NL2005620C2 - Plants with increased tolerance to metal ions. - Google Patents

Description

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PLANTS WITH INCREASED TOLERANCE TO METAL IONS FIELD OF THE INVENTION

The present invention relates generally to the field of plant molecular biology and 5 plants, especially genetically altered or genetically evolved plants, plant parts, progeny, subsequent generations and reproductive material having cells exhibiting an altered metal cation level tolerance compared to a non-genetically altered or selectively evolved plant are provided. Furthermore, the present invention also relates to genetic and proteinaceous agents capable of modulating or altering the tolerance to metal salts, in the soil and/or 10 water supply in a cell, group of cells, organelle, part or reproductive portion of a plant.

BACKGROUND OF THE INVENTION

Bibliographic details of references provided in the subject specification are listed at the end of the specification.

Environmental stress due to salinity is one of the most serious factors limiting the 15 productivity of agricultural crops, which are predominantly sensitive to the presence of high concentrations of metal salts in the soil. Large terrestrial areas of the world are affected by levels of metal salts which inhibit plant growth. It is estimated that a large portion of the land presently under irrigation is already affected by salinity, not including the regions classified as arid and desert lands, which comprises about a quarter of the total 20 land of our planet. Salinity has been an important factor in human history and in the life spans of agricultural systems. Large areas of the Indian subcontinent have been rendered unproductive through salt accumulation and poor irrigation practices. In this century, other areas, including vast regions of Australia, Europe, southwest USA, the Canadian prairies and others have seen considerable declines in crop productivity.

25 Although there is engineering technology available to combat this problem, through drainage and supply of high quality water, these measures are cumbersome and require high energy input. In most of the cases, due to the increased need for extensive agriculture, neither improved irrigation efficiency nor the installation of drainage systems is applicable. Moreover, in the arid and semi-arid regions of the world water evaporation exceeds 30 precipitation. These soils are inherently high in salt and require vast amounts of irrigation to become productive. Since irrigation water contains dissolved salts and minerals, an application of water is also an application of salt that compounds the salinity problem.

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The presence of especially sodium (Na+) salts in the soil is damaging to most plant species because salt excess prevents proper water absorption and has disastrous consequences on plant growth and productivity. Therefore, the sensitivity of most species to salt stress is limiting the diversity of crops that can be cultivated in salty areas. The latter 5 are not only coastal areas, where salt damage comes from the soil and from the salty sprays brought by the wind, but also areas where salt is put on streets, sidewalks and driveways in massive amounts for ice control, and cultivated regions where irrigation water has relatively high salt content.

The strong demand for renewable energy sources and the consequent recent actions 10 by governments to speed up production and commercialization of biofuels (Gura, 2009) have strongly increased the demand for agricultural space, which should now be obtained from areas that were so far not exploited in order to not subtract land from the areal for food production.

A further phenomenon that calls for higher salt resistance is soil salinization due to 15 irrigation. This has been recognized in the last years as a serious problem under conditions were the diluting effect of rain is absent, for instance in tunnels and under greenhouse conditions, or irrigated arid areas. Production of vegetables and flowers in protected conditions, on marginal land or in the presence of salt containing water can be economically convenient, but the impact of this practice on the environment, which 20 includes soil enrichment for a variety of solutes, cannot be neglected. The repeated application of greenhouse cultivation for instance causes increasing stress to the plants, typically leading to drop in production.

Plants suitable for cultivation on salt-rich soil and/or with salt containing water are the obvious goal of several breeding programs, but these were so far not too successful. 25 Species naturally resistant to rather high salt concentration are often not crops, but wild species that grow in coastal areas or other scarcely vegetated regions. One of the difficulties for improving this trait in crops comes from its multifactorial character. The homeostasis of ions inside a living cell involves multiple different cellular mechanisms controlled by several genes. The identification of a minimum number of factors/genes 30 necessary to achieve such change in the cellular balance of ions is essential to finally get to the goal, which has so far not been successfully achieved.

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Similarly, copper, iron, zinc, cobalt, nickel, and manganese metal cations, when present at high concentrations, along with cadmium, mercury, silver, and lead, can become extremely toxic, since they can cause oxidative damage or compete with other essential ions. The heavy metal cations are typically present in soil as natural components or as a 5 result of human activity. The primary sources of metal pollution are the burning of fossil fuels, mining and smelting of metalliferous ores, downwash from power lines, municipal wastes, fertilizers, pesticides, and sewage. Most existing remediation methods involve physicochemical technologies, such as chemical reduction/oxidation, soil washing and excavation, and are useful for intensive in- or ex-situ treatment of relatively highly 10 polluted sites, and not suitable for the remediation of vast, diffusely polluted areas where pollutants occur only at relatively low concentrations and/or superficially.

Accordingly, it would be desirable to have metal accumulator crops that could accumulate the metal ions in their tissue, which through harvesting and incinerating would allow removal of these metals from the soil, and hence also effective bioremediation.

15 The characterization of several families of Na+ transporters in plants, (Zhu, 2000) and yeast (Munoz-Mayor et al., 2008) has briefly given the hope that their over-expression might result in sodium resistant plants (Gisbert et al., 2000). Unfortunately, the activity of these transporters is post-transcriptionally regulated by the conditions in the cellular compartment where they localize (vacuole or cytoplasm). While all mentioned ion 20 transporters are able to translocate sodium ions across a membrane in exchange of another ion (H+ or K+ in the best characterized examples), their activity requires the simultaneous expression of several proteins, making the system therefore unpredictable and usually resulting in defects that prohibit growth. The SOS system, for example, requires the contemporary activity of SOS1, SOS2 and SOS3, of which SOS1 is the actual plasma 25 membrane Na+/H+ exchanger, while the other two are respectively a Ser/Thr kinase (which phosphorylates SOS1, (Quintero et al., 2002)) and a calcium binding protein. The S02 protein, (or another similar kinase) also seems to be involved in the regulation of the vacuolar Na+/H+ exchanger NHX (Qiu et al., 2003). The activity of the pumps SOS1 and NHX is also modulated by the presence of a proton gradient across the membrane on 30 which these transporters are localized, as they use such gradient as a driving force to translocate the sodium anions.

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In a first attempt to engineer salt-tolerant Arabidopsis lines, a 35S:NHX transgene was overexpressed. Transgenic plants that showed increased amounts of NHX protein were tested for their capability to grow on salt and accumulate sodium (Apse et al., 1999 and 2006). The performance of these plants was slightly improved, with sodium 5 accumulation increasing from 2% to 2.5% of the dry weight, and plants were still rather normally growing at a concentration of 200mM NaCl, while untransformed controls started showing delayed growth at 150mM NaCl. These results however indicate that a higher level of the NHX transporter is not sufficient for a strong increase in salt tolerance.

Overexpression of the plasma membrane H+-ATPase PMA4 (either full size or a 10 constitutively active form that lacks the regulatory C-terminal region) in tobacco would in theory provide a larger proton gradient accross the plasma membrane and stimulate SOS translocation activity. Unfortunately, the overexpression of these proton pumps gives rise to various developmental defects, making this strategy not feasible (Gévaudant et al., 2007).

15 The plant H+PPase are single peptide pumping system and therefore it is relatively simple to generate transgenic plants with increased H+PPase activity. Unfortunately, the overexpression of H+PPase results in heavy defects due to changes in auxin transport and distribution (Li et al, 2005).

Furthermore, plants that extrude the toxic compound/ion and therefore manage to 20 grow in disadvantageous conditions still do not solve the problem of the increasing

salinization of the soil due to irrigation or other sources of salt. For this reason, it would be highly advantageous to cultivate plants adapted to high salt concentration, and able to sequestrate the salt in different plant parts such as seeds, leaves, petals, and fruit including berries and other easy to harvest material. It would furthermore be beneficial if the salt 25 resistance in plants could be increased without impairing the function of the plant cell. SUMMARY OF THE INVENTION

Accordingly, in a first aspect, the present invention relates to a transgenic plant comprising a gene construct, a fragment, homolog or variant thereof encoding PHI and PH5 and optionally, a vacuolar metal cation antiporter that is activated through a proton 30 gradient, wherein the gene construct is operably linked to a plant promoter so that it ectopically over expresses PHI, PH5 and the optional vacuolar metal cation antiporter, and wherein the plant has an increased tolerance to one or more metal salt(s) as compared to a -5- corresponding progenitor plant which does not contain the gene construct, wherein the metal salt tolerance is defined as a lower rate of deformation and/or morbidity than that of the progenitor plant under identical metal salt limiting growth conditions.

Preferably, viral promoters are employed to give ectopic expression. However, 5 where only selective expression in part of the plant material is desired, promoters for organ specific or cell-type specific expression may be conveniently employed, for instance to avoid salt accumulation in edible parts.

Preferably, the proteins encoded by PHI, PH5 and the optional metal cation antiporter gene construct are residing in the membrane of the vacuole. Preferably, PHI 10 encodes a vacuolar P3BATPase. Preferably, PH5 encodes a vacuolar P3AATPase proton pump.

The metal cations that may be transported into the vacuole by the vacuolar metal cation antiporter that is activated through a proton gradient preferably include sodium, magnesium, copper, iron, zinc, cobalt, nickel, and/or manganese metal cations. Preferably, 15 this also works in the presence of cadmium, mercury, silver, and/or lead. The vacuolar metal cation antiporter may be one already present in a plant cell, or preferably may be a an additional metal cation antiporter, exogenous or from a different plant of the same species.

The vacuolar metal cation antiporter is preferably selected from one or more of a 20 vacuolar sodium/proton antiporter (NHX), a copper transporter (CTR), a cation diffusion facilitator (CDF), a zinc-iron permease (ZIP), and/or a cation exchanger (CAX).

