WO2004090141A2 - Method to increase stress tolerance in plants - Google Patents

Abstract

A method is presented for selecting and isolating nucleic acids capable of conferring tolerance or resistance to environmental stress conditions in plants or yeast. Furthermore, nucleic acids, the proteins they encode and their use for the production of plants or yeast with enhanced environmental stress resistance is disclosed.

Description

Stress Tolerance

Field of the invention

The present invention concerns a method for identifying and obtaining nucleic acids capable of modifying stress tolerance, particularly cold tolerance, in plants The invention also concerns isolated nucleic acids so obtained The invention further concerns a method for obtaining plants having modified stress tolerance and to plants obtained by the methods according to the invention The invention also relates to a yeast strain having modified tolerance to cold stress

Background

Environmental stress conditions, such as shortage or excess of solar energy, water or nutrients, high salinity and pollution (e g , heavy-metal pollution), can have a major impact on plant growth and can significantly reduce plant yield Osmotic stress, a type of environmental stress, may be induced by conditions of excess salinity, drought, excessive heat, cold or freezing

Cold stress may be induced by temperatures below the range which allow optimal growth for a particular plant species Each plant species or variety has an optimal growth temperature at which the growth rate is maximal, the further the deviation from this optimal growth temperature, the greater the stress on the plants Many plant species, especially from tropical or subtropical regions, are sensitive to cold For example, it has been estimated that the worldwide rice production would decrease by 40% if the worldwide mean temperature dropped only between 0 5 to 1 0°C (Salisbury and Ross, Plant Physiology 4¹" ed Wadsworth Publishing Company, Belmont, CA, 1992) Plants from temperate regions however have the ability to adapt their metabolism and to survive freezing temperatures after undergoing a process of adaptation to low but non-freezing temperatures, a process called cold acclimation For instance non-acclimated rye typically does not survive temperatures below -5°C, but after cold acclimation it can withstand temperatures as low as -30°C The process of cold acclimation involves altered expression of many genes Plants may differ in their ability to withstand cold, which could lead to periodic but significant losses in plant productivity As a consequence, the areas in which crops or horticultural plants can be cultivated is determined by assessing the risk of lower temperatures, relative to typical growth temperatures for any given plant

The most prominent changes during cold acclimation include a reduction or cessation of growth, reduction of tissue water content (Levitt, Responses of Plants to Environmental Stresses, Vol 1 2nd edn Academic Press New York, NY 1980), transient increase in abscisic acid (ABA) levels (Chen ef al , Plant Physiology 71 , 362 - 365, 1983), changes in membrane lipid composition (Lynch and Steponkus, Plant Physiology 83, 761 - 767, 1987, Uemura and Steponkus, Plant Physiology 104, 479 - 496, 1994), the accumulation of compatible osmolytes such as proline, betaine, polyols and soluble sugars, and increased levels of antioxidants (Koster and Lynch, Plant Physiology 98, 108 - 113, 1992, Kishitani ef al , Plant, Cell and Environment 17, 89 - 95, 1994, Murelli ef al , Physiologia Plantarum 94, 87 - 93 1995, Nomura ef al , Euphytica 83, 247 - 250, 1995, Dόrffling θt al , Plant Molecular Biology 23, 221 - 225, 1997, Tao ef al , Cryobiology 37, 38 - 45, 1998)

Various methods for the identification and isolation of genes or proteins differentially expressed during cold stress are known For example, mapping techniques allow determination of chromosome locations of genes involved in cold tolerance (Pan ef al , Theoretical and Applied Genetics 89, 900 - 910, 1994, Gahba ef a/ , Theoretical and Applied Genetics 90, 1174 - 1179, 1995) Another approach involves mutational analysis in which mutants that have an altered response to cold tolerance are isolated and characterized For example, βskimol, conferring improved freezing tolerance of 2°C over acclimated wild-type plants, was isolated from a collection of 800000 Ethyl Methyl Sulphonate (EMS)- mutagenised Arabidopsis lines that were screened for constitutively freezing-tolerant mutants (Xin and Browse, PNAS 95, 7799 - 7804, 1998) Conversely, plant lines were screened for mutants defective in cold acclimation (Warren et al , Plant Physiology 111 , 1011 - 1019, 1996, Knight ef a/ , Plant Cell 8, 489 - 503, 1996) cos-, los- and hos-mutants (for respectively constitutive, low and high expression of osmotically responsive genes) were isolated using a combination of mutagenesis and reporter gene activation (Ishitam ef al , Plant Cell 9, 1935 - 1949, 1997, Ishitani et al , Plant Cell 10, 1151 - 1161 , 1998, Lee βt al , Plant Journal 17, 301 - 308, 1999) One of the drawbacks of mapping and the mutant analysis strategy is that they do not directly result in the isolation of nucleic acids coding for cold-induced genes Another strategy, using differential screening of cDNA libraries and related techniques, has in the past yielded several cold induced genes from different plant species (reviewed in Xin and Browse, Plant, Cell and environment 23, 893 - 902, 2000) Many of those genes have known functions and can be grouped as being involved in drought stress, in signal transduction pathways, or as being related to heat shock proteins, molecular chaperones, "antifreeze proteins" or regulatory proteins Several of the genes are highly expressed during cold stress and are commonly referred to as COR (COId Regulated) genes (Tomashow, Annual Review of Plant Physiology and Plant Molecular Biology 50, 571 - 599, 1999)

Strategies used to engineer cold resistant plants include accumulation of osmoprotectants such as mannitol (US 6,416,985), proline (US 6,239,332), trehalose (US 6,323,001 ) or glycine- betaine (Hayashi et al , Plant Journal 12, 133 - 142, 1997, US 6,281 ,411) Other approaches involve manipulating the signal transduction pathway controlling the stress response (WO 01/77355), including use of transcription factors (WO 01/77311 , US 6,417,428, WO 02/44389, US 5,891,859) Furthermore a number of genes have been used to enhance cold resistance Examples are members of the COR group (COR15a US 5,296,462, US 5,356,816), a cell cycle related gene (WO 01 /77354), protein kinase related proteins (WO 01/77356), the LEA- like protein CAP85 (US 5,837,545) and use of a phospholipid binding protein (WO 02/00697) Nevertheless, signal transduction pathways leading to cold acclimation and the identity of the genes that confer resistance to cold stress in plants remain largely unknown

Yeast has been used for screening plant genes that confer resistance to salt stress For example, a salt-sensitive yeast strain (JM26) has previously been transformed with a cDNA library from salt-stressed sugar beet and used to screen for clones having increased salt tolerance (WO 02/52012) The transformed yeast cells were grown on a rich medium (YPD) or on a synthetic medium plus methionine and leucine (SD), supplemented with 0 15 M NaCI or with 20 mM LiCI Putative positive clones showing better growth on the selective media compared to the non-transformed yeast strain were isolated and further characterised However, the use of yeast for identifying plant genes involved in cold stress has not been used before A recent study in haploid yeast by de Jesus Ferreira et al (2001 ), in which trans poson mutagenesis was employed, identified 10 different yeast genes responsive to cold tolerance, which upon mutation caused a growth stop at 15 °C The identified genes include a gene coding for a glutamate synthase (YDL171C), a GTP binding protein (YML121W), a GSK-3 Ser/Thr protein kinase (YNL307C) and a component of TFIID (YLR399C) Three of the genes were previously described as cold responsive (YLR399C, YML121W, YNL307C) and four of the isolated genes were also involved in resistance to salt stress

Summary of the invention

The present invention provides a novel screening method for nucleic acids involved in stress responses in a plant, which method involves screening in diploid yeast for plant genes involved in modifying tolerance/resistance to temperature stress The present invention also provries new plant genes identified by this screen and polypeptides encoded by these genes Also provided are methods for producing plants having modified tolerance or resistance to environmental stress conditions, comprising introduction of the above-mentioned genes into plants Also provided are plants having modified tolerance or resistance to environmental stress conditions, which plants are transformed with the gene according to the invention Detailed description of the invention

According to a first embodiment of the present invention, there is provided a screening method for identifying nucleic acids capable of modifying tolerance or resistance to cold stress conditions in plants or yeast, which method comprises the steps of (i) providing a cDNA library of coding sequences from an organism,

(n) introducing these coding sequences in an expressible format into wild type yeast cells, (in) growing the yeast cells of (u) under conditions of cold stress, (iv) identifying differences between transgenic yeast cells and wild type yeast cells, preferably identifying differences in growth rate,

(v) isolating nucleic acids from the transgenic yeast cells that differ from the wild type yeast cells Preferably the wild type yeast cells are wild type diploid Saccharomycβs cβ visiaβ yeast cells, more preferably wild type Saccharomycβs cerβvisiaβ W303 yeast cells Furthermore preferably the organism is a plant, which plant preferably is a salt treated plant, more preferably a salt treated halophytic plant or a part thereof, most preferably salt treated Beta vulgaπs or a part thereof

The use of yeast cells for identifying genes involved in salt or osmotic stress is known in the art Yeast is a good model organism for testing genes conferring tolerance to osmotic or salt stress because suitable mutants are known that allow for complementation WO 02/052012 teaches a screening method wherein a mutant salt sensitive yeast strain is transformed with cDNA isolated from salt stresses sugar beet The method resulted in the identification of genes that may contribute to increased tolerance to salt, drought or osmotic stress in plants Here, for the first time yeast was used for screening a plant cDNA library involving the application of cold stress In contrast to WO 02/052012, where a mutation causing salt sensitivity is to be complemented by an introduced plant cDNA, wild type yeast was used in the present invention instead of a mutant, the wild type yeast was diploid to avoid any effects of recessive chromosomal mutations that would eventually result in cold tolerance of the yeast host The present invention demonstrates that, since no suitable cold sensitive mutants exist, wild type yeast cells transformed with cDNA from salt stressed plants can be used to isolate genes capable of conferring tolerance against cold stress in plants The terms "tolerance" and "resistance" are used interchangeably herein

The first step of the screening method involves providing a cDNA library of coding sequences from any organism, such as plants animals or fungi According to a preferred feature of the present invention, the cDNA library is made from a plant, preferably a salt treated plant, further preferably a salt treated halophytic plant or a part thereof, more preferably from a salt treated sugar beet plant or a part thereof, most preferably from leaves of salt treated Sefa vulgaπs plants Sugar beet (Beta vulgaπs), a relatively halophytic crop plant, provides a potentially good source of cold tolerance genes Although the present invention is exemplified by use of a sugar beet cDNA library, rt is to be understood that other halophytic plants could equally serve the same purpose The preparation of cDNA libraries is a routine technique well known in the art The cDNA library preferably comprises copies of essentially all mRNA of the plant cell Advantageously, coding sequences alone are sufficient

The second step of the screening method involves introducing the coding sequences into yeast cells Methods for transformation of yeast, such as electroporation or treatment with Lithium Acetate, and for expressing genes in yeast, including yeast vectors, such as pYES, are well known in the art (see e g Current Protocols in Molecular Biology, Unit 13 (Ausubel ef al , 1994) and the Guide to Yeast Genetics and Molecular Biology (Guthne and Fink, 1991)) Advantageously, coding sequences may be introduced and expressed in yeast using any of several known methods, with the aim of testing tolerance or resistance to stress conditions According to a preferred feature of the present invention, a vector based on the λ phage is employed, more preferably λPG15 is used for introducing and expressing coding sequences in yeast Phage λPG15 comprises the excisable expression plasmid pYPGE15 which may be used directly for both Escheπchia coli and yeast complementation (Brunelli and Pall, Yeast 9, 1309 - 1318, 1993) A plasmid cDNA library can be recovered from XPG15 using the cre-lox recombinase system (Brunelli and Pall, Yeast 9, 1309 - 1318, 1993) Preferably, the yeast cells are Saccharomyces cerevisiae, more preferably the diploid wild type strain W303 and its diploid mutant deficient for glycerol phosphate dehydrogenase (gpdl) The yeast strain W303 has the genotype MATa/MATα, ADE2/ade2, CAN1/can1-100, CYH2/cyh2, hιs3-11,15/hιs3- 11 ,15, LEU1/leu1-c, LEU2/leu2-3,112, trp1-1 URA3 trp1-3'D /trp1-1 , ura3-1/ura3-1, and originates from the parent strains W303-1A and W303-1 B (Pπmig et al , Nat Genet 26, 415 - 423, 2000) The W303 gpdl mutant was unexpectedly more cold tolerant than the W303 wild type strain (see Figure 1) For this reason the wild type strain was used in the screening, while the gpdl mutant strain served as a standard for comparison It was thus expected that nucleic acids conferring cold tolerance would enhance the growth of the wild type yeast cells to a comparable or better level to that of the gpdl mutant

Advantageously, the gpdl gene can be used for enhancing cold tolerance of yeast, for example baker's yeast Yeast is known to be sensitive to cold stress Freezing stress in particular has a negative impact on the quality of yeast as a leaven Yeast cells that have been mutated or engineered such that the glycerol phosphate dehydrogenase (gpdl) gene is inactivated (using techniques known in the art) are surprisingly more tolerant to cold and/or freezing stress than wild type yeast This trait can be of benefit in, for example, the baking or brewing industries The present invention thus also provides a method for increasing cold tolerance of yeast cells, comprising dowπregulating expression in yeast of a nucleic acid encoding a glycerol phosphate dehydrogenase and/or inhibiting activity of a glycerol phosphate dehydrogenase The invention furthermore provides for the use of a gpdl gene for altering the stress tolerance of yeast by downregulatmg its expression The stress-tolerant yeast cells thus obtained can be used in purified form (for example leaven) or in compositions (for example dough)

