WO2002083911A1 - Production of plants with increased tolerance to drought stress or with increased transpiration - Google Patents

Abstract

Described is a method for producing transgenic and mutant plants having an increased tolerance to drought stress due to a reduced activity of an ABC transporter which is expressed in guard cells. This reduction of ABC transporer activity can be achieved by introducing a suitable nucleic acid molecule into the plant genome, for example, for inducing an antisense, co-supression or like effect or for inactivating the gene encoding the ABC transporter by T-DNA or transposon insertion. Furthermore described are transgenic and mutant plants obtainable by the above-mentioned method as well as transgenic plant cells and propagation and harvestable material and corresponding uses of suitable nucleic acid molecules. Also described is a method for producing transgenic and mutant plants having an increased transpiration due to an increased activity of an ABC transporter which is expressed in guard cells. This increase of ABC transporter activity can be achieved by overexpressing the ABC transporter. Furthermore described are transgenic and mutant plants obtainable by the above-mentioned method as well as transgenic plant cells and propagation and harvestable material and corresponding uses of suitable nucleic acid molecules.

Description

PRODUCTION OF PLANTS WITH INCREASED TOLERANCE TO DROUGHT STRESS OR WITH INCREASED TRANSPIRATION

The present invention relates to a method for producing transgenic and mutant plants having an increased tolerance to drought stress due to a reduced activity of an ABC transporter which is expressed in guard cells. According to the present invention, the reduction of ABC transporter activity can, in a preferred embodiment, be achieved by introducing a suitable nucleic acid molecule into the plant genome, for example to induce an antisense, co-suppression or like effect or for inactivating the gene encoding said ABC transporter by T-DNA or transposon insertion. The present invention furthermore relates to transgenic and mutant plants obtainable by the above-mentioned method as well as to corresponding plant cells, propagation and harvestable material and to corresponding uses of suitable nucleic acid molecules.

The present invention furthermore relates to a method for producing transgenic and mutant plants having an increased transpiration due to an increased activity of an ABC transporter which is expressed in guard cells. According to the present invention, the increase of ABC transporter activity can, in a preferred embodiment, be achieved by introducing a suitable nucleic acid molecule into the plant genome, for example for overexpressing said ABC transporter. The present invention furthermore relates to transgenic and mutant plants obtainable by the above-mentioned method as well as to corresponding plant cells, propagation and harvestable material and to corresponding uses of suitable nucleic acid molecules.

Environmental stresses such as drought are major factors in limiting plant growth and productivity. For example, the worldwide loss in yield of the three major cereal crops rice, maize and wheat due to water stress (drought) has been estimated to be over 10 billion dollars annually. In addition to this, the world is presently faced with an increasing fresh water scarcity that has been identified as one of the principal global problems in the new century (Postel, Science 271 (1996), 785-788) and plant account for around 65% of global fresh water use. Recent progress in plant transformation techniques and the availability of potentially useful genes from different sources make it possible to generate stress-tolerant crops using transgenic approaches. Various strategies have been taken in the past to attain this goal such as the attempt to produce drought stress-tolerant plants by enhancing the endogenous accumulation of low molecular weight osmolytes (see, e.g., Tarczynski, Science 259 (1993), 508-510). Another direction follows the aim to influence the water balance in plants at the stomatal pores. The review article Schroeder (Nature 410 (2001), 327-330) summarizes the latest knowledge available on the signalling cascade of abscisic acid (ABA)-triggered stomatal closing and some implications on engineering drought-resistant plants. The guard cells, arranged in pairs, surround the stomatal pores present in plant leaves and control with the opening and closure of the stomata the influx of CO₂ and the transpiration of water through the pores. The plant hormone ABA which is synthesized in plants upon drought stress triggers closure of the stomata thereby reducing water loss. Mechanically, the closure is facilitated by a reduction of the internal pressure (turgor) in the guard cells which is achieved by a concerted efflux of potassium ions and anions, sucrose removal and malate-conversion into osmotically inactive starch. It has been discussed that the increasing molecular understanding of the guard cell signal transduction network opens possibilities for controlling stomatal responses to plant water loss. For instance, the deletion of the ERA1 farnesyltransferase β-subunit being a negative regulator of ABA-mediated stomatal closing resulted in plants showing reduced wilting under drought conditions (Pei, Science 282 (1998) 287-290). Equivalent results were obtained with the intragenic suppressor mutant abi1-1R of the ABA-insensitive Arabidopsis mutant abi1-1 (Gosti, Plant Cell 11 (1999), 1897-1910). However, in both cases, the mutant alleles displayed additional growth and developmental phenotypes. Therefore, there is still a need for improved plants that show wide stomatal opening when water is available and efficiently close stomata during drought periods in order to combine a maximal yield with an efficient protection against desiccation. It appears as if in conventionally bred high-yield varieties breeders have selected against such a robustness to drought because genes controlling guard cell signalling are also expressed in other tissues and control other yield parameters (cf., e.g., Cutler, Science 273 (1996), 1239-1241 and Pei (1998), loc. cit.).

Therefore, the technical problem underlying the present invention is the provision of methods for producing plants having an increased resistance to drought stress and/or a reduced need for water. This technical problem is solved by the provision of the embodiments as characterized in the claims.

Accordingly, the present invention relates to a method for producing transgenic or mutant plants having an increased tolerance to drought stress comprising the step of providing transgenic or mutant plants having a reduced activity of an ABC transporter which is expressed in guard cells. Preferably, the activity of MRP5 is reduced to non-detectability.

The present invention is based on the surprising finding that Arabidopsis thaliana plants in which the gene encoding the ABC transporter MRP5 is inactivated due to the insertion of a T-DNA tag showed a pronounced resistance against water stress as compared to corresponding wild-type plants (see Example 8). In the context of the present invention, the term "tolerance to (or synonymously used "resistance against") water stress" (herein also referred to as "drought stress") means that plants produced by the method of the present invention survive a longer time period of insufficient water supply than corresponding source plants. The term "corresponding source plant" refers to plants which have been taken as starting point for applying the method of the present invention and which have a normal ABC transporter activity in guard cells, i.e. corresponding to that of wild-type plants. The term "source plant" also encompasses transgenic or mutant plants in which other traits than the activity of an ABC transporter expressed in guard cells is genetically modified. Preferred, however, are wild-type plants. The term "insufficient water supply" refers to amounts of water the plant is supplied with that are too low for the plant to be maintained viable. For instance, the water supply is insufficient when plants that have no access to natural water resources are not watered. It is preferred that the time period that plants produced according to the method of the invention survive is extended by at least one day, preferably two days, more preferably by at least four days or even longer or may, advantageously, be increased by at least 50%, preferably by at least 100%, more preferably by at least 150% or particularly preferred by at least 2- or at least 3-fold compared to the time period that corresponding source plants survive a drought period. However, water stress also manifests itself, prior to death of a plant, in a wilty state, i.e. in a state where a plant or parts thereof have a reduced turgor compared to the state where water is sufficiently available to the plant. Thus, in this regard, plants produced by a method according to the invention are less wilty than corresponding source plants when exposed to drought stress for the same time period. Preferably they have the full turgor when a conventional plant is wilty or, which is preferred, already irreversibly dehydrated. The term "irreversibly dehydrated" refers to plants which or parts of which die as a consequence of drought stress. As it has been shown in the experiments underlying the present invention, the increased tolerance to drought stress correlates in mutant plants deficient of the relevant ABC transporter with a reduction of transpiration and water uptake (see Examples 9 and 10). Thus, the method of the present invention is, in addition, suited for producing transgenic or mutant plants having a reduced need for water. This may lead to a reduction of costs in agriculture that are to be spent for watering. Furthermore, this may reduce the negative effect of continuous irrigation which often leads to soil salinity, an additional limiting factor for many crop plants. Likewise, tolerance to drought stress may be expressed in terms of a reduced transpiration rate and/or water uptake. Methods for measuring these parameters are common to the person skilled in the art and can be taken from the literature or, as it is preferred, can be carried out as described in the Examples. For example, the transpiration rate of a plant produced by the method of the invention is reduced when the mean volume of water loss per time unit and dry weight is significantly below, preferably by a factor of at least 1.2, more preferably by at least 1.5, still more preferably at least 2 and most preferably by a factor of at least 3, compared to the corresponding source plant. Accordingly, the same values of reduction may refer to the effect on water uptake, e.g. the mean volume of water entry into the roots per time unit and dry weight. For determining the extent of tolerance to drought stress, e.g. an experimental setup for measuring water uptake may be modified in that drought stress may be mimicked, e.g. by applying a suitable amount of abscisic acid (ABA) or an artificial change in the water potential may be introduced, e.g. by adding an osmotically active substance such as polyethylene glycole (PEG). Thereby, for example plants being produced by the method of the invention show an inhibition of water entry both upon mimicked water stress and/or upon change of water potential which is significantly weaker than that of conventional plants, preferably by a factor of at least 1.2, more preferably of at least 1.5, still more preferably of at least 2 and most preferably of at least 3. The term "ABC transporter" refers to plant proteins which belong to the super-family of ABC (ATP-binding cassette) transporters. Detailed descriptions of structure and function of plant ABC transporters are, for example, given in Rea (1998), Rea (1999), Theodoulou (2000) and Martinoia (2000). ABC transporters are characterized by the presence of two kinds of basic structural elements, the integral membrane spanning domains (MSD) and the ATP-binding folds (NBF) oriented towards the cytoplasm. In plants, in most cases at least one MSD and NBF is fused in various combinations. The so-called full-size ABC transporters contain two of each kind of structural elements either in the arrangement MSD-NBF-MSD-NBF as in multi drug resistance (MDR, also interchangeably referred to as P-glycoprotein, PGP) and multi drug resistance associated proteins (MRP) or in the arrangement NBF-MSD-NBF-MSD as in pleiotropic drug resistance (PDR5)-like proteins. The so-called half-size ABC transporters occur in the arrangement MSD-NBF or NBF- MSD in half-size proteins (Higgins, 1992). Full-size ABC transporters are preferred in connection with the method of the invention.

ABC transporters are structurally characterized by the presence of three conserved sequence motifs at least in one NBF: an ATP-binding site constituted by a Walker A (GXXGXG) and a Walker B box (T/IYLLD) (Walker et al., 1982) separated by approximately 120 amino acids. Walker motifs are also known from many nucleotide- binding enzymes, such as myosin adenylate kinase or protein kinases. The third conserved sequence motif is a specific ABC signature situated between the two Walker boxes, consisting of the amino acids [LIVMFY]-S-[SG]-G-X(3)-[RKA]-[LIVMYA]-X- [LIVMF]-[AG] (Higgins 1992). Moreover, among different ABC transporters, the highest similarity is observed in the NBFs, where an identity of 30 - 40% over a span of about 200 - 400 amino acid residues can be found.

The term "activity of an ABC transporter" relates to an ATP-dependent transport of solutes across a membrane which is independent of a proton gradient at the membrane. The term "ATP-dependent" is often also called "directly energized" in contrast to the "indirectly energized" transport driven by proton gradients. The term "solutes" refers to any compounds which are transported by ABC transporters which includes, for example, organic anions such as glutathione conjugates or auxin-conjugates, chlorophyll catabolytes, glycosylated compounds, glucuronides, sulfated flavonoids, glucuronited flavonoids, bile acids, sulphonated compounds, lipids, metal complexes and steroid compounds (Rea et al. 1998, Martinoia et al. 2000). Preferably such transport processes take place at the tonoplast (vacuolar influx) or the plasma membrane (extracellular efflux). Preferably, transport of such compounds is involved in a detoxification of the cell. Further features that distinguish the ABC transporter activity from other cellular transport processes include

(i) a direct dependence on MgATP;

(ii) that ATP can be substituted by other nucleotides, preferably GTP; and/or (iii) that it can be inhibited by micromolar concentrations of vanadate. Preferably this property is tested for transport processes across the vacuolar membrane (tonoplast) since there, in contrast to the plasma membrane, the possibly interfering H⁺-ATPase activity is not vanadate-inhibited and can therefore easily be distinguished. The above-described activities should be measured at those parts of the transgenic or mutant plant where the relevant ABC transporter is predominantly expressed in wild-type plants, preferably at the guard cells. Techniques for detecting such transport activities are known by the person skilled in the art and are described in the literature such as by Li et al. (1996) and Tommasini et al. (1996).

The "activity of an ABC transporter" also encompasses other activities than ATP- dependent solute transport which have been reported. Such activities include, for instance, ion transport or its regulation concerning for example potassium channels (e.g. ABC-type sulfonylurea receptors, SUR, Bryan (1999)) or chloride channels (e.g. cystic fibrosis trans membrane conductance regulators, CFTR, Akabas (2000)). Techniques for measuring ion channel activities are likewise well known in the art (see for instance Hille (1992) or Hedrich (1995)). Other ABC transporter activities involve the regulation of plant development, wherein specific examples are described by Sidler (1998), Møller (2001) and Kushnir (2001). In Example 5 of the present specification, the experiments showed that root growth in a T-DNA insertion mutant of AtMRPδ differed from control plants. In particular, root elongation of AtMRPδ-deficient seedlings was reduced and the lateral and secondary roots were initiated earlier than by wild-type seedlings. The results concerning reduced root growth as well as the behaviour of these plants upon glibenclamide treatment (see infra) have already been reported in conference talks (Martinoia, at the Universitat Gieβen, December 18, 2000; Martinoia, at the Universitat Wϋrzburg, January 25, 2001; Mϋller-Rober, at the 14. Tagung Molekularbiologie der Pflanzen, Dabringhausen, March 1, 2001; Martinoia, at the meeting "ABC Proteins: From genetic disease to multi-drug resistance", Grosau, March 5, 2001). All of these activities are within the scope of the present invention to be reduced so as to obtain drought-resistant plants, however, the above described ATP-dependent solute transport activity is preferred in this context.

The term "reduced activity of an ABC transporter" refers to a significant reduction of the ATP-dependent solute transport activity of an ABC transporter in guard cells compared to the corresponding source plant. Preferably said reduction amounts to at least 20%, preferably 50% and most preferably 100% of the corresponding activity in the source plant. Likewise, a reduction of said ABC transporter activity may be determined by measuring the amount of the corresponding ABC transporter transcript or protein in the plant tissue where it is normally expressed such as in mature leaves, preferably in guard cells. Accordingly, transgenic or mutant plants produced according to the method of the invention are characterized by a reduction of the said transcript by at least 20%, preferably by at least 50% and most preferably by at least 90% compared to the corresponding amount of transcript in the source plant or, on the protein level, by a reduction of at least 20%, preferably by at least 50% and most preferably by at least 90% of the corresponding ABC transporter polypeptide compared to the corresponding source plant. The above-described reductions of ABC transporter activity, transcript and/or protein level provide for an efficient increase of tolerance to drought stress in transgenic or mutant plants. In a preferred embodiment, activity and/or transcript amount and/or protein amount of the ABC transporter is reduced to non-detectability.

In a preferred embodiment, transgenic or mutant plants produced in accordance with the method of the invention, having a reduced activity of an ABC transporter expressed in guard cells as defined above, is characterized by the feature that stomata of these plants do not open upon treatment with sulfonylurea compound which is an inhibitor of stomatal closure, preferably with glibenclamide. This assay may be carried out according to methods described in the literature or, most reliably, as described in Example 7, infra.

