WO2005048693A2

WO2005048693A2 - Plant with improved drought tolerance

Info

Publication number: WO2005048693A2
Application number: PCT/IB2004/052440
Authority: WO
Inventors: István Papp; Csaba Koncz; Ferenc Nagy
Original assignee: Agricultural Biotechnology Center
Current assignee: Agricultural Biotechnology Center
Priority date: 2003-11-19
Filing date: 2004-11-16
Publication date: 2005-06-02
Anticipated expiration: 2006-05-19
Also published as: HU0303778D0; WO2005048693A3; HUP0303778A2

Abstract

The present invention relates to a plant with improved drought tolerance that is transgenic and/or mutagenized chemically and/or physically, wherein the function of the Cap Binding Protein (CBP20) gene or a homologue thereof is decreased or blocked due to genetic manipulation and/or mutagenization. The invention further relates to constructs used for the manipulation and/or mutagenezis, as well as methods for the production of the plant with improved drought tolerance.

Description

Description Plant with improved drought tolerance

[1] The present invention relates to a plant with improved drought tolerance that is transgenic and/or mutagenized chemically and/or physically, wherein the function of the Cap Binding Protein (CBP20) gene or a homologue thereof is decreased or blocked due to genetic manipulation and/or mutagenization. The invention further relates to constructs used for the manipulation and/or mutagenezis, as well as methods for the production of the plant with improved drought tolerance. Background Art

[2] Agronomicallyimportant genes can be isolated in several different ways. A generally used strategy is direct mapping of genes responsible for favorable traits in crop species, followed by map based cloning. This approach is, however, hampered by the relatively scarce information available in these species.

[3] Similar strategies may be followed in model plant systems such as Arabidopsis thaliana as well. Due to available sequence information and sophisticated genetic techniques, advances in these model species can be made at a faster pace. Considerable drawbacks are, however, that results must be adapted to crop species, and a number of valuable traits may not be present in the few model plants. Nevertheless, a large amount of information becomes available as a result of these efforts.

[4] Activation or inactivation of an important signaling pathway in a plant may cause multiple phenotypic changes beside the primary biochemical or physiological defect (pleiotropy). These changes may affect the plant's stature, growth, developmental phases or other parameters that can be scored relatively easily. Based on these subtle changes, mutants often can be spotted easily in a mutant plant population. In a functional genomic screen these mutants may be tested further to reveal the primary nature of the mutation, with special emphasis on the scientifically or agronomically useful traits they may carry. Practically, this means a second round of rigorous screening, where the selected mutants are tested under a number of different stress conditions. The usefulness of this screening strategy has been validated recently in the literature [Boyes, D.C., Zayed, A.M., Ascenzi, R., McCaskill, A.J., Hoffman, N.E., Davis, K.R., Gorlach, J., Growth stage-based phenotypic analysis of Arabidopsis: a model for high throughput functional genomics in plants., Plant Cell 13, pp. 1499-1510 (2001)]. Known biochemical mutants were scrutinized and found different from the wild type plant in a number of morphological characteristics that have no apparent connection to the primary biochemical defect.

[5] In agriculture there is a great need for the development of new plant varieties with improved parameters, involving tolerance to environmental stresses. A particularly important stress factor is tolerance against drought. This implies the continuous demand for breeding varieties with improved drought tolerance. [6] The physiological background of the drought tolerance phenotype is extremely complex. Improvement of this trait could be achieved by several ways:

[7] A method that has been employed to this end is conventional breeding, where crossings and selection is used to improve the characteristics of the particular variety (see e.g. Hungarian Patent No. 218 309 and US Patent No. P9885).

[8] With the development of molecular biological methods, transgenic techniques are used more and more often to tailor the physiology of a plant to resist environmental stresses, such as drought. A few examples for this method: overproduction of a molybdenum-cofactor sulphurase, which yields the cofactor of the last enzyme of abscisic acid (ABA) biosynthesis (US patent application No. 2003/0084485); increasing the raffϊnose content of a plant by transgenic means (US patent application No. 2003/0074696A1); overproduction of the ABI5 protein - which is a stress-induced transcription factor - (US patent application No. 2002/0174454A1).

[9] Another way of improving tolerance to water deprivation is to modulate the function of the nuclear cap binding complex (nCBC) of a plant. nCBC binds to the 5' cap structure of nascent mRNAs transcribed by RNA polymerase II. Most data are available on the structure and function of nCBC in yeast and animals. Here the core of the nCBC complex consists of the Cap Binding Proteins CBP80, CBP20 (with molecular weights of 80 and 20 kDa, respectively) bound by an additional handful of proteins, including eIF4G [McKendrick, L., Thompson, E., Ferreira, J., Morley, S.J., Lewis, J.D., Interaction of eukaryotic translation initiation factor 4G with the nuclear cap-binding complex provides a link between nuclear and cytoplasmic functions of the m⁷guanosine cap., Mol. Cell. Biol. 21, pp. 3632-3641 (2001)]. [We note that the abbreviation CBP20 has also been used in the literature to designate the chitin binding protein, unrelated to the subject of the present invention; see Ponstein A.S. et al., Plant Physiology 104(1), pp. 109-118 (1994)]. CBP80 and CBP20 are able to bind the 5' cap structure together. The full biological function of nCBC however is probably achieved in a complex interaction with all the proteins participating in the complex. CBP20 is the subunit that binds the 5' cap directly, and the details of this interaction are well documented [Calero G. et al., Nat. Struct. Biol. 9(12), pp. 912-917 (2002)]. nCBC is implicated in mRNA splicing, 3' end maturation and in the export of U snRNA from the nucleus [Izaurralde, E., Lewis, J., McGuigan, C, Jankowska, M., Darzynkiewicz, E., Mattaj, I.W., A nuclear cap binding protein complex involved in pre-mRNA splicing., Cell 78, pp. 657-668 (1994); Flaherty, S., Fortes, P., Izaurralde, E., Mattaj, I.W., Gilmartin, G:M., Participation of the nuclear cap binding complex in pre-mRNA 3' processing., Proc. Natl. Acad. Sci. USA 94, pp. 11893-11898 (1997)].

