for use in the present invention.

SEQ ID No 3 : Amino acid sequence of an alfalfa (Medicago satvia) aldose reductase homologous enzyme for use in the present invention.

SEQ ID No 4 .and 5 are those of primers referred to in Example 3.

FIGURES Figure 1 : is a comparison of the amino acid sequence of the alfalfa MsALRh protein to human, animal and plant aldose and aldehyde reductase proteins.

Figure 2 shows the results of Southern hybridisation of genomic DNA isolated from alfalfa. 20μg of genomic DNA was digested with restriction enzymes indicated in the figure below each lane. The restriction fragments were separated on 1% agarose gel and blotted onto Hybond-N membrane. P-32 labelled coding region fragment of the MsALRh CDNA was used as hybridization probe.

Figure 3 shows the results of Northern hybridisation experiments with the MsALRh cDNA. Total RNA was isolated from alfalfa A2 cell suspension treated with 150μM ABA (abscisic acid) hormone (3a), 10% polyethylene glycol (PEG 4000, 3b) and cadmium chloride (3c), 20μg total RNA was electrophoresed on a 1% formaldehyde-agarose gel and transferred onto Hybond-N membrane, and subsequently probed with P-32 labelled coding region of the MsALRh cDNA fragment. C in each case is untreated control. Figure 4 depicts a Western hybridisation analysis of the MsALRh gene expression. 4a shows the level of protein in different alfalfa tissues with longer (upper) and shorter expression time. 4b shows the change of protein level during 10% PEG, 250μM cadmium chloride and ImM hydrogen peroxide treatments.

Figure 5 is a map of a vector of pGEX origin (pGEX-5X-3) suitable for the expression of the cDNA encoding the enzyme. The vector expresses the enzyme as a glutathion-S-transferase fusion protein in E. coli cells.

Figure 6 shows purification of MsALRh-GST fusion protein on glutathione- Sepharose.

Figure 7 shows the effect of ammonium sulphate treatment on the activity of the MsALRh-GST recombinant fusion protein. Enzymatic activity on lOmM DL- glyceraldehyde substrate was measured spectrophotometrically by determining the measure of NADPH oxidation at 340 run. The standard reaction mixture comprised 5 μg enzyme and 0.15 mM NADPH. Before starting the activity measurement, the enzyme was preincubated with ammonium-sulfate for 3 minutes. Figure 8 shows the Western hybridisation detection of the aldose reductase- homologous protein in cell extracts isolated from the leaves of transgenic tobacco plants.

Figure 9a and 9b show the results of light induced fluorescence measurements of control and transgenic plant leaf discs during 20μM paraquat (9a) and 250μM cadmium chloride (9b) treatments.

Figures 10a, 10b, 10c and lOd demonstrate the results of germination tests of control (SRI) and transgenic (1/1, 1/5, 1/9) plants in control 0 conditions (10a), in the presence of 15mM hydrogen peroxide (10b), 150μM cadmium chloride (10c) and 250mM sodium chloride (lOd).

Figure 11 shows results of light induced fluorescence measurements during water deficient growth of control (SRI) and transgenic (TR1/4, 1/7, 1/1, 1/5 and 1/9) plants.

Figure 12a shows a photograph of control and transgenic plants after 10 days of rehydration after a 35 day severe drought treatment. 12b shows detection of the aldose reductase-homologous protein in cell extracts made from leaves of control and transgenic plants shown in 12a after the aforesaid drought tolerance experiment .

Figure 13 illustrates the photsynthetic efficiency measured as Gentry-parameter of chlorophyll fluorescence in tobacco leaves before (day 1), during (days 2-4) and after (day 8 and day 15) UV-B irradiation. Full circles represent average data for plants 1/1, 1/5, 1/8, 1/9 and 1/10. Empty circles represent data from plants 1/4 1/7 and control SRI.