Moreover, the term "reduced activity of an ABC transporter" encompasses that the properties of the relevant ABC transporter regarding the interaction with other proteins involved in stomatal regulation of transpiration may be modified. This may lead to a reduced transpiration rate with the effect of an enhanced tolerance to drought stress. Such modified properties may be independent of the transport activity of said ABC transporter or, which is preferred, may concur with a reduced transport activity as defined above. The interactions which are meant herein may influence the activity and/or stability and/or conformation and/or localization of the other protein. Preferably, the interaction of said ABC transporter with another protein is decreased, for instance by at least 50% compared to the corresponding interaction the corresponding wild-type ABC transporter has, preferably it is totally abolished. Potential interacting proteins include for instance potassium channels. Whether an ABC transporter having a reduced activity shows a modified interaction with other proteins relevant for stomatal regulation can be determined by methods known in the art. Preferably, such methods are applied in vivo, for example, using the bioluminescence resonance energy transfer (BRET) method (Angers, Proc. Natl. Acad. Sci. U.S.A. 97 (2000), 3684-3689) or the fluorescence resonance energy transfer (FRET) method (Gadella, Trends in Plant Science 4 (1999), 287-291). The interaction between said ABC transporter and another protein may likewise be determined by protein-binding assays which are known in the art as for instance by the surface plasmon resonance method (Hall, Anal. Biochem 288 (2001), 109-125; Rich, J. Mol. Recognition 13 (2000), 388-407; Fivash, Curr. Op. Biotechnol. 9 (1998), 97-101). Preferably, such measurements include determining the activity of the interacting protein depending on the interaction with said ABC transporter.

From plants several ABC transporters are known in the art that may be of use for the present method if they are expressed in guard cells. In Arabidopsis, for instance, a total of 48 genes can be found that encode full-size ABC transporters of the three subclusters PDR5 (pleiotropic drug resistance), AtMRP (multidrug resistance associated protein) and AtPGP (P-glycoprotein) (AGI, 2000). In addition, half-size transporters including the groups ' of putative mitochondrial heme transporters, homologues to peroxisomal ABC transporters and to the ABC1 transporter class make a total number of 56. This class of putative transporters consists of one hydrophobic domain - the majority contains 4-6 alpha-helices - an ATP binding site and an ABC signature.

A phylogenetic analysis revealed the relationships of the Arabidopsis full-size ABC transporters with their counterparts of non-plant origin (Figure 2). This analysis showed that the ABC transporters of each of the three Arabidopsis subclusters are more closely related to non-plant counterparts than to ABC transporters of other Arabidopsis subclusters which allows for a clear distinction between the subclusters. Accordingly, the AtPGP genes are grouped with their human homologues and TAP1 (transporter associated with antigen processing). MDRs have also been cloned from barley (Davies et al. 1997) and potato (Wang et al. 1996). A class of putative mitochondrial half-size transporters that show homologies to the human ABC7 genes and the yeast ATM1 gene are closely related. This yeast protein is known to transport iron-sulfur cluster precursors from the mitochondria to the cytosol. The Arabidopsis mutant starik, which is defective in the ATM1 gene due to a T-DNA insertion leads to dwarfism and chlorosis (Kushnir et al., 2001). The second group of full-size ABC transporters is formed by the AtPDR-5 like genes. These proteins are characterized in fungal systems as efflux transporters for cytotoxic compounds (Andrade et al., 2000). A group of half-size transporters is related to these full-size transporters, i.e. the pigment transporters "white" from Drosophila and their homologues found in Arabidopsis.

In a preferred embodiment, the method of the invention relates to ABC transporters which are multidrug resistance associated proteins (MRP). This third group shown in Figure 2 is characterized by its closer relationship to SUR and CFTR ABC transporters than the other plant ABC transporter subclusters MDR PGP and PDR-5. The arrangement of the structural elements MSD and NBF can be used as a further distinct feature between MRPs and PDR5-like ABC transporters (see supra). On the other hand, MDRs and MRPs exhibit the same arrangement but differ at the NH₂-terminus where an extremely hydrophobic N-terminal extension (NTE) of around 220 amino acids is observed for the MRPs but not for MDRs. A careful study of the membrane topology of human MRP employing glycosylation-site mutants indicates that the hydrophobic NTE forms a third MSD of five transmembrane -helices and that the NH₂-terminus is most likely situated in the extracytoplasmic side of the membrane bilayer (Hipfner et al. 1997). Hydropathy plots of animal and plant MRPs are extremely similar, suggesting a similar arrangement of the membrane spanning domains in animals and plants.

AtMRP ABC transporters are known to function as vacuolar sequesters of glutathionylated compounds, malonylated chlorophyll catabolites and glucuronides. In a phylogenetic analysis of all full-size transporter genes (Figure 2), the order of sequences in the phylogenetic tree suggests the grouping of AtMRPI, 2, 11 and 12 in a closely related group. In a sister group thereof, AtMRPδ is assembled with AtMRP3, 6, 7, 9 and, in a more distant relationship, AtMRP4, 14, 8 and 10. Finally, AtMRP13 represents an MRP gene distinct from both MRP subgroups. Interestingly, these phylogenetic data concur with the intron-exon structure. Expression studies revealed that most of the AtMRP genes are expressed in leaves, roots, flowers, stems and siliques. Only a few genes showed increased accumulation of mRNA in a specific tissue. Some AtMRPs exhibit increased transcript levels when plants are treated with xenobiotics or salicylic acid (Tommasini et al. 1997, Sanchez-Fernandez, 1998). Beside transport measurements for glutathione conjugates (see below), the only report for an MRP-like ABC transporter from other plants than Arabidopsis was the demonstration that antibodies raised against an AtMRP homologue from wheat upregulated by safener treatment recognized a 170 kD polypeptide in the vacuolar fraction of these plants (Theodoulou et al., 1998).

Three members belonging to the subfamily of the MRPs, AtMRPI, At RP2₁ and AtMRP3 have been shown to be glutathione conjugate transporters (Lu et al. 1997, Lu et al. 1998, Tommasini et al. 1998). In addition, AtMRP2 and AtMRP3 are also able to transport chlorophyll catabolites produced during senescence.

As it has been explained above, the method of the invention refers to ABC transporters expressed in guard cells. The term "expressed in guard cells" means in the context of the present invention that the relevant ABC transporter is present in guard cells which can be determined by measuring its activity, amount of transcript and/or amount of protein in guard cells which can be carried out as described above. For determining whether an ABC transporter is expressed in guard cells, the skilled person can choose among several techniques which are described in the prior art. For example, this can be carried out by fusions of suitable transcriptional regulatory sequences such as a promoter taken from a candidate gene with a reporter gene and determining whether reporter gene expression is detectable in guard cells of transgenic plants transformed with said fusion (as described in Example 3). Other possibilities include, e.g., single cell RT-PCR (reverse transcription-polymerase chain reaction), in situ hybridization, in situ-PCR and RNA expression profiling (i.e. transcriptome analysis; see, e.g., Schaffer, Curr. Op. Biotechnol. 11 (2000), 162-167). On the protein level, ABC transporter expression can be determined by suitable techniques such as immunolocalization. Preferably, such measurements are undertaken with guard cells being isolated, for instance, according to methods as mentioned above. Preferably said ABC transporter is present and active in guard cells.

In a furthermore preferred embodiment, the method of the present invention relates to a reduction of the activity of the ABC transporter MRP5 which is known to be expressed in guard cells. An example for MRP5 is the Arabidopsis thaliana AtMRPδ the cDNA of which having the nucleotide sequence shown under SEQ ID NO:1 or obtainable from the Gen Bank/EMBL data base entry Y11250 has been isolated in connection with the present invention. The corresponding deduced amino acid sequence of AtMRPδ is shown under SEQ ID NO:2. In the context of the present invention, the term "MRP5" refers to AtMRPδ and any plant homologues thereof having MRP5 activity and wherein a reduction of this activity leads to an increase of tolerance to drought stress. The term "MRPδ activity" refers to ATP-dependent solute transport activity as defined above for ABC transporters in general. In particular, MRPδ activity is characterized in that its glutathione conjugate transport activity, which can, for instance, be measured in a complementation assay with ycfl -deficient yeast strains as described in Example 2, is (i) inhibited by vanadate; and (ii) insensitive to compounds disrupting the pH gradient generated by proton pumps such as bafilomycin A1 or NH₄CI. Furthermore, MRPδ activity is characterized by its ATP-dependent transport activity of estradiol-17-(β-D- glucuronide) (E₂17G) which may be tested using yeast strains having a reduced glucuronide transport activity such as YYA4 (see Example 2). Preferably, in such measurements, E₂17G transport is insensitive to reduced glutathione, oxidized glutathione and/or dinitrobenzene glutathione and/or is, on the other hand, sensitive to, i.e. significantly reduced in the presence of, other organic anions such as estradiol-3 sulfate, luteolin-7-O-diglucuronide-4'-O-glucuronide and/or glycocholate and/or is sensitive to glibenclamide. Most preferably, the E₂17G transport of an MRPδ transporter as referred to in the present preferred embodiment shows the characteristics depicted in Table 5, supra.

In a preferred embodiment, such AtMRPδ homologues are encoded by a polynucleotide selected from the group consisting of:

(a) polynucleotides encoding a polypeptide having the amino acid sequence depicted in SEQ ID NO: 2; (b) polynucleotides comprising the nucleotide sequence depicted in SEQ ID NO:

1 ;

(c) polynucleotides hybridizing to the complementary strand of the polynucleotide of (a) or (b); and

(d) polynucleotides the nucleotide sequence of which deviates from the nucleotide sequence of a polynucleotide of (c) due to the degeneracy of the genetic code.

The term "hybridizing" refers in this context to hybridization under conventional hybridization conditions, preferably under stringent conditions, as for instance described in Sambrook at al., Molecular Cloning, A Laboratory Manual, 2^nd edition (1989) Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY. In an especially preferred embodiment the term "hybridizing" means that hybridization occurs under the following conditions:

Hybridization buffer: 2 x SSC; 10 x Denhardt solution (Fikoll 400 + PEG +

BSA; ratio 1:1:1); 0.1% SDS; δ mM EDTA; δO mM Na₂HPO₄;

2δ0 μg/ml of herring sperm DNA; δO μg/ml of tRNA; or

0.2δ M of sodium phosphate buffer, pH 7.2;

1 mM EDTA

7% SDS Hybridization temperature T = 60°C

Washing buffer: 2 x SSC; 0.1% SDS

Washing temperature T = 60°C.

For identifying genes encoding MRP5 from plant specifies being more distantly related with Arabidopsis such as cereal plants, less stringent hybridization conditions might be necessary than that outlined above. Such less stringent hybridizations may for instance be carried out as described in Amasino (Anal. Biochem. 1δ2 (1986), 304-307) using PEG buffer with 2δ-δ0% formamide, with a hybridization temperature of 42°C and washing with 3x SSC, 0,5% SDS at42°C. Advantageously, polynucleotides encoding an MRPδ have a nucleotide sequence of at least 60%, preferably of at least 70%, more preferably of at least 80%, still more preferably 90% and most preferably of at least a 9δ% identity to the nucleotide sequence of SEQ ID NO:1. Likewise, such polynucleotides encode a polypeptide having an amino acid sequence of at least 60%, preferably of at least 70%, more preferably of at least 80%, still more preferably of at least 90% and most preferably of at least a 9δ% identity to the amino acid sequence of SEQ ID NO:2.

In the context of the present invention, the term "transgenic" means that the plants contain cells in which the genome structurally deviates from that of corresponding source plants in such a way that the activity of an ABC transporter which is expressed in guard cells is reduced as explained above. Such a structural difference preferentially refers to the gene encoding this ABC transporter, which includes for instance the inactivation due to a deletion. The prior art provides means and methods for providing transgenic plants wherein the activity of a specific protein is reduced.

The invention refers in a preferred embodiment to a method, wherein providing of transgenic plants having a reduced activity of said ABC transporters comprises the steps of

(a) introducing into a plant cell a nucleic acid molecule the presence of which in the genome of said plant leads to a reduced activity of said ABC transporter in plant cells;

(b) regenerating from transformed cells produced in step (a) plants; and optionally

(c) producing progeny from the plants produced in step (b).

Accordingly, the term "presence of a nucleic acid molecule" as used herein refers to any foreign nucleic acid molecule that is present in cells of a transgenic plant produced in accordance with the invention but absent from the cells of the corresponding source plant. Thereby encompassed are nucleic acid molecules, e.g. gene sequences, which differ from the corresponding nucleic acid molecule in the source plant cell by at least one mutation (substitution, insertion, deletion, etc. of at least one nucleotide), wherein such a mutation inhibits the expression of the affected gene or reduces the activity of the gene product. Furthermore encompassed by the term "foreign" are nucleic acid molecules which are homologous with respect to the source plant cell but are situated in a different chromosomal location or differ, e.g., by way of a reversed orientation for instance to the promoter.

In principle, the nucleic acid molecule to be introduced in step (a) may be of any conceivable origin, e.g. eukaryotic or prokaryotic. It may be of any organism which comprises such molecules. Furthermore, it may be synthetic or derived from naturally occurring molecules by, e.g., modification of its sequence, i.e. it may be a variant or derivative of a naturally occurring molecule. Such variants and derivatives include but are not limited to molecules derived from naturally occurring molecules by addition, deletion, mutation of one or more nucleotides or by recombination. It is, e.g., possible to change the sequence of a naturally occurring molecule so as to match the preferred codon usage of plants, in particular of those plants in which the nucleic acid molecule shall be expressed.

It is furthermore preferred that the nucleic acid molecule introduced into a plant cell in step (a) has to be expressed in the transgenic plant in order to exert the reducing effect upon ABC transporter activity. The term "expressed" means for such a nucleic acid molecule that it is at least transcribed, and for some embodiments also translated into a protein, in at least some of the cells of the plant. Preferred examples of such nucleic acid molecules relate to those embodiments of the method of the invention wherein said reduced ABC transporter activity is achieved by an antisense, co-suppression, ribozyme or RNA interference effect or by the expression of antibodies or other suitable (poly)peptides capable of specifically reducing said activity or by the expression of a dominant-negative mutant. These methods are further explained in the following.

Accordingly, the use of nucleic acid molecules encoding an antisense RNA which is complementary to transcripts of a gene encoding a plant ABC transporter expressed in guard cells is a preferred embodiment of the present invention. Thereby, complementarity does not signify that the encoded RNA has to be 100% complementary. A low degree of complementarity may be sufficient as long as it is high enough to inhibit the expression of such an ABC transporter protein upon expression of said RNA in plant cells. The transcribed RNA is preferably at least 90% and most preferably at least 9δ% complementary to the transcript of the nucleic acid molecule encoding MRPδ. In order to cause an antisense effect during the transcription in plant cells such RNA molecules have a length of at least 1δ bp, preferably a length of more than 100 bp and most preferably a length or more than δOO bp, however, usually less than δOOO bp, preferably shorter than 2600 bp. Exemplary methods for achieving an antisense effect in plants are for instance described by Mϋller-Rόber (EMBO J. 11 (1992), 1229-1238), Landschϋtze (EMBO J. 14 (1995), 660-666), D'Aoust (Plant Cell 11 (1999), 2407-2418) and Keller (Plant J. 19 (1999), 131-141) and are herewith incorporated in the description of the present invention. Likewise, an antisense effect may also be achieved by applying a triple-helix approach, whereby a nucleic acid molecule complementary to a region of the gene, encoding the relevant transporter, designed according to the principles for instance laid down in Lee (Nucl. Acids Res. 6 (1979), 3073); Cooney (Science 241 (1998), 456) or Dervan (Science 2δ1 (1991), 1360) may inhibit its transcription.