[10] Interestingly, mammalian nCBC was found to function dependent on stresses and growth factor stimulation. It seems, therefore, that its role in mRNA metabolism is not a 'house keeping' function but a checkpoint for posttranscriptional regulation [Wilson, K.F., Fortes, P., Singh, U.S., Ohno, M., Mattaj, I.W., Cerione, R.A., The nuclear cap- binding complex is a novel target of growth factor receptor-coupled signal transduction., J. Biol. Chem. 274, pp. 4166-4173 (1999)]. nCBC itself is known to be regulated posttranscriptionally by protein phosphorylation in mammals.

[11] In plants, there are in vivo data available on the function of only one of the proteins participating in nCBC. A mutant in a CBP80 homolog (abhl) has been described by Hugouvieux et al 2001 in Arabidopsis [Hugouvieux, V., Kwak, J.M., Schroeder, J., An mRNA cap binding protein, ABH1, modulates early abscisic acid signal transduction in Arabidopsis., Cell 106, pp. 477-487 (2001)]. ABH1 is expressed in all organs of the plant according to a complicated, temporally and spatially regulated pattern, abhl was selected in a biochemical screen for ABA sensitivity at germination. Stomata in abhl are hypersensitive to ABA, its water loss is reduced compared to wild type.

[12] The rice homolog for the CBP80 protein is detailed in WO02/081696. It is therefore likely that, at least structurally, nCBC is evolutionally conserved in dicotyledonous and monocotyledonous plants and animals (see references above), as well as in yeast [e.g. see Colot et al., The yeast splicing factor Mudl3p is a commitment complex component and corresponds to CBP20, the small subunit of the nuclear cap-binding complex., Genes Dev. 10, pp. 1699-1708 (1996)].

[13] A possible mechanism for nCBC function is to influence alternative splicing. Clark et al. have investigated the effects of different mutations on alternative splicing in yeast by splicing specific microarrays [Clark, T.A., Sugnet, C.W. and Ares, M., Genom wide analysis of mRNA processing in yeast using splicing-specifϊc microarrays., Science 296, pp. 907-910 (2002)]. According to their data, the gcr3 and mudl3 yeast mutants (corresponding to cbp80 and cbp20, respectively) do not have the same effect on the accumulation of the spliced RNA set of the yeast. Consequently the two proteins may have at least partially different functions in the cell.

[14] Expression of CBP20 in Arabidopsiήs regulated by developmental and stress factors as revealed by microarray experiments [Zimmermann P., Hirsch-Hoffmann M., Hennig L., Gruissem W., GENEVESTIGATOR. Arabidopsis Microarray Database and Analysis Toolbox., Plant Physiol. 136, pp. 2621-2632 (2004)]. The regulation of CBP20 is generally more pronounced and not necessarily parallel with that of the other members of the nCBC complex (e.g. CBP80). These data suggest that CBP20 expression might be a possible key regulatory element for nCBC function.

[15]

[16] As genetic analysis discovered, the Arabidopsis mutant line with improved drought tolerance made by the present inventors carried a lesion in the CBP20 gene, which is likely a member of the nCBC complex. We have isolated the gene in a 'reverse' manner, that is first selected the mutant line based on its pleiotropic phenotype, then cloned the gene responsible. For the purpose of easier reference, the nucleotide and amino acid sequence of CBP20 gene is presented on SEQ. ID. NOs. 1 and 2, respectively. [ 17] We also investigated the inducibility of CBP20 expression after different stresses, and concordant with the microarray data, found that bacterial expression may down regulate it. This may lead to changes in certain mRNA levels leading to adaptive responses of the plant (such as closing of stomata). Our data suggest that regulation of CBP20 expression may be part of a natural system for stress adaptation. More research is needed to establish whether regulation also takes place in the posttranscriptional level (e.g. protein phosphorylation).

[18] As genetic analysis discovered, the Arabidopsis mutant line with improved drought tolerance made by the present inventors carried a lesion in the CBP20 gene, which is likely a member of the nCBC complex. We have isolated the gene in a 'reverse' manner, that is first selected the mutant line based on its pleiotropic phenotype, then cloned the gene responsible. For the purpose of easier reference, the nucleotide and amino acid sequence of CBP20 gene is presented on SEQ. ID. NOs. 1 and 2, respectively.

[19] Our work on the identification of the gene responsible for the improved drought tolerance phenotype yielded substantially new results in the following:

[20] There had been no in vivo data regarding the function of the protein coded by the gene At5g44200, homologous to other CBP20 genes. There had been no indication whether beside its possible involvement in nCBC the same protein may fulfill other functions as well. Multifunctional proteins are common in plants. The only recognizable protein domain in At5g44200 (RRM type RNA binding domain) can be found in diverse proteins with different functions.

[21] On the other hand, it is generally known that important functions are often fulfilled redundantly by more than one proteins. Proteins with similar structure to CBP20 are present in the Arabidopsis genome (e.g. BAB08317, NP198588, AAG52402). It could not be predicted whether or not any of them would take over the lost function in the mutant even partially.

[22] Nevertheless, in vitro was surprisingly found that the mutation isolated from the T- DNA mutagenized population, showing morphological traits as well as improved drought tolerance, is due to a defective CBP20 gene. In the above mentioned references, only the role of the large subunit of the nCBC complex is emphasized and supported by in vivo data, thus suggesting being the only key component in producing the drought tolerant plant. In the contrary, the present invention proves that tolerance to water stress can be improved by manipulating the expression of the CBP20 gene as well.

[23] Since the Arabidopsis mutant cbp20 displays only mild pleiotropic morphological abnormalities, these will not prevent the agricultural use of similar mutated versions of crop plants.

[24] 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 inter- pretation of the present invention, it shall be understood that the terms defined below 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.