The present invention further relates to an isolated cell, plant or part of a plant or progeny thereof according to the invention, wherein the cell, plant or part comprises an elevated PHI or PHI homolog, an elevated PH5 or PH5 homolog and an elevated gene 25 construct or homo log encoding the vacuolar metal cation antiporter. Elevated herein means a gene with elevated expression. In the cell, plant or part, the metal salt concentration in a vacuole of the cell or cells of the plant or plant parts is altered relative to a non-genetically modified plant when exposed to elevated metal cation concentrations, in for instance the soil, in the growth medium or in the water source.

30 Preferably, the plant parts include flower, fruit, vegetable, nut, root, stem, leaf and seed.

The subject invention further relates to a method of producing a plant having an -6- increased metal salt tolerance, comprising: a) identifying a plant that optionally ectopically, expresses a combination of PHI, PH5, and preferably a vacuolar metal cation antiporter from among plants having a gene construct comprising a gene encoding PHI, PH5 and preferably a vacuolar metal cation antiporter operably linked to a plant promoter 5 so that they are ectopically overexpressed in plants, b) screening the plant overexpressing PHI, PH5 and preferably a vacuolar metal cation antiporter for an improved metal salt tolerance under limiting growth conditions, and c) selecting the plant having an increased metal salt tolerance; wherein the metal salt tolerance is defined as a lower rate of deformation and/or morbidity 10 than that of the progenitor plant under identical metal salt limiting growth cond itions.

The plant according to the invention may advantageously expresses the combination of these genes outside the flowers, more preferably the expression may occur only in vegetative parts, in fruits, in roots and/or in tubers instead of the whole plant. This will permit to selectively increase metal cations in certain parts of the plant, which may be 15 easier to harvest. This is particularly interesting where perennial plants are to be grown for bioremediation.

The plants according to the invention advantageously comprise perennial plants , i.e. plants that lives for more than two years, but equally annual and biennial plants. Plants include perennial applies specifically to winter hardy herbaceous plants including but no 20 limited to woody plants like shrubs and trees, graminaceous plants such as switchgrass, Miscanthus, sugarcane, com, Arianthus, sorghum, other cereals and other forage and turf grasses. Other plants include fruit crops, such as tomatoes and relatives of Solanum lycopersicum, Grapes or any other suitable plant/ A perennial plant or "perennial" is a plant that produces flowers and seeds more 25 than once in its lifespan, and therefore lives for more than one year. As used herein, this term applies to all plants which flowers and produces seeds more than once. A plant that flowers and produces seeds only once in its lifetime is called a "monocarp". These include annual plants, which flower in their first living year, then die, or biennial plants, which flower in their second season. Some monocarp plants can live for many years before 30 flowering (and dying) as bamboo and agave.

Herbaceous perennials are plants that do not form permanent woody tissue. In warmer and more clement climates they may grow continuously. In seasonal climates, -7- their growth pattern is adapted to the growing season. In cooler temperate regions they generally grow and bloom during the warm part of the year, and the foliage dies back every winter. Regrowth is from their existing tissue or root-stock rather than from seed, as with annuals and biennials. In some cases, these perennials may retain their foliage all year 5 round, even in seasonal climates. Herbaceous perennials that retain their foliage all year round may be called evergreen perennials. Others are called deciduous. Woody perennials (ie. trees and shrubs) retain their woody structure permanently, but may lose their foliage in seasonal climates.

Perennial plants live more than 2 years and are usually grouped into two categories: 10 herbaceous perennials and woody perennials. Herbaceous perennials have soft, nonwoody stems that generally die back to the ground each winter. New stems grow from the plant's crown each spring. Trees and shrubs, on the other hand, have woody stems that withstand cold winter temperatures. They are referred to as woody perennials. There are many perennial plants important to human food production including many herbs, shrubs, and 15 trees.

The present invention further relates to a method of producing a transgenic plant having an increased metal salt tolerance, comprising: a) transforming at least one plant cell with an exogenous nucleic acid encoding an elevated PHI or PHI homolog, an elevated PH5 or PH5 homolog, and preferably an elevated gene construct or homolog encoding the 20 vacuolar metal cation antiporter, wherein the metal salt concentration in a vacuole of the cell or cells of the plant or plant parts is altered relative to a non-genetically modified plant, and b) regenerating the transformed cell into a plant having an increased metal salt tolerance, wherein the metal salt tolerance is defined as a lower rate of deformation and/or morbidity than that of the progenitor plant under identical metal salt limiting growth 25 conditions.

In a further preferred aspect, the present invention relates to a method for growing a plant having an increased metal salt tolerance, comprising: a) providing a genetically modified, or selectively evolved plant as set out above, and b) growing the plant on soil and/or with water under metal salt concentrations that are growth inhibiting for a 30 corresponding progenitor plant which does not contain the gene construct.

The present method further preferably comprises harvesting at least in part the plant material obtained in step (b).

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The present invention thus relates to plants, preferably either selectively evolved or genetically modified plants that are able to grow and/or bioremediate soil in the presence of elevated metal salt concentrations. In particular, these plants can advantageously remove metal salt from the soil and/or the water source and accumulate it in plant material such as 5 leaves and roots. In a preferred embodiment, the plants may then be harvested, and in the case of for instance of increased sodium metal cation tolerance, may preferably be fed to cattle as salt containing feed, or simply removed in the case of other metal salts toxic or noxious to cattle. In a preferred embodiment of the present invention, the metal salt, preferably sodium, does not accumulate in the fruits of the plant.

10 In a further preferred aspect, the method according to the present invention further comprises extracting the metal salts from the harvested plant material. This may advantageously be executed by a process for generating renewable energy, wherein the harvested plant material is subjected to one ore more fermentation step and/or a thermo chemical treatment, preferably torrefaction and/or pyrolysis and/or gasification, 15 resulting in energy ad useful products, as well as ash comprising the metal cations. The metal salts may then advantageously be extracted from the ash or the useful products.

The present invention further preferably relates to a method for modulating the metal salt concentration in a vacuole of a plant cell, comprising introducing into the plant cell or a parent or relative of the plant cell or modulating a modulating levels of protein from 20 PHI ,PH5 and a vacuolar metal cation antiporter, for the purposes of increasing the metal salt tolerance of the plant cell, and culturing the plant cell or plant comprising the cell or parent or relative of the cell under conditions to permit expression of the proteins.

The present invention further preferably relates to a method for producing a plant tolerant to increased metal salt levels, comprising stably transforming a cell of a suitable plant with 25 a nucleic acid sequence as described herein above under conditions permitting the eventual expression of the nucleic acid sequence, regenerating a transgenic plant from the cell and growing said transgenic plant for a time and under conditions sufficient to permit the expression of the nucleic acid sequence and optionally generating genetically modified progeny thereof.

30 The present invention further preferably relates to a method for producing a plant with reduced indigenous or existing pH modulating or altering activity, comprising stably transforming a cell of a suitable plant with a nucleic acid molecule as described herein -9- above which is antisense or sense to a sequence encoding PHI, PH5 and the sequence encoding the vacuolar metal cation antiporter, regenerating a transgenic plant from the cell and where necessary growing said transgenic plant under conditions sufficient to permit the expression of the nucleic acid and optionally generating genetically modified progeny 5 thereof.

The present invention further preferably relates to a gene construct, a fragment, homo log or variant thereof encoding PHI, PH5 and preferably a vacuolar metal cation antiporter comprising the Seq. ID. No:...

The subject invention further preferably relates to the use of the nucleic acid 10 molecule and/or corresponding polypeptide to generate genetic agents or constructs or other molecules which manipulate the metal cation uptake in a cell, groups of cells, organelles, parts or reproductions of a plant.

The present invention preferably also provides a nucleic acid molecule derived, obtainable or from plants encoding a polypeptide having pH modulating or pH altering 15 activity as well as a metal cation antiporter activity in the tonoplast membrane.

The present invention preferably also provides for the use of the sequences of PHI and PH5 to identify plants present in nature which do naturally express a combination of PHI and PH5, or preferably PHI and PH5 and a vacuolar ion transporter outside the flower and/or petals, and that show increased level of resistance to metal ions when 20 compared to the related crops without the expression.. SNPs or other molecular markers may then be advantageously generated to be able to follow the presence of these particular alleles. Such plants coming from the wild may then advantageously be used in breeding programs and the different steps of selection in the progeny will be assisted by marker assisted breeding.

25 The present invention further preferably relates to the use of the gene construct as described herein above for the purposes of increasing the metal salt tolerance of plant cells. Nucleotide and amino acid sequences are referred to by a sequence identifier number (SEQ ID NO:). The SEQ ID numbers correspond numerically to the sequence identifiers <400>1 (SEQ ID NO:l), <400>2 (SEQ ID NO:2), etc. A summary of the sequence identifiers is 30 provided in Table 1.

