METHOD FOR ENHANCING DROUGHT TOLERANCE IN PLANTS

FIELD OF THE INVENTION

The present invention relates to a method for increasing the tolerance to cold and salt stress of a plant by altering the expression of the gene RCI5. In particular, the present invention provides a transgenic plant has an increased level of the protein RCI5 regarding a non-transgenic plant, and the use of compositions containing TMAO to improved salt stress tolerance of plants affected by the application of solutions containing said compound by any means which contact the composition of the invention in any part of the plant and/or seeds.

STATE OF THE ART

Freezing temperatures are one of the major environmental factors that limit the growth, productivity and geographical distribution of plants (Boyer, 1982). Many temperate species have acquired along the evolution an adaptive response that allows them to cope with freezing temperatures. By this response, known as cold acclimation (Levitt, 1980), plants acquire a tolerance to freezing temperatures after an exposure time of non-freezing low temperatures. The cold acclimation is a representative example of the interaction of plants with their environment and how this interaction has influenced the evolution of some species. The elucidation of the molecular mechanisms that control this adaptive process is not only interesting from a basic point of view, to understand how to grow and develop the plants, but also has great biotechnological potential in obtaining molecular tools for improving the tolerance to the freezing of important crops. It should be noted that various studies have shown that acclimation to low temperatures also increases plant tolerance to other abiotic stresses such as dehydration and salt stress. Therefore, the study of cold acclimation should provide essential information on how plants respond in a coordinated manner to different adverse environmental conditions

The cold acclimation process is very complex and involves several changes to biochemical and physiological level, including changes in the membrane lipid (Uemura et al., 1995), synthesis of new proteins (Kawamura and Uemura, 2003), increased ABA endogenous content (Lang et al., 1994) and the accumulation of compatible solutes (Junttila and Wanner, 1999). There is growing evidence showing that Most of these changes are controlled through an extensive reprogramming of gene expression (Salinas 2002, Yamaguchi-Shinozaki and Shinozaki, 2006; Welling and Palva, 2006). In fact, recent studies using microarray technology have revealed that the expression of hundreds of genes changes in response to low temperatures, indicating that cold acclimation is mediated by multiple pathways (Fowler and Thomashow, 2002; Kreps et al. 2002, Seki et al., 2002, Maruyama et al., 2004, Vogel et al., 2005, Lee et al., 2005; Oono et al., 2006, Matsui et al. 2008; Chawade et al., 2007). Many of these genes are also regulated by dehydration and salinity (Matsui et al., 2008), confirming at the molecular level close relationship in the responses of the plant to these abiotic stresses. As an example this serves a group of genes that contain the motif CCGAC in its promoter, that is the central sequence of the regulatory element CRT/DRE (C-repeat/dehydration-responsive element) (Baker et al., 1994; Yamaguchi-Shinozaki and Shinozaki, 1994). CCGAC is sufficient to mediate the response of gene expression to cold, dehydration and salt stress (Baker et al. 1994; Yamaguchi-Shinozaki and Shinozaki, 1994), and interacts specifically with the family of transcription factors called CBF/DREB (C-repeat Binding Factor/DRE Binding protein) (Stockinger et al., 1997, Liu et al., 1998). Three genes of this family (CBF1/DREB1 B, CBF2/DREB1C, CBF3/DREB1A) are rapidly induced by low temperatures (Gilmour et al. 1998; Liu et al. 1998; Medina et al., 1999), whereas the expression of other two (DREB2A, DREB2B) is mainly due to dehydration and salt stress (Liu et al., 1998). Unfortunately, however, despite the large number of genes identified whose expression is regulated by cold, the metabolic cellular functions of most of them and their involvement in cold acclimation, are still unknown. The sequence analysis of some of these genes suggests that they should be involved in protecting the cell producing functional proteins, while other genes encode proteins with a role in the regulation of gene expression and in the transmission of the signal (Fowler and Thomashow, 2002; Kreps et al., 2002, Seki et al., 2002, Maruyama et al., 2004, Vogel et al., 2005, Lee et al., 2005; Oono et al., 2006, Matsui et al., 2008; Chawade et al., 2008). Functional proteins include enzymes for the production of compatible osmolyte, enzymes related with the quenching of reactive oxygen species (ROS), chaperones that protect proteins and membranes, and ion and water channels to maintain ion and water homeostasis. Among the regulatory proteins, as well as transcription factors, are RNA-binding proteins, calcium binding, protein kinases and phosphatases, enzymes involved in recycling phosphoinositol and other signaling molecules (Matsui et al., 2008). The characterization of the function of cold-regulated genes and their involvement in the acclimatization process should contribute to the understanding of the mechanisms that govern this process adaptive. Environmental abiotic stresses, such as freezing temperatures and salt stress are among the most important issues for sustainable agriculture worldwide. In recent years, great efforts have been made to understand at the molecular level the response to these stresses, which facilitated the identification of genes involved in tolerance to freezing temperatures and salt stress (Hasegawa et al., 2000; Zhu et al., 2007).

DESCRIPTION OF THE INVENTION
Brief Description

One aspect of the invention consists of a transgenic plant tolerant to cold and salt stress in transgenic plant later RCI5 of the invention, comprising a nucleotide sequence that allows the expression of a protein with monooxygenase (FMO), selected from the following group: i.—a gene nucleotide sequence RCI5 of SEQ ID NO: 1 or a fragment thereof, and ii.—a nucleotide sequence analogous to the sequence defined in a).

A particular aspect of the present invention consists of a transgenic plant of the present invention in which the sequence of nucleotides, which allows the expression of a protein with monooxygenase (FMO) is the RCI5 gene of SEQ ID NO: 1. A particular embodiment of this is the plant Arabidopsis transgenic RCI5-OE generated in the present invention (see Example 1.2 and Materials and Methods).

Another aspect of the invention consists of a method of producing transgenic plant RCI5, hereinafter method of producing a transgenic plant of the invention, which consists in the introduction into a plant a nucleotide sequence consisting of: i.—A nucleotide sequence encoding protein with monooxygenase (FMO), selected from the following group:—the sequence of nucleotides RCI5 gene of SEQ ID NO: 1 or a fragment thereof, or a nucleotide sequence analogous to the sequence defined in a), and ii.—a nucleotide sequence and regulates promoter allowing the expression of the sequence i.—and the appearance of a protein with monooxygenase activity inside the cell.

A particular embodiment of the present invention consists of a method for obtaining plants of the invention comprising the following steps: a) obtaining an expression vector containing a gene nucleotide sequence RCI5 SEQ ID NO: 1, b) obtaining microorganism carrying the vector a), c) transforming plants with the vector from a) or the microorganism b).

Another aspect of the invention consists of a nucleotide sequence, hereinafter sequence of the present invention, which allows the expression of a protein with monooxygenase activity in the plant cells and is constituted by one of the following possibilities: a) a nucleotide sequence encoding a protein with activity monooxygenase (FMO), selected from the following group: nucleotide sequence of RCI5 gene of SEQ ID NO: 1 or a fragment thereof, or a nucleotide sequence analogous to the sequence defined in a), with or without b) a promoter nucleotide sequence that enables and regulates the expression of the sequence of a) and the appearance of a protein with monooxygenase activity inside the cell. Another particular aspect of the present invention consists of a nucleotide sequence of the invention in which the sequence of nucleotides encoding a protein with monooxygenase activity consists of RCI5 nucleotide sequence of SEQ ID NO: 1, a fragment of the same or a nucleotide sequence analogous to the sequence defined above.