[25] The term 'gene' refers to a nucleic acid fragment that expresses mRNA, functional RNA, or specific protein, including its regulatory sequences. The term 'native gene' refers to gene as found in nature. A 'transgene' refers to a gene that has been introduced into the genome by transformation and is stably maintained. Transgenes may include, for example, genes that are either heterologous or homologous to the genes of a particular plant to be transformed. Additionally, transgenes may comprise native genes inserted into a non-native organism, or chimeric genes. The term 'endogenous gene' refers to a native gene in its natural location in the genome of an organism.

[26] The term 'coding sequence' refers to a DNA or RNA sequence that codes for a specific amino acid sequence and excludes the non-coding sequences. The terms 'open reading frame' and 'ORF' refer to the amino acid sequence encoded between translation initiation and termination codons of a coding sequence. The terms 'initiation codon' and 'termination codon' refer to units of three adjacent nucleotides ('codon') in a coding sequence that specifies initiation and chain termination, respectively, of protein synthesis (mRNA translation).

[27] The terms 'regulatory sequences' refers to nucleotide sequences located upstream (5' non-coding sequences), within, or downstream (3' non-coding sequences) of a coding sequence, and which influence the transcription, RNA processing or stability, or translation of the associated coding sequence. Regulatory sequences include enhancers, promoters, translation leader sequences, introns, and polyadenylation signal sequences. They include natural and synthetic sequences, as well as sequences which may be a combination of synthetic and natural sequences. Some regulatory sequences useful in the present invention will include, but are not limited to constitutive plant promoters, plant tissue-specific promoters, plant developmental stage-specific promoters, inducible plant promoters and viral promoters.

[28] The '3' region' means the 3' non-coding regulatory sequences located downstream of a coding sequence. This DNA can influence the transcription, RNA processing or stability, or translation of the associated coding sequence.

[29] The term 'promoter' refers to a nucleotide sequence, usually upstream (5') to its coding sequence, which controls the expression of the coding sequence by providing the recognition for RNA polymerase and other factors required for proper transcription. The term 'promoter' includes a minimal promoter that is a short DNA sequence comprised of a TATA-box and other sequences that serve to specify the site of transcription initiation, to which regulatory elements are added for control of expression. 'Promoter' also refers to a nucleotide sequence that includes a minimal promoter plus regulatory elements that is capable of controlling the expression of a coding sequence or functional RNA. This type of promoter sequence consists of proximal and more distal upstream elements, the latter elements often referred to as enhancers. Accordingly, an 'enhancer' is a DNA sequence which can stimulate promoter activity and may be an innate element of the promoter or a heterologous element inserted to enhance the level or tissue specificity of a promoter. It is capable of operating in both orientations (normal or flipped), and is capable of functioning even when moved either upstream or downstream from the promoter. Both enhancers and other upstream promoter elements bind sequence-specific DNA-binding proteins that mediate their effects. Promoters may be derived in their entirety from a native gene, or be composed of different elements derived from different promoters found in nature, or even be comprised of synthetic DNA segments. A promoter may also contain DNA sequences that are involved in the binding of protein factors which control the effectiveness of transcription initiation in response to physiological or developmental conditions.

[30] The term 'homologue' or 'variant' of a nucleic acid sequence refers to a sequence having at least 50%, preferably at least 70%, more preferably at least 80%, even more preferably at least 90%, and still more preferably at least 95% sequence identity with the said sequence. To determine whether the sequences are, say, at least 50% identical, the FastDB program of EMBL or SWISSPROT data bases can be used. Other algorithms and computerized embodiments thereof well known in the art may also be used for the determination of this homology.

[31] The term 'homologue' or 'variant' of an amino acid sequence is defined similarly to that of nucleic acid sequences. Therefore, the term 'homologue' or 'variant' of an amino acid sequence refers to a sequence having at least 70%, preferably at least 80%, more preferably at least 90%, even more preferably at least 95%, and still more preferably at least 98% sequence identity with the said sequence. To determine whether the sequences are homologous, the above-mentioned programs and algorithms may be used.

[32] '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.

[33] 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.

[34] 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.

[35] The terms 'transformed', 'transformant' and 'transgenic' refer to plants or calli that have been through the transformation process and contain a foreign gene integrated into their chromosome. The term 'untransformed' refers to normal plants that have not been through the transformation process.

[36] As used herein, 'transgenic plant' includes reference to a plant, which comprises within its genome a heterologous polynucleotide. Generally, the heterologous polynucleotide is stably integrated within the genome such that the polynucleotide is passed on to successive generations. The heterologous polynucleotide may be integrated into the genome alone or as part of a recombinant expression cassette. 'Transgenic' is used herein to include any cell, cell line, callus, tissue, plant part or plant, the genotype of which has been altered by the presence of heterologous nucleic acid including those transgenics initially so altered as well as those created by sexual crosses or asexual propagation from the initial transgenic. The term 'transgenic' as used herein does not encompass the alteration of the genome (chromosomal or extra- chromosomal) by conventional plant breeding methods or by naturally occurring events such as random cross-fertilization, non-recombinant viral infection, non- recombinant bacterial transformation, non-recombinant transposition, or spontaneous mutation.

[37] The function of a gene is 'decreased', 'inhibited' or 'blocked' if the expression or transcription of the gene, the splicing of the RNA transcribed from the gene, or the transport, membrane integration, activation or activity of the protein encoded by the gene, or any other molecular, cellular or plant level process having a role in the expression or function of the gene or the product encoded, is lowered, such as about 75%), preferably about 50%, more preferably about 25%, even more preferably about 10%, more preferably even about 5%, or most preferably completely blocked, that is about 0%, compared to the same function of the wild type plant.

[38]

[39] The present invention relates to a plant with improved drought tolerance that is transgenic and/or mutagenized chemically and/or physically, wherein the function of the Cap Binding Protein (CBP20) gene or a homologue thereof is decreased or blocked due to genetic manipulation and/or mutagenization.

[40] In a further embodiment, the present invention relates to a plant wherein the function of the CBP20 gene or a homologue thereof is decreased or blocked by addition, deletion and/or substitution of one or more nucleotide within the regulatory and/or coding region of the gene.

