# Patent

## Title
Transcriptional activators involved in abiotic stress tolerance

## Abstract

The present invention provides compositions and methods for regulating expression of nucleotide sequences in a plant. Compositions comprise novel nucleic acid sequences encoding a transcription factor involved in modulating gene expression in response to abiotic stress such as cold or drought. Methods for expressing the nucleic acid sequence in a plant and improving cold and/or drought tolerance of plants are also provided.

## Background
This is a continuation-in-part application of U.S. Ser. No. 11/114,672, filed Apr. 26, 2005 now U.S. Pat. No. 7,253,000, which claims priority to and incorporates by reference U.S. provisional application 60/565,430, filed Apr. 26, 2004.


FIELD OF THE INVENTION
The present invention relates to the field of plant molecular biology, more particularly to regulation of gene expression in plants.
BACKGROUND OF THE INVENTION
Stresses to plants may be caused by both biotic and abiotic agents. For example, biotic causes of stress include infection with a pathogen, insect feeding, parasitism by another plant such as mistletoe, and grazing by ruminant animals. Abiotic stresses include, for example, excessive or insufficient available water, temperature extremes, synthetic chemicals such as herbicides, and excessive wind. Yet plants survive and often flourish, even under unfavorable conditions, using a variety of internal and external mechanisms for avoiding or tolerating stress. Plants' physiological responses to stress reflect changes in gene expression.
Insufficient water for growth and development of crop plants is a major obstacle to consistent or increased food production worldwide. Population growth, climate change, irrigation-induced soil salinity, and loss of productive agricultural land to development are among the factors contributing to a need for crop plants which can tolerate drought. Drought stress often results in reduced yield. In maize, this yield loss results in large part from plant failure to set and fill seed in the apical portion of the ear, a phenomenon known as tip kernel abortion.
Low temperatures can also reduce crop production. A sudden frost in spring or fall may cause premature tissue death.
Physiologically, the effects of drought and low-temperature stress may be similar, as both result in cellular dehydration. For example, ice formation in the intercellular spaces draws water across the plasma membrane, creating a water deficit within the cell. Thus, improvement of a plant's drought tolerance may improve its cold tolerance as well.
CBF genes (for C-repeat/DRE binding factor) encode proteins which may interact with a specific cis-acting element of certain plant promoters. (U.S. Pat. Nos. 5,296,462 and 5,356,816; Yamaguchi-Shinozaki, et al., The Plant Cell 6:251-264 (1994); Baker, S. S., et al., Plant Mol. Biol. 24:701-713 (1994); Jiang, C., et al., Plant Mol. Biol. 30:679-684 (1996)) The cis-acting element is known as the C-repeat/DRE and typically comprises a 5-base-pair core sequence, CCGAC, present in one or more copies.
CBF proteins comprise a CBF-specific domain and an AP2 domain and have been identified in various species, including Arabidopsis (Stockinger et al., Proc. Natl. Acad. Sci. 94:1035-1040, 1997; Liu et al., Plant Cell 10:1391-1406, 1998); Brassica napus, Lycopersicon esculentum, Secale cereale, and Triticum aestivum (Jaglo et al., Plant Phys. 127:910-917, 2001) and Brassica juncea, Brassica oleracea, Brassica rapa, Raphanus sativus, Glycine max, and Zea mays (U.S. Pat. No. 6,417,428).
Overexpression of CBF in plants has been shown to improve tolerance to drought, cold, and/or salt stress (Jaglo-Ottosen et al., Science 280:104-106, 1998; Kasuga et al., Nature Biotechnology 17:287-291, 1999; Hsieh et al., Plant Phys. 129:1086-1094, 2002; Hsieh et al., Plant Phys. 130:618-626, 2002; Dubouzet et al., Plant J. 33:751-763, 2003). While CBF transcription factors may be useful in transgenic approaches to regulate plant response to stress, constitutive expression of CBF results in negative pleiotropic effects. Controlled expression of CBF in selected tissues and/or under stress conditions is of interest.
SUMMARY OF THE INVENTION
Compositions and methods for regulating gene expression in a plant are provided. Compositions comprise isolated polynucleotides encoding a transcription factor involved in modulating gene expression in response to cold and/or drought. Further compositions of the invention comprise a polynucleotide of the sequence set forth in SEQ ID NO: 1 or SEQ ID NO: 3, and operable fragments of each. The compositions of the invention further comprise polynucleotides having at least 85% identity to either of the complete sequences set forth in SEQ ID NO: 1 and SEQ ID NO: 3, and polynucleotides which hybridize under stringent conditions to SEQ ID NO: 1 or to SEQ ID NO: 3 or to a complement of either.
In one embodiment of the invention, a DNA construct comprises an isolated polynucleotide of the invention operably linked to a promoter sequence, wherein the promoter is capable of driving expression of the nucleotide sequence in a plant cell. The promoter sequence may be heterologous to the linked nucleotide sequence. In some embodiments, said promoter sequence is inducible by an exogenous agent or environmental condition. In some embodiments, said promoter initiates transcription preferentially in certain tissues or organs.
Also provided are expression cassettes comprising said DNA construct; vectors containing said expression cassette; transformed plant cells, transformed plants, and transformed seeds comprising the novel sequences of the invention.
Further embodiments comprise methods for expressing a polynucleotide of the invention in a plant. The methods comprise stably incorporating into the genome of a plant cell an expression cassette comprising a promoter sequence operably linked to a polynucleotide of the invention, wherein the promoter is capable of initiating transcription of said polynucleotide in a plant cell. Certain embodiments of the present invention comprise methods for modulating the development of a transformed plant under conditions of stress.



BRIEF DESCRIPTION OF THE DRAWINGS
FIG. 1 provides an alignment of several CBF polypeptides: AtCBF3 (SEQ ID NO: 11-12), RyeCBF31 (SEQ ID NO: 10), ZmCBF1 (SEQ ID NO: 3-4), ZmCBF2 (SEQ ID NO: 1-2). The boxed region outlined by the dotted line is the CBF-specific domain, and boxed region outlined by the solid line is the AP2 domain.
FIG. 2 provides photosynthesis data for transgenic and non-transgenic plants as described in Example 12.
FIG. 3 provides average fresh weight under chilling stress for transgenic and non-transgenic seedlings as described in Example 10.
FIG. 4 is a table of 66 genes upregulated more than two-fold in maize plants expressing UBI::ZmCBF1.



