Stress tolerant plants

The present invention relates to methods for improving environmental stress tolerance of plants and plants with such improved stress tolerance. More particularly, the invention relates to the finding that the expression of a flavoprotein such as flavodoxin within plant cells is beneficial to plants which are subjected to environmental stress.

BACKGROUND OF THE INVENTION

Environmental stress is a major limiting factor for plant productivity and crop yield. Many of the deleterious processes undergone by plants exposed to adverse environmental conditions are mediated by reactive oxygen species (ROS) which are generated in chloroplasts through the faulty performance of the photosynthetic apparatus (Foyer, C. H. et al. (1994) Plant Cell Environ. 17,507-523, Hammond-Kosack, K. E., and Jones, J. D. G. (1996) Plant Cell 8, 1773-1791, Allen, R. (1995) Plant Physiol. 107, 1049-1054).

Auto-oxidation of components of the photosynthetic electron transport chain leads to the formation of superoxide radicals and their derivatives, hydrogen peroxide and hydroxyl radicals. These compounds react with a wide variety of biomolecules (most conspicuously, DNA), causing cell stasis and death.

To cope with the damaging effects of reactive oxygen species (ROS), aerobic organisms have evolved highly efficient antioxidant defense systems which are made up of both enzymatic and non-enzymatic constituents. In different tissues and organisms, antioxidants play different and often complementary protective functions, such as direct scavenging of ROS, replacement of damaged oxidant sensitive biomolecules and DNA repair activities (Fridovich, I. (1997). J. Biol. Chem. 272,1851-1857). At least part of the cellular response against oxidative stress is of an adaptive nature and involves de novo synthesis of committed members of the antioxidant barrier. Various multigenic responses have been recognized in the facultative aerobic bacterium

Escherichia coli

, including those modulated by the soxRS and oxyR regulons (Hidalgo, E., and Demple, B. (1996). In Regulation of Gene Expression in

Escherichia coli

, Molecular Biology Intelligence Unit Series (E. C. C. Lin and A. S. Lynch, eds.), pp. 434-452, Austin, Tex.: R. G. Landis).

The soxRS response appears to be specifically tailored to face the challenges imposed by exposure of the cells to superoxide radicals or to nitric oxide. Many different components of the response have been identified, including two soluble flavoproteine: FAD-containing ferredoxin-NADP+ reductase (FNR), and its electron partner substrate flavodoxin (Liochev et al. (1994) Proc. Natl Acad. Sci. U.S. Pat. No. 91,1328-1331, Zheng, M. et al (1999) J. Bacteriol. 181,4639-4643).

Flavodoxins are small monomeric proteins (Mw 18,800) containing one molecule of non-covalently bound FMN (Razquin, P. et al (1988) J. Bacteriol. 176, 7409-7411). FNR is able to use, with roughly similar efficiencies, both flavodoxin and the iron-sulfur protein ferredoxin as substrates for its NADP(H) oxidoreductase activity. In cyanobacteria, flavodoxin expression is induced under conditions of iron deprivation, when ferredoxin cannot be synthesized.

As part of the soxRS response of

E. coli

, both FNR and flavodoxin levels increase over twenty times upon treatment of the bacteria with superoxide-propagating compounds such as the redox cycling herbicide methyl viologen (MV), whereas ferredoxin amounts are not affected (Rodriguez, R. E. et al (1998) Microbiology 144,2375-2376). Unlike FNR and ferredoxins, which are widely distributed among plastids, mitochondria and bacteria, flavodoxin occurrence appears to be largely restricted to bacteria. Flavodoxins have not been isolated from plant tissues, and no flavodoxin homologue has been recognized in the

Arabidopsis thaliana

genome (The Arabidopsis Genome Initiative (2000) Nature 408,796-815).

The present invention relates to the finding that plant lines which have been engineered to express a flavoprotein such as flavodoxin display highly enhanced tolerance compared to control, untreated plants, when exposed to a plethora of adverse environmental conditions.

SUMMARY OF THE INVENTION

In various aspects, the present invention provides nucleic acids and vectors suitable for use in methods of producing stress tolerant plants. In preferred embodiments, such nucleic acids and vectors provide for the accumulation of flavoprotein within the choloroplasts of plant cells transformed therewith. In some embodiments of the invention, accumulation within the chloroplasts is achieved by fusing the flavoprotein to a chloroplast targeting polypeptide.

A first aspect of the present invention provides an isolated nucleic acid encoding a fusion polypeptide comprising a flavoprotein polypeptide and a chloroplast targeting peptide.

A nucleic acid may encode a fusion polypeptide comprising a flavoprotein polypeptide and a chloroplast targeting peptide.

A flavodoxin polypeptide may be a bacterial flavodoxin polypeptide, for example a cyanobacterial flavodoxin polypeptide such as the flavodoxin of the cyanobacterium Anabaena PCC7119 (Fillat M. et al (1991) Biochem J. 280 187-191). Other suitable flavodoxin polypeptides include flavodoxins from photosynthetic anoxigenic bacteria, enterobacteria, diazotrophs and algae. Examples of flavodoxin polypeptides suitable for use according to the present invention are exemplified in Table 1. Whilst a wild type flavodoxin polypeptide is preferred, a flavodoxin polypeptide may also be a fragment, mutant, derivative, variant or allele of such a wild type sequence.

Suitable fragments, mutants, derivatives, variants and alleles are those which encode a protein which retain the functional characteristics of the polypeptide encoded by the wild-type flavoprotein gene, especially the ability to act as an anti-oxidant. Changes to a sequence, to produce a mutant, variant or derivative, may be by one or more of addition, insertion, deletion or substitution of one or more nucleotides in the nucleic acid, leading to the addition, insertion, deletion or substitution of one or more amino acids in the encoded polypeptide. Of course, changes to the nucleic acid which make no difference to the encoded amino acid sequence are included.

Flavodoxin polypeptides are monomeric hydrophillic flavoproteins of a molecular mass of less than 20 kDa, containing one mole of non covalently bound flavin mononucleotide (FMN) per molecule of apoprotein. The flavin group can be reversibly dissociated by mild acid treatment.

Flavodoxin polypeptides engage in one-electron transfer reactions with several electron partners such as FNR, pyruvate-flavodoxin reductase and photosystems, replacing ferredoxin in most of its activities. Even though flavodoxin can in principle exchange two electrons, it behaves as an obligatory one-electron carrier. Contrary to other flavoproteins, the half-reduced semiquinone and the fully reduced hydroquinone are the most stable species, and these are the forms relevant for flavodoxin functions.

A polypeptide which is a member of the Flavodoxin family or which is an amino acid sequence variant, allele, derivative or mutant thereof may comprise an amino acid sequence which shares greater than about 30% sequence identity with the sequence of Anabaena PCC7119 flavodoxin, greater than about 35%, greater than about 40%, greater than about 45%, greater than about 55%, greater than about 65%, greater than about 70%, greater than about 80%, greater than about 90% or greater than about 95%. The sequence may share greater than about 30% similarity with Anabaena PCC7119 flavodoxin, greater than about 40% similarity, greater than about 50% similarity, greater than about 60% similarity, greater than about 70% similarity, greater than about 80% similarity or greater than about 90% similarity.

In certain embodiments, a flavodoxin polypeptide may show little overall homology, say about 20%, or about 25%, or about 30%, or about 35%, or about 40% or about 45%, with the Anabaena PCC7119 flavodoxin sequence, even though it possesses the same anti-oxidation activity. However, in functionally significant domains or regions, the amino acid homology may be much higher. For example, a flavodoxin polypeptide comprises an FMN-binding domain which has high homology to the flavodoxin FMN binding domain (a flavodoxin-like domain). Putative functionally significant domains or regions can be identified using processes of bioinformatics, including comparison of the sequences of homologues.

Sequence similarity and identity is commonly defined with reference to the algorithm GAP (Genetics Computer Group, Madison, Wis.). GAP uses the Needleman and Wunsch algorithm to align two complete sequences that maximizes the number of matches and minimizes the number of gaps. Generally, default parameters are used, with a gap creation penalty=12 and gap extension penalty=4.

Use of GAP may be preferred but other algorithms may be used, e.g. BLAST (which uses the method of Altschul et al. (1990)

J. Mol. Biol

. 215: 405-410), FASTA (which uses the method of Pearson and Lipman (1988)

PNAS USA

85: 2444-2448), or the Smith-Waterman algorithm (Smith and Waterman (1981)

J. Mol Biol

. 147: 195-197), or the TBLASTN program, of Altschul et al. (1990) supra, generally employing default parameters. In particular, the psi-Blast algorithm (Nucl. Acids Res, (1997) 25 3389-3402) may be used.

Similarity allows for “conservative variation”, i.e. substitution of one hydrophobic residue such as isoleucine, valine, leucine or methionine for another, or the substitution of one polar residue for another, such as arginine for lysine, glutamic for aspartic acid, or glutamine for asparagine. Particular amino acid sequence variants may differ from a known flavodoxin polypeptide sequence as described herein by insertion, addition, substitution or deletion of 1 amino acid, 2, 3, 4, 5-10, 10-20 20-30, 30-50, or more than 50 amino acids.

