HU227099B1 - Amelioration of stress tolerance of plants - Google Patents

Abstract

The present invention provides an isolated nucleic acid molecule comprising a sequence encoding a protein that, upon expression in a plant, confers tolerance to osmotic stress. Also provided transformation vectors, transgenic plants and plant propagation materials comprising the said sequence, and a method for producing a transgenic plant that is tolerant to osmotic stress conditions and/or has improved germination. The use of a nucleic acid molecule according to the invention is also provided.

Description

The present invention relates to an isolated nucleic acid molecule comprising a protein coding sequence which, when expressed in a plant, produces tolerance to osmotic stress. The invention also relates to transformation vectors, transgenic plants, plant propagation material containing the sequence, and to a method of producing a transgenic plant which is tolerant to osmotic stress and / or has improved germination. The invention also relates to the use of a nucleic acid molecule according to the invention.

Environmental stress activates the expression of plant genes through abscisic acid (ABA) -dependent and ABA-independent signaling pathways. The hormone ABA plays an important role in regulating the resting state of the nucleus, the osmotic stress response, and water loss. ABA affects seed drying, maturation, salt, cold and drought tolerance (Leung and Giraudat, 1998, Finkelstein et al., 2002). Several mutants and genes that influence ABA signaling in model Arabidopsis and other plants have been described in the last decade (Finkelstein and Rock, 2002). Characterized genes include type 2C protein phosphatases (ABI1 and ABI2: Leung et al., 1994, 1997, Meyer et al., 1994), transcription factors (ABI3: Giraudat et al., 1992, ABI4: Finkelstein et al., 1998, ABI5: Finkelstein and Lynch, 2000), GTP-binding proteins (GPA1: Wang et al., 2001), FIERY inositol polyphosphate phosphatase (Xiong et al., 2001a) and ABA-dependent protein kinases (e.g. AAPK: Li et al., 2000).

In recent years, several RNA-binding proteins have been shown to play a role in ABA-mediated stress response or seed germination. CAP binding ABH1 protein negatively regulates the ABA response of stoma cells (Hugouvieux et al., 2001, 2002). SAD1 is similar to Sm proteins involved in messenger RNA maturation (Xiong et al., 2001b). The HYL1 protein has a dsRNA-binding property and regulates ABA, cytokinin and auxin-induced gene expression via micro RNAs (Lu and Fedoroff, 2000, Han et al., 2004).

Several patents disclose plant genes controlling resistance to environmental stress. These include ABH1 (WO0196585), ABI4 (WO9955840, WO00149850), ABI5 (WO02077163), ABF genes (US Patent Nos. 6194559, 6245905, 6218527, 6232461). ), the osmotic stress and ABA-induced endokitinase gene family (EC 3.2.1.14) (U.S. Patent 5,656,474), and the farnesyl transferase gene (WO9906580).

A specific group of RNA-binding proteins are those composed of pentatricopeptide domains (PPRs) characterized by a 35 amino acid repeat sequence (Small and Peeters, 2000). PPR sequences are recognized as RNA-binding domains that play a role in RNA stabilization and splicing of RNA (Bárkán et al., 1994, Manthey et al., 1995, Small and Peeters, 2000). Around 450 such genes encoding PPR domain proteins have been identified in the Arabidopsis genome. However, very little information is available on the function of genes or proteins. In the last few years some publications have been published describing the properties and functions of some PPR genes and proteins. Different PPR genes may play a role in cytoplasmic male sterility (Desloire et al., 2003, Nakamura, et al., 2003, WO002088179), in the regulation of circadian clock (Oguchi et al., 2004) in the transcription and translation of genes encoded in plastis (Bárkán et al. ., 1994, Meierhoff et al., 2003, Williams and Bárkan, 2003). Embryo lethality, reduced growth, and low fertility have been observed in the insertion mutant of several PPR genes, suggesting that these genes are involved in important developmental processes (Lurin et al., 2004). PPR proteins are localized in mitochondria and chloroplasts and may play a role in the regulation of RNA metabolism in organelles (Meierhof et al., 2003, Nakamura et al., 2003, Williams and Bárkán, 2003, Lurin et al., 2004).