The third step of the screen involves growing the yeast cells under stressed conditions Yeast cells transformed with cDNA of the salt stressed sugar beet were plated onto a suitable medium and grown under cold stress A temperature of 10°C was chosen, as this still allowed a minimal growth of the yeast strain, but a person skilled in the art may choose any other temperature below the optimal growth temperature After a certain period of time colonies that were able to grow under these conditions of cold were selected and their cold tolerance was retested by growing the transgenic cells again under cold stress conditions Advantageously, the cDNA from salt treated plants may also be a suitable basis for finding genes capable for conferring tolerance against other stresses This may be achieved simply by growing the yeast cells in step (in) above in conditions of stress determined by the type of gene sought For example, in order to identify genes conferring tolerance or resistance to heat stress, the yeast cells would be grown in conditions of heat According to a preferred feature of the invention, the stress is preferably cold stress It was then determined whether the stress tolerance onginated from the transgene and not from a mutation in the host genome To this end, the plasmid comprising the transgene was cured from a transgenic cold tolerant yeast clone and it was verified whether the cold tolerance had disappeared too, secondly the plasmid comprising the transgene was isolated from a transgenic cold tolerant yeast clone and re introduced into a non-transgenic yeast strain, whereafter the cold tolerance of the newly transformed yeast strain was compared to the non-transformed yeast strain

The fourth step in the screening method is the identification of fast growing yeast cells Yeast cells transformed with a plant nucleic acid conferring stress resistance were identified based on their ability to grow faster under stress conditions than yeast cells not transformed with such a nucleic acid, although other selection criteria may also be used, depending on the type of stress that is applied Finally, in the last step of the screening method nucleic acids conferring stress tolerance are isolated from the yeast host and characterised Methods for isolating nucleic acids from yeast and sequencing these nucleic acids are known to those skilled in the art

The present invention also encompasses the use of the screening method described above for identifying nucleic acids encoding proteins capable of conferring cold stress tolerance to plant cells or yeast cells

The screening method described above yielded several nucleic acids encoding proteins that increase cold stress tolerance of the yeast strain, hereafter named CRYO genes and CRYO proteins The proteins encoded by these nucleic acids all relate to vesicle trafficking to the vacuole or plasma membrane Response to stress requires an adaptation of metabolism, including transport of proteins and other components between different organelles, in particular between the Golgi apparatus and the vacuole, but also between the plasma membrane and the vacuole Plant vacuoles perform different functions, depending on the cell type in which they occur They play an important role in cell growth or function as storage organelles for proteins, ions, secondary metabolites and metabolic waste products In this last aspect, vacuoles also resemble lysosomes They contain many hydrolytic enzymes for degradation of damaged or redundant cell material Adaptation to changing environmental conditions or to stress involves not only synthesis of new cellular components, but also degradation of cellular material These degradation processes require an extensive trafficking of material via membrane bound vesicles such as endosomes Also hydrolytic enzymes are delivered to the vacuole via endosomes

CRY04 (SEQ ID NO 8) is a protein with homology to At1g72160, a cytosolic factor in Arabidopsis tha ana, and it further has significant homology with yeast SEC14 (=YMR079W) This yeast protein is a cytosolic phosphatidylinositol/phosphatidylchohne transfer protein and is required for the transport of secretory proteins from the Golgi complex and for protein secretion (Bankaitis et al , (1990) Nature 347, 561-562) In yeast it is associated with the Golgi complex as a peripheral membrane protein and forms a link between phosphohpid metabolism and vesicle trafficking (Li et al , (2000) Mol Biol Cell 11 , 1989-2005) It catalyses the transfer of phosphatidylinositol and phosphatidylcholme between membranes in vitro and is essential for viability and secretion (Tschopp et al , (1984) J Bacteπol 160, 966-970)

CRY05 (SEQ ID NO 10) is a protein with a RING-domain RING-domain proteins are known to be involved in biological processes such as transcnptional and translational regulation, and in targeted proteolysis The RING-domain mediates protein-protein interactions and is a C3HC4 type zinc-finger domain of 40 to 60 ammo acids long Various proteins with a RING finger domain exhibit binding to E2 ubiquitin-conjugatmg enzymes (Ubc's) (Freemont (2000) Curr Biol 10, R84-87) The above domain Zf-RING finger is different from that found in yeast CLASS E vacuolar sorting protein VPS27 However, there may be some functional conservation since VPS27 has also been linked to ubiquitination processes and protein turnover, and the Zf-RING finger domain present in CRI05 is usually found in proteins that are also involved in ubiquitination and proteasome protein degradation

The other proteins of the present invention belong to the group of Class-E vacuolar trafficking mutants Certain mutants in yeast, known as "Class-E" mutants (Jones ef al , In Yeast III, Cold Spring Harbor Laboratory Press, p363 - 470, 1997), are unable to perform a correct sorting of proteins to the vacuole Microscopical analysis reveals that these mutants contain large aberrant endosomal structures (Raymond ef al , Molecular Biology of the Cell 3, 1389, 1992), filled with proteins that are normally transited to the vacuole CRY01 , CRY02 and CRY03 all have a SNF7 domain (Pfam PF03357/IPR005024, Pfa database, Bateman et al , (2004) Nucleic Acids Research Database Issue 32, D138-D141 ) Structurally, the three proteins belong to the family of CHMP proteins (Howard et al , (2001) J Cell Sci 114, 2395-2404) CRY01 (SEQ ID NO 2) and CRY2 (SEQ ID NO 4) are isoforms of each other CRY01 and its plant homologues have not yet been functionally characterised, but they are related to yeast SNF7 (=DID1 =VPS32 =YLR025W) SNF7 mutants belong to the group of class E vacuolar trafficking mutants (Jones ef al , In Yeast III, Cold Spring Harbor Laboratory Press, p363 - 470, 1997) The SNF7 mutant accumulates a prominent organelle distinct from the vacuole, containing large amounts of enzymes which are normally present in the vacuole such as the hydrolases CpY, PrA & PrB The protein is involved in derepression of SUC2 in response to glucose limitation SNF7 mutants show a decrease in invertase derepression, a growth defect on raffinose, temperature-sensitive growth on glucose, and a sporulation defect in homozygous diploids The SNF7 sugar related phenotype could be due to an altered turnover of a glucose sensor These and other data suggest that the protein transport from the Golgi network and from the plasma membrane to the vacuole is interfered with SNF7 forms a family of coiled-coil-forming proteins with vps20 and mos10 The proteins are involved in same trafficking step, endosome-to-vacuole transport, but probably participate in different cargo-specific events (Kranz et al , 2001 )

SEQ ID NO 6 (CRY03) is the plant homologue of yeast DID2 (=FT11 =YKR035W-A), another member of the class E vacuolar trafficking proteins DID2 is related to SNF7, it has similar structural features, it may have a comparable function and possibly belongs to the same protein complex in yeast (Ameπk ef al , Molecular Biology of the Cell, 11 , 3365 - 3380, 2000) A human orthologue, CHMP1, is reported to be involved in membrane trafficking and localises to early endosomes (Howard et al , 2001) CHMP1 also localises to the nuclear matrix, thereby affecting chromatm structure and cell-cycle progression and furthermore interacts with the PcG protein Polycomblike (Pel) (Stauffer et al , 2001 J Cell Sci 114, 2383-93)

Besides modifying tolerance to cold stress, the proteins may also be involved in protein transport and sorting (CRY01 [SEQ ID NO 1/2], CRY02 [SEQ ID NO 3/4], CRY03 [SEQ ID NO 5/6] and CRY04 [SEQ ID NO 7/8]), vacuole formation, development or functioning (CRY01 , CRY02, CRY03), in transcription and translation (CRY03, CRY05), in membrane fluidity (CRY04) and in protein turnover (CRY05)

The proteins encoded by the nucleic acids identified by the screening method according to the present invention were hitherto unknown Therefore, the invention also provides an isolated CRYO protein, (a) comprising the sequence as given in SEQ ID NO 2, 4, 6, 8 or 10,

(b) comprising a sequence having at least i 76 %, alternatively 80%, preferably 90%, more preferably 95%, 96%, 97%, 98% or 99% sequence identity to the full length sequence as given in SEQ ID NO 2, ii 55 %, alternatively 60%, 70%, 80%, preferably 90%, more preferably 95%, 96%, 97%, 98% or 99% sequence identity to the full length sequence as given in SEQ ID NO 4, in 90 5 %, alternatively 906%, 90 7%, 90 8%, 90 9%, preferably 91%, 92%, 93%,

94% more preferably 95%, 96%, 97%, 98% or 99% sequence identity to the full length sequence as given in SEQ ID NO 6, iv 50 %, alternatively 60%, 70%, 80%, preferably 90%, more preferably 95%,

96%, 97%, 98% or 99% sequence identity to the full length sequence as given

v 50 %, alternatively 60%, 70%, 80%, preferably 90%, more preferably 95%,

96%, 97%, 98% or 99% sequence identity to the full length sequence as given in SEQ ID NO 10,

(c) comprising a substitution variant or insertion variant of (a),

(d) according to any of (a) to (c), comprising substitutions with corresponding naturally or non-naturally altered a mo acids,

A CRYO gene according to the present invention is any nucleic acid encoding a CRYO protein as defined above "Homologues" of a protein encompass peptides, oligopeptides, polypeptides, proteins and enzymes having ammo acid substitutions, deletions and/or insertions relative to the unmodified protein in question and having similar biological and functional activity as the unmodified protein from which they are derived To produce such homologues, ammo acids of the protein may be replaced by other ammo acids having similar properties (such as similar hydrophobicity, hydrophi city, antigenicity, propensity to form or break α-helical structures or β- sheet structures) Conservative substitution tables are well known in the art (see for example Creighton (1984) Proteins W H Freeman and Company) The homologues useful in the methods according to the invention have in the case of CRY04 or CRY05 at least 50% sequence identity or similarity (functional identity) to the unmodified protein, alternatively at least 60% sequence identity or similarity to an unmodified protein, or alternatively at least 70% sequence identity or similarity to an unmodified protein Typically, the homologues of CRY04 or CRY05 have at least 80% sequence identity or similarity to an unmodified protein, preferably at least 85% sequence identity or similarity, further preferably at least 90% sequence identity or similarity to an unmodified protein, most preferably at least 95% sequence identity or similarity to an unmodified protein In the case of CRY02, the homologues useful in the methods according to the invention have at least 55% sequence identity or similarity (functional identity) to the unmodified protein, alternatively at least 60% sequence identity or similarity to an unmodified protein, or alternatively at least 70% sequence identity or similarity to an unmodified protein Typically, the homologues of CRY02 have at least 80% sequence identity or similarity to an unmodified protein, preferably at least 85% sequence identity or similarity, further preferably at least 90% sequence identity or similarity to an unmodified protein, most preferably at least 95% sequence identity or similarity to an unmodified protein In the case of CRY01 , the homologues useful in the methods according to the invention have at least 76% sequence identity or similarity (functional identity) to the unmodified protein Typically, the homologues of CRY01 have at least 80% sequence identity or similarity to an unmodified protein, preferably at least 85% sequence identity or similarity, further preferably at least 90% sequence identity or similarity to an unmodified protein, most preferably at least 95% sequence identity or similarity to an unmodified protein In the case of CRY03, the homologues useful in the methods according to the invention have at least 90 5% sequence identity or similarity (functional identity) to the unmodified protein Typically, the homologues of CRY03 have at least 91% sequence identity or similarity to an unmodified protein, preferably at least 92% sequence identity or similarity, further preferably at least 93% sequence identity or similarity to an unmodified protein, most preferably at least 95% sequence identity or similarity to an unmodified protein Furthermore, homologues of a CRYO protein according to the present invention are capable of increasing the cold stress tolerance of yeast in an assay as outlined above Two special forms of homology, orthologous and paralogous, are evolutionary concepts used to describe ancestral relationships of genes The term "paralogous" relates to gene- dup cations within the genome of a species leading to paralogous genes The term "orthologous" relates to homologous genes in different organisms due to ancestral relationship The term "homologues" as used herein also encompasses paralogues and orthologues of the proteins useful in the methods according to the invention

Orthologues in other plant species may easily be found by performing a so-called reciprocal blast search This may be done by a first blast involving blasting the sequence in question

(any one of SEQ ID NO 1 to 10) against any sequence databases, such as the publicly available NCBI database which may be found at http //www ncbi nlm nih gov If orthologues in rice are sought, the sequence in question would be blasted against, for example, the 28,469 full-length cDNA clones from Oryza sativa Nipponbare available at NCBI BLASTn may be used when starting from nucleotides or TBLASTX when starting from the protein, with standard default values The blast results may be filtered The full-length sequences of either the filtered results or the non-filtered results are then blasted back (second blast) against the sequence in question The results of the first and second blasts are then compared True orthologues are those that match again with the query gene In the case of large families, ClustalW and the neighbour joining method is used to construct a phylogenetic tree to help visualize the clustering

Homologous proteins can be grouped in "protein families" A protein family can be defined by functional and sequence similarity analysis, such as, for example, Clustal W A neighbour- joining tree of the proteins homologous to a protein of interest can be generated by the Clustal W program and gives a good overview of their structural and ancestral relationships

The CRY01 , CRY02 or CRY3 proteins comprise a SNF7 domain and can be regarded as members of the same protein family as human CHMP1 and yeast SNF7 The SNF7 domain (Pfam PF03357) occurs in a group of proteins involved in protein sorting and transport from the endosome to the vacuole/lysosome in eukaryotic cells In the Interpro database, the SNF7 family is described as a family of eukaryotic proteins which are variously described as either hypothetical protein, developmental protein or related to yeast SNF7 The family contains human CHMP1 CHMP1 (CHromatin Modifying Protein, CHarged Multivesicular body Protein) is encoded by an alternative open reading frame in the PRSM1 gene and is conserved in both complex and simple eukaryotes CHMP1 contains a predicted bipartite nuclear localisation signal and distributes as distinct forms to the cytoplasm and the nuclear matrix in all cell lines tested Human CHMP1 is strongly implicated in multivesicular body formation A multivesicular body is a vesicle-filled endosome that targets proteins to the interior of lysosomes Immunocytochemistry and biochemical fractionation localise CHMP1 to early endosomes and CHMP1 physically interacts with SKD1/VPS4, a highly conserved protein directly linked to multivesicular body sorting in yeast Similar to the action of a mutant SKD1 protein, over expression of a fusion derivative of human CHMP1 dilates endosomal compartments and disrupts the normal distribution of several endosomal markers Genetic studies in Saccharomyces cerβvisiaθ further support a conserved role of CHMP1 in vesicle trafficking Deletion of CHM1 , the budding yeast homolog of CHMP1 , results in defective sorting of carboxypeptidases S and Y and produces abnormal, multi-lamellar prevacuolar compartments This phenotype classifies CHM1 as a member of the class E vacuolar protein sorting genes In this CHMP family other CHMP proteins can be found, such as the yeast proteins Chml p, Chm2p, Vps24p, Chmδp and Chm6p, or the human proteins AF281064, AF042384, AF151842, AF19226, AF161483, AF132968 and AW965590 (Howard et al , 2001 ) All these proteins constitute a family of structurally related proteins with a similar size and charge distribution (basic N-termmus and acidic C-terminus) and sequence conservation throughout the complete protein sequence (Howard et al , 2001) This structural conservation indicates also a functional conservation CHM gene products are involved in correct sorting of carboxypeptidase Y and can be assayed in a pulse chase experiment as described by Howard et al (2001)