- 10- TABLE 1 Summary of sequence identifiers SEQ Name Type of Description ID Sequence NO:____ I PHI ATG-XhoI F Nucleotide Primer ~2 PHI STOP-Sall R Nucleotide Primer 3 PHI bp2230gDNA F Nucleotide Primer 4 PHI bp3360 gDNA R Nucleotide Primer 5 PHI ATG Kpnl F Nucleotide Primer 6 PHI stop XbaIR Nucleotide Primer PHI ATG-Clal F Nucleotide Primer 8 PHI STOP+XhoI R Nucleotide Primer 9 PHI ATG-PstI F Nucleotide Primer 10 PHI bp760(intronl) F Nucleotide Primer II PHI bp865(intron 1) R Nucleotide Primer 12 PHl+Kpnl F Nucleotide Primer 13 PhPHl+Notl R Nucleotide Primer 14 PhPHl-stop+Notl R Nucleotide Primer 15 PhPHl+stop+Notl R Nucleotide Primer 16 PHI-stop (R27) R Nucleotide Primer 17 PHl-ATG+Bgl IIF Nucleotide Primer 18 PHI ATG+Xhol F Nucleotide Primer 19 PHl_Tail+BglII R Nucleotide Primer 20 PH5/PPM-1 F Nucleotide Primer 21 PH5PPM-lEcoRI F Nucleotide Primer "~22 PH5/PPM-1 STOP R Nucleotide Primer "~23 PH5/PPM-1ATG (Ncol) F Nucleotide Primer 24 PH5 exon 12 F Nucleotide Primer 25 PH5 exon 12 R Nucleotide Primer 26 PH5 exon 3 R Nucleotide Primer 27 PH5ex-STOPNcoI R Nucleotide Primer 28 PH5 exon 14 F Nucleotide Primer 29 PH5 exon 13 F Nucleotide Primer 30 PH5 5TJTRF Nucleotide Primer ~Tl PH5 ATG + Sail F Nucleotide Primer 32 PH5 exon2 R Nucleotide Primer 33 PH5 exon2 F Nucleotide Primer 34 PH5 rose+petunia F Nucleotide Primer - 11 -

Table (continued)___ SEQ Name Type of Description ID Sequence NO:____ 35 PH5 rose+petunia R Nucleotide Primer 36 PH5 ATG+Bglll F Nucleotide Primer 37 PH5Stop+Xba/PvuII R Nucleotide Primer 38 PH5Stop-3aa+Xba/PvuII R Nucleotide Primer 39 PH5 ATG TOPO F Nucleotide Primer 40 PH5-stop R Nucleotide Primer 41 PH5 tail R Xho R Nucleotide Primer 42 PH5 aa838 Topo F Nucleotide Primer 43 PH5 stop R Nucleotide Primer 44 PH5 aa80(+stop) R Nucleotide Primer 45 PH5 aal51 Topo F Nucleotide Primer 46 PH5 aa235(+stop) R Nucleotide Primer 47 PH5 aa305 Topo F Nucleotide Primer 48 PH5 aa635 (+stop) R Nucleotide Primer 49 PH5 aa303 Ncol F Nucleotide Primer 50 PH5 aa635 EcoRl R Nucleotide Primer ~51 PH5(MSGW) F Nucleotide Primer 52 PH5MSG R Nucleotide Primer 53 PH5ATG+attBl F Nucleotide Primer 54 PH5-stop+attB2 R Nucleotide Primer 55 PhNHXl F Nucleotide Primer 56 PhNHXl-Fw2 F Nucleotide Primer 57 PhNHXl-Fw3 F Nucleotide Primer 58 PhNHXl-Fw4F Nucleotide Primer 59 PhNHXl-Rvl R Nucleotide Primer 60 PhNHXl-Rv2 R Nucleotide Primer 61 AMINHX1.1 miR-s Nucleotide Primer 62 AMINHX1.1 miR-a Nucleotide Primer 63 AMINHX1.1 miR*s Nucleotide Primer 64 AMINHX1.1 miR*s Nucleotide Primer 65 AMINHX1.2 miR-s Nucleotide Primer 66 AMINHX1.2 miR-a Nucleotide Primer 67 AMINHX1.2 miR*s Nucleotide Primer 68 AMINHX1.2 miR*a Nucleotide Primer 69 PhNHXl-Rv3 R Nucleotide Primer - 12-

Table (continued)___ SEQ Name Type of Description ID Sequence NO:____ 70 PhNHXl-Rv4R Nucleotide Primer ~Tl PhNHXl-Fw5 +CACC F Nucleotide Primer 72 PhNHXl-Rv5 stopR Nucleotide Primer 73 PhNHXl-Fw6F Nucleotide Primer 74 PhNHX tail-Xba-Rev R Nucleotide Primer 75 NHX-attBlFwF Nucleotide Primer 76 NHX-attB2Rev R Nucleotide Primer 77 NHX-exon3Fw F Nucleotide Primer 78 NHX-exon4FwF Nucleotide Primer 79 NHXexon3-revR Nucleotide Primer 80 PhNHX-3'UTR-R R Nucleotide Primer 81 magnesium/proton exchanger AtMHX Nucleotide Nucleotide sequence of Arabidopsis

Thaliana magnesium/proton __exchanger_ 82 magnesium/proton exchanger AtMHX Amino Acid Amino Acid sequence of

Arabidopsis Thaliana magnesium/proton exchanger_ 83 ZAT Nucleotide Nucleotide sequence of Arabidopsis

Thaliana ZAT (ZINC

____TRANSPORTER)_ 84 ZAT Amino acid Amino Acid sequence of

Arabidopsis Thaliana ZAT (ZINC

____TRANSPORTER)_ 85 Fe(II) transport protein (IRT1) Nucleotide Nucleotide sequence of Arabidopsis

Thaliana Zinc iron permease_ 86 Fe(II) transport protein (IRT1) Amino acid Amino Acid sequence of

Arabidopsis Thaliana Zinc iron __permease_ 87 CAX9 (CATION EXCHANGER 9) Nucleotide Nucleotide sequence of Arabidopsis

Thaliana cation transmembrane transporter/ cationxation antiporter/ manganese ion transmembrane transporter/ potassium ion transmembrane transporter/ sodium ion transmembrane transporter 88 CAX9 (CATION EXCHANGER 9) Amino acid Amino Acid sequence of

Arabidopsis Thaliana cation transmembrane transporter/ cationxation antiporter/ manganese ion transmembrane transporter/ potassium ion transmembrane transporter/ sodium ion transmembrane_transporter - 13 -

Table (continued)___ SEQ Name Type of Description ID Sequence NO:____ 89 ZIP7 (ZINC TRANSPORTER 7 Nucleotide Nucleotide sequence of Arabidopsis PRECURSOR) Thaliana cation transmembrane transporter/ metal ion transmembrane transporter/ zinc ion transmembrane transporter 90 ZIP7 (ZINC TRANSPORTER 7 Amino acid Amino Acid sequence of PRECURSOR) Arabidopsis Thaliana cation transmembrane transporter/ metal ion transmembrane transporter/ zinc ion transmembrane transporter 91 PhNHXl cDNA V30 start-stop Nucleotide Nucleotide sequence of Arabidopsis ____NHX_ 92 PhNHXl genomic DNA R27 start-stop Nucleotide Nucleotide sequence of Arabidopsis ____NHX_ 93 PhNHXl R27 protein sequence Amino acid Amino acid sequence of ____Arabidopsis NHX_ 94 PhPHl cDNA R27 start-stop Nucleotide Nucleotide sequence of petunia ____PHI_ 95 PhPHl R27 protein sequence Amino acid Amino acid sequence of petunia ____PHI_ 96 PhPH5 cDNA R27 start-stop Nucleotide Nucleotide sequence of petunia ____PH5_ 97 PhPH5 R27 protein sequence Amino acid Amino acid sequence of petunia ___ PH5_ - 14-

BRIEF DESCRIPTION OF THE FIGURES

Some figures contain color representations or entities. Color photographs are available from the Patentee upon request or from an appropriate Patent Office.

Figure lis a diagrammatic representation of the genomic PCR fragment containing 5 the complete coding sequence (from ATG to STOP codon) of PHI. P1025: genomic fragment containing the full cds of the PHI petunia gene (including all introns between ATG and STOP). The vector is pB7WG2,0. The reference signs in Figure 1 are listed in Table la:

Table la: Reference Sisns of Figure 1

Reference Sign Reference Sign

Exon 1 1 Bar 26

Exon 2 2 LB 27

Exon 3 3 Sm/SpR 28

Exon 4 4 RB 29

Exon 5 5 p35S 30

Exon 6 6 attB 1 31

Topo

overhang(C

Exon 7 7 ACC) 32

Primer 4001

Exon 8 8 Fw 33

Primer 3917

Exon 9 9 rev 34

TOPO

attB2 24 binding site 35 T35S 25

Figure 2 (1027 35S.PHI rose gDNA in pK2GW7) is a diagrammatic representation of the rose PHI genomic fragment derived from the construct in described in Figure 5 10 - 15 - following cloning into the expression vector pK2GW7 between the 35S promoter and the 35S terminator. This construct confers resistance to Kanamicin in plant cells, p 836: genomic fragment of the PH5 petunia gene containing the full coding sequence between ATG and STOP and including all introns. The vector is pK2GW7,0.

5 The reference signs in Figure 2 are listed in Table 2a:

Table 2a: Reference Signs of Figure 2

Reference Sign Reference Sign

Exon 1 1 Exon 15 15

Exon 2 2 Exon 16 16

Exon 3 3 Exon 17 17

Exon 4 4 Exon 18 18

Exon 5 5 Exon 19 19

Exon 6 6 Exon 20 20

Exon 7 7 Exon 21 21

Exon 8 8 Pr. 0629 (rev) 38

Exon 9 9 nptll CDS 39

Exon 10 10 Primer 2285 Rev 40

Exon 11 11 0027 Fw 41

Exon 12 12 0628Fw 42

Exon 13 13 attBl 31

Exon 14 14 Toes 44

Figure 3 (1028 35S:PH1 rose gDNA in pB7WG2.0) is a diagrammatic representation of the rose PHI genomic fragment derived from the construct in described 10 in Figure 5 following cloning into the expression vector pB7GW2.0 between the 35S promoter and the 35S terminator. This construct confers resistance to the herbicide Basta in plant cells. (Construct for the localization of NHX as GFP fusion pi 165; genomic - 16- fragment containg the coding sequence of the petunia NHX gene between ATG and STOP including introns. The vector is pH7WG2,0).