For the purposes of the specification and claims, the technical terms are used in accordance with the following definitions. For the purposes of the present invention, it is obvious that the terms defined below are used in the sense of the definitions, even if these definitions are not fully consistent with the usual interpretation of the technical term.

By "gene" is meant a nucleic acid fragment that expresses mRNA, functional RNA, or a specific protein, including regulatory sequences. By "native gene" is meant a gene found in nature. By "transgene" is meant a gene that is introduced into the genome by transformation and stably stays there. For example, transgenes may be either heterologous or homologous to the genes of the particular plant to be transformed. In addition, transgenes may contain native genes for delivery to a non-native organism. By "endogenous gene" is meant a native gene in its natural place in the genome of the body.

By "coding sequence", "structural gene" or "structural region" is meant a DNA or RNA sequence that encodes a specific amino acid sequence and does not contain non-coding sequences. By "open reading phase" and "ORF" is meant the coding sequence between the translation start and end codons. The terms "start codon" and "end codon" are understood to mean three contiguous nucleic acid units ("codons") that determine the initiation and termination of protein synthesis (mRNA translation).

"Regulatory sequences" and "corresponding regulatory sequences are all within, within, or equal to the reading from the coding sequence upstream of (5 'non-coding sequence)

Nucleotide sequences in the direction of Β1 (3 'non-coding sequence) which affect the transcription, RNA maturation and stability, or translation of the associated coding sequence are meant. Regulatory sequences include enhancers, promoters, translational leader sequences, introns, and polyadenylation signal sequences. There may be natural and synthetic sequences as well as sequences that are a combination of synthetic and natural sequences. As noted above, the term "appropriate regulatory sequences" is not limited to promoters alone, but non-limiting examples of suitable regulatory sequences suitable for use in the invention include constitutive plant promoters, plant tissue-specific promoters, plant-stage specific promoters, inducible plant promoters and viral promoters.

By "3'region" is meant the 3'non-coding control sequences downstream of the coding sequence. This DNA may influence the transcription, RNA maturation and stability, or translation of the associated coding sequence (e.g., recombinase, transgene, etc.).

By "promoter" is meant a nucleotide sequence, generally upstream of the coding sequence (5 '), that regulates expression of the coding sequence by providing recognition of RNA polymerase and other factors necessary for correct transcription. A "promoter" may be a minimal promoter, which is a short DNA sequence containing a TATA box and other sequences for determining the origin of transcription to which regulatory elements for expression control are attached. By "promoter" is also meant a nucleotide sequence comprising a minimal promoter with regulatory elements capable of regulating the expression of the coding sequence or functional RNA. This type of promoter sequence consists of proximal and distal upstream elements, often referred to as enhancers. Accordingly, an "enhancer" is a DNA sequence that can stimulate promoter activity and may be a natural part of a promoter or may be a heterologous element inserted to enhance promoter levels or tissue specificity. The enhancer is operable in both orientations (normal or reverse) and works when positioned either upstream or downstream of the promoter. Both enhancers and other promoter elements bind sequence-specific DNA binding proteins that mediate their activity. Promoters may be entirely derived from a native gene, may consist of different elements from different naturally occurring promoters, or may even contain synthetic DNA sequences. A promoter may also contain DNA sequences involved in binding protein factors that regulate the efficiency of initiation of transcription, under physiological or developmental conditions.

By "homologue" or "variant" of a nucleic acid sequence is meant a sequence which is at least 50%, preferably at least 70%, more preferably at least 80%, more preferably 90%, and most preferably at least 95% identical to the sequence. That at least 50% of the sequences are homologous to each other can be determined, for example, by the FastDB program of the EMBL or SWISSPROT database. Of course, other algorithms known in the art and their computer implementation may be used to determine this homology.

An "homologue" or "variant" of an amino acid sequence is defined similarly to a nucleic acid sequence. Thus, a homologue or variant of an amino acid sequence refers to a sequence which is at least 70%, preferably at least 80%, more preferably at least 90%, more preferably 95%, and most preferably at least 98% identical to the sequence of interest. That the sequences are homologous to each other can be determined using the above mentioned programs and algorithms.