CRY04 belongs to the same family as yeast SEC14 CRY04 comprises a SEC14 domain as defined in the SMART database (Schultz et al (1998) Proc Natl Acad Sci USA 95, 5857- 5864, Letunic et al (2004) Nucleic Acids Res 32, D142-D144) the SEC14 domain is found in homologues of a S cerevisiae phosphatidylmositol transfer protein (Sec14ρ) and in RhoGAPs, RhoGEFs and the RasGAP, neurofibromin (NF1) It is also reported to be a lipid-binding domain The SEC14 domain of Dbl is known to associate with G protein beta/gamma subunits This domain is also described in the Interpro database (Mulder et al , (2003) Nucl Acids Res 31 , 315-318 ) (IPR001251 ), where the proteins comprising this domain are grouped as a family of various retinaldehyde/retinal-bindmg proteins that may be functional components of the visual cycle in animals Cellular retinaldehyde-bmding protein (CRALBP) carries 11-cιs-retιnol or 11-cιs-retιnaldehyde as endogenous hgands and may function as a substrate carrier protein that modulates interaction of these retinoids with visual cycle enzymes The multidomain protein Trio binds the LAR transmembrane tyros e phosphatase, contains a protein kinase domain, and has separate rac-specific and rho-specific guanme nucleotide exchange factor domains Trio is a multifunctional protein that integrates and amplifies signals involved in coordinating actm remodeling, which is necessary for cell migration and growth Other members of the family are transfer proteins that include, guanme nucleotide exchange factor that may function as an effector of RAC1 , phosphatidylinositol/phosphatidylcholine transfer protein that is required for the transport of secretory proteins from the Golgi complex and alpha-tocopherol transfer protein that enhances the transfer of the ligand between separate membranes Homologues useful in the present invention comprise a SEC14 domain and exhibit lipid transfer activity (a suitable assay is described by Jouannic et al , Eur J Biochem 258, 402-410 (1998)) and include for example the Arabidopsis proteins At1g72160, At4g09160, At1g22530, At1g72150, At3g51670, At1g30690, the rice protein BAB86220 and the maize protein encoded by AY107978

CRY05 comprises a RING type zinc finger domain in the carboxy-terminal part of the protein, in addition there is a conserved sequence spanning ammo acid (AA) 15 to 305 of SEQ ID NO 10 that corresponds to a Pfam-B_23829 domain (which domain is postulated to be associated with the C3HC4 type Zn-finger domain) and a sequence from AA 425 to 473, corresponding to Pfam-B_2377 The CRY05 sequence corresponding to this Pfam-B_2377 domain comprises a conserved stretch of ammo acids that is also found in other homologous plant proteins and includes of the sequence HDQHRDMRLDI DNMSYEELLALEERIG, in which less than 7 mismatches may occur among the various homologues Plant homologues of CRY05 constitute a new class of proteins (CRY05-lιke proteins) characteπsed by the presence of a serme rich region in the N-terminal half of the protein, an acidic region that comprises the conserved sequence signature hereabove and the RING finger domain Exemplary homologues include sequences NP_196626, NP_974832, NP_568462 2, and the protein encoded by AK066069

Advantageously, homologous proteins belonging to these families and/or comprising one or more of these domains may be useful in the methods of the present invention for conferring abiotic stress tolerance and in particular cold stress tolerance to plant cells or yeast

Two polypeptides or nucleic acids are said to be "identical" if the sequence of ammo acid residues or nucleotides, respectively, in the two sequences is the same when optimally aligned Sequence comparisons between two (or more) polypeptide or nucleic acids are typically performed by companng sequences of the two sequences over a "comparison window" to identify and compare local regions of sequence similarity A "comparison window", as used herein, refers to a segment of at least about 20 contiguous positions, usually about 50 to about 200, more usually about 100 to about 150, in which a sequence may be compared to a reference sequence of the same number of contiguous positions after the two sequences are optimally aligned Optimal alignment of sequences for comparison may be conducted by the local homology algorithm of Smith and Waterman (Adv Appl Math 2, 482, 1981), by the homology alignment algorithm of Needleman and Wunsch (J Mol Biol 48 443, 1970), by the search for similarity method of Pearson and Lipman (Proc Nat Acad Set 85, 2444, 1988), by computerized implementations of these algoπthms (GAP, BESTFIT, BLAST, FASTA, and TFASTA in the Wisconsin Genetics Software Package, Genetics Computer Group (GCG), 575 Science D , Madison, Wl), or by inspection When the Needleman-Wunsch algorithm is used, a gap opening penalty of 10 or 11, a gap extension penalty of 0 5 or 1 is used and where possible, full-length sequences are compared to each other

The term "derivatives" refers to peptides, oligopeptides, polypeptides, proteins and enzymes which may comprise substitutions, deletions or additions of naturally and non-naturally occurring ammo acid residues compared to the ammo acid of a naturally-occurring form of the proteins as presented in SEQ ID NO 2, 4, 6, 8 or 10 "Derivatives" of a protein comprise proteins in which ammo acid residues are substituted by corresponding naturally or non- naturally altered ammo acids "Derivatives" of a protein encompass peptides, oligopeptides, polypeptides, proteins and enzymes which may comprise naturally occurring altered, (such as glycosylated, acylated, myπstoylated or phosphorylated ammo acids) or non-naturally occurring ammo acid residues (such as biotmylated ammo acids, or ammo acids modified after CNBr treatment) compared to the ammo acid of a naturally-occurring form of the polypeptide "Derivatives" of a protein also encompass proteins carrying post-translational modifications A derivative may also comprise one or more non-ammo acid substituents compared to the ammo acid from which it is derived, for example a reporter molecule or other ligand, covalently or non-covalently bound to the am o acid such as, for example, a reporter molecule which is bound to facilitate its detection, and non-naturally occurring ammo acid residues relative to the ammo acid of a naturally-occurring protein "Substitutional variants" of a protein are those in which at least one residue in an ammo acid has been removed and a different residue inserted in its place Ammo acid substitutions are typically of single residues, but may be clustered depending upon functional constraints placed upon the polypeptide, insertions will usually be of the order of about 1-10 ammo acid residues, and deletions will range from about 1-20 residues Preferably, ammo acid substitutions comprise conservative ammo acid substitutions "Insertional variants" of a protein are those in which one or more ammo acid residues are introduced into a predetermined site in said protein Insertions can comprise ammo-terminal and/or carboxy-terminal fusions as well as intra-sequence insertions of single or multiple ammo acids Generally, insertions within the ammo acid will be smaller than ammo- or carboxy- terminal fusions, in the order of about 1 to 10 residues Examples of ammo- or carboxy- terminal fusion proteins or peptides include the binding domain or activation domain of a transcπptional activator as used in the yeast two-hybrid system, phage coat proteins, (hιstιdιne)₆-tag, glutathione S-transferase-tag, protein A, maltose-binding protein, dihydrofolate reductase, Tag 100 epitope, c-myc epitope, FLAG^®-epιtσpe, lacZ, CMP (calmodulin-binding peptide), HA epitope, protein C epitope and VSV epitope "Functional fragments" or "deletion variants" of a protein are characterised by the removal of one or more ammo acids from the protein, such that the remaining fragment still retains the biological activity of the unmodified protein, for example the capacity of conferπng cold stress to yeast in an assay detailed in examples 2 and 3 Such "functional fragments" of a protein encompasses at least fifteen contiguous ammo acid residues of a protein, in case of a functional fragment the minimum size being a sequence of sufficient size to provide this sequence with at least a comparable function and/or activity to the original sequence which was truncated, while the maximum size is not critical Typically, the truncated ammo acid will range from about 5 to about 60 ammo acids in length "Immunologically active" refers to molecules or specific fragments thereof, such as specific epitopes or haptens, that are recognised by (i e that bind to) antibodies Specific epitopes may be determined using, for example, peptide-scanning techniques as described in Geysen ef al , ( Chem Biol 3, 679-688, 1996) Functional fragments can also include those comprising an epitope which is specific for the proteins according to the invention

Preferably, the derivatives, functional fragments, substitution, deletion or insertion variants of a CRYO protein have at least the same or better functional activity than the unmodified protein, such as the capability of increasing the cold stress tolerance of yeast The functional activity can be tested with for example the screening method as described above or with methods described for related proteins

Ammo acid variants of a protein may readily be made using peptide synthetic techniques well known in the art, such as solid phase peptide synthesis and the like, or by recombinant DNA manipulations The manipulation of DNA sequences to produce substitution, insertion or deletion variants of a protein are well known in the art For example, techniques for making substitution mutations at predetermined sites in DNA are well known to those skilled in the art and include M13 mutagenesis, T7-Gen in vitro mutagenesis (USB, Cleveland, OH), QuickChange Site Directed mutagenesis (Stratagene, San Diego, CA), PCR-mediated site- directed mutagenesis or other site-directed mutagenesis protocols

Another embodiment of the present invention provides nucleic acids obtainable by the screening method according to the present invention, which nucleic acids can be used to modify stress tolerance or resistance in plants and/or yeast The screening method according to the invention identified several nucleic acids hitherto unknown The present invention therefore also provides an isolated nucleic acid encoding a protein as defined above, the complement thereof or a part thereof

The terms "nucleic acιd(s)", "nucleotide sequence(s)", "gene(s)", "polynucleotιde(s)" and "nucleic acid molecule(s)" are used herein interchangeably to refer to πbonucleotides or deoxynbonucleotides or a combination of both, in a polymeric form of any length The terms also include double-stranded and single-stranded DNA and RNA Also included are known nucleotide modifications such as methylation, cyc zation and 'caps' and substitution of one or more naturally occurring nucleotides with an analogue such as inosme The terms also encompass peptide nucleic acids (PNAs)

Advantageously, the nucleic acids according to the invention may be produced using recombinant or synthetic means, such as, for example, PCR cloning mechanisms Generally, such techniques as defined herein are well known in the art, for example as described in Sambrook et al (Molecular Cloning a Laboratory Manual, 2001) Polynucleotides may also be synthesized by well-known techniques as described in the technical literature See, e g , Caruthers et al , Cold Spring Harbor Symp Quant Biol 47, 411-418 (1982), and Adams et al , J Am Chem Soc 105, 661 (1983) Double stranded DNA fragments may then be obtained either by synthesizing the complementary strand and annealing the strands together under appropriate conditions, or by adding the complementary strand using DNA polymerase with an appropriate primer sequence

A nucleotide sequence encoding a protein (gene, coding sequence, open reading frame or ORF) is a nucleotide sequence that can be transcribed into mRNA and/or translated into a polypeptide when present in an expressible format, i e when the coding sequence or ORF is placed under the control of appropriate control sequences or regulatory sequences A coding sequence or ORF is bounded by a 5' translation start codon and a 3' translation stop codon A coding sequence or ORF can include, but is not limited to RNA, mRNA, cDNA, recombinant nucleotide sequences, synthetically manufactured nucleotide sequences or genomic DNA The coding sequence or ORF can be interrupted by intervening nucleic acids

By "expressible format" is meant that the isolated nucleic acid molecule is in a form suitable for being transcribed into mRNA and/or translated to produce a protein, either constitutively or following induction by an tracellular or extracellular signal, such as an environmental stimulus or stress (mitogens, anoxia, hypoxia, temperature, salt, light, dehydration, etc) or a chemical compound such as IPTG (isopropyl-β-D-thiogalactopyranoside), or such as an antibiotic (tetracyclme, ampicillm, rifampicm, kanamycin), hormone (e g gibberellm, auxin, cytokmin, glucocorticoid, brassinosteroid, ethylene, abscisic acid etc), hormone analogue (indolacetic acid (IAA), 2,4-D, etc), metal (zinc, copper, iron, etc), or dexamethasone, amongst others As will be known to those skilled in the art, expression of a functional protein may also require one or more post-translational modifications, such as glycosylation, phosphorylation, dephosphorylation, or one or more protein-protein interactions, amongst others All such processes are included within the scope of the term "expressible format"

