WO2003023038A1 - Isolation and use of the desiccation-specific promoter from ds2 gene - Google Patents

Abstract

The present invention relates to an isolated single stranded or double stranded nucleic acid molecule comprising at least one regulatory gene region of plant origin that is drought-inducible and ABA-, salt- and cold-insensitive. In particular, the invention provides an isolated nucleic acid molecule comprising, in part or in its entirety, the 5' regulatory sequence region of the DS2 gene of a Solanaceae species, or orthologues, homologues or functional variants thereof. The present invention further provides transformation vectors, transgenic plants or any reproducible part thereof, and plant propagation materials, as well the use of the nucleic acid molecules as probe in screening methods, and a method for gene expression in plants, comprising expression of a gene in a drought-inducible and ABA-, salt- and cold-insensitive manner.

Description

Isolation and use of the desiccation-specific promoter from DS2 gene

The present invention relates to an isolated single stranded or double stranded nucleic acid molecule comprising at least one regulatory gene region of plant origin that is drought-inducible and ABA-, salt- and cold-insensitive. In particular, the invention provides an isolated nucleic acid molecule comprising, in part or in its entirety, the 5' regulatory sequence region of the DS2 gene of a Solanaceae species, or orthologues, homologues or functional variants thereof. The present invention further provides transformation vectors, transgenic plants or any reproducible part thereof, and plant propagation materials, as well as a method for gene expression in plants, comprising expression of a gene in a drought-inducible and ABA-, salt- and cold-insensitive manner.

With respect to the present specification and claims, the foregoing technical terms will be used in accordance with the below given definitions. With regard to the interpretation of the present invention, it shall be understood that the below defined terms are used in accordance with the given definitions even if said definitions might not be in perfect harmony with the usual interpretation of said technical term.

A "homologue" or a "variant" of a nucleic acid sequence is defined as a sequence that is at least 50%, preferably at least 60%, more preferably at least 70% or 75% even more preferably at least 80% or 85, highly preferably at least 90%, 95%, 96%, 97%, 98% or 99% identical to the sequence in question.

A "functional variant" of a sequence is a variant of that sequence having the same type of biological activity even if the measure of the biological activity of the functional variant is significantly different from that of the original sequence (e.g., the transcriptional activity of the functional variant of a promoter can either be smaller or larger than that of the original promoter). The terms "orthologues" or "orthologous genes" or "a gene orthologous to" refer to genes related by common phylogenetic descent. Orthologous genes are those genes from one species which corresponds to a gene in another species that is related via a common ancestral species (a homologous gene), but which has evolved to become different from the gene of the other species.

The coding sequence of DS2 gene in any species will have about 70% identity with those sequences illustrated in Figure 2 (SEQ ID NO: 2) or fragments thereof . That is, 70% of the residues are the same. Advantageously, a DS2 gene will have greater than 80% homology, preferably greater than 85% or 90% homology, more preferably greater than 95%, 96%, 97%, 98% or 99% homology, and most preferably the coding sequence of a DS2 gene will be essentially identical to those sequences illustrated in Figure 2 (SEQ ID NO: 2) or fragments thereof. "Isolated" means altered "by the hand of man" from natural state. If an "isolated" composition or substance occurs in nature, it has been changed or removed from its original environment, or both. For example, a polynucleotide or a polypeptide naturally present in a living animal is not "isolated", but the same polynucleotide or polypeptide separated from the coexisting materials of its natural state is "isolated", as the term employed herein. A nucleic acid molecule is regarded "hybridizable" with another nucleic acid molecule if it can specifically be bound to the other molecule (i.e., the binding can give rise to a signal that is distinguishable from the background noise and from the signal caused by the non-specific binding of any random sequenced nucleic acid molecule), preferably a nucleic acid molecule is regarded as hybridizable if it specifically binds to another nucleic acid molecule under stringent conditions.

A regulatory sequence is "operably linked" to a structural gene within a DNA construct if the regulatory sequence is able to influence the expression rate or manner of said structural gene under conditions suitable for the expression of said structural gene and for the functioning of said regulatory sequence. Plants experience and have to cope with a wide range of adverse biotic and abiotic environmental conditions. Drought, heat, cold and flooding are common abiotic stresses and plants have evolved an array of physiological and metabolic responses to recognize and respond to them. This process results in the expression of numerous stress-specific genes. These genes are regulated by several different signal transduction mechanisms. Cross-talk between different pathways is common [for review, see Ingram and Bartels, Annual Review of Plant Physiology and Plant Molecular Biology 47, 377-403 (1996); Tabaeizadeh, International Review of Cytology 182, 193-247 (1998); Knight and Knight, TRENDS in Plant Science 6, 262-267 (2001)].

Signal transduction of environmental stresses has been a subject of intense research, and several mechanisms are already well characterized. The plant hormone abscisic acid (ABA) has long been known to be involved in stress signalling. ABA is considered to be the main mediator of dehydration [reviewed by Giraudat et al., Plant Molecular Biology 26, 1557-1577 (1994); Bonetta and McCourt, Trends Plant Sciences 3, 231-235 (1998); Busk and Pages, Plant Molecular Biology 37, 425-435 (1998)]. ABA-mediated signalling pathways cross-talk with other pathways such as cold [Shinozaki and Yamaguchi-Shinozaki, Plant Physiology 115, 327-334 (1997)] or ethylene [Beaudoin et al., Plant Cell 12, 1103-1115 (2000); Ghassemian et al., Plant Cell 12, 1117-1126 (2000)] responsive mechanisms. On the other hand, non-ABA-mediated regulation of drought responsive genes is also known. However, this kind of regulation is significantly less clearly understood [Shinozaki and Yamaguchi-Shinozaki, Current Opinion in Plant Biology 3, 217-223 (2000)]. The role of ABA in dehydration responsive gene expression is well described as well as in interaction between drought and cold signalling. There are at least four pathways of drought response: two of them are ABA-mediated and two of them are not. C/s-elements such as MYCR/MYBR and ABREs and the corresponding transcription factors MYC/MYB and bZip, respectively, of ABA-mediated pathways have already been characterized. In addition, further upstream signalling genes, abi1-2, eral have also been analyzed. Another pathway regulates genes such as rd29A, Iti78 and cor15a in an ABA-independent manner, and these genes have DRE/CRT elements in their promoter regions. This pathway converges with a cold-inducible pathway and their transcription factors DREB1-2 and CBF and many signalling genes are also characterised (see Shinozaki & Yamaguchi-Shinozaki 2000, supra, for review). There is another pathway, however, that is independent not only of ABA but also of cold signalling. Expression of the Arabidopsis genes erdl [Kiyosue et al., Biochemical and Biophysical Research Communication 196, 1214-1220 (1993)], rd19 and rd21 [Koizumi et al., Gene 30, 175-182 (1993)] are regulated by this pathway. Erd1 encodes a Clp protease regulatory subunit while rd19 and rd21 encode different thiol proteases. A 0.9-kb promoter region of the erdl gene was tested in a GUS reporter assay. This sequence was sufficient for driving GUS expression in a dehydration and salt stress-specific manner. Furthermore, expression of erdl was developmentally up-regulated by senescence [Nakashima et al., Plant Journal 12, 851-856 (1997)]. However, neither c/s-elements nor transcription factors and further upstream signalling elements of this particular pathway are known (Shinozaki & Yamaguchi-Shinozaki 2000, supra).