A similar effect as with antisense techniques can be achieved by producing transgenic plants expressing suitable constructs in order to mediate an RNA interference (RNAi) effect. Thereby, the formation of double-stranded RNA leads to an inhibition of gene expression in a sequence-specific fashion. More specifically, in RNAi constructs, a sense portion comprising the coding region of the gene to be inactivated (or a part thereof, with or without non-translated region) is followed by a corresponding antisense sequence portion. Between both portions, an intron not necessarily originating from the same gene may be inserted. After transcription, RNAi constructs form typical hairpin structures. In accordance with the method of the present invention, the RNAi technique may be carried out as described by Smith (Nature 407 (2000), 319-320) or Marx (Science 288 (2000), 1370-1372).

Also DNA molecules can be employed which, during expression in plant cells, lead to the synthesis of an RNA which reduces the expression of the gene encoding the ABC transporter in the plant cells due to a co-suppression effect. The principle of co- suppression as well as the production of corresponding DNA sequences is precisely described, for example, in WO 90/12084. Such DNA molecules preferably encode an RNA having a high degree of homology to transcripts of the target gene. It is, however, not absolutely necessary that the coding RNA is translatable into a protein. The principle of the co-suppression effect is known to the person skilled in the art and is, for example, described in Jorgensen, Trends Biotechnol. 8 (1990), 340-344; Niebel, Curr. Top. Microbiol. Immunol. 197 (199δ), 91-103; Flavell, Curr. Top. Microbiol. Immunol. 197 (1996), 43-36; Palaqui and Vaucheret, Plant. Mol. Biol. 29 (1996), 149-169; Vaucheret, Mol. Gen. Genet. 248 (1995), 311-317; de Borne, Mol. Gen. Genet. 243 (1994), 613-621 and in other sources.

Likewise, DNA molecules encoding an RNA molecule with ribozyme activity which specifically cleaves transcripts of a gene encoding the relevant ABC transporter can be used. Ribozymes are catalytically active RNA molecules capable of cleaving RNA molecules and specific target sequences. By means of recombinant DNA techniques, it is possible to alter the specificity of ribozymes. There are various classes of ribozymes. For practical applications aiming at the specific cleavage of the transcript of a certain gene, use is preferably made of representatives of the group of ribozymes belonging to the group I intron ribozyme type or of those ribozymes exhibiting the so-called "hammerhead" motif as a characteristic feature. The specific recognition of the target RNA molecule may be modified by altering the sequences flanking this motif. By base pairing with sequences in the target molecule these sequences determine the position at which the catalytic reaction and therefore the cleavage of the target molecule takes place. Since the sequence requirements for an efficient cleavage are low, it is in principle possible to develop specific ribozymes for practically each desired RNA molecule. In order to produce DNA molecules encoding a ribozyme which specifically cleaves transcripts of a gene encoding the relevant ABC transporter, for example a DNA sequence encoding a catalytic domain of a ribozyme is bilaterally linked with DNA sequences which are complementary to sequences encoding the target protein. Sequences encoding the catalytic domain may for example be the catalytic domain of the satellite DNA of the SCMo virus (Davies, Virology 177 (1990), 216-224 and Steinecke, EMBO J. 11 (1992), 1526-1630) or that of the satellite DNA of the TobR virus (Haseloff and Gerlach, Nature 334 (1988), δδδ-691). The expression of ribozymes in order to decrease the activity of certain proteins in cells is known to the person skilled in the art and is, for example, described in EP-B1 0 321 201. The expression of ribozymes in plant cells is for example described in Feyter (Mol. Gen. Genet. 260 (1996), 329-338).

Furthermore, nucleic acid molecules encoding antibodies specifically recognizing the relevant ABC transporter in a plant, i.e. specific fragments or epitopes of such a protein, can be used for inhibiting the activity of this protein. These antibodies can be monoclonal antibodies, polyclonal antibodies or synthetic antibodies as well as fragments of antibodies, such as Fab, Fv or scFv fragments etc. Monoclonal antibodies can be prepared, for example, by the techniques as originally described in Kohler and Milstein (Nature 256 (1975), 496) and Galfre (Meth. Enzymol. 73 (1981) 3), which comprise the fusion of mouse myeloma cells to spleen cells derived from immunized mammals. Furthermore, antibodies or fragments thereof to the aforementioned peptides can be obtained by using methods which are described, e.g., in Harlow and Lane "Antibodies, A Laboratory Manual", CSH Press, Cold Spring Harbor, 1988. Expression of antibodies or antibody-like molecules in plants can be achieved by methods well known in the art, for example, full-size antibodies (During, Plant. Mol. Biol. 15 (1990), 281-293; Hiatt, Nature 342 (1989), 469-470; Voss, Mol. Breeding 1 (1996), 39-50), Fab- fragments (De Neve, Transgenic Res. 2 (1993), 227-237), scFvs (Owen, Bio/Technology 10 (1992), 790-794; Zimmermann, Mol. Breeding 4 (1998), 369-379; Tavladoraki, Nature 366 (1993), 469-472; Artsaenko, Plant J. 8 (1996), 745-760) and variable heavy chain domains (Benvenuto, Plant Mol. Biol. 17 (1991), 866-874) have been successfully expressed in tobacco, potato (Schouten, FEBS Lett. 416 (1997), 235- 241) or Arabidopsis, reaching expression levels as high as 6.8% of the total protein (Fiedler, Immunotechnology 3 (1997), 205-216).

Moreover, also nucleic acid molecules encoding (poly)peptides capable of reducing the activity of the relevant ABC transporter other than antibodies can be used in the present context. Examples of suitable (poly)peptides that can be constructed in order to achieve the intended purpose can be taken from the prior art and include, for instance, binding proteins such as lectins.

In addition, nucleic acid molecules encoding a mutant form the relevant ABC transporter can be used to interfere with the activity of the wild-type protein. Such a mutant form preferably has lost its biological activity, e.g. its ATP-dependent transport function, and may be derived from the corresponding wild-type protein by way of amino acid deletion(s), substitution(s), and/or additions in the amino acid sequence of the protein. Mutant forms of such proteins may show, in addition to the loss of transport or ATPase activity, an increased substrate affinity and/or an elevated stability in the cell, for instance, due to the incorporation of amino acids that stabilize proteins in the cellular environment. These mutant forms may be naturally occurring or, as preferred, genetically engineered mutants. In another preferred embodiment, the nucleic acid molecule introduced into a plant cell in step (a) does not require its expression to exert its reducing effect on ABC transporter activity. Correspondingly, preferred examples relate to methods wherein said reduced ABC transporter activity is achieved by in vivo mutagenesis or by the insertion of a heterologous DNA sequence in the gene encoding the ABC transporter. The term "in vivo mutagenesis", relates to methods where the sequence of the gene encoding the relevant ABC transporter is modified at its natural chromosomal location such as for instance by techniques applying homologous recombination. This may be achieved by using a hybrid RNA-DNA oligonucleotide ("chimeroplast") which is introduced into cells by transformation (TIBTECH 15 (1997), 441-447; WO96/15972; Kren, Hepatology 25 (1997), 1462-1468; Cole-Strauss, Science 273 (1996), 1386- 1389). Part of the DNA component of the RNA-DNA oligonucleotide is homologous to the target ABC transporter gene sequence, however, displays in comparison to this sequence a mutation or a heterologous region which is surrounded by the homologous regions. The term "heterologuous region" corresponds to any sequence that can be introduced and encompasses, for instance, also sequences from the same ABC transporter gene but from a different site than that which is to be mutagenized. By means of base pairing of the homologous regions with the target sequence followed by a homologous recombination, the mutation or the heterologous region contained in the DNA component of the RNA-DNA oligonucleotide can be transferred to the corresponding gene of the plant cell. By means of in vivo mutagenesis, any part of the gene encoding the ABC transporter can be modified as long as it results in a decrease of the activity of said ABC transporter. Thus, in vivo mutagenesis can for instance concern, the promoter, e.g. the RNA polymerase binding site, as well as the coding region, in particular those parts encoding the ATP binding site or a signal sequence directing the protein to the appropriate cellular compartment.

The term "heterologous DNA sequence" refers to any DNA sequences which can be inserted into the target gene via appropriate techniques other than those described above in connection with in vivo mutagenesis. The insertion of such a heterologous DNA sequence may be accompanied by other mutations in the target gene such as the deletion, inversion or rearrangement of the sequence located at the insertion site. This embodiment of the method of the invention includes that the introduction of a nucleic acid molecule in step (a) leads to the generation of a pool, i.e. a plurality, of transgenic plants in the genome of which the nucleic acid molecule, i.e. the heterologous DNA sequence, is randomly spread over various chromosomal locations, and that step (c) is followed by selecting those transgenic plants out of the pool which show the desired genotype, i.e. an inactivating insertion in the relevant ABC transporter gene and/or the desired phenotype, i.e. a reduced ABC transporter activity and/or tolerance to drought stress. Suitable heterologous DNA sequences that can be taken for such an approach are described in the literature and include, for instance vector sequences capable of self- integration into the host genome or mobile genetic elements. Particularly preferred in this regard are T-DNA or transposons which are well-known to the person skilled in the art from so-called tagging experiments used for randomly knocking out genes in plants. The production of such pools of transgenic plants can for example be carried out as described in Jeon (Plant J. 22 (2000), 661-670) or Parinov (Curr. Op. Biotechnol. 11 (2000), 157- 161).

Another example of insertional mutations that may result in gene silencing includes the duplication of promoter sequences which may lead to a methylation and thereby an inactivation of the promoter (Morel, Current Biology 10 (2000), 1591-1594). Furthermore, it is immediately evident to the person skilled in the art that the above- described approaches, such as antisense, ribozyme, co-suppression, in-vivo mutagenesis, RNAi, expression of antibodies, other suitable (poly)peptides or dominant- negative mutants and the insertion of heterologous DNA sequences, can also be used for the reduction of the expression of genes that encode a regulatory protein such as a transcription factor, that controls the expression of the relevant ABC transporter or, e.g., proteins that are necessary for the ABC transporter to become active. It is also evident from the disclosure of the present invention that any combination of the above-identified strategies can be used for the generation of transgenic plants, which due to the one or more of the above-described nucleic acid molecules in their cells display a reduced activity of the relevant ABC transporter compared to corresponding source plants. Such combinations can be made, e.g., by (co-)transformation of corresponding nucleic acid molecules into the plant cell, plant tissue or plant or by crossing transgenic or mutant plants that have been generated by different embodiments of the method of the present invention. Likewise, the plants obtainable by the method of the present invention can be crossed with plants, e.g. transgenic plants having other desired traits, so as to achieve a combination of an increased tolerance to drought stress, with other traits, such as for example increased yield or an improved quality of the harvested products. Another possibility relates to the combination with the trait of a modified, i.e. decreased, stomatal density as, for instance, described by Berger (Genes and Development 14 (2000), 1119- 1131).

As already explained above, for some embodiments, the nucleic acid molecule to be introduced has to be expressed in the resulting transgenic plant. It is in principle possible that the nucleic acid molecule is expressed in all or substantially all cells of the plant. However, it is also possible that it is only expressed in certain parts, organs, cell types, tissues etc. Moreover, it is possible that the expression of the nucleic acid molecule only takes place upon induction or only at a certain developmental stage. In a preferred embodiment, the nucleic acid is expressed in guard cells.

In order to be expressed, the nucleic acid molecule that is introduced into a plant cell according to the method of the invention is preferably operatively linked to a regulatory sequence, e.g. a promoter, active in plant cells. The term "operatively linked", as used throughout the present description, refers to a linkage between a regulatory sequence, and the nucleic acid molecule to be expressed in such a way that expression is achieved under conditions compatible with the regulatory sequence.

The promoter may be homologous or heterologous to the plant. Suitable promoters are for instance the promoter of the 35S RNA of the Cauliflower Mosaic Virus (see for instance US-A-5,3δ2,60δ) and the ubiquitin-promoter (see for instance US-A-δ,614,399) which lend themselves to constitutive expression, the patatin gene promoter B33 (Rocha- Sosa et al., EMBO J. 8 (1989), 23-29) which lends itself to a tuber-specific expression in potatoes or a promoter ensuring expression in photosynthetically active tissues only, for instance the ST-LS1 promoter (Stockhaus et al., Proc. Natl. Acad. Sci. USA 84 (1987), 7943-7947; Stockhaus et al., EMBO, J. 8 (1989) 2446-2461), the Ca/b-promoter (see for instance US-A-5,656,496, US-A-5,639,952, Bansal et al., Proc. Natl. Acad. Sci. USA 89 (1992), 3654-3668) and the Rubisco SSU promoter (see for instance US-A-5,034,322; US-A-4,962,028) or the glutelin promoter from wheat which lends itself to endosperm- specific expression (HMW promoter) (Anderson, Theoretical and Applied Genetics 96, (1998), 568-576, Thomas, Plant Cell 2 (12), (1990), 1171-1180), the glutelin promoter from rice (Takaiwa, Plant Mol. Biol. 30(6) (1996), 1207-1221, Yoshihara, FEBS Lett. 383 (1996), 213-218, Yoshihara, Plant and Cell Physiology 37 (1996), 107-111), the shrunken promoter from maize (Maas, EMBO J. 8 (11) (1990), 3447-3452, Werr, Mol. Gen. Genet. 202(3) (1986), 471-475, Werr, Mol. Gen. Genet. 212(2), (1988), 342-350), the USP promoter, the phaseolin promoter (Sengupta-Gopalan, Proc. Natl. Acad. Sci. USA 82 (1985), 3320-3324, Bustos, Plant Cell 1 (9) (1989), 839-863) or promoters of zein genes from maize (Pedersen et al., Cell 29 (1982), 1016-1026; Quatroccio et al., Plant Mol. Biol. 15 (1990), 81-93). However, promoters which are only activated at a point in time determined by external influences can also be used (see for instance WO 93/07279). In this connection, promoters of heat shock proteins which permit simple induction may be of particular interest. Likewise, artificial and/or chemically inducible promoters may be used in this context. Moreover, seed-specific promoters such as the USP promoter from Vicia faba which ensures a seed-specific expression in Vicia faba and other plants may be used (Fiedler et al., Plant Mol. Biol. 22 (1993), 669-679; Baumlein et al., Mol. Gen. Genet. 225 (1991), 459-467). Moreover, fruit-specific promoters, such as described in WO 91/01373 may be used too. Preferred are promoters which ensure constitutive expression. In a preferred embodiment, the nucleic acid molecule is operatively linked to a promoter which is inducible upon drought stress. Furthermore preferred are promoters mediating expression in guard cells such as the promoter of the AtMRPδ gene as described in the appended Examples. Guard cell-specific promoters are for instance described by Plesch (Gene 249 (2000), 83-89) and Mϋller-Rober (Plant Cell 6 (1994), 601-612).

Moreover, the nucleic acid molecule may be linked to a termination sequence, which serves to terminate transcription correctly and to add a poly-A-tail to the transcript, which is believed to have a function in the stabilization of the transcripts. Such elements are described in the literature (see for instance Gielen et al., EMBO J. 8 (1989), 23-29) and can be replaced at will.