Experiments In the experimental examples below the inventors demonstrate the isolation of a gene encoding an alfalfa (Medicago sativa) aldose reductase-homologous protein, in cDNA form. Its nucleotide sequence and the deduced amino acid sequence verified that the isolated cDNA codes for a novel plant aldose reductase-homologous protein that has not been identified so far. The isolated aldose reductase cDNA was cloned into a plant expression vector and by the means of a generally used gene introduction method transformed tobacco plants were produced. Beyond the molecular biological characterization of the transgenic tobacco plants their increased tolerance to different stress conditions has also been shown.

Example 1.

Isolation of the alfalfa aldose reductase-homologous cDNA (MsALRh) from a gene bank and prediction of the encoded amino acid sequence

For the preparation of the alfalfa cDNA library and the identification of the aldose reductase-homologous cDNA widely used molecular biology methods were applied according to the protocols of Sambrook, J. et al. (Sambrook, J. et al : Molecular cloning, A Laboratory Manual, 2nd edition, Cold Spring Harbour N.Y., 1989, incorporated herein by reference). The details of the procedure can be summarised as the following:

The alfalfa cDNA library was produced as follows. Total cellular RNA was isolated from in vitro cultivated auxin shock activated alfalfa tissues, which contained dedifferentiated callus tissues and high number of somatic embryos. Stress activation (auxin shock) was performed by using 2,4- dichloro-phenoxyacetic acid (an auxin analog) applying a method known per se so as to enhance the concentration of stress induced mRNAs. The mRNA isolation method applied is detailed in Cathala et al. (1983 DNA 2: 329-335) incorporated herein by reference.

The mRNA molecules were then separated from the total RNA by the means of oligo-dT cellulose chromathography according to the method of Aviv, H. and Leder, P. (1972 Proc Natl. Acad. Sci. USA 69: 1408-1412) incorporated herein by reference. The first cDNA strand was then synthesised on the isolated poly-A⁺ (mRNA) fraction with an oligo-dT primer using AMV reverse transcriptase enzyme. Synthesis of the second DNA strand was done using DNA polymerase I enzyme, removal of the excess of template mRNA was performed via RNase H treatment. Synthesis of poly- dC tails to the end of the synthesised cDNA and poly-dG tails to Pstl digested pGEM2 vector (Promega) end was done using terminal transferase enzyme. After annealing and ligation using T4 DNA ligase the constructs were transformed into MCI 061 E. coli strain and the transformation mixture was plated out onto 100 mg/1 ampicillin containing selective LB-plates. Using 25 μg DNA, 2.5X10⁵ primary transformant colonies were obtained. It was shown that approx. 96% of the clones contained inserts of detectable sizes. The preparation procedure of the cDNA library followed widely used protocols of the art and was especially based on that of De Loose et al. (1988 Gene 70: 13-23) incorporated herein by reference.

The nucleotide sequence of about a hundred individual clones of this cDNA library (i.e. cDNA clones prepared from a stress induced mRNA isolate) was determined. Before sequencing, the inserts were subcloned into plasmid pUC 19 and the sequencing reaction was carried out on double stranded DNA using the dideoxy chain termination method with α-35-S-dATP labelling. For the sequencing reactions the T7 Sequencing Kit (Pharmacia) and Sequenase 2.0 Kit (US Biochem) were used, according to the manufacturers' protocols. Using the determined cDNA sequence, Genebank and ΕMBL nucleotide sequence databases were homology searched. The result showed that the amino acid sequence derived from the clone later designated MsALRh has homology to both human, rat and plant aldo-keto reductase proteins. The length of the MsALRh cDNA is 1231 base pairs and the coding region can be located between 34 and 975 nucleotides (SΕQ ID no 2). This region codes for a 313 amino acid protein. The amino acid sequence of the alfalfa aldose reductase-homologous protein showed 44.3% identity with the known and best characterised monocot barley aldose reductase-homologous protein and it has 46.2% and 46.1% identity with human aldehyde reductase and pig aldose reductase enzymes, respectively (Fig. 1). It further showed a 42% amino acid identity to the dicotyledonous soybean NADPH-dependent oxidoreductase enzyme. The observed level of amino acid homology led us to conclude that the cloned alfalfa cDNA codes for a novel enzyme of the aldo-keto reductase superfamily. Example 2.