The ABA-independent pathways, as described above, all cross-talk with other stress signalling pathways (genes such as rd29A, Iti78 and cor15a are cold-inducible, rd19 and rd21 are salt stress-specific). Despite the long felt need, no truly selective drought-inducible promoter has been identified. Earlier work in our laboratory examined the drought specificity of the DS2 gene [Silhavy et al., Plant Molecular Biology 27, 587-595 (1995)]. In that publication, experiments were carried out on a wild Solanaceae species, Solanum chacoense, and the coding sequence of the gene is disclosed. Some limited investigation on the specificity of expression was also carried out. Another publication [Jenes et al., In: Use of Agriculturally Important Genes in Biotechnology (ed. G. Hrazdina), IOS Press, pp. 7-13 (2000)] describes planned and preliminary work using a DS2 promoter from an unidentified potato cultivar, and the expression of a reporter gene from a recombinant vector comprising the promoter in rice protoplasts. Though the applicability of the promoter in a monocot plant is significant, the presented results were preliminary, and the use of the DS2 regulatory sequences in a transgenic plant was not disclosed. Since the potato cultivar is unidentified in the work of Jenes et al. (2000, supra), the skilled person would have to consider the risk of testing possibly a huge number of potato cultivars and varieties [over 228 wild potato species are known to date and the European Cultivated Potato Database lists over 4000 cultivated potato varieties. See e.g.: J.G. Hawkes: Origins of cultivated potatoes and species relationships, In: Potato Genetics, eds. Bradshaw and Mackay, CAB International, (1994)], with an uncertain outcome. The heterogeneity of potato is very well known and it is quite common that a gene is differently regulated in different Solanum species or even differently regulated copies of a gene exists within the potato genome [see e.g.: Mignery et al., Gene 62, 27-44 (1988), Fu and Park, Plant Cell 7, 1369-1385 (1995, Banfalvi et al., Plant Science, 147, 81-88 (1999), Molnar et al., Plant Molecular Biology, 46, 301-311, (2001)]. Further, as it is clearly outlined in the examples below, the skilled person would have faced technical difficulties (e.g. constitutive expression of a reporter gene fused to the DS2 promoter in culture) when trying to obtain and apply a promoter on the basis of teaching by Jenes et al (2000, supra). Based on the above, it has been unexpected and surprising to find that the regulatory sequences of the DS2 gene in transgenic plants are highly specific, namely, they are not only drought-inducible and ABA-insensitive, but also contrary to all known regulation schemes, salt- and cold-insensitive. The promoters and other regulatory sequences of said genes will fulfill the requirements of a very specific plant expression system.

There are a lot of factors influencing the advantageous applications of transgenic plants, one of the most important of which - with respect to the establishment of new technologies - is the appropriate choice of the so called "agronomic" genes (genes causing positive effects) and the regulatory elements directing their expression (e.g., promoters, introns and 3' regulatory regions). A very narrow range of promoters has been used so far for the expression of foreign genes in plants. The cauliflower mosaic virus derived CaMv 35S promoter is most often used for the construction of plant expression vectors. With regard to the limitations of such expression systems there is a rapidly growing need for the preparation of further expression systems based on tissue specific or inducible promoters. In a wide range of possible applications it would be advantageous to use drought inducible vector constructions ensuring a suitable or, preferably, a high expression rate of the desired product in the transformed plant only if certain conditions are met. This is especially important when the product itself or its high level is toxic to the host. Also, it is particularly advantageous where the recombinant expression of the product can be controlled by means of changing the particular conditions that induce the production of the desired products. It would also be preferred to use plant promoters instead of virus-derived promoters to avoid possible biohazard.

It is, thus, an object of the present invention to obtain a highly specific, drought-inducible gene regulation element (promoter). A further object of the invention is to provide plant gene expression system using the regulatory element. The present invention therefore relates to an isolated single stranded or double stranded nucleic acid molecule comprising, in part or in its entirety, the 5' regulatory sequence region of the DS2 gene of a dycotiledon, preferably of a Solanaceae, more preferably of Solanum sp. and most preferably Solanum tuberosum.

In particular, the present invention provides for an isolated single stranded or double stranded nucleic acid molecule comprising a 5' regulatory sequence region from a dicotyledonous plant that is operable in a drought inducible and ABA, salt and cold insensitive manner, or a functional part thereof, wherein said regulatory region or functional part thereof is at least 50% homologous or hybridizable to the regulatory region of a DS2 gene, or a functional part thereof, of a species of the Solanaceae family, preferably of any of the Solanum or Nicotiana species, more preferably to a sequence of the regulatory region of LeDS2 of tomato or DS2 of Solanum tuberosum. In preferred embodiments, the homology between the said regulatory region or functional part thereof and the regulatory region of the said DS2 gene, or a functional part thereof is at least 55% or 60%, more preferably is at least 65%, 70% or 75%, even more preferably is at least 80%, 85% or 90%, highly preferably is at least 95%, 96%, 97%, 98%, end most preferably is at least 99% or more.

In a preferred embodiment of the present invention, the said regulatory region or functional part thereof in the isolated single stranded or double stranded nucleic acid molecule according to the invention is from a Solanaceae species. In particular, the said regulatory region is from any of the species of the Solanum, Lycopersicum or Nicotiana genus.

In another preferred embodiment of the present invention, the said regulatory region or functional part thereof in the isolated single stranded or double stranded nucleic acid molecule according to the invention is at least 70% homologous, more preferably is at least 75% or 80%, even more preferably is at least 85% or 90% homologous, highly preferably is at least 95%, 96%,

97%, 98% homologous, end most preferably is at least 99% homologous to a sequence of the regulatory region, or functional part thereof, of the eDS2 of tomato orDS2 of Solanum tuberosum.

In a preferred embodiment the present invention further relates to an isolated single stranded or double stranded nucleic acid molecule comprising, in part or in its entirety, the 5' regulatory sequence region of the DS2 gene of potato {Solanum tuberosum) as presented in SEQ

ID NO: 1.

The 1140-bp upstream sequence (Figure 1, SEQ ID NO: 1, EMBL database accession number AJ320154) is not homologous to any known sequences. Sequence analysis was performed using the Wisconsin GCG package [Devereux et al., Nucleic Acids Research 12, 387- 395 (1984)].