Another particular aspect of the present invention consists of a nucleotide sequence of the invention in which the sequence of nucleotides promoter is any promoter nucleotide sequence capable of regulating gene expression in plants such as, by way of illustration and without limiting the scope of the invention, a strong constitutive promoter such as the promoter mosaic virus 35S promoter, or a promoter that is expressed in response to various abiotic stresses, such as the RD29A gene (Kasuga et al, 1999). The invention also relates to an expression vector comprising the nucleotide sequence of the invention RCI5, hereinafter expression vector of the present invention, and that allows the transformation or transfection of cells and microorganisms and the subsequent production of transgenic plants of the invention. As particular embodiments of the expression vector of the invention, the vectors were generated GST::RCI5 (see Example 1) and the vector 35S::RCI5 (see example 2). Another aspect of the invention consists of a microorganism or cell, hereinafter cell of the invention, which contains the RCI5 nucleotide sequence of the invention or the expression vector of the invention. Another more particular aspect of the invention consists of a seed of a plant comprising a nucleotide sequence encoding a protein with monooxygenase activity, comprising RCI5 nucleotide sequence of SEQ ID NO: 1, a fragment thereof or nucleotide sequence analogous to the sequence defined above. Finally, another aspect of the present invention is the use of nucleotide sequences, expression vectors, transformed cells, and the method of the present invention for the production of transgenic plants of commercial interest.

Furthermore, another aspect of the invention is based on the use of a composition eg aqueous, comprising the compound trimethylamine oxide (TMAO) or a derivative thereof, hereinafter use of a composition of the invention, to induce tolerance salt and cold stress in plants and/or seeds.

Another particular aspect of the present invention is based on the use of a composition of the invention where the aqueous composition comprising the compound TMAO in a concentration between 1 μM and 100 mM, preferably between 50 μM and 1 mM, and more preferably 100 μM.

A particular embodiment of the present invention is the use of a composition of the invention in which the concentration of TMAO is 100 μM.

The concentration of the active components of the compositions will depend on the plant type, stage of development of the same, as well as the frequency and mode of application of the compositions.

Another particular aspect of the invention is the use of a composition of the invention to induce tolerance to salt stress in plants by applying a spray its top part.

The physiological transport of compounds absorbed by the leaves by spraying, are similar to those entering the absorption by the plants through the roots, however, the movement of compounds applied on the leaves is not the same in time and that is performed from the roots to the rest of the plant. Foliar absorption is most effective when the conditions of absorption from the soil are adverse for example in case of drought, salt stress, extreme temperatures or other stresses. Furthermore, it is much easier to obtain uniform distribution, unlike the application of granulated or physical mixtures. Another particular aspect of the present invention is the use of a composition of the invention to induce tolerance to cold and salt stress in plants by applying the stem injection.

The application of aqueous compositions by means of the immersion of the root portion of the plant and seed is performed for a time and at a concentration which depends on the plant type, stage of development and on the frequency and mode of application of the compositions. In the case of seeds which require special treatment or disposal scarification for easy germination, the immersion in the aqueous compositions of the present invention, can be made more effectively after the elimination of covers facilitate the absorption of the active compounds. Likewise, also the seeds may be immersed in any stage of germination.

Application forms that have been mentioned so far do not limit other applications of compositions containing TMAO plants.

Uses TMAO composition described above may further comprise an additive selected from organic or inorganic fertilizers, insecticides, nematicides, fungicides, bactericides and herbicides. Thus, by applying compositions containing TMAO together with additives, either to provide nutrients or to treat certain infections or pests, is achieved not increase the costs of treatments to do so coordinated.

DETAILED DESCRIPTION

The present invention addresses the problem of providing new transgenic plants able to adapt to environmental limiting situations, preferably in their tolerance to salt stress and cold, as well as need for new compositions for improving the tolerance of the plant stress.

The solution provided by this invention is based on that the inventors have isolated, identified and characterized a novel gene encoding a flavin-containing monooxygenase (FMO), hereinafter RCI5 (SEQ ID NO: 1), ie a new gene RC/, which is induced by salt stress and cold. Constitutive overexpression of the protein RCI5 (SEQ ID NO: 2) in Arabidopsis resulted in increased expression of different stress-inducible genes, including the CBF regulon and genes encoding proteins involved in the elimination of reactive oxygen species, whereby it is causes an improvement of the tolerance to the freezing and salt stress.

In animals, monooxygenases (FMOs) are involved in the synthesis of trimethylamine oxide (TMAO) that functions as an important osmoprotectant (Yancey et al., 2004). Additionally, data presented demonstrate for the first time, the plants also contain RCI5 TMAO and endogenous protein involved in biosynthesis of TMAO which is capable of oxidizing TMA to TMAO endogenous metabolite. Accordingly, the levels of TMAO in Arabidopsis RCI5 increased when the protein is overexpressed in response to lower temperatures and to NaCl. The RCI5 gene was identified looking for new genes in Arabidopsis involved in cold acclimation. Like other genes induced by cold, RCI5 gene is subject to complex regulation. Under controlled conditions during early developmental stages of Arabidopsis, its expression is very low and is limited to the vascular tissue of all tissues. In adult plants, however, only RCI5 is expressed at very low levels and, in the veins of the leaves. In response to low temperatures, the expression levels are induced RCI5 intensively, mainly in the leaves vasculature. This induction was not affected in mutants deficient in ABA or not expressing CBFs plants, indicating that the regulation of cold RCI5 is performed by signal transduction pathways CBFs and ABA independent. RCI5 is also induced by treatment with NaCl through an ABA-independent pathway. Expression levels induced by NaCl are identical to those caused by low temperatures. It is interesting that the effect of NaCl is not due to osmotic component but is specific ion. Dehydration, however, has no effect on the expression of RCI5. The Arabidopsis genome contains more than 50 genes whose expression is regulated, as RCI5, by low temperatures and NaCl but not by dehydration (Fowler and Thomashow, 2002; Kreps et al. 2002; Seki et al. 2002, Maruyama et al., 2004, Vogel et al., 2005, Lee et al., 2005; Oono et al.; 2006 Matsui et al., 2008), demonstrating the existence of a significant interaction pathways cold and salt signaling.

RCI5 constitutive overexpression significantly increases the freezing tolerance in Arabidopsis, before and after cold acclimatization, strongly suggesting that RCI5 positively regulates both the response to cold acclimation and constitutive freezing tolerance. Moreover, transgenic Arabidopsis plants overexpressing the protein RCI5 are significantly more tolerant to high concentrations of NaCl and LiCl that original wild plants, demonstrating that RCI5 acts as a positive regulator of salt stress tolerance in Arabidopsis. By contrast, it appears that the protein RCI5 is not involved in the tolerance to stress caused by the dehydration and treatment with mannitol. Therefore, the results of the present invention indicate that RCI5 not play a general role in the development of tolerance to abiotic stress in plants, but has a more specific function in tolerance to cold and salt stress, whichever corresponds with the fact that the expression of the protein RCI5 is induced in response to low temperature, NaCl and LiCl, but not by dehydration or by mannitol. Also, data of the present invention demonstrate that the protein RCI5 upregulates gene expression induced stress. In fact, under control conditions and/or stress (cold, salt stress) RCI5-OE plants show high levels of transcripts corresponding to different stress-inducible genes, among which are the CBFs, KIN1, LTI78, COR15A, COR47, CAT2 and SOD2. In contrast, the expression of RAB18 and RCI1A is not altered in RCI5-OE lines, demonstrating once again that the protein RCI5 operates not as a general positive regulator of gene expression induced by stress. On the other hand, it is important to be described that the expression of CBFs, KIN1, LTI78, COR15A, COR47, CAT2 and SOD2 is able to improve the survival to abiotic stress (Jaglo-Ottosen et al., 1998, Liu et al., 1998, Kasuga et al. 1999; Gilmour et al. 2000; Catala et al., 2003, Maruyama et al. 2004; Gilmour et al. 2004; Steer et al. 2004; McKersie et al., 1996; Van Breusegem et al. 1998; Apel and Hirt, 2004). Therefore, the increase of the tolerance to freezing temperatures before and after acclimation to cold, and salt stress shown by RCI5-OE plants can be attributed to high expression of these genes.