When polypeptide expression from said nucleic acid molecule is required, there exists in principle the possibility that the synthesized protein can be localized in any compartment of the plant cell (e.g. in the cytosol, plastids, vacuole, mitochondria) or the plant (e.g. in the apoplast). In order to achieve the localization in a particular compartment, the coding region must, where necessary, be linked to DNA sequences ensuring localization in the corresponding compartment. The signal sequences used must each be arranged in the same reading frame as the DNA sequence encoding a polypeptide that is used for reducing the activity of the relevant ABC transporter. A localization in the vacuole or cytosol or in the membranes surrounding these compartments or in the apoplast is preferred, as it is necessary for the intended application.

In order to ensure the location in the plastids it is conceivable to use one of the following transit peptides: of the plastidic Ferredoxin: NADP+ oxidoreductase (FNR) of spinach which is enclosed in Jansen et al. (Current Genetics 13 (1988), 517-522). In particular, the sequence ranging from the nucleotides -171 to 165 of the cDNA Sequence disclosed therein can be used which comprises the 5^' non-translated region as well as the sequence encoding the transit peptide. Another Example is the transit peptide of the waxy protein of maize including the first 34 amino acid residues of the mature waxy protein (Klδsgen et al., Mol. Gen. Genet. 217 (1989), 156-161). It is also possible to use this transit peptide without the first 34 amino acids of the mature protein. Furthermore, the signal peptides of the ribulose bisphosphate carboxylase small subunit (Wolter et al., Proc. Natl. Acad. Sci. USA 86 (1988), 846- 860; Nawrath et al., Proc. Natl. Acad. Sci. USA 91 (1994), 12760-12764), of the NADP malat dehydrogenase (Gallardo et al., Planta 197 (1995), 324-332), of the glutathion reductase (Creissen et al., Plant J. 8 (1996), 167-175) or of the R1 protein (Lorberth et al. Nature Biotechnology 16, (1998), 473-477) can be used. In order to ensure the location in the vacuole, it is conceivable to use one of the following transit peptides: the N-terminal sequence (146 amino acids) of the patatin protein (Sonnewald et al., Plant J. 1 (1991), 95-106) or the signal sequences described by Matsuoka and Neuhaus (Journal of Experimental Botany 50 (1999), 165-174); Chrispeels and Raikhel (Cell 68 (1992), 613-616); Matsuoka and Nakamura (Proc. Natl. Acad. Sci. USA 88 (1991), 834-838); Bednarek and Raikhel (Plant Cell 3 (1991), 1195-1206); and Nakamura and Matsuoka (Plant Phys. 101 (1993), 1-5).

In order to ensure the location in the mitochondria, it is for example conceivable to use the transit peptide described by Braun (EMBO J. 11 , (1992), 3219-3227). In order to ensure the location in the apoplast, it is conceivable to use one of the following transit peptides: signal sequence of the proteinase inhibitor ll-gene (Keil et al., Nucleic Acid Res. 14 (1986), 5641-5650; von Schaewen et al., EMBO J. 9 (1990), 30-33), of the levansucrase gene from Erwinia amylovora (Geier and Geider, Phys. Mol. Plant Pathol. 42 (1993), 387-404), of a fragment of the patatin gene B33 from Solanum tuberosum, which encodes the first 33 amino acids (Rosahl et al., Mol Gen. Genet. 203 (1986), 214- 220) or of the one described by Oshima et al. (Nucleic Acid Res. 18 (1990), 181). The method according to the invention relates to transgenic plants which may, in principle, be plants of any plant species, that is to say they may be both monocotyledonous and dicotyledonous plants. Preferably, the plants are useful plants cultivated by man for nutrition or for technical, in particular industrial, purposes. They are preferably sugar storing and/or starch-storing plants, for instance cereal species (rye, barley, oat, wheat, maize, millet, sago etc.), rice, pea, marrow pea, cassava, sugar cane, sugar beet and potato; tomato, rape, soybean, hemp, flax, sunflower, cow pea or arrowroot, fiber-forming plants (e.g. flax, hemp, cotton), oil-storing plants (e.g. rape, sunflower, soybean) and protein-storing plants (e.g. legumes, cereals, soybeans). The method of the invention also relates to fruit trees, palms and other trees or wooden plants being of economical value such as in forestry. Moreover, the method of the invention relates to forage plants (e.g. forage and pasture grasses, such as alfalfa, clover, ryegrass) and vegetable plants (e.g. tomato, lettuce, chicory) and ornamental plants (e.g. roses, tulips, hyacinths).

According to the method of the invention, transgenic plants can be prepared by introducing a nucleic acid molecule into plant cells and regenerating the transformed cells to plants by methods well known to the person skilled in the art.

A plurality of techniques is available by which DNA can be inserted into a plant host cell. These techniques include the transformation of plant cells by T-DNA using Agrobacterium tumefaciens or Agrobacterium rhizogenes as a transforming agent, the fusion of protoplasts, injection, electroporation of DNA, insertion of DNA by the biolistic approach and other possibilities.

The use of the Agrobacteria-mediated transformation of plant cells has been extensively investigated and sufficiently described in EP 120 516; Hoekema, In: The Binary Plant Vector System, Offsetdrukkerij Kanters B.V., Alblasserdam (1985), Chapter V; Fraley et al, Crit. Rev. Plant Sci. 4 (1993), 1-46 and An et al., EMBO J. 4 (1985), 277-287. Regarding the transformation of potatoes see for instance Rocha-Sosa et al. (EMBO J. 8 (1989), 29-33).

The transformation of monocotyledonous plants by means of Agrobacterium-based vectors has also been described (Chan et al., Plant Mol. Biol. 22 (1993), 491-506; Hiei et al., Plant J. 6 (1994) 271-282; Deng et al, Science in China 33 (1990), 28-34; Wilmink et al, Plant Cell Reports 11 (1992), 76-80; May et al., Bio/Technology 13 (1995), 486-492; Conner and Dormisse, Int. J. Plant Sci. 153 (1992), 550-655; Ritchie et al. Transgenic Res. 2 (1993), 252-265). An alternative system for transforming monocotyledonous plants is the transformation by the biolistic approach (Wan and Lemaux,. Plant Physiol. 104 (1994), 37-48; Vasil et al., Bio/Technology 11 (1993), 1553-1558; Ritala et al., Plant Mol. Biol. 24 (1994) 317-325; Spencer et al., Theor. Appl. Genet. 79 (1990), 625-631), protoplast transformation, electroporation of partially permeabilized cells, insertion of DNA via glass fibers. The transformation of maize in particular has been repeatedly described in the literature (see for instance WO 95/06128, EP 0 513 849, EP 0 465 875, EP 29 24 35; Fromm et al, Biotechnology 8, (1990), 833-844; Gordon-Kamm et al., Plant Cell 2, (1990), 603-618; Koziel et al., Biotechnology 11 (1993), 194-200; Moroc et al., Theor. Appl. Genet. 80, (1990), 721-726). The successful transformation of other types of cereals has also been described for instance of barley (Wan and Lemaux, supra; Ritala et al., supra, Krens et al., Nature 296 (1982), 72-74), wheat (Nehra et al., Plant J. 5 (1994), 285-297) and rice.

According to another preferred embodiment of the invention, the above-described method for producing mutant plants having an increased tolerance to drought stress comprises the step of determining those plants out of a pool of plant mutants the stomata of which do not open upon the administration of a sulfonylurea compound which is an inhibitor of stomatal closure. Preferably this sulfonylurea compound is glibenclamide or tolbutamide.

This embodiment is based on the surprising finding that knock-out plants for AtMRPδ do not open the stomata in response to glibenclamide treatment, indicating that this ABC transporter is involved in the control of ion fluxes (see Example 7). Glibenclamide is an inhibitor of sulfonylurea receptor (SUR) ABC proteins and also inhibits both S-type anions channels and K⁺ efflux channels thereby abolishing stomatal closure triggered by ABA or external Ca²⁺ (Leonhardt (1999)). In addition to glibenclamide, other sulfonylurea compounds can be used that inhibit stomatal closure such as tolbutamide (Leonhardt, 1997). The skilled person is able to identify further sulfonylurea compounds having an inhibitory effect on stomata closure, for instance, by methods described in Leonhardt (1997; 1999). The present embodiment refers to "mutant plants" (or "plant mutants"), i.e. plants the genotype of which is modified compared to the corresponding source plants by other means than genetic engineering, i.e. the introduction of an exogenous nucleic acid molecules into plant cells. Such "mutant plants" may be provided by methods known in the art, e.g. produced under the influence of a suitable dosis of ionizing radiation (e.g. x- rays, gamma or neutron radiation) or by the effect of suitable mutagens (e.g. EMS, MMS, etc.). Furthermore encompassed are mutant plants wherein the mutation occurs naturally.

The administration of a suitable sulfonylurea compound and the determination whether stomata open may be carried out according to the methods described in the prior art or, as it is preferred, in the Example 7, infra.

Plants that do not show stomatal opening upon sulfonylurea treatment may be further examined for a reduced activity of an ABC transporter expressed in guard cells and/or for the presence of one or more loss-of-function mutations in the gene encoding said ABC transporter in accordance with the explanations given above. Such mutations comprise additions, substitutions, deletions, inversions and the like. Suitable methods for detecting the mutations in a gene are well known to the person skilled in the art and encompass, for example, PCR amplification and subsequent DNA-sequencing as it is, e.g., described in Example 4. Preferably, said method of determining mutant plants is preceded by the step of pre-selecting said pool of plant mutants for the tolerance to drought stress.

In a further aspect, the present invention relates to transgenic or mutant plants obtainable by the method for producing transgenic or mutant plants having an increased tolerance to drought stress as described herein above. Preferably, these transgenic plants contain a nucleic acid molecule as defined above, i.e. a nucleic acid molecule that is introduced in a plant cell and the presence of which in the genome of said plant leads to a reduced activity of an ABC transporter which is expressed in guard cells, stably integrated into the genome. As regards the properties of these transgenic or mutant plants and the modes how to produce them, everything that is said in connection with the method of the invention applies.

Furthermore, the invention also refers to transgenic plant cells in which the activity of an ABC transporter which is expressed in guard cells is reduced. The above explanations and definitions made in connection with the method and the transgenic plants of the present invention apply, where appropriate. Thus, it is preferred that these transgenic plant cells contain the nucleic acid molecule as defined above stably integrated into their genome. Likewise, this embodiment relates to plant cells wherein the gene encoding said ABC transporter is inactivated, i.e. carries a mutation that abolishes its expression or the proper activity of a polypeptide encoded by its coding sequence.

The invention also relates to propagation material of the plants of the invention comprising plant cells according to the invention. The term "propagation material" comprises those components or parts of the plant which are suitable to produce offspring vegetatively or generatively. Suitable means for vegetative propagation are instance cuttings, callus cultures, rhizomes or tubers. Other propagation material includes for instance fruits, seeds, seedlings, protoplasts, cell cultures etc. The preferred propagation materials are tubers and seeds. The invention also relates to harvestable parts of the plants of the invention such as, for instance, fruits, seeds, tubers, rootstocks, leaves or flowers.

In addition, the present invention pertains to the use of a nucleic acid molecule suitable for the introduction in a plant genome and, therein, having a reducing effect on the activity of an ABC transporter which is expressed in guard cells as described above in connection with the method of the invention.

The present invention relates in a further embodiment to a method for producing transgenic or mutant plants having an increased transpiration comprising the step of providing transgenic or mutant plants having an increased activity of an ABC transporter which is expressed in guard cells.

Based on the properties observed for AtMRPδ, it is conceivable that an increase of ABC transporter activity in guard cells results in an increase of transpiration. The terms "ABC transporter" as well as "MRP" and "MRPδ" as preferred ABC transporters in the present method have been defined above. This also applies to the "activity of an ABC transporter". The term "increased activity of an ABC transporter" refers to a significant increase of the ABC transporter activity in guard cells, preferably as it relates to the ATP-dependent transport activity, compared to the corresponding source plant. Preferably, said increase amounts to an increase of at least 20%, preferably at least 60% and most preferably at least 100% of the corresponding activity in the source plant. Likewise, an increase of said ABC transporter activity may be determined by measuring the amount of the corresponding ABC transporter transcript or protein in the plant tissue where it is normally expressed such as in mature leaves, preferably in guard cells. Accordingly, transgenic or mutant plants produced according to the present method of the invention are characterized by an increase of the said transcript by at least 20%, preferably by at least 50%) and most preferably by at least 90% compared to the corresponding amount of transcript in the source plant or, on the protein level, by an increase of at least 20%, preferably by at least 50% and most preferably by at least 90% of the corresponding ABC transporter polypeptide compared to the corresponding source plant. The above- described increases of ABC transporter activity, transcript and/or protein level provide for an efficient increase of transpiration in transgenic or mutant plants. The term "increase of transpiration" refers to a significantly higher loss of water by evaporation, in particular through the stomata, in plants produced in accordance with the present method compared to corresponding source plants. Transpiration, or the amount of water uptake which directly correlates with transpiration, can be determined as described above.

Preferably, the increase of transpiration amounts to at least 20%, more preferably at least 50%, still more preferably at least 80% and most preferably to at least 100% of the value of a corresponding source plant. Various uses of transgenic or mutant plants obtainable by the method of the present embodiment are conceivable such as in phytoremidiation, i.e. the accumulation of generally damaging substances such as heavy metals from the soil. It is furthermore envisaged to apply the present method to plants which are meant for sites where usually water supply is not limiting. In such cases an increased transpiration may lead to a higher biomass production. Further effects arising from an increased stomatal opening may include an increase of CO₂ entry, thereby potentially augmenting yield of photosynthesis and plant growth, and, furthermore, maintenance of leaf temperatures at low values due to an enhanced vaporization. Methods for producing transgenic or mutant plants having an increased activity and/or gene expression level of an ABC transporter are well known in the state of the art and described in the literature.

In a preferred embodiment, said providing of transgenic plants having an increased activity of said ABC transporter comprises the steps of

(a) introducing into a plant cell a nucleic acid molecule the presence of which in the genome of said plant leads to an increase of the activity of said ABC transporter in plant cells;

(b) regenerating from transformed cells produced in step (a) plants; and optionally

(c) producing progeny from the plants produced in step (b).

Herein, all the explanations and references given above in connection with the method for producing transgenic plants having a reduced ABC transporter activity apply, where appropriate.

Accordingly, in a particularly preferred embodiment, the nucleic acid molecule introduced in the plant cells is expressed. Preferably, the increase of ABC transporter activity is achieved by overexpressing said ABC transporter. The term "overexpressing" refers to expressing an ABC transporter encoded by the introduced nucleic acid molecule to an extent that its transcript and/or protein level significantly exceeds that of the corresponding endogenous wild-type ABC transporter.

In yet another preferred embodiment of the method for producing transgenic plants having an increased transpiration, the nucleic acid molecule is operatively linked to a regulatory sequence active in plants. With regard to suitable regulatory sequences, the explanations given above in connection with the method that leads to a reduced ABC transporter activity are herewith incorporated. Preferably, the regulatory sequence is a constitutive promoter.

An increase of ABC transporter expression may likewise be achieved by in vivo mutagenesis methods as described above. Accordingly, a naturally occurring ABC transporter gene may for example be modified such that its transcription and/or translation is enhanced. Other approaches may aim at modifying the protein activity or other protein functions such as the properties related to the interaction with other proteins involved in stomatal regulation as for instance potassium channels.