"Isolated" means a change from the natural state "by a human hand." If any "isolated" preparation or substance occurs in nature, it has been altered or removed from its original environment, or both. For example, a polynucleotide or polypeptide that is naturally present in a living animal is not "isolated," but said polynucleotide or polypeptide, which is separated from its co-existing material, is "isolated" as used herein.

A nucleic acid molecule is said to be "hybridizable" with another nucleic acid molecule if it is capable of specifically binding to the other molecule (i.e., binding produces a signal that is distinguishable from background noise and is unspecific to any randomly sequenced nucleic acid molecule); preferably, a nucleic acid molecule is considered hybridizable when it specifically binds to another nucleic acid molecule under stringent conditions.

A regulatory sequence is "operably linked" to a structural gene in a DNA construct if the regulatory sequence is capable of influencing the rate or mode of expression of the structural gene under conditions suitable for expression of the structural gene and operation of the regulatory sequence.

The terms "transformed", "transformant" and "transgenic" refer to plants or corns that have been subjected to genetic manipulation and contain a foreign gene integrated into their chromosome. The term "untransformed" refers to normal plants that have not undergone transformation.

As used herein, a "transgenic plant" is a plant that contains a heterologous polynucleotide in its genome. In general, the heterologous polynucleotide is stably integrated into the genome such that the polynucleotide is passed on to subsequent generations. The heterologous polynucleotide can integrate alone

EN 227 099 Β1 as part of the genome or recombinant expression cassette. As used herein, the term "transgenic" includes any cell, cell line, callus, tissue, plant, or plant part whose genotype has been altered in the presence of a heterologous nucleic acid, including transgenic plants that were originally modified in this way, and those by sexual crossing or was propagated from the initial transgenic plant. As used herein, the term "transgenic" does not include the genome (chromosomal or extra-chromosomal) by conventional plant breeding procedures or naturally occurring events such as random cross-fertilization, non-recombinant virus infection, non-recombinant transformation, non-recombinant transposition or spontaneous mutation.

The function of a gene is "increased", "overexpressed", or "upregulated" when its expression, transcription, splicing, translation or transport of the gene encoded protein, its incorporation into the membrane, its activation, its activity, or any other molecular, cellular, or whole plant level activity involved in the expression and function of the gene and the gene product encoded by it, relative to that of a wild type plant, such as about 120%, preferably about 150%, more preferably about 200%, more preferably above about 200%.

In plants, it is the result of adverse environmental conditions leading to "water osmotic stress". Drought and the high salinity of the soil are the primary causes of osmotic stress, which causes disturbance of water balance. During drought, the lack of water causes water scarcity, while high salt concentration prevents water uptake due to its high osmotic potential (Skriver and Mundy, 1990, Zhu et al., 1997, Hare et al., 1998, Wang et al. , 2003).

Environmental stress negatively affects plant growth and development. Adverse effects on seed germination inhibition, reduced growth and small size, morphological changes, etc. lead. Seed germination is particularly sensitive to environmental conditions, and germination rates are often used to characterize the stress sensitivity of a particular plant species, mutant or other genetic variant. The germination rate is usually expressed as the percentage of seeds germinated at a given time. The term "improved germination" as used herein refers to the germination of a larger number of seeds under the conditions described herein.

The present invention relates to an isolated nucleic acid molecule comprising a sequence encoding a protein of SEQ ID NO: 2 or a protein having at least 90% amino acid sequence identity, which protein expressed tolerance to osmotic stress.

In another embodiment, the present invention provides a nucleic acid molecule comprising at least 70% sequence identity to SEQ ID NO: 1.

In another preferred embodiment, the invention relates to an isolated nucleic acid molecule from a dicotyledonous plant. In another embodiment, the invention relates to an isolated nucleic acid molecule comprising a sequence derived from an Arabidopsis species.

In other preferred embodiments of the invention, the isolated nucleic acid molecules have a sequence of mitochondrial origin.

In another embodiment, the invention relates to a transformation vector operably linked to regulatory sequences of the above sequence.

In another embodiment, the present invention provides a transgenic plant that overproduces the nucleic acid molecule of the invention and which plant is tolerant to osmotic stress and / or has improved germination.

In another embodiment, the invention relates to a plant propagation material prepared from or using a plant of the invention comprising the nucleotide sequence of the invention.