Molecular biological characterisation of the alfalfa aldose reductase-homologous gene The thorough molecular biological characterisation of the alfalfa aldose reductase-homologous gene according to the invention showed that its behaviour differed from the known plant aldose reductase-homologues. The copy number of the gene in the alfalfa genome was analysed with Southern hybridisation; the expression of the gene was analysed by Northern hybridisation on the mRNA level and by Western hybridisation on the protein level.

Genomic DNA was isolated from alfalfa cells and the purified DNA was digested with restriction endonucleases. The MsALRh cDNA fragment carrying full length coding sequence was used as probe for the Southern hybridisation experiments after radioactive isotope labelling using the "random priming" method (Freinberg, A.P. and Vogelstein, B. 1983. Anal. Biochem. 137: 266-267). The hybridisation was carried out in Rapid-hyb buffer (Amersham) at 65°C. The result of the hybridisation (Fig. 2) showed that the aldose reductase-homologous gene has a low copy number but faint additional bands on the autoradiogram present at al restriction digestion might indicate the presence of other gene homologues in the alfalfa genome.

The expression of the gene was analyzed by Northern hybridisation using generally known methods [Sambrook, J. et al : "Molecular cloning, A Laboratory Manual" (2^nd edition, Cold Spring Harbour N.Y., 1989.) incorporated herein by reference]. The analysis can be detailed as follows:

Total RNA was purified from alfalfa A2 cell suspension according to the method of Maes (1992. Nucl. Acids Res. 20: 4374) incorporated herein by reference. After separation on formaldehyde-agarose gel the RNA was transferred onto Hybond- N membrane (Amersham). Full length coding region of the MsALRh gene was radioactively labeled using the "random priming" method (Freinberg, A.P. and Vogelstein, B. 1983. Anal. Biochem. 137: 266-267, incorporated herein by reference) and hybridizations were carried out at 65°C in Rapid-hyb hybridisation buffer (Amersham). The tissue specificity experiments revealed that the alfalfa aldose reductase-homologous gene is expressed in each tissue in contrary to the barley aldose reductase-homologue, which was expressed only temporally and only in embryos. The alfalfa cell suspension was exposed to different hormone and stress treatments. The results indicated that the studied gene is homoinduced. Treatment with the plant stress hormone abscisic acid (ABA) significantly enhanced the RNA level 4 hours after its initiation (Fig. 3). The importance of this finding lies in the fact that hormone ABA is a key compound in the regulation of several genes that play important role in the adaptation to environmental stresses such as drought and low temperature.

Treatment with polyethylene-glycol results in an osmotic stress in plant cells. Expression of the MsALRh gene could be induced by such an osmotic stress as well as by cadmium-chloride treatment (heavy metal stress; Fig. 3). Cadmium ion is known to induce oxidative stress beside its photosynthetic inhibitory property .and in these cases the level of lipid peroxides increases in the plant cells. As it was also disclosed in the introduction, the decomposition products of the lipid peroxides are those lipid aldehydes that can serve as substrates of the aldo-keto reductases in the detoxification reactions.

Example 3.

Biochemical characteristics of the alfalfa aldose reductase-homologous protein

In order to characterise the substrate specificity of the enzyme, a recombinant protein was produced and purified in a prokaryotic protein expression system.

The cDNA of the MsALRh gene was cloned into pGEX 4T-1 prokaryotic expression vector (Pharmacia) and the GST (gluthathion-S-transfarase) containing fusion protein was produced in and purified from E. coli. Using the purified fusion protein as antigen, rabbit polyclonal antibody was raised by conventional techniques. This antibody was used to analyse, by Western hybridization, the effects of different stress treatments on the production of the MsALRh protein and to detect the protein in different tissues of the alfalfa plant (Fig. 4).

The coding region (975 bp) of the MsALRh cDNA was PCR amplified, using

"primerl" (5'-cgaactcgagatggccacagcaatcaagttt-3') as upper and "primer2" (5'- ccgagctctacttcaccatcccagag-3 ' ) as lower primer (Xhol restriction sites in the primers are underlined) according the method of Mullis and Faloona (1987 Meth. Enzymol. 155: 335) incorporated herein by reference. The PCR product was digested with Xhol restriction endonuclease and the digested fragment was cloned into the Xhol site of the pGEX 4T-1 vector (Pharmacia). The nucleotide sequence of the cloned product was verified in order to avoid PCR generated errors.