In another preferred embodiment of the present method, said providing comprises the step of determining those plants out of a pool of plant mutants the stomata of which do not close upon the administration of a K⁺ channel opener (KCO) which are compounds that induce stomatal closure in the light in the source plant. Advantageously, said KCO is RP49356 or cromakalim.

KCOs physiologically act as antagonists to sulfonylurea compounds such as glibenclamide (Leonhardt et al., 1997). Thus, it is justified to assume that a modified sensitivity of the stomata to KCOs can be used for a screening method to identify plants with an increased transpiration due to an increased activity of an ABC transporter expressed in guard cells. For instance, plant mutants that can be identified with the present method may show a modified interaction between said ABC transporter and a K⁺ channel.

Furthermore, the present invention relates to transgenic plants obtainable by the method for producing transgenic or mutant plants having an increased transpiration. Preferably, these plants contain a nucleic acid molecule as defined above for carrying out said method stably integrated into the genome.

In another embodiment the present invention relates to transgenic plant cells in which the activity of an ABC transporter which is expressed in guard cells is increased and to propagation material or harvestable parts of a transgenic plant obtainable by the method for producing transgenic plants with an increased transpiration containing such transgenic plant cells.

Finally, the present invention relates to the use of a nucleic acid molecule as defined above, i.e. suitable for increasing the activity of an ABC transporter expressed in guard cells, for the production of plants having an increased transpiration.

These and other embodiments are disclosed and obvious to a skilled person and embraced by the description and the examples of the present invention. Additional literature regarding one of the above-mentioned methods, means and applications, which-can be used within the meaning of the present invention, can be obtained from the state of the art, for instance from public libraries for instance by the use of electronic means. This purpose can be served inter alia by public databases, such as the "medline", which are accessible via internet, for instance under the address http://www.ncbi.nlm.nih.gov/PubMed/medline.html. Other databases and addresses are known to a skilled person and can be obtained from the internet, for instance under the address http://www.lycos.com. An overview of sources and information regarding patents and patent applications in biotechnology is contained in Berks, TIBTECH 12 (1994), 352- 364.

All of the above cited disclosures of patents, publications and database entries are specifically incorporated herein by reference in their entirety to the same extent as if each such individual patent, publication or entry were specifically and individually indicated to be incorporated by reference.

The figures show:

Figure 1 Sequence and genomic structure of AtMRPδ. (A) The predicted AflVIRPδ protein sequence. Small letters of the gene sequence indicate the δ^' non- translated sequence. Putative transmembrane-spanning domains identified using the TMpred program (Hofmann and Stoffel, Biol. Chem. Hoppe- Seyler 347 (1993), 166) are underlined; the ABC signature and the Walker motifs A and B, as well as motif C are boxed and given in bold letters. The methionine incorrectly annotated in gene F20D22.11 (Ace. no. AC002411) to be the first amino acid, is boxed. (B) Genomic organisation of the AtMRPδ gene as deduced from the cDNA and a corresponding genomic sequence located on BAG F20D22. The promoter (arrow), as well as 5^' and 3^' untranslated regions are shown as black boxes; exons are presented as dark grey, introns as light grey boxes. Exon and intron sizes are given in bold and standard letters, respectively. The insertion site and the orientation of the T-DNA in the mrpδ-1 mutant is indicated. Figure 2 Phylogenetic comparison of full-size ABC transporter genes from Arabidopsis thaliana with representative members of the ABC transporter superfamily from yeast, Drosophila and humans.

Sequences encoding full-size ABC transporters in the Arabidopsis genome have been identified in two ways: in one approach all annotated protein sequences in MATDB (http://mips.qsf.de/proi/thal/db) with ABC signatures were examined on their structural similarity or sequence homology toward characterized full-size ABC transporters. In another way several MRP, PDR and PGP amino acid sequences from Arabidopsis were used for BLAST searches against its entire genomic sequences deposited in GENBANK. An overview over the Arabidopsis ABC transporter gene sequences used in the present phylogenetic analysis is presented by Table 1. The unrooted phylogenetic tree shown bases on a multiple alignment of 7 full length polypeptide sequences of ABC transporters produced by the CLUSTAL program in the DNASTAR DNA analysis software package. Distance matrix, phylogenetic tree and boot strap values were calculated with CLUSTALW. Bootstrap analysis (1000 replicates) confirmed the structure of the tree. The majority of values were over 90%.

The two letters preceding the protein names describe the organisms the sequences were derived from. At, Arabidopsis thaliana; Dm, Drosophila melanogaster; Hs, Homo sapiens; Sc, Saccharomyces cerevisiae.

Figure 3 Histochemical localization of GUS activity. (A) Seedling 7 dag showing GUS expression in cotyledones and vascular tissue in the tip of primary leaves. (B) Leaf of an Arabidopsis plant 21 dag exhibiting GUS expression in lower and higher order veins. (C) Dark-field observation of a cross section of an adult leaf with GUS expression in vascular tissue, epidermal cells and weakly in mesophyll cells. (D) The abaxial epidermis of an adult leaf exhibits strong GUS staining in guard cells. (E) Flower petals showing GUS expression in guard cells. (F) Root of a seedling 11 dag. GUS expression is present in the central cylinder but not in root tips. (G) GUS- staining in pollen sacs is present along the central vascular strand of the filament and in connecting tissue. (H) GUS staining at the pod attachment site.

Figure 4 DNA blot analysis of six singular F2 plants of the mφδ-1 ANs-2 crossing exhibiting mrpδ-1/mrpδ-1 (lanes 1, 2 in B and C), mrpδ-1ΛNs-2 (lanes 3, 4) and Ws-2/Ws-2 (lanes 5, 6) genotypes (A - C) and RT-PCR analysis of mrpδ-1/mrpδ-1 plants (D). (A) Schematic view of the 5.6 kb genomic sequence of AtMRPδ and of the 17 kb T-DNA construct 3850:1003 (Schulz et al., 1995) inserted in mφδ-1 (triangle) with predicted restriction sites of enzymes EcoRI (E), Hindlll (H) and BamHI (B) (numbers indicate relative positions in kb). The position of the gene-specific probe and a probe specific for the T-DNA left border are indicated as boxes denoted A and T, respectively. (B) DNA blot analysis of genomic DNA digested with EcoRI, Hindlll and BamHI probed with the gene-specific probe A. (C) same as (B) using the T-DNA probe T. The arrow highlights the 15 kb band visible after restriction with BamHI. (D) RT-PCR analysis of AtMRPδ and S16 expression in Ws-2ΛΛ/s-2 and mφδ-1/mφδ-1 plants.

Figure 5 The mφδ-1 mutant displays a reduction in root growth. (A) Light-grown mφδ-1 (upper row) and Ws-2 (lower row) seedlings 8 dag grown vertically on 1/2 x MS/1% sucrose plates. (B) Single mφδ-1 plant at higher magnification exhibiting lateral roots. (C) Comparison of Ws-2 (closed circles) and mrp5-1 (open circles) primary root length. Each data point represents the average of 20 seedlings. (D) Reaction of seedling growth after change of the gravitropic angle. Three mφδ-1 (upper row) and three Ws-2 (lower row) seedlings 11 dag grown on vertical plates. Plates were turned 4 dag. (E) 26 d old plants grown on vertical plates in the light. The three mrp5-1 seedlings on the left exhibit bushy roots due to the presence of more root branches when compared to the three Ws-2 plants on the right. (F) Seedlings 10 dag grown on a medium corresponding to 1 x MS which was made by mixing the following components separately: 99 mg x ml^"1 myo-inositol, 1% (w/v) sucrose, 2.5 mM Mes/KOH pH 5.8, 2.6 mM NaH₂PO4, 2.25 mM CaCI₂, 0.73 mM MgSO4, 19mM KNO3, 1 mM NaNO₃, 1 x micronutrient solution (Sigma M-0529), 0.8% (w/v) agar. (A, B, D) length of squares = 2 cm, (E, F) bar = 1 cm.

Figure 6 Stomata of mrpδ-1 are insensitive towards the sulfonylurea glibenclamide. (A) The change in stomatal aperture was measured as the difference between aperture values in the presence and absence of 8 μM glibenclamide. Each column represents the mean of δ independent experiments (+ SEM) each conducted on 5 plants. The aperture of 60 stomata was determined per experiment. Individual stomata exposed for 3 h are illustrated in the respective columns. (B) A representative experiment showing that application of glibenclamide for 3 h in the dark produces a dose-dependent increase in stomatal aperture in the wild-type plant (open square) but not in the mφδ-1 plants (solid circle). Half-maximal opening of stomata is at 0.8 μM glibenclamide.

Figure 7 Shape of mrp5-1 mutant plants (left-hand) and Ws-2 wild-type plants (right hand) after 48 hours drought stress.

Figure 8 Similar growth of Ws-2 wild-type plants (left-hand) and mrpδ-1 mutant plants (right-hand) when grown on soil under the same conditions without drought stress.

Figure 9 The shape of Ws-2 wild type plants (left-hand) shows a pronounced wilted state six days after having stopped irrigation in contrast to the mrp5-1 mutant plants (right-hand).

Figure 10 Growth of plants eleven days after having stopped irrigation. Most Ws-2 wild-type plants (left-hand) were dead, while the mrp5-1 mutant plants (right-hand) still showed good viability.

Figure 11 Transpiration rate of Ws-2 wild-type plants (filled dots) and mrp5-1 mutant plants (open dots).

Figure 12 Stomata frequency of Ws-2 wild-type plants (filled dots) and mrp5-1 mutant plants (open dots).

Figure 13 Schematic drawing of a potometer constructed for measuring the water uptake of Arabidopsis plants. This device is common in plant physiology. Corresponding applications are for example described by Weyers (1990), In: Methods in Stomatal Research eds. Longman Scientific & Technical, London.

Figure 14 Diagram showing the water uptake of Ws-2 wild-type plants (filled dots) and mrpδ-1 mutant plants (open squares) when grown under light. The inset shows the effect of drought stress imposed by the addition of 10% of PEG on the respective water uptake curves.

Figure 15 Diagram showing the water uptake of Ws-2 wild-type plants (filled dots) and mrp5-1 mutant plants (open squares) after addition of 10 μM ABA. In the dark (hours 6.6 to 22.6) the water uptake stops.

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Table 1 : List of full-size ABC transporters in Arabidopsis

All characterized or putative ABC transporters in Arabidopsis are listed, ordered by homology to AtPGPs, AtMRPs, AtPDR5-like proteins and others. In the second and third column the protein entry codes are shown together with the chromosomal locations and the accession numbers of the genomic sequence (fourth and fifth column). The last column contains cDNAs entries of the different transporter genes (where available). Accession numbers of cDNAs are given in those cases where either the full length cDNA sequence or single EST sequences are available. In cases that different ESTs together with annotation data are assembled to tentative contigs (TC) the corresponding TC number is shown (Quackenbusch, Nucl. Acid Res. 29 (2001), 159-164). Putative pseudogenes and aberrant proteins are included in this list. AtPGPδ represents a truncated AtPGP isolog whose annotated amino acid sequence is partially confirmed by one EST. The same holds true for AtPGP23. Here the predicted protein sequence consists only of a single nucleotide binding domain. Therefor the function of this protein as a ABC transporter is not very probable. Although an EST for AtPGP23 has been found, this gene encodes possibly a transcribed pseudogene. In the cases of AtMRPIδ and AtMRP16 both genes represent pseudogenes. The genomic sequence of AtMRP15 is highly homologous to AtMRP9 (>99 % identity). Comparing the first exon of AtMRP9 and the annotated 5^'-UTR of AtMRPIδ, both sequences are nearly identical with two major exceptions: the AtMRPIδ sequence shows 2 single base pair deletions and two insertion of one base pair and 230 bp length. These sequence alterations destroy the open reading frame and lead to a truncation of the predicted peptide of AtMRPIδ by 400 amino acids. Thus, AtMRPIδ rather looks like a truncated version of AtMRP9. In the case of AtMRP16 the annotated coding sequence would encode 324 amino acids. No ABC signature could be found in this genomic region, which makes a function as ABC transporter unlikely. PDRδ-2 has been annotated as two different genes, but new analysis of the sequence data in comparison to other members of this gene family led to the conclusion that this locus encodes just one gene (Kolukisaoglu, pers. comm.)

The following Examples serve to further illustrate the invention. In the Examples the following materials and methods were used.

1. Molecular biological techniques

Unless stated otherwise in the examples, all recombinant DNA techniques are performed according to protocols as described in Sambrook et al. (1989), Molecular Cloning : A Laboratory Manual. Cold Spring Harbor Laboratory Press, NY or in Volumes 1 and 2 of Ausubel et al. (1994), Current Protocols in Molecular Biology, Current Protocols. Standard materials and methods for plant molecular work are described in Plant Molecular Biology Labfase (1993) by R.D.D. Cray, jointly published by BIOS Scientific Publications Ltd (UK) and Blackwell Scientific Publications (UK).

2. Plant material and growth conditions

If not stated otherwise, plants were grown on soil under a 16 h light 8 h dark regime. For epidermal strip experiments, plants were grown individually in pots of sand watered with half-strength Hoagland's solution in a growth chamber with 8 h light/16 h dark. For the analysis of the phenotype of mrpδ-1, surface-sterilized and vernalized seeds (48 h at 4° C) were germinated on half-strength Murashige and Skoog (MS) salts (Duchefa, M0233, NL) with 1 % sucrose under continuous light. For growth of the plants shown in Figure 6F, a modified MS medium was used (see Figure legend).

3. Cloning of AtMRPδ cDNA

A nested PCR was performed on an expressed sequence tag (EST; GenBank Ace. no. W43620), encoding a putative ABC transporter from Arabidopsis using the primers CFTRAt-1 (SEQ ID NO:13), CFTRAMn (SEQ ID NO:14), CFTRAt-2 (SEQ ID NO:15) and CFTRAt-2n (SEQ ID NO:16). The resulting DNA fragment was used to isolate a full-size cDNA of 5.1 kb by screening 1 x 10⁶ plaque-forming units of a hypocotyl cDNA library. The transcriptional start site of the AtMRPδ gene was determined by RACE-PCR. Sequence similarities were identified by using default parameters of the BESTFIT program. AtMRPδ cDNA sequence has been deposited in the GenBank™ EMBL Data Bank (accession no. Y11260; SEQ ID NO:1).

4. Northern and Southern blot analysis

For Northern blots RNA was isolated from different tissues of A. thaliana after Chomczynski et al., 1987. Northern (40μg total RNA) and Southern (10 μg DNA) blots were performed following standard protocols (Sambrook et al., 1989). Northern blots were hybridized with an 0.7 kb EcoRI/ Nhe\ fragment of the 5' region of the AtMRPδ cDNA. DNA gel blots performed to analyze the PCR reactions in reverse genetic screens and to investigate the genotype of mφδ-1 mutant plants were hybridized with a probe generated by PCR using Ws-2 genomic DNA and primers MRPδAn-sense and MRPδAn-antisense (see Table 2). T-DNA specific probes were a 6.5 kb and a 3.5 kb Hind\\\ fragment containing the left and right border of 3850:1003, respectively (Jones et al., 1987). For RT-PCR analysis of Ws-2 and mφδ-1, total RNA from seedlings grown in liquid cultures under mixotrophic conditions (1 x MS, 1 % sucrose; constant light) for 7 days was prepared using the RNeasy Plant Kit (Qiagen). Oligo-dT primed cDNA from 1 μg of total RNA was synthesized using the Reverse Transcription system (Promega). MRP5 and 40S ribosomal protein S16-specific cDNAs were amplified by PCR for 30 or 25 cycles, respectively, at 52°C. RT-PCR primers used were: S16-upper 5' ggc gac tea ace age tac tga (SEQ ID NO.3), S16-lower 5' egg taa etc ttc tgg taa cga (SEQ ID NO.4), and MRPδD-sense, MRP5D-anti (Table 2) for S16 and AfVlRPδ, respectively. Table 2 Primers used for the identification and verification of the mφδ-1 mutant, for the generation of probes and RT-PCR analysis. LB2 and RB2 represent primers specific for the T-DNA left and right border, respectively.