The invention further relates to a method for producing a plant which is tolerant to osmotic stress and / or has improved germination, comprising introducing into a plant an expressable form of the nucleic acid molecule of the invention.

In addition, the present invention provides the use of a nucleic acid molecule of the invention for the production of a transgenic plant which is tolerant to osmotic stress and / or has improved germination.

We identified a T-DNA-labeled Arabidopsis mutant, designated salt 1, ABA, and sugar-sensitive (sasi) mutant, which results in hypersensitivity to treatment with high concentrations of salt, ΑΒΑ, glucose, and sucrose. T-DNA insertion disrupted the At3g16890 gene, which encodes an unknown mitochondrial protein (SEQ ID NO: 2) consisting of 14 repetitive pentatricopeptides (PPRs). The full-length cDNA of the SASI gene (SEQ ID NO: 1) was cloned and inserted into various plant expression vectors. In the sasi mutant, overexpression of SASI cDNA complemented the salt and ABA hypersensitivity of the mutant. Constitutive expression of the SASI gene increased the tolerance of transgenic Arabidopsis plants to high salt and ABA concentrations. Improved germination efficiency can be important for agricultural crops on salt soils or in other adverse environmental conditions. The results show that the SASI gene of Arabidopsis thaliana is important for adaptation to conditions under osmotic stress, and increased expression of the gene may help germination, growth and yield of the plant on saline soils.

The PPR gene family is particularly diverse in plants. In Arabidopsis, 441 PPR proteins have been identified but few have been characterized in detail (Lurin et al., 2004). The genome is sequenced4

EN 227 099 és1 and available EST data suggest that PPR proteins are present in similar numbers in other plants. None of the PPR proteins has been linked to stress responses so far. However, based on the specific details disclosed in the present invention, homologues of the SASI gene can be more easily searched and tested for the phenotype of the invention.

In preferred embodiments, a salt tolerant plant of the invention can be produced by introducing the SASI gene or by increasing the expression of the gene in the plant. Delivery of the SASI gene or enhancement of its gene expression can be achieved by introducing various DNA constructs into the plant. This is preferably accomplished using biologically functional transformation vectors.

Thus, the present invention provides a biologically operable transformation vector comprising regulatory sequences operably linked to a structural gene of the invention. Transformation into a host cell requires an independent vector capable of amplification, where an appropriate vector may be used depending on the host cell, the transformation mechanism, the function and the size of the DNA. A wide variety of vectors, as well as methods and expressions known to those skilled in the art, have been described and / or practiced in the art, see, 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 low molecular weight and may contain selectable genes that result in a readily recognizable phenotype in the cell, allowing for easy selection of vector-containing and vector-free host cells. To achieve high yields of DNA and related gene product, the vector must contain gene amplification signals and regulatory sequences. In addition, the origin of replication is important for autonomous vector replication. Polyadenylation sites are responsible for the correct processing of splice signals of mRNA and RNA transcripts. When phages, viruses, or virus particles are used as vectors, packaging signals control the DNA packaging of the vector.

As will be apparent to those skilled in the art, these regulatory sequences may be native or non-native to either the nucleic acid of the invention or the plant into which they are introduced. Although native sequences may have further advantages, it is routine practice to employ certain conventional regulatory elements known in the art to construct transformation vectors. Such elements have well characterized properties such as being constitutive or inducible, tissue or organ specific, germination specific, etc. These properties of non-native regulatory elements allow specific control of the transformation vector of the invention.

Numerous methods exist and are known to those skilled in the art for introducing DNA constructs into plant host cells [Potrykus I., Annu. Port. Plant Physiol.

Plant Mole. Biol. 42, 205-225. (1991). These methods include DNA transformation using Agrobacterium tumefaciens or A. rhizogenes as transforming agents, electroporation, particle acceleration, and the like. (see, for example, EP 295 959 and EP 138 341). Especially preferred is the use of binary vectors containing the TiI1R plasmid of Agrobacterium species. Ti-derived plasmids transform a wide variety of higher plants, including monocotyledonous and dicotyledonous plants such as soybean, cotton, rape, tobacco, and rice (Pacciotti et al., 1985); Byrne et al., 1987; Sukhapinda et al., 1987; Lorz et al., 1985; Potrykus et al., 1985; Park et al., 1995; Hiei et al., 1994). The use of T-DNA for the transformation of plant cells has been extensively studied and extensively described in EP 120 516; Hoekema, 1985; Knauf et al., 1983; and An et al. (1985).