The reference signs in Figure 3 are listed in Table 3a:

Table 3a: Reference Signs of Figure 3

Reference Sign. Reference Sign

Exon 1 1 Exon 12 12

Exon 2 2 Exon 13 13

Exon 3 3 Exon 14 14

Exon 4 4 2285 R 47

Exon 5 5 4496 F 48

Exon 6 6 Hyg 49

Exon 7 7 4497 R 50

Exon 8 8 3818 Rev 51

Exon 9 9 3767 Rev 52

Exon 10 10 3817Rev 53

Exon 11 11 3769 Rev 54 5

Figure 4 is a diagrammatic representation of construct 0836 (893) for expression of Petunia hybrida PH5 in plants containing 35S: petunia PH5:35S expression cassette in a binary transformation vector.

The reference signs in Figure 4 are listed in Table 4a: - 17-

Table 4a: Reference Sisns of Figure 4

Reference Sign Reference Sign

Exon 1 1 Exon 12 12

Exon 2 2 Exon 13 13

Exon 3 3 kan 57

Exon 4 4 Egfp 58

Exon 5 5 Primer 1664 59

Exon 6 6 START 60

Exon 7 7 4005 Fw 61

Exon 8 8 3766 Fw 62

Exon 9 9 4054 Fw 63

Exon 10 10 4006 Rev 64

Exon 11 11 STOP 65

Figure 5 and 6 is a diagrammatic representation of amiRNA constructs for the silencing of NHX expression in petunia plants. In 5 the construct pl004 and in 6 pl005.

5 The reference signs in Figures 5 and 6 are listed in Tables 5a and 6a, respectively: - 18-

Table 5a: Reference Sims of Figure 5

Reference Sign Reference Sign

Nco I (53) 68 NHX 1,1 miRs 75

Nco I (443) 69 Nco I (1531) 76 35S Promoter 70 Bam HI (1539) 77

Pst I (1048) 71 35S terminal 78

Eco R1 (1115) 72 Pst I (1823) 79

Eco R1 (1125) 73 Pst I (1843) 80 NHX 1,1 miR^s 74 Clal(5097 81

Table 6a: Reference Signs of Figure 6

Reference Sign Reference Sign attB2 24 Primer 2285 Rev 40 T35S 25 0027 Fw 41 LB 27 0628Fw 42

Sm/SpR 28 kan 57 RB 29 NHX l,2mi 84 p35S 30 NHX l,2n 85 attBl 31 0629 rev 86 5 Figure 7 is a photographic and graphical representation of different tonoplast proteins on vacuolar pH and sodium transport. A) In petal cells, PH5 is necessary to build a proton gradient across the vacuolar membrane. The activity of this pump quickly creates a large electrochemical gradient against which PH5 cannot pump, unless the transporter PHI lowers the positive charge of the vacuolar lumen by pumping out Mg++ ions. Both 10 transporters use ATP as energy source. B) When NaCl is present, NHX uses (part of) the - 19- H+ gradient to sequester sodium into the vacuolar lumen. For this reason no damages are visible on flowers when high NaCl concentration gives necrosis on leaves of he same plant (C). This whole mechanism can be reproduced in leaves by ectopic expression of PHI and PH5. (D) The pH of the crude leaf extract of these transgenics is indeed lower than that of 5 control plants. We have shown that the proton gradient built by PHI and PH5 in leaves is consumed when NHX is expressed ectopically from the CaMV35S promoter. We think that the large proton gradient resulting from PH5 and PHI expression in leaves, energizes the tonoplast for a stronger activity of NHX (E).

The reference signs in Figure 7 are listed in Table 7a: 10

Table 7a: Reference Signs ofFieure 7

Reference Sign Reference Sign WT 88 Mg++ 96 200nM NaCl 89 H+ 97 35S:PH1 35S:PH5 90 Na+ 98 cytoplasm 91 pH 5.5 100 vacuolar lumen 92 pH 6.2 100’ PHI 93 pH 6 100” PH5 94 pH 5.5 100’” NHX 95 pH? 101

Figure 8 is a photographic and graphical representation of NHX localization in 15 petal epidermal protoplasts. The presence of sects in the membrane marked by the GFP signal and the presence of the nucleus outside this membrane indicates that this is the tonoplast.

DETAILED DESCRIPTION

The present invention particularly relates to transgenic plants that are expressing, 20 advantageously overexpressing, more advantageously ectopically, PHI and PH5 and a -20- preferably optionally a vacuolar metal cation antiporter that is activated through a proton gradient, i.e. genes encoding respectively a vacuolar P3BATPase, a vacuolar P3AATPase proton pump and the vacuolar antiporter, all of which reside in the tonoplast. The vacuolar P3BATPase is highly similar to bacterial Mg2+ transporters.

5 Vacuoles occupy a large part of the plant cell volume and play a crucial role in the maintenance of cell homeostasis. In mature cells, these organelles can approach 90% of the total cell volume, and can store a large variety of molecules ranging from ions, organic acids, sugar, enzymes, storage proteins to different types of secondary metabolites. The vacuoles also serve as reservoirs of protons and other metabolically important ions.

10 Different transporters on the membrane of the vacuoles regulate the accumulation of solutes in this compartment and drive the accumulation of water producing the turgor pressure of the cell. These structurally simple organelles play a wide range of essential roles in the life of a plant and this requires their internal environment to be tightly regulated. Storage of metal salts in the vacuoles would therefore be an ideal solution 15 overcome toxicity of these metal salts without negatively affecting the plant growth.

Moreover, since through ectopical expression in the entire plant material, as compared to only in petals, the entire plant would be able to store the metal salts, the metal cations would be removed permanently from the soil and/or the water source, and could advantageously be recovered or extracted from harvested plant material. This could 20 advantageously be performed by incineration of harvested plant material to generate renewable energy, preferably followed by a metal extraction from the obtained ash.

With respect to the storage of sodium cations, this would result in a truly halophytic response to salinity in plants.

Applicants have found that the compartmentation of metal cations, such as Na+ into 25 vacuoles may provide an efficient mechanism to avert the toxic effects of the metal cations, such as Na+ in the cytosol, without affecting the other properties of the plant, such as healthy growth and leaf deformation. Without being bound to any particular theory, it is believed that the transport of Na+ into the vacuoles as mediated by for instance the NHX Na+/H+ antiporter is driven by the electrochemical gradient of protons generated by 30 overexpressing vacuolar H+-translocating enzymes PHI and PH5, namely a vacuolar P3BATPase and a vacuolar P3AATPase proton pump. Similarly, other vacuolar metal cation antiporters will have the same activity increase, resulting in increased levels of metal -21 - cation capture in the vacuoles.

Suitable metal cation antiporters include NHX, a copper transporter (CTR), a cation diffusion facilitator (CDF), zinc-iron permease (ZIP), and/or cation exchanger (CAX).

There are several cation/H+ exchanger that are expected to be fiield by a steep 5 proton gradient across the tonoplast. Therefore, any transporter of this type would be energized to transport more of the ion it exchanges for the protons. Others include CTR transporters, which are constituted by transmembrane polypeptides, containing several copper-binding sequences of functional and/or regulatory value, and assembling as trimers. The CTR family copper transporters suitable according to the present invention are 10 included within the channel-type facilitators. Typically, it is assumed that Copper is transported down a concentration gradient since intracellular copper is immediately sequestered, notably by Cu chaperones. CTR family copper transporters were first discovered in yeast, where Ctrlp and Ctr3p, localized to the plasma membrane, are functionally redundant. A third CTR type transporter, Ctr2p, mobilizes stored copper from 15 the vacuole under conditions of copper deficiency. CTR transporters according to the invention also include those present in animal and plant cells. Six CTR members (named AtCOPTl-6) were for instance identified in A. thaliana, of which AtCOPTl, 2, 3, and 5 have been functionally characterized. Similarly, three genes encoding CTR transporters have been identified and characterized from the alga C. reinhardtii.

20 Furthermore, CAX can transport Ca2+ as well as several other ions like cadmium, mercury, manganese, zinc, nickel, and silver (K. D. Hirschi, R. Zhen, K. W. Cunningham, P. A. Rea, and G. R. Fink. (1996) CAX1, an H+/Ca2+ antiporter from Arabidopsis. Proceedings of the National Academy of Sciences, USA 93:8782-8786); MHX is atransporter of Magnesium, Zinc and Iron ions (Berezin et al. 2008: Functional Plant 25 Biology 35, 15-25 and Elbaz et al 2006. Plant Cell and Environment 29,1179-1190); CHX describes a large family of transporters. For most of them the transported ion is unknown, but some of the members of this family are involved in K+ transport. (Pardo et al.J. Exp. Bot. (March 2006) 57 (5): 1181-1199).

The activity of any one of these transporters is controlled by de steepness of the 30 proton gradient across the membrane of the organell on which the pump is located. Therefore, the PHI /PH5 acidification machinery will speed up the activity of these proteins when it localizes on the same membrane on which they are active.

-22-

With respect to the transporter NHX, the fact that it uses the proton pool contained inside the same compartment which PHI and PH5 acidify, has been proven by comparison of a wild type plant having a red flower due to a low pH of the petal extract, which had been transformed with a 35S:PhNHX construct. Among the transgenics that resulted from 5 this transformation, those that show the highest expression of the NHX transgene, also had blue flowers and a high pH.

Applicants have advantageously found that a two-protein system that drives acidification of the central vacuole in petal cells of pigmented flowers could be also employed for the purpose of selecting, or genetically modifying plants to increase the 10 tolerance to certain metal cations. When ectopically expressed in plant cells, the proton pump turns out to be advantageous to drive metal cation transporters that are also based in the tonoplast, this allowed the import of metal cations into the vacuole.