The regulatory sequence of the invention may comprise shorter segments that are capable to exert the same or similar functionality. Therefore these shorter segments form a further aspect of the invention. The existence of such shorter segments in other promoters is well known in the art. The functional segments of promoters are usually relatively short, well defined sequences. Sequence analysis, however, did not reveal any of the previously described ABA- and stress- inducible cv^'s-elements such as ABRE, CE, DRE, Myc and Myb elements within the promoter region of the invention. Promoter sequence analysis was performed by programs at the Plant Cis-Acting Regulatory DNA Elements (PLACE) database [Higo et al., Nucleic Acids Research 27, 297-300 (1999)] and at PlantCARE, another plant cis-acting regulatory element database [Rombauts et al., Nucleic Acids Research 27, 295-296 (1999)]. In the case of the Myb element, the sequence given in Busk and Pages, ..Regulation of abscisic acid-induced transcription", Plant Mol Biol 37: 425-435 (1998) was used instead of the sequence in the PLACE database.

Accordingly, shorter fragments of the regulatory sequences of the invention form an important aspect of the invention. Therefore, the present invention further provides an isolated single stranded or double stranded nucleic acid molecule comprising no more than 498 nucleotides of the 3' end of the sequence presented in SEQ ID NO: 1.

In certain embodiments, the shorter fragments of the regulatory sequences of the invention are at least 100, 200, 300, 400, 500, 600, 700, 800, 900, 100 or 1100 nucleotides in length, including any intermediate integer values. Preferred embodiments of the present invention are even shorter fragments of the inventive sequences. On the basis of the present disclosure a skilled person will be able to identify suitable fragments within the regulatory sequences of the invention, which are responsible for their functional specificity. Standard methods for identification of cis regulatory elements include footprinting [Sasse-

Dwight and Gralla, Methods Enzymol. 208, 146 (1991)], electrophoresis mobility shift analysis (EMSA)[Schneider et al. Nucleic Acids Res. 14, 1303 (1986)], gain-of-function analyses of DS2 promoter fragments fused to a minimal CaMV 35S promoter in transgenic plants, etc. Therefore, these identified regulatory elements within the inventive sequences form a preferred aspect of the invention.

The present invention moreover relates to an isolated single stranded or double stranded nucleic acid molecule comprising, in part or in its entirety, the 5" regulatory sequence region of the DS2 gene of a Solanaceae species, or a sequence that is at least 80% homologous, preferably at least 85% homologous, more preferably at least 90% homologous, even more preferably at least 95% homologous and even more preferably at least 96%, 97%, 98% or 99% homologous to, or hybridizable to the said regulatory sequence region, especially to the sequence as presented in SEQ ID NO: 1 , or any orthologues or homologues thereof.

Whether sequences have at least 80% or at least 95% homology to the sequences according to the invention, can be determined e.g. by means of the Wisconsin GCG package (Devereux et al, 1984, supra).

In another embodiment of the present invention there is provided an isolated nucleic acid molecule hybridizable with a nucleic acid molecule according to the invention, in particular with the sequence as presented in SEQ ID NO: 1 , or any orthologues or homologues thereof.

In another embodiment of the present invention, orthologues, homologues or functional variants of the above-identified sequences are used.

The nucleotide sequences of the invention can be obtained by first determining the genomic coding nucleotide sequence of the DS2 gene of potato (Solanum tuberosum), then, based on the gained sequence information, by determining the location of the 5' promoter region and by testing its specificity. Accordingly, the present invention also relates to an isolated single stranded or double stranded nucleic acid molecule comprising at least one regulatory gene region of plant origin that is drought-inducible and ABA-, salt- and cold-insensitive, said nucleic acid molecule being obtainable by

(1) screening a genomic library generated from a plant with a nucleic acid comprising the coding sequence of the DS2 gene of potato (Solanum tuberosum) as presented in SEQ ID NO: 2, or orthologues, homologues, functional variants or parts thereof;

(2) identifying gene(s) hybridizing with the said nucleic acid that is (are) expressed in a drought-inducible and ABA-, salt- and cold-insensitive manner; and (3) isolating a nucleic acid molecule comprising an upstream regulatory region of any of the identified gene(s) that is (are) expressed in a drought-inducible and ABA-, salt- and cold-insensitive manner from the genome of said plant.

The methods for generating genomic libraries from plants, screening them and selecting suitable candidate genes for further functional testing are well known to the person skilled in the art, and one scenario is demonstrated in Example 2.

Functional characterization of the identified genes can be carried out according to the present disclosure, or any alternative methods that can assess the drought-inducible and ABA-, salt- and cold-insensitive expression of the identified gene. As the last step, the upstream regulatory region of the gene is isolated from the genomic library using known methods in the art.

In certain cases, the isolated single stranded or double stranded nucleic acid molecule as described above may be used as probe for identifying additional sequences that are orthologues, homologues or functional variants of the sequences according to the invention. It will be readily recognized by the person skilled in the art that these newly identified inventive sequences will function in organisms, which are related to the organisms from which they have been isolated, more efficiently to identify additional inventive sequences than the original sequences of the invention.

Accordingly, the present invention further relates to an isolated single stranded or double stranded nucleic acid molecule comprising at least one regulatory gene region of plant origin that is drought-inducible and ABA-, salt- and cold-insensitive, wherein said molecule is obtainable by

(1) screening a genomic library generated from a plant with a nucleic acid comprising the coding sequence of the DS2 gene of potato (Solanum tuberosum) as presented in SEQ ID NO: 2, or orthologues, homologues, functional variants or parts thereof; (2) identifying gene(s) hybridizing to the said nucleic acid that is (are) expressed in a drought-inducible and ABA-, salt- and cold-insensitive manner;

(3) isolating a nucleic acid molecule identified in step (2), or parts or mutated variants thereof that optionally contains only selected parts of an upstream regulatory region or coding sequence or both; (4) screening a genomic library generated from another plant with a nucleic acid obtained in step (3);

(5) identifying gene(s) hybridizing to the nucleic acid of step (4) that is (are) expressed in a drought-inducible and ABA-, salt- and cold-insensitive manner; and

(6) isolating a nucleic acid molecule comprising an upstream regulatory region of any of the gene(s) identified in step (5) that is (are) expressed in a drought-inducible and ABA-, salt- and cold- insensitive manner from the genome of said plant.

All said above about the steps leading to the isolation of orthologues, homologues or functional variants of the sequences according to the invention are also valid for these newly identified inventive sequences. ln a preferred embodiment of the present invention there is provided an isolated single stranded or double stranded nucleic acid molecule obtained by screening a genomic library generated from a Solanum species with a nucleic acid comprising the coding sequence of the DS2 gene of potato (Solanum tuberosum) as presented in Figure 2 or orthologues, homologues, functional variants or parts thereof.