In order to induce the production of the N-terminal GST containing fusion protein (MsALRh-GST protein, later on) the transformant E. coli cells were treated with 0.5 mM isopropyl-β-D-galactopyranoside (IPTG) at 25°C for 3 hours. Transformants expressing the fusion protein were identified according to the method of Smith and Johnson (1988 Gene 67: 31-40) incorporated herein by reference. The MsALRh-GST fusion protein was purified on glutathione-Sepharose 4B columns following the instructions of the manufacturer's protocol (Pharmacia).

The produced and purified MsALRh-GST fusion protein (100-150 μg in 0.5 ml) was thoroughly mixed with the same volume of complete Freund's adjuvant (Sigma) and rabbit was immunised with this emulsion. Two subsequent immunisations were made with the emulsion of the antigen and incomplete Freund's adjuvant after 3 and 6 weeks, respectively. The blood serum was checked in Western analysis. The specificity of the serum for the MsALRh protein was tested in a competition experiment. For the Western blot analysis, proteins of the alfalfa cell suspension extracts were separated on a 12% SDS-polyacrylamide gel. After separation, the proteins were transferred onto nitrocellulose membrane. The blood serum raised against the fusion protein was used as first antibody (in 1 :2000 dilution); anti-rabbit-IgG antibody conjugated with peroxidase was used as second antibody in these experiments. The antigen-antibody complex was detected using Super Signal^R™ CL-HRP Substrate System (Pierce), a high sensitivity chemiluminescent detection method.

The presence of the MsALRh protein in different tissue types of the alfalfa plant was examined. The result clearly indicated (Fig. 4a) that the protein can be found in each tested plant organ. This result is absolutely in contrast with those published by Bartels, D. et al. (1991. The EMBO J. 10: 1037-1043) because the barley aldose reductase-homologous protein was not present in detectable amounts in vegetative tissues. Our result showed the difference of the alfalfa aldose reductase-homologous protein from the previously isolated plant aldose reductases and may indicate it's different physiological role, as well. The increase in the amount of the MsALRh protein during osmotic stress induced by PEG treatment or during heavy metal stress induced by cadmium-chloride treatment as well as due to toxic free radical production by hydrogen peroxide treatment was also detected by the Western hybridisation technique (Fig. 4b). These results showed that the increase of the MsALRh protein level during osmotic and heavy metal stress correlated with the observed increase of the mRNA level of the gene and showed that the oxidative stress-inducing hydrogen-peroxide treatment also increased the MsALRh protein level in alfalfa cells. These results strongly support a role for the MsALRh protein in plant defence reactions against the toxic effect of reactive free radicals. The substrate specificity tests of the MsALRh enzyme - besides the amino acid homology - proved that the MsALRh gene codes for an enzyme belonging to the aldoketo reductase superfamily and, at the same time, it also has numerous different characteristics as compared to the so far characterised plant aldose reductase- homologues. Enzymatic activity of the MsALRh-GST fusion protein on different substrates was measured photometrically: measuring the decrease of the concentration of the NADPH cofactor at 340 nm wavelength at 25°C during a 5 minutes reaction time. One ml of the reaction mixture contained 50 mM sodium-phosphate buffer (pH=7.0), 0.1 mM NADPH as cofactor and different concentrations of the substrates. One enzyme activity unit is expressed as the enzyme amount (in mg) which is necessary for the oxidation of 1 μmol NADPH in one minute in the presence of the substrate. Aldose sugars, DL-glyceraldehyde and 4-hydroxy-nonenal (ICN Biochemicals) were used as substrates in these specificity measurements, and the results (as Km kinetic constants) are shown in Tables 1-3 below. According to our results the purified enzyme is able to reduce aldehyde substrates in the presence of NADPH cofactor. High enzymatic activities could only be measured on DL- glyceraldehyde (known as the best substrate for plant aldose reductase-homologues) and on 4-hydroxy-nonenal. The MsALRh enzyme could reduce D-xylose with much less activity and showed no activity on other aldose substrates (such as D-glucose, D- galactose, D-mannose or D-ribose) even at very high (400 mM) substrate concentrations. According to the results (Table 1 below) the alfalfa enzyme showed very similar activity on glyceraldehyde substrate as compared to the animal aldehyde reductases. At the same time, it works less effectively in the presence of 4-hydroxy- nonenal (Table 2).