Genes and coding sequences essentially encoding the same protein but isolated from different sources can consist of substantially divergent nucleic acids Reciprocally, substantially divergent nucleic acids can be designed to effect expression of essentially the same protein These nucleic acids are the result of e g the existence of different alleles of a given gene, or of the degeneracy of the genetic code or of differences in codon usage Differences in preferred codon usage are illustrated in http //www kazusa or jp/codon Allelic variants are further defined as to comprise single nucleotide polymorphisms (SNPs) as well as small insertion/deletion polymorphisms (INDELs, having a size of usually less than 100 bp) SNPs and INDELs form the largest set of sequence variants in naturally occurring polymorphic strains of most organisms Additionally or alternatively, in particular conventional breeding programs, such as for example marker assisted breeding, it is sometimes practical to introduce allelic variation in the plants by mutagenic treatment of a plant One suitable mutagenic method is EMS mutagenesis Identification of allelic variants then takes place by, for example, PCR This is followed by a selection step for selection of superior allelic variants of the sequence in question and which give rise to altered growth characteristics Selection is typically earned out by monitoring growth performance of plants containing different allelic variants of the sequence in question (for example SEQ ID NO 1, 3, 5, 7 or 9) Monitoring growth performance can be done in a greenhouse or in the field Further optional steps include crossing plants in which the supenor allelic variant was identified with another plant This could be used, for example, to make a combination of interesting phenotypic features According to another aspect of the present invention, advantage may be taken of the nucleotide sequence capable of modulating expression of a nucleic acid encoding a CRYO protein (such as SEQ ID NO 2, 4, 6, 8 or 10) in breeding programs For example, in such a program, a DNA marker is identified which may be genetically linked to the gene capable of modulating the activity of a protein of interest (for example SEQ ID NO 2, 4, 6, 8 or 10) in a plant (which gene can be the gene encoding a protein of interest or another gene capable of influencing the activity of a protein of interest) This DNA marker is then used in breeding programs to select plants having altered growth characteristics Many techniques are nowadays available to identify SNPs and/or INDELs Also within the scope of the present invention are nucleic acids which are alternative splice variants of a CRYO protein encoded by any one of SEQ ID NO 1, 3, 5, 7 or 9 The term "alternative splice variant" as used herein encompasses variants of a nucleic acid encoding a CRYO protein in which selected introns and exons have been excised, replaced or added, optionally in response to specific signals Methods for the determination of mtron and exon positions in a genomic sequence of a gene are known in the art Splice variants all originate from one and the same pre-mRNA The splicing occurs after transcnption of the gene but before mRNA translation, and is usually regulated in a tissue specific or temporal way (for example, such that the mRNA has tissue-specific expression, see for example Burge et al , (1999) Splicing of precursors to mRNAs by the spliceosomes In The RNA World II, Gesteland, Cech, and Atkins, eds (Cold Spring Harbor, NY, Cold Spring Harbor Laboratory Press), pp 525-560) Preferred variants will be ones in which the biological activity of the protein remains unaffected, which can be achieved by selectively retaining functional segments of the protein Methods for making such splice variants are well known in the art, for example by RNAi (Celotto and Graveley (2002) RNA 8, 718-724), with πbozymes, by introducing mutations in the gene, by modifying the spliceosome or by modifying the signal transduction pathways inducing alternative splicing

The invention furthermore encompasses nucleic acids that are capable of hybridising with a nucleic acid encoding a protein as represented by SEQ ID NO 2, 4, 6, 8 or 10 The term "hybridisation" as used herein is the process wherein substantially homologous complementary nucleotide sequences anneal to each other The hybridisation process can occur entirely in solution, i e both complementary nucleic acids are in solution Tools in molecular biology relying on such a process include the polymerase chain reaction (PCR, and all methods based thereon), sub tractive hybridisation, random primer extension, nuclease S1 mapping, primer extension, reverse transcription, cDNA synthesis, differential display of RNAs, and DNA sequence determination The hybridisation process can also occur with one of the complementary nucleic acids immobilised to a matrix such as magnetic beads, Sepharose beads or any other resin Tools in molecular biology relying on such a process include the isolation of poly (A+) mRNA The hybridisation process can furthermore occur with one of the complementary nucleic acids immobilised to a solid support such as a nitro-cellulose or nylon membrane or immobilised by e g photolithography to e g a siliceous glass support (the latter known as nucleic acid arrays or microarrays or as nucleic acid chips) Tools in molecular biology relying on such a process include RNA and DNA gel blot analysis, colony hybridisation, plaque hybridisation, in situ hybridisation and microarray hybridisation In order to allow hybridisation to occur, the nucleic acid molecules are generally thermally or chemically denatured to melt a double strand into two single strands and/or to remove hairpins or other secondary structures from single stranded nucleic acids The stπngency of hybndisation is influenced by conditions such as temperature, salt concentration and hybridisation buffer composition High stringency conditions for hybridisation include high temperature and/or low salt concentration (salts include NaCI and Na₃-cιtrate) and/or the inclusion of formamide in the hybridisation buffer and/or lowering the concentration of compounds such as SDS (detergent) in the hybndisation buffer and/or exclusion of compounds such as dextran sulphate or polyethylene glycol (promoting molecular crowding) from the hybridisation buffer Conventional hybridisation conditions are described in, for example, Sambrook (2001) Molecular Cloning a laboratory manual (3rd Edition Cold Spring Harbor Laboratory Press, CSH, New York), but the skilled craftsman will appreciate that numerous different hybridisation conditions can be designed in function of the known or the expected homology and/or length of the nucleic acid Typical conditions for "stringent hybridisation" are for example hybridising at a temperature of 60°C followed by washes in 2XSSC, 0 1XSDS, and 1X SSC, 0 1X SDS

Advantageously, the method according to the present invention may also be practised using portions of a DNA or nucleic acid, which portions encode polypeptides that retain CRYO activity, i e a similar biological function to those encoding proteins represented in SEQ ID NO 2, 4, 6, 8 or 10 Portions of a DNA sequence refer to a piece of DNA derived or prepared from an original (larger) DNA molecule, which DNA portion, when expressed in a plant, gives rise to plants having modified growth characteristics The portion may comprise many genes, with or without additional control elements, or may contain just spacer sequences etc

The term "part of a sequence" means a truncated sequence of the original sequence referred to The truncated nudeic acid sequence can vary widely in length, the minimum size being a sequence of sufficient size to provide a sequence with at least a comparable function and/or activity or the original sequence referred to, while the maximum size is not critical In some applications, the maximum size usually is not substantially greater than that required to provide the desired activity and/or functιon(s) of the original sequence Typically, the truncated nucleotide sequence will range from about 15 to about 180 nucleotides in length More typically, however, the sequence will be a maximum of about 150 nucleotides in length, preferably a maximum of about 180 nucleotides It is usually desirable to select sequences of at least about 30, 36 or 45 nucleotides, up to a maximum of about 60 or 75 nucleotides

DNA sequences as defined in the current invention can also be interrupted by intervening sequences With "intervening sequences" is meant any nucleic acid which disrupts a coding sequence in the DNA sequence of interest or which disrupts the expressible format of a DNA sequence comprising the DNA sequence of interest Removal of intervening sequences restores the coding sequence or said expressible format Examples of intervening sequences include introns and mobilisable DNA sequences such as transposons With "mobilisable DNA sequence" is meant any DNA sequence that can be mobilized as the result of a recombination event

The present invention also relates to a recombinant genetic construct comprising a nucleic acid according to the invention The genetic constructs facilitate the introduction and/or expression and/or maintenance of a nucleotide sequence as defined above into a plant cell, tissue or organ Preferably, the genetic construct comprises (i) an isolated nucleic acid encoding a plant protein

(a) comprising the sequence as given in SEQ ID NO 2, 4, 6, 8 or 10,

(b) comprising a sequence having at least 50 %, alternatively 60%, 70%, 80%, preferably 90%, more preferably 95%, 96%, 97%, 98% or 99% sequence identity to the full length sequence as given in SEQ ID NO 2, 4, 6, 8 or 10, (c) comprising a substitution variant or insertion vaπant of (a),

(d) according to any of (a) to (c), comprising substitutions with corresponding naturally or non-naturally altered ammo acids, (n) a regulatory element operably linked to the nucleic acid of (i), which regulatory element is a plant and/or yeast expressible promoter, and optionally (HI) a transcription termination sequence

The nucleic acid construct can be an expression vector wherein the nucleic acid is operably linked to one or more regulatory elements allowing expression in prokaryotic and/or eukaryotic host cells The vector may be commercially available, suitable for transforming into plants and suitable for expression of the gene of interest in the transformed cells

Advantageously, any nucleic acid obtainable by the screening method according to the present invention can be used in the construct, preferably a nucleic acid as defined in any of (a) to (d) above is used

The term "operably linked" as used herein refers to a functional linkage between the regulatory element and the gene of interest, such that the regulatory element is able to initiate transcription of the gene of interest

As used herein, the term "plant-expressible promoter" refers to a promoter that is capable of dπving transcription in a plant cell This not only includes any promoter of plant origin, such as the natural promoter of the transcribed DNA sequence, but also any promoter of non-plant origin which is capable of directing transcription in a plant cell The promoter may also be an artificial or synthetic promoter The term "plant-expressible promoter" includes, but is not restricted to, constitutive, mducible, organ-, tissue- or cell-specific and/or developmental ly regulated promoters The terms "regulatory element", "control sequence", "promoter" are all used herein interchangeably and, taken in a broad context, refer to regulatory nucleic acids capable of effecting expression of the sequences to which they are ligated

Advantageously, any type of promoter may be used to drive expression of the nucleic acid encoding a CRYO protein in a plant More specifically, a constitutive promoter can be, but is not restricted to, one of the following a 35S promoter (Odell ef al , Nature 313, 482 - 493, 1985), a 35S'3 promoter (Hull and Howell, Virology 86, 482 - 493, 1987), the promoter of the nopalme synthase gene ("PNOS") of the Ti-plasmid (Herrera-Estrella, Nature 303, 209 - 213, 1983) or the promoter of the octopiπe synthase gene ("POCS", De Greve et al , J Mol Appl Genet 1 , 499 - 511 , 1982) It is clear that other constitutive promoters can be used to obtain similar effects A meπstem-specific promoter, such as the rnr (πbonucleotide reductase), cdc2a promoter and the cyc07 promoter, could be used to effect expression in all growing parts of the plant, thereby increasing cell proliferation, which in turn would increase yield or biomass If the desired outcome would be to influence seed characteristics, such as the storage capacity, seed size, seed number, biomass etc , then a seed-specific promoter, such as p2S2, pPROLAMIN, pOLEOSIN could be selected An aleurone-specific promoter may be selected in order to increase growth at the moment of germination, thereby increasing the transport of sugars to the embryo An inflorescence-specific promoter, such as pLEAFY, may be utilised if the desired outcome would be to modify the number of flower organs To produce male-sterile plants one would need an anther specific promoter To impact on flower architecture for example petal size, one could choose a petal -specific promoter If the desired outcome would be to modify growth and/or developmental characteristics in particular organs, then the choice of the promoter would depend on the organ to be modified For example, use of a root-specific promoter would lead to increased growth and/or increased biomass or yield of the root and/or phenotypic alteration of the root This would be particularly important where it is the root itself that is the desired end product, such crops include sugar beet, turnip, carrot, and potato A fruit-specific promoter may be used to modify, for example, the strength of the outer skin of the fruit or to increase the size of the fruit A green tissue-specific promoter may be used to increase leaf size A cell wall-specific promoter may be used to increase the rigidity of the cell wall, thereby increasing pathogen resistance An anther-specific promoter may be used to produce male-sterile plants A vascular-specific promoter may be used to increase transport from leaves to seeds A nodule-specific promoter may be used to increase the nitrogen fixing capabilities of a plant, thereby increasing the nutrient levels in a plant A stress- mducible promoter may also be used to drive expression of a nucleic acid to increase membrane integrity during conditions of stress A stress mducible promoter such as the water stress induced promoter WSI18, the drought stress induced Trg-31 promoter, the ABA related promoter rab21 or any other promoter which is induced under a particular stress condition such as temperature stress (cold, freezing, heat) or osmotic stress, or drought stress or oxidative stress or biotic stress can be used to drive expression of a CRYO gene

If the desired outcome would be to influence the cold tolerance of a plant under adverse conditions, then a cold-mducible promoter such as, for example, prd29, pws18 or pcor15 could be selected

Similarly, the term "yeast-expressible promoter" refers to a promoter that is capable of driving transcription in a yeast cell and encompasses natural yeast promoters as well as other promoter sequences capable of driving expression in yeast cells Suitable promoters for expression in yeast are known in the art, see for example Current Protocols in Molecular Biology, Unit 13 (Ausubel et al , 1994) and the Guide to Yeast Genetics and Molecular Biology (Guthne and Fink, 1991)

The recombinant genetic construct according to the present invention may include further regulatory or other sequences from other genes Encompassed are transcriptional regulatory sequences derived from a classical eukaryotic genomic gene (including the TATA box which is required for accurate transcription initiation, with or without a CCAAT box sequence) and additional regulatory elements (i e upstream activating sequences, enhancers and silencers) which alter gene expression in response to developmental and/or external stimuli, or in a tissue-specific manner Also included is a transcriptional regulatory sequence of a classical prokaryotic gene, in which case it may include a -35 box sequence and/or -10 box transcriptional regulatory sequences Regulatory elements also encompass a synthetic fusion molecule or denvative which confers, activates or enhances expression of a nucleic acid molecule in a cell, tissue or organ

Optionally, one or more terminator sequences may also be used in the construct introduced into a plant The term "terminator" encompasses a control sequence which is a DNA sequence, at the end of a transcriptional unit, which signals 3' processing and polyadenylation of a primary transcript and termination of transcription Additional regulatory elements may include transcriptional as well as translational enhancers Those skilled in the art will be aware of terminator and enhancer sequences which may be suitable for use in performing the invention Furthermore, the recombinant nucleic acid can be constructed and employed to target the gene product of the nucleic acid of the invention to a specific intracellular compartment within a plant cell or to direct a protein to the extracellular environment This can generally be obtained by operably joining a DNA sequence encoding a transit or signal peptide to the recombinant nucleic acid

The genetic constructs of the invention may further include an origin of replication sequence which is required for maintenance and/or replication in a specific cell type, for example a bacterial cell, when the genetic construct is required to be maintained as an episomal genetic element (e g plasmid or cosmid molecule) in a cell Preferred origins of replication include, but are not limited to, the f1 -oπ and colE1 origins of replication

The genetic construct may optionally comprise a selectable marker gene As used herein, the term "selectable marker gene" includes any gene which confers a phenotype on a cell in which it is expressed to facilitate the identification and/or selection of cells which are transfected or transformed with a genetic construct of the invention or a derivative thereof Suitable markers may be selected from markers that confer antibiotic or herbicide resistance Cells containing the recombinant DNA will thus be able to survive in the presence of antibiotic or herbicide concentrations that kill untransformed cells Examples of selectable marker genes include the bar gene which provides resistance to the herbicide Basta, the ampicillin resistance gene