The present invention further relates to a biologically functional transformation vector comprising regulatory sequences of the invention operably linked to a structural gene. For transformation into host cells, an independent vector capable of amplification necessary, wherein, depending on the host cell, transformation mechanism, task and size of the DNA molecule, a suitable vector can be used. A large number of different vectors, described and/or used in the art, are known to the skilled person, as well as all the techniques and terms used in this specification, cf. e.g. Sambrook et al., Molecular Cloning: A Laboratory Manual, 2nd Edition, Cold Spring Harbor Laboratory Press, Cold Spring Harbor (1989). Preferably, the vector has a small molecular mass and should comprise selectable genes so as to lead to an easily recognizable phenotype in a cell so thus enable an easy selection of vector-containing and vector-free host cells. To obtain a high yield of DNA and corresponding gene products, the vector should comprise the regulatory sequences according to the present invention, gene amplification signals and other regulatory sequences. For an autonomous replication of the vector, furthermore, a replication origin is important. Polyadenilation sites are responsible for correct processing of the mRNA and splice signals for the RNA transcripts. If phages, viruses or virus particles are used as vectors, packaging signals will control the packaging of the vector DNA.

In a particularly preferred embodiment the present invention relates to a transformation vector suitable for transforming plant cells.

A person skilled in the art will recognize that the expression level and regulation of a transgene in a plant can vary significantly from line to line. Thus, the skilled person will advantageously test several lines to find one with an appropriate expression level and regulation.

A variety of techniques are available and known to those skilled in the art for introduction of constructs into a plant cell host. These techniques include transformation with DNA employing A. tumefaciens or A. rhizogenes as the transforming agent, electroporation, particle acceleration, etc. (See for example, EP 295 959 and EP 138 341). It is particularly preferred to use the binary type vectors of Ti and Ri plasmids of Agrobacteήum spp. Ti-derived vectors transform a wide variety of higher plants, including monocotyledonous and dicotyledonous plants, such as soybean, cotton, rape, tobacco, and rice [Pacciotti et al., Bio/Technology 3, 241 (1985); Byrne et al., Plant Cell. Tissue and Organ Culture 8, 3 (1987); Sukhapinda et al., Plant Mol. Biol. 8, 209-216 (1987); Lorz et al., Mol. Gen. Genet. 199, 178 (1985); Potrykus, Mol. Gen. Genet. 199, 183 (1985); Park et al., J. Plant Biol. 38, 365-71 (1995); Hiei et al., Plant J. 6, 271-282 (1994)]. The use of T-DNA to transform plant cells has received extensive study and is amply described [EP 120 516; Hoekema, In: The Binary Plant Vector System, Offset-drukkerij Kanters B.V.; Alblasserdam (1985), Chapter V, Knauf, et al., Genetic Analysis of Host Range Expression by Agrobacterium In: Molecular Genetics of the Bacteria-Plant Interaction, Puhler, A. ed., Springer- Verlag, New York, 1983, p. 245; and An, et al., EMBO J. 4, 277-284 (1985)].

Other transformation methods are available to those skilled in the art, such as direct uptake of foreign DNA constructs (see EP 295 959), techniques of electroporation [see Fromm et al., Nature 319, 791 (1986)] or high-velocity ballistic bombardment with metal particles coated with the nucleic acid constructs [see Kline el al., Nature 327, 70 (1987); and US4,945,050]. Once transformed, the cells can be regenerated by those skilled in the art. Of particular relevance are the recently described methods to transform foreign genes into commercially important crops, such as rapeseed [see De Block et al., Plant Physiol. 91, 694-701 (1989)], sunflower [Everett et al., Bio/Technology 5, 1201 (1987)], soybean [McCabe et al., Bio/Technology 6, 923 (1988); Hinchee et al., Bio/Technology 6, 915 (1988); Chee et al., Plant Physiol. 91, 1212-1218 (1989); Christou et al., Proc. Natl. Acad. Sci USA 86, 7500-7504 (1989); EP 301 749], rice [Hiei et al., Plant J. 6, 271- 282(1994)], and corn [Gordon-Kamm et al., Plant Cell 2, 603-618 (1990); Fromm et al., Biotechnology 8, 833-839 (1990)]. Transgenic plant cells are then placed in an appropriate selective medium for selection of transgenic cells, which are then grown to callus. Shoots are grown from callus and plantlets generated from the shoot by growing in rooting medium. The various constructs normally will be joined to a marker for selection in plant cells. Conveniently, the marker may be resistance to a biocide (particularly an antibiotic such as kanamycin, G418, bleomycin, hygromycin, chloramphenicol, herbicide, or the like). The particular marker used will allow for selection of transformed cells as compared to cells lacking the DNA, which has been introduced. Components of DNA constructs of the present invention may be prepared from sequences, which are native (endogenous) or foreign (exogenous) to the host. By "foreign" it is meant that the sequence is not found in the wild-type host into which the construct is introduced. Heterologous constructs will contain at least one region, which is not native to the gene from which the transcription-initiation- region is derived.

To confirm the presence of the transgenes in transgenic cells and plants, a Southern blot analysis can be performed using methods known to those skilled in the art. Expression products of the transgenes can be detected in any of a variety of ways, depending upon the nature of the product, and include Western blot and enzyme assay. One particularly useful way to quantitate protein expression and to detect replication in different plant tissues is to use a reporter gene, such as GUS. Once transgenic plants have been obtained, they may be grown to produce plant tissues or parts having the desired phenotype. The plant tissue or plant parts may be harvested, and/or the seed collected. The seed may serve as a source for growing additional plants with tissues or parts having the desired characteristics.

Accordingly, the present invention provides a transgenic plant or any reproducible part thereof comprising a nucleic acid molecule according to the invention.

Additionally, a further important aspect of the invention is a plant propagation material prepared from the above transgenic plant or any reproducible part thereof, or plant propagation material prepared by using a transgenic plant described above or any reproducible part thereof, wherein said plant propagation material comprises a nucleotide sequence of the invention.

In another embodiment of the present invention it is provided the use of an isolated single stranded or double stranded nucleic acid molecule of the invention as a probe for identifying a 5' regulatory sequence region of plant origin that is operable in a drought inducible and ABA, salt and cold insensitive manner, or a functional part thereof. As outlined above, these probes are useful in the identification and isolation of homologues, orthologues or functional variants of the regulatory sequences of the invention.

According to another preferred embodiment the present invention relates to a method for gene expression in plants, said method comprising the expression of a gene in a drought-inducible and ABA-, salt- and cold-insensitive manner, wherein a nucleic acid molecule according to the invention encoding for at least one regulatory gene region is operably linked to said gene.

The invention is further illustrated by the attached figures the short description of which is as follows:

Figure 1. The 5' sequence region of the DS2 gene of potato (Solanum tuberosum).

Figure 2. The nucleotide sequence of the coding region of the DS2 gene of potato (Solanum tuberosum).