The gene encodes a protein RCI5 monooxygenase activity as evidenced by the characterization of the recombinant protein obtained and the analysis of the oxidation of NADPH in extracts of transgenic RCI5-OE plants. However, this protein contains all the characteristic motifs typical of FMO enzymes including a FAD binding point, a source identifier FMO and NADPH binding domain (Cashman, 1994). FMOs motifs have been described in various organisms, from bacteria to humans, eukaryotic origin being the best characterized. This motif binds FMO FAD cofactor and catalyzes the oxygenation of endogenous metabolites containing nucleophilic nitrogen, phosphorus, sulfur, or selenium at the expense of NADPH. In plants, has been described that FMOs proteins play a role in auxin biosynthesis in petunia and Arabidopsis (Zhao et al. 2001; Tobeña-Santamaria et al., 2002, Cheng et al., 2007). Furthermore, in Arabidopsis, the FMOs are required in the response of defense against pathogens (Bartsch et al., 2006, Koch et al., 2006; Mishina and Zeier, 2006) and have been implicated in glucosinolate biosynthesis (Hansen et al., 2007). The results described herein clearly indicate that these domains FMOs of the protein of the invention in RCI5 plants have a role in responses to abiotic stress, particularly in response to cold stress or salt stress. Finally, RCI5 presents a functional property of the plant responses to pathogens but should be investigated in the future. Lastly, exogenous application of compound TMAO partially reproduces the gene expression profiles induced by low temperatures and salt stress, and the phenotypes caused by the overexpression of RCI5 (see Example 3), indicating that the TMAO is a new signaling molecule in mediates the plants tolerance to cold and salt stress through the positive regulation of the expression of genes induced by stress. Note that the tolerance phenotypes induced by TMAO are very similar to those expressed by the transgenic plants RCI5-OE, as might be expected RCI5 to be involved in TMAO biosynthesis. In summary, the results presented in this invention are the first evidence that plants also contain TMAO metabolite, whose synthesis is induced by the protein RCI5 and transgenic plants RCI5-OE containing higher levels are more tolerant RCI5 cold and salt stress than wild plants and the application of the compound exogenous plants TMAO protects from cold and salt stress. Thus, one aspect of the invention consists of a transgenic plant tolerant to cold and salt stress in transgenic plant later RCI5 of the invention, comprising a nucleotide sequence that allows the expression of a protein with monooxygenase (FMO) selected from the following group: i.—RCI5 gene nucleotide sequence of SEQ ID NO: 1 or a fragment thereof, and ii.—a nucleotide sequence analogous to the sequence defined in a). In the sense used in this description, the term “analogous” intends to include any nucleotide sequence that may be isolated or constructed based on the sequence of nucleotides shown in SEQ. ID NO1, for example, by means of introducing conservative nucleotide substitutions or non-conservative, including the insertion of one or more nucleotides, the addition of one or more nucleotides at either end of the molecule or the deletion of one or more nucleotides at any end or inside the sequence.

Overall, a similar nucleotide sequence is substantially homologous to the nucleotide sequence identified as SEQ ID NO: 1. In the sense used in this description, the expression “substantially homologous” means that the nucleotide sequences in question have a degree of identity, at the nucleotide level, of at least 60%, preferably at least 85%, or more preferably at least 95%. A particular aspect of the present invention consists of a transgenic plant of the present invention in which the sequence of nucleotides, which allows the expression of a protein with monooxygenase (FMO) is the RCI5 gene of SEQ ID NO: 1. A particular embodiment of this is the Arabidopsis transgenic plant RCI5-OE generated in the present invention (see Example 1.2 and Materials and Methods). Furthermore, the present invention relates to a method of obtaining improvement of the plant tolerance to salinity and cold through genetic engineering of plants. The technique was developed in the plant model Arabidopsis thaliana and can be applied to plants of agronomic and commercial interest—including, by way of illustration and without limiting the scope of the present invention: rice, wheat, soy, corn, tomato, tobacco, bean as well as different species of fruit (orange, lemon, etc.)—by various genetic engineering techniques known to one skilled in the art. This is possible because the nucleotide sequences of orthologous or homologous to the sequence described in the present invention (SEQ ID NO: 1) are conserved in plants of agronomic interest and adequate technology for isolation and use in plant genetic engineering.

Nucleotide sequences homologous to the one described in this invention (SEQ ID NO: 1) can be present as homologous forms in other species of higher plants of commercial interest which may be naturally or otherwise, could also be the result of a genic transformation process wherein the transformed organism reproduces said DNA molecules. These homologous forms of the invention can be isolated, by conventional techniques from the DNA of any plant that hold back and by the use of probes or oligonucleotides, prepared thanks to the information of the nucleotide sequences of said DNA molecules provided herein, one of ordinary skill in the art.

Thus, another aspect of the invention consists of a procedure for obtaining transgenic plant RCI5, hereinafter method of producing a transgenic plant of the invention, which consists in the introduction into a plant a nucleotide sequence consisting of: i.—a nucleotide sequence encoding the protein with monooxygenase (FMO), selected from the following group:

- The sequence of nucleotides RCI5 gene of SEQ ID NO: 1 or a fragment thereof, or a nucleotide sequence analogous to the sequence defined in a), and ii.—A nucleotide promoter sequence that enables and regulates the expression of the sequence of i.—and the appearance of a protein with monooxygenase activity inside the cell.

The nucleotide sequences, expression vectors and transformed cells or microorganisms, developed and necessary for putting into practice the method of obtaining transgenic plants of the present invention as well as their use for the production of such plants, constitute additional aspects of the present invention.

Thus, another aspect of the present invention consists of a nucleotide sequence, hereinafter sequence of the present invention, which allows the expression of a protein with monooxygenase activity in the plant cells and is constituted by one of the following possibilities: b) a nucleotide sequence encoding a protein with activity monooxygenase (FMO), selected from the following group: nucleotide sequence Ia RCI5 gene of SEQ ID NO: 1 or a fragment thereof, or a nucleotide sequence analogous to Ia sequence defined in a), with or without b) a promoter nucleotide sequence that enables and regulates the expression of the sequence of a) and the appearance of a protein with monooxygenase activity inside the cell.

Another particular aspect of the present invention consists of a nucleotide sequence of the invention in which the sequence of nucleotides encoding a protein RCI5 with monooxygenase activity consists in nucleotide sequence of SEQ ID NO: 1, a fragment of the same or a nucleotide sequence analogous to the sequence defined above. Another particular aspect of the present invention consists of a nucleotide sequence of the invention in which the sequence of nucleotides promoter is any promoter nucleotide sequence capable of regulating gene expression in plants such as, by way of illustration and without limiting the scope of the invention, a strong constitutive promoter such as the promoter mosaic virus 35S promoter, or a promoter that is expressed in response to various abiotic stresses, such as the RD29A gene (Kasuga et al, 1999)

RCI5 the nucleotide sequence of the invention can be used, in general, in the generation of an expression vector that allows the expression of these proteins in a wide range of host cells.

Therefore, the invention also relates to an expression vector comprising the nucleotide sequence of the invention RCI5, hereinafter expression vector of the present invention, and that allows the transformation or transfection of cells and microorganisms and the subsequent obtaining transgenic plant of the invention. As particular embodiments of the expression vector of the invention, the vectors were generated GST::RCI5 (see Example 1) and the vector 35S::RCI5 (see example 2). In general, the expression vector of the present invention comprises at least a sequence of the invention and, at least, a promoter that directs the transcription of the gene of interest (in this case indicated as gene cDNA nucleotide sequence representative coding gene itself) that is operatively linked, and other necessary or appropriate sequences for the transcription of the gene of interest and proper regulation in time and place, for example, start and stop signals, splice sites, polyadenylation signal, replication origin, transcriptional activators (enhancers), transcriptional silencers (silencers), etc. Examples of appropriate expression vectors can be selected according to the conditions and needs of each individual case between plant expression plasmids (Rothstein et al, 1987; Potrykus, I, 1991), viruses (“Plant Virology Protocols” From Virus Isolation to Transgenic Resistance Edited by Gary D. Foster University of Bristol, Bristol, UK Published: 1998), which can also contain a bacterial or yeast origin of replication so that it can be amplified in bacteria or yeasts, as well as a marker usable for selecting the transfected cells different from the gene or genes of interest. For the transformation of plants can be used various methods—plasmid vectors, liposomes, electroporation, microinjection, etc.—described in various manuals (see for example: (Plant Gene Transfer and Expression Protocols Jones, Heddwyn University of Hertfordshire, Hatfield, UK. Human Press Publishers). The choice of vector will depend on the host cell and the plant in which it is to be introduced later.