(Amp^r), the tetracyclme resistance gene (Tc^r), the bacterial kanamycm resistance gene (Kan^r), the phosphinothπcin resistance gene, the neomycin phosphotransferase gene (nptll), the hygromycin resistance gene, and the chloramphemcol acetyltransferase (CAT) gene Visual markers, such as the Green Fluorescent Protein (GFP, Haseloff et al , Nature 334, 585-591 ,

1997), β-glucuromdase (GUS), and luciferase, may also be used as selectable markers

According to another embodiment, the present invention relates to the use of the nucleic acids encoding a protein of the present invention or the use of such a protein as selectable marker gene in plants or other organisms More preferably, the present invention also relates to the use of a gene coding for a CRYO protein as defined above as selectable marker gene, selection taking place by treating with a stress condition such as a sub-optimal growth temperature

The nucleic acids obtainable by the screening method as described herein encode proteins that support faster growth of yeast under stress conditions, relative to wild type yeast, therefore it is likely, since these nucleic acids originate from plants, that modulation of expression of these nucleic acids upon introduction into plants, will also support faster growth of plants under stress conditions when compared to corresponding wild type plants Therefore the present invention provides a method for increasing abiotic stress tolerance of plants, comprising the steps of introducing a genetic modification in these plants and selecting for modulated expression in these plants of a nucleic acid sequence encoding a protein

(a) comprising the sequence as given in SEQ ID NO 2, 4, 6, 8 or 10,

(b) comprising a sequence having at least 50 %, alternatively 60%, 70%, 80%, preferably 90%, more preferably 95%, 96%, 97%, 98% or 99% sequence identity to the full length sequence as given in SEQ ID NO 2, 4, 6, 8 or 10, (c) comprising a substitution variant or insertion variant of (a),

(d) according to any of (a) to (c), comprising substitutions with corresponding naturally or non-naturally altered ammo acids,

(e) comprising a functional fragment of any of (a) to (d)

Preferably this protein increases cold stress tolerance of yeast Similarly, the present invention provides a method for increasing stress tolerance of yeast, preferably to cold stress, comprising modulating expression in plants of a nucleic acid sequence encoding a CRYO protein and/or modulating activity of a CRYO protein

Advantageously, proteins homologous to a CRYO protein can also be used in the methods of the present invention The invention thus provides a method for increasing abiotic stress tolerance of plants, comprising the steps of introducing a genetic modification in these plants and selecting for modulated expression in these plants of a nucleic acid sequence encoding a protein chosen from the group of (i) proteins belonging to the family of CHMP proteins (n) proteins comprising a SEC14 domain and exhibiting hpid transfer activity

(in) CRY05 like plant proteins comprising a RING finger domain, a serme rich domain and an acid domain which comprises the signature "HDQHRDMRLDTDNMSYFELLALEERIG", in which no more than 6 substitutions may occur

"Increased stress tolerance" as used herein comprises, for any given stress, increasing tolerance in plants or yeast to that particular stress, whether those plants or yeast already have some degree of tolerance to the particular stress or whether that plant or yeast is being provided with tolerance to that stress anew

Preferably, the increased tolerance is to at least one of temperature stress, osmotic stress, drought stress, salt stress or oxidative stress, more preferably cold stress The terms "tolerance" and "resistance" as used herein encompass protection against stress ranging from a delay to substantially a complete inhibition of alteration in cellular metabolism, reduced cell growth and/or cell death caused by environmental stress conditions Advantageously, transgenic plants or yeasts obtained by the methods of the present invention are tolerant or resistant to environmental stress conditions

The term "environmental stress" as used herein encompasses stress factors such as drought stress (water, dehydration), osmotic stress, salt stress, temperature stress (due to for example heat or frost) "Temperature stress" which includes "cold stress", "chilling stress", "freezing stress" or "heat stress" is a stress induced by sub-optimal or supra-optimal growth temperatures for a particular organism Optimal growth temperature ranges may be readily determined or would be known to those skilled in the art "Osmotic stress" is any stress associated with or induced by loss of water, reduced turgor or reduced water content of a cell, tissue, organ or whole plant "Drought stress" refers to any stress which is induced by or associated with the deprivation of water or reduced supply of water to a cell, tissue, organ or organism The term "salt-stress" refers to any stress which is associated with or induced by elevated concentrations of salt or ions in general and which result in a perturbation in the osmotic potential of the intracellular or extracellular environment of a cell "Oxidative stress" occurs in situations of cold stress combined with intensive light, in situations of ozone stress, in cases of necrosis as a result of pathogen infection or wounding, in cases of senescence and due to application of certain herbicides (like atrazme or paraquat)

According to a preferred feature of the invention, the stress is cold stress Advantageously the results of testing for tolerance or resistance to environmental conditions in the yeast cells give a reliable measure of the capability of the inserted coding sequence or gene to induce tolerance or resistance to environmental stress in plants The capacity of an isolated nucleic acid to confer tolerance or resistance to environmental stress tolerance to plants can be tested according to methods well-known in the art, see for example, Physical Stresses in Plants Genes and Their Products for Tolerance S Gnllo (Editor), A Leone (Editor) (June 1996),

Springer Verlag, ISBN 3540613471 , Handbook of Plant and Crop Stress Mohammad

Peassarak (Editor), Marcel Dekker, ISBN 0824789873, The Physiology of Plants Under

Stress, Abiotic Factors Erik T Nilsen, David M Orcutt (Contributor), Eric T Nilsen 2^nd edition

(October 1996), John Wiley & Sons, ISBN 047131526, Drought, Salt, Cold and Heat Stress Molecular Responses in Higher Plants (Biotechnology Intelligence Unit) Kazuo Shinozaki

(Editor), Kazuko Yamaguchi-Shinozaki (Editor) (1999) R G Landes Co, ISBN 1570595631 ,

Plants Under Stress Biochemistry, Physiology and Ecology and Their Application to Plant Improvement (Society for Experimental Biology Seminar Sene) Hamlyπ G Jones, T J Flowers, M B Jones (Editor) (September 1989) Cambridge Univ Pr (Short), ISBN 0521344239, Plant Adaptation to Environmental Stress Leslie Fowden, Terry Mansfield, John Stoddart (Editor) (October 1993) Chapman & Hall, ISBN 0412490005, or the appended examples Similar methods exist for yeast, see for example The molecular and cellular biology of the yeast Saccharomyces cerevisiae Pnngle, Jones, Broach and Strathern, Cols Spring Harbor laboratory press, 1992 (New York), Guide to yeast Genetics and Mollecular and Cell Biology (Volum 350 and 351 of Methods in enzymology) (Guthπe and Fmf Eds), Academic Press (2002) San Diego, Yeast Gene Analysis (Brown and Tuite) (Volume 26 of Methods in Microbiology) Academic press (San Diego), Yeast Stress Responses (Ed Hohmann and Mager) Springer Verlag, Heidelberg 1997

The methods of the present invention encompass a genetic modification of a plant or a plant cell The term "genetic modification" refers to a change by human intervention in the genetic content of a cell compared to a wild type cell and includes techniques like genetic engineeπng, breeding or mutagenesis The change in genetic content comprises modifications of the genome and includes addition, deletion and substitution of genetic material in the chromosomes of a plant cell as well as in episomes The term also encompasses the addition of extrachromosomal information to a plant cell Preferably, the genetic modification results in modulated expression of a nucleic acid The methods of the present invention also encompass a subsequent step of selection, during which plants with the desired characteristics are selected The selection step may be based on monitoring the presence or absence of modified growth characteristics, or on monitoring the presence or absence of selectable or screenable marker genes linked an introduced nucleic acid of interest

Modulation (enhancing or decreasing) of expression of a nucleic acid encoding a CRYO protein or modulation of a CRYO protein itself encompasses altered expression of a gene or altered levels of a gene product, namely a polypeptide, in specific cells or tissues, which gene or gene-product influences CRYO gene expression or protein activity

The nucleic acids obtained by the screening method according to the invention will have the capacity to modify tolerance to cold stress in plants or yeast This effect may also be obtained by applying the proteins encoded by the nucleic acids as defined above, directly to the plants or yeast

Preferably, modulation of expression of a nucleic acid encoding a CRYO protein and/or modulation of activity of the CRYO protein itself is effected by recombinant means Such recombinant means may comprise a direct and/or indirect approach for modulation of expression of a nucleic acid and/or for modulation of the activity of a protein

For example, an indirect approach may comprise introducing, into a plant, a nucleic acid capable of modulating activity of the protein in question (a CRYO protein) and/or expression of the gene in question (a gene encoding a CRYO protein) The CRYO gene or the CRYO protein may be wild type, i e the native or endogenous nucleic acid or polypeptide Alternatively, it may be a nucleic acid derived from the same or another species, which gene is introduced as a transgene, for example by transformation This transgene may be substantially modified from its native form in composition and/or genomic environment through deliberate human manipulation Also encompassed by an indirect approach for modulating activity of a CRYO protein and/or expression of a CRYO gene is the inhibition or stimulation of regulatory sequences that drive expression of the native gene or transgene Such regulatory sequences may be introduced into a plant

A direct and more preferred approach comprises introducing into a plant or yeast a nucleic acid encoding a protein, or a functional fragment thereof, as defined above The nucleic acid may be introduced by, for example, transformation The nucleic acid may be derived (either directly or indirectly (if subsequently modified)) from any source provided that the sequence, when expressed in a plant or yeast, leads to modulated expression of a CRYO nucleic acid/gene or modulated activity of a CRYO protein

Preferably, the nucleic acid is isolated from a halophytic plant, more preferably from Sefa vulgans Most preferably, the nucleic acid capable of modulating expression of a CRYO gene or activity of a CRYO protein in a plant is a nucleic acid as represented by SEQ ID NO 1, 3, 5, 7, 9, or homologues, derivatives or functional fragments thereof, or a nucleic acid encoding a protein represented by SEQ ID NO 2, 4, 6, 8 or 10, or homologues, derivatives or functional fragments thereof

However, it should be clear that the applicability of the invention is not limited to use of a nucleic acid represented by SEQ ID NO 1 , 3, 5, 7, 9 nor to the nucleic acid encoding the protein of SEQ ID NO 2, 4, 6, 8 or 10, but that other nucleic acids encoding homologues, derivatives or functional fragments of SEQ ID NO 1 to 10 may be useful in the methods of the present invention Advantageously, the method according to the present invention serves to confer tolerance or resistance to environmental stress conditions in plants and parts thereof, or in yeast Modulating the activity of a nucleic acid/gene can be achieved for example by inhibiting or stimulating control elements that dπve expression of a native gene or of a transgene, such regulatory sequences may be introduced into a plant or yeast The "nucleic acid" or "protein" may be wild type, i e the native or endogenous nucleic acid or polypeptide Alternatively, the gene may be a heterologous nucleic acid derived from the same or another species, which gene is introduced as a transgene, for example, by transformation This transgene may be substantially modified from its native form in composition and/or genomic environment through deliberate human manipulation Modulating gene expression also encompasses altered transcript level of a gene, which can be sufficient to induce certain phenotypic effects

According to a preferred feature of the present invention, enhanced or increased expression of a nucleic acid is envisaged Methods for obtaining enhanced or increased expression of genes or gene products are well documented in the art and include, for example, overexpression dnven by a strong promoter, the use of transcription enhancers or translation enhancers

However downregulation of the expression of a nucleic acid may also give rise to modified stress tolerance in a plant or yeast Advantageously, plants having modified stress tolerance may be obtained by expressing a nucleic acid encoding a CRYO protein in either sense or antisense orientation Techniques for downregulation are well known in the art Similar and other approaches for downregulation expression in yeast are known in the art (for example interruption of the ORF with a gene complementing a metabolic defect of the host strain or with a gene from bacteria conferring tolerance to the antibiotics Kanamycin or Geπeticin)

Another embodiment of the invention provides host cells comprising a nucleic acid molecule encoding a CRYO protein Preferred host cells are plant cells or yeast The polypeptides of the present invention may also be produced by recombinant expression in prokaryotic and eukaryotic engineered cells other than plant cells, such as bacteria, fungi or animal cells Suitable expression systems are known to those skilled in the art

The invention extends to plants or yeast tolerant to abiotic stress, preferably cold stress, which plants or yeast have elevated levels of a protein as defined above compared to corresponding wild type plants or yeast The present invention thus also encompasses plants obtainable by the methods according to the present invention The present invention therefore provides plants obtainable by the method according to the present invention, which plants have increased stress tolerance and which plants have altered CRYO protein activity and/or altered expression of a nucleic acid encoding a CRYO protein In particular, the present invention provides plants with increased tolerance to abiotic stress, preferably cold stress, which plants have increased expression of a nucleic acid encoding a protein, or a functional fragment thereof, chosen from the group of (ι) proteins belonging to the family of CHMP proteins, (n) proteins comprising a SEC14 domain and exhibiting lipid transfer activity, (in) CRY05 like plant proteins comprising a RING finger domain, a serme πch domain and an acid domain which comprises the signature "HDQHRDMRLDIDNMSYEELLALEERIG", in which no more than 6 substitutions may occur, which increased tolerance is relative to corresponding wild type plants

Furthermore, the present invention provides plants with increased tolerance to abiotic stress, preferably cold stress, which plants have increased expression of a nucleic acid encoding a protein

(a) comprising the sequence as given in SEQ ID NO 2, 4, 6, 8 or 10,

(b) comprising a sequence having at least 50 %, alternatively 60%, 70%, 80%, preferably 90%, more preferably 95%, 96%, 97%, 98% or 99% sequence identity to the full length sequence as given in SEQ ID NO 2, 4, 6, 8 or 10,

(c) comprising a substitution variant or insertion variant of (a),

(d) according to any of (a) to (c), comprising substitutions with corresponding naturally or non-naturally altered ammo acids, (e) comprising a functional fragment of any of (a) to (d), when compared to corresponding wild type plants