Table 1 Comparison of Km kinetic parameters on DL-glyceraldehyde substrate of plant aldose reductase-homologues and mammalian aldehyde reductases

Table 2

Km kinetic parameters of aldose reductase enzymes on 4-hydroxy-nonenal substrate

Table 3

Alteration of the activity of aldose and aldehyde reductases as a consequence of a treatment with 0.3 M (NH₄)₂SQ₄

We have examined whether the MsALRh enzyme could reduce the aldehyde group of 4-hydroxy-nonenal (HNE), which was mentioned earlier as one of the degradation products of the lipid peroxides, thus eliminating its previously disclosed toxic effect. Our results showed that the HNE is better substrate for the MsALRh than the DL-glyceraldehyde, formerly known to be the best substrate for plant aldose reductases. The Km kinetic constant value for HNE is approx. 700 μM that is about 1/3 of the value having been measured on glyceraldehyde. These measurements were first carried out using the ALRh-GST fusion protein but in the subsequent experiments measurements were repeated with protein from which the GST-tag was removed via a cleavage by thrombin at the thrombin recognition site present between the MsALRh and the GST moieties. The Km values for both DL-glyceraldehyde and 4-hydroxy-nonenal were the same in both cases thus we could conclude that the GST fusion partner did not alter the substrate specificity of the MsALRh enzyme.

The S0 ^2" ions are known to alter the enzymatic activity of rat aldose and aldehyde reductases in a different manner. In our case the resulted twofold increase in the enzymatic activity in the presence of 0.3 M ammonium-sulfate is a characteristic of the aldose reductases (Fig. 7). An important property of the MsALRh enzyme is that it's activity increases by 100% even in the presence of low concentration (45 mM) of ammonium-sulfate. This result may indicate that the advantageous effect of the enzyme can be increased in the transgenic plants by ammonium-sulfate treatment. Based on these results we can conclude that the MsALRh enzyme, as well, as the barley aldose reductase-homologous enzyme, has also similar characteristics to both the animal aldose and aldehyde reductases but it possesses specific different characteristics, too.

Example 4. Insertion of the alfalfa aldose reductase-homologous (MsALRh) cDNA into tobacco plants to produce the aldose reductase protein in the vegetative organs of transgenic plants

Foreign genes can be introduced into plant genomes by the Agrobacterium- based transformation system (for details see: Hiche, E. et al. "Plant Cell and Tissue Culture" 1994. pp. 231-270, ed. Vasil, I.K. and Thorpe, T.A., Kluwer Academic Publisher) incorporated herein by reference. The cDNA studied was fused to the viral CaMV35S promoter disclosed in Benfey et al 1989. EMBO J. 8: 2195-2202 incorporated herein by reference.

The produced binary vector construct was mobilized into an Agrobacteήum strain, tobacco leaves were infected by scratching, co-cultivation was performed and kanamycin resistant plants were regenerated according to Claes et al. (1991. The

Plant J. 1: 15-26) incorporated herein by reference. Transformant plants and seeds obtained by self-pollinating were used in the germination experiments.

The functioning of the alfalfa aldose reductase gene in transgenic tobacco plants was proved by immunological methods.