Another aspect of the invention consists of a microorganism or cell, hereinafter cell of the invention, which contains the RCI5 nucleotide sequence of the invention or the expression vector of the invention.

Another more particular aspect of the invention consists of a seed of a plant comprising a nucleotide sequence encoding a protein with monooxygenase activity, comprising RCI5 nucleotide sequence of SEQ ID NO: 1, a fragment thereof or nucleotide sequence analogous to the sequence defined above. Finally, another object of the present invention is the use of the nucleotide sequences, expression vectors, transformed cells, and the method of the present invention for the production of transgenic plants of commercial interest. Furthermore, another aspect of the invention is based on the use of a composition eg aqueous, comprising the compound trimethylamine oxide (TMAO) or a derivative thereof, hereinafter use of a composition of the invention, to induce tolerance salt and cold stress in plants and/or seeds. Another particular aspect of the present invention is based on the use of a composition of the invention where the aqueous composition comprising the compound TMAO in a concentration between 1 μM and 100 mM, preferably between 50 μM and 1 mM, and more preferably at 100 μM. A particular embodiment of the present invention is the use of a composition of the invention in which the concentration of TMAO is 100 μM.

The concentration of the active components of the compositions will depend on the plant type, stage of development of the same, as well as the frequency and mode of application of the compositions.

Another particular aspect of the invention is the use of a composition of the invention to induce tolerance to salt stress in plants by applying a spray its top part.

Another particular aspect of the present invention is the use of a composition of the invention to induce tolerance to cold and salt stress in plants by applying the stem injection.

Another particular aspect of the present invention is the use of a composition of the invention which is applied to the soil or other growing media, irrigation water (or culture solution) or by dipping the root system of plants and/or seeds. The application of aqueous compositions by means of the immersion of the root portion of the plant and seed is performed for a time and at a concentration which depends on the plant type, stage of development and on the frequency and mode of application of the compositions. In the case of seeds which require special treatment or disposal scarification some roofs for easy germination, the immersion in the aqueous compositions of the present invention, can be made more effectively after the elimination of covers facilitate the absorption of the active compounds. Likewise, also the seeds may be immersed in any stage of germination.

Application forms that have been mentioned so far do not limit other applications of compositions containing TMAO plants.

Uses

TMAO composition described above may further comprise an additive selected from organic or inorganic fertilizers, insecticides, nematicides, fungicides, bactericides and herbicides. Thus, by applying compositions containing TMAO together with additives, either to provide nutrients or to treat certain infections or pests, is achieved not increase the costs of treatments to do so coordinated.

DESCRIPTION OF THE FIGURES

FIG. 1. RCI5 is transcriptionally regulated by cold and NaCl in leaves and flowers by ABA-independent pathway and CBFs.

In Northern hybridizations of the probe was used RCI5 and GUS. Each lane contained 20 μg of total RNA. As treatment control and loading corresponding probes were used to kin1 and 18S rRNA genes respectively. a) RNAs from plants grown for 3 weeks 2O° C. (C), or additionally exposed to 4° C. the times indicated. b) RNAs from roots, leaves, stems, flowers and siliques from plants grown at 8 weeks 2O° C. (C), or exposed additional 1 day at 4° C. c) RNAs from Ler plants, aba1-1, Col, CBF2, CBF1-AS3 3 weeks, grown to 2O° C. (C), further exposed to 1 day 4° C. (4° C.) or treated with NaCl 25O mM collecting the material one days later (NaCl). d) RNAs from three weeks plants grown at 2O° C. (C), exposed an additional day at 4° C. (4° C.), irrigated with NaCl 25O mM 24 h after collecting the material (NaCl), dehydrated to lose 50% of their fresh weight (D), irrigated with 2O mM LiCl (LiCl) or 50O mM mannitol (Mannitol) collecting the material at 24 h. e) RNAs from RCI5::GUS transgenic plants 3 weeks grown at 2O° C. (C), further exposed to 1 day 4° C. (4° C.) and watered with 25O mM NaCl (NaCl) collecting the material at 24 h. f) Location Ia histochemical GUS activity in seeds, seedlings and various organs of transgenic plants::GUS RCI5 8 weeks grown at 2O° C. (C), or exposed to an additional day 4° C. (4° C.). Dehydration in the response to the same pattern was obtained GUS activity than the control plants (C). In the case of NaCl treatment was obtained the same pattern as shown in plants exposed to 4° C. (4° C.).

FIG. 2. RCI5 has monooxygenase activity. a) Coomassie staining of SDS-PAGE gel at 12% containing the different fractions obtained by purifying RCI5. The first lane includes a bacterial extract containing the protein expression vector (pGEX), the second lane includes a bacterial extract containing the expression plasmid pGEX::RCI5 (pGEX-RCI5), the third lane includes the fusion protein GST::RCI5 after being purified through a glutathione sepharose 4B column (GST-RCI5), and the last lane contains RCI5 the protein purified from GST::RCI5 by digestion with thrombin (RCI5). Each lane contained 20 μg of protein. b) Measures monooxygenase activity protein fractions shown in panel (a). The graph shows the pmol of NADPH consumed per hour at 24° C. In all cases, experiments were carried out with 100 ug of protein. Data are mean±SE of three independent experiments. c) RCI5 mRNAs levels in transgenic 35S::RCI5. Northern blot hybridizations were performed with total RNA (20 ng) isolated from Arabidopsis plants 3 weeks and four independent transgenic lines (Q1-Q4) containing the fusion 35S::RCI5 (RCI5-OE) grown in control conditions. As loading control was used Ia probes corresponding to the 18S rRNA gene. d) The graph shows the NADPH consumed per hour at 24° C. in controlling Arabidopsis plants (WT) and four transgenic lines (1T-4T) containing the fusion 35S::RCI5 (RCI5-OE) grown in control conditions.

FIG. 3. 35S::RCI5 plants have increased tolerance to the freezing and salt stress that Col. a) Percentage of Col plants and transgenic 35S::RCI5 (1T1 line) grown for 2 weeks at 2O° C. to survive after being exposed to 6 h different freezing temperatures. Data are mean±SE of three independent experiments with a minimum of 50 plants of each genotype per experiment. b) Percentage of transgenic plants and 35S::Col RCI5 (1T1 line) grown for 2 weeks at 2O° C. and acclimated for 7 days at 4° C., surviving 6 h after being exposed to different freezing temperatures. Data are mean±SE of three independent experiments with a minimum of 50 plants of each genotype per experiment. c) transgenic plants and 35S::Col RCI5 (Line 1T1) grown 2 weeks at 2O° C. after being frozen at −6° C. for 6 h. d) and transgenic plants 35S::Col RCI5 (Line 1T1) grown 2 weeks at 2O° C. and acclimated for 7 days at 4° C., after being frozen at −8° C. for 6 h. e) Tolerance to NaCl, LiCl and mannitol. Number of green leaves and percentage of initial fresh weight in plants transgenic Col and 35S::RCI5 (1T1 line) exposed to increasing concentrations of NaCl (50 mM-200 mM), LiCl (5 mM-20 mM) or mannitol (100 mM-400 mM) during 2 days at each concentration with respect to the same parameters Col plants to which they have been applied no treatment. Data are mean±SE of three independent experiments, with a minimum of 20 plants of each genotype per experiment. f) Col plants and transgenic 35S::RCI5 (1T1 line) after exposure to increasing concentrations of NaCl (50 mM-200 mM), LiCl (5 mM-20 mM) or mannitol (100 mM-400 mM) for 2 days at each concentration.

FIG. 4. RCI5 Ia is involved in biosynthesis of TMAO in Arabidopsis.