This may advantageously be employed to grow selectively evolved or genetically modified plants under conditions that are growth limiting for non-evolved or genetically 15 unmodified progenitor plants.

The two major structural genes required for vacuolar acidification were found to be PHI (unpublished, manuscript in preparation) and PH5 (Verweij et al., 2008). The expression of both genes is controlled by the transcription regulators AN1, AN11, PH3 and PH4.

20 PHI and PH5 preferably encode respectively a PseATPase that is highly similar to bacterial Mg2+ transporters and a P3AATPase proton pump closely related to plant plasma membrane proton pumps, which both reside in the tonoplast of epidermal petal cells.

Accordingly, genetic agents and proteinaceous agents are provided which increase the salt tolerance of plant cells, The agents include nucleic acid molecules such as cDNA 25 and genomic DNA or parts or fragments thereof, antisense, sense or RNAi molecules or complexes comprising same, ribozymes, peptides and proteins. In a particular embodiment, the cell salt storage and transport is altered by twofold manipulation to the vacuolar pH, by manipulation of PHI in combination with PH5 and a sodium-potassium antiporter for the purposes of altering the salt concentration in the vacuole of plant cells including seeds, 30 leafs and other reproductive and vegetative material.

Accordingly, genetic agents and proteinaceous agents are provided which increase the salt resistance of plant cells, and which in particular regulate the level of sodium -23- acidity or alkalinity in a plant cell. The agents include nucleic acid molecules such as cDNA and genomic DNA or parts or fragments thereof, antisense, sense or RNAi molecules or complexes comprising same, ribozymes, peptides and proteins. In a particular embodiment, the cell salt storage and transport is altered by twofold manipulation to the 5 vacuolar pH, by manipulation of PHI in combination with PH5 and a metal cation antiporter for the purposes of altering the ion concentration in the vacuole of plant cells including seeds, leafs and other reproductive and vegetative material. In the case of sodium cations, the antiporter preferably is a sodium-proton antiporter such as NHX.

The nucleic acid molecules referred to herein as “PHI”, “PH5 ” and “metal cation 10 antiporter” includes their homo logs, orthologs, paralogs, polymorphic variants and derivatives from a range of plants and/or animals.

As used herein, "operably linked" can refer to a situation wherein the components described are in a relationship permitting them to function in their intended manner. For instance, a control sequence "operably linked" to a coding sequence is ligated in such a 15 manner that expression of the coding sequence is achieved under conditions compatible with the control sequence.

The present invention preferably relates to a method of producing a plant having an increased metal salt tolerance, comprising: a) identifying a plant that ectopically, or in one or more plant parts expresses a 20 combination of PHI and PH5 from among plants having a gene construct comprising a gene encoding PHI and PH5 operably linked to a plant promoter so that they are overexpressed in plants, b) screening the plant overexpressing PHI, PH5 for an improved metal salt tolerance under limiting growth conditions, and 25 c) selecting the plant having an increased salt tolerance; wherein the metal salt tolerance is defined as a lower rate of deformation and/or morbidity than that of the progenitor plant under identical metal salt limiting growth conditions. The gene construct comprising a gene encoding PHI and PH5 operably linked to a plant promoter thereby can advantageously serve as a marker gene that permits 30 selection of salt tolerant plants from wild type plasts as wella s from transgene plants.

-24-

In a transgenic plant that expresses the polynucleotide according to the present invention claim 1, at least a part of the transgenic plant has an altered trait as compared to a non-transgenic plant or wild-type plant.

Preferably, a PHI is selected from a plant which:

5 (i) comprises a nucleotide sequence which has at least 50% identity to SEQ ID

NO: 94 after optimal alignment; (ii) comprises a nucleotide sequence which is capable of hybridizing to SEQ ID NO:94 or its complement; (iii) encodes an amino acid sequence which has at least 50% similarity to SEQ 10 IDNO:95 after optimal alignment.s.

Preferably, PH5 is selected from a plant which: (i) comprises a nucleotide sequence which has at least 50% identity to SEQ ID NO:96 after optimal alignment;

(ii) comprises a nucleotide sequence which is capable of hybridizing to SEQ ID

15 NO:96 or its complement; (iii) encodes an amino acid sequence which has at least 50% similarity to SEQ IDNO:97 after optimal alignment.

The PH5 gene is disclosed in Verweij et al, Nature Cell Biology 10:1456-1462, 2008 and in International Patent Application Nos. PCT/AU2006/000451 and 20 PCT/AU2007/000739.

Preferably, the metal cation antiporter is selected from a plant which: (i) comprises a nucleotide sequence which has at least 50% identity to SEQ ID NOs: 91, 92, 81, 83, 85, 87 and/or 89 after optimal alignment; (ii) comprises a nucleotide sequence which is capable of hybridizing to SEQ ID 25 NOs: 91, 92, 81, 83, 85, 87 and/or 89 or its complement; (iii) encodes an amino acid sequence which has at least 50% similarity to SEQ ID NOs:93, 82, 84, 86, 88 and/or 90 after optimal alignment.

Preferably, the transgene further comprises a promoter sequence operably linked to the first nucleic acid sequence. More preferably, the promoter is a constitutive promoter or 30 an inducible promoter. Advantageously, the promoter may be a CaMV35 S promoter (from Cawliflower Mosaic Virus) or any organ- plant part-, tissue- or cell type-specific promoter.

-25-

Altemative percentage similarities and identities (at the nucleotide or amino acid level) encompassed by the present invention include at least about 60% or at least about 65% or at least about 70% or at least about 75% or at least about 80% or at least about 85% or at least about 90% or above, such as about 95% or about 96% or about 97% or 5 about 98% or about 99%, such as at least about 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100%.

For the purposes of determining the level of stringency to define nucleic acid 10 molecules capable of hybridizing to SEQ ID NO:94 or 96 or 91 or 92 or 81 or 83 or 85 or 87 or 89 reference herein to a low stringency includes and encompasses from at least about 0% to at least about 15% v/v formamide and from at least about 1M to at least about 2 M salt for hybridization, and at least about 1 M to at least about 2 M salt for washing conditions. Generally, low stringency is from about 25-30°C to about 42°C. The 15 temperature may be altered and higher temperatures used to replace the inclusion of formamide and/or to give alternative stringency conditions. Alternative stringency conditions may be applied where necessary, such as medium stringency, which includes and encompasses from at least about 16% v/v to at least about 30% v/v formamide and from at least about 0.5 M to at least about 0.9 M salt for hybridization, and at least about 20 0.5 M to at least about 0.9 M salt for washing conditions, or high stringency, which includes and encompasses from at least about 31% v/v to at least about 50% v/v formamide and from at least about 0.01 M to at least about 0.15 M salt for hybridization, and at least about 0.01 M to at least about 0.15 M salt for washing conditions. In general, washing is carried out Tm = 69.3 + 0.41 (G+C)% (Marmur and Doty, J. Mol. Biol 5: 109, 25 1962). However, the Tm of a duplex DNA decreases by 1°C with every increase of 1% in the number of mismatch base pairs (Bonner and Laskey, Eur. J. Biochem. 46: 83, 1974). Formamide is optional in these hybridization conditions. Accordingly, particularly preferred levels of stringency are defined as follows:low stringency is 6 x SSC buffer, 1.0% w/v SDS at 25-42°C; a moderate stringency is 2 x SSC buffer, 1.0% w/v SDS at a 30 temperature in the range 20°C to 65°C; high stringency is 0.1 x SSC buffer, 0.1% w/v SDS at a temperature of at least 65°C.

-26-

Another aspect of the present invention provides a nucleic acid molecule comprising a sequence of nucleotides encoding or complementary to a sequence encoding an amino acid sequence substantially as set forth in SEQ ID NO:82, 84, 86, 88, 90, 93, 95, or 97, or an amino acid sequence having at least about 50% similarity thereto after optimal 5 alignment.

The term similarity as used herein includes exact identity between compared sequences at the nucleotide or amino acid level. Where there is non-identity at the nucleotide level, similarity includes differences between sequences which result in different amino acids that are nevertheless related to each other at the structural, functional, 10 biochemical and/or conformational levels. Where there is non-identity at the amino acid level, similarity includes amino acids that are nevertheless related to each other at the structural, functional, biochemical and/or conformational levels. In a particular embodiment, nucleotide sequence comparisons are made at the level of identity and amino acid sequence comparisons are made at the level of similarity.

15 Terms used to describe sequence relationships between two or more polynucleotides or polypeptides include "reference sequence", "comparison window", "sequence similarity", "sequence identity", "percentage of sequence similarity", "percentage of sequence identity", "substantially similar" and "substantial identity". A "reference sequence" is at least 12 but frequently 15 to 18 and often at least 25 or above, 20 such as 30 monomer units, inclusive of nucleotides and amino acid residues, in length. Because two polynucleotides may each comprise (1) a sequence (i.e. only a portion of the complete polynucleotide sequence) that is similar between the two polynucleotides, and (2) a sequence that is divergent between the two polynucleotides, sequence comparisons between two (or more) polynucleotides are typically performed by comparing sequences of 25 the two polynucleotides over a "comparison window" to identify and compare local regions of sequence similarity. A "comparison window" refers to a conceptual segment of typically 12 contiguous residues that is compared to a reference sequence. The comparison window may comprise additions or deletions (i.e. gaps) of about 20% or less as compared to the reference sequence (which does not comprise additions or deletions) for optimal 30 alignment of the two sequences. Optimal alignment of sequences for aligning a comparison window may be conducted by computerized implementations of algorithms (GAP, BESTFIT, FASTA, and TFASTA in the Wisconsin Genetics Software Package Release -27- 7.0, Genetics Computer Group, 575 Science Drive Madison, WI, USA) or by inspection and the best alignment (i.e. resulting in the highest percentage homology over the comparison window) generated by any of the various methods selected. Reference also may be made to the BLAST family of programs as, for example, disclosed by Altschul et 5 al, (Nucl. Acids Res. 25: 3389-3402, 1997). A detailed discussion of sequence analysis can be found in Unit 19.3 of Ausubel et al, Current Protocols in Molecular Biology John Wiley & Sons Inc, 1994-1998, Chapter 15, 1998.