Other transformation techniques are available to those skilled in the art, such as direct uptake of foreign DNA constructs (EP 295 959), electroporation techniques (Fromm et al., 1986), or high-speed ballistic bombardment of metal particles coated with nucleic acid constructs (Kline et al. , 1987, and U.S. Patent No. 4,945,050). The foreign DNA construct may be incorporated into the host plant chromosome during transformation procedures, but insertion of any gene into the plant genome may also be used to insert into the organelles, such as chloroplast, mitochondria, or any other organoleptic (see, e.g., Maligaat et al., 2003). , 2001).

There are many ways to deliver a nucleic acid molecule of the invention to a host cell under conditions that allow stable gene expression and expression. For example, expression cassettes may be prepared which contain a nucleotide construct of interest linked to a transcriptional and translational regulatory signal for expression of the nucleotide constructs and a nucleotide sequence homologous to, and thus integrated into, a nucleotide sequence that is homologous to and reservation occurs.

The primary cells containing the construct are then selected by the amplifiable gene or other marker present in the construct. The presence of the marker gene ensures the presence and integration of the construct into the host genome. If desired, the occurrence of the homologous recombination event may be determined by PCR and either by sequencing the resulting amplified DNA sequences or by determining the appropriate length of the PCR fragment when DNA from the correct homologous integrants is present and only cells containing such fragments are propagated.

HU 227,099 Β1

One skilled in the art will recognize that the expression level and regulation of a transgene in a plant may be significantly different for each line. In this way, it is advantageous for one skilled in the art to test a number of lines in order to find one that is appropriately expressed and regulated.

Once the DNA construct is introduced into the cell, it may be necessary to propagate and select the resulting transformed cells. To do this, the transgenic plant cells are placed in an appropriate selective medium to select the transgenic cells, which are then grown to callus. Shoots are grown from the callus and small plants are grown from the shoot, grown in rooting medium. Transformation can also be accomplished by direct transformation of certain organs (e.g., buds) of the plant with Agrobacterium, which can then be selected from the resulting seeds. The various constructs can generally be linked to a marker for selection in plant cells. Suitably, the marker may be biocidal resistance (especially antibiotic such as kanamycin, G418, bleomycin, hygromycin, chloramphenicol, herbicide, or the like). The marker used allows selection of transformed cells as compared to cells without the introduced DNA. Components of DNA constructs can be prepared from native (endogenous) or foreign (exogenous) sequences for the host. By "foreign" we mean that the sequence is not found in the wild type host into which the construct is introduced. Heterologous constructs contain at least one region that is not native to the gene from which the transcription initiating region is derived.

Southern blot analysis of transgenes in transgenic cells and plants can be performed using methods known to those skilled in the art. Expression products of transgenes can be detected in many ways, depending on the nature of the product, and include Western blot and enzymatic assays. A particularly suitable method for quantifying protein expression and detecting replication in various plant tissues is the use of reporter genes such as GUS and its enzymatic detection. Once transgenic plants are created, they can be grown into plants bearing plant tissues or portions of the desired phenotype. The plant tissues or portions may be harvested and / or the seeds collected. In the appropriate genetic environment, the tissues and organs involved may exhibit the desired phenotype. DNA constructs introduced into the plant host cell by the above methods may be transferred to suitable plants by conventional crossing.

Once transformed, the person skilled in the art can regenerate the cells. Of particular interest are the recently discovered methods for introducing foreign genes into commercially important crops, such as rape [De Block et al., Plant Physiol. 91.694-701. sunflower (Everett et al., Bio / Technology 5, p. 1201 (1987)), soybean (McCabe et al., Bio / Technology 6, p. 923 (1988)); Hinchee et al., Bio / Technology 6, p. 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] to rice (Hiei et al., Plant J. 6, 271-282). (1994)] and corn (Gordon-Kamm et al., Plant Cell 2, 603-618). p. (1990); Fromm et al., Biotechnology 8, 833-839. (1990).