The graphs show the TMAO pmol per kg of fresh weight. Data are mean±SE of five independent experiments and each experiment included at least 50 plants. a) Col plants grown at 20° C. for 3 weeks and 1 days exposed additional 4° C., irrigated with NaCl 25O mM collecting the material after 1 day or dehydrated to lose 50% of their fresh weight. b) Col plants and transgenic 35S::RCI5 grown at 2O° C. for 3 weeks. c) Plants transgenic cabbage and 35S::RCI5 grown at 20° C. and exposed for 3 weeks 1 day at 4° C. or irrigated with 250 mM NaCl collecting the material after one day. d) pmol of TMAO obtained by a reaction in vitro using different concentrations (50, 100 and 200 μM) of purified RCI5.

FIG. 5. The TMAO mediates the gene expression response to low temperatures. Northern blot hybridizations using probes specific for (a and c) kin1, LTI78, COR15A, COR47, RAB18, RCHA, CAT2, SOD2 and RCI5 (the latter only in c), (b and d) CBFJ, CBF2 and CBF3 and GUS. Each lane contained 20 μg of total RNA. In all cases, specific probes were used for each gene. As the load control was used for the probe to 18S rRNA. a) Northern blot hybridizations with probes aforementioned samples from Col plants (WT) and transgenic 35::RCI5 {RCI5-OE) grown for 3 weeks at 20° C. (C), further exposed to 1 day 4° C. 250 mM NaCl treated or collecting the material after 1 day. b) Northern blot hybridizations with probes aforementioned samples from Col plants (WT) and transgenic 35::RCI5 (RCI5-OE) grown for 3 weeks at 20° C. (C), exhibited an additional 3 hours at 4° C. 250 mM NaCl or treated with the material collected after 3 hours. c) Northern blot hybridizations with probes aforementioned samples from Col plants grown for 3 weeks at 20° C. (C), treated with 100 μM TMAO (TMAO), exposed at 4° C. and treated with 250 mM NaCl collecting the material After 1 day. d) Northern blot hybridizations with probes aforementioned samples from Col plants grown for 3 weeks at 20° C. (C), treated with 100 μM TMAO (TMAO), exposed at 4° C. and treated with 250 mM NaCl collecting the material After 3 hours.

FIG. 6. The TMAO increases the freezing tolerance Ia and salt stress. a) Percentage of cabbage plants grown at 2O° C. (C) and treated with 100 μM TMAO 3 days before the freezing (TMAO), non-acclimated (NA) and acclimated 7 days at 4° C. (A), which survive after being exposed 6 hours at −6° C. or −8° C., respectively. Data are mean±SE of three independent experiments with a minimum of 50 plants of each genotype per experiment. b) Percentage of number of green leaves in Col plants grown in MS (C) and Col grown on MS supplemented with 100 μM TMAO (TMAO), subsequently exposed to increasing concentrations of NaCl (50 mM-200 mM), LiCl (5 mM-20 mM) or mannitol (100 mM-400 mM) for 2 days at each concentration, relative to the number of green leaves in Col plants to which no treatment has been applied. Data are mean±SE of three independent experiments, with a minimum of 20 plants of each genotype per experiment. c) Col plants grown at 20° C. (C) and treated with 100 μM TMAO 3 days before the freezing (TMAO), non-acclimated (NA) and acclimated 7 days at 4° C. (A), which survive after being exposed 6 hours at −6° C. or −8° C., respectively. d) Col plants grown in MS (C) or MS supplemented 100 μM TMAO (TMAO) after exposure to increasing concentrations of NaCl (50 mM-200 mM), LiCl (5 mM-20 mM) or mannitol (100 mM-400 mM) for 2 days at each concentration.

FIG. 7. Genomic organization RCI5 a) Restriction map of the genomic DNA fragment containing the gene RCI5. Indicates the cleavage sites of the restriction enzymes EcoRI (E), XbaI (X) and Hind III (H). ATG indicates the start of translation RCI5 and gray rectangles their coding regions. Line denotes the region used as a probe. b) Southern blot hybridization with 5 μg of Arabidopsis genomic DNA digested with EcoRI (E), Xba I (X) and HindIII (H) using the probe of RCI5. To the right are indicated the sizes (in kbp) of the bands obtained.

FIG. 8. Measures of TMAO in Arabidopsis plants exposed to cold or salt stress

Representative graphs showing TMAO pmol per kg fresh weight of plants grown Col three weeks at 20° C. and exposed at 4° C. for different times (0, 6, 12, 24, 48 and 72 hours) (a) or exposed to different concentrations of NaCl (50, 100, 150, 200, 250, and 300 mM) for 1 day (b).

FIG. 9. TMAO measures in plants treated with TMAO

Representative graphs showing TMAO pmol per kg fresh weight of plants grown Col three weeks at 20° C. and treated with various concentrations of TMAO during one day (0, 10, 50, 100 and 200 μM) (a) or TMAO 100 μM treated for different times (0, 6, 12, 24, 48, and 76 hours).

EXAMPLES OF THE INVENTION
Example 1
Isolation and Identification of Gene and the Protein of the Invention RCI5
1.1. Identification of a cDNA Encoding the Protein RCI5 Induced in Response to Low Temperature and Salt Stress

RCI5 (At1g12140) was isolated by screening a library of cDNAs prepared from the Arabidopsis etiolated seedlings cold acclimated (4° C., for 3 days) with a subtractive probe enriched in cold-induced transcripts. Comparing this cDNA (SEQ ID NO: 1), hereinafter RCI5 with other genomic sequences revealed that the RCI5 gene contains five exons and four introns (SEQ ID NO: 3). The analysis of the expression of this gene in Arabidopsis etiolated seedlings by Northern hybridizations with specific probe confirmed that, in fact, its expression is induced in response to low temperatures (FIG. 1a). RCI5 transcripts accumulate transiently during the exposure to 4° C., peaking at 12 h after the onset of treatment. In seedlings grown under control conditions, the RCI5 transcript levels are very low (FIG. 1a). The specificity of the probe was determined by Southern hybridizations (FIG. 7b). When we studied the expression of RCI5 in mature plants (FIG. 1b), at 4° C. the induction was detected mainly in leaves but there was a little induction of flowers. RCI5 messenger accumulation experienced no apparent changes in either roots or stems or siliques exposed to cold (FIG. 1b). In non-stress conditions, were detected very low levels of transcripts RCI5 in all organs except siliques (FIG. 1b). RCI5 induction in response to low temperatures was not affected in aba1-1, a mutant deficient in ABA (Koornneef et al., 1982), cbf2-1, a null mutant for the gene CBF2 (Steer et al. 2004), and CBF1-AS3, a transgenic line of Arabidopsis that do show cold induction of CBF1 or CBF3 (Novillo et al., 2007) (FIG. 1c), suggesting that RCI5 is regulated by low through an signalling pathway independent of ABA and CBFs. Since, as mentioned above, many cold-inducible genes also respond to other abiotic stresses, we examined the effect of the dehydration and salt stress in the expression of RCI5. Accumulation of RCI5 mRNAs was observed only after treatment with NaCl (FIG. 1d). To determine if this accumulation was caused by ionic component or osmotic of the treatment with NaCl, RCI5 expression was studied in Arabidopsis plants exposed to LiCI, since Li⁺ is a cation closely related toxic Na⁺ ion, and mannitol, which mimics the osmotic stress without ionic stress toxicity. The results revealed that RCI5 was induced by LiCI but not mannitol (FIG. 1d), indicating that the ionic component was responsible for the accumulation of RCI5 transcripts in response to salt stress. As in the case of low temperatures, the induction of RCI5 in response to NaCl appears to be mediated through an ABA-independent pathway (FIG. 1c). KIN1 was used as a positive control in all experiments.