DETAILED DESCRIPTION OF THE INVENTION
In accordance with the invention, isolated polynucleotides are provided that encode transcription initiation factors involved in stress-induced gene expression, particularly drought or cold stress.
By “recombinant expression cassette” or “expression cassette” is meant a nucleic acid construct, generated recombinantly or synthetically, comprising a series of specified nucleic acid elements which permit transcription of a particular nucleic acid in a host cell. The recombinant expression cassette can be incorporated into a plasmid, chromosome, mitochondrial DNA, plastid DNA, virus, or nucleic acid fragment. Typically, the expression cassette portion of an expression vector includes, among other sequences, a promoter and a nucleic acid to be transcribed.
By “heterologous nucleotide sequence” is intended a sequence that is not naturally occurring with another sequence. For example, a nucleotide sequence encoding a transcription factor may be heterologous to the promoter sequence to which it is operably linked. Further, the coding sequence and/or the promoter sequence may be native or foreign to the plant host.
By “operable fragment” is meant a truncated or altered form of a particular polynucleotide or polypeptide which is sufficient to perform or provide the relevant function. For example, where the goal is to interfere with gene function, a truncated form of a polynucleotide may be sufficient for purposes of co-suppression or anti-sense regulation. Where the goal is to initiate transcription, a promoter or transcription factor which is less than the full length known, or which comprises minimal internal deletions or alterations, may still function appropriately. Promoter sequences provided, or one or more fragments thereof, may be used either alone or in combination with other sequences to create synthetic promoters. In such embodiments, the fragments (also called “cis-acting elements” or “subsequences”) confer desired properties on the synthetic promoter.
By “promoter” is intended a region of DNA upstream from the start of transcription and involved in recognition and binding of RNA polymerase and other proteins to initiate transcription. A promoter usually comprises a TATA box capable of directing RNA polymerase II to initiate RNA synthesis at the appropriate transcription initiation site for a particular coding sequence. A promoter can additionally comprise other recognition sequences generally positioned upstream or 5′ to the TATA box, referred to as upstream promoter elements, which influence the transcription initiation rate. Thus a promoter region may be further defined by comprising upstream regulatory elements such as those responsible for tissue and temporal expression of the coding sequence, enhancers, and the like. In the same manner, the promoter elements which enable expression in the desired tissue can be identified, isolated, and used with other core promoters.
A “plant promoter” is a promoter capable of initiating transcription in plant cells whether or not its origin is a plant cell. Exemplary plant promoters include, but are not limited to, those that are obtained from plants, plant viruses, and bacteria which comprise genes expressed in plant cells, such as Agrobacterium or Rhizobium. Examples of promoters under developmental control include tissue-preferred promoters, which preferentially initiate transcription in certain tissues, such as leaves, roots, or seeds, and those promoters driving expression when a certain physiological stage of development is reached, such as senescence. Promoters which initiate transcription only in certain tissue are referred to as “tissue-specific.” A “cell-type-preferred” promoter primarily drives expression in certain cell types in one or more organs, for example, vascular tissue in roots or leaves. An “inducible” or “repressible” promoter is a promoter which is under environmental control. Examples of environmental conditions that may effect transcription by inducible promoters include anaerobic conditions or the presence of light. Certain promoters are induced by unfavorable environmental conditions, for example, rab17 (exemplified by SEQ ID NO: 5; see also Busk et al., Plant J 11:1285-1295 (1997)), rd29A (exemplified by SEQ ID NO: 6; see also GenBank D13044 and Plant Cell 6:251-264, 1994), rip2 (exemplified by SEQ ID NOS: 7 and 8; see also GenBank L26305 and Plant Phys. 107(2):661-662 (1995)), and mlip15 (exemplified by SEQ ID NO: 9; see also GenBank D63956; Mol. Gen. Gen. 248(5):507-517 (1995). Tissue-specific, tissue-preferred, cell-type-preferred, and inducible promoters are members of the class of “non-constitutive” promoters. A “constitutive” promoter is a promoter which is active in all or nearly all tissues, at all or nearly all developmental stages, under most environmental conditions.
It is recognized that to increase transcription levels, enhancers can be utilized in combination with promoter regions to increase expression. Enhancers are known in the art and include the SV40 enhancer region, the 35S enhancer element, and the like.
A “subject plant” or “subject plant cell” is one in which genetic alteration, such as transformation, has been effected as to a gene of interest, or is a plant or plant cell which is descended from a plant or plant cell so altered and which comprises the alteration. A “control” or “control plant” or “control plant cell” provides a reference point for measuring changes in the subject plant or plant cell.
A control plant or control plant cell may comprise, for example: (a) a wild-type plant or plant cell, i.e., of the same genotype as the starting material for the genetic alteration which resulted in the subject plant or subject plant cell; (b) a plant or plant cell of the same genotype as the starting material but which has been transformed with a null construct (i.e. with a construct which has no known effect on the trait of interest, such as a construct comprising a marker gene); (c) a plant or plant cell which is a non-transformed segregant among progeny of a subject plant or subject plant cell; (d) a plant or plant cell genetically identical to the subject plant or subject plant cell but which is not exposed to conditions or stimuli that would induce expression of the gene of interest; or (e) the subject plant or subject plant cell itself, under conditions in which the gene of interest is not expressed.
The term “isolated” refers to material, such as a nucleic acid or a protein, which is: (1) substantially or essentially free from components which normally accompany or interact with it as found in its natural environment. The isolated material optionally comprises material not found with the material in its natural environment; or (2) if the material is in its natural environment, the material has been synthetically altered or synthetically produced by deliberate human intervention and/or placed at a different location within the cell. The synthetic alteration or creation of the material can be performed on the material within or apart from its natural state. For example, a naturally-occurring nucleic acid becomes an isolated nucleic acid if it is altered or produced by non-natural, synthetic methods, or if it is transcribed from DNA which has been altered or produced by non-natural, synthetic methods. The isolated nucleic acid may also be produced by the synthetic re-arrangement (“shuffling”) of a part or parts of one or more allelic forms of the gene of interest. Likewise, a naturally-occurring nucleic acid (e.g., a promoter) becomes isolated if it is introduced to a different locus of the genome.
A polynucleotide may be single- or double-stranded, depending on the context, and one of skill in the art would recognize which construction of the term is appropriate.
The Zea mays sequences of the invention can be used to isolate corresponding sequences from other organisms, particularly from other plants, more particularly from other monocotyledonous plants. Methods such as PCR, hybridization, and the like can be used to identify such sequences based on their similarity to a sequence set forth herein. In hybridization techniques, all or part of a known nucleotide sequence is used as a probe that selectively hybridizes to other corresponding nucleotide sequences present in a population of cloned genomic DNA fragments or cDNA fragments (i.e., genomic or cDNA libraries) from a chosen organism. The hybridization probes may be genomic DNA fragments, cDNA fragments, RNA fragments, or other oligonucleotides, and may be labeled with a detectable group such as 32P, or any other detectable marker. Thus, for example, probes for hybridization can be made by labeling synthetic oligonucleotides based on the sequences of the invention. For example, an entire sequence disclosed herein, or one or more portions thereof, may be used as a probe capable of specifically hybridizing to corresponding sequences. To achieve specific hybridization under a variety of conditions, such probes include sequences that are distinctive and are at least about 10 nucleotides in length. The well-known process of polymerase chain reaction (PCR) may be used to isolate or amplify additional sequences from a chosen organism or as a diagnostic assay to determine the presence of corresponding sequences in an organism. Hybridization techniques include hybridization screening of plated DNA libraries (either plaques or colonies; see, for example, Sambrook et al., supra; see also Innis et al., eds., (1990) PCR Protocols, A Guide to Methods and Applications, Academic Press). Methods for preparation of probes for hybridization and for construction of cDNA and genomic libraries are generally known in the art and are disclosed in Sambrook et al. (1989) Molecular Cloning: A Laboratory Manual (2d ed., Cold Spring Harbor Laboratory Press, Plainview, N.Y.) and Ausubel et al., eds. (1995) Current Protocols in Molecular Biology, Chapter 2 (Greene Publishing and Wiley-Interscience, New York).