Sequence comparison may be made over the full-length of the relevant sequence described herein, or may more preferably be over a contiguous sequence of about or greater than about 20, 25, 30, 33, 40, 50, 70, 120, 170 or more amino acids or nucleotide triplets, compared with the relevant amino acid sequence or nucleotide sequence as the case may be.

Other methods suitable for use in identifying flavodoxin polypeptides are well-known in the art.

In other embodiments, the isolated nucleic acid may encode a fusion polypeptide which comprises an FNR polypeptide and a heterogeneous chloroplast targeting peptide.

Many ferredoxin-NADP(+) reductase (FNR) polypeptides are known in the art and an FNR polypeptide suitable for use in accordance with embodiments of the present invention may readily identified by a skilled person. Other suitable FNR polypeptides may be found on the NCBI database at www(dot)ncbi(dot)nlm(dot)nih(dot)gov(forward slash)entrez(forward slash), for example, FNR polypeptide sequences having database accession numbers NP 418359, NP 312876, P28861, and AAG59117. Suitable PNR polypeptides have the anti-oxidant and electron transfer activity of wild type ferredoxin-NADP(+) reductase.

A chloroplast targeting peptide suitable for use in accordance with certain embodiments of the present invention may be any peptide sequence which directs a polypeptide to the chloroplast of a plant cell. Suitable peptides may readily be identified by a skilled person and some examples are shown in Table 2. Other examples may be found on the NCBI database (www(dot)ncbi(dot)nlm(dot)nih(dot)gov(forward slash)entrez(forward slash)). In some preferred embodiments, a peptide may have the chloroplast transit polypeptide of the pea FNR, which has the sequence shown in FIG.

6

.

In other embodiments of the present invention, flavoprotein may accumulate within chloroplasts as a result of expression within the chloroplast of nucleic acid encoding the polypeptide following direct transformation of the chloroplast. There is no requirement for a targeting or transit peptide in such embodiments.

Particle bombardment methods (Ruf, S. et al. (2001)

Nature Biotechnol.

19, 870-875) are particularly suitable for direct chloroplast transformation. With suitable plant regulatory elements, the transformed DNA may be transcribed within the plastid and translated into polypeptide in stromal ribosomes.

A nucleic acid encoding any flavoprotein polypeptide as defined above may be used in accordance with the present invention with any suitable chloroplast targeting peptide as defined above. The particular choice of flavoprotein polypeptide and targeting peptide is not critical to the practice of the present invention. Preferably, the flavoprotein polypeptide is not fused to a targeting peptide with which it is naturally associated i.e. it is fused to a heterogeneous targeting polypeptide. Flavodoxin polypeptides, which are not found in plants, are not naturally associated with chloroplast targeting signals.

In some preferred embodiments, a fusion polypeptide comprising a flavodoxin polypeptide and a chloroplast targeting peptide may have the sequence shown in

FIG. 4. A

suitable nucleic acid molecule encoding such a fusion polypeptide may have the sequence shown in FIG.

3

.

The present invention also provides a nucleic acid construct or vector which comprises a nucleic acid encoding a fusion polypeptide comprising a flavodoxin polypeptide and a chloroplast targeting peptide, preferably a construct or vector from which the fusion polypeptide encoded by the nucleic acid sequence can be expressed. The construct or vector is preferably suitable for transformation into and/or expression within a plant cell.

A construct or vector comprising nucleic acid according to this aspect of the present invention need not include a promoter or other regulatory sequence, particularly if the vector is to be used to introduce the nucleic acid into cells for recombination into the genome.

However, in one aspect the present invention provides a nucleic acid construct comprising a nucleic acid sequence encoding a flavodoxin polypeptide operably linked to a plant specific regulatory sequence, such as a promoter. Such constructs are particularly useful in embodiments in which chloroplasts are directly transformed with nucleic acid, which is subsequently expressed therein under the control of the plant specific regulatory element.

A plant specific regulatory sequence or element is a sequence which preferentially directs the expression (i.e. transcription) of a nucleic acid within a plant cell relative to other cell types. For example, expression from such a sequence is reduced or abolished in non-plant cells, such as bacterial or mammalian cells. A suitable regulatory sequence may for example be derived from a plant virus such as Cauliflower Mosaic Virus 35S. A regulatory sequence may be inducible, as described further below. The present invention also encompasses vectors comprising such a nucleic acid sequence.

Another aspect of the present invention provides the use of a nucleic acid as described herein in the production of a transgenic plant. Such a method may be for improving the tolerance of a plant to stress, in particular environmental stress, such as oxidative stress. Such stress may be biotic, abiotic or xenobiotic in nature and may include herbicide exposure, ultraviolet AB radiation, extreme temperatures, infection, for example with a necrotizing pathogen such as a bacterium or fungus and/or high doses of irradiation.

Nucleic acid may of course be double- or single-stranded, cDNA or genomic DNA, or RNA. The nucleic acid may be wholly or partially synthetic, depending on design. Naturally, the skilled person will understand that where the nucleic acid according to the invention includes RNA, reference to the sequence shown should be construed as reference to the RNA equivalent, with U substituted for T. The present invention also encompasses the expression product of any of the nucleic acid sequences disclosed and methods of making the expression product by expression from encoding nucleic acid therefore under suitable conditions in suitable host cells.

Those skilled in the art are well able to construct vectors and design protocols for recombinant gene expression, for example in a microbial or plant cell. Suitable vectors can be chosen or constructed, containing appropriate regulatory sequences, including promoter sequences, terminator fragments, polyadenylation sequences, enhancer sequences, marker genes and other sequences as appropriate. For further details see, for example,

Molecular Cloning: a Laboratory Manual

: 2nd edition, Sambrook et al, 1989, Cold Spring Harbor Laboratory Press.

Many known techniques and protocols for manipulation of nucleic acid, for example in preparation of nucleic acid constructs, mutagenesis, sequencing, introduction of DNA into cells and gene expression, and analysis of proteins, are described in detail in

Protocols in Molecular Biology

, Second Edition, Ausubel et al. eds., John Wiley & Sons, 1992. Specific procedures and vectors previously used with wide success upon plants are described by Bevan, Nucl. Acids Res. (1984) 12, 8711-8721), and Guerineau and Mullineaux, (1993) Plant transformation and expression vectors. In: Plant Molecular Biology Labfax (Croy RRD ed) Oxford, BIOS Scientific Publishers, pp 121-148.

A nucleic acid sequence as described herein, for example a sequence encoding a flavodoxin polypeptide, may be under operative control of a regulatory sequence active in plants for control of expression. It is indeed preferred that the coding sequence is operably linked to one or more regulatory sequences which may be heterologous or foreign to the gene (i.e. a non-bacterial sequence), for example a plant regulatory sequence. Such regulatory sequences may provide for efficient expression within a plant cell.

The term “heterologous” may be used to indicate that the gene/sequence of nucleotides in question have been introduced into said cells of the plant or an ancestor thereof, using genetic engineering or recombinant means, i.e. by human intervention. Nucleotide sequences heterologous, or exogenous or foreign, to a plant cell may be non-naturally occurring in cells of that type, variety or species. For example, there are no reports of flavodoxins in plant cells and nucleic acid encoding a flavodoxin polypeptide is therefore “heterologous” to a plant cell transformed therewith.

A nucleic acid construct which comprises a nucleic acid sequence encoding a flavoprotein such as flavodoxin, may include an inducible promoter operatively linked to the nucleic acid sequence. Such a promoter may be a stress inducible promoter. As discussed, this allows control of expression, for example, in response to an environmental stress. The invention also provides plants transformed with said gene construct and methods including introduction of such a construct into a plant cell and/or induction of expression of a construct within a plant cell, e.g. by application of a suitable stimulus, which may be an environmental stress stimulus such as a change in external conditions.

The term “inducible” as applied to a promoter is well understood by those skilled in the art. In essence, expression under the control of an inducible promoter is “switched on” or increased in response to an applied stimulus (which may be generated within a cell or provided exogenously). The nature of the stimulus varies between promoters. Whatever the level of expression is in the absence of the stimulus, expression from any inducible promoter is increased in the presence of the correct stimulus The preferable situation is where the level of expression increases upon in the presence of the relevant stimulus by an amount effective to alter a phenotypic characteristic i.e. to enhance stress tolerance. Thus an inducible (or “switchable”) promoter may be used which causes a basic level of expression in the absence of the stimulus which level is too low to bring about the desired stress tolerant phenotype (and may in fact be zero). Upon application of the stimulus, which may for example, be an increase in environmental stress, expression is increased (or switched on) to a level which causes enhanced stress tolerance.

Many examples of inducible promoters will be known to those skilled in the art.

Other suitable promoters may include the Cauliflower Mosaic Virus 35S (CaMV 35S) gene promoter that is expressed at a high level in virtually all plant tissues (Benfey et al, (1990) EMBO J 9: 1677-1684); the cauliflower meri 5 promoter that is expressed in the vegetative apical meristem as well as several well localised positions in the plant body, e.g. inner phloem, flower primordial branching points in root and shoot (Medford, J. I. (1992)

Plant Cell

4, 1029-1039; Medford et al, (1991)

Plant Cell

3, 359-370) and the

Arabidopsis thaliana

LEAFY promoter that is expressed very early in flower development (Weigel et al, (1992)

Cell

69, 843-859).