RCI5 expression at the tissue level during the development of the plant and in response to different treatments was studied by the analysis of Arabidopsis plants containing a fusion of a 782 by fragment RCI5 promoter (from −770 to +12 from the ATG) to the uidA reporter gene (GUS). Examined five independent transgenic lines containing a single copy of the transgene homozygous. In all cases, the accumulation of mRNAs of GUS in leaves exposed to 4° C. or NaCl showed a very similar pattern to that observed for RCI5 endogenous transcripts (FIG. 1e). These results suggest that the isolated promoter fragment contains all the cis regulatory elements involved in the induction of RCI5 in response to low temperature and salt stress, and that this induction is regulated at the transcriptional level. The histochemical analysis of GUS activity showed no staining in the transgenic seeds (FIG. 1f). Under not stress only very low levels of GUS staining during germination and early stages of development in the vascular bundles of roots, hypocotyls, cotyledons and leaves were detected (FIG. 1f). In fully developed plants, GUS staining was slightly noticeable in the veins of the leaves (FIG. 1f). When transgenic plants were exposed to low temperatures or germination in NaCl, GUS staining was as in unstressed plants but significantly stronger (FIG. 1f). Only adult transgenic plants showed high levels of GUS activity in the vascular tissue, although slight staining on flowers (base and veins of the sepals) and siliques (abscission) (FIG. 1f). In transgenic lines subjected to dehydration, the pattern of GUS activity was, in all cases, as obtained under control conditions (FIG. 1f). Taken together, these data demonstrate that the expression of RCI5 is regulated during development of Arabidopsis and is induced in response to cold, salt stress, especially in the vascular tissue of the leaves.

1.2.—RCI5 the Gene Encodes a Protein with Flavin Monooxygenase Activity

The RCI5 cDNA sequence (SEQ ID NO: 1) encodes a polypeptide of 459 amino acids (SEQ ID NO: 2), with an estimated molecular weight of 52 kDa and a pI of 6.1. Database searches revealed that RCI5 said polypeptide or protein contains motifs typical of an enzyme flavin-containing monooxygenase (FMO) (Cashman, 1994). In fact, RCI5 has a highly conserved FAD-binding site, identifying a domain FMO and NADPH binding domain that is characteristic of the enzymes FMO.

RCI5 protein is an enzyme with FMO which was analyzed firstly investigating its ability to oxidize NADPH, having been expressed in Escherichia coli and purified (FIG. 2a). The results showed that, in fact, RCI5 has monooxygenase activity. Purified GST::RCI5 fusion protein exhibited enhanced ability to oxidize NADPH than extracts from E. coli containing empty plasmid or RCI5 cDNA (2 and 8 times, respectively) (FIG. 2b). Subsequently RCI5 monooxygenase activity was analyzed in vivo using transgenic Arabidopsis plants constitutively overexpressing the protein RCI5 (transgenic plants RCI5-OE). To determine the monooxygenase activity four separate homozygous lines with a single copy of the transgene and that showed high expression levels of RCI5 in control conditions were identified (FIG. 2c). RCI5 overexpressing plants grew and developed in an identical manner to the wild plants under normal conditions. In all cases, these extracts RCI5-OE transgenic lines showed an ability to oxidize NADPH significantly higher than wild plant extracts (FIG. 2d). Thus, we conclude that the protein is an enzyme RCI5 FMO activity.

Example 2
RCI5 Transgenic Plants of the Invention Exhibit Increased Tolerance to Cold and Salt Stress Ia which is Mediated by Increased Levels of the Protein Produced by TMAO RCI5
2.1.—The Overexpression of the Protein RCI5 Increases Tolerance of Plants, Arabidopsis, to Cold Stress and Saline

The freezing tolerance of two weeks-old OE-RCI5 and wild-type plants before and after acclimatization to cold (4° C., 7 d), was quantified as the percentage of surviving plants after having been exposed for 6 h at different temperatures freezing. Dehydration was induced maintaining 10 days-old transgenic and wild-type seedlings on a filter paper for 2 days. Stress tolerance was quantified as the percentage of initial fresh weight (FW) remaining after treatment. Finally, tolerance to salt stress was estimated by determining the fresh weight and the number of leaves of 10 day-old OE-RCI5 transgenic plants and wild-type cultivated for 8 days in media with increasing concentrations of NaCl (50 to 200 mM). RCI5 overexpression significantly increased freezing tolerance of Arabidopsis to both nonacclimated and acclimated plants 7 days at 4° C., and this increase is very similar in both cases. As all RCI5-OE transgenic lines showed similar tolerance phenotypes, only the results of the line Q1 are shown (FIGS. 3a, b). LT50 values (temperature which causes a 50% mortality) of non-acclimated plant and transgenic wild-OE RCI5 estimates were −5.4 and −6.5° C., respectively (FIG. 3a). LT₅₀values of wild plants and RCI5-OE acclimated to cold were −7.5 and −8.7° C., respectively (FIG. 3b). Increased tolerance of RCI5-OE lines before and after acclimatization was very apparent at the phenotypic level (FIG. 3c, d). There was no difference between wild plants and RCI5-OE when analyzed dehydration tolerance (data not shown). However, all of RCI5 overexpressing lines showed more tolerance to salt stress than wild plants. Tolerance phenotypes displayed by different RCI5-OE lines were nearly indistinguishable due to which only shows the results corresponding to line 1T (FIG. 3e). The RCI5-OE transgenic plants exposed to salt stress had a greater number of green leaves and wild FW unstressed plants (eg, about 120% in both cases; FIG. 3e), indicating that the overexpression of the RCI5 gene is sufficient to overcome the stress caused by NaCl treatment. In addition, wild plants were only 80 and 70% of green leaves and fresh weight in control conditions presented (FIG. 3e). The same results were obtained when wild type and transgenic RCI5-OE plants exposed to increasing concentrations of LiCl (5 mM, 20 mM, FIG. 3e). By contrast, RCI5-OE transgenic and wild-type plants exposed to increasing concentrations of mannitol (100 mM-400 mM) showed very similar values of number of green leaves and FW (FIG. 3e). The differences found between tolerance of RCI5-OE transgenic and wild-type plants to NaCl and LiCl treatments were very clear at the phenotypic level (FIG. 3f). Taken together, these results demonstrate that constitutive overexpression of the RCI5 gene increases Arabidopsis tolerance to freezing and salt stress, due to its ionic component suggesting that the RCI5 protein acts as a positive regulator of the response of a cold Arabidopsis salt stress.

2.2.—The RCI5 Protein of the Invention (SEQ ID NO: 2) is Involved in Biosynthesis of Trimethylamine Oxide (TMAO)

The resulting question was how the protein RCI5 in Arabidopsis could regulate responses to cold stress or saline. Numerous studies indicate that one of the physiological functions of the FMO is osmoregulación. It could be as in animals have shown that these enzymes oxidize endogenous metabolite trimethylamine (TMA) to TMAO (Ziegler, 1993; Schelenk Larsen, 2001), an osmolyte powerful facilitating the adaptation of various marine organisms to environments with saline and freezing temperatures (Yancey et al. 1982; Schelenk Larsen, 2001; Seibel and Walsh, 2002; Treberg et al., 2005; Strambini and Gonnelli 2008). Therefore we decided to investigate whether the RCI5 protein was involved in biosynthesis TMAO. Thus, TMAO was detected in Arabidopsis growing under controlled conditions. The results revealed that, in fact, unstressed Arabidopsis plants have very low, but detectable (˜5 mol kg ^″1FW) of TMAO (FIG. 4a). Is noteworthy, however, that when plants were exposed to salt stress and low temperatures, the two treatments that induce the expression of the gene RCI5 (FIG. 1d), the TMAO content increases significantly. Thus the exposure to 4° C. or 250 mM for 24 h resulting in an accumulation of TMAO to values close to 25 to 35 mol kg ^″1FW, respectively (FIG. 4a), which are maximum levels of endogenous TMAO is detected in the stress conditions (FIG. 8). The dehydration stress, which has no effect on the expression of RCI5 (FIG. 1d), does not affect the levels of TMAO (FIG. 4a).

The identification of endogenous TMAO in Arabidopsis and the existence of a correlation between the expression of the protein RCI5 and TMAO content was analyzed by determining the contents of this molecule in the transgenic lines RCI5-OE. Clearly, all plants overexpressing the gene RCI5 (RCI5-OE) showed significantly higher levels of TMAO (˜2 fold) than the original wild-type plants (FIG. 4b), indicating that the RCI5 protein is involved in TMAO biosynthesis. These levels, however, were lower (3-4 times) than those determined in wild plants exposed to cold or salt treatment (FIG. 4a), which strongly suggests that other FMO enzymes in addition to RCI5 have to be involved in TMAO biosynthesis in response to these stresses. Likewise, transgenic plants under RCI5-OE 4° C. or 250 mM NaCl for 24 hours exhibited higher (˜2 fold) amounts of TMAO than wild plants exposed to the same treatment (FIG. 4c).