[41] In another embodiment the invention relates to a plant wherein the function of the CBP20 gene or a homologue thereof is decreased or blocked by introduction of a re- combinant construct that leaves the regulatory and/or coding region of the gene intact.

[42] In preferred embodiments the said plant is dicotyledonous, preferably the plant belongs to the order of Brassicales, and more preferably to the family of Brassicaceae. In a highly preferred embodiment, the plant is Arabidopsis.

[43] In other preferred embodiments, the plant is monocotyledonous.

[44] The invention also relates to a cell, tissue, organ, part, seed, fruit, product, progeny or propagation material of the plant according to the invention.

[45] In a specific embodiment, the invention relates to a recombinant nucleic acid construct comprising a nucleic acid inhibiting or blocking the CBP20 gene upon its expression, operably linked to a regulatory sequence capable of directing transcription in a plant.

[46] In a preferred embodiment, the construct according to the invention comprises a nucleic acid sequence encoding the CBP20 gene or a homologue thereof in its entirety or a part thereof in antisense direction with respect to the regulatory sequence.

[47] In another preferred embodiment, the construct according to the invention comprises a nucleic acid sequence encoding the CBP20 gene or a homologue thereof in its entirety or a part thereof in sense direction with respect to the regulatory sequence.

[48] In another preferred embodiment, the construct according to the invention comprises a nucleic acid sequence encoding the CBP20 gene or a homologue thereof in its entirety or a part thereof in an inverted repeat arrangement.

[49] The invention further relates to a method for the production of a plant with improved drought tolerance comprising the preparation a mutagenized plant population by random mutagenezis; selection of the mutagenized plants by molecular biological techniques capable of recognizing mutations within the CBP20 gene or homologues thereof; and optional sexual or asexual propagation of the resulting plants with improved drought tolerance.

[50] In another embodiment, the invention relates to a method for the production of a plant with improved drought tolerance comprising the preparation a mutagenized plant population by introducing a nucleic acid construct suitable for the mutagenesis or functional blocking of the CBP20 gene or homologue thereof; selection of the mutagenized plants with improved drought tolerance; and optional sexual or asexual propagation of the resulting plant with improved drought tolerance.

[51] In a preferred embodiment, the invention relates to a method wherein a construct according to the invention is used for the mutagenesis or functional blocking of the gene.

[52] In another preferred embodiment, the invention relates to a method wherein a wherein the mutation within the CBP20 gene or homologue thereof is preferably detected hybridization, sequencing, PCR, SSCP, and/or TILLING.

[53] In other preferred embodiments, the invention also relates to a method wherein during the selection step the plant population is screened in advance for visible pleiotropic features being characteristic of plants comprising a mutant CBP20 gene or a homologue thereof. Brief description of the drawings

[54] Figure 1 shows wild type (on the left) and cbp20 mutant (on the right) plants and leaves.

[55] Figure 2 shows leaves of cbp20 and serrate (se) plants, and their FI progeny.

[56] Figure 3 shows the result of T-DNA specific PCR on F2 plants with wild type (panel A) and mutant (panel B) phenotype.

[57] Figure 4 shows Northern hybridization on total RNA samples prepared from wild type (on the left) and cbp20 mutant (on the right) plants.

[58] Figure 5 shows wild type and cbp20 mutant plants following 8 days of water deprivation.

[59]

[60] In a preferred embodiment, drought resistant plant according to the invention can be obtained by damaging (i.e. inhibiting or completely blocking) the CBP20 gene or gene expression. Damaging of the CBP20 gene or gene expression can be achieved by introducing DNA constructs into the plant.

[61] There are several ways known to the person skilled in the art to introduce foreign genes into plant host cells [Potrykusl., Annu. Rev. Plant Physiol. Plant Mol. Biol. 42, pp. 205-225 (1991)]. These methods include DNA transformation using Agrobacterium tumefaciens or A. rhizogenes as transforming agent, electroporation, particle bombardment, etc. (see e.g. EP 295 959 and EP 138 341). The application of Ti- or Ri-plasmid containing binary vectors is especially preferred. Ti derived plasmids transform a wide range of higher plants, including monocot and dicot plants such as soybean, cotton, rape, tobacco and rice [Pacciotti et al., Bio/Technology 3, p. 241 (1985); Byrne et al., Plant Cell, Tissue and Organ Culture 8, p. 3 (1987); Sukhapinda et al., Plant Mol. Biol. 8, pp. 209-216 (1987); Lorz et al., Mol. Gen. Genet. 199, p. 178 (1985); Potrykus et al., Mol. Gen. Genet. 199, p. 183 (1985); Park et al., J. Plant Biol. 38, pp. 365-371 (1995); Hiei et al., Plant J. 6, pp. 271-282 (1994)]. The application of T-DNA for the transformation of plant cells is well established and described [EP 120 516; Hoekema, The Binary Plant Vector System, Chapter 5, Offset-drukkerij Kanters B. V., Alblasserdam (1985); Knauf et al., Molecular Genetics of the Bacteria-Plant Interaction, p. 245, Puhler, A. (ed.), Springer- Verlag, New York (1983); and An et al., EMBO J. 4, pp. 277-284 (1985)].

[62] Other methods of transformation are also available for the person skilled in the art, such as direct uptake of foreign DNA constructs (EP 295 959), electroporation methods [Fromm et al., Nature 319, p. 791 (1986)], or high speed ballistic bombardment with metal particles coated with nucleic acid constructs [Kline et al., Nature 327, p. 70 (1987), and US patent No. 4 945 050]. Foreign DNA constructs may be inserted into the nuclear chromosome of the target plants, but may also be inserted into the genome of other organelles, such as chloroplast, mitochondria or other subcellular compartments [See e.g. Maliga P., Trends Biotechnol. 21, pp. 826-827 (2003), Ruf et al., Nat. Biotechnol. 19, pp. 826-827 (2001)].