The present invention also relates to a method for the production of transgenic plants, plant cells or plant tissues, comprising introduction of a nucleic acid molecule of the invention in an expressible format or a genetic construct as defined above into a plant, plant cell or plant tissue Therefore, according to a further embodiment of the present invention there is provided a method for producing transgenic plants having modified tolerance to stress, relative to corresponding wild type plants, which method comprises (i) introducing into a plant cell a nucleic acid encoding a CRYO protein or a family member of a CRYO protein or a functional fragment thereof, and

(n) regenerating and/or growing a mature plant from said plant cell

Preferably, the stress is at least one of cold stress, salt stress, osmotic stress, drought stress or oxidative stress More preferably, the stress is cold stress

The present invention extends to any plant cell, plant or plant part or yeast cell obtained by any of the methods described herein, and to all plant parts, including harvestable parts of a plant, seeds and propagules thereof The present invention also encompasses a plant or a part thereof compnsmg a plant cell transformed with a nucleic acid according to the invention The present invention extends further to encompass the progeny of a primary transformed or traπsfected cell, tissue, organ or whole plant that has been produced by any of the aforementioned methods, the only requirement being that progeny exhibit the same genotypic and/or phenotypic characterιstιc(s) as those produced in the parent by the methods according to the invention

The term "plant" as used herein encompasses whole plants, ancestors and progeny of the plants, plant parts, plant cells, tissues and organs The term "plant" also therefore encompasses suspension cultures, embryos, menstematic regions, callus tissue, leaves, flowers, fruits, seeds, rhizomes, bulbs, roots (including tubers), shoots (including stem cultures), gametophytes, sporophytes, pollen, and microspores Plants that are particularly useful in the methods of the invention include all plants which belong to the superfamily Vindiplantae, in particular monocotyledonous and dicotyledonous plants including a fodder or forage legume, ornamental plant, food crop, tree, or shrub selected from the list comprising Acanthaceae, Aceraceae, Acoraceae, Adiantaceae, Agavaceae, Aizoaceae, A smataceae, Alliaceae, Aloeaceae, Alstroemeriaceae, Amaranthaceae, Amaryllidaceae, Anacardiaceae, Anemiaceae, Angiopteπdaceae, Annonaceae, Apocynaceae, Aponogetonaceae, Aquifoliaceae, Araceae, Araliaceae, Araucaπaceae, Arecaceae, Anstolochiaceae, Asparagaceae, Aspleniaceae, Asteliaceae, Asteraceae, Balsaminaceae, Basellaceae, Bataceae, Begoniaceae, Berbeπdaceae, Betulaceae, Bignoniaceae, Bixaceae, Blechnaceae, Bombacaceae, Boraginaceae, Brassicaceae Alliaπa petiolata, Arabidopsis thaliana, Arabis petiolans, Arabis pumila, Arabis sp , Berteroa incana, Biscutella laevigata, Brassicajunceae, Brassica napus, Brassica napus var napus, Brassica nigra, Brassica oleracea, Brassica oleracea var gongylo, Capsella bursa-pastons, Cardamme pratensis, Cochleana offiαnalis, Deπtana lacmiata, Descuraima pinnata, Draba asprella, Draba vema, Draba, Erysimum aspβrum, Erysimum asperu , Erysimum capitatum, Lepidiu flavum, Lepidium virginicum, Lesquerella argyraea, Lesquerella densiflora, Lesquerella rubicundula, Lesquerella sp , Lobulaπa manti a, Lunaπa annua, Lunaπa rediviva, Neobeckia aquatica, Nensyrenia camporum, Physana chambersii, Raphanus sativus, Sinapis alba, Stanleya pinnata, Streptanthus cordatus, Thlaspi arvense, Thlaspi rotundifolium, Bromeliaceae, Buddlejaceae, Burseraceae, Buxaceae, Cabombaceae, Cactaceae, Caesalpmiaceae, Callitnchaceae, Calochortaceae, Calyceraceae, Campanulaceae, Cannabaceae, Cannaceae, Capparaceae, Capπfoliaceae, Cancaceae, Caryophyllaceae, Casuannaceae, Celastraceae, Chenopodiaceae, Cistaceae, Clusiaceae, Cneoraceae, Cochlospermaceae, Combretaceae, Commelmaceae, Convallanaceae, Convolvulaceae, Comaceae, Corylaceae, Crassulaceae, Crossosomataceae, Cucurbitaceae, Cunoniaceae, Cupressaceae, Cuscutaceae, Cyatheaceae, Cycadaceae, Cyperaceae, Cynllaceae, Dennstaedtiaceae, Dicksoniaceae, Didiereaceae, Dilleniaceae, Dioscoreaceae, Dipsacaceae, Dipterocarpaceae, Droseraceae, Dryoptendaceae, Ebenaceae, Ehretiaceae, Elaeagnaceae, Elaeocarpaceae, Elat aceae, Empetraceae, Epacπdaceae, Ephedraceae, Equisetaceae, Ericaceae, Eπocaulaceae, Erythroxylaceae, Escalloniaceae, Euphorbiaceae, Eupomatiaceae, Fabaceae, Fagaceae, Flacourtiaceae, Fouquienaceae, Frankeniaceae, Fumanaceae, Gentianaceae, Geraniaceae, Gesnenaceae, Gmkgoaceae, Globulanaceae, Goodeniaceae, Grossulanaceae, Gunneraceae, Haemodoraceae, Haloragaceae, Hamamelidaceae, He coniaceae, Hippocastanaceae, Hyacmthaceae, Hydrangeaceae, Hydrophyllaceae, Hypencaceae, Indaceae, Isoetaceae, Juglandaceae, Juncaceae, Koeberliniaceae, Krameπaceae, Lamiaceae, Lauraceae, Lecythidaceae, Lemnaceae, Lentibulanaceae, Liliaceae, Limnanthaceae, Limnochaπtaceae, Linaceae, Loasaceae, Lobe aceae, Loganiaceae, Lomandraceae, Lomanopsidaceae, Loranthaceae, Lycopodiaceae, Lythraceae, Magnoliaceae, Malpighiaceae, Malvaceae, Marantaceae, Marcgraviaceae, Marsileaceae, Martyniaceae, Mayacaceae, Melanthiaceae, Melastomataceae, Meliaceae, Mehanthaceae, Menispermaceae, Menyanthaceae, Mimosaceae, Monimiaceae, Monotropaceae, Moraceae, Musaceae, Myoporaceae, Myπcaceae, Mynsticaceae, Myrsmaceae, Myrtaceae, Nelumbonaceae, Nyctaginaceae, Nymphaeaceae, Nyssaceae, Ochnaceae, Oenotheraceae, Oleaceae, Oliniaceae, Onagraceae, Ophioglossaceae, Orchidaceae, Orobanchaceae, Osmundaceae, Oxalidaceae, Paeoniaceae, Pandanaceae, Papaveraceae.Passifloraceae, Pedaliaceae, Philydraceae, Phormiaceae, Phytolaccaceae, Pinaceae, Piperaceae, Pittosporaceae, plantaginaceae, Platanaceae, Plumbaginaceae, Poaceae, Podocarpaceae, Podophyllaceae, Polemoniaceae, Polygalaceae, Polygonaceae, Polypodiaceae, Pontedeπaceae, Portulacaceae, Pnmulaceae, Proteaceae, Pteπdaceae, Punicaceae, Pyrolaceae, Rafflesiaceae, Ranunculaceae, Resedaceae, Restioπaceae, Rhamnaceae, Rosaceae, Rubiaceae, Ruscaceae, Rutaceae, Salicaceae, Salvmiaceae, Santalaceae, Sapmdaceae, Sapotaceae, Sarraceniaceae, Saururaceae, Saxifragaceae, Scrophulanaceae, Selagmellaceae, Simaroubaceae, Smilacaceae, Solanaceae, Sparganiaceae, Sterculiaceae, Stre tziaceae, Styracaceae, Taccaceae, Tamaπcaceae, Taxaceae, Taxodiaceae, Theaceae, Thelypteπdaceae, Thymelaeaceae, Tiliaceae, Trapaceae, Tremandraceae, Trilliaceae, Trochodendraceae, Tropaeolaceae, Tumeraceae, Typhaceae, Ulmaceae, Urticaceae, Valeπanaceae, Verbenaceae, Veronicaceae, Violaceae, Viscaceae, Vitaceae, Welwitschiaceae, Winteraceae, Xanthorrhoeaceae, Xerophyllaceae, Xyndaceae, Zamiaceae, Zingiberaceae, and Zygophyllaceae According to a preferred feature of the present invention, the plant is a monocotyledonous or dicotyledonous plant, such as a crop plant selected from rice, maize, wheat, barley, soybean, sunflower, canola, alfalfa, millet, barley, rapeseed and cotton Additional species such as amaranth, artichoke, asparagus, broccoli, Brussels sprouts, cabbage, carrot, cauliflower, celery, collard greens, flax, kale, lentil, oilseed rape, okra, onion, potato, sugar beet, sugar cane, tomato, squash, and tea, trees and algae are not excluded Further advantageously, plants obtained by the methods according to the invention enable crops to be grown with improved yield, growth, development and productivity under stress conditions, preferably under conditions of cold stress The present invention also enables crops to be grown in areas which would otherwise not be possible

The gene of interest is preferably introduced into a plant by transformation The term "transformation" as referred to herein encompasses the transfer of an exogenous polynucleotide into a host cell, irrespective of the method used for transfer The polynucleotide may be transiently or stably introduced into a host cell and may be maintained non-integrated, for example, as a plasmid, or alternatively, may be integrated into the host genome The resulting transformed plant cell can then be used to regenerate a transformed plant in a manner known to persons skilled in the art Transformation of a plant species is now a fairly routine technique Advantageously, any of several transformation methods may be used to introduce the gene of interest into a suitable ancestor cell Transformation methods include the use of liposomes, electroporation, chemicals that increase free DNA uptake, injection of the DNA directly into the plant, particle gun bombardment, transformation using viruses or pollen and microprojection Methods may be selected from the calcium/polyethylene glycol method for protoplasts (Krens ef al , Nature 296, 72 - 74, 1982, Negrutiu I et al , Plant Mol Biol 8, 363 - 373, 1987), electroporation of protoplasts (Shillito et al , Bio/Technol 3, 1099 - 1102, 1985), microinjectioπ into plant material (Crossway ef al , Mol Gen Genet 202, 179 - 185, 1986), DNA or RNA-coated particle bombardment (Klein ef al , Nature 327, 70 1987) infection with (non-integrative) viruses and the like, Λgrooacferavm-mediated transformation (Cheng ef al 1997 - WO 97/48814, Hansen 1998 - WO 98/54961 , Hiei et al 1994 - WO 94/00977, Hiei et al 1998 - WO 98/17813, Riknshi ef al 1999 - WO 99/04618, Saito ef al 1995 - WO 95/06722), including the 'flower dip' transformation method (Bechtold and Pelletier, Methods Mol Biol 82, 259 - 266, 1998, Tπeu ef al , Plant J 22, 531 - 541 , 2000)

Generally after transformation, plant cells or cell groupings are selected for the presence of one or more markers which are encoded by plant-expressible genes co-transferred with the gene of interest, following which the transformed material is regenerated into a whole plant A whole organism may be regenerated from a single transformed or transfected cell, using methods known in the art Plant tissue capable of subsequent clonal propagation, whether by organogenesis or embryogenesis, may be transformed with a genetic construct of the present invention and a whole plant regenerated therefrom The particular tissue chosen will vary depending on the clonal propagation systems available for, and best suited to, the particular species being transformed Exemplary tissue targets include leaf disks, pollen, embryos, cotyledons, hypocotyls, megagametophytes, callus tissue, existing menstematic tissue (e g , apical meπstem, axillary buds, and root meπstems), and induced menstem tissue (e g , cotyledon menstem and hypocotyl menstem)

Following DNA transfer and regeneration, putatively transformed plants may be evaluated, for instance using Southern analysis, for the presence of the gene of interest, copy number and/or genomic organisation Alternatively or additionally, expression levels of the newly introduced DNA may be undertaken using Northern and/or Western analysis, both techniques being well known to persons having ordinary skill in the art

The generated transformed plants may be propagated by a variety of means, such as by clonal propagation or classical breeding techniques For example, a first generation (or T1 ) transformed plant may be selfed to give homozygous second generation (or T2) transformants, and the T2 plants further propagated through classical breeding techniques

The generated transformed organisms may take a variety of forms For example, they may be chimeras of transformed cells and non-transformed cells, clonal transformants (e g , all cells transformed to contain the expression cassette), grafts of transformed and untransformed tissues (e g , in plants, a transformed rootstock grafted to an untransformed scion)

The invention also provides a method for producing a transgenic yeast cell having modified tolerance to abiotic stress relative to corresponding wild type yeast cells, which method comprises introducing into a yeast cell a nucleic acid encoding a CRYO protein as defined above In particular, the present invention provides a method for producing transgenic yeast with increased tolerance to abiotic stress, preferably cold stress, relative to corresponding wild type yeast, which method comprises introducing into a yeast cell a nucleic acid encoding a protein, or a functional fragment thereof, chosen from the group of (i) proteins belonging to the family of CHMP proteins, (u) proteins comprising a SEC14 domain and exhibiting lipid transfer activity,

(in) CRY05 like plant proteins comprising a RING finger domain, a senne rich domain and an acid domain which comprises the signature "HDQHRDMRLDIDNMSYEELLALEERIG", in which no more than 6 substitutions may occur

Furthermore, the present invention provides a method for producing transgenic yeast with increased tolerance to abiotic stress, preferably cold stress, relative to corresponding wild type yeast, which method comprises introducing into a yeast cell a nucleic acid encoding a protein, or a functional fragment thereof