BRIEF DESCRIPTION OF THE DRAWINGS The accompanying drawings are briefly described below

Figure 1 shows the insertion position and phenotype of the T-DNA localized in the sasi mutant. A) The At3g16890 gene is cleaved by repeated insertion of the T-DNA region of the pTAc16 vector at the 312 bp position of the gene, resulting in a 4 bp deletion. In the genomic sequences adjacent to the T-DNA, the deleted region was highlighted. The position of the PCR primers used to control the segregation of T-DNA is indicated by arrows. B) Control of At3g16890 gene expression in homozygous Sasi mutant and wild-type (Col-0) plants. The ubiquitin 10 gene was used as a control. C) Phenotype of 14-day in vitro germinated mutant (sasi) and wild-type (Col-0) plants. D) Morphology of 4-week-old mutant (sasi) and wild-type (Col-0) plants. E) 10-week-old mutant (sasi) and wild-type (Col-0) plants.

Figure 2 shows that Sasi mutant seedlings show increased sensitivity to salt, high sugar, and ABA treatment. A) Germination efficiency of wild-type (Col-0) and mutant (sasi) plants is high salt (100 mM, 150 mM, 200 mM NaCl), sugar (100 mM, 200 mM, 300 mM glucose), and ABA (0.1). μΜ, 0.2 μΜ in the presence of 0.5 μΜ). The percentage of germinated plants was recorded for 10 days. B) Phenotype of wild-type and mutant plants germinated on high salt, sugar and ABA medium.

Figure 3 shows the stress sensitivity of the Sasi mutant. A) Salt sensitivity of root growth of wild type and mutant plants. The seedlings were maintained on vertical agar plates and the root growth was recorded daily. The average daily root growth during the 10-day observation is shown in the figure. B) In vitro treated

Changes in fresh weight of plants at 100 mM, 150 mM, and 200 mM NaCl.

Figure 4 shows the expression of the SASI gene in wild-type Arabidopsis plants. By quantitative RT-PCR analysis, the SASI transcript was tested on cDNA templates from total RNA isolated from various organs.

Figure 5 shows the amino acid sequence of the SASI protein. The mitochondrial target polypeptide is underlined and the peptide cleavage site is framed (qryis). The sequence of the computer-labeled 14 PPR domains was aligned to indicate the conserved amino acids and the consensus sequence (CON). B) Structure of the protein domain of SASI. PPR domains have been highlighted. C) Expression of the pCaMV35S: HA-SAS1 and pCaMV35S :: SAS1-HA genes in transgenic Arabidopsis plants. Northern analysis was performed with a radiolabeled full length SASI cDNA probe. The Ubiquitin10 probe was used as a control. D) Localization of SASI-HA protein in mitochondria. T: Total protein extract, Mt: Mitochondrial protein extract, Ch: Chloroplast protein extract. The HA epitope-labeled SASI protein was detected by Ha-specific antibody by Western analysis.

Figure 6 shows the germination efficiency of the sasi mutant, the complement mutant and the SASI overexpressing lines. Germination efficiency was tested in control 1 / 2MS medium (A), 0.5 μΜ ΑΒΑ (Β), 200 mM NaCl (C) and 300 mM glucose (D). The percentage of germinated seeds is shown in the figure. Symbols:

Col / BC7: transgenic Col-0 overexpressing p35SCaMV35S :: SAS1-HA,

Col / BN5: transgenic Col-0 overexpressing p35SCaMV35S :: HA-SAS1,

Col-0: wild-type Col-0 plants, sas1 / BC5: complemented sasi overexpressing p35SCaMV35S :: SAS1-HA, sas1 / BN6: complemented sasi overexpressing p35SCaMV35S :: HA-SAS1, sasi: sasi mutant.

The invention will now be illustrated in more detail by the experimental examples described below, but it is in no way intended to limit the scope of the invention to the embodiments shown in the examples.

Example 1: Isolation of the Sasi mutant

As a result of our T-DNA insertion mutagenesis program, several insertion mutants with altered morphology and different reactions to environmental influences were isolated. In line h036, a small mutant was identified that was linked to the T-DNA insertion localized on chromosome 3 (Figure 1). The insertion inactivated the At3g16890 gene (see www.arabidopsis.org) and led to a loss of function mutation. RT-PCR analysis revealed that the At3g16890 gene transcript is present in wild-type plants but is absent in the mutant (Figure 1). The mutant was 50% smaller than the wild-type Col-0 plant at early stages of development (seedling, rosette), but the difference in size at the flowering stage became smaller or disappeared (Figure 1). The mutants were fertile and had similar seed yields to wild-type plants.