Figure 3. Expression analysis of DS2 in S. tuberosum cv. Desiree leaves under different environmental conditions. D, air dried leaves; U, untreated; W, distilled water; NaCI, 200 mM NaCI for 8 h; 02-, hypoxia by flooding for 12 h; PQT, 100 μM paraquat for 4 h; ABA, 200 μM ABA in darkness for 24 h; cold, 4 °C for 48 h; heat, 42 °C for 6 h; PEG, 20% PEG solution for 24 h.

Figure 4. (a) Genomic organisation of the DS2 gene in potato. Sequences corresponding to the DS2 cDNA clone are represented by black bars, the sequence corresponding to the intron is represented by an empty box. Restriction sites used for cloning are indicated above the lines. ATG and STOP indicate the translational initiation and termination codons, respectively, (b) Chimeric plant transformation constructs containing DS2 promoter fragments derived from the potato genomic clones (prStDS2), GUS reporter gene (GUS) and the Nos terminator sequence (NosT).

Lengths of the DS2 promoter fragments are given in base pairs, (c) Effect of treatments on GUS activities of transgenic potato leaves carrying different constructs. U, untreated; drought, water withdrawn for one week; PEG, 20% PEG for 48 h; ABA, 200 μM ABA for 24 h; cold, 4 °C for 48 h.

Untransformed Desiree plants were used as negative-, and plants containing the 35S::GUS construct as positive controls. The bars show the average data obtained from two independent transgenic lines of JGUS and HGUS constructs. The experiments were repeated three times. Error bars represent the standard error. Specific GUS activity is expressed as pmol 4-methylumbelliferyl- β-D-glucuronide mg^"1 protein min^"1.

Figure 5. Expression analysis of LeDS2 in L. esculentum leaves under different environmental conditions, (a) L esculentum cv. Korall "wild type" (b) L. esculentum "sitiens", ABA^" mutant. D, air dried leaves; U, untreated; W, distilled water; NaCI, 200 mM NaCI for 8 h; 02-, hypoxia by flooding for 12 h; PQT, 100 μM paraquat for 4 h; ABA, 200 μM ABA in darkness for 24 h; cold, 4 °C for 48 h; heat, 42 °C for 6 h; PEG, 20% PEG solution for 24 h.

Figure 6. Activity of the 498-bp DS2 promoter region fused to the GUS reporter gene

(HGUS construct). GUS activity is detected by histochemical staining, (a) Dried leaf of a transgenic potato plant, (b) Transient assay in tomato by Agrobacterium infiltration: the leaf on the left is untreated, the leaf on the right is dried, (c) Transient assay in tobacco by Agrobacterium infiltration: the leaf on the left is untreated, the leaf on the right is dried.

Figure 7. Detection of DS2-type genes in S. tuberosum cv. White Lady (S.t.WL.) and Desiree (S.t.D.), in S. brevidens (S.b.), in L esculentum cv. Korall (L.e.K.) and K262 (L.e.2), and in N. benthamiana (N.b.). EcoRI (E) or H/ndlll (H) or Xba\ (X) digested genomic DNA of both species was blotted and hybridised with DS2-specific probe.

The present invention is further illustrated by the experimental examples described below, however, the scope of the invention will by no means be limited to the specific embodiments described in the examples.

Example 1: Response of DS2 expression to different environmental stimuli

Solanum tuberosum cv. Desiree was cultivated in pots in the greenhouse or vegetatively propagated from cuttings on MS medium [Murashige and Skoog, Physiologia Plantarum 15, 473- 497 (1962)].

Drought stress was achieved by wilting detached leaves at room temperature until 30% weight loss. Chemical treatments were applied by placing compound leaves into the appropriate solutions with their petioles submerged. Leaf samples were induced by PEG for 24 h, NaCI for 4 h and 8 h and by ABA for 24 h. For oxidative stress intact plants were sprayed (ca. 15 ml/plant) by paraquat and samples were taken after 90 min and 4 h, according to Pert-Treves and Galun [Plant Molecular Biology 17, 745-760 (1991)]. Hypoxic stress was applied by flooding pots for 6 h and 12 h. Heat (42 °C) and cold (4 °C) stresses were performed in phytotron at 99-100% relative humidity in order to prevent water loss. Samples were taken after 3 h and 6 h from heat-treated plants and after 48 h from cold-treated plants. Expression of DS2 in S. tuberosum in response to dehydration and other treatments was studied by northern hybridization. Total RNA was extracted according to Stiekema et al. [Plant Molecular Biology 11 , 255-269 (1988)] and 20 μg were separated on 1% agarose gels containing formaldehyde [Logemann et al., Analytical Biochemistry 163, 16-20 (1987)]. Nucleic acids were blotted onto nylon membranes in 10xSSC buffer (Sambrook et al, 1989, supra) and fixed by an UV cross-linker.

In order to avoid any cross-hybridization the insert of pDS12 was used as hybridisation probe. This is a truncated version of DS2 (68-440 bp) corresponding to the non-ASR like insert within DS2 [Silhavy et al., Plant Molecular Biology 27, 587-595 (1995)] with no homology to any known sequences. Hybridizations were performed in the hybridization buffer of Church and Gilbert [Proc. Natl. Acad. Sci. USA 81, 1991-1995 (1984)] for 20 hours at 65 °C. Isolated DNA fragments were labeled with α-³²P-dCTP by random priming (Sambrook et al., 1989, supra). The filters were then washed twice for 30 min with 0.1% SDS, 2xSSC at 65 °C. Membranes were exposed to autoradiography films with intensifying screens at -70 °C. Images were captured digitally and reformatted for publication with Adobe Photoshop (Adobe Systems, San Jose, CA, USA). Results

Expression of DS2 in S. tuberosum was tested by northern hybridization. DS2 transcript was not detectable in leaves of well-watered plants while a very strong induction occurred in air- dried leaves. Compared to the drastic and rapid accumulation of DS2 transcript by water loss, ABA treatment could generate only a very slight increase in DS2 expression (Figure 3). These data suggest that regulation of DS2 expression in S. tuberosum is drought-inducible and not primarily dependent on ABA.

The effect of stresses other than drought on DS2 expression was also tested. According to the northern hybridization shown in Figure 3 neither salt, cold and heat stresses nor hypoxia and oxidative stress caused substantial increase in DS2 expression. The same result was obtained when treatments were applied for shorter periods as in the experiment shown in Figure 3. Quite to the contrary, dehydration by PEG treatment, like dehydration by drying resulted in accumulation of high amount of DS2 mRNA (Figure 3). Thus the DS2 gene of potato can be assessed to a similar category of drought-inducible genes as erdl, (Nakashima et al., 1997, supra), however, with even higher specificity since it is not inducible by salt.

The cross-talk between different signalling pathways is so common that it questions whether there are any truly specific abiotic stress signalling responses (Knight & Knight, 2001 , supra). The invention probably discloses one of the rare examples of such highly specific responses.