A complete and definitive evidence of involvement of RCI5 protein in TMAO biosynthesis was obtained by in vitro assays which analyzed the capacity of RCI5 to oxidize TMA to TMAO. The results confirmed that the protein of the invention RCI5 purified from E. coli was capable of oxidizing TMA in a dose dependent manner, generating substantial amounts of TMAO (FIG. 4d). These data demonstrate that Arabidopsis plants contain constitutive levels of TMAO, that these levels are significantly increased in response to low-temperature stress and saline, and that this increase is due in part by the induction of the expression of RCI5.

3.—Compound Average TMAO Cold and Salt Tolerance in Arabidopsis
3.1.—Compound Average TMAO Cold and Salt Tolerance in Arabidopsis by the Positive Regulation of the Expression of Stress-Responsive Genes

The results described so far suggest a role of the protein RCI5 in osmoregulation through biosynthesis of TMAO. However, greater accumulation of TMAO in Arabidopsis subjected to cold stress or salt (25-35 mol Kg ^″1FW) appears to mediate an adjustment low osmotic in these conditions. Indeed, this concentration is approximately three times of magnitude lower than that described for TMAO in animals (Larsen and Schlenk, 2001, Seibel and Walsh, 2002) and for different osmolytes as proline and glycine betaine, in plants (Wood et al. 1996; Russell et al. 1998; Sakamoto and Murata, 2000.) is proposed that, at low concentrations, certain osmolytes can exert its protective role against abiotic stress through different mechanisms osmoprotection (Chen and Murata, 2002). For example, it has been suggested that proline and glycine betaina could regulate gene expression (Rajendrakumar et al. 1997; lyer and Caplan, 1998; Kavi Kishor et al., 2005; Mattioli et al., 2008). Therefore, we examined the possibility that the TMAO could act as a signaling molecule mediating the induction of gene expression in response to stress.

RCI5 since the protein is involved in the synthesis of TMAO (see above), we first studied the levels of transcripts from different genes whose expression is induced by stress in the transgenic lines RCI5-OE grown in control conditions or exposed to low temperatures or salt stress. Among the genes studied included KIN1, LTI78, COR15A and COR47, which are induced in response to various abiotic stresses through dependent and independent pathways of CBFs (Gilmour et al., 2004, Maruyama et al., 2004), RAB18 and RCI1A, which are upregulated by cold separate means of SFBC (Lang and Palva, 1992; Jarillo et al. 1994; Gilmour et al., 2004, Maruyama et al., 2004, Lopez-Cobollo and Salinas, unpublished results), and CAT2 and SOD2 two genes whose expression increases in response to various stresses and encoding proteins involved in detoxification of ROS (Refs; Kliebestein et al. 1998; Chevalier et al., 1992). Also studied genes levels CBFs (CBF1-3), whose expression is specifically promoted by low temperatures (Gilmour et al., 1998, Liu et al., 1998, Medina et al., 1999). As the expression patterns of all RCI5-OE transgenic lines analyzed were very similar only the results obtained with line Ia 1T (FIGS. 5a, b). Under control conditions, the accumulation of transcripts corresponding to kin1, COR15A, COR47, and especially those of CAT2 and SOD2 were considerably higher in RCI5-OE than in wild-type plants (FIG. 5a). The expression of the other genes studied were not affected by RCI5-OE (FIGS. 5a, b). The induction of all genes, except RAB18 and RCI1A in response to cold was higher in plants overexpressing RCI5 (FIG. 5a, b). Under salt stress, all NaCl-responsive genes (eg, kin1, LTI78, COR15A, COR47, CAT2 and SOD2, see these genes in Ia FIG. 5a) showed a greater induction RCI5-OE plants (FIG. 5a,b). These expression patterns fully justify the increasing in freezing tolerance before and after cold acclimation and salt stress of RCI5-OE lines (FIG. 3). Therefore, we conclude that the RCI5 protein upregulates the response of stress gene expression, suggesting that TMAO could indeed mediate the induction of stress gene expression and, therefore, tolerance of Arabidopsis the freezing and salt stress.

3.2.—The External Application by Spraying of TMAO Induces Tolerance to Cold and Salt Stress

If the TMAO acts as a signaling molecule and half the induction of gene expression by stress, exogenous application of Arabidopsis plants under control conditions should induce the stress-regulated gene expression. This hypothesis was verified by examining the accumulation of transcripts corresponding to the previously analyzed genes in Arabidopsis wild plants sprayed with 100 uM TMAO, a concentration that after 24 h of treatment, causes an accumulation of endogenous TMAO in the same range detected in plants exposed to low temperatures and salt stress (30-35 mol kg ^″1FW; FIG. 9). Except in the case of RAB18 and RCI1A, TMAO exogenous activated the expression of all genes to levels similar to those reached after the exposure to 4° C. and NaCl (FIGS. 5c, d), confirming the TMAO induction of stress gene expression. It was also studied RCI5 mRNA accumulation in response to TMAO. Results obtained indicate that the induction by cold and salt of RCI5 gene is not mediated by TMAO (FIG. 5c). Finally, it was studied if TMAO also mediated tolerance of Arabidopsis to the freezing and salt stress. For this, wild Arabidopsis plants were sprayed with 100 uM was determined TMAO and the freezing tolerance before and after acclimatization to cold and the tolerance to dehydration and to salt stress. Consistent with what has been described previously, the exogenous application of TMAO significantly increased tolerance of Arabidopsis to freezing, both as non-acclimated and acclimated plants, salt stress (FIGS. 6a, b), but had no effect on Arabidopsis ability to tolerate the dehydration. It was also studied LiCl and mannitol tolerance of Arabidopsis plants treated with TMAO. TMAO increased tolerance of Arabidopsis to LiCI but not mannitol (FIG. 6b), indicating that, as the protein RCI5 is specifically involved in ionic component tolerance of salt stress. Phenotypic differences between treated and untreated plants TMAO are evident (FIG. 6c, d). Taken together, these results indicate that the molecule TMAO acts as a signal mediating the tolerance of Arabidopsis to salt stress freezing and upregulating the induction of gene expression.

Materials and Methods

Plant Material, Growth Conditions and Treatments

In this study was used as plant Arabidopsis thaliana (L.) ecotype Columbia (Col) and Landsberg erecta (Ler), mutants aba1-1 (ref) and CBF2-1 (Steer et al., 2004), as well as CBF1-AS3, a transgenic line of Arabidopsis CBF1 and CBF3 Ia which are induced by cold (Steer et al., 2007). For obtaining seedlings, seeds were planted in sterile conditions in Petri dishes with MS medium (Murashige and Skoog, 1962) supplemented with 250 mg/1 MES, solidified with 0.8% agar (w/v). The plants were grown in pots containing a mixture of the organic substrate with vermiculite (3:1, v/v), and irrigated with water and a mineral solution (Haughn and Somerville, 1986). Both seedlings and plants, is growing at 21±1° C. in long-day conditions (16 hours of white fluorescent light, photon flux of 70-90 mol m^{“2 sec” 1}).

Expression analyzes were performed using three weeks plants, except in the analysis in different organs of plants which used 8 weeks. Low temperature processing plant was transferred to a growth chamber at 4±1° C. for different periods under conditions of light and photoperiod described above. For treatments with NaCl, LiCl and mannitol plants were irrigated with NaCl 25O mM, 2O mM 50O mM LiCl and mannitol respectively, and collected one day later. Dehydration was induced leaving plants, previously separated from their root systems, lose 50% of its FW. For TMAO treatments, plants were sprayed with 100 μM TMAO and collected one day later. Control TMAO treatment plant was obtained by spraying with water. In all cases, plants were immediately frozen with N₂after treatment and stored at −8O° C. until use.