The At3g16890 gene consists of an exon, no intron, the 1980bp long coding region encodes a 659 amino acid 74 kDa protein. The structure of the SASI protein is similar to that of a large family of proteins containing the pentatricopeptide (PPR) domain. In the SASI protein, 14 PPR domains have been identified by bioinformatics tools (Figure 2), suggesting that the protein has RNA binding capacity. Further computer manipulation of tissue manipulation suggests mitochondrial or chloroplast localization.

Our T-DNA insertion mutagenesis program thus led to the discovery of a novel gene encoding the PPR domain protein. It was shown that the insertion resulted in a complete loss of function in the mutant.

Example 2: Sasi Mutant Hypersensitive to High Salt, Sugar and ABA Treatment The germination of the sasi mutant is slightly delayed in wild-type plants. In in vitro germination tests, germination of the Sasi mutant was much more sensitive to high salt, high sugar, or ABA treatments (Figure 2). Treatment with A150 mM NaCl delayed germination of wild-type plants by an average of 1.5 days and germination of the mutant by 3 days. 200 mM NaCl completely blocked the germination of the Sasi mutant, whereas most wild-type seeds germinated under these conditions after 10 days. Similarly, high sugar treatment inhibited the germination of the mutant better. 0.5 μΜ ABA treatment only slightly reduced the germination of wild-type Arabidopsis seeds, while inhibiting the germination of the mutants for nearly one week. The other growth hormones (auxin, cytokinin, ethylene, salicylic acid) and other environmental influences (light, dark, high temperature, heavy metal, oxidative stress) did not affect the germination capacity of the mutants.

The stress sensitivity of the plants was also tested in vegetative growth stage (rosette). The growth rate of the mutant roots was slightly below that of wild type plants in the 1/2 MS medium used. Treatment with 100 mM and 150 mM NaCl resulted in 65% and 75% less root growth for the mutants, respectively. 200 mM NaCl completely blocked mutant roots but only partially inhibited the growth of wild-type roots

EN 227 099 Β1 (Figure 3). Fresh weight gain in mutants was also more inhibited by saline treatment (Figure 3).

The stomatal closure of leaves is regulated by ABA signals, which depend on the water balance. In the case of water deprivation (decaying plants), the stomas are closed by the accumulation of ABA. The ABA sensitivity of mutants and wild-type stoma strains was compared in epidermal extracts. The diameter of the openings of stomas exposed to different concentrations of ABA treatment was measured. The stoma aperture of untreated mutants and wild-type plants was similar. After ABA treatment, the mutants had a smaller stoma opening than wild-type plants, i.e., mutant cells were more sensitive to ABA treatment than wild-type plants (Figure 3). These data confirmed that the Sasi mutant is more sensitive to ABA than wild-type plants, not only in the seedling state but also during vegetative growth.

In summary, the insertion mutant of Sasi has a lower, but normal, fertility than the wild-type plant. As a result of the mutation, the plant responds differently to different environmental stress factors. The germination and growth of the mutant is much more sensitive to high salt and sugar stress and to abscisic acid treatment.

Example 3: Expression of the SASI gene in wild-type plants

At3g16890 gene expression was first tested by Northern hybridization. However, the level of the At3g16890 transcript was so low that this technique could not be detected (data not shown). However, the more sensitive RT-PCR technique was able to detect the SASI transcript in the plant tissues tested. The SASI transcript was highest in green fruits and seedlings, but lower in roots, leaves, stems, and flowers. High salt and sugar treatment doubled or tripled the level of the SASI transcript (Figure 4). The activity of the SASI gene was not altered for auxin, cytokinin, gibberellin, brassinosteroid or salicylic acid treatment (data not shown).