Hybridization of such sequences may be carried out under stringent conditions. By “stringent conditions” or “stringent hybridization conditions” is intended conditions under which a probe will hybridize to its target sequence to a detectably greater degree than to other sequences (e.g., at least 2-fold over background). Stringent conditions are target-sequence-dependent and will differ depending on the structure of the polynucleotide. By controlling the stringency of the hybridization and/or washing conditions, target sequences that are 100% complementary to the probe can be identified (homologous probing). Alternatively, stringency conditions can be adjusted to allow some mismatching in sequences so that lower degrees of similarity are detected (heterologous probing).
Typically, stringent conditions will be those in which the salt concentration is less than about 1.5 M Na ion, typically about 0.01 to 1.0 M Na ion concentration (or other salts) at pH 7.0 to 8.3 and the temperature is at least about 30° C. for short probes (e.g., 10 to 50 nucleotides) and at least about 60° C. for long probes (e.g., greater than 50 nucleotides). Stringency may also be adjusted with the addition of destabilizing agents such as formamide. Exemplary low stringency conditions include hybridization with a buffer solution of 30 to 35% formamide, 1 M NaCl, 1% SDS (sodium dodecyl sulphate) at 37° C., and a wash in 1× to 2×SSC (20×SSC=3.0 M NaCl/0.3 M trisodium citrate) at 50 to 55° C. Exemplary moderate stringency conditions include hybridization in 40 to 45% formamide, 1 M NaCl, 1% SDS at 37° C., and a wash in 0.5× to 1×SSC at 55 to 60° C. Exemplary high stringency conditions include hybridization in 50% formamide, 1 M NaCl, 1% SDS at 37° C., and a wash in 0.1×SSC at 60 to 65° C. The duration of hybridization is generally less than about 24 hours, usually about 4 to about 12 hours.
Specificity is typically the function of post-hybridization washes, the critical factors being the ionic strength and temperature of the final wash solution. For DNA-DNA hybrids, the Tm can be approximated from the equation of Meinkoth and Wahl (1984) Anal. Biochem. 138:267-284: Tm=81.5° C.+16.6 (log M)+0.41 (% GC)−0.61 (% form)−500/L; where M is the molarity of monovalent cations, % GC is the percentage of guanine and cytosine nucleotides in the DNA, % form is the percentage of formamide in the hybridization solution, and L is the length of the hybrid in base pairs. The Tm is the temperature (under defined ionic strength and pH) at which 50% of a complementary target sequence hybridizes to a perfectly matched probe. Tm is reduced by about 1° C. for each 1% of mismatching; thus, Tm, hybridization, and/or wash conditions can be adjusted to hybridize to sequences of the desired identity. For example, if sequences with ≧90% identity are sought, the Tm can be decreased 10° C. Generally, stringent conditions are selected to be about 5° C. lower than the thermal melting point (Tm) for the specific sequence and its complement at a defined ionic strength and pH. However, severely stringent conditions can utilize a hybridization and/or wash at 1, 2, 3, or 4° C. lower than the thermal melting point (Tm); moderately stringent conditions can utilize a hybridization and/or wash at 6, 7, 8, 9, or 10° C. lower than the thermal melting point (Tm); low stringency conditions can utilize a hybridization and/or wash at 11, 12, 13, 14, 15, or 20° C. lower than the thermal melting point (Tm). Using the equation, hybridization and wash compositions, and desired Tm, those of ordinary skill will understand that variations in the stringency of hybridization and/or wash solutions are inherently described. If the desired degree of mismatching results in a Tm of less than 45° C. (aqueous solution) or 32° C. (formamide solution), it is preferred to increase the SSC concentration so that a higher temperature can be used. An extensive guide to the hybridization of nucleic acids is found in Tijssen (1993) Laboratory Techniques in Biochemistry and Molecular Biology—Hybridization with Nucleic Acid Probes, Part I, Chapter 2 (Elsevier, N.Y.); and Ausubel et al., eds. (1995) Current Protocols in Molecular Biology, Chapter 2 (Greene Publishing and Wiley-Interscience, New York). See also Sambrook et al. (1989) Molecular Cloning: A Laboratory Manual (2d ed., Cold Spring Harbor Laboratory Press, Plainview, N.Y.). Thus, isolated sequences that retain the function of the invention and hybridize under stringent conditions to the sequences disclosed herein, or to their complements, or to fragments of either, are encompassed by the present invention. Such a sequence will usually be at least about 85% identical to a disclosed sequence. That is, the identity of sequences may range, sharing at least about 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity.
Methods of alignment of sequences for comparison are well-known in the art. 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. Sci. 85: 2444 (1988); by computerized implementations of these algorithms, including, but not limited to: CLUSTAL in the PC/Gene program by Intelligenetics, Mountain View, Calif.; GAP, BESTFIT, BLAST, FASTA, and TFASTA in the GCG® Wisconsin PackageTM from Accelrys, Inc., San Diego, Calif.
The CLUSTAL program is well described by Higgins and Sharp, Gene 73: 237-244 (1988); Higgins and Sharp, CABIOS 5: 151-153 (1989); Corpet, et al., Nucleic Acids Research 16: 10881-90 (1988); Huang, et al., Computer Applications in the Biosciences 8: 155-65 (1992), and Pearson, et al., Methods in Molecular Biology 24: 307-331 (1994). A description of BLAST (Basic Local Alignment Search Tool) is provided by Altschul, S. F., et al., (1993) J. Mol. Biol. 215:403-410.
Identity to the sequence of the present invention would mean a polynucleotide sequence having at least 85% sequence identity, wherein the percent sequence identity is based on the entire length of SEQ ID NO: 1 or SEQ ID NO: 3.
Variants of the nucleotide sequences disclosed herein are also encompassed by the present invention. By “variants” is intended substantially similar sequences wherein one or more bases have been modified, removed or added. For nucleotide sequences, naturally-occurring variants can be identified with the use of well-known molecular biology techniques, as, for example, with polymerase chain reaction (PCR) and hybridization techniques as outlined above. Variant nucleotide sequences also include synthetically-derived nucleotide sequences, such as those generated, for example, by using site-directed mutagenesis. Generally, variants of a particular nucleotide sequence of the invention will have at least about 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to that particular nucleotide sequence as determined by sequence alignment programs described elsewhere herein, using default parameters.
The AP2 domain is highly conserved among CBF genes, and some species share an additional conserved region bracketing the AP2 domains. (Jaglo et al., Plant Phys. 127:910-917, 2001). Thus one of skill in the art would recognize that variants most likely to retain function are those in which the AP2 domain is undisturbed. Surprisingly, however, the current application provides a novel sequence in which the AP2 domain is altered. FIG. 1 provides a multiple sequence alignment of several CBF polypeptides.
The expression cassette may also include, at the 3′ terminus of the heterologous nucleotide sequence of interest, a transcriptional and translational termination region functional in plants. The termination region can be native with the promoter nucleotide sequence present in the expression cassette, can be native with the DNA sequence of interest, or can be derived from another source. Convenient termination regions are available from the Ti-plasmid of A. tumefaciens, such as the octopine synthase and nopaline synthase termination regions. See also, Guerineau et al. (1991) Mol. Gen. Genet. 262:141-144; Proudfoot (1991) Cell 64:671-674; Sanfacon et al. (1991) Genes Dev. 5:141-149; Mogen et al. (1990) Plant Cell 2:1261-1272; Munroe et al. (1990) Gene 91:151-158; Ballas et al. 1989) Nucleic Acids Res. 17:7891-7903; Joshi et al. (1987) Nucleic Acid Res. 15:9627-9639.
The expression cassettes can additionally contain 5′ leader sequences. Such leader sequences can act to enhance translation. Translation leaders are known in the art and include: picornavirus leaders, for example, EMCV leader (Encephalomyocarditis 5′ noncoding region), Elroy-Stein et al. (1989) Proc. Nat. Acad. Sci. USA 86:6126-6130; potyvirus leaders, for example, TEV leader (Tobacco Etch Virus), Allison et al. (1986); MDMV leader (Maize Dwarf Mosaic Virus), Virology 154:9-20; human immunoglobulin heavy-chain binding protein (BiP), Macejak et al. (1991) Nature 353:90-94; untranslated leader from the coat protein mRNA of alfalfa mosaic virus (AMV RNA 4), Jobling et al. (1987) Nature 325:622-625); tobacco mosaic virus leader (TMV), Gallie et al. (1989) Molecular Biology of RNA, pages 237-256; and maize chlorotic mottle virus leader (MCMV) Lommel et al. (1991) Virology 81:382-385. See also Della-Cioppa et al. (1987) Plant Physiology 84:965-968. The cassette can also contain sequences that enhance translation and/or mRNA stability such as introns.
In those instances where it is desirable to have the expressed product of the heterologous nucleotide sequence directed to a particular organelle, particularly the plastid, amyloplast, or to the endoplasmic reticulum, or secreted at the cell's surface or extracellularly, the expression cassette can further comprise a coding sequence for a transit peptide. Such transit peptides are well known in the art and include, but are not limited to, the transit peptide for the acyl carrier protein, the small subunit of RUBISCO, plant EPSP synthase, and the like.
In preparing the expression cassette, the various DNA fragments can be manipulated so as to provide for the DNA sequences in the proper orientation and, as appropriate, in the proper reading frame. Toward this end, adapters or linkers can be employed to join the DNA fragments, or other manipulations can be involved to provide for convenient restriction sites, removal of superfluous DNA, removal of restriction sites, or the like. For this purpose, in vitro mutagenesis, primer repair, restriction digests, annealing, and resubstitutions, such as transitions and transversions, can be involved.
As noted herein, the present invention provides vectors capable of expressing the claimed sequences under the control of an operably linked promoter. In general, the vectors should be functional in plant cells. At times, it may be preferable to have vectors that are functional in E. coli (e.g., production of protein for raising antibodies, DNA sequence analysis, construction of inserts, obtaining quantities of nucleic acids). Vectors and procedures for cloning and expression in E. coli are discussed in Sambrook et al. (supra).
The transformation vector, comprising a sequence of the present invention operably linked to a promoter in an expression cassette, can also contain at least one additional nucleotide sequence for a gene to be cotransformed into the organism. Alternatively, the additional sequence(s) can be provided on another transformation vector.
Vectors that are functional in plants can be binary plasmids derived from Agrobacterium. Such vectors are capable of transforming plant cells. These vectors contain left and right border sequences that are required for integration into the host (plant) chromosome. At a minimum, between these border sequences is the gene to be expressed under control of an operably-linked promoter. In preferred embodiments, a selectable marker and a reporter gene are also included. For ease of obtaining sufficient quantities of vector, a bacterial origin that allows replication in E. coli is preferred.
Reporter genes can be included in the transformation vectors. Examples of suitable reporter genes known in the art can be found in, for example, Jefferson et al. (1991) in Plant Molecular Biology Manual, ed. Gelvin et al. (Kluwer Academic Publishers), pp. 1-33; DeWet et al. (1987) Mol. Cell. Biol. 7:725-737; Goff et al. (1990) EMBO J. 9:2517-2522; Kain et al. (1995) BioTechniques 19:650-655; and Chiu et al. (1996) Current Biology 6:325-330.
Selectable marker genes for selection of transformed cells or tissues can be included in the transformation vectors. These can include genes that confer antibiotic resistance or resistance to herbicides. Examples of suitable selectable marker genes include, but are not limited to, genes encoding resistance to chloramphenicol, Herrera Estrella et al. (1983) EMBO J. 2:987-992; methotrexate, Herrera Estrella et al. (1983) Nature 303:209-213; Meijer et al. (1991) Plant Mol. Biol. 16:807-820; hygromycin, Waldron et al. (1985) Plant Mol. Biol. 5:103-108; Zhijian et al. (1995) Plant Science 108:219-227; streptomycin, Jones et al. (1987) Mol. Gen. Genet. 210:86-91; spectinomycin, Bretagne-Sagnard et al. (1996) Transgenic Res. 5:131-137; bleomycin, Hille et al. (1990) Plant Mol. Biol. 7:171-176; sulfonamide, Guerineau et al. (1990) Plant Mol. Biol. 15:127-136; bromoxynil, Stalker et al. (1988) Science 242:419423; glyphosate, Shaw et al. (1986) Science 233:478-481; phosphinothricin, DeBlock et al. (1987) EMBO J. 6:2513-2518.
Other genes that could serve utility in the recovery of transgenic events but might not be required in the final product would include, but are not limited to, examples such as GUS (β-glucuronidase), Jefferson (1987) Plant Mol. Biol. Rep. 5:387); GFP (green fluorescence protein), Chalfie et al. (1994) Science 263:802, and Gerdes (1996) FEBS Lett. 389:44-47; DSred (Dietrich et al. (2002) Biotechniques 2(2):286-293); luciferase, Teeri et al. (1989) EMBO J. 8:343; KN1 (Smith et al. (1995) Dev. Genetics 16(4):344-348); Sugary1, Rahman et al. (1998) Plant Physiol. 117:425-435; James et al. (1995) Plant Cell 7:417-429 and GenBank Accession U18908; and systems utilizing the maize genes encoding enzymes for anthocyanin production, including CRC, P (Bruce et al. (2000) Plant Cell 12(1):65-79, and R (Ludwig et al. (1990) Science 247:449).
The transformation vector comprising an isolated polynucleotide of the present invention, operably linked to a promoter sequence in an expression cassette, can be used to transform any plant. In this manner, genetically modified plants, plant cells, plant tissue, seed, and the like can be obtained. Transformation protocols can vary depending on the type of plant or plant cell targeted for transformation, e.g., monocot or dicot. Suitable methods of transforming plant cells include microinjection, Crossway et al. (1986) Biotechniques 4:320-334; electroporation, Riggs et al. (1986) Proc. Natl. Acad. Sci. USA 83:5602-5606; Agrobacterium-mediated transformation, see for example, Townsend et al. U.S. Pat. No. 5,563,055; direct gene transfer, Paszkowski et al. (1984) EMBO J. 3:2717-2722; and ballistic particle acceleration, see for example, Sanford et al. U.S. Pat. No. 4,945,050; Tomes et al. (1995) in Plant Cell, Tissue, and Organ Culture: Fundamental Methods, ed. Gamborg and Phillips (Springer-Verlag, Berlin); and McCabe et al. (1988) Biotechnology 6:923-926. Also see Weissinger et al. (1988) Annual Rev. Genet. 22:421-477; Sanford et al. (1987) Particulate Science and Technology 5:27-37 (onion); Christou et al. (1988) Plant Physiol. 87:671-674 (soybean); McCabe et al. (1988) Bio/Technology 6:923-926 (soybean); Datta et al. (1990) Biotechnology 8:736-740 (rice); Klein et al. (1988) Proc. Natl. Acad. Sci. USA 85:4305-4309 (maize); Klein et al. (1988) Biotechnology 6:559-563 (maize); Klein et al. (1988) Plant Physiol. 91:440-444 (maize); Fromm et al. (1990) Biotechnology 8:833-839; Hooydaas-Van Slogteren et al. (1984) Nature (London) 311:763-764; Bytebier et al. (1987) Proc. Natl. Acad. Sci. USA 84:5345-5349 (Liliaceae); De Wet et al. (1985) in The Experimental Manipulation of Ovule Tissues, ed. G. P. Chapman et al. (Longman, N.Y.), pp. 197-209 (pollen); Kaeppler et al. (1990) Plant Cell Reports 9:415-418; and Kaeppler et al. (1992) Theor. Appl. Genet. 84:560-566 (whisker-mediated transformation); D. Halluin et al. (1992) Plant Cell 4:1495-1505 (electroporation); Li et al. (1993) Plant Cell Reports 12:250-255 and Christou et al. (1995) Annals of Botany 75:407-413 (rice); Osjoda et al. (1996) Nature Biotechnology 14:745-750 (maize via Agrobacterium tumefaciens); all of which are herein incorporated by reference.
The cells that have been transformed can be grown into plants in accordance with conventional ways. See, for example, McCormick et al. (1986) Plant Cell Reports 5:81-84. These plants can then be pollinated with the same transformed strain or different strains. The resulting plants having expression of the desired characteristic can then be identified. Two or more generations can be grown to ensure that the desired phenotypic characteristic is stably maintained and inherited under conditions of interest.
In certain embodiments the nucleic acid sequences of the present invention can be used in combination (“stacked”) with other polynucleotide sequences of interest in order to create plants with a desired phenotype. The polynucleotides of the present invention may be stacked with any gene or combination of genes, and the combinations generated can include multiple copies of any one or more of the polynucleotides of interest. The desired combination may affect one or more traits; that is, certain combinations may be created for modulation of gene expression involved in plant response to stress. Other combinations may be designed to produce plants with a variety of desired traits, including but not limited to traits desirable for animal feed such as high oil genes (e.g., U.S. Pat. No. 6,232,529); balanced amino acids (e.g. hordothionins (U.S. Pat. Nos. 5,990,389; 5,885,801; 5,885,802; and 5,703,409); barley high lysine (Williamson et al. (1987) Eur. J. Biochem. 165:99-106; and WO 98/20122); and high methionine proteins (Pedersen et al. (1986) J. Biol. Chem. 261:6279; Kirihara et al. (1988) Gene 71:359; and Musumura et al. (1989) Plant Mol. Biol. 12: 123)); increased digestibility (e.g., modified storage proteins (U.S. application Ser. No. 10/053,410, filed Nov. 7, 2001); and thioredoxins (U.S. application Ser. No. 10/005,429, filed Dec. 3, 2001)), the disclosures of which are herein incorporated by reference. The polynucleotides of the present invention can also be stacked with traits desirable for insect, disease or herbicide resistance (e.g., Bacillus thuringiensis toxic proteins (U.S. Pat. Nos. 5,366,892; 5,747,450; 5,737,514; 5,723,756; 5,593,881; Geiser et al (1986) Gene 48:109); lectins (Van Damme et al. (1994) Plant Mol. Biol. 24:825); fumonisin detoxification genes (U.S. Pat. No. 5,792,931); avirulence and disease resistance genes (Jones et al. (1994) Science 266:789; Martin et al. (1993) Science 262:1432; Mindrinos et al. (1994) Cell 78:1089); acetolactate synthase (ALS) mutants that lead to herbicide resistance such as the S4 and/or Hra mutations; inhibitors of glutamine synthase such as phosphinothricin or basta (e.g., bar gene); and glyphosate resistance (EPSPS gene)); and traits desirable for processing or process products such as high oil (e.g., U.S. Pat. No. 6,232,529); modified oils (e.g., fatty acid desaturase genes (U.S. Pat. No. 5,952,544; WO 94/11516)); modified starches (e.g., ADPG pyrophosphorylases (AGPase), starch synthases (SS), starch branching enzymes (SBE) and starch debranching enzymes (SDBE)); and polymers or bioplastics (e.g., U.S. Pat. No. 5,602,321; beta-ketothiolase, polyhydroxybutyrate synthase, and acetoacetyl-CoA reductase (Schubert et al. (1988) J. Bacteriol. 170:5837-5847) facilitate expression of polyhydroxyalkanoates (PHAs)), the disclosures of which are herein incorporated by reference. One could also combine the polynucleotides of the present invention with polynucleotides affecting agronomic traits such as male sterility (e.g., see U.S. Pat. No. 5,583,210), stalk strength, flowering time, or transformation technology traits such as cell cycle regulation or gene targeting (e.g. WO 99/61619; WO 00/17364; WO 99/25821), the disclosures of which are herein incorporated by reference.
These stacked combinations can be created by any method, including but not limited to cross breeding plants by any conventional or TopCross methodology, or genetic transformation. If the traits are stacked by genetically transforming the plants, the polynucleotide sequences of interest can be combined at any time and in any order. For example, a transgenic plant comprising one or more desired traits can be used as the target to introduce further traits by subsequent transformation. The traits can be introduced simultaneously in a co-transformation protocol with the polynucleotides of interest provided by any combination of transformation cassettes. For example, if two sequences will be introduced, the two sequences can be contained in separate transformation cassettes (trans) or contained on the same transformation cassette (cis). Expression of the sequences of interest can be driven by the same promoter or by different promoters. In certain cases, it may be desirable to introduce a transformation cassette that will suppress the expression of a polynucleotide of interest. This may be accompanied by any combination of other suppression cassettes or over-expression cassettes to generate the desired combination of traits in the plant.
The transformed plants of the invention may be used in a plant breeding program. The goal of plant breeding is to combine, in a single variety or hybrid, various desirable traits. For field crops, these traits may include, for example, resistance to diseases and insects, tolerance to heat, cold, and/or drought, reduced time to crop maturity, greater yield, and better agronomic quality. With mechanical harvesting of many crops, uniformity of plant characteristics such as germination and stand establishment, growth rate, maturity, and plant and ear height, is desirable. Traditional plant breeding is an important tool in developing new and improved commercial crops. This invention encompasses methods for producing a maize plant by crossing a first parent maize plant with a second parent maize plant wherein one or both of the parent maize plants is a transformed plant, as described herein.
Plant breeding techniques known in the art and used in a maize plant breeding program include, but are not limited to, recurrent selection, bulk selection, mass selection, backcrossing, pedigree breeding, open pollination breeding, restriction fragment length polymorphism enhanced selection, genetic marker enhanced selection, doubled haploids, and transformation. Often combinations of these techniques are used.
The development of maize hybrids in a maize plant breeding program requires, in general, the development of homozygous inbred lines, the crossing of these lines, and the evaluation of the crosses. There are many analytical methods available to evaluate the result of a cross. The oldest and most traditional method of analysis is the observation of phenotypic traits. Alternatively, the genotype of a plant can be examined.
A genetic trait which has been engineered into a particular maize plant using transformation techniques, could be moved into another line using traditional breeding techniques that are well known in the plant breeding arts. For example, a backcrossing approach is commonly used to move a transgene from a transformed maize plant to an elite inbred line, and the resulting progeny would then comprise the transgene(s). Also, if an inbred line was used for the transformation then the transgenic plants could be crossed to a different inbred in order to produce a transgenic hybrid maize plant. As used herein, “crossing” can refer to a simple X by Y cross, or the process of backcrossing, depending on the context.
The development of a maize hybrid in a maize plant breeding program involves three steps: (1) the selection of plants from various germplasm pools for initial breeding crosses; (2) the selfing of the selected plants from the breeding crosses for several generations to produce a series of inbred lines, which, while different from each other, breed true and are highly uniform; and (3) crossing the selected inbred lines with different inbred lines to produce the hybrids. During the inbreeding process in maize, the vigor of the lines decreases. Vigor is restored when two different inbred lines are crossed to produce the hybrid. An important consequence of the homozygosity and homogeneity of the inbred lines is that the hybrid created by crossing a defined pair of inbreds will always be the same. Once the inbreds that give a superior hybrid have been identified, the hybrid seed can be reproduced indefinitely as long as the homogeneity of the inbred parents is maintained.
Transgenic plants of the present invention may be used to produce a single cross hybrid, a three-way hybrid or a double cross hybrid. A single cross hybrid is produced when two inbred lines are crossed to produce the F1 progeny. A double cross hybrid is produced from four inbred lines crossed in pairs (A×B and C×D) and then the two F1 hybrids are crossed again (A×B)×(C×D). A three-way cross hybrid is produced from three inbred lines where two of the inbred lines are crossed (A×B) and then the resulting F1 hybrid is crossed with the third inbred (A×B)×C. Much of the hybrid vigor and uniformity exhibited by F1 hybrids is lost in the next generation (F2). Consequently, seed produced by hybrids is consumed rather than planted.
The following examples are offered by way of illustration and not by way of limitation.
EXAMPLES
Example 1
Expression of Transgenes in Monocot Cells
A plasmid vector is constructed comprising the full-length polynucleotide of SEQ ID NO: 1 or SEQ ID NO: 3, operably linked to a heterologous promoter, such as a stress-responsive promoter, for example rab17, rd29A, rip2, or mlip15. This construct can then be introduced into maize cells by the following procedure.
Immature maize embryos are dissected from developing caryopses. The embryos are isolated 10 to 11 days after pollination when they are 1.0 to 1.5 mm long. The embryos are then placed with the axis-side facing down and in contact with agarose-solidified N6 medium (Chu et al. (1975) Sci. Sin. Peking 18:659-668). The embryos are kept in the dark at 27° C. Friable embryogenic callus, consisting of undifferentiated masses of cells with somatic proembryoids and embryoids borne on suspensor structures, proliferates from the scutellum of these immature embryos. The embryogenic callus isolated from the primary explant can be cultured on N6 medium and sub-cultured on this medium every 2 to 3 weeks.
The plasmid p35S/Ac (Hoechst Ag, Frankfurt, Germany) or equivalent may be used in transformation experiments in order to provide for a selectable marker. This plasmid contains the Pat gene (see European Patent Publication 0 242 236) which encodes phosphinothricin acetyl transferase (PAT). The enzyme PAT confers resistance to herbicidal glutamine synthetase inhibitors such as phosphinothricin. The pat gene in p35S/Ac is under the control of the 35S promoter from Cauliflower Mosaic Virus (Odell et al. (1985) Nature 313:810-812) and the 3′ region of the nopaline synthase gene from the T-DNA of the Ti plasmid of Agrobacterium tumefaciens. 
The particle bombardment method (Klein et al. (1987) Nature 327:70-73) may be used to transfer genes to the callus culture cells. According to this method, gold particles (1 μm in diameter) are coated with DNA using the following technique. Ten μg of plasmid DNA are added to 50 μL of a suspension of gold particles (60 mg per mL). Calcium chloride (50 μL of a 2.5 M solution) and spermidine free base (20 μL of a 1.0 M solution) are added to the particles. The suspension is vortexed during the addition of these solutions. After 10 minutes, the tubes are briefly centrifuged (5 sec at 15,000 rpm) and the supernatant removed. The particles are resuspended in 200 μL of absolute ethanol, centrifuged again and the supernatant removed. The ethanol rinse is performed again and the particles resuspended in a final volume of 30 μL of ethanol. An aliquot (5 μL) of the DNA-coated gold particles can be placed in the center of a Kapton flying disc (Bio-Rad Labs). The particles are then accelerated into the corn tissue with a Biolistic PDS-1000/He (Bio-Rad Instruments, Hercules Calif.), using a helium pressure of 1000 psi, a gap distance of 0.5 cm and a flying distance of 1.0 cm.
For bombardment, the embryogenic tissue is placed on filter paper over agarose-solidified N6 medium. The tissue is arranged as a thin lawn and covers a circular area of about 5 cm in diameter. The petri dish containing the tissue can be placed in the chamber of the PDS-1000/He approximately 8 cm from the stopping screen. The air in the chamber is then evacuated to a vacuum of 28 inches of Hg. The macrocarrier is accelerated with a helium shock wave using a rupture membrane that bursts when the He pressure in the shock tube reaches 1000 psi.
Seven days after bombardment the tissue can be transferred to N6 medium that contains gluphosinate (2 mg per liter) and lacks casein or proline. The tissue continues to grow slowly on this medium. After an additional 2 weeks the tissue can be transferred to fresh N6 medium containing gluphosinate. After 6 weeks, areas of actively growing callus about 1 cm in diameter can be identified on some of the plates containing the glufosinate-supplemented medium. These calli may continue to grow when sub-cultured on the selective medium.
Plants can be regenerated from the transgenic callus by first transferring clusters of tissue to N6 medium supplemented with 0.2 mg per liter of 2,4-D. After two weeks the tissue can be transferred to regeneration medium (Fromm et al. (1990) Bio/Technology 8:833-839).
Example 2
Expression of Transgenes in Dicot Cells
Soybean embryos are bombarded with a plasmid comprising a CBF polynucleotide operably linked to a promoter, as follows. To induce somatic embryos, cotyledons of 3-5 mm in length are dissected from surface-sterilized, immature seeds of the soybean cultivar A2872, then cultured in the light or dark at 26° C. on an appropriate agar medium for six to ten weeks. Somatic embryos producing secondary embryos are then excised and placed into a suitable liquid medium. After repeated selection for clusters of somatic embryos that multiplied as early, globular-staged embryos, the suspensions are maintained as described below.
Soybean embryogenic suspension cultures can be maintained in 35 ml liquid media on a rotary shaker, 150 rpm, at 26° C. with fluorescent lights on a 16:8 hour day/night schedule. Cultures are subcultured every two weeks by inoculating approximately 35 mg of tissue into 35 ml of liquid medium.
Soybean embryogenic suspension cultures may then be transformed by the method of particle gun bombardment (Klein et al. (1987) Nature (London) 327:70-73, U.S. Pat. No. 4,945,050). A DuPont Biolistic PDS1000/HE instrument (helium retrofit) can be used for these transformations.
A selectable marker gene that can be used to facilitate soybean transformation is a transgene composed of the 35S promoter from Cauliflower Mosaic Virus (Odell et al. (1985) Nature 313:810-812), the hygromycin phosphotransferase gene from plasmid pJR225 (from E. coli; Gritz et al. (1983) Gene 25:179-188), and the 3′ region of the nopaline synthase gene from the T-DNA of the Ti plasmid of Agrobacterium tumefaciens. The expression cassette comprising the sequence of interest operably linked to a promoter can be isolated as a restriction fragment. This fragment can then be inserted into a unique restriction site of the vector carrying the marker gene.
To 50 μl of a 60 mg/ml 1 μm gold particle suspension is added (in order): 5 μl DNA (1 μg/μl), 20 μl spermidine (0.1 M), and 50 μl CaCl2 (2.5 M). The particle preparation is then agitated for three minutes, spun in a microfuge for 10 seconds and the supernatant removed. The DNA-coated particles are then washed once in 400 μl 70% ethanol and resuspended in 40 μl of anhydrous ethanol. The DNA/particle suspension can be sonicated three times for one second each. Five microliters of the DNA-coated gold particles are then loaded on each macro carrier disk.
Approximately 300-400 mg of a two-week-old suspension culture is placed in an empty 60×15 mm petri dish and the residual liquid removed from the tissue with a pipette. For each transformation experiment, approximately 5-10 plates of tissue are normally bombarded. Membrane rupture pressure is set at 1100 psi, and the chamber is evacuated to a vacuum of 28 inches mercury. The tissue is placed approximately 3.5 inches away from the retaining screen and bombarded three times. Following bombardment, the tissue can be divided in half and placed back into liquid and cultured as described above.
Five to seven days post bombardment, the liquid media may be exchanged with fresh media, and eleven to twelve days post-bombardment with fresh media containing 50 mg/ml hygromycin. This selective media can be refreshed weekly. Seven to eight weeks post-bombardment, green, transformed tissue may be observed growing from untransformed, necrotic embryogenic clusters. Isolated green tissue is removed and inoculated into individual flasks to generate new, clonally propagated, transformed embryogenic suspension cultures. Each new line may be treated as an independent transformation event. These suspensions can then be subcultured and maintained as clusters of immature embryos or regenerated into whole plants by maturation and germination of individual somatic embryos.
Example 3
Identification of the Gene from a Computer Homology Search
Gene identities can be determined by conducting BLAST (Basic Local Alignment Search Tool; Altschul, S. F., et al., (1993) J. Mol. Biol. 215:403-410; see also information available from NCBI (National Center for Biotechnology Information, U.S. National Library of Medicine, 8600 Rockville Pike, Bethesda, Md. 20894)) searches under default parameters for similarity to sequences contained in the BLAST “nr” database (comprising all non-redundant GenBank CDS translations, sequences derived from the 3-dimensional structure Brookhaven Protein Data Bank, the last major release of the SWISS-PROT protein sequence database, EMBL, and DDBJ databases). The cDNA sequences are analyzed for similarity to all publicly available DNA sequences contained in the “nr” database using the BLASTN program. The DNA sequences are translated in all reading frames and compared for similarity to all publicly available protein sequences contained in the “nr” database using the BLASTX program (Gish, W. and States, D. J. Nature Genetics 3:266-272 (1993)) provided by the NCBI. In some cases, the sequencing data from two or more clones containing overlapping segments of DNA are used to construct contiguous DNA sequences.
Sequence alignments and percent identity calculations can be performed using software such as GAP, BestFit, PileUp or Pretty, available as part of the GCG® Wisconsin PackageTM from Accelrys, Inc., San Diego, Calif. Default parameters for pairwise alignments of polynucleotide sequences using GAP and BestFit are Gap Creation Penalty=50, Gap Extension Penalty=3; nwsgapdna.cmp is the scoring matrix. Default parameters for pairwise alignments for polypeptide sequences using GAP and BestFit are Gap Creation Penalty=8, Gap Extension Penalty=2; BLOSUM62 is the scoring matrix. There is no penalty for gaps at ends of polynucleotide or polypeptide alignments.
Default parameters for polynucleotide sequence comparison using PileUp and Pretty are: Gap Creation Penalty=5, Gap Extension Penalty=1. Default parameters for polypeptide sequence comparison using PileUp or Pretty are Gap Creation Penalty=8, Gap Extension Penalty=2; BLOSUM62 is the scoring matrix.
Sequence alignments can also be accomplished with the Megalign program of the LASERGENE bioinformatics computing suite (DNASTAR Inc., Madison, Wis.). Multiple alignment of the sequences can be performed using the Clustal method of alignment (Higgins and Sharp (1989) CABIOS. 5:151-153) with the default parameters (GAP PENALTY=10, GAP LENGTH PENALTY=10). Default parameters for pairwise alignments using the Clustal method are KTUPLE 1, GAP PENALTY=3, WINDOW=5 and DIAGONALS SAVED=5.
Other pairwise comparison tools are also available and known to those of skill in the art.
Example 4
Time Course of Cold-Induced Gene Expression in B73 Seedling Shoots
Seeds of maize inbred B73 were germinated and grown in the greenhouse under optimal temperatures of approximately 28° C. during the day and 25° C. during the night. After ten days of growth they were moved into growth chambers set at a constant chilling temperature of 10° C. under 16-hour (h) day lengths. Seedling shoots were harvested at various time points after exposure to the chilling temperature, starting at Oh (which served as control) and continuing at 0.5 h, 1 h, 4 h, 8 h and 24 h. At the end of the 24 h period of cold exposure, seedlings were allowed to recover at optimal temperatures of 25° C./22° C. (16 h day/8 h night) and were harvested after 48 hours to constitute the treatment ‘48 h Rec’ (48 hours of recovery).
At harvest, seedling tissues above the coleoptile, comprising leaves and stems, were ground in liquid nitrogen and stored at −80° C. before submission for RNA extraction and Agilent profiling using the Agilent Oligo Microarray Kit (Protocol Version 5.0, June 2003) and the Agilent Fluorescent Linear Amplification Kit (Protocol Version 3.0, June 2002) (both, Agilent Technologies, Palo Alto, Calif.). RNA expression levels at each time point were compared against the control (zero time-point). Thus, expression ratios were obtained for 0.5 h, 1 h, 4 h, 8 h and 24 h of cold treatment and 48 h of recovery. Results are shown in Table 1.
The time course of cold-induced gene expression provided an understanding of genes expressed early upon stress exposure, those that are expressed later during stress exposure, and those that express after the stress has been relieved and the plant has been allowed to recover. A CBF-like gene (SEQ ID NO: 1-2) showed its second highest expression level at the earliest measured time point (0.5 h relative to zero time), and its highest expression level at 1 hour from the start time. This shows a very early induction pattern of CBF upon exposure to stress. This is in accordance with published reports on the time-course of CBF induction. It is also consistent with the role of CBF acting as a transcription factor to induce the simultaneous expression of several stress-responsive genes.







TABLE 1







Expression of ZmCBF2 with exposure to chilling










Time
Relative to zero level at start












0.5
h
3.42


1
h
4.46


4
h
3.3


8
h
−1.49


24
h
−2.5


48
h Rec
−17.77









Example 5
Transient Activity Assay for CBF
A transient assay was designed to check for CBF activity using the expression of the maize CRC gene from the RAB17 promoter as a reporter for CBF activity. The objective was to identify a CBF protein with the best activity which would then be expressed in transgenic plants under the stringent control of a stress-inducible promoter. This would allow the high activity of CBF to be prevalent only during times of stress, thus preventing any pleiotropic effects from the highly active transcription factor.
The RAB17 promoter comprises CRT/DRE elements to which CBF binds (see SEQ ID NO: 5). Consequently, re-transformation of transgenic embryos already comprising the RAB17::CRC construct, with a CBF construct, should allow activation of RAB17 in the first construct. This activation will produce reporter gene (CRC) expression, which can be visualized through red/purple anthocyanin pigmentation in cultured embryos or callus tissue.
Maize plants, hemizygous for a single-copy Rab17::CRC event previously confirmed as showing CRC expression upon induction by drought or ABA treatment, were pollinated with inbred GS3, which enhances transformation with Agrobacterium. The resulting embryos segregated in a 1:1 ratio of transgenic:non-transgenic embryos. Immature embryos were isolated 9 to 10 days after pollination (DAP) and cultured with Agrobacterium suspension comprising a CBF construct of interest, following the standard Agrobacterium transformation protocol as provided in Example 8.
After infection with Agrobacterium suspension, the embryos were incubated at 20° C. for 3 days and then moved to a 26° C. culture room for continuous culture. CRC expression was monitored at 7-8 days after infection, and the final score (counts of CRC-expressing embryos) for CRC expression was determined 10-11 days after infection.
ZmCBF1 (SEQ ID NO: 3-4), ZmCBF2 (SEQ ID NO: 1-2) and RyeCBF31 (SEQ ID NO: 10) were tested in this transient expression system. Each CBF gene was operably linked to the ubiquitin promoter. Certain constructs further comprised the C1 activation domain (C1 AD) from maize (Goff et al. (1991) Genes Dev. 5:298-309; Singer et al. (1998) Genet. Res. 71(2):127-132), fused to either the N or C terminus of each CBF gene.
Results are shown in Table 2 for the ZmCBF2 and RyeCBF31 proteins in combination with the C1 activation domain. The activation percent represents the % of embryos displaying CRC, with 50% being the expected maximum in the segregating population.









TABLE 2





Promoter
Coding Region
Activation (%)

















Ubiquitin
C1 AD + Rye-CBF31
9.1


Ubiquitin
C1 AD + Zm-CBF2
26.4


Ubiquitin
Rye-CBF31 + Cq AD
19.5


Ubiquitin
Zm-CBF2 + C1 AD
35.2









In this assay system, ZmCBF2 produces a higher activation % than Rye CBF31. Further, the fusion of the C1 activation domain at the C-terminus of CBF produces higher activity levels than does fusion of the C1 activation domain at the N-terminus.

Example 6
Transient Activity Assay for CBF Under Cold Stress
The transient assay system of Example 5 was also used to test ZmCBF1 (SEQ ID NO: 3) and RyeCBF31 (SEQ ID NO: 10) in constructs comprising the mlip15 promoter (SEQ ID NO: 9).
After infection with Agrobacterium suspension, the embryos were incubated at 20° C. For cold treatment to induce ZmLIP15, 25 to 30 infected embryos were transferred to a fresh plate at 4 to 5 days after infection. These were kept at 4° C. for 4 hours, and then returned to 26° C. for monitoring CRC activity as in Example 5.
Results are shown in Table 3.












TABLE 3







Promoter
Coding Region
Activation (%)
Treatment





















Ubiquitin
Zm-CBF1
51.8
None



Zm-Cyclo1
GUS
0.0
None



Zm-LIP15
Zm-CBF1
0.0
None



Zm-LIP15
Zm-CBF1
23.3
Cold



Zm-LIP15
Zm-CBF2
0.0
None



Zm-LIP15
Zm-CBF2
37.0
Cold










The construct containing ZmCBF1, without a C1 activation domain fusion, and expressed from the ubiquitin promoter, produced the fastest and strongest CRC expression in transient assays. This expression is relative to other constructs that either contained a C1 activation domain fusion or were linked to stress-inducible promoters. Constructs containing ZmCBF2 and Rye CBF31 expressed from the ubiquitin promoter are awaiting test in the transient assay system.

Example 7
Activity of CBF on Reporter Gene Expression in Stable Transformants
Transgenic plants containing ZmCBF1, ZmCBF2 or Rye CBF31, expressed from constitutive or stress-inducible promoters, are crossed with plants containing the reporter construct RAB17::CRC to create progeny that will show stable expression of the reporter gene resulting from the activity of CBF on the RAB17 promoter.
Transgenic maize plants comprising Rab17::CRC, a construct previously confirmed to show CRC expression upon induction by drought or ABA treatment, are crossed with transgenic plants comprising a Rab17::ZmCBF2 construct, such as proprietary Pioneer plasmid PHP22621, or other construct comprising a CBF coding sequence operably linked to a constitutive promoter, such as ubiquitin, or a stress-inducible promoter, such as rd29A, rip2 or mlip15. The construct further comprises LTP2::DS-RED2 (Kalla et al., Plant J (1994) 6:849-860; Dietrich et al. (2002) Biotechniques 2(2):286-293) which allows visualization of segregation among kernels on the ear. Red-fluorescing kernels are selected and planted. Progeny are evaluated for expression of the reporter gene as well as tolerance to cold or drought stress.
Example 8
Standard Agro Transformation Protocol as Referred to in Example 5
For Agrobacterium-mediated transformation of maize, the method of Zhao is employed (U.S. Pat. No. 5,981,840, and PCT patent publication WO98/32326, the contents of which are hereby incorporated by reference). Briefly, immature embryos are isolated from maize and the embryos immersed in an Agrobacterium suspension, where the bacteria are capable of transferring the gene of interest to at least one cell of at least one of the immature embryos (step 1: the infection step). The embryos are then co-cultured for a time with the Agrobacterium on solid medium (step 2: the co-cultivation step). During the co-cultivation step infected embryos are cultured at 20° C. for 3 days, and then at 26° C. for 4 days. Following this co-cultivation period an optional “resting” step is contemplated in which the embryos are incubated in the presence of at least one antibiotic known to inhibit the growth of Agrobacterium, without the addition of a selective agent for plant transformants (step 3: resting step). Transient expression based on a color marker can be monitored during the co-cultivation and the resting steps. Next, inoculated embryos are cultured on medium containing a selective agent and growing transformed callus is recovered (step 4: the selection step). Finally, calli grown on selective medium are cultured on solid medium to regenerate transformed plants (step 5: the regeneration step).
Example 9
Native Expression of ZmCBF1 in B73 Seedlings
Seeds of maize inbred B73 were germinated and grown in the greenhouse under optimal temperatures of approximately 28° C. during the day and 25° C. during the night. After ten days of growth they were moved into growth chambers set at a constant chilling temperature of 10° C. under 16-hour (h) day lengths. Seedling shoots from the V1 stage were harvested at various time points after exposure to the chilling temperature, starting at 0 h (which served as control) and continuing at 0.25 h, 1 h, 4 h, 8 h and 24 h. At the end of the 24 h period of cold exposure, seedlings were allowed to recover at optimal temperatures of 23° C. (16 h day/8 h night) and were harvested after 48 hours under the optimal temperature to constitute the treatment ‘48 h Rec’ (48 hours of recovery).
At harvest, seedling tissues above the coleoptile, comprising leaves and stems, were ground in liquid nitrogen and stored at −80° C. before RNA extraction and RT-PCR using gene-specific primers for ZmCBF1 (SEQ ID NO: 3) over a 35-cycle period. Expression of ZmCBF was not detectable at the zero point or at 0.25 hours. Expression was detected after 1 hour of cold treatment, peaking at 4 hours of cold treatment. Expression at the 8 h and 24 h points was less than the level indicated at 1 h. Expression was not detected at the 48-hour recovery point. This is in accordance with published reports on the time-course of CBF induction. It is also consistent with the role of CBF acting as a transcription factor to induce the simultaneous expression of several stress-responsive genes.
Example 10
Chilling Tolerance Exhibited by ZmCBF1 in Maize Seedlings
As described in Example 1, maize was transformed with a construct comprising a maize, Arabidopsis, or rye CBF coding region, driven by a constitutive (ubiquitin) or stress-inducible promoter, such as rab17, rd29a, mlip15, or the native promoter of a CBF gene. Certain constructs further comprised the C1 activation domain (Goff et al. (1991) Genes Dev. 5:298-309; Singer et al. (1998) Genet. Res. 71(2):127-132) or the OP2 activation domain (Schmitz et al. (1997) Nucleic Acids Res. 25(4):756-763), fused to either the N or C terminus of the CBF coding region.
Regenerated plants were backcrossed with pollen from recurrent parent, and the resulting T2 seeds were planted and grown to V1-V2 (10 days from sowing) at optimal growth conditions of 28° C.-26° C. day-night temperatures in a 16 h-8 h day-night regime. A total of 18 seeds were planted with an expectation of observing 9 transgene-positive plants and 9 transgene-negative plants.
Ten-day-old seedlings were leaf-painted with 1% Liberty® (a.i., glufosinate) to determine transgenic positive and negative plants, and were marked as such based on the leaf-painting results. At 14 days after sowing, they were subjected to a low-temperature stress of 13° C.-7° C. day-night temperatures for the next 14 days. At the end of the 14-day stress period, plants were cut at the base and whole-shoot fresh weight was determined.
For five constructs shown in Table 4, PHP17421, PHP18990, PHP18991, PHP18992 and PHP19021, transgenic plants within several events showed increased growth under low-temperature stress, relative to their non-transgenic sib plants. As an example, fresh weight data for PHP18990 seedlings are provided in FIG. 3.









TABLE 4





PHP #
Promoter
Coding Region







17421
Ubiquitin
Zm-CBF1


18990
Rd29a
ZmCBF1


18991
Rd29a
ScCBF31


18992
Mlip15
ZmCBF1


19021
Mlip15
ScCBF31









Example 11
Freezing Tolerance in Maize Seedlings Overexpressing ZmCBF1
As described in Example 1, maize was transformed with a construct comprising the ubiquitin promoter driving ZmCBF1. Regenerated plants were backcrossed with pollen from recurrent parent, and the resulting T2 seeds were planted in 4′′ pots, using 3 seeds/pot (18 seeds/event) and allowed to grow at optimal growth conditions in the greenhouse. After plants had reached the V1-V2 stage (10 days after planting), leaves were painted with 1.0% Liberty® (a.i., glufosinate), to screen for the presence or absence of the transgene. After 4 days, when visible leaf scorching could be observed as a result of the herbicide application, individual plants were scored as either transgene positive or negative.
The flats containing the pots with the 14-day-old seedlings were moved to growth chambers where the temperature was lowered to 4° C. overnight to allow complete chilling of the potting medium. Thereafter, the temperature was lowered to −4° C. and retained for 3 hours. At the end of the freezing period, plants were allowed to recover at 4° C. overnight, and then moved to the optimal temperature conditions of the greenhouse. The plants were scored for survival 3-4 days after the freezing treatment.
Screening of 19 events of the UBI::ZmCBF1 construct revealed three events that were freezing tolerant. Results are shown in Table 5.











TABLE 5






No. of survivors/

No. of survivors/



Event
total no. of trans-
%
total no. of trans-
%


number
genic positives
survival
genic negatives
survival



















8
8/11
73
0/7
0


16
5/8 
63
 0/10
0


23
6/11
55
0/7
0









Example 12
Evaluation of Rd29a::RyeCBF31 Transgenic Plants for Photosynthetic Performance
Maize plants transgenic (hybrid material which contained three doses of the recurrent parent that was crossed to elite inbred, and then selfed) for a construct comprising rd29a::ryeCBF31 were subjected to drought stress at different points during the growth period, i.e. at 2, 5, 8, 10 and 12 weeks after sowing. Water was withheld at these specified time points and plants were monitored for photosynthesis from the time of withholding water until photosynthesis was reduced to zero. Photosynthesis measurements were recorded using a LI-COR 6400 (LI-COR, Lincoln, Nebr.) portable photosynthesis machine connected to a fluorescence chamber. Standard protocols specified by the manufacturer were used to record photosynthesis and chlorophyll fluorescence simultaneously. At the point when photosynthesis was lowered to zero, re-watering was initiated.
Results are shown in FIG. 2. The photosynthesis readings are the average of three transgenic plants and three non-transgenic plants, where the stress was imposed at 8 weeks after sowing and 10 weeks after sowing. Transgenic plants are indicated by ‘tng’ and non-transgenic plants or wild-type are indicated by ‘wt’.
Example 13
Overexpression of ZmCBF1 in Arabidopsis (Construct PHP  17421-UBI::ZmCBF1 or PHP18990-RD29A::ZmCBF1)
Transgenic Arabidopsis thaliana (Ecotype Columbia) seeds heterozygous for the CBF construct PHP17421 (UBI::ZmCBF1) or PHP18990 (RD29A::ZmCBF1) were germinated on medium comprising 0.5×MS salts (Murashige and Skoog (1962) Physiol. Plant. 15:473) with no sucrose, and seedlings were grown for 20 days. The plates were then subjected to a freezing temperature of −7° C. for 4 hours and thereafter allowed to recover at room temperature (22° C.) for 3 to 4 days. The number of surviving plants was counted after the recovery period.
Eight of twelve events of the construct expressing ZmCBF1 from the stress-inducible Arabidopsis promoter, RD29A, showed improved freezing tolerance over the wild-type. Results are shown in Table 6.











TABLE 6







Total no. of
No. of
%


Construct
Event
plants
Survivors
Survival



















18990
1
10
8
80%


18990
2
9
4
44%


18990
3
12
9
75%


18990
4
10
5
50%


18990
5
9
4
44%


18990
6
11
2
18%


18990
7
11
1
 9%


18990
8
18
1
 6%


18990
9
19
2
11%


18990
10
14
11
79%


18990
11
13
12
92%


18990
12
20
16
80%


18990
WT1
13
2
15%


18990
WT2
17
6
35%


18990
WT3
10
1
10%


18990
WT4
19
3
16%









The construct expressing ZmCBF1 from the UBI promoter showed 5 events out of a total of 12 that had improved freezing tolerance relative to the wildtype.
Transgenic Arabidopsis thaliana (Ecotype Columbia) heterozygous for the CBF construct PHP17421 (UBI::ZmCBF1) was also evaluated for tolerance to drought stress. Pots were filled with Metro-Mix 200 with 90 g of Osmocote® controlled release fertilizer (18-6-12) per 2.8 cu ft bag of soil. At planting the potting medium was watered to saturation with water plus 20-10-20 fertilizer and allowed to drain. Arabidopsis seeds were sown at nine seeds per pot, and allowed to germinate and grow under 17-hour day length at a temperature of 22° C. during the day, 20° C. during the night, under light intensities of  ̃200 uE. During the first two weeks, watering was done without added fertilizers. At two weeks water was withheld. Effect of the stress was evident as the plants entered the reproductive phase. The trend towards complete wilting was monitored by digital imaging. In one of twelve events tested, wildtype plants were completely wilted at 36 days after stress, while transgenic plants survived. This event was then subjected to a second test and the drought tolerance effect was confirmed.
Example 14
Constitutive Expression of ZmCBF1 in Maize
Constitutive expression of ZmCBF1 from the UBI promoter in construct PHP17421 caused severe reduction in plant height of transgenic events, as observed by visual observation throughout the entire growth cycle. We overcame this pleiotropic effect by using stress-inducible promoters including RD29A and RAB17.
Example 15
Expression Analysis of Transgenic Maize Expressing UBI::ZmCBF1
Seedling shoots of transgenic maize plants (V4) expressing UBI::ZmCBF1, event number 7, were harvested, ground in liquid nitrogen and extracted for mRNA. Using the Agilent chip system (see Example 4), the RNA expression in the transgenic plants was compared with that of non-transgenic plants. Several genes were found to be upregulated in the transgenic plants relative to the control, with a major proportion of these being unknown genes. From amongst the upregulated genes, a list of all public maize genes with known or putative function that showed upregulation above the two-fold limit was compiled. To this list was added a small selection of maize homologs of known genes from other species that were known to be stress-regulated. Thus a total list of 66 is presented in the table (FIG. 4) as those that were upregulated above the two-fold level. Of these, 57 are known maize genes, while 9 are maize homologs of known stress-responsive genes in other species, primarily, rice. In Arabidopsis, homologs of several of these 66 are known to be either stress-responsive or part of the CBF regulon (Seki et al. 2001. Plant Cell 13: 61-72; Zhang et al., 2004. Plant J 39: 905-919).
The above examples are provided to illustrate the invention but not to limit its scope. Other variants of the invention will be readily apparent to one of ordinary skill in the art and are encompassed by the appended claims.
All publications and patent applications cited in the specification are indicative of the level of skill of those in the art to which this invention pertains. All publications, patents, patent applications, and computer programs cited herein are incorporated by reference to the same extent as if specifically and individually indicated to be incorporated by reference.

## Claims

1. A method for increasing plant tolerance to abiotic stress due to low temperature or dehydration, comprising the steps of:
(a) transforming a plant with a transformation vector comprising an isolated polynucleotide encoding a transcription factor of SEQ ID NO: 4; and
(b) expressing said polynucleotide encoding said transcription factor, wherein said expression results in increased tolerance to low temperature or dehydration stress relative to that of said plant prior to transformation.

2. The method of claim 1, wherein said polynucleotide is operably linked to a promoter which drives expression in a stress-responsive or tissue-preferred manner.