[63] Once the cells are transformed, the person skilled in the art may regenerate plants from them. Recently developed methods to deliver transgenes into important crop plants are: De Block et al., Plant Physiol. 91, pp. 694-701 (1989)] into rape, Everett et al., Bio/Technology 5, p. 1201 (1987) into sunflower, McCabe et al., Bio/Technology 6, p. 923 (1988); Hinchee et al., Bio/Technology 6, p. 915 (1988); Chee et al., Plant Physiol. 91, pp. 1212-1218 (1989); Christou et al., Proc. Natl. Acad. Sci. USA 86, pp. 7500-7504 (1989); EP 301 749 into soybean, Hiei et al, Plant J. 6, pp. 271-282 (1994) into rice, Gordon-Kamm et al., Plant Cell 2, pp. 603-618 (1990); Fromm et al., Biotechnology 8, pp. 833-839 (1990) into maize.

[64] There are a number of methods available for the person skilled in the art to interfere with the expression of the targeted gene by using foreign DNA constructs. Some examples are given in the next sections.

[65] Antisense inhibition.According to the sequence of the gene or a homolog thereof targeted to be silenced, a nucleic acid construct is designed covering the full or partial sequence of the target gene (may include exons, introns, 5' or 3' untranslated regions of mRNA) in antisense orientation, operably linked to a promoter suitable in the plant manipulated. A minimum of 40-50 base pair homology is generally thought to be required to achieve silencing, in general practice a ~300-400 base pair long DNA sequence is used. Having introduced into the target cell, antisense RNA is synthesized from this construct that may disturb the expression of the target gene at different levels. The nucleic acid delivered to the cell may be antisense DNA or RNA oligonucleotide as well. In the target cell, the antisense nucleic acid may hybridize with the target mRNA, yielding double stranded RNA that is degraded by the host cell, preventing the expression of the gene [Matzke and Matzke, Plant Physiology 107, pp. 679-685 (1995), Vaucheret et al., The Plant Journal 16, pp. 651-659 (1998), Mlotshwa et al., Plant Cell 14 Suppl, pp. 289-301 (2002)]. The silencing construct need not be fully homologous with the target sequence, moreover one construct may silence more than one target genes.

[66] Sense suppression.The target gene or a part of its sequence is overexpressed in the plant, driven by a promoter that is functional in the plant. In some cases this leads to silencing of the introduced and endogenous genes, or any homolog sequences in the genome. Presumably there is a control mechanism in the cell degrading the mRNA species in excess [Taylor C.B., Plant Cell 9, pp. 1245-1249 (1997), Jorgensen et al., Trends in Genetics 15, pp. 11-12 (1999), Fagard and Vaucheret, Annu. Rev. Plant Physiol. Plant Mol. Biol. 51, pp. 167-194 (2000)].

[67] Gene silencing based on inverted repeat sequences. The principle of the method is similar to that of antisense suppression, so as its probable mechanism. This technique is based on the observation that a sequence is capable of silencing more efficiently in inverted repeated orientation (for example palindrome or hairpin structures). Silencing is even more efficient with a small (preferably intron derived) spacer sequence inserted between the two parts of the inverted repeat [Wesley et al., Construct design for efficient, effective and high-throughput gene silencing in plants., Plant J. 27, pp. 581-590 (2001)]. The silencing construct may contain the promoter of the gene, exons, introns, 5' or 3' non-translatable regions or coding sequences of the target gene or parts of these elements [Brummell et al., Inverted repeat of a heterologous 3'-nontranslated region for high-efficiency, high-throughput gene silencing., Plant J. 33, pp. 793-800 (2003); Aufsatz et al., RNA-directed DNA methylation in Arabidopsis., Proc. Natl. Acad. Sci. USA 99 Suppl 4, pp. 16499-16506 (2002)]. The size of the silencing construct required is similar to that of used in the antisense method, there is no need for full homology, and multiple genes can be targeted at once, as well.

[68] The regulatory (promoter) elements controlling the expression of the above constructs may work constitutively, but can also be regulated. Stress induced transgene expression is described in response to wounding by Rizhsky L. and Mittler R. [Plant Mol. Biol. 46, pp. 313-323 (2001)], pathogen attack [Strittmatter G. et al., Bio/ Technology 13, pp. 1085-1089 (1995)], drought or ABA induction [Su J. et al., Plant Physiol. 117, pp. 913-922 (1998)]. Regulated expression has the obvious advantage that any secondary effect that would reduce the fitness of the whole plant is less pronounced.

[69] Similarly favorable way of transgene expression is tissue specific expression. Promoter sequences have been described that drive gene expression specifically in cambium tissue by Tuominen H. et al. [Plant Physiol. 123, pp. 531-541 (2000)], in phloem by Ordiz M.I. et al. [Proc. Natl. Acad. Sci. USA 99, pp. 13290-13295 (2002)], or in the guard cells of stomata (KAT1 promoter) by Nakamura R.L. et al. [Plant Physiol 109(2), pp. 371-374 (1995)]. The advantage of the method is again the reduction of undesired secondary effects to the non targeted parts of the plant.

[70] Homologous recombination-According to the known genomic sequence, a DNA construct can be designed that is capable of specific integration into the target region bringing about targeted mutation in the gene. By this method several favorable kind of mutations may be created, such as loss of function, dominant negative, altered substrate specificity, etc. Apart from the exon and intron sequences, 5' and 3' noncoding regions, as well as other regulatory regions (promoter, enhancer or other) may be used. In plants so far the efficiency of this method is low, however it is expected that it can be improved in the future [Offriga et al., Transgenic Res. 1, pp. 114-123 (1992), Vergunst and Hooykaas, Crit. Rev. In Plant Sci. 18, pp. 1-31 (1999)].

[71] Oligonucleotide chimeras.Targeted mutation can be obtained by introducing short DNA or RNA nucleotides into the cell that are homologous to the target sequence. Efficiency of this method is also low at present [Hohn and Puchta, Proc. Natl. Acad. Sci. USA 96, pp. 8321-8323 (1999)].