Example 2: Isolation and analysis of DS2 genomic clones

Total DNA was isolated from S. tuberosum according to Chung et al. [Plant Molecular Biology Reporter 12, 304-309 (1994)]. Genomic library was prepared in the replacement vector λ DASH II (Stratagene, La Jolla, CA, USA) according to the manufacturer's instruction. Plated phage particles were screened as described by Sambrook et al., 1989, supra) using the insert of pDS12 as a probe. Two independent DS2 containing phage clones were isolated, one with a 498-bp and the other with a 1140-bp promoter fragment located upstream of the translational start codon.

Sequencing of the cloned genomic DNA fragments were performed by the dideoxy chain termination method (Sambrook et al., 1989, supra) using Sequenase T7 DNA polymerase (USB, Cleveland, OH, USA), or by the DNA Sequencing Laboratory of the Biological Research Centre of the Hungarian Academy of Sciences (Szeged, Hungary).

Sequence analysis was performed using the Wisconsin GCG package (Devereux et al, 1984, supra). Promoter sequence analysis was also performed by programs at the Plant Cis-Acting Regulatory DNA Elements (PLACE) database (Higo et al., 1999, supra) and at PlantCARE, another plant c/s-acting regulatory element database (Rombauts et al., 1999, supra). In the case of the Myb element, the sequence given in Busk and Pages [1998, supra] was used instead of the sequence in the PLACE database. Results Two overlapping recombinant phage clones were isolated from the genomic library of S. tuberosum that hybridized to the DS2-specific probe. DNA sequence analysis revealed that the coding region of the DS2 isolated from S. tuberosum (Figure 2) is almost identical to the sequence of the cDNA clone DS2 isolated previously from S. chacoense (accession number U12439). The overall homology at DNA level was 99.35%. At amino acid level this caused an S-R and a K-Q replacement at positions 42 and 119, respectively. The coding region of the DS2 gene is interrupted by an intron of 263 nucleotides at position 621 (Figure 4a).

The two genomic clones contained 498 and 1140 nucleotides (Figure 1, SEQ ID NO: 1 , EMBL database accession number AJ320154) upstream of the translation initiation codon. No homology of the promoter fragments to any known sequences was detected. Sequence analysis did not reveal any of the previously described ABA- and stress-inducible cs-elements such as ABRE, CE, DRE, Myc and Myb elements within the putative promoter region.

Example 3: Construction of DS2 promoter::Gl/S fusions and use for detection of promoter activity in transgenic potato plants In order to verify the transcriptional activity of the isolated sequence the two promoter fragments were fused to the GUS::NosT reporter construct. This resulted in two plant transformation vectors: pHGUS and pJGUS with 498-bp and 1140-bp DS2 promoter regions, respectively (Figure 4b).

The β-glucuronidase (GUS) gene with Nos terminator sequence was isolated from the plasmid pDV437 (G. Dallmann, unpublished) as a BamH\-Not\ fragment and cloned into the binary vector pCP60, kindly provided by P. Ratet (CNRS Institut des Sciences Vegetales, Gif-sur-Yvette, France). This cloning resulted the plasmid pCP/GUS that contained the 35S promoter fused upstream to the GUS gene. The Xba\ site from pDV437 had been previously eliminated by filling with Klenow polymerase and re-ligation in order to maintain the Xba\ site of pCP/GUS that originated from pCP60 unique.

From the genomic phage clone containing the 498-bp promoter region a 3-kb H/ndlll fragment hybridizing to the DS2 cDNA was cloned into the pBluescript II KS vector (Stratagene, La Jolla, CA, USA.). Using the primer DS2PROM (5'- GATGTI 1 1 1 1 GGTGATAATT-3') extending from -1 to -20 bp with respect to the translational start site and the T3 primer specific to pBluescript, the promoter region of 498 bp was amplified and cloned as a Smal-H/ndlll fragment into the cloning vector pGEM7 (Promega, Madison.WI, USA). From this clone the insert was isolated and ligated into the pCP/GUS upstream of the GUS gene using Xba\ and HindW restriction sites replacing thereby the 35S promoter. The resulting binary vector was designated pHGUS. From the other genomic phage clone with an 1140-bp promoter region a 2.2-kb EcoRl- Pvull fragment was cloned into the pBluescript II KS vector. Using the same pair of primers as for construction of pHGUS the 1140-bp promoter region was amplified and cloned into the Smal site of pBluescript II KS. The 35S promoter from pCP/GUS was removed by H/πdlll and BamHI digestions. The H/ndlll site was filled with Klenow polymerase and the DS2 promoter was ligated upstream to the GUS gene as a Smal-SamHI fragment. This clone was designated pJGUS.

Transgenic potato lines carrying either JGUS or HGUS constructs and the npfll gene suitable for selection on kanamycin containing media were generated via Λgroftacterum-mediated transformation. Agrobacteήum tumefaciens strain C58C1 containing pGV2260 [Deblaere et al., Nucleic Acids Research 13, 4777-4788 (1985)] was used to introduce the constructs HGUS and JGUS into S. tuberosum by the leaf transformation method of Dietze et al. [Agrobacterium- mediated transformation of potato (Solanum tuberosum), in: Gene Transfer to Plants, (eds. I. Potrykus & G. Spangenberg) pp. 24-29, Springer- Verlag, Berlin (1995)]. Results 18 JGUS and 97 HGUS transgenic potato lines resistant for kanamycin were obtained.

These lines were individually tested for DS2 promoter activity by histochemical staining of detached leaves after 20% PEG treatment for 48 h with X-Gluc substrate as described by [Jefferson, Plant Molecular Biology Reporter 5, 387-405 (1987)].

Two of the JGUS lines and 7 HGUS lines showed apparent GUS activity even in in vitro culture, in MS medium [Murashige and Skoog, 1962, supra]. Histochemical staining was detected mainly in leaves, especially in veins, but in some cases it was extended to the stem and root system. This result suggested a constitutive expression driven by the isolated DS2 promoter fragments, while the DS2 gene in the genome was previously found to be drought inducible. An obvious explanation of this phenomenon was that neither the promoter fragment of 498 bp nor that of the 1140 bp carries the cis regulatory elements necessary for drought-specific expression. These elements might be negative elements locating 5' upstream to the isolated region. In lack of the negative elements the promoter fragments H and J provide constitutive expression to the GUS structural gene resulting in detection of GUS activity in tissue culture. Thus, these results were negative in respect of drought specificity. Apparently, a longer nucleic acid region should have been isolated.