Constructs and vectors may further comprise selectable genetic markers consisting of chimaeric genes that confer selectable phenotypes such as resistance to antibiotics such as kanamycin, hygromycin, phosphinotricin, chlorsulfuron, methotrexate, gentamycin, spectinomycin, imidazolinones and glyphosate.

When introducing a chosen gene construct into a cell, certain considerations must be taken into account, well known to those skilled in the art. The nucleic acid to be inserted should be assembled within a construct which contains effective regulatory elements which will drive transcription. There must be available a method of transporting the construct into the cell. Once the construct is within the cell membrane, integration into the endogenous chromosomal material either will or will not occur. Finally, as far as plants are concerned, the target cell type must be such that cells can be regenerated into whole plants.

Techniques well known to those skilled in the art may be used to introduce nucleic acid constructs and vectors into plant cells to produce transgenic plants of the appropriate stress tolerant phenotype.

Agrobacterium transformation is one method widely used by those skilled in the art to transform dicotyledonous species. Production of stable, fertile transgenic plants in almost all economically relevant monocot plants is also now routine:(Toriyama, et al. (1988)

Bio/Technology

6, 1072-1074; Zhang, et al. (1988)

Plant Cell Rep

. 7, 379-384; Zhang, et al. (1988)

Theor Appl Genet

76, 835-840; Shimamoto, et al (1989)

Nature

338, 274-276; Datta, et al. (1990)

Bio/Technology

8, 736-740; Christou, et al. (1991)

Bio/Technology

9, 957-962; Peng, et al. (1991) International Rice Research Institute, Manila, Philippines 563-574; Cao, et al. (1992)

Plant Cell Rep

. 11, 585-591; Li, et al. (1993)

Plant Cell Rep

. 12, 250-255; Rathore, et al. (1993)

Plant Molecular Biology

21, 871-884; Fromm, et al. (1990)

Bio/Technology

8, 833-839; Gordon-Kamm, et al. (1990)

Plant Cell

2, 603-618; D'Halluin, et al. (1992)

Plant Cell

4, 1495-1505; Walters, et al. (1992)

Plant Molecular Biology

18, 189-200; Koziel, et al. (1993)

Biotechnology

11, 194-200; Vasil, I. K. (1994)

Plant Molecular Biology

25, 925-937; Weeks, et al. (1993)

Plant Physiology

102, 1077-1084; Somers, et al. (1992)

Bio/Technology

10, 1589-1594; WO92/14828). In particular, Agrobacterium mediated transformation is now a highly efficient alternative transformation method in monocots (Hiei et al. (1994)

The Plant Journal

6, 271-282).

The generation of fertile transgenic plants has been achieved in the cereals rice, maize, wheat, oat, and barley (reviewed in Shimamoto, K. (1994)

Current Opinion in Biotechnology

5, 158-162.; Vasil, et al. (1992)

Bio/Technology

10, 667-674; Vain et al., 1995

, Biotechnology Advances

13 (4): 653-671; Vasil, 1996

, Nature Biotechnology

14 page 702). Wan and Lemaux (1994)

Plant Physiol

. 104: 37-48 describe techniques for generation of large numbers of independently transformed fertile barley plants.

Other methods, such as microprojectile or particle bombardment (U.S. Pat. No. 5,100,792, EP-A-444882, EP-A-434616), electroporation (EP 290395, WO 8706614)), microinjection (WO 92/09696, WO 94/00583, EP 331083, EP 175966, Green et al. (1987)

Plant Tissue and Cell Culture

, Academic Press) direct DNA uptake (DE 4005152, WO 9012096, U.S. Pat. No. 4,684,611), liposome mediated DNA uptake (e.g. Freeman et al.

Plant Cell Physiol

. 29: 1353 (1984)), or the vortexing method (e.g. Kindle,

PNAS U.S.A

. 87: 1228 (1990d)) may be preferred where Agrobacterium transformation is inefficient or ineffective.

Physical methods for the transformation of plant cells are reviewed in Oard, 1991

, Biotech. Adv

. 9: 1-11.

Alternatively, a combination of different techniques may be employed to enhance the efficiency of the transformation process, e.g. bombardment with Agrobacterium coated microparticles (EP-A-486234) or microprojectile bombardment to induce wounding followed by co-cultivation with Agrobacterium (EP-A-486233).

Following transformation, a plant may be regenerated, e.g from single cells, callus tissue or leaf discs, as is standard in the art. Almost any plant can be entirely regenerated from cells, tissues and organs of the plant. Available techniques are reviewed in Vasil et al.,

Cell Culture and Somatic Cell Genetics of Plants, Vol I, II and III, Laboratory Procedures and Their Applications

, Academic Press, 1984, and Weissbach and Weissbach,

Methods for Plant Molecular Biology

, Academic Press, 1989.

The particular choice of a transformation technology will be determined by its efficiency to transform certain plant species as well as the experience and preference of the person practising the invention with a particular methodology of choice. It will be apparent to the skilled person that the particular choice of a transformation system to introduce nucleic acid into plant cells is not essential to or a limitation of the invention, nor is the choice of technique for plant regeneration.

A further aspect of the present invention provides a method of producing a cell which includes incorporating an isolated nucleic acid sequence encoding a flavoprotein polypeptide such as a flavodoxin polypeptide or an FNR polypeptide or a nucleic acid vector comprising such a sequence into the cell by means of transformation. Such a method of producing a cell may include recombining the nucleic acid with the cell genome nucleic acid such that it is stably incorporated therein. A plant may be regenerated from one or more cells transformed as described.

The flavoprotein polypeptide, the encoding nucleic acid, and/or the vector comprising the nucleic acid are preferably heterogeneous i.e. exogenous or foreign to the plant cell transformed therewith.

A method of producing a plant cell may include expressing the nucleic acid and causing or allowing the accumulation of flavoprotein polypeptide expressed thereby in the chloroplast-of said plant cell.

A suitable flavoprotein polypeptide for use in such methods may be an FNR polypeptide or a flavodoxin polypeptide.

A method of making such a plant cell may include the introduction of such a nucleotide sequence or a suitable vector including the sequence into a plant cell and causing or allowing recombination between the vector and the plant cell genome to introduce the nucleic acid sequence into the genome.

A method may further include sexually or asexually propagating or growing off-spring or a descendant of the plant regenerated from said plant cell.

The invention further encompasses a host cell transformed with a nucleic acid sequence or vector as set forth above, i.e. containing a nucleic acid or vector as described above, especially a plant cell, for example a higher plant cell, or a microbial cell. Thus, a host cell, such as a plant cell, including a nucleotide sequence as herein indicated is provided. Within the cell, the nucleotide sequence may be incorporated within the chromosome or may be extra-chromosomal. There may be more than one heterologous nucleotide sequence per haploid genome. This, for example, enables increased expression of the gene product compared with endogenous levels, as discussed below. A nucleic acid sequence comprised within a plant cell may be placed under the control of an externally inducible gene promoter, either to place expression under the control of the user or to provide for expression in response to stress.

A nucleic acid which is stably incorporated into the genome of a plant is passed from generation to generation to descendants of the plant, cells of which descendants may express the encoded flavoprotein polypeptide and so may have enhanced stress or pathogen tolerance.

A plant cell may contain a nucleic acid sequence encoding a flavoprotein polypeptide as a result of the introduction of the nucleic acid sequence into an ancestor cell.

In some embodiments, the flavoprotein polypeptide may be expressed within the plant cell as part of a fusion polypeptide which also comprises a chloroplast targeting peptide.

A plant cell as described herein may be comprised in a plant, a plant part or a plant propagule, or an extract or derivative of a plant as described below.

Plants which include a plant cell as described herein are also provided, along with any part or propagule thereof, seed, selfed or hybrid progeny and descendants. Particularly provided are transgenic higher plants, especially crop plants, which have been engineered to carry genes identified as stated above. Examples of suitable plants include tobacco, cucurbits, carrot, vegetable brassica, melons, capsicums, grape vines, lettuce, strawberry, oilseed brassica, sugar beet, wheat, barley, maize, rice, soyabeans, peas, sorghum, sunflower, tomato, potato, pepper, chrysanthemum, carnation, poplar, eucalyptus and pine.

A plant according to the present invention may be one which does not breed true in one or more properties. Plant varieties may be excluded, particularly registrable plant varieties according to Plant Breeders Rights. It is noted that a plant need not be considered a “plant variety” simply because it contains stably within its genome a transgene, introduced into a cell of the plant or an ancestor thereof.

In addition to a plant, the present invention provides any clone of such a plant, seed, selfed or hybrid progeny and descendants, and any part or propagule of any of these, such as cuttings and seed, which may be used in reproduction or propagation, sexual or asexual. Also encompassed by the invention is a plant which is a sexually or asexually propagated off-spring, clone or descendant of such a plant, or any part or propagule of said plant, off-spring, clone or descendant.

The present invention also encompasses the polypeptide expression product of a nucleic acid molecule according to the invention as disclosed herein. Such an isolated polypeptide may consist of a fusion polypeptide which comprises or consists of a flavoprotein polypeptide and a chloroplast targeting peptide, for example a fusion polypeptide which comprises or consists of a flavodoxin polypeptide and a chloroplast targeting peptide. The chloroplast targeting peptide may be heterogeneous to i.e. foreign or not normally or naturally associated with the flavoprotein polypeptide.