The terms "sequence similarity" and "sequence identity" as used herein refers to the extent that sequences are identical or functionally or structurally similar on a nucleotide-10 by-nucleotide basis or an amino acid-by-amino acid basis over a window of comparison. Thus, a "percentage of sequence identity", for example, is calculated by comparing two optimally aligned sequences over the window of comparison, determining the number of positions at which the identical nucleic acid base (e.g. A, T, C, G, I) or the identical amino acid residue (e.g. Ala, Pro, Ser, Thr, Gly, Val, Leu, lie, Phe, Tyr, Trp, Lys, Arg, His, Asp, 15 Glu, Asn, Gin, Cys and Met) occurs in both sequences to yield the number of matched positions, dividing the number of matched positions by the total number of positions in the window of comparison (i.e., the window size), and multiplying the result by 100 to yield the percentage of sequence identity. For the purposes of the present invention, "sequence identity" will be understood to mean the "match percentage" calculated by the DNASIS 20 computer program (Version 2.5 for windows; available from Hitachi Software engineering Co., Ltd., South San Francisco, California, USA) using standard defaults as used in the reference manual accompanying the software. Similar comments apply in relation to sequence similarity.

The nucleic acid sequences contemplated herein also encompass oligonucleotides 25 useful as genetic probes for amplification reactions or as antisense or sense molecules capable of regulating expression of the corresponding PHI, PH5 and metal cation antiporter genes in a plant.

Sense molecules include hairpin constructs, short double stranded DNAs and RNAs and partially double stranded DNAs and RNAs which one or more single stranded 30 nucleotide over hangs.

An antisense molecule as used herein may also encompass a genetic construct comprising the structural genomic or cDNA gene or part thereof in reverse orientation -28- relative to its own or another promoter. It may also encompass a homologous genetic sequence. An antisense or sense molecule may also be directed to terminal or internal portions of the PHI, PH5 and metal cation antiporter genes such that the expression of the genes is reduced or eliminated.

5 With respect to this aspect, there is provided an oligonucleotide of 5-50 nucleotides such as 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20,21,22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80 having substantial similarity to a part or region of a molecule 10 with a nucleotide sequence set forth in SEQ ID NO:91 and 92, 94 or 96 or 81 or 83, 85, 87, or 89 or which hybridizes to a complementary strand of SEQ ID NO:94, 96, 91, 92, 81, 83, 85, 87, or 89 under low stringency conditions.

By substantial similarity or complementarity in this context is meant a hybridizable similarity under low, alternatively and preferably medium and alternatively and most 15 preferably high stringency conditions specific for oligonucleotide hybridization (Sambrook et al, Molecular Cloning: A Laboratory Manual, 2nd edition, Cold Spring Harbor Laboratories, Cold Spring Harbor, NY, USA, 1989). Such an oligonucleotide is useful, for example, in screening for pH modulating or altering genetic sequences from various sources or for monitoring an introduced genetic sequence in a transgenic plant.

20 In one embodiment, the nucleic acid sequence encoding PHI or various functional derivatives thereof is used to reduce the level of an endogenous PHI (e.g. via cosuppression or antisense-mediated suppression) or other post-transcriptional gene silencing (PTGS) processes including RNAi or alternatively the nucleic acid sequence encoding this enzyme or various derivatives or parts thereof is used in the sense or antisense orientation 25 to reduce the level of a pH modulating or altering protein. The use of sense strands, double or partially single stranded such as constructs with hairpin loops is particularly useful in inducing a PTGS response. In a further alternative, ribozymes, minizymes or DNAzymes could be used to inactivate target nucleic acid sequences.

Still a further embodiment encompasses post-transcriptional inhibition to reduce 30 translation into PHI, PH5 and optionally metal cation antiporter polypeptide material. Still yet another embodiment involves specifically inducing or removing methylation.

-29-

Reducing PHI levels or activity leads to an increase in pH leading to alkaline conditions.

Reference herein to the changing of a pH modulating or altering activity relates to an elevation or reduction in activity of up to 30% or more preferably of 30-50%, or even 5 more preferably 50-75% or still more preferably 75% or greater above or below the normal endogenous or existing levels of activity. Such elevation or reduction may be referred to as modulation or alteration of PHI. Often, modulation is at the level of transcription or translation of PHI. Alternatively, changing pH modulation is measured in terms of degree of alkalinity or acidity and/or an ability to complement a PHI mutant plant such as & phi 10 petunia mutant.

It is proposed that PHI in combination with PH5, and optionally a metal cation antiporter which uses proton gradients to transport ions, such as NHX (which exchanges protons for Na+ or K+) promotes a higher level of metal cation, preferably sodium sequestration in the vacuolar lumen.

15 The nucleic acids of the present invention encoding or controlling PHI, PH5 and the metal cation antiporter may be a ribonucleic acid or deoxyribonucleic acids, single or double stranded and linear or covalently closed circular molecules. Generally, the nucleic acid molecule is cDNA.

The term gene is used in its broadest sense and includes cDNA corresponding to 20 the exons of a gene. Accordingly, reference herein to a gene is to be taken to include:- (i) a classical genomic gene consisting of transcriptional and/or translational regulatory sequences and/or a coding region and/or non-translated sequences (i.e. introns, 5'- and 3'- untranslated sequences); or (ii) mRNA or cDNA corresponding to the coding regions (i.e. exons) and 5'- and 3'-25 untranslated sequences of the gene.

The term gene is also used to describe synthetic or fusion molecules encoding all or part of an expression product. In particular embodiments, the term nucleic acid molecule and gene may be interchangeably used.

The nucleic acid or its complementary form may encode the full-length PHI, PH5 30 and/or the metal cation antiporter enzyme or a part or derivative thereof. By "derivative" is meant any single or multiple amino acid substitutions, deletions, and/or additions relative to the naturally occurring enzyme and which retains a pH modulating or altering activity -30- and/or an ability to complement a PHI mutant plant or plant tissue such as a petunia phi mutant plant. In this regard, the nucleic acid includes the naturally occurring nucleotide sequence encoding a pH modulating or altering activity or may contain single or multiple nucleotide substitutions, deletions and/or additions to the naturally occurring sequence.

5 The nucleic acid of the present invention or its complementary form may also encode a "part" of the pH modulating or altering protein, whether active or inactive, and such a nucleic acid molecule may be useful as an oligonucleotide probe, primer for polymerase chain reactions or in various mutagenic techniques, or for the generation of antisense molecules.

10 Reference herein to a "part" of a nucleic acid molecule, nucleotide sequence or amino acid sequence, preferably relates to a molecule which contains at least about 10 contiguous nucleotides or five contiguous amino acids, as appropriate.

Amino acid insertional derivatives of the pH modulating or altering protein of the present invention include amino and/or carboxyl terminal fusions as well as intra-sequence 15 insertions of single or multiple amino acids. Insertional amino acid sequence variants are those in which one or more amino acid residues are introduced into a predetermined site in the protein although random insertion is also possible with suitable screening of the resulting product. Deletional variants are characterized by the removal of one or more amino acids from the sequence. Substitutional amino acid variants are those in which at 20 least one residue in the sequence has been removed and a different residue inserted in its place.

Where PHI, PH5 and/or metal cation antiporter protein is derivatized by amino acid substitution, the amino acids are generally replaced by other amino acids having like properties, such as hydrophobicity, hydrophilicity, electronegativity, bulky side chains and 25 the like. Amino acid substitutions are typically of single residues. Amino acid insertions will usually be in the order of about 1-10 amino acid residues and deletions will range from about 1-20 residues. Generally, deletions or insertions are made in adjacent pairs, i.e. a deletion of two residues or insertion of two residues.

The amino acid variants referred to above may readily be made using peptide 30 synthetic techniques well known in the art, such as solid phase peptide synthesis (Merrifield, J. Am. Chem. Soc. 55:2149, 1964) and the like, or by recombinant DNA manipulations. Techniques for making substitution mutations at predetermined sites in -31 - DNA having known or partially known sequence are well known and include, for example, M13 mutagenesis. The manipulation of DNA sequence to produce variant proteins which manifest as substitutional, insertional or deletional variants are conveniently described, for example, in Sambrook et al, 1989 supra.

5 Other examples of recombinant or synthetic mutants and derivatives of Where PHI, PH5 and/or metal cation antiporter described herein include single or multiple substitutions, deletions and/or additions of any molecule associated with the enzyme such as carbohydrates, lipids and/or proteins or polypeptides.

The terms "homologs", "orthologs", "paralogs", "polymorphic variants" and 10 "derivatives" also extend to any functional equivalent of PHI, PH5 and/or METAL CATION ANTIPORTER and also to any amino acid derivative described above. For convenience, reference to PHI, PH5 and/or METAL CATION ANTIPORTER herein includes reference to any functional mutant, derivative, part, fragment or homo log thereof.

A nucleic acid sequence is described herein encoding PHI, PH5 and/or METAL 15 CATION ANTIPORTER may be introduced into and expressed in a transgenic plant in either orientation thereby providing a means to modulate or alter the vacuolar pH by either reducing or eliminating endogenous or existing pH modulating or altering protein activity thereby allowing the vacuolar pH to increase.