In spite of the above negative result, the present inventors propagated the 2 JGUS and 2 HGUS lines with the highest GUS activity in tissue culture in vitro and transferred them to pots in the greenhouse. The plants were watered regularly. After 2 months, GUS activity of untreated, dried and PEG treated leaves were measured by fluorimetric assay (Figure 4c). It was surprisingly found that in contrast to the results obtained in tissue culture the transgenic lines grown in pots without treatment showed only slightly higher fluorimetric activity then the untransformed Desiree plants. Though the explanation to this phenomenon is not known, it may be guessed that the high activity of the promoter fragments in tissue culture was not due to the lack of drought-specific cis elements but rather due to the presence of unknown inducing factor(s) under in vitro conditions. After dehydration achieved either by withdrawing water from the plants for one week or by 20% PEG treatment of detached compound leaves for 48 h, GUS activity of all four DS2 promoter- containing transgenic lines increased markedly. This demonstrates that both promoter regions examined carry the c/s-elements for drought-induced transcriptional activation. On the other hand, GUS activity did not change after 12 h ABA or 48 h cold treatments. Thus both promoter fragments are sufficient to maintain the specific nature of DS2 regulation. Transgenic potatoes containing a CaMV 35S::GUS construct were used as positive controls that showed very high GUS expression levels during all experiments (Figure 4c).

Silhavy et al. (1995, supra) published that the coding region of DS2 is partly homologous to the ABA, stress and ripening induced ASR genes isolated from tomato [lusem et al., Plant Physiology 102, 1353-1354 (1993)]. Despite of sequence homology between the two structural genes promoter sequences of DS2 and ASR genes [Rossi et al., Molecular and General Genetics 258, 1-8 (1998)] are unrelated and there is a marked difference in the specificity of the two promoters.

Example 4: Response of LeDS2 expression to different environmental stimuli

A homologue gene to DS2 of potato was detected in the closely related Solanaceous species, tomato (Lycopersicum esculentum) [Silhavy et al. (1995), supra], and designated LeDS2.

Expression of LeDS2 was studied in the tomato cultivars K262 and Korall, as well as in the ABA deficient mutant line, sitiens. The plants were cultivated in pots in the greenhouse. All treatments, RNA isolation and hybridizations were carried out in the same way as in the case of potato described in Example 1.

Results

Expression of LeDS2 was tested by northern hybridisation. Like DS2 gene of potato, LeDS2 was not expressed under optimal growth conditions but it was strongly induced by drought or PEG treatment. The effect of water-loss could not be mimicked by ABA treatment. Other abiotic stresses than drought did not induce the expression of LeDS2 (Figure 5a).

The role of ABA in LeDS2 regulation was further tested by studying an ABA biosynthesis (aba) mutant tomato line, sitiens. As a consequence of ABA deficiency these plants show a wilty phenotype even under well-watered conditions. A high level of LeDS2 expression was detected not only in air-dried plants but also in well-watered plants (Figure 5b). It appears that the wilty phenotype itself, caused by the lack of ABA, sufficient to induce the drought specific DS2 signaling. Nevertheless, the high level of LeDS2 expression in an ABA-free plant strongly supports the idea that regulation of LeDS2 expression is independent of ABA.

Example 5: Analysis of DS2 promoter activity in tomato and tobacco

Functionality of the DS2 promoter sequence active in potato was tested in two other species of the family Solanaceae, in tomato (L esculentum) and tobacco (Nicotiana benthamiana), using a transient gene-expression assay based on the method of Kapila et al. [Plant Sci. 122, 101- 108 (1997)]. A. tumefaciens strains were grown in YEB medium [Dietze et al. 1995, supra] of pH 5.6 supplemented with acetosyringone to logarithmic phase (OD₂₆₀=0,8). The cells were then centrifuged and resuspended in MMA medium [Kapila et al. 1997, supra] to a final OD₂₆o of 2.4. The Agrobacterium suspension was kept at 22°C for 1-2 h and then injected by a syringe into the adaxial side of the leaves. A darkening of the leaf surface colour indicated successful suspension uptake.

Results

Agrobacterium suspension containing the HGUS construct (described in details in Example 3) was infiltrated into tomato and tobacco leaves. In accordance to the publication of Kapila et al. [1997, supra] the suspension could be easily introduced into N. benthamiana leaves. Three tomato lines (K262, Korall, and Mobil) were tested for feasibility of suspension uptake but only Mobil plants turned out to be suitable for infiltration. The samples were then either dried or well-watered for two days. Activity of the GUS gene driven by the 498-bp DS2 promoter was visualized by X-Gluc staining. In both species, like in potato transgenic plants carrying the same construct, the blue coloration in the dried samples was much more intense than in the well-watered samples (Figure 6). These results suggest that the signal transduction mechanism regulating DS2 expression is conserved in solanaceous species.

Example 6. Conservation of the DS2 gene in related and non-related species Conservation of the DS2 coding region and the copy number of the DS2 gene was tested in potato and in three related species, the non-tuberising Solanum brevidens, and in the closer and farther relatives, tomato and tobacco, respectively. Genomic DNA were isolated and digested with three different restriction enzymes. The resulted fragments were separated by electrophoresis.in an agarose gel and blotted to Hybond N⁺ filter for Southern hybridization. Figure 7 shows that the DS2-specific probe prepared from the repeated region of the DS2 cDNA [Silhavy et al., 1995, supra] detected one major hybridising band in each species and cultivars. The only exception was the S. tuberosum cv. Desiree in which three bands with equal intensity appeared on the blot when the DNA was digested with either EcoRI or Hind\\\. These findings together indicate that DS2-type genes are widely present in solanaceous species. The intensity of hybridization to the potato DS2 probe, however, was much lower in the farther relative, tobacco, than in the closer relative, tomato, reflecting lower sequence conservation of DS2 in tobacco than in tomato. The presence of a single hybridising band suggests that the DS2-type gene is a single-, or low copy number gene in most of the solanaceous plants. Although, the example of the S. tuberosum cv. Desiree points out the possibility of higher copy number or greater polymorphism even within a species between different cultivars.

Conservation of the DS2 coding region in non-related plant species was tested by computer assisted DNA sequence comparison in the EMBL and SwissProt Databanks. No significant homologs of the DS2 cDNA were detected even in Arabidopsis whose entire genome sequence is available in databanks. Conservation of the DS2 promoter region was also tested by sequence comparison in the EMBL Databank. Sequence homology to the DS2 promoter was detected in tomato. In case of tomato homology was found to the EST sequence EMBL Databank No. A933909, and 5 other ESTs almost identical to it. The clone A933909 carried 27 bps 5' non-coding region and 608 bps coding region with only 2 bps alteration in the non-coding-, and with 91% identity in the coding region compared to the DS2 gene of potato. Since only one hybridizing band to the DS2-specific probe was detected in tomato by Southern hybridization it is fairly likely that the detected band corresponds to the sequenced ESTs. Moreover, sequence similarities of the promoter regions support the result of northern hybridization that the DS2 gene of potato and the LeDS2 gene of tomato are regulated in the same way.