A preferred polypeptide includes the amino acid sequence shown in FIG.

4

. Such a fusion polypeptide may be encoded by a nucleic acid sequence as described herein, for example the nucleic acid sequence shown in FIG.

3

.

Also provided are methods of making such an expression product by expression from a nucleotide sequence encoding therefore under suitable conditions in suitable host cells e.g.

E. coli

. Those skilled in the art are well able to construct vectors and design protocols and systems for expression and recovery of products of recombinant gene expression.

The invention further provides a method of enhancing improving or increasing the stress tolerance of a plant which includes expressing a nucleic acid sequence encoding a flavoprotein polypeptide (i.e. causing or allowing transcription from a nucleic acid) within cells of the plant.

Suitable flavoprotein polypeptides include FNR polypeptides and flavodoxin polypeptides as described herein.

Improved stress tolerance may include enhanced or increased tolerance to environmental stresses such as ultraviolet UV radiation, extreme temperatures, irradiation, and/or pathogen infection, for example bacterial or fungal infection, in particular necrotizing pathogens, relative to normal, untreated plants.

The ability of a plant to tolerate stress may be increased by expression from a nucleotide sequence encoding a flavoprotein polypeptide such as a flavodoxin polypeptide within cells of a plant (thereby producing the encoded polypeptide), following an earlier step of introduction of the nucleotide sequence into a cell of the plant or an ancestor thereof. Such a method may raise the plants tolerance to stress and/or resistance to pathogen.

Preferably such a method includes causing or allowing the accumulation of the flavoprotein polypeptide within the chloroplasts of said cells, for example by expressing the nucleic acid within the chloroplasts of said cells or providing for the transport of the expressed protein into the chloroplasts. The level of flavoprotein in chloroplasts is increased or enhanced over the normal, endogenous levels of the flavoprotein as a result of such expression.

In some embodiments, chloroplast accumulation is achieved by expressing a fusion protein which comprises the flavodoxin polypeptide and a chloroplast targeting peptide.

Control experiments may be performed as appropriate in the methods described herein. The performance of suitable controls is well within the competence and ability of a skilled person in the field.

The disclosures of all documents mentioned herein are incorporated herein by reference.

Various further aspects and embodiments of the present invention will be apparent to those skilled in the art in view of the present disclosure.

Certain aspects and embodiments of the invention will now be illustrated by way of example and with reference to the figure described below,

BRIEF DESCRIPTION OF THE FIGURES AND TABLES

FIG. 1

shows that flavodoxin expression increases resistance to methyl viologen toxicity in transgenic tobacco plants.

FIG. 2

shows a scheme of the binary vector pCAMBIA 2200 containing a fragment of the in-frame fusion between the sequences encoding pea FNR transit peptide and the flavodoxin gene. The cassette inserted in the Eco RI site of the pCAMBIA 2200 was previously constructed in pDH51. This Eco RI fragment contained the CaMV 35S promoter, the flavodoxin chimeric gene and the CaMV35S polyadenylation signal.

FIG. 3

shows the nucleotide sequence of the in-frame fusions of the pea FNR transit peptide with the flavodoxin gene (SEQ ID NO:1). The initiation codon (ATG) of the transit peptide and the initial codon of flavodoxin (ATG) are indicated in bold, and the stop codon (TAA) is underlined.

FIG. 4

shows the predicted protein sequence of the transit peptide and flavodoxin protein (SEQ ID NO:2).

FIG. 5

shows the nucleic acid sequences of the chloroplast transit peptide of pea FNR (SEQ ID NO:3).

FIG. 6

shows the amino acid sequence of the chloroplast transit peptide of pea FNR (SEQ ID NO:4).

Table 1 shows the database details of known flavodoxin genes from a variety of microbes.

Table 2 shows the database details of known chloroplast targeting peptides.

Experimental

Materials and Methods

Construction of Ti Vectors for Flavodoxin Expression

A DNA fragment encoding Anabaena PCC7119 flavodoxin was obtained by PCR amplification of the cloned gene (Fillat, M. F. et al(1991) Biochem. J. 280, 187-191) from primers 5′-GACGAGCTCTCATA

ATG

TCAAAG-3′ (SEQ ID NO:5), and; 5′-ACTGTCGACTTT

TTA

CAAACCAAAT-3′ (SEQ ID NO:6), complementary to positions-14 to 9 and 515 to 540, respectively.

To facilitate further manipulations, a SacI recognition site (GAGCTC) was introduced in the 5′ end primer and a SalI site (GTCGAC) in the 3′ end primer The PCR conditions were 30 cycles of 60 s at 94° C., 90 s at 52° C. and 90 s at 72° C., using 1 ng of template DNA and 50 pmol of each primer in a medium containing 10 mM Tris-HCl pH 8.4, 5 mM KCl, 1.5 MM MgCl

2

, 0.2 mM of each dNTP and 5 units of Taq DNA polymerase. After the 30 cycles were completed, the reactions were incubated at 72° C. for 10 min. A purified PCR fragment of the predicted length (540 bp) was digested with SacI and SalI. The fragment was cloned into compatible sites of a pUC9-derived recombinant plasmid encoding the entire FNR precursor (Ceccarelli, E. A. et al (1991) J. Biol. Chem. 266, 14283-14287) between BamHI and SalI restriction sites, and from which a DNA fragment encoding the mature region of pea FNR had been removed by digestion with SacI and SalI. This generated an in-frame fusion of the chloroplast transit peptide derived from FNR with the flavodoxin protein.

The sequence of the chimeric gene was determined on both strands, and excised from the corresponding plasmid by digestion with BamHI and SalI. The 710-bp fragment was then cloned between the CaMV 35S promoter and polyadenylation regions of pDH51 (Pietrzcak, M. et al (1986) Nucleic Acids Res. 14, 5857-5868). The entire cassette was further isolated as an EcoRI fragment and inserted into the EcoRI site of the binary vector pCAMBIA 2200 (Hajdukiewiez, P. et al (1994) Plant Mol Biol. 25, 989-994). The construct was finally mobilized into

Agrobacterium tumefasciens

strain LBA 4404 by electroporation (Ausubel, F. M. et al (1987) Current Protocols in Molecular Cloning. New York, N.Y.: John Wiley and Sons).

Plant Transformation and Characterization

Tobacco leaf disc transformation was carried out using conventional techniques (Gallois, P. and Marinho, P. (1995) Plant gene transfer and expression protocols. In Methods in Molecular Biology (H. Jones, ed.), vol, 49, pp. 39-49. Humana Press Inc., Totowa, N.J.), and the progenies of kanamycin-resistant transformants were analyzed further. The presence of the flavodoxin gene in the transgenic lines was evaluated by Southern blot hybridization, using standard procedures. Primary transformants expressing high levels of bacterial flavodoxin, as evaluated by SDS-PAGE and immunoblotting (Krapp, A. R. et al (1997) Eur. J. Biochem. 249, 556-563), were self-pollinated and all subsequent experiments were carried out with the homozygous progeny.

Seeds of control and transgenic plants were germinated on Murashige-Skoog (MS) solid medium supplemented with 2% (w/v) sucrose and, in the case of transformants, 100 μg ml

−1

kanamycin. After two weeks at 25° C. and 200 μmol quanta m

−2

s

−1

(14 h light/10 h dark), seedlings were transplanted to fresh MS medium in Magenta vessels. When required, four-week old plantlets were placed on soil or grown hydroponically in nutrient medium (Geiger, M. et al. (1999) Plant Cell Environ. 22, 1177-1199). MV was included in the watering solution. Leaf discs of 12 mm diameter were punched from young fully expanded leaves of two-month old tobacco plants grown on soil. Discs were weighted and floated individually, top side up, on 1 ml sterile distilled water containing the indicated amounts of MV in 24-well plates, infiltrated in vacuum, and incubated in the dark for 2 h at 25° C. to allow diffusion of the MV into the leaf. Wells were illuminated with a white light source at 500 μmol quanta m

−2

s

−1

. Controls were kept in the dark. Electrolyte leakage of the leaf discs during Mv stress was measured as conductivity of the medium with an Horiba model B-173 conductivity meter.

To evaluate the tolerance of flavodoxin-expressing transformants to extreme temperatures, two-week old plantlets were exposed to 500 μmol quanta m

−2

s

−1

plantlets at either 8° C. or 40° C. for 12 h. Light treatments were carried out on four-week old plants, by focusing a light beam (B cm diameter) of 2000 μmol quanta m

−2

s

−1

on the upper surface of the third or fourth fully expanded leaf with the aid of a light cannon for 18 h at at 25° C. Tolerance to ultraviolet AB radiation was assayed by exposing four-week old plants to a combination of UV-A (315-400 nm at 2.2 W m

−2

), UV-B (250-315 nm at 1.0 W m

−2

) and photosynthetic active radiation (67 μmol quanta m

−2

s

−1

) for 24 h at 25° C. The UV lamps were wrapped in cellulose acetate foil (0,076 mm thick) to screen out any UV-C radiation (<280 nm). UV intensities were measured with UV-AB radiometer (Macam photometric LTD, Scotland).