(a) comprising the sequence as given in SEQ ID NO 2, 4, 6, 8 or 10,

(b) comprising a sequence having at least 50 %, alternatively 60%, 70%, 80%, preferably 90%, more preferably 95%, 96%, 97%, 98% or 99% sequence identity to the full length sequence as given in SEQ ID NO 2, 4, 6, 8 or 10,

(c) comprising a substitution variant or insertion variant of (a),

(d) according to any of (a) to (c), comprising substitutions with corresponding naturally or non-naturally altered ammo acids, (e) comprising a functional fragment of any of (a) to (d)

The invention furthermore provides a transgenic yeast cell obtained by the methods of the invention Preferably, the transgenic yeast cell with increased expression of a nucleic acid encoding a protein as defined here above, or a functional fragment thereof, is tolerant to abiotic stress, preferably cold stress when compared to corresponding wild type yeast cells

Furthermore, the invention also relates to the use of a nucleic acid encoding a CRYO protein as defined above or of a CRYO protein itself, to modify stress tolerance of plants or parts thereof or of plant cells The sequences as depicted in SEQ ID NO 1 to SEQ ID NO 10 are revealed to be involved in important processes leading to stress tolerance, as exemplified by plants having altered stress tolerance, which plants have been transformed with a nucleic acid sequence encoding a CRYO protein as defined above Similarly, the invention also relates to the use of a nucleic acid encoding a CRYO protein or of a CRYO protein itself, to modify stress tolerance of yeast Preferably, the stress is at least one of temperature stress, osmotic stress, drought stress salt stress or oxidative stress

The present invention encompasses also the use of homologues of a CRYO protein Therefore the invention relates to the use of a protein or an active fragment thereof, or of a nucleic acid encoding that protein or active fragment thereof, which protein is chosen from (i) proteins belonging to the family of CHMP proteins,

(n) proteins comprising a SEC14 domain and exhibiting lipid transfer activity, (in) CRY05 like plant proteins comprising a RING finger domain, a senne rich domain and an acid domain which comprises the signature "HDQHRDMRLDIDNMSYEELLALEERIG", in which no more than 6 substitutions may occur to modify stress tolerance of plants or parts thereof or of plant cells Preferably, the stress is at least one of temperature stress, osmotic stress, drought stress salt stress or oxidative stress The present invention encompasses also the use of homologues of a CRYO protein Therefore the invention relates to the use of a protein or of a nucleic acid encoding that protein, which protein (a) comprises the sequence as given in SEQ ID NO 2, 4, 6, 8 or 10,

(b) comprises a sequence having at least 50 %, alternatively 60%, 70%, 80%, preferably 90%, more preferably 95%, 96%, 97%, 98% or 99% sequence identity to the full length sequence as given in SEQ ID NO 2, 4, 6, 8 or 10,

(c) comprises a substitution variant or insertion variant of (a), (d) according to any of (a) to (c), comprises substitutions with corresponding naturally or non-naturally altered ammo acids, (e) comprises a functional fragment of any of (a) to (d),

Furthermore, the characteristic of the transgenic plants of the present invention to display tolerance to cold stress conditions can be combined with other approaches to confer cold stress tolerance to plants, e g , use of osmotic protectants such as mannitol, proline, glycme- betaine, water-channeling proteins, etc Thus, the approach of the present invention to confer tolerance to environmental stress conditions to plants can be combined with known approaches which include introduction of various stress tolerance genes Combination of these approaches may have additive and/or synergistic effects in enhancing tolerance or resistance to environmental stress

The methods of the present invention to create plants with enhanced tolerance to stress can also be combined with other traits of interest, for example

(i) herbicide tolerance (DE-A 3701623, Stalker, Science 242 (1988), 419), (II) insect resistance (Vaek, Plant Cell 5 (1987), 159-169),

(in) virus resistance (Powell, Science 232 (1986), 738-743, Pappu, World Journal of Microbiology & Biotechnology 11 (1995), 426-437, Lawson, Phytopathology 86 (1996) 56 suppl ),

(iv) ozone resistance (Van Camp, Biotechnol 12 (1994), 165-168), (v) improving the preserving of fruits (Oeller, Science 254 (1991 ), 437-439),

(vi) improvement of starch composition and/or production (Stark, Science 242 (1992), 419, Visser, Mol Gen Genet 225 (1991), 289-296), (vn) altering lipid composition (Voelker, Science 257 (1992), 72-74), (VIM) production of (bιo)polymers (Poirer, Science 256 (1992), 520-523), (ix) alteration of the flower colour, e g , by manipulating the anthocyanm and flavonoid biosynthetic pathway (Meyer, Nature 330 (1987), 667-678, WO90/12084), (x) resistance to bacteria, insects and fungi (Dueπng, Molecular Breeding 2 (1996), 297-305, Stπttmatter, Bio Technology 13 (1995), 1085-1089, Estruch, Nature Biotechnology 15 (1997), 137-141),

(xi) alteration of alkaloid and/or cardia glycoside composition, (xii) inducing maintaining male and/or female sterility (EP-A1 0 412 006, EP-A1 0 223

399, W093/25695),

(xin) higher longevity of the inflorescences/flowers, and (xiv) abiotic stress resistance, other than temperature stress

Description of the figures:

The present invention will now be illustrated with reference to the following figures

Fig 1 Cold sensitivity of the wild type (wt) yeast strain compared to the gpdl mutant The yeast cells were grown on YPD (top row) or on SD medium (bottom row) at 30°C (control, left column), at 10°C (middle column) or at 15 °C (top right) The WT strain showed reduced growth compared to the gpdl strain

Fig 2 Cold tolerance of the wild type yeast strain transformed with the CRY01 , CRY02, CRY03, CRY04 or CRY05 gene, compared to the wt yeast strain transformed with an empty vector (ρYPGE@) (a) Enhanced growth after 10 days at 10°C of the yeast cells transformed with the CRY01, CRY02 or CRY03 genes, or after 14 days for yeast cells transformed with the CRY04 gene (b) Enhanced growth of the yeast cells transformed with the CRY05 gene compared to wild type yeast transformed with an empty vector (pYPGE) The two left panels are controls grown at 30°C on YPD medium or SD medium The two right panels show growth of the same yeast strains grown at 10°C on YPD medium or SD medium

Fig 3 Alignment between sequences of CRY01 and CRY02 from sugar beet and their homologues in Arabidopsis and yeast At= Arabidopsis thaliana, Bv= Beta vulgans and Sc= Sacchaωmyces cerevisiae

Fig 4 Alignment between the sequences of CRY03 and homologous proteins from various organisms, showing a high degree of conservation among the different species At= Arabidopsis thaliana, Bv= Beta vulgans, Mm= Mus musculus, Hs= Homo sapiens, Sp= Schizosaccharomyces pombe

Fig 5 Southern blot with a CRY01 and CRY02 probe on genomic sugar beet DNA Enzymes used were Ba YW, Hιnd\\\ and EcoRI Fig 6 a) Northern blot with a CRY02 probe Different timepoints (in hrs) after treating the sugar beet plants with 250 mM NaCI are indicated α₃-tubulιn was used as control b) Northern blot with a CRY02 probe Different timepoints (in hrs) after treating the sugar beet plants with 100 μM ABA are indicated α₃-tubulιn was used as control

Fig 7 Growth of wild type yeast (upper row) and yeast transformed with the CRY05 gene (bottom row) on YPD (left panel) and YPD supplemented with 1 mM tert-butyl hydroperoxide (right panel)

Examples

Unless stated otherwise in the Examples, all recombinant DNA techniques are carried out according to standard protocols as described in Sambrook et al (2001) Molecular Cloning A Laboratory Manual, Third Edition, Cold Spring Harbor Laboratory Press, NY and in volumes 1 and 2 of Ausubel et al (1994) Current Protocols in Molecular Biology, Current Protocols, USA Standard materials and methods for plant molecular work are described in Plant Molecular Biology Labfax (1993) by R D D Cray, jointly published by BIOS Scientific Publications Ltd (UK) and Blackwell Scientific Publications, UK Example 1: Construction of a sugar beet cDNA library induced by salt stress:

Sugar beet seeds (Sefa vulgans cv Dita) were sown in pots containing a mixture of sand and vermiculite (1 1 w/w) The plants were grown under greenhouse conditions (8 h at 20°C, 16 h at 25°C with supplementary lighting to ensure a minimum of 12 h photopeπod) The plants were periodically irrigated with a nutrient solution (24 g/l Ca(N03)₂4H₂0, 1 g/l KN0₃, 1 g/l MgSO, 7H₂0, 0 3 g/l KH₂P0₄, 5 6 mg/l Fequelate (Kelantren, Bayer), 1 1 mg/l ZnS0₄7H₂0, 3 3 mg/l Mn0₄ H₂0, 0 3 mg/l CuS0₄5H₂0, 3 8 mg/l H₃B0₃, 0 18 mg/l (NH₄)6Mo₇4H₂0) For the construction of the cDNA library, 3-week old plants were irrigated with 200 mM NaCI for 24 h before harvesting Directional cDNAs were synthesized (cDNA synthesis kit, Stratagene) with poly(A)+ RNA prepared from leaves of salt-treated sugar beet plants cDNAs were ligated into phage ΛPG15 vector and packaged with Gigapack III gold packaging extract (Stratagene) A plasmid cDNA library was recovered from PG15 by the cre-lox recombinase system (Brunelli and Pall, 1993)

Example 2: Setup of a screening assay:

The yeast strains used in this work were the wild type diploid strain W303/W303 (can1-100,hιs 3-11, 15,leu2-3, 112, trp1-1,ura3-1,GAL+) (WT) and a mutant deficient for glycerol phosphate dehydrogenase (gpdl) A diploid strain from two gpdl mutant strains (YRA111(W303-1A mat a gpdi 77?Pf) and YRA114 (W303-1A gpdf TRP1 mat α)) was constructed The diploid strains were used because these prevent the isolation of recessive chromosomal mutations which might give tolerance to cold stress The strains were grown on YPD medium (2% glucose, 2% peptone, and 1% of yeast extract) or on SD medium (2% glucose, 0 7% yeast nitrogen base (Difco) without am o acids, 50 mM MES [2-(N-morpholιno)-ethanesulfonιc acid] adjusted to pH 5 5 with Tπs, and the required am o acids, puπne and pyπmidine bases) In a first step, the sensitivity to cold of the WT diploid strain was compared with that of the gpdl mutant strain It was assumed that the production of glycerol could be a response against cold stress Growth was monitored under cold stress conditions Yeast strains were grown until stationary phase and 20 μl of 1/10, 1/100 and 1/1000 dilutions of the culture were spot on both YPD and on SD medium at a temperature of 15 or 10°C for 10 to 14 days, and at 30°C for 2 days as a control 10°C was the lowest temperature measured that allowed growth of the WT strain Surprisingly, the gpdl strain was shown to be cold tolerant whereas the wild type was cold sensitive (Fig 1) This allowed the use of the WT strain for screening genes that could confer cold tolerance, while the gpdl strain could serve as a standard cold tolerant yeast strain for comparative studies

In a second step, the best conditions for transformation were determined At the end the best protocol was 300 ml YPD medium was inoculated with 30 μl of a saturated preculture of WT cells The culture was grown overnight until an OD₆₆o≡0 8, and centrifuged at 2000 rpm The cells were subsequently washed with water and AcLiTE solution (0 1 M lithium acetate, 10mM Tπs-HCI pH 7 6 and 1 mM EDTA (Ethylene diammetetraacetic acid, disodium salt)) Next, the pellet of cells was resuspended in 2 ml of AcLiTE solution, incubated during 15 minutes at 30°C while shaking, whereafter 200 μl of ssDNA (10 mg/ml) was added The cell suspension was divided in 110 μl a quots in an Eppendorf tubes, and 200 ng of cDNA library was added The heat shock transformation according to Gietz ef al (Nucleic Acids Res 20, 1425, 1992) was used in brief, 500 μl of PEG-AcLiTE solution (AcLiTE solution with 40% w/w of PEG (Polyethyleneglycol) 4000) was added to each aliquot After mixing, the ahquots were incubated for 30 minutes at 30°C and next for twenty minutes at 42°C, then the cells were harvested and resuspended in 200 μl of 1 M sorbitol Two ahquots were plated in 14 cm Petπ dishes with SD agar and all the necessary supplements except tryptophan (marker for the gpdl mutation), and uracil (marker for the plasmid) To quantify the efficiency of the transformation, four 55 μl ahquots were kept separately from the original cell pellet and were inoculated with 0, 10, 50 and 100 ng of cDNA library Then the same transformation protocol was applied, at the end the cells were resuspended in 100 μl of sorbitol and plated in 7 cm Petn dish containing the same SD medium The average yield was about 60 colonies per ng of cDNA In addition it was observed that transformation with competent cells that had been frozen, or transformation in one large-scale reaction instead of many small-scale reactions dramatically decreased the yield of the transformation

Example 3: Isolation of CRYO genes:

The cDNA library constructed in pYPGE15 was used to transform the yeast WT strain W303 by the LiCI method (Nucleic Acids Res 20, 1425, 1992) Transformants were selected on SD plates with leucine and adenine by uracil prototrophy Three days after transformation colonies appeared in the Petn dishes The colonies were harvested in sterile water and the number of cells quantified by plating different dilutions On average a 10-fold higher concentration of cells than recovered from the transformation plates was plated on YPD and SD medium Then the plates were left in a 10°C incubator and colonies able to grow after eight days were selected Next the tolerance of the colonies isolated in the first round was re-checked and those not giving significant tolerance were discarded From the remaining colonies, the plasmid was eliminated by selection in minimal medium for analysing whether the tolerance was dependent on the plasmid As a final confirmation, the plasmid was recovered from the colonies that were able to pass the previous controls, transformed into a wild type strain and again a selection for those clones giving tolerance was performed The results obtained are summarised in Table 1

Table 1 Summary of the screening procedure for the selection of cold tolerant yeast transformants on YPD or SD medium

The reconfirmed positive clones were sequenced and it was shown that they encoded different genes, among those were the genes named CRY01, CRY02, CRY03, CRY04 and CRY05 (for cryo-tolerant) An overview is given in Table 2