Experimental part 20 The present invention is further described by the following non-limiting Examples.

In relation to these Examples, the following methods and agents are employed.

In general, the methods followed were as described in Sambrook et al, 1989 supra or Sambrook and Russell, Molecular Cloning:A Laboratory Manual 3rd edition, Cold Spring Harbor Laboratories, Cold Spring Harbor, NY, USA, 2001 or Plant Molecular 25 Biology Manual (2nd edition), Gelvin and Schilperoot (eds), Kluwer Academic Publisher, The Netherlands, 1994 or Plant Molecular Biology Labfax, Croy (ed), Bios scientific Publishers, Oxford, UK, 1993.

HPLC and TLC analysis HPLC analysis was as described in de Vetten et al, Plant Cell 11(8):1433-1444, 30 1999. TLC analysis was as described in van Houwelingen et al, Plant J. 13(1):39-50, 1998.

Construction of transgenic plants -32-

Constructs for the expression in plants of PHI, PH5 and NHX habe been obtained by GateWay technology. For all three genes, we have used the full size genomic fragment containing the coding sequence (from ATG to STOP codon) including the introns.

Although for NHX it might be possible to make expression constructs using the cDNA, 5 this last option is surely not applicable to the PHI and PH5 coding sequence.

Plasmids containing the cDNA of PHI and PH5 result indeed to be extremely unstable in E. coli, making it practically impossible to obtain correct constructs in this way.

In appendix 1, 2 and 3 are the maps of the constructs for expression in plant of PHI, PH5 and NHX respectively.

10 The plasmids were then used to transform petunia leaf disks by Agrobacterium tumefaciens mediated procedure as disclosed in Verwij et al. 2008.

DNA and RNA technology

Extractions of DNA and total RNA were performed as described previously (de Vetten et al., 1997). Genomic DNA amplification, RT-PCR and preparation of fragments 15 for the introduction in expression vectors, was performed with gene specific primers listed in the table hereunder. Also the primers used for the construction of the NHX amiRNA constructs are listed in the table.

Analysis of nucleotide and predicted amino acid sequences

Unless otherwise stated, nucleotide and predicted amino acid sequences were 20 analyzed with the program Vector NTI (Registered Trademark) application (version 6.5.3) (Oxford Molecular Ltd., Oxford, England). Multiple sequence alignments were produced with a we b-based version of the program ClustalW (http://dot.imeen.bcrnimc.edu:9331 /multi-aligm/muIti-align.html) using default parameters (Matrix = blossom; GAPOPEN = 0, GAPEXT = 0, GAPDIST = 8, MAXDIV = 40). 25 Phylogenetic trees were built with PHYLIP (bootstrap count = 1000) via the same website, and visualized with Treeviewer version 1.6.6 (htfp://taxonomv.zoolo gv.gla.ac.uk/rod/rod.html).

Homology searches against Genbank, SWISS-PROT and EMBL databases were performed using the FAST A and TFASTA programs (Pearson and Lipman, Proc. Natl. 30 Acad. Sci. USA 85(8): 2444-2448, 1988) or BLAST programs (Altschul et al., J. Mol. Biol. 215(3): 403-410, 1990). Percentage sequence identities and similarities were obtained using LALIGN program (Huang and Miller, Adv. Appl. Math. 12: 373-381, 1991) or -33-

ClustalW program (Thompson et al., Nucleic Acids Research 22: 4673-4680, 1994) within the MacVector (Registered Trademark) application (Oxford Molecular Ltd., England) using default parameters.

RNA isolation and RT-PCR

5 RNA isolation and RT-PCR analysis were carried out as described by de Vetten et al, 1997 supra. Rapid amplification of cDNA (3') ends (RACE) was done as described by Frohman et al, PNAS 85:8998-9002, 1988.

General procedure: pH measure of crude tissue extract.

One flower corolla or two young leaves where ground in 6ml autoclaved distilled water. 10 The pH of the extract was then immediately measured (within 1 minute to avoid atmosferic CO2 to alter the pH) with a portable pH meter (pH electrode Checker, Hanna instruments) (Verweij, 2008).

EXAMPLE 1 Salt accumulation in leaves of plants ectopicallv expressing NHX. PHI and 15 PH5

Applicants have determined the concentration of sodium in leaves of plants expressing the ectopically PHI, PH5, NHX or a combination of the three and compared it to untransformed plants of the same genotype.

To measure the concentration of Na per mg of dry weight we have used AAS (Atomic 20 Absorption Spectrography) as described in (Ghandilyan et al., 2009). The plants were normally grown in the greenhouse and watered with water from the town line. The results of such analysis are reported in table

Cuttings from perfectly isogenic plants M1XV30 (Wild Type) and J2060 (ph3 mutant) were allowed to produce roots in half concentration Hoagland medium. 25 Furthermore, Mutants P7049-17 (ph3-+35S-NHX+35S-PHl+35S-PH5, according to invention), P7034-6 (ph3-+35S-NHX mutant, not according to invention), P7035-4 (WT+ 35S-NHX mutant, not according to invention) and M7110-2 (ph3- +35S-PH1+35S-PH5, alos according to invention) were equally subjetcted to the same treatment.

The NaCl addition starting from two weeks after the production of the cuttings 30 (when roots of at least 10cm length were present).

Although the plants were not exposed to a high NaCl concentration, the accumulation of sodium is clearly different in the distinct transgenics compared to the -34- untransformed controls. The leaves of plants expressing all three transgenes show the highest sodium content, while the lowest is detected in untransformed leaves. No damages where visible in any of the analyzed plants.

5 Table 4: pH of the crude extract and concentration of sodium in leaves of plants ectopically expressing PHI, PH5, NHX or a combination of those, compared to untransformed controls

Plant (genotype) pH in leaves [Na] (nMol/mg dry ___weight)_ P7049-17 6.2 2035 (ph3-+35S-NHX+35S-PH1 +35S-PH5, according to invention)___ M7110-2 (ph3- +35S-PH1+35S-PH5, according to 5.5 1528 invention)___ P7034-6 (ph3-+35S-NHX mutant, not according to 6.0 1763 invention)___ P7035-4 (WT+35S-NHX mutant, not according to 6.0 1748 invention)___ M1XV30 (Wild Type, not according to invention)__53)__1284_ J2060 (ph3 mutant, not according to invention)__CO__1225_ 10 The results show that plants that ectopically express a combination of PHI and PH5 already perform better than the wild types, while those expressing NHX, PHI and PH5 are able to accumulate an even a higher concentration of sodium in the leaves. Furthermore, it also shows that plants that ectopically express a combination of NHX alone show only a slightly increased activity.

15 Experiments during which the same plants were exposed to different NaCl concentrations were performed, and showed that a better performance of the plants when exposed to high sodium concentration.

Example 2: NaCl tolerance test in petunia

The same plants as used in Example 1, with the exception of a control set of 5 20 plants which were kept at OmM NaCl for the whole experiment was supplied with water comprising 25mM NaCl. After a week a set of 5 plants was kept at 25mM NaCl, while the medium for the remainder was brought to 50mM NaCl.

At the end 6 sets each of 5 plants were exposed respectively to 0, 25, 50, 100, 150 and 200mM NaCl. The medium was refreshed twice per week and the experiment was run -35- up to when the plants exposed to 150 and 200 mM NaCl had totally stopped growing and some even died (after about 3 months).

Example 3: Damage assessment of Petunia plants grown in NaCl-rich medium

Petunia Wild Type plants of the FI hybrid M1XV30 were selected to study their 5 behaviour when exposed to different concentrations of NaCl in the growing medium.

To this end cuttings of M1XV30 plants were allowed to root in 0.5x Hoagland medium for about two weeks, during which time the roots reached 10-15cm of length. Then the plantlets were divided in groups of 5 plants. Each group was exposed to increasing concentration of NaCl, starting from 25mM upward. Twice a week the medium 10 was refreshed and the concentration was increased step-wise to obtain within 2 weeks up to 200 mM for the last group (one control group was kept to 0 mM NaCl).

The plants exposed to 50mM NaCl showed already rather serious necrosis on the leaves and a slower growth than that of plants growing on 0 mM NaCl. 25mM NaCl had also a clear effect on the appearance of necrosis and growth, although less pronounced.

15 Plants kept at 200mM NaCl showed severe necrosis in leaves, very slow growth if at all, and they generally died within 3 months since the beginning of the experiment while the control plants growing on 0 mM NaCl were still looking perfectly healthy.

Some flowers of plants exposed to high concentration of NaCl (starting from lOOmM in some cases) turned blue within two weeks after the beginning of the experiment 20 (see Figure 7A). Also plants exposed to 25mM NaCl showed some bluing of the flowers after three weeks (Figure 7B). On the contrary of what observed in leaves, no necrosis was detectable in petals.

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The subject invention further provide for the expression of PHI, PH5 and preferably metal cation antiporter, more preferably NHX in the case of Sodium cations, in leaves and other parts of the plants without causing any deleterious effect as the plants grow normally and are perfectly fertile, in sharp contrast to the overexpression of other 5 proton pumps which results in severe growth defects (Gévaudant et al., 2007).

The genes characterized from petunia can advantageously be used to transform other species for ectopic expression or in selected parts to produce transgenic plants with improved salt resistance. In a different preferred embodiment, the plant parts may be used as markers to find wild type plants that have ectopic or selective expression in some plant 10 parts, and to then selectively breed plants having these genes and exhibit higher salt tolerance, thereby avoiding the use genetically modified material for legal for ethical reasons.