Expression of the aldose reductase gene in the transgenic tobacco plants was verified by Western blot analysis using the polyclonal antibody raised against the MsALR-GST fusion protein. Ten tobacco lines were analysed (Fig. 8). Protein extracts obtained from the leaves of these lines were tested according to the western hybridisation protocol detailed previously except that the protein samples were separated on 10% SDS-polyacrylamide denaturing gel and alkaline phosphatase conjugated anti-IgG antibody was used as second antibody. The antibody-protein complex was detected by BCIP (5-bromo-4-chloro-3-indolyl-phosphate) and NBT (2,2'-di-p-nitrophenyl-3,3'-[3,3'-dimethoxi-4,4'-diphenylene]-ditetrazolium-chloride) substrates. Synthesis of considerable amount of the MsALRh protein could be detected in five of the ten examined transgenic lines, the other transgenic lines showed no expression of the MsALRh gene and no signal could be detected in the control wild type (SRI) plant, either.

Example 5.

Transformant tobacco plants expressing alfalfa aldose reductase-homologous protein show elevated level of tolerance against different stress treatments, such as herbicide, heavy metal, hydrogen peroxide and sodium chloride

In order to verify the concept of the invention, the behaviour of the transformants were tested in the presence of paraquat (PQ) herbicide and cadmium (Cd). Leaf discs were cut from the leaves of control SRI and transformants and treated with paraquat (20 μM) and cadmium chloride (250 μM) solutions in Petri- dishes. The functional damage of the photosynthetic apparatus was monitored by light induced fluorescence measurements using PAM fiuorimeter according to Vass. I. et al. (Biochem. 1996. 35: 8964-8973). The decrease of the fluorescence intensity in the case of the control SRI plant showed considerable damage after a 24 hour treatment and this effect was more pronounced after 48 hours. In contrast, the intensity decreased much less in the case of the transgenic lines and thus showed a significantly reduced loss of photosynthetic functions (Fig. 9). Germination tests were also used to check the stress resistance of the transformants. After self-pollination, seeds from the transformants were grown on culture media containing 15 mM hydrogen peroxide, 150 μM cadmium chloride and 250 mM sodium chloride, respectively. The seeds were germinated under controlled conditions in the light. As Fig. 10 shows every transformant line is more resistant to the applied stress treatments than the control SRI tobacco plants. The protective effect of the expressed MsALRh protein clearly appears to be concentration dependent as transgenic line 1/5 was found to be the most tolerant against these treatments and this line expressed the transgene at the highest level. Example 6.

Transgenic tobacco lines producing the alfalfa aldose reductase-homologous protein show resistance against water deficiency

Transformant tobacco plants were grown in soil under controlled greenhouse conditions for 5 weeks and the water content of the soil was measured at the beginning of the experiment. The damage of the photosynthetic system was monitored during the experiment by light induced fluorescence measurements as detailed in the above experiments. Prior to the beginning of the experiment we have selected plants with similar developmental stage from the transgenic tobacco lines and the SRI control plants and leaves with the same age were chosen on each plant for the fluorescence measurements to be made at given time points during the experiment.

At the beginning of the experiment we stopped the watering of the tested plants. Between days 32 and 35 of the water deficient growth the Fv/Fm fluorescence values showing viability of the plants dropped intensively in the case of the SRI plant and lines 1/4 and 1/7 (Fig. 11), while did not decrease significantly in the case of the lines 1/1, 1/5 and 1/9. The tested plants on day 45 of the experiment are shown on Fig. 12. After the desiccation period the humidity of the soil was determined. At this point the plants were rehydrated for 10 days and the light induced fluorescence values of the pre-selected leaves were measured again. In the case of lines 1/1, 1/5 and 1/9 these values were about the same as the day 45 values or slightly better, however, in the case of the SRI control and the non-expressing 1/4 and 1/7 lines the Fv/Fm value continued to decrease. After rehydration, protein extracts were isolated from the leaves and the MsALRh protein level was determined (Fig. 13). The result shows that in SRI, 1/4 and 1/7 plants the protein was not detectable, whereas in 1/1, 1/5 and 1/9 lines the MsALRh protein was present. Thus we could conclude that only those plants could tolerate desiccation that expressed the MsALRh protein.

In order to prove that this increased drought resistance was not due to the different water storage capability of the leaves, the dehydration experiment was repeated on single leaves. The results showed, that the speed of water loss was approximately the same in both MsAlRh protein producing and control plants. Example 7.

UV-B irradiation protection.