Exposure to Plant Pathogens

X campestris

pv.

vesicatoria

cells were grown to OD

600

-1.3 in PYDAC medium (Vernière, C. et al (1991) Fruits 46, 162-170). The third and fourth leaves above hypocotyl from eight-week old control and transgenic tobacco plants were inoculated with a suspension of these bacteria in 0.85% (w/v) NaCl.

Isolates from

Alternaria alternata

strain CEREMIC 1333 were grown for six days in potato dextrose agar. Exposure of tobacco leaves to the pathogen was carried out using two different methods. In one of the procedures, discs of 7 mm were excised from fungal cultures and layered on the surface of cut leaves placed on 1.5% (w/v) agar in Petri dishes. Plates were incubated at 25° C. essentially as described by Deák et al. (Deák, M. et al. (1999) Nature Biotechnol. 17, 192-196). Alternatively, conidia from a saturated culture were re-suspended in 500 μl of sterile distilled water and infiltrated on the upper surface of the corresponding leaves with the aid of a needle-less syringe.

In all cases, symptoms were evaluated by estimating the diameters of necrotized leaf areas.

Analytical Procedures

Carbon dioxide assimilation in response to changes in incident PPFD were measured on the second fully expanded leaf of 4 four-week old transgenic and control plants, using a Qubit Systems Inc. infra-red gas analyzer (IRGA) Package (Kingston, Canada). Chlorophyll contents in leaves and plastids were determined using standard methods (Lichtenthaler, H. K. (1987) Methods Enzymol. 148, 350-382).

Results

Expression of Anabaena PCC 7119 Flavodoxin in Transgenic Tobacco Chloroplasts

To express cyanobacterial flavodoxin in tobacco plastids, a chimeric gene was prepared in which the flavodoxin coding region (Fillat, M. F. et al (1991) Biochem. J. 280, 187-191) was fused in-frame to DNA sequence encoding the chloroplast transit peptide of pea FNR (Newman, B. J., and Gray, J. C. (1988) Plant Mol. Biol. 10, 511-520) (for details, see Methods). The construct was cloned into an Agrobacterium binary vector under the control of the constitutive CaMV 35S gene promoter, and delivered into tobacco cells via Agrobacterium-mediated leaf disc transformation.

Kanamycin-resistant plants were recovered from tissue culture and evaluated for flavodoxin accumulation by immunoblotting.

Proteins extracted from sampled primary transformants (pfl5-pfl12) or from a wild-type tobacco specimen (PH) were resolved by SDS-PAGE, and either stained with Coomassie Brilliant Blue, or blotted onto nitrocellulose membranes and probed with antisera raised against Anabaena PCC7119 flavodoxin using standard techniques (Krapp, A. R. et al. (1997) supra). Proteins corresponding to 16 mm

2

of foliar tissue were loaded onto each lane of the gel. Typical homozygous (pfl5-8 and pfl5-6), heterozygous (pfl5-7) and segregant (pfl5-22) plants were recognised based on their flavodoxin contents.

A mature-sized reactive band could be detected at various levels in leaf extracts obtained from several transformants, suggesting plastid import and processing of the expressed flavoprotein. Immunodetection of flavodoxin in purified chloroplast fractions from transformed plants confirmed that the bacterial protein was targeted to plastids.

Primary transformants displaying high levels of flavodoxin expression, and containing a single transgene insertion locus per genome as assessed by Southern blot hybridization, were selfed, and homozygous lines were further selected by flavodoxin dosage.

Chloroplast-Targeted Bacterial Flavodoxin Promotes Tolerance to Methyl Viologen Toxicity in Transgenic Tobacco

Two-week old transgenic (pfl5-8) or wild-type (PH) plantlets were cultured on MS agar broth containing 15 μM or 30 μM MV and illuminated at 300 μmol quanta m

−2

s

−1

for 24 h at 25° C. Four-week old plants grown under hydroponic conditions were exposed to 30 μM MV in the nutrient solution, using the incubation time and light regime reported above.

Two-week old plantlets expressing flavodoxin were observed to survive treatment with 30-50 μM of the superoxide radical propagator MV in illuminated agar plates, whereas non-transformed tobacco controls were extensively bleached under the same conditions. Four week old plants were more tolerant to MV, but significant differences were still evident between transgenic plants and their wild-type siblings (FIG.

1

).

Experiments were performed to show the tolerance of flavodoxin-proficient leaf discs to MV as described above. Leaf tissue bleaching was perceived visually in the control discs, reflecting increased chlorophyll degradation. Conductance values were corrected for ion leakage occurring in the dark under the same conditions. The ion leakage values of each sample were expressed as a percentage of the total ion content (maximal value obtained after autoclaving the leaf disks at the end of the MV treatment). Chlorophyll contents were expressed as the fraction of the total chlorophyll content of leaf disks incubated under the same conditions in the absence of Mv. The heights of the vertical bare represent the averages of four experiments with SE lower than 15% (FIG.

1

).

Over-production of flavodoxin was observed to provide protection against MV-induced ion leakage (which is indicative of cell membrane deterioration) and bleaching of leaf discs from two-month old plants. In all cases, the extent of damage decreased as the levels of chloroplastic flavodoxin were raised.

Flavodoxin Protects Tobacco Plants against Extreme Temperatures and Irradiation

Transformants of the F

1

generation (pfl5-8) and their wild-type siblings (PH) were exposed to 40° C., high fluence rates, or ultraviolet UV-AB radiation, as described under Methods. Four-week old plants were used in all cases, except for the heating experiments, in which two-week old specimens were employed.

To investigate differences in chilling sensitivity of wild-type and flavodoxin-expressing tobacco plants, seeds of control and transgenic plants were germinated on MS solid medium supplemented with 2% (w/v) sucrose for two weeks at 25° C. and 200 μmol quanta m

−2

s

−1

(14 h light/10 h dark). Seedlings were transferred to 9° C. under the same light regime for other two weeks allowing plants to acclimate. Thereafter, they were continuously illuminated (500 μmol quanta m

−2

s

−1

) for one more week at 9° C.

To investigate water-deficit tolerance of transgenic tobacco expressing flavodoxin, plants were grown for two weeks in MS as described above and then transferred to soil and daily irrigated with nutrient solution The water-deficit stress was applied to two month-old plants by withholding water for up to 3 days. All other growing conditions were the same as described in Methods

Transformants exhibited increased tolerance to this drought regime, whereas damage in control plants was reflected by extensive leaf withering and/or bleaching, with concomitant decreases in chlorophyll contents and photosynthetic capacities.

Young tobacco plantlets accumulating flavodoxin were found to survive prolonged illumination (500 μmol quanta m

−2

s

−1

for 12 h) at 40° C. and exhibited increased tolerance to chilling. Under similar conditions, wild-type control seedlings were severely damaged.

Exposure of the plants to ultraviolet AB (UV-AB) radiation, extremely high light intensities or water deficiency yielded essentially the same results as above.

The damage caused by these treatments in control tobacco plants was reflected by extensive bleaching of the leaf tissue, with concomitant decreases in chlorophyll contents and photosynthetic capacities.

Reduced Damage in Tobacco Transformants Expressing Flavodoxin Exposed to Necrotrophic Pathogens

Control and flavodoxin-expressing tobacco plants, grown in the greenhouse for eight weeks were inoculated with a suspension of the pathogenic bacterium

Xanthomonas campestris

pv.

vesicatoria

, known to induce the hyper-sensitive response in tobacco (Baker, C. J., and Orlandi, E. W. (1995) Ann. Rev. Phytopathol. 33, 299-321). The number of necrotic symptoms was drastically reduced in transformants expressing the cyanobacterial protein, with a negative correlation between the extent of damage and the flavodoxin levels accumulated in the corresponding tissue.

Fungal infection with

A. alternata

was done using two different procedures. Firstly, discs of 7 mm excised from fungal cultures were layered on the surface of cut leaves placed on 1.5% (w/v) agar in Petri dishes. Plates were incubated at 25° C. essentially as described by Deák et al (Deák et al (1999) supra) for 10 days. Secondly, a disc of filter paper imbibed in conidia from a saturated culture (around 20,000 conidia) was placed on the upper surface of the corresponding leaves. Symptoms on the leaves were observed after 18 days of fungal infection.

Transgenic plants were observed to be more tolerant than their wild-type siblings to necrosis induced by infection with the pathogenic fungus

Alternaria alternata.

To investigate necrotization of tobacco wild-type and transgenic leaves after infection by tobacco necrotic virus, plants were planted in soil and grown for three weeks at 25° C. and 300 μmol quanta m

−2

s

−1

with 14 h light/10 h dark as described in Methods, The third leaves position above hypocotyl were inoculated with a suspension of the tobacco necrotic virus, with an abrasive and kept under the same growing conditions. Lesions were examined after one week of inoculation.

Transgenic plants were observed to be more tolerant than their wild-type siblings to necrosis induced by infection with the tobacco necrotic virus.

Expression of flavodoxin in plant cells was thus observed to protect transgenic plants against pathogens that cause necrotic lesions.

TABLE 1

Accession

No.