Example 4: Overexpression of HA-SAS1 in wild type or mutant background

The full-length cDNA clone of the SASI gene was generated by PCR and verified by sequencing. The SASI protein was linked to the HA epitope to detect the protein by immunoassay. The HA-SAS1 construct was inserted into a pPCV-type transformation vector with the pCaMV35S promoter (Koncz et al., 1994, Figure 5). The vector carrying the gene construct was transformed by Agrobacterium into wild-type Arabidopsis and Sasi mutant plants (Bechtold and Pelletier, 1998). Northern hybridization in transgenic plants revealed elevated levels of the SASI transcript in 70% of the transgenic plants. Several transgenic plants expressing elevated levels were used for further studies. The HA epitope-labeled SASI protein was detected by Western blot analysis and chemiluminescence detection in transgenic plants containing the C-terminal HA epitope-labeled SASI protein (SASI-HA construct). No signal was obtained for the N-terminal HA-SAS1 gene construct.

Preliminary computer analysis suggested the mitochondrial or chloroplast localization of the SASI protein. The use of the HA epitope-tagged SASI protein allowed for localization testing. Whole cell protein extracts showed weak Western signaling in mitochondria, while chloroplasts showed no signal in the detection of C-terminal SASI-HA protein (Figure 5). The N-terminal construct did not produce any signal in the mitochondria either. As a result of Western analysis, it can be stated that the SAS 'protein has mitochondrial localization.

Example 5: Overexpression of the SASI gene improves stress resistance The stress sensitivity of SASI overexpressing plants was tested by seedling tests. Seeds of transgenic lines were germinated on different salt, sugar and ABA media and germination was recorded daily for 14 days. The slower germination ability of the Sasi mutant was fully complemented by SASI overexpression, the germination dynamics of the overexpression lines were similar to the wild type (Figure 6). The position of the HA epitope (N- or C-terminal) did not affect germination efficiency. The germination efficiency and speed of the overexpressing lines on the high-salt, sugar- and ABA-containing media far exceeded that of both the mutant and wild type plants. While 200 mM salt completely blocked germination of the Sasi mutant, SASI overexpressing plants showed better germination ability than wild-type Col-0 plants: such seeds germinated faster and in higher percentages. The improved germination ability suggests that SASI protein positively influences mitochondrial function under certain environmental stresses.

The presented data demonstrate that SASI cDNA is fully complementary to the stress susceptibility of the Sasi mutant. Overproduction of SASI results in ABA-insensitivity and an increased germination capacity of transgenic seeds. Our data clearly demonstrate that the SASI gene is a negative regulator of ABA-mediated stress responses and is important for adapting to certain adverse environmental effects.

The germination capacity of plants is the first and critical phase of plant development. Adverse environmental influences adversely affect the germination ability of crop plants, so any treatment or genetic modification that improves the germination capacity of these crops is of great importance for future crop yields. The SASI gene is overexp8

EN 227 099 Β1 has such a positive effect and positively influences the germination ability of crop plants.

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Claims (10)

PATENT CLAIMS An isolated nucleic acid molecule comprising a sequence encoding a protein of SEQ ID NO: 2 or having a protein having at least 90% amino acid sequence identity, which protein expressed tolerance to osmotic stress. 2. The nucleic acid molecule of claim 1, wherein the nucleic acid molecule is SEQ ID NO: 1 or at least 70% identical. The nucleic acid molecule of claim 1 or 2, which is derived from a dicotyledonous plant. 4. A nucleic acid molecule according to any one of claims 1 to 5, which is derived from an Arabidopsis species. 5. A nucleic acid molecule according to any one of claims 1 to 6, which is a mitochondrial sequence. Transformation vector which is 1-5. A nucleic acid molecule according to any one of claims 1 to 6 operably linked to regulatory sequences. 7. A transgenic plant which is selected from the group consisting of 1-6. A nucleic acid molecule according to any one of claims 1 to 6 which is tolerant to osmotic stress and / or has improved germination. The plant propagation material obtained from or using the plant of claim 7, which is from 1 to 6. A nucleotide sequence according to any one of claims 1 to 6. 9. A process for the production of a plant which is tolerant to osmotic stress and / or has improved germination. An expressable form of the nucleic acid molecule of claims 1 to 6 is introduced into the plant. 10. Use of a nucleic acid molecule according to any one of claims 1 to 6 for the production of a transgenic plant which is tolerant to osmotic stress and / or has improved germination.