[72] Other methods that may be used to perturb gene expression are discussed by Jen and Gewirtz [Stem Cells 18, pp. 307-319 (2000)]. These include triple helix forming oligonucleotides, DNA binding molecules such as lexitropins or polyamines, used to block the transcription of a gene; DNAzymes, hammerhead or hairpin ribozymes that bind and cut the mRNA sequence specifically [Xicai et al., Proc. Natl. Acad. Sci. USA 94, pp. 4861-4865 (1997)].

[73] There are a number of alternative ways to abolish the expression or function of a gene. These include blocking or inactivating a regulator (usually a transcription factor) of the gene, or inhibit a modificator (e.g. kinase, phosphatase) of the protein targeted. Blocking agents (peptides or else) can be introduced into the cell thereby preventing the targeted protein from function. Dominant negative forms of a protein can also be expressed [Larsen and Cancel, Plant J. 34, pp. 709-718 (2003), Jin et al., Plant Cell 15, pp. 2357-2369 (2003); Pimpl et al., Plant Cell 15, pp. 1242-1256 (2003)]. A dominant negative variant of a protein may disturb the function of the target polypeptide by binding to it [Ferrario S. et al., Plant Cell 16, pp. 1490-1505 (2004)], or alternatively, similar effect can be achieved by binding or changing the substrate of the protein [Niki T. et al., Plant Physiol. 126, pp. 965-972 (2001)].

[74] Recombinant antibodies may also be expressed in plant cells [Conrad and Manteuffel, Trends in Plant Sci. 6, pp. 399-402 (2001)], where the antibody can recognize and inactivate the antigene that may be a protein or other.

[75] Once the DNA construct intended to abolish the expression of a target gene has been introduced into the plant cell, it may be needed to propagate and select those cells. To achieve this, the transgenic cells are selected on appropriate media, then grown into calli by tissue culture methods. Shoot development is induced from the calli on appropriate media, followed by regeneration of the whole plant. Certain parts of the plant (e.g. buds) can be transformed directly by Agrobacterium at a competent developmental stage. In this case, seeds are selected to obtain transgenic progeny. Transgene constructs may be linked to selectable markers in order to differentiate between transformed and wild type genotypes. Useful markers are different antibiotics (e.g. kanamycin, G418, bleomycin, hygromycin, chloramfenicol, etc.) or some herbicides (e.g. BASTA). Components of DNA constructs introduced may derive from the same host plant (endogen), or from foreign organism (exogen origin). 'Foreign' means that the DNA sequence is not found in the genome of the host plant. Heterologous constructs contain at least one region of foreign origin.

[76] The presence of a transgene in the plant can be verified by Southern blot experiments, by using methods known to the skilled artisan. The protein product of the gene expression can be detected in several ways, among them by Western blot and by enzymatic assays. An especially preferred method to quantify gene expression and to detect replication is the use of marker genes, such as GUS, followed by enzymatic detection. Once transgenic plants are obtained, they can be propagated and harvested, and their seeds may be collected. Heritable alterations have been made in their genome that all offspring will carry. In appropriate genetic context, the tissues and organs affected will show the desired phenotype. The introduced transgenes and genetic alterations may be transferred into appropriate recipients by conventional crossings.

[77] In addition to the targeted transgenic constructs, destruction of a gene can be achieved by random mutagenesis as well. With some of these methods it is easier to introduce mutations, but this advantage is balanced by the more difficult selection procedures to obtain the desired, in our case drought tolerant, mutants. Methods for random mutagenesis are well known to the person skilled in the art. Non-limiting examples include irradiation, chemical mutagenesis, treatment with modified nucleotides, etc. [Negrutiul., In vitro mutagenesis., pp. 19-38., in: Plant Cell Line Selection, Dix, P.J. (ed.), VCH Verlag GmbH. (1990)]. Random mutagenesis can be done by using mutator DNA constructs with known sequence. A well known example for this is T-DNA mutagenesis, where large scale plant transformation is used to generate a mutant population [e.g. Chen et al., Distribution and characterization of over 1000 T-DNA tags in rice genome., Plant J. 36, pp. 105-113 (2003)]. According to the nature of the individual integration events and the introduced T-DNA, this population may contain mutants with loss of function, gain of function, or mutants with altered functions [Alonso et al., Genome-wide insertional mutagenesis of Arabidopsis thaliana., Science 301, pp. 653-657 (2003), Sessions et al., Ahigh- throughput Arabidopsis reverse genetics system., Plant Cell 14, pp. 2985-2994 (2002), Jeong et al., T-DNA insertional mutagenesis for activation tagging in rice., Plant Physiol. 130, pp. 1636-1644 (2002)]. Similarly, transposons may be used to obtain mutant populations. In this case, the transposons may be endogenous or foreign (heterolog) to the species, and can be introduced either by one of the transformation methods outlined above or traditional sexual crossing [May et al., Maize-targeted mutagenesis: A knockout resource for maize., Proc. Natl. Acad. Sci. USA 100, pp. 11541-11546 (2003), Parinov and Sundaresan, Functional genomics in Arabidopsis: large-scale insertional mutagenesis complements the genome sequencing project., Curr. Opin.Biotechnol. 2, pp. 157-161 (2000)].

[78] Selection of the mutant with improved drought tolerance from the random mutagenized population can be achieved by direct screening for the trait, or in one of the following ways. If we already possess an induced transgenic mutant obtained by any of the ways described above, it may help in the selection procedure. As we mentioned, in addition to the improved drought tolerance, the morphology of the cbp20 mutant is also slightly changed compared to wild type. These visible pleiotropic traits may aid in selecting the mutant from a large mutant population, with more effective screening of the visible phenotype. Pleiotropic effects may vary among species, which can be tested by a transgenic mutant created by silencing. While similar pleiotropic phenotypes may result from different mutant backgrounds (see serrate and cbp20 mutants in example 1), more than one mutants are expected after this screening. This results, however, in a small mutant population which is easier to examine thoroughly than the original, large population. This method is also usable if the random mutagenesis was carried out in a traditional, non transgenic way. The drought tolerant mutant selected from this population has high commercial value as it contains no foreign DNA, which makes public acceptance easier.