Gene

Species

NP_358768

gi|15903218

Flavodoxin

Streptococcus

pneumoniae R6

NP_345761

gi|15901157

Flavodoxin

Streptococcus

pneumoniae TIGR4

NP_311794

gi|15833021

flavodoxin 2

Escherichia coli

O157:H7]

NP_311593

gi|15832820

putative flavodoxin

Escherichia coli

O157:H7

NP_308742

gi|15829969

flavodoxin 1

Escherichia coli

O157:H7

CAC92877

gi|15980620

flavodoxin 1

Yersinia pestis

CAC89737

gi|15978964

flavodoxin 2

Yersinia pestis

NP_350007

gi|15896658

Flavodoxin

Clostridium

acetobutylicum

NP_349066

gi|15895717

Flavodoxin

Clostridium

acetobutylicum

NP_347225

gi|15893876

Flavodoxin

Clostridium

acetobutylicum

NP_346845

gi|15893496

Flavodoxin

Clostridium

acetobutylicum

NP_348645

gi|15895296

Predicted flavodoxin

Clostridium

acetobutylicum

NP_347225

gi|15893876

Flavodoxin

Clostridium

acetobutylicum

NP_346845

gi|15893496

Flavodoxin

Clostridium

acetobutylicum

NP_282528

gi|15792705

Flavodoxin

Campylobacter jejuni

AAK28628

gi|13507531

Flavodoxin

Aeromonas hydrophila

NP_268951

gi|15674777

putative flavodoxin

Streptococcus

pyogenes

NP_266764

gi|15672590

Flavodoxin

Lactococcus lactis

subsp.

lactis

NP_207952

gi|15645775

flavodoxin (fldA)

Helicobacter pylori

26695

NP_232050

gi|15642417

flavodoxin 2

Vibrio cholerae

NP_231731

gi|15642099

flavodoxin 1

Vibrio cholerae

NP_219360

gi|15639910

Flavodoxin

Treponema pallidum

NP_24012

gi|15616909

flavodoxin 1

Buchnera sp. APS

NP_214435

gi|15607053

Flavodoxin

Aquifex aeolicus

FXAVEP

gi|625194

flavodoxin

Azotobacter

vinelandii

S38632

gi|481443

flavodoxin

-Synechocystis sp.

(strain PCC 6803)

FXDV

gi|476442

flavodoxin

Desulfovibrio

vulgaris

A34640

gi|97369

flavodoxin

Desulfovibrio

salexigens

S24311

gi|97368

flavodoxin

Desulfovibrio gigas

(ATCC 19364)

A37319

gi|95841

flavodoxin A

Escherichia coli

S06648

gi|81145

flavodoxin

red alga (

Chondrus

crispus

)

S04600

gi|79771

flavodoxin

Anabaena variabilis

A28670

gi|79632

flavodoxin

Synechococcus sp

S02511

gi|78953

flavodoxin

Klebsiella

pneumoniae

FXDVD

gi|65884

flavodoxin

Desulfovibrio

|

desulfuricans

(ATCC

29577)

FXCLEX

gi|65882

flavodoxin

Clostridium sp

FXME

gi|65881

flavodoxin

Megasphaera elsdenii

NP_071157

gi|11499913

flavodoxin, putative

Archaeoglobus

fulgidus

BAA17947

gi|1653030

flavodoxin

Synechocystis sp.

PCC 6803

BAB61723

gi|14587807

flavodoxin 2

Vibrio fischeri

BAB61721

gi|14587804

flavodoxin 1

Vibrio fischeri

AAK66769

gi|14538018

flavodoxin

Histophilus ovis

P57385

gi|11132294

FLAVODOXIN

AAC7593

gi|1789262

flavodoxin 2

Escherichia coli

K12

AAC73778

gi|1786900

flavodoxin 1

Escherichia coli

K12

AAC75752

gi|1789064

putative flavodoxin

Escherichia coli

K12

F69821

gi|7429905

flavodoxin homolog

Bacillus subtilis

yhcB

QQKBFP

gi|2144338

pyruvate

Klebsiella

(flavodoxin)

pneumoniae

dehydrogenase nifJ

S16929

gi|95027

flavodoxin A

Azotobacter

chroococcum

F71263

gi|7430914

probable flavodoxin

Syphilis spirochete

A64665

gi|7430911

flavodoxin

Helicobacter

pylori

(strain 26695

JE0109

gi|7430907

flavodoxin

Desulfovibrio

vulgaris

S42570

gi|628879

flavodoxin

Desulfovibrio

desulfuricans

(ATCC

27774)

BAB13365

gi|10047146

flavodoxin

Alteromonas sp. O-7

AAF34250

gi|6978032

flavodoxin

Desulfovibrio gigas

CAB73809

gi|6968816

flavodoxin

Campylobacter jejuni

D69541

gi|7483302

flavodoxin homolog

Archaeoglobus

fulgidus

F70479

gi|7445354

flavodoxin

Aquifex aeolicus

S55234

gi|1084290

flavodoxin isoform I

Chlorella fusca

S18374

gi|2117434

flavodoxin

Anabaena sp. (PCC

7119)

S55235

gi|1084291

flavodoxin isoform

Chlorella fusca

II

C64053

gi|1074088

flavodoxin A

Haemophilus

influenzae (strain

Rd KW20)

A61338

gi|625362

flavodoxin

Clostridium

pasteurianum

A39414

gi|95560

flavodoxin

Enterobacter

agglomerans plasmid

pEA3

AAD08207

gi|2314319

flavodoxin (fldA)

Helicobacter pylori

26695

CAB37851

gi|4467982

flavodoxin

Rhodobacter

capsulatus

AAC65882

gi|3323245

flavodoxin

Treponema pallidum

AAB88920

gi|2648181

flavodoxin, putative

Archaeoglobus

fulgidus

AAB65080

gi|2289914

flavodoxin

Klebsiella

pneumoniae

AAB53659

gi|710356

flavoprotein

Methanothermobacter

thermautotrophicus

AAB51076

gi|1914879

flavodoxin

Klebsiella

pneumoniae

AAB36613

gi|398014

flavodoxin

Azotobacter

chroococcum

AAB20462

gi|239748

flavodoxin

Anabaena

AAA64735

gi|142370

flavodoxin (nifF)

Azotobacter

vinelandii

BAA35341

gi|1651296

Flavodoxin

Escherichia coli

BAA35333

gi|1651291

Flavodoxin

Escherichia coli

AAA27288

gi|415254

flavodoxin

Synechocystis sp.

AAA27318

gi|154528

Flavodoxin

Synechococcus sp.

AAC45773

gi|1916334

putative flavodoxin

Salmonella

typhimurium

AAC07825

gi|2984302

flavodoxin

Aquifex aeolicus

AAC02683

gi|2865512

flavodoxin

Trichodesmium

erythraeum

TABLE 2

Accession

No

Gene

Species

P32260

gi|12644209

CYSTEINE SYNTHASE,

Spinacia

CHLOROPLAST

oleracea

PRECURSOR

AAG59996

gi|12658639

ferredoxin:sulfite

Glycine

reductase precursor

max

S10200

gi|100078

carbonate dehydratase

Pisum

precursor

sativum

CAB89287

gi|7672161

chloroplast FtsZ-like

Nicotiana

protein

tabacum

P17067

gi|115471

CARBONIC

Pisum

ANHYDRASE,

sativum

CHLOROPLAST

PRECURSOR

(CARBONATE

DEHYDRATASE)