Primer name Primer sequence

RB2 5'-TCCTTCAATCGTTGCGGTTCTGTCAGTTC-3' (SEQ ID NO:5)

LB2 5'-GATGCACTCGAAATCAGCCAATTTTAGAC-3' (SEQ ID NO:6)

MRP35A-sense 5'-TGTGGYACMGTTGGCTCTGGRAAATC-3' (SEQ ID NO:7)

MRP35A-anti 5'-GTGTGTGCATCMASAGCRCTAAAAGG-3' (SEQ ID NO:8)

MRP5An-sense 5'-CTTGCATCCTAGGGGAAATCCCAAAAA-3' (SEQ ID NO:9)

MRP5An-anti 5'-TAATGCCCTTGCAAGTTGTACACGC-3' (SEQ ID NO: 10)

MRP5D-sense 5'-GCGCATAGGAGATTACTCATGGTATC-3' (SEQ ID NO: 11 )

MRP5D-anti 5'-CCGAAGTGGCTCCTGAAGAATACAGA-3' (SEQ ID NO: 12)

5. Isolation of the AtMRPδ promoter and GUS expression analysis

The structural organization of the AtMRPδ gene was deduced from genomic Southern blots (not shown) and the sequence of BAC clone F20D22 (GenBank Ace. no. AC002411). Partial digestion of Arabidopsis genomic DNA with HindlW BglW and XbaU Bgl\\ yielded fragments of 3 kb and 1.8 kb which were fused to produce promoter-β-glucuronidase constructs. A. thaliana (col-0) plants were transformed using Agrobacterium and vacuum infiltration (Bechthold et al., 1993). Selected transformants were assayed for β-glucuronidase (GUS) activity (δ h incubation at 37°C, unless stated otherwise) using 5-bromo-4-chloro-3-indoIyl-β-D- glucuronic acid (X-Gluc; Duchefa) as substrate. For microscopic analysis, GUS- stained plant tissue was embedded in Technovit 7100 (Kulzer, Wertheim, Germany) and 8 μm sections were cut with a microtome (RM2165, Leica, Germany). Specimens were stained with δ % Fuchsin (Sigma) for 20 min before microscopy (Olympus AX-70 microscope). Expression of AtMRPδ in yeast, preparation of yeast microsomes, uptake experiments and analysis of cadmium sensitivity

The AtMRPδ cDNA was cloned into pNEV (Tommasini et al. 1996) to give pN- Afl RPδ. pNEV and pN-AflVIRPδ were introduced into yeast strains DTY168 (Szczypka et al., 1994) and YYA4 (Mat a, Δycfl r.loxP-KAN-loxP, Δyhl03δ::HIS3, ade2-1, his3-11,-1δ, leu2-3, 112 tφ1-1, ura3-1, can1-100). Microsomes for transport analysis were isolated as described (Tommasini et al., 1996). Uptake of 40 μM [¹ C]-DNB-GS or 10 μM [³H]-E₂17G was measured by rapid filtration using nitrocellulose (0.45 μm pore size) or Durapore® filters (0.22μm pore size; Millipore GmbH, Eschbom, Germany), respectively (Tommasini et al. 1996; Klein et al. 1998). Analysis of Cd²⁺ tolerance of yeast strains DTY168, DTY7, DTY168-pN- A.MRP5 or DTY168-pNev was performed as described (Tommasini et al., 1996).

Screening for the Arabidopsis mutant mrpδ-1

In this study, a collection of 4120 T-DNA transformed lines from seed transformation (Forsthoefel et al., 1992) arranged in pool sizes of 20, 100 and 500 independent lines was screened. DNA from individual lines was isolated following a cetyltrimethylammonium bromide (CTAB) DNA extraction procedure (Murray -and Thompson, 1980). PCR reactions (50 μl) on DNA pools and single lines contained 20 mM Tris-HCI (pH 8.4), 50 mM KCI, 2.5 mM MgCI₂, 0.2 mM deoxyribonucleotide triphosphate, 0.5 μM of each primer (forward or reverse and left border or right border primer, see Table 2), 0.1-1 μg genomic DNA, and 1U Taq DNA polymerase. The PCR started with an initial 2 min incubation at 94° C, followed by 35 cycles of PCR (94° C 1 min, 58° C 1 min, 72° C 2 min 30 sec and an elongation step of 5 min at 72° C).

Primers used for the identification of the null-mutation of AtMRPδ and for the subsequent characterization are shown in Table 2. Gene-specific primers (MRP35A-sense and MRP35A-anti) used for the initial screen on DNA pools were slightly degenerate to recognize both genes, AtMRPδ and AtMRP3 (Tommasini et al., 1997). For AtMRPδ, MRP35A-sense and MRP35A-anti amplify a 485 bp long genomic and a 377 bp long cDNA fragment, respectively. PCRs were first performed on DNA pools representing 500 lines followed by gel separation and hybridization using a probe described in materials and methods. To confirm insertion of T-DNA in the gene of interest, PCR reactions that yield hybridisation signals in the first round of screening were repeated using one of the AflvlRPδAn primers in combination with the corresponding border primer. PCR products which were readily visible in agarose gels and showed hybridization with A/MPRδ specific probes were gel-purified, subcloned into pGEM T-easy (Promega) and sequenced. One cloned fragment indicated the presence of a T-DNA insertion in AtMRPδ. Subsequently, PCRs were performed on DNA pools of 100 and 20 lines. To identify single mφδ mutant plants in pools of 20 lines, leaf material from 385 plants arranged in five trays of 77 individuals was pooled using a two-dimensional sampling strategy.

An individual T-DNA insertion mutant named mφδ-1 was identified by PCR and confirmed by subsequent sequencing of the PCR product. To analyze both gene regions flanking the T-DNA integration site in AtMRPδ, PCRs were performed on genomic DNA of the isolated mutant plant using one of the A-MRPδD-primers in combination with border primers. PCR products were again subcloned and sequenced. For primer sequences see Table 2, above.

8. Extraction of plant material and auxin measurement

Roots (8 to 10 dag; 20 to 80 mg FW) were immersed in 1 ml of methanol with 30 pmol of [²H₂]-IAA and incubated for 60 min at 37°C and 1-2 h at room temperature. The methanolic extract was concentrated to dryness in a stream of nitrogen. The residue was redissolved in 100 μl of diethylether and applied to 30 μl bed volume Bondesil NH₂ (Varian, Darmstadt). After washing with chloroform: isopropanol (2:1 , 100μl) the compounds were eluted with 200 μl of acidic diethylether (2% formic acid). This fraction was again dried, redissolved in 50% aqueous methanol and then applied to 10 μl bed volume ENV+ (1ST, Mid Glamorgan U.K.). The liquid was removed from the solid phase by a stream of nitrogen. Compounds were eluted with 100 μl etheral diazomethane, dried and dissolved in 5μl chloroform. An aliquot of 1μl was subjected to GC-MS using a Varian Saturn 2000 ion trap mass spectrometer. All spectra were recorded on this spectrometer by operating in CI-MRM mode (chemical ionisation, methanol) with the following settings: GC, splitless injection of 1μl (injector temp. 260°C) using a ZB-50 fused silica capillary column (30m, 0.2δ mm i.d., 0,25 μm film thickness; Phenomenex) with 1 ml/min He carrier gas flow; chromatographic conditions: 1 min 50° C, linear ramp at 20°C/min to 260°C; transfer line 260°C; mass spectrometric conditions: 2 scans (100-200 amu) s^"1. Endogenous IAA was detected by collecting m/z 190 [M+H]⁺ of IAA methyl ester (R_t = 11.02 min) and observing the fragment ion m/z 130. The internal standard [²H₂]-IAA was determined analysing the corresponding ions m/z 192 and m/z 132 (R_t = 11.01 min). The amounts of endogenous IAA were calculated from the signal ratios of the unlabelled m/z 130 over the isotopically labelled mass fragment m/z 132.

9. Growth analysis and epidermal strip experiments involving glibenclamide treatment

Primary root length was measured with seedlings grown on vertical plates every 24 h starting 4 dag. Leaves from 4- to δ-week old plants were harvested in the dark at the end of the night period and placed with their abaxial side onto a transparent medical adhesive coated cover slip. The adaxial epidermis and mesophyll were removed using a razor blade. The cover slip was then placed in a Petri dish containing 10 mM KCI, 30 mM KOH, 26 mM iminodiacetate and 10 mM Mes pH 6.5 at 20°C. After 30 min in the dark, glibenclamide prepared as described (Leonhardt et al., 1997) was added to the solution and measurements of stomatal apertures for Ws-2 and mprδ-1 mutant plants were performed after 3 h in the dark. Only "mature stomata" whose ostiole length was higher than one third the length of stoma were analyzed. For each treatment at least 60 stomatal apertures were measured. All experiments were repeated five times.

Example 1: Isolation of a cDNA encoding AtMRPδ

An EST database analysis and cDNA library screening approach succeeded in identifying a 5.1 kb long cDNA (SEQ ID _,NO:1) that encodes a novel ABC transporter protein (AflvlRPδ) from A. thaliana (Gaedeke (1999), Dissertation zur Eriangung des Doktorgrades, Freie Universitat Berlin, Germany). Additional sequence information corresponding to the 5' untranslated region of the AtMRPδ mRNA was obtained by RACE-PCR. The AtMRPδ cDNA contains an open reading frame (ORF) which encodes a protein that spans 1614-amino acids with a predicted molecular weight of 167 kDa (Figure 1A and SEQ ID NO:2). An in-frame stop codon upstream of the start ATG indicated that the complete A.MRP5 coding region was present on the cDNA (SEQ ID NO:1).

The alignment of the AtMRPδ cDNA with BAC sequence F20D22.11 (Ace. no. AC002411) allowed it to deduce the genomic organization of the AtMRPδ gene as well as its intron-exon structure. (Figure B). The gene maps on chromosome 1 of A. thaliana and consists of 11 exons and 10 introns, with the first intron of 192 bp located within the δ' untranslated region. The two ATP-binding cassettes of AtMRPδ are similar to those conserved in the ABC superfamily proteins (Higgins, 1992), each consisting of a domain of about 200 amino acids and comprising the ATP-binding motifs Walker A (GXXGXG) and Walker B (T/IYLLD) (Walker et al. 982) and the ABC signature ([LIVMFY]-S-[SG]-G- X(3)-[RKA]-[LIVMYA]-X-[LIVMF]-[AG]) (Higgins, 1992). The N-terminal ATP-binding cassette of AtMRPδ contains two well-conserved A and B Walker motifs with a typical ABC signature, whereas the COOH-terminal ATP-binding cassette contains a degenerated Walker B motif (ILVLD).

A phylogenetic analysis of AtMRPδ (Figure 2) reveals that this gene forms a subcluster with AtMRP3 to AtMRP9 while AtMRPI and 2 cluster in a separate branch (Martinoia et al., 2000). AtMRP3, which is at present the only biochemically characterized AflvlRP of this subgroup (Tommasini et al. 1997, Tommasini et al. 1998), exhibits 60% identity and 71% similarity to AtMRPδ. Interestingly, the phylogenetic tree indicates that the AflvlRPδ subcluster is at least as closely related to the sulfonylurea receptor (SUR) and CFTR as to human MRPs and yeast YCF1. This is not the case for AtMRPI and AtMRP2. In order to find out to what extent the results obtained for Arabidopsis in the context of the present invention can be transferred to other plant species, a search through the database material so far available has been conducted. As a result, a comparison of sequences coding for Arabidopsis ABC transporters with sequences from other plants revealed that they contain AtMRP homologs and that these homologs can be assigned a specific identity on the basis of their sequence to a particular AtMRP or to members of a particular AtMRP subgroup. AtMRPI AtMRP2 AtMRP3 AtMRP4 AtMRPδ AtMRP6 AtMRP7 AtMRPδ AtMRP9

% ι % s % ι % s % ι % s % ι % s % ι % s % ι % s % ι % s % ι % s % ι % s

100 100 88 93 52 71 54 73 56 74 50 66 54 70 52 69 52 69 AtMRPI

100 100 54 73 54 74 58 75 52 67 54 70 52 68 53 69 AtMRP2 100 100 62 81 75 86 59 78 85 92 83 90 65 81 AtMRP3 100 100 61 78 65 78 63 83 61 80 61 80 AtMRP4 100 100 63 76 68 81 69 82 69 82 AtMRPδ 100 100 43 60 49 66 47 65 AtMRP6 100 100 91 95 60 72 AtMRP7 C3 100 100 66 78 AtMRPδ 100 100 AtMRPΘ

Table 3: Alignments between the C-terminal portions of AtMRPs 1-9. Shown are the % identities (%l) and % similarities (%S) for the sequences encompassed by the second NBF and the C-terminal domain. For AtMRPs 1 , 2, 3, 4 and δ, the C- terminal sequences starting at residues 1246, 1246, 1280, 1284 and 1280 were employed for the comparisons; for AtMRPs 6, 7, 8 and 9, the last 250 amino acid residues of the putative translation product were employed for the comparisons.

AtMRPI AtMRP2 AtMRP3 AtMRP4 AtMRP5 AtMRPβ AtMRP7 AtMRPδ AtMRP9

% ! % S % l % S % l % S % l % S % l % S % l % S % l % S % l % S % l % S

AW092564 84 92 85 91 54 71 56 77 58 76 40 55 54 71 52 69 54 74

AW219577 82 93 82 91 45 63 50 66 51 69 50 64 48 61 48 62 49 66

AW216929 51 68 51 68 85 93 64 81 70 83 57 74 83 90 82 90 69 82

AW276374 51 74 52 74 60 81 83 92 63 83 57 81 60 81 58 79 60 80

AI668454 55 71 57 66 74 86 60 77 88 97 31 50 68 82 70 84 70 82

AW034253 44 66 46 67 58 80 61 77 57 75 80 88 58 78 55 76 55 70

AW396018 51 68 54 70 63 78 57 74 68 78 51 68 66 81 61 77 82 94

Table 4: Alignments between the C-terminal portions of AtMRPs 1-9 and those of the putative translation products of ETSs from plant species other than Arabidopsis. The same C-terminal AtMRP sequences as those listed in Table 3 were used as query sequences for BLAST searches of the EST databases. The results shown are for ESTs from tomato (AW092564,AW219δ77, AW216929, AW034263), soybean (AW278374, AW396018) and corn (AI668454).

The analyses summarized in Tables 3 and 4 represent an inventory of the nine known AtMRPs, their alignments with each other (Table 3) and a sample of the results of EST database searches for MRP homologs in other plant species (Table 4). The sequences from individual AtMRPs were compared with each other and then used as query sequences to not only identify AtMRP homologs but also gain an indication of the particular type of AtMRP identified in the target organism. To simplify the search and preclude double hits for a given gene, the C-terminal portions of the AtMRPs, corresponding to the second NBF and the C-terminal domain, were used for the searches. These results show that with a known Arabidopsis sequence the respective orthologue of other plants can be found.