Isolation of a longer promoter region of LeDS2 was attempted by PCR. Ten different overlapping primers corresponding to the promoter region of DS2 from -599 to -491 were used in 11 PCR with a primer designed to the repeated region of the LeDS2 structural gene. However, only 3 primer combinations resulted in multiplication of a tomato fragment similar in size to that obtained in potato. Nested primers based again on the potato sequence were designed and used in a consecutive reaction with the 3 tomato fragments as templates. As a result of this experiment PCR products were obtained from all 3 templates. Since two consecutive primers were designed to the promoter region of DS2 and two primers to the coding region of LeDS2 it is fairly likely that the resulted fragments involve a region of the LeDS2 promoter. At the same time, however, these results indicate differences in sequence of DS2 and LeDS2 promoters since 8 out of 11 primer combinations failed to multiply the LeDS2 promoter. In spite of these differences the two promoters should contain the same cis regulatory elements since the expression pattern of the two genes are the same. These cis elements, however, have not been identified yet.

According to Southern hybridization the sequence homology of DS2 in tobacco is much lower than in tomato. Although, the cis elements of the DS2 promoter and the signal transduction pathway leading to the expression of DS2 should be conserved since the HGUS construct is active not only in tomato but also in tobacco. These cis elements, however, are not necessary connected to the coding region with weak homology to DS2 in tobacco. This is the same for example in the case of ASR genes that share homology with DS2 but are regulated in a different way. It is even possible that the cis and trans elements of DS2 regulation are conserved in other species than those of the family Solanaceae. If it is so, or at least the trans elements necessary for recognition and activation of the DS2 promoter are conserved, the DS2 promoter can be applied for transgene expression in a wide variety of different plant species.

The cross-talk between different signaling pathways is so common that it questions whether there are any truly specific abiotic stress signaling responses (Knight & Knight, 2001). The invention discloses one of the rare examples of such highly specific responses.

A 1140-bp putative promoter region of DS2 was isolated from S. tuberosum. This sequence is not homologous to any known sequences and does not contain any described dehydration-specific c/s-regulatory motifs. In transgenic potato plants a 498-bp sequence upstream ofthe translafion initiation codon is able to drive GUS expression in a drought-specific manner, and this sequence is not responsive to ABA or cold or salt.

Claims

CLAIMS:

1. Isolated single stranded or double stranded nucleic acid molecule comprising a 5' regulatory sequence region from a dicotyledonous plant that is operable in a drought inducible and ABA, salt and cold insensitive manner, or a functional part thereof, wherein said regulatory region or functional part thereof is at least 50% homologous or hybridizable to the regulatory region of a DS2 gene, or a functional part thereof, of a species of the Solanaceae family, preferably of any of the Solanum or Nicotiana species, more preferably to a sequence of the regulatory region of LeDS2 of tomato or DS2 of Solanum tuberosum.

2. Isolated single stranded or double stranded nucleic acid molecule according to claim 1 in which the regulatory region is from a Solanaceae species.

3. Isolated single stranded or double stranded nucleic acid molecule according to claim 1 or 2, in which said regulatory region or functional part thereof is at least 70% homologous to a sequence of the regulatory region, or functional part thereof, of the LeDS2 of tomato or DS2 of Solanum tuberosum.

4. Isolated single stranded or double stranded nucleic acid molecule according to claim 1 obtainable by

(a) screening a genomic library generated from a plant with a nucleic acid comprising the sequence of the DS2 gene of potato (Solanum tuberosum) as presented in SEQ ID NO: 2, or the sequence of its tomato or tobacco homologues; (b) identifying gene(s) hybridizing with the said nucleic acid that is (are) expressed in a drought-inducible and ABA-, salt- and cold-insensitive manner; and

(c) isolating a nucleic acid molecule comprising an upstream regulatory region of any of the identified gene(s) that is (are) expressed in a drought-inducible and ABA-, salt- and cold-insensitive manner from the genome of said plant.

5. Isolated single stranded or double stranded nucleic acid molecule according to claim 1 obtainable by

(a) screening a genomic library generated from a plant with a nucleic acid comprising the sequence of the DS2 gene of potato (Solanum tuberosum) as presented in SEQ ID NO: 2, or the sequence of its tomato or tobacco homologues; (b) identifying gene(s) hybridizing to the said nucleic acid that is (are) expressed in a drought-inducible and ABA-, salt- and cold-insensitive manner;

(c) isolating a nucleic acid molecule identified in step (b), or parts or mutated variants thereof that optionally contains only selected parts of an upstream regulatory region or coding sequence or both; (d) screening a genomic library generated from another plant with a nucleic acid obtained in step (c);

(e) identifying gene(s) hybridizing to the nucleic acid of step (d) that is (are) expressed in a drought-inducible and ABA-, salt- and cold-insensitive manner; and (0 isolating a nucleic acid molecule comprising an upstream regulatory region of any of the gene(s) identified in step (e) that is (are) expressed in a drought-inducible and ABA-, salt- and cold- insensitive manner from the genome of said plant.

6. Isolated single stranded or double stranded nucleic acid molecule according to claim 4 or 5, wherein, in step (a), a genomic library generated from a Solanaceae species is screened.

7. Isolated single stranded or double stranded nucleic acid molecule comprising, in part or in its entirety, the 5' regulatory sequence region of the DS2 gene of a Solanaceae species, or a sequence that is at least 80% homologous or hybridizable to the said regulatory sequence region.

8. Isolated single stranded or double stranded nucleic acid molecule according to claim 7, comprising, in part or in its entirety, the 5' regulatory sequence region of the DS2 gene of potato

(Solanum tuberosum) as presented in SEQ ID NO: 1 , or any orthologues or homologues thereof.

9. Isolated single stranded or double stranded nucleic acid molecule according to claim 8, comprising no more than 1140, or no more than 498 nucleotides of the 3' end of the sequence presented in SEQ ID NO: 1.

10. Transformation vector comprising sequences according to any of claims 1 to 9 operably linked to a structural gene.

11. Transformation vector according to claim 10, suitable for transforming plant cells.

12. Transgenic plant or any reproducible part thereof comprising a nucleic acid molecule according to any of claims 1 to 11.

13. Plant propagation material prepared from or by using a plant according to claim 12, said plant comprising a nucleotide sequence as claimed in any of claims 1 to 9.

14. Use of an isolated single stranded or double stranded nucleic acid molecule according to any of claims 1 to 9 as a probe for identifying a 5' regulatory sequence region of plant origin that is operable in a drought inducible and ABA, salt and cold insensitive manner, or a functional part thereof.

15. Method for gene expression in plants, comprising expression of a gene in a drought- inducible and ABA-, salt- and cold-insensitive manner, said gene containing a nucleic acid molecule according to any of claims 1 to 9 encoding at least one regulatory gene region, wherein said regulatory region is operably linked to said gene.