AAD22109

gi|4530595

heme oxygenase 2

Arabi-

dopsis

thaliana

AAD22108

gi|4530593

heme oxygenase 1

Arabi-

dopsis

thaliana

AAC50035

gi|450235

APS kinase

Arabi-

dopsis

thaliana

AAC12846

gi|1051180

phytoene desaturase

Zea mays

AAB87573

gi|2645999

chlorophyll a/b binding

Panax

protein of LHCII type I

ginseng

precursor

CAA47329

gi|312944

cysteine synthase

Spinacia

oleracea

CAA31137

gi|141201

O-acetylserine

Escheri-

sulfhydrylase

chia

coli

AAA82068

gi|1079732

cpFtsZ

Arabi-

dopsis

thaliana

T06368

gi|7489040

photosystem II oxygen-

Lycoper-

evolving complex

sicon

protein 1 precursor

esculen-

tum

S71750

gi|7488813

import intermediate-

Pisum

associated 100K protein

sativum

precursor

S71749

gi|7459239

DCL protein precursor,

Lycoper-

chloroplast

sicon

esculen-

tum

15825883

gi|15825883

Chain B, Structure Of

Arabi-

Threonine Synthase

dopsis

thaliana

15825882

gi|15825882

Chain A, Structure Of

Arabi-

Threonine Synthase

dopsis

thaliana

T09543

gi|7488970

deoxyxylulose synthase

Capsicum

TKT2 precursor

annuum

JC5876

gi|7447856

early light-inducible

Glycine

protein precursor

max

P24493

gi|1170215

DELTA-AMINOLEVULINIC

Spinacia

ACID DEHYDRATASE

oleracea

PRECURSOR

S47966

gi|1076532

probable lipid transfer

Pisum

protein M30 precursor

sativum

A44121

gi|322404

ribosomal protein S1

Spinacia

precursor

oleracea

S01056

gi|81896

early light-induced

Pisum

protein precursor

sativum

O22773

gi|7388292

THYLAKOID LUMENAL 16.5

Arabi-

KDA PROTEIN,

dopsis

CHLOROPLAST

thaliana

PRECURSOR

P80470

gi|6093830

PHOTOSYSTEM II CORE

Spinacia

COMPLEX PROTEINS PSBY

oleracea

PRECURSOR

P55195

gi|1709930

PHOSPHORIBOSYL-

Vigna

AMINOIMIDAZOLE

aconiti-

CARBOXYLASE,

folia

CHLOROPLAST PRECURSOR

P11970

gi|1709654

PLASTOCYANIN B,

Populus

CHLOROPLAST PRECURSOR

nigra

P00299

gi|1709651

PLASTOCYANIN A,

Populus

CHLOROPLAST PRECURSOR

nigra

P80484

gi|1709608

PERIDININ-

Amphidi-

CHLOROPHYLL A

nium

PROTEIN 1 PRECURSOR

carterae

P08823

gi|134102

RUBISCO SUBUNIT

Triticum

BINDING-PROTEIN ALPHA

aestivum

SUBUNIT PRECURSOR

P04045

gi|130173

ALPHA-1,4 GLUCAN

Solanum

PHOSPHORYLASE, L-1

tuberosum

ISOZYME, CHLOROPLAST

PRECURSOR

S30897

gi|7427677

3-isopropylmalate

Solanum

dehydrogenase precursor

tuberosum

TXSPM

gi|7427615

thioredoxin m precursor

Spinacia

oleracea

FEKM

gi|7427604

ferredoxin [2Fe-2S]