Example 2: Characterization of AtMRPδ as an organic anion transporter

It has been shown that YCF1, which confers resistance to cadmium, also functions as a glutathione-conjugate transporter in yeast (Li et al., 1996, Tommasini et al., 1996). In order to investigate whether AfMRPδ is also a glutathione-conjugate pump, the full-length cDNA of AtMRPδ was cloned into a yeast expression vector and the resulting recombinant plasmid pN-AtMRPδ was transformed into the cadmium hypersensitive yeast strain DTY168, in which the YCF1 coding sequence had been deleted (Szczypka et al., 1994). AflvlRPδ could partially complement glutathione conjugate transport activity in Aycfl (not shown). The transport activities were typical for ABC transporters: i) inhibition by vanadate, ii) insensitivity to bafilomycin A1 , a specific inhibitor of V-type H⁺- ATPases, and NH4CI, which disrupts the pH gradient generated by proton pumps. These findings indicate that the transport mechanism is independent of the electrochemical potential generated by proton pumps. It should be mentioned that independent transformants exhibited different transport activities and in some preparations no transport activity could be observed. Yeast ycfl mutants transformed with AtMRPδ did not restore cadmium tolerance, confirming that in spite of the rather broad substrate specificity of investigated MRP's the affinity towards a given substrate may vary among these transporters (Kόnig et al., 1999).

ATP-dependent uptake of estradiol-17-(β-D-glucuronide) (E₂17G) and a rye flavonoid glucuronide was reported for vacuoies from rye and barley (Klein et al., 1998, 2000). Yeasts exhibit a low transport activity for glucuronides, but up to now yeast glucuronide transporters have not been identified. The construct pN-AtMRPδ was introduced into the YYA4 yeast strain exhibiting a reduced glucuronide transport activity. The yeast mutant transformed with AtMRPδ was able to transport E₂17G when compared to the empty vector control. The transport revealed typical characteristics of an ABC-type transporter protein as described above for glutathione conjugates (Table δ) Reduced glutathione, oxidized glutathione as well as dinitrobenzene glutathione had no effect on E₂17G uptake. However, the AtMRPδ dependent transport activity of E₂17G was severely influenced by other organic anions such as estradiol-3-sulfate, the natural flavone- glucuronide luteolin-7-O-diglucuronide-4'-O-glucuronide, glycocholate and by the sulfonylurea glibenclamide. For unknown reasons and as observed for GS-X transport activity, transport rates of the complemented YYA4 strain strongly differed from one preparation to another.

Table δ Characteristics of the ATP-dependent β-estradiol 17-(β-D-glucuronide) uptake into vesicles isolated from YYA4 yeasts transformed with pN-AflvlRPδ. Yeast vesicles were incubated with 10 μM [³H]-E₂17G in the presence of δ mM MgATP and the inhibitors and potential competitors indicated. After 8 min, uptake was terminated by transfer of three aliquots on Durapore filters. Values are corrected for corresponding controls with vesicles isolated from YYA4 yeasts transformed with the empty NEV vector. Due to the variability of the uptake activities in different preparations, uptake rates were standardized to 100% which correspond to 7 to 2δ pmol E₂17G x (mg protein x min)^"1. The different inhibitors and competitors where always tested using the same vesicle preparation.

Condition % of control

Control 100.0

1 mM vanadate 14.6 ± 6.4

0.1 μM bafilomycin A1 8δ.δ ± 1.6 δ mM NH₄CI 90.0 ± 6.0

160 μM glibenclamide 10.6 + 6.1

0.2 mM luteolin 7-O-diglucuronide (4'-0-glucuronide) 33.4 ± 7.5

0.2 mM estradiol-sulfate 0.2 ± 3.5

0.2 mM glycocholate 13.9 ± 6.4

3 mM GSH 80.7 ± 4.6

3 mM GSSG 71.6 ± 4.8

0.2 mM dinitrobenzene-GS 74.4 ± 6.9

Example 3: Expression studies showing that AtMRPδ is mainly expressed in vascular tissues and epidermis

In order to understand the physiological function of the AtMRPδ, its expression pattern was analyzed by RNA gel blot analysis and promoter studies were conducted using transgenic plants expressing AtMRPδ promoter-GUS fusion constructs. In RNA blot as well as in RT-PCR experiments, AtMRPδ mRNA accumulation was detected in seedlings, flowers, roots, siliques, and leaves (data not shown). For the promoter-Gl/S fusion experiments two different promoter fragments of AtMRPδ of 1.8 kb and 3 kb length have been isolated. Both promoter fragments comprised the complete 5^' untranslated leader of the corresponding cDNA including the nucleotides encoding the first six amino acids of the AtMRPδ protein. The two AtMRPδ promoter fragments were joined to the β- glucuronidase coding region. More than six lines were analyzed for each promoter- reporter gene construct. No significant difference of the expression pattern was detected between the lines carrying the two promoters, indicating that all c/s-elements relevant for AtMRPδ expression are present on the shorter 1.8-kb promoter fragment. GUS gene expression driven by the AtMRPδ promoter was tested in seedlings and mature plants by staining with X-Gluc (Figure 3). In seedlings grown on sterile culture medium, strong GUS staining was observed in cotyledons and roots (Figure 3A, G). In roots of seedlings and mature plants, GUS activity was restricted to the central cylinder and was absent from the root cortex and the root tip (Figure 3G). In mature leaves the AtMRPδ gene appeared to be most strongly expressed in the vascular tissue of leaves. Almost all vascular strands of lower and higher order veins were stained in strongly expressing lines (Figure 3B, C). GUS staining in weakly expressing lines was most prominent in vascular anastomoses (data not shown). GUS staining in vascular tissue was not restricted to individual cells, but was seen in almost every cell (with the exception of xylem cells) of the vascular strand (Figure 3C). Considerable GUS activity was also detected in leaf epidermal cells including mature guard cells of leaves and flower petals (Figure 3C-E). In some cases weak staining was seen in parenchyma cells. In anthers, high GUS activity was concentrated along the central vascular strand of the filament and in the tissue connecting the pollen sacs (Figure 3F). Interestingly, intense GUS staining was also visible at the silique attachment site of the pedicel, as seen in Figure 3H, indicating that the AtMRPδ gene may have a defined function during silique ripening or abscision.

Example 4: Isolation and characterization of a T-DNA knockout mutant for the gene encoding AtMRPδ

In plants MRPs are considered to play a role in detoxification. However, the large number of these transporters and specific expression patterns indicate that they have specific functions. The analysis of deficiency mutants is a valuable tool to help to discover the role of a particular gene's role in physiological and developmental functions in plants. In this context, a screen has been carried out in a collection of 4120 T-DNA transformed lines from seed transformation (Forsthoefel et al., 1992) using a reverse-genetic PCR-based screening strategy. Thereby, a T-DNA knockout mutant for AtMRPδ called mrp5-1 could be identified. Sequence analysis of a PCR fragment amplified with the primer combination MRP3δA-anti (sequence-specific)/RB2(T-DNA primer) (see Table 2) on genomic DNA of mφδ-1 revealed a T-DNA inserted into AtMRPδ at position +1420. For further characterizaton of the T-DNA insertion, two additional primers, flanking the site of the T-DNA insertion, were designed. One of these primers, MRPδD-sense, was used in combination with the T-DNA primers LB2 and RB2 to amplify the junction sequence of the T-DNA and the 3^' region of AtMRPδ in mφδ-1. Sequencing of the resulting PCR fragment amplified with LB2 and MRPδD-sense showed the insertion of T-DNA with left border sequences facing the MRPδ locus at position +1471. This analysis revealed that T-DNA integration into the first exon of MRPδ occurred with intact left border and right border sequences and resulted in a deletion of 62 bp at the integration site (Figure 1 B). Two findings suggested that mrpδ-1 isolated from the screen was homozygous for the T- DNA insertion: (i) All PCRs carried out on genomic DNA isolated from more than 20 individuals of the offspring of the isolated plant resulted in PCR products of correct size with the combination of primers specific for AtMRPδ and the T-DNA. In contrast, all PCRs performed with the MRPδD-sense/MRPδD-anti primer combination gave no fragment amplification. This indicated that all plants carried the T-DNA and none possessed the wild-type allele. (ii) More than 100 seeds of the primary mrpδ-1 plant isolated were tested for segregation of the kanamycine resistance marker of the 3860:1003 T-DNA construct (Velten and Schell, Nucl. Acids Res. 13 (1986), 6981-6998). All exhibited a resistant phenotype in the presence of this antibiotic which is expected for a homozygous T-DNA transformant (Table 6).

Table 6 Segregation of the kanamycine resistance marker of mrp5-1 T-DNA mutant crosses into the Wassilewskia wild-type (Ws-2). In the F3, seeds of single F2 parents that were found to represent mφδ-1/mφδ-1, mφδ-1ΛNs- 2 and Ws-2/Ws-2 genotypes due to southern analysis (see Figure 3) were analyzed.

Selection on kanamycine

Generation n plants n Kan^R (%) n Kan^s (%)

P (mφδ-1 parent) 109 109 (100) 0 (0)

F2 mφδ-1 xWs-2 2δδ 199 (78.0) 56 (22.0)

F3 mφδ-1 x Ws-2 of a mφδ-11 mφδ-1 F2 278 278 (100) 0 (0) parent

F3 mφδ- 1 x Ws-2 of a mφδ- 1N\ls-2 F2 parent 311 226 (72.4) 86 (27.6)

F3 mφδ-1 x Ws-2 of a Ws-2/Ws-2 F2 parent 243 0 (0) 243 (100)

Feldmann (1991) concluded that the average number of independent T-DNA inserts in this collection is about 1.6 per diploid genome. In order to analyze the genotype of mφδ-1 and to select homozygous mutant plants carrying a T-DNA insertion at a single insertion locus, the mrpδ-1 plant was crossed with the Wassilewskia (Ws-2) wild-type. After self- pollination of the F1 generation, genomic DNA of 90 resulting F2 plants was subjected to restriction digestion and DNA gel blot analysis using probes for the AtMRPδ gene and the T-DNA borders. In Figure 4 corresponding results are depicted for genomic DNA isolated from six single plants with different genotypes. A shift in size of hybridizing bands after hybridization with a gene-specific probe allowed the identification of plants that were homozygous for the T-DNA insertion. The expected size for hybridizing bands from insertion alleles could be calculated on the basis of the physical map of the AflvlRPδ locus in conjunction with the physical map of the integrated T-DNA (Figure 4A). Furthermore, hemizygous plants could be identified by the presence of hybridizing bands that showed the same shift in size as the bands in plants that were homozygous for the T-DNA insertion. In addition to these signals, the hybridization pattern of wild-type plants is also present in hemizygous plants (Figure 4B). Hybridization signals with a probe specific for T-DNA left border sequences were detected only in hemizygous plants and plants homozygous for the.T-DNA insertion (Figure 4C). Single bands found exclusively in hemizygous and homozygous mutants were also detected when a right border T-DNA fragment was used as a probe (data not shown).

On selective medium, the F2 generation of the mφδ-1N\ls-2 backcross segregated in a ratio of approximately 3:1 for the kanamycin marker (Table 6). The F3 generation of homozygous mutants selected through the results of the DNA blot analysis was 100% resistant, while hemizygous mutants again segregated in a 3:1 ratio. Thus, DNA blot analysis and the segregation of the kanamycine marker in the mφδ-1l\Ns-2 backcross proved that mrpδ-1 plants carry a single T-DNA insertion in the AMRPδ gene.

Table 7 Characteristic features of germination and development of wild-type (Ws-2) and mrp5-1 mutant seedlings. The growth of 20 plants was analyzed using a time-lapse video system. 1 min on the video corresponded to 80 min real time. Sterile seeds were grown on 1/2 x MS with 1 % sucrose and 0.8 % agar after 48 h vernalization at 4° C in the laboratory with constant light coming from the side (neon light). In (A) typical features of a vertical growth test are reported. Plant growth was recorded over a total time of 300 h. In (B) the average dimensions of mutant and wild-type grains are given. 20 grains were measured using a scanning electron microscope.

A Feature Ws-2 mφ5-1

Appearance of primary root / first seedling 29 h 30 h

Germination 10 seedlings 35 h 33 h all seedlings 41 h 45 h

Primary root length 2 mm first seedling 45 h 39 h

10 seedlings 49 h 52 h all seedling 56 h 59 h

Root growth velocity after arriving at 2 5 h 40 min per mm 12 h 16 min mm length per mm

First lateral roots appearing 191 h 136 h

First secondary roots appearing 192 h 141 h n plants with lateral roots after 220 h no lateral roots 12 2

Growth 1 lateral root 5 9 >2 lateral roots 3 9 n plants with secondary roots after 220 4 18 h growth

Appearance of cotyledons 10 seedlings 60 h 62 h Primary leaves visible 10 seedlings 187 h 180 h

B

Dimensions of grains in μm 452±39 x 267±19 473±35 In order to investigate AtMRPδ transcript levels RT-PCR was performed on total RNA isolated from homozygous mrpδ-1 knockout plants using the primer pair MRPδD-sense and MRPδD-anti (Table 2, supra) which would amplify the gene region where the T-DNA insertion is located. While RT-PCR with RNA isolated from wild-type Ws-2 plants clearly confirmed the presence of AtMRPδ transcripts, no mRNA was detectable in knockout plants (Figure 4D).

Example δ: Observations on root morphology showing that mrpδ-1 mutants exhibit a strongly reduced root growth

Since heterologously expressed AtMRPδ mediated organic anion transport in yeast (Table δ), special interest lied in the question whether the transtonoplast transport of a glutathione conjugate (GS-X) was reduced in adult mrpδ-1 plants. Monochlorobimane is readily converted in the cytosol to form the fluorescent bimane-GS conjugate followed by vacuolar transfer of the dye via MRP-like ABC transporters (Coleman et al., 1997). The incubation of leaf mesophyll protoplasts from Ws-2 and mrpδ-1 plants showed no significant difference in the vacuolar fluorescence (data not shown). However, since the accumulation was variable between one cell and another in the wild-type as well as in the mutants, it cannot be excluded that uptake of GS-X was reduced in some cell types of the mutant normally exhibiting stronger expression of AtMRPδ.

Seeds of homozygous mrpδ-1 were surface-sterilised and germinated on vertical plates with sterile O.δ x MS medium supplemented with 1 % sugar in a 16/8 h light/dark cycle or under continuous light. Up to four days after germination (dag), all seedlings showed typical wild-type morphology when compared to Ws-2 plants. Starting with day δ, the root elongation of mrpδ-1 seedlings was strongly reduced and mutant plants initiated the lateral and secondary roots earlier than wild-type seedlings (Figure δA-C). In all our experiments, hypocotyl length and leaf morphology were not visibly affected in mrpδ-1 seedlings (data not shown).

The morphology of the entire root system of mrp5-1 seedlings grown vertically under continuous light for 24 d appeared to be more branched than Ws-2 seedlings, (Figure 5E). In contrast, the development of root hairs in mrp5-1 was normal (Figure 5B). The aerial parts of adult mφδ-1 plants grown on soil either in a 10/14 h or in a 16/8 h light/dark cycle exhibited wild-type morphology at all stages of development (data not shown).

Under high nutrient culture conditions (1xMS medium) the effect on root growth appeared inversed: mrpδ-1 plants produced short but visible roots, while root growth of Ws-2 was extremely reduced (Figure 6F).