[79] The desired mutant may be selected from the mutant population created by random mutagenizer DNA constructs by several way. PCR reactions may be performed based on the sequences of the target gene (including promoter and other regulatory regions) and that of the mutator sequence [for example see: Krysan et al., Identification of transferred DNA insertions within Arabidopsis genes involved in signal transduction and ion transport., Proc. Natl. Acad. Sci. USA 93, pp. 8145-8150 (1996)]. Screening may be accomplished with degenerate primers if the exact sequence of the target gene is unknown, but a characteristic motif is chosen (e.g. RNA binding, kinase domain, etc.) [Young et al., Efficient screening of Arabidopsis T-DNA insertion lines using degenerate primers., Plant Physiol. 125, pp. 513-518 (2001)]. In the individuals of the mutant population, sequences flanking the mutator DNA may be amplified by PCR. From among these sequences the gene of interest can be selected. For an overview of the method see Maes et al., Plant tagnology Trends in Plant Sci. 4, pp. 90-96 (1999). For examples see Jones et al., UGT73C6 and UGT78Dl-glycosyltransferases involved in flavonol glycoside biosynthesis in Arabidopsis thaliana., J. Biol. Chem., Aug 4 [Epub] (2003), Schumann and Wanner, AthPEXlO, a nuclear gene essential for peroxisome and storage organelle formation during Arabidopsis embryogenesis., Proc. Natl. Acad. Sci. USA 100, pp. 9626-9631 (2003)].

[80] A further possibility to select a particular mutant from a mutant population is the use of the TILLING method [(McCallum et al., Targeting Induced Local Lesions IN Genomes (TILLING) for plant functional genomics., Plant Physiol. 123, pp. 439-442 (2000)]. In this case, the targeted gene is specifically amplified from individuals of a random mutagenized population, then a wild type target sequence hybridized. With the help of an enzyme specifically cutting mismatched heteroduplex DNA, individuals carrying point mutations or small sequence changes in the gene of interest can be selected.

[81] Other methods to distinguish between mutant and wild type allels of a particular gene on large number of samples are: single strand conformation polymorphism (SSCP), carbodiimides, denaturing gradient gel elecfrophoresis, methods using RNaseA, etc. [for a review see: Prosser, J., Detecting single base mutations, TIBTECH 11, pp. 238-246 (1993)].

[82] The methods presented here may be used in any plant where a functional variant of the CBP20 gene is present.

[83] The present invention will be further exemplified by way of the following non- limiting examples. Example 1

[84] Isolation, genetic analysis, and drought resistance of the cbp20 mutant

[85] Wild type Arabidopsis (ecotype Columbia) have been randomly mutagenized by Agrobacterium infiltration with a T-DNA construct [pPCV6NFHyg; Koncz, C, Mayerhofer, R., Koncz-Kalman, Z., Nawrath, C, Reiss, B., Redei, G.P. έs Schell, J., Isolation of a gene encoding a novel chloroplast protein by T-DNA tagging in Arabidopsis thaliana., EMBO J . 9, pp. 1337-1346 (1990)], creating a mutant plant population. Hygromycin resistant transformants were grown, their seeds were harvested. Using phenotypic screening of approx. 10,000 T2 progeny, a plant line has been selected showing more compact stature than wild type, slightly delayed development and serrated leaf margins (Figure 1). The mutant was fertile and in most characteristics very similar to the wild type.

[86] A mutant with similar leaf morphology had already been described in the literature [serrate; Prigge, M.J. and Wagner, D.R., The Arabidopsis SERRATE gene encodes a zinc-finger protein required for normal shoot development., The Plant Cell 13, pp. 1263-1279 (2001)]. By crossing the two mutants, however, wild type progenies were observed in the FI generation (Figure 2). Consequently the two mutations were assessed nonallelic.

[87] In order to further characterize the mutant genetically, it was backcrossed with wild type Arabidopsis. The FI generation consisted of plants with wild type phenotype, while in the F2 generation the plants segregated at a 38(mutant):188(wild type) ratio. Under the conditions used, wild type Arabidopsis germinated at 95%, while the homozygous mutant at 65% frequency. These data corresponded to a single recessive mutation, causing the phenotype observed. To follow the inheritance of the T-DNA, genomic DNA was prepared from the F2 plants by the method of Edwards [NAR 19, p. 1349 (1991)] and T-DNA specific PCR reactions were performed.

[88] To amplify an appropriate piece of the T-DNA the following primers were used:

[89] 5'-CCTGAACTCACCGCGAC-3' (SEQ. ID. NO. 3)

[90] 5'-GCTCATCGAGAGCCTGC-3' (SEQ. ID.NO. 4)

[91] All mutants and 2/3 ^rd of the phenotypically wild type plants tested (24 out of 24 and 16 out of 24 plants, respectively) contained the T-DNA insertion (Figure 3). Thus the cpb20 mutant carried a single T-DNA insertion which co-segregated with the mutant phenotype.

[92] The plant genomic sequences flanking the T-DNA have been cloned by cutting the genomic DNA with an appropriate restriction enzyme (Hindlll), religating and transforming it into competent Escherichia coli. The plasmid construct rescued has been sequenced with T-DNA specific primers. This way in vitro was found out that the T-DNA was inserted into the second exon of gene At5g44200 on chromosome 5, which codes Cap Binding Protein 20. The T-DNA substituted a 36 base sequence between base pairs 62147 and 62183 of the MLNl PI clone. The integrated large piece of T-DNA disrupts the transcription and translation of the gene at this point. In the mutant only the first exon and part of the second exon can be transcribed. The produced truncated mRNA is degraded, we could not detect it in our Northern experiment (Figure 4).