Chlamy-

precursor

domonas

reinhardtii

CCKM6R

gi|2144284

cytochrome c6 precursor

Chlamy-

domonas

reinhardtii

S30145

gi|419757

ketol-acid

Arabi-

reductoisomerase

dopsis

precursor

thaliana

DEMZMC

gi|319840

malate dehydrogenase

Zea mays

(NADP+) precursor

S20510

gi|81676

3-isopropylmalate

Brassica

dehydrogenase precursor

napus

S17180

gi|81509

ketol-acid

Spinacia

reductoisomerase

oleracea

precursor

Q9SEL7

gi|15214049

PROTEASE HHOA,

Arabi-

CHLOROPLAST PRECURSOR

dopsis

thaliana

O23403

gi|13959580

THYLAKOID LUMENAL 21.5

Arabi-

KDA PROTEIN,

dopsis

CHLOROPLAST PRECURSOR

thaliana

P82281

gi|12644689

PUTATIVE L-ASCORBATE

Arabi-

PEROXIDASE,

dopsis

CHLOROPLAST

thaliana

PRECURSOR

O22609

gi|9910645

PROTEASE DO-LIKE,

Arabi-

CHLOROPLAST PRECURSOR

dopsis

thaliana

P48417

gi|1352186

ALLENE OXIDE SYNTHASE,

Linum

CHLOROPLAST PRECURSOR

usitatis-

simum

P49080

gi|1351905

BIFUNCTIONAL

Zea mays

ASPARTOKINASE/

HOMOSERINE

DEHYDROGENASE 2,

CHLOROPLAST PRECURSOR

P31853

gi|461595

ATP SYNTHASE B′ CHAIN,

Spinacia

CHLOROPLAST PRECURSOR

oleracea

P10933

gi|119905

FERREDOXIN--NADP

Pisum

REDUCTASE, LEAF

sativum

ISOZYME PRECURSOR

P52422

gi|14917033

PHOSPHORIBOSYL-

Arabi-

GLYCINAMIDE

dopsis

FORMYLTRANSFERASE,

thaliana

CHLOROPLAST PRECURSOR

P49077

gi|14917032

ASPARTATE

Arabi-

CARBAMOYLTRANSFERASE

dopsis

PRECURSOR

thaliana

O50039

gi|14917022

ORNITHINE

Arabi-

CARBAMOYL-

dopsis

TRANSFERASE,

thaliana

CHLOROPLAST PRECURSOR

P55229

gi|14916987

GLUCOSE-1-

Arabi-

PHOSPHATE

dopsis

ADENYLYLTRANSFERASE

thaliana

LARGE SUBUNIT 1,

CHLOROPLAST PRECURSOR

Q96291

gi|14916972

2-CYS PEROXIREDOXIN

Arabi-

BAS1, CHLOROPLAST

dopsis

PRECURSOR

thaliana

Q9ZT00

gi|14916690

RIBULOSE BISPHOSPHATE

Zea mays

CARBOXYLASE/

OXYGENASE

ACTIVASE, CHLOROPLAST

PRECURSOR

Q9LZX6

gi|14547977

DIHYDRODIPICOLINATE

Arabi-

SYNTHASE 1,

dopsis

CHLOROPLAST

thaliana

PRECURSOR

O64903

gi|12644076

NUCLEOSIDE DIPHOSPHATE

Arabi-

KINASE II, CHLOROPLAST

dopsis

PRECURSOR

thaliana

O04130

gi|3122858

D-3-PHOSPHOGLYCERATE

Arabi-

DEHYDROGENASE

dopsis

PRECURSOR

thaliana

O24364

gi|3121825

2-CYS PEROXIREDOXIN

Spinacia

BAS1, CHLOROPLAST

oleracea

PRECURSOR

P49107

gi|1709825

PHOTOSYSTEM I REACTION

Arabi-

CENTRE SUBUNIT N

dopsis

PRECURSOR

thaliana

P49132

gi|1352199

TRIOSE

Flaveria

PHOSPHATE/PHOSPHATE

trinervia

TRANSLOCATOR,

CHLOROPLAST PRECURSOR

P37107

gi|586038

SIGNAL RECOGNITION

Arabi-

PARTICLE 54 KDA

dopsis

PROTEIN, CHLOROPLAST

thaliana

PRECURSOR

Q04836

gi|464662

31 KDA

Arabi-

RIBONUCLEOPROTEIN,

dopsis

CHLOROPLAST PRECURSOR

thaliana

Q01909

gi|461551

ATP SYNTHASE GAMMA

Arabi-

CHAIN 2, CHLOROPLAST

dopsis

PRECURSOR

thaliana

P14671

gi|136251

TRYPTOPHAN SYNTHASE

Arabi-

BETA CHAIN 1 PRECURSOR

dopsis

thaliana

P07089

gi|132144

RIBULOSE BISPHOSPHATE

Flaveria

CARBOXYLASE

trinervia

SMALL CHAIN

PRECURSOR

P22221

gi|130384

PYRUVATE, PHOSPHATE

Flaveria

DIKINASE PRECURSOR

trinervia

P22178

gi|126736

NADP-DEPENDENT MALIC

Flaveria

ENZYME, CHLOROPLAST

trinervia

PRECURSOR

P26259

gi|118241

DIHYDRODIPICOLINATE

Zea mays

SYNTHASE, CHLOROPLAST

PRECURSOR

P23577

gi|118044

APOCYTOCHROME

Chlamy-

F PRECURSOR

domonas

reinhardtii

Q42522

gi|14195661

GLUTAMATE-1-

Arabi-

SEMIALDEHYDE 2,1-

dopsis

AMINOMUTASE

thaliana

2 PRECURSOR

Q96242

gi|13878924

ALLENE OXIDE SYNTHASE

Arabi-

PRECURSOR

dopsis

thaliana

P46312

gi|13432148

OMEGA-6 FATTY ACID

Arabi-

DESATURASE,

dopsis

CHLOROPLAST

thaliana

PRECURSOR

P34802

gi|13432144

GERANYLGERANYL

Arabi-

|

PYROPHOSPHATE

dopsis

SYNTHETASE,

thaliana

CHLOROPLAST

PRECURSOR

P50318

gi|12644295

PHOSPHOGLYCERATE

Arabi-

KINASE, CHLOROPLAST

dopsis

PRECURSOR

thaliana

P46309

gi|12644273

GLUTAMATE--CYSTEINE

Arabi-

LIGASE, CHLOROPLAST

dopsis

PRECURSOR

thaliana

P21276

gi|12644157

SUPEROXIDE DISMUTASE

Arabi-

[FE], CHLOROPLAST

dopsis

PRECURSOR

thaliana

O23787

gi|6094476

THIAZOLE BIOSYNTHETIC

Citrus

ENZYME, CHLOROPLAST

sinensis

PRECURSOR

P93407

gi|3915008

SUPEROXIDE DISMUTASE

Oryza

[CU-ZN], CHLOROPLAST

sativa

PRECURSOR

Q96255

gi|3914996

PHOSPHOSERINE

Arabi-

AMINOTRANSFERASE,

dopsis

CHLOROPLAST PRECURSOR

thaliana

O24600

gi|3914826

DNA-DIRECTED RNA

Arabi-

POLYMERASE,

dopsis

CHLOROPLAST

thaliana

PRECURSOR

O49937

gi|3914665

50S RIBOSOMAL PROTEIN

Spinacia

L4, CHLOROPLAST

oleracea

PRECURSOR

Q42915

gi|3914608

RIBULOSE BISPHOSPHATE

Manihot

CARBOXYLASE SMALL

esculenta

CHAIN PRECURSOR

Q39199

gi|2500098

DNA REPAIR PROTEIN

Arabi-

RECA, CHLOROPLAST

dopsis

PRECURSOR

thaliana

Q96529

gi|2500026

ADENYLOSUCCINATE

Arabi-

SYNTHETASE PRECURSOR

dopsis

thaliana

P55826

gi|2495184

PROTOPORPHYRINOGEN

Arabi-

OXIDASE, CHLOROPLAST

dopsis

PRECURSOR

thaliana

Q42496

gi|2493687

CYTOCHROME B6-F

Chlamy-

COMPLEX 4

domonas

KDA SUBUNIT,

reinhardtii

CHLOROPLAST PRECURSOR

P52424

gi|1709925

PHOSPHORIBOSYL-

Vigna

FORMYLGLY

unguicu-

CINAMIDINE CYCLO-

lata

LIGASE, CHLOROPLAST

PRECURSOR

P49572

gi|1351303

INDOLE-3-GLYCEROL

Arabi-

PHOSPHATE SYNTHASE,

dopsis

CHLOROPLAST PRECURSOR

thaliana

P48496

gi|1351271

TRIOSEPHOSPHATE

Spinacia

ISOMERASE, CHLOROPLAST

oleracea

PRECURSOR

P25269

gi|1174779

TRYPTOPHAN SYNTHASE

Arabi-

BETA CHAIN 2 PRECURSOR

dopsis

thaliana

P46225

gi|1174745

TRIOSEPHOSPHATE

Secale

ISOMERASE, CHLOROPLAST

cereale

PRECURSOR

P46283

gi|1173345

SEDOHEPTULOSE-1,7-

Arabi-

BISPHOSPHATASE,

dopsis

CHLOROPLAST PRECURSOR

thaliana

P32069

gi|418134

ANTHRANILATE SYNTHASE

Arabi-

COMPONENT I-2

dopsis

PRECURSOR

thaliana

P29450

gi|267120

THIOREDOXIN F-TYPE,

Pisum

CHLOROPLAST PRECURSOR

sativum

Q9ZTN9

gi|13878459

PHYTOENE

Oryza

DEHYDROGENASE

sativa

PRECURSOR

Q9SHI1

gi|13627881

TRANSLATION INITIATION

Arabi-

FACTOR IF-2,

dopsis

CHLOROPLAST PRECURSOR

thaliana

Q9LR75

gi|13431553

COPROPORPHYRINOGEN III

Arabi-

OXIDASE, CHLOROPLAST

dopsis

PRECURSOR

thaliana

Q9ZNZ7

gi|12643970

FERREDOXIN-DEPENDENT

Arabi-

GLUTAMATE SYNTHASE 1,

dopsis

CHLOROPLAST PRECURSOR

thaliana

Q9SZ30

gi|12643854

BIFUNCTIONAL HISTIDINE

Arabi-

BIOSYNTHESIS PROTEIN

dopsis

HISHF, CHLOROPLAST

thaliana

PRECURSOR

Q9SJE1

gi|12643848

MAGNESIUM-CHELATASE

Arabi-

SUBUNIT CHLD

dopsis

PRECURSOR

thaliana

Q42624

gi|12643761

GLUTAMINE

Brassica

SYNTHETASE,

napus

CHLOROPLAST PRECURSOR

Q38933

gi|12643749

LYCOPENE BETA CYCLASE,

Arabi-

CHLOROPLAST PRECURSOR

dopsis

thaliana

Q42435

gi|12643508

CAPSANTHIN/CAPSORUBIN

Capsicum

SYNTHASE, CHLOROPLAST

annuum

PRECURSOR

6

1

706

DNA

Artificial Sequence

Description of Artificial Sequence Nucleic
acid molecule encoding fusion polypeptide

1
ggatccatca tcaacaacaa caacaaacat ggctgctgca gtaacagccg cagtctcctt 60
gccatactcc aactccactt cccttccgat cagaacatct attgttgcac cagagagact 120
tgtcttcaaa aaggtttcat tgaacaatgt ttctataagt ggaagggtag gcaccatcag 180
agctctcata atgtcaaaga aaattggttt attctacggt actcaaactg gtaaaactga 240
atcagtagca gaaatcattc gagacgagtt tggtaatgat gtggtgacat tacacgatgt 300
ttcccaggca gaagtaactg acttgaatga ttatcaatat ttgattattg gctgtcctac 360
ttggaatatt ggcgaactgc aaagcgattg ggaaggactc tattcagaac tggatgatgt 420
agattttaat ggtaaattgg ttgcctactt tgggactggt gaccaaatag gttacgcaga 480
taattttcag gatgcgatcg gtattttgga agaaaaaatt tctcaacgtg gtggtaaaac 540
tgtcggctat tggtcaactg atggatatga ttttaatgat tccaaggcac taagaaatgg 600
caagtttgta ggactagctc ttgatgaaga taatcaatct gacttaacag acgatcgcat 660
caaaagttgg gttgctcaat taaagtctga atttggtttg taaaaa 706

2

233

PRT

Artificial Sequence

Description of Artificial Sequence Predicted
protein sequence of transit peptide and flavodoxin
protein

2
Asp Pro Ser Ser Thr Thr Thr Thr Asn Met Ala Ala Ala Val Thr Ala
1 5 10 15
Ala Val Ser Leu Pro Tyr Ser Asn Ser Thr Ser Leu Pro Ile Arg Thr
20 25 30
Ser Ile Val Ala Pro Glu Arg Leu Val Phe Lys Lys Val Ser Leu Asn
35 40 45
Asn Val Ser Ile Ser Gly Arg Val Gly Thr Ile Arg Ala Leu Ile Met
50 55 60
Ser Lys Lys Ile Gly Leu Phe Tyr Gly Thr Gln Thr Gly Leu Thr Glu
65 70 75 80
Ser Val Ala Glu Ile Ile Arg Asp Glu Phe Gly Asn Asp Val Val Thr
85 90 95
Leu His Asp Val Ser Gln Ala Glu Val Thr Asp Leu Asn Asp Tyr Gln
100 105 110
Tyr Leu Ile Ile Gly Cys Pro Thr Trp Asn Ile Gly Glu Leu Gln Ser
115 120 125
Asp Trp Glu Gly Leu Tyr Ser Glu Leu Asp Asp Val Asp Phe Asn Gly
130 135 140
Lys Leu Val Ala Tyr Phe Gly Thr Gly Asp Gln Ile Gly Tyr Ala Asp
145 150 155 160
Asn Phe Gln Asp Ala Ile Gly Ile Leu Glu Glu Lys Ile Ser Gln Arg
165 170 175
Gly Gly Lys Thr Val Gly Tyr Trp Ser Thr Asp Gly Tyr Asp Phe Asn
180 185 190
Asp Ser Lys Ala Leu Arg Asn Gly Lys Phe Val Gly Leu Ala Leu Asp
195 200 205
Glu Asp Asn Gln Ser Asp Leu Thr Asp Asp Arg Ile Lys Ser Trp Val
210 215 220
Ala Gln Leu Lys Ser Glu Phe Gly Leu
225 230

3

162

DNA

Pisum sativum

3
atggctgctg cagtaacagc cgcagtctcc ttgccatact ccaactccac ttcccttccg 60
atcagaacat ctattgttgc accagagaga cttgtcttca aaaaggtttc attgaacaat 120
gtttctataa gtggaagggt aggcaccatc agagctctca ta 162

4

54

PRT

Pisum sativum

4
Met Ala Ala Ala Val Thr Ala Ala Val Ser Leu Pro Tyr Ser Asn Ser
1 5 10 15
Thr Ser Leu Pro Ile Arg Thr Ser Ile Val Ala Pro Glu Arg Leu Val
20 25 30
Phe Lys Lys Val Ser Leu Asn Asn Val Ser Ile Ser Gly Arg Val Gly
35 40 45
Thr Ile Arg Ala Leu Ile
50

5

23

DNA

Artificial Sequence

Description of Artificial Sequence Primer

5
gacgagctct cataatgtca aag 23

6

25

DNA

Artificial Sequence

Description of Artificial Sequence Primer

6
actgtcgact ttttacaaac caaat 25

Stress tolerant plants

Information

Patent Number

Date Filed

Date Issued

Inventors

Original Assignees

Examiners

Agents

CPC

US Classifications

Field of Search

US

International Classifications

Term Extension

Abstract

Description

Claims

Non-Patent Literature Citations (5)

Entry
Paltnik et al., Plant Physiol., 1997, vol. 115, pp. 1721-1727.*
Dupree et al. , Curr. Res. Photosyn. 1990, vol. III, pp. 625-628.*
Espinosa-Ruiz et al, “Arabidopsis thaliana AtHAL3: a flavoprotein related to salt and osmotic tolerance and plant growth”, Plant Journal 20(5):529-539 (1999).
Lin Chentao et al, “Expression of an Arabidopsis cryptochrome gene in transgenic tobacco results in hypersensitivity to blue. UV-A, and green light”, PNAS 92(18):8423-8427 (1995).
Kitamura Masaya et al, “Cloning and expression of the gene encoding flavodoxin from Desulfovibrio vulgaris (Miyazaki F)”, Journal of Biochemistry 123(5):891-898 (1998).