Table 2

The genes encoding CRY01 to CRY05 proteins conferred cold stress tolerance when transferred into yeast (Fig 2)

CRY01 was found to be homologous to the yeast DID1 protein and, upon further analysis of the homology data, was shown to have significant homology (<90%) to the Arabidopsis thaliana putative proteins gι/15233464 and gι/15224854 (shown in Fig 3) According to the invention, these putative Arabidopsis proteins were named AtCRYOI and AtCRY02 respectively

CRY03 is also conserved in Arabidopsis, with three putative proteins annotated in the database as At1g73030, At1g17730 and At4g17680, sharing more than 90% of homology According to the invention, these putative Arabidopsis proteins were named AtCRY03, AtCRY03 2 and AtCRY03 3 respectively CRY03 is also conserved in humans and mice, as shown in the pile-up of Fig 4

Example 4: Southern blotting reveals more than one isoform in sugar beet

In order to confirm the presence of CRY01 and CRY02 in the sugar beet genome and to estimate the number of genes encoding the haemoglobin in this plant species, a Southern blot analysis was performed Genomic DNA was prepared from leaves of 3-week old sugar beet leaves (Rogers SO and Bendich AJ, Extraction of total cellular DNA from plants, algae and fungi (Eds) Plant molecular biology manual, Kluwer Academic Publishers, Dordrecht, Netherlands, 1994) 5 mg of DNA were digested with Sa/nHI, Hιnd\\\ or EcoRI, electrophoresed in 0 8% agarose gel and blotted onto a nylon membrane filter (Hybond N+, Amersham Life Science) The membrane filter was hybridised with ³²P-labelled probes for CRY01 and CRY02 Hybridisation and washes were carried out under high stringency conditions (65°C) (Church GM and Gilbert W , Proc Nat Acad Sci USA 81 , 1991-1995, 1984) The presence of several hybridisation fragments in all lanes, independent of the restriction endonucleases used to digest the genomic DNA, suggests that there are several isoforms in the genome, especially for CRY02 (Figure 5)

Example 5: CRY02 is not induced by NaCI and ABA in sugar beet

In order to investigate whether CRY02 also participates in the response of sugar beet plants to salt stress, the accumulation of CRY02 mRNA in response to various exposure times to NaCI was analysed using northern blot analysis Total RNA was isolated from control, 250 mM Na* or 100 mM ABA -treated sugar beet leaves as described by Davis et al (Basic methods in Molecular Biology Elsevier Amsterdam pp 143-146 1986) 30 mg of total RNA were separated on a 1% agarose gel containing 22% formaldehyde and blotted onto a nylon membrane filter (Hybond N, Amersham Life Science) Hybridization using the above described probe The CRY02 specific probe showed only one band that corresponded to the size of the CRY02 cDNA The filter was washed twice with 4X SSC buffer (06 M NaCI, 0 06 M tπsodium citrate adjusted to pH 7 with HCI), 0 1% SDS for 5 minutes and twice with 04X SSC, 0 1% SDS for five minutes at 65°C The same filter was re-hybridized with a 1 9 kb EcoRI fragment comprising the a₃-tubulin gene of Arabidopsis thaliana (Ludwig ef al , PNAS 84, 5833-5837, 1987) As shown in Figure 6a the CRY02 mRNA did not accumulate with time upon NaCI treatment Similarly, no induction of CRY02 after 3 hours of ABA treatment was observed (Figure 6b)

Example 6: CRYOS gives also tolerance to oxidative stress.

A dilution series of W303 pYPGECRYOδ and wt yeast (control) was plated on YPD medium with 1 mM tert-butyl hydroperoxyde (t-BOOH) and tested for tolerance to oxidative stress after 2 and 4 days

The yeast clone with CRY05 had a strong t-BOOH tolerance phenotype and the phenotype was very reproducible at a concentration of 1 mM t-BOOH, control yeast cells did not grow at all, whilst yeast cells overexpressing CRY05 did (Fig 7)

The definition of a strong phenotype is based on drop test experiments Different dilutions of saturated cultures (1 10, 1 100, 1 1000) were made and these were grown on selective media (YPD with 1 mM t-BOOH) "Strong phenotypes" were those clones that grew well in all the dilutions assayed With "no strong phenotypes" is meant that the clone does not grow in all dilutions The control cells expressing the empty plasmid did not grow at all in the selective media

Example 7: construction of cold tolerant plants:

Plants are transformed with at least one of the CRYO genes in an expressible format under control of a constitutive or mducible promoter, using standard techniques

Example 8: Testing of cold tolerant plants:

Transformed plants are tested by subjecting the plants to cold stress during a sufficiently long time period When compared to the same untransformed plant line, the transformed lines show a better growth during stress conditions and/or better recovery after stress conditions and/or higher yield (biomass and/or harvestable parts)

Claims

Claims

1 Method for increasing abiotic stress tolerance of plants, comprising the steps of introducing a genetic modification in said plants and selecting for modulated expression in said plants of a nucleic acid sequence encoding a protein chosen from the group of (i) proteins belonging to the family of CHMP proteins,

(n) proteins comprising a SEC14 domain and exhibiting lipid transfer activity, (in) CRY05 like plant proteins compπsmg a RING finger domain, a senne rich domain and an acid domain which comprises the signature "HDQHRDMRLDIDNMSYEELLALEERIG", in which no more than 6 substitutions may occur

2 A method for increasing abiotic stress tolerance of plants or yeast, comprising the steps of introducing a genetic modification in said plant or yeast and selecting for modulated expression in said plant or yeast of a nucleic acid encoding a protein, and/or modulated activity in a plant or yeast of a protein, which protein

(a) comprises the sequence as given in SEQ ID NO 2, 4, 6, 8 or 10,

(b) comprises a sequence having at least 50 %, alternatively 60%, 70%, 80%, preferably 90%, more preferably 95%, 96%, 97%, 98% or 99% sequence identity to the full length sequence as given in SEQ ID NO 2, 4, 6, 8 or 10, (c) comprises a substitution variant or insertion variant of (a),

(d) according to any of (a) to (c), comprises substitutions with corresponding naturally or non-naturally altered ammo acids,

(e) comprises a functional fragment of any of (a) to (d)

3 Method of claim 1 or 2, wherein said genetic modification comprises introducing into a plant or yeast an isolated nucleic acid encoding said protein or a functional fragment thereof

4 Method of any of claims 1 to 3, wherein said abiotic stress is at least one of temperature stress, osmotic stress, drought stress, salt stress or oxidative stress, preferably said abiotic stress is cold stress

5 A method for producing a transgenic plant having increased tolerance to abiotic stress relative to corresponding wild type plants, which method comprises the steps of (i) introducing into a plant cell a nucleic acid encoding a protein as listed in claim 1 or 2 or a functional fragment thereof, and (n) regenerating and/or growing a mature plant from said plant cell A plant, plant part or plant cell obtained by the method according to any of claims 1 to 5

Plants with increased tolerance to abiotic stress, preferably cold stress, which plants have increased expression of a nucleic acid encoding a protein, or a functional fragment thereof, chosen from the group of

(i) proteins belonging to the family of CHMP proteins, (n) proteins comprising a SEC14 domain and exhibiting lipid transfer activity, (in) CRY05 like plant proteins comprising a RING finger domain, a senne rich domain and an acid domain which comprises the signature "HDQHRDMRLDIDNMSYEELLALEERIG", in which no more than 6 substitutions may occur, when compared to corresponding wild type plants

Plants with increased tolerance to abiotic stress, preferably cold stress, which plants have increased expression of a nucleic acid encoding a protein as listed in claim 2, when compared to corresponding wild type plants

A harvestable part, organ, tissue, propagation material, seed, and ancestors or progeny of a plant according to any of claims 6 to 8

A method for producing a transgenic yeast cell having increased tolerance to abiotic stress relative to corresponding wild type yeast cells, which method comprises introducing into a yeast cell a nucleic acid encoding a protein as listed in claim 1 or 2, or a functional fragment thereof

A transgenic yeast cell obtained by the method of claim 10

Transgenic yeast cell with increased tolerance to abiotic stress, preferably cold stress, which yeast cell has increased expression of a nucleic acid encoding a protein as listed in claim 1 or 2, or a functional fragment thereof, when compared to corresponding wild type yeast cells

The use of a nucleic acid encoding a protein as listed in claim 2 for modifying abiotic stress tolerance in yeast, a plant, plant part or plant cell, wherein said abiotic stress is at least one of temperature stress, osmotic stress, drought stress, salt stress or oxidative stress, preferably said abiotic stress is temperature stress, more preferably said abiotic stress is cold stress

The use of a nucleic acid encoding a protein, or a functional fragment thereof, chosen from the group of

(i) proteins belonging to the family of CHMP proteins, (n) proteins comprising a SEC14 domain and exhibiting lipid transfer activity, (in) CRYOδ like plant proteins comprising a RING finger domain, a senne rich domain and an acid domain which comprises the signature "HDQHRDMRLDIDNMSYEELLALEERIG", in which no more than 6 substitutions may occur, for modifying abiotic stress tolerance in yeast, a plant, plant part or plant cell, wherein said abiotic stress is at least one of temperature stress, osmotic stress, drought stress, salt stress or oxidative stress, preferably said abiotic stress is temperature stress, more preferably said abiotic stress is cold stress

The use of a protein according to claim 2 for modifying abiotic stress tolerance in yeast, a plant, plant part or plant cell, wherein said abiotic stress is at least one of temperature stress, osmotic stress, drought stress salt stress or oxidative stress, preferably said abiotic stress is temperature stress, more preferably said abiotic stress is cold stress

The use of a protein, or a functional fragment thereof, chosen from the group of

(i) proteins belonging to the family of CHMP proteins, (n) proteins comprising a SEC14 domain and exhibiting lipid transfer activity, (in) CRY05 like plant proteins comprising a RING finger domain, a senne rich domain and an acid domain which comprises the signature "HDQHRDMRLDIDNMSYEELLALEERIG", in which no more than 6 substitutions may occur, for modifying abiotic stress tolerance in yeast, a plant, plant part or plant cell, wherein said abiotic stress is at least one of temperature stress, osmotic stress, drought stress salt stress or oxidative stress, preferably said abiotic stress is temperature stress, more preferably said abiotic stress is cold stress

The use of a nucleic acid encoding a protein as listed in claim 1 or 2, or the use of a protein as listed in claim 1 or 2 as a selectable marker in plants or other organisms

An isolated protein (a) comprising the sequence as given in SEQ ID NO 2, 4, 6, 8 or 10,

(b) comprising a sequence having at least i 76 %, alternatively 80%, preferably 90%, more preferably 95%, 96%, 97%, 98% or 99% sequence identity to the full length sequence as given in SEQ ID NO 2, ii 55 %, alternatively 60%, 70%, 80%, preferably 90%, more preferably 95%, 96%, 97%, 98% or 99% sequence identity to the full length sequence as given in SEQ ID NO 4, in 90 5 %, alternatively 90 6%, 90 7%, 90 8%, 90 9%, preferably 91 %, 92%, 93%, 94% more preferably 95%, 96%, 97%, 98% or 99% sequence identity to the full length sequence as given in SEQ ID NO 6, iv 50 %, alternatively 60%, 70%, 80%, preferably 90%, more preferably 95%, 96%, 97%, 98% or 99% sequence identity to the full length sequence as given in SEQ ID NO 8, v 50 %, alternatively 60%, 70%, 80%, preferably 90%, more preferably

95%, 96%, 97%, 98% or 99% sequence identity to the full length sequence as given in SEQ ID NO 10,

(c) comprising a substitution variant or insertion variant of (a),

(d) according to any of (a) to (c), comprising substitutions with corresponding naturally or non-naturally altered ammo acids

An isolated nucleic acid encoding a protein according to claim 19, the complement thereof or a part thereof

A genetic construct comprising

(i) an isolated nucleic acid encoding a plant protein

(a) comprising the sequence as given in SEQ ID NO 2, 4, 6, 8 or 10,

(b) comprising a sequence having at least 50 %, alternatively 60%, 70%, 80%, preferably 90%, more preferably 95%, 96%, 97%, 98% or 99% sequence identity to the full length sequence as given in

SEQ ID NO 2, 4, 6, 8 or 10,

(c) comprising a substitution variant or insertion variant of (a),

(d) according to any of (a) to (c), comprising substitutions with corresponding naturally or non-naturally altered ammo acids, (n) a regulatory element operably linked to the nucleic acid of (i), which regulatory element is a plant and/or yeast expressible promoter, and optionally (MI) a transcription termination sequence

A host cell compnsmg an isolated nucleic acid encoding a protein according to claim 18, wherein said host cell is a bacterial, yeast, fungal, plant or animal cell

Screening method for identifying nucleic acids capable of modifying tolerance or resistance to cold stress conditions in plants or yeast, said method comprising the steps of

(i) providing a cDNA library of coding sequences from an organism, (n) introducing these coding sequences in an expressible format into wild type yeast cells, (in) growing the yeast cells of (n) under conditions of cold stress, (iv) identifying differences between the transgenic yeast cells and wild type yeast cells, preferably identifying differences in growth rate, (v) isolating nucleic acids from the transgenic yeast cells that differ from the wild type yeast cells

Method according to claim 22, wherein said wild type yeast cells are wild type Sacchammyces cerevisiae yeast cells, preferably wild type Sacchammyces cerevisiae W303 yeast cells

Method according to claim 22, wherein said organism is a plant, preferably a salt treated plant, more preferably a salt treated halophytic plant or a part thereof, most preferably salt treated Beta vulgaπs or a part thereof

Use of the screening method according to any of claims 22 to 24 for identifying nucleic acids encoding proteins capable of conferring cold stress tolerance to plant cells or yeast cells

Method for increasing cold tolerance of yeast cells, comprising downregulatmg expression in yeast of a nucleic acid encoding a glycerol phosphate dehydrogenase and/or inhibiting activity of a glycerol phosphate dehydrogenase