[93] The mutant plants were tested under different biotic and abiotic stresses. They showed considerable differences from wild type under water deprivation stress. Mutant and wild type plants were sawn and grown under normal conditions for 8 weeks then they were left without watering. The phenotype of the mutants were markedly different from wild type after approx. 6-8 days of drought (Figure 5); they tolerated water deprivation better. As mutants develop somewhat slower than wild type, in the above experiment we used plants of approx. the same developmental stage, but at slightly different age. By using plants of exactly the same age however, similar results could be achieved. Example 2

[94] Production of drought tolerant plant by T-DNA mutagenesis

[95] In a plant species with a functional homolog of CBP20 (identified e.g. by known genomic or EST sequence), large scale T-DNA mutagenesis is performed. Sequences flanking T-DNAs are determined, and integration into a CBP20 homologous gene (or into its regulatory region, e.g. promoter) is selected. Among the progenies of the selected plant lines homozygous cbp20 mutants are found. They are tested whether they indeed show improved drought tolerance. Example 3

[96] Production of drought tolerant plant by gene silencing based on inverted repeats

[97] From a plant species with a functional homolog of CBP20 (identified e.g. by known genomic or EST sequence), an appropriate piece of the homologous gene is cloned. From this sequence an inverted repeated construct is made. This construct is functionally linked to a promoter, which is active in the target species. The activity of the promoter may be constitutive, tissue specific (e.g. in the guard cells of stomata) or stress induced (e.g. by drought). Plants are transformed with the construct, and CBP20 silenced mutants are produced. The mutant is characterized whether it displays pleiotropic phenotypic traits, whether it is suitable for cultivation in the greenhouse or in the field, and whether it indeed shows improved drought tolerance. Example 4

[98] Production of transgene-free drought tolerant plant by random mutagenesis and targeted selection

[99] If in a plant species a functional homolog of CBP20 can be identified, and the cbp20 mutant phenotype is known in this species and it is discemable from wild type, then transgene-free cbp20 mutant can be obtained.

[100] Random mutagenesis is performed by conventionally used mutagens (e.g. irradiation, chemical mutagenesis). The mutant population screened phenotypically for the visible traits of the cbp20 mutant, whereby a small number of mutants will be selected. This small population is screened extensively for water stress resistance, yielding the transgene-free drought tolerant mutant. Example 5

[101] Production of drought tolerant plant by random mutagenesis and targeted selection by molecular method

[102] In a plant species with a functional homolog of CBP20 (identified e.g. by known genomic or EST sequence) random chemical or irradiation mutagenesis is performed resulting in a mutant plant population. DNA is prepared from individuals of this population. From these DNA samples the CBP20 homologous gene is amplified by PCR reactions, based on the sequence of the gene. Homozygous or heterozygous cbp20 mutant plants are selected from these samples by the TILLING method. Among the progenies of the selected lines will be homozygous cbp20 mutant plants with drought resistance phenotype.

Claims

[I] A plant with improved drought tolerance that is transgenic and/or mutagenized chemically and/or physically, wherein the function of the Cap Binding Protein (CBP20) gene or a homologue thereof is decreased or blocked due to genetic manipulation and/or mutagenization.

[2] The plant according to claim 1, wherein the function of the CBP20 gene or a homologue thereof is decreased or blocked by addition, deletion and/or substitution of one or more nucleotide within the regulatory and/or coding region of the gene.

[3] The plant according to claim 1, wherein the function of the CBP20 gene or a homologue thereof is decreased or blocked by introduction of a recombinant construct that leaves the regulatory and/or coding region of the gene intact.

[4] The plant according to any of claims 1 to 3, wherein the said plant is dicotyledonous.

[5] The plant according to any of claims 1 to 4, wherein the said plant belongs to the order of Brassicales.

[6] The plant according to any of claims 1 to 5, wherein the said plant belongs to the family of Brassicaceae.

[7] The plant according to any of claims 1 to 6, wherein the said plant is Arabidopsis

[8] The plant according to any of claims 1 to 3, wherein the said plant is mono- cotyledonous. [9] A cell, tissue, organ, part, seed, fruit, product, progeny or propagation material of the plant according to any of claims 1 to 8. [10] A recombinant nucleic acid construct comprising a nucleic acid inhibiting or blocking the CBP20 gene upon its expression, operably linked to a regulatory sequence capable of directing transcription in a plant.

[I I] The construct according to claim 10 comprising a nucleic acid sequence encoding the CBP20 gene or a homologue thereof in its entirety or a part thereof in antisense direction with respect to the regulatory sequence.

[12] The construct according to claim 10 comprising a nucleic acid sequence encoding the CBP20 gene or a homologue thereof in its entirety or a part thereof in sense direction with respect to the regulatory sequence.

[13] The construct according to claim 10 comprising a nucleic acid sequence encoding the CBP20 gene or a homologue thereof in its entirety or a part thereof in an inverted repeat arrangement.

[14] A method for the production of a plant with improved drought tolerance comprising the preparation a mutagenized plant population by random mu- tagenezis; selection of the mutagenized plants by molecular biological techniques capable of recognizing mutations within the CBP20 gene or homologues thereof; and optional sexual or asexual propagation of the resulting plants with improved drought tolerance. [15] A method for the production of a plant with improved drought tolerance comprising the preparation a mutagenized plant population by introducing a nucleic acid construct suitable for the mutagenesis or functional blocking of the CBP20 gene or homologue thereof; selection of the mutagenized plants with improved drought tolerance; and optional sexual or asexual propagation of the resulting plant with improved drought tolerance. [16] The method according to claim 15, wherein a construct according to any of claims 10 to 13 is used for the mutagenesis or functional blocking of the gene. [17] The method according to any of claims 14 to 16, wherein the mutation within the CBP20 gene or homologue thereof is preferably detected hybridization, sequencing, PCR, SSCP, and/or TILLING. [18] The method according to any of claims 14 to 17, wherein during the selection step the plant population is screened in advance for visible pleiotropic features being characteristic of plants comprising a mutant CBP20 gene or a homologue thereof.