In order to investigate the phenotype of mrpδ-1 plants in more detail, the development of Ws-2 and mrpδ-1 was continuously recorded using a time-lapse video system (Table 7, see supra). In a first experiment, simple vertical growth of mrpδ-7 and Ws-2 seedlings was recorded for 300 hrs. During germination, onset of primary roots as well as cotyledons and primary leaves differed insignificantly. Later the root growth rate of mrpδ-1 was only half of the rate observed for Ws-2 seedlings. In comparison to Ws-2, lateral and secondary roots initiated about 60 hrs earlier in mrpδ-1 seedlings. 220 hrs after germination, only 40% of Ws-2 seedlings developed a lateral root, while 90% of mrpδ-1 seedlings showed formation of one or more lateral roots. In most seed batches root growth was inhibited about 60%, however, it must be mentioned that in a minority of seed batches root growth was reduced only 20 to 30%.

In a second experiment, the gravitropic reaction of mrp5-1 roots was analysed. Seedlings grown for six days were turned at a 90° angle and the direction of root growth was recorded. No difference in the gravitropic response was observed between wild-type and the mutant.

Example 6: Determination of auxin levels in roots of mrpδ-1 plants

The phenotype observed for mrp5-1 plants grown in standard O.δ x MS/1% sucrose medium suggested that under these conditions increased auxin levels could inhibit primary root growth but induce lateral root development. Indeed, auxin levels were increased by a factor of about two in roots of mutant plants (Table 8). Since auxin levels of plants grown on different plates differed, probably due to slight differences in light intensity, the ratio of auxin in mutant and wild type plants was always calculated for plants grown on the same plate. Table 8 The level of free auxin is increased in roots of the mφδ-1 T-DNA mutant. For the analysis of free auxin 40 to 80 whole roots of mφδ-1 or Ws-2 plants grown vertically for 10 d were cut, removed and after determination of the fresh weight extracted with MeOH in the presence of [²H₂]-IAA as a standard. Auxin was measured by GC-MS. Each experiment consisted of three or four independent determinations analysing various seed batches and different positions of the seedlings on the plate. Due to variations between different plates the ratios were determined from mφδ-1 and Ws-2 samples grown on the same plate. The absolute IAA content of all independent determinations in Ws-2 roots ranged between 107 and 279 fmol x mg FW^"1.

Ratio of IAA per mg FW in mφδ-1 vs. Ws-2 roots (n)

Experiment 1 2.11 ± 0.63 (3)

Experiment 2 1.67 ± 0.31 (3)

Experiment 3 2.02 ± 0.12 (4)

Example 7: AtMRPδ controls giibenclamide-dependent stomata opening

The high similarity of AtMRPδ to CFTR and SUR (see Example 1 , supra), together with the facts that AtMRPδ was strongly expressed in guard cells and that its transport activity was inhibited by glibenclamide prompted to investigate whether AtMRPδ might control ion fluxes in guard cells. Stomatal movement is mediated by anion and K⁺ fluxes from guard cells and it has previously been shown that glibenclamide, a well-known modulator of K-ATP channels and CFTR chloride channels (Schmid-Antomarchi et al., 1987; Sheppard and Welsh, 1992) is involved in the regulation of ionic channels in guard cells (Leonhardt et al., 1997; 1999). As already demonstrated in other species, glibenclamide triggered stomatal opening in darkness in Arabidopsis wild-type plants in a dose- dependent manner. In contrast, stomatal opening induced by glibenclamide was completely abolished in mφδ-1 (Figure 6). The insensitivity of stomata from mutant plants to glibenclamide suggests that AtMRPδ controls either K⁺ or anion channels. However, it cannot be excluded that AtMRPδ itself acts as a channel or is a member of the signal transduction pathway leading to stomata opening.

Example 8: Growth experiments showing a higher tolerance of Atmrpδ^" mutants to drought stress

Arabidopsis knock-out Atmrpδ mutants (mrpδ-1) and wild-type plants with the same genetic background (Ws-2) were grown either on sand or on soil (Mauser, Zurich, Switzerland). Plants grown on sand were watered with half-strength Hoagland's solution in a growth chamber (8 h light period, 22°C; 16 h dark period, 21°C; relative humidity 70%). Light (260-300 μmol m^"2s^"1 PAR) was supplied by halogen lamps (HQI-TS, 160W/NDL, Osram, Mϋnchen, Germany). When 4- to δ- week-old plants were deprived of water in the phytotron, a water stress developed after 48 hours. Whereas Ws-2 plants exhibited a strong wilty phenotype, Atmrpδ mutant plants were little or non-affected (Figure 7). Growth conditions for plants on soil were 16/8 h light dark cycle at 21 °C and a relative humidity of 60 to 70%. Growth of wild-type plants and mrpδ-1 mutants under control conditions was similar (Figure 8). After 4 to δ weeks irrigation was stopped. After 6 days wild-type plants showed strong symptoms of drought stress (Figure 9). In contrast, mrpδ-1 mutant plants behaved still well. After 11 days, most wild type plants were dead while mrpδ-1 mutants were still green and survived the imposed drought stress (Figure 10).

These results show that independent of the growth conditions, the knockout of the AtMRPδ gene confers a higher resistance to drought stress to the plant.

Example 9: Determination of transpiration rates being decreased in the knock-out mutant Atmrpδ^"

Arabidopsis thaliana plants were grown in sand watered with half-strength Hoagland's solution (Epstein, 1972) in a growth chamber (8 h light period, 22°C; 16 h dark period, 21 °C; relative humidity 70%). Light (260-300 μmol m^"2s^"1 PAR) was supplied by halogen lamps (HQI-TS, 160W/NDL, Osram, Mϋnchen, Germany). Twelve independent experiments were conducted, six for wild-type and six for mrpδ-1 mutant plants. These experiments were strictly carried out under standard conditions as described by Weyers and Meidner (1990, In: Methods in Stomatal Research, eds. Longman Scientific & Technical, London). Briefly, one rosette from a 3- to δ-weeks old plant was harvested in the phytotron at the end of the night period when the leaves were in a state of full turgor. The cut petiole was rapidly and lightly smeared with Vaseline. The rosette was immediately fixed head downwards by the petiole on a hook of a wire support. The whole setup was then placed, on a precision balance. This setup allows avoiding any contact between the leaves of the rosette and the balance since such a contact would have limited the plant water loss during the experiment. The balance setup with fully turgid rosette was kept in the dark under a hood during the whole experiment. In these experiments, the water loss is expressed as: g(t) - g(t0)

DW where: g(t) is the mass of the rosette measured at time t, g(to) is the mass of the rosette measured at the beginning of the experiment

DW is the dry weight of the rosette

Figure 11 illustrates a typical experiment showing the kinetics of water loss from excised mrp5-1 or WS-2 rosettes sowed the same day. After a 1-hour period of equilibration, the kinetics of water lost by wild-type plants was strongly higher than that of mrpδ-1 plants. After 6-hours, Ws-2 plants lost almost twice the amount of water compared to mrpδ-1 plants.

When all experiments are considered, the mean volume of water loss per hour and per g of dry weight was 426 ± 160 μl.h^"1.g^"1. It was found that the water lost by Ws-2 was by a factor of 1.66 ± 0.62 higher than that of mrpδ-1 plants.

One possibility to explain why mrpδ-1 mutant plants lose less water than wild-type plants would be that the mutants exhibit a lower stomatal density. In order to check this hypothesis, the stomatal density of Ws-2 and mrpδ-1 mutant plants was compared. Leaves from 4- to δ-week old plants were harvested in darkness at the end of the night period. Paradermal sections of abaxial epidermis obtained in dim green light were immediately placed cuticle up in Petri dishes containing 10 mM KCI, 16 mM K₂IDA, 10 mM Mes (pH 6.1δ with KOH) in a thermoregulated water-bath (Lasceve et al., Plant Cell & Environment 20 (1997), 360-368). Stomatal frequencies were measured according to the method of Lasceve et al. (1997, loc cit.) with an optical microscope fitted with a camera lucida and a digitizing table linked to a personal computer. Round-shaped immature stomata being frequently observed, only fully developed stomata whose pore width was more than one third of the stomata length and without underlying mesophyll were measured. All experiments were repeated at least δ times. Error bars represent standard error to the mean (SEM) with a confidence interval of 9δ %. Plot and curve fitting (non-linear regression by the Levenberg-Marquardt method) were done using Origin δ.O (MicroCal Software Inc., Northampton, MA, USA). Six different plants for each genotype were used and stomata were counted from epidermal strips of 3 different leaves per plant. Based on 2600 stomata observed for each genotype, a stomatal frequency of 160 and 177 stomata per mm² was obtained for mrpδ-1 and Ws-2 plants, respectively (Figure 12). These values were not significantly different demonstrating that the low sensitivity of mrpδ-1 mutant plants to water stress is not supported by a lower stomatal density.

Example 10: Measurements of water uptake which is reduced in Atmrpδ mutants compared to wild type plants

A homemade potometer was designed according to the diagram presented in Figure 13. A 4- to δ- week old plant was disposed in the setup, the root and shoot compartments being isolated by the application of an inert synthetic mastic at the crown level. The nutrient solution reservoir and all circulation tubes were free of air spaces. All experiments were conducted in the phytotron (parameters as described in Example 8) in order to maintain the plants in standard conditions. Rates of water flow are measured in a capillary by the movement of an air bubble. The measurements reflect values of, and change in, total conductance related to ambient conditions (light supply, wind speed, application of hormones, etc.) The data are represented by the curve describing the evolution of the volume of water entry (ml) per gram of dry weight versus time. In order to compare different conditions/experiments, a quantitative determination of the slope of this curve was calculated. In the phytotron under light, the mean volume of water entry into the root compartment was 1912 and 2660 μl.h^"1.g^"1 of dry weight for mrpδ-1 and Ws-2 plants, respectively. In ten independent experiments (δ for each genotype), it was observed that Ws-2 plants absorbed 1.48 ± 0.3δ fold more water than mrpδ-1 mutant plants (Figure 14). To mimic a water stress or a change in the soil water potential, the same kind of experiments was conducted after addition, in the root compartment, of either 10 μM abscisic acid (ABA) or 10% polyethylene glycol (PEG 3360). The addition of 10% PEG led to a strong inhibition of water entry in the Ws-2 plants (1476 → 276 μl.h^"1.g^"1 DW) (Figure 14), this inhibition being weaker with mrpδ-1 mutant plants (947 → 498 μl.h^"1.g^"1 DW). Figure 1 δ illustrates the effect of a water stress. After a 24 hour application of 10 μM ABA, the kinetics of water entry was reduced by more than two fold in Ws-2 plants (2977 → 1370 μl.h^"1.g^"1 DW) whereas it was only slightly reduced in mrpδ-1 mutant plants (1488 -> 1160μl.h^"1.g^"1 DW). When an osmotic stress was applied the effect was even more drastic.

Taken together, these experiments demonstrate that the water need of mrpδ-1 mutant plants is lower than that of Ws-2 plants, in standard but also in different stress conditions.

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Claims

1. A method for producing transgenic or mutant plants having an increased tolerance to drought stress comprising the step of providing transgenic or mutant plants having a reduced activity of an ABC transporter which is expressed in guard cells.

2. The method of claim 1 , wherein the activity of said ABC transporter is reduced to non-detectability.

3. The method of claim 1 or 2, wherein said ABC transporter is a multidrug resistance associated protein (MRP).

4. The method of any one of claims 1 to 3, wherein said ABC transporter is MRP5.

δ. The method of any one of claims 1 to 4, wherein said providing of transgenic plants having a reduced activity of said ABC transporter comprises the steps of

(a) introducing into a plant cell a nucleic acid molecule the presence of which in the genome of said plant leads to a reduced activity of said ABC transporter in plant cells;

(b) regenerating from transformed cells produced in step (a) plants; and optionally

(c) producing progeny from the plants produced in step (b).

6. The method of any one of claims 1 to δ, wherein said reduced activity is achieved by an antisense, co-suppression, ribozyme or RNA interference effect or by the expression of antibodies or other suitable (poly)peptides capable of specifically reducing said activity or by the expression of a dominant-negative mutant.

7. The method of any one of claims 1 to δ, wherein said reduced activity is achieved by in-vivo mutagenesis or by the insertion of a heterologous DNA sequence in the gene encoding MRPδ.

8. The method of claim 7, wherein the heterologous DNA sequence is T-DNA or a transposon.

9. The method of claim 5 or 6, wherein the nucleic acid molecule is operatively linked to a regulatory sequence active in plants.

10. The method of claim 9, wherein the regulatory sequence is a constitutive promoter.

11. The method of any one of claims 1 to 4, wherein said providing comprises the step of determining those plants out of a pool of plant mutants the stomata of which do not open upon the administration of a sulfonylurea compound which is an inhibitor of stomatal closure.

12. The method of claim 11, wherein said sulfonylurea compound is glibenclamide or tolbutamide.

13. The method of claim 10 or 11, wherein said pool is pre-selected for tolerance to drought stress.

14. A transgenic or mutant plant obtainable by the method of any one of claims 1 to 13.

15. The transgenic plant of claim 14, containing a nucleic acid molecule as defined in any one of claims 5 to 10 stably integrated into the genome.

16. Transgenic plant cells in which the activity of an ABC transporter which is expressed in guard cells is reduced.

17. Propagation material or harvestable parts of a transgenic plant of claim 14 or 16 containing transgenic plant cells of claim 16.

18. Use of a nucleic acid molecule as defined in any one of claims δ to 10 for the production of plants having an increased tolerance to drought stress.

19. A method for producing transgenic or mutant plants having an increased transpiration comprising the step of providing transgenic or mutant plants having an increased activity of an ABC transporter which is expressed in guard cells.

20. The method of claim 19, wherein said ABC transporter is a multidrug resistance associated protein (MRP).

21. The method of claim 19 or 20, wherein said ABC transporter is MRPδ.

22. The method of any one of claims 19 to 21, wherein said providing of transgenic plants having an increased activity of said ABC transporter comprises the steps of

(a) introducing into a plant cell a nucleic acid molecule the presence of which in the genome of said plant leads to an increase of the activity of said ABC transporter in plant cells;

(b) regenerating from transformed cells produced in step (a) plants; and optionally

(c) producing progeny from the plants produced in step (b).

23. The method of any one of claims 19 to 22, wherein said increased activity is achieved by overexpressing said ABC transporter in guard cells.

24. The method of claim 22 or 23, wherein the nucleic acid molecule is operatively linked to a regulatory sequence active in plants.

26. The method of claim 24, wherein the regulatory sequence is a constitutive promoter.

26. The method of claim 19 or 20, wherein said providing comprises the step of determining those plants out of a pool of plant mutants the stomata of which do not close upon the administration of a K⁺ channel opener which is an inducer of stomatal closure in the light.

27. The method of claim 26, wherein said K⁺ channel opener is RP 49366 or Cromakalim.

28. A transgenic or mutant plant obtainable by the method of any one of claims 19 to 27.

29. The transgenic plant of claim 28, containing a nucleic acid molecule as defined in any one of claims 22 to 26 stably integrated into the genome.

30. Transgenic plant cells in which the activity of an ABC transporter which is expressed in guard cells is increased.

31. Propagation material or harvestable parts of a transgenic plant of claim 28 or 29 containing transgenic plant cells of claim 30.

32. Use of a nucleic acid molecule as defined in any one of claims 22 to 26 for the production of plants having an increased transcription.