Furthermore, the subject invention preferably provides for markers to select salt resistant plants in breeding programs. Preliminary studies in rose and grape already 15 showed that expression of PHI and PH5 in green parts of the plants can be found among the natural variation of species, to identity and selectively breed alleles to be used in breeding programs by the simple screening of different sources of alleles.

The present invention accordingly also preferably relates to a method for providing a plant increased tolerance to metal cations present in the growth medium, with 20 independently segregating transgenes comprising the steps of: a) introducing a nucleotide sequence into a plant cell, wherein the sequence comprises a nucleotide, preferably DNA construct comprising a first Agrobacterium Ti plasmid border region linked to first nucleotide region containing at least one transgene of agronomic interest, in particular higher salt tolerance, linked to a second Agrobacterium Ti 25 plasmid border region, linked to a second nucleotide region containing a positive selectable marker transgene comprising a nucleotide sequence expressing PHI, PH5 and optionally a metal cation antiporter, or a protein encoded by PHI, PH5 and optionally a metal cation antiporter linked to at least one plasmid maintenance element, whereby the nucleotide molecule provides for the integration of one or both of the nucleotide regions into a plant 30 genome; and b) regenerating the plant cell into a transgenic plant by positive selection provided by expression of the positive selectable marker transgene; and c) selecting the transgenic plant for the presence of the transgene of agronomic interest; and d) screening -37- the transgenic plant by a DNA detection method that identifies the linkage of the transgene of agronomic interest and the positive selectable marker transgene; and e) growing said transgenic plant into a fertile plant.

In a different preferred embodiment relating to non-transgenic plants, the present 5 invention accordingly also relates to a method for providing a plant increased tolerance to metal cations present in the growth medium, comprising the steps of: a) selecting a plant for the presence of a nucleotide sequence expressing PHI, PH5 and optionally a metal cation antiporter, or a protein encoded by PHI, PH5 and optionally a metal cation antiporter; and b) screening the plant by a DNA detection method that 10 identifies the linkage of the nucleotide sequence expressing PHI, PH5 and optionally a metal cation antiporter, or a protein encoded by PHI, PH5 and optionally a metal cation antiporter as selectable marker genes; and c) growing the plant into a fertile plant.

The possibility of changing the vacuolar pH value in different cell type by the simple expression of two proteins PHI and PH5 advantageosuly may open also other 15 possibilities of applications related to radical absorption properties, tolerance to other ions (like metals etc.) for which transporters are known that are energized by the proton gradient between tonoplast and cytoplasm, and the compartmentation of useful metabolites (e.g. tannins) as well as toxic ones. In the case of tannins (for instance) it has been shown that their accumulation in the seed coat is driven by a MATE type of transporter 20 (Debeaujon et al., 2001), which is dependent from the proton gradient across the tonoplast to drive transport. Seeds of both petunia and arabidopsis are uncolored (proanthocyanins are not accumulated) when the proton pumps that acidicy the vacuole of seed coat cells are mutated (Baxter et al., 2005) and (Verweij et al., 2008).

Because of the large varieties of cellular processes that depend on proton 25 concentration differences in different endocellular compartment, the possibility to manipulate this parameter, could result is a wide range of applications.

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Those skilled in the art will appreciate that the invention described herein is susceptible to variations and modifications other than those specifically described. It is to be understood that the invention includes all such variations and modifications. The invention also includes all of the steps, features, compositions and compounds referred to or indicated in 5 this specification, individually or collectively, and any and all combinations of any two or more of said steps or features.

Claims (23)

A transgenic plant, a gene construct, a fragment, a homologue, or a variant thereof comprising coding for PH1 and PH5 as described above, wherein the gene construct is operatively linked to a plant promoter in such a way that it PH1 and PH5 expresses ectopically or in one or more plant parts, and that the plant thereby exhibits an increased tolerance with respect to one or more metal salts in comparison with a corresponding original stem plant that does not contain the gene construct, the tolerance with respect to the metal salt is defined as a lower deformation rate and / or morbidity than that of the original stem plant under identical conditions with regard to the growth restriction due to the metal salt. The plant of claim 1, further comprising a gene construct, a fragment, a homologue, or a variant thereof that encodes a vacuolar metal cation anti-carrier which is activated by a proton gradient over a tonoplast of a vacuole of a planted. The plant according to claim 1 or claim 2, wherein the proteins encoded by PHI, PH5 and the metal cation-anti-carrier gene construct are present in the membrane of the vacuole. 4. Plant according to any one of claims 1-3, wherein PH1 encodes a vacuolar P3BATP ase. 5. Plant according to any one of claims 1-4, wherein PH5 codes for a vacuolar Ρ3ΑΑΤΡ-ase proton pump. Plant according to any one of claims 1-5, wherein the metal cations comprise sodium, magnesium, copper, iron, zinc, cobalt, nickel and / or manganese metal cations, preferably in the presence of cadmium, mercury, silver, and / or lead. The plant of any one of claims 1-6, wherein the vacuolar metal cation anti-carrier is selected from a vacuolar sodium / proton anti-carrier (NHX), a copper carrier (CTR), a cation diffusion mediator (CDF), a zinc-iron permease ( ZIP), and / or a cation exchanger (CAX). An isolated cell, plant, or part of plant, or a descendant thereof, from a plant according to any of claims 1-7, wherein the cell, the plant, the part, or the descendant comprises an elevated PHI or PH1 homologue , as well as an increased PH5 or jP / YJ homologue, wherein the concentration of metal salt in a vacuole of the cell or of cells of the plant or of plant parts is changed relative to a plant that has not been genetically modified. The isolated cell, plant, or part of a plant, or a descendant thereof, from a plant according to claim 8, wherein the cell, the plant, the part, or the descendant further comprises a raised gene construct or homologue encoding a 20 vacuolar metal cation anti-carrier. Plant part according to claim 8 or claim 9, selected from a flower, fruit, vegetable, nut, carrot, stem, and / or seed. A method for producing a plant with an increased tolerance to metal salt, comprising: a. Identifying a plant ectopically or in one or more plant parts expressing a combination of PH1 and PH5, from plants having a gene construct that comprises a gene coding for PH1 and PH5, and which is operatively linked to a plant promoter, in such a way that they are excessively expressed in plants, b. screening the plant that over-expresses PHI, PH5 for improved tolerance to metal salt in growth-limiting conditions, and c. selecting the plant with an increased salt tolerance; 5 wherein the tolerance to metal salt is defined as a lower deformation rate and / or morbidity than that of the original stem plant in identical conditions with regard to the growth restriction due to the metal salt. The method of claim 11, wherein the plant additionally excessively expresses a vacuolar metal cation antimicrobially operatively connected to a plant promoter so that it too is expressed excessively. 13. A method for producing a transgenic plant with an increased tolerance to metal salt, comprising: a. Transforming at least one plant cell with an exogenous nucleic acid encoding an increased PH1 or PH1 homolog, an increased PB5 or Pi / 5 homologue, wherein the concentration of the metal salt in a vacuole of the cell or of cells of the plant or parts of plants is changed relative to a plant that has not been genetically modified, and b. regenerating the transformed cell in a plant with a high tolerance to metal salt, wherein the tolerance to the metal salt is defined as a lower strain rate and / or morbidity than that of the original stem plant in identical conditions with regard to the 25 growth restriction due to the metal salt. 14. A method according to claim 13, wherein the plant cell is transformed with an exogenous nucleic acid encoding an elevated gene construct or an elevated homologue encoding a vacuolar metal cation anti-carrier which is activated by a proton gradient. A method for growing a plant with an increased tolerance to metal salt, comprising: a. Providing a plant according to any of claims 1-9, or selected or cultivated according to claims 11 to 14, and 5 b. growing the plant in soil and / or with water at concentrations of metal salt that limit growth for a corresponding original stem plant that does not contain the gene construct. The method of claim 15, further comprising at least partial harvesting of the plant material obtained in step b. The method of claim 15, further comprising extracting the metal salts from the harvested plant material. A method for modulating the metal salt concentrations in a vacuole of a plant cell, the method comprising introducing into the plant cell or into a parent or relative of the plant cell, or modulating the protein levels of PH1 and PH5, with the in view of increasing the resistance of the plant cell to metal salt, and cultivating the plant cell or of the plant containing the cell or the parent or relative of the cell, under conditions that allow the expression of the proteins. 19. A method according to claim 18, comprising introducing a vacuolar metal cation anti-carrier into the plant cell or a parent or relative of the plant cell, or modulating a protein level of the vacuolar metal cation anti-carrier, with a view to increasing the resistance of the plant cell to metal salt. 20. A method for producing a plant that can withstand elevated levels of metal salts, comprising stably transforming a cell of a suitable plant using a nucleic acid sequence according to any of claims 1-9, under conditions allowing the final expression of the nucleic acid sequence, regenerating a transgenic plant from the cell, and culturing the transgenic plant for a specified time and under conditions sufficient to allow the expression of the nucleic acid sequence, and optionally generating genetically modified descendants thereof. A method of producing a plant with reduced native or existing pH modulating or altering activity, the method comprising stably transforming a cell of a suitable plant using a nucleic acid molecule 10 according to any of claims 1-9, which is antisense of sense to a sequence encoding PH1, PH5, and optionally the sequence encoding the vacuolar metal cation anti-carrier, regenerating a transgenic plant from the cell, and, where necessary, culturing the transgenic plant under conditions which are sufficient to allow the expression of the nucleic acid, and optionally generate genetically modified descendants thereof. 22. Gene construct, fragment, homologue, or variants thereof which encode PH1 and PH5, and which encode the Seq. ID. No. 94 and 96. Use of the gene construct according to claim 22, with a view to increasing the tolerance of plant cells to metal salt.