Tobacco plants were kept in a ventilated growth chamber for five test days at 26°C. Photosynthetically active radiation was provided from fluorescent tubes (Tungsten 40W) at 60-80μmol m^"2 s^"2 intensity from above and from the side between 8am and 6pm daily. UV-B (280-320nm) irradiation was provided from UV-B 313 tubes (Q- panel, USA) at 7.5μmol m^" s^" total UV-B intensity from above between 9am and 3pm daily, for 4 days starting from the second day of the test. To exclude the effect of possible UV-C (λ < 280nm) irradiation, one layer of cellulose acetate filter (Courtaulds Chemicals UK) was placed between the plants and the UV-B source. During the recovery period plants were kept in the green house under normal growth conditions.

For chlorophyll fluorescence measurement plants were transferred to the laboratory. Measurement was performed in the dark and was completed in ca. 45 minutes including the initial 15 minutes dark adaptation. After this plants were transferred back to the growth chamber or (during the recovery period) to the green house. All chlorophyll fluorescence measurements were performed in the afternoon between ca 3 and 5pm. Untreated plants were measured in the afternoon of the first test day, before starting UV-B irradiation at the second test day. UV-B irradiated plants were measured on the 2^nd-5^th test days, 30 minutes after cessation of irradiation.

Chlorophyll fluorescence was measured with a PAM Chlorophyll Fluorometer (WALZ Company, Germany) using weak (<1 μmol m^" s^" ) modulated red light . Steady state (F_s) and maximal (F_m') fluorescence yields were measured before and upon a 0.5s saturating white light pulse (2500 μmol m^" s^" ), respectively, in 11.5μmol m^"2 s^"2 actinic red light. Efficiency of the photosynthetic electron transport was characterised with the Gentry-parameter, calculated as

Two leaves were tested on each plant and two chlorophyll fluorescence measurements were performed on each leaf, on the adaxial side, symmetrically on both sides of the midrib. Results: Plant leaves showed visible symptoms of UV-B damage, ie. spots with damage pigmentation, from the third day of the test, after two days of UV-B irradiation. These became more intense during further irradiation and remained on the leaves even during the recovery period. Chlorophyll fluorescence indicated decline of photosynthetic efficiency (Gentry parameter) from the firstUV-B irradiation day. In the case of plants 1/1, 1/5, 1/8, 1/9 and 1/10 this recovered to ca. 90% of original after one week recovery in the greenhouse. In the control SRI, 1/4 and 1/7 plants there was no marked recovery.

Photosynthetic efficiency was measured as the Gentry-parameter of chlorophyll fluorescence in tobacco leaves before (day 1), during (days 2-4) and after (days 8 and

15) UV-B irradiation. In Figure 13 full circles represent average data obtained using plants 1/1, 1/5, 1/8, 1/9 and 1/10. Empty circles represent average data measured in plants 1/4, 1/7 and SRI.

Summarising the results of the above-detailed experiments and considering the biochemical characteristics of the enzyme we can conclude that:

(1) The isolated alfalfa aldose reductase-homologous gene codes for an enzyme of novel characteristics, which can react with the toxic lipid aldehyde 4- hy droxy-nonenal . (2) Transgenic plants expressing the alfalfa aldose reductase-homologous gene were produced to make use of these and other characteristics of the enzyme and the produced plants were proven to be resistant against a wide range of harmful stress treatments.

(3) The described examples clearly prove the usefulness of the concept of the invention, which was also supported by theoretical considerations, namely, plants overproducing the alfalfa aldose reductase-homologous enzyme have an increased resistance against numerous stress conditions of diverse origin.

As the fundamentally novel approach of the invention does not comprise any species specific essential feature, the skilled person can reasonably conclude that plant cells of any species oveφroducing aldose reductase-homologous protein of any origin will have an enhanced resistance against numerous different stress conditions. Based on the above demonstrated results, considering that the general protecting effect of the aldose reductase-homologous protein is most probably based on the disclosed basic cell biological processes, one can also reasonably conclude that the approach of the invention is also useful for developing resistance against further stress conditions, for example against high or low temperature, UV-B irradiation, and plant pathogen action.