TMAO always endogenous plant was measured 3 weeks. Plants were sprayed with 100 μM TMAO and collected after different times of treatment, or sprayed with different concentrations (10, 50, 100 and 200 μM) of TMAO and collected one day later. Well watered plants with different concentrations (50, 100, 150, 200, 250, and 300 mM) NaCl and harvested the following day. The effect of low temperatures on the content of endogenous TMAO exposing plants to study 4° C. for various times. After collecting them, always plants were frozen in N₂and used immediately.

Freezing experiments were conducted with plants 2 weeks as described previously (Novillo et al., 2004). Tolerance was established as the capacity of plants to grow after 14 days of recovery in control conditions. Salt tolerance was analyzed transferring seedlings or days 1 medium supplemented new plates with increasing concentrations of NaCl (50, 100, 150, and 20O mM) or LiCl (5, 10, 15 and 2O mM). Osmotic stress imposed transferring seedlings 10 days media plates supplemented with increasing concentrations of mannitol (100, 200, 300, and 40O mM). In all cases, the plants were kept 2 days at each concentration. Tolerance to these treatments was evaluated by determining the fresh weight and number of green leaves at the end of treatment. Dehydration was induced by removing the seedlings 10 days of the growth medium and placing them into a wet filter paper, and allowed to grow for 2 days without water. Dehydration was estimated as the percentage of initial FW after treatment. To analyze the effect of TMAO in the freezing tolerance. Plants were sprayed with 100 μM 2 weeks TMAO 1 h before freezing or cold acclimated as described previously. The effect of TMAO on Arabidopsis tolerance to NaCl, LiCI, mannitol and dehydration germinating seeds was studied in medium plates supplemented with 100 μM TMAO. Then 10 days the seedlings were processed as described above.

Methods of Molecular Biology

RCI5 was isolated by screening a cDNA library prepared from Arabidopsis etiolated seedlings of cold acclimated with a subtractive probe enriched in cold-inducible transcripts following protocols previously described (Jarillo et al. 1994). For sequence similarity analysis program was used Basic Local Alignment Search Tool (BLAST) of the National Centre for Biotechnology Information (Altschul et al., 1997). Digestion with restriction enzymes, the clonings, the extraction of total proteins in plants, and RNA and DNA hybridizations were performed according to standard procedures (Sambrook et al., 1989). Total RNA was isolated as has previously been reported (Logemann et al., 1987).

RCI5 the complete cDNA was obtained by RT-PCR of total RNA from Arabidopsis ecotype Col plants exposed to 4° C. for 24 hours, using the primers SEQ ID NO: 4 and SEQ ID NO: 5. The resulting fragment was filled in with Klenow and ligated into the Sma1 site of pBluescript SK (+) (Stratagene Cloning Systems, La Jolla, USA) to obtain plasmid pBSRCIδ. For the fusion GST::RCI5, pBSRCIδ plasmid was digested with BamHI and SalI. Then, the fragment containing the cDNA RCI5 filled in with Klenow and ligated into the SmaI site of the pGEX-4T1 vector (GE Healthcare Bio-Sciences, Piscataway, N.J.).

To express RCI5 in E. coli, transformed the strain BL21 (DE3) pLysS (Invitrogen, Carlsbad, Calif., USA) with the pGEX-4T1 vector containing the fusion GST RCI5. RCI5 purification of E. coli was performed according to protocol Ia commercial house, inducing the expression in culture for 4 hours at 28° C. with 0.1 mM IPTG. PGEX-4T1 vector was used as control.

RCI5 specific probe was generated by PCR on Arabidopsis ecotype Col cDNA using primers SEQ ID NO: 6 and SEQ ID NO: 7. Kin1 specific probes, LTI78, COR47, COR15A, RCHA, RAB18, CBF1, CBF2, CBF3, GUS and SOD2 were obtained as previously described (Kliebenstein et al. 1998; Steer et al., 2007;). CAT2 specific probe was obtained by PCR on Arabidopsis genomic DNA using primers SEQ ID NO: 8 and SEQ ID NO: 9. As loading control was used EcoRI fragment of 18S rRNA (Tremousaygue et al., 1992). RNA samples from each experiment were analyzed in at least two separate membranes, and each experiment was repeated at least 3 times.

Obtaining Transgenic Plants

PCR amplified a 0.77 kb fragment immediately prior to the ATG RCI5 Ia coding region, using as template genomic DNA of Arabidopsis ecotype Col and primers SEQ ID NO: 10 and SEQ ID NO: 11. Promoter fragment was cloned into the Sma1 site of pBluescript SK (+). Subsequently, the plasmid was digested with SalI and BamHI, and the fragment obtained was ligated into the corresponding restriction sites of the plasmid pBI101 (Clontech, Palo Alto, Calif., USA) for the fusion RCI5..GUS. PBSRCIδ plasmid (see above) was digested with Sma1 and RCI5 cDNA was ligated into the Sma1 site pROK2 vector under the control of the CaMV35S promoter (Baulcombe et al., 1986) for obtaining the construction 35S..RCI5.

The recombinant plasmids, once verified by sequencing construction Ia, Ia introduced into Agrobacterium tumefaciens strain C58C1 (Deblaere et al., 1985). To transform Arabidopsis ecotype Col plants followed the floral dip method (Clough and Bent, 1998). Ia histochemical localization of GUS activity

Ia histochemical analysis of GUS activity in transgenic Arabidopsis plants containing the fusion RCI5..GUS was performed according to previously described protocols (Medina et al., 2001).

Quantification of the Monooxygenase Activity

The quantification of the monooxygenase activity was performed according to previously published protocols essentially modified (Agustsson and Strom, 1981), (Lang et al., 1998). Briefly, 100 ul of PBS buffer (pH 7.4) containing 100 mg of proteins from bacterial extracts, 100 ug of purified protein E. coli, or 100 ug of total protein plants 3 week 2 ml were added to a reaction mixture consisting of 2.5 nmol standard FAD, NADPH 100 νmol, 40 μmol 6 μmol MgCl₂and potassium pyrophosphate (pH 8.2). The reaction was initiated with 300 nmol TMA. After 20 min at 24° C., the reaction was stopped by adding HClO₄to a final concentration of 1%. Monooxygenase activity was quantified as the amount of NADPH consumed per hour at 24 or V”.

Content Determination of TMAO

TMAO content was quantified by reducing TMAO to TMA with TiCl₃following the protocol described previously (Bystedt et al., 1959) with some modifications. Ten grams of frozen green tissue was homogenized with 7.5%. TCA. After 2 h incubation at room temperature, samples were centrifuged 15 min at 2O° C. and the supernatant was filtered through Whatman No 1. TMA content was estimated by adding 1 ml of 20% formaldehyde, 3 ml of 30% KOH and 10 mL 1 ml of toluene to the filtered supernatant. This mixture was stirred vigorously for 15 s and left to stand 5 min, toluene phase containing the TMA was recovered and transferred to a fresh tube containing 1 mg of Na₂SO₄. By gentle agitation an aqueous phase was obtained free of toluene as used in the spectrophotometric analysis. Five ml of the toluene extract was incubated with 5 ml of 0.02% picric acid for 30 s at room temperature. Following incubation in the presence of TMA is developing a yellow color whose concentration was proportional to the concentration of TMA, which was directly proportional to the absorbance at 410 nm. The TMAO was reduced by adding 132 ul of 15% TiCl₃to 4 ml of the filtered supernatant. The mixture was incubated at 8O° C. for 9O s and cooled immediately. TMA content in the mixture after the reduction of TMAO was determined as TMA samples. The values obtained are referred to a calibration curve generated with several solutions with different concentrations of commercial TMA (Sigma, St. Louis, Mo.). TMAO content was calculated as the difference in TMA before and after the reduction.

To evaluate the ability to generate TMAO RCI5, monooxygenase activity was quantified, using 0, 50, 100 and 200 ug of purified protein E. coli as described previously (Quantification Monooxygenase activity). Then, the determination of TMAO was performed as described above by taking 1 ml of the final reaction to measure the TMA and 1 mi to reduce TMAO TMA.

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	Number	Date	Country
Parent	14142285	Dec 2013	US
Child	14203261		US

METHOD FOR ENHANCING DROUGHT TOLERANCE